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High-grade serous ovarian cancer (HGSC) is the most lethal histotype of ovarian cancer and is highly prone to metastasis, with the majority of patients presenting at advanced stages of disease. There are few targeted treatment options for HGSC patients, especially for women with high metastatic tumor burdens. In order to identify potential genetic targets in metastatic disease, we characterized the genomic and transcriptomic alterations of metastatic tumors in HGSC patients. In this study, matched normal tissue, primary, and metastatic tumors from 23 HGSC short-term (ST) survivors (overall survival [OS] 5 years) underwent whole exome and RNA sequencing. We compared somatic mutations, copy number alternations, mutational burdens, differential expression, immune cell fractions, and gene fusion predictions between the primary and metastatic tumors of the ST and LT survival groups. We confirmed that tumors of LT survivors were more likely to have BRCA1 alterations and more copy-altered segments when compared to tumors of ST survivors. There was a higher percentage of shared variants between the primary and metastatic tumors for ST survivors compared to LT survivors. ST survivors also had a greater mutational burden and more predicted gene fusions than LT. We observed few differentially expressed genes between primary and metastatic tumors, but we did observe significant differences between the transcriptomes of LT and ST survivors' tumors. Overall, we observed that HGSC primary and metastatic tumors had similar genomic and transcriptomic landscapes, but we identified genetic characteristics specific to metastatic tumors that correlate with survival. Citation Format: Emilee N. Kotnik, Nicholas C. Spies, Tiandao Li, Matthew Inkman, Jin Zhang, Fernanda Martins-Rodrigues, Ian S. Hagemann, Carolyn K. McCourt, Premal H. Thaker, Andrea R. Hagemann, Matthew A. Powell, David G. Mutch, Christopher A. Maher, Christopher A. Miller, Katherine C. Fuh. Genetic characterization of metastasis and prognosis in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2202.

Background: Evidence suggests that inducing homologous recombination deficiency (HRD) can be a therapeutic target in endometrial cancer (EC). Given this, we determined whether AVB-500, an AXL inhibitor, could induce HRD measured by a reduction of RAD51 foci in response to DNA damage in a uterine serous cancer (USC). Methods: Baseline homologous recombination (HR) status was determined after inducing DNA damage by radiating USC cell lines (ARK1 and ARK4) with 10Gy radiation treatment (RT) and identifying RAD51 foci by immunofluorescence (IF). Both radiated and non-radiated cells were quantified in both cell lines. Cell lines were considered to be HR proficient if over 30% of the cells expressed RAD51 (>5 foci per cell). AVB-500 (Aravive Biologics, Houston, TX) was given prior to radiation after baseline HR status was established. IF was conducted using a Leica confocal microscope and foci were quantified using FociCounter. Results: Both ARK1 and ARK4, showed HR proficiency at baseline. After radiation, there was an increase in gamma-H2Ax indicative of DNA damage and an appropriate increase in RAD51 foci in response. However, when cells were treated with AVB-500 prior to radiation, there was a decrease in RAD51 foci when compared to radiation alone in both ARK1 (2.7 foci for AVB + RT vs 7.3 foci for RT only, p=0.0003) and ARK4 (6.3 foci for AVB + RT vs 13.0 foci for RT only, p=0.0054). Furthermore, after treatment with AVB-500 followed by RT, the proportion of ARK1 cells expressing RAD51 decreased to 21%, indicating HR deficiency. Conclusions: ARK1 and ARK4 are HR proficient at baseline. Pretreatment with AVB500 followed by RT led to a decrease in RAD51 foci suggestive of inducing HRD. Additional therapeutic and mechanistic experiments are ongoing. Citation Format: Michael D. Toboni, Barbara Blachut, Elena Lomonosova, Dineo Khabele, Lindsay Kuroki, Andrea Hagemann, Carolyn McCourt, Premal Thaker, David Mutch, Matthew Powell, Katherine Fuh. Inducing homologous recombination deficiency in uterine serous cancer (USC) using AVB-500, a receptor tyrosine kinase AXL inhibitor [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO002.

