French, Amy J.

Abstract Background: The microbiome in colorectal cancer (CRC) tumors is now recognized as a potent facilitator of tumorigenesis. Identification of the intra-tumoral microbiome may provide an avenue to enhance prediction of adverse outcomes and ultimately lead to improved survival of CRC, the second leading cause of cancer death. Methods: We performed targeted sequencing of tumor and matched normal DNA samples for 3695 colorectal cancer cases within the Genetics and Colorectal Cancer Consortium (GECCO) and the Colon Cancer Family Registry (CCFR). Tumor DNA was extracted primarily from formalin fixed paraffin embedded (FFPE) or from fresh frozen tissue. FFPE slides were dissected to ensure >70% tumor content. Normal DNA was primarily extracted from blood and otherwise normal surrounding colorectal tissue. We designed probes for bacterial pathogens including Bacteroides fragilis (ETBF), polyketide synthase positive E. coli (pks+ EC), enterotoxigenic and Fusobacterium nucleatum (Fn). Logistic regression was used to assess tumor-associated bacteria and various clinical parameters, tumor molecular characteristics, and tumor stage adjusted for age, sex, and study and a Cox promotional hazard to assess association with CRC-specific survival adjusted for age at diagnosis, sex, mutational burden and microsatellite instability (MSI) and stratified baseline hazards by study. Survival data were available for 2357 patients. Results: The prevalence of patients with bacteria in tumors varied for ETBF (5.2%), pks+ EC (12.9%), and Fn (10.4%). Men had a lower probability of tumors with Fn (odds ratio (OR)=0.8, 95% confidence interval (CI)=0.64-0.99,p=0.04) but higher odds of tumors with pks+ EC (OR=1.38, CI=1.14-1.68,p=0.001). Patients with proximal tumors had higher odds of carrying Fn (OR=1.76, CI=1.38-2.26, p<0.001) as did patients with MSI high tumors (OR 2.79, CI=2.08-3.74) p<0.001). Patients with MSI-high tumors had lower odds of carrying pks+ EC (OR=0.63, CI=0.44-0.88, p=0.008) when compared to patients with microsatellite stable tumors. Among non-hypermutated tumors, we observed that tumors positive for pks+ EC were associated with better CRC-specific survival (OR=0.73, CI=0.57-0.93,p=0.01 whereas in patients with ETBF positive tumors had shorter survival (OR=1.99,CI=1,18-3.36,p=0.009) as did patients with tumors positive for Fn (OR=1.33, CI=1.04-1.72, p=0.03). Conclusions: In this largest investigation of intra-tumoral bacteria in CRC, we demonstrate that inclusion of pathogenic intra-tumoral microbiome may enhance prediction of survival and can identify a subset of patients with CRC that may benefit from targeted therapies that modulate the tumor microbiome. Citation Format: Meredith A. J. Hullar, Keith R. Curtis, Tabitha A. Harrison, Yi Lin, Robert S. Steinfelder, Sonja I. Berndt, Daniel D. Buchanan, Andrew T. Chan, David Drew, Jane C. Figueiredo, Amy J. French, Ellen L. Goode, Mark A. Jenkins, Yohannes A. Melaku, Victor Moreno, Shuji Ogino, Stephen N. Thibideau, Amanda I. Phipps, Ulrike Peters. Evaluation of intra-tumoral pathogenic bacteria pks+ E. coli, enterotoxigenic B. fragilis and fusobacterium nucleatum in 3695 colorectal cancer cases [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr PR008.

