Ogino, Shuji

Background: Considering the pathophysiological importance of the intestine-passing microbiota, we hypothesized that the mutational and microbial profiles of colorectal cancer might gradually change along detailed anatomical locations and that these trends might differ by the age of diagnosis and the microsatellite instability (MSI) status. Methods: We assessed somatic mutations and selected bacterial species in colorectal carcinomas along the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum, utilizing targeted sequencing data ( N = 5685) from the Genetics and Epidemiology of Colorectal Cancer Consortium and tissue microbial data ( N = 1389) from the Health Professionals Follow-up Study and Nurses’ Health Study. Results: There were decreasing trends from the cecum to rectum in the prevalence of gene mutations in the IGF/PI3K, receptor tyrosine kinase (RTK)/RAS, TGFB, and WNT signaling pathways (all P trend < 0.0001), as well as high levels of Bacteroides fragilis ( P trend < 0.0001) and high levels of Fusobacterium nucleatum ( P trend = 0.0029) (but not enterotoxigenic Bacteroides fragilis or pks+ Escherichia coli ). Conversely, TP53 pathway mutations showed an increasing cecum-to-rectum trend ( P trend < 0.0001). Similar to later-onset cases diagnosed at ages 50 years and older, in early-onset cases diagnosed under the age of 50 years, we found generally decreasing cecum-to-rectum trends of IGF/PI3K, RTK/RAS, TGFB, and WNT pathway mutations and an increasing cecum-to-rectum trend in TP53 pathway mutations. By contrast, the cecum-to-rectum prevalence-changing trends in IGF/PI3K, RTK/RAS, TGFB, and TP53 pathway mutations were present in non–MSI-high tumors but not in MSI-high tumors (all P interaction < 0.0052). Conclusion: Non–MSI-high colorectal cancers exhibited decreasing cecum-to-rectum trends of IGF/PI3K, RTK/RAS, and TGFB pathway mutations (and an increasing trend of TP53 pathway mutations), in contrast to MSI-high tumors that demonstrated no such trends. Our study highlights the importance of detailed colorectal subsite information combined with tumor molecular and microbial characteristics in colorectal cancer research.

163 Background: Being physically inactive is an independent predictor of poor outcomes in patients with stage III colon cancer. Whether physical activity can mitigate the oncologic risk of postoperative MRD is unknown. Methods: We concluded a post hoc analysis of a phase III trial evaluating celecoxib vs. placebo and 3 vs. 6 months of adjuvant chemotherapy in patients with resected stage III colon cancer. Self-reported physical activity was assessed in standardized measures of metabolic equivalent hours per week (MET-h/wk) at 2 timepoints: 1) midway through and 2) 6 months after completing adjuvant chemotherapy, modeled as a time-weighted average. MRD was determined via a clinically validated, tumor-informed circulating tumor DNA (ctDNA) assay (Signatera, Natera, Inc.). Disease-free survival (DFS) and overall survival (OS) were estimated using flexible parametric survival models. Results: Among 763 patients (median follow-up 6.0 years), 24% were highly active (≥18 MET-h/wk) and 32% were inactive (<3 MET-h/wk); 18% were ctDNA positive. Adjusting for covariates, DFS improved in patients who were ctDNA negative and moderately active (Ref: inactive; 3-17.9 MET-h/wk; HR=0.62, 95% CI [0.40-0.97]) or highly active (HR = 0.53 [0.30-0.94]). No benefit was seen among patients who were ctDNA positive (Ref: inactive; moderately active: HR = 1.20 [0.75-1.92]; highly active: HR=0.83 [0.45-1.54]). The interaction was not significant (Pint=0.269). Physical activity did not impact OS regardless of ctDNA status. The interaction of physical activity and adjuvant celecoxib use is shown in Table. Conclusions: Among patients who were ctDNA negative, physical activity was associated with improved DFS, and may demonstrate increased survival benefit alongside adjuvant celecoxib. The additive effects of both interventions were inconclusive among those who were ctDNA positive. Support: U10CA180821, U10CA180882, U24CA196171; https://acknowledgments.alliancefound.org; Natera, Pfizer; NCT01150045. Disease-free survival by physical activity level and celecoxib use, stratified by ctDNA status. 3-yr event # at risk Adjusted HR (95% CI) P-value Pint ctDNA Negative 0.258 Inactive + placebo 23 182 Ref Active + placebo 17 132 0.74 (0.43-1.28) 0.283 Inactive + celecoxib 23 167 0.92 (0.57-1.48) 0.720 Active + celecoxib 10 146 0.40 (0.22-0.79) 0.007 ctDNA Positive 0.221 Inactive + placebo 26 33 Ref Active + placebo 15 20 0.96 (0.50-1.84) 0.903 Inactive + celecoxib 34 54 0.62 (0.37-1.05) 0.076 Active + celecoxib 14 29 0.34 (0.18-0.64) 0.001 ctDNA, circulating tumor DNA; HR, hazard ratio; CI, confidence interval. Active = ≥9 MET-h/week, inactive = <9 MET-h/week. Celecoxib use defined by assignment in CALGB/SWOG 80702. Models adjusted by T and N stage, age, sex, race, assigned cycles of fluorouracil, leucovorin, and oxaliplatin, KRAS and BRAF status, BMI, and Western diet.

