Serum gastrin in Zollinger-Ellison syndrome: II. Prospective study of gastrin provocative testing in 293 NIH patients and comparison with 537 cases from the literature. Evaluation of diagnostic criteria, proposal of new criteria and correlations with clinical and tumoral features

Introduction

In the accompanying paper ³⁰, we analyzed the role of fasting serum gastrin (FSG) and confirmed its importance in providing relevant information for diagnosis and management of ZES. However, like others ^{39,80,136,140,168,184,196,199,201,205,247,266,306,341,343,380}, we found in the majority of patients, the determination of FSG, alone or in combination with acid secretion studies, cannot establish the diagnosis of ZES ³⁰. In these patients, gastrin provocative tests are needed. Of the several tests developed, only secretin, calcium and meal stimulation are widely used in the today ^{78,110,111,165,165,167,196,219,238,250,397}. However, the exact role of these provocative tests, the optimal testing procedures and criteria for positive responses remain controversial, so that some studies even question their overall usefulness ^304,343,344. This has occurred because most existing studies have one or multiple limitations including small patient numbers, retrospective nature, non-standardized radioimmunoassay (RIA), different criteria for positivity, different testing procedures and lack of stratification for FSG < or > 10x normal^{110,120,152,165,167,196,248,249,295,344,360,381,397}.

To address these issues, we analyzed the results of secretin, calcium and meal provocative tests in 293 ZES patients from a 31-year prospective NIH study. FSG was determined using well-established gastrin RIAs ³⁰. Provocative tests were performed using well-established time points and standardized stimulant conditions ^110,111. Because of the prospective nature of our study with regular follow-ups, including detailed imaging studies and surgical exploration ^{121,279,283,319,320,349}, correlations of test results with clinical, laboratory and tumoral parameters were possible. We compared our data to results from 537 ZES patients from the literature. Furthermore, we determined the specificity of provocative tests by analyzing data from published non-ZES patients. This approach allowed us to determine the optimal criteria for positive secretin and calcium tests and to propose a new criterion for the secretin test with high sensitivity/specificity. In conjunction with the accompanying report ³⁰, this extensive analysis allowed us to identify important guidelines for gastrin provocative tests in the clinical management of ZES patients.

Case History

The detailed history and clinical findings of this 38-year-old man with a 12-year history of recurrent upper gastrointestinal symptoms resistant to standard medical treatment are reported in the accompanying paper ³⁰. The patient was hospitalized for an acute abdomen requiring surgical exploration for a perforated duodenal ulcer with an oversew as well as a vagotomy/pyloroplasty. His FSG was 221 pg/ml (normal <100) while taking omeprazole and he had an elevated serum calcium (2.41 mM, normal 2.05–2.50 mM). The possibility of ZES was raised and because no secretin was available, a calcium infusion study was performed. The calcium test results supported the diagnosis with his FSG increasing from 215 pg/ml to 1010 pg/ml after 180 minutes (Figure 1, middle panel). Subsequently, the patient was referred to the NIH where FSG levels were 200–250 pg/ml (normal <100) and basal acid output (BAO) was 9 mEq/hr 2 weeks after stopping omeprazole. A secretin test with 2-unit/kg bolus of GIH secretin showed an increase from 217 to 1150 pg/ml (Figure 1, left panel) and a meal test showed an increase of only 30% over basal (Figure 1, right panel), supporting the diagnosis of ZES. Imaging revealed three pancreatic lesions while further biochemical studies showed hyperparathyroidism and MEN1 (see accompanying paper, Figure 1³⁰). A parathyroidectomy was performed, followed by an abdominal exploration with resection of pancreatic/duodenal tumors and metastatic tumor in lymph nodes. Postoperatively, the secretin test was negative (i.e. <200 pg/ml increase) ^110,238,397, but after three years became positive. The patient has remained asymptomatic with no evidence of recurrent tumor on imaging studies, however, the secretin test has remained positive.

Comment:

This case illustrates the difficulty in making the diagnosis of ZES with the increasing use of potent acid suppressants such as proton pump inhibitors (PPI’s). Therefore, the correct assessment of FSG and gastrin provocative tests have become essential in the diagnosis of ZES ^{67,144,160,231,244,257}. In this patient, the diagnosis was complicated because of an initial FSG elevation of only 2-fold (characteristically increased >10-fold in 40–60% of ZES patients) ^{110,111,162,165,250}. Furthermore, this moderate hypergastrinemia was detected while the patient was taking omeprazole, which can cause FSG elevations in patients without ZES ^{67,80,156,187} and the BAO was only moderately increased to 9 mEq/hr (mean 16–26 mEq/hr post gastric surgery in ZES patients)^{160,165,250,315,319}. This case illustrates the importance of the secretin test in the diagnosis of ZES and shows that PPI’s should be discontinued prior to FSG determination. As false positive secretin tests have been reported in achlorhydric patients ^98,141,219, gastrin provocative testing may not be reliable in patients treated with PPI’s, providing another reason PPI’s should be carefully stopped to determine the diagnosis of ZES. Lastly, this case illustrates the importance of assessing both the secretin test and the FSG level to determine cure ^102,283,284. This patient underwent a gastrinoma resection and a parathyroidectomy, both of which can affect the FSG ^{29,72,123,143,157,159,197,234,263,276,281}, and subsequently his FSG levels became normal. However, with time, although the FSG level remained normal, the secretin test became positive showing persistent ZES. This latter finding illustrates the particular importance of secretin testing in MEN1 patients post resection or post parathyroidectomy. Numerous patients with MEN1/ZES in the literature have been reported cured long-term. However, recent studies in MEN/1 ZES patients who underwent repetitive secretin testing show that the long-term cure rate is very low in this patient group (i.e. <1–2%) ^{210,274,283,286,333}.

MATERIALS AND METHODS

All patients admitted to the National Institutes of Health (NIH) with a diagnosis of ZES over a 31-year period (1974–2005) who agreed to participate in the initial and follow-up evaluations were eligible for this study. Details on study organization, diagnostic criteria for ZES or MEN1 and the FSG determination have been described in the accompanying study ³⁰ and the literature ^{15,16,117,123,220,224,283,320}.

FSG levels were determined as described previously ^{21,30,56,102,110,319}. All gastrin provocative tests were performed prior to any gastrinoma treatment in patients with diffuse metastases who subsequently underwent chemotherapy ³⁷⁹, treatment with α-interferon ²⁹⁸ or somatostatin analogues ³³⁴. FSG and provocative tests were performed prior to gastrinoma resection in all but 13 patients and were usually performed on the initial NIH admission. Furthermore, because correction of hypercalcemia by parathyroidectomy can affect both the FSG level and gastrin provocative test results ^{72,123,143,157,159,197,234,263,276}, all patients with ZES and MEN1 had provocative testing performed prior to any treatment for hyperparathyroidism.

FSG determinations after stimulation with secretin were carried out in all patients, whereas calcium and standard meal tests were carried out routinely until 1995. Results of serum gastrin responses after stimulation were expressed as the relative rise over the pre-test value ($\mathrm{\Delta }\%=\frac{\text{gastrin}\text{max}-\text{FSG}}{\text{FSD}}\times 100$), or the absolute increase expressed in pg/ml (Δ=gastrin max-FSG). For some criteria, the maximal FSG level after provocative stimulation was considered.

Secretin testing was performed using an intravenous bolus injection (2 U/kg body weight) of Secretin-Kabi (Ferring AB, Malmo, Sweden) ¹¹⁰. FSG concentrations were measured at −15, −5 and 0 minutes before as well as 2, 5, 10, 15, 20 and 30 minutes after secretin injection. Secretin test results were analyzed using the criterion of McGuigan and Wolfe ^110,238 (Δ≥200 pg/ml); the criterion of Deveney and colleagues ⁷⁸ (Δ≥110 pg/ml); another criterion of Deveney ⁷⁷ (Δ>100 pg/ml); the criterion of Lamers and van Tongeren ¹⁹⁶ (Δ>50%); the criterion of Modlin and colleagues ²⁶⁰ (Δ≥100%); the criterion of Malageleda ²¹⁹, (gastrin_max≥335 pg/ml); and one of the criteria proposed by Poynard and Bonfils ³⁰¹ (gastrin_max≥186 pg/ml).

The calcium infusion provocative test was performed as described previously ^78,110 with calcium gluconate (10%) (5 mg calcium/kg per hr)(54 mg/kg per hr of calcium gluconate) given by intravenous infusion for 3 hours. FSG and calcium levels were measured simultaneously at 30 or 15 min before, immediate prior to as well as at 30, 60, 90, 120, 150 and 180 minutes after the calcium infusion. A rise in serum calcium ≥1.5 mEq/l was required for the test to be considered valid. Patients with hypercalcemia, renal disease, cardiac disease or unstable clinical condition did not undergo calcium testing. Calcium test results were analyzed using the criterion of Deveney ⁷⁸, (Δ≥395 pg/ml); a second criterion of Deveney ⁷⁷ (Δ>450 pg/ml); the criterion of Lamers and van Tongeren ¹⁹⁶ (Δ>50%); the criterion proposed by Modlin and colleagues ²⁶⁰ (Δ≥100%); and the criterion of Malageleda ²¹⁹, (gastrin_max≥326 pg/ml).