Objectives: We determined whether inhibition of GAS6/AXL with AVB-500 in combination with olaparib could improve response in HR-proficient (HRP) uterine serous cancer (USC). Methods: Two USC cell lines (ARK1 & ARK4) were radiated with 10Gy, and RAD51 foci were identified by immunofluorescence (IF). These were treated with AVB-500 (Aravive Biologics, Houston, TX) in combination with the poly ADP ribose polymerase (PARP) inhibitor, olaparib. Colony forming assays were assessed after 4 days of treatment with either AVB-500 alone, olaparib alone or combination treatment (olaparib + AVB-500). Colonies were stained and absorbance was obtained to calculate relative cell survival using Graph Pad Prism. In vivo studies were performed using NOD-SCID mice injected with 1 × 107 ARK1 cells intraperitoneally followed by treatment q3 days for 14-day treatment period. Treatment groups were vehicle control, AVB-500 alone, olaparib alone and olaparib with AVB-500. Results: ARK1 and ARK4 cells were found to have an increase in gamma-H2AX and RAD51 foci after radiation that was consistent with HRP. In clonogenic assays, colonies were stained and absorbance was obtained for each experimental arm. Olaparib + AVB-500 had significantly less absorbance than the olaparib only group for ARK1s (0.417nm vs 0.756nm, p=0.001) as well as in ARK4s (0.186nm vs 0.641nm, p=0.003). In an intraperitoneal model with ARK1 tumors, the olaparib + AVB-500 treated group had less tumor weight than those treated with olaparib alone (0.008g vs 0.138g, p=0.002) and AVB-500 alone (0.008g vs 0.145g, p=0.0006) after a 14-day treatment period. Conclusions: AVB-500 in combination with olaparib demonstrates an improved response than olaparib alone with a greater decrease in tumor burden. This was demonstrated in two HRP cell lines. Additional therapeutic and mechanistic experiments are ongoing. Citation Format: Michael D. Toboni, Mary Mullen, Jo'an Tankou, Hollie Noia, Alyssa Oplt, Daniel Wilke, Dineo Khabele, Lindsay Kuroki, Andrea Hagemann, Carolyn McCourt, Premal Thaker, David Mutch, Matthew Powell, Katherine Fuh. AVB-500, a receptor tyrosine kinase AXL inhibitor, improves response to olaparib in uterine serous cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO047.

Background Metastasis via pelvic and/or para-aortic lymph nodes is a major risk factor for endometrial cancer. Lymph-node resection ameliorates risk but is associated with significant co-morbidities. Incidence in patients with stage I disease is 4–22% but no mechanism exists to accurately predict it. Therefore, national guidelines for primary staging surgery include pelvic and para-aortic lymph node dissection for all patients whose tumor exceeds 2cm in diameter. We sought to identify a robust molecular signature that can accurately classify risk of lymph node metastasis in endometrial cancer patients. 86 tumors matched for age and race, and evenly distributed between lymph node-positive and lymph node-negative cases, were selected as a training cohort. Genomic micro-RNA expression was profiled for each sample to serve as the predictive feature matrix. An independent set of 28 tumor samples was collected and similarly characterized to serve as a test cohort. Results A feature selection algorithm was designed for applications where the number of samples is far smaller than the number of measured features per sample. A predictive miRNA expression signature was developed using this algorithm, which was then used to predict the metastatic status of the independent test cohort. A weighted classifier, using 18 micro-RNAs, achieved 100% accuracy on the training cohort. When applied to the testing cohort, the classifier correctly predicted 90% of node-positive cases, and 80% of node-negative cases (FDR = 6.25%). Conclusion Results indicate that the evaluation of the quantitative sparse-feature classifier proposed here in clinical trials may lead to significant improvement in the prediction of lymphatic metastases in endometrial cancer patients. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3604-y) contains supplementary material, which is available to authorized users.

Thilo S Lange1,2, Carolyn McCourt2, Rakesh K Singh2, Kyu Kwang Kim2, Ajay P Singh3, Brian S Luisi4, Onur Alptürk5, Robert M Strongin6, Laurent Brard21Division of Biology and Medicine Brown University, Providence, R1, USA; 2Molecular Therapeutics Laboratory, Program in Women’s Oncology, Department of Obstetrics and Gynecology, Women and Infants’ Hospital of RI, Warren Alpert Medical School of Brown University, Providence, RI, USA; 3Department of Plant Biology, Rutgers University, New Brunswick, NJ, USA; 4Department of Chemistry, Brown University, Providence, RI, USA; 5Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; 6Department of Chemistry, Portland State University, Portland, OR, USAAbstract: The cytotoxicity of organometallic compounds iron(III)-, cobalt(III)-, manganese(II)-, and copper(II)-salophene (-SP) on platinum-resistant ovarian cancer cell lines was compared. Fe-SP displayed selective cytotoxicity (IC50 at ∼1 μM) against SKOV-3 and OVCAR-3 cell lines while Co-SP caused cytotoxic effects only at higher concentrations (IC50 at 60 μM) and Cu-SP effects were negligible. High cytotoxicity of Mn-SP (30–60 μM) appeared to be nonspecific because the Mn-chloride salt reduced cell viability similarly. The effect of Fe-SP at 1 μM proved to be ovarian cancer cell selective when compared to a panel of cell lines derived from different tumors. The first irreversible step in the induction of cell death by Fe-SP occurred after 3 hrs as indicated by the mitochondrial transmembrane potential (ΔΨm) and was mainly linked to apoptotic, not necrotic events. To evaluate the toxicity of Fe-SP in vivo we conducted an acute toxicity study in rats. The LD50 of Fe-SP is >2000 mg/kg orally and >5.5 mg/kg body weight by intraperitoneal injection. An ovarian cancer animal model showed that the chemotherapeutic relevant dose of Fe-SP in rats is 0.5–1 mg/kg body weight. The present report suggests that Fe-SP is a potential therapeutic drug to treat ovarian cancer.Keywords: iron(III)-salophene, chemotherapeutic properties, p38 MAPK, ovarian cancer animal model

