Discovery of Biomarkers for Clinical Response to Radiotherapy in HCC via Deep Immunoprofiling of Peripheral Blood

Abstract

Introduction: Hepatocellular carcinoma (HCC) is a highly malignant primary liver tumour which ranks third as the cause of cancer-related deaths worldwide. Yttrium-90 (Y90)-radioembolisation (RE) is a form of localised radiotherapy recommended for intermediate and inoperable liver tumors. Y90-RE has been shown to downsize tumors and delay disease progression. Despite its short half-life (∼64 hrs), Y90 induces sustained therapeutic effects months after delivery, which may be linked to a systemic immune response. Method: Tumour tissues from treatment-naïve (control) or Y90 treated HCC patients (n=7 each) were subjected to Next-Generation-Sequencing (NGS). Tumor-infiltrating leukocytes (TILs) and isolated peripheral blood mononuclear cells (PBMCs) obtained at various time-points post-treatment were analysed using time-of-flight mass cytometry (CyTOF). Random Forests predictive model based on CyTOF data was used to predict response outcome for each patient. Result: CyTOF data from TILs isolated post-Y90-RE suggested presence of local immune activation, observed by the increased expression of granzyme B and infiltration of CD8+T cells, CD56+NK cells and CD8+CD56+NKT cells. NGS data revealed that post-Y90-RE tumors upregulated genes involved in innate and adaptive immune activation and also chemokines, CCL5 and CXCL16, which corresponded with recruitment of activated GB+CD8+ T cells to the Y90-RE-treated tumors. Comparing PBMCs obtained pre- and post-Y90-RE, an increase in TNFα on both the CD8+ and CD4+T cells as well as an increase in percentage of antigen-presenting cells after Y90-RE was observed, which suggest at systemic immune activation. Y90-RE responders exhibited higher percentage of PD-1+/Tim-3+CD8+T cells co-expressed homing receptors, CCR5 and CXCR6. A prediction model was built to identify sustained responders to Y90-RE based on the immune profiles from pre-treatment PBMCs. Conclusion: Deep-immunophenotyping of tumour and systemic environment revealed immune activation that corresponded to sustained response to Y90-RE observed in HCC patients. Potential biomarkers associated with a positive clinical response were also identified and used to build a prediction model.