Thibodeau, Steven

Purpose The Sangre Por Salud (SPS) Biobank was established to facilitate biomedical research opportunities for the Latino community by creating an easily accessible prospective cohort for scientists interested in studying health conditions and health disparities in this population. Participants Individuals self-identifying as Latino, aged 18–85 years, were prospectively recruited from the primary care Internal Medicine clinic at Mountain Park Health Center in Phoenix, Arizona. After obtaining informed consent, detailed medical history questionnaires were captured, and blood samples were obtained for common laboratory tests. Participants authorised the research team to access their electronic health records for research purposes. In addition, participants had serum, plasma and DNA samples isolated and stored at the Mayo Clinic Arizona Biorepository Laboratory for long-term storage and future access. As part of the study, participants consented and agreed to be contacted for potential participation in future research studies. Findings to date 3756 participants provided informed consent, of whom 3733 completed all study questionnaires, an oral glucose tolerance test and had blood collected and stored. The SPS cohort is predominantly composed of females (72%), with a median age at time of consent of 42 years. All participants self-identified as Hispanic/Latino, 45% were married, 53% were employed for wages and 60% had less than a high school degree. Around 25% of participants met diagnostic criteria for overweight (BMI 25–29 kg/m 2 ), and 49% met for obesity (BMI≥30 kg/m 2 ). At time of recruitment, hypertension, hyperlipidaemia and depression affected 22%, 20% and 13% of the cohort, respectively. Future plans We plan to regularly update the participants’ electronic health records and self-reported health data to longitudinal research. Additionally, we plan to obtain a more comprehensive genomic analysis on the entire cohort, ensuring greater research interest and investigation into the underlying genetic factors that contribute to disease susceptibility in this cohort.

Abstract Background: Colorectal cancer (CRC) incidence has increased in those 50 years or younger compared to older individuals in recent decades. While the intra-tumoral microbiome is a potent facilitator of tumorigenesis, the role of the microbiome in early onset (eo)CRC is unclear. Identification of the intra-tumoral microbiome may enhance prediction of adverse outcomes to improve survival of CRC overall age groups and eoCRC. Methods: We performed targeted sequencing of tumor and matched normal DNA samples from 3695 colorectal cancer cases within the Genetics and Colorectal Cancer Consortium (GECCO) and the Colon Cancer Family Registry (CCFR). Tumor DNA was extracted from formalin fixed paraffin embedded (FFPE) or fresh frozen tissue. Normal tissue DNA was extracted from blood or normal surrounding colorectal tissue. We designed probes for the bacterial pathogens enterotoxigenic Bacteroides fragilis (ETBF), polyketide synthase positive E. coli (pks+ EC), and Fusobacterium nucleatum (Fn). We used logistic regression to assess tumor-associated bacteria and 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, overall and in eoCRC, adjusted for age at diagnosis, sex, mutational burden, and microsatellite instability (MSI) and stratified baseline hazards by study. Survival data were available for 2357 and 308 patients, overall and in eoCRC, respectively. Results: The prevalence of different bacteria in tumors differed slightly overall vs. in eoCRC tumors: ETBF (5.2%, 6.3%), pks+ EC (12.9%, 14.8%), and Fn (10.4%, 8.2%). Overall, 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), compared to women. The odds of prevalent Fn were higher for proximal vs distal tumors (OR=1.76, CI=1.38-2.26, p<0.001), but there was no association between tumor site and pks+ EC or ETBF. MSI-high tumors were more likely to carry Fn (OR=2.79, CI=2.08-3.75, p<0.001) and less likely to carry pks+ EC (OR=0.63, CI=0.44-0.88, p=0.008) when compared to patients with MSS tumors. In patients with non-hypermutated tumors, we observed that pks+ EC positive tumors were associated with better CRC-specific survival (HR=0.73, CI=0.57-0.93, p=0.01), whereas ETBF positive tumors (HR=1.99, CI=1,18-3.36,p=0.009) and Fn positive tumors (HR=1.33, CI=1.04-1.72, p=0.03) were associated with poorer survival. In eo-CRC patients, Fn positive status was associated with poorer survival (HR=2.4, CI= 1.056 - 5.492, p=0.026). Conclusions: We showed that the intra-tumoral microbiome differs in distribution across patient populations and patient survival over all age groups and, specifically, in eoCRC. Citation Format: Meredith A. Hullar, Keith R. Curtis, Tabitha Harrison, Yi Lin, Robert Steinfelder, Sonja I. Berndt, Daniel D. Buchanan, Andrew T. Chan, David A. Drew, Jane Figueiredo, Amy J. French, Tabitha A. Harrison, Mark A. Jenkins, Johannes Melaku, Victor Moreno, Tomotaka Ugai, Shuji Ogino, Conghui Qu, Chenxu Qu, Steven Thibodeau, Syed Zaidi, Amanda I. Phipps, Ulrike Peters, on behalf of GECCO and CCFR investigators. Evaluation of intra-tumoral pks+ E. coli, enterotoxigenic B. fragilis and Fusobacterium nucleatum, overall and in early onset disease, in colorectal cancer cases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3039.

