Juha P. Väyrynen

The RNF43 p.G659fs ( RNF43 ^659mut ) mutation occurs in 8% of colorectal cancers (CRCs) and is enriched in microsatellite instability–high (MSI-H) tumors. Although RNF43 ^659mut promotes tumor growth through phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin (PI3K/AKT/mTOR) activation independent of WNT signaling, its immunologic effects are poorly defined. We show that RNF43 ^659mut shapes an immunosuppressive natural killer (NK) cell landscape in MSI-H CRC. Single-cell RNA sequencing and multiplex immunohistochemistry revealed increased NK cell infiltration in RNF43 ^659mut tumors, yet these cells displayed inhibitory phenotypes. Bulk RNA sequencing and flow cytometry of NK cells cocultured with RNF43 ^659mut isogenic lines and MSI-H patient-derived organoids demonstrated reduced functional NK subsets, decreased activation markers, increased inhibitory receptors, and impaired cytotoxicity. Mechanistically, RNF43 ^659mut -mediated PI3K/AKT activation induced HLA-E expression, suppressing NK cell function. Pharmacological or small interfering RNA–mediated PI3K/AKT inhibition restored NK cell activity and enhanced tumor cell killing. Spatial analysis of MSI-H patient tumors revealed close proximity between NKG2A ⁺ NK cells and HLA-E ⁺ tumor cells, which were themselves adjacent to PI3K/AKT-activated tumor cells, highlighting a localized immunosuppressive niche. These findings uncover an immune evasion mechanism in MSI-H CRC, implicating the PI3K/AKT-HLA-E/NKG2A axis as a promising therapeutic target to overcome immunotherapy resistance.

Background: Postoperative circulating tumor DNA (ctDNA) is a highly prognostic marker of residual disease among patients with stage III colon cancer, and physical activity has been associated with improved survival after curative resection. Whether physical activity predicts outcomes according to postoperative ctDNA status remains unknown. Methods: We conducted a post hoc analysis of Cancer and Leukemia Group B/SWOG 80702, a phase III study evaluating adjuvant celecoxib vs placebo and 3 vs 6 months of chemotherapy. Total physical activity volume was assessed using validated questionnaires and quantified in metabolic equivalent task hours per week (MET-h/week). Circulating tumor DNA status was determined using a clinically validated, tumor-informed assay (Signatera, Natera, Inc.). Results: Among 763 patients (median follow-up = 6.0 years), 32.3% were inactive ( .05 for all), though interactions were not statistically significant. Among patients who were physically active (≥9.0 MET-h/week) and received adjuvant celecoxib, improved DFS was observed in ctDNA-negative (HR = 0.41, 95% CI = 0.22 to 0.77) and ctDNA-positive (referent: inactive, placebo; HR = 0.29, 95% CI = 0.15 to 0.57) subgroups. Conclusions: Greater physical activity was associated with improved DFS among patients who were ctDNA-negative. Physical activity and adjuvant celecoxib were associated with favorable survival outcomes regardless of ctDNA status. Trial identifier: ClinicalTrials.gov NCT01150045

Colon cancer is a major global malignancy with significant mortality. Right-sided (RCC) and left-sided colon cancers (LCC) exhibit distinct clinicopathological characteristics. Tumor microenvironment score (TMS) combines Klintrup-Mäkinen grade, tumor stroma percentage, and tumor budding, classifying cases into 4 groups (TMS 0-3). Evaluation of TMS is an integrative method for prognostic classification; however, further validation is needed, and differences between RCC and LCC remain unclear. A discovery cohort (n=940) and a validation cohort (n=613) were retrospectively included. TMS was evaluated from hematoxylin-eosin-stained slides. Five-year disease-specific survival (DSS) was analyzed using life-table methods and Cox regression. Associations with clinicopathological variables were assessed. In both the discovery and validation cohorts, higher TMS was associated with worse 5-year DSS in univariable analyses for both RCC and LCC. In multivariable analysis, high TMS (TMS3 vs. TMS0) remained independently associated with survival in the discovery cohort (RCC—HR: 2.47, 95% CI: 1.36-4.46, P =0.003; LCC—HR: 3.08, 95% CI: 1.46-6.45, P =0.003). In the validation cohort, the prognostic impact of TMS was more pronounced in RCC (HR: 4.35, 95% CI: 2.32-8.14, P P =0.12). TMS also appeared to provide improved prognostic stratification compared with the Glasgow microenvironment score (combination of Klintrup-Mäkinen grade and tumor stroma percentage). In conclusion, TMS serves as an integrative histopathological marker with potential to improve prognostic stratification in colon cancer, particularly in right-sided tumors.

Supplementary Figure S2. Cutpoint selection plots for SMI and SMD

Background:Low skeletal muscle index (SMI) and skeletal muscle density (SMD) are associated with poor postoperative outcomes in colorectal cancer. However, standardized diagnostic thresholds remain undefined. Methods:In total, 927 patients with stage I to IV colorectal cancer were included. Preoperative computed tomography was used to determine body composition parameters. Optimal stratification was applied to identify the sex-specific and body mass index (BMI)–specific SMD and SMI cutoffs. SMD and SMI were analyzed as continuous variables relative to clinicopathologic features, systemic inflammatory markers, and sex-specific quartiles in survival analyses using Kaplan–Meier and Cox regression methods. Results:Optimal stratification did not yield reproducible cutoff values for SMI or SMD. In multivariable analysis, low SMD and SMI were associated with female sex, older age, and BMI, whereas tumor-related factors were not associated with either measure. Low SMD was associated with systemic inflammation, including elevated circulating C-reactive protein and interleukin 6 (IL6) levels, whereas low SMI showed only a nominal association with elevated IL6 levels after adjustment. Low SMD independently predicted poorer cancer-specific survival [multivariable hazard ratio (HR) for lowest vs. highest SMD = 4.29; 95% confidence interval (CI), 1.86–9.91; P = 0.001] and overall survival (HR for lowest vs. highest SMD = 3.42; 95% CI, 1.62–7.25; P = 0.003) in women but not in men, whereas SMI was not associated with survival. Conclusions:Low SMD is a sex-specific prognostic factor. Incorporating SMD assessment into preoperative risk stratification may enhance individualized management, particularly for female patients. Impact:Survival-based cutoff values for SMD or SMI in colorectal cancer could not be established. Moreover, preoperative body composition reflects patient-related factors rather than tumor-induced changes.

Supplementary Table S7. Multivariable Cox regression models for CSS, all variables, stages I-III

Figure S1 illustrates the patient selection process for the study

Supplementary Table S2. Correlations between SMD/SMI and circulating inflammatory factors

Supplementary Table S3. Sex-specific quartiles for SMI and SMD.

Supplementary Table S5. Multivariable Cox regression models for OS, all variables