Small noncoding microRNAs (miRNAs) have been shown to be abnormally expressed in every tumor type examined. The importance of miRNAs as potential cancer prognostic indicators is underscored by their involvement in the regulation of basic cellular processes such as cell proliferation, differentiation, and apoptosis. In this study, miRNA expression profiles of head and neck squamous cell carcinoma (HNSCC) tumor and adjacent normal tissue were examined by microarray analysis and validated by quantitative TaqMan real-time polymerase chain reaction. Using TaqMan real-time polymerase chain reaction we measured the quantitative associations between a subset of miRNAs identified on microarrays in primary tumors at diagnosis and cancer survival in a cohort of 104 HNSCC patients undergoing treatment with curative intent. The majority of miRNAs exhibiting altered expression in primary human HNSCC tumors (including miR-1, miR-133a, miR-205, and let7d) show lower expression levels relative to normal adjacent tissue. In contrast, hsa-miR-21 is frequently overexpressed in human HNSCC tumors. Using univariate and multivariable statistical models we show that low levels of hsa-miR205 are significantly associated with loco-regional recurrence independent of disease severity at diagnosis and treatment. In addition , combined low levels of hsa-miR-205 and hsa-let-7d expression in HNSCC tumors are significantly associated with poor head and neck cancer survival Our results show that miRNA expression levels can be used as prognostic markers of head and neck cancer.

Detection of human papillomavirus in head and neck cancer has therapeutic implications. In-situ hybridization and immuno-histochemistry for p16 are used by surgical pathologists. We compared the sensitivity and specificity of three popular commercial tests for human papillomavirus detection in head and neck squamous cell carcinomas to a “gold standard” human papillomavirus PCR assay. One hundred-and-ten prospectively collected, formalin fixed tumor specimens were compiled onto tissue microarrays and tested for human papillomavirus DNA by in-situ hybridization with two probe sets: a biotinylated probe for high-risk human papillomavirus types 16/18 (Dako, CA), and a probe cocktail for 16/18 plus 10 additional high-risk types (Ventana, AZ). P16INK4 expression was also assessed using a Pharmingen immuno-histochemistry antibody (BD Biosciences, CA). Tissue microarrays were stained and scored at expert laboratories. Human papillomavirus DNA was detected by MY09/11-PCR using Gold AmpliTaq and dot-blot hybridization on matched fresh frozen specimens in a research laboratory. Human papillomavirus 16 E6 and E7-RNA expression was also measured using RT-PCR. Test performance was assessed by receiver operating characteristic analysis. High-risk human papillomavirus DNA types 16, 18 and 35 were detected by MY-PCR in 28% of tumors, with the majority (97%) testing positive for type 16. Compared to MY-PCR, the sensitivity and specificity for high-risk human papillomavirus DNA detection with Dako in-situ hybridization was 21% (95%CI:7–42) and 100% (95%CI:93–100), respectively. Corresponding test results by Ventana in-situ hybridization were 59% (95%CI:39–78) and 58% (95%CI:45–71), respectively. P16 immuno-histochemistry performed better overall than Dako (p=0.042) and Ventana (p=0.055), with a sensitivity of 52% (95%CI:32–71) and specificity of 93% (95%CI:84–98). Compared to a gold standard human papillomavirus PCR assays, HPV detection by in-situ hybridization was less accurate for head and neck squamous cell carcinoma on tissue microarrays than p16 immuno-histochemistry. Further testing is warranted before these assays should be recommended for clinical human papillomavirus detection.

Small, noncoding microRNAs (miRNAs) have been shown to be abnormally expressed in every tumor type examined. We used comparisons of global miRNA expression profiles of head and neck squamous cell carcinoma (HNSCC) samples and adjacent normal tissue to rank those miRNAs that were most significantly altered in our patient population. Rank Consistency Score analysis revealed miR-375 to have the most significantly lowered miRNA levels in tumors relative to matched adjacent nonmalignant tissue from the same patient among 736 miRNAs that were evaluated. This result has been previously observed by other groups; however, we extend this finding with the unique observation that low miR-375 expression levels correlate significantly with cancer survival and distant metastasis. In a study of 123 primary HNSCC patients using multivariable Cox proportional hazard ratios (HR) and 95% confidence intervals (CI), both death from disease (HR: 12.8, 95% CI: 3 to 49) and incidence of distant metastasis (HR: 8.7, 95% CI: 2 to 31) correlated with lower expression levels of miR-375 regardless of the site or stage of the tumor. In addition, we found that oral cavity tumor cell lines (eg, UMSCC1 and UMSCC47) overexpressing miR-375 were significantly less invasive in vitro than their matched empty vector controls. We conclude that miR-375 represents a potential prognostic marker of poor outcome and metastasis in HNSCC and that it may function by suppressing the tumor's invasive properties.

While its prognostic significance remains unclear, p16INK4a protein expression is increasingly being used as a surrogate marker for oncogenic human papillomavirus (HPV) infection in head and neck squamous cell carcinomas (HNSCC). To evaluate the prognostic utility of p16 expression in HNSCC, we prospectively collected 163 primary tumor specimens from histologically confirmed HNSCC patients who were followed for up to 9.4 years. Formalin fixed tumor specimens were tested for p16 protein expression by immunohistochemistry. HPV type-16 DNA and RNA was detected by MY09/11-PCR and E6/E7 RT-PCR on matched frozen tissue, respectively. P16 protein expression was detected more often in oropharyngeal tumors (53%) as compared with laryngeal (24%), hypopharyngeal (8%), or oral cavity tumors (4%; P<0.0001). With respect to prognosis, p16-positive oropharyngeal tumors exhibited significantly better overall survival than p16-negative tumors (log-rank test p=0.04), whereas no survival benefit was observed for non-oropharyngeal tumors. However, when both p16 and HPV DNA test results were considered, concordantly positive non-oropharyngeal tumors had significantly better disease-specific survival than concordantly negative non-oropharyngeal tumors after controlling for sex, nodal stage, tumor size, tumor subsite, primary tumor site number, smoking, and drinking (adjusted hazard ratio [HR]=0.04, 0.01–0.54). Compared with concordantly negative non-oropharyngeal HNSCC, p16(+)/HPV16(-) non-oropharyngeal HNSCC (n=13, 7%) demonstrated no significant improvement in disease-specific survival when HPV16 was detected by RNA (adjusted HR=0.83, 0.22–3.17). Our findings show that p16 immunohistochemistry alone has potential as a prognostic test for oropharyngeal cancer survival, but combined p16/HPV testing is necessary to identify HPV-associated non-oropharyngeal HNSCC with better prognosis.