Journal of Experimental & Clinical Cancer Research, 2015
Uterine and ovarian carcinosarcomas (CS) are rare but highly aggressive gynecologic tumors which ... more Uterine and ovarian carcinosarcomas (CS) are rare but highly aggressive gynecologic tumors which carry an extremely poor prognosis. We evaluated the expression levels of EpCAM and the in vitro activity of solitomab, a bispecific single-chain antibody construct which targets epithelial-cell-adhesion-molecule (EpCAM) on tumor cells and also contains a CD3 binding region, against primary uterine and ovarian CS cell lines. EpCAM expression was evaluated by flow cytometry in a total of 5 primary CS cell lines. Sensitivity to solitomab-dependent-cellular-cytotoxicity (ADCC) was tested against the panel of primary CS cell lines expressing different levels of EpCAM in standard 4 h (51)Cr release-assays. The proliferative activity, activation, cytokine secretion (i.e., Type I vs Type II) and cytotoxicity of solitomab in autologous tumor-associated-T cells (TAL) in the pleural fluid of a CS patient were also evaluated by CFSE and flow-cytometry assays. Surface expression of EpCAM was found in 80.0 % (4 out of 5) of the CS cell lines tested by flow cytometry. EpCAM positive cell lines were found resistant to NK or T-cell-mediated killing after exposure to peripheral blood lymphocytes (PBL) in 4-h chromium-release assays (mean killing ± SEM = 1.1 ± 1.6 %, range 0-5.3 % after incubation of EpCAM positive cell lines with control BiTE®). In contrast, after incubation with solitomab, EpCAM positive CS cells became highly sensitive to T-cell-cytotoxicity (mean killing ± SEM of 19.7 ± 6.3 %; range 10.0-32.0 %; P < 0.0001). Ex vivo incubation of autologous TAL with EpCAM expressing malignant cells in pleural effusion with solitomab, resulted in a significant increase in T-cell proliferation in both CD4+ and CD8+ T cells, increase in T-cell activation markers (i.e., CD25 and HLA-DR), and a reduction in number of viable CS cells in the exudate (P < 0.001). Solitomab may represent an effective treatment for patients with recurrent/metastatic and/or chemo-resistant CS overexpressing EpCAM.
HER2/neu gene amplification and PIK3CA driver mutations are common in uterine serous carcinoma (U... more HER2/neu gene amplification and PIK3CA driver mutations are common in uterine serous carcinoma (USC), and may represent ideal therapeutic targets against this aggressive variant of endometrial cancer. We examined the sensitivity to neratinib, taselisib and the combination of the two compounds in in vitro and in vivo experiments using PIK3CA mutated and PIK3CA-wild type HER2/neu amplified USC cell lines. Cell viability and cell cycle distribution were assessed using flow-cytometry assays. Downstream signaling was assessed by immunoblotting. Preclinical efficacy of single versus dual inhibition was evaluated in vivo using two USC-xenografts. We found both single agent neratinib and taselisib to be active but only transiently effective in controlling the in vivo growth of USC xenografts harboring HER2/neu gene amplification with or without oncogenic PIK3CA mutations. In contrast, the combination of the two inhibitors caused a stronger and long lasting growth inhibition in both USC xeno...
We evaluated the role of PIK3CA-mutations as mechanism of resistance to trastuzumab in primary HE... more We evaluated the role of PIK3CA-mutations as mechanism of resistance to trastuzumab in primary HER2/neu-amplified uterine-serous-carcinoma (USC) cell lines. Fifteen whole-exome-sequenced USC cell lines were tested for HER2/neu-amplification and PIK3CA-mutations. Four HER2/neu-amplified USC (2-harbouring wild-type-PIK3CA-genes and 2-harbouring oncogenic-PIK3CA-mutations) were evaluated in in vitro dose-titration-proliferation-assays, cell-viability and HER2 and S6-protein-phosphorylation after exposure to trastuzumab. USC harbouring wild-type-PIK3CA were transfected with plasmids encoding oncogenic PIK3CA-mutations (i.e., H1047R/R93Q) and exposed to trastuzumab. Finally, trastuzumab efficacy was tested by using two USC xenograft mouse models. Seven out of fifteen (46%) of the USC cell lines were HER2/neu-amplified by fluorescence in situ hybridisation. Within these tumours four out of seven (57%) were found to harbour oncogenic PIK3CA-mutations vs two out of eight (25%) of the HER2/n...
American Journal of Obstetrics and Gynecology, 2015
Uterine serous carcinoma (USC) is an aggressive form of endometrial cancer which carries an extre... more Uterine serous carcinoma (USC) is an aggressive form of endometrial cancer which carries an extremely poor prognosis. Solitomab is a novel bispecific single-chain antibody construct which targets epithelial-cell-adhesion-molecule (EpCAM) on tumor cells and also contains a CD3 binding region. We evaluated the expression levels of EpCAM and the in vitro activity of solitomab against primary USC cell lines in vitro and ex-vivo in the ascites of USC patients. To determine the frequency of expression of EpCAM on uterine serous carcinoma cell lines as well as the ability of solitomab to modulate immune responses (T-cell proliferation, activation, cytokine production, and tumor killing) to tumor cells when it is combined with lymphocytes and EpCAM positive cell lines or EpCAM positive acitic fluid in vitro. EpCAM expression was evaluated by flow cytometry in a total of 14 primary USC cell lines. Sensitivity to solitomab-dependent-cellular-cytotoxicity (ADCC) was tested against a panel of primary USC cell lines expressing different levels of EpCAM in standard 4h (51)Cr release-assays. The proliferative activity, activation, cytokine secretion (i.e., Type I vs Type II) and cytotoxicity of solitomab in autologous tumor-associated-T cells (TAL) in the ascitic fluid of USC patients was also evaluated by CFSE and flow-cytometry assays. Differences in EpCAM expression, ADCC levels were analyzed using upaired t test. T-cell activation marker increase and cytokine release were analyzed by paired t test. Surface expression of EpCAM was found in 85.7% (12 out of 14) of the USC cell lines tested by flow cytometry. EpCAM positive cell lines were found resistant to NK or T-cell-mediated killing after exposure to peripheral blood lymphocytes (PBL) in 4-hour chromium-release assays (mean killing ± SEM, 2.7 ± 3.1% after incubation of EpCAM positive cell lines with control BiTE®). In contrast, after incubation with solitomab, EpCAM positive USC cells became highly sensitive to T cell cytotoxicity (mean killing ± SEM of 25.7 ± 4.5%; P < 0.0001) by PBL. Ex vivo incubation of autologous tumor associated lymphocytes (TAL) with EpCAM expressing malignant cells in ascites with solitomab, resulted in a significant increase in T-cell proliferation in both CD4+ and CD8+ T cells, increase in T-cell activation markers (i.e., CD25 and HLA-DR), and a reduction in number of viable USC cells in ascites (P < 0.001). Solitomab induces robust immunologic responses in vitro resulting in increased T-cell activation, proliferation, production of cytokines, and direct killing of tumor cells. These finding suggest that solitomab may represent a novel, potentially effective agent for treatment of recurrent/metastatic and/or chemo-resistant USC overexpressing EpCAM.
Around 7-10% of endometrial carcinomas are characterized by polymerase-ε-(POLE) exonuclease-domai... more Around 7-10% of endometrial carcinomas are characterized by polymerase-ε-(POLE) exonuclease-domain-mutations, an ultra-mutated-phenotype and a favorable prognosis. It is currently unknown whether POLE ultra-mutated-tumors are more immunogenic when compared to the other groups of endometrial cancers. We used autologous-dendritic-cells (DC) pulsed with whole-tumor-extracts to assess the level of CD8+ and CD4+ T-cell-activation induced by POLE-ultramutated (+) and POLE wild-type (-) endometrial cancer cells in vitro. T-lymphocyte-proliferations were evaluated using CFSE and/or ([3H])thymidine-incorporation-assays while the ability to specifically kill autologous-tumor-cells by cytotoxic-T-lymphocyte (CTL) was tested in standard 4-h-(51)Cr-cytotoxicity-assays. In order to correlate cytotoxic activity and proliferation by CD4+ and CD8+ T-lymphocytes, respectively, with a particular lymphoid subset, two-color-flow-cytometric analysis of intracellular-cytokine-expression (IFN-γ vs IL-4) at the single cell level was also performed. DC-pulsed with tumor extracts were able to induce CTL-responses against autologous-tumor-cells in both POLE (+) and POLE (-) cancer patients (P=0.305). These CD8+ T-cell-populations were cytotoxic against tumor-cells but they did not lyse PHA-stimulated-autologous-lymphocytes or autologous-EBV-transformed-lymphoblastoid-control-cell-lines. In contrast, only POLE (+) tumor-lysate-pulsed-DC were able to induce significant proliferation and high IFN-γ expression (i.e., Th1-cytokine-bias) in autologous in vitro DC-stimulated CD4+ T-cells as well as naïve CD4+ and CD8+ T-cells from patients-peripheral-blood (P<0.05). POLE ultra-mutated-tumors are significantly more immunogenic when compared to POLE (-) tumors, in particular to the helper arm of the immune system. These data lend support to the hypothesis that the better prognosis of patients with POLE (+) tumors may at least in part be linked to their enhanced immunogenicity.
Journal of Experimental & Clinical Cancer Research, 2015
Uterine and ovarian carcinosarcomas (CS) are rare but highly aggressive gynecologic tumors which ... more Uterine and ovarian carcinosarcomas (CS) are rare but highly aggressive gynecologic tumors which carry an extremely poor prognosis. We evaluated the expression levels of EpCAM and the in vitro activity of solitomab, a bispecific single-chain antibody construct which targets epithelial-cell-adhesion-molecule (EpCAM) on tumor cells and also contains a CD3 binding region, against primary uterine and ovarian CS cell lines. EpCAM expression was evaluated by flow cytometry in a total of 5 primary CS cell lines. Sensitivity to solitomab-dependent-cellular-cytotoxicity (ADCC) was tested against the panel of primary CS cell lines expressing different levels of EpCAM in standard 4 h (51)Cr release-assays. The proliferative activity, activation, cytokine secretion (i.e., Type I vs Type II) and cytotoxicity of solitomab in autologous tumor-associated-T cells (TAL) in the pleural fluid of a CS patient were also evaluated by CFSE and flow-cytometry assays. Surface expression of EpCAM was found in 80.0 % (4 out of 5) of the CS cell lines tested by flow cytometry. EpCAM positive cell lines were found resistant to NK or T-cell-mediated killing after exposure to peripheral blood lymphocytes (PBL) in 4-h chromium-release assays (mean killing ± SEM = 1.1 ± 1.6 %, range 0-5.3 % after incubation of EpCAM positive cell lines with control BiTE®). In contrast, after incubation with solitomab, EpCAM positive CS cells became highly sensitive to T-cell-cytotoxicity (mean killing ± SEM of 19.7 ± 6.3 %; range 10.0-32.0 %; P < 0.0001). Ex vivo incubation of autologous TAL with EpCAM expressing malignant cells in pleural effusion with solitomab, resulted in a significant increase in T-cell proliferation in both CD4+ and CD8+ T cells, increase in T-cell activation markers (i.e., CD25 and HLA-DR), and a reduction in number of viable CS cells in the exudate (P < 0.001). Solitomab may represent an effective treatment for patients with recurrent/metastatic and/or chemo-resistant CS overexpressing EpCAM.
HER2/neu gene amplification and PIK3CA driver mutations are common in uterine serous carcinoma (U... more HER2/neu gene amplification and PIK3CA driver mutations are common in uterine serous carcinoma (USC), and may represent ideal therapeutic targets against this aggressive variant of endometrial cancer. We examined the sensitivity to neratinib, taselisib and the combination of the two compounds in in vitro and in vivo experiments using PIK3CA mutated and PIK3CA-wild type HER2/neu amplified USC cell lines. Cell viability and cell cycle distribution were assessed using flow-cytometry assays. Downstream signaling was assessed by immunoblotting. Preclinical efficacy of single versus dual inhibition was evaluated in vivo using two USC-xenografts. We found both single agent neratinib and taselisib to be active but only transiently effective in controlling the in vivo growth of USC xenografts harboring HER2/neu gene amplification with or without oncogenic PIK3CA mutations. In contrast, the combination of the two inhibitors caused a stronger and long lasting growth inhibition in both USC xeno...
We evaluated the role of PIK3CA-mutations as mechanism of resistance to trastuzumab in primary HE... more We evaluated the role of PIK3CA-mutations as mechanism of resistance to trastuzumab in primary HER2/neu-amplified uterine-serous-carcinoma (USC) cell lines. Fifteen whole-exome-sequenced USC cell lines were tested for HER2/neu-amplification and PIK3CA-mutations. Four HER2/neu-amplified USC (2-harbouring wild-type-PIK3CA-genes and 2-harbouring oncogenic-PIK3CA-mutations) were evaluated in in vitro dose-titration-proliferation-assays, cell-viability and HER2 and S6-protein-phosphorylation after exposure to trastuzumab. USC harbouring wild-type-PIK3CA were transfected with plasmids encoding oncogenic PIK3CA-mutations (i.e., H1047R/R93Q) and exposed to trastuzumab. Finally, trastuzumab efficacy was tested by using two USC xenograft mouse models. Seven out of fifteen (46%) of the USC cell lines were HER2/neu-amplified by fluorescence in situ hybridisation. Within these tumours four out of seven (57%) were found to harbour oncogenic PIK3CA-mutations vs two out of eight (25%) of the HER2/n...
