Advancing Education, Research, and Quality of Care for the Head and Neck oncology patient.
Background: Patients with head and neck squamous cell carcinoma of unknown primary (HNSCCUP) present with metastatic squamous cell carcinoma in one or more lymph nodes of the neck, without a clearly identifiable primary source of disease. Transoral robotic surgery (TORS) is often successful in identifying the primary site of disease in >80% of cases which arise most often in the oropharyngeal lymphoepithelium. However, very little is known about tumor biology driving the unique clinical presentation of HNSSCUP. This study aims to compare the gene expression profiles of HNSCCUP tumors with traditional macroscopically apparent HNSCC tumors.
Methods: Formalin-fixed paraffin-embedded (FFPE) archival tissue sections from patients with HNSCCUP (n=7), traditional macroscopic oropharyngeal HNSCC (“macro tumors”, n=6) and normal tonsil controls (n=3) were obtained. Laser-capture microdissection was performed on primary tumor and metastatic lymph nodes, targeting tumor epithelial and tumor microenvironment regions separately. RNA-seq was performed using a specialized in-house technique designed and optimized for FFPE tissue. A total of 108 biologically-unique gene expression libraries were prepared and sequenced. Raw sequencing reads were mapped to the human genome with STAR-aligner. After filtering for quality, 102 gene expression libraries were analyzed. Differential gene expression analysis and gene set enrichment analysis were performed with DESeq2 and MSigDB gene sets respectively.
Results: Clustering with UMAP showed distinct clusters of tumor epithelial, tumor microenvironment, normal epithelial and normal-adjacent microenvironment gene profiles, confirming the purity of microdissection. Consistent with a malignant phenotype, HNSCCUP tumor epithelial regions were enriched for hallmark processes such as E2F targets (NES=2.16, p-adj < 0.0001) and G2M checkpoint (NES=1.89, p-adj < 0.0001), when compared to normal epithelium.
Within the tumor epithelial compartment, HNSCCUP tumors were highly enriched for immune processes including regulation of immune response (NES=2.09, p-adj < 0.0001), when compared to macro tumors. In contrast, macro tumors were enriched for epithelial mesenchymal transition (NES=2.49, p-adj < 0.0001), compared to HNSCCUP. In the microenvironment compartment, HNSCCUP tumors had significantly higher deconvoluted immune microenvironment scores compared to macro tumors (mean score 0.815 vs 0.571, p = 0.0070).
Conclusion: HNSCCUP tumors are significantly enriched for immune processes and disenriched for epithelial mesenchymal transition. These preliminary biological insights are consistent with the behavior of HNSCCUP tumors and provide potential pathways for targeted treatment. Our full cohort of 40 pairs of HNSCCUP and macro tumors will provide further evidence for a distinct HNSCCUP biological phenotype.