PURPOSE In breast cancer, bevacizumab increased pCR rate but not long-term survival and no predictive markers are available to identify patients with long-term benefit from the drug. EXPERIMENTAL DESIGN We profiled 289 pretherapeutic formalin-fixed, paraffin-embedded (FFPE) biopsies of HER2-negative patients from the GeparQuinto trial of neoadjuvant chemotherapy ± bevacizumab by exome-capture RNA-sequencing (RNA-seq). In a prospectively planned study, we tested molecular signatures for response prediction. IHC validation was performed using tissue microarrays. RESULTS We found strong agreement of molecular and pathologic parameters as hormone receptors, grading, and lymphocyte infiltration in 221 high-quality samples. Response rates (49.3% pCR overall) were higher in basal-like (68.9%) and HER2-enriched (45.5%) than in luminal B (35.7%), luminal A (17.9%), and normal-like (20.0%) subtypes. T-cell (OR = 1.60; 95% confidence interval, 1.21-2.12; P = 0.001), proliferation (OR = 2.88; 95% CI, 2.00-4.15; P < 0.001), and hypoxia signatures (OR = 1.92; 95% CI, 1.41-2.60; P < 0.001) significantly predicted pCR in univariate analysis. In a prespecified multivariate logistic regression, a small hypoxia signature predicted pCR (OR = 2.40; 95% CI, 1.28-4.51; P = 0.006) with a significant interaction with bevacizumab treatment (P = 0.020). IHC validation using NDRG1 as marker revealed highly heterogenous expression within tissue leading to profound loss of sensitivity in TMA analysis, still a significant predictive value for pCR was detected (P = 0.025). CONCLUSIONS Exome-capture RNA-seq characterizes small FFPE core biopsies by reliably detecting factors as for example ER status, grade, and tumor-infiltrating lymphocytes levels. Beside molecular subtypes and immune signatures, a small hypoxia signature predicted pCR to bevacizumab, which could be validated by IHC. The signature can have important applications for bevacizumab treatment in different cancer types and might also have a role for novel combination therapies of bevacizumab with immune checkpoint inhibition.

中文翻译：

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在新辅助GeparQuinto乳腺癌试验中，一个小的低氧信号可预测对贝伐单抗的pCR反应。

目的在乳腺癌中，贝伐单抗可提高pCR率，但不能提高长期生存率，并且尚无可预测的标志物来识别可长期受益于该药物的患者。实验设计我们通过外显子组捕获RNA测序（RNA-seq）对来自GeparQuinto新辅助化疗±贝伐单抗试验的HER2阴性患者的289例治疗前福尔马林固定的石蜡包埋（FFPE）活检进行了分析。在一项前瞻性计划研究中，我们测试了用于预测反应的分子标记。使用组织微阵列进行IHC验证。结果我们在221份高质量样品中发现了分子和病理学参数（如激素受体，分级和淋巴细胞浸润）的一致性。基底样（68.9％）和富含HER2的应答率（总体pCR为49.3％）和富含HER2的应答率（45.5％）高于管腔B（35.7％），腔A（17.9％）和正常类（20.0％）亚型。T细胞（OR = 1.60; 95％置信区间，1.21-2.12; P = 0.001），增殖（OR = 2.88; 95％CI，2.00-4.15; P <0.001）和低氧信号（OR = 1.92; 95 ％CI，1.41-2.60； P <0.001）在单变量分析中显着预测了pCR。在预先指定的多元逻辑回归中，一个小的低氧信号预测了pCR（OR = 2.40; 95％CI，1.28-4.51; P = 0.006），与贝伐单抗治疗有显着相互作用（P = 0.020）。使用NDRG1作为标记物进行的IHC验证揭示了组织内高度异质的表达，导致TMA分析中灵敏度的严重丧失，但仍检测到pCR的重要预测值（P = 0.025）。结论外显子捕获RNA-seq通过可靠地检测诸如ER状态，级别和肿瘤浸润淋巴细胞水平。除了分子亚型和免疫特征外，一个小的低氧信号也预测pCR为贝伐单抗，这可以通过IHC验证。该标记对于在不同癌症类型中的贝伐单抗治疗可能具有重要的应用，并且对于具有免疫检查点抑制作用的贝伐单抗的新型联合疗法也可能具有作用。