The standard meal test was performed as described previously ¹¹¹ and consisted of 30 g of protein, 20 g of fat and 25 g of carbohydrate ^111,196. FSG levels were measured at −15, 0, 30, 60, 90 and 120 min ¹¹¹. Test results were analyzed using the criterion of Lamers and van Tongeren ¹⁹⁶ (Δ>50%) and the criterion of Malageleda ²¹⁹ (gastrin_max≥500 pg/ml). Meal tests were not performed if the patient had a prior total gastrectomy or if they could not take oral feedings.

Basal acid output (BAO), maximal acid output (MAO) and assessment of acid secretory control by anti-secretory medication were measured ^{30,223,225,228,245,310,319,355}. Patient histories ^{30,123,320,409}, laboratory investigations ^{30,117,319,320}, endoscopy ^{30,36,122,320} and imaging studies using both conventional ^{109,206,227,292,297,332,353,354,382} and functional^{56,85,185,256,288,357} localization methods were performed. Based on the imaging studies and surgical exploration, patients were stratified into those with primary tumor only, with lymph node metastases and those with liver metastases. Lymph node and liver metastases were established by biopsy in all patients ^350,384,409.

Surgical exploration was performed in all patients with ZES without MEN1, without diffuse liver metastases or an illness limiting life-expectancy and in patients with MEN1 with imageable lesions >2.5 cm as described previously ^{3,30,103,104,112,273,275,277,279,280,282,286,287,349,387}. Patients with limited hepatic metastases considered resectable also underwent surgical exploration and resection ^{52,274,278,289,290,387}. For the purpose of analysis of the effect of primary tumor location, patients were divided into those who had only a duodenal primary; a pancreatic primary; a lymph node primary; a primary in another non-duodenal, non-pancreatic, non-lymph node location; or a primary tumor in an unknown location. A primary tumor of the lymph node was defined as occurring in a patient who was disease-free [normal fasting gastrin, negative provocative test and negative imaging ¹⁰²] post-resection of only a lymph node gastrinoma as described previously ^14,273,283. Other non-duodenal-pancreatic-lymph node primary locations were defined as occurring in patients who were disease-free post resection of a gastrinoma from these sites ^283,402,403.

Computed tomography or an MRI of the sella turcica and determinations of serum prolactin, calcium (total and ionized) and parathyroid hormone concentrations (using both an assay for the intact PTH molecule and with an antibody directed against the mid-portion of PTH) were also performed to determine if associated MEN1 was present as described previously ^{16,29,123,283}.

Results

Discussion

In this study, we prospectively analyzed the increase in serum gastrin after provocative tests in 293 ZES patients and compared our results to data from 537 patients from the literature. In the accompanying paper ³⁰, we found that the determination of FSG levels alone cannot establish the diagnosis in two-thirds of ZES patients, because FSG levels of ZES patients significantly overlap with those seen in idiopathic peptic disease or other non-ZES conditions ^{78,151,152,217}. Therefore, several gastrin provocative tests have been developed. These tests use different stimuli including calcium infusion or bolus injection ^{196,295,318,380}, infusion or bolus injection of secretin ^{31,153,192,196,247,252}, administration of a meal ^31,196,348 and injection of glucagon ^{26,69,153,183,183,265,343,360} or bombesin ^22–24,158. Only the secretin (bolus), calcium (infusion) and meal tests are generally used today ^{78,110,111,167,196,219,238,250,397}. Due to methodological inconsistencies in previous studies, the criteria for each of these tests remain an area of controversy and some studies even question their overall usefulness.

Since the initial description of the ability of secretin to stimulate an exaggerated serum gastrin increase in ZES patients ^137,153, the secretin test has been increasingly used in the diagnosis of ZES ^{110,238,394,397}. Although some studies have proposed that this increased response represents an exaggerated normal response ^40,44,45, there is strong evidence that secretin can stimulate gastrin release by direct interaction with gastrinoma cells. Secretin receptors occur on gastrinoma cells ⁸⁴, secretin stimulates gastrin release from isolated gastrinoma cells ^{58,93,129,129,147} and this effect can be inhibited by somatostatin ⁵⁸.

