Uterine leiomyosarcoma: Are novel therapeutics on the horizon?

Abstracts / Gynecologic Oncology 145 (2017) 2–220

Conclusion: We observed accumulation of gene expression variation depending on progression of recurrence. This phenomenon has shown that ovarian cancer continuously transformed its characteristic via adaptation of chemotherapy. Although we did not analyze the clinical information about patients' chemo regimen, it could be highly feasible that the repetitive use of uniform or simple chemotherapy induced recurrence of ovarian cancer. doi:10.1016/j.ygyno.2017.03.207

180 - Poster Session RNA expression patterns in human ascites-derived ovarian cancer cells allow molecular characterization and identification of potential therapeutic targets M.D. Toboni, T.B. Turner, H.J. Smith, L.A. Norian, J.M. Straughn Jr, R.C. Arend. University of Alabama at Birmingham, Birmingham, AL, USA Objective: Recent studies suggest that immunotherapy may have activity in patients with ovarian cancer. We evaluated ovarian cancer ascites cells for known immunogenic expression patterns and examined response to epigenetic therapy. Method: Ascites cells from 10 patients with papillary serous ovarian cancer were isolated and plated. Cells were treated with a DNA methyltransferase inhibitor (5-azacytidine), a histone deacetylase inhibitor (entinostat), or both. Cells were collected, mRNA extracted, and expression patterns of 16 genes evaluated using the NanoString nCounterÒ Gene Expression Assay. Target genes included B7-H3, B7H4, EZH2, PD-1, PD-L1, PD-L2, CD3D, and MHC II complex genes. Results: Untreated ascites from 4 patients showed high levels of MHC II complex genes, but only higher HLA-DRB6 expression was correlated with increased progression-free survival (PFS) (P = .0105). Treatment with both 5-azacytidine and entinostat resulted in a decrease in B7-H4 expression, and global downregulation of HLA genes, except the inhibitory gene HLA-DOB. The absolute-fold change relative to untreated controls was greatest with combination treatment (2.5), compared to entinostat (0.96), and 5-azacytidine (2.0) alone when averaged across all genes. Average PD-1 expression was increased with all 3 treatments, but was greatest with combination therapy (7.8-fold increase compared to untreated controls). Expression of the ligand PD-L1 was increased 3.9-fold with combination therapy. There was no significant correlation between changes in gene expression after treatment and PFS. There was an association between improved PFS and B7-H4 downregulation, which approached statistical significance (P = 0.06). Conclusion: Changes in RNA expression on human ascites-derived ovarian cancer cells after epigenetic therapy were identified using the NanoString technology. Increased PD-1 and PD-L1 have been shown to correlate with response to anti-PD-1 checkpoint inhibitor therapy. This method has the potential to identify which patients would benefit from treatment with epigenetic therapy to enhance immunotherapeutic efficacy. doi:10.1016/j.ygyno.2017.03.208

181 - Poster Session Interleukin-6 and leukemia inhibitory factor are expressed in both ovarian cancer tumor cells and stroma and may impact response to anti-estrogen therapy C.A. McIlwain, L. Tan, A. Sciallis, J. Erba, H.C. Crawford, K. McLean. The University of Michigan Hospitals, Ann Arbor, MI, USA

