Public-data reanalysis identifies cell-cycle signal in sinonasal cancer

A multi-platform reanalysis of public transcriptomic data found extensive gene-expression differences between sinonasal squamous cell carcinoma (SNSCC) and normal nasal tissue, with cell-cycle programs showing the largest standardized shift in bulk samples. The study combined single-nucleus RNA sequencing, bulk RNA sequencing, paired microarray data and a tumor-only context cohort, then examined four candidate biomarkers in SNSCC cell lines. CD74, CCL5, IL32 and HLA-DRA were consistently elevated across major data layers, and preliminary laboratory assays supported their overexpression in cancer cells. The findings remain exploratory because the single-nucleus comparison included only one healthy library and one tumor library, while the available data did not support comparisons by human papillomavirus status or clinical outcome.

The investigation centered on four public datasets. The single-nucleus RNA subset contained healthy nasal mucosa library HC1 and SNSCC tumor library CA5. Bulk RNA sequencing data contained seven SNSCC tumors and seven normal nasal mucosa samples, while a microarray dataset supplied five matched tumor-adjacent-normal pairs. A separate set of 21 nonkeratinizing sinonasal cancer samples was used only as tumor-only context and was not treated as independent tumor-normal validation. The researchers did not substitute head-and-neck cancer data from The Cancer Genome Atlas for missing SNSCC material and did not make an HPV-positive versus HPV-negative inference. After quality control and doublet detection, the single-nucleus dataset contained 14,479 nuclei: 6,817 from healthy tissue and 7,662 from the tumor.

The retained nuclei were annotated into 11 biological cell types, including epithelial, secretory epithelial, basal epithelial, ciliated epithelial, tuft, endothelial, perivascular, fibroblast, myeloid, T and NK, and plasma cells. The largest observed compositional difference was a 44.7-percentage-point increase in epithelial cells in the tumor library relative to the healthy library. Secretory epithelial cells showed the largest decrease, at 56.2 percentage points. These figures describe the two available libraries and do not establish how cell populations change in patients generally. The authors also reported Shannon diversity values of 1.444 for the healthy library and 1.285 for the tumor library, but did not treat cell-level nuclei as independent patient replicates when calculating program scores or reporting comparisons.

Bulk RNA sequencing provided the study’s principal evidence for tumor-versus-normal differential expression. An edgeR robust quasi-likelihood model evaluated 17,677 genes across the 14 bulk libraries and identified 5,732 genes at a false-discovery-rate threshold below 0.05. In an analysis of nine predefined gene programs, cell cycle showed the largest mean standardized shift, increasing by 1.727 units in tumors relative to normal samples. Running-enrichment analysis gave an enrichment score of 0.926 for an interferon-response program and 0.733 for a fibroblast extracellular-matrix program, whereas a myeloid-activation program had a score of −0.424. Despite enrichment of individual interferon-response genes, the composite antigen-presentation score was lower in tumor samples, at −0.238 versus 0.238 in normal tissue, indicating that the elevated genes did not amount to uniform induction of that entire program.

Integration between the bulk and single-nucleus layers showed a moderate correspondence. Exact gene-symbol matching was possible for 16,148 genes, and bulk log fold changes correlated with observed single-nucleus pseudobulk shifts at a Spearman rho of 0.543. Among 1,615 genes in the top 10% by absolute single-nucleus shift, 975 also belonged to the bulk set meeting the false-discovery-rate criterion. Program-level effects across nine predefined sets showed a cross-scale Spearman rho of 0.467. CD74, CCL5, IL32 and HLA-DRA were highlighted because their observed directions were consistent across the layers. Their patterns involved different cellular compartments: CD74 was enriched in myeloid cells in the single-nucleus atlas, while CCL5 and IL32 were concentrated in immune-active populations and HLA-DRA was associated with MHC class II expression.

The paired microarray dataset supplied additional context without extending the claims beyond its measurement limits. A paired limma model tested 60,755 probes and identified 2,620 features at an adjusted probability below 0.05 across the five tumor-adjacent-normal pairs. Because the platform annotation did not permit reliable genome-wide conversion to gene symbols, the researchers retained probe identifiers and did not present these results as gene-level validation of the four candidates. Expression distributions in the 21 tumor-only nonkeratinizing sinonasal cancer samples were also treated as context rather than evidence of a tumor-normal difference or replication of the bulk findings. The cross-layer candidate rankings were filtered for measured expression, bulk statistical significance and concordant direction, making the nominated panel exploratory rather than a comprehensive marker-discovery assay.

