Tumour hypoxia status provides prognostic informa‑
tion and predicts response to hypoxia‑modifying treatments. A
previous study by our group derived a 24‑gene signature to
assess hypoxia in bladder cancer. The objectives of the present
study were to compare platforms for generating signature
scores, identify cut‑off values for prospective studies, assess
intra‑tumour heterogeneity and confirm hypoxia relevance.
Briefly, RNA was extracted from prospectively collected diag‑
nostic biopsies of muscle invasive bladder cancer (51 patients),
and gene expression was 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. The
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 was run on custom TLDA cards.
Hypoxia scores (HS) values showed good agreement between
platforms: Clariom S vs. TLDA (r=0.72, P<0.0001; concor‑
dance 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. HS values were higher in bladder cancer
cells exposed to hypoxia compared with normoxia (P<0.02).
In conclusion, the present study revealed that application of
the 24‑gene bladder cancer hypoxia signature was platform
agnostic, cut‑off values determined prospectively can be used
in a clinical trial, intra‑tumour heterogeneity was low and the
signature was sensitive to changes in oxygen levels in vitro.