Microsatellite instability (MSI) and K-ras mutation are independent predictors of survival in colorectal cancer

M1346

Co nt r~-~il e aoti vity

20

Octreotide Improves Reperfusiou-luduced Oxidative Organ Damage in Acute Abdominal Hypertension

t--t. t ....... I|~um t t

Ayhan Kacmaz, All Polat, Yilmaz User, Metro Tilki, Sirri Ozkan, Goksel Sener

M1344

Acute abdominal hypertension (AAH) may lead to abdominal compartment syndrome, which has high morbidity and mortality. Ischaemia and reperfusion injury should play an important role in worsening this pathological condition. The main goal of the management of abdominal compartment syndrome is to lower the intra-abdominal pressure despite reperfusion injury. Octreotide (OCT) lowers the splanchnic perfnsion. The aim of this study was to investigate whether OCT improves the reperfusion injury after decompression of acute abdominal hypertension or not. Wistar albino rats of both sexes were used for the experiments. Under ketamine anesthesia, intraperitoneal cannulas were inserted percutaneonsly and'then exposed to intra-abdominal hypertension by air insufflation where pressure was increased to 20 mmHg (Group A) for 1 hour. In group B, rats were allowed 1 hour of reperfusion period by decompressing of the intra-abdominal pressure. OCT (Sandostatin-Novartis) was administered (50 micro g/kgip; Group C) jnst before the intra-abdominal pressure was decompressed. Control groups of rats had no manipulation. At the end of the experiment liver and intestinal tissues levels of malondialdehyde (MDA) -an index of lipid peroxidation- and glutathione (GSH) -a key to antioxidant- were estimated. The results demonstrate that tissue levels of MDA were elevated, while GSH levels were reduced in both group A and B. On the other hand, in group C, OCT reduced the levels of MDA and increased the GSH levels significantly. Our results implicate tl'Lat AAH causes oxidative damage to the both liver and intestinal tissues. On the other hand, OCT, by reducing splanchnic perfusion could improve the reperfusion-induced oxidative damage by contronmg the reperfusion of abdominal organs. Therefore, its therapeutic role as a "reperfusion injury-limiting" agent must be further elucidated in AAH-induced abdominal organ injury.

Melatonin Ameliorates Oxidative Intestinal Damage Induced by Acute lntraabdominal Compartment Syndrome in the Rats

M1347

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10

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1

2

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4

5

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Goksel Sener, Yilmaz User, Ayhan Kacmaz, Berrak C. Yegen

Short Bowel Syndrome and Crohn's Disease

Acutely increased intraabdominal pressure (IP) can lead to multiple organ failure, including the intestinal tissue. Since blood flow to intraabdominal organs is reduced by high venous resistance, ischaemia+reperfnsion (1R) injury plays an important role in the pathogenesis of nitraabdominal compartment syndrome (ICS). Melatonin, a secretory product of the pineal gland, is shown to have free radical scavenging and antioxidative properties in several oxidative processes. The objective of this study was to examine the potential protective properties of melatonin on the oxidative intestinal injury in a rat model of ICS. Under ketamine anesthesia, an arterial catheter was inserted intrapenoneally and using an aneroid manometer connected to the catheter, IP was kept at 20 mmHg (ischemia group; [) for 1 hour. In the IR group, IP applied for an hour was decompressed and 1-hour of reperfusion period was allowed. In another group of IR, melatonin was administered (10 mg/kg, ip) immediately before the decompression of IP. Control rats had normal IP. The levels of malondialdehyde (MDA) -an index of lipid peroxidation- and glutathione (GSH) -a key to antioxidant- were estimated in the intestinal tissues. The degree of tissue neutrophil infiltration was determined by measuring myeloperoxidase (MPO) activity. The results demonstrate that tissue levels of MDA and MPO activity were elevated, while GSH levels were reduced in both I and IR groups (p<0.05-0.001). Melatonin treatment to I/R group reversed these changes (p<0.01-0.001). Our results implicate that melatonin, with its antioxidant and free radical scavenging properties, ameliorates the reperfusion-induced oxidative damage of the intestinal tissue. In conclusion, the results of the present study suggest that the therapeutic value of melatonin as a "reperfusion injury-limiting" agent must be considered in ICS.

