Sequential vs. cross-sectional measurements of 6-mercaptopurine metabolite levels in children with inflammatory bowel disease

using a Mulitspect camera with high-resolution collimators at 1 and 3 hours. At least 48 hr later, an egg meal labeled with 1 mCi Hqn DTPA was consumed with images taken using medium energy collimators at 24 and 48 hr later. The % marker retained in the following regions was determined: ascending colon/hepatic flexure (AS), transverse colon (IV), descendingcolon (DS) and rectosigmoid/rectum (RS). Results: During active IBD, the enema is dispersed into the RS and DS in 75% of patients by 3 hours, with the same pattern seen in remission. During active disease, only 50% of patients had migration of the oral marker into the OS by 48 hours, with virtually no label in the RS where disease was most active. The pattern of colonic distribution of the oral marker changed during remission, with 100% of patients having distribution into the OS, and 75% of patients having movement of colonic contents into the RS by 48 hours post ingestion. Summary: Topical enemas are distributed to the primary area of disease in left-sided IBD. Oral markers move into the DS colon during disease in approximately 50% of patients, but advance into the RS during remission in 75% of subjects. Conclusions: With topically active drugs, enematherapy provides the best distribution in active left-sided IBD, but in remission, either oral or rectal therapy distributes medication to the area of prior inflammation. Fundedby an unrestricted educationalgrant from Solvay Pharmaceuticals.

18 Red Cell Mean Corpuscular Volume (MCV) Correlates With 6 Thioquanine liucleotide (6TG) Levels During Azathioprine Or 6-MP Therapy For Crohn's Disease Brenda Jobson, Austin Garza, Charles A. Sninsky, Vanderbilt Univ, Nashville, TN Background:Azathioprine(Aza)and 6-MP are effective at inducing and maintaining remission in Crohn's disease. There are substantial differences between patients regarding oral bioavailability and metabolism of these drugs. Our clinical experience supported by reports in the literature led us to postulate that the red cell MCV may be a reliable and inexpensive method to optimize dose titration in patients with normal B12 and folate levels in Crohn's disease. Interestingly, a recent study reports that the change in MCV correlates with 6-TG levels in patients with various immune disorders (j Lab Clio Med 135:256,'00). The exact mechanism responsible for the change in MCV is not understood. Methods: The aim of our study was to perform a retrospective analysis, in 18 consecutive patients treated with Aza or 6MP for Crohn's disease, to examine whether there was a correlation between red cell MCV (fL) and 6TG levels (pmoV8 x lOS red cells}. CBCs and 6TG levels were performed while on a stable dose of Aza or 6MP in patients not deficient in iron, folate or B12. Data are expressed as the mean + SEM. Correlation coefficient was determined by linear regression. Results:The mean values for the individual paramenters were as follows: WBC 8.4 + 0,7; Hgb 12.5 + 0.4; MCV 94+3; %lymphocytes 14+2; total lymphocyte count 1101 +166 and 6TG level 296+64. We found a statistically significant correlation (r = 0.62; p< 0.02) between the MCV and 6TG tevets. There was no significant correlation between 6TG levels and the WBC, %lymphocytes or total lymphocyte count. In a separate group of 64 Crohn's patients treated with Aza or 6MP, in whom 6TG levels were not measured, we determined that the MCV increased by ->9 in 64% of patients in remission off steroids. In contrast, only 23% of patients who were still symptomtic or in whom steroids could not be tapered had an MCV increase ->9. Conclusion: There is a significant correlation between red cell MCV and 6TG levels in Crohn's patients taking kza or 6MP, No correlation could be identified with 6TG levels and WBC or total lymphocyte count. Our results taken together with data from the literature indicates that a ~MCV of ---9 can be used to anticipate a favorable therapeutic response and this probably reflects a 6TG level of ->235. In that MCV changes typically don't occur for 3 to 6 months, 6TG levels may be necessary during this early period and later on to monitor compliance and toxicity. It is also possible that the L~MCVreflects a therapeutic mechanism separatefrom 6TG.