Abstract Background Smoking is associated with colorectal cancer (CRC) risk. Previous studies suggested this association may be restricted to certain molecular subtypes of CRC, but large-scale comprehensive analysis is lacking. Methods A total of 9789 CRC cases and 11 231 controls of European ancestry from 11 observational studies were included. We harmonized smoking variables across studies and derived sex study–specific quartiles of pack-years of smoking for analysis. Four somatic colorectal tumor markers were assessed individually and in combination, including BRAF mutation, KRAS mutation, CpG island methylator phenotype (CIMP), and microsatellite instability (MSI) status. A multinomial logistic regression analysis was used to assess the association between smoking and risk of CRC subtypes by molecular characteristics, adjusting for age, sex, and study. All statistical tests were 2-sided and adjusted for Bonferroni correction. Results Heavier smoking was associated with higher risk of CRC overall and stratified by individual markers (Ptrend < .001). The associations differed statistically significantly between all molecular subtypes, which was the most statistically significant for CIMP and BRAF. Compared with never-smokers, smokers in the fourth quartile of pack-years had a 90% higher risk of CIMP-positive CRC (odds ratio = 1.90, 95% confidence interval = 1.60 to 2.26) but only 35% higher risk for CIMP-negative CRC (odds ratio = 1.35, 95% confidence interval = 1.22 to 1.49; Pdifference = 2.1 x 10-6). The association was also stronger in tumors that were CIMP positive, MSI high, or KRAS wild type when combined (Pdifference < .001). Conclusion Smoking was associated with differential risk of CRC subtypes defined by molecular characteristics. Heavier smokers had particularly higher risk of CRC subtypes that were CIMP positive and MSI high in combination, suggesting that smoking may be involved in the development of colorectal tumors via the serrated pathway.

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: Smoking has been associated with colorectal cancer (CRC) risk; but limited evidence has shown the association between smoking and molecular subtypes of CRC. Methods: We analyzed 9,422 CRC cases and 9,950 controls from 10 observational studies. Multinomial logistic regression analysis was performed to assess the association between sex-study-specific quartiles of pack years of smoking and risk of CRC molecular subtypes, using non-smokers as reference group, adjusting for age, sex, and study. Known oncogenic mutations in four somatic colorectal tumor markers were assessed individually and in combination, including BRAF mutations, KRAS mutations, CpG island methylator phenotype (CIMP), and microsatellite instability (MSI) status. Case-only analysis was also performed to estimate heterogeneity in risk of molecular subtypes of CRC. Results: Compared with controls, higher pack years of smoking were statistically significantly associated with increased CRC risk when stratified, individually, by all four markers, and the associations got stronger with higher quartiles pack years (p-trend<0.001). Associations between smoking and CRC risk also differed significantly among molecular subtypes. Compared to nonsmokers, the risk of BRAFmut CRC was 83% higher for smokers within the highest quartile of pack-years (OR=1.83; 95% CI: 1.50, 2.25), and 29% higher for BRAFwt CRC (OR=1.29; 95% CI: 1.17, 1.43; Ratio of ORs (ROR)=1.45; 95% CI: 1.18, 1.17; p=3.45x10-4). Similarly, heavy pack-years of smoking was associated with almost two times higher risk of CIMP-high CRC (OR=1.93; 95% CI: 1.60, 2.31), but only 33% higher risk of CIMP-low/negative CRC (OR=1.33; 95% CI: 1.19, 1.48; ROR=1.49; 95% CI: 1.24, 1.79; p=1.72x10-5). The association between smoking and CRC was also stronger in MSI-high tumors (ORMSI-H=1.65; 95% CI: 1.36, 2.00; ORMSI-L/MSS=1.37; 95% CI: 1.23, 1.52; ROR=1.22; 95% CI: 1.00, 1.48; p=0.046). In contrast, the association between smoking and CRC risk was stronger for KRASwt (OR=1.43; 95% CI: 1.27, 1.60), than KRASmut tumors (OR=1.18; 95% CI: 1.02, 1.37; ROR=0.83; 95% CI: 0.71, 0.97; p =0.016). When combining tumor markers, smoking was found to be significantly associated with higher risk of colorectal tumors from the serrated pathway. Conclusion: In this largest study with a total of 19,372 subjects, we found that heavier pack years of smoking was associated with increased risk of all CRC molecular subtypes. Smokers with heavier pack-years of smoking had particularly higher risk of CRC subtypes with BRAF mutation and CIMP-high, suggesting smoking may be particularly involved in the development of these subtypes of colorectal tumor. Citation Format: Xiaoliang Wang, Efrat Amitay, Barbara L. Banbury, Hermann Brenner, Daniel D. Buchanan, Peter T. Campbell, Jenny Chang-Claude, Steven J. Gallinger, Graham G. Giles, Tabitha A. Harrison, John L. Hopper, Mark A. Jenkins, Yi Lin, Reiko Nishihara, Polly A. Newcomb, Shuji Ogino, Lori C. Sakoda, Robert E. Schoen, Martha L. Slattery, Steven N. Thibodeau, Bethany Van Guelpen, Michael O. Woods, Li Hsu, Michael Hoffmeister, Ulrike Peters. Smoking is associated with risks of molecular subtypes of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 630.