In the literature, some ZES patients have no increase in FSG after a bolus injection of secretin ^{73,94,219,229,230,233,247,318,341,343,344,351,360,405,406}. The percentage of these patients varies from 0% in most studies ^{78,150,157,189,196,240,255,315} to 7–75% ^{229,247,318,343,344,360}. In larger series however, most patients had a significant increase in FSG post secretin (median 262–1103 pg/ml in 10 large studies ^{66,78,100,150,196,218,232,315,341,412}). In our study, only 1.4% of literature or NIH ZES patients had no increase in serum gastrin after secretin, demonstrating that >98% respond to secretin. Our results demonstrate that in ZES patients, no response to secretin injection is very uncommon.

Numerous studies have proposed criteria for a positive secretin test ^{77,78,110,196,219,260,301}. However, these criteria vary widely, which is primarily due to multiple methodological differences. Some studies are limited by small patient numbers (<25 patients) and patients with FSG< or >10-fold increased are usually considered together, although provocative tests are of clinical relevance only in the former group ^{78,110,152,196,342,344,360}. Moreover, different protocols and different secretin preparations have been used (Boots, GIH, synthetic), which is relevant because false positive secretin tests have been reported with Boots secretin ^41,238,398. Different modes for secretin administration have been employed (infusion, bolus injection) ^{238,247,249,250,252,301} and different doses used. The secretin dose could be relevant because some studies ^{153,229,238,405}, but not others ^{153,229,329,405}, report that different doses may give different results. Our systematic analysis of secretin test results in patients receiving different doses of secretin (1 U/kg, 75 U total, 2 U/kg, ≥3 U/kg) showed no dose-response effect (Table 9). These results are similar to some studies ^189,219,380, but not others ^{153,229,329,405}, and led us to conclude that there is no need to increase the usual 2 units/kg dose or to repeat a secretin study with a higher dose if the initial study was negative, as suggested by others ^229,405. Furthermore, secretin test protocols frequently varied in the time points for collection of blood samples. This is important because some studies did not measure gastrin 2 minutes after secretin injection ^196,255. In one study ¹¹⁰ however, 6% of ZES patients had their only positive response 2 minutes post injection. Moreover, these studies were further limited by small patient numbers (<25 patients) and the fact that patients with FSG< or >10-fold increased were considered together, although gastrin provocative tests are of clinical relevance only in the former group ^{78,110,152,196,342,344,360}.

Three different types of criteria for a positive secretin test have been proposed: 3 criteria based on the absolute increase in FSG post secretin (>100, ≥110, ≥200 pg/ml) ^77,78,110, 2 criteria based on the relative increase (i.e. >50%, >100%) ^196,260 and 2 criteria based on the maximal FSG level post secretin (i.e. ≥186 or ≥335 pg/ml) ^219,301. Based on a review of 122 cases from the literature ²³⁸, the criterion of ≥200 pg/ml increase post secretin has been most widely used since 1980 in the United States ^{110,119,166,397}. Because we had a large number of patients, we could systematically assess the sensitivity of the various proposed criteria, both in all patients and the clinically important subset of ZES patients with FSG<10-fold increased ^{78,110,152,196,342,344,360}. In NIH and literature ZES patients, 3 criteria had sensitivities of 90–100% (Δ>100 pg/ml, Δ≥110pg/ml, gastrin max. ≥186 pg/ml) and 3 had sensitivities of 85–89% (Δ≥200 pg/ml, Δ>50%, gastrin max.≥335 pg/ml). One proposed criterion, Δ>100%, had a low sensitivity of only 60–62%, suggesting it is not generally useful. Because ZES is an uncommon condition (1–3 cases/year/million population) and hypergastrinemia due to H. pylori infection or other conditions is much more frequent ^{78,90,151,152,203,217,236,246,337}, it is very important to limit false positive responses, so that the specificity may be the most important factor in determining an optimal secretin test criterion. To assess the specificity of the six different secretin test criteria with high sensitivity (85–100%), we analyzed the results from 489 non-ZES patients’ responses from the literature (Table 10), including 462 without and 27 subjects with achlorhydria. Because there was an unacceptably high false positive rate varying from 7% to 100% in achlorhydric patients, our analysis confirms the proposal that secretin test results are not reliable in these subjects ^{42,98,141,192,219}. After exclusion of achlorhydric patients, the 3 absolute increase criteria (i.e.Δ >100, ≥110, ≥ 200 pg/ml) gave very low false positive rates (0–0.5%) resulting in a specificity of 99.8–100%, whereas the remaining highly sensitive criteria (Δ>50%, gastrin max. ≥186 pg/ml, gastrin max. ≥ 335 pg/ml) gave higher false positive rates of 4–13%. Our analysis demonstrated that the proposed criteria with the highest sensitivity and specificity are Δ≥100 pg/ml [95%, 99.8%], Δ≥110 pg/ml [94%, 100%] and Δ≥200 pg/ml [87%, 100%]. Because the sensitivity of the Δ>100 and Δ≥110 criteria was significantly higher than that of Δ≥200 pg/ml, we tried to identify a new criterion with maximal sensitivity and 100% specificity. This criterion is a Δ≥120 pg/ml, which has 94% sensitivity and a specificity of 100%. Both the Δ≥120 and Δ≥130 pg/ml criteria had a 100% specificity and each had a significantly higher sensitivity than the commonly used Δ≥200 pg/ml criterion (Table 11). The Δ≥120 pg/ml criterion had the same sensitivity as the previously proposed Δ≥110 criterion ⁷⁸ (i.e. 94%) and its sensitivity was not significantly different from that of the Δ>100 criterion ⁷⁷. However, in the non-ZES patients without achlorhydria, one patient had an increase in serum gastrin after secretin of 101 pg/ml and 4 patients had an increase of 100 pg/ml. Therefore, we conclude that Δ≥120 pg/ml was the criterion with maximal sensitivity and 100% specificity. Criteria of ≥110 and ≥120 pg/ml both had equal sensitivity and 100% specificity, but because of the importance of limiting a false positive result to the greatest extent, we recommend a criterion of Δ≥120 pg/ml rather than 110 pg/ml as recommended by others ⁷⁸.