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COX-2 Expression in Fecal Colonocytesfrom Patients wHh InflammMow Dowel Disease(IBD) Sara A. Lagerholm, JHU Sch of Hygiene and Public Health, Sinai Hosp, Baltimore, MD; Sedhir K. Dutta, Sinai Hosp, Univ of Maryland Sch of Medicine, Baltimore, MD; Nipun B. Merchant, Sinai Hospital/Johns Hopkins Sch of Medicine, Baltimore, MD; Padmanabhan P. Nair, JHU Sch of Hygiene and Public Health, Baltimore, MD Background and aims: IBD (Idiopathic ulcerative colitis and Crohn's disease) is a eflrooJc disorder with frequent acute recurrences and associated complications. Diagnosis of IBD is based on clinical, radiological, endoscopic and histologic evidence and is often difficult to make. Repeated evaluations for IBD are cumbersome and expensive, hence a noninvasive method for diagnosis of IBD is critically needed. COX-2, an inducible form of cyclooxygenase, is an enzyme involved in prostaglandin synthesis (Oncogene1999,18:7908). Recent evidence suggests that COX-2 expression is increased in colonic mucosal biopsies obtained from patients with IBO (AJG 1997, 92:1170; Gastroenterology 1998; 115:297. We hypothesized that COX-2 expression in patients with IBD can be detected in fecal colonosytes, and can be used as a noninvasive marker for the presence of IBD. Methods: Stool samples were collected 3 months apart from 8 patients with IBD. Stool samples were also collected from 4 healthy control subjects. Colonocytes were isolated from stool samples by a modified procedure adapted from our earlier work (FASEB 1991, 5:2856; Int J Cancer 52:347). mRNA, isolated from these cells was used to generate first strand cDNA by RT-PCR, followed by PCR amplification of COX-2 using primers yielding a 276bp fragment. Similarily, as a reference marker for epithelial cells, cytokeratin-19 was amplified. All PCR products were analyzed by gel electrophoresis. Results: Marked COX-2 expression was observed in all 8 patients with IBD while no expression of COX-2 was seen in healthy control subjects. Three months after initial sampling, expression of COX-2 was still persistent in fecal colonocytes from all IBO patients. In a~)subjects, the presence of epithelial cells was confirmed by the ~roonstrated expression of cytokeretin-19. Conclusion: These observations suggest that COX-2is expressed in fecal colonocytes in patients with IBD. This expression, in fecal colonocytes, reflects an inflammatory response in the GI tract and may serve as a noninvasive marker for IBD

19 VEGF Is RqMMod by the WNT Pathway in Colon Cancer Xiaopo Zhang, John P. Gaspard, Daniel C. Chung, MAGen Hosp, Boston, MA Background:Anoiooenesis plays an essential role in tumor progression, and the angiogenic growth factor VEGF is critical for this process. The precise molecular mechanisms underlying the upregulation of VEGF in colon cancer are not well described. The Wnt signaling pathway is activated in the majority of colonic tumors. All the relevant targets of the Wnt pathway have not yet been identified, and we sought to determine the role of Wnt signaling in the regulation of VEGFexpression. Methods: RT-PCRand immunohistochemistry were performed to determine expression levels of VEGFmRNA and protein. Luciferase activity of VEGFreporter plasmids was measured 48 hours after transient transfection. Site-directed mutagenesis was performed ofif~ng the Quick-Change protocol (Strafagene) and mutations were confirmed by direct sequencing. Results: VEGF is upregulated in premalignant colonic adenomas. In colon cancer cell lines with activated Wnt signaling, dominant negative TCF4 inhibited the expression of both VEGF and a 2.8 kb VEGF promoter construct by 55-80%. Similarly, reintroduction of wild-type APC suppressed VEGF expression by 45%. In Hela cells with an intact Writ pathway, constitutively active ~-catenin upregulated the VEGF promoter as much as 30-fold, and this stimulation was blocked by dominant negativeTCF4.Site-directed mutagenesis identified two key TCF4-binding elements in the proximal VEGF promoter that mediated this effect. Concurrent expression of mutant K-ras enhanced the effects of/3-catenin on the VEGF promoter, and this enhancementwas blocked by dominant negative TCF4. Conclusions: VEGF is upregulated early in colon cancer development, at the stage of the benign adenoma. Wnt signaling upregulates VEGFthrough TCF4-binding elements in its promoter. Furthermore, K-ras enhances Wnt pathway stimulation of VEGF expression. VEGF is thus a novel gene target of the Wnt signaling pathway, and this implies an important role for angiogenesis in the premalignant stages of colon cancer.

