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Chemoprevention of colorectal cancer — The mechananism of NSAID effect is multifactorial
April 1998
Gastrointestinal Oncology A571
aneuploidy is a harbinger of future dysplasia. To further understand the underlying widespread genomic instability that occurs in UC tumorigenesis we studied chromosomal changes using fluorescencein-situ hybridization (FISH). METHODS: Nine patients with UC-dysplasia or cancer were studied. Colonic epithelium was purified by cell sorting (aneuploid sites) or by epithelial shake-off (diploid sites). Cells were hybridized with probes from chromosome 8q, l l q (Cyclin DI), 17p (p53) and 18q, each matched with a corresponding centromeric probe in another color. At least 100 nuclei were counted per probe per site. RESULTS: Patient
13Y 24Y 4Z 12Z
121J 128J l13J 142J 131J
Histology Indefinite Indefinite LGD Cancer LGD LGD Negative Negative Cancer Indefinite Indefinite HGD Negative Indefinite LGD HGD Indefinite HGD HGD LGD = Low Grade
Ploidy Abnormal Normal Diploid 17 Diploid 11, 17 Diploid 11 8, 17, 18 Aneuploid 8, 11, 17 N/A 8, 11, 17, 18 Aneuploid 17 Aneupl0i d 17 N/A 8 11,17, 18 Aneuploid 8, 11, 17,18 Diploid 8, 11, 17, 18 Diploid 11, 18 8, 17 Diploid 11 8 Diploid 17 8 Diploid 11 8, 17, 18 Diploid 11, 17, 18 8 Diploid 11, 18 8 Diploid 11 N/A 8, 17, 18 Diploid 17 Dysplasia HGD = High Grade Dysplasia
CONCLUSION: Chromosomal aberrations were frequent in all histologic grades of tissue in patients with UC neoplasia. Aberrations were present in negative and indefinite diploid tissue and persisted in higher grades of dysplasia found within the same patient. These findings suggest that colonwide genomic instability occurs before the formation of aneupl0idy and/or histologic neoplastic change in UC. Chromosomal instability could potentially be a sensitive predictor of increased risk of neoplastic progression in UC. • G2336 CHEMOPREVENTION OF COLORECTAL CANCER - THE MECHANISM OF NSAID EFFECT IS MULTIFACTORIAL. W. Brown, S. Skinner, D. Vogiagis, C. Malcontenti-Wilson, T. deJong, I. Van Driel, K. Farmer, P. O'Brien. Monash University Department of Surgery, Alfred Hospital, MELBOURNE, 3181. AUSTRALIA. Nonsteroidal anti-inflammatory drugs (NSAIDs) have been found to reduce the incidence of colorectal cancers in humans and rodent models. This study aims to compare the relative effectiveness of standard NSAIDs, selective cyclooxygenase-2 inhibitors and agents with no or indeterminate cyclooxygenase activity, in their ability to prevent colon cancer precursors, aberrant crypt loci (AC loci). Methods; AC foci were induced in six week old male Sprague-Dawley Rats by orogastric administration of 2 doses of the carcinogen 1,2-dimethylhydrazine. We studied the standard NSAIDs indomethacin, sulindac; selective cyclooxygenase-2 inhibitor meloxicam; a drug with no known prostaglandin inhibitory activity sulindac sulphone; and drugs with minimal ability to block prostaglandins olsalazine & 5-aminosalicylic acid (5-ASA). Drugs were administered in equivalent antiinfiammatory doses, starting one day after the first dose of carcinogen for a period of 21 days. All animals were then killed, and the number of AC foci in the colon counted under light microscopy. Results: There was a significant reduction in the number of AC loci in the groups treated with indomethacin, sulindac, meloxicam, sulindac sulphone and 5-ASA. Drug
Dose
mean # AC foci ± Std Dev Control Carder 1 ml/kg 10 216 ± 20 Indomethacin 2 mg/kg/d 127 ± 37 10 Sulindac 20 mg/kg/d 117±76 7 Meloxicam 0.6 mg/kg/d 10 118 -+ 20 Sulindac Sulphone 40 mg/kg/d 10 140 ± 22 5 ASA 20 mg/kg/d 10 132 ± 35 Olsalazine 25 mg/kg/d 10 221 ± 98 *analysis of raw and lognormal data by ANOVA and Scheffe's.
