Detection of tumor cells in peripheral blood and bone marrow in patients with pancreatic cancer

GASTROENTEROLOGY Vol. 114, No. 4

A512 AGA ABSTRACTS G2085

REGULATION OF TRANSCRIPTION OF THE TWO CYCLOOXYGENASE ISOFORMS IN PANCREAS ACINI. Alexander Zabel-Langhennig, Babett Holler, Kurt Engeland, and Joachim Mt~ssner Medizinische Klinik II, University of Leipzig, Philipp-Rosenthal-Str. 27, D-04301 Leipzig, Germany, moej @medizin.uni-leipzig.de Background and Aims: In earlier experiments we have shown that exogenous application of prostaglandin E2 inhibits secretagogue stimulated enzyme secretion in isolated rat pancreatic acini. Also specific receptors for prostaglandin E2 were characterized on acinar cell membrane (Am. J. Physiol. 1991, 260, G711-19). Prostaglandins as mediators of inflammation are synthesized by the key enzyme cyclooxygenase (Cox or prostaglandin H synthase), of which two isoforms have been identified, Cox-1 and Cox-2. We are interested in two aspects of regulation of cell function through prostaglandins: Modulation of prostaglandin synthesis dependent on diet and through induction of pancreatitis in an animal model. Since a number of prostaglandins might be involved in these processes it appears reasonable to look at the regulation of the two cyclooxygenase isoforms by which all prostaglandins have to be synthesized. M e t h o d s and Results: We have developed an RT-PCR method to determine changes in the expression of Cox-1 and Cox-2 isoforms. Pancreatitis was induced in rats by different concentrations of cerulein. An optimal concentration for the induction of pancreatitis was determined. Pancreatic acini were isolated employing a collagenase digestion method. Total RNA was isolated from these acini from cerulein treated and untreated rats. We could detect both, Coxl and Cox-2, mRNA in isolated rat pancreatic acini. Amounts of Cox mRNA were standardized by comparison with GAPDH and actin expression. We also looked at and could exclude leukocyte infiltration in these tissue preparations. We will present data an induction and repression of cyclooxygenase transcription dependent on induced pancreatitis. We will discuss the correlation of enzyme expression, synthesis of prostaglandins and a potential cytoprotective role of prostaglandins. Conclusion: Cox-1 and Cox-2 are expressed in acinus cells of the pancreas with pancreatitis and in normal acini. These enzymes synthesize prostaglandins which may play a roll in mediation of pancreatitis. G2086

IL-6 AND CRP LEVELS AS PREDICTIVE FACTORS FOR POST-ERCP PANCREATITIS. T~ Zfig0ni, L. Pr6nai, K. Papik, A. Kt~pe, A. Nrmeth, Zs. Tulassay; 2nd Dept. Internal Medicine, Semmelweis Medical University, Hungarian Academy of Science, Clinical GastroenterologyResearch Unit, Budapest Background: The suggested prevalence of post-ERCP acute pancreatitis is 2-3%, however elevated serum amylase levels can be observed in 40% of cases. The significance of elevated amylase levels is unclear. On the other hand, elevation of IL-6 and CRP suggested to be good predictive factors for acute pancreatitis and show a close correlation with the severity of the attacks. They play a significant role in multiorgan damage eg. ARDS. Aims: The present study was designed to determine the prevalence of postERCP acute pancreatitis by concomitant measurement of IL-6, Creactive protein (CRP) and amylase levels in concordance with the clinical picture (pain, tenderness, leukocytosis, fever) prior to and following ERCP. Methods: Eighty-one patients (31 male, mean age 59 y.o., range 24-85 and 50 female, mean age 64 y.o., range 31-89) who undervent routine ERCP were involved in the study. IL-6, CRP and amylase were measured from venous blood prior to, and 1 and 24 hours after the ERCP. Clinical symptoms were assessed in each cases. Acute pancreatitis was suspected if serum amylase levels elevated at least two-fold and clinical symptoms appeared. We investigated those patients in whom a significant elevation of IL-6 and/or CRP levels could be detected. Results: IL-6 (max. 1680 pg/ml) and/or CRP (max. 1150 mg/1) were elevated in 29 patients, while remained unaltered in 52 cases. Clinical symptoms in concordance with acute pancreatitis developed only in 10 of these 29 patients. (Pancreatography was performed in 9, EST in 3 of these cases). Serum amylase was significantly elevated in 9 out of 10 patients (max. 3194 IU/1). Conclusion: IL-6 and CRP are good early predictive factors of post-ERCP acute pancreatitis. They show a good correlation with the appearance of clinical symptoms and hyperamylasemia. This work was supported in part by OTKA Grant No: F023788 G2087