Abstract Background Obesity is an established risk factor for colorectal cancer (CRC), but the evidence for the association is inconsistent across molecular subtypes of the disease. Methods We pooled data on body mass index (BMI), tumor microsatellite instability status, CpG island methylator phenotype status, BRAF and KRAS mutations, and Jass classification types for 11 872 CRC cases and 11 013 controls from 11 observational studies. We used multinomial logistic regression to estimate odds ratios (OR) and 95% confidence intervals (CI) adjusted for covariables. Results Higher BMI was associated with increased CRC risk (OR per 5 kg/m2 = 1.18, 95% CI = 1.15 to 1.22). The positive association was stronger for men than women but similar across tumor subtypes defined by individual molecular markers. In analyses by Jass type, higher BMI was associated with elevated CRC risk for types 1-4 cases but not for type 5 CRC cases (considered familial-like/Lynch syndrome microsatellite instability-H, CpG island methylator phenotype-low or negative, BRAF-wild type, KRAS-wild type, OR = 1.04, 95% CI = 0.90 to 1.20). This pattern of associations for BMI and Jass types was consistent by sex and design of contributing studies (cohort or case-control). Conclusions In contrast to previous reports with fewer study participants, we found limited evidence of heterogeneity for the association between BMI and CRC risk according to molecular subtype, suggesting that obesity influences nearly all major pathways involved in colorectal carcinogenesis. The null association observed for the Jass type 5 suggests that BMI is not a risk factor for the development of CRC for individuals with Lynch syndrome.

AbstractDiabetes is an established risk factor for colorectal cancer. However, colorectal cancer is a heterogeneous disease and it is not well understood whether diabetes is more strongly associated with some tumor molecular subtypes than others. A better understanding of the association between diabetes and colorectal cancer according to molecular subtypes could provide important insights into the biology of this association. We used data on lifestyle and clinical characteristics from the Colorectal Cancer Family Registry (CCFR) and the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO), including 9756 colorectal cancer cases (with tumor marker data) and 9985 controls, to evaluate associations between reported diabetes and risk of colorectal cancer according to molecular subtypes. Tumor markers included BRAF and KRAS mutations, microsatellite instability and CpG island methylator phenotype. In the multinomial logistic regression model, comparing colorectal cancer cases to cancer‐free controls, diabetes was positively associated with colorectal cancer regardless of subtype. The highest OR estimate was found for BRAF‐mutated colorectal cancer, n = 1086 (ORfully adj: 1.67, 95% confidence intervals [CI]: 1.36‐2.05), with an attenuated association observed between diabetes and colorectal cancer without BRAF‐mutations, n = 7959 (ORfully adj: 1.33, 95% CI: 1.19‐1.48). In the case only analysis, BRAF‐mutation was differentially associated with diabetes (Pdifference = .03). For the other markers, associations with diabetes were similar across tumor subtypes. In conclusion, our study confirms the established association between diabetes and colorectal cancer risk, and suggests that it particularly increases the risk of BRAF‐mutated tumors.

20023 Background: EGFR is a transmembrane receptor tyrosine kinase belonging to the ErbB family of proteins. Anti-EGFR antibodies have shown efficacy as monotherapy and can enhance cytotoxic chemotherapy in patients with advanced colorectal cancers. We studied EGFR expression in primary colon cancers and its association with, microsatellite instability (MSI), clinicopathological variables and patient survival rates. Methods: Archival colon carcinomas were studied from patients with high risk Dukes’ stage B2 (n = 78) and C (n = 230) patients enrolled in a prior adjuvant trial of 5-fluorouracil, leucovorin plus standard dose or high-dose levamisole (NCCTG 91–46–53). EGFR expression (anti-EGFR H11 antibody, DAKO) was analyzed by immunohistochemistry (IHC) and intensity (0–3+), extent and their product (weighted score) were determined. MSI was analyzed using BAT26 and expression of hMLH1, hMSH2 and hMSH6 proteins by IHC. Patients were censored at 5 years after randomization for DFS and censored at 8 years post study randomization for overall survival (OS) data. Results: EGFR protein expression was detected in 216 of 308 (70%) of Dukes’ B2 and C primary colon carcinomas from patients and localized to the plasma membrane. EGFR expression variables (intensity, extent, weighted score) were increased in poor/undifferentiated compared to moderate/well differentiated tumors (p ≤ 0.036). Similar EGFR expression levels were found in MSI-H (n = 27) versus MSS (n = 203) colon cancers and for other clinicopathological variables. EGFR intensity was more likely moderate (2+) or strong (3+) in Dukes’ C versus B2 tumors (p = 0.088). In addition, higher EGFR intensity (2+, 3+) showed a trend toward worse disease-free survival (5 yr DFS %: 62.2 vs. 71.7; p = 0.095), but not overall survival (p = 0.14). Conclusions: EGFR overexpression is associated with increased tumor aggressiveness as indicated by poor tumor cell differentiation and reduced DFS in colon cancer patients receiving adjuvant 5-FU and levamisole. These findings indicate that EGFR may be an important therapeutic target in the adjuvant setting. [Table: see text]

