Abstract LB062: Profiling of pediatric neuroblastoma reveals a dynamic and clinically significant tumor immune microenvironment
Authors: Masih Katherine E. , Islam Zahin , Aiyetan Paul , Kuznetsov Igor B. , Hewitt Stephen M. , Catchpoole Daniel , Wei Jun S. , Bocik William , Khan Javed 
Published: June 2022

Abstract
Background: Neuroblastoma (NB) is the 3rd most common childhood cancer and accounts for 15% of all pediatric cancer deaths. Recently, immunotherapy using monoclonal antibodies targeting GD2 have improved survival rates for some patients with NB. Unfortunately, this response is not uniform across patients, which suggests an incomplete understanding of the underlying immune biology of this disease. Large-scale sequencing efforts of patient tumors have suggested that NB has diverse immune microenvironments (TMEs), which are associated with MYCN-amplification (A) and patient outcomes. While this is strong evidence, these results need to be further validated, specifically to determine whether the infiltrating immune cells can interact with tumor cells and if the TME is impacted by evolutionary pressures. We hypothesized the TME is dynamic, changing with therapy and metastasis, influenced by molecular subtype, and associated with patient outcomes.
Methods: To better understand the heterogeneity seen in NB TMEs, we obtained 93 clinically annotated tumors from 72 pediatric patients with neuroblastoma, consisting of high-risk primary and metastatic tumors both pre- and post- chemotherapy. We designed two highly multiplexed antibody panels targeting immune cells and performed either imaging mass cytometry (IMC) (n = 46) or NanoString GeoMx DSP (n = 47).
Results: We confirmed that MYCN-non amplified (NA) tumors display higher frequencies of lymphocytes including CD4 (p &lt; 0.003) and CD8 (p &lt; 0.005) T-cells. Using nearest neighbor analysis, we found that not only are both CD4 and CD8 T-cells more frequent in MYCN-NA samples, but they are significantly closer to tumor cells compared to MYCN-A tumors (p &lt; 2.2E-16), suggesting increased interactions. We then investigated the effects of exposure to chemotherapy on the TME and discovered that MYCN-NA tumors displayed higher frequencies of T-cells (p &lt; 0.0041) and B-cells (p = 0.047) prior to exposure to chemotherapy. We also revealed increased frequencies of macrophages (p = 0.0193) and antigen presentation in tumors post-treatment. Interestingly, we saw increased expression of the immune checkpoints CTLA-4 (p = 0.0432) and TIM-3 (p = 2.05E-5), but not PD-1, PD-L1, or PD-L2, suggesting targeted checkpoint blockade could improve response to therapy in these patients. Notably, high expression of CD56, a marker for both NK cells and NB, was associated with increase overall survival, indicating a potential role of NK cells in improving outcomes.
Conclusions: Using two independent protein-based profiling methods, we investigated the TME in clinically annotated patient NBs. We find that the TME in NB varies with tumor subtype and changes dynamically with chemotherapy. These results can inform future trials to optimize the timing and specificity of novel immunotherapeutic approaches for these high-risk patients.
Citation Format: Katherine E. Masih, Zahin Islam, Paul Aiyetan, Igor B. Kuznetsov, Stephen M. Hewitt, Daniel Catchpoole, Jun S. Wei, William Bocik, Javed Khan. Profiling of pediatric neuroblastoma reveals a dynamic and clinically significant tumor immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB062.