Molecular Evidence for Olfactory Neuroblastoma as a Tumor of Malignant Globose Basal Cells

Abstract

Olfactory neuroblastoma (ONB, esthesioneuroblastoma) is a sinonasal cancer with an underdeveloped diagnostic toolkit, and is the subject of many incidents of tumor misclassification throughout the literature. Despite its name, connections between the cancer and normal cells of the olfactory epithelium have not been systematically explored and markers of olfactory epithelial cell types are not deployed in clinical practice. Here, we utilize an integrated human-mouse single-cell atlas of the nasal mucosa, including the olfactory epithelium, to identify transcriptomic programs that link ONB to a specific population of stem/progenitor cells known as olfactory epithelial globose basal cells (GBCs). Expression of a GBC transcription factor NEUROD1 distinguishes both low- and high-grade ONB from sinonasal undifferentiated carcinoma, a potential histologic mimic with a distinctly unfavorable prognosis. Furthermore, we identify a reproducible subpopulation of highly proliferative ONB cells expressing the GBC stemness marker EZH2, suggesting that EZH2 inhibition may play a role in the targeted treatment of ONB. Finally, we study the cellular states comprising ONB parenchyma using single-cell transcriptomics and identify evidence of a conserved GBC transcriptional regulatory circuit that governs divergent neuronal-versus-sustentacular differentiation. These results link ONB to a specific cell type for the first time and identify conserved developmental pathways within ONB that inform diagnostic, prognostic, and mechanistic investigation.

Keywords: esthesioneuroblastoma; head and neck cancer; olfactory epithelium; olfactory neuroblastoma; transcriptomics.

Figure 1.. Transcriptomic characterization of ONB vis-à-vis the normal human olfactory mucosa and stages of olfactory epithelial neurogenesis

A Volcano plot of differential gene expression between ONB and human OM assessed via bulk tissue RNAseq. Cutoffs for Log2 fold change and statistical significance are indicated with solid black lines. To each side, gene ontology networks for the indicated sets of differentially expressed genes. Select terms are annotated. Node size is proportional to the number of genes annotated with each term. Edges indicate the presence of shared genes between terms. Node color indicates communities of similar GO annotations (29). B Top; integrated UMAP embedding of msGBCs, npGBCs, and iOSNs from human and mouse single cell transcriptomic data sets. Number of cells per cell type and species of origin is reported. Lower left; cell clusters correspond to known cell types according to established marker expression. Lower right; pseudotime trajectory and ordering of cells. C Top; expression of two gene expression modules that vary as a function of pseudotime. Rather than coinciding with discrete clusters and annotated cell types, these signatures are maximal at transition points between two cell types along the trajectory; thus, they are regarded as differentiation signatures. The number of genes that comprise each module is indicated. Bottom; gene set enrichment testing for the above-mentioned differentiation modules in ONB versus the human olfactory mucosa.

Figure 2.. Projection of ONB single cells onto the nasal mucosal cell atlas.

Top left; uniform manifold approximation projection (UMAP) of 51,005 nasal mucosal cells generated via the standard analytic pipeline. The integrated data set contains 28,830 human cells from Durante et al. (31) and 22,175 mouse cells. Cell type annotations were assigned to clusters according to marker gene expression and tissue of origin. Labels are as follows: msGBC, multipotent stem GBC; npGBC, neuronal progenitor GBC; iOSN, immature olfactory sensory neuron; mOSN, mature olfactory sensory neuron; Olf.HBC, olfactory horizontal basal cell; Sus, sustentacular cell; Olf.DG, olfactory epithelial duct/gland cell; Resp.BC, respiratory basal cell; Cili.REcyte, ciliated respiratory epithelial cell; Secr.REcyte, secretory respiratory epithelial cell; Resp.DG, respiratory epithelial duct/gland cell; Lymph.WBC, lymphoid leukocyte; Myelo.WBC, myeloid leukocyte; OEC, olfactory ensheathing cell. Bottom left; UMAP of nasal mucosal cells generated by limiting the principal component analysis step to transcription factors. Right column; projection of single ONB tumor cells into the gene expression space defined by nasal mucosal cells. ONB cells, leukocytes, and stromal cells are color-coded and labeled according to analysis of the ONB single cell data set (see Supplementary Figure 2A). O; ONB cells. S; stromal cells. L; leukocytes. Axis numbering indicates UMAP coordinates. Dashed contours surround identical regions.

Figure 3.. NEUROD1 immunohistochemistry on patient samples of ONB and sinonasal undifferentiated carcinoma.

A Histopathologic assessment of ONB and SNUC. Slides were stained with hematoxylin and eosin (H&E, top row) or processed for immunoperoxidase labeling with 3,3’-diaminobenzidine. Representative staining patterns for NEUROD1 (middle row) and KI67 (bottom row) are shown. B Higher magnification of the representative images in (A). C Results from semi-quantitative scoring of tissue samples. Tissue categories are as follows: HG-ONB, high-grade ONB; LG-ONB, low-grade ONB; SNUC, sinonasal undifferentiated carcinoma.

Figure 4.. Expression of EZH2 in the proliferating compartment of ONB.

A Scatterplot of normalized EZH2 expression and mitotic rate for ONB tumors in the Classe et al. cohort (18). B Left; representative images of paired EZH2 and KI67 immunolabeling. Right; semi-quantitative scoring of EZH2 and KI67 immunolabeling across 15 ONB tumors. Linear fit with 95% confidence interval is indicated (red line and grey region, respectively). Spearman’s rho indicates the correlation between the two percentages. Visualization was generated by introducing Gaussian noise to resolve overplotted points. C Expression of NEUROD1, EZH2, and MKI67 in the ONB single cell data set. Colors indicate the proliferating cell cluster versus non-cycling ONB cells. D Representative image of NEUROD1 immunofluorescence in cryopreserved ONB tissue. E Representative image of dual-labeled EZH2 and KI67 immunofluorescence in cryopreserved ONB tissue used as input for the CellProfiler analytic pipeline. F Median-normalized output from CellProfiler for all five ONB tumors under analysis. Total number of cells analyzed from each tumor is noted. Universal threshold for calling KI67 positivity is indicated with a dashed line. G Quantification of the data in (F) by decile binning. Bar height and error bars indicate the mean and standard deviation of n=3 technical replicates. Asterisks indicate statistical significance: * p < 0.05; ** p < 0.01; *** p < 0.001 by the two-sided Student’s t-test.

Figure 5.. Single cell analysis of divergent differentiation in olfactory neuroblastoma

A Left; UMAP embedding and clustering of ONB parenchymal cell types after the influence of non-cell-identity signatures were regressed out (see Supplementary Figure 2B, 2C). Right; violin plots depicting expression of NEUROD1 and diagnostic neuroendocrine markers in each of the ONB cell states. The color of the violin indicates statistical significance (adjusted p-value < 0.05) in a one-versus-all fashion. Red; significantly upregulated in that cluster. Black; significantly downregulated in that cluster. Grey; no statistically significant difference in expression. B Differentially expressed transcription factors and cytokeratin species between ONB cell states. The top ten transcription factors for each cluster are shown. C Diffusion map embedding of the three ONB cell types with and without superimposed RNA velocity streams. D Ranking of normal nasal mucosal cell types according to how strongly they express the ONB cell type signatures. E Expression of ONB cell type signatures in nasal mucosal cells. Top ranking cell types from (C) are indicated across panels.