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The effect of short-course preoperative radiotherapy on the vascularity of rectal cancer
April 1998
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.
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.