p value*
< 0.05 <0.05 < 0.05 < 0.05 < 0.05 ns
The degree of inhibition of AC foci did not correlate with the established efficacy of these drugs against prostaglandin synthesis. The selective cyclooxygenase-2 inhibitor was as effective as standard NSAIDs. 5-ASA, which is absorbed in the proximal gastrointestinal tract, effectively inhibited AC foci whereas the drug olsalazine, which predominantly acts topically in the colon, was ineffective. This suggests that this drug work by systemic
rather than local mechanisms. These data indicate that the efficacy of NSAIDs cannot be by pure cyclooxygenase-2 inhibition or by broad cyclooxygenase inhibition alone. • G2337 ASPIRIN REVERSIBLY SUPPRESSES TRANSFORMING GROWTH FACTOR-ALPHA EXPRESSION AND PROSTAGLANDIN BIOSYNTHESIS IN RECTAL MUCOSA IN PATIENTS WITH A HISTORY OF ADENOMATOUS POLYPS. D. Buckelew. G. Maze, W.E. Hardman, I.L. Cameron, K.V. Speeg, M. Lee, University of Texas Health Science Center, San Antonio, TX. There is ample laboratory and epidemiologic evidence that supports the inverse association between the use of aspirin (ASA) and other non-steruidal anti-inflammatory drugs (NSAIDs) and the risk of colorectal cancer. PURPOSE: This study was designed to determine whether ASA would reduce colorectal mucosal hyperproliferation and suppress mucosal prostaglandin (PG) biosynthesis. METHODS: Patients with a history of adenomatous polyps that had been removed endoscopically were enrolled in a randomized, placebo-controlled, double-blind study. Rectal biopsies were obtained by flexible sigmoidoscopy from these subjects at baseline, after 3 months of ASA treatment, and after 3 months of placebo washout period (following ASA treatment). Colorectal mucosal proliferation was assessed by immunohistochemical localization and quantitation of various putative biomarkers (including proliferating cell nuclear antigen (PCNA), transforming growth factor-alpha (TGFcc), and k-ras) as described previously (Cancer Epidem Biomark Prev 6:633, 1997). PGE 2 biosynthesis was assessed by measuring PGE2 content (pg/mg) and synthesis (pg/mg/min) as described previously (Dig Dis Sci 37:1282, 1992). RESULTS (n = 7, means ± SEM): Assav Basglin¢ After 3 mos of ASAa After 3 mos of placebo TGFetb 0.4 ± 0.2 0.2 ± 0.1 c 0.3 ± 0.1 PGE2 synthesis 19.7 ± 9.5 7.9 ± 5.4d 31.7 ± 14.3 aFive patients received 81 mg/day orally while the other 2 patients received 325 mg/day, bpercent of colonic crypts with positive immunoreactivity. cp < 0.05 vs baseline values, up < 0.05 vs baseline and after-placebo values. There were no significant changes in expression of PCNA, K-ras and PGE2 contents. CONCLUSIONS: ASA reversibly suppresses TGFct and PGE2 in rectal polyps. These findings suggest TGFet and PGE 2 may be used as surrogate end point biomarkers in chemoprevention trials. Support in part by NIH research grants (ROI-CA67847 and P30-CA54174). • G2338 ADENOCARCINOMA INVOLVING COLONIC HYPERPLASTIC POLYPS IS ASSOCIATED WITH LOSS OF DNA REPAIR ENZYME MLH-1. LJ Burgart. KP Batts, L Wang, PC Roche, DJ Tester, SN Thibodeau. Mayo Clinic, Rochester, MN. Hyperplastic polyps (HPPs) are not thought to be premalignant lesions, yet 3 recent cases demonstrated an intimate admixture of HPP (nondysplastic), areas of dysplastic transformation and adjacent invasive adenocarcinoma (AdCA). In each case the AdCA had histopathologic and clinical features suggesting the loss of DNA repair enzyme activity (implying the presence of microsatellite instability). AIM: To determine DNA repair