Richard V. Benya

International Union of Pharmacology. LXVIII. Mammalian Bombesin Receptors: Nomenclature, Distribution, Pharmacology, Signaling, and Functions in Normal and Disease States

The mammalian bombesin receptor family comprises three G protein-coupled heptahelical receptors: the neuromedin B (NMB) receptor (BB1), the gastrin-releasing peptide (GRP) receptor (BB2), and the orphan receptor bombesin receptor subtype 3 (BRS-3) (BB3). Each receptor is widely distributed, especially in the gastrointestinal (GI) tract and central nervous system (CNS), and the receptors have a large range of effects in both normal physiology and pathophysiological conditions. The mammalian bombesin peptides, GRP and NMB, demonstrate a broad spectrum of pharmacological/biological responses. GRP stimulates smooth muscle contraction and GI motility, release of numerous GI hormones/neurotransmitters, and secretion and/or hormone release from the pancreas, stomach, colon, and numerous endocrine organs and has potent effects on immune cells, potent growth effects on both normal tissues and tumors, potent CNS effects, including regulation of circadian rhythm, thermoregulation; anxiety/fear responses, food intake, and numerous CNS effects on the GI tract as well as the spinal transmission of chronic pruritus. NMB causes contraction of smooth muscle, has growth effects in various tissues, has CNS effects, including effects on feeding and thermoregulation, regulates thyroid-stimulating hormone release, stimulates various CNS neurons, has behavioral effects, and has effects on spinal sensory transmission. GRP, and to a lesser extent NMB, affects growth and/or differentiation of various human tumors, including colon, prostate, lung, and some gynecologic cancers. Knockout studies show that BB3 has important effects in energy balance, glucose homeostasis, control of body weight, lung development and response to injury, tumor growth, and perhaps GI motility. This review summarizes advances in our understanding of the biology/pharmacology of these receptors, including their classification, structure, pharmacology, physiology, and role in pathophysiological conditions.

Phase IIA Clinical Trial of Curcumin for the Prevention of Colorectal Neoplasia

Curcumin is derived from the spice tumeric and has antiinflammatory and antineoplastic effects in vitro and in animal models, including preventing aberrant crypt foci (ACF) and adenomas in murine models of colorectal carcinogenesis. Inhibiting the production of the procarcinogenic eicosanoids prostaglandin E2 (PGE2) and 5-hydroxyeicosatetraenoic acid (5-HETE) can suppress carcinogenesis in rodents. Curcumin reduces mucosal concentrations of PGE2 (via inhibition of cyclooxygenases 1 and 2) and 5-HETE (via inhibition of 5-lipoxygenase) in rats. Although preclinical data support curcumin activity in many sites, the poor bioavailability reported for this agent supports its use in the colorectum. We assessed the effects of oral curcumin (2 g or 4 g per day for 30 days) on PGE2 within ACF (primary endpoint), 5-HETE, ACF number, and proliferation in a nonrandomized, open-label clinical trial in 44 eligible smokers with eight or more ACF on screening colonoscopy. We assessed pre- and posttreatment concentrations of PGE2 and 5-HETE by liquid chromatography tandem mass spectroscopy in ACF and normal-tissue biopsies; ACF number via rectal endoscopy; proliferation by Ki-67 immunohistochemistry; and curcumin concentrations by high-performance liquid chromatography in serum and rectal mucosal samples. Forty-one subjects completed the study. Neither dose of curcumin reduced PGE2 or 5-HETE within ACF or normal mucosa or reduced Ki-67 in normal mucosa. A significant 40% reduction in ACF number occurred with the 4-g dose (P < 0.005), whereas ACF were not reduced in the 2-g group. The ACF reduction in the 4-g group was associated with a significant, five-fold increase in posttreatment plasma curcumin/conjugate levels (versus pretreatment; P = 0.009). Curcumin was well tolerated at both 2 g and 4 g. Our data suggest that curcumin can decrease ACF number, and this is potentially mediated by curcumin conjugates delivered systemically. Cancer Prev Res; 4(3); 354–64. ©2011 AACR.

