A comprehensive evaluation of human papillomavirus positive status and p16INK4a overexpression as a prognostic biomarker in head and neck squamous cell carcinoma

Abstract

Head and neck squamous cell carcinoma (HNSCC) patients with human papillomavirus (HPV) infection have better prognosis than those without HPV infection. Although p16(INK4a) expression is used as a surrogate marker for HPV infection, there is controversy as to whether p16(INK4a) reliably indicates HPV infection. Here, to evaluate the accuracy of p16(INK4a) expression for determining HPV infection and the prognostic value of HPV infection and p16(INK4a) expression for HNSCC survival, especially oropharyngeal squamous cell carcinoma (OPSCC) survival, 150 fresh-frozen HNSCC samples were analyzed for HPV DNA, E6/E7 mRNA and p16(INK4a) expression by polymerase chain reaction and immunohistochemistry. p16(INK4a) expression was scored from 0 to 4 according to the percentage of p16(INK4a)-positive cells, with overexpression defined as >40% positive cells. Of the 150 tumor samples tested, 10 tumors were nasopharyngeal, 53 oropharyngeal, 39 hypopharyngeal, 24 laryngeal and 24 were located in the oral cavity. HPV DNA was detected in 47 (31.3%) samples, but only 21 also exhibited HPV mRNA expression. Inter-rater agreement was low between p16(INK4a) expression and HPV DNA presence and between p16(INK4a) expression and HPV mRNA expression, but was good between the combination of HPV DNA status and p16(INK4a) overexpression and HPV mRNA expression. Three-year recurrence-free survival was significantly higher for OPSCC patients who were HPV DNA-positive than for OPSCC patients who were HPV DNA-negative (P=0.008) and for OPSCC patients overexpressing p16(INK4a) than for without overexpressing p16(INK4a) (P=0.034). Multivariate analysis revealed that T1-3 stage and the combination of HPV DNA positivity and p16(INK4a) overexpression predicted significantly better recurrence-free survival. This combination is a more accurate marker for active HPV infection in HNSCC than HPV DNA status or general p16(INK4a)-positive status alone and offers a useful and reliable method for detecting and determining the prognosis of HPV-related HNSCC.

Figure 1.

Immunohistochemical evaluation of p16^INK4a expression and scoring in HNSCC. p16^INK4a immunoreactivity was scored as 0 (no staining), 1 (1–10% of the tumor cells positive), 2 (11–40% of the tumor cells positive), 3 (40–70% of the tumor cells positive) or 4 (>70 of the tumor cells positive). Each micrograph shows the typical p16^INK4a immunoreactivity pattern corresponding to each score (A–F, ×100; bar, 100 μm). Sections were also stained with hematoxylin and eosin (H&E).

Figure 2.

Kaplan-Meier curves of recurrence-free survival in OPSCC patients according to (A) HPV DNA, (B) HPV mRNA and (C) p16^INK4a overexpression. Recurrence-free survival was significantly better in HPV DNA-positive OPSCC patients than HPV DNA-negative OPSCC patients and in OPSCC patients with p16^INK4a overexpression than in OPSCC patients without p16^INK4a overexpression.

Figure 3.

Kaplan-Meier curves of recurrence-free survival in OPSCC patients according to the combination of HPV DNA status and p16^INK4a overexpression. OPSCC patients with both HPV DNA-positive status and p16^INK4a overexpression showed significantly better recurrence-free survival compared to other patients (HPV DNA-positive without p16^INK4a overexpression, HPV DNA-negative with p16^INK4a overexpression and HPV DNA-negative without p16^INK4a overexpression (P=0.034).