Abstract 4706: Multispectral immunofluorescence analysis of the olfactory neuroblastoma tumor immune microenvironment reveals macrophage and polymorphonuclear leukocyte stroma localization and tumor parenchyma exclusion
Authors: Larkin Riley , Lopez Diana , Robbins Yvette , Lassoued Wiem , Gallia Gary , Allen Clint T. , London Nyall R. 
Published: April 2023

Abstract
Background: Olfactory neuroblastoma (ONB), also known as esthesioneuroblastoma, is a rare malignancy of the nasal cavity and anterior skull base. Multispectral immunofluorescence (mxIF), allows for comprehensive evaluation of tumor immune cell spatial relationships and thorough characterization of the tumor immune microenvironment (TIME). The objective of this study was to comprehensively define the ONB myeloid cell TIME with mxIF.
Methods: A tissue microarray including 47 clinically annotated human ONB samples was obtained, in addition to IRB approval, from our tertiary care hospital. A myeloid specific panel was validated in ONB tissue as well as controls. The panel stained for CD15, CD68, CD11b, CD14, HLA-DR, synaptophysin, and DAPI. Phenotypes of interest included CD11b+/CD15+/CD14−/HLA-DRlow/− polymorphonuclear leukocytes (PMNs), CD11b+/CD14+/CD15−/HLA-DR low/−monocytes, and CD68+ Pan Macrophages. HALO image analysis v3.4 was used to objectively quantify immune cell spatial relationships. A retrospective chart review was performed from these clinically annotated specimens with collection of patient demographics, stage, Hyams grade, dural infiltration status, and outcomes.
Results: Three samples were excluded based on too little available tissue for meaningful analysis. Of the remaining 44, 38 were primary and 6 were recurrent tumors. PMNs, monocytes, and macrophages are present in the TIME of ONB at 22 cells per mm2, 2.0 cells per mm2, and 34 cells per mm2 respectively. Increased PMN and macrophage cell densities were noted in the stroma when compared to the tumor parenchyma (28 cells per mm2 vs 14 cells per mm2, p&lt;0.0001 and 46 cells per mm2 vs 21 cells per mm2, p&lt;0.0001). A similar trend was seen in monocytes (3.5 cells per mm2 vs 0.80 cells per mm2, p=0.0588) but it did not reach statistical significance. When we compared by Kadish Stage, higher stage (C/D) had more tumor infiltrating PMNs than low stage (A/B) (median of 1.3 cells per mm2 vs median 0 cells per mm2, p=0.0011). No further differences in cell distributions were noted when comparing for Hyams grade, stage, dural infiltration, recurrent disease, or other clinical factors. Nearest neighbor analysis demonstrates that macrophages are closer to tumor cells than PMNs or monocytes (19.68 µm vs 27.78 µm, p&lt;0.0001, and 25.77 µm, p=0.0026, respectively).
Conclusion: This study is the first to comprehensively define the ONB myeloid TIME using mxIF. Our study demonstrates the ONB TIME contains abundant myeloid populations. As these cells have been shown to be immunosuppressive, thereby fostering tumor cell escape and host evasion, in other human malignancies we posit they play a similar role in ONB. The functional study of myeloid populations in ONB, and whether they represent targets for therapeutic intervention, is warranted.
Citation Format: Riley Larkin, Diana Lopez, Yvette Robbins, Wiem Lassoued, Gary Gallia, Clint T. Allen, Nyall R. London. Multispectral immunofluorescence analysis of the olfactory neuroblastoma tumor immune microenvironment reveals macrophage and polymorphonuclear leukocyte stroma localization and tumor parenchyma exclusion. [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 4706.