Frequency and prognostic significance of p16(INK4A) protein overexpression and transcriptionally active human papillomavirus infection in laryngeal squamous cell carcinoma

Abstract

Background: Human papillomavirus (HPV) infection is a powerful prognostic biomarker in a subset of head and neck squamous cell carcinomas, specifically oropharyngeal cancers. However, the role of HPV in non-oropharyngeal sites, such as the larynx, remains unconfirmed.

Methods: We evaluated a cohort of 324 laryngeal squamous cell carcinoma (LSCC) patients for the expression of p16(INK4A) (p16) protein by immunohistochemistry (IHC) and for high-risk HPV E6 and E7 mRNA transcripts by RNA in situ hybridisation (ISH). p16 expression and HPV status were correlated with clinicopathological features and outcomes.

Results: Of 307 patients assessable for p16 IHC, 20 (6.5%) were p16 positive. Females and node-positive patients were more likely to be p16 positive (P<0.05). There were no other significant clinical or demographic differences between p16-positive and -negative cases. There was no difference in overall survival (OS) between p16-positive and -negative patients with 2-year survival of 79% in each group (HR=0.83, 95% CI 0.36-1.89, P=0.65). There was no statistically significant difference in failure-free survival (FFS) with 2-year FFS of 79% and 66% for p16-positive and -negative patients, respectively (HR=0.60, 95% CI 0.26-1.36, P=0.22). Only seven cases were found to be HPV RNA ISH positive, all of which were p16 IHC positive. There was no statistically significant difference in OS between patients with HPV RNA ISH-positive tumours compared with -negative tumours with 2-year survival of 86% and 71%, respectively (HR=0.76, 95% CI 0.23-2.5, P=0.65). The 2-year FFS was 86% and 59%, respectively (HR=0.62, 95% CI 0.19-2.03, P=0.43).

Conclusions: p16 overexpression is infrequent in LSCC and the proportion of cases with high-risk HPV transcripts is even lower. There are no statistically significant correlations between p16 IHC or HPV RNA ISH status and OS or disease outcomes.

Figure 1

Examples of p16 immunohistochemical staining. (A) A p16-negative case with no detectable p16 expression. (B) A p16-positive case with strong and diffuse p16 expression in tumour cells. Images taken with a × 20 objective lens.

Figure 2

Examples of HPV RNA ISH. (A) A p16/HPV-positive case showing strong p16 overexpression (1) and brown punctate cytoplasmic signals for HPV RNA ISH (2). The RNA ISH-positive control (3) confirms the presence of intact RNA. (B) a p16-positive/HPV-negative case showing strong p16 overexpression (4) but no signals for HPV RNA ISH (5). The RNA ISH-positive control (6) confirms the presence of intact RNA. (C) a p16-negative/HPV-negative case showing no p16 expression (7) and no HPV RNA ISH signals (8). The RNA ISH-positive control (9) confirms the presence of intact RNA. Images 1, 4 and 7= × 20 objective lens, images 2, 3, 5, 6, 8 and 9= × 60 objective lens.

Figure 3

Kaplan–Meier plots for overall survival (OS) and failure-free survival (FFS). (A) OS by p16 protein expression determined by immunohistochemistry; (B) OS for HPV status determined by RNA in situ hybridisation; (C) FFS for p16; (D) FFS for HPV.