Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing
Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing
复制标题
DOI：
10.1038/srep20944
复制
发表时间：
2016-02-11
影响因子：
4.6
通讯作者：
Manaresi, Nicolo
中科院分区：
文献类型：
--
作者：
Bolognesi, Chiara;Forcato, Claudio;Manaresi, Nicolo
肿瘤学中的精确医学需要对肿瘤分子轮廓进行准确的表征，以便对患者进行分层。尽管靶向深度测序是检测体细胞序列变异的有效工具，但相当数量的患者样本不能满足常规临床应用的要求。正常细胞的污染和固有的肿瘤异质性，加上处理福尔马林固定石蜡包埋标本(FFPE)中常见的微量组织和DNA损伤的挑战，阻碍了分析。在这里，我们提出了一种使用DEPArray(TM)系统的创新工作流程，这是一种基于微芯片的数字分选器，可以从FFPE样本中获得100%纯净、同质的细胞亚群。通过荧光标记的抗体和DNA含量来区分细胞。解决肿瘤异质性的能力使得能够明确地确定真阳性序列变异、杂合性丧失以及拷贝数变异。建议的策略克服了在从混合人群中检测基因变异时在敏感性和特异性之间的内在权衡，从而挽救了即使是较小的肿瘤细胞密度较低的临床样本进行分析。
Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification. Though targeted deep sequencing is an effective tool to detect the presence of somatic sequence variants, a significant number of patient specimens do not meet the requirements needed for routine clinical application. Analysis is hindered by contamination of normal cells and inherent tumor heterogeneity, compounded with challenges of dealing with minute amounts of tissue and DNA damages common in formalin-fixed paraffin-embedded (FFPE) specimens. Here we present an innovative workflow using DEPArray(TM) system, a microchip-based digital sorter to achieve 100% pure, homogenous subpopulations of cells from FFPE samples. Cells are distinguished by fluorescently labeled antibodies and DNA content. The ability to address tumor heterogeneity enables unambiguous determination of true-positive sequence variants, loss-of-heterozygosity as well as copy number variants. The proposed strategy overcomes the inherent trade-offs made between sensitivity and specificity in detecting genetic variants from a mixed population, thus rescuing for analysis even the smaller clinical samples with low tumor cellularity.