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Objective: In epithelial ovarian cancer, clinical response rates to antiestrogen therapy are lower than predicted in estrogen receptor (ER)-positive tumors, suggesting modifiers of response. Cytokines interleukin-6 (IL-6) and leukemia ihibitory factor (LIF) signal through the GP130/JAK/STAT pathway. We sought to assess primary ovarian cancers for IL-6 and LIF expression in tumor cells and stroma and to evaluate the relationship between cytokine expression and clinical outcome. In addition, we sought to identify evidence of cross talk between estrogen signaling and IL-6/LIF signaling in vitro. Method: Epithelial ovarian cancer cases of patients treated with antiestrogen therapy were identified, and immunohistochemical (IHC) staining of IL-6 and LIF expression was performed and scored. Progression-free interval (PFI) on antiestrogen therapy and overall survival were assessed by the Kaplan-Meier method, and outcomes compared using the log rank test. Ovarian cancer cell lines were treated with IL-6 and/or LIF, and estrogen-response element (ERE)driven reporter activity was quantified. Immunoblotting for ERα was performed following treatment with IL-6 and/or LIF. Cell viability assays were performed following antiestrogen treatment with tamoxifen, fulvestrant, or letrozole with or without the JAK2 inhibitor ruxolitinib. Results: Of the 40 cases evaluated by IHC, IL-6 expression was observed in tumor cells of 34 (85%) cases and in stroma of 29 (73%) cases. LIF was observed in tumor cells of 34 (85%) cases and in stroma of 16 (40%) cases. Cytokine expression in 39 of 40 cases prevented assessment of an association with clinical outcome. IL-6 and LIF treatment in ovarian cancer cell lines resulted in greater than 2-fold increase in ERE activity over baseline in multiple cell lines. Immunoblotting showed increased ERα expression following IL-6 and LIF treatment. Antiestrogen therapy in combination with ruxolitinib resulted in at least additive cell killing compared to either treatment alone. Conclusion: IL-6 and LIF are expressed in both epithelial ovarian cancer tumor cells and stroma. IL-6 and LIF activate estrogen signaling in ovarian cancer cell lines, and improved killing is achieved with blockade of both cytokine and estrogen pathways. Thus, inhibition of IL-6/LIF signaling may improve response to antiestrogen therapy in patients. doi:10.1016/j.ygyno.2017.03.209

182 - Poster Session Uterine leiomyosarcoma: Are novel therapeutics on the horizon? S. Graboscha, M.M. Boisena, T. Mab, B. Philipsc, M. Stranged, E. Elishaeva, G. Tsengb, R.P. Edwardsa, S.E. Taylora, M. Huange. aMageeWomens Hospital of UPMC, Pittsburgh, PA, USA, bUniversity of Pittsburgh, Pittsburgh, PA, USA, cUniversity of Pittsburgh Cancer Institute, Pittsburgh, PA, USA, dMagee-Womens Research Institute, Pittsburgh, PA, USA, eUniversity of Miami Sylvester Comprehensive Cancer Center, Miami, FL, USA Objective: Uterine leiomyosarcoma (uLMS) portends a poor prognosis with dismal responses to chemotherapy. Targeted therapy for a variety of cancers has grown exponentially, but there is limited knowledge of driver mutations in uLMS. We performed molecular profiling on uLMS tissue samples to identify pathways involved in development and progression. Method: Institutional review board approval was obtained. Clinical data were abstracted from medical records. Paraffin-embedded tissue from patients with uLMS (n = 12) and leiomyoma controls (n = 12) were deparaffinized and RNA was extracted. Nanostring analysis was utilized to identify the top differentially expressed (DE) genes. Ingenuity Pathway Analysis (IPA) was performed to further characterize the top canonical pathways involved.

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Abstracts / Gynecologic Oncology 145 (2017) 2–220

Results: For controls, average age was 51 ± 10 uears; 100% were Caucasian; 33% had a history of tobacco use; and average BMI was 34.7 ± 5.9. In uLMS patients, average age was 52 ± 10 years; 67% were Caucasian and 33% African-American; 42% had a history of tobacco use; and average BMI was 29.7 ± 5. Cancer stage was as follows: stage I (33%), stage II (25%), stage III (17%), and stage IV (25%). Nanostring identified 130 DE genes among uLMS samples versus controls with 37 genes tightly clustered as upregulated, 38 genes conserved and downregulated, and 55 generally upregulated but with more variability (Fig. 1). Among the top pathways were cell cycle functions including estrogen-mediated (E2F1, CDK2), DNA damage-induced (BRCA1/2, AKT3, CCNB1, P53), GADD45 signaling, and cell cycle control (CDKN2C, CDK2, CHEK2). Conclusion: DE genes were successfully identified using the Nanostring platform. IPA identified and confirmed canonical pathways involving those DE genes. As we continue to expand our sample size, we anticipate more significant gene clustering and the ability to identify pathways correlated with disease stage. In turn, this will translate into the ability to identify actionable targets for clinical trial development.

Method: With institutional board approval, 70 endometrial tumors with clinical outcomes were identified representing a spectrum of grade and histology. Immunohistochemistry (IHC) was performed for PD-L1 and B7-H4 and scored according to guidelines developed at Jounce Therapeutics. Microsatellite instability (MSI) status was assessed for endometrioid tumors using the institutional clinical IHC assay for expression of the mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2. Results: We identified 40 low-grade endometrioid tumors, 10 highgrade endometrioid tumors, 10 uterine serous carcinomas, and 10 carcinosarcomas with complete clinical information. While not significantly different between the high-grade and low-grade cohorts (57% vs 35%, P = 0.07), PD-L1 expression was associated with MSI status in the low-grade subset (both P b 0.01). The high-grade cohort had similar rates of PD-L1 expression compared to low-grade MSI tumor (57% and 62%, respectively), and both were distinct from low-grade MSS tumors (22%, P b 0.05). While high (3+) B7-H4 expression was observed in both high- and low-grade carcinomas (33% and 31%, respectively), linear modeling revealed B7-H4 expression to be positively correlated with grade (P b 0.05). Conclusion: These data suggest that expression of immune regulatory molecules PD-L1 and B7-H4 are most commonly associated with MSI status in low-grade endometrioid and high-grade endometrial tumors of all histologies. The development and testing of multiagenttargeted therapeutics targeting the PD1 pathway and B7-H4 may be a novel strategy for this subset of endometrial tumors. doi:10.1016/j.ygyno.2017.03.211

184 - Poster Session Augmenting the MHC II antigen-presentation pathway with epigenetic therapy in epithelial ovarian cancer T.B. Turner, S. Meza-Perez, J.E. Peabody, H.J. Smith, L.A. Norian, D.J. Buchsbaum, J.M. Straughn Jr, T. Randall, R.C. Arend. University of Alabama at Birmingham, Birmingham, AL, USA

Fig. 1. Heatmap of 130 Differentially Expressed (pb 0.05) Genes in Leiomyosarcoma Samples. 130 differentially expressed (DE) genes (pb 0.05) were identified in leiomyosarcoma samples when compared to leiomyoma controls. The top portion represents 37 genes that appear consistently upregulated versus controls, the bottom demonstrates 38 genes downregulated versus controls, and the middle section contains a variable area of 55 genes that are generally upregulated versus controls.

doi:10.1016/j.ygyno.2017.03.210

183 - Poster Session Characterization of immune regulatory molecules B7-H4 and PD-L1 in low- and high-grade endometrial tumors A.J. Bregara,b, A. Deshpandec, H. Hirschc, T. Zic, J. Reevesc, S. Sathyanarayananc, B.R. Ruedab, W.B. Growdona. aHarvard Medical School, Boston, MA, USA, bMassachusetts General Hospital, Boston, MA, USA, cJounce Therapeutics, Cambridge, MA, USA Objective: The objective of this investigation was to characterize the expression landscape of immune regulatory molecules programmed death-ligand-1 (PD-L1, B7-H1) and B7-H4 in a cohort of endometrial tumors across the spectrum of grade and histology.

Objective: Despite advances in surgery, chemotherapy, and genetic testing, ovarian cancer remains a deadly disease. The known immunogenicity of ovarian cancer and the recent advances in immunotherapy in other solid tumors have created enthusiasm for evaluating immunotherapeutic targets. Previous studies have demonstrated that increased expression of the MHC class II antigen presentation pathway in ovarian cancer correlates with prolonged survival. We investigated epigenetic modifiers as induction agents for expression of the MHC II pathway on ovarian cancer cells in vivo and in vitro. Method: Murine epithelial ovarian cancer cells (ID8) and human ovarian cancer cells (A2780.IP2, A2780.CP20, SKOV3) were treated with 2 types of epigenetic modulators: histone deacetylase inhibitors (HDACi) and a DNA methyltransferase inhibitor (DNMTi). mRNA and protein expression of MHC II pathway genes were evaluated after treatment with HDACi (entinostat and panobinostat) and a DNMTi (5-azacytidine (5AZA)) by qPCR and flow cytometry. MHC II pathway mRNA expression in a patient-derived xenograft (PDX) model of ovarian cancer was assessed after 3 weeks of treatment with entinostat and 5AZA alone and in combination. Results: Treatment with entinostat and 5AZA in ID8 and human ovarian cancer cells increased mRNA levels of CD74, CIITA, and MHC II. MHC II and CD74 protein expression was increased after treatment with either agent alone. A dose-dependent response in mRNA and protein expression was seen with entinostat. Combination treatment showed higher MHC II protein expression than single-agent treatment. In the PDX model of ovarian cancer, CIITA, CD74, and MHC II