enzyme status in these combination AdCA/dysplasia/HPP lesions. METHODS: Each lesion underwent immunoperoxidase (IP) staining using antibodies to human DNA repair enzymes MLH1 (Pharmingen) and MSH2 (Oncogene Science) using standard labeled streptavidin-biotin method. IP staining pattern was closely correlated with H&E morphology for interpretation. Clinical records were reviewed. RESULTS: All 3 patients were women, ages 67, 74 and 78. Two of the three patients had a history of a previous colon AdCA, each within the last 2 years. All lesions were located proximal to the splenic flexure. T h e HPP/dysplasia/AdCA lesions were 2.0, 1.0 and 0.8 cm in diameter. In each case the AdCA and dysplastic areas stained negatively for MLH1 while retaining MSH2 immunoreactivity. The majority of the HPP in each case stained positively for MLH1 and MSH2. However, in 2 cases discrete areas within the HPP demonstrated complete loss of staining for MLH1, retaining MSH2 positivity. One case showed 3 such foci, 2 involving 1-2 crypts (each with over 100 confluent negative epithelial cells) with the third focus representing the interface between HPP and dysplasia where residual nondysplastic crypts stained negatively. The second case showed a 2 crypt focus (>150 confluent cells) within the HPP portion with loss of MLH1 staining. SUMMARY: Three lesions characterized by an intimate association of HPP, dysplasia and AdCA demonstrated significant loci of MLH1 negativity within the HPP as well as complete absence of MLH1 expression in the AdCA and dysplasia. CONCLUSION: In patients at risk of developing colon AdCA related to DNA repair enzyme loss, HPPs may provide a fertile substrate for development of dysplasia and malignancy.
Gastrointestinal Oncology A571
aneuploidy is a harbinger of future dysplasia. To further understand the underlying widespread genomic instability that occurs in UC tumorigenesis we studied chromosomal changes using fluorescencein-situ hybridization (FISH). METHODS: Nine patients with UC-dysplasia or cancer were studied. Colonic epithelium was purified by cell sorting (aneuploid sites) or by epithelial shake-off (diploid sites). Cells were hybridized with probes from chromosome 8q, l l q (Cyclin DI), 17p (p53) and 18q, each matched with a corresponding centromeric probe in another color. At least 100 nuclei were counted per probe per site. RESULTS: Patient
13Y 24Y 4Z 12Z
121J 128J l13J 142J 131J
Histology Indefinite Indefinite LGD Cancer LGD LGD Negative Negative Cancer Indefinite Indefinite HGD Negative Indefinite LGD HGD Indefinite HGD HGD LGD = Low Grade
Ploidy Abnormal Normal Diploid 17 Diploid 11, 17 Diploid 11 8, 17, 18 Aneuploid 8, 11, 17 N/A 8, 11, 17, 18 Aneuploid 17 Aneupl0i d 17 N/A 8 11,17, 18 Aneuploid 8, 11, 17,18 Diploid 8, 11, 17, 18 Diploid 11, 18 8, 17 Diploid 11 8 Diploid 17 8 Diploid 11 8, 17, 18 Diploid 11, 17, 18 8 Diploid 11, 18 8 Diploid 11 N/A 8, 17, 18 Diploid 17 Dysplasia HGD = High Grade Dysplasia
CONCLUSION: Chromosomal aberrations were frequent in all histologic grades of tissue in patients with UC neoplasia. Aberrations were present in negative and indefinite diploid tissue and persisted in higher grades of dysplasia found within the same patient. These findings suggest that colonwide genomic instability occurs before the formation of aneupl0idy and/or histologic neoplastic change in UC. Chromosomal instability could potentially be a sensitive predictor of increased risk of neoplastic progression in UC. • G2336 CHEMOPREVENTION OF COLORECTAL CANCER - THE MECHANISM OF NSAID EFFECT IS MULTIFACTORIAL. W. Brown, S. Skinner, D. Vogiagis, C. Malcontenti-Wilson, T. deJong, I. Van Driel, K. Farmer, P. O'Brien. Monash University Department of Surgery, Alfred Hospital, MELBOURNE, 3181. AUSTRALIA. Nonsteroidal anti-inflammatory drugs (NSAIDs) have been found to reduce the incidence of colorectal cancers in humans and rodent models. This study aims to compare the relative effectiveness of standard NSAIDs, selective cyclooxygenase-2 inhibitors and agents with no or indeterminate cyclooxygenase activity, in their ability to prevent colon cancer precursors, aberrant crypt loci (AC loci). Methods; AC foci were induced in six week old male Sprague-Dawley Rats by orogastric administration of 2 doses of the carcinogen 1,2-dimethylhydrazine. We studied the standard NSAIDs indomethacin, sulindac; selective cyclooxygenase-2 inhibitor meloxicam; a drug with no known prostaglandin inhibitory activity sulindac sulphone; and drugs with minimal ability to block prostaglandins olsalazine & 5-aminosalicylic acid (5-ASA). Drugs were administered in equivalent antiinfiammatory doses, starting one day after the first dose of carcinogen for a period of 21 days. All animals were then killed, and the number of AC foci in the colon counted under light microscopy. Results: There was a significant reduction in the number of AC loci in the groups treated with indomethacin, sulindac, meloxicam, sulindac sulphone and 5-ASA. Drug
Dose
mean # AC foci ± Std Dev Control Carder 1 ml/kg 10 216 ± 20 Indomethacin 2 mg/kg/d 127 ± 37 10 Sulindac 20 mg/kg/d 117±76 7 Meloxicam 0.6 mg/kg/d 10 118 -+ 20 Sulindac Sulphone 40 mg/kg/d 10 140 ± 22 5 ASA 20 mg/kg/d 10 132 ± 35 Olsalazine 25 mg/kg/d 10 221 ± 98 *analysis of raw and lognormal data by ANOVA and Scheffe's.
p value*
< 0.05 <0.05 < 0.05 < 0.05 < 0.05 ns
The degree of inhibition of AC foci did not correlate with the established efficacy of these drugs against prostaglandin synthesis. The selective cyclooxygenase-2 inhibitor was as effective as standard NSAIDs. 5-ASA, which is absorbed in the proximal gastrointestinal tract, effectively inhibited AC foci whereas the drug olsalazine, which predominantly acts topically in the colon, was ineffective. This suggests that this drug work by systemic
rather than local mechanisms. These data indicate that the efficacy of NSAIDs cannot be by pure cyclooxygenase-2 inhibition or by broad cyclooxygenase inhibition alone. • G2337 ASPIRIN REVERSIBLY SUPPRESSES TRANSFORMING GROWTH FACTOR-ALPHA EXPRESSION AND PROSTAGLANDIN BIOSYNTHESIS IN RECTAL MUCOSA IN PATIENTS WITH A HISTORY OF ADENOMATOUS POLYPS. D. Buckelew. G. Maze, W.E. Hardman, I.L. Cameron, K.V. Speeg, M. Lee, University of Texas Health Science Center, San Antonio, TX. There is ample laboratory and epidemiologic evidence that supports the inverse association between the use of aspirin (ASA) and other non-steruidal anti-inflammatory drugs (NSAIDs) and the risk of colorectal cancer. PURPOSE: This study was designed to determine whether ASA would reduce colorectal mucosal hyperproliferation and suppress mucosal prostaglandin (PG) biosynthesis. METHODS: Patients with a history of adenomatous polyps that had been removed endoscopically were enrolled in a randomized, placebo-controlled, double-blind study. Rectal biopsies were obtained by flexible sigmoidoscopy from these subjects at baseline, after 3 months of ASA treatment, and after 3 months of placebo washout period (following ASA treatment). Colorectal mucosal