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, suggesting an incomplete understanding of the underlying immune biology. Large-scale sequencing of patient tumors have suggested that NB has diverse immune microenvironments (TMEs), which are associated with MYCN-amplification (A) and patient outcomes. 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 therapeutic pressures. We hypothesized the TME is dynamic, changing with therapy, influenced by molecular subtype, and associated with patient outcomes. Methods: To better understand the NB TME, we obtained 93 clinically annotated tumors from 72 pediatric patients with NB, consisting of high-risk primary and metastatic tumors both pre- and post- chemotherapy. We designed multiplexed antibody panels targeting immune cells and performed either imaging mass cytometry (IMC) (n = 46) or NanoString GeoMx DSP (n = 47). Results: We identified 4 different immune cell types (CD8, CD4, myeloid, and NK cells) and confirmed that MYCN-non amplified (NA) tumors display higher frequencies of lymphocytes including CD4 (p < 0.003) and CD8 (p < 0.005) T-cells. Using nearest neighbors’ analysis, we found that CD4 cells are closer to tumor (p = 6.24E-16) and CD8 T-cells (p = 7.55E-11) in MYCN-NA tumors compared to MYCN-A. However, in MYCN-NA tumors, myeloid cells are also closer to tumor, (p = 1.71E-68), CD4 (p = 4.72E-58), and CD8 T-cells (p = 1.27E-70). Furthermore, we find that all immune cell subsets are further away from tumor cells independently of subtype following exposure to chemotherapy, and that there is a decrease (p = 0.0091) in the number of myeloid cells in MYCN-NA tumors, but not MYCN-A. Interestingly, we also observed an increased expression of the immune checkpoints CTLA-4 (p = 0.043) and the exhaustion marker TIM-3 (p = 2.05E-5), but not PD-1, PD-L1, or PD-L2, post chemotherapy, suggesting blockade of CTLA-4 or TIM-3 could improve response to therapy in these patients. Notably, high expression of CD56, a marker for both NK cells and NB, was associated with increased 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 discovered that the TME in NB varies with tumor subtype and changes with chemotherapy. We find that there is an upregulation of immune check point markers post 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, Ben J. Somerville, Igor B. Kuznetsov, William Bocik, Daniel R. Catchpoole, Jun S. Wei, Javed Khan. Discovery of subtype-specific and therapy associated effects on the tumor immune microenvironment in pediatric neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1199.
<div>Abstract<p><b>Purpose:</b> Amplified <i>MYCN</i> oncogene defines a subgroup of neuroblastomas with poor outcome. However, a substantial number of <i>MYCN</i> single-copy neuroblastomas exhibits an aggressive phenotype similar to that of <i>MYCN</i>-amplified neuroblastomas even in the absence of high <i>MYCN</i> mRNA and/or protein levels.</p><p><b>Experimental Design:</b> To identify shared molecular mechanisms that mediate the aggressive phenotype in <i>MYCN</i>-amplified and single-copy high-risk neuroblastomas, we defined genetic programs evoked by ectopically expressed <i>MYCN in vitro</i> and analyzed them in high-risk versus low-risk neuroblastoma tumors (<i>n</i> = 49) using cDNA microarrays. Candidate gene expression was validated in a separate cohort of 117 patients using quantitative PCR, and protein expression was analyzed in neuroblastoma tumors by immunoblotting and immunohistochemistry.</p><p><b>Results:</b> We identified a genetic signature characterized by a subset of MYCN/MYC and E2F targets, including <i>Skp2</i>, encoding the F-box protein of the SCF<sup>Skp2</sup> E3-ligase, to be highly expressed in high-risk neuroblastomas independent of amplified <i>MYCN</i>. We validated the findings for <i>Skp2</i> and analyzed its expression in relation to <i>MYCN</i> and <i>E2F-1</i> expression in a separate cohort (<i>n</i> = 117) using quantitative PCR. High <i>Skp2</i> expression proved to be a highly significant marker of dire prognosis independent of both <i>MYCN</i> status and disease stage, on the basis of multivariate analysis of event-free survival (hazard ratio, 3.54; 95% confidence interval, 1.56-8.00; <i>P</i> = 0.002). Skp2 protein expression was inversely correlated with expression of p27, the primary target of the SCF<sup>Skp2</sup> E3-ligase, in neuroblastoma tumors.</p><p><b>Conclusion:</b> Skp2 may have a key role in the progression of neuroblastomas and should make an attractive target for therapeutic approaches.</p></div>
Neuroblastoma (NB) is a heterogeneous pediatric tumor. To better understand the biological pathways involved in the development of high-risk neuroblastoma, we performed parallel global protein and mRNA expression profiling on NB tumors of stage 4 MYCN-amplified (4+) and stage 1 MYCN-not-amplified (1−) using isotope-coded affinity tags (ICAT) and Affymetrix U133plus2 microarray, respectively. A total of 1461 proteins represented by 2 or more peptides were identified from the quantitative ICAT analysis, of which 433 and 130 proteins are up- or down-regulated, respectively, in 4+ tumor compared to the 1− tumor. Pathway analysis of the differentially expressed proteins showed the enrichment of glycolysis, DNA replication and cell cycle processes in the up-regulated proteins and cell adhesion, nervous system development and cell differentiation processes in the down-regulated proteins in 4+ tumor; suggesting a less mature neural and a more invasive phenotype of 4+ tumor. Myc targets and ribosomal proteins are overrepresented in the 4+ tumors as expected; functional gene sets reported to be enriched in neural and embryonic stem cells are significantly enriched in the 4+ tumor, indicating the existence of a stemness signature in MYCN-amplified stage 4 tumor. In addition, protein and mRNA expression are moderately correlated (r = 0.51, p < 0.0001), as approximately half of the up-regulated proteins in 4+ tumor have elevated mRNA level (n = 208), and one-third of down-regulated proteins have lower mRNA expression (n = 47). Further biological network analysis revealed that the differentially expressed proteins closely interact with other proteins of known networks; the important role of MYCN is confirmed and other transcription factors identified in the network may have potential roles in the biology of NB tumor. We used global genomic and proteomic analysis to identify biologically relevant proteins and pathways important to NB progression and development that may provide new insights into the biology of advanced neuroblastoma.
Despite advances in the management of osteosarcoma (OSA) and other solid tumors, the development of metastasis continues to be the most significant problem and cause of death for cancer patients. To define genetic determinants of pulmonary metastasis, we have applied cDNA microarrays to a recently described murine model of OSA that is characterized by orthotopic tumor growth, a period of minimal residual disease, spontaneous pulmonary metastasis, and cell line variants that differ in metastatic potential. Microarray analysis defined 53 genes (of 3166 unique cDNAs) that were differentially expressed between the primary tumors of the more aggressive (K7M2) and less aggressive (K12) OSA models. By review of the literature, these differentially expressed genes were assigned to six nonmutually exclusive metastasis-associated categories (proliferation and apoptosis, motility and cytoskeleton, invasion, immune surveillance, adherence, and angiogenesis). Functional studies to evaluate K7M2 and K12 for differences in each of these metastasis-associated processes revealed enhanced motility, adherence, and angiogenesis in the more aggressive K7M2 model. For this reason, 10 of the 53 differentially expressed genes that were assigned to the motility and cytoskeleton, adherence, and angiogenesis categories were considered as most likely to define differences in the metastatic behavior of the two models. Ezrin, a gene not described previously in OSA, with functions in motility, invasion, and adherence, was 3-fold overexpressed in K7M2 compared with K12 by microarray. Differential expression for RNA was confirmed by Northern analysis and for protein by immunostaining. Alterations in ezrin protein levels and concomitant cytoskeletal changes in our model confirmed predictions from the arrays. The potential relevance of ezrin in OSA was suggested by its expression in five of five human OSA cell lines. This work represents a rationale approach to the evaluation of microarray data and will be useful to identify genes that may be causally associated with metastasis.
