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Can clinical and histological parameter predict prognosis in patients with metastatic neuroendocrine gastroentetropancreatic (GEP) tumors?
GASTROENTEROLOGY Vol. 118, No.4
A514 AGA ABSTRACTS
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POST-OPERATIVE SERUM MUTANT K-RAS· A SENSITIVE MARKER OF DISEASE RECURRENCE IN PATIENTS WITH COLORECTAL CANCER.
PROGNOSTIC FACTORS IN PATIENTS WITH NEUROENDOCRINE GASTROENTEROPANCREATIC (GEP) TUMORS.
Barbara M. Ryan, Ross McManus, Jacqueline S. Daly, Francois LeFort, Napoleon Keeling, Donald G. Weir, Dermot Kelleher, St James's Hosp and Trinity Coli, Dublin, Ireland. Background: It has been shown that mutant k-ras can be detected in the plasma or serum of patients with colorectal (CRC) and other cancers. It is not known whether this mutant DNA can also be detected in the circulation of CRC patients post-operatively, and if so, whether it correlates with disease recurrence. Aims: We followed a cohort of CRC patients longitudinally post-operatively, to determine whether serum mutant k-ras could be detected post-op and if so, whether this predicts or predates clinical recurrence of disease. Methods: Serum or plasma was collected at intervals post-operatively in a total of 92 CRC patients with disease ranging from dysplastic tubulovillous adenoma to Duke's C cancer. DNA was extracted as described previously to this meeting. Mutant k-ras was detected using an enriched-PCR-RFLP method, and confirmed by direct sequencing. Detailed clinical, radiological and endoscopic follow-up was carried out in all patients. Results: 47 of the 92 patients (51 %) were k-ras mutation-positive in the primary tumor. Of these 47 patients, 15 (32%) have become serum k-ras mutant-positive during the post-operative follow-up period and 5 patients (33%) in this group have developed overt clinical and histological recurrence. In one case, serum mutant k-ras positivity pre-dated clinical recurrence by I year. No patient who was mutation-positive in the primary tumor and who remains serum mutant- negative has developed clinical recurrence. Of the 45 patients who were k-ras mutation-negative in the primary tumor, 6 (13%) have developed clinically recurrent disease, but all remained persistently serum mutant k-ras negative during the follow-up period. Conclusions: We demonstrate that serum mutant k-ras can be detected in the plasma/serum post-operatively, in a proportion of patients with CRC. 33% of these patients have developed clinical recurrence during the follow-up period, and in one case serum mutant-positivity pre-dated clinical recurrence by I year. These findings suggest that serum mutant k-ras may prove a clinically useful, early, sensitive and specific marker of disease recurrence in patients with CRC, possibly detecting disease at the micrometastatic stage.
2742 RELATIONSHIP BETWEEN FAS LIGAND EXPRESSION, DUKES STAGE AND SURVIVAL IN HUMAN COLORECTAL CANCER. Katherine M. Sheehan, Gillian Fitzmaurice, Tony O'Grady, Deirdre G. O'Donovan, Ronan M. Conroy, Diarmuid P. O'Donoghue, Kieran Sheahan, Elaine Kay, Frank E. Murray, Clin Pharmacology, Royal Coli of Surgeons in Ireland, Dublin, Ireland; Pathology Dept, Royal Coli of Surgeons in Ireland, Dublin, Ireland; Gastroenterology Dept, Beaumont Hosp, Dublin, Ireland; Epidemiology Dept, Royal Coli of Surgeons in Ireland, Dublin, Ireland; Gastroenterology Dept, St Vincents Hosp, Dublin, Ireland; Pathology Dept, St Vincents Hosp, Dublin, Ireland. Fas Ligand (FasL) is an important mediator of immune function and induces apoptosis by binding to its receptor Fas on sensitized cells. It has recently been shown that malignancies may express FasL and acquire immune privilege by inducing apoptosis of lymphocytes. Acquired resistance to Pas-mediated apoptosis is known to be an early event in carcinogenesis. The aim of this study was to determine the extent of FasL expression in various Dukes stage tumors in patients with colorectal cancer. Methods: 68 patients (median age 66 yrs) with colorectal cancer were evaluated whose diagnosis was made between 1988 and 1991, and in whom long-term follow up was available. The tumors were of varying stages at diagnosis (8 Dukes A, 28 Dukes B, 23 Dukes C, 9 Dukes D). The expression of FasL was immunohistochernically detected with a rabbit polyclonal IgG using the DAKO EnVision +System. Relationship with Dukes stage was determined using Kendall s r-b correlation. Overall survival was estimated using Kaplan Meier product limit curves. Differences in observed survival were tested for statistical significance using the Mantel-Haenszel log rank test. Both the extent and intensity of staining were graded by a blinded observer. Results: FasL was predominantly expressed in tumor epithelial cells in 93% of the cases. Positive staining of tumors varied in both intensity and extent. FasL-binding specificity was confirmed by pre-incubation of the antibody with the immunizing peptide prior to staining. FasL staining was higher in earlier Dukes stage tumors in that the extent of FasL staining negatively correlated with Dukes stage (Kendall T-b= -0.22, p=0.038). Consistent with this, overall survival was better with greater extent of FasL expression (log-rank = 10.1, p=0.OO2). No relationship was detected between Fas intensity and outcome. Conclusion: FasL is widely expressed in colorectal cancers. Overexpression of FasL occurs early during tumor invasiveness and corresponds with improved survival in patients with colorectal cancer. The mechanism of this upregulation is currently unknown.
