- Email: [email protected]
BL6 mice in vivo
and extra-coloinc cancer profiles were compared between the MLH1 and MSH2 mutationpositive families, families with MSH2 gene mutatiorts were found to have significantly more extra-colonic cancers compared to families with mutations in the MLHI gene (p<0.001).
43 Early Detection of Experimental Colon Carcinogenesis Utilizing Light-Scattering Spectroscopy (LSS) Hemant K. Roy, Ramesh K. Wall, Young L. Kim, Yang Lin, Michael J. Goldberg, Alexei K. Kromine, Kun Chen, Vadim 8ackman
Conclusions: Clinical phenotypes appear to be different in HNPCC families depending on the specific mismatch repair gene mutated. Mutations in MLH1 tend to lead to families predominantly affected with colorectal cancer, whereas mutations in MSH2 are more frequently associated with extracolonic tumors. Sequential analysis of specific mismatch repair genes based on family history features may provide a more streamlined and cost-effective approach to genetic testing in HNPCC families.
The suboptimal nature of current biomarkers .makes exploitation of the "field effect" for colon cancer screening and risk-stratification impractical. LSS is a powerful novel technology for in vivo measurement of cellular architecture, sensitive to submicron structures 20-50 times smaller than visualized with conventional microscopy. Members of our group have pioneered the use of LSS for identification of dysplastic cells (Nature 2000;406:35-6) including colonocytes (Nat Meal 2001;?:1245-8). In this study, we investigated the ability of LSS to detect neoplastic transformation prior to the development of standard hiomarkers. Methods: Fisher F344 rats were injected twice with azoxymethane (AOM) 15 mg/kg s.c. or saline, separated by one weeks. Rats were sacrificed at 2, 8 and 12 weeks after second injection. We bulk a novel apparatus to rapidly analyze the spectral (X range 450 - 700 nm), scattering angle and azimuthal properties of the light scattering from colonic mucosa. Quantitative information (LSS signatures) about nano- and microseale tissue architecture as well as its composition was derived. Aberrant crypt loci (ACF) were detected through examination of methylene blue stained colons. Results: ACF were detectable at 8 and 12 weeks, predominantly in the distal colon (tumors typically develop 40 weeks after AOM). AOM treatment resuhed in marked qualitative and quantitative LSS signature differences versus the timematched control rats at 2 weeks and these alterations were magnified at both 8 and 12 weeks. At the early time points (2 and 8 weeks), these changes were distally confined, with no statistically sigmficant changes noted in the proximal colon. These alterations encompassed all facets of LSS (spectral, scattering angle, and azimuthal properties). Moreover, in the AOM-treated group, there were large differences in multiple LSS parameters such as the fractal dimension of tissue microarchitectnre, ( degree of mutual correlation between the intra- and intercellular components) and the average roundness of intracelhilar structures (40% reduction, p<0.0001). Conclusions: We demonstrate dramatic LSS signatures differences prior to the time when morphological and genetic markers are detectable. The sigmture's relevance is underscored by the temporal and regional correlation with AOM's carcinogenic ability. Future studies will explore the biological and clinical implications of these striking alterations in micro-architecture during the initiation of colon carcinogenesis.
