Mass-spectrometry-based quantitation of Her2 in gastroesophageal tumor tissue: comparison to IHC and FISH.
Mass-spectrometry-based quantitation of Her2 in gastroesophageal tumor tissue: comparison to IHC and FISH.
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DOI：
10.1007/s10120-015-0566-0
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发表时间：
2016-10
期刊：
影响因子：
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通讯作者：
Hembrough T
中科院分区：
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作者：
Catenacci DVT;Liao WL;Zhao L;Whitcomb E;Henderson L;O'Day E;Xu P;Thyparambil S;Krizman D;Bengali K;Uzzell J;Darfler M;Cecchi F;Blackler A;Bang YJ;Hart J;Xiao SY;Lee SM;Burrows J;Hembrough T
曲妥珠单抗对Her2阳性胃食道癌(GEC)有生存益处。免疫组织化学(IHC)和荧光原位杂交(FISH)目前确定了曲妥珠单抗治疗的资格。然而，这些低通量分析经常产生不一致或模棱两可的结果。我们建立了一种基于选择性反应监测质谱仪(SRM-MS)和定量Her2-SRM蛋白在细胞系(n=27)和GEC组织(n=139)中的定量水平(amol/ug)的靶向蛋白质组分析方法。我们将Her2-SRM蛋白的表达与IHC/FISH进行比较，试图确定最佳的SRM蛋白表达界值，以确定HER2基因的扩增。在展示了分析的发展、精密度和稳定性后，观察到Her2-SRM蛋白测量与HER2/CEP17比率高度一致，特别是在调整了SRM-Met、EGFR、HER3和HER2-异质性协变量及其相互作用的多元回归模型中(细胞系R2=0.9842；FFPER2=0.7643)。在GEC组织中，Her2-SRM蛋白阳性率为71.2%。ROC曲线显示，HER2-SRM蛋白水平对HER2-FISH扩增肿瘤的诊断具有高度的特异性(100%)和敏感性(75%)，上界值为750amol/μg。一个450-750 amol/ug的Her2-SRM蛋白的“模棱两可的区域”类似于“IHC2+#x2019；”，但代表较少的病例(9-16%的病例比36-41%)。与IHC相比，靶向SRM-Her2蛋白质组学提供了更客观和定量的Her2表达，具有良好的HER2/CEP17 FISH相关性和较少的模棱两可的病例。除了对其他相关癌蛋白的多重表达能力外，这些结果还证明了一种用于临床应用的改进的HER2蛋白表达分析方法。
Trastuzumab showed survival benefit for Her2-positive gastroesophageal cancers (GEC). Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) currently determine eligibility for trastuzumab-based therapy. However, these low-throughput assays often produce discordant or equivocal results. We developed a targeted proteomic assay based on selected reaction monitoring mass spectrometry (SRM-MS) and quantified levels (amol/ug) of Her2-SRM protein in cell lines (n=27) and GEC tissues (n=139). We compared Her2-SRM protein expression with IHC/FISH, seeking to determine optimal SRM protein expression cut-offs to identify HER2 gene amplification. After demonstrating assay development, precision, and stability, Her2-SRM protein measurement was observed to be highly concordant with HER2/CEP17 ratio, particularly in a multivariate regression model adjusted for SRM-expression of Met, Egfr, Her3, and HER2-heterogeneity covariates, and their interactions (cell lines r2=0.9842; FFPE r2=0.7643). In GEC tissues, Her2-SRM protein was detected in 71.2% of cases. ROC curves demonstrated Her2-SRM protein levels to have high specificity (100%) at an upper-level cut-off of >750 amol/μg and sensitivity (75%) at lower-level cut-off of <450 amol/ug to identify HER2 FISH amplified tumors. An ‘equivocal-zone’ of 450-750 amol/ug of Her2-SRM protein was analogous to ’IHC2+#x2019;, but represented fewer cases (9-16% of cases versus 36-41%). Compared to IHC, targeted SRM-Her2 proteomics provided more objective and quantitative Her2 expression with excellent HER2/CEP17 FISH correlation and fewer equivocal cases. Along with the multiplex capability for other relevant oncoproteins, these results demonstrated a refined HER2 protein expression assay for clinical application.