INTRADUCTAL PAPILLARY MUCINOUS TUMORS OF THE PANCREAS (IPMTP): PARTICULARITIES OF MALIGNANT FORMS. Zamora C, Garcia D, Barthet M, Seleznieff I, Sastre B, Payan M, Bernard JP, Sahel J. H6pital Sainte-Marguerite, Service d'hrpato-gastroentrrologie, Service de chirnrgie digestive, 270 Bd Sainte-Marguerite, 13009 Marseille, France. IPMTP are rare pancreatic tumors including ductectatic mucinous tumors and villous tumors of the main pancreatic duct. We studied the differences between malignant and benign IPMTP in order to determine risk factors. Among 26 patients presenting with IPMTP, 19 patients were treated surgically with histological confirmation. Ultrasonography was performed in 25, CT scan in 23, endoscopic retrograde cholangio pancreatography (ERCP)

in 23 and endo-ultrasonography in 6 patients. The mean follow-up duration was 26 months. Among the 19 surgically treated patients, 5 had malignant and 14 had benign tumors. Malignant forms were significantly associated with greater age (mean difference 7 years). There was no difference between malignant and benign forms in clinical presentation particularly concerning weight loss and presence of a biliary stenosis. During ERCP, more frequent issue of mucus through the papilla was noted in malignant forms. Histology analysis (cytology, brushing, mierobiopsy) was performed in 11 cases including 4 malignant forms, but never permitted the diagnosis of malignant character of the the IMPTP. The surgical treatment was segmental pancreatectomy in all cases. Resection margins were free of tumoral process in all benign IPMTP, but in one case a malignant relapse was observed 14 months after surgery. The mean follow up of the 5 patients presenting with malignant IMPTP was 23 months and a relapse occured in the single patient presenting neoplastic process in margins of resection. The malignant characters of IPMTP are difficult to diagnose from clinical and radiological arguments. However, malignant IMPTP are generally resectable with prolongated survival without relapse. • G2088

ADHESION MOLECULE ICAM-1 IS PRESENT AND FUNCTIONS IN RAT PANCREATIC ACINAR CELLS. V.Zaninovic. A. S. Gukovskaya, I.Gukovsky, S.Kim, S.J.Pandol. CURE: Digestive Diseases Research Center, Department of Medicine, UCLA & VAMC West Los Angeles, CA. Neutrophil infiltration is one of the characteristics of experimental pancreatitis. Neutrophil adhesion in various inflammatory diseases is mediated by intracellular adhesion molecule (ICAM)-I. Earlier we showed that ICAM-1 is upregulated in experimental pancreatitis induced by supramaximal doses of cerulein. The aim of this study was to determine if ICAM-1 is present in pancreatic acinar cells, if it is upregulated by the hormone, and what is its functional role. Dispersed rat pancreatic acini were isolated by a standard collagenase digestion technique and incubated in 199 medium for up to 6 h with or without cerulein (1 pM to 100 nM). RT-PCR demonstrated mRNA expression for ICAM-1 in acinar cells. Immunoprecipitation followed by Western blot analysis showed the presence of ICAM-1 95 kD protein. Immunofluoreseence performed on acinar cell preparations localized ICAM-1 mostly to basolateral membrane. Cerulein upregulated ICAM-1 in acinar cells in a dose- and time-dependent manner. With 3 h incubation, upregulation of ICAM-1 protein was detected at 10 pM cemlein and reached maximum at 100 nM. ICAM-1 upregulation in cells treated with the supramaximal dose of the hormone (100 nM) was seen at 1 h and reached maximum (3-fold increase) at 6 h incubation. To assess the functional role of ICAM-1 on acinar cells we tested if ICAM-1 mediated neutrophil adhesion to these cells. Dispersed pancreatic acini were incubated for 2 h with or without a neutralizing ICAM-1 antibody and then plated out on polylysine-coated slides. Neutrophils were isolated from rat blood, suspended in RPMI 1640, and labded with a fluorescent antineutrophil antibody. The layer of acinar cells on the slide was covered with labeled neutrophils, and the slides were incubated for 30 min with shaking. Unattached cells were then vigorously washed out, the remaining cells were fixed, and the number of attached neutrophils per 1,000 acinar cells was counted under fluorescence microscope. The results demonstrated neutrophil attachment to pancreatic acinar cells. Preincubation with neutralizing ICAM-1 antibody decreased the number of neutrophils attached to acinar cells by 6 -+ 1 times (n=3). The results show that the adhesion molecule ICAM-1 is present on pancreatic acinar ceils and functions by mediating neutrophil attachment to these cells. This direct interaction between neutrophils and acinar ceils may play a role in the development of inflammatory response in pancreatitis. • G2089 D E T E C T I O N OF T U M O R CELLS IN PERIPHERAL BLOOD AND

