Salivary High-Risk Human Papillomavirus (HPV) DNA as a Biomarker for HPV-Driven Head and Neck Cancers

Abstract

High-risk human papillomavirus (HR-HPV) infection is a major risk factor of head and neck cancers (HNCs). Despite the rising prevalence of HPV-driven HNC (HPV-HNC), biomarkers for detection, prognostication, and disease monitoring are lacking. To evaluate the capacity of salivary HR-HPV DNA as a biomarker of HPV-HNC, the salivary HR-HPV statuses of 491 and 10 patients with primary and recurrent HNC, respectively, were determined at diagnosis, using quantitative real-time PCR and MassARRAY. Tumor cyclin-dependent kinase inhibitor 2A (p16) expression was determined by IHC analysis. Patients with oropharyngeal cancer (OPC) (n = 215) were followed up for ≤5 years. Survival characteristics were evaluated in terms of event-free and cause-specific survival. Of the primary-HNC cohort, 43.2% were positive for salivary HR-HPV DNA, with most having OPC. Salivary HR-HPV DNA was detected in 81.4% of tumor p16-positive OPC patients at diagnosis. Prognosis in salivary HR-HPV-positive OPC patients was favorable compared with that in salivary HR-HPV-negative patients (event-free survival, hazard ratio = 0.42 [95% CI, 0.21-0.81, P = 0.010]; cause-specific survival, hazard ratio = 0.39 [95% CI, 0.18-0.86, P = 0.019]). In the recurrent-HNC cohort, salivary HR-HPV DNA was detected in 83.3% of those who previously had tumor p16-positive HNC. These findings indicate that this liquid biopsy-based, noninvasive biomarker can play an essential role in the detection and management of HPV-HNC.

Figure 1

Kaplan-Meier estimates of OPC patient survival by salivary HR-HPV and tumor p16. EFS (A, C, and E) and CSS (B, D, and F), by salivary HR-HPV (A and B), tumor p16 (C and D), and salivary HR-HPV and tumor p16 (E and F) status. Survival differences were evaluated using the log-rank test.

Supplemental Figure S1

Study flow chart. A total of 523 HNC patients were recruited for the study from Princess Alexandra Hospital and Royal Brisbane and Women's Hospital (Brisbane, QLD, Australia). After the exclusion of 22 patients due to unsatisfactory salivary DNA yield, 491 primary-HNC patients and 10 recurrent-HNC patients were considered in the study. Tumor p16 expression was evaluated as a part of the routine clinical investigations. Saliva samples were collected before the initiation of the treatment. Considering that HPV16 is the HPV type that most commonly causes HPV-HNC, all of the salivary samples were initially tested for HPV16 DNA. Samples that were salivary HPV16 DNA negative and p16 positive were tested for other HR-HPV types. The OPC cohort (n = 268) considered in the study was followed up for up to 5 years and 215 OPC patients were considered in the survival analysis after the exclusion of patients who refused the treatment, died from other causes, and/or were lost to follow-up.

Supplemental Figure S2

Kaplan-Meier estimates of OPC patient survival, by American Joint Committee on Cancer (AJCC) stage. EFS (A) and CSS (B), by AJCC stage. Survival differences were evaluated using the log-rank test.

Supplemental Figure S3

Associations between salivary HPV16 copy number and T stage (A), N stage of the tumor-node-metastasis (TNM) staging system (B), and American Joint Committee on Cancer (AJCC) stage (C).

Supplemental Figure S4

Kaplan-Meier estimates of OPC patient survival, by salivary HPV16 copy number. EFS (A) and CSS (B), by salivary HPV16 copy number. Survival differences were evaluated using the log-rank test.

Supplemental Figure S5

Salivary HPV16 physical status by OPC site. BOT, base of the tongue.

Supplemental Figure S6

Kaplan-Meier estimate of OPC patient survival, by salivary HPV16 physical status. EFS (A) and CSS (B), by salivary HPV16 physical status. Survival differences were evaluated using the log-rank test.

Supplemental Figure S7

Salivary HR-HPV status and tumor p16 status, by OPC site. Salivary HR-HPV status (A) and tumor p16 status (B), by OPC site. BOT, base of the tongue.

Supplemental Figure S8

Kaplan-Meier estimates of OPC patient survival, by age category. EFS (A) and CSS (B), by age category. Survival differences were evaluated using the log-rank test.