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:As degradation of formalin-fixed paraffin-embedded (FFPE) samples limits the ability to profile mRNA expression, we explored factors predicting the success of mRNA expression profiling of FFPE material and investigated an approach to overcome the limitation.Methods:Bladder (n=140, stored 3–8 years) and cervix (n=160, stored 8–23 years) carcinoma FFPE samples were hybridised to Affymetrix Exon 1.0ST arrays. Percentage detection above background (%DABG) measured technical success. Biological signal was assessed by distinguishing cervix squamous cell carcinoma (SCC) and adenocarcinoma (AC) using a gene signature. As miR-205 had been identified as a marker of SCC, precursor mir-205 was measured by Exon array and mature miR-205 by qRT–PCR. Genome-wide microRNA (miRNA) expression (Affymetrix miRNA v2.0 arrays) was compared in eight newer FFPE samples with biological signal and eight older samples without.Results:RNA quality controls (QCs) (e.g., RNA integrity (RIN) number) failed to predict profiling success, but sample age correlated with %DABG in bladder (R=−0.30, P<0.01) and cervix (R=−0.69, P<0.01). Biological signal was lost in older samples and neither a signature nor precursor mir-205 separated samples by histology. miR-205 qRT–PCR discriminated SCC from AC, validated by miRNA profiling (26-fold higher in SCC; P=1.10 × 10−5). Genome-wide miRNA (R=0.95) and small nucleolar RNA (R=0.97) expression correlated well in the eight newer vs older FFPE samples and better than mRNA expression (R=0.72).Conclusion:Sample age is the best predictor of successful mRNA profiling of FFPE material, and miRNA profiling overcomes the limitation of age and copes well with older samples.

Purpose: Intratumoral hypoxia and immunity have been correlated with patient outcome in various tumor settings. However, these factors are not currently considered for treatment selection in head and neck cancer (HNC) due to lack of validated biomarkers. Here we sought to develop a hypoxiaimmune classifier with potential application in patient prognostication and prediction of response to targeted therapy.Experimental Design: A 54-gene hypoxia-immune signature was constructed on the basis of literature review. Gene expression was analyzed in silico using the The Cancer Genome Atlas (TCGA) HNC dataset (n ¼ 275) and validated using two independent cohorts (n ¼ 130 and 123). IHC was used to investigate the utility of a simplified protein signature. The spatial distribution of hypoxia and immune markers was examined using multiplex immunofluorescence staining.Results: Unsupervised hierarchical clustering of TCGA dataset (development cohort) identified three patient subgroups with distinct hypoxia-immune phenotypes and sur-vival profiles: hypoxia low /immune high , hypoxia high /immune low , and mixed, with 5-year overall survival (OS) rates of 71%, 51%, and 49%, respectively (P ¼ 0.0015). The prognostic relevance of the hypoxia-immune gene signature was replicated in two independent validation cohorts. Only PD-L1 and intratumoral CD3 protein expression were associated with improved OS on multivariate analysis. Hypoxia low / immune high and hypoxia high /immune low tumors were overrepresented in "inflamed" and "immune-desert" microenvironmental profiles, respectively. Multiplex staining demonstrated an inverse correlation between CA-IX expression and prevalence of intratumoral CD3 þ T cells (r ¼ À0.5464; P ¼ 0.0377), further corroborating the transcription-based classification.Conclusions: We developed and validated a hypoxiaimmune prognostic transcriptional classifier, which may have clinical application to guide the use of hypoxia modification and targeted immunotherapies for the treatment of HNC.

Purpose: Hypoxia-inducible factor (HIF)-1␣ expression was studied retrospectively in locally advanced carcinoma of the cervix in relation to other methods for measuring/assessing tumor hypoxia and outcome after radiotherapy.Experimental Design: HIF-1␣ expression was examined in formalin-fixed tumor biopsies using a semiquantitative scoring system and correlated with measurements of hypoxia obtained using oxygen electrodes, pimonidazole staining, and carbonic anhydrase 9.Results: High HIF-1␣ expression showed a weak correlation with low pO 2 (r ‫؍‬ ؊0.26; P ‫؍‬ 0.030; n ‫؍‬ 72). Weak significant correlations were found between HIF-1␣ and pimonidazole staining (r ‫؍‬ 0.34; P ‫؍‬ 0.040; n ‫؍‬ 36) and carbonic anhydrase IX (r ‫؍‬ 0.27; P ‫؍‬ 0.001; n ‫؍‬ 160). There was no relationship with surviving fraction at 2 Gy. The relationship between HIF-1␣ expression and radiotherapy outcome was examined in 99 patients. HIF-1␣ expression did not correlate with disease stage, grade, tumor size, and patient age. HIF-1␣ alone was not a significant prognostic factor for disease-free survival, metastasis-free survival, or local recurrence-free survival. High HIF-1␣ expression tended to be associated with poor outcome in small tumors but good outcome in large tumors, with statistically significant interactions between HIF-1␣ and tumor size for survival (P ‫؍‬ 0.046) and local control (P ‫؍‬ 0.009).Conclusions: In this study, HIF-1␣ had no prognostic significance in locally advanced carcinoma of the cervix. The possible switch in large tumors for an association between high HIF-1␣ expression and good outcome might relate to tumor size-related changes in the balance of genes up-regulated by HIF-1␣. Whereas angiogenesis-promoting genes might be preferentially up-regulated in small tumors, proapoptotic genes might be induced in large tumors. This hypothesis needs testing in future work.

The aim of the study is to evaluate the pattern and level of expression of glucose transporter-1 (GLUT-1) in rectal carcinoma in relation to outcome as a potential surrogate marker of tumour hypoxia. Formalin-fixed tumour sections from 43 patients with rectal carcinoma, who had undergone radical resection with curative intent, were immunohistochemically stained for GLUT-1. A mean of three sections per tumour (range 1 -12) were examined. Each section was semiquantitatively scored; 0, no staining; 1, o10%; 2, 10 -50%; 3, 450% and a score given for the whole section, the superficial (luminal) and deep (mural) part of the tumour. Staining was seen in 70% of tumours. Increased staining was noted adjacent to necrosis and ulceration. A diffuse and patchy pattern of staining, with and without colocalisation to necrosis was seen. Patients with high GLUT-1-expressing tumours (score 3 vs 0 -2) had a significantly poorer overall survival (P ¼ 0.041), which was associated with poorer metastasis-free survival with no difference in local control. No significant correlation was seen with other prognostic factors. There was a strong correlation between the score for the superficial and deep parts of the tumour (r ¼ 0.81), but a significant relationship with outcome was only found in the deep part (P ¼ 0.003 vs P ¼ 0.46). In conclusion, increased GLUT-1 expression in rectal tumours was an adverse prognostic factor and is worth further evaluation as a predictive marker of response to therapy.

Hypoxia-inducible factor-1 (HIF-1)a expression was studied in the gastric carcinogenesis sequence and as a prognostic factor in surgically resected gastric and gastro-oesophageal junction tumours. Protein expression was examined using immunohistochemistry on formalin-fixed biopsies of normal mucosa (n ¼ 20), Helicobacter pylori associated gastritis (n ¼ 24), intestinal metaplasia (n ¼ 24), dysplasia (n ¼ 12) and intestinal (n ¼ 19) and diffuse (n ¼ 21) adenocarcinoma. The relationship between HIF-1a expression and prognosis was assessed in resection specimens from 177 patients with gastric and gastro-oesophageal junction adenocarcinoma. Hypoxia-inducible factor-1a expression was not observed in normal gastric mucosa but increased in density (Po0.01) and intensity (Po0.01) with progression from H. pylori-associated gastritis, intestinal metaplasia, dysplasia to adenocarcinoma. The pattern of staining in the resection specimens was focally positive in 49 (28%) and at the invasive tumour edge in 41 (23%). Invasive edge expression was associated with lymph node metastases (P ¼ 0.034), advanced TNM stage (P ¼ 0.001) and was an adverse prognostic factor for cancer-specific survival (P ¼ 0.019). In univariate analysis and in comparison with tumours not expressing HIF-1a, invasive edge staining was associated with a hazard ratio of 1.6 (95% CI 1.0À2.5) and focally positive staining a hazard ratio of 0.7 (95% CI 0.5À1.2). Hypoxia-inducible factor-1a lost prognostic significance in multivariate analysis. The results suggest HIF-1a is involved in gastric carcinogenesis and disease progression, but is only a weak prognostic factor for survival.

Receptor tyrosine kinase pathways are potential therapeutic targets in gastric adenocarcinoma patients. We evaluated HER2 and cMet protein expression, and FGFR2 gene amplification to assess their prognostic significance, and downstream mediators pS6 and pERK for their potential utility as pharmacodynamic biomarkers in patients with gastric adenocarcinoma. Tissue microarrays were constructed from resection samples of 184 patients who underwent surgery for gastric/gastro-oesophageal junction adenocarcinoma. Tissue cores were obtained from the tumour body (TB), luminal surface (LS) and invasive edge (IE), and immunohistochemical and fluorescence in situ hybridisation (FGFR2) analysis was performed. FGFR2 amplification was identified in 2 % of cases and associated with worse survival (P = 0.005). HER2 overexpression was observed in 10 % of cases and associated with increased survival (P = 0.041). cMet overexpression was observed in 4 % of cases and associated with worse survival (P < 0.001). On multivariate analysis, only cMet retained significance (P = 0.006). pS6 and pERK expression were observed in 73 % and 30 % of tumours, respectively, with no association with survival. HER2 (P = 0.004) and pERK (P = 0.001) expression differed between tumour regions with HER2 expression increased in the LS compared with the TB and IE. These findings confirm subpopulations in gastric adenocarcinoma with poor outcome that may benefit from specific therapeutic strategies. However, we found heterogeneous HER2, pS6 and pERK overexpression, which presents challenges for their use as predictive biomarkers in gastric biopsies. The potential downstream pharmacodynamic markers pS6 and pERK were expressed across tumour regions, providing evidence that resections and biopsies would yield comparative results in clinical trials.

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