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Soluble Epcam Levels in Ascites Correlate with Positive Cytology And

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Soluble Epcam Levels in Ascites Correlate with Positive Cytology And Seeber et al. BMC Cancer (2015) 15:372 DOI 10.1186/s12885-015-1371-1 RESEARCH ARTICLE Open Access Soluble EpCAM levels in ascites correlate with positive cytology and neutralize catumaxomab activity in vitro Andreas Seeber1,2,3†, Agnieszka Martowicz1,4†, Gilbert Spizzo1,2,5, Thomas Buratti6, Peter Obrist7, Dominic Fong1,5, Guenther Gastl3 and Gerold Untergasser1,2,3* Abstract Background: 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). Methods: 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. Results: 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. Conclusion: 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. Keywords: Ascites, Catumaxomab, Cytology, Elisa, Liver cirrhosis, Peritoneal carcinomatosis, Soluble EpCAM * Correspondence: [email protected] †Equal contributors 1Experimental Oncogenomics, Tyrolean Cancer Research Institute, Innsbruck, Austria 2Oncotyrol – Center for Personalized Cancer Medicine, Innsbruck, Austria Full list of author information is available at the end of the article © 2015 Seeber et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Seeber et al. BMC Cancer (2015) 15:372 Page 2 of 12 Background suffering from malignant ascites. Catumaxomab is a tri- In cancer patients, abnormal accumulation of fluid within functional antibody binding to EpCAM on epithelial- the peritoneal cavity results in ascites formation and is fre- like tumor cells and CD3+ T cells and can activate with quently accompanied by cancer cell accumulation. Such its Fc part monocytes and NK cells. Catumaxomab can malignancy-related ascites accounts for 10% of all ascites lower ascites level and prolong the puncture-free sur- cases [1]. A disturbed equilibrium between fluid production vival of cancer patients and increase their quality of life and drainage due to lymphatic vessel obstruction, hyper- [26]. Actually, there are no reliable data on the levels of permeability and fluid overproduction is the main reason sEpCAM in patients with malignancy-related ascites. for accumulation of ascites fluid [2,3]. There are different Therefore, we analyzed and quantified soluble EpCAM mechanisms how cancer cells can cause ascites. Some (sEpCAM) in a series of ascites samples. To guarantee malignancies such as ovarian cancer tend to form peri- specificity and accuracy we developed a sandwich ELISA toneal carcinomatosis [4]. In contrast, colon, gastric, breast system based on human recombinant EpCAM spiked and pancreatic cancer patients frequently form ascites due into ascites as biological matrix. The ELISA system was val- to massive liver metastases with or without peritoneal idated according to “Guidance for Industry - Bioanalytic carcinomatosis [5,6]. In the clinical routine, cytological Method Validation” for measurement in ascites as bio- analysis and biochemical tests are used to confirm periton- logicalmatrix.OurdatagaveevidencethatsEpCAM eal carcinomatosis or portal hypertension as primary cause can be found in 39% of ascites samples with positive for ascites [7]. Malignant ascites caused by peritoneal car- cytology in concentrations significantly higher than in cinomatosis is associated with a poor outcome [8,9]. In ascites samples with negative cytology or specimens the last decade, the understanding of the biology of ma- from patients with liver cirrhosis. lignant ascites has evolved. Markers, such as Carcinoem- To study effects of sEpCAM on catumaxomab activity, bryonic Antigen (CEA) or Vascular Endothelial Growth we performed antibody-dependent cell-mediated cyto- Factor (VEGF) have been described to play a role in the toxicity (ADCC) assays in vitro.Ourin vitro assay con- pathogenesis of malignancy-related ascites [10-12]. Fre- firmed that sEpCAM concentrations found in the C+ quently, these markers correlate with malignancy and cohort of patients are able to neutralize catumaxomab- increase the sensitivity of cytology analysis, which is ap- dependent cell-mediated cytotoxicity. proximately 58 to 75% to predict peritoneal carcinoma- tosis [13,14]. Methods EpCAM is a tumor-associated membrane marker Patients and specimens overexpressed in various epithelial malignancies and it Ascites specimens from 102 patients from the period has been reported to enhance tumor signaling and pro- between 2011 and 2013 were retrieved from the local liferation [15-18]. EpCAM can undergo regulated intra- bio-bank repository. Ascites samples without anticoag- membranous proteolysis (RIP) thereby, translocating ulants were centrifuged at 2,000 x g for 10 minutes to the intracellular domain to the nucleus and shedding separate cellular components from the fluid and cell the extracellular domain to the extracellular compart- free supernatants (plasma) were stored at −20°C. This ment [17]. Consequently, the soluble variant of the retrospective analysis was approved by the ethic com- extracellular domain of EpCAM (EpEX) has been found mittee of Merano (I) after oral and written informed in sera of cancer patients [19]. EpEX has been shown consents of patients (ethics protocol Nr. 16/2011). to support invasion processes of breast carcinoma cells by supporting c-jun signaling [20]. EpEX has been de- Generation of lentiviral expression plasmids tected by different ELISA-systems in serum samples of The plasmids EpCAM-YFP, EpICD-YFP and YFP in the healthy and cancer patients, but no accurate reference pEYFP-N1 vector backbone were a generous gift of Dr. levels have been determined so far [21,22]. Due to its Olivier Gires and are described by Maetzel etal.[17] broad expression in most frequent epithelial cancers, The extracellular part of EpCAM with membrane an- EpCAM became an attractive antigen for targeted ther- chor (EpEX-TM) was cloned by amplifying the frag- apies. However, most EpCAM-targeting agents did not ment by the use of KOD polymerase (Novagen) and hold promise [23] and further studies on predictive bio- specific forward (5-TTA GTG AAC CGT CAG ATC markers are necessary to privilege the right patient col- CGC TAG C) and reverse primers (5-GGC GAC CGG lectives for EpCAM-targeted therapies [24]. EpCAM TGA AAT AAC CAG CAC AAC). PCR fragments were gene expression has been observed in cancer cells of digested with Nhe-I, Age-I (NEB) and ligated into the approximately 75% of patients with malignant ascites predigested p-EYFP-N1 vector (Nhe-I, Age-I, NEB) by [25] and therefore, the EpCAM-targeting antibody the use of the Quick ligation Kit (NEB). Open reading catumaxomab was approved in 2009 by the European frames were cut out by Nhe-I and Not-I (NEB), polished Union for the intraperitoneal treatment of patients with Klenow (NEB) and ligated into the pENTR-11 Seeber et al. BMC Cancer (2015) 15:372 Page 3 of 12 Gateway vector (Invitrogen), predigested with Xmn-I protein production transfected HEK293FT cells were and Eco RV (NEB) and polished with Klenow. All amp- cultivated in serum-free Ex-cell ACF medium (SIGMA lified and purified pENTR-11 vectors were sequenced for Biochemicals) for 4 days. Supernatants of HEK293FT correct orientation and exclusion of incorporated muta- were prepared by high speed centrifugation (10,000 x g, tions. pENTR-11 vectors were site-specifically recombined 20 min, 4°C) and sterile filtered by the use of a 0.2 μm with the pLenti6-V5 DEST vector (Invitrogen) using the filter (Millipore). Next,
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