41
Impact of Immunochemical Versus Guaiac Fecal Occult Blood Tests (FOBT) for Life Years Gained (LYG) and Cost A G. Zauber, M. Van Balleguoijen, M. Brown, R. Boer, D. Habbema, F. Loeve, D. Schrag, E Feuer, S. Winawer BACKGROUND. FOBT screening with Hemoccult II reduced colorectal cancer mortality (CRC) in 3 randomized controlled trials. Hemoccult Sensa and immunochemical FOBTs were developed with the intent to improve sensitivity (Se) but maintain high specificity (Sp) relative to the non-rehydrated Hemoccuh II. We explored whether FOBTs with better Se or Sp can increase life years gained while maintaining acceptable overall net costs. METHODS. We simulate life histories of CRC screening in those age > = 65 with no prior CRC screening, 100% compliant with screening FOBT, all positive tests followed by a diagnostic colonoscopy (CO) and surveillance CO for those with adenomas detected. The base ease uses the Medicare test payment of $4.50 per FOBT in a program of annual FOBT screening where Sp=98% and Se is 30% for CRC, 5% for adenomas > = 1.0 cm, and 2% for adenomas < 1.0 cm. OE (3% discount)for base case is $9,350FLYG. We consider higher Se and lower Sp to provide a range of test performance consistent with that reported for sensitive guatac and immunocbemical FOBTs. Using a threshold analysis, we determine the FOBT payment that would provide cost effectiveness equivalent to the base case, for tests with the range of Se/ Sp characteristics. RESULTS. Tests with improved Se increase LY's gained and still maintain cost-effectiveness with base case even if priced dramatically higher per test ($62 - $85) than base ($4.50) (Table). A test with lower Sp (95%) than base (98%) and comparable Se results in comparable health benefits but at much higher net cost. CONCLUSION. FOBTs that provide high Se and Sp can deliver marked benefits in life-years gained with acceptable costs effectiveness ratios, even at substantially higher test payment levels and with increases tn CO's. The expected increase in benefits and the impact of higher unit-costs of newer tests on cost-effectiveness is highly dependent on the estimated relative sensitivity and specificitylevels. These results emphasize the need to establish the performance characteristics of newer tests in well characterized studies in order to appropriately assess the benefit. Irapactof FOBTStl~llk~ and ~lltlvlty ,m ToUl C o l ~ , Corn ~ ~ulvlku,4 OlE Scnmd~ Sw.mdty % II~
Samdllvl~% co'z(teee) 30_05_02 60_30_10 70_60_20 30_08_02 60_30_10 70,60.20
524 907 1,066 1.033 1.243 1~398
56 Daily Intake of Sulforaphane-Rlch Broccoli Sprouts Prevents Progression of High Salt Diet-Induced Gastric Atrophy in H. py/ori-lnfected C57/BL6 Mice In Vivo Akinori Yanaka, Songhua Zhang, Masafumi Tauchi, Hideo Suzuki, Takeshi Shibahara, Hirofumi Matsui, Akira Nakahara, Naomi Tanaka
LifeY m GalMd, Coal, a ~ ~
Background: Sulforaphane, shown to be abundantly present in broccoli sprouts, possesses anticarcinogenic activity, since it strongly induces phase 2 enzymes, such as glutathione transferase and NADPH:quinone reductase, which exert as free radical scavengers, thereby protecting cells from oxidative injury (PNAS, 94:10367, 1997). Recent studies have also shown that sulforaphane has strong bactericidal activity against Helicobc~ter pylori (Hp) in vitro (PNAS 99:7610, 2002). We have previously shown that high salt diet accelerates progression of gastric atrophy in Hp-infected mice in vivo. (Gastroenterology 122: A1, 2002) Based on these backgrounds, we examined if 1) sulforaphane inhibits Hp colonization in vivo, and if 2) sulforaphane prevents progression of high salt diet-induced gastric atrophy in Hp-infected gastric mucosa in vivo. Methods: Standardized mice model of Hp infection were set up by inoculating C57BL/6 female mice with Hp Sydney Strain; SS1. Mice were maintained with high salt diet (7.5% NaC1), supplememed with or without 2.5 mmol sulforaphane or homogenate of broccoli sprouts, which contains 2.5 mmol sulforaphane. Mice were sacrificed at 8 wks after starting the treatment. Degree of gastritis and atrophy was evaluated by updated Sydney system. DNA damage was estimated by measuring mucosal level of 8-OHdG. Expressions of TNF-u, IL-113, IL-8, and COX-2 were analyzed by real time PCR. Results: 1. Treatment with either sulforaphane or broccoli sprouts decreased Hp colonization in gastric mucosa, but failed to achieve complete eradication of Hp in vivo. 2. Sulforaphane or broccoli sprouts attenuated mucosal expressions of TNF-ct, 1L-113, IL-8, and COX-2, mitigated gastritis and DNA damage, and prevented progression of high salt diet-induced gastric atrophy. 3. The protective effects of sulforaphane or broccoli sprouts on gasmc mucosa were in parallel with their effects on inhibition of Hp-coloinzation. However, the protective effects of sulforaphane were observed even in the mice in which Hp-colonization was not inhibited. Conclusion: Daily intake of sulforaphane-rich broccolisprouts attenuates gastritis and prevents progression of gastric atrophy in Hp-infected mice fed with high salt diet, effects appear to be mediated in part by suppressing Hp colonization, and in part by enhancmg activity of gastric mucosal chemoprotection against oxidative stress during Hp infection.
