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Analysis of the immune cell composition in serous ovarian cancer
Abstracts / Gynecologic Oncology 133 (2014) 2–207
extracted from vaginal tampons. With further development, this technology holds promise for screening for this deadly disease.
117
trastuzumab demonstrated significant synergy in the HER2 geneamplified models. Dual targeting of HER2 may be a promising avenue for future investigation in HER2-amplified UPSC.
doi:10.1016/j.ygyno.2014.03.307 doi:10.1016/j.ygyno.2014.03.308 288 - Poster Session A Dual HER2 targeting impedes growth of HER2 gene-amplified uterine papillary serous carcinoma xenografts W.B. Growdon1, J.W. Groeneweg1, V.F. Byron1, S.F. Hernandez1, D.R. Borger1, R. Tambouret1, J.O. Schorge2, M.G. Del Carmen2, R. Foster1, B.R. Rueda1. 1Massachusetts General Hospital, Boston, MA, USA, 2Massachusetts General Hospital/Harvard University, Boston, MA, USA. Objectives: Uterine papillary serous carcinoma (UPSC) is an aggressive tumor with a high rate of HER2 gene amplification. Since combination anti-HER2 therapy has shown efficacy in other HER2amplified cancers, we measured the combined effect of lapatinib and trastuzumab on the growth of tumor xenografts derived from human UPSC cell lines and primary human UPSCs. Methods: Human UPSC cell lines (ARK2, SPEC2) and prospectively collected tissue from two UPSC patients undergoing primary surgery (ENCA1, ENCA2) were obtained. HER2 protein expression and gene amplification were assessed in all samples. ARK2, SPEC2, ENCA1, and ENCA2 cells were injected subcutaneously into female NOD/SCID mice. Tumor formation was monitored regularly and mice were randomized into four treatment arms (5 mice/arm) once tumor volume reached 200 to 400 mm3. Lapatinib (100 mg/kg) was dosed by oral gavage 6 out of every 7 days, and 10 mg/kg trastuzumab was administered by intraperitoneal injection 3 times a week. Tumor size was monitored every 3 days. Posttreatment analysis of xenografts was carried out by immunohistochemistry and Western blot. Wilcoxon ranksum testing was used to compare xenograft growth across treatment arms. Results: ARK2- and ENCA1-derived xenografts exhibited HER2 gene amplification and 3+ protein expression. SPEC2- and ENCA2-derived xenografts were disomic for HER2 with 1+ protein expression. In all xenografts, trastuzumab exhibited no single-agent antitumor efficacy. Dual administration of trastuzumab and lapatinib resulted in significant synergistic activity (P b 0.01) only in those xenografts exhibiting HER2 gene amplification. Effective anti-HER2 therapy was associated with decreased Ki67 expression and decreased phosphorylation of AKT and ERK. In the non-HER2 amplified models, no alterations in downstream signaling were observed and no tumor response was manifested. Conclusions: Our data suggested that single-agent trastuzumab has no antitumor activity in UPSC, even in the setting of HER2 gene amplification. When administered in concert with lapatinib, however,
289 - Poster Session A Use of SRC pathway activation in predicting dasatinib activity in ovarian cancer A. Wallace1, D. Corcoran2, M. Lopez1, D.G. Teoh3, D. Adams1, L. Grace1, A.A. Secord1. 1Duke University Medical Center, Durham, NC, USA, 2Duke Institute for Genome Sciences and Policy, Durham, NC, USA, 3 University of Minnesota, Minneapolis, MN, USA. Objectives: To determine if genomic biomarkers, including SRC pathway activation, can predict dasatinib antiproliferative activity in ovarian cancer. Methods: Eighteen ovarian cancer cell lines were treated with singleagent dasatinib and dose-response curves were constructed. The cell lines were stratified into dasatinib-sensitive and dasatinib-resistant cohorts based on median inhibition concentration (IC50) values. Pretreatment gene expression profiles were used to assess for associations between SRC pathway activation as well as other genomic biomarkers and dasatinib’s antiproliferative effect. SRC pathway expression was determined from the normalized array data by an 85-gene SRC signature decomposed into three factors based on singular value decomposition. A Bayesian probit regression model was fit to the three factors (Monte Carlo Markov Chain algorithm) and the SRC pathway activation level was scored. Results: Seven cell lines were considered dasatinib-sensitive (IC50 b 400 nmol), while 11 were dasatinib-resistant (IC50 N 400 nmol) (IC50 range, 0.1–5000 nmol). SRC pathway activation ranged from 15% to 83%. There was a marginal association between dasatinib activity and SRC pathway activation (P = 0.1). Specifically, cell lines that exhibited very low SRC activation (lowest quartile) were more likely to be sensitive to dasatinib. In contrast, cell lines with the highest SRC pathway activation (top quartile) were likely to be dasatinib-resistant. There was no association between dasatinib antiproliferative activity and other previously reported molecular markers for dasatinib activity (annexin1, IGFBP2, SRC, YES1, LYN, EPHA2, CAV1/2, or MSN). Conclusions: SRC pathway activation may be indicative of dasatinib activity in ovarian cancer. However, further evaluation is needed to identify the gene(s) within this pathway that are most predictive of dasatinib activity. doi:10.1016/j.ygyno.2014.03.309
290 - Poster Session A Analysis of the immune cell composition in serous ovarian cancer C.J. Stashwick, A.F. Haggerty, G. Kari, T. Garrabrant, A. Best, K.S. Tan, W.T. Hwang, G. Coukos, D.J. Powell Jr. University of Pennsylvania, Philadelphia, PA, USA. Objectives: To assess the immune cell composition in serous ovarian cancer by flow cytometry and its association with progression-free survival. Methods: Fresh tumor samples from an institutional review boardapproved human ovarian cancer tumor bank were enzymatically digested and analyzed by flow cytometry. A total of 89 samples (83 tumor and 6 ascites) from primary and recurrent ovarian cancer were analyzed for frequency of tumor cells and leukocytes among viable cells using 7AAD, EpCAM, CD45, CD3, CD19, and CD14. Descriptive statistics,
118
Abstracts / Gynecologic Oncology 133 (2014) 2–207
Wilcoxon rank sum tests, Kaplan–Meier curves, log-rank test, and Cox regression models for progression-free survival were performed using Stata statistical software. Results: Across all samples (n = 89), the median frequency of EpCAM+ cells was 20.7% (range, 0% to 78%), and among CD45+ leukocytes, the median frequency of T-cells was 36.7% (range, 5% to 87%), of B-cells was 3.3% (range, 0% to 28%), and of monocytes was 19.1% (range, 0% to 77%). Compared to the matched solid tumor sample from the same patient, ascites fluid gave higher frequencies of T-cells but lower frequencies of EpCAM+ cells and monocytes. Of the patients undergoing primary surgery (n = 50), those who had received neoadjuvant chemotherapy (NACT) (n = 6) had much lower frequencies of EpCAM + cells (0.8% vs 27.8%, P b 0.001) and monocytes (7.6% vs 23.5%, P b 0.05) compared to those who had not received NACT (n = 44). The frequency of T-cells was not statistically different between these groups. The same pattern was seen when recurrent and primary cancer samples previously exposed to chemotherapy were combined (n = 39) and compared to samples not exposed to chemotherapy (n = 44) (EpCAM+: 16.8% vs 27.8%, P = 0.03 and monocytes: 16.9% vs 23.5%, P = 0.02). Trends toward worse progression-free survival with lower frequencies of T-cells was observed and met statistical significance comparing the lowest 10% percentile Tcell group with the upper 90% percentile group (HR 3.66, 95% CI 1.19– 11.29, P = 0.024) (Figure). Conclusions: Tumor and immune cell frequency analysis by flow cytometry was feasible and gave information pertinent to immunebased therapy. While intratumoral EpCAM + cells and monocytes were decreased in patients exposed to chemotherapy, T-cell frequencies were preserved. Furthermore, this analysis suggests worse progression-free survival in patients with low T-cell frequencies.
