NF-κB over-activation portends improved outcomes in HPV-associated head and neck cancer

Evolving understanding of head and neck squamous cell carcinoma (HNSCC) is leading to more specific diagnostic disease classifications. Among HNSCC caused by the human papilloma virus (HPV), tumors harboring defects in <em>TRAF3 or CYLD</em> are associated with improved clinical outcomes and maintenance of episomal HPV. TRAF3 and CYLD are negative regulators of NF-&kappa;B and inactivating mutations of either leads to NF-&kappa;B overactivity. Here, we developed and validated a gene expression classifier separating HPV+ HNSCCs based on NF-&kappa;B activity. As expected, the novel classifier is strongly enriched in NF-&kappa;B targets leading us to name it the NF-&kappa;B Activity Classifier (NAC). High NF-&kappa;B activity correlated with improved survival in two independent cohorts. Using NAC, tumors with high NF-&kappa;B activity but lacking defects in <em>TRAF3</em> or <em>CYLD</em> were identified; thus, while <em>TRAF3 or CYLD</em> gene defects identify the majority of tumors with NF-&kappa;B activation, unknown mechanisms leading to NF-kB activity also exist. The NAC correctly classified the functional consequences of two novel <em>CYLD</em> missense mutations. Using a reporter assay, we tested these CYLD mutations revealing that their activity to inhibit NF-kB was equivalent to the wild-type protein. Future applications of the NF-&kappa;B Activity Classifier may be to identify HPV+ HNSCC patients with better or worse survival with implications for treatment strategies.