A. Machchhar3, E. Cochran1, J. Jacobson1, S.N. Fitzlaff2, B. Mobley2, J. Qiao1, D.H. Chung1,3 1University Of Texas Southwestern Medical Center, Pediatric Surgery, Dallas, TX, USA 2Vanderbilt University Medical Center, Cancer Biology, Nashville, TN, USA 3Children’s Health, Research Department, Dallas, TX, USA
Introduction: High-risk neuroblastoma presents a significant challenge in pediatric oncology due to frequent relapse and poor outcomes. The amplification of the oncogene MYCN is a notable genomic aberration found to be associated with poor prognosis. It promotes a non-inflamed and T-cell infiltration-poor tumor microenvironment, which poses challenges for treatment. Immunotherapy, particularly with immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 pathway, has been demonstrated to improve survival rates in cancer patients, including those with high-risk neuroblastoma. However, the regulatory mechanisms governing PD-L1 expression and its correlation with tumor malignancy and therapeutic resistance remain largely unexplored in neuroblastoma. This study aims to investigate PD-L1 expression in neuroblastoma and its association with clinical outcomes, potentially leading to novel therapeutic targets for high-risk neuroblastoma patients, improving clinical outcomes, and providing insights into immunotherapy resistance mechanisms.
Methods: We examined the expression of PD-L1, PD-L2, and their receptor PD-1 in neuroblastoma using publicly available gene expression analysis tools and Genomics Analysis and Visualization Platform (R2). We analyzed 110 neuroblastoma tissue samples, which were constructed into four tissue microarrays (TMAs). These TMAs were subjected to Spatial Digital Profiling (SDP) using GeoMx DSP technology from NanoString. Additionally, immunoblotting was performed to assess total protein level in neuroblastoma cells, and flow cytometry analysis was used to detect the protein expression level on the cell surface.
Results: Our analysis of data from 649 neuroblastoma patients using the R2 platform revealed higher endogenous PD-L1 expression in MYCN non-amplified samples compared to MYCN amplified samples. We identified an inverse relationship between intrinsic PD-L1 expression and MYCN amplification in neuroblastomas. Conversely, PD-L2 and PD-L1 did not show any correlation with N-Myc levels. Through SDP analysis, we observed lower PD-L1 protein levels in poorly differentiated neuroblastoma tumors. In vitro analysis of neuroblastoma cell lines showed higher intrinsic PD-L1 expression in MYCN non-amplified cells, as determined by both immunoblotting and flow cytometry. Notably, interferon-gamma (IFNg) increased PD-L1 expression in MYCN amplified and MYCN non-amplified cells. Our results show differential endogenous PD-L1 expression in MYCN amplified and MYCN non-amplified neuroblastoma cells.
Conclusion: PD-L1, as an immune checkpoint protein, plays a crucial role in tumor cell immune evasion and metastasis in neuroblastoma. Our findings contribute to a better understanding of immunotherapy resistance mechanisms in neuroblastoma, which may guide the development of more effective treatment approaches.