Ion S. Thompson, Kishore R. lyer, John K. Dibaise, Renee L. Young, Cindy R. Brown, Alan N. Langnas Background: Patients with Crohn's disease are at high risk for recurrent disease and often undergo multiple operations. Current surgical and medical management aims at preventing extensive resection. Our aim was to evaluate surgical management and outcome of patients with Crohn's disease who develop short bowel syndrome(SBS). Methods: We reviewed the records of 160 adult patients with SBS evaluated over a 20-year period. 28(18%) had Crohn's disease. SBS was defined as an intestinal remnant < 180 cm with associated malabsorption. Initial presentation, time to SBS, surgical therapy and nutritional support were evaluated. Results: There were 18 women and 10 men ranging from 18 to 62 years of age. 17(61%) presented initially with ileocolonic disease, 6(21%) with colonic disease and 5(18%) with small intestinal disease. The interval from initial diagnosis to development of SBS ranged from 2 to 32 years, with 20(71%) patients having an interval >15 years. The number of resections leading to SBS varied from 2 to 12 with 23(82%) patients having < 4 resections. 17(61%) patients had an ostomy. Remnant length was <~ 60 cm in 9 patients, 60-120 cm in 6 patients, and > 120 cm in 13 patients. Only one patient underwent stricturoplasty prior to developing SBS. Four patients were initially diagnosed as ulcerative colitis and underwent either an ileoanal procedure(n = 3) or a continent ileostomy(n = 1) which were subsequently resected. 19(68%) patients require parenteral nutrition. Three patients have undergone reversed intestinal segment to slow intestinal transit. Two patients underwent intestinal transplantation. Two patients have died. Conclusion: Crohn's disease remains a common cause of SBS. SBS develops over a long period of time after multiple resections. Error in initial diagnosis and aggressive resectional therapy contributes to development of SBS in these patients. Selected patients are candidates for surgical therapy for SBS.

M1345

Afferent Intestinal Mechanosensitivity During Postoperative lleus In The Mouse Jejunum Mario Mueller, Martin Kreis, Joerg Glatzle, Wolfgang Kunae, David Grundy

M1348

Introduction: Nonobstructive liens, signifying the impairment of coordinated propulsive intestinal motility, remains a frequently documented and almost inevitable consequence of open abdominal surgery and sepsis. Despite the frequency and major impact of liens on morbidity and mortality, the underlying mechanisms are still ill defined. Animal models suggest that both neuronal and local inflammatory responses contribute to intestinal liens. (Bauer AJ et at. 2002). Altered sensory mechanisms are believed to contribute but this has not been investigated directly. Here we describe the results of an in vitro electrophysiological study of mesenteric afferent sensitivity in jejunal segments from mice with postoperative ileus. Method: C 57 BL/6 mice were anaesthetized with enflurane inhalation and at laparotomy the small bowel was either manipulated to induce ileus or left untouched (sham controls). 24h later, 3-4 cm segments of jejunum with mesenteric arcade attached were removed and bathed in Krebs buffer at 34~ Extracellular muhninit mesenteric afferent recordings were made from one paravascular nerve bundle during luminal distension with saline (0.5 ml/ min to a max. pressure of 60mmHg). Data were quoted as mean increases above over _+ SEM and were compared with Students' t-tests as appropriate. Results: Segments of ileus bowel were dilated and flaccid while sham controls showed phasic increases in intraluminal pressure. There was a significant difference between/lens and sham segments in spontaneous discharge (17_+ 1 vs. 12_+2 imp/s, P=O.02, N=6), but not in the peak response to ramp distension (D at 60mmHg = 85_+ 6.0 vs. 70-+ 4 imp/s respectively, P = 0.07). However, the afferent response profile was different in ileus compared to sham control with a significantly increased response at low distending pressures (2-20 mmHg) compared to sham control (9 + 2 vs 18 _+ 2imp/s, P<0.05). The distending volume at 60 mmHg was greater in postoperative liens animals than in controls (0.44 +- 0.08 vs 0.36 -+ 0.02ml). Conclusion: Small bowel manipulation leads to an inhibition of jejunal motility and augmented afferent sensitivity particularly to low distending pressures. The increased sensitivity to low threshold distension might be due to local inflammatory processes or secondary to altered compliance. Supported by the fortuene-program University of Tuebingen

