John Waldron

The incidence of human papillomavirus‐positive (HPV+) oropharyngeal cancer (OPC) has increased rapidly, and HPV early antigen serology has been proposed as a scalable and cost‐effective early detection test. HPV seropositivity can precede clinical presentation of OPC by several years, so additional surveillance procedures may be necessary to optimize early cancer detection. The potential for HPV circulating tumor DNA (ctDNA) to confirm a diagnosis of OPC in seropositive individuals is poorly understood. Here, we assess the relationship between HPV serology and HPV ctDNA with disease burden in a large cohort of HPV+ OPC. We analyzed baseline blood samples from 262 patients using a multiplex serology assay and an HPV‐targeted deep sequencing assay (HPV‐seq), and HPV‐seq results were validated by an orthogonal droplet digital PCR assay. Both assays identified HPV16 as the most prevalent genotype (84%), with serology results indicating a total of 4 HPV genotypes and HPV ctDNA results indicating a total of 6 HPV genotypes. HPV ctDNA results demonstrated higher sensitivity and lower cross‐reactivity between HPV types compared with HPV serology results. Furthermore, HPV ctDNA but not HPV16 E6 antibody levels were positively associated with disease burden as determined by N‐category, overall stage, and by tumor volume (ctDNA vs. volume, r = 0.48 p = 6.4e‐12; E6 vs. volume, r = −0.079 p = 0.26). Overall, this is the largest cohort to compare HPV serology and HPV ctDNA results in OPC, and these findings highlight the potential for ctDNA to augment future strategies for blood‐based early detection of HPV+ OPC.

Table S1: Patient Radiotherapy Information and Multivariate Analysis. Figure S1: Workflow of evaluation of T cell receptor (TCR) rearrangement and ESTIMATE immune score from RNA-sequencing data, and the expression of tumor-infiltrating lymphocytes (TILs) in NPC patients. Figure S2: The correlation of ESTIMATE immune score with rearranged TCR reads and CD8+ TILs. Figure S3: The correlation of CIBERSORT and TIMER 2.0 with rearranged TCR reads and CD8+ TILs. Figure S4: The distribution of TILs in NPC samples. Figure S5: Clinical impact of CD8+ TILs in the combined cohort of NPC patients.

Table S1: Patient Radiotherapy Information and Multivariate Analysis.

Purpose:Tumor-infiltrating lymphocytes (TIL) are immune cell populations found within tumors, critical in the antigen-specific host immune response. In this study, we aimed to elucidate the prognostic significance of CD3+, CD4+, and CD8+ TILs in nasopharyngeal cancer (NPC).Experimental Design:Immune cell infiltration was quantified in NPC samples (n = 50) using RNA-sequencing (RNA-seq) data based on rearranged T-cell receptor (TCR) reads and the Estimation of Stromal and Immune cells in malignant tumors using expression data (ESTIMATE) immune score tool. The differential abundances of TIL subset populations were also characterized through IHC staining of formalin-fixed, paraffin-embedded samples from a training cohort (n = 35), which was a subset of the RNA-seq cohort (n = 50).Results:In the RNA-seq cohort, patients with higher rearranged TCR reads experienced superior 5- and 10-year overall survival (OS; P < 0.001), and disease-free survival (DFS; P < 0.001). Similarly, patients with higher ESTIMATE immune scores experienced superior 5- and 10-year OS (P = 0.024) and DFS (P = 0.007). In the training cohort, high abundances of CD8+ TILs were significantly associated with improved 5- and 10-year OS (P = 0.003) and DFS (P = 0.005). These findings were corroborated in an independent validation cohort (n = 84), and combined analysis of the training and validation cohorts [n = 119 (35+84)], which further demonstrated improved 5- and 10-year survival in terms of locoregional control (P < 0.001) and distant metastasis (P = 0.03).Conclusions:Taken together, our study highlights the prognostic value of CD8+ TILs in NPC, and the potential of future investigations into cellular-based immunotherapies employing CD8+ lymphocytes.

Table S1: Patient Radiotherapy Information and Multivariate Analysis. Figure S1: Workflow of evaluation of T cell receptor (TCR) rearrangement and ESTIMATE immune score from RNA-sequencing data, and the expression of tumor-infiltrating lymphocytes (TILs) in NPC patients. Figure S2: The correlation of ESTIMATE immune score with rearranged TCR reads and CD8+ TILs. Figure S3: The correlation of CIBERSORT and TIMER 2.0 with rearranged TCR reads and CD8+ TILs. Figure S4: The distribution of TILs in NPC samples. Figure S5: Clinical impact of CD8+ TILs in the combined cohort of NPC patients.

Table S1: Patient Radiotherapy Information and Multivariate Analysis.

Table S1: Patient Radiotherapy Information and Multivariate Analysis.

Table S1: Patient Radiotherapy Information and Multivariate Analysis. Figure S1: Workflow of evaluation of T cell receptor (TCR) rearrangement and ESTIMATE immune score from RNA-sequencing data, and the expression of tumor-infiltrating lymphocytes (TILs) in NPC patients. Figure S2: The correlation of ESTIMATE immune score with rearranged TCR reads and CD8+ TILs. Figure S3: The correlation of CIBERSORT and TIMER 2.0 with rearranged TCR reads and CD8+ TILs. Figure S4: The distribution of TILs in NPC samples. Figure S5: Clinical impact of CD8+ TILs in the combined cohort of NPC patients.

Purpose:Tumor-infiltrating lymphocytes (TIL) are immune cell populations found within tumors, critical in the antigen-specific host immune response. In this study, we aimed to elucidate the prognostic significance of CD3+, CD4+, and CD8+ TILs in nasopharyngeal cancer (NPC).Experimental Design:Immune cell infiltration was quantified in NPC samples (n = 50) using RNA-sequencing (RNA-seq) data based on rearranged T-cell receptor (TCR) reads and the Estimation of Stromal and Immune cells in malignant tumors using expression data (ESTIMATE) immune score tool. The differential abundances of TIL subset populations were also characterized through IHC staining of formalin-fixed, paraffin-embedded samples from a training cohort (n = 35), which was a subset of the RNA-seq cohort (n = 50).Results:In the RNA-seq cohort, patients with higher rearranged TCR reads experienced superior 5- and 10-year overall survival (OS; P < 0.001), and disease-free survival (DFS; P < 0.001). Similarly, patients with higher ESTIMATE immune scores experienced superior 5- and 10-year OS (P = 0.024) and DFS (P = 0.007). In the training cohort, high abundances of CD8+ TILs were significantly associated with improved 5- and 10-year OS (P = 0.003) and DFS (P = 0.005). These findings were corroborated in an independent validation cohort (n = 84), and combined analysis of the training and validation cohorts [n = 119 (35+84)], which further demonstrated improved 5- and 10-year survival in terms of locoregional control (P < 0.001) and distant metastasis (P = 0.03).Conclusions:Taken together, our study highlights the prognostic value of CD8+ TILs in NPC, and the potential of future investigations into cellular-based immunotherapies employing CD8+ lymphocytes.