Importance Observational studies have associated use of aspirin and selective cyclooxygenase inhibitors with decreased recurrence and improved survival in patients with colon cancer. While randomized clinical trials have not shown benefit across all patients, these findings suggest that select subgroups may benefit from their use. Despite the well-established prognostic value of circulating tumor DNA (ctDNA), its role in guiding treatment remains unclear. Objective To investigate the predictive value of postoperative ctDNA for survival outcomes with adjuvant celecoxib alongside conventional chemotherapy in patients with stage III colon cancer. Design, Setting, and Participants This was a post hoc analysis of the phase 3 Cancer and Leukemia Group B (now Alliance)/Southwest Oncology Group 80702 randomized clinical trial (2010-2015) assessing adjuvant celecoxib vs placebo and 3 vs 6 months of adjuvant 5-fluorouracil, leucovorin, and oxaliplatin for stage III colon cancer. Patients consented to biospecimen collection and had ctDNA analysis performed. Data analysis was performed from September 2024 to June 2025. Exposures Postoperative ctDNA positivity was determined using a clinically validated, tumor-informed 16-plex-polymerase chain reaction-next-generation sequencing assay (Signatera; Natera Inc) performed between surgery and initiation of adjuvant therapy. Main Outcomes and Measures Disease-free survival (DFS) and overall survival (OS). Survival by ctDNA status and adjuvant celecoxib use were assessed as part of a post hoc companion study with prespecified statistical analysis plan. Results Among 940 patients (mean [SD] age, 60.9 [10.8] years; 426 female [45.3%] and 515 male [54.7%] individuals; 222 [23.6%] with prior low-dose aspirin use; and median follow-up of 6.0 [95% CI, 6.0-6.0] years), 767 (81.6%) were ctDNA negative and 173 (18.4%) were ctDNA positive. ctDNA positivity was highly prognostic of worse DFS (reference, ctDNA negativity; adjusted hazard ratio [aHR], 6.12; 95% CI, 4.66-8.03) and OS (aHR, 5.86; 95% CI, 4.19-8.19). In patients with ctDNA positivity, celecoxib was associated with improved DFS (aHR, 0.61; 95% CI, 0.42-0.89) and OS (aHR, 0.62; 95% CI, 0.40-0.96) compared to placebo. Among patients with ctDNA negativity, celecoxib did not provide survival benefit (DFS: aHR, 0.76; 95% CI, 0.53-1.09; OS: aHR, 0.85; 95% CI, 0.54-1.36), although the interaction was not significant ( P for interaction, .41 and .33 for DFS and OS, respectively). These findings persisted when stratifying patients by microsatellite instability status and PIK3CA mutational status. Conclusion and Relevance The findings of this post hoc analysis suggest that ctDNA status has the potential to inform clinical decision-making among patients with stage III colon cancer who should consider adjuvant celecoxib in addition to conventional chemotherapy. Trial Registration ClinicalTrials.gov Identifier: NCT01150045