Based on these results, we suggest that a secretin test should be performed with 2 units GIH or synthetic secretin per kg body weight, it should include an assessment of serum gastrin before secretin injection, and 2, 5, 10, 15 and 20 minutes ¹¹⁰ after secretin. An absolute increase in serum gastrin ≥ 120 pg/ml should be the criterion for positivity because it has the highest sensitivity with 100% specificity (Table 11). When this new criterion is applied to the 453 NIH and literature ZES patients in our study with FSG<10-fold increased, it allows the correct diagnosis of 37 ZES patients that would have had a negative test with the commonly used the 200 pg/ml criterion.

Numerous studies show serum calcium levels can greatly influence FSG in ZES patients with or without MEN1. Intravenous administration of calcium augments basal hypergastrinemia ^{69,78,157,181,197,212,237,295,311,361,368,368,386}, whereas a reduction of calcium levels by EDTA administration ^157,388 or correction of hyperparathyroidism by parathyroidectomy ^{59,87,88,127,153,159,276,361,368,369,393} diminishes hypergastrinemia in ZES patients. This calcium-induced augmentation of FSG in ZES patients is the basis of the calcium provocative test ^{69,197,262,295,360,368}. Subsequent studies have shown that this effect is likely due to a direct action of calcium on gastrinoma cells ^{2,33,91,93,399,400}. In fact, calcium can increase gastrin release from isolated gastrinoma cells ^{2,33,91,93,399,400}. Furthermore, these cells possess both calcium sensing receptors and various calcium channels ^126,155,378 whose activation could stimulate gastrin secretion.

In previous reports ^{69,78,192,196,197,219,255,295,343,360}, very few ZES patients did not respond to an intravenous calcium infusion ^195,233,351 and the median FSG increase was 438–4325 pg/ml in 8 larger series ^{23,78,110,196,232,262,318,341}. In our study, 6 NIH and 3 literature ZES patients did not respond to calcium, demonstrating that calcium evokes an increase in serum gastrin in >97% of ZES patients. The median increase was 850 pg/ml in NIH and 994 pg/ml in literature patients.

