Supplementary Table S7. Confounding analyses for the association between the interaction of the TGF-B-Polygenic Risk Score and red meat intake in relation to colorectal cancer risk.

Supplementary Figure S1, LocusZoom plots.

BACKGROUND:Fusobacterium nucleatum (Fn) is a bacterium typically found in the human oral cavity that has been implicated in the development and progression of colorectal cancer (CRC). OBJECTIVE:This study examines the association between prevalence of Fn in tumor tissue and CRC-specific mortality in a heterogeneous United States population. METHODS:The present study includes 233 participants with stage I-III CRC who died of CRC, matched to 458 participants with CRC who did not die from their disease, within the Translational Research Program in Cancer Differences across Populations. Genomic DNA was extracted from formalin-fixed paraffin-embedded tumor tissue samples. Droplet digital polymerase chain reaction was used to assess the prevalence of Fn. The authors fit logistic regression models for the association between Fn positivity and CRC-specific mortality, adjusting for age, year of diagnosis, sex, stage, tumor site, population group, and tumor macrodissection status. RESULTS:Presence of Fn in CRC tumor tissue was associated with 79% greater odds (95% CI, 1.20-2.67) of CRC-specific mortality. This association was more pronounced in participants with rectal (odds ratio [OR], 7.73; 95% CI, 2.13-34.79) than proximal colon (OR, 1.17; 95% CI, 0.65-2.08) or distal colon tumors (OR, 1.51; 95% CI, 0.74-3.04, p value for interaction = .023), and more pronounced with respect to early-onset (age <50 years; OR, 10.30; 95% CI, 2.09-70.53) than later-onset CRC (age ≥50 years; OR, 1.51; 95% CI, 0.98-2.33, p value for interaction = .010). CONCLUSIONS:These findings support an association between presence of Fn in CRC tumor tissue and CRC-specific mortality, particularly for rectal tumors and early-onset CRC, in a heterogeneous population.

Abstract The immune microenvironment is a critical regulator of colorectal cancer (CRC) biology and represents a clinically targetable tumor feature in a subset of patients. T cells are one of the most common cell types in the CRC immune microenvironment and exist in a broad variety of subtypes and activation states. Proliferation status represents a general measure of T cell activity that is applicable to many subtypes. Evidence from other tumor types suggests that proliferating cytotoxic T cells may mediate adaptive immunity in cancer. However, T cell proliferation in CRC is incompletely characterized. To investigate the role of proliferating T cells in CRC, we developed a multiplexed immunofluorescence assay assessing CD3, CD4, CD8, FOXP3, PTPRC (CD45RA and CD45RO), MKI67 (Ki67), and KRT (cytokeratin) expression, and assessed 1,610 primary resected CRC specimens from four US/Canadian cohort studies. Digital imaging and pathologist-supervised machine learning were used to decompose image data into single-cell level information. The Kaplan-Meier method was used to describe CRC-specific survival (CSS) and multivariable Cox proportional hazards models were used to examine associations of proliferating and non-proliferating T cell densities with CSS. Multivariable ordinal logistic regression was used to adjust for confounding factors (age, cancer site, sex, and study batch) in analyses of T cell densities and tumor features. Across one million T cells, 4.5 % were classified as proliferating, as defined by positive MKI67 expression. While higher densities of both proliferating and non-proliferating T cells were positively associated with microsatellite instability (MSI), a CpG island methylator high phenotype, BRAF mutant status and KRAS wildtype status, these associations were stronger for proliferating T cells (e.g., adjusted odds ratio (OR) 2.09, CI 1.62-2.70, p<0.0001 for MSI and higher proliferative T cell density versus adjusted OR 1.40, CI 1.08-1.81, p=0.01 for MSI and higher non-proliferative T cell density). Higher proliferative and non-proliferative T cell densities were both positively associated with CSS (adjusted hazard ratio (HR) 0.75, CI 0.55-1.03, comparing highest versus lowest quartile of proliferative T cell densities (Ptrend=0.018) and adjusted HR 0.42, CI 0.31-0.57, comparing highest versus lowest quartile of non-proliferative T cell densities (Ptrend<0.001). Despite comprising a minor subset of total T cell infiltration in the CRC microenvironment, proliferative T cells are more strongly associated with specific molecular subtypes and are also associated with CSS. These results, across a large series of primary human tumors, suggest that proliferation marks a subset of functionally distinct subset of T cells in the CRC microenvironment. Citation Format: Yasutoshi Takashima, Claire Elizabeth Thomas, Andressa Dias Costa, Sushma Thomas, Evertine Wesselink, Conghui Qu, Steven Gallinger, Robert C. Grant, Li Hsu, Marios Giannakis, Jeroen Huyghe, Daniel D. Buchanan, Shuji Ogino, Riki (Ulrike) Peters, Amanda I. Phipps, Jonathan A. Nowak. Proliferative T cell infiltration is associated with colorectal cancer molecular subtypes and cancer specific survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6571.

