Mitochondrial DNA (mtDNA) expression as used to define metabolic and immune states in colorectal cancer (CRC).

2647 Background: CRC exhibits metabolic reprogramming driven by the Warburg effect; however, active mitochondria and oxidative phosphorylation (OXPHOS) often remain crucial for tumor growth. mtDNA encodes critical OXPHOS components and influences the balance between OXPHOS and glycolysis, with effects on tumor microenvironment and response to immune checkpoint inhibitors (ICIs). We evaluated whether mtDNA gene expression predicts metabolic phenotype, immune contexture and benefit from ICIs. Methods: 30,887 CRC cases with DNA/RNA sequencing were analyzed from Caris Life Sciences. Expression of mtDNA-encoded OXPHOS genes ( MT-ND1–6, MT-ND4L, MT-CO1–3, MT-ATP6, MT-CYB ) was summarized as a composite Z-score due to correlation (r>0.9). Tumors were stratified into quartiles (n=7,722 each), mtDNA-high (MT-H, top quartile) and mtDNA-low (MT-L, bottom quartile) cohorts. Overall Survival (OS) was calculated in months (m) from first treatment to last contact. Hazard ratios (HRs) were calculated using Cox proportional hazards models and p-values by log-rank tests. Gene set enrichment analysis (GSEA) was performed to evaluate pathway differences. Results: MT-H tumors were enriched for Consensus Molecular Subtype (CMS) 2 compared to MT-L (44.2% vs 21.9%) and CMS3 (24.5% vs 8.7%), whereas CMS4 was markedly enriched in MT-L tumors (MT-H 16.5% vs MT-L 52.2%); all p<0.001. GSEA showed a trend toward higher OXPHOS activity in MT-H tumors (NES 1.19, FDR q=0.472), while glycolysis was significantly downregulated (NES -2.43, FDR q=0.002), with low immune/inflammatory signaling (interferon signaling and inflammatory response, among others) and reduced immune cell infiltration. These associations persisted in microsatellite stable (MSS) CRC, including CMS (CMS2/CMS3 46.3%/24.9% in MT-H vs CMS4 55.6% in MT-L) and immune signatures (all q<0.05). MT-L was prognostic for improved OS vs MT-H (median OS [mOS] 30.1 vs 27.2 m; HR 0.87, 95% CI 0.84-0.91, p<0.001). In ICI-treated patients (pts), MT-L showed amplified effect (mOS 24.3 vs 13.5 m; HR 0.69, 95% CI 0.58-0.83, p<0.001), including in CMS1 (mOS 39.3 vs 26.0 m; HR 0.71, 95% CI 0.53-0.96, p=0.024), CMS4 (mOS 19.3 vs 10.7 m; HR 0.61, 95% CI 0.40-0.94, p=0.024), MSS (mOS 13.8 vs 9.02 m; HR 0.67, 95% CI 0.54-0.84, p<0.001) and in pts with liver metastases (mOS 13.4 vs 7.93 m; HR 0.65, 95% CI 0.44-0.97, p=0.034). Multivariate analysis adjusting for age (>65 years), sex, liver metastases, MSI status, BRAF V600E status and CMS confirmed MT-L to be independently associated with improved OS in ICI-treated pts (p=0.01). No survival association was observed in pts treated with other therapies. Conclusions: mtDNA-encoded OXPHOS expression defines biologically distinct CRC subsets with distinct metabolic states and immune infiltration, with MT-L linked to improved OS and enhanced benefit from immunotherapy, including in MSS and liver metastases where ICI sensitivity is limited.