Cancer Stem Cell Characterization in Olfactory Neuroblastoma Tissue

Key Points

• Cancer stem cells are uncommon in olfactory neuroblastoma

• When present, cancer stem cells are more likely to be PD‐L1‐positive

1. Introduction

Olfactory neuroblastoma (ONB), also known as esthesioneuroblastoma, is a rare malignant neoplasm arising from the olfactory neuroepithelium. Treatment typically consists of surgical resection, when feasible, followed by adjuvant radiation with chemotherapy being reserved for advanced or metastatic cases. While the 5‐year survival rate for ONB is high, patients with recurrent or metastatic disease have a poorer prognosis with limited treatment options [1, 2].

Cancer stem cells (CSCs) have been identified in multiple cancer types and are self‐renewing oncogenic cells that drive tumor progression, proliferation, and recurrence [3]. Interestingly, CSCs in chordoma were found to have higher programmed death‐ligand 1 (PD‐L1) expression than non‐CSCs [4], potentially offering a method to specifically target CSCs. However, the presence and role of CSCs in ONB has not yet been evaluated. The objective of this study was to characterize CSC density and PD‐L1 expression of CSCs in ONB using multispectral immunofluorescence and evaluate whether this cell population is associated with clinical correlates of ONB disease severity.

2. Methods

This study was approved by the Institutional Review Board at Johns Hopkins. Formalin‐fixed, paraffin‐embedded (FFPE) tissue microarray (TMA) slides including 47 clinically annotated human ONB samples in triplicate were obtained. Twenty‐nine samples were included in the final analysis, as eighteen samples had insufficient tissue for CSC analysis. A retrospective chart review was performed from these clinically annotated specimens with a collection of patient demographics, dural infiltration, orbital infiltration, Hyams grade, Kadish stage, recurrence, and survival.

Leica BOND Rx autostainer was used for deparaffinization and staining of all tissue. The dilution of each antibody specific to the FFPE ONB tissue type was determined first using monoplex immunohistochemistry (IHC) and then monoplex immunofluorescence (IF). The CSC multiplex‐IF panel used Tyramide signal amplification method and opals (Akoya) and stained for CD15 (BD Biosciences [HI98], #555400, 1:1500), CD24 (Novus biologicals [ML5], #NB100‐77903, 1:1000), ALDH1A1 (Abcam [EP1933Y], #ab52492, 1:400), PDL1 (Cell Signaling [E1L3N(R)], #13684S, 1:300), synaptophysin (Abcam [YE269], #ab32127, 1:2500), and DAPI (Figure S1). Whole slide scanning was performed using the PhenoImager (Akoya) at 40X magnification. Characterization of the TMA slide was manually performed using the HALO image analysis platform v3.5 to objectively identify CSCs. All statistical analysis was done using GraphPad Prism version 10. Mann–Whitney U test was used to compare CSC densities among groups by clinical factors of interest. A p‐value <0.05 was considered statistically significant.

3. Results

The average age at diagnosis was 56 years with 17/29 (59%) of patients being male. Ten out of 29 patients had high Hyams grade tumors (III/IV) and 26 out of 29 patients had Kadish stage C/D tumors. Twenty‐four percent of patients went on to have disease recurrence, 57% had dural involvement, and 13% had orbital involvement.

The putative CSC phenotype was established based on common CSC surface markers in other tumors and was defined by cells that were CD15⁺/CD24⁺/ALDH1A1^{+ 4‐7}. On average, 0.37% of ONB tumor cells were putative CSCs (Figure 1A). The median CSC density was 1.13 cells per mm² of tumor parenchyma (Figure 1B). Interestingly, there was a significant difference in the median number of PD‐L1⁺ CSCs versus PD‐L1⁻ CSCs (0.97 vs. 0.39 cells per mm², p < 0.001) (Figure 1C). Additionally, while ONB PD‐L1 expression is relatively low, tumor PD‐L1 expression moderately correlated with the presence of uncommon putative CSCs (r = 0.527, p = 0.003) (Figure 1D).

FIGURE 1.

