Data from: Whole transcriptome RNA-Seq analysis of breast cancer recurrence risk using formalin-fixed paraffin-embedded tumor tissue

RNA biomarkers discovered by RT-PCR-based gene expression profiling of archival formalin-fixed paraffin-embedded (FFPE) tissue form the basis for widely used clinical diagnostic tests; however, RT-PCR is practically constrained in the number of transcripts that can be interrogated. We have developed and optimized RNA-Seq library chemistry as well as bioinformatics and biostatistical methods for whole transcriptome profiling from FFPE tissue. The chemistry accommodates low RNA inputs and sample multiplexing. These methods both enable rediscovery of RNA biomarkers for disease recurrence risk that were previously identified by RT-PCR analysis of a cohort of 136 patients, and also identify a high percentage of recurrence risk markers that were previously discovered using DNA microarrays in a separate cohort of patients, evidence that this RNA-Seq technology has sufficient precision and sensitivity for biomarker discovery. More than two thousand RNAs are strongly associated with breast cancer recurrence risk in the 136 patient cohort (FDR < 10%). Many of these are intronic RNAs for which corresponding exons are not also associated with disease recurrence. A number of the RNAs associated with recurrence risk belong to novel RNA networks. It will be important to test the validity of these novel associations in whole transcriptome RNA-Seq screens of other breast cancer cohorts.

通过对存档福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded, FFPE）组织开展基于逆转录聚合酶链反应（reverse transcription-polymerase chain reaction, RT-PCR）的基因表达谱分析所发现的RNA生物标志物，是当前广泛应用的临床诊断检测的基础；然而，RT-PCR在可检测的转录本数量层面存在实际应用局限。我们开发并优化了适用于FFPE组织全转录组表达谱分析的RNA测序（RNA-Seq）文库构建试剂流程，以及配套的生物信息学与生物统计学分析方法。该体系可兼容低起始RNA投入量样本与样本多重复用。这些方法不仅能够重现针对136名患者队列开展RT-PCR分析时所鉴定出的疾病复发风险RNA生物标志物，还可识别出在另一独立患者队列中通过DNA微阵列（DNA microarrays）此前发现的绝大多数复发风险标志物，这一结果证实该RNA-Seq技术具备足够的精度与灵敏度以支撑生物标志物发现研究。在该136名患者的队列中，超过两千条RNA与乳腺癌复发风险呈显著相关（错误发现率False Discovery Rate, FDR < 10%）。其中多数为内含子RNA，其对应的外显子并未与疾病复发存在关联。诸多与复发风险相关的RNA隶属于全新的RNA调控网络。在其他乳腺癌队列的全转录组RNA-Seq筛选中验证这些全新关联的有效性，将具备重要的研究价值。