Cediranib is a multi-tyrosine kinase inhibitor targeting vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) receptors. This phase II study was conducted to assess activity and tolerability of single-agent cediranib in recurrent/persistent endometrial cancer.Eligible patients had recurrent or persistent endometrial cancer after receiving one or two prior cytotoxic regimens, measurable disease, and Gynecologic Oncology Group (GOG) performance status of ≤2 (≤1 if two prior cytotoxic regimens given). Cediranib 30mg orally daily for a 28daycycle was administered until disease progression or prohibitive toxicity. Microvessel density (MVD) was measured in tumor tissue from initial hysterectomy specimens and correlated with clinical outcome. Primary endpoints were tumor response and surviving progression-free for six months without subsequent therapy (6-month event-free survival [EFS]).Of 53 patients enrolled, 48 were evaluable for cediranib efficacy and toxicity. Median age was 65.5 years, 52% of patients had received prior radiation, and 73% of patients received only one prior chemotherapy regimen. A partial response was observed in 12.5%. Fourteen patients (29%) had six-month EFS. Median progression-free survival (PFS) was 3.65 months and median overall survival (OS) 12.5 months. No grade 4 or 5 toxicities were observed. A trend towards improved PFS was found in patients whose tumors expressed high MVD.Cediranib as a monotherapy treatment for recurrent or persistent endometrial cancer is well tolerated and met protocol set objectives for sufficient activity to warrant further investigation. MVD may be a useful biomarker for activity.

Objective The objective of this study was to examine SLN evaluation alone in women with squamous cell carcinoma (SCC) of the vulva and evaluate the inguinal recurrence and complication rates. Methods An IRB approved prospective study enrolled patients with SCC of the vulva. Peritumoral injection of Tc-99 sulfur colloid and blue dye was used to identify SLNs intraoperatively. Patients with negative SLN for metastasis were followed clinically without further treatment. Patients with metastasis to a SLN underwent full groin node dissection followed by standard treatment protocols. Results A total of 73 women were enrolled onto protocol with 69 patients undergoing SLN dissection. Mean age was 66.9years (range: 29–91) with 47 stage I, 12 stage II, 9 stage III, 2 stage IV and 3 unstaged patients. SLN dissections were successful in 63 patients. Of the 111 groins evaluated with a SLN dissection 93% had a SLN identified with an average of 2 SLN per groin. There were 92 groins with negative SLN and 11 groins with positive SLN. 57 patients had negative SLN and underwent conservative management with the median follow-up of 58.3months. Three patients experienced groin recurrences (2 unilateral, 1 bilateral) for a recurrence rate of 5.2% (3/57). The complication rate for the inguinal incisions was 17.5% (1 cellulitis, 1 abscess, 2 lymphoceles, 5 lymphedema and leg pain). Conclusions Isolated SLN dissection alone has a low inguinal recurrence rate with decreased complications and should be considered as an option for women with SCC of the vulva.

Background In this pioneer study to the biological activity of organometallic compound Iron(III)-salophene (Fe-SP) the specific effects of Fe-SP on viability, morphology, proliferation, and cell-cycle progression on platinum-resistant ovarian cancer cell lines were investigated. Methodology/Principal Findings Fe-SP displayed selective cytotoxicity against SKOV-3 and OVCAR-3 (ovarian epithelial adenocarcinoma) cell lines at concentrations between 100 nM and 1 µM, while the viability of HeLa cells (epithelial cervix adenocarcinoma) or primary lung or skin fibroblasts was not affected. SKOV-3 cells in contrast to fibroblasts after treatment with Fe-SP revealed apparent hallmarks of apoptosis including densely stained nuclear granular bodies within fragmented nuclei, highly condensed chromatin and chromatin fragmentation. Fe-SP treatment led to the activation of markers of the extrinsic (Caspase-8) and intrinsic (Caspase-9) pathway of apoptosis as well as of executioner Caspase-3 while PARP-1 was deactivated. Fe-SP exerted effects as an anti-proliferative agent with an IC50 value of 300 nM and caused delayed progression of cells through S-phase phase of the cell cycle resulting in a complete S-phase arrest. When intra-peritoneally applied to rats Fe-SP did not show any systemic toxicity at concentrations that in preliminary trials were determined to be chemotherapeutic relevant doses in a rat ovarian cancer cell model. Conclusion/Significance The present report suggests that Fe-SP is a potent growth-suppressing agent in vitro for cell lines derived from ovarian cancer and a potential therapeutic drug to treat such tumors in vivo.