Abstract Background Physical activity is associated with lower colorectal cancer (CRC) risk, but its association with molecular subtypes defined by genetic and epigenetic alterations of the disease is unclear. Such information may enhance the understanding of the mechanisms related to the benefits of physical activity. Methods Pooled observational (cases: n = 5386; controls: n = 6798; studies n = 5) and genome-wide association data (cases: n = 8178; controls: n = 10 472; studies n = 5) were used. We used multivariable logistic regression models and Mendelian randomization to assess the association between physical activity and the risk of CRC subtypes defined by individual tumor markers (and marker combinations), namely microsatellite instability status, CpG island methylator phenotype status, and BRAF and KRAS mutations. We used case-only analysis to test for differences between molecular subtypes. We applied Bonferroni correction to account for multiple tests. Results In the pooled observational analysis, higher levels of physical activity were associated with lower CRC risk (Obs-per 1SD, odds ratio [OR] = 0.94, 95% confidence interval [CI] = 0.90 to 0.97), with an association that was stronger in males (Obs-per 1SD, OR = 0.91, 95% CI = 0.87 to 0.96) than in females (Obs-per 1SD, OR = 0.97, 95% CI = 0.91 to 1.03; Pinteraction = .04). Higher physical activity was associated with a lower risk of CRC across all molecular subtypes, especially in males. There was no difference in the associations by subtypes by pooled observational or Mendelian randomization analyses. The findings did not differ by study design, anatomical site, and early or late age onset of CRC. Conclusions Our findings suggest that physical activity is not differentially associated with the 4 major molecular subtypes involved in colorectal carcinogenesis, indicating that its benefits extend broadly across colorectal cancer pathogenesis.

Abstract Recent research suggests higher circulating lymphocyte counts may protect against colorectal cancer (CRC). However, the role of specific lymphocyte subtypes and activation states remain unclear. CD4+ T cells—a highly dynamic lymphocyte subtype—undergo gene expression changes upon activation that are critical to their effector function. Previous studies using bulk tissue have limited our understanding of their role in CRC risk to static associations. We applied Mendelian randomization (MR) and genetic colocalisation to evaluate causal relationships of gene expression on CRC risk across multiple CD4+ T cell subtypes and activation states. Genetic proxies were obtained from single-cell transcriptomic data, allowing us to investigate the causal effect of expression of 1,805 genes across CD4+ T cell activation states on CRC risk (78,473 cases; 107,143 controls). Analyses were stratified by CRC anatomical subsites and sex, with sensitivity analyses assessing whether the observed effect estimates were likely to be CD4+ T cell-specific. We identified 6 genes—FADS2, FHL3, HLA-DRB1, HLA-DRB5, RPL28, and TMEM258—with strong evidence for a causal role in CRC development (FDR-P &lt; 0.05; colocalisation H4 &gt; 0.8). Causal estimates varied by CD4+ T cell subtype, activation state, CRC subsite and sex. However, many of genetic proxies used to instrument gene expression in CD4+ T cells also act as eQTLs in other tissues, highlighting the challenges of using genetic proxies to instrument tissue-specific expression changes. We demonstrate the importance of capturing the dynamic nature of CD4+ T cells in understanding CRC risk, and prioritize genes for further investigation in cancer prevention.

Abstract Background Physical activity (PA) is an established protective factor for colorectal cancer (CRC), but it is unclear if genetic variants modify this effect. To investigate this possibility, we conducted a genome-wide gene–PA interaction analysis. Methods Using logistic regression and two-step and joint tests, we analyzed interactions between common genetic variants across the genome and PA in relation to CRC risk. Self-reported PA levels were categorized as active (≥ 8.75 MET-h/wk) vs. inactive (< 8.75 MET-h/wk) and as study- and sex-specific quartiles of activity. Results PA had an overall protective effect on CRC (OR [active vs. inactive] = 0.85; 95%CI = 0.81–0.90). The two-step GxE method identified an interaction between rs4779584, an intergenic variant near the GREM1 and SCG5 genes, and PA for CRC risk (p-interaction = 2.6×10 − 8 ). Stratification by genotype at this locus showed a significant reduction in CRC risk by 20% in active vs. inactive participants with the CC genotype (OR = 0.80; 95%CI = 0.75–0.85), but no significant PA–CRC association among CT or TT carriers. When PA was modeled as quartiles, the 1-d.f. GxE test identified that rs56906466, an intergenic variant near the KCNG1 gene, modified the association between PA and CRC (p-interaction = 3.5×10 − 8 ). Stratification at this locus showed that increase in PA (highest vs. lowest quartile) was associated with a lower CRC risk solely among TT carriers (OR = 0.77; 95%CI = 0.72–0.82). Conclusions In summary, we identified two genetic variants that modified the association between PA and CRC risk. One of them, related to GREM1 and SCG5 , suggests that the bone morphogenetic protein (BMP)-related, inflammatory, and/or insulin signaling pathways may be associated with the protective influence of PA on colorectal carcinogenesis.