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A. Sprenger, M. Wied, H. H. Mueller, A. Rickenbach, W. Mathias, R. Funk, R. Arnold, Philipps Univ, Marburg, Germany. Aim: To better understand the spontaneous course of the disease in patients with GEP tumors we analyzed patients and tumor variables as possible prognostic factors. Methods: Data from 301 patients with neuroendocrine GEP tumors (178 with non-functioning tumors, 75 with carcinoid syndrome, 32 with gastrinoma, 11 with insulinoma and 5 with other functionally active pancreatic tumors)have been analyzed retrospectively. 233 out of 301 patients (77%) had metastatic disease at diagnosis. Using univariate and multivariate Cox analyses the following parameters obtained at time of diagnosis have been tested as possible variables influencing outcome: gender, age (;::,50 years), functional activity, localization of the primary, resection of the primary, metastases present at diagnosis, carcinoid heart disease at diagnosis in patients with carcinoid syndrome, plasma chromogranin A (>400 UII). Results: Using univariate analysis significant hazard ratios (95%CI) were found for: presence of metastases at diagnosis: 9.97 (4.31-23.07; p=O.OOOI); localization of the primary in the stomach: 9.05 (2.79-29; p=0.OO2); carcinoid heart disease: 2.89 (1.06-7.88; p= 0.038); age >50 years: 0.65 (0.44-0.97; p=O.03); resection of the primary tumor: 0.51 (0.35-0.75; p=0.OOO6). With the exception of age these variables have been confirmed by multivariate Cox regression analysis. Functional activity and plasma chromogranin A levels did not prove as significant prognostic variables. Conclusion: These data indicate that resection of the primary and age >50 years at diagnosis of a GEP tumor influence the course of the disease favourably. Data from this retrospective study should initiate multicenter, multinational trials to validate and to extent these findings which could influence future therapeutic strategies.
2744 ANGIOGENESIS AT THE SITE OF DEEPEST PENETRATION PREDICTS LYMPH NODE METASTASIS OF SUBMUCOSAL COLORECTAL CANCER. Shinji Tanaka, Hirotoki Oh-e, Ken Haruma, Shiro Oka, Yasuhiko Kitadai, Masaharu Yoshihara, Koji Sumii, Goro Kajiyama, Fumio Shimamoto, Hiroshima Univ Sch of Med, Hiroshima, Japan. The site of deepest penetration of colorectal cancer (CRC) is considered to be the part that ultimately will invade, spread locally, and metastasize. We had previously reported that histologic grade, cellular proliferative activity, and MUC- I expression at the site of deepest penetration of CRCs closely correlated with their malignant potential (Oncology 1993, 1995, 1998; Cancer 1994; Dis Colon Rectum 1998, etc). The AIM of this study is to investigate the relation between microvessel count (MVC), which has been reported as a useful prognostic factor in patients with cancer of various organs, and lymph node metastasis in submucosal CRe. METHODS: MVC was estimated in 254 invasive tumors that had been resected from patients with submucosal CRC. The depth of submucosal invasion was defined as the distance (um) between the lower edge of the muscularis mucosae and the deepest margin of submucosal invasion. Immunohistochemical studies were performed on paraffin-embedded sections using the labeled streptavidin-biotin method (Dako LSAB kit; Dako Japan Co., Kyoto, Japan). Anti-CD34 monoclonal antibody (Nichirei, Tokyo, Japan) was applied, and MVC were estimated based on the average count of three 400x fields in the most vascular area at the site of deepest submucosal penetration. RESULTS: MVC ranged from 10 to 98 with a median of 40. MVC correlated significantly with tumor size (p < 0.05), depth of submucosal invasion (p < 0.0l), histologic grade (p < 0.05), presence of adenoma (p < 0.01), vessel involvement (p < 0.01), and lymph node metastasis (p < 0.0l). The incidence of lymph node metastasis increased as MVC increased (MVC<20: 3.0%; 20-39: 7.2%; 40-59: 19.3%; ;::,60: 27.8%). Lesions with low MVC « 40) and submucosal invasion up to 1500 JoLm had no lymph node metastasis, regardless of histologic grade (0/79; 0%). In multivariate analysis, MVC was an independent risk factor for lymph node metastasis in submucosal CRe. CONCLUSIONS: MVC at the site of deepest submucosal penetration can be one of the quite useful predictors for lymph node metastasis and may broaden the indications of curative endoscopic treatment for submucosal CRe.
2745 CAN CLINICAL AND HISTOLOGICAL PARAMETER PREDICT PROGNOSIS IN PATffiNTS WITH METASTATIC NEUROENDOCRINE GASTROENTETROPANCREATIC (GEP) TUMORS? M. Wied, A. Sprenger, P. S. Barth, H. H. Mueller, R. Arnold, Philipps Univ, Marburg/Lahn, Germany. Aim: To analyze clinical and histological parameters in patients with metastatic GEP tumors as possible prognostic factors influencing patients coutcome. Method: Clinical data and tumor histologies were available from 67 patients (26 with carcinoid syndrome, 38 with non-functioning tumors, 2 with gastrinoma, 1 with glucagonoma). Clinical variables included: age (;::,50 years), site of the primary tumor, presence of liver metastases at diagnosis, resection of the primary, presence of carcinoid syndrome. Pathomorphological parameters included: proliferation index (MIBI), expression of non-mutated p53, number of mitoses, nuclear polymorphism, presence of necroses and bleeding, tumor infiltration into lymphatic and blood vessseis. Multivariate Cox regression analysis was performed to
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examine the prognostic relevance with regard to survival. Results: Statistically significant hazard ratios (95%CI) were found for: the presence of goblet cell carcinoids in the appendix: 5.60 (1.07-29.37; p=0.04); nonmutated p53 expression: 3.29 (1.35-8.01; p=0.OOO9); MIBI: 3.15 (1.337.43; p=0.0009). The presence of liver metastases at diagnosis did not reach significance: 3.00 (1.0-9.05; p=0.051). Expression ofp53 and MIBI were closely related. The other variables failed to prove as independant prognostic factors influencing survival. Conclusion: Histologic proof of an increased proliferative activity and atypical appendiceal carcinoids seem to be associated with an unfavourable prognosis in patients with metastatic neuroendocrine GEP tumors.