LY Galn~l FOBT~ for Coetz(mlglo.) ~slutCJE (1000) 37 64 73 49 65 73
$213 $128 $94 $371 $331 $299
$4.50 $62,00 $85.00 417.00 $16.00 $39.00
42 1"he Loss of Stool DNA Mutation Abnormalities in Colorectal Neoplasia After Treatment Sapna Syngal, Daniel Chung, Christopher Willet, David Schoetz, Paul Schroy, Elena Stoffel, Deepa Jagadeesh, Kathleen Morel, Michael Ross Background: DNA abnormalities can be detected from the stool of patients with colorectal neoplasia. The effect of surgical resection and treatment on such changes has not been previously reported. Methods: Patients with newly diagnosed colorectal cancer or advanced adenomas requiring surgical resection provided a single stool sample at least two weeks after endoscopy, but prior to therapy. Additional stool samples were obtained approximately one and six months after surgery with or without chemoradiation therapy. Stool was homogenized and DNA isolated and analyzed as previously described (Gastro. 2000;119:1219). The stool-based assay(PreGen-PlusTM)includes 21 mutations (MuMu) in p53, K-ras and APC, a microsatelfite instability marker (BAT-26), and the DNA Integrity Assay (DIA~M), a marker of apoptosis. If any individual marker was positive, the overall panel result was considered positive. Results: 129 patients have been enrolled to date. Of 72 baseline samples currently analyzed (56 with cancer, 16 with advanced adenomas), 34 had a DNA mutation and 20 had an abnormal DIA. The overall sensitivity on baseline samples was 55.5%, including 62.5% for patients with invasive cancer. 52 samples have been analyzed 1-3 months following surgery; of these, 38 patients had no detectable DNA abnormalities, 9 were positive (1 with mutations, 8 positive by DIA) and 5 were technical failures. Of the 8 samples positive by DIA, 2 had been positive at baseline, and 6 had been negative 12 samples have been analyzed 6-8 months following surgery; of these, 11 patients had no detectable DNA abnormalities and 1 was positive with a p53 mutation. Conclusion: Mutational abnormalities in APC, k-ras, p53, and BAT26 detected in stool arise from colorectal neoplastic lesions and resolve with removal of the primary lesion. The etiology of abnormalities in apoptotic markers is unclear, however, and may be due to more pervasive field effects, take longer to resolve, or be affected by chemoradiation or surgical therapy These results support the utility of DNA analysis as a non-invasive option for colorectal cancer screening and provide evidence to support the potential use of mutational abnormalities to monitor colorectal cancer patients after treatment.
57 Deficiency of P27 Leads to Increased Gastric Epithelial and Inflammatory Responses to Helicobacter Pylori Noriyoshi Knzushita, Bassam I. Aswad, Min J. Park, Steven F. Moss Background: p27 is a cyclin-dependent kinase inhibitor that also acts as a tumor suppressor gene. Additionally, p27 may regulate apoptotic and inflammatory responses. H. pylori reduces expression of p27 in human gastric epithelial cells in vivo and in vitro, a potential mechanistic link between H. pylon and gastric carcinogenesis. Aim: To assess the role of reduced gastric p27 in modulating the gastric epithelial and inflammatory response to H. pylori, by comparing the effects of experimental H. pylori infection in wild type and p27 knockout mice. Methods: Six to 8 week-old wild-type (WT) C57 BL/6 mice and p27-/- C57 BL/6 mice were in parallel gavaged with 109 H. pylori SS1 strain, on three occasions over 6 days. Ten mice of each type were sacrificed at 15 and 30 weeks, and 6 of each type at 45 and 60 weeks postinfection. Formalin-fixed, paraffin embedded, H&E stained gastric tissues were scored for inflammatory and epithelial cell responses (modified Sydney system). Proliferating cells were
A-5
AGA
Abstracts
43 Early Detection of Experimental Colon Carcinogenesis Utilizing Light-Scattering Spectroscopy (LSS) Hemant K. Roy, Ramesh K. Wall, Young L. Kim, Yang Lin, Michael J. Goldberg, Alexei K. Kromine, Kun Chen, Vadim 8ackman
Conclusions: Clinical phenotypes appear to be different in HNPCC families depending on the specific mismatch repair gene mutated. Mutations in MLH1 tend to lead to families predominantly affected with colorectal cancer, whereas mutations in MSH2 are more frequently associated with extracolonic tumors. Sequential analysis of specific mismatch repair genes based on family history features may provide a more streamlined and cost-effective approach to genetic testing in HNPCC families.