Quantitative Immunohistochemistry by Measuring Cumulative Signal Strength Using Commercially Available Software Photoshop and Matlab

Currently available techniques for performing quantitative immunohistochemistry (Q-IHC) rely upon pixel-counting algorithms and therefore cannot provide information as to the absolute amount of chromogen present. We describe a novel algorithm for true Q-IHC based on calculating the cumulative signal strength, or energy, of the digital file representing any portion of an image. This algorithm involves subtracting the energy of the digital file encoding the control image (i.e., not exposed to antibody) from that of the experimental image (i.e., antibody-treated). In this manner, the absolute amount of antibody-specific chromogen per pixel can be determined for any cellular region or structure.

A prospective study of intraoperative methods to diagnose and resect duodenal gastrinomas

ObjectiveThis study determined, prospectively, whether duodenotomy (DX) should be routinely performed in explorations for patients with Zollinger-Ellison syndrome (ZES). Summary Background DataDuodenal gastrinomas are now being found with increasing frequency in patients with Zollinger-Ellison syndrome. The surgical approach used to detect these tumors is controversial. Some recommend intraoperative endoscopy with transillumination (IOE) at surgery, while others recommend routine DX. MethodsBeginning in 1989, the authors prospectively compared the ability of palpation, intraoperative ultrasound (IOUS), IOE, and DX (in that sequence) to detect gastrinomas in 35 consecutive patients with ZES. Each patient also underwent preoperative localization studies. ResultsThirty-three of 35 patients (94%) had tumor detected and excised; duodenal gastrinomas were excised in 27 patients (77%). The average size of the duodenal tumors was 0.8 cm, significantly smaller (p < 0.005) than the pancreatic and lymph node tumors in this series. Standard palpation after a Kocher maneuver identified 19 of the 31 duodenal tumors (61 %) in the 27 patients. IOUS revealed only eight duodenal tumors (26%) and no new lesions. IOE identified 20 duodenal gastrinomas (64%) and 6 new lesions. DX identified 31 duodenal tumors (100%) and 5 additional tumors. The morbidity rate was 17%. One patient had a duodenal fistula after operation (2.8%) and subsequently recovered. No patient died. ConclusionsThese results demonstrate that the duodenum is the most common location for gastrinoma in patients with ZES (77%) and that DX to detect and remove duodenal gastrinomas should be routinely performed in all explorations for patients with ZES.

Assessment and Prediction of Long-term Cure in Patients with the Zollinger-Ellison Syndrome: The Best Approach

