BackgroundThe purpose of this study was to determine prospectively both human papillomavirus (HPV) load and physical status in different types of head and neck squamous cell carcinoma (HNSCC).MethodsHPV DNA, E6/E7 mRNA expression, viral load, and physical status of 184 patients with HNSCC were examined simultaneously by polymerase chain reaction (PCR)‐based methods.ResultsThe HPV genome was detected in 54 HNSCC samples (29.3%), particularly in tonsillar carcinomas (69.6%). Compared with nonoropharyngeal HNSCC, oropharyngeal carcinoma harbored a relatively higher viral load, especially in tonsillar carcinoma. Although integrated or mixed status was observed in 75.6% of HPV‐16–positive samples, E6/E7 mRNA transcripts were detected in only 27.5% of HPV DNA‐positive cases. High HPV‐16 load correlated significantly with E6/E7 mRNA expression.ConclusionE6/E7 mRNA expression in patients with HNSCC with low viral load remains low even in cases of integration to the host genome. Tonsillar carcinomas were significantly associated with HPV among various types of HNSCC. © 2012 Wiley Periodicals, Inc. Head Neck, 2012

Head and neck squamous cell carcinoma (HNSCC) patients with human papillomavirus (HPV) infection have better prognosis than those without HPV infection. Although p16INK4a expression is used as a surrogate marker for HPV infection, there is controversy as to whether p16INK4a reliably indicates HPV infection. Here, to evaluate the accuracy of p16INK4a expression for determining HPV infection and the prognostic value of HPV infection and p16INK4a 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 p16INK4a expression by polymerase chain reaction and immunohistochemistry. p16INK4a expression was scored from 0 to 4 according to the percentage of p16INK4a-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 p16INK4a expression and HPV DNA presence and between p16INK4a expression and HPV mRNA expression, but was good between the combination of HPV DNA status and p16INK4a 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 over-expressing p16INK4a than for without overexpressing p16INK4a (P=0.034). Multivariate analysis revealed that T1-3 stage and the combination of HPV DNA positivity and p16INK4a 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 p16INK4a-positive status alone and offers a useful and reliable method for detecting and determining the prognosis of HPV-related HNSCC.

To clarify the synergistic influence of human papillomavirus (HPV) status and squamous cell carcinoma antigen (SCCA) mRNA expression on head and neck squamous cell carcinoma (HNSCC) prognosis, HPV DNA presence and SCCA1 and SCCA2 mRNA expression were determined by PCR and quantitative real-time RT-PCR, respectively, in 121 patients with primary HNSCC who were receiving curative treatment. HPV DNA was detected in 28.1% (34/121) of HNSCC cases, and only high-risk types were observed. Positive HPV status showed a significantly better prognosis than negative HPV status (P = 0.022). An elevated SCCA2/SCCA1 mRNA ratio was an independent predictor of disease recurrence (P = 0.004). In addition, HPV-negative patients with a high SCCA2/SCCA1 ratio (>0.27) had a significantly lower recurrence-free survival rate than HPV-negative patients with a low SCCA2/SCCA1 ratio (P < 0.011). Our findings revealed that both HPV status and the SCCA2/SCCA1 mRNA ratio are independently associated with prognosis in HNSCC. Patients with both a HPV-negative status and a high SCCA2/ SCCA1 ratio might need intensified treatment and rigorous follow up after treatment because of the high risk of recurrence. (Cancer Sci 2012; 103: 2127-2134 H ead and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer, accounting for more than 600 000 new cases annually.(1) The two most important risk factors for the development of HNSCC are heavy smoking and extensive alcohol consumption.(2) Recently, the strongest correlation between human papillomavirus (HPV) and HNSCC has been found in oropharyngeal squamous cell carcinoma (SCC), particularly tonsillar carcinoma, with HPV DNA present in up to 70% of studied patients. (3)(4)(5)(6)(7)(8) Furthermore, many studies have demonstrated that patients with HPV-positive oropharyngeal carcinoma have a better prognosis than those with HPV-negative oropharyngeal carcinoma. (3,9) Squamous cell carcinoma antigen (SCCA), originally isolated from SCC tissue of the uterine cervix, (10) is a member of the family of serine protease inhibitors that map to the serine protease inhibitor (serpin) cluster at chromosome 18q21.3. (11) Molecular studies demonstrate that SCCA is transcribed by two almost identical genes (SCCA1 and SCCA2). Using column isoelectric focusing, SCCA was shown to contain at least 14 subfractions that were divided arbitrarily into two groups: acidic fractions (pI < 6.25) corresponding to SCCA2 and neutral fractions (pI > 6.25) corresponding to SCCA1. (12) SCCA2 inhibits chymotrypsin-like proteinases cathepsin G and mast cell chymase, (13) whereas SCCA1 inhibits cysteine proteinases, such as cathepsins K, L and S. (14) The serum SCCA level usually decreases after tumor resection and increases with the recurrence of tumors arising from the uterine cervix and head and neck. (15,16) The increased SCCA2/SCCA1 mRNA ratio in a primary tumor is also associated with recurrence of uterine cervical cancer and HNSCC.(17,18) However, there is no report on the mutual interaction between HPV status a...

ObjectiveTo 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 MaterialsHPV 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.ResultsOf 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).ConclusionsOur 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.

Frequent promoter hypermethylation in association with prognosis, and growth suppression after re-expression, supports the hypothesis that GALR2 may act to suppress tumor activity. GALR2 is a potentially significant therapeutic target and prognostic factor for this cancer type.