and their genetic and phenotypic stability for gynecologic cancer including ovarian, endometrial and cervical cancer. Methods: Small pieces (3 × 3 × 3 mm) of human gynecologic cancer tissue (n = 94) were meticulously grafted under renal capsules of female BALB/C-nude mice within 2 h of surgical removal. Grossly visible tumor tissues serially transplanted for 2–5 generations. After the development of tumor in mice, phenotypic and genetic comparisons were performed between primary tumor and corresponding transplantable xenografts using H&E, Ion Torrent (AmpliSeq Cancer Panel), and array-comparative genomic hybridization (aCGH) analysis. Results: Total tumor tissue engraftment rate was 37.2% (35/94) including ovarian cancer 32.8% (21/64), cervical cancer 47.6% (10/21) and endometrial cancer 44.4% (4/9). The mean time to the development of first generation in mice was 6.6 months in ovarian, 5.5 months in cervical and 4 months in endometrial cancer. Comparison of primary and Avatar tumor tissues showed highly similar histopathologic features. Moreover, analysis of Ion Torrent and aCGH indicated that all examined mutation and genomic alterations found in primary cancer tissues were precisely replicated in the corresponding Avatar tumors. Conclusions: Avatar mice for human gynecologic cancer can be developed as a method of subrenal capsule implantation and have very similar phenotypic and genetic alteration of the original tissues. This has the potential to provide a very effective tool for future personalized therapy and for conducting translational gynecologic cancer research. doi:10.1016/j.ygyno.2014.03.311
292 - Poster Session A The microenvironmental effects of endometriosis on VCAM1 and IL-10 expression in early stage epithelial ovarian cancer J. Nakayama1, L. Duska1, K. Atkins2, J.M. Scalici3, M. Smolkin4, M. Conaway5, J. Slack-Davis5. 1University of Virginia Health System, Charlottesville, VA, USA, 2University of Virginia Medical Center, Charlottesville, VA, USA, 3University of South Alabama, Mobile, AL, USA, 4University of Virginia School of Medicine, Charlottesville, VA, USA, 5 University of Virginia, Charlottesville, VA, USA.
doi:10.1016/j.ygyno.2014.03.310
291 - Poster Session A Patient-derived tumor xenograft model (“avatar mice”) for gynecologic cancer J.W. Lee1, Y.J. Cho2, S.Y. Song1, B.G. Kim3, D.S. Bae1. 1Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea, 2Samsung Medical Center, Seoul, South Korea, 3Sungkyunkwan University School of Medicine, Seoul, South Korea. Objectives: Patient-derived tumor xenograft model (Avatar mice) may provide more accurate and reliable information about individual patients' tumor biology when compared with established cell line model. This study was designed to study the development of Avatar mice
Objectives: We have previously shown that vascular cellular adhesion molecule-1 (VCAM1) is overexpressed in the peritoneum of advanced epithelial ovarian cancer (EOC) patients but not in stage I EOC. VCAM1 is also highly expressed in endometriosis. Interleukin (IL)-10 is thought to promote EOC development and inhibit endometriosis. We hypothesized that VCAM1 and IL-10 are involved in the development of endometriosis-associated EOC. Methods: Seventeen patients with stage I EOC and endometriosis were identified. Tissue samples were stained with a VCAM1-specific antibody and compared to samples from 24 patients with only stage I EOC and 25 with only endometriosis. Specimens from 10 patients with EOC and endometriosis, 6 with endometriosis only, and 8 with EOC only were stained with an IL-10-specific antibody. All samples were read by one blinded pathologist. VCAM1 was scored as positive or negative and IL-10 as the percentage of positive cells. The effect of group on VCAM1 and IL-10 were evaluated with a regression and the Jonckherre–Terpstra test (for IL-10). VCAM1's effect on IL-10 was evaluated with a regression. Results: VCAM-1 was positive in 56% of endometriosis, 29% of EOC, and 24% of EOC and endometriosis samples. The frequency of VCAM1 expression was higher in patients with only endometriosis compared to those with only EOC (P = 0.042) or endometriosis and EOC (P = 0.049). There was no difference in VCAM1 expression between patients with EOC only and those with EOC and endometriosis. The median percentage of IL-10 positive cells was 18% in endometriosis, 30% in EOC, and 85% in endometriosis and EOC. IL-10 expression was higher in patients with endometriosis and EOC compared with
extracted from vaginal tampons. With further development, this technology holds promise for screening for this deadly disease.
117
trastuzumab demonstrated significant synergy in the HER2 geneamplified models. Dual targeting of HER2 may be a promising avenue for future investigation in HER2-amplified UPSC.
doi:10.1016/j.ygyno.2014.03.307 doi:10.1016/j.ygyno.2014.03.308 288 - Poster Session A Dual HER2 targeting impedes growth of HER2 gene-amplified uterine papillary serous carcinoma xenografts W.B. Growdon1, J.W. Groeneweg1, V.F. Byron1, S.F. Hernandez1, D.R. Borger1, R. Tambouret1, J.O. Schorge2, M.G. Del Carmen2, R. Foster1, B.R. Rueda1. 1Massachusetts General Hospital, Boston, MA, USA, 2Massachusetts General Hospital/Harvard University, Boston, MA, USA. Objectives: Uterine papillary serous carcinoma (UPSC) is an aggressive tumor with a high rate of HER2 gene amplification. Since combination anti-HER2 therapy has shown efficacy in other HER2amplified cancers, we measured the combined effect of lapatinib and trastuzumab on the growth of tumor xenografts derived from human UPSC cell lines and primary human UPSCs. Methods: Human UPSC cell lines (ARK2, SPEC2) and prospectively collected tissue from two UPSC patients undergoing primary surgery (ENCA1, ENCA2) were obtained. HER2 protein expression and gene amplification were assessed in all samples. ARK2, SPEC2, ENCA1, and ENCA2 cells were injected subcutaneously into female NOD/SCID mice. Tumor formation was monitored regularly and mice were randomized into four treatment arms (5 mice/arm) once tumor volume reached 200 to 400 mm3. Lapatinib (100 mg/kg) was dosed by oral gavage 6 out of every 7 days, and 10 mg/kg trastuzumab was administered by intraperitoneal injection 3 times a week. Tumor size was monitored every 3 days. Posttreatment analysis of xenografts was carried out by immunohistochemistry and Western blot. Wilcoxon ranksum testing was used to compare xenograft growth across treatment arms. Results: ARK2- and ENCA1-derived xenografts exhibited HER2 gene amplification and 3+ protein expression. SPEC2- and ENCA2-derived xenografts were disomic for HER2 with 1+ protein expression. In all xenografts, trastuzumab exhibited no single-agent antitumor efficacy. Dual administration of trastuzumab and lapatinib resulted in significant synergistic activity (P b 0.01) only in those xenografts exhibiting HER2 gene amplification. Effective anti-HER2 therapy was associated with decreased Ki67 expression and decreased phosphorylation of AKT and ERK. In the non-HER2 amplified models, no alterations in downstream signaling were observed and no tumor response was manifested. Conclusions: Our data suggested that single-agent trastuzumab has no antitumor activity in UPSC, even in the setting of HER2 gene amplification. When administered in concert with lapatinib, however,
289 - Poster Session A Use of SRC pathway activation in predicting dasatinib activity in ovarian cancer A. Wallace1, D. Corcoran2, M. Lopez1, D.G. Teoh3, D. Adams1, L. Grace1, A.A. Secord1. 1Duke University Medical Center, Durham, NC, USA, 2Duke Institute for Genome Sciences and Policy, Durham, NC, USA, 3 University of Minnesota, Minneapolis, MN, USA. Objectives: To determine if genomic biomarkers, including SRC pathway activation, can predict dasatinib antiproliferative activity in ovarian cancer. Methods: Eighteen ovarian cancer cell lines were treated with singleagent dasatinib and dose-response curves were constructed. The cell lines were stratified into dasatinib-sensitive and dasatinib-resistant cohorts based on median inhibition concentration (IC50) values. Pretreatment gene expression profiles were used to assess for associations between SRC pathway activation as well as other genomic biomarkers and dasatinib’s antiproliferative effect. SRC pathway expression was determined from the normalized array data by an 85-gene SRC signature decomposed into three factors based on singular value decomposition. A Bayesian probit regression model was fit to the three factors (Monte Carlo Markov Chain algorithm) and the SRC pathway activation level was scored. Results: Seven cell lines were considered dasatinib-sensitive (IC50 b 400 nmol), while 11 were dasatinib-resistant (IC50 N 400 nmol) (IC50 range, 0.1–5000 nmol). SRC pathway activation ranged from 15% to 83%. There was a marginal association between dasatinib activity and SRC pathway activation (P = 0.1). Specifically, cell lines that exhibited very low SRC activation (lowest quartile) were more likely to be sensitive to dasatinib. In contrast, cell lines with the highest SRC pathway activation (top quartile) were likely to be dasatinib-resistant. There was no association between dasatinib antiproliferative activity and other previously reported molecular markers for dasatinib activity (annexin1, IGFBP2, SRC, YES1, LYN, EPHA2, CAV1/2, or MSN). Conclusions: SRC pathway activation may be indicative of dasatinib activity in ovarian cancer. However, further evaluation is needed to identify the gene(s) within this pathway that are most predictive of dasatinib activity. doi:10.1016/j.ygyno.2014.03.309
290 - Poster Session A Analysis of the immune cell composition in serous ovarian cancer C.J. Stashwick, A.F. Haggerty, G. Kari, T. Garrabrant, A. Best, K.S. Tan, W.T. Hwang, G. Coukos, D.J. Powell Jr. University of Pennsylvania, Philadelphia, PA, USA. Objectives: To assess the immune cell composition in serous ovarian cancer by flow cytometry and its association with progression-free survival. Methods: Fresh tumor samples from an institutional review boardapproved human ovarian cancer tumor bank were enzymatically digested and analyzed by flow cytometry. A total of 89 samples (83 tumor and 6 ascites) from primary and recurrent ovarian cancer were analyzed for frequency of tumor cells and leukocytes among viable cells using 7AAD, EpCAM, CD45, CD3, CD19, and CD14. Descriptive statistics,
118
Abstracts / Gynecologic Oncology 133 (2014) 2–207
Wilcoxon rank sum tests, Kaplan–Meier curves, log-rank test, and Cox regression models for progression-free survival were performed using Stata statistical software. Results: Across all samples (n = 89), the median frequency of EpCAM+ cells was 20.7% (range, 0% to 78%), and among CD45+ leukocytes, the median frequency of T-cells was 36.7% (range, 5% to 87%), of B-cells was 3.3% (range, 0% to 28%), and of monocytes was 19.1% (range, 0% to 77%). Compared to the matched solid tumor sample from the same patient, ascites fluid gave higher frequencies of T-cells but lower frequencies of EpCAM+ cells and monocytes. Of the patients undergoing primary surgery (n = 50), those who had received neoadjuvant chemotherapy (NACT) (n = 6) had much lower frequencies of EpCAM + cells (0.8% vs 27.8%, P b 0.001) and monocytes (7.6% vs 23.5%, P b 0.05) compared to those who had not received NACT (n = 44). The frequency of T-cells was not statistically different between these groups. The same pattern was seen when recurrent and primary cancer samples previously exposed to chemotherapy were combined (n = 39) and compared to samples not exposed to chemotherapy (n = 44) (EpCAM+: 16.8% vs 27.8%, P = 0.03 and monocytes: 16.9% vs 23.5%, P = 0.02). Trends toward worse progression-free survival with lower frequencies of T-cells was observed and met statistical significance comparing the lowest 10% percentile Tcell group with the upper 90% percentile group (HR 3.66, 95% CI 1.19– 11.29, P = 0.024) (Figure). Conclusions: Tumor and immune cell frequency analysis by flow cytometry was feasible and gave information pertinent to immunebased therapy. While intratumoral EpCAM + cells and monocytes were decreased in patients exposed to chemotherapy, T-cell frequencies were preserved. Furthermore, this analysis suggests worse progression-free survival in patients with low T-cell frequencies.
