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Prognostic factors in patients with neuroendocrine gastroenteropancreatic (GEP) tumors
GASTROENTEROLOGY Vol. 118, No.4
A514 AGA ABSTRACTS
2741
2743
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
A514 AGA ABSTRACTS
2741
2743
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.
¥
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