10022 Background: Patients with colon cancers displaying high frequency microsatellite instability (MSI-H) are reported to have a favorable prognosis compared to microsatellite stable (MSS) tumors. However, prognostic factors underlying this observation are poorly understood. We studied apoptotic and proliferative indices and their relationship to MSI status, clinicopathological features, and patient survival rates. Methods: Archival Dukes’ stage B2 (n=83) and C (n=246) primary colon adenocarcinomas from patients enrolled in five 5-fluorouracil-based adjuvant therapy trials were analyzed for MSI using a PCR-based assay (MSI-H: ≥30% of loci with instability), and expression of hMLH1, hMSH2 and p53 proteins by immunostaining. Apoptosis was analyzed by the TUNEL assay and the proliferative index (PI: S phase + G2M) and DNA ploidy by flow cytometry. Correlations between markers and associations with overall survival (OS) censored at 8 yrs were sought. Results: MSI-H (n=58.18%) tumors were more likely proximal, diploid, high grade, p53 negative (vs. MSS/MSI-L; all p<0.0001), and from women (p=0.002). Of 58 MSH-H cases tested, 54 showed loss of hMLH1 (n=50) or hMSH2 (n=4) proteins. Median proliferative index (PI) [12.6 vs. 17.4; p=0.0002] was reduced in MSI-H versus MSS/MSI-L tumors. Lower PI was associated with diploidy (p<0.0001) and negative p53 expression (p=0.003). Apoptotic indices (AIs) were increased in MSI-H cancers (vs. MSS/MSI-L, p=0.082), as was the AI/PI ratio (p=0.0065). Interestingly, AI (p=0.02) and AI/PI (p<0.0001) were significantly increased in diploid versus nondiploid tumors, and after removal of MSI-H cases, relationships held for PI and AI/PI with ploidy. Better OS was related to MSI-H (p=0.032), loss of hMLH1 or hMSH2 (p=0.024), diploidy (p=0.0015), and lower PI (p=0.078), but not AI, AI/PI, nor p53 (adjusting for stage, grade, treatment and stratifying by study). Conclusions: MSI-H tumors are characterized by reduced proliferative indices and a higher ratio of apoptosis to proliferation, reflecting slower tumor growth rates compared to MSS/MSI-L tumors. These features may contribute to their better survival. Lower PI and increased AI/PI are also features of diploid MSS cancers. [Table: see text]

10019 Background: Colon cancers with microsatellite instability (MSI) display resistance to 5-fluorouracil (5-FU), and in vitro resistance can be reversed by restoring DNA mismatch repair proficiency. 5-FU inhibits the thymidylate synthase (TS) enzyme and TS may predict clinical outcome after 5-FU-based chemotherapy. To define molecular predictors of prognosis, we analyzed TS, p53, chromosome 17p allelic imbalance (AI), and patient survival stratified by MSI status. Methods: Primary colon carcinomas from patients enrolled in five 5-FU-based adjuvant therapy trials were analyzed for MSI and 17p AI using 11 microsatellite markers (MSI-H: ≥ 30% of the loci demonstrating instability). For 17p AI, markers included D17S261 and TP53 at or near the p53 locus. Expression of DNA mismatch repair (hMLH1, PharMingen; hMSH2; Oncogene), TS (TS106, Zymed), and p53 (D07, Novacastra) proteins were analyzed by immunohistochemistry. Correlations between markers and associations with overall survival (OS) were determined. Patients were censored at 5 years for DFS and at 8 years post study randomization for overall survival (OS) data. Results: Of 320 Dukes’ stage B2 and C cancers studied, 60 of 320 (19%) were MSI-H. TS expression variables (intensity, extent, weighted score) were similar in MSI-H and MSI stable/low frequency (MSS/MSI-L) cancers; similar results were found using DNA mismatch repair (dMMR) proteins. MSI-H tumors had lower stage (p= 0.0007), fewer metastatic lymph nodes (p= 0.004), and improved OS (vs. MSS/MSI-L tumors; p= 0.01). Loss of dMMR proteins was also associated with better OS (p= 0.006). None of the TS variables were prognostic for OS. Histologic grade (p= 0.0008) and nodal status (p= 0.0002) were associated with OS in contrast to 17p LOH or p53. Only MSI status or dMMR, histologic grade, and tumor stage were independent markers for OS. Conclusions: MSI-H tumors show earlier stage at presentation and better stage-adjusted survival rates. MSI status and TS expression were unrelated and TS was not prognostic, suggesting that TS levels cannot explain therapeutic resistance to 5-FU reported in MSI-H colon cancers. [Table: see text]