BONE MARROW IN PATIENTS WITH PANCREATIC CANCER. K. Z'~,ra~en. B.A. Centeno*, J. Werner, A.L. Warshaw, R.E. Jimenez, S. Kupa, C. Fernandez-del Castillo, Departments of Surgery and Pathology*, Massachusetts General Hospital and Harvard Medical School, Boston, MA The detection of tumor cells in the peripheral blood and bone marrow of patients with pancreatic cancer may correlate to advanced tumor stages. Methods: We collected blood from 136 patients, 78 with pancreatic cancer and 58 with benign diseases (pancreatic adenomas - 19, chronic pancreatitis - 18, acute pancreatitis - 4, non pancreatic controls - 17). In 30 of the patients with pancreatic cancer a bone marrow sample was obtained simultaneously. Immunocytochemistry was performed on cytospins of the mononuclear cell population using a monoclonal antibody against cytokeratins (AEI/AE3). Slides were analyzed in a blinded fashion by a cytopathologist. Two-tailed Fisher exact tests were used for statistical analysis. Results: Thirty~one percent of pancreatic cancer patients were found to have circulating tumor cells. The specificity of the AEI/AE3 assay in blood was 96% (1/58 false positive = 1.7%). Ninety-two percent of patients with cell-positive blood had unresectable cancer. Carcinomas confined to the pancreas (T1, T2) and those with a lower histologic grade (G1, G2) had a lower prevalence of circulating tumor cells in blood (10%, 9% respectively) than advanced (27%; p= 0.22)