Gastrinoma is the most common functional pancreatic endocrine tumor [1-3]. Because of autonomous gastrin release by the gastrinoma, patients develop gastric acid hypersecretion as part of the Zollinger-Ellison syndrome [1, 4]. In recent years, effective medical therapies have been developed to control gastric acid hypersecretion, which previously was the leading cause of death in patients with the Zollinger-Ellison syndrome [5-7]. Increasingly, the malignant nature of the tumor is becoming the primary determinant of long-term survival [2, 8], and therefore patients with the Zollinger-Ellison syndrome are increasingly being considered for possible gastrinoma resection [1, 2, 9, 10]. However, considerable disagreement has arisen about the possible benefit of resection to the patient [11]. This difference in opinion has occurred primarily because of widely ranging disease-free rates (4% to 90%) in different series [1, 12-14]. Further, even without surgery, many patients have an excellent long-term prognosis because of the slow growth of the gastrinoma [15, 16]. Also, many patients experience complications from peptic ulcer disease (for example, perforation) before diagnosis of the syndrome; such complications increase the surgical risk. Thus, the possible long-term benefit of surgery in terms of disease-free survival must be studied to determine whether it outweighs the risk. Disease-free rates have varied markedly among studies for several reasons, including differences in sample size, follow-up, selection criteria for surgery, and operative approach. For example, surgery in these studies has ranged from a detailed exploration with removal of only gastrinomas identified at surgery to blind proximal pancreaticoduodenectomy in cases in which no tumor was found but selective venous sampling showed gastrin positivity in this area [1, 12, 14, 17-21]. However, the primary reason for the variation in the disease-free rate in different series is the lack of agreement on what evaluations should be routinely done after operation to establish disease-free status. Many studies only determined fasting serum gastrin levels [10, 17, 22-24]; however, fasting hypergastrinemia can be caused by achlorhydria, which is frequently seen in patients receiving gastric acid antisecretory drugs, and thus hypergastrinemia may not always represent a disease recurrence or a failure to render the patient disease free [25]. Further, some patients with the Zollinger-Ellison syndrome have a normal fasting serum gastrin level but a positive secretin or calcium provocative test result [26]; other patients have been described who only had a positive calcium provocative test or only abnormal imaging studies [27, 28]. To address this problem, we did a study to determine the best method to assess disease-free status after surgery and to predict long-term disease-free status during follow-up. Methods Our investigation included 81 consecutive patients who underwent surgical exploration for possible curative gastrinoma resection between December 1980 and December 1991 as part of the ongoing National Institutes of Health prospective study of patients with the Zollinger-Ellison syndrome. Thus, many of these patients have been described previously [4, 18, 19, 28-39]. Patients were considered to have a confirmed diagnosis of the Zollinger-Ellison syndrome if they met at least two of the following criteria: an elevated fasting serum gastrin level; a basal acid output of more than 15 mEq/h if the patient had not undergone previous gastric surgery or of more than 5 mEq/h if the patient had undergone previous gastric surgery; an increase in the serum gastrin level of 200 pg/mL after the intravenous administration of secretin; and an increase in the serum gastrin level of 395 pg/mL after an intravenous calcium infusion. The secretin provocative test was done as described previously [28], with Secretin-Kabi (Ferring AB, Malmo, Sweden) given by intravenous bolus injection (2 U/kg body weight). The calcium provocative test was done as described previously [28], with calcium gluconate (10%) (54 mg/kg per hour [5 mg calcium/kg per hour]) given by continuous intravenous infusion for 3 hours. Serum gastrin levels were determined by Bioscience Laboratories (New York, New York). All samples were diluted as necessary to give values that fell on the midportion of the standard curve as described previously [28]. Patients with elevated gastrin levels had repeated determinations. All patients had upper gastrointestinal endoscopy, computed tomography [29], ultrasonography [30], and selective angiography [31] that included selective injections of the hepatic, gastroduodenal, splenic, and superior mesenteric arteries before operation. In addition, patients evaluated after 1987 also underwent magnetic resonance imaging [32]. Before surgery, basal and maximal acid outputs were measured as described previously [33, 34]. Maximal acid output was measured after pentagastrin stimulation (6 g/kg subcutaneously). Sufficient oral antisecretory medication was given to reduce gastric acid output to less than 10 mEq/h before the next dose of medication for patients without previous gastric surgery and to less than 5 mEq/h for those who had previous gastric surgery [4, 33]. The minimal dose of cimetidine or ranitidine given by continuous infusion or of omeprazole given by intermittent intravenous bolus injection to reduce gastric acid secretion to 10 mEq/h or less was determined in all patients as described previously [35, 36, 39], and this dose was administered at surgery and during the postoperative period to control gastric acid secretion. At surgery, an extensive exploration, including mobilization of the duodenum, was done as described previously [18, 19]. In addition, patients who had surgery after 1986 also underwent transduodenal endoscopic transillumination [37] and duodenotomy [19] to localize duodenal gastrinomas and intraoperative ultrasonography as described previously [19, 38]. Specific Protocol After surgery, before discharge from the hospital, fasting serum gastrin concentrations were determined on at least 3 separate days and a secretin provocative test was done. Each patient was discharged on the same dose of oral antisecretory drug that he or she was taking before operation, as described previously [18, 19]. If tumor was resected, patients were reevaluated 3 to 6 months after surgery to determine disease-free status and to assess control of gastric acid hypersecretion. Thereafter, patients who had had tumor resection were reassessed yearly. Patients who did not undergo resection were evaluated yearly. Tumor recurrence after resection was assessed by noninvasive imaging studies (ultrasonography, computed tomography, and magnetic resonance imaging) functional studies, which included determination of fasting serum gastrin levels on 3 different days, and provocative tests with secretin and calcium. Gastric acid antisecretory drugs were withdrawn and gastric acid output was determined to ensure that fasting serum gastrin determinations and secretin provocative tests were done on days when patients were not achlorhydric to avoid physiologic hypergastrinemia. As shown in Figure 1, elevation of the fasting serum gastrin level may be secondary to drug-induced achlorhydria. Therefore, to adequately assess the importance of either an elevated fasting serum gastrin level or a positive secretin or calcium provocative test, all gastric acid antisecretory agents had to be withdrawn before these studies. Figure 1. Relation between the fasting serum gastrin concentration and basal acid output in a patient assessed for recurrence 6 months after resection of a gastrinoma. Disease-free status was defined by amelioration of all symptoms of the Zollinger-Ellison syndrome after operation; a significant reduction in gastric acid output at follow-up, no significant increase in basal acid output (that is, <50% increase) at follow-up, and no significant increase in gastric acid antisecretory drug requirements after operation as described previously [34]; a normal fasting serum gastrin concentration (<110 pg/mL) [19, 34]; negative results on provocative testing with secretin (an increase of less than 200 pg/mL in gastrin level) and calcium (an increase of less than 395 pg/mL in gastrin level) [27]; and no evidence of tumor on imaging studies. Further, these criteria were used at all follow-up times so that the persistence of these findings further established disease-free status. In patients who were considered disease-free at any postoperative visit, recurrence was defined by the development of an elevated fasting serum gastrin level (>110 pg/mL) when achlorhydria was not present, a positive result on a calcium or secretin provocative test, abnormal imaging studies, or a significant increase in basal acid output during follow-up, increased drug requirements, or clinical symptoms of Zollinger-Ellison syndrome that persisted during subsequent visits. Patients who had a recurrence (n = 11) were divided into two groups: One group included eight patients who had a recurrence within 36 months after surgery (early recurrence), and the other group included three patients who had a recurrence 36 to 72 months after surgery (late recurrence). Statistics The proportion of patients remaining disease free after surgery was plotted using the Kaplan-Meier method [40], and this curve was used to estimate the probability that a patient would remain disease free [41]. The sensitivity, specificity, and predictive values [42] for the fasting serum gastrin determination and the secretin provocative test were calculated using the results at 3-year follow-up for the 46 patients who remained in the study to that point. To compare differences in recurrence rates, the McNemar test was used [40]. We used Rothmans method [41] to calculate 95% CIs for the probability of being disease free at 3 years and Brown and Hollanders [42

