Characterizing the heterogeneity of triple-negative breast cancers using RNA Sequencing

Triple-negative breast cancers (TNBCs) are a heterogeneous set of tumors defined by an absence of actionable therapeutic targets (ER, PR, and HER-2). Microdissected normal ductal epithelium from healthy volunteers represents a novel comparator to reveal insights into TNBC heterogeneity and to inform drug development. Using RNA-sequencing data from our institution and The Cancer Genome Atlas (TCGA) we compared the transcriptomes of 94 TNBCs, 20 microdissected normal breast tissues from healthy volunteers from the Susan G. Komen for the Cure Tissue Bank, and 10 histologically normal tissues adjacent to tumor. Pathway analysis comparing TNBCs to optimized normal controls of microdissected normal epithelium versus classic controls composed of adjacent normal tissue revealed distinct molecular signatures. Differential gene expression of TNBC compared with normal comparators demonstrated important findings for TNBC-specific clinical trials testing targeted agents; lack of over-expression for negative studies and over-expression in studies with drug activity. Next, by comparing each individual TNBC to the set of microdissected normals, we demonstrate that TNBC heterogeneity is attributable to transcriptional chaos, is associated with non-silent DNA mutational load, and explains transcriptional heterogeneity in addition to known molecular subtypes. Finally, chaos analysis identified 146 core genes dysregulated in >90 % of TNBCs revealing an over-expressed central network. In conclusion, use of microdissected normal ductal epithelium from healthy volunteers enables an optimized approach for studying TNBC and uncovers biological heterogeneity mediated by transcriptional chaos. Overall design: RNA sequencing data from 10 TNBC tumors is included in this submission. No data from the 84 TCGA TNBCs or normal breast tissues are included in this submission. Reference dbGaP accession phs000644.v1.p1 for data from normal tissues.

三阴性乳腺癌（Triple-negative breast cancers, TNBCs）是一类异质性肿瘤群，其定义为缺乏可靶向治疗靶点——雌激素受体（ER）、孕激素受体（PR）及人表皮生长因子受体2（HER-2）。来自健康志愿者的显微切割正常导管上皮，是一种新型对照样本，可用于解析三阴性乳腺癌的异质性并为药物研发提供参考。本研究结合本院及癌症基因组图谱（The Cancer Genome Atlas, TCGA）的RNA测序数据，比较了94例三阴性乳腺癌、20份来自苏珊·G·科曼乳腺癌防治组织组织库的健康志愿者显微切割正常乳腺组织，以及10份肿瘤旁组织学正常组织的转录组。将三阴性乳腺癌与显微切割正常上皮这一优化正常对照，以及由肿瘤旁正常组织构成的传统对照进行通路分析，结果显示二者存在显著不同的分子特征。三阴性乳腺癌与正常对照样本的差异基因表达分析，为靶向药物相关的三阴性乳腺癌专属临床试验提供了重要参考：靶点未过表达对应临床试验阴性结果，靶点过表达则对应药物活性研究的阳性结果。随后，通过将每一例三阴性乳腺癌与上述显微切割正常组织样本集进行比对，本研究证实三阴性乳腺癌的异质性可归因于转录紊乱，且与非同义DNA突变负荷相关，该因素在已知分子亚型之外进一步解释了转录组异质性。最后，通过转录紊乱分析，本研究在超过90%的三阴性乳腺癌样本中鉴定出146个失调的核心基因，揭示了一个过表达的核心调控网络。综上，采用健康志愿者的显微切割正常导管上皮作为对照样本，为三阴性乳腺癌研究提供了优化的研究策略，并揭示了由转录紊乱介导的生物学异质性。研究整体设计：本次提交的数据包含10例三阴性乳腺癌肿瘤的RNA测序数据，未包含84例癌症基因组图谱三阴性乳腺癌样本及正常乳腺组织的相关数据。正常组织数据可参考基因型与表型数据库（dbGaP）注册号phs000644.v1.p1获取。