Supplementary Table S3. Weights used for polygenic risk score estimation and coordinates from single nucleotide polymorphisms considered in the study.

Background Asian American (AsA) and Native Hawaiian and Pacific Islander (NHPI) populations are underrepresented in U.S. health studies. Objectives The MOSAAIC (Multi-ethnic Observational Study in American Asian and Pacific Islander Communities) is a cohort study designed to improve understanding of disparities in cardiovascular disease and other health conditions affecting AsA and NHPI groups. Methods Through broad eligibility criteria and outreach tailored to each study community, MOSAAIC will include 11,500 adults aged 18+ years living in 5 field center regions: New York, Philadelphia, Chicago, San Francisco Bay Area, and Honolulu. The protocol specifies sample-size targets to allow valid comparisons among persons having personal or family origins in 4 geographically defined regions, including East Asia (the largest groups being Chinese and Korean), South Asia (Indian), Southeast Asia (Vietnamese and Filipino) and Oceania (NHPI). Measurements to be obtained at an in-person examination include clinical laboratory tests, medical history, medication use, spirometry, cognitive and physical function, anthropometry, and electrocardiogram. Surveys in multiple languages to assess medical, behavioral, lifestyle, social and environmental influences on health were developed by a multistep process to ensure equivalency between translations and cultural appropriateness. Biospecimens are stored for future studies. Long-term follow-up will be conducted to ascertain and adjudicate major health events including myocardial infarction, stroke, heart failure, and mortality. Conclusions The National Institutes of Health established MOSAAIC as a resource for wide-ranging epidemiologic investigation of factors related to cardiometabolic, pulmonary, or mental health in persons of diverse AsA and NHPI background.

Abstract Colorectal cancer (CRC) is the third most commonly diagnosed cancer and has the second-highest mortality rate overall in the United States (US). Appreciable population differences in incidence and mortality rates persist in the US. These differences are particularly pronounced among Alaska Native peoples who, for over 40 years, have experienced the highest incidence and mortality rates, despite dedicated screening efforts. The reasons underlying these high rates are not understood. A key knowledge gap is the lack of studies characterizing the molecular features of colorectal tumors in Alaska Native patients. This study aims to identify tumor and tumor microenvironment (TME) features present at time of diagnosis that are associated with CRC mortality, and that may contribute to the elevated mortality rate.We analyzed treatment-naïve tumor tissue samples from 217 Alaska Native participants with stage I-III CRC diagnosed at the Alaska Native Medical Center from 2000 to 2017. Participants who died from CRC (n=60) were matched 1:2 to participants who did not die from CRC and who lived at least as long after diagnosis as the participant they were matched to (n=157). Matching was based on age at diagnosis, sex, year of diagnosis, and tumor site and stage. The present study included 139 females (64.1%) and 78 males (35.9%). The median age at diagnosis was 68 years (range: 36-85). At diagnosis, 33 participants (15.2%) had stage I, 93 (42.9%) had stage II, and 91 (41.9%) had stage III CRC. We constructed tissue microarrays (TMAs) from formalin-fixed paraffin-embedded (FFPE) tumor tissue blocks (2 tumor center cores and 1 invasive margin core per tissue) and performed spatial transcriptomics profiling using the GeoMx DSP platform and the Whole Transcriptome Atlas panel. We measured gene expression separately in epithelial and stromal tissue compartments. After quality control, adjustment for batch effects, and normalization, we used DESeq2 to find genes associated with CRC mortality. Analysis was stratified by tissue compartment and tumor location. We performed gene set enrichment analyses using the fgsea R package. Differential gene expression results showed significant downregulation of SPINK4 (3.2-fold decrease, false discovery rate [FDR]-adjusted p=4.72e-3) and MUC2 (2.6-fold decrease, FDR-adjusted p=1.37e-3) genes in the tumor center epithelial compartment in participants who died from CRC. Gene set enrichment analyses showed that upregulation of interferon gamma response hallmark genes was associated with favorable prognosis across tissue compartments and tumor locations, while upregulation of epithelial-mesenchymal transition hallmark genes in the tumor margin was associated with worse prognosis (all FDR-adjusted p<0.05). Additional ongoing work includes cell type deconvolution analyses and expansion of the study size to over 400 Alaska Native participants with CRC. Citation Format: Garrick Chang, Diana Redwood, Amanda L. Koehne, Matthew Fitzgibbon, Conghui Qu, Hang Yin, Elizabeth Donato, Mingang Lin, Cecilia Yeung, James J. Tiesinga, Sushma Thomas, Li Hsu, Christopher I. Li, Timothy K. Thomas, Riki (Ulrike) Peters, Jeroen Huyghe. Spatial transcriptomics profiling of colorectal cancer tumors in Alaska Native peoples: Discovery of prognostic biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6749.