Zollinger-Ellison syndrome can be the initial endocrine manifestation in patients with multiple endocrine neoplasia-type I

PURPOSE To determine whether patients with multiple endocrine neoplasia type I (MEN-I) can initially present with Zollinger-Ellison syndrome (ZES), and to learn whether ZES exhibits any distinguishing features when it occurs as a first manifestation of MEN-I. PATIENTS AND METHODS Sixty patients who had been referred to a clinical research center with ZES were examined by cohort analysis. Twenty-eight had MEN-I and 32 did not. In patients with MEN-I, we analyzed the temporal relationships between the clinical and biochemical manifestations of ZES and the other endocrinopathies associated with the neoplasia. To determine whether patients who had ZES as a first manifestation of MEN-I (n = 8) had any distinguishing clinical characteristics, we compared them to a cohort of patients with established sporadic ZES (n = 32) matched for age, sex, and time since the onset of symptoms consistent with ZES. RESULTS Of the 28 patients with ZES and MEN-I, 11 initially presented with ZES and hyperparathyroidism (HP) and 1 with evidence only for pituitary disease. Eight patients (29%) presented with features of ZES and developed clinical and biochemical evidence for HP later, while the same number developed these 2 endocrinopathies in the opposite order. In whichever order ZES and HP occurred, the time from the diagnosis of the first to the diagnosis of the second was similar. It ranged from 9 to 177 months in patients who presented with ZES first, and from 12 to 264 months in patients who presented with HP first. At the time of initial diagnosis, the patients who presented with ZES as a manifestation of MEN-I had no distinguishing ZES-related symptoms, biochemical assays, or tumor imaging results compared to the cohort of patients who had the syndrome sporadically. CONCLUSION Patients with MEN-I can initially present with a symptomatic pancreatic endocrine tumor syndrome without any other disease manifestations. In patients with ZES and MEN-I, up to one third may present with ZES without evidence of any other endocrinopathy. Consequently, patients with presumed sporadic ZES should undergo continual biochemical screening for other endocrinopathies characteristic of MEN-I and, in the future, genetic studies for the MEN-I gene.