Observational studies have demonstrated that aspirin and/or PTSG2 inhibitor (e.g., celecoxib) use, before or after colon cancer (CC) diagnosis, is associated with a lower risk of recurrence. However, a randomized trial of adjuvant celecoxib therapy in stage III CC did not show improved survival compared to placebo treatment. We therefore hypothesized that immune microenvironment features may identify patient subgroups with stronger survival benefits from anti-inflammatory celecoxib treatment. Drawing from an NCI-Intergroup trial (Cancer and Leukemia Group B [now Alliance for Clinical Trials in Oncology]/SWOG 80702) for patients with stage III resected CC that compared 3 versus 6 months of adjuvant fluorouracil, leucovorin, and oxaliplatin ± 3 years of celecoxib, we conducted tissue-based tumor microenvironment profiling on 1,098 resected tumors using a custom 9-plex, T-cell multiplex immunofluorescence assay coupled with digital image analysis and supervised machine learning. The Kaplan-Meier method was used to describe disease-free survival, based on T cell density, and log-rank testing was performed. Cox proportional hazards models were used to examine unadjusted and adjusted associations between T cell density and disease-free survival (DFS). Two-sided P values ≤0.05 were considered statistically significant. We found that higher CD3+ T cell density was associated with longer DFS with an adjusted hazard ratio (HR) of 0.69 (95% confidence interval [CI] 0.52-0.91) for patients with densities in the highest tertile as compared the lowest tertile (P=0.01). T cell subset analysis showed that stromal regulatory T helper cells (HR 0.42 comparing highest to lowest tertile, CI 0.31-0.58, P&lt;0.001) and stromal memory cytotoxic T cells (HR 0.55 comparing highest to lowest tertile, CI 0.42-0.74, P&lt;0.001) were most strongly associated with longer DFS. Compared to the placebo group, adjuvant celecoxib use associated with improved DFS for patients with low but not high stromal CD3+ T cell density (adjusted HRs of 0.45 [CI 0.31-0.65] for lowest tertile and 1.13 [CI 0.73-1.74] for highest tertile; Pinteraction=0.01). This association was also seen when the analysis was restricted to microsatellite stable tumors (HR 0.50 [CI 0.32-0.80] for lowest density tertile and 1.50 [CI 0.87-2.59] for highest density tertile; Pinteraction=0.01). T cell subset analysis showed that DFS benefit for celecoxib use most strongly associated with stromal memory helper T cell density (HR 0.38 [CI 0.26-0.56] for lowest density tertile and 1.26 [CI 0.80-1.97] for highest density tertile; Pinteraction&lt;0.001). Together, these results show that while higher T cell infiltration is associated with improved DFS, lower T cell infiltration is associated with improved DFS for patients treated with celecoxib. This unexpected finding may inform immunomodulatory treatment strategies for CC. Citation Format: Yasutoshi Takashima, Chao Ma, Qian Shi, Andressa Dias Costa, Tyler Twombly, Juha P. Väyrynen, Melissa Zhao, Ardaman Shergill, Pankaj Kumar, Felix Couture, Philip Kuebler, Smitha Krishnamurthi, Benjamin Tan, Eileen M. O'Reilly, Anthony F. Shields, Shuji Ogino, Charles S. Fuchs, Jeffrey A. Meyerhardt, Jonathan A. Nowak. Prognostic and predictive role of T cell infiltration in stage III colon cancer (CC) treated with celecoxib: CALGB/SWOG 80702 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5858.

