Epstein-Barr virus and human papillomavirus infections and genotype distribution in head and neck cancers

Abstract

Objective: To investigate the prevalence, genotypes, and prognostic values of Epstein-Barr virus (EBV) and human papillomavirus (HPV) infections in Japanese patients with different types of head and neck cancer (HNC).

Methods and materials: HPV and EBV DNA, EBV genotypes and LMP-1 variants, and HPV mRNA expression were detected by PCR from fresh-frozen HNC samples. HPV genotypes were determined by direct sequencing, and EBV encoded RNA (EBER) was examined by in situ hybridization.

Results: Of the 209 HNC patients, 63 (30.1%) had HPV infection, and HPV-16 was the most common subtype (86.9%). HPV E6/E7 mRNA expression was found in 23 of 60 (38.3%) HPV DNA-positive cases detected. The site of highest prevalence of HPV was the oropharynx (45.9%). Among 146 (69.9%) HNCs in which EBV DNA was identified, 107 (73.3%) and 27 (18.5%) contained types A and B, respectively, and 124 (84.9%) showed the existence of del-LMP-1. However, only 13 (6.2%) HNCs were positive for EBER, 12 (92.3%) of which derived from the nasopharynx. Co-infection of HPV and EBER was found in only 1.0% of HNCs and 10.0% of NPCs. Kaplan-Meier survival analysis showed significantly better disease-specific and overall survival in the HPV DNA+/mRNA+ oropharyngeal squamous cell carcinoma (OPC) patients than in the other OPC patients (P = 0.027 and 0.017, respectively). Multivariate analysis showed that stage T1-3 (P = 0.002) and HPV mRNA-positive status (P = 0.061) independently predicted better disease-specific survival. No significant difference in disease-specific survival was found between the EBER-positive and -negative NPC patients (P = 0.155).

Conclusions: Our findings indicate that co-infection with HPV and EBV is rare in HNC. Oropharyngeal SCC with active HPV infection was related to a highly favorable outcome, while EBV status was not prognostic in the NPC cohort.

Figure 1. PCR results for EBV subtyping from the EBNA-3C gene (upper) and for the 30 bp deletion LMP-1 variant (middle), and sequence analysis results for wild-type LMP-1 and the 30 bp deletion LMP-1 variant (lower Upper panel: the primers generated a specific 153 bp fragment from specimens containing type A EBV (samples 1, 2, 4, 5, and positive control PC-2) and a specific 246 bp fragment for specimens containing type B EBV (samples 3, 6, 7, and PC-1).

Middle panel: a specific 316 bp band showed wild-type LMP-1 (samples 3, 4, 6, 7, and positive control PC-1) and a specific 286 bp band indicated the 30 bp deletion LMP-1 (samples 1, 2, 5, and PC-2). The products of positive controls were confirmed by direct sequencing. Lower panel: the 30 bp deletion sequence of del-LMP-1 from wild-type LMP-1 by sequence analysis.

Figure 2. EBV encoded RNA in in situ hybridization.

Micrograph A: most of the neoplasm cells were positive (nasopharyngeal carcinoma, ×100, bar = 100 µm), as indicated by the arrow at high magnification (×200). Micrograph B: although some positive lymphocytes are apparent (arrow), no neoplasm cells were positive. Micrographs C and D: Sections stained with hematoxylin and eosin (HE).

Figure 3. Kaplan-Meier curves of disease-specific survival (A) and overall survival (B) according to HPV status in all OPC patients.

Disease-specific survival and overall survival rates were significantly better in HPV DNA-positive/mRNA-positive OPC patients than in HPV DNA-positive/mRNA-negative and HPV DNA-negative/mRNA-negative OPC patients. However, no significant differences in disease-specific survival or overall survival were found between HPV DNA-positive and HPV DNA-negative OPC patients.

Figure 4. Kaplan-Meier curves of recurrence-free survival and disease-specific survival rates according to HPV status in OPC patients receiving curative treatment.

After curative treatment, HPV DNA-positive OPC patients showed significantly better recurrence-free survival and disease-specific survival rates compared to HPV DNA-negative OPC patients. Similar results were found in HPV mRNA-positive OPC patients.

Figure 5. Kaplan-Meier curves of disease-specific survival according to EBER status in NPC patients.

No significant differences in the rates of disease-specific survival were found between EBER-positive (tumor EBV infection) and EBER-negative NPC patients.