21046 Background: The Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor tyrosine kinase that is important for colon cancer cell growth and represents a therapeutic target. We studied EGFR expression in relation to rates of apoptosis and cell proliferation. Methods: TNM stage II and III colon carcinomas (n= 361) from patients treated in a 5-FU-based adjuvant trial were studied. EGFR expression (EGFR pharmDx™) and extent were analyzed and their relationship to rates of cell turnover, histologic grade, mismatch repair (MMR) status (defective if instability at BAT 26 locus plus loss of hMLH1, hMSH2 or hMSH6 proteins) and clinical outcome were determined. Results: EGFR plasma membrane staining was detected in 214 (59.3 %) colon carcinomas. Increased EGFR intensity (2+, 3+) was associated with a higher number of caspase-3- (p= 0.02) and Ki-67- (p< 0.001)positive tumor cells. Increased EGFR intensity (p= 0.02) and extent (p= 0.03) were associated with poor differentiation, and did not depend upon MMR status (Breslow-Day test p ≥ 0.46). Diffuse (vs. focal) EGFR extent was increased in tumors with defective MMR [22 of 32 (69%)] compared to those with intact MMR [136 of 271 (50%)] (p= 0.047). EGFR intensity was unrelated to MMR status. Neither EGFR intensity nor extent was significantly associated with patient survival. Conclusions: Stage II and III colon cancers with EGFR overexpression show adverse prognostic variables, i.e., increased rates of cell turnover and poor histologic grade, suggesting that EGFR may be an important target for adjuvant therapy. *Chi-square test for categorical data, Wilcoxin rank-sum test for continuous data [Table: see text] No significant financial relationships to disclose.

10529 Background: BAX mutations are associated with defective DNA mismatch repair (MMR) in human colon cancers. However, the impact of BAX inactivation upon tumor cell apoptosis and proliferation in vivo remain unknown. We analyzed and compared caspase-3 and Ki-67 expression in tumors with and without BAX mutations. Methods: TNM stage II and III (n= 377) colon carcinomas were studied from participants in a 5-FU-based adjuvant therapy trial. Archival tumors were analyzed for instability at the BAT26 mononucleotide locus using polymerase chain reaction and hMLH1, hMSH2 and hMSH6 by immunohistochemistry (IHC). Frameshift mutations in a tract of eight deoxyguanosines within BAX were analyzed. Expression of caspase-3 and Ki-67 proteins were analyzed by IHC. Results: Thirty-nine of 377 (10%) tumors showed defective MMR defined as instability at BAT26 and loss of either hMLH1, hMSH2 and/or hMSH6 proteins. BAX mutations were found in 20 of 37 (54%) MMR deficient tumors and in 1 of 50 (2%) tumors with intact MMR. Mean and median number of caspase-3-positive cells were increased in tumors with defective MMR (p= 0.04), but did not differ based upon BAX status [ Table ]. However, tumors with BAX mutations showed higher Ki-67 labeling indices compared to those with wild type BAX (p= 0.01)[ Table ]. Neither BAX mutations nor caspase-3- positive cells were prognostic in a univariate analysis. Tumors with lower Ki-67 extent had improved overall survival (p=0.06), but not DFS (p=0.24). Defective MMR (vs intact) was associated with better DFS in a multivariate analysis (p= 0.03). Conclusion: MMR deficient colon cancers show frequent BAX inactivation, yet have increased apoptotic rates as indicated by increased caspase-3 expressing tumor cells. BAX mutation was associated with hyperproliferation suggesting a growth advantage compared to wild type tumors. [Table: see text] No significant financial relationships to disclose.