SSAT Abstracts

MicrosateUite Instability (MSI) and K-ras Mutation Are Independent Predictors of Survival in Colorectal Cancer Garrett M. Nash, Mark Gimbel, Daniel R. Nathanson, Alfred T. Culfiford IV, Mathew J. D'Alessio, Zhaoshi Zeng, Philip B. Paty INTRODUCTION: Microsatellite instability (MSI) and mutation of the K-ras gene are two well-described genetic alterations in colorectal adenocarcinoma (CRC) which may be useful in estimating prognosis and managing patients following surgical resection. We examined the relationship of these markers to metastatic disease progression and survival. METHODS: Using polymerase chain reaction and figase detection reaction (PCR/LDR), K-ras mutation status in codon 12 and 13 was assessed in 330 patients after colon resection for CRC. MSI was assessed in a subset (n=265) of those patients with available tissue using fluorescent detection of PCR products. All clinical stages were represented (I; 48 patients, ll; 106, III; 71, IV; 105). Median age was 67 years (31-93). Median follow up was 4 years (0-12.4). RESULTS: MSI and K-ras mutation were detected in 11% and 35% of cases, respectively. There was a lower prevalence of MSI in patients presenting with stage IV cancer compared to stages 1-11I (2 v. 16%, p = 0.001) and improved disease specific survival for patients with MS1 tumors compared to those with microsatellite stability (MSS) tumors (81 v. 58%, p = 0.01). Prevalence of K-ras mutation did not vary with stage (I; 25%, II; 37%, Ill; 34%, IV; 37%); however, it was a marker of poor survival (5-year survival 52 v. 65%, p = 0.006) and remained a sigmficant marker independent of clinical stage (p = 0.007). In the entire cohort, both markers were independent predictors of survival using Cox regression analysis (K-ras relative risk 1.7 (1.1-2.4), p = 0.01 and MSI relative risk 0.34 (0.14-0.84), p = 0.02). Among younger patients (age ~ 60, n = 94), both markers remained highly informative. No young patient with an MSI tumor died (p = 0.005) and 5 year survival for young patients with wild-type K-ras tumors (stages 1-11I)was 95% (p = 0.007). CONCLUSIONS: MSI and K-ras are independent and informative markers for predicting metastatic potential and clinical outcome in colorectal cancer. Genetic markers hold trer~endous potential to enhance clinical staging and provide targets for gene-directed therapy.

A-800

M1351

Rleugt|

MSI

~

all pMlents prevalence% 5-yr surviva/%

11 81

89 58

age < 60

13 1O0 16

87 SO 84

p = Or~5

84

86

p = ns

Itage I.III

prevalence% 5-yrsurvival% prevaleflce%

5-yr survival % MUT=mutaUon WT=wtld-type

p = 0.02

~ MUT 35 52 30 38 33 74

Immunohistochemistry in the Evaluation of Response and Prognosis in Distal Rectal Cancer Patients Treated by Neo-adjuvant Radio/Chemotherapy Ulysses Ribeiro Jr., Venancin F. A. AIves, Pedro M. S. Souza, Carlos E. P. Corbett, Adriana V. Safaile-Ribeiro, Viviane Rawer, Sueli Nonogaki, Joaquim Gama-Rodrigues, Angelita Habr-Gama

K-ru 65 65 70 64 67 88

p = 0206

p = 0.004

Preoperative radio/chemotherapy may be used to treat distal rectal cancer patients. It is desirable to better predict the biologic behavior and the therapeutic response. Aim: To determine the relationship between immunoexpression of molecular markers with: clinicalpathological parameters, treatment responsiveness and prognosis. Methods: The patients underwent preoperative radiotherapy (5,040 cGy) and chemotherapy (5-fluorouracil + leucovorin). The complete responders were followed without surgical treatmefit, while resection was performed for the partial responders. Histologic sections of the tumors from 72 patients were immunostained for p53, Ki-67, CD34, thymidylate synthase (TS) and p16INK4a. The median follow-up was 62 months. Results: Response to radio/chemotherapy was classified as partial in 77.8% and complete in 22.2%. p53 immnnoexpression was detected in 51.4% of the tumors; Ki-67 proliferative index had a media of 50.3 + 19.6 (10 to 90%); the microvascular density (CD34) revealed a media of 48.3 + 23 vessels (17 to 120); immunostaining for TS was negative or weak in 28.7%; while p161NK4a was negative in 24.6%. There was a statistical association between Ki-67 and CD34 with histology; and between p'16INK4a and the rectal wall infiltration (T), and lymph node involvement (N). Intense TS immunoreactivity was associated with the presence of residual tumor after radio/chemotherapy. High proliferative index and p53 immunopositivity were associated with disease recurrence. After multivariate analysis by Cox and logistic regression it was observed that, besides TNM stage, the immunoexpression of p53 and Ki-67 were independent variables for recurrence, with relative risk of 12.7 tbr high Ki-67 index, 5.98 for p53 and 3.75 for stage IlL Based on estimation model of recurrence, when the patient was in the stage IIl, with p53 positive and high Ki-67 index, the recurrence probability was 97.2%; when the patient was in stage [, with negative p53 and low Ki-67 index, the estimated recurrence rate was 1.9%. p53 positive patiems and high Ki-67 index had diminished disease-free interval and overall survival. Conclusions: 1. TS immunoexpression predicted the response to radio/chemotherapy; 2. p53 e Ki-67 were of prognostic value; 3. The use of immunohistochemica] panel permitted the construction of algorithm with potential impact in the therapeutic management and evolutive surveillance of patients with distal rectal adenocarcinoma.

