OncoPlex Diagnostics, Inc., Rockville, MD 20850, USA.
J Mol Diagn. 2013 Jul;15(4):454-65. doi: 10.1016/j.jmoldx.2013.03.002. Epub 2013 May 11.
One of the critical gaps in the clinical diagnostic space is the lack of quantitative proteomic methods for use on formalin-fixed, paraffin-embedded (FFPE) tissue. Herein, we describe the development of a quantitative, multiplexed, mass spectrometry-based selected reaction monitoring (SRM) assay for four therapeutically important targets: epidermal growth factor receptor, human EGF receptor (HER)-2, HER3, and insulin-like growth factor-1 receptor. These assays were developed using the Liquid Tissue-SRM technology platform, in which FFPE tumor tissues were microdissected, completely solubilized, and then subjected to multiplexed quantitation by SRM mass spectrometry. The assays were preclinically validated by comparing Liquid Tissue-SRM quantitation of FFPE cell lines with enzyme-linked immunosorbent assay/electrochemiluminescence quantitation of fresh cells (R(2) > 0.95). Clinical performance was assessed on two cohorts of breast cancer tissue: one cohort of 10 samples with a wide range of HER2 expression and a second cohort of 19 HER2 IHC 3+ tissues. These clinical data demonstrate the feasibility of quantitative, multiplexed clinical analysis of proteomic markers in FFPE tissue. Our findings represent a significant advancement in cancer tissue analysis because multiplexed, quantitative analysis of protein targets in FFPE tumor tissue can be tailored to specific oncological indications to provide the following: i) complementary support for anatomical pathological diagnoses, ii) patient stratification to optimize treatment outcomes and identify drug resistance, and iii) support for the clinical development of novel therapies.
在临床诊断领域，一个关键的空白是缺乏可用于福尔马林固定、石蜡包埋（FFPE）组织的定量蛋白质组学方法。在此，我们描述了一种定量、多重、基于质谱的选择反应监测（SRM）测定法的开发，用于四个治疗上重要的靶标：表皮生长因子受体、人表皮生长因子受体（HER）-2、HER3 和胰岛素样生长因子-1 受体。这些测定法是使用液体组织-SRM 技术平台开发的，其中 FFPE 肿瘤组织被微切割、完全溶解，然后通过 SRM 质谱进行多重定量。通过将 FFPE 细胞系的液体组织-SRM 定量与新鲜细胞的酶联免疫吸附测定/电化学发光定量进行比较，对这些测定法进行了临床前验证（R(2)＞0.95）。在两个乳腺癌组织队列中评估了临床性能：一个队列是 10 个具有广泛 HER2 表达的样本，另一个队列是 19 个 HER2 IHC 3+组织。这些临床数据证明了在 FFPE 组织中定量、多重分析蛋白质标志物的可行性。我们的发现代表了癌症组织分析的重大进展，因为 FFPE 肿瘤组织中蛋白质靶标的多重、定量分析可以针对特定的肿瘤学适应症进行定制，以提供以下支持：i）对解剖病理学诊断的补充支持，ii）患者分层以优化治疗效果并识别耐药性，以及 iii）支持新型治疗方法的临床开发。
