A hypoxia biomarker does not predict benefit from giving chemotherapy with radiotherapy in the BC2001 randomised controlled trial

Background: BC2001 showed combining chemotherapy (5-FU + mitomycin-C) with radiotherapy improves loco-regional disease-free survival in patients with muscle-invasive bladder cancer (MIBC). We previously showed a 24-gene hypoxia-associated signature predicted benefit from hypoxia-modifying radiosensitisation in BCON and hypothesised that only patients with low hypoxia scores (HSs) would benefit from chemotherapy in BC2001. BC2001 allowed conventional (64Gy/32 fractions) or hypofractionated (55Gy/20 fractions) radiotherapy. An exploratory analysis tested an additional hypothesis that hypofractionation reduces reoxygenation and would be detrimental for patients with hypoxic tumours. Methods: RNA was extracted from pre-treatment biopsies (298 BC2001 patients), transcriptomic data generated (Affymetrix Clariom-S arrays), HSs calculated (median expression of 24-signature genes) and patients stratified as hypoxia-high or -low (cut-off: cohort median). Primary endpoint: invasive loco-regional control (ILRC); secondary overall survival. Findings: Hypoxia affected overall survival (HR = 1.30; 95% CI 0.99–1.70; p = 0.062): more uncertainty for ILRC (HR = 1.29; 95% CI 0.82–2.03; p = 0.264). Benefit from chemotherapy was similar for patients with high or low HSs, with no interaction between HS and treatment arm. High HS associated with poor ILRC following hypofractionated (n = 90, HR 1.69; 95% CI 0.99–2.89 p = 0.057) but not conventional (n = 207, HR 0.70; 95% CI 0.28–1.80, p = 0.461) radiotherapy. The finding was confirmed in an independent cohort (BCON) where hypoxia associated with a poor prognosis for patients receiving hypofractionated (n = 51; HR 14.2; 95% CI 1.7–119; p = 0.015) but not conventional (n = 24, HR 1.04; 95% CI 0.07–15.5, p = 0.978) radiotherapy. Interpretation: Tumour hypoxia status does not affect benefit from BC2001 chemotherapy. Hypoxia appears to affect fractionation sensitivity. Use of HSs to personalise treatment needs testing in a biomarker-stratified trial. Funding: Cancer Research UK, NIHR, MRC.