Numerous authors have proposed different criteria for a positive calcium test ^{77,78,196,219,260}. With the exception of two studies advocating a bolus injection of calcium ^318,380, these studies used very similar protocols (dose of calcium, time of infusion, time points for blood collection). Nevertheless, their results are limited by small patient numbers and the fact that patients with a FSG<10 and >10-fold increased are considered together. Three different types of criteria have been proposed for a positive calcium test: 2 criteria based on the absolute increase in FSG post calcium (≥450, ≥395 pg/ml) ^77,78, 2 criteria based on the relative increase (>50%, >100%) ^196,260 and 1 criterion based on the maximum FSG level post calcium (≥326 pg/ml) ²¹⁹. In the United States, the criteria of ≥395 pg/ml and >50% are widely used ^78,110,196. We found that 1 criterion had >90% sensitivity (gastrin max. ≥326 pg/ml), 1 criterion had 79–90% sensitivity (Δ>50% increase) and 3 criteria had 57–76% sensitivity (Δ≥450, ≥395 pg/ml, >100%). In patients with <10-fold increase in FSG the sensitivities of the two criteria based on absolute increases dropped 5–13%, whereas there was no relevant decrease in sensitivity for the other criteria. For the two criteria generally used in the United States (Δ≥395 pg/ml or >50% increase), the sensitivities in the NIH/literature patients were 54/70% and 78/91%, respectively. As discussed above, avoiding false positive tests is especially important. An analysis of the available data from 105 non-ZES patients who underwent calcium testing allowed us to assess the specificity of the different criteria. As the calcium test may be falsely positive in up to 50 % of non-ZES patients with achlorhydria ^196,219, only non-ZES patients without achlorhydria were included in this analysis. The Δ>50% and Δ>100% criteria gave an unacceptably high number of 10–17% false positives, whereas only one non-ZES patient exceeded the gastrin max. ≥326 pg/ml criterion. However, this last criterion is not clinically useful because most patients with a FSG<10-fold increased (64% in our study) have a fasting serum gastrin ≥326 pg/ml prior to any stimulation. No non-ZES patient without achlorhydria in the literature had an increase in serum gastrin post calcium ≥ 395 pg/ml, so that the Δ≥450 and Δ≥395 pg/ml criteria have 100% specificity. The ≥395 pg/ml criterion had a greater sensitivity than the ≥450 pg/ml criterion, although this difference did not quite reach statistical significance (p>0.1). Nevertheless, it could be clinically relevant because the calcium test is generally used as second-line test in patients with strong clinical suspicion of ZES but a negative secretin test. Therefore, maximal sensitivity, along with 100% specificity, is crucial for choosing the optimal criterion. Taking this into account, we conclude that Δ≥395 pg/ml should be used. However, even with this criterion, the sensitivity of the calcium test is significantly lower than that of the secretin test (63 vs. 94%, p<0.01). Furthermore, unlike with secretin, numerous side-effects are reported with the infusion of calcium, including abdominal pain, nausea, vomiting, headache, phlebitis, shortening of Q-T interval on ECG, paresthesias, diuresis, severe fatigue, palpitations, arrhythmias and a rise in blood pressure ^{110,177,196,255,318,380}. These complications lead to early termination of the infusion in some patients ³¹⁸. Moreover, the calcium infusion test takes longer than the secretin test (3 hr. vs. 30 min) and requires a physician’s attendance. In addition, the secretin test may be superior to calcium testing in distinguishing antral from tumoral hypergastrinemia ^{182,190,196,347}. For these reasons, we conclude the calcium test should not be used routinely as the first-line test in patients suspected of having ZES. However, in one previous study ¹¹⁰, 33% of ZES patients with a negative secretin test had a positive calcium test. Two smaller series report secretin-negative ZES patients with a positive response to calcium infusion ³¹⁸ or injection ³⁸⁰, whereas another study reports negative calcium tests in all secretin-negative ZES patients ¹⁹⁶. In our study, 38% of NIH and 50% of literature ZES patients with a negative secretin test (applying the Δ≥120 pg/ml criterion) had a positive calcium test (by the Δ≥395 pg/ml criterion). Therefore, we conclude that the calcium test is useful in patients with a clinical suspicion of ZES but a negative secretin test.

Ingestion of food is a physiological stimulus for the release of gastrin from the antral and duodenal mucosa ³¹. In patients with hypergastrinemia of antral origin, i.e. atrophic gastritis or antral G-cell hyperfunction, a condition reported to occur in H. pylori infection ⁹, feeding induces an increase >100% in FSG^{31,83,114,158,176,204,300}. In ZES patients, gastrin produced by the tumor is the main source of serum gastrin and early studies reported no major increase in FSG after ingestion of a test meal, suggesting that feeding does not stimulate gastrin release from gastrinomas ^{31,152,158,190,195,264,347}. Therefore, it was proposed that a standard meal test could differentiate hypergastrinemia of antral and tumoral origin ^31,347. This is an important distinction because antral G-cell hyperplasia/hyperfunction can mimic ZES clinically and cause hyperchlorhydria/hypergastrinemia ^{6,7,167,186,194,204,299,397}. However, other studies have reported ZES patients with large increases in FSG after feeding and questioned the value of the meal test ^{69,111,196,343}. The mechanism of postprandial FSG increase in ZES patients is controversial. Some studies suggest the gastrin could be of antral origin in patients having both ZES and antral syndromes ¹⁰⁶, while other authors propose the gastrin release is mediated by the tumor ¹¹¹, possibly involving release of a gastrin-stimulating agent, e.g. secretin, from the duodenum ^359,360. Some reports suggest that previous gastric surgery could influence the increase in gastrin post meal in ZES patients ^{69,111,195,360}. The small number of ZES patients investigated, the different proportion of patients with prior gastric surgery and differences in tumor characteristics could partly explain the large discrepancies between some of these studies (Table 12). In our study, 39% of NIH and 20% of literature patients had no increase in serum gastrin post meal. In the remaining patients, the median increase was 132 pg/ml among NIH and 212 pg/ml among literature ZES patients. In five larger series, the median increase in FSG post meal was 55–2269 pg/ml ^{69,158,196,198,360}. In our present study, meal test results did not differ in ZES patients with or without prior acid-reducing surgery (median increase 175 vs. 223 pg/ml, p=0.2).

