恶性软组织肉瘤(STS)是罕见、异质性的肿瘤，需要生物标志物来辅助治疗决策。我们之前制定了一种预后肿瘤微环境分类器(24基因低氧特征)。本研究开发/验证了一种可用于临床的检测方法。我们比较了靶向检测(Taqman低密度阵列、NanoString)在28个预选的福尔马林固定、石蜡包埋(FFPE) 活检样本中的技术表现。使用HIF-1α/CAIX免疫组化法(IHC)验证了NanoString检测方法的生物学准确性。曼彻斯特(165例)和涡流相III试验(203例)队列用于临床验证。主要结果为总生存期（OS）。两种检测方法都表现出良好的复现性。 NanoString检测方法在体外检测到24基因特征在低氧环境下的上调，并且在体内高CAIX表达的肿瘤组织中，16/24个低氧基因上调。在曼彻斯特队列(HR 3.05, 95% CI 1.54-5.19, P = 0.0005)和涡流相III试验队列(HR 2.13, 95% CI 1.19-3.77, P = 0.009)中，低氧肿瘤患者预后较差。在合并队列中，该方法可独立预测总生存期(HR 2.24, 95% CI 1.42-3.53, P = 0.00096)，并且与更严重的局部复发无关(HR 2.17, 95% CI 1.01-4.68, P = 0.04)。 本研究全面验证了一种适合FFPE STS活检样品的微环境分类器。未来，该方法可用于：(1)为围手术期化疗选择高危患者；(2)基于生物标志物的低氧靶向疗法试验。©2023年，作者。

Soft tissue sarcomas (STS) are rare, heterogeneous tumours and biomarkers are needed to inform management. We previously derived a prognostic tumour microenvironment classifier (24-gene hypoxia signature). Here, we developed/validated an assay for clinical application.Technical performance of targeted assays (Taqman low-density array, nanoString) was compared in 28 prospectively collected formalin-fixed, paraffin-embedded (FFPE) biopsies. The nanoString assay was biologically validated by comparing to HIF-1α/CAIX immunohistochemistry (IHC) in clinical samples. The Manchester (n = 165) and VORTEX Phase III trial (n = 203) cohorts were used for clinical validation. The primary outcome was overall survival (OS).Both assays demonstrated excellent reproducibility. The nanoString assay detected upregulation of the 24-gene signature under hypoxia in vitro, and 16/24 hypoxia genes were upregulated in tumours with high CAIX expression in vivo. Patients with hypoxia-high tumours had worse OS in the Manchester (HR 3.05, 95% CI 1.54-5.19, P = 0.0005) and VORTEX (HR 2.13, 95% CI 1.19-3.77, P = 0.009) cohorts. In the combined cohort, it was independently prognostic for OS (HR 2.24, 95% CI 1.42-3.53, P = 0.00096) and associated with worse local recurrence-free survival (HR 2.17, 95% CI 1.01-4.68, P = 0.04).This study comprehensively validates a microenvironment classifier befitting FFPE STS biopsies. Future uses include: (1) selecting high-risk patients for perioperative chemotherapy; and (2) biomarker-driven trials of hypoxia-targeted therapies.© 2023. The Author(s).