Simple SummaryRadiation therapy (RT) is an important treatment for cancer. Advances in technology over the last 100 years have helped to deliver radiation to tumours (and avoid normal tissues) with increasing accuracy. Comparatively little progress has been made in adjusting radiation treatment based on genetic differences from one person to another. Techniques proposed for adjusting dose have been assessed in clinical datasets, but studies have not considered how technical aspects affect predictions or gene signature specificity to radiation. This work shows that preprocessing has a large influence on model predictions and demonstrates a lack of evidence for radiation specificity in existing gene expression-based models.AbstractTranscriptomic personalisation of radiation therapy has gained considerable interest in recent years. However, independent model testing on in vitro data has shown poor performance. In this work, we assess the reproducibility in clinical applications of radiosensitivity signatures. Agreement between radiosensitivity predictions from published signatures using different microarray normalization methods was assessed. Control signatures developed from resampled in vitro data were benchmarked in clinical cohorts. Survival analysis was performed using each gene in the clinical transcriptomic data, and gene set enrichment analysis was used to determine pathways related to model performance in predicting survival and recurrence. The normalisation approach impacted calculated radiosensitivity index (RSI) values. Indeed, the limits of agreement exceeded 20% with different normalisation approaches. No published signature significantly improved on the resampled controls for prediction of clinical outcomes. Functional annotation of gene models suggested that many overlapping biological processes are associated with cancer outcomes in RT treated and non-RT treated patients, including proliferation and immune responses. In summary, different normalisation methods should not be used interchangeably. The utility of published signatures remains unclear given the large proportion of genes relating to cancer outcome. Biological processes influencing outcome overlapped for patients treated with or without radiation suggest that existing signatures may lack specificity.