Prospective technical validation and assessment of intra-tumour heterogeneity of a low density array hypoxia gene profile in head and neck squamous cell carcinoma

Journal article

Background and purpose: Tumour hypoxia is associated with a poor prognosis in head and neck squamous cell carcinoma (HNSCC), however there is no accepted method for assessing hypoxia clinically. We aimed to conduct a technical validation of a hypoxia gene expression signature using the TaqMan Low Density Array (TLDA) platform to investigate if this approach reliably identified hypoxic tumours. Materials and methods: Tumour samples (n = 201) from 80 HNSCC patients were collected prospectively from two centres. Fifty-three patients received pimonidazole prior to surgery. TaqMan Low Density Array-Hypoxia Scores (TLDA-HS) were obtained by quantitative real-time PCR (qPCR) using a 25-gene signature and customised TLDA cards. Assay performance was assessed as coefficient of variation (CoV). Results: The assay was sensitive with linear reaction efficiencies across a 4log10 range of inputted cDNA (0.001-10 ng/μl). Intra- (CoV = 6.9%) and inter- (CoV = 2.0%) assay reproducibility were excellent. Intra-tumour heterogeneity was lower for TLDA-HS (23.2%) than for pimonidazole (67.2%) or single gene measurements of CA9 (62.2%), VEGFA (45.0%) or HIG2 (39.4%). TLDA-HS in HNSCC cell lines increased with decreasing pO2. TLDA-HS correlated with Affymetrix U133 Plus 2.0 microarray HS (p < 0.01) and positive pimonidazole scores (p = 0.005). Conclusions: Gene expression measurements of hypoxia using a 25-gene signature and TLDA cards are sensitive, reproducible and associated with lower intra-tumour heterogeneity than assaying individual genes or pimonidazole binding. The approach is suitable for further assessment of prognostic and predictive capability in clinical trial material. © 2012 Elsevier Ltd. All rights reserved.

Authors: Betts, G; Eustace, A; Patiar, S; Valentine, H; Irlam, J

• Journal: European Journal of Cancer
• Volume: 49
• Issue: 1
• Pages: 156-165
• Publication date: 2013-01-01
• DOI: 10.1016/j.ejca.2012.07.028
• EISSN: 1879-0852
• ISSN: 0959-8049
• Language: English
• Keywords: Hypoxia; Carbonic anhydrase IX; Pimonidazole; Head and neck cancer; Gene expression; TaqMan Low Density Array