Abstract 2014: Immune exclusion signatures identify patients at high risk of relapse despite adjuvant chemotherapy in stage 2 colon cancer in the TOSCA phase 3 trial: PROSIT study

Background: The decision to administer adjuvant chemotherapy (ACT) in stage II colorectal cancer (CRC) remains challenging. Clinical and histopathological criteria, even when integrated with ctDNA-based assessment of residual disease, are still suboptimal ACT benefit predictors. Immune cell infiltration has been repeatedly associated with better outcome, but the prognostic value of transcriptional immune signatures has not been assessed in prospective trial cohorts in a systematic way. We aimed to identify a prognostic immune transcriptional signature for high-risk stage II CRC patients undergoing ACT in the TOSCA trial. TOSCA tested the noninferiority of 3 vs 6 months of FOLFOX/CAPOX, and, with a median follow-up of more than 6 years, is an ideal patient set to test outcome-associated variables. Methods: We built a case-control cohort nested in the TOSCA trial database, matching 62 patients with high-risk stage II CRC who relapsed (R) with 62 who did not (NR). Matching criteria included recruitment center, pT stage, ACT type and duration. Whole transcriptome sequencing (WTS) was conducted on formalin-fixed, paraffin-embedded (FFPE) blocks using stranded ribodepletion library prep. MMR status was derived from WTS by majority report of three well-established methods. Immune gene signatures were assessed by the Miracle and ssGSEA packages. Differentially expressed signatures between R and NR were evaluated with the Log Rank test. Covariates included in the multivariable Cox regression models included MMR status, ACT duration (3 vs 6 months) and type (FOLFOX vs CAPOX). Results: Out of 124 stage II CRC samples, 72 were of sufficient quality for RNAseq and were included in the analysis (“final cohort”). No key matching variable was differentially represented in the final cohort. Median follow-up was 70.9 months. The median age was 62.5, 40% of cases were right-sided, 60% received FOLFOX, 54% received 3 months of ACT, and 19% were MSI-high. We tested 67 different signatures. Immune constant of rejection and immune-exclusion signatures (IES) were the only ones with significant adjusted p-values. IES-high patients (∼27%) had significantly shorter disease-free survival (41 months vs median not reached, HR 2.5, p-value=0.02). IES signature remained significant after adjusting for known covariates. Longer (6 months) ACT duration did not show any advantage over 3 month ACT in neither IES-high nor -low patients. Conclusion: IES signature identifies patients at high risk of relapse despite receiving standard ACT, irrespective of ACT duration. IES can be exploited to design novel adjuvant trials for treatment intensification in specific patient subgroups. Funding: Italian Ministry of Health Ricerca Corrente di Rete (ACCORD) 2022-2024, AIRC GRANT IG 2018 Citation Format: Luca Mazzarella, Alberto Puccini, Lorenzo Ferrando, Gianmaria Frige', Eliana Rulli, Anna Maria Pessino, Alessandro Pastorino, Valentino Martelli, Anna Garuti, Davide Bedognetti, Alberto Ballestrero, Valter Torri, Roberto Labianca, Matteo Fassan, Sara Lonardi, Gabriele Zoppoli, Alberto Sobrero. Immune exclusion signatures identify patients at high risk of relapse despite adjuvant chemotherapy in stage 2 colon cancer in the TOSCA phase 3 trial: PROSIT study [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 2014.