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Sequential vs. Cross-sectional Measurements of 6-Mercaptopudne Metabolite Levels in Children with Inflammatory Bowel Disease Puneet Gupta, Ranjana Gokhale, Barbara S. Kirschner, Univ of Chicago, Chicago, IL Background & Aims: Some authors suggest that efficacy of 6-mercaptopurine (6-MP) in patients with inflammatory bowel disease (IBD) correlates with circulating 6-thioguanine levels >235 picomoles per 8 x 10~ RBC. We evaluated the relationship between 6-MP metapolite levels and diseaseactivity in children and adolescentswith IBD. Methods: Therapeutic response (as assessed by PCDAI for CD and Truelove and Witts index for UC), hematologic and hepatic parameters were determined in 101 children with IBD from a single center and compared with 6-MP metabolite levels. Results: Cross-sectional analysis did not show a significant correlation between 6TG levels and clinical response (P=0.18, Figure 1). The likelihood of therapeutic response did not increase significantly at 6TG levels >235 (Odds Ratio 1.7, P = 0.11). However, sequential measurementsof 6-MP metabolite levels in 50 patients showed that increasing 6TG levels correlated significantly with higher likelihood of remission(P = 0.04). Leukopeniawas significantly associated with high 6-TG levels (P = 0.03) but not with clinical response (P =0.2). 6MMP levels >5700 were not significantly associated with hepetotoxicity (P= 0.2). Conclusions: Our data suggest that there may not be a consistent range of target 6TG levels in pediatric patients with 180. However sequential monitoring of 6MP metabolite levels in individual patients should allow dose escalation and induction of remission while minimizing the risk of toxicity.

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2o Tumor-Host Interaction Modulates Production Of Antiangiogenic Factors From Human Pancreatic Cancer Yoshikazu Tsuzuki, Dai Fukumura, Rakesh K. Jain, MAGen Hosp, Boston, MA BACKGROUND& AIMS: There is growing body of evidencethat host organ microenvironmant affects gene expression and physiological functions in solid tumors. We have recently found that tumor-host interaction in pancreas promotes VEGFexpression and tumor growth. Balance of pro- and anti- angiogeni¢ factors governs angiogenesis and tumor growth. It has been hypothesizedthat anti-angiogenic factors suppress angiogenesisand tumor growth at secondary site due to their longer half lives. In this study, we investigated the effect of tumorhost interaction in pancreas on circulatory endogenous pro- and anti-angiogenic factors, angiogenesis, and tumor growth at a secondary site using pancreatic edenocarcinoma (PAl xenoOraff models at odhotopic and ectopic sites. MATERIALS & METHODS: HPAC, a poorly differentiated human PA, was xenografted into the pancreas (odhotopic site) or left abdominal wall (ectopic site) of immunodeficient Rag2 knockout mice. First, tumor growth kinetics for both sites were monitored for eight weeks. Secondary HPAC was inoculated at right flank whenprimarytumor reachedapproximately 2O0mm3and was monitored for 4 weeks. Vascular density, vessel diameter were measured in the secondary tumors by fluorescence intrevital microscopy and vascular volume density was calculated, VEGF, bFGF, Endostatin, and TGF/]1 in plasma from tumor-bearing mice were measured by ELISA. Angiostatin and thrombosponifin-1 (TSP-1) in plasma was measured by Western blot. RESULTS: Tumors in orthotopic site grew faster than those in ectopic site beyond five weeks. The orthotopic not ectopic primary HPACtumor significantly suppressed the secondary tumor growth and angiogenesis

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