For a study, researchers sought to understand that although some cases were caused by human papillomavirus (HPV) infection, HPV-negative HNSCC was more common and had a worse prognosis. Surgery, chemoradiation, targeted therapy, or immune checkpoint inhibition may treat advanced HPV-negative HNSCC (ICI). Predictive biomarkers were desperately needed for these treatments. Defects in DNA repair capacity and loss of cell-cycle checkpoints make tumors more susceptible to cytotoxic therapies. They can contribute to phenotypes like elevated tumor mutation burden (TMB) associated with ICI response. In HPV-negative HNSCC, mutations in the tumor suppressors and checkpoint mediators TP53 and CDKN2A were common. Investigators analyzed genomic data from 1,669 HPV-negative HNSCC tumors with multiple criteria proposed for assessing the damaging effect of TP53 mutations to gain insight into the interaction of TP53 and CDKN2A mutations with TMB in HNSCC. Data analysis revealed the TP53 and CDKN2A mutation profiles in specific anatomic subsites and suggested that specific categories of TP53 mutations were more likely to be associated with CDKN2A mutations or high TMB depending on the tumor subsite. Surprisingly, the pattern of hotspot mutations in TP53 differed depending on whether or not a cooccurring CDKN2A mutation was present. These findings highlighted the importance of tumor subsite in evaluating mutational profiles in HNSCC, and they linked TP53 and CDKN2A defects to elevated TMB levels in some tumor subgroups.