Laboratory experiments offered preliminary support for the four leading candidates. The investigators compared two SNSCC cell lines, RPMI 2650 and Detroit 562, with normal bronchial epithelial BEAS-2B cells using quantitative reverse-transcription PCR and enzyme-linked immunosorbent assays. Relative to BEAS-2B cells, RPMI 2650 cells showed mean messenger RNA increases of 4.27-fold for CD74, 3.15-fold for CCL5, 3.68-fold for IL32 and 3.82-fold for HLA-DRA, with all reported P values below 0.01. Detroit 562 cells also showed significant increases, ranging from 2.46-fold for CCL5 to 3.53-fold for CD74, with all P values below 0.01. ELISA measurements showed significantly elevated concentrations of all four proteins in both cancer lines, including a 4.68-fold increase in CD74 protein in RPMI 2650 cells, with P below 0.001. These were cancer-versus-normal cell-culture comparisons, not tests of diagnostic performance or treatment response.

The biological interpretations remain bounded by the design. CCL5 is a chemokine associated with recruitment of T cells, NK cells and other immune effectors, while IL32 is linked to inflammatory signaling and has been implicated in antitumor immune activity. CD74 encodes the MHC class II invariant chain involved in antigen processing, and HLA-DRA is an MHC class II gene, so their coordinated elevation is compatible with selected immune-related responses. However, the assays did not measure immunotherapy response, and neither RPMI 2650 nor Detroit 562 was an established HPV-positive model. The experiments therefore cannot show whether the candidates differ by HPV subtype or predict sensitivity to immunotherapy. Likewise, enrichment of interferon-response genes in the bulk comparison does not establish a clinical response to checkpoint blockade.

Several technical and clinical gaps constrain the conclusions. The single-nucleus atlas represents one library per condition, so its thousands of nuclei cannot substitute for independent patient replication, and library-level differences may reflect factors beyond disease status. Ambient RNA was not estimated because empty-droplet matrices were unavailable, raising the possibility that some low-level signals, including ribosomal signals, were influenced by technical contamination. HPV annotations came from deposited metadata rather than new viral sequencing, E6 or E7 detection, or p16 staining. The immune signatures grouped cells at broad lineage level rather than resolving CD4 and CD8 T-cell subsets, regulatory T cells, distinct B-cell populations, macrophages, dendritic cells or myeloid-derived suppressor cells. Independent cohorts, spatial tissue studies, immunohistochemistry and appropriately powered HPV-matched analyses would be needed to confirm the origins and clinical significance of the signals.

Overall, the reanalysis provides a descriptive map of tumor-normal remodeling in SNSCC and nominates CD74, CCL5, IL32 and HLA-DRA as candidate markers for further study. Its strongest supported result is the widespread bulk differential-expression landscape, including a pronounced cross-layer cell-cycle signal supported by concordant observations in the smaller single-nucleus comparison. The four candidates additionally showed concordant elevation in public transcriptomic layers and increased messenger RNA and protein abundance in two SNSCC cell lines. These results do not establish diagnostic performance, HPV-specific biology or immunotherapy utility. Instead, they identify experimentally tractable signals for validation in larger independent tissue cohorts, with particular attention to immune-cell localization, spatial organization and relationships to HPV status and treatment outcome.

Subject of Research: Exploratory multi-platform transcriptomic analysis of tumor-normal dysregulation in sinonasal squamous cell carcinoma

Article Title: Reanalysis of public bulk and single-nucleus transcriptomes identifies widespread tumor-normal dysregulation and a cross-layer cell-cycle signal in sinonasal squamous cell carcinoma

Article References: Yuan, J., Wang, Y., Lin, K., & Sun, L. (2026). Reanalysis of public bulk and single-nucleus transcriptomes identifies widespread tumor-normal dysregulation and a cross-layer cell-cycle signal in sinonasal squamous cell carcinoma. Frontiers in Genetics, 17, Article 1918021. https://doi.org/10.3389/fgene.2026.1918021

Image Credits: AI Generated

DOI: 10.3389/fgene.2026.1918021

Keywords: SNSCC, single-nucleus RNA sequencing, bulk RNA sequencing, cell-cycle regulation, CD74, CCL5, IL32, HLA-DRA, tumor immunology, biomarker discovery, transcriptomics, Reanalysis