proliferation was assessed by immunohistochemical localization and quantitation of various putative biomarkers (including proliferating cell nuclear antigen (PCNA), transforming growth factor-alpha (TGFcc), and k-ras) as described previously (Cancer Epidem Biomark Prev 6:633, 1997). PGE 2 biosynthesis was assessed by measuring PGE2 content (pg/mg) and synthesis (pg/mg/min) as described previously (Dig Dis Sci 37:1282, 1992). RESULTS (n = 7, means ± SEM): Assav Basglin¢ After 3 mos of ASAa After 3 mos of placebo TGFetb 0.4 ± 0.2 0.2 ± 0.1 c 0.3 ± 0.1 PGE2 synthesis 19.7 ± 9.5 7.9 ± 5.4d 31.7 ± 14.3 aFive patients received 81 mg/day orally while the other 2 patients received 325 mg/day, bpercent of colonic crypts with positive immunoreactivity. cp < 0.05 vs baseline values, up < 0.05 vs baseline and after-placebo values. There were no significant changes in expression of PCNA, K-ras and PGE2 contents. CONCLUSIONS: ASA reversibly suppresses TGFct and PGE2 in rectal polyps. These findings suggest TGFet and PGE 2 may be used as surrogate end point biomarkers in chemoprevention trials. Support in part by NIH research grants (ROI-CA67847 and P30-CA54174). • G2338 ADENOCARCINOMA INVOLVING COLONIC HYPERPLASTIC POLYPS IS ASSOCIATED WITH LOSS OF DNA REPAIR ENZYME MLH-1. LJ Burgart. KP Batts, L Wang, PC Roche, DJ Tester, SN Thibodeau. Mayo Clinic, Rochester, MN. Hyperplastic polyps (HPPs) are not thought to be premalignant lesions, yet 3 recent cases demonstrated an intimate admixture of HPP (nondysplastic), areas of dysplastic transformation and adjacent invasive adenocarcinoma (AdCA). In each case the AdCA had histopathologic and clinical features suggesting the loss of DNA repair enzyme activity (implying the presence of microsatellite instability). AIM: To determine DNA repair enzyme status in these combination AdCA/dysplasia/HPP lesions. METHODS: Each lesion underwent immunoperoxidase (IP) staining using antibodies to human DNA repair enzymes MLH1 (Pharmingen) and MSH2 (Oncogene Science) using standard labeled streptavidin-biotin method. IP staining pattern was closely correlated with H&E morphology for interpretation. Clinical records were reviewed. RESULTS: All 3 patients were women, ages 67, 74 and 78. Two of the three patients had a history of a previous colon AdCA, each within the last 2 years. All lesions were located proximal to the splenic flexure. T h e HPP/dysplasia/AdCA lesions were 2.0, 1.0 and 0.8 cm in diameter. In each case the AdCA and dysplastic areas stained negatively for MLH1 while retaining MSH2 immunoreactivity. The majority of the HPP in each case stained positively for MLH1 and MSH2. However, in 2 cases discrete areas within the HPP demonstrated complete loss of staining for MLH1, retaining MSH2 positivity. One case showed 3 such foci, 2 involving 1-2 crypts (each with over 100 confluent negative epithelial cells) with the third focus representing the interface between HPP and dysplasia where residual nondysplastic crypts stained negatively. The second case showed a 2 crypt focus (>150 confluent cells) within the HPP portion with loss of MLH1 staining. SUMMARY: Three lesions characterized by an intimate association of HPP, dysplasia and AdCA demonstrated significant loci of MLH1 negativity within the HPP as well as complete absence of MLH1 expression in the AdCA and dysplasia. CONCLUSION: In patients at risk of developing colon AdCA related to DNA repair enzyme loss, HPPs may provide a fertile substrate for development of dysplasia and malignancy.