Tumor-intrinsic and immune-related features associated with treatment failure in human papillomavirus-related oropharyngeal cancer

Abstract

Background: Limited understanding of the biology predisposing certain human papillomavirus-related (HPV+) oropharyngeal squamous cell carcinomas (OPSCCs) to relapse impedes therapeutic personalization. We aimed to identify molecular traits that distinguish recurrence-prone tumors.

Methods: Fifty HPV+ OPSCCs that later recurred (cases) and 50 nonrecurrent controls matched for stage, therapy, and smoking history were RNA-sequenced. Groups were compared by gene set enrichment analysis, and select differences were validated by immunohistochemistry. Features discriminating groups were scored in each tumor using gene set variation analysis, and scores were evaluated for recurrence prediction ability.

Results: Cases downregulated pathways linked to antitumor immunity (FDR-adjusted P < .05) and contained fewer tumor-infiltrating lymphocytes (P < .001), including cytotoxic T-cells (P = .005). Cases also upregulated pathways related to cell division and other aspects of tumor progression. Upregulated and downregulated pathways were respectively used to define a tumor progression score (TPS) and immune suppression score (ISS) for each tumor. Correlation between TPS and ISS (r = .603, P < .001) was potentially explained by observed upregulation of DNA repair pathways in cases, which might enhance their progression directly and by limiting cytosolic DNA-induced inflammation. Accordingly, cases contained fewer double-strand breaks based on staining for phospho-RPA32 (P = .006) and γ-H2AX (P = .005) and downregulated the cytosolic DNA sensing pathway. A combined score derived from TPS and ISS optimized recurrence prediction and stratified survival in a manner generalizable to 3 external cohorts.

Conclusions: We describe a potential link in HPV+ OPSCCs between reduced DNA damage and other tumor-intrinsic and immune-related contributors to recurrence risk, opening opportunities to detect and target this high-risk biology.

Figure 1.

Process for selecting case and matched control tumors from the total cohort. A) Kaplan–Meier analysis of OS in total cohort by clinical (left) and pathologic (right) 8th edition American Joint Committee on Cancer (AJCC) stage. B) Kaplan–Meier analysis of OS in total cohort by smoking-related risk group (low risk: ≤10 pack-years and/or AJCC 7th ed. <cN2b, intermediate risk: >10 pack-years and AJCC 7th edition ≥cN2b) and OS by smoking history alone (right). C) CONSORT diagram outlining selection of cases and controls. *A single control was duplicated as a match for 2 cases in molecular analyses. P-values were calculated for the log-rank test. Abbreviation: OS = overall survival; RT = radiation therapy; TORS = transoral robotic surgery.

Figure 2.

Evidence of a cold immune microenvironment in the HPV+ OPSCCs predisposed to recur. A) Statistically significantly downregulated pathways (FDR-adjusted P < .05) in cases compared groupwise to controls using GSEA. Significance was determined using the Benjamini and Hochberg False Discovery method. B) Percent CD3+ and CD8+ cells per tumor area by IHC. C) Immunoscore derived from average of percent CD3 and CD8 cells per tumor area (left) and ROC curve for Immunoscore separating cases from controls (right). D) Percent CD45+ cells per tumor area in cases vs controls (left) and ROC curve for separation of cases from controls by TIL content (right). Images are 40×, bar = 100µm. E) Combined positive score (CPS) based on PD-L1 IHC (left) and frequency of cases and controls in CPS categories (right) with P-value calculated for 2-way comparison using Fisher exact and 3-way comparison using Sidak’s procedure. P-values for all other 2 group comparisons were calculated using unpaired t test with Welch’s correction. P-values for AUCs were calculated from the z ratio using the normal distribution. Abbreviations: AUCs = area under the ROC curve; CI = confidence interval; FDR = false discovery rate; GSEA = gene set enrichment analysis; HPV+ = human papillomavirus; IHC = immunohistochemistry; OPSCCs = oropharyngeal squamous cell carcinomas; OR = odds ratio; OS = overall survival; TIL = tumor-infiltrating leukocyte.

Figure 3.

Pathways related to tumor progression and immune suppression are coordinately regulated. A) Statistically significantly upregulated pathways (FDR-adjusted P < .05) in cases compared groupwise to controls using GSEA. Significance was determined using the Benjamini and Hochberg False Discovery method. B) Comparison of mitochondrial mass expressed as MTCO1 to β2M ratio measured using DNA qPCR. P-value was calculated using unpaired t test with Welch’s correction (left) and ROC curve distinguishing cases from controls by mitochondrial mass (right). P-value for the left panel was calculated using unpaired t test with Welch’s correction. P-value for the right panel was calculated from the z ratio using the normal distribution. C) Flow chart illustrating the process of scoring immune suppression and tumor progression. D) Correlation plot of immune suppression score and tumor progression score across individual cases and controls. Pearson correlation coefficients were used to calculate r values, and P-values were determined by t distribution. Abbreviation: GSEA = gene set enrichment analysis.

Figure 4.

Intact DNA repair and reduced double-strand breaks in HPV+ OPSCCs predisposed to recur. A) Normalized HPV16 transcript alignments in cases and controls. E6* indicates all spliced E6 forms. Adjusted P-values were calculated using Bonferroni and Sidak’s procedure. B) DNA repair signaling pathways in the Protein Interaction Database compared between cases and controls using GSEA. P-values were calculated using the Benjamini and Hochberg False Discovery method. C) Percent γH2AX-positive nuclei (top) and percent pRPA32-positive nuclei (bottom) per tumor area by IHC. Images are 40× with bar = 100µm. P-values were calculated using unpaired t test with Welch’s correction. D) Expression of each gene in the KEGG cytosolic DNA Sensing Pathway with pro-inflammatory functions compared between case and control groups. P-value for each comparison was calculated using unpaired t test. Abbreviations: HPV+=human papillomavirus; IHC=immunohistochemistry; OPSCCs=oropharyngeal squamous cell carcinomas.

Figure 5.

Prediction of recurrence and survival stratification using combined score. A) ROC curves for predicting recurrence (cases vs controls) using TPS, ISS, and combined core. B) ROC curves for TPS, ISS, and combined score in the UNC cohort (top). Kaplan–Meier survival curves for RFS and OS stratified using a Youden index cutpoint for combined score in the UNC cohort (bottom). C) Kaplan–Meier survival curves for RFS and OS stratified using a Youden index cutpoint for combined score in the JHU cohort (top) and the TCGA cohort (bottom). Abbreviations: CI = confidence interval; ISS = immune suppression score; OR = odds ratio; OS = overall survival; RFS = recurrence-free survival; TPS = tumor progression score.