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Background Curative treatment of the most aggressive solid tumors utilizes radiation therapy, either as a monotherapy or combined with surgery and/or chemotherapy. Previously, we linked the expression of PD-L1 with clinical radioresistance; however, the relationship between outcome following radiation and immune function is unclear. Methods We used two well-annotated cohorts to examine clinical outcomes. The discovery cohort included 94 patients diagnosed with Head and Neck squamous cell carcinoma (HNSCC) who were treated uniformly with surgery and adjuvant radiation. The validation cohort consisted of 97 patients with similar treatment. Immune infiltrates in tumors were derived from RNAseq gene expression in tumor tissues using xCell. The association between each immune cell type and clinical outcomes was tested using Cox proportional hazards models. Immune cell types significantly associated with overall survival (OS), distant metastasis (DM), or locoregional recurrence (LRR) in the discovery cohort were examined in the independent validation cohort. Genes that carry nonsynonymous somatic mutations or have mRNA expression that is associated with the abundance of CD4 + T helper 2 (Th2) cell infiltrate in tumors were also identified in the full TCGA HNSCC cohort as well as non-overlapping cohorts from the International Cancer Genome Consortium (ICGC) and the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC). Results In the discovery cohort, we identified intratumoral cell types significantly associated with OS (monocytes, CD4 + memory T cells & Th2 cells), DM (CD4 + memory T cells, sebocytes, and epithelial cells) or LRR (common lymphoid progenitor (CLP) cells, CD4 + memory T cells, Th2 cells, and immature dendritic cells (iDCs). However, only a positive association between high levels of Th2 cells and LRR was identified in the validation cohort. We also identified CREBBP/EP300 and CASP8 among the genes that carry somatic mutations significantly associated with the presence of Th2 cells. Additionally, pathway analysis of genes correlated with Th2 cells revealed potential repression of the antitumor immune response and activation of BRCA1-associated DNA damage repair in multiple cohorts. Conclusions Th2 infiltrate is enriched in HPV-negative HNSCC tumors and is associated with LRR in two independent cohorts of patients following surgery and radiation. Th2 infiltrate is associated with mutations in CASP8 and CREBBP/EP300 and pathways previously shown to impact the response to radiation. Further investigation is warranted to investigate the mechanisms underlying the role of Th2 cells in mediating radioresistance in HPV-negative HNSCC.

Background Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) leads to the death of over 360,000 patients annually worldwide. Curative therapy for this disease commonly consists of surgery followed by radiation therapy. Previously, we linked the expression of PD-L1 with clinical radioresistance in HNSCC; however, the relationship between outcome following radiation and immune function is unclear. Methods We used two well-annotated cohorts to examine clinical outcomes. The discovery cohort included 94 patients diagnosed with HPV-negative HNSCC who were treated uniformly with surgery and adjuvant radiation. The validation cohort consisted of 97 patients with similar treatment. Immune infiltrates in tumors were derived from RNAseq gene expression in tumor tissues using xCell. The association between each immune cell type and clinical outcomes was tested using Cox proportional hazards models. Immune cell types significantly associated with overall survival (OS), distant metastasis (DM), or locoregional recurrence (LRR) in the discovery cohort were examined in the independent validation cohort. Genes that carry nonsynonymous somatic mutations or have mRNA expression that is associated with the abundance of CD4 + T helper 2 (Th2) cell infiltrate in tumors were also identified in the full TCGA HPV-negative HNSCC cohort as well as nonoverlapping cohorts from the International Cancer Genome Consortium (ICGC) and the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC). Results In the discovery cohort, we identified intratumoral cell types significantly associated with OS (monocytes, CD4 + memory T cells & Th2 cells), DM (CD4 + memory T cells, sebocytes and epithelial cells) or LRR (common lymphoid progenitor (CLP) cells, CD4 + memory T cells, Th2 cells, and immature dendritic cells (iDCs). However, only a positive association between high levels of Th2 cells and LRR was identified in the validation cohort. We also identified CREBBP/EP300 and CASP8 among the genes that carry somatic mutations significantly associated with the presence of Th2 cells. Additionally, pathway analysis of genes correlated with Th2 cells revealed potential repression of the antitumor immune response and activation of BRCA1-associated DNA damage repair in multiple cohorts. Conclusions Th2 infiltrate is enriched in HPV-negative HNSCC tumors and is associated with LRR in two independent cohorts of patients following surgery and radiation. Th2 infiltrate is associated with mutations in CASP8 and CREBBP/EP300 and pathways previously shown to impact the response to radiation. Further investigation is warranted to investigate the mechanisms underlying the role of Th2 cells in mediating radioresistance in HPV-negative HNSCC.

Squamous cell carcinoma driven by human papillomavirus (HPV) is more sensitive to DNA-damaging therapies, such as radiation, than its HPV-negative counterpart. Here we show that p16, the clinically utilized surrogate for HPV positivity, renders cells more sensitive to radiation via a ubiquitin-dependent signaling pathway, linking high levels of this protein to increased activity of the transcription factor SP1, increased HUWE1 transcription and degradation of ubiquitin-specific protease 7 (USP7). Activation of this pathway in HPV-positive disease leads to an absence of TRIP12, decreased DNA damage repair, improved response to radiation and better clinical outcomes. Conversely, repression of this pathway in HPV-negative disease is druggable via USP7 inhibitors under clinical development, resulting in potentiation of radiation response. Our findings may lead to improved outcomes for patients with HPV-negative radioresistant tumors, while allowing decreased intensity of therapy for patients with HPV-positive tumors.

Human papillomavirus (HPV) drives the development of squamous cell carcinoma at several sites, including the oropharynx. Generally, the presence of HPV renders a tumor more sensitive to DNA-damaging therapies such as radiation; however, the mechanism behind this phenomenon is elusive. Previous studies have shown that p16, the clinically utilized surrogate for HPV tumor positivity, can render cells more sensitive to radiation. In the current manuscript, using a combination of immunoprecipitation mass spectrometry (IP/MS), in vivo and in vitro modulation and clinical tumor profiling, we identify a novel ubiquitin-dependent signaling pathway linking p16 to increased activity of the transcription factor SP1 leading to increased HUWE1 transcription and degradation of ubiquitin-specific protease 7 (USP7). This pathway is activated in HPV-positive tumor cells, leading to an absence of TRIP12, decreased DNA damage repair and increased mitotic death following radiation. As USP7 inhibitors are currently in clinical trials, this pathway provides a novel means by which radioresistant tumors may be targeted to increase response and improve outcome.