A sarcoma hypoxia signature (nanoString (R) assay) validates in the phase III VorteX radiotherapy trial

Abstract

Purpose or Objective Soft tissue sarcomas (STS) are a rare group of tumours encompassing ~50 malignant, heterogeneous subtypes. Localised disease is managed with surgery ± radiotherapy. Neoadjuvant radiotherapy response is varied and despite high metastatic recurrence rates, there is no definite overall survival benefit from the addition of systemic chemotherapy. Each subtype is very rare; therefore, biomarkers of adverse biological features present across subtypes might be more successful in selecting high-risk patients for clinical trials. Tumour hypoxia is associated with poor radiotherapy response and metastasis. We previously derived a 24-gene STS hypoxia signature that was prognostic in two independent cohorts. The study aims were (1) signature validation in a new whole transcriptome cohort (2) assay development for pretreatment biopsies (3) assay validation. Material and Methods Three cohorts were used (1) VorteX n=203 (2) single centre retrospective n=165 (3) MCRC Biobank n=28. VorteX was a randomised phase III trial comparing radiotherapy volumes in adults with localised extremity STS. Validation cohorts included mainly high-risk patients (85% high-grade). Whole transcriptome RNA-sequencing data (Illumina HiSeq4000) were generated from fresh frozen tumour samples for VorteX. Two targeted assays (Taqman® array cards and nanoString®) were compared in formalin-fixed, paraffin-embedded (FFPE) biopsies. Prognostic validation of the nanoString® assay was performed in FFPE samples from the retrospective and VorteX cohorts. Intra-tumour heterogeneity was assessed in patients (n=10) with multiple biopsies and compared with CAIX expression. Results In the RNA-sequencing VorteX cohort, data were generated for 125/140 patients with available tissue. There was a trend towards poor disease-free survival (DFS) in patients with hypoxic tumours (HR 1.68 95% CI 0.95-2.98 p=0.061). Taqman® array cards and nanoString® demonstrated excellent pass rates and reproducibility. The nanoString® was more sensitive, required lower input and can measure more genes simultaneously. The gene signature showed considerably less intra-tumour heterogeneity than a single marker (figure 1). The nanoString® assay produced a result for 126/127 (99.2%) and 154/159 (96.9%) of patients with sufficient RNA yield and classified 41.3% and 44.8% of samples as hypoxic from the retrospective and VorteX cohorts, respectively. In a univariate analysis patients with hypoxic tumours had worse disease free survival in both cohorts (retrospective HR 2.302 95% CI 1.182-4.486 p=0.0083, VorteX HR 1.687 95% CI 1.013-2.81 p=0.0377) Conclusion A hypoxia signature nanoString® assay with excellent technical performance is prognostic in two independent cohorts of patients with high-grade tumours, including the phase III VorteX radiotherapy trial. The assay could identify high-risk patients for adjuvant chemotherapy trials.