Multiomic Selection of Cancer-Testis Antigens as Precision Immuno-oncologic Targets in Head and Neck Cancer.

Importance: Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignant neoplasm with an increasing need for precision therapeutics. Cancer-testis antigens (CTAs) represent promising targets given their aberrant tumor expression and otherwise localized expression to immune-privileged tissues with no (testis-restricted) or minimal (testis-selective) expression in other human body sites. Despite their potential, limited studies rigorously evaluate CTAs as therapeutic targets in HNSCC. Objective: To orthogonally validate and present specific CTAs as potential precision immuno-oncologic targets in both previously untreated (de novo) and recurrent HNSCC tumors. Design, Setting, and Participants: This was a cross-sectional study conducting multiomic analyses on a single academic tertiary care center's tumor registry from 2018 to 2023, with validation using publicly available transcriptomic datasets. A total of 33 tumor samples from patients with HNSCC, including both de novo and radiation-recurrent tumors, were analyzed. Data were analyzed from August to December 2024. Main Outcomes and Measures: The primary outcome was the identification and rigorous validation of specific CTAs with tumor-specific expression in HNSCC. Results: This study analyzed 33 HNSCC institutional tumor specimens, including 25 de novo and 8 radiation-recurrent tumors. Of 33 included patients, 25 (76%) were male, 8 (24%) were female, and the median (range) age was 61 (29-87) years. Tumor subsites included the oral cavity (24 [73%]), larynx (7 [21%]), and oropharynx (2 [6%]). Tumors were primarily T4a (23 [70%]), with nodal involvement in 16 (48%). Initial analysis of bulk RNA-sequenced institutional data and single-cell RNA-sequenced external data identified several CTAs (DKKL1, SPANXB1, SPANXD, and ACTL8) upregulated in recurrent tumors. An expanded reanalysis revealed that CTAs were expressed across HNSCCs, including robust expression not only in radiation-recurrent disease but also in de novo tumors. Immunohistochemistry was performed on ACTL8 to confirm transcriptional level findings at the protein level, which showed moderate focal cytoplasmic staining in tumor tissue. To refine the tumor-specific CTA list, an exclusionary analysis using single-cell RNA-sequenced data from normal oral mucosa was conducted, removing any CTAs with any expression in normal tissue. This resulted in a final list of 23 testis-restricted and 44 testis-selective CTAs specific to HNSCC, of which 14 CTAs overlapped across all transcriptomic datasets. Finally, using CopyKAT, CTAs were specifically enriched in malignant epithelial populations compared with benign populations within the same patients with HNSCC. Conclusions and Relevance: In this study, a set of 23 testis-restricted and 44 testis-selective CTAs were orthogonally validated in multiple tumor datasets. Of these, a core set of 14 CTAs were consistently detected across all datasets. Their tumor specificity and broad expression bolster their potential as promising precision immuno-oncologic targets for future T-cell receptor engineering efforts.