Multiomic data analysis poses statistical challenges. We evaluated statistical models for our multiplex immunofluorescence study of T cell subset densities in colorectal cancer. Using 1235 cases, we compared 7 models-ordinal logistic regression, Poisson, quasi-Poisson, quadratic negative binomial (NB), linear NB, zero-inflated NB, and hurdle NB models-assessing associations with a strong (microsatellite instability, MSI) and a weak (calcium intake) exposure. Simulation studies assessed type I error and power. Effect estimates were generally consistent for the strong exposure (MSI) but varied for the weaker exposure (calcium). Simulations revealed inflated false-positive rates for the Poisson and NB-based models, including quadratic NB, zero-inflated, and hurdle, but not for ordinal logistic regression or the linear NB model. The quasi-Poisson model showed modest inflation of low P-values, but the overall P-value distribution remained approximately uniform under the null. Ordinal logistic, linear NB, and quasi-Poisson models achieved the best or near-best power across a range of zero proportions, dispersion levels, and distributions. The ordinal logistic, linear NB, and quasi-Poisson models are useful and robust options for epidemiologic analyses of overdispersed, right-skewed multiomic data with a nontrivial proportion of zero counts.

Supplemental Figure 4 shows the m2CLR distribution (boxplots) of the phylum Fusobacteriota, the genera Fusobacterium and Leptotrichia, stratified by population group. Due to high percentages of zeros the median of all boxplots is zero.

Supplementary Table S5. Results from multinomial logistic regression models to evaluate pathway-based polygenic risk score interactions with red meat and processed meat intake by topological tumor location.

Abstract Background: The microbiome and tumor immune response are important and inter-related components that are implicated in colorectal cancer (CRC) prognosis. However, associations between these components and potential joint effects on CRC mortality remain unclear. Methods: We included 366 participants with CRC (106 African American, 161 Alaska Native, 91 Hispanic, 8 non-Hispanic White) from the Translational Research Program in Cancer Differences across Populations (TRPCDP). 241 participants who did not die of CRC were matched to 125 participants who died of CRC during follow-up by age, sex, tumor site, tumor stage, year of diagnosis, and population group. We sequenced microbial DNA from the V4 region of the 16S rRNA bacterial gene and sequenced RNA using the Illumina TruSeq RNA Exome kit from formalin-fixed paraffin embedded (FFPE) tumor tissue. We calculated the T-cell inflamed gene expression profile (GEP) score as a weighted sum of log2-transformed transcripts per million of 18 genes: CCL5, CD27, CD274 (PD-L1), CD276 (B7-H3), CD8A, CMKLR1, CXCL9, CXCR6, HLA-DQA1, HLA-DRB1, HLA-E, IDO1, LAG3, NKG7, PDCD1LG2 (PDL2), PSMB10, STAT1, and TIGIT. We dichotomized the T-cell inflamed GEP score into high and low groups using the top tertile as a threshold. Using logistic regression, we estimated odds ratios (OR) and 95% confidence intervals (CI) for associations between bacterial presence and dichotomized T-cell inflamed GEP score, as well as interaction effects of bacteria and dichotomized T-cell inflamed GEP score on CRC-specific mortality, adjusting for matching factors. Results: Among 48 genera tested, Anaerococcus was associated with lower odds of high T-cell inflamed GEP score (OR=0.34, 95% CI 0.20-0.58) and Leptotrichia was associated with higher odds of high T-cell inflamed GEP score (OR=2.93, 95% CI 1.66-5.22). When combined, the joint effect of tumors being Leptotrichia positive and low T-cell inflamed GEP score was associated with over four times the odds of CRC mortality (OR=4.41, 95% CI 1.86-10.83) compared to tumors that were Leptotrichia negative and high T-cell inflamed GEP score. Conclusions: The joint effect of Leptotrichia presence and low T-cell inflamed GEP score resulted in markedly higher odds of CRC death. Understanding the influence of this immune-microbiota interaction may improve CRC prognostic stratification and enable the discovery of new treatment targets to improve CRC prognosis. Citation Format: Claire Elizabeth Thomas, Hang Yin, Jeroen Huyghe, Nicole Catalina Lorona, Scott D. Labrie, Keith R. Curtis, Orsalem Kahsai, Sosun Nayemi, Ningxin Ma, Timothy Randolph, Conghui Qu, Sushma Thomas, Li Hsu, Amanda L. Koehne, Heather Green-Mantrana, Marc Matrana, James J. Tiesinga, William M. Grady, Diana Redwood, Christopher I. Li, Li Li, Riki (Ulrike) Peters, Jane C. Figueiredo, Timothy K. Thomas, Amanda I. Phipps, Meredith A. Hullar. Interaction between T-cell inflamed gene expression profile score and tumor-associated microbiome on colorectal cancer mortality in a heterogeneous patient population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2867.

