Multispectral Immunofluorescence Analysis of the Esthesioneuroblastoma Tumor Immune Microenvironment Reveals T-Cell Stroma Localization and Tumor Parenchyma Exclusion

 

Background: Esthesioneuroblastoma (ENB), also known as olfactory neuroblastoma, is a rare malignancy of the anterior skull base. The mainstay of treatment is surgery followed by adjuvant radiation; however, treatment options are limited for recurrent or metastatic disease. Multispectral immunofluorescence (mxIF) allows for comprehensive evaluation of tumor immune cell spatial relationships with seven channels simultaneously and thorough characterization of the tumor immune microenvironment (TIME). The objective of this study was to comprehensively define the ENB T cell TIME with mxIF to identify new immunotherapeutic treatment strategies to improve ENB patient outcomes.

Methods: A tissue microarray (TMA) including 47 clinically annotated human ENB samples in triplicate was obtained, in addition to IRB approval, from our tertiary care hospital. A T-lymphocyte specific panel was validated in ENB tissue and implemented. The panel stained for CD4, CD8, FOXP3, PD-1, Ki67, synaptophysin, and DAPI. Phenotypes of interest included FOXP3+CD4+ regulatory T cells, CD4+ helper T cells, and CD8+ cytotoxic T cells. Ki67 expression was used to assess mitotic activity. Segmentation of tumor and stroma was manually performed and HALO image analysis platform v3.4 was used to objectively quantify T cell immune cell spatial relationships in the tumor and stroma. A retrospective chart review was performed from these clinically annotated specimens with collection of patient demographics, stage, Hyam's grade, dural infiltration, and outcomes.

Results: Eight samples were excluded based on too little available tissue for meaningful analysis. Of the remaining 39 tumors, 33 were primary and 6 were recurrent tumors. 54% of patients were male and average age at presentation was 54. 33% (13/39) were high Hyam's grade (III/IV). Kadish stages were predominantly stage C (28). Of note, 13 patients had dural infiltration and 14 experienced posttreatment recurrent disease. Higher T cell densities were noted in the stroma compared with the tumor parenchyma including T_reg cells (12.5 cells per mm² vs 1.90 cells per mm², p = 0.05), helper T cells (276 cells per mm² vs. 43.7 cells per mm2, p = 0.023), cytotoxic T cells (117 cells per mm² vs. 33.0 cells per mm², p < 0.0001), PD-1+ CD8+ cytotoxic T cells (44.5 cells per mm² vs 14.9 cells per mm2, p = 0.002) and Ki67+ proliferating CD8+ cytotoxic T cells (12.8 vs. 6.84, p = 0.05; [Fig. 1]). As expected, increased Ki67+ tumor cells were noted in high Hyam's grade tumors (1,502 cells per mm² vs. 556.9 cells per mm², p = 0.01). No significant differences were noted when comparing the T cell TIME for Hyam's grade, stage, dural infiltration, recurrent disease, or other clinical factors.

Conclusion: This study is the first to comprehensively define the ENB T lymphocyte TIME using mxIF. These data demonstrate that T cells are highly excluded from the ENB tumor parenchyma and largely restricted to the stromal compartment. Furthermore, Treg cells in proximity to cytotoxic T cells in the tumor stroma may play an immunosuppressive role and aid in tumor immune evasion. This study suggests that immunotherapeutic strategies that improve T cell tumor parenchyma infiltration, target Treg immunosuppressive activity, or inhibit the PD-1/PD-L1 pathway may be of benefit for ENB.

Publication History

Article published online:
01 February 2023

© 2023. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany