Overexpression of EpCAM in uterine serous papillary carcinoma: implications for EpCAM-specific immunotherapy with human monoclonal antibody adecatumumab (MT201)

Abstract

We evaluated the expression of epithelial cell adhesion molecule (EpCAM) and the potential of MT201 (adecatumumab), a human monoclonal antibody against EpCAM, in uterine serous papillary carcinoma (USPC). EpCAM expression was evaluated by real-time PCR and immunohistochemistry in a total of 56 USPC fresh-frozen biopsies and paraffin-embedded tissues. EpCAM surface expression was also evaluated by flow cytometry and immunohistochemistry in six USPC cell lines. Sensitivity to MT201 antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity was tested against a panel of primary USPC cell lines expressing different levels of EpCAM in standard 5-h (51)Cr release assays. EpCAM transcript was significantly overexpressed in fresh-frozen USPC when compared with normal endometrial cells (NEC). Median (minimum-maximum) copy number was 943.8 (31.5-1568.3) in tumor samples versus 12.9 (1.0-37.0) in NEC (P < 0.001). By immunohistochemistry, EpCAM expression was found in 96% (26 out of 27) of USPC samples with significantly higher expression compared with NECs (P < 0.001). High surface expression of EpCAM was found in 83% (five out of six) of the USPC cell lines tested by flow cytometry. EpCAM-positive cell lines were found highly sensitive to MT201-mediated antibody-dependent cellular cytotoxicity in vitro, whereas primary USPC cell lines were resistant to natural killer cell-dependent cytotoxicity. Human plasma IgG did not significantly inhibit MT201-mediated cytotoxicity against USPC. EpCAM is highly expressed in uterine serous carcinoma at mRNA and protein levels, and primary USPC are highly sensitivity to MT201-mediated cytotoxicity. MT201 might represent a novel therapeutic strategy in patients harboring advanced/recurrent or metastatic USPC refractory to standard treatment modalities.

Figure 1

Figure 1a. EpCAM mRNA expression levels in primary and metastatic/recurrent USPC compared to normal endometrial tissues (NEC). EpCAM transcript was found to have significantly higher expression in primary and metastatic/recurrent carcinoma when compared pairwise to fresh-frozen normal endometrial tissues (P values <0.0001) Figure 1b. Representative image of normal endometrium showing negative EpCAM immunohistochemical reaction (Upper Panel, 200 x), and a representative primary USPC showing strong (3+) EpCAM expression by immunohistochemistry (Lower Panel, 400 x).

Figure 1

Figure 1a. EpCAM mRNA expression levels in primary and metastatic/recurrent USPC compared to normal endometrial tissues (NEC). EpCAM transcript was found to have significantly higher expression in primary and metastatic/recurrent carcinoma when compared pairwise to fresh-frozen normal endometrial tissues (P values <0.0001) Figure 1b. Representative image of normal endometrium showing negative EpCAM immunohistochemical reaction (Upper Panel, 200 x), and a representative primary USPC showing strong (3+) EpCAM expression by immunohistochemistry (Lower Panel, 400 x).

Figure 2

Dose titration cytotoxicity experiments using MT201 against USPC ARK-5 (upper panel) and USPC ARK-6 (middle panel) cell lines at 25 : 1 effector-target cell ratio. ADCC was found to plateau at a dose of 5 µg/ml to 10 µg/ml in multiple experiments. Lower Panel: example of ADCC in USPC ARK-6 cell line at different effector to target cell ratios in presence or absence of MT201 in 5-h chromium release cytotoxicity assay. High levels of ADCC MT201 induced cytotoxicity was evident against USPC ARK-6 cells while negligible cytotoxicity was detectable in the absence of MT201 or in the presence of rituximab.

Figure 3

Figure 3a. USPC cell lines were challenged by adding human plasma diluted 1:2 (with or without heat inactivation) in the presence or absence of the effector cells and MT201 to standard 5-h ⁵¹Cr release assays. The addition of PBLs with or without plasma (untreated or heat-inactivated) was not able to induce significant cytotoxicity against USPC ARK-2 cell line (Upper Panel). Addition of physiological concentrations of IgG (i.e., heat-inactivated plasma diluted 1 : 2) to PBL in the presence of MT201 did not significantly alter the degree of ADCC achieved in the presence of MT201 (lower panel). In contrast, addition of untreated plasma (diluted 1:2) to PBL in the presence of MT201 consistently increased MT201-mediated cytotoxicity against USPC (p < 0.03) (lower panel). Figure 3b. The effect of low doses of interleukin-2 (IL-2) in combination with MT201 (5 µg/ml) on ADCC against USPC cell lines. PBL from healthy donors were incubated for 5 to 72 h in the presence of 100 IU/ml of IL-2. MT201-mediated ADCC was significantly increased in the presence of low doses of IL-2. No significant increase in cytotoxicity was detected after 5-h IL-2 treatment in the absence of MT201 or in the presence of rituximab isotype control mAb.

Figure 3

Figure 3a. USPC cell lines were challenged by adding human plasma diluted 1:2 (with or without heat inactivation) in the presence or absence of the effector cells and MT201 to standard 5-h ⁵¹Cr release assays. The addition of PBLs with or without plasma (untreated or heat-inactivated) was not able to induce significant cytotoxicity against USPC ARK-2 cell line (Upper Panel). Addition of physiological concentrations of IgG (i.e., heat-inactivated plasma diluted 1 : 2) to PBL in the presence of MT201 did not significantly alter the degree of ADCC achieved in the presence of MT201 (lower panel). In contrast, addition of untreated plasma (diluted 1:2) to PBL in the presence of MT201 consistently increased MT201-mediated cytotoxicity against USPC (p < 0.03) (lower panel). Figure 3b. The effect of low doses of interleukin-2 (IL-2) in combination with MT201 (5 µg/ml) on ADCC against USPC cell lines. PBL from healthy donors were incubated for 5 to 72 h in the presence of 100 IU/ml of IL-2. MT201-mediated ADCC was significantly increased in the presence of low doses of IL-2. No significant increase in cytotoxicity was detected after 5-h IL-2 treatment in the absence of MT201 or in the presence of rituximab isotype control mAb.