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Angiogenesis as a marker of recurrence in rectal carcinoma
A608 AGA ABSTRACTS • G2498 DETECTION OF K-ras/p53 MUTATIONS IN STOOL SPECIMENS OF PATIENTS WITH COLORECTAL CANCER. Takashi Hattori, Norihirn Kaminaga, Yutaka Endo, Hiroshi Takahashi, Rikiya Fujita. Division of Gastroenterology, Department of Intenal Medicine, Fujigaoka Hospital, Showa University, Kanagawa, Japan Background & Aims Fecal occult blood (FOB) testing has been the mainstay as a screening test for colorectal cancer. However, the efficacy of FOB testing remains controversial, and the development of more reliable markers for colorectal cancer are needed. In the present study, we evaluated whether or not the analysis of K-ras mutations in DNAs isolated from the stool of patients with colorectal cancer and expression of the p53 protein in tumor cells collected from the stool of these patients can be useful for detecting colorectal cancer. Methods K-ras mutations in codon 12, 13 were analysed using mutant-allele-specific amplification (MASA) in the stools and tissues of 54 persons with colorectal cancer and 20 without gastrointestinal disease. The expression of p53 was evaluated immunohistochemically in 54 paraffinembedded tissues and smeared stool specimens by an automated smear processor. The status of p53 was assesed in a coded manner by a surgical pathologist without knowledge of the clinical and pathological features of the case or the clinical outcome. At the outset, samples were to be regarded as positive for p53 when at least one more than the tumor cells which were over Class III b in the Papanicolaou classification in the smeared stool specimens were immunoreactive. The protocol empolyed in this investigation has been approved by Showa University's Institutional Review Board. Permission forms are signed by cancer patients or their next of kin. Results The sensitivity of K-ras screening alone in this study was 30% (16/54) and the specificity was 100% (20•20). The sensitivity of p53 screening alone in this study was 50% (27154) and the specificity was 100% (20120). Of three patients who were negative for p53 screening, K-ras mutations in the stool were evident in three patients. The sensitivity of K-ras/p53 screening in this study was 56% (30154). Conclusions The screening of stool K-ras/p53 mutations deserves evaluation as a screening test for the detection of colorectal cancer. • G2499 SUPERFICIAL DEPRESSED COLORECTAL ADENOCARCINOMAS AND ADENOMAS: CLINICOPATHOLOGICAL STUDY. M. Hay~kawa, K. Kusugami#, Y. Nishio#, M. Sugihara, Y. Morooka, T. Fujita, and M. Nakamura. Department of Gastroenterology, Meitetsu Hospital, and First Department of Internal Medicine, Nagoya University School of Medicine#, Nagoya, Japan. Introduction: In the present study, we analyzed the endoscopic findings, dissecting microscopic features, histopathological characteristics, and positivity of p53 immunostaining in the superficial depressed type (superficial) early carcinomas and adenomas in the colon and rectum. Methods: Dissecting stereo-microscopy was used to ascertain the depression, size, and pit pattern of the lesions removed by endoscopic mucosal resection. Immunohistochemical staining of p53 was performed with an antigen retrieval system using monoclonal antibody to p53. ResultS: We encountered 50 lesions of depressed colorectal tumors (early adenocarcinomas, n=20 and adenomas, n=30) during 7898 colonoscopic examinations. Most tumors were detected as a slightly reddish or discolored depression with (IIa+Ilc) or without a marginal elevation (IIc) on colonoscopic examination. In the stereomicroscopic study of specimens resected by endoscopic mucosal resection, the depressed early adenocarcinomas showed almost exclusively irregular small round pits except for four lesions with moderate submucosal invasion in which the pit structure was absent. In contrast, the depressed adenomas had either regular small round (29130 lesions) or oval pits (1/30 lesions). Although the incidence of cellular atypia was comparable between the depressed early adenocarcinomas and adenomas, the structural atypia was frequently found in the former (75% or more), but this was not true in the latter. The incidence of p53 positivity was 75% in the depressed adenocarcinomas and 7% in the adenomas of similar morphological type. In the former, p53 positivity was observed more frequently in the carcinomas invading the submucosal layer (100%) than those confined to the mucosal layer (64%). The area with p53 expression corresponded to the portion with irregular small round pits. Conclusion: The depressed early colorectal adenocarcinomas, especially those with submucosal invasion, may bear more malignant potential than the protruding ones. We should keep in mind that such lesions can be detected by careful colonoscopic examination.