Pancreatic Disorders A513

April 1998 and high-grade tumors (32%; p= 0.06). The prevalence of AE1/AE3-positive cells in blood was 55% in patients with distant metastases and 21% in patients with apparently localized disease (p = 0.01). When both blood and bone marrow were available for analysis, AEl/AE3-positive cells were found in 60% (18/30) of patients, 43% in blood and 30% in bone marrow. The prevalence of cytokeratin positive ceils was lower in patients with resectable cancer (blood: 0 (0/7), bone marrow: 14% (1/7) than in patients with unresectable or metastatic cancer (blood: 56% (13/23, p =0.01); bone marrow: 35% (8/23, p = 0.24)). In patients with metastatic disease circulating tumor cells were detected more frequently in peripheral blood than in bone marrow samples (70% vs. 10%; p = 0.01) Conclusion: The prevalence of cytokeratinpositive cells in blood and bone marrow of pancreatic cancer patients is similar. However, peripheral blood and bone marrow analysis are complementary in the detection of tumor cells. The presence of positive cells in blood but not in bone marrow correlates to the presence of metastatic disease. The significance of positive cells in bone marrow in the absence of circulating tumor cells in the blood remains to be established. • G2090 EFFECT OF FATTY ACID, VITAMIN E AND GLUTATHIONE ON THE EXPRESSION OF ARYL HYDROCARBON RECEPTOR (AhR) IN PANCREATIC DUCTAL CELL (TAKA-1). J. Zhang. V.A. Tan, D. Heber, V.L.W. Go. UCLA Center for Human Nutrition, Los Angeles, CA 90095 AhR and its related proteins have been suggested as integral parts of the electrophile response element-binding transcriptional complex associated with oxidative stress and carcinogenesis. We previously reported that AhR is expressed by an immortalized hamster pancreatic ductal cell, TAKA-1 (Gastroenterology, Vol. 110,No.4, pA444, 1996). In the current study, we investigated the effect of adding antioxidants vitamin E and glutathione and decreasing fatty acid on the expressions of AhR and certain cytochrome P450s in TAKA-1 cells. TAKA-1 cells were grown in culture under the following conditions: 1) RPMI-1640 medium with 10% FBS (control); 2) control medium with 10% delipided FBS; 3) control medium with 0.1 mM Vitamin E and 7 mM Glutathione. The cells were harvested after 48 h and cytosolic protein was extracted. A hepatoma cell line (Hepa-lclc7) was used as a positive control. AhR and cytochrome P450 were determined by Western Blot. Results: Cytochrome P450s, including CYP1A1 and 4A, were not affected in the absence or presence of antioxidants. However, AhR decreased in TAKA-1 cells cultured in the presence of antioxidants as well as in cells treated with low fatty acid medium. The glutathione S-transferase increased slightly in the treatment groups (2 & 3). These results suggest that expression of AhR is an indicator of oxidative stress and can be modified either in the presence of an increased level of antioxidants or decreased level of lipid peroxidation. Conclusion: AhR and its related phase I and II enzymes, such as glutathione S-transferase, may play a major role in the metabolism and detoxification of a variety of xenobiotics in pancreas. Supported by NIH 5T32DK07688 and CA42170.

G2091 LONG-TERM ENDOSCOPIC MANAGEMENT OF OBSTRUCTIVE PANCREATITIS. D. S. Zimmon St. Vincent's Hospital, New York, NY Unrelieved pancreatic duct obstruction leads inevitably to pancreatic fibrosis, atrophy and diabetes. In patients with sclerosing pancreatitis the duct is too narrow for surgical anastomosis. The only therapeutic options are long term endoscopic stenting or resection with loss of endocrine and exocrine function. Another indication for endoscopic therapy is a contraindication to surgery. Five such patients were managed for a mean of 8.2 years (range 10 to 5) with pigtail-flap pancreatic stents to maintain duct patency, relieve pain and prevent recurrent attacks of pancreatitis or pancreatic sepsis. Stent exchanges were initially performed every three months and then extended or reduced to prevent pain, attacks of pancreatitis or infection. Patients were encouraged to seek stent exchange when symptoms recurred. Each developed a unique pattern. Stent length extend beyond the most proximal stenosis. Stent diameter was selected to accommodate the duct diameter of 5 or 7F. When possible two side by side 5F stents were used to enlarge the lumen without distorting the gland or exerting pressure on the unstrictured duct. There were no complications of stent insertion or stents lost into the pancreatic duct. Attempts to remove stents resulted in return of symptoms, recurrent infection or recurrent obstruction. In contrast to the majority of patients treated with stents, these patients had minimal duct remodeling or enlargement from the initial radiographic presentation. A 43 year old male with obstructive pancreatitis from biliary tract disease with bile duct stenosis required 5F stent exchanges every 6 weeks over a 9 year period (total 75) to prevent recurrent pancreatitis with maintenance of endocrine and exocrine function. A 58 year old male with chronic pancreatitis associated with ethanol abuse, portal vein thrombosis, hepatic jejunostomy for bile duct obstruction, gastrojejunostomy for duodenal stenosis and a distal pancreatic duct stenosis required stent exchanges every 4 months for 10 years to prevent recurrent pancreatic sepsis. After multiple episodes of monilial sepsis controlled by stent exchanges every 2 months over 9 years, a 72 year old female with intestinal pseudo obstruction died from monilia sepsis. Two other patients required stent exchanges every 3 months for 8 years and 5 years respectively. Obstructive pancreatitis is a subtle complication of acute or chronic pancreatitis that may present as infection or recurrent pancreatitis and lead to death or progressive destruction of the gland. Therapy of pancreatic duct obstruction by endoscopic stenting in patients where alternatives are precluded can be successful in relieving symptoms, preventing infection and preserving pancreatic function. The pigtail-flap pancreatic stent is patented by D.S. Zimmon and licensed to Wilson-Cook Medical Inc.