Lamers and van Tongeren proposed a postprandial increase <50% to be characteristic of ZES ¹⁹⁶. In another study, a maximal serum gastrin value post meal >500 pg/ml was suggested as criterion. However, this latter criterion is of little clinical value because it does not differentiate antral hypergastrinemia from ZES and is not applicable to ZES patients with a FSG >500 pg/ml. In our study, 57% of NIH and 67% of literature ZES patients had an increase in serum gastrin post meal <50%. These numbers change to 54 and 77%, among patients with FSG<10-fold increased. However, 20% of NIH and 15% of literature ZES patients had an increase in FSG post meal ≥100%, a value reported to be characteristic of antral G-cell hyperplasia ^{7,9,108,111,158,167,204,336,347}. Because of this overlap of meal test results in patients with hypergastrinemia of antral or tumoral origin, we conclude, in accordance with others ^69,111,219, that the meal test is not useful in the diagnosis of ZES.

Only very limited information is available on correlations of gastrin provocative test results with clinical, laboratory or tumoral features in ZES. However, these correlations might be useful because they could define a subgroup of clinically relevant patients. Moreover, such correlations could identify patients in whom provocative test results are not conclusive because they are biased by different factors. In accordance with previous studies ^196,318, we found a positive correlation (r=0.52–0.56) between the absolute increase in FSG after secretin and calcium (Figure 5). This correlation remained unchanged if the relative (%) increases were correlated, suggesting that the correlation is not due to the influence of FSG levels on both variables, but rather reflects common factors may be involved in determining the response to both stimulants. Our study did not identify these factors, but they could include the response of the gastrinoma to external stimuli, the amount of gastrin stored or shared pathways of stimulation. Similar to other studies ^42,315, we found that secretin and calcium test results were both correlated with FSG values when the absolute increases post stimulation (Δ) were considered (Figure 2). However, this correlation was not significant after correction for higher FSG values by correlating the relative increases. We found a correlation of secretin and calcium test results with BAO levels and with some clinical findings (duration of medical treatment, diarrhea) associated with gastric hyperacidity ³¹⁹ (Tables 4–6 and Figure 3). Most interestingly, MEN1 patients, who frequently have hypercalcemia due to hyperparathyroidism ^29,123,221, did not show a significantly greater serum gastrin response to secretin (Table 5). This finding is surprising because hypercalcemia facilitates secretin-induced gastrin release in ZES patients ^157,196,318. In our study, we found that larger tumors are associated with a higher response to secretin or calcium (Tables 4–6, Figures 3, 4). The size of the largest tumor correlates better with FSG increases post secretin than primary tumor size. This correlation is even stronger in patients without liver metastases, suggesting that the absolute tumor mass rather than the characteristics of the primary tumor influences the response to secretin. While these clinical, laboratory or tumor variables had some influence on the magnitude of the serum gastrin response to secretin, they had little effect on the positivity of the provocative test results. This finding has clinical importance because it shows that clinical, laboratory or tumor features do not bias provocative test results with secretin or calcium and that these tests can therefore be performed in subgroups of ZES patients with different characteristics.