Quantitative Immunohistochemistry by Measuring Cumulative Signal Strength Accurately Measures Receptor Number

We previously demonstrated that quantitative immunohistochemistry (Q-IHC) performed by measuring the cumulative signal strength of the digital file encoding an image can be used to determine the absolute amount of chromogen present per pixel. We now show that Q-IHC so performed can be used to accurately determine the amount of peptide hormone receptor of interest in archived tissues. To do this we transfected Balb 3T3 fibroblasts with the cDNA encoding the human receptor for gastrin-releasing peptide (GRP), and selected six cell lines stably expressing between 102 and 106 receptors/cell. These cell lines were fixed in formalin, embedded in paraffin, and treated with antipeptide antibodies against the GRP receptor, followed by DAB chromogen to identify bound antibody. Images were acquired using a 4.9 million pixel digital scanning 24-bit RGB camera, saved in TIFF format, and used for subsequent analysis. Q-IHC was performed after digitally dissecting out the relevant portion of the image for analysis, and processing using a program written in C (available at http://www.uic.edu/com/dom/gastro/Freedownloads.html). Under the conditions defined here, chromogen quantity as determined by Q-IHC tightly correlated with GRP receptor number (r2=0.867) in these cell lines. Using the conversion factor identified as a result of these studies, we then determined GRP receptor number on eight randomly selected, archived human colon cancers. Overall GRP receptor expression in colon cancer depended on the degree to which cells within any particular tumor were differentiated, with well-differentiated cells expressing the greatest numbers of receptors (∼55,000 ± 10,000 sites/cell). These studies indicate that Q-IHC can be used to determine receptor quantity in archived tissues and other samples of limited quantity.

Aberrant expression of gastrin-releasing peptide and its receptor by well-differentiated colon cancers in humans

Epithelial cells lining the adult human colon do not normally express gastrin-releasing peptide (GRP) or its receptor (GRPR). In contrast, approximately one-third of human colon cancers and cancer cell lines have been shown to express GRP-binding sites. Because GRPR activation causes the proliferation of many cancer cell lines, GRP has been presumed to act as a clinically significant growth factor. Yet GRP has not been shown to be expressed by colon cancers in humans nor has the effect of GRP and/or GRPR coexpression on tumor behavior been investigated. We therefore determined GRP and GRPR expression by immunohistochemistry in 50 randomly selected colon cancers resected between 1980 and 1997, all 37 associated lymph node and liver metastases, and 20 polyps. Tumor sections studied were those that contained the margin and adjacent nonmalignant epithelium. Overall, 84% of cancers aberrantly expressed GRP or GRPR, with 62% expressing both ligand and receptor, whereas expression was not observed in adjacent normal epithelium. Consistent with the previously established mitogenic capabilities of GRP, tissues coexpressing GRP and GRPR were more likely to express proliferating cell nuclear antigen than tissues not expressing both ligand and receptor. Yet GRP/GRPR coexpression was seen with equal frequency in stage A as in stage D cancers and was only detected in 1 in 37 metastases. Furthermore, Kaplan-Meier analysis did not reveal any difference in patient survival between those whose tumors did or did not express GRP/GRPR. In contrast, GRP/GRPR coexpression was found in all well-differentiated tumor regions, whereas poorly differentiated tissues never coexpressed GRP/GRPR. Overall, these data indicate that, although GRP is a mitogen, it is not a clinically significant growth factor in human colon cancers. Rather, the strong association of GRP/GRPR coexpression with tumor differentiation raises the possibility that these proteins primarily act in vivo as morphogens.Epithelial cells lining the adult human colon do not normally express gastrin-releasing peptide (GRP) or its receptor (GRPR). In contrast, approximately one-third of human colon cancers and cancer cell lines have been shown to express GRP-binding sites. Because GRPR activation causes the proliferation of many cancer cell lines, GRP has been presumed to act as a clinically significant growth factor. Yet GRP has not been shown to be expressed by colon cancers in humans nor has the effect of GRP and/or GRPR coexpression on tumor behavior been investigated. We therefore determined GRP and GRPR expression by immunohistochemistry in 50 randomly selected colon cancers resected between 1980 and 1997, all 37 associated lymph node and liver metastases, and 20 polyps. Tumor sections studied were those that contained the margin and adjacent nonmalignant epithelium. Overall, 84% of cancers aberrantly expressed GRP or GRPR, with 62% expressing both ligand and receptor, whereas expression was not observed in adjacent normal epithelium. Consistent with the previously established mitogenic capabilities of GRP, tissues coexpressing GRP and GRPR were more likely to express proliferating cell nuclear antigen than tissues not expressing both ligand and receptor. Yet GRP/GRPR coexpression was seen with equal frequency in stage A as in stage D cancers and was only detected in 1 in 37 metastases. Furthermore, Kaplan-Meier analysis did not reveal any difference in patient survival between those whose tumors did or did not express GRP/GRPR. In contrast, GRP/GRPR coexpression was found in all well-differentiated tumor regions, whereas poorly differentiated tissues never coexpressed GRP/GRPR. Overall, these data indicate that, although GRP is a mitogen, it is not a clinically significant growth factor in human colon cancers. Rather, the strong association of GRP/GRPR coexpression with tumor differentiation raises the possibility that these proteins primarily act in vivo as morphogens.