The suboptimal nature of current biomarkers .makes exploitation of the "field effect" for colon cancer screening and risk-stratification impractical. LSS is a powerful novel technology for in vivo measurement of cellular architecture, sensitive to submicron structures 20-50 times smaller than visualized with conventional microscopy. Members of our group have pioneered the use of LSS for identification of dysplastic cells (Nature 2000;406:35-6) including colonocytes (Nat Meal 2001;?:1245-8). In this study, we investigated the ability of LSS to detect neoplastic transformation prior to the development of standard hiomarkers. Methods: Fisher F344 rats were injected twice with azoxymethane (AOM) 15 mg/kg s.c. or saline, separated by one weeks. Rats were sacrificed at 2, 8 and 12 weeks after second injection. We bulk a novel apparatus to rapidly analyze the spectral (X range 450 - 700 nm), scattering angle and azimuthal properties of the light scattering from colonic mucosa. Quantitative information (LSS signatures) about nano- and microseale tissue architecture as well as its composition was derived. Aberrant crypt loci (ACF) were detected through examination of methylene blue stained colons. Results: ACF were detectable at 8 and 12 weeks, predominantly in the distal colon (tumors typically develop 40 weeks after AOM). AOM treatment resuhed in marked qualitative and quantitative LSS signature differences versus the timematched control rats at 2 weeks and these alterations were magnified at both 8 and 12 weeks. At the early time points (2 and 8 weeks), these changes were distally confined, with no statistically sigmficant changes noted in the proximal colon. These alterations encompassed all facets of LSS (spectral, scattering angle, and azimuthal properties). Moreover, in the AOM-treated group, there were large differences in multiple LSS parameters such as the fractal dimension of tissue microarchitectnre, ( degree of mutual correlation between the intra- and intercellular components) and the average roundness of intracelhilar structures (40% reduction, p<0.0001). Conclusions: We demonstrate dramatic LSS signatures differences prior to the time when morphological and genetic markers are detectable. The sigmture's relevance is underscored by the temporal and regional correlation with AOM's carcinogenic ability. Future studies will explore the biological and clinical implications of these striking alterations in micro-architecture during the initiation of colon carcinogenesis.
