Comparison of triple-negative breast cancer molecular subtyping using RNA from matched fresh-frozen versus formalin-fixed paraffin-embedded tissue.
Comparison of triple-negative breast cancer molecular subtyping using RNA from matched fresh-frozen versus formalin-fixed paraffin-embedded tissue.
复制标题
DOI：
10.1186/s12885-017-3237-1
复制
发表时间：
2017-04-04
期刊：
影响因子：
3.8
通讯作者：
Lehmann BD
中科院分区：
文献类型：
--
作者：
Jovanović B;Sheng Q;Seitz RS;Lawrence KD;Morris SW;Thomas LR;Hout DR;Schweitzer BL;Guo Y;Pietenpol JA;Lehmann BD
三阴性乳腺癌（TNBC）是一种异质性疾病，缺乏可以指导治疗决策的统一分子改变。我们先前使用冷冻肿瘤标本在微阵列平台上生成的基因表达数据鉴定了TNBC的不同分子亚型（TNBC型）。代表所鉴定亚型的肿瘤和细胞系在生物学相关转录物中具有明显的富集，对标准化疗和靶向药物具有不同的敏感性。自从我们最初的发现以来，RNA测序（RNA-seq）已经发展成为测量转录本丰度的灵敏和定量工具。为了证明TNBC亚型在平台之间是相似的，我们比较了来自癌症基因组图谱（TCGA）的微阵列和RNA-seq分析的匹配标本的基因表达。在TNBC患者的临床护理中，通过福尔马林固定和石蜡包埋（FFPE）处理为诊断目的收集的肿瘤标本。因此，为了使TNBC型最终具有广泛和实际的临床实用性，我们在新鲜冷冻（FF）和FFPE肿瘤标本之间进行了RNA-seq基因表达和分子分类比较。TCGA分析显示91%的可评估样品之间的一致亚型调用，表明TNBC亚型在微阵列和RNA-seq平台上的保守性。我们比较了在21对FF和FFPE TNBC标本上进行的RNA-seq，并评估了基因组比对、转录覆盖率、差异转录富集和TNBC分子亚型调用的一致性。我们证明了匹配的FF和FFPE样品之间的亚型准确性随着测序深度和与单个TNBC亚型的相关强度而增加。从FFPE样本中可靠地鉴定出TNBC亚型，如果样本小于4年，则准确度最高，并且可重复的亚型分型随着测序深度而增加。为了使用基因表达可重复地分型肿瘤，选择不因平台类型、组织处理或RNA分离方法而变化的基因至关重要。匹配的FF和FFPE样品之间的大部分差异表达的转录本可以归因于通过RNA富集方法选择的转录本。虽然差异表达的转录本不影响TNBC亚型，但它们将为确定在实施基因集大小减少策略时应避免哪些转录本提供指导。 NCT 00930930 07/01/2009。 本文的在线版本（doi：10.1186/s12885-017-3237-1）包含补充材料，可供授权用户使用。
Triple negative breast cancer (TNBC) is a heterogeneous disease that lacks unifying molecular alterations that can guide therapy decisions. We previously identified distinct molecular subtypes of TNBC (TNBCtype) using gene expression data generated on a microarray platform using frozen tumor specimens. Tumors and cell lines representing the identified subtypes have distinct enrichment in biologically relevant transcripts with differing sensitivity to standard chemotherapies and targeted agents. Since our initial discoveries, RNA-sequencing (RNA-seq) has evolved as a sensitive and quantitative tool to measure transcript abundance. To demonstrate that TNBC subtypes were similar between platforms, we compared gene expression from matched specimens profiled by both microarray and RNA-seq from The Cancer Genome Atlas (TCGA). In the clinical care of patients with TNBC, tumor specimens collected for diagnostic purposes are processed by formalin fixation and paraffin-embedding (FFPE). Thus, for TNBCtype to eventually have broad and practical clinical utility we performed RNA-seq gene expression and molecular classification comparison between fresh-frozen (FF) and FFPE tumor specimens. Analysis of TCGA showed consistent subtype calls between 91% of evaluable samples demonstrating conservation of TNBC subtypes across microarray and RNA-seq platforms. We compared RNA-seq performed on 21-paired FF and FFPE TNBC specimens and evaluated genome alignment, transcript coverage, differential transcript enrichment and concordance of TNBC molecular subtype calls. We demonstrate that subtype accuracy between matched FF and FFPE samples increases with sequencing depth and correlation strength to an individual TNBC subtype. TNBC subtypes were reliably identified from FFPE samples, with highest accuracy if the samples were less than 4 years old and reproducible subtyping increased with sequencing depth. To reproducibly subtype tumors using gene expression, it is critical to select genes that do not vary due to platform type, tissue processing or RNA isolation method. The majority of differentially expressed transcripts between matched FF and FFPE samples could be attributed to transcripts selected for by RNA enrichment method. While differentially expressed transcripts did not impact TNBC subtyping, they will provide guidance on determining which transcripts to avoid when implementing a gene set size reduction strategy. NCT00930930 07/01/2009. The online version of this article (doi:10.1186/s12885-017-3237-1) contains supplementary material, which is available to authorized users.