Abstract Background: Waist circumference (WC) and its allometric counterpart, “a body shape index” (ABSI), are risk factors for colorectal cancer; however, it is uncertain whether associations with these body measurements are limited to specific molecular subtypes of the disease. Methods: Data from 2,772 colorectal cancer cases and 3,521 controls were pooled from four cohort studies within the Genetics and Epidemiology of Colorectal Cancer Consortium. Four molecular markers (BRAF mutation, KRAS mutation, CpG island methylator phenotype, and microsatellite instability) were analyzed individually and in combination (Jass types). Multivariable logistic and multinomial logistic models were used to assess the associations of WC and ABSI with overall colorectal cancer risk and, in case-only analyses, to evaluate heterogeneity by molecular subtype, respectively. Results: Higher WC (ORper 5 cm = 1.06, 95% confidence interval, 1.04–1.09) and ABSI (ORper 1-SD = 1.07, 95% confidence interval, 1.00–1.14) were associated with elevated colorectal cancer risk. There was no evidence of heterogeneity between the molecular subtypes. No difference was observed regarding the influence of WC and ABSI on the four major molecular markers in proximal colon, distal colon, and rectal cancers, as well as in early- and late-onset colorectal cancers. Associations did not differ in the Jass-type analysis. Conclusions: Higher WC and ABSI were associated with elevated colorectal cancer risk; however, they do not differentially influence all four major molecular mutations involved in colorectal carcinogenesis but underscore the importance of maintaining a healthy body weight in colorectal cancer prevention. Impact: The proposed results have potential utility in colorectal cancer prevention.

LBA14 Background: CALGB/SWOG 80702 previously showed that the addition of celecoxib to standard adjuvant chemotherapy with FOLFOX did not significantly improve disease-free survival (DFS) in patients with stage III colon cancer. Here, we evaluated the prognostic and predictive value of ctDNA in identifying a subpopulation of patients who may benefit from celecoxib. Methods: In the subset of patients with adequate biospecimens who participated in CALGB/SWOG 80702, a randomized phase III trial of 3 versus 6 months of adjuvant 5-FU, leucovorin, oxaliplatin (FOLFOX) +/- celecoxib, ctDNA assessment was performed using a clinically validated, tumor-informed 16-plex mPCR-NGS assay (Signatera(TM), Natera, Inc.) after surgery and before the start of adjuvant therapy (baseline). The Kaplan-Meier method was used to describe the distribution of survival time based on ctDNA positivity and log-rank testing was performed. Cox proportional hazards models were used to examine unadjusted associations between ctDNA positivity and disease-free (DFS) and overall survival (OS). Two-sided P values equal to or less than 0.05 were considered statistically significant, except for interaction P values (tested with a likelihood ratio). Results: In total, 1,011 of the 2,526 patients who participated in 80702 had ctDNA testing results from baseline; 189 were ctDNA positive (18.7%). ctDNA positivity associated with male sex, higher T stage, and N2 (versus N1) stage. ctDNA positivity was significantly associated with worse DFS (hazard ratio [HR] 6.52 [95% confident interval (CI) 5.09-8.34; p<0.0001] and OS (HR 6.28 [95% CI 4.63-8.51; p<0.0001]). Three-year DFS was 86.6% in ctDNA-negative cases and 36.8% in ctDNA-positive cases. Among patients who were ctDNA negative, celecoxib use was not significantly associated with worse DFS as compared to placebo (HR 0.75 [95% CI 0.54-1.05; p=0.095]) with a three-year DFS of 87.7% versus 85.5%, respectively. Among ctDNA-positive patients, celecoxib significantly improved DFS compared to placebo (HR 0.59 [95% CI 0.42-0.85; p=0.004]) with a three-year DFS of 44.1% versus 26.6% (P interaction = 0.25). Similar results were seen for overall survival with a hazard ratio of 0.86 (95% CI 0.56-1.33; p=0.49) for ctDNA negative cases and 0.63 (95% CI 0.41-0.96; p=0.028) for ctDNA-positive cases for celecoxib versus placebo (P interaction = 0.28). Conclusion: In a randomized phase III adjuvant therapy trial for stage III colon cancer, ctDNA was highly prognostic of DFS and OS. ctDNA positivity also appeared predictive of the benefit of adjuvant celecoxib for DFS and OS. These results suggest a potential role for ctDNA in determining which patients should consider celecoxib in addition to standard FOLFOX adjuvant therapy. Clinical trial information: NCT01150045 .