Expression of Vitamin D Receptor and 25-Hydroxyvitamin D3-1α-Hydroxylase in Normal and Malignant Human Colon

Considerable evidence exists to support the use of vitamin D to prevent and/or treat colorectal cancer. However, the routine use of bioactive vitamin D, 1,25-dihydroxyvitamin D3, is limited by the side effect of toxic hypercalcemia. Recent studies, however, suggest that colonic epithelial cells express 25-hydroxyvitamin D3-1α-hydroxylase, an enzyme that converts nontoxic pro-vitamin D, 25-hydroxycholecalciferol [25(OH)D3], to its bioactive form. Yet, nothing is known as to the cellular expression of 1α-hydroxylase and the vitamin D receptor (VDR) in the earliest histopathologic structures associated with malignant transformation such as aberrant crypt foci (ACF) and polyps [addressing the possibility of using nontoxic 25(OH)D3 for chemoprevention], nor is anything known as to the expression of these proteins in colorectal cancer as a function of tumor cell differentiation or metastasis [relevant to using 25(OH)D3 for chemotherapy]. In this study, we show that 1α-hydroxylase is present at equal high levels in normal colonic epithelium as in ACFs, polyps, and colorectal cancer irrespective of tumor cell differentiation. In contrast, VDR levels were low in normal colonic epithelial cells; were increased in ACFs, polyps, and well-differentiated tumor cells; and then declined as a function of tumor cell de-differentiation. Both 1α-hydroxylase and VDR levels were negligible in tumor cells metastasizing to regional lymph nodes. Overall, these data support using 25(OH)D3 for colorectal cancer chemoprevention but suggest that pro-vitamin D is less likely to be useful for colorectal cancer chemotherapy.

Currently used doses of omeprazole in Zollinger-Ellison syndrome are too high

The efficacy of omeprazole increases during the first few days of administration, suggesting that long-term maintenance dose requirements in patients with Zollinger-Ellison syndrome may be lower than those initially established by upward titration. Long-term maintenance doses of omeprazole were prospectively reduced in 37 patients who had been taking omeprazole for 22 +/- 4 months. Successful reduction was defined as reduction to 20 mg once or twice daily with an absence of symptoms, endoscopy without evidence of active acid-peptic disease, and a gastric acid output of < 10 mEq/h. Sixty-eight percent of patients (25/37) were successfully reduced to 20 mg of omeprazole once (18/24) or twice daily (7/13). Ninety-five percent of patients (20/21) without multiple endocrine neoplasia type I, severe gastroesophageal reflux disease, or previous partial gastrectomy had safe reductions of doses. It is concluded that the currently used omeprazole maintenance doses in patients with Zollinger-Ellison syndrome are too high and advocated that the initial dose still be established by acute daily upward titration followed by gradual reduction once control of acid output has been achieved.