Abstract Background: Different molecules within coffee may be pro-carcinogenic, while others may have anti-oxidant and anti-neoplastic effects. Hence, it can be hypothesized that coffee may have differing risk associations with specific colorectal cancer (CRC) subtypes. The aim of this study was to evaluate associations between coffee consumption and incident CRC overall, by anatomic subsite, and by molecularly defined tumor subtypes, in the population-based Iowa Women's Health Study (IWHS). Methods: IWHS participants, ages 55-69 years, provided dietary intake estimates using self-reported food frequency questionnaires at baseline (1986). From 35,216 women included the final analysis, 1298 developed CRC (673 with proximal CRC, 597 with distal CRC) through December 31, 2002 (end of the IWHS molecular epidemiology project study period). Archived, paraffin-embedded tissue specimens for 732 representative CRC cases were collected and analyzed to determine if coffee consumption (none or &lt;1 cup/month, &lt;1 cup/week, 1 cup/day, 2-3 cups/day and ≥4 cups/day) was associated with microsatellite instability (MSI-L/MSS or MSI-H), CpG island methylator phenotype (CIMP-negative or CIMP-positive), BRAF mutation status (BRAF-wildtype or BRAF-mutated), KRAS mutation status (KRAS-wildtype and KRAS-mutated), p53 protein expression level (p53 absent, low or high) and estrogen receptor β protein expression level (ERβ absent, low or high). Multivariable Cox regression models were fit to estimate relative risks (RR) and 95% confidence intervals (CI) with adjustment for age, smoking, alcohol use, diabetes, body mass index and waist-hip ratio, physical activity, estrogen use, dietary intake of fat, calcium, vitamin E, sucrose, red meat and methionine. P-values were not adjusted for multiple testing. Results: There was no significant difference in the risk of overall (RR, 1.05; 95% CI, 0.85-1.29; Ptrend=0.50), proximal (RR, 0.91; 95% CI, 0.68-1.21; Ptrend=0.57) or distal CRC (RR, 1.18; 95% CI, 0.86-1.64; Ptrend=0.19) in women who consumed ≥4 cups of coffee/day (caffeinated or decaffeinated), as compared to non-consumers. By CRC molecular subtypes for these same two exposure groups, women with ≥4 cups of coffee/day were more likely to develop ERβ-low CRC (RR, 2.98; 95% CI, 1.04-8.51; Ptrend=0.06), though no significant associations were observed for caffeinated or decaffeinated coffee specifically. In contrast, women who consumed ≥4 cups of decaffeinated coffee/day were less likely to develop MSI-L/MSS (RR, 0.57; 95% CI, 0.36-0.92; Ptrend=0.04) and BRAF-WT (RR, 0.69; 95% CI, 0.44-1.06; Ptrend=0.06) CRCs than non-consumers. Women who consumed ≥4 cups of caffeinated coffee/day were also less likely to develop p53-low CRCs (RR, 0.53; 95% CI, 0.24-1.15; Ptrend=0.02). Null associations were observed between decaffeinated coffee consumption and risk of CIMP-, KRAS- and p53- defined CRC subtypes, as well as between caffeinated coffee consumption and risk of MSI-, BRAF-, CIMP- or KRAS-defined CRC subtypes. Conclusion: In this prospective cohort study of older women, though coffee consumption was not associated with risk of overall or site-specific CRC, unique associations were observed with molecularly defined CRC subtypes, supporting a need for further investigation of the potential pro- and anti-carcinogenic effects of coffee constituents on biologic targets involved in colorectal carcinogenesis. Citation Format: Siddharth Singh, Robert Vierkant, Lori Tillmans, Alice Wang, Daniel Weisenberger, Peter Laird, Maki Inoue-Choi, Andrea Mariani, Charles Lynch, Kristin Anderson, Amy French, Robert Haile, Lisa Harnack, John Potter, Susan Slager, Thomas Smyrk, Stephen Thibodeau, James Cerhan, Paul Limburg. Coffee consumption and colorectal cancer risk by molecularly defined subtypes in a prospective cohort study of older women. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr A44.