Abstract 2019: The relationship between pSMAD3 expression, clinicopathological features, tumor microenvironment and survival in operable colorectal cancer patients

Abstract Background: Consensus molecular subtype 4 (CMS4) colorectal cancer (CRC) is associated with prominent Transforming Growth Factor-β (TGF-β) dysregulation & poorer prognosis. The TGF-β pathway has context-dependent tumor suppressor & promoting activity regulated by SMAD transcriptional factors. Increasingly, SMAD4 loss is shown to lead to tumor suppression loss in CRC (1). Evidence around other SMADs is lacking. We examined the relationship between phosphorylated SMAD3 (pSMAD3) expression, clinicopathological features, gene expression & survival in a large cohort of CRC patients. Methods: Patients who underwent curative resection of primary CRC from 1997 to 2012 at 3 hospitals in Glasgow, UK, were included. pSMAD3 protein immunohistochemistry (abcam Ab52903) was performed on a tissue microarray. Tumor/stromal & subcellular pSMAD3 expression histoscores were quantified on Qupath. High/low expression thresholds were determined by maximally selected ranked statistics. Expression was correlated with clinicopathological factors & survival. Gene set enrichment analyses (MSigDB Hallmarks) were performed on bulk transcriptomic (TempO-Seq, Bioclavis) data in 318 patients (2). Results: 1236 patients were eligible for analysis. High tumor cytoplasmic, nuclear & stromal cytoplasmic & nuclear pSMAD3 groups comprised 77.8%, 50.6%, 34.9% & 54.6% of the cohort respectively. High pSMAD3 expression across all compartments was associated with favourable tumor features: high tumor cytoplasmic, & stromal cytoplasmic & nuclear scores related to lower T-stage (all p<0.05). High tumor cytoplasmic & nuclear, & stromal cytoplasmic scores related to absence of venous invasion (all p<0.01). High tumor nuclear & stromal cytoplasmic scores also related to a strong Klintrup-Makinen immune microenvironment score (both p<0.01). High pSMAD3 was associated with TGF-β pathway gene set enrichment in CMS4 tumors alone. Within CMS4, high pSMAD3 was also associated enriched MYC & epithelial mesenchymal transition (EMT) pathways. Conversely, in non-CMS4 tumors, high pSMAD3 relates to suppressed EMT. High pSMAD3 across all compartments relates to better cancer specific survival (CSS) on univariate analyses (all p<0.05). On multivariate analysis, high stromal nuclear pSMAD3 retained independent prognostic significance (HR0.69, 95%CI 0.5-0.95, p=0.023). This survival relationship persisted in T2-3 & stromal poor tumors but not in T4 or stromal rich tumors. Conclusion: High pSMAD3 expression relates to favourable tumor features & stage-independent improved CSS, indicative of preserved TGF- β tumor suppression. In contrast, within CMS4 tumors, high pSMAD3 relates to enriched tumor promoter pathways, suggesting contextual TGF-β pleiotropy. References 1Voorneveld et al. Transl Oncol 2015;8(1):18-24 2Liberzon et al. Cell Syst 2015;1(6):417-425 Citation Format: Chia Yew Kong, Natthamon Sonthi, Leonor Schubert Santana, Lily V. Hillson, Kathryn Pennel, Peter Alexander, Noori Maka Joanne Edwards, Colin W. Steele, Campbell S. Roxburgh. The relationship between pSMAD3 expression, clinicopathological features, tumor microenvironment and survival in operable colorectal cancer patients [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 2019.