41
Impact of Immunochemical Versus Guaiac Fecal Occult Blood Tests (FOBT) for Life Years Gained (LYG) and Cost A G. Zauber, M. Van Balleguoijen, M. Brown, R. Boer, D. Habbema, F. Loeve, D. Schrag, E Feuer, S. Winawer BACKGROUND. FOBT screening with Hemoccult II reduced colorectal cancer mortality (CRC) in 3 randomized controlled trials. Hemoccult Sensa and immunochemical FOBTs were developed with the intent to improve sensitivity (Se) but maintain high specificity (Sp) relative to the non-rehydrated Hemoccuh II. We explored whether FOBTs with better Se or Sp can increase life years gained while maintaining acceptable overall net costs. METHODS. We simulate life histories of CRC screening in those age > = 65 with no prior CRC screening, 100% compliant with screening FOBT, all positive tests followed by a diagnostic colonoscopy (CO) and surveillance CO for those with adenomas detected. The base ease uses the Medicare test payment of $4.50 per FOBT in a program of annual FOBT screening where Sp=98% and Se is 30% for CRC, 5% for adenomas > = 1.0 cm, and 2% for adenomas < 1.0 cm. OE (3% discount)for base case is $9,350FLYG. We consider higher Se and lower Sp to provide a range of test performance consistent with that reported for sensitive guatac and immunocbemical FOBTs. Using a threshold analysis, we determine the FOBT payment that would provide cost effectiveness equivalent to the base case, for tests with the range of Se/ Sp characteristics. RESULTS. Tests with improved Se increase LY's gained and still maintain cost-effectiveness with base case even if priced dramatically higher per test ($62 - $85) than base ($4.50) (Table). A test with lower Sp (95%) than base (98%) and comparable Se results in comparable health benefits but at much higher net cost. CONCLUSION. FOBTs that provide high Se and Sp can deliver marked benefits in life-years gained with acceptable costs effectiveness ratios, even at substantially higher test payment levels and with increases tn CO's. The expected increase in benefits and the impact of higher unit-costs of newer tests on cost-effectiveness is highly dependent on the estimated relative sensitivity and specificitylevels. These results emphasize the need to establish the performance characteristics of newer tests in well characterized studies in order to appropriately assess the benefit. Irapactof FOBTStl~llk~ and ~lltlvlty ,m ToUl C o l ~ , Corn ~ ~ulvlku,4 OlE Scnmd~ Sw.mdty % II~
Samdllvl~% co'z(teee) 30_05_02 60_30_10 70_60_20 30_08_02 60_30_10 70,60.20
524 907 1,066 1.033 1.243 1~398
56 Daily Intake of Sulforaphane-Rlch Broccoli Sprouts Prevents Progression of High Salt Diet-Induced Gastric Atrophy in H. py/ori-lnfected C57/BL6 Mice In Vivo Akinori Yanaka, Songhua Zhang, Masafumi Tauchi, Hideo Suzuki, Takeshi Shibahara, Hirofumi Matsui, Akira Nakahara, Naomi Tanaka
LifeY m GalMd, Coal, a ~ ~
Background: Sulforaphane, shown to be abundantly present in broccoli sprouts, possesses anticarcinogenic activity, since it strongly induces phase 2 enzymes, such as glutathione transferase and NADPH:quinone reductase, which exert as free radical scavengers, thereby protecting cells from oxidative injury (PNAS, 94:10367, 1997). Recent studies have also shown that sulforaphane has strong bactericidal activity against Helicobc~ter pylori (Hp) in vitro (PNAS 99:7610, 2002). We have previously shown that high salt diet accelerates progression of gastric atrophy in Hp-infected mice in vivo. (Gastroenterology 122: A1, 2002) Based on these backgrounds, we examined if 1) sulforaphane inhibits Hp colonization in vivo, and if 2) sulforaphane prevents progression of high salt diet-induced gastric atrophy in Hp-infected gastric mucosa in vivo. Methods: Standardized mice model of Hp infection were set up by inoculating C57BL/6 female mice with Hp Sydney Strain; SS1. Mice were maintained with high salt diet (7.5% NaC1), supplememed with or without 2.5 mmol sulforaphane or homogenate of broccoli sprouts, which contains 2.5 mmol sulforaphane. Mice were sacrificed at 8 wks after starting the treatment. Degree of gastritis and atrophy was evaluated by updated Sydney system. DNA damage was estimated by measuring mucosal level of 8-OHdG. Expressions of TNF-u, IL-113, IL-8, and COX-2 were analyzed by real time PCR. Results: 1. Treatment with either sulforaphane or broccoli sprouts decreased Hp colonization in gastric mucosa, but failed to achieve complete eradication of Hp in vivo. 2. Sulforaphane or broccoli sprouts attenuated mucosal expressions of TNF-ct, 1L-113, IL-8, and COX-2, mitigated gastritis and DNA damage, and prevented progression of high salt diet-induced gastric atrophy. 3. The protective effects of sulforaphane or broccoli sprouts on gasmc mucosa were in parallel with their effects on inhibition of Hp-coloinzation. However, the protective effects of sulforaphane were observed even in the mice in which Hp-colonization was not inhibited. Conclusion: Daily intake of sulforaphane-rich broccolisprouts attenuates gastritis and prevents progression of gastric atrophy in Hp-infected mice fed with high salt diet, effects appear to be mediated in part by suppressing Hp colonization, and in part by enhancmg activity of gastric mucosal chemoprotection against oxidative stress during Hp infection.