Supplemental Figure 5 shows the m2CLR distribution (boxplots) of two MetaCyc pathways of interest stratified by population group.

Colorectal cancer (CRC) is a leading cause of cancer-related death, with incidence rising substantially among individuals under 50 years of age. Polygenic risk scores (PRS) hold promise for identifying high-risk individuals; when combined with lifestyle factors, they substantially improve prediction accuracy compared with models based on lifestyle factors alone. However, few clinical tools currently exist that facilitate this integrated, PRS-enhanced risk assessment. To bridge this gap, we developed MyGeneRisk Colo n, a publicly accessible web portal that delivers individualized CRC risk prediction by incorporating genetic, demographic, family history, and lifestyle factors. This paper details the development of the underlying risk prediction model, the portal's architecture and data security, our reporting framework, and engagement with a community advisory panel. Designed as a user-friendly platform, MyGeneRisk Colon aims to effectively communicate personalized CRC risk profiles and educate users and healthcare providers about prevention strategies.

Cancer prevention is recognized as a key strategy for reducing disease incidence, mortality, and the overall burden on individuals and society. However, determining when to begin preventive interventions presents a significant challenge: starting too early may lead to more interventions and increased lifetime burdens due to repeated administrations, while delaying may miss opportunities to prevent cancer. Evidence-based recommendations require a benefit-burden analysis that weighs life-years gained against the burden of interventions. With the growing availability of large-scale observational data, there is now an opportunity to empirically evaluate these trade-offs. In this paper, we propose a causal framework for assessing the benefit and burden of cancer prevention, using an illness-death model with semi-competing risks. Extensive simulations demonstrate that the proposed estimators are unbiased, with robust inference across realistic scenarios. We apply this approach to a benefit-burden analysis of the preventive screening for colorectal cancer, utilizing data from the large-scale Women's Health Initiative. Our findings suggest that initiating screening at age 50 years achieves the highest life-year gains with an acceptable incremental burden-to-benefit ratio compared to no screening, contributing valuable real-world evidence to the field of preventive cancer interventions.

