Supplementary Material for: Immune and histopathological biomarkers for prognosis and neoadjuvant immunotherapy response in intrahepatic cholangiocarcinoma

Introduction: The tumor microenvironment (TME) plays a critical role in determining the clinical outcomes in patients with intrahepatic cholangiocarcinoma (iCCA). This study investigates the prognostic significance of intratumoural CD8+ T cells and regulatory T cells (Tregs), their relationship to histopathological features and their potential as predictive biomarkers for neoadjuvant immunotherapy (NAT) response. Methods: Immunofluorescence analysis was conducted on a cohort of 34 resected iCCA cases to assess CD8+ T cell and Treg densities. Statistical correlations with survival and histopathological features were examined. An independent cohort of 95 iCCA pre-neoadjuvant therapy (NAT) biopsy specimens was analyzed to evaluate the potential of these biomarkers in predicting therapeutic response. Results: In the cohort of resected iCCA cases, immunofluorescence revealed that increased CD8+ T cell infiltration is associated with improved survival (P = 0.018), underscoring their role in anti-tumor immunity. Conversely, higher density of Tregs is linked to poorer survival (P = 0.038), suggesting its tumor-promoting, immunosuppressive effects. Intriguingly, CD8+ T cells and Tregs are associated with distinct histopathological features, with CD8+ T cells correlating with dense tumor infiltrating lymphocytes (TIL, Pearson’s R = 0.498; P = 0.004) and Tregs being more prevalent in regions of tumor budding (TB, Pearson’s R = 0.382; P = 0.029). These observations were validated in an independent pre-NAT cohort with (n = 95), whereby higher TIL density, particularly increased CD8+ T cells and reduced Treg, and lower TB predict favorable response to NAT, as shown by improved overall and disease-free survival. Conclusion: These findings provide critical insights for stratifying patients and highlight the potential for optimizing immune-targeted therapies in patients with iCCA.