Overall survival according to clinical characteristics.

(DOCX)</p

Consort diagram of the study methodology.

Trial flow diagram of the study population. Among 85 patients with head and neck cancer, 81 patients with OPSCC were enrolled. Based on tissue availability, 10 patients were excluded from multiplexed-IHC analysis.</p

Immune cell proportion according to the HPV infection status.

(A and B) Immune cell distribution according to p16 immunohistochemistry (IHC) analysis in the tumor region (A) and the stroma region (B). Data are represented as dot plots (bar: mean ± SE). P value (p) was calculated using two-sided Student’s t-test. (C) Pearson-correlation matrix of immune cells in the tumor and stromal regions according HPV status. HPV status was determined by p16 IHC. According to the results of the p16 IHC analysis, the distribution of other immune cells did not differ.</p

Immune profile of oropharyngeal squamous cell carcinoma (OPSCC).

(A) Formalin-fixed paraffin-embedded (FFPE) sections of representative OPSCC tissues were analyzed by the panels of lineage-selective antibodies to identify lymphoid lineages. Representative multispectral images of OPSCC. Left panels; Tissue segmentation by InForm image analysis software (T: tumor; S: stroma). Middle panels; Cell segmentation using counterstaining with DAPI. Right panel; Multiplex image (cytokeratin: cyan, CD8: green, CD4: yellow, CD20: orange, CD68: magenta, FOXP3: red) (B) The landscape of patients with OPSCC. Upper panel: clinical characteristics, Lower panel: heatmap of immune cell proportion in 71 patients with OPSCC. Each column represents patient. (C) Immune cell distribution in the tumor and stroma. Data are represented as dot plots (bar: mean ± SE). P value (p) was calculated using two-sided Student’s t-test. (D) Pearson-correlation matrix of immune cells in the tumor and stromal regions.</p

Relapse-free survival according to immune cell proportions.

(DOCX)</p

Baseline characteristics of patients with oropharyngeal squamous cell carcinoma.

Baseline characteristics of patients with oropharyngeal squamous cell carcinoma.</p

Univariate and multivariate analyses of prognostic factors for survival.

Univariate and multivariate analyses of prognostic factors for survival.</p

Relapse-free survival according to clinical characteristics.

(DOCX)</p

Kaplan-Meier analyses of OPSCC stratified by Treg proportion in the tumor region.

(A) Relapse-free survival (RFS) and (B) overall survival (OS) in patients with OPSCC stratified by Treg cells in the tumor region. RFS and OS in relation to low (< median) or high frequencies (≥ median) of Treg cells were plotted by Kaplan-Meier survival curves. Statistical significance was determined by log-rank (Mantel-Cox) regression analysis.</p

Immune cell distribution between tonsillar and BOT OPSCC.

(A and B) Immune cell distribution according to the site of OPSCC (tonsil vs. BOT) in the tumor region (A) and the stromal region (B). Data are represented as dot plots (bar: mean ± SE). P value (p) was calculated using two-sided Student’s t-test. (C) Pearson-correlation matrix of immune cells in the tumor and stromal regions according the site of OPSCC (tonsil vs. BOT). The distribution of immune cells differed between tonsillar and BOT OPSCC.</p