and their genetic and phenotypic stability for gynecologic cancer including ovarian, endometrial and cervical cancer. Methods: Small pieces (3 × 3 × 3 mm) of human gynecologic cancer tissue (n = 94) were meticulously grafted under renal capsules of female BALB/C-nude mice within 2 h of surgical removal. Grossly visible tumor tissues serially transplanted for 2–5 generations. After the development of tumor in mice, phenotypic and genetic comparisons were performed between primary tumor and corresponding transplantable xenografts using H&E, Ion Torrent (AmpliSeq Cancer Panel), and array-comparative genomic hybridization (aCGH) analysis. Results: Total tumor tissue engraftment rate was 37.2% (35/94) including ovarian cancer 32.8% (21/64), cervical cancer 47.6% (10/21) and endometrial cancer 44.4% (4/9). The mean time to the development of first generation in mice was 6.6 months in ovarian, 5.5 months in cervical and 4 months in endometrial cancer. Comparison of primary and Avatar tumor tissues showed highly similar histopathologic features. Moreover, analysis of Ion Torrent and aCGH indicated that all examined mutation and genomic alterations found in primary cancer tissues were precisely replicated in the corresponding Avatar tumors. Conclusions: Avatar mice for human gynecologic cancer can be developed as a method of subrenal capsule implantation and have very similar phenotypic and genetic alteration of the original tissues. This has the potential to provide a very effective tool for future personalized therapy and for conducting translational gynecologic cancer research. doi:10.1016/j.ygyno.2014.03.311
292 - Poster Session A The microenvironmental effects of endometriosis on VCAM1 and IL-10 expression in early stage epithelial ovarian cancer J. Nakayama1, L. Duska1, K. Atkins2, J.M. Scalici3, M. Smolkin4, M. Conaway5, J. Slack-Davis5. 1University of Virginia Health System, Charlottesville, VA, USA, 2University of Virginia Medical Center, Charlottesville, VA, USA, 3University of South Alabama, Mobile, AL, USA, 4University of Virginia School of Medicine, Charlottesville, VA, USA, 5 University of Virginia, Charlottesville, VA, USA.
doi:10.1016/j.ygyno.2014.03.310
291 - Poster Session A Patient-derived tumor xenograft model (“avatar mice”) for gynecologic cancer J.W. Lee1, Y.J. Cho2, S.Y. Song1, B.G. Kim3, D.S. Bae1. 1Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea, 2Samsung Medical Center, Seoul, South Korea, 3Sungkyunkwan University School of Medicine, Seoul, South Korea. Objectives: Patient-derived tumor xenograft model (Avatar mice) may provide more accurate and reliable information about individual patients' tumor biology when compared with established cell line model. This study was designed to study the development of Avatar mice
Objectives: We have previously shown that vascular cellular adhesion molecule-1 (VCAM1) is overexpressed in the peritoneum of advanced epithelial ovarian cancer (EOC) patients but not in stage I EOC. VCAM1 is also highly expressed in endometriosis. Interleukin (IL)-10 is thought to promote EOC development and inhibit endometriosis. We hypothesized that VCAM1 and IL-10 are involved in the development of endometriosis-associated EOC. Methods: Seventeen patients with stage I EOC and endometriosis were identified. Tissue samples were stained with a VCAM1-specific antibody and compared to samples from 24 patients with only stage I EOC and 25 with only endometriosis. Specimens from 10 patients with EOC and endometriosis, 6 with endometriosis only, and 8 with EOC only were stained with an IL-10-specific antibody. All samples were read by one blinded pathologist. VCAM1 was scored as positive or negative and IL-10 as the percentage of positive cells. The effect of group on VCAM1 and IL-10 were evaluated with a regression and the Jonckherre–Terpstra test (for IL-10). VCAM1's effect on IL-10 was evaluated with a regression. Results: VCAM-1 was positive in 56% of endometriosis, 29% of EOC, and 24% of EOC and endometriosis samples. The frequency of VCAM1 expression was higher in patients with only endometriosis compared to those with only EOC (P = 0.042) or endometriosis and EOC (P = 0.049). There was no difference in VCAM1 expression between patients with EOC only and those with EOC and endometriosis. The median percentage of IL-10 positive cells was 18% in endometriosis, 30% in EOC, and 85% in endometriosis and EOC. IL-10 expression was higher in patients with endometriosis and EOC compared with