Abstract Aberrantly methylated genes represent attractive and broadly informative markers for colorectal cancer (CRC). As demographics, numerous ingestants, environmental exposures, and obesity variably influence both gene methylation rates and CRC incidence, it is important to understand the effects of such covariates on the clinical performance of CRC screening tests that incorporate assay of methylated gene markers. Based on a recent multicenter study (Ahlquist et al. Gastroenterology 2012 (in press)), stool assay of carefully selected methylated gene marker candidates (NDRG4, BMP3, vimentin, TFPI2), alone or in combination, yield high detection rates of both CRC and large adenomas. Aim: To assess the impact of demographic, exposure, body mass, and other patient variables on stool levels of the above 4 methylated gene markers. Methods: We studied buffered stools collected within 3 years of a normal colonoscopy from 500 patients undergoing average-risk screening or polyp surveillance (median age 64 (range 44-85); 53% women). Upon receipt, stools were promptly homogenized, aliquoted, and frozen at –80°C. On supernatants from thawed/spun aliquots, target genes were purified by hybrid capture, bisulfite treated, and assayed using the analytically-sensitive QuARTS method (quantitative allele-specific real-time target and signal amplification), as described (above citation). The reference gene β-actin was assayed along with methylation of NDRG4, BMP3, vimentin, TFPI2. Log-converted data were normalized to allow comparison of effects between markers, and effect size was expressed as % change relative to standard deviation for each marker (standardized relative change (SRβ)). Results: The only patient characteristic that significantly influenced all methylated marker levels in stool was age (p<0.0001 for each marker); SRΔ was greatest with TFPI2 at +91.3 (p<0.001 vs other markers), least with BMP3 at +29.7 (p<0.001 vs others), and intermediate with vimentin at +46.0 and NDRG4 at +45.1. In contrast to methylation markers, levels of β-actin did not change across age. The other demographic variables of sex, race, and geographic residence had no effect on methylation markers. Marker levels were also not affected by exposure variables (smoking, alcohol consumption, or analgesic use), by family history of CRC or polyps, or by personal history of polyps. Finally, neither overweight status (n=176) nor obesity (n=148) had an effect on marker levels. Conclusions: Age significantly affects stool levels of tumor-associated methylated gene markers in colonoscopy-normal patients, and the degree of this age effect differs across markers. In contrast, other key patient variables had no effect on stool marker levels. Comment: Optimal use of these methylation markers for stool screening of CRC may require age-adjustment of normal cutoff levels to maximize test specificity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3572. doi:1538-7445.AM2012-3572

Abstract Colon cancer is the result of a multi-step process involving the accumulation of genetic and epigenetic alterations leading to the transformation of normal colon epithelium to adenocarcinoma. Identification of characteristic genetic alterations is of critical importance to increase our understanding of the transformation process. We focused our analysis on the most common molecular sub-type of colon cancers, microsatellite stable (MSS) and CpG island methylator phenotype (CIMP)-negative, to identify characteristic copy number alteration (CNA) events and associated gene expression profiles using high-resolution genome-wide microarrays. DNA and RNA were extracted from 41 fresh-frozen paired colon tumors and adjacent normal tissue collected by the Colon Cancer Family Registry. Genomic profiles, such as CNAs and loss of heterozygosity (LOH), were identified with the Affymetrix Genome-Wide Human SNP 6.0 array for both tumor and adjacent normal tissue. Gene expression profiles of tumors and adjacent normal tissue were identified with the Affymetrix GeneChip Human Exon 1.0 ST array. Partek Genomics Suite software was used for analysis and data integration. We identified recurrent (>25%) CNAs in several chromosomal regions: gain in 1q, 7p, 7q, 8q, 13q, 20p, 20q, Xp, Xq and loss in 5q, 8p, 14q, 16p, 17p, 18p, and 18q. In addition, based on the allele information from the SNP arrays, we found a subset of these recurrent CNAs were associated with LOH. Preliminary results from the integration of CNA and gene expression data in tumors indicate that a subset of these recurrent CNAs is associated with the disruption of gene expression. For example, we observed recurrent copy number gain in 8q24.21, the genomic region containing the c-MYC gene, and associated up-regulation of c-MYC gene expression in tumors compared to adjacent normal tissue. In another example, recurrent copy number loss at 5q22.2 was associated with down-regulation of APC gene expression; loss of function of this tumor suppressor is associated with both hereditary and sporadic colon cancer. This genome-wide characterization of both genomic alterations and gene expression may help identify key genes and pathways that are disrupted in this common molecular subtype of colon cancer. We will present detailed results from this integrated and comprehensive genetic profiling of MSS and CIMP-negative colon cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2145.