As discussed above, our data suggest that the meal test is generally not helpful in the diagnosis of ZES. However, correlations of the increase in serum gastrin post meal in ZES patients with clinical, laboratory and tumor features might allow some insight into ongoing controversies concerning meal-stimulated gastrin release. Some authors propose that the postprandial gastrin release in ZES patients could be mediated by the release of a gastrin-stimulating agent, such as secretin, from the duodenum ^359,360. If secretin were involved in postprandial gastrin release, patients with increases in serum gastrin post meal might have a proportional response to secretin. Therefore a significant correlation might exist between secretin and meal test results. However, we did not find such a correlation (Figure 5). Because Billroth II resection results in markedly diminished meal-stimulated secretin release ^268,269, our finding of similar meal test results in ZES patients with or without previous Billroth II resection further invalidates the hypothesis of a secretin-mediated mechanism for postprandial gastrin release in ZES patients. It has been suggested that the increase in serum gastrin post meal observed in some ZES patients could be due to coexisting antral G-cell hyperplasia/hyperfunction ¹⁰⁶. Because the meal response is markedly affected by gastrinoma resection in ZES patients¹¹¹, this proposal is unlikely. MEN1/ZES patients are reported to have an increased incidence of antral syndromes in some studies ¹⁰⁶, but not in others ¹¹¹. Our data do not support this proposal, because we found that prior antrectomy does not influence meal test results in ZES patients. However, our data did show that NIH MEN1/ZES patients had a significantly higher median FSG increase post meal than sporadic ZES patients (Table 7) and a significantly higher percentage of these patients had a ≥50% postprandial FSG increase. This difference could be related to other factors than an increased occurrence of antral syndromes, such as concomitant hyperparathyroidism with hypercalcemia, which could affect meal-stimulated hormone release. It has been suggested that duodenal gastrinomas are associated with higher postprandial FSG increases than pancreatic tumors ¹⁹⁶, whereas another large study did not find such a correlation ¹¹¹. In our study, we found that patients with duodenal gastrinomas have larger postprandial serum gastrin increases (Table 7). However, this did not affect meal test results, i.e. the percentage of patients with ≥ 50% increase in serum gastrin post meal. Therefore our result is similar to another study ¹¹¹ and suggests that the meal test is not useful in localizing duodenal gastrinomas.

Our results for secretin testing in general show similarities to most other series including ≥10 patients, however, they also show some important differences (Table 12). Specifically, we found the most widely used criterion of ≥200 pg/ml does not have a sensitivity of 100% as reported in a number of studies ^{66,238,341,412} or a low sensitivity of 70–80% as reported in other studies^78,232. Our results agree with a number of other series reporting a sensitivity of 83–86% ^100,315 (Table 12). Similar to most studies, we found the criterion of Δ≥110 pg/ml to have a higher sensitivity (92–100%) and the >50% criterion to be less sensitive than either the Δ≥110 or Δ≥200 pg/ml criteria (Table 12). For the calcium test, a number of studies reported a higher sensitivity than we found, especially for the 50% criterion ^{23,78,232,318,341}, while others had results comparable to those of our study ²⁶² (Table 12). This difference in sensitivity cannot be due to a difference in the amount of calcium infused because in most studies, 5 mg/kg/hr of calcium was infused for 3 hours and all patients had at least a 1.5 mEq/l increase in serum calcium. The meal test results in our study are comparable to results reported in some series, ^69,347,360, but markedly differ from results of other studies which reported no ZES patients with a ≥100% increase in serum gastrin post meal ^157,196,216 (Table 12).

The systematic analysis of gastrin provocative test results in 293 NIH and 537 literature ZES patients presented in this paper allowed us to draw several important conclusions which are summarized in Table 13. We could show a new criterion, Δ≥120%, which had the highest sensitivity and specificity. When this new criterion is applied, the secretin test is highly sensitive (94%) and specific (100%) and therefore play a crucial role in the diagnosis of ZES in the two-thirds of patients with non-diagnostic FSG values. This new criterion had greater sensitivity (p<0.003) than the usually used criterion of Δ≥200 pg/ml with equal specificity (i.e. 100%) and allowed the detection of 37 more ZES patients in the NIH and literature groups than the Δ≥200 pg/ml criterion. We applied the same analysis to calcium test results and could confirm the usefulness of the Δ≥395 pg/ml criterion proposed by others ⁷⁸. However, even with this criterion, the calcium test has only a 62% sensitivity. Furthermore, multiple side-effects have been reported with intravenous calcium infusion. Therefore, the calcium test should not be used as first-line provocative test. However, the calcium test may be useful in patients with a negative secretin test, but strong clinical suspicion of ZES because it will be positive in 38–50% of these ZES patients. We could not confirm the utility of the meal test, because we found significant overlap of meal test results in patients with ZES and antral syndromes. We found the magnitude of the gastrin response to secretin and calcium to correlate with BAO values and some clinical findings reflecting hyperchlorhydria as well as with tumor size and extent. However, the results (i.e. positive or negative) of these tests were only minimally influenced by these factors. The magnitude of the meal-induced gastrin response correlated with some clinical findings, including MEN1 status, and with tumor location. However, meal test results were only minimally influenced by these factors, so that the meal test is not useful for tumor localization.