American Journal of Obstetrics and Gynecology, 2015
Uterine serous carcinoma (USC) is an aggressive form of endometrial cancer which carries an extre... more Uterine serous carcinoma (USC) is an aggressive form of endometrial cancer which carries an extremely poor prognosis. Solitomab is a novel bispecific single-chain antibody construct which targets epithelial-cell-adhesion-molecule (EpCAM) on tumor cells and also contains a CD3 binding region. We evaluated the expression levels of EpCAM and the in vitro activity of solitomab against primary USC cell lines in vitro and ex-vivo in the ascites of USC patients. To determine the frequency of expression of EpCAM on uterine serous carcinoma cell lines as well as the ability of solitomab to modulate immune responses (T-cell proliferation, activation, cytokine production, and tumor killing) to tumor cells when it is combined with lymphocytes and EpCAM positive cell lines or EpCAM positive acitic fluid in vitro. EpCAM expression was evaluated by flow cytometry in a total of 14 primary USC cell lines. Sensitivity to solitomab-dependent-cellular-cytotoxicity (ADCC) was tested against a panel of primary USC cell lines expressing different levels of EpCAM in standard 4h (51)Cr release-assays. The proliferative activity, activation, cytokine secretion (i.e., Type I vs Type II) and cytotoxicity of solitomab in autologous tumor-associated-T cells (TAL) in the ascitic fluid of USC patients was also evaluated by CFSE and flow-cytometry assays. Differences in EpCAM expression, ADCC levels were analyzed using upaired t test. T-cell activation marker increase and cytokine release were analyzed by paired t test. Surface expression of EpCAM was found in 85.7% (12 out of 14) of the USC cell lines tested by flow cytometry. EpCAM positive cell lines were found resistant to NK or T-cell-mediated killing after exposure to peripheral blood lymphocytes (PBL) in 4-hour chromium-release assays (mean killing ± SEM, 2.7 ± 3.1% after incubation of EpCAM positive cell lines with control BiTE®). In contrast, after incubation with solitomab, EpCAM positive USC cells became highly sensitive to T cell cytotoxicity (mean killing ± SEM of 25.7 ± 4.5%; P < 0.0001) by PBL. Ex vivo incubation of autologous tumor associated lymphocytes (TAL) with EpCAM expressing malignant cells in ascites with solitomab, resulted in a significant increase in T-cell proliferation in both CD4+ and CD8+ T cells, increase in T-cell activation markers (i.e., CD25 and HLA-DR), and a reduction in number of viable USC cells in ascites (P < 0.001). Solitomab induces robust immunologic responses in vitro resulting in increased T-cell activation, proliferation, production of cytokines, and direct killing of tumor cells. These finding suggest that solitomab may represent a novel, potentially effective agent for treatment of recurrent/metastatic and/or chemo-resistant USC overexpressing EpCAM.
Around 7-10% of endometrial carcinomas are characterized by polymerase-ε-(POLE) exonuclease-domai... more Around 7-10% of endometrial carcinomas are characterized by polymerase-ε-(POLE) exonuclease-domain-mutations, an ultra-mutated-phenotype and a favorable prognosis. It is currently unknown whether POLE ultra-mutated-tumors are more immunogenic when compared to the other groups of endometrial cancers. We used autologous-dendritic-cells (DC) pulsed with whole-tumor-extracts to assess the level of CD8+ and CD4+ T-cell-activation induced by POLE-ultramutated (+) and POLE wild-type (-) endometrial cancer cells in vitro. T-lymphocyte-proliferations were evaluated using CFSE and/or ([3H])thymidine-incorporation-assays while the ability to specifically kill autologous-tumor-cells by cytotoxic-T-lymphocyte (CTL) was tested in standard 4-h-(51)Cr-cytotoxicity-assays. In order to correlate cytotoxic activity and proliferation by CD4+ and CD8+ T-lymphocytes, respectively, with a particular lymphoid subset, two-color-flow-cytometric analysis of intracellular-cytokine-expression (IFN-γ vs IL-4) at the single cell level was also performed. DC-pulsed with tumor extracts were able to induce CTL-responses against autologous-tumor-cells in both POLE (+) and POLE (-) cancer patients (P=0.305). These CD8+ T-cell-populations were cytotoxic against tumor-cells but they did not lyse PHA-stimulated-autologous-lymphocytes or autologous-EBV-transformed-lymphoblastoid-control-cell-lines. In contrast, only POLE (+) tumor-lysate-pulsed-DC were able to induce significant proliferation and high IFN-γ expression (i.e., Th1-cytokine-bias) in autologous in vitro DC-stimulated CD4+ T-cells as well as naïve CD4+ and CD8+ T-cells from patients-peripheral-blood (P<0.05). POLE ultra-mutated-tumors are significantly more immunogenic when compared to POLE (-) tumors, in particular to the helper arm of the immune system. These data lend support to the hypothesis that the better prognosis of patients with POLE (+) tumors may at least in part be linked to their enhanced immunogenicity.
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