p = 0.002

M1349 Inhibition of Phosphatidylinositol 3-Kinase Attenuates the Severity Of Dextran Sulfate Sodium Induced Intestinal Inflammation Carlos A. Muriilo, Qing-Ding Wang, Buckminster Farrow, Wesley Marquant, B. Mark Evers inflammatory bowel disease (IBD) is a chronic, inflammatory condition of the intestinal mucosa characterized by acute exacerbations with varying periods of quiescence; the etiology and signaling pathways contributing to IBD are poorly defined. Phosphatidylinositol 3kinase (P13K), a ubiquitous lipid kmase, composed of a regulatory subunit (p85) and a catalytic subunit (p110a, b, d, or g), regulates a number of important cellular processes; P13K, acting through Akt/PKB, may also contribute to inflammatory conditions. The purpose of this study was to determine whether PI3K is activated in IBD and, conversely, whether PI3K inhibition decreases inflammation. METHODS. 1BD was induced in 2-month-old C57/ bl mice using a 5% dextran sulfate sodium (DSS) solution, given ad libitum for 7 days. Mice were separated into three groups: control, 5% DSS, and 5% DSS with the PI3K inhibitor wortmannin (1.5 mg/kg, po). Mice were sacrificed after the 7-day treatment period and the small and large bowel excised for histology and extracted for protein. Protein was analyzed by Western blots for PI3K isozymes (p85a and p110g), Akt, phosphorylated (ie, activated) Akt (pAkt), and GSK-3 (a downstream target of Akt). RESULTS. Administration of 5% DSS resulted in generalized bowel wall edema of the small and large bowel, with evidence of inu'aluminal hemorrhage in the colon; the lesions are characterized by leukocyte infiltration, mucossl hyperplasia, and submucosal edema. Treatment with wortmannin decreased DSSinduced intestinal inflammation and leukocyte infiltration. Interestingly, wortmannin alone caused mild bowel wall edema v~thout hemorrhage and leukocyte infiltration. Inflammation induced by 5% DS8 increased the levels of the p 1 lOg isoform of PI3K and pAkt; wortmannin decreased the induction of these proteins. CONCLUSIONS. IBD is associated with increased PI3K/Akt activation; inhibition of PI3K attenuates the intestinal inflammation. Our results would also suggest that the response is dependent on specific P13K isoforms since wortmannin alone can produce a mild inflammation. These findings suggest that selective inhibition of PI3K isoforms may represent a novel strategy to decrease the inflammation associated with ]BD.

M1352 Modified Salmonella, a Potential Agent For in Situ Colon Cancer Therapy, Delays Growth of Established Tumors and Induces an Increase in the Number of T Cells lrena Kirman, Nataha Poltoratskaia, Richard L Whelan VNP20009, an attenuated strain of Salmonella typhimurium, has been modified such that it does not produce lipoplysacharide. It has been shown to accumulate in tumor tissue and, therefore, may be useful as an oral-cancer agent. The goal of the present work was to evaluate the effect of VNP20009 on the growth of established CT26 murine colocarcinomas and on immune cells in tumor bearing animals. Methods: Syngeneic CT26 murine colon cancer cells, 10(5)/mouse were injected s/c into 30 female BALB/c mice and allowed to grow. Two weeks later, 26 mice with equal tumor sizes (0.07-0.08 cm2) were selected and randomly distributed to either the VNP20009(n = 15) or the control group(n = 11 ). VNP20009 group mice received peritumora[ injections of 2x10(7) modified salmonella in saline on two occasions ] week apart. Control group mice received saline injections per the same schedule. Two weeks after tumor establishment, the animals were sacrificed and their tumors excised, measured, and assayed for salmonella. The splenic cells were recovered and the number of T- and B- cells determined via flow cytometry using specific antiboides. Statistical Analysis was performed using the Mann-Whimey test. Results: Mean tumor volumes were significantly lower in the VNF20009 group mice (0 75 + A0.49 cm3) than in the control group (3.44 +/1.48 cm3, p<0.0001). On average, there were 1.92+A1.70x10(7) Salmonella per neoplasm.Total number of splenic T cells was significantly higher in VNP20009 mice,12.63 + A 2.81x10(7) compared to control mice,6.06 4/-1.29 x10(7). The number of B cells in treated and control mice was comparable. Conclusions: Multiple peritumoral injections of VNP20009 significantly inhibit the growth of established tumors in this murine model. This effect may depend, in part, on a salmonella induced T cell proliferative response. The overall duration of this effect and the efficacy of other routes of administration need to be determined.

