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.

通过基于逆转录聚合酶链反应（RT-PCR）的基因表达谱分析对存档福尔马林固定石蜡包埋（FFPE）组织开展RNA生物标志物筛选，已成为当前临床广泛应用的诊断检测的核心依据；但RT-PCR在可检测的转录本数量方面存在实际局限。本研究开发并优化了适用于FFPE组织全转录组谱分析的RNA测序（RNA-Seq）建库化学方法，以及配套的生物信息学与生物统计学分析流程。该建库体系可兼容低起始RNA投入量与多样本复合测序。依托该方法体系，本研究不仅成功复现了此前通过对136例患者队列进行RT-PCR分析所鉴定的疾病复发风险RNA生物标志物，还在另一独立患者队列中鉴定出了此前通过DNA微阵列技术发现的绝大多数复发风险标志物，这证明本RNA-Seq技术具备足够的精度与灵敏度以用于生物标志物筛选。在该136例患者队列中，有超过2000种RNA与乳腺癌复发风险呈强相关性（错误发现率FDR < 10%）。其中多数为内含子RNA，其对应的外显子区域并未与疾病复发风险存在关联。部分与复发风险相关的RNA隶属于全新的RNA调控网络。在其他乳腺癌患者队列的全转录组RNA-Seq筛选中验证这些全新关联的有效性，将是后续研究的关键方向。