Affymetrix U133plus2 GeneChips were used to profile 59 head and neck squamous cell cancers. A hypoxia metagene was obtained by analysis of genes whose in vivo expression clustered with the expression of 10 well-known hypoxiaregulated genes (e.g., CA9, GLUT1, and VEGF). To minimize random aggregation, strongly correlated up-regulated genes appearing in >50% of clusters defined a signature comprising 99 genes, of which 27% were previously known to be hypoxia associated. The median RNA expression of the 99 genes in the signature was an independent prognostic factor for recurrence-free survival in a publicly available head and neck cancer data set, outdoing the original intrinsic classifier. In a published breast cancer series, the hypoxia signature was a significant prognostic factor for overall survival independent of clinicopathologic risk factors and a trained profile. The work highlights the validity and potential of using data from analysis of in vitro stress pathways for deriving a biological metagene/gene signature in vivo. [Cancer Res 2007;67(7):3441-9]

BACKGROUND:Hypoxia is an important mechanism of treatment resistance in head and neck squamous cell carcinoma (HNSCC). MicroRNAs are short noncoding RNAs that regulate multiple mRNAs and are frequently dysregulated in cancer. The authors have investigated the role of 3 microRNAs, including the hypoxia-induced hsa-miR-210, as potential markers of hypoxia or prognosis. METHODS: Three hypoxia-related microRNAs, hsa-miR-210, hsa-miR-21, and hsa-miR-10b, were measured in 46 samples from patients with HNSCC. Expression levels were correlated with clinicopathological variables and other markers of hypoxia: a published 99-gene hypoxia metagene, individual hypoxia-related genes such as TWIST1, and immunohistochemical expression of hypoxia-inducible factor 1 and its target gene carbonic anhydrase 9. We then performed survival analyses to investigate the prognostic significance of these microRNAs. RESULTS: Only the level of hsa-miR-210 was significantly correlated with other markers of hypoxia, including the 99-gene hypoxia metagene (rho ¼ 0.67, P < .001). We found no association between hsa-miR-210, hsamiR-21, or hsa-miR-10b and clinicopathological variables such as tumor size, differentiation, and stage. However, high levels of hsa-miR-210 were associated with locoregional disease recurrence (P ¼ .001) and short overall survival (P ¼ .008). hsa-miR-21 and hsa-miR-10b had no prognostic significance. CONCLUSIONS: Expression of hsa-miR-210 in head and neck cancer correlates with other approaches for assessing hypoxia and is associated with prognosis. This warrants further study as a classification marker of patients for therapies involving modulation of hypoxia.

Mice and cattle injected with plasmids encoding bovine herpesvirus 1 (BHV-1) glycoproteins developed gene-specific antibody responses capable of neutralizing BHV-1. The ability of animals to respond serologically to DNA injections was in part dependent on the quantity of DNA injected and was also negatively affected by carrier DNA. Calves injected with a plasmid encoding BHV-1 gIV developed significant antibody titers to gIV and shed less virus than did the control calf after challenge. This report indicates the potential of DNA injection as a method of vaccination.

BACKGROUND.Hypoxia promotes tumorigenesis through the hypoxia‐inducible factor (HIF) pathway. There are 2 main homologues of the regulatory proteins, HIF‐1α and HIF‐2α, which have different effects in genetic knock‐out experiments. Anemia may contribute to hypoxia by reducing oxygen delivery, but it is not known whether this influences HIF‐α expression in tumors.METHODS.The expression of HIF‐1α, HIF‐2α, carbonic anhydrase‐9 (CA‐9), and peripheral hemoglobin (Hb) levels in 151 patients who underwent surgery for head and neck squamous cell carcinoma (HNSCC) were analyzed and related to outcome.RESULTS.High HIF‐1α was expressed in 45 of 140 tumors (30%), HIF‐2α was expressed in 21 of 139 tumors (14%), and CA‐9 was expressed in 56 of 149 tumors (62%). There was a positive correlation between HIF‐1α expression and HIF‐2α expression (P = .0001). HIF‐1α alone was associated with a worse disease‐specific survival (DSS) (P = .05) and disease‐free survival (DFS) (P = .03) in multivariate analyses. Nine percent of tumors expressed both high HIF‐1α and high HIF‐2α. High HIF‐1α/high HIF‐2α expression was an independent prognostic factors in DSS (P = .04) and DFS (P = .005) in multivariate analyses. There was no correlation noted between Hb and HIF‐1α, HIF‐2α, or CA‐9.CONCLUSIONS.HIF‐1α alone was correlated with DSS and DFS. The additive effect of HIF‐2α on poor prognosis suggested that different pathways may be regulated by HIF‐2α. Anemia that was not related to HIF‐α expression suggests that tumor intrinsic factors regulate HIF‐α; therefore, anemia may be a surrogate marker for other factors that affect outcome. Cancer 2006. © 2006 American Cancer Society.

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