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

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).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.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).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

Co-infection of HPV and EBV in head and neck cancers, according to PCR for HPV DNA and ISH for <i>EBER</i>.

<p>HPV, human papillomavirus; EBV, Epstein-Barr virus; PCR, polymerase chain reaction; ISH, in situ hybridization; HNC, head and neck cancer; NPC, nasopharyngeal cancer; <i>EBER</i>, Epstein-Barr virus encoded RNA.</p><p>Co-infection of HPV and EBV in head and neck cancers, according to PCR for HPV DNA and ISH for <i>EBER</i>.</p

Descriptive statistics of the main variables concerning patients and neoplasm parameters.

1<p>Brinkman index: daily cigarettes × years.</p>2<p>Light drinker ≤50 g alcohol per day; heavy drinker>50 g alcohol per day.</p>3<p>HPV, human papillomavirus.</p>4<p><i>EBNA</i>, Epstein-Barr nuclear antigen.</p><p>Descriptive statistics of the main variables concerning patients and neoplasm parameters.</p

Detection results of HPV DNA, mRNA, <i>EBNA</i>-3C, the 30 bp deletion LMP-1 variant, and <i>EBER</i> in different types of head and neck cancer.

<p>HPV, human papillomavirus; <i>EBNA</i>, Epstein-Barr nuclear antigen; <i>LMP-1</i>, latent membrane protein-1; <i>EBER</i>, Epstein-Barr virus encoded RNA.</p><p>* Positive rate of the variables in different subsites of head and neck cancer.</p>#<p>Positive rate of HPV mRNA in HPV DNA-positive patients; 3 cases with HPV DNA-positive derived from oropharynx, hypopharynx, and larynx were not sufficient to be examined for RNA extraction and <i>E6/E7</i> mRNA expression.</p><p>Detection results of HPV DNA, mRNA, <i>EBNA</i>-3C, the 30 bp deletion LMP-1 variant, and <i>EBER</i> in different types of head and neck cancer.</p

EBV encoded RNA in in situ hybridization.

<p>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).</p

Kaplan-Meier curves of disease-specific survival according to <i>EBER</i> status in NPC patients.

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

PCR results for EBV subtyping from the <i>EBNA-3C</i> gene (upper) and for the 30 bp deletion <i>LMP-1</i> variant (middle), and sequence analysis results for wild-type <i>LMP-1</i> and the 30 bp deletion <i>LMP-1</i> 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).

<p>Middle panel: a specific 316 bp band showed wild-type <i>LMP-1</i> (samples 3, 4, 6, 7, and positive control PC-1) and a specific 286 bp band indicated the 30 bp deletion <i>LMP-1</i> (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 <i>del-LMP-1</i> from wild-type LMP-1 by sequence analysis.</p