Background: TNM-8 staging separates oropharyngeal squamous cell carcinomas (OPSCC) into human papillomavirus (HPV)-mediated and-unrelated OPSCC based on p16INK4a overexpression (p16þ), as surrogate marker for HPV. However, OPSCC is histologically and clinically heterogenous including tonsillar and base of tongue squamous cell carcinomas (TSCC and BOTSCC respectively), and carcinomas of soft palate and walls (otherOPSCC). The significance of HPV is established in TSCC/BOTSCC, while its role in otherOPSCC is unclear, which is not considered in TNM-8. Here, p16þ was therefore evaluated in relation to overall survival (OS) and tumor stage per OPSCC subsite. Patients and methods: All 932 patients, treated with curative intent in Stockholm 2000e2016 with OPSCC, previously analyzed for p16 expression, were included. Clinical data, including stage and OS, was collected retrospectively. Results: Patients with p16þ otherOPSCC had significantly poorer OS compared to patients with p16þ TSCC/BOTSCC (p Z 0.005) and their survival was similar to that of patients with p16-otherOPSCC/TSCC/BOTSCC. Moreover, patients with TNM-8 stage I-II and p16þ otherOPSCC had a significant poorer OS compared to patients with p16þ TSCC/BOTSCC

Human papillomavirus (HPV) is a major etiological factor for tonsillar and the base of tongue cancer (TSCC/BOTSCC). HPV-positive and HPV-negative TSCC/BOTSCC present major differences in mutations, mRNA expression and clinical outcome. Earlier protein studies on TSCC/BOTSCC have mainly analyzed individual proteins. Here, the aim was to compare a larger set of cancer and immune related proteins in HPV-positive and HPV-negative TSCC/BOTSCC in relation to normal tissue, presence of HPV, and clinical outcome. Fresh frozen tissue from 42 HPV-positive and 17 HPV-negative TSCC/BOTSCC, and corresponding normal samples, were analyzed for expression of 167 proteins using two Olink multiplex immunoassays. Major differences in protein expression between TSCC/BOTSCC and normal tissue were identified, especially in chemo- and cytokines. Moreover, 34 proteins, mainly immunoregulatory proteins and chemokines, were differently expressed in HPV-positive vs HPV-negative TSCC/BOTSCC. Several proteins were potentially related to clinical outcome for HPV-positive or HPV-negative tumors. For HPV-positive tumors, these were mostly related to angiogenesis and hypoxia. Correlation with clinical outcome of one of these, VEGFA, was validated by immunohistochemistry. Differences in immune related proteins between HPV-positive and HPV-negative TSCC/BOTSCC reflect the stronger activity of the immune defense in the former. Angiogenesis related proteins might serve as potential targets for therapy in HPV-positive TSCC/BOTSCC.

Anticancer immunotherapies have revolutionized cancer management, yet the effect of systemic anti‐programmed cell death protein 1 (PD‐1) treatment is predominantly studied in tumor‐infiltrating lymphocytes (TILs). Its impact on PD‐1 expressing cells in tumor‐draining lymph nodes (TDLNs) is not well understood and yet to be explored. Thus, further research aiming for better understanding of the PD‐1 pathway not only in tumor tissue but also in TDLNs is warranted. In this study, we investigated the expression of PD‐1, CD69, and HLA‐DR on CD4+ and CD8+ T cells by flow cytometry analysis of peripheral blood mononuclear cells (PBMCs), TDLNs, and tumor samples from patients with oral squamous cell carcinoma (OSCC). Our data showed that both helper and cytotoxic T lymphocytes in OSCC tissue were highly activated and expressed high level of PD‐1 (over 70% positivity). Lymphocytes in TDLNs and peripheral blood expressed significantly lower levels of PD‐1 and other activation markers compared to TILs. Moreover, we demonstrated that a significant fraction of PD‐1 negative TILs expressed high levels of human leukocyte antigen – DR isotype and CD69. In contrast, PD‐1 negative cells in TDLNs and PBMCs scarcely expressed the aforementioned activation markers. Furthermore, we proved that patients with a high percentage of CD3+ PD‐1+ cells in tumor‐draining lymph nodes had significantly lower disease‐free and overall survival rates (log‐rank test P = .0272 and P = .0276, respectively). Taken together, we proved that flow cytometry of lymph nodes in OSCC is feasible and may be used to investigate whether PD‐1 levels in TDLNs correspond with survival and potentially with response to anti‐PD‐1 therapy. Such knowledge may ultimately help guide anti‐PD‐1 treatment.