• G2500 PIT PATTERN DIAGNOSIS OF DEPRESSED TUMORS OF THE COLON. S. Havashi.* Y. Suzuki,~ M. Kobayasi,* T. Honmat, R. Narisawa,t and H. Asakura.* *Dept. of Gastroenterology, Niigata Rinkoh General Hospital, Niigata, Japan; and tThird Dept. of Internal Medicine, Niigata University School of Medicine, Niigata, Japan Background and aim: Recent advances of colonoscopic diagnostics have allowed the identification of small depressed tumors with greater frequency. It is, however, occasionally difficult to distinguish depressed adenomas from cancers. The aim of this study is to clarify the difference of the two in terms
GASTROENTEROLOGY Vol. 114, No. 4 of pit patterns. Materials and methods: 141 depressed adenomas and 19 depressed cancers resected endoscopically or surgically were studied. All resected lesions were fixed in 10% buffered formaldehyde solution and stained with methylen blue. Then, the pit patterns of the surface of the lesions were analyzed using dissecting microscope in view of the shapes and the distribution patterns of neoplastic pits, comparing with their histological sections. Results: Tubular and roundish pits of various sizes were seen in 99% of 141 adenomas and branched pits were seen in only one adenoma. On the other hand, irregular pits were seen in 79% of 19 depressed cancers. Moreover, they had no adenomatous components. The distribution of the neoplastic pits at the margin of the depressed tumors were also examined. Grossly, distribution patterns of the neoplastic pits were classified into two types. The first type was characterized by complete separation of the area of neoplastic pits and non-neoplastic pits. The second type was characterized by a distribution of neoplastic pits lying between the covering epithelial areas of non-neoplastic pits. The first type consisted of 15 cancers without adenomatous components. The second type consisted of 141 adenomas and 4 cancers with adenomatous components. Conclusions: Analyzing the shapes and the distribution patterns of pits were useful not only to distinguish depressed adenomas from cancers but also distinguish cancers without adenomatous components from those with adenomatous components. • G2501 K-ras AND p53 GENE MUTATIONS IN COLONIC LAVAGE AND p53 ANTI-BODIES IN SERUM IN LONG-STANDING INFLAMMATORY BOWEL DISEASE. M. Heinzlmann, D.F. Stratakis, S.M. Lang, Ch. Folwaczny, S. Urban*, S. Borgmann*, H. Fricke*, K. Loeschke, Medizinische Klinik Innenstadt, Ludwig Maximilians University, Depts. of Gastroenterology and Clinical Immunology*, Munich, Germany. Patients with long-standing inflammatory bowel disease were screened to improve the detection of malignant transformation at an early stage. DNA extracted from endos-copically obtained lavage solution was analysed for p53 and K-ras gene mutations, and serum for p53 antibodies. Methods: 34 patients (22f, 12m; 26-70yrs.) with inflammatory bowel disease of > 7 years duration were studied. Patients had endoscopicaUy diagnosed pancolitis (pc, n=13), left-sided or distale ulcerative colitis (de, n=6) or Crohn's disease (cd, n=15). DNA was extracted from endoscopically obtained colonic lavage fluid. Exon 5-8 of the p53 gene and Exon 1 and 2 of the K-ras gene were amplified by PCR. p53 mutations were analysed by SSCP using vertical electrophoresis and visualization by silver staining. Kras mutations were detected by hybridization with digoxigenin-labeled oligonucleotides. Positive controls were K-ras mutations of colorectal cancer biopsies and p53 mutations of the HaCaT cell line, respectively. Leucocyte DNA from healthy volunteers served as negative control. Serum p53 antibodies were detected by ELISA and Western blot techniques. Results: Mutations of the K-ras gene were detected in 3 patients (pc, n=2, cd, n=l) in codon 12 of the first exon (2 cases gly to asp and 1 case gly to val). Mutations of the p53 gene were found in 3 patients (pc, n=2, cd, n=l) in exon 5 (2 cases) and exon 6 (1 case). Three patients had p53 serum antibodies (pc, n--l, de, n=l, cd, n=l). Discussion: Purified DNA could be recovered from lavage solution in all patients. With optimizied PCR and DNA detection techniques K-ras and p53 mutations (3 cases each) could be identified in 6 of 34 patients (17.6 %) with long-standing disease. One of 3 patients with p53 serum antibodies, with resection of breast cancer one year previously to the study, also had a K-ras mutation in the lavage fluid. There was no clinical evidence of relapse of breast cancer in this patient. None of the 34 patients had dysplasia in serial colonic biopsies. Conclusion: Search for p53 and K-ras mutations in endoseopically obtained lavage fluid and for p53 antibodies in serum may be useful for cancer surveillance in long-standing inflammatory bowel disease. This research was supported by Wilhelm Vaillant-Stiftung, Munich, Ferring GmbH, Kiel and Dr. Falk GmbH, Freiburg, Germany. • G2502 ANGIOGENESIS AS A MARKER OF RECURRENCE IN RECTAL CARCINOMA. AG Heriot. S Edwards*, C Finlayson*, D Kumar. Departments of Colorectal Surgery and *Histopathology, St. George's Hospital, London, UK. Aim. Angiogenesis is important in the development and spread of malignant tumours. The aim of this study was to investigate the significance of tumour microvascular density (MVD) in the recurrence of rectal carcinoma following curative surgery. Method. 4um sections stained with anti-factor VIII antibody were obtained from tumour blocks from the resection specimens of 97 patients who had undergone "curative' resection of rectal carcinoma. 49 patients developed tumour recurrence and 48 patients remained tumour free 5 year post surgery. Anti-Factor VIII antibody stains endothelial cells and allows identification of blood vessels. Microvessel counts at the invading tumour edge were made in six 200 X fields (20 X objective and 10 X ocular) by two independent observers. Results. Overall, MVD increased with tumour stage and was significantly higher in patients developing recurrence. In the no recurrence group, there
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Gastrointestinal Oncology A609
was an increase in MVD from Dukes' stage A to B to C, but the difference between A and B was lost in the recurrence group. There was no difference in tumour MVD between local and distant recurrence. Microvascular Density Overall (n = 97) Dukes' A (n = 13) Dukes' B (n = 38) Dukes' C (n = 46)
No Recurrence mean (S.D.) 27.5 (7.9) 21.4 (4.8) 26.9 (7.8) 30.9 (7.5)
Recurrence mean (S.D.) 33.7 (9.6) 27.8 (4.3) 29.5 (7.0) 37.2 (10.2)
T Test (unpaired) 0.0007 0.034 0.30 0.029
Conclusion. This study demonstrates that microvascular density increases with turnout stage and is a significant marker of tumour recurrence in Dukes' stages A and C. The behavior of Dukes' A stage tumours above a certain microvascular density begins to parallel that of Dukes' B stage tumours in terms of recurrence. The mechanism of local recurrence may be similar to that of distant recurrence in cases where local recurrence cannot be accounted for by residual turnout at the resection margins.
G2503 ASSESSMENT OF PERFUSION OF COLORECTAL TUMOURS USING TECHNETIUM-LABELED HMPAO AND SPECT IMAGING. AG Heri0t, M Masoomi*, C Demetrescu**, A Britton*, AEA Joseph*, VR McCready, D Kumar. Departments of Colorectal Surgery, *Nuclear Medicine, and **Cardiology, St. George's Hospital, London, UK. Aim. Perfusion of tumours may play a significant role in the uptake of antibodies or chemotherapy. Technetium-99m labeled hexamethylpropyleneamineoxime (HMPAO) and single photon emission computed tomography (SPECT) has demonstrated accurate measurement of tumour and regional perfusion in breast, lung and brian turnouts, and in conjunction with measurement of cardiac output, allows calculation of turnout and regional blood flow. The aim of this study was to explore the relationship between colorectal turnout perfusion and antibody uptake. Method, 5 patients with primary colorectal carcinomas were recruited and underwent a cardiac doppler scan to measure their cardiac output at rest using assessment of stroke volume. Whilst at rest patients were given a peripheral intravenous injection of 370MBq of Tc-99m labeled HMPAO. Twenty minutes later, after voiding, the patient underwent dual planar and SPECT imaging to assess tumour perfusion. A standard source was also scanned for calibration. The tomograms of the turnout area were reconstructed using a Harming filter and were corrected for attenuation. Results. Four patients had rectal tumours and one patient had a tumour in the ascending colon. The mean cardiac output was 5 l/rain. (0.57). The turnout uptake of HMPAO was not significantly greater than that of the background and in all but the largest turnout, it was impossible to distinguish the turnout from the background either on the simple antero-posterior view or on the reconstructed image. In one case, it was possible to distinguish the tumour uptake from the background but the turnout margins were too indistinct to permit quantitative analysis of HMPAO uptake, and hence perfusion. Conclusion. Unlike tumour perfusion measurement in the lung and breast, colorectal tumour perfusion is sufficiently similar to the background perfusion for the tumour to be indistinguishable from the background by uptake of HMPAO. For successful tumour studies of the pelvis, techniques with higher sensitivities and resolution are required. • G2504 THE EFFECT OF SHORT-COURSE PREOPERATIVE RADIOTHERAPY ON THE VASCULARITY OF RECTAL CANCER. AG Hcriot, S Edwards*, C F'mlayson*, J Glees**, D Kumar. Departments of Colorectal Surgery, *Histopathology, and **Radiotherapy, St. George's Hospital,London, UK. Short-course preoperative radiotherapy (25Gy/5 fractions) has demonstrated a survival advantage in rectal cancer, though the mechanism is unknown. Tumour microvascular density (MVD) has been shown to correlate with recurrence and inversely with survival for a variety of tumours, including rectal carcinoma. The aim of this study was to assess the effect of preoperative radiotherapy on the vascularity of rectal tumours. Method. 4urn sections stained with anti-factor VIII antibody were obtained prospectively from tumour blocks from the resection specimens of 13 patients with rectal carcinoma who had undergone preoperative radiotherapy and surgery. Control sections were obtained retrospectively from 97 patients who had undergone 'curative' resection of rectal carcinoma without radiotherapy. 49 of these developed tumour recurrence and 48 remained tumour free 5 year post surgery. Anti-Factor VIII antibody stains endothelial cells and allows identification of blood vessels. Microvessel counts at the invading tumour edge were made in six 200 X fields (20 X objective and 10 X ocular) by two independent observers. Results. MVD increases across Dukes' stages A to C in both the radiotherapy group and in the control group but the increase was substantially less pronounced in the radiotherapy group. In Dukes' C tumours, the MVD was significantly lower in the radiotherapy group (n=6; 23.4(3.8): mean (S.D.)) compared to the control group overall (n=46; 34.7(9.6): p=0.007, unpaired t-test) and to the control group subdivided by recurrence (no recurrence, n=18, 30.9(7.5), p=0.03; recurrence, n=28, 37.2(10.7), p=0.003). Aim.
Conclusion. Preoperative radiotherapy
reduces the increase in tumour microvascular density which occurs with increasing stage across all Dukes' stages, with a significant reduction in Dukes' C. The mechanism of action of short-course preoperative radiotherapy in rectal cancer may be through a reduction in tumour vascularity. G2505 THE INFLUENCE OF PRIMARY COLORECTAL CANCER ON IMMUNE FUNCTION. AG Heriot, JB Marriott, AG Dalgleish, D Kumar. Depts. of Colorectal Surgery and *Oncology, St. George's Hospital, London, UK. Aim. Malignant turnouts appear to influence the immune system. The aim of
this study was to assess the effect of surgical resection on the immune status of patients with colorectal carcinoma. Immune function can be assessed by production of TNF-a, IL-10, and IFN-T by lipopolysaccharide(LPS) stimulated whole blood which preferentially activates monocyte macrophages. Method. Heparinised venous blood samples were taken from 22 patients with colorectal cancer prior to surgery and from 8 age-matched controls. Serial samples were obtained postoperatively. Blood was diluted in RPMI medium (1:9) and stimulated with LPS (llag/ml) by incubation at 37°C for 24 hours. Cell-free supematants were collected and stored at -70°C. Supernatants were assayed for IFN-~/, TNF-a, and IL-10 by ELISA. Results. The levels of TNF-~ and of IFN-7 in patients with colorectal carcinoma prior to surgery (TNF-ct, 9548{7615}, IFN-7, 173{559}; mean{S.D.}) were both significantly reduced compared to controls (TNF-a, 21757{10649}, p=0.0065, IFN-y, 840{678},p=0.0076. Mann-Whitney). The levels of TNF-¢t and IFN-T in patients 10 to 32 weeks after surgery had increased (TNF-a: 15438{10973}; IFN-3,: 744{1690}; mean{S.D.}) and become similar to that of the controls ((TNF-a, p=0.16; IFN-'/, p=0.14). No differences were seen in IL-10 levels. Conclusion. Monocytes in patients with colorectal carcinoma, compared to age-matched controls, are refractory to LPS stimulation as reflected by reduction in TNF-a and IFN-7 production. This suppression disappears following surgical resection of the tumour. This provides evidence for turnout induced suppression of the immune system in patients with colorectal cancer and identifies a potential therapeutic avenue. G2506
CHROMOSOMAL ABERRATIONS DETECTED BY COMPARATIVE GENOMIC HYBRIDIZATION IN COLORECTAL ADENOMAS FROM PATIENTS WITH MULTIPLE TUMORS. MAJA H~rmsen, MM Weiss, GA Meijer, I Weiss, PJF Snijders, JMM Walboomers, SGM Meuwissen, JPA Bank. Depts. of Pathology and Gastroenterology, Free University Hospital, Amsterdam, The Netherlands. Colorectal cancer is the second leading cause of cancer death in the Western world. Since there is little substantial progress in curation, most benefit is expected from secondary prevention in high-risk groups. Adenoma bearing patients constitute the major risk group. However, only an estimated 5% of these patients actually develop metachronous carcinoma. Therefore, it is important to identify those adenoma bearing patients that are truly at high risk of developing cancer. The aim of the present study is to find those genetic abnormalities in adenomas which are associated with malignancy. In addition, we investigated whether adenomas in patients with multiple neoplasias had specific chromosomal changes in common. To date, we studied a total of 22 adenomas from: 2 patients with multiple adenomas (n=7), 5 patients with synchronous adenoma and carcinoma (n=9) and 6 patients with an adenoma and carcinoma region within one tumor (malignant polyp) (n=6). After microdissecting the adenomatous mucosa from 10 ~tm paraffin sections, DNA was collected and analysed by comparative genomic hybridization (CGH). This technique allows the detection of amplifications and deletions on the chromosomal level throughout the whole genome in a single experiment. On average, the 22 adenomas showed 3 losses and 4.5 gains, most of which, however, appeared to be randomly distributed, with frequencies around 20%. Gain of chromosome 7 and 13q occurred at higher frequencies: 33 and 38%, respectively. We found the chromosomal changes in multiple lesions within the same patient to be genetically independent. Based on our previous work and literature data, we defined the following chromosomal regions as "bad events': gain of 8q, 13q and 20q, and loss of 17p and 18q. These events occur at high frequency in colorectai carcinomas. We found that the number of these "bad events' were higher in the villous types, in the severe dysplastic cases, and that they were significantly correlated with increasing adenoma size (p=0.005). So, generally the CGH results are in agreement with the histological characteristics of the adenomas. In addition, the adenoma parts of the malignant polyps appeared to harbour more "bad events' than the adenomas synchronous to carcinoma, and tumors from patients with multiple adenomas. This suggests that adenomas from patients with multiple lesions in themselves do not necessarily constitute a high malignant potential, in contrast to the adenomatous parts of malignant polyps.
• G2500 PIT PATTERN DIAGNOSIS OF DEPRESSED TUMORS OF THE COLON. S. Havashi.* Y. Suzuki,~ M. Kobayasi,* T. Honmat, R. Narisawa,t and H. Asakura.* *Dept. of Gastroenterology, Niigata Rinkoh General Hospital, Niigata, Japan; and tThird Dept. of Internal Medicine, Niigata University School of Medicine, Niigata, Japan Background and aim: Recent advances of colonoscopic diagnostics have allowed the identification of small depressed tumors with greater frequency. It is, however, occasionally difficult to distinguish depressed adenomas from cancers. The aim of this study is to clarify the difference of the two in terms
GASTROENTEROLOGY Vol. 114, No. 4 of pit patterns. Materials and methods: 141 depressed adenomas and 19 depressed cancers resected endoscopically or surgically were studied. All resected lesions were fixed in 10% buffered formaldehyde solution and stained with methylen blue. Then, the pit patterns of the surface of the lesions were analyzed using dissecting microscope in view of the shapes and the distribution patterns of neoplastic pits, comparing with their histological sections. Results: Tubular and roundish pits of various sizes were seen in 99% of 141 adenomas and branched pits were seen in only one adenoma. On the other hand, irregular pits were seen in 79% of 19 depressed cancers. Moreover, they had no adenomatous components. The distribution of the neoplastic pits at the margin of the depressed tumors were also examined. Grossly, distribution patterns of the neoplastic pits were classified into two types. The first type was characterized by complete separation of the area of neoplastic pits and non-neoplastic pits. The second type was characterized by a distribution of neoplastic pits lying between the covering epithelial areas of non-neoplastic pits. The first type consisted of 15 cancers without adenomatous components. The second type consisted of 141 adenomas and 4 cancers with adenomatous components. Conclusions: Analyzing the shapes and the distribution patterns of pits were useful not only to distinguish depressed adenomas from cancers but also distinguish cancers without adenomatous components from those with adenomatous components. • G2501 K-ras AND p53 GENE MUTATIONS IN COLONIC LAVAGE AND p53 ANTI-BODIES IN SERUM IN LONG-STANDING INFLAMMATORY BOWEL DISEASE. M. Heinzlmann, D.F. Stratakis, S.M. Lang, Ch. Folwaczny, S. Urban*, S. Borgmann*, H. Fricke*, K. Loeschke, Medizinische Klinik Innenstadt, Ludwig Maximilians University, Depts. of Gastroenterology and Clinical Immunology*, Munich, Germany. Patients with long-standing inflammatory bowel disease were screened to improve the detection of malignant transformation at an early stage. DNA extracted from endos-copically obtained lavage solution was analysed for p53 and K-ras gene mutations, and serum for p53 antibodies. Methods: 34 patients (22f, 12m; 26-70yrs.) with inflammatory bowel disease of > 7 years duration were studied. Patients had endoscopicaUy diagnosed pancolitis (pc, n=13), left-sided or distale ulcerative colitis (de, n=6) or Crohn's disease (cd, n=15). DNA was extracted from endoscopically obtained colonic lavage fluid. Exon 5-8 of the p53 gene and Exon 1 and 2 of the K-ras gene were amplified by PCR. p53 mutations were analysed by SSCP using vertical electrophoresis and visualization by silver staining. Kras mutations were detected by hybridization with digoxigenin-labeled oligonucleotides. Positive controls were K-ras mutations of colorectal cancer biopsies and p53 mutations of the HaCaT cell line, respectively. Leucocyte DNA from healthy volunteers served as negative control. Serum p53 antibodies were detected by ELISA and Western blot techniques. Results: Mutations of the K-ras gene were detected in 3 patients (pc, n=2, cd, n=l) in codon 12 of the first exon (2 cases gly to asp and 1 case gly to val). Mutations of the p53 gene were found in 3 patients (pc, n=2, cd, n=l) in exon 5 (2 cases) and exon 6 (1 case). Three patients had p53 serum antibodies (pc, n--l, de, n=l, cd, n=l). Discussion: Purified DNA could be recovered from lavage solution in all patients. With optimizied PCR and DNA detection techniques K-ras and p53 mutations (3 cases each) could be identified in 6 of 34 patients (17.6 %) with long-standing disease. One of 3 patients with p53 serum antibodies, with resection of breast cancer one year previously to the study, also had a K-ras mutation in the lavage fluid. There was no clinical evidence of relapse of breast cancer in this patient. None of the 34 patients had dysplasia in serial colonic biopsies. Conclusion: Search for p53 and K-ras mutations in endoseopically obtained lavage fluid and for p53 antibodies in serum may be useful for cancer surveillance in long-standing inflammatory bowel disease. This research was supported by Wilhelm Vaillant-Stiftung, Munich, Ferring GmbH, Kiel and Dr. Falk GmbH, Freiburg, Germany. • G2502 ANGIOGENESIS AS A MARKER OF RECURRENCE IN RECTAL CARCINOMA. AG Heriot. S Edwards*, C Finlayson*, D Kumar. Departments of Colorectal Surgery and *Histopathology, St. George's Hospital, London, UK. Aim. Angiogenesis is important in the development and spread of malignant tumours. The aim of this study was to investigate the significance of tumour microvascular density (MVD) in the recurrence of rectal carcinoma following curative surgery. Method. 4um sections stained with anti-factor VIII antibody were obtained from tumour blocks from the resection specimens of 97 patients who had undergone "curative' resection of rectal carcinoma. 49 patients developed tumour recurrence and 48 patients remained tumour free 5 year post surgery. Anti-Factor VIII antibody stains endothelial cells and allows identification of blood vessels. Microvessel counts at the invading tumour edge were made in six 200 X fields (20 X objective and 10 X ocular) by two independent observers. Results. Overall, MVD increased with tumour stage and was significantly higher in patients developing recurrence. In the no recurrence group, there
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Gastrointestinal Oncology A609
was an increase in MVD from Dukes' stage A to B to C, but the difference between A and B was lost in the recurrence group. There was no difference in tumour MVD between local and distant recurrence. Microvascular Density Overall (n = 97) Dukes' A (n = 13) Dukes' B (n = 38) Dukes' C (n = 46)
No Recurrence mean (S.D.) 27.5 (7.9) 21.4 (4.8) 26.9 (7.8) 30.9 (7.5)
Recurrence mean (S.D.) 33.7 (9.6) 27.8 (4.3) 29.5 (7.0) 37.2 (10.2)
T Test (unpaired) 0.0007 0.034 0.30 0.029
Conclusion. This study demonstrates that microvascular density increases with turnout stage and is a significant marker of tumour recurrence in Dukes' stages A and C. The behavior of Dukes' A stage tumours above a certain microvascular density begins to parallel that of Dukes' B stage tumours in terms of recurrence. The mechanism of local recurrence may be similar to that of distant recurrence in cases where local recurrence cannot be accounted for by residual turnout at the resection margins.
G2503 ASSESSMENT OF PERFUSION OF COLORECTAL TUMOURS USING TECHNETIUM-LABELED HMPAO AND SPECT IMAGING. AG Heri0t, M Masoomi*, C Demetrescu**, A Britton*, AEA Joseph*, VR McCready, D Kumar. Departments of Colorectal Surgery, *Nuclear Medicine, and **Cardiology, St. George's Hospital, London, UK. Aim. Perfusion of tumours may play a significant role in the uptake of antibodies or chemotherapy. Technetium-99m labeled hexamethylpropyleneamineoxime (HMPAO) and single photon emission computed tomography (SPECT) has demonstrated accurate measurement of tumour and regional perfusion in breast, lung and brian turnouts, and in conjunction with measurement of cardiac output, allows calculation of turnout and regional blood flow. The aim of this study was to explore the relationship between colorectal turnout perfusion and antibody uptake. Method, 5 patients with primary colorectal carcinomas were recruited and underwent a cardiac doppler scan to measure their cardiac output at rest using assessment of stroke volume. Whilst at rest patients were given a peripheral intravenous injection of 370MBq of Tc-99m labeled HMPAO. Twenty minutes later, after voiding, the patient underwent dual planar and SPECT imaging to assess tumour perfusion. A standard source was also scanned for calibration. The tomograms of the turnout area were reconstructed using a Harming filter and were corrected for attenuation. Results. Four patients had rectal tumours and one patient had a tumour in the ascending colon. The mean cardiac output was 5 l/rain. (0.57). The turnout uptake of HMPAO was not significantly greater than that of the background and in all but the largest turnout, it was impossible to distinguish the turnout from the background either on the simple antero-posterior view or on the reconstructed image. In one case, it was possible to distinguish the tumour uptake from the background but the turnout margins were too indistinct to permit quantitative analysis of HMPAO uptake, and hence perfusion. Conclusion. Unlike tumour perfusion measurement in the lung and breast, colorectal tumour perfusion is sufficiently similar to the background perfusion for the tumour to be indistinguishable from the background by uptake of HMPAO. For successful tumour studies of the pelvis, techniques with higher sensitivities and resolution are required. • G2504 THE EFFECT OF SHORT-COURSE PREOPERATIVE RADIOTHERAPY ON THE VASCULARITY OF RECTAL CANCER. AG Hcriot, S Edwards*, C F'mlayson*, J Glees**, D Kumar. Departments of Colorectal Surgery, *Histopathology, and **Radiotherapy, St. George's Hospital,London, UK. Short-course preoperative radiotherapy (25Gy/5 fractions) has demonstrated a survival advantage in rectal cancer, though the mechanism is unknown. Tumour microvascular density (MVD) has been shown to correlate with recurrence and inversely with survival for a variety of tumours, including rectal carcinoma. The aim of this study was to assess the effect of preoperative radiotherapy on the vascularity of rectal tumours. Method. 4urn sections stained with anti-factor VIII antibody were obtained prospectively from tumour blocks from the resection specimens of 13 patients with rectal carcinoma who had undergone preoperative radiotherapy and surgery. Control sections were obtained retrospectively from 97 patients who had undergone 'curative' resection of rectal carcinoma without radiotherapy. 49 of these developed tumour recurrence and 48 remained tumour free 5 year post surgery. Anti-Factor VIII antibody stains endothelial cells and allows identification of blood vessels. Microvessel counts at the invading tumour edge were made in six 200 X fields (20 X objective and 10 X ocular) by two independent observers. Results. MVD increases across Dukes' stages A to C in both the radiotherapy group and in the control group but the increase was substantially less pronounced in the radiotherapy group. In Dukes' C tumours, the MVD was significantly lower in the radiotherapy group (n=6; 23.4(3.8): mean (S.D.)) compared to the control group overall (n=46; 34.7(9.6): p=0.007, unpaired t-test) and to the control group subdivided by recurrence (no recurrence, n=18, 30.9(7.5), p=0.03; recurrence, n=28, 37.2(10.7), p=0.003). Aim.
Conclusion. Preoperative radiotherapy
reduces the increase in tumour microvascular density which occurs with increasing stage across all Dukes' stages, with a significant reduction in Dukes' C. The mechanism of action of short-course preoperative radiotherapy in rectal cancer may be through a reduction in tumour vascularity. G2505 THE INFLUENCE OF PRIMARY COLORECTAL CANCER ON IMMUNE FUNCTION. AG Heriot, JB Marriott, AG Dalgleish, D Kumar. Depts. of Colorectal Surgery and *Oncology, St. George's Hospital, London, UK. Aim. Malignant turnouts appear to influence the immune system. The aim of
this study was to assess the effect of surgical resection on the immune status of patients with colorectal carcinoma. Immune function can be assessed by production of TNF-a, IL-10, and IFN-T by lipopolysaccharide(LPS) stimulated whole blood which preferentially activates monocyte macrophages. Method. Heparinised venous blood samples were taken from 22 patients with colorectal cancer prior to surgery and from 8 age-matched controls. Serial samples were obtained postoperatively. Blood was diluted in RPMI medium (1:9) and stimulated with LPS (llag/ml) by incubation at 37°C for 24 hours. Cell-free supematants were collected and stored at -70°C. Supernatants were assayed for IFN-~/, TNF-a, and IL-10 by ELISA. Results. The levels of TNF-~ and of IFN-7 in patients with colorectal carcinoma prior to surgery (TNF-ct, 9548{7615}, IFN-7, 173{559}; mean{S.D.}) were both significantly reduced compared to controls (TNF-a, 21757{10649}, p=0.0065, IFN-y, 840{678},p=0.0076. Mann-Whitney). The levels of TNF-¢t and IFN-T in patients 10 to 32 weeks after surgery had increased (TNF-a: 15438{10973}; IFN-3,: 744{1690}; mean{S.D.}) and become similar to that of the controls ((TNF-a, p=0.16; IFN-'/, p=0.14). No differences were seen in IL-10 levels. Conclusion. Monocytes in patients with colorectal carcinoma, compared to age-matched controls, are refractory to LPS stimulation as reflected by reduction in TNF-a and IFN-7 production. This suppression disappears following surgical resection of the tumour. This provides evidence for turnout induced suppression of the immune system in patients with colorectal cancer and identifies a potential therapeutic avenue. G2506
CHROMOSOMAL ABERRATIONS DETECTED BY COMPARATIVE GENOMIC HYBRIDIZATION IN COLORECTAL ADENOMAS FROM PATIENTS WITH MULTIPLE TUMORS. MAJA H~rmsen, MM Weiss, GA Meijer, I Weiss, PJF Snijders, JMM Walboomers, SGM Meuwissen, JPA Bank. Depts. of Pathology and Gastroenterology, Free University Hospital, Amsterdam, The Netherlands. Colorectal cancer is the second leading cause of cancer death in the Western world. Since there is little substantial progress in curation, most benefit is expected from secondary prevention in high-risk groups. Adenoma bearing patients constitute the major risk group. However, only an estimated 5% of these patients actually develop metachronous carcinoma. Therefore, it is important to identify those adenoma bearing patients that are truly at high risk of developing cancer. The aim of the present study is to find those genetic abnormalities in adenomas which are associated with malignancy. In addition, we investigated whether adenomas in patients with multiple neoplasias had specific chromosomal changes in common. To date, we studied a total of 22 adenomas from: 2 patients with multiple adenomas (n=7), 5 patients with synchronous adenoma and carcinoma (n=9) and 6 patients with an adenoma and carcinoma region within one tumor (malignant polyp) (n=6). After microdissecting the adenomatous mucosa from 10 ~tm paraffin sections, DNA was collected and analysed by comparative genomic hybridization (CGH). This technique allows the detection of amplifications and deletions on the chromosomal level throughout the whole genome in a single experiment. On average, the 22 adenomas showed 3 losses and 4.5 gains, most of which, however, appeared to be randomly distributed, with frequencies around 20%. Gain of chromosome 7 and 13q occurred at higher frequencies: 33 and 38%, respectively. We found the chromosomal changes in multiple lesions within the same patient to be genetically independent. Based on our previous work and literature data, we defined the following chromosomal regions as "bad events': gain of 8q, 13q and 20q, and loss of 17p and 18q. These events occur at high frequency in colorectai carcinomas. We found that the number of these "bad events' were higher in the villous types, in the severe dysplastic cases, and that they were significantly correlated with increasing adenoma size (p=0.005). So, generally the CGH results are in agreement with the histological characteristics of the adenomas. In addition, the adenoma parts of the malignant polyps appeared to harbour more "bad events' than the adenomas synchronous to carcinoma, and tumors from patients with multiple adenomas. This suggests that adenomas from patients with multiple lesions in themselves do not necessarily constitute a high malignant potential, in contrast to the adenomatous parts of malignant polyps.