2746 EXPRESSION OF THYMIDINE PHOSPHORYLASE AND VASCULAR ENDOTHELIAL GROWTH FACTOR IN CARCINOMA OF THE PAPILLA OF VATER. Bin Zhao, Kimura Wataru, Masatoshi Makuuchi, Shyoki Yazaki, Tokyo Univ Sch of Medicine, Tokyo, Japan; Yamagata Univ Sch of Medicine, Yamagata, Japan; T; Tohoku Univ Sch of Medicine, Tokyo, Japan. Background. Thymidine phosphorylase (TP) and vascular endothelial growth factor (VEGF) are important angiogenic factors which have been reported to be associated with tumor progression in malignant tumors. There has been no study of expression of angiogenic factors and neovascularization in carcinoma of the papilla of Vater. We investigated the expression of both TP and VEGF and the correlation with neovascularization and clinicopathological factors as well as clinical outcome in this malignancy. Methods. Fifty nine cases of carcinoma of the papilla of Vater were studied. All patients were surgically-treated at Department of Surgery, the University of Tokyo, from 1980 to 1997. There were 37 man and 22 women. The average age was 64 years (range 42 to 85). Tumor staging was according to TNM classification. An immunohistochemical method was used to evaluate the expression ofTP and VEGF as well as to quantify microvessel density (MVD) with formalin-fixed paraffin-embedded sections. MVD was defined as the microvessel count in a field with the most highest density of microvessels under a high magnification (about I ILmz in size). Results. TP expression was demonstrated in tumor cells in 62.7 % (37/59) of the cases. The expression was mainly cytoplasmic. A significant correlation was observed between TP expression and regional lymph node metastasis (p=0.006). TP-positive tumors were more advanced in stage as compared with TP-negative tumors (p=O.03). Moreover, the expression of TP correlated significantly with a poor prognosis (p=0.023). As regard to VEGF, 72.9% (43/59) of the cases was positive. The expression of VEGF was significantly correlated with venous invasion (p=0.042), and tumor ulceration (p=0.05). However, no survival significance was found (p=0.750). MVD in TP-positive tumors (27.6::':: 10.1) was significantly higher than that in TP-negative tumors (20.4::':: 10.0, p=O.OI). VEGF alone was not correlated with MVD. However, when TP and VEGF were combined, tumors positive for both had a significantly higher MVD (27.7::'::9.6) than that in tumors negative for both (18.2::'::8.5, p=O.OII). Conclusions. In carcinoma of the papilla of Vater, TP enhances tumor progression, and is a useful prognostic factor. Neovascularization in this malignancy is regulated by both TP and VEGF.
2747 OMEGA·3 FATTY ACIDS (03FA) ENHANCE CHEMOSENSITIV· ITY OF THE COLON CARCINOMA CELL LINE CACO·2 TO 5·FLUOROURACIL (5FU). Bora Akoglu, Angela Jordan, Wolfgang F. Caspary, Juergen Stein, J W Goethe Univ, Frankfurt, Germany; 2nd Dept Medicine, JW Goethe Univ, Frankfurt, Germany. Background/Aim: The use of 5-Fluorouracil (5FU), a chemotherapeutic agent for the treatment of colorectal carcinoma, is limited because of increasing antitumor drug resistance. We have recently shown (Gastroenterology 1999, G1880). that fish oil rich in omega-S fatty acids (03FA) inhibits growth of colorectal tumor cells. The objective of the present investigation was to determine the effect of an 03FA containing lipid emulsion on growth and chemosensitivity to 5FU in the colon carcinoma cell line, Caco-2. Methods: Caco-2 cells were exposed to two different lipid emulsions (Omegaven-lO%®on fish oil basis (FO) containing 2.1 g/Ioo mL eicosapentanoic acid (EPA); Lipovenos-l O'io PLR® on basis of soybean oil (SO), no omega-3-fatty acids ), alone or in combination with 5FU. Cell proliferation was determined by cell counting (crystal violet assay) and BrdU uptake after 24, 48 and 72 h. Cell cycle was assessed by flow cytometry. Results: Treatment of Caco-2 cells with FO induced a time- and dose-dependent decrease in cell counts and BrdU uptake. Cell counts:[% control after 24 h: -10::'::8(*); 48 h: -33::'::7(*); 72 h: -53::'::4(*)], BrdU uptake: [% control after 24 h: -67::'::2(**); 48 h: -86::':: 1(**); 72 h: -88::'::1(**)] at 15.5 ILI/loo ILl medium corresponding to 100 ILM EPA. Incubation with SO resulted in a less prominent effect. Cell counts [ % control after 24 h: -4::'::8(ns); 48 h: -1::'::12(*); 72 h: -23::'::6(*)], BrdV uptake: [% control after 24 h: -14::':: 11(*); 48 h: -64::'::8 (**); 72 h: -65±7(**)] at the same dose [(ns)=not significant, (*)=p<0.05, (**)=p
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stance vs. combination). Conclusions: Fish oil based lipid emulsion (FO) has a potent antiproliferative effect on the colon carcinoma cell line Caco-2, due to cell cycle arrest. Addition of 5FU has an additive and/or synergistic growth inhibitory effect. (Supported by the Else Kroner Fresenius Stiftung)
2748 PHASE IIII TRIAL WITH lllIN·PENTETREOTIDE IN PATIENTS WITH ADVANCED MALIGNANCIES. Athanassios Argiris, Kathy Peccerillo, John R. Murren, Eugene Cornelius, Irvin M. Modlin, Yale Vniv Sch of Medicine, New Haven, CT. Radiolabeled somatostatin analogs can be utilized to deliver therapeutic doses of radiation to neuroendocrine and other tumors expressing somatostatin receptors in high density. We conducted a phase 11I1 trial with 111 Indium-labeled DTPA-D-Phe-I-octreotide IIIn-pentetreotide) in patients (pts) with advanced malignancies with positive uptake on baseline diagnostic IIIIn-pentetreotide scan. 1llln-pentetreotide was administered intravenously every 3 weeks for a total of 4 cycles. at 3 dose levels: 160, 225, and 300 mCi/cycie. From 12196 to 8199 30 pts were enrolled: 25 with carcinoid or other gastroenteropancreatic endocrine tumors (GEPT), I small-cell lung cancer, I chordoma, I Hodgkin's disease, I thymoma. and I giant cell tumor (dose levels 11I11I1 : 6/4/20 pts). Mean age was 52 (range 27- 78); 18 malesl12 females. All pts were symptomatic with extensive metastatic disease; 11130 had prior chemotherapy. 4 pts who derived clinical benefit received 2 additional cycles (maximum cumulative dose 1.800 mCi). A total of 123 cycles of 111- In-pentetreotide have been delivered. Mild/moderate myelosuppression was common, and sometimes prolonged. At dose level III grade I or 2 thrombocytopenia developed in 11120 pts (55%), grade 3 leukopenia in 2 pts (10%), and grade I or 2 leukopenia in 6 pts (30%). Other non-hematologic toxicities, possibly unrelated to therapy, included acute renal failure due to dehydration (1), reversible neuropathy (1), psoriasis flare (1), and new onset diabetes (1). 6/23 evaluable pts with GEPT (26%) had symptomatic improvement (dose levels II and III), and 15/23 (65%) had stable symptomatology; 2 minor radiographic responses (dose levels II and III) and I partial response (PR) (level III) were observed. 5/18 evaluable pts (28%) had a > 50% decrease of at least one hormonal marker. In pts with other malignancies (N=5), I pt with Hodgkin's disease achieved a PR (level III). Radiographic responses were delayed, occurring 2-5 months after treatment initiation. Median time to progression was 6 months. IIIIn-pentetreotide could be escalated to a cumulative dose of at least 1,200 mCi with acceptable hematologic toxicities, and produced clinical benefit in patients with endstage malignancies.
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NFKB INHIBITION IN PANCREATIC CARCINOMA CELLS TRIGGERS APOPTOSIS INDUCED BY VP16 OR ADRIAMYCIN. Alexander Arlt, Anke Huenermann-Jansen, Ulrich R. Foelsch, Wolfgang E. Schmidt, Heiner Schaefer, Ist Dept of Medicine, Kiel, Germany; Dept of Medicine I. Bochum, Germany. Treatment of cancer with genotoxic agents is hampered by resistance to apoptosis exhibited by many tumors, including those of the gastrointestinal tract. Since NFKB mediated cellular responses are supposed to confer anti-apoptotic properties we investigated whether blockade of NFKB activity enhances the cytoxic effect of etoposide (VPI6) or adriamycin on pancreatic carcinoma cells. Amongst various pancreatic cancer cell lines, some (BxPC-3, PT45-1) were highly susceptible to apoptosis induced by VPI6 and adriamycin, whereas others were less sensitive or almost resistant (Capan-I, 818-4). These distinct apoptotic responses were verified by annexin-VIPI labelling and FACS analysis as well as by PARP cleavage analysis. Gel shift assays demonstrated that treatment with VPI6 (20 ILM), but not with adriamycin (0,3 ILM), elicits a rapid increase (I h) in NFKB binding activity in all cell lines, but the duration of this NFKBresponse and its basal activity significantly varied. In BxPc3- and PT45-1 cells, basal NFKB binding was nearly undetectable and the VPl6-induced activity rapidly declined. In Capan-l- and 818-4 cells, basal NFKB activity was strongly elevated and the period of NFKB activation lasted up to 24 h. By means of NFKB-Iuciferase assay, this distinct pattern of NFKB activity could be verified on the level of its transactivating capacity. Blockade of NFKB with agents that interfere with NFKB activation at distinct levels (Gliotoxin, MG132 and Sulfasalazine) not only abolished NFKB activation but also enhanced the apoptotic effects of VPI6 and adriamycin - particularly in those cell lines being resistant to these drugs. Our results indicate that the lack of a cytotoxic effect of chemotherapy in some tumors is due to constitutive NFKB activity rather than a transient induction of NFKB. Thus, blockade of NFKB activity by some well established immunosuppressive drugs obviously reduces resistance of GI-cancer cells.(Supported by the Deutsche Forschungsgemeinschaft: SFB 415,and by the IZKF: Immunobiology of Malignant Disease)
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POST-OPERATIVE SERUM MUTANT K-RAS· A SENSITIVE MARKER OF DISEASE RECURRENCE IN PATIENTS WITH COLORECTAL CANCER.
PROGNOSTIC FACTORS IN PATIENTS WITH NEUROENDOCRINE GASTROENTEROPANCREATIC (GEP) TUMORS.
Barbara M. Ryan, Ross McManus, Jacqueline S. Daly, Francois LeFort, Napoleon Keeling, Donald G. Weir, Dermot Kelleher, St James's Hosp and Trinity Coli, Dublin, Ireland. Background: It has been shown that mutant k-ras can be detected in the plasma or serum of patients with colorectal (CRC) and other cancers. It is not known whether this mutant DNA can also be detected in the circulation of CRC patients post-operatively, and if so, whether it correlates with disease recurrence. Aims: We followed a cohort of CRC patients longitudinally post-operatively, to determine whether serum mutant k-ras could be detected post-op and if so, whether this predicts or predates clinical recurrence of disease. Methods: Serum or plasma was collected at intervals post-operatively in a total of 92 CRC patients with disease ranging from dysplastic tubulovillous adenoma to Duke's C cancer. DNA was extracted as described previously to this meeting. Mutant k-ras was detected using an enriched-PCR-RFLP method, and confirmed by direct sequencing. Detailed clinical, radiological and endoscopic follow-up was carried out in all patients. Results: 47 of the 92 patients (51 %) were k-ras mutation-positive in the primary tumor. Of these 47 patients, 15 (32%) have become serum k-ras mutant-positive during the post-operative follow-up period and 5 patients (33%) in this group have developed overt clinical and histological recurrence. In one case, serum mutant k-ras positivity pre-dated clinical recurrence by I year. No patient who was mutation-positive in the primary tumor and who remains serum mutant- negative has developed clinical recurrence. Of the 45 patients who were k-ras mutation-negative in the primary tumor, 6 (13%) have developed clinically recurrent disease, but all remained persistently serum mutant k-ras negative during the follow-up period. Conclusions: We demonstrate that serum mutant k-ras can be detected in the plasma/serum post-operatively, in a proportion of patients with CRC. 33% of these patients have developed clinical recurrence during the follow-up period, and in one case serum mutant-positivity pre-dated clinical recurrence by I year. These findings suggest that serum mutant k-ras may prove a clinically useful, early, sensitive and specific marker of disease recurrence in patients with CRC, possibly detecting disease at the micrometastatic stage.
2742 RELATIONSHIP BETWEEN FAS LIGAND EXPRESSION, DUKES STAGE AND SURVIVAL IN HUMAN COLORECTAL CANCER. Katherine M. Sheehan, Gillian Fitzmaurice, Tony O'Grady, Deirdre G. O'Donovan, Ronan M. Conroy, Diarmuid P. O'Donoghue, Kieran Sheahan, Elaine Kay, Frank E. Murray, Clin Pharmacology, Royal Coli of Surgeons in Ireland, Dublin, Ireland; Pathology Dept, Royal Coli of Surgeons in Ireland, Dublin, Ireland; Gastroenterology Dept, Beaumont Hosp, Dublin, Ireland; Epidemiology Dept, Royal Coli of Surgeons in Ireland, Dublin, Ireland; Gastroenterology Dept, St Vincents Hosp, Dublin, Ireland; Pathology Dept, St Vincents Hosp, Dublin, Ireland. Fas Ligand (FasL) is an important mediator of immune function and induces apoptosis by binding to its receptor Fas on sensitized cells. It has recently been shown that malignancies may express FasL and acquire immune privilege by inducing apoptosis of lymphocytes. Acquired resistance to Pas-mediated apoptosis is known to be an early event in carcinogenesis. The aim of this study was to determine the extent of FasL expression in various Dukes stage tumors in patients with colorectal cancer. Methods: 68 patients (median age 66 yrs) with colorectal cancer were evaluated whose diagnosis was made between 1988 and 1991, and in whom long-term follow up was available. The tumors were of varying stages at diagnosis (8 Dukes A, 28 Dukes B, 23 Dukes C, 9 Dukes D). The expression of FasL was immunohistochernically detected with a rabbit polyclonal IgG using the DAKO EnVision +System. Relationship with Dukes stage was determined using Kendall s r-b correlation. Overall survival was estimated using Kaplan Meier product limit curves. Differences in observed survival were tested for statistical significance using the Mantel-Haenszel log rank test. Both the extent and intensity of staining were graded by a blinded observer. Results: FasL was predominantly expressed in tumor epithelial cells in 93% of the cases. Positive staining of tumors varied in both intensity and extent. FasL-binding specificity was confirmed by pre-incubation of the antibody with the immunizing peptide prior to staining. FasL staining was higher in earlier Dukes stage tumors in that the extent of FasL staining negatively correlated with Dukes stage (Kendall T-b= -0.22, p=0.038). Consistent with this, overall survival was better with greater extent of FasL expression (log-rank = 10.1, p=0.OO2). No relationship was detected between Fas intensity and outcome. Conclusion: FasL is widely expressed in colorectal cancers. Overexpression of FasL occurs early during tumor invasiveness and corresponds with improved survival in patients with colorectal cancer. The mechanism of this upregulation is currently unknown.
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A. Sprenger, M. Wied, H. H. Mueller, A. Rickenbach, W. Mathias, R. Funk, R. Arnold, Philipps Univ, Marburg, Germany. Aim: To better understand the spontaneous course of the disease in patients with GEP tumors we analyzed patients and tumor variables as possible prognostic factors. Methods: Data from 301 patients with neuroendocrine GEP tumors (178 with non-functioning tumors, 75 with carcinoid syndrome, 32 with gastrinoma, 11 with insulinoma and 5 with other functionally active pancreatic tumors)have been analyzed retrospectively. 233 out of 301 patients (77%) had metastatic disease at diagnosis. Using univariate and multivariate Cox analyses the following parameters obtained at time of diagnosis have been tested as possible variables influencing outcome: gender, age (;::,50 years), functional activity, localization of the primary, resection of the primary, metastases present at diagnosis, carcinoid heart disease at diagnosis in patients with carcinoid syndrome, plasma chromogranin A (>400 UII). Results: Using univariate analysis significant hazard ratios (95%CI) were found for: presence of metastases at diagnosis: 9.97 (4.31-23.07; p=O.OOOI); localization of the primary in the stomach: 9.05 (2.79-29; p=0.OO2); carcinoid heart disease: 2.89 (1.06-7.88; p= 0.038); age >50 years: 0.65 (0.44-0.97; p=O.03); resection of the primary tumor: 0.51 (0.35-0.75; p=0.OOO6). With the exception of age these variables have been confirmed by multivariate Cox regression analysis. Functional activity and plasma chromogranin A levels did not prove as significant prognostic variables. Conclusion: These data indicate that resection of the primary and age >50 years at diagnosis of a GEP tumor influence the course of the disease favourably. Data from this retrospective study should initiate multicenter, multinational trials to validate and to extent these findings which could influence future therapeutic strategies.
2744 ANGIOGENESIS AT THE SITE OF DEEPEST PENETRATION PREDICTS LYMPH NODE METASTASIS OF SUBMUCOSAL COLORECTAL CANCER. Shinji Tanaka, Hirotoki Oh-e, Ken Haruma, Shiro Oka, Yasuhiko Kitadai, Masaharu Yoshihara, Koji Sumii, Goro Kajiyama, Fumio Shimamoto, Hiroshima Univ Sch of Med, Hiroshima, Japan. The site of deepest penetration of colorectal cancer (CRC) is considered to be the part that ultimately will invade, spread locally, and metastasize. We had previously reported that histologic grade, cellular proliferative activity, and MUC- I expression at the site of deepest penetration of CRCs closely correlated with their malignant potential (Oncology 1993, 1995, 1998; Cancer 1994; Dis Colon Rectum 1998, etc). The AIM of this study is to investigate the relation between microvessel count (MVC), which has been reported as a useful prognostic factor in patients with cancer of various organs, and lymph node metastasis in submucosal CRe. METHODS: MVC was estimated in 254 invasive tumors that had been resected from patients with submucosal CRC. The depth of submucosal invasion was defined as the distance (um) between the lower edge of the muscularis mucosae and the deepest margin of submucosal invasion. Immunohistochemical studies were performed on paraffin-embedded sections using the labeled streptavidin-biotin method (Dako LSAB kit; Dako Japan Co., Kyoto, Japan). Anti-CD34 monoclonal antibody (Nichirei, Tokyo, Japan) was applied, and MVC were estimated based on the average count of three 400x fields in the most vascular area at the site of deepest submucosal penetration. RESULTS: MVC ranged from 10 to 98 with a median of 40. MVC correlated significantly with tumor size (p < 0.05), depth of submucosal invasion (p < 0.0l), histologic grade (p < 0.05), presence of adenoma (p < 0.01), vessel involvement (p < 0.01), and lymph node metastasis (p < 0.0l). The incidence of lymph node metastasis increased as MVC increased (MVC<20: 3.0%; 20-39: 7.2%; 40-59: 19.3%; ;::,60: 27.8%). Lesions with low MVC « 40) and submucosal invasion up to 1500 JoLm had no lymph node metastasis, regardless of histologic grade (0/79; 0%). In multivariate analysis, MVC was an independent risk factor for lymph node metastasis in submucosal CRe. CONCLUSIONS: MVC at the site of deepest submucosal penetration can be one of the quite useful predictors for lymph node metastasis and may broaden the indications of curative endoscopic treatment for submucosal CRe.
2745 CAN CLINICAL AND HISTOLOGICAL PARAMETER PREDICT PROGNOSIS IN PATffiNTS WITH METASTATIC NEUROENDOCRINE GASTROENTETROPANCREATIC (GEP) TUMORS? M. Wied, A. Sprenger, P. S. Barth, H. H. Mueller, R. Arnold, Philipps Univ, Marburg/Lahn, Germany. Aim: To analyze clinical and histological parameters in patients with metastatic GEP tumors as possible prognostic factors influencing patients coutcome. Method: Clinical data and tumor histologies were available from 67 patients (26 with carcinoid syndrome, 38 with non-functioning tumors, 2 with gastrinoma, 1 with glucagonoma). Clinical variables included: age (;::,50 years), site of the primary tumor, presence of liver metastases at diagnosis, resection of the primary, presence of carcinoid syndrome. Pathomorphological parameters included: proliferation index (MIBI), expression of non-mutated p53, number of mitoses, nuclear polymorphism, presence of necroses and bleeding, tumor infiltration into lymphatic and blood vessseis. Multivariate Cox regression analysis was performed to
April 2000
examine the prognostic relevance with regard to survival. Results: Statistically significant hazard ratios (95%CI) were found for: the presence of goblet cell carcinoids in the appendix: 5.60 (1.07-29.37; p=0.04); nonmutated p53 expression: 3.29 (1.35-8.01; p=0.OOO9); MIBI: 3.15 (1.337.43; p=0.0009). The presence of liver metastases at diagnosis did not reach significance: 3.00 (1.0-9.05; p=0.051). Expression ofp53 and MIBI were closely related. The other variables failed to prove as independant prognostic factors influencing survival. Conclusion: Histologic proof of an increased proliferative activity and atypical appendiceal carcinoids seem to be associated with an unfavourable prognosis in patients with metastatic neuroendocrine GEP tumors.
2746 EXPRESSION OF THYMIDINE PHOSPHORYLASE AND VASCULAR ENDOTHELIAL GROWTH FACTOR IN CARCINOMA OF THE PAPILLA OF VATER. Bin Zhao, Kimura Wataru, Masatoshi Makuuchi, Shyoki Yazaki, Tokyo Univ Sch of Medicine, Tokyo, Japan; Yamagata Univ Sch of Medicine, Yamagata, Japan; T; Tohoku Univ Sch of Medicine, Tokyo, Japan. Background. Thymidine phosphorylase (TP) and vascular endothelial growth factor (VEGF) are important angiogenic factors which have been reported to be associated with tumor progression in malignant tumors. There has been no study of expression of angiogenic factors and neovascularization in carcinoma of the papilla of Vater. We investigated the expression of both TP and VEGF and the correlation with neovascularization and clinicopathological factors as well as clinical outcome in this malignancy. Methods. Fifty nine cases of carcinoma of the papilla of Vater were studied. All patients were surgically-treated at Department of Surgery, the University of Tokyo, from 1980 to 1997. There were 37 man and 22 women. The average age was 64 years (range 42 to 85). Tumor staging was according to TNM classification. An immunohistochemical method was used to evaluate the expression ofTP and VEGF as well as to quantify microvessel density (MVD) with formalin-fixed paraffin-embedded sections. MVD was defined as the microvessel count in a field with the most highest density of microvessels under a high magnification (about I ILmz in size). Results. TP expression was demonstrated in tumor cells in 62.7 % (37/59) of the cases. The expression was mainly cytoplasmic. A significant correlation was observed between TP expression and regional lymph node metastasis (p=0.006). TP-positive tumors were more advanced in stage as compared with TP-negative tumors (p=O.03). Moreover, the expression of TP correlated significantly with a poor prognosis (p=0.023). As regard to VEGF, 72.9% (43/59) of the cases was positive. The expression of VEGF was significantly correlated with venous invasion (p=0.042), and tumor ulceration (p=0.05). However, no survival significance was found (p=0.750). MVD in TP-positive tumors (27.6::':: 10.1) was significantly higher than that in TP-negative tumors (20.4::':: 10.0, p=O.OI). VEGF alone was not correlated with MVD. However, when TP and VEGF were combined, tumors positive for both had a significantly higher MVD (27.7::'::9.6) than that in tumors negative for both (18.2::'::8.5, p=O.OII). Conclusions. In carcinoma of the papilla of Vater, TP enhances tumor progression, and is a useful prognostic factor. Neovascularization in this malignancy is regulated by both TP and VEGF.
2747 OMEGA·3 FATTY ACIDS (03FA) ENHANCE CHEMOSENSITIV· ITY OF THE COLON CARCINOMA CELL LINE CACO·2 TO 5·FLUOROURACIL (5FU). Bora Akoglu, Angela Jordan, Wolfgang F. Caspary, Juergen Stein, J W Goethe Univ, Frankfurt, Germany; 2nd Dept Medicine, JW Goethe Univ, Frankfurt, Germany. Background/Aim: The use of 5-Fluorouracil (5FU), a chemotherapeutic agent for the treatment of colorectal carcinoma, is limited because of increasing antitumor drug resistance. We have recently shown (Gastroenterology 1999, G1880). that fish oil rich in omega-S fatty acids (03FA) inhibits growth of colorectal tumor cells. The objective of the present investigation was to determine the effect of an 03FA containing lipid emulsion on growth and chemosensitivity to 5FU in the colon carcinoma cell line, Caco-2. Methods: Caco-2 cells were exposed to two different lipid emulsions (Omegaven-lO%®on fish oil basis (FO) containing 2.1 g/Ioo mL eicosapentanoic acid (EPA); Lipovenos-l O'io PLR® on basis of soybean oil (SO), no omega-3-fatty acids ), alone or in combination with 5FU. Cell proliferation was determined by cell counting (crystal violet assay) and BrdU uptake after 24, 48 and 72 h. Cell cycle was assessed by flow cytometry. Results: Treatment of Caco-2 cells with FO induced a time- and dose-dependent decrease in cell counts and BrdU uptake. Cell counts:[% control after 24 h: -10::'::8(*); 48 h: -33::'::7(*); 72 h: -53::'::4(*)], BrdU uptake: [% control after 24 h: -67::'::2(**); 48 h: -86::':: 1(**); 72 h: -88::'::1(**)] at 15.5 ILI/loo ILl medium corresponding to 100 ILM EPA. Incubation with SO resulted in a less prominent effect. Cell counts [ % control after 24 h: -4::'::8(ns); 48 h: -1::'::12(*); 72 h: -23::'::6(*)], BrdV uptake: [% control after 24 h: -14::':: 11(*); 48 h: -64::'::8 (**); 72 h: -65±7(**)] at the same dose [(ns)=not significant, (*)=p<0.05, (**)=p
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stance vs. combination). Conclusions: Fish oil based lipid emulsion (FO) has a potent antiproliferative effect on the colon carcinoma cell line Caco-2, due to cell cycle arrest. Addition of 5FU has an additive and/or synergistic growth inhibitory effect. (Supported by the Else Kroner Fresenius Stiftung)
2748 PHASE IIII TRIAL WITH lllIN·PENTETREOTIDE IN PATIENTS WITH ADVANCED MALIGNANCIES. Athanassios Argiris, Kathy Peccerillo, John R. Murren, Eugene Cornelius, Irvin M. Modlin, Yale Vniv Sch of Medicine, New Haven, CT. Radiolabeled somatostatin analogs can be utilized to deliver therapeutic doses of radiation to neuroendocrine and other tumors expressing somatostatin receptors in high density. We conducted a phase 11I1 trial with 111 Indium-labeled DTPA-D-Phe-I-octreotide IIIn-pentetreotide) in patients (pts) with advanced malignancies with positive uptake on baseline diagnostic IIIIn-pentetreotide scan. 1llln-pentetreotide was administered intravenously every 3 weeks for a total of 4 cycles. at 3 dose levels: 160, 225, and 300 mCi/cycie. From 12196 to 8199 30 pts were enrolled: 25 with carcinoid or other gastroenteropancreatic endocrine tumors (GEPT), I small-cell lung cancer, I chordoma, I Hodgkin's disease, I thymoma. and I giant cell tumor (dose levels 11I11I1 : 6/4/20 pts). Mean age was 52 (range 27- 78); 18 malesl12 females. All pts were symptomatic with extensive metastatic disease; 11130 had prior chemotherapy. 4 pts who derived clinical benefit received 2 additional cycles (maximum cumulative dose 1.800 mCi). A total of 123 cycles of 111- In-pentetreotide have been delivered. Mild/moderate myelosuppression was common, and sometimes prolonged. At dose level III grade I or 2 thrombocytopenia developed in 11120 pts (55%), grade 3 leukopenia in 2 pts (10%), and grade I or 2 leukopenia in 6 pts (30%). Other non-hematologic toxicities, possibly unrelated to therapy, included acute renal failure due to dehydration (1), reversible neuropathy (1), psoriasis flare (1), and new onset diabetes (1). 6/23 evaluable pts with GEPT (26%) had symptomatic improvement (dose levels II and III), and 15/23 (65%) had stable symptomatology; 2 minor radiographic responses (dose levels II and III) and I partial response (PR) (level III) were observed. 5/18 evaluable pts (28%) had a > 50% decrease of at least one hormonal marker. In pts with other malignancies (N=5), I pt with Hodgkin's disease achieved a PR (level III). Radiographic responses were delayed, occurring 2-5 months after treatment initiation. Median time to progression was 6 months. IIIIn-pentetreotide could be escalated to a cumulative dose of at least 1,200 mCi with acceptable hematologic toxicities, and produced clinical benefit in patients with endstage malignancies.
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NFKB INHIBITION IN PANCREATIC CARCINOMA CELLS TRIGGERS APOPTOSIS INDUCED BY VP16 OR ADRIAMYCIN. Alexander Arlt, Anke Huenermann-Jansen, Ulrich R. Foelsch, Wolfgang E. Schmidt, Heiner Schaefer, Ist Dept of Medicine, Kiel, Germany; Dept of Medicine I. Bochum, Germany. Treatment of cancer with genotoxic agents is hampered by resistance to apoptosis exhibited by many tumors, including those of the gastrointestinal tract. Since NFKB mediated cellular responses are supposed to confer anti-apoptotic properties we investigated whether blockade of NFKB activity enhances the cytoxic effect of etoposide (VPI6) or adriamycin on pancreatic carcinoma cells. Amongst various pancreatic cancer cell lines, some (BxPC-3, PT45-1) were highly susceptible to apoptosis induced by VPI6 and adriamycin, whereas others were less sensitive or almost resistant (Capan-I, 818-4). These distinct apoptotic responses were verified by annexin-VIPI labelling and FACS analysis as well as by PARP cleavage analysis. Gel shift assays demonstrated that treatment with VPI6 (20 ILM), but not with adriamycin (0,3 ILM), elicits a rapid increase (I h) in NFKB binding activity in all cell lines, but the duration of this NFKBresponse and its basal activity significantly varied. In BxPc3- and PT45-1 cells, basal NFKB binding was nearly undetectable and the VPl6-induced activity rapidly declined. In Capan-l- and 818-4 cells, basal NFKB activity was strongly elevated and the period of NFKB activation lasted up to 24 h. By means of NFKB-Iuciferase assay, this distinct pattern of NFKB activity could be verified on the level of its transactivating capacity. Blockade of NFKB with agents that interfere with NFKB activation at distinct levels (Gliotoxin, MG132 and Sulfasalazine) not only abolished NFKB activation but also enhanced the apoptotic effects of VPI6 and adriamycin - particularly in those cell lines being resistant to these drugs. Our results indicate that the lack of a cytotoxic effect of chemotherapy in some tumors is due to constitutive NFKB activity rather than a transient induction of NFKB. Thus, blockade of NFKB activity by some well established immunosuppressive drugs obviously reduces resistance of GI-cancer cells.(Supported by the Deutsche Forschungsgemeinschaft: SFB 415,and by the IZKF: Immunobiology of Malignant Disease)