To determine predictive factors for the presence of malignant transformation in ovarian endometriotic cysts.This was an IRB approved, case control study analyzing patient data from 2004 to 2013. Pathology database records were searched to identify patients with benign endometrioma and ovarian carcinoma arising in the background of endometriosis. Inclusion criteria required each patient to have a preoperative diagnosis of adnexal mass and no other findings concerning for malignancy. Patient clinical records were queried for preoperative symptoms, serum CA125 levels and radiologic findings. Pathologic data were collected including histology, tumor grade and stage.A total of 138 patients met inclusion criteria; 42 women with ovarian cancer arising in the background of endometriosis and 96 women with benign endometrioma. Women diagnosed with ovarian cancer were significantly older than women with endometriosis (53.6 vs. 39.2 years). There was no difference in presence of symptoms between the two groups. Women with malignant tumors were found to have significantly larger cysts (14 cm vs. 7.5 cm; p0.0001) that were more often multilocular (45.7% vs. 12.2%; p0.0001), and contained solid components (77.1% vs. 14.5%; p0.0001). Among patients that were observed prior to surgery there was a significant difference in the change in size of the mass over time with 4.2 cm increase for cases vs. 1.0 cm increase for controls (p=0.02). Multiple logistic regression analysis indicated that for every 5 years increase in age there was an adjusted OR of 2.17 (p=0.003). An age of 49 years or greater had an 80.6% sensitivity (95% CI: 62.5-92.5%) and an 82.9% specificity (95% CI: 67.9-92.8%) for malignancy, and solid component on imaging had an adjusted OR of 23.7 (p0.0001). Serum CA125 levels tended to be higher in patients with malignant tumors but did not reach statistical significance with a mean of 204.9 vs. 66.9 (p=0.1).Significant predictors for malignant transformation of endometriosis include cyst characteristics and age. Women above the age of 49 with multilocular cysts and solid components are at high risk for malignant transformation of endometriosis. Serum CA125 level is not a significant predictor of malignant transformation.

Objectives: PTEN mutations are frequently observed in endometrioid uterine cancer and lead to hyperactivation of the PI3K/Akt pathway. However, the role of BRCA1 and 2 mutations, leading to homologous recombination deficiency, is not known in this disease. We sought to determine the frequency and clinical significance of BRCA mutations in patients with uterine cancer and PTEN mutations. Methods:We assessed PTEN, BRCA1, and BRCA2mutation status for 248 uterine tumor samples using level 3 data fromThe Cancer GenomeAtlas (TCGA). We performed mutation analysis for PTEN, BRCA1, and BRCA2 for an additional 251 samples from TCGA using ANNOVAR. A total of 499 patientswere included in the analysis. Clinical data, including stage, tumor histology, and overall survival, were also extracted. Fisher's exact tests were used to identify the association betweenmutation status and histologic subtype. Kaplan–Meier plots were constructed to demonstrate survival differences between groups. Results: Among the samples analyzed, 335 (67%) had endometrioid histology, 76 (15%) had serous histology, 18 (44%) had mixed histology, and histology was unknown in the remainder of cases. Of the patients with endometrioid histology, 260 (78%) had a PTEN mutation, 23 (7%) had both BRCA1 and 2 mutations, 4 (1%) had mutations in BRCA1 only, and 25 (8%) hadmutations in BRCA2 only. A total of 52/335 patients (16%) had mutations in either BRCA1 or 2; 18/52 (35%) of these had stage II or above disease and the remainder had stage I disease. No survival advantage was detected in patients with BRCA1 mutations over wildtype BRCA1, independent of PTEN mutation status (P=0.16 and 0.19, respectively). Patients with both PTEN and BRCA2 mutations had improved overall survival compared to patients with wild-type PTEN and BRCA2 (P=0.03). A singlemutation in BRCA1, BRCA2, or PTEN did not confer a significant survival advantage compared towild-type (P=0.15). Conclusions: BRCA1 and 2 mutations are prevalent in patients with endometrioid uterine cancer, and PTEN/BRCA2 double mutations are associated with prolonged patient survival. These findings may have significant clinical and treatment implications.