BACKGROUND Formalin-fixed, paraffin-embedded (FFPE) tissues for RNA-seq have advantages over fresh frozen tissue including abundance and availability, connection to rich clinical data, and association with patient outcomes. However, FFPE-derived RNA is highly degraded and chemically modified, which impacts its utility as a faithful source for biological inquiry. METHODS True archival FFPE breast cancer cases (n = 58), stored at room temperature for 2-23 years, were utilized to identify key steps in tissue selection, RNA isolation, and library choice. Gene expression fidelity was evaluated by comparing FFPE data to public data obtained from fresh tissues, and by employing single-gene, gene set and transcription network-based regulon analyses. RESULTS We report a single 10 μm section of breast tissue yields sufficient RNA for RNA-seq, and a relationship between RNA quality and block age that was not linear. We find single-gene analysis is limiting with FFPE tissues, while targeted gene set approaches effectively distinguish ER+ from ER- breast cancers. Novel utilization of regulon analysis identified the transcription factor KDM4B to associate with ER+ disease, with KDM4B regulon activity and gene expression having prognostic significance in an independent cohort of ER+ cases. CONCLUSION Our results, which outline a robust FFPE-RNA-seq pipeline for broad use, support utilizing FFPE tissues to address key questions in the breast cancer field, including the delineation between indolent and life-threatening disease, biological stratification and molecular mechanisms of treatment resistance.

中文翻译：

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来自存档 FFPE 乳腺癌样本的 RNA-seq：分子途径保真度和新发现。

背景 用于 RNA-seq 的福尔马林固定石蜡包埋 (FFPE) 组织比新鲜冷冻组织具有优势，包括丰度和可用性、与丰富临床数据的连接以及与患者结果的关联。然而，FFPE 衍生的 RNA 被高度降解和化学修饰，这影响了其作为生物学研究的忠实来源的实用性。方法 利用在室温下保存 2-23 年的真实档案 FFPE 乳腺癌病例 (n = 58) 来确定组织选择、RNA 分离和文库选择的关键步骤。通过将 FFPE 数据与从新鲜组织获得的公共数据进行比较，并采用单基因、基因集和基于转录网络的调节子分析来评估基因表达保真度。结果我们报告了乳腺组织的单个 10 μm 切片产生了足够的 RNA 用于 RNA-seq，并且 RNA 质量和块年龄之间的关系不是线性的。我们发现单基因分析对于 FFPE 组织有限制，而靶向基因组方法可以有效区分 ER+ 和 ER- 乳腺癌。调节子分析的新用途确定了转录因子 KDM4B 与 ER+ 疾病相关，KDM4B 调节子活性和基因表达在独立的 ER+ 病例队列中具有预后意义。结论 我们的结果概述了一个强大的可广泛使用的 FFPE-RNA-seq 流程，支持利用 FFPE 组织来解决乳腺癌领域的关键问题，包括惰性疾病和危及生命的疾病之间的划分、生物分层和治疗的分子机制反抗。