Seeber Andreas, Martowicz Agnieszka, Spizzo Gilbert, Buratti Thomas, Obrist Peter, Fong Dominic, Gastl Guenther, Untergasser Gerold
Experimental Oncogenomics, Tyrolean Cancer Research Institute, Innsbruck, Austria.
Oncotyrol - Center for Personalized Cancer Medicine, Innsbruck, Austria.
BMC Cancer. 2015 May 7;15:372. doi: 10.1186/s12885-015-1371-1.
EpCAM is highly expressed on membrane of epithelial tumor cells and has been detected as soluble/secreted (sEpCAM) in serum of cancer patients. In this study we established an ELISA for in vitro diagnostics to measure sEpCAM concentrations in ascites. Moreover, we evaluated the influence of sEpCAM levels on catumaxomab (antibody)--dependent cellular cytotoxicity (ADCC).
Ascites specimens from cancer patients with positive (C+, n = 49) and negative (C-, n = 22) cytology and ascites of patients with liver cirrhosis (LC, n = 31) were collected. All cell-free plasma samples were analyzed for sEpCAM levels with a sandwich ELISA system established and validated by a human recombinant EpCAM standard for measurements in ascites as biological matrix. In addition, we evaluated effects of different sEpCAM concentrations on catumaxomab-dependent cell-mediated cytotoxicity (ADCC) with human peripheral blood mononuclear cells (PBMNCs) and human tumor cells.
Our ELISA showed a high specificity for secreted EpCAM as determined by control HEK293FT cell lines stably expressing intracellular (EpICD), extracellular (EpEX) and the full-length protein (EpCAM) as fusion proteins. The lower limit of quantification was 200 pg/mL and the linear quantification range up to 5,000 pg/mL in ascites as biological matrix. Significant levels of sEpCAM were found in 39% of C+, 14% of C- and 13% of LC ascites samples. Higher concentrations of sEpCAM were detectable in C+ (mean: 1,015 pg/mL) than in C- (mean: 449 pg/mL; p = 0.04) or LC (mean: 326 pg/mL; p = 0.01). Soluble EpCAM concentration of 1 ng/mL significantly inhibited ADCC of PBMNCs on EpCAM overexpressing target cells.
Elevated concentrations of sEpCAM can be found in a subgroup of C+ and also in a small group of C- patients. We consider that sEpCAM levels in different tumor entities and individual patients should be evaluated prior to applying anti-EpCAM antibody-based cancer therapies, since sEpCAM neutralizes catumaxomab activity, making therapy less efficient.
上皮细胞黏附分子（EpCAM）在上皮肿瘤细胞表面高度表达，且在癌症患者血清中可检测到可溶性/分泌型（sEpCAM）。在本研究中，我们建立了一种用于体外诊断的酶联免疫吸附测定（ELISA）法来检测腹水中sEpCAM的浓度。此外，我们评估了sEpCAM水平对卡妥索单抗（抗体）依赖性细胞毒性（ADCC）的影响。
收集细胞学检查阳性（C+，n = 49）和阴性（C-，n = 22）的癌症患者腹水标本以及肝硬化（LC，n = 31）患者的腹水。使用通过人重组EpCAM标准品建立并验证的夹心ELISA系统，对所有无细胞血浆样本进行sEpCAM水平分析，以腹水作为生物基质进行测量。此外，我们用人外周血单核细胞（PBMNCs）和人肿瘤细胞评估了不同sEpCAM浓度对卡妥索单抗依赖性细胞介导的细胞毒性（ADCC）的影响。
通过稳定表达细胞内（EpICD）、细胞外（EpEX）和全长蛋白（EpCAM）作为融合蛋白的对照HEK293FT细胞系测定，我们的ELISA法对分泌型EpCAM具有高特异性。在作为生物基质的腹水中，定量下限为200 pg/mL，线性定量范围高达5000 pg/mL。在39%的C+、14%的C-和13%的LC腹水样本中发现了显著水平的sEpCAM。在C+中可检测到的sEpCAM浓度（均值：1015 pg/mL）高于C-（均值：449 pg/mL；p = 0.04）或LC（均值：326 pg/mL；p = 0.01）。1 ng/mL的可溶性EpCAM浓度显著抑制了PBMNCs对过表达EpCAM的靶细胞的ADCC。
在一部分C+患者以及一小部分C-患者中可发现sEpCAM浓度升高。我们认为，在应用基于抗EpCAM抗体的癌症治疗之前，应评估不同肿瘤实体和个体患者的sEpCAM水平，因为sEpCAM会中和卡妥索单抗的活性，使治疗效果降低。