Abstract Background: The incidence of colorectal cancer (CRC) has been rising globally among adults aged under 50 years, particularly those born after the 1950s. We hypothesized that the polygenic risk score (PRS) may show stronger associations with CRC risk in more recent birth cohorts, reflecting interactions between genetic susceptibility and increasingly prevalent life-time environmental exposures such as obesity and sedentary behaviors. Methods: Among 37,313 CRC cases and 35,891 controls with available birth year information and genetically defined European ancestry from the Genetics and Epidemiology of Colorectal Cancer Consortium, we calculated the PRS as the weighted sum of risk alleles across 205 CRC-associated genetic variants identified so far. We used multivariable logistic models to estimate odds ratios (ORs) and 95% confidence intervals (CIs) of CRC risk associated with PRS across different birth cohorts. We assessed the interaction between PRS and birth cohorts using Wald test and performed subgroup analysis according to age at diagnosis, family history, endoscopic screening, and CRC molecular subtypes. Results: PRS showed a generally stronger association with risk of CRC across successive birth cohorts (p for interaction = 0.005). The OR for CRC per 1-standard deviation increase in PRS was 1.62 (95% CI: 1.48, 1.78) among individuals born before 1920, 1.55 (1.49, 1.60) for 1920-1929, 1.60 (1.55, 1.65) for 1930-1939, 1.67 (1.61, 1.74) for 1940-1949, 1.64 (1.55, 1.73) for 1950-1959, and 1.79 (1.65, 1.95) for those born in 1960 or later. The birth cohort effect was primarily observed among participants without a first-degree family history of CRC, with ORs of 1.62 (1.45, 1.80) for those born before 1920 and 1.91 (1.73, 2.12) for 1960 or later (p for interaction <0.001). The birth cohort effect did not differ by age at diagnosis or history of screening. Across CRC subsites, the birth cohort effect was observed for distal (p for interaction = 0.003) and rectal CRC (p = 0.002) but not proximal CRC (p = 0.82). For tumor molecular subtypes, we observed no interactions between PRS and birth cohorts for subtypes classified by microsatellite instability, BRAF and KRAS mutations, or CpG island methylator phenotype, although only 9.7-12.3% CRC cases had molecular data, limiting statistical power. Conclusions: PRS is more strongly associated with CRC risk in more recent birth cohorts, suggesting the role of gene-environment interaction in the rise in early-onset CRC and highlighting the increasing utility of genetic risk stratification in contemporary populations. Citation Format: Xinyu Wang, Constance Turman, Yufeng Chen, Conghui Qu, Li Hsu, William J. Gauderman, John D. Potter, Ulrike Peters, Mingyang Song. Germline genetic impact on risk of colorectal cancer according to birth cohorts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6816.

Supplementary Table S6. Associations between red meat/processed meat intake with colorectal cancer risk stratified by quartiles of pathway-based polygenic risk scores.

Supplemental Table 1. Associations of continuous (m2CLR) 16S microbiome with CRC-specific mortality (N=581)

Sickle cell trait (SCT) is the heterozygous carrier state for the HBB missense variant which causes sickle cell disease (SCD). SCT has been associated with increased risk of venous thromboembolism and chronic kidney disease as well as alterations in clinical laboratory parameters. To investigate differential gene expression in SCT, we used RNA sequencing of whole blood samples collected from 805 African American female participants (143 SCT; 660 controls) from the Women's Health Initiative Long Life Study (mean age = 76). We identified 226 differentially expressed genes (DEGs) in SCT compared to non-carriers (FDR < 0.05). Enriched pathways included those related to erythropoiesis, hemoglobin synthesis, and proteasomal degradation. Many of the SCT-associated DEGs were previously reported as differentially expressed in blood from individuals with SCD. Among the DEGs associated with SCT, we observed enrichment of upregulated ubiquitin-related genes normally downregulated during the later stages of erythroid differentiation, a pattern previously reported in SCD. Several of the SCT-associated DEGs highlight mechanisms that potentially link hemolysis or erythropoiesis to hypoxic kidney tubular injury. Future investigation of these genes using single cell transcriptomic analysis in relevant tissues may be useful in understanding mechanisms for adverse health outcomes in individuals with SCT.

Susceptibility transcription factors (TF) whose DNA bindings are altered by genetic variants regulating colorectal cancer (CRC) risk genes remain poorly defined. Using generalized linear mixed models, we analyze 218 TF ChIP-Seq datasets alongside GWAS data from 100,204 CRC cases and 154,587 controls of East Asian and European ancestries. We identify 51 TFs and TF-cofactor interactions, including VDR-cofactors, as key regulators of CRC risk. Integrating these TF insights with transcriptome-wide association studies (TWAS), we further evaluate associations between genetically predicted gene expression, alternative splicing, and alternative polyadenylation with CRC risk, using RNA-seq data from 364 Asian-ancestry and 707 European-ancestry individuals. Multi-ancestry TWAS identify 222 risk genes, including 95 novel genes and 48 potentially druggable targets. Single-cell analysis provides additional functional evidence supporting ~45% of these genes, and experimental validation confirms oncogenic roles for RHPN2, IRS2, and TXN. Our findings elucidate key TF-gene regulatory networks and uncover novel CRC risk genes.