Disclosure: F. Sharafath Yoosuf: None. C. Magne De: None. C.D. Clyne: None. S. Chu: None. P.J. Fuller: None. Breast cancer is the most common cancer in women, with over 2 million cases diagnosed annually worldwide. Two-thirds of breast cancers are estrogen receptor positive (ER+), and may be treated with hormone therapy such as tamoxifen, to block estrogen’s effects on residual cancer cells and reduce recurrence risk. However, ∼30% of women develop endocrine resistance or fail to respond to treatment. Limited alternative therapies exist for endocrine-resistant disease, leaving patients at higher risk of relapse. This creates an urgent need to uncover and characterize other molecular drivers of breast cancer to develop novel therapeutic strategies. Apart from steroid receptors such as the ER and progesterone receptor (PR), the role of other nuclear receptors (NR) in breast cancer remains underexplored. Our previous research investigating NR expression in breast cancer revealed that NR2F6, also known as EAR2 (V-Erb-A related protein 2), is uniquely upregulated in both ER+ and ER- breast cancers. In contrast, most other NR are universally downregulated, suggesting that EAR2 may drive breast cancer proliferation.In this study we investigated the role of EAR2 in breast cancer cell proliferation, invasion and migration using three breast cancer cell lines with modulated EAR2 expression. EAR2 was inducibly overexpressed in MCF7 cells, constitutively overexpressed in Hs578T cells and knocked down using CRISPR/Cas9 in T47D cells. Proliferation/viability were assessed using the xCELLigence Real Time Cell Analyser SP, MTS assay and Incucyte Live Cell Imaging. Cell migration was studied using the Incucyte. RNA-seq analysis was conducted using DNBSEQ technology and selected differentially expressed genes were validated using RT-qPCR. EAR2 overexpression increased proliferation while knockdown decreased proliferation and increased cell size. Migration was enhanced with EAR2 overexpression. In MCF7 cells overexpressing EAR2, highly expressed