Comparison of multiple gene expression platforms for measuring a bladder cancer hypoxia signature

Tumour hypoxia status provides prognostic information and predicts response to hypoxia-modifying treatments. We previously derived a 24-gene signature to assess hypoxia in bladder cancer. The objectives here were to: compare platforms for generating signature scores, identify cut-off values for prospective studies, assess intra-tumour heterogeneity and confirm hypoxia relevance.
RNA was extracted from prospectively-collected diagnostic biopsies of muscle invasive bladder cancer (51 patients) and gene expression measured using customised Taqman Low Density Array (TLDA) cards, NanoString and Clariom S arrays. Cross-platform transferability of the gene signature was assessed using regression and concordance analysis. Cut-off values were the cohort median expression values. Intra- and inter-tumour variability were determined in a retrospective patient cohort (n=51) with multiple blocks (2-18) from the same tumour. To demonstrate relevance, bladder cancer cell lines were exposed to hypoxia (0.1% oxygen, 24 h), and extracted RNA run on custom TLDA cards.
Hypoxia scores showed good agreement between platforms: Clariom S vs TLDA (r=0.72, p<0.0001; concordance 73%); Clariom S vs NanoString (r=0.84, p<0.0001; 78%); TLDA vs NanoString (r=0.80, p<0.0001; 78%). Cut-off values were 0.047 (TLDA), 7.328 (NanoString) and 6.667 (Clariom S). Intra-tumour heterogeneity in gene expression and HS (coefficient of variation 3.9%) was less than inter-tumour (7.9%) variability. Hypoxia scores were higher in bladder cancer cells exposed to hypoxia vs normoxia (p<0.02).
Application of the 24-gene bladder cancer hypoxia signature is platform agnostic, cut-off values determined prospectively can be used in a clinical trial, intra-tumour heterogeneity is low and the signature is sensitive to changes in oxygen levels in vitro.