M1350 Identification of Differentially Expressed Genes in invasive and Central Colorectal Cancer Maya Heinze, Joern Groene, Birgit Weber, Irina Klaman, Henrik Kinnemann, Thomas Braemmendorf, Heinz J. Buhr, Benno Mann Purpose: It has been shown, that gene expression varies within one tumor entity. Combining chiparray-technology and Laser Capture Microdisseetion (LCM) enables expression analysis of thousands of genes of different, well defined tumor areas. Aim: To identify and compare differentially expressed genes (DEGs)in vital tumor cells of the invasion front and the central tumor. M&M: Homogeneous normal epithelial cells (E) and carcinoma cells (invasion front (IF)and central tumor (RT)) from fresh frozen colorectal tumor and corresponding normal tissue from 7 patients undergoing colorectal surgery in our department were isolated by LCM. Extraction of Poly(A) mRNAs, linear amplification and hybridization of Affymetrix Genechips. Selection of DEGs in IF vs. E and RT vs. E using statistical ranks. Validation of sequences (CPA, MTN, rt-PCR, IHC). Results: 34 DEGs were identified in the invasion front and 11 DEGs in the central tumor following reduction and selection of the 6117 chip sequences. 23 DEGs were found to be differentially expressed in both, IF and RT. Further classification of probesets was achieved by data mining and public data banks(Tab.i). Conclusion: The majority of DEGs identified in this study were found either in IF or RT representing the heterogenons gene expression within tumor tissue LCM is essential in chiparray based gene expression analysis for specific tasks dependend on defined areas within the inhomgeneons tumor tissue (e.g. neoangiogenesis, protelysis). Further investigation has to evaluate clinical and therapeutic relevance of these differences. DEGsIn Invukm front =nd c m l

r

A Better Cell Cycle Target for Gene Therapy of Colorectal Cancer: Cyclin G Rodrigo Perez, Nancy Wu, Adam A. Klipfel, Robert W. Beart Jr

tumor

b'msmebrlneou$

IF 57 20% 5% 21%

RT 134 8% 3% 14%

IF&RT 23 8% 4% 17%

IdnNu

8%

14%

8%

phosphatHe= unknown

2% 44%

6% 55%

5% 58%

DEG$ lecr~Kl

M1353

PURPOSE: To evaluate the expression or over expression of cyclin G in colorectal neoplasia, which may be a more frequent event than cyclin D1 during the cell cycle and thus may have a more enhanced therapuetic and diagnostic potential in treating colorectal cancer. METHODS: Ninety formalin-fixed, paraffin-embedded human colon and rectal specimens were obtained from the Pathology Department of Norris Cancer Center/University of Southern California (Los Angeles, Ca). The tissues had been obtained after surgical resection between 1995 and 2001 and had been processed by routine clinical histopathologic methods. RESULTS: 91% of colorectal tumors had cyclin G over expression. These cyclin positive patients were evenly distributed between men and women, and between tumor location, 36% rectal tumors, 34% right sided tumors. 32% were well differentiated, and 66% were moderately differentiated. Thirty patients (38%) had stage I disease, sixteen (20%) had stage II disease, twenty-five (32%) had stage If[ and seven patients (9%) had stage IV disease. Eight patients (10%) in this group had recurrent disease during follow-up. There was no correlation between Cycfin G over expression and clinical and pathological characteristics. Cyclin D1 over expression was found to be present m only 42% of colorectal adenocarcinomas. There was no correlation between cycfin D 1 over expression and clinical and pathological

A-801

SSAT Abstracts