(A) ONB tumor cells were identified as synaptophysin positive and nuclei were stained with DAPI. Cancer stem cells were triple positive for CD15, CD24, and ALDH1A1 and the white arrows indicate a representative CSC. (B) The median number of CSCs found in the ONB was 1.13 cells per mm² of tumor parenchyma, with an interquartile range of 0 to 9.12. (C) For patients with CSCs present, there were more significantly more PD‐L1⁺ CSCs compared to PD‐L1⁻ CSCs per patient tumor sample. (D) Tumor PD‐L1 expression moderately correlated with the presence of uncommon CSCs (r = 0.527, p = 0.003).

Next, we evaluated whether the quantity of putative ONB CSC is associated with clinical correlates of ONB disease severity. While more ONB CSC were observed in high Hyams Grade tumors (median 3.18 cells per mm²) versus low Hyams Grade tumors (median 0.67 cells per mm²), this did not reach statistical significance (p = 0.08) (Figure 2A). There were no other statistically significant differences noted in other comparisons including demographics, Kadish stage, recurrence, survival, or dural or orbital involvement (Figure 2B). Next, we compared PD‐L1⁺ and PD‐L1⁻ ONB CSC and did not note any differences in these clinical correlate comparisons (Figure 2C,D).

FIGURE 2.

(A) There was no significant difference in the median CSCs when comparing low versus high Hyams grade. (B) The Mann–Whitney test results between the CSC median and the clinical correlates of interest were all nonsignificant. (C) There was no significant difference in the median PD‐L1⁺ CSCs when comparing low versus high Hyams grade. (D) The Mann–Whitney test results between the PD‐L1⁺ CSC median and the clinical correlates of interest were all nonsignificant.

4. Discussion

While CSCs are present in ONB, they are uncommon, comprising only 0.37% of ONB tumor cells. Similar to what our group has observed in chordoma, a significant proportion of ONB CSCs are positive for PD‐L1 [4]. With the propensity of ONB CSC to express PD‐L1 this may also allow for methods to specifically target the CSC population by harnessing immunotherapeutic treatment methods that target PD‐L1 [4]. This may be particularly suited for Hyams grade III/IV tumors as more ONB CSC were observed in high Hyams Grade tumors (median 3.18 cells per mm²) versus low Hyams Grade tumors (median 0.67 cells per mm²). However, this did not reach statistical significance (p = 0.08) and should be further evaluated in larger multi‐institutional studies.

PD‐L1, which may be expressed on cancer cells, is known to be critical for tumor cell evasion of immune surveillance. PD‐L1 positivity in colorectal carcinoma was also found to promote the expansion of CSCs and was correlated with chemoresistance through activation of the HMGA1‐signaling pathway [5]. Given the known importance of CSCs in tumor progression, treatment resistance, and disease recurrence these may be important targets to further investigate [3].

There are limitations to this study. Reliable human in vitro or in vivo models of ONB have yet to be established. Thus, the clonogenic and self‐renewal potential of CD15⁺/CD24⁺/ALDH1A1⁺ ONB CSCs cannot yet be verified in vitro or in vivo through methods of cell sorting and evaluation of self‐renewal capabilities of this cell population and why we have referred to these cells as putative CSCs. However, these markers were selected as they have been shown to be key markers of CSCs in multiple other tumor types [4, 6–8]. In the future, evaluating the cancer stem‐like properties of CD15⁺/CD24⁺/ALDH1A1⁺ ONB cells such as clonogenicity and self‐renewal properties may be feasible after the establishment of human in vitro and in vivo models of ONB. Additionally, single‐cell RNA sequencing and spatial transcriptomic approaches may also allow for further characterization and evaluation of these putative CSC populations. These approaches will also help to overcome the limitations of the number of targets that can be simultaneously evaluated using multispectral immunofluorescence.

Conflicts of Interest

N. London received research funding from Merck Sharp & Dohme, LLC regarding HPV‐associated sinonasal carcinomas not relevant to the present manuscript. All other authors declare no conflicts of interest.

Supporting information

Supporting Figure 1: alr23620‐supinfo‐0001‐Figures_1.docx

Ramolia S., Larkin R., Robbins Y., et al. “Cancer Stem Cell Characterization in Olfactory Neuroblastoma Tissue.” International Forum of Allergy & Rhinology 15, no. 9 (2025): 15, 1008–1011. 10.1002/alr.23620