Editor's Note: Yttrium-90 (Y90) resin microsphere selective internal radiotherapy (Y90-RE) is a minimally invasive local treatment method that can effectively control tumor progression in hepatocellular carcinoma (HCC) patients who are ineligible for resection or ablation, with compensated liver function and good overall condition. With the advancement of precision medicine, the clinical understanding of immune activation by Y90-RE has deepened, revealing the potential of pre-Y90-RE peripheral blood mononuclear cell (PBMC) immune profiling in predicting patient prognosis. Hepatology Digest invited Dr. Lin Zhang from Beijing Tsinghua Changgung Hospital to interpret a study published in Gut (IF=23), which explores the immune activation mechanisms of Y90-RE.Research Methods
The study included tumor or blood samples from 41 HCC patients in Singapore, with some undergoing Y90 radioembolization (Y90-RE). Tumor-infiltrating lymphocytes (TILs) were categorized from HCC tissue samples of 14 patients. Additionally, PBMCs were collected from 31 patients before and at 1, 3, and 6 months after Y90-RE. Tumor responses were assessed, defining sustained responders (SRs) as patients with no disease progression (PD) at 6 months post-Y90-RE. Non-responders (NRs) had no minimal response or stable disease (SD) by the third month, and transient responders (TRs) showed a response by the third month but progressed by the sixth month. Mass cytometry (CyTOF) was used to analyze TILs, PBMCs, and tumor tissue, with next-generation sequencing (NGS) and quantitative PCR performed. A random forest algorithm was used to construct a predictive model for Y90-RE clinical response, which was then compared with actual outcomes to plot receiver operating characteristic (ROC) curves.
Research Results
Y90-RE Activates Local Immune Response
The study designed an in-depth analysis workflow based on CyTOF and NGS to investigate the immune phenotypes of TILs, tumor tissues, and PBMCs before and after Y90-RE in HCC patients. TILs were obtained from tumor samples of patients who had undergone Y90-RE and those who had not received any treatment (control group, Ctl), and analyzed using CyTOF to identify immune subpopulations with differential expression after Y90-RE. Notably, specific CD56+ NK cells, CD8+CD56+ NKT cells, CD8+ T cells, and CD4+ T cells were enriched in TILs isolated post-Y90-RE. Additionally, Y90-RE group TILs exhibited higher expression of Granzyme B (GB) and Tim-3 compared to the control group, with a higher proportion of GB+CD8+ T cells and Tim-3+CD8+ T cells. The Y90-RE group also showed a higher overall expression of GB in CD56+ NK cells and CD8+CD56+ NKT cells, as well as a higher proportion of CXCR3-expressing CD4+CD45RO+ T cells. In contrast, the control group TILs had a higher proportion of immunosuppressive Treg cells.
Y90-RE Induces Immune Activation Pathways in Liver Cancer Tissue
NGS analysis of advanced HCC in the Y90-RE and control groups revealed multiple differentially expressed genes, with 88% of highly expressed genes enriched in the Y90-RE group. These enriched genes were mostly associated with innate or adaptive immune responses, while genes enriched in the control group tumors were unrelated to immune pathways.
Y90-RE Induces Chemotaxis of CD8+ T Cells to the Liver Cancer Immune Microenvironment
Analysis of gene responses enriched post-Y90-RE suggested chemotactic activity involving upregulation of CXCL16 and CCL5. qPCR of tumor samples from the same patients confirmed increased expression of CCL5 and CXCL16, chemokines that bind CCR5 and CXCR6, respectively. These chemokines were positively correlated with the percentage of activated GB+CD8+ T cells in TILs, indicating that Y90-RE treatment remodels the HCC immune microenvironment by inducing tumor cell apoptosis and recruiting and activating T cells.
Y90-RE Induces Early and Late Immune Responses in Liver Cancer
To capture the systemic immune response induced by Y90-RE, 31 HCC patients were categorized into SRs and TRs/NRs, and PBMCs were collected at different time points before and after Y90-RE. CyTOF analysis of PBMCs in SRs before and one month after Y90-RE showed increased expression of TNF-α on CD8+Tim-3+ T cells and CD4+ T cells. Notably, TNF-α expression on these T cell subpopulations remained higher in SRs compared to NRs/TRs at three months post-treatment. The proportion of CD14+HLADR+ antigen-presenting cells (APCs) in PBMCs was significantly higher at three months post-Y90-RE, particularly in the SRs group. At six months post-treatment, differences in immune cell subpopulations in PBMCs, aside from APCs, were not significant, consistent with increased tumor antigen presentation observed in NGS results after Y90-RE.
High Expression of PD-1 and Tim-3 Predicts Sustained Response to Y90-RE
The study highlighted a comparison of systemic immune characteristics between SRs and TRs/NRs before Y90-RE and at three months post-treatment. SRs exhibited specific CD4+ and CD8+ T cell profiles, with higher PD-1 and Tim-3 expression and a higher proportion of PD-1 and Tim-3-expressing CD8+ T cells before and three months after Y90-RE. This higher expression of exhaustion markers may indicate that higher levels of T cell activation before and three or six months after treatment are specific to SRs, potentially mediating a sustained response to Y90-RE.
Predictive Model for Sustained Response Based on Pre-Y90-RE PBMC Immune Profiling
Differences in immune marker expression in peripheral blood could distinguish Y90-RE SRs from TRs/NRs. The study established a predictive model based on the single-cell immune profiling of pre-Y90-RE PBMCs to predict SR. The model was constructed using a training dataset (randomly selected training cohort, n=22) and tested on a test dataset (remaining samples, n=8) to provide probability scores for prediction. Cross-validation of the model in the training cohort achieved an accuracy of 95.5%, and independent testing in the test cohort showed an accuracy of 75%.
Discussion
The study provided a detailed analysis of immune characteristics in tumor tissues and PBMCs of HCC patients, strongly demonstrating that Y90-RE induces both local and systemic immune responses. It identified specific immune cell subpopulations associated with a sustained response to Y90-RE and developed a predictive model based on pre-Y90-RE PBMC immune profiling for prognosis.
The in-depth immune phenotyping of TILs showed significant immune activation in the local tumor microenvironment. The Y90-RE group had a higher percentage of activated or GB-expressing CD56+ NK cells, CD8+CD56+ NKT cells, and CD8+ T cells, with a lower percentage of Treg cells. NGS analysis of tumor tissues also provided strong evidence of enhanced activation of T cells, NK cells, and NKT cells.
By analyzing PBMCs before and after Y90-RE using CyTOF, the study captured the systemic immune response triggered by treatment and identified potential biomarkers for predicting clinical outcomes. Tim-3 is an exhaustion marker on immune cells, associated with the progression of various cancers, including HCC. Co-expression of PD-1 and Tim-3 can enhance T cell dysfunction and is related to tumor progression. In mouse cancer models, targeting the Tim-3 and PD-1 pathways can reverse T cell exhaustion and restore anti-tumor immune responses. Based on this, sequential use of immune checkpoint inhibitors targeting the PD-1/PD-L1 or Tim-3 pathways after Y90-RE may enhance clinical efficacy in HCC patients. Currently, clinical trials of radiotherapy combined with immunotherapy in multiple cancers are showing promising results.
This study also revealed that the co-expression of Tim-3 and homing receptors CCR5 and CXCR6 on CD8+ T cells indicates their migration towards CCL5 and CXCL16, a mechanism through which Y90-RE recruits T cells to the tumor microenvironment, enhancing the clinical efficacy of Y90-RE. Indeed, the importance of chemokines in recruiting cytotoxic immune cells to the tumor microenvironment is widely reported, and the infiltration of cytotoxic immune cells in immunotherapy is well-known for controlling tumor progression.
In conclusion, this study, through in-depth immune phenotyping and transcriptomics, demonstrated that Y90-RE induces both local and systemic immune activation in the tumor microenvironment of HCC patients, leading to a sustained response to treatment. The study identified immune activation induced by Y90-RE and potential biomarkers in peripheral blood that can predict sustained clinical response, providing a basis for guiding future integrated treatment options for HCC patients.
Expert Commentary
Over the past 20 years, Y90-RE has shown good efficacy and safety, earning recommendations from multiple authoritative guidelines worldwide. The mechanism by which Y90-RE kills tumor cells is complex, with currently accepted mechanisms including direct DNA damage to tumor cells by β-rays, indirect tumor cell damage through reactive oxygen species, and sustained effects through immune system activation.
This study provides a detailed exploration of immune system activation following Y90-RE treatment in liver cancer. Although Y90 has a short half-life in the body, it can induce sustained tumor remission through immune system activation. The study demonstrates that Y90-RE can induce both local and systemic immune responses, activating T cells, NK cells, and NKT cells, triggering a series of immune responses in TILs and PBMCs. Increased CD8+ cells, NK cells, and NKT cells in TILs post-Y90-RE, along with activated immune subgroups and antigen-presenting cells (APCs) in PBMCs three months after treatment, play a lasting anti-tumor role, elucidating the mechanism of Y90-RE-induced local and systemic immune activation.
The study shows that the sustained response subgroup post-Y90-RE had higher PD-1 and Tim-3 expression at three months, with a higher proportion of PD-1 and Tim-3-expressing CD8+ T cells at three and six months. Based on this, the researchers suggest that sequential use of PD-1/PD-L1 or Tim-3 pathway inhibitors after Y90-RE may yield better results. A recent retrospective study in the United States involving 1,664 advanced patients showed that combining immunotherapy with SIRT doubled overall survival (OS) in HCC patients compared to immunotherapy alone (19.8 months vs. 9.5 months) . Several small-sample studies are also exploring the effectiveness of Y90-SIRT combined with immunotherapy, with preliminary results achieved . Moreover, the study suggests that the observed decrease in the percentage of CCR5 and CXCR6 on CD8+ T cells and Tim-3+CD8+ T cells one month post-Y90-RE may indicate that combining immune checkpoint inhibitors one month after surgery might be more appropriate.
Another highlight of this study is the discovery of biomarkers that can effectively predict the prognosis of Y90-RE and the development of a predictive model. By selecting sustained response patients through this model based on the immune profile of PBMCs before treatment, accuracy exceeded 75%. In the era of precision medicine, effectively screening the most suitable patients is key to maximizing patient benefits. We look forward to larger-scale studies to further validate this model and the improvement of accessibility and affordability of relevant testing methods to help enhance HCC treatment standards.
Under the guidance of Academician Jiahong Dong, our team at Beijing Tsinghua Changgung Hospital has become the center with the most Y90 treatment cases, the widest range of tumor types, the most complex and difficult cases, and the most innovative techniques in China. In the future, our center will conduct comprehensive and systematic research on the regulation of the immune microenvironment and integrated treatment of liver cancer by Y90 to maximize the value of Y90 technology in integrated tumor treatment.