LY Galn~l FOBT~ for Coetz(mlglo.) ~slutCJE (1000) 37 64 73 49 65 73
$213 $128 $94 $371 $331 $299
$4.50 $62,00 $85.00 417.00 $16.00 $39.00
42 1"he Loss of Stool DNA Mutation Abnormalities in Colorectal Neoplasia After Treatment Sapna Syngal, Daniel Chung, Christopher Willet, David Schoetz, Paul Schroy, Elena Stoffel, Deepa Jagadeesh, Kathleen Morel, Michael Ross Background: DNA abnormalities can be detected from the stool of patients with colorectal neoplasia. The effect of surgical resection and treatment on such changes has not been previously reported. Methods: Patients with newly diagnosed colorectal cancer or advanced adenomas requiring surgical resection provided a single stool sample at least two weeks after endoscopy, but prior to therapy. Additional stool samples were obtained approximately one and six months after surgery with or without chemoradiation therapy. Stool was homogenized and DNA isolated and analyzed as previously described (Gastro. 2000;119:1219). The stool-based assay(PreGen-PlusTM)includes 21 mutations (MuMu) in p53, K-ras and APC, a microsatelfite instability marker (BAT-26), and the DNA Integrity Assay (DIA~M), a marker of apoptosis. If any individual marker was positive, the overall panel result was considered positive. Results: 129 patients have been enrolled to date. Of 72 baseline samples currently analyzed (56 with cancer, 16 with advanced adenomas), 34 had a DNA mutation and 20 had an abnormal DIA. The overall sensitivity on baseline samples was 55.5%, including 62.5% for patients with invasive cancer. 52 samples have been analyzed 1-3 months following surgery; of these, 38 patients had no detectable DNA abnormalities, 9 were positive (1 with mutations, 8 positive by DIA) and 5 were technical failures. Of the 8 samples positive by DIA, 2 had been positive at baseline, and 6 had been negative 12 samples have been analyzed 6-8 months following surgery; of these, 11 patients had no detectable DNA abnormalities and 1 was positive with a p53 mutation. Conclusion: Mutational abnormalities in APC, k-ras, p53, and BAT26 detected in stool arise from colorectal neoplastic lesions and resolve with removal of the primary lesion. The etiology of abnormalities in apoptotic markers is unclear, however, and may be due to more pervasive field effects, take longer to resolve, or be affected by chemoradiation or surgical therapy These results support the utility of DNA analysis as a non-invasive option for colorectal cancer screening and provide evidence to support the potential use of mutational abnormalities to monitor colorectal cancer patients after treatment.
57 Deficiency of P27 Leads to Increased Gastric Epithelial and Inflammatory Responses to Helicobacter Pylori Noriyoshi Knzushita, Bassam I. Aswad, Min J. Park, Steven F. Moss Background: p27 is a cyclin-dependent kinase inhibitor that also acts as a tumor suppressor gene. Additionally, p27 may regulate apoptotic and inflammatory responses. H. pylori reduces expression of p27 in human gastric epithelial cells in vivo and in vitro, a potential mechanistic link between H. pylon and gastric carcinogenesis. Aim: To assess the role of reduced gastric p27 in modulating the gastric epithelial and inflammatory response to H. pylori, by comparing the effects of experimental H. pylori infection in wild type and p27 knockout mice. Methods: Six to 8 week-old wild-type (WT) C57 BL/6 mice and p27-/- C57 BL/6 mice were in parallel gavaged with 109 H. pylori SS1 strain, on three occasions over 6 days. Ten mice of each type were sacrificed at 15 and 30 weeks, and 6 of each type at 45 and 60 weeks postinfection. Formalin-fixed, paraffin embedded, H&E stained gastric tissues were scored for inflammatory and epithelial cell responses (modified Sydney system). Proliferating cells were
A-5
AGA
Abstracts