Biliary Disorders • G2092 IMPAIRED CI-/HCO3" EXCHANGER ACTIVITY, ASSOCIATED WITH DEFECTIVE CI° CONDUCTANCE, CAN BE RESTORED BY PURINERGIC STIMULATION IN A CYSTIC FIBROSIS PANCREATIC DUCT CELL LINE (CFPAC-1). A. Zsemberv. M. Strazzabosco*, J. Graf. Department of General and Experimental Pathology, Univ. of Vienna, Austria, *Inst. of Internal Medicine, Univ. of Padova, Italy. Background and aims. The cAMP-dependent regulation of CI- conductance is defective in cystic fibrosis (CF). In CF patients pancreatic HCO3- secretion is reduced. This defect is attributed to derangement of the concerted action of CI-/HCO3- anion exchanger (AE) and CI- conductance in the apical membrane of pancreatic duct cells. In order to test for this cooperative function we compared measurements of AE activity in normal and CF pancreatic cell lines (PANC-1 and CFPAC-1 respectively) with parallel measurements of membrane ion conductance in whole cell patch clamp experiments. Methods: Intracellular pHi was measured by BCECF microfluorimetry. AE activity was assessed from the rate of pHi recovery. Data are presented as ApHi/min. Whole cell currents were measured after intemal perfusion with KC1 rich solution at different test potentials between -100mV and +100mV. Data are presented as chord conductance (nS/pF). Results: The basal pHi was higher in CFPAC-1 than in PANC-1 cells (7.32 ±0.08 n=6 vs. 7.15-+ 0.15 n=6) and basal AE activity was higher in PANC-1 than in CFPAC-1 cells (0.133 ± 0.025 n=6 vs. 0.088 ± 0.044 n=7). Raising intracellular cAMP levels [forskolin, IBMX, DBcAMP (cAMPmix)] caused a 55% increase in AE activity in normal but not in CF cells. Blocking C1- channels with 10taM

NPPB or cell depolarization ([K÷]e=70 mM) prevented the stimulatory effect of cAMPmix in PANC-1 cells, indicating that conductive CI- flux is required for AE activation. In CF cells, purinergic stimulation (administration of 10pM ATP) or raising [Ca2+]i to approx, llaM with ionomycin, resulted in an increase of AE activity by 53% and 62% respectively, indicating a cAMPindependent alternative pathway for AE activation. In PANC-1 ceils, cAMPmix increased the membrane chord conductance at both negative and positive clamping voltages (between -100mV and -80mV: from 0.041 ± 0.026 n=8 to 0.078 ± 0.025 n=5; between +80mV and +100mV: 0.96 ± 0.61 n=8 to 4.60 ± 2.73 n=5). No effects of cAMPmix on membrane conductance were detected in CF cells, but in these cells administration of 10pM ATP or internal perfusion with l~tM Ca2+ increased the conductance at both, negative and positive clamping voltages and shifted Erev towards Ecv In addition, PANC-1 ceils exhibit a large voltage-dependent outwardly rectifying current which was blocked by administration of 5mM Ba2÷ in the bath or by substitution of K÷ in the pipette solution. Conclusions: PANC-1 (control) and CFPAC-1 (CF) cells are both endowed with the CI-[HCO3- anion exchanger. Activity of AE requires the presence of a parallel CI- conductance. In PANC-1 cells this conductance is provided by the cAMP-activated CFTR gene product. In CFrR-deficient CF ceils, a purinergic stimulated and [Ca2÷]i-activated C1conductance is available to substitute for the CFTR defect. An additional role of K÷ conductance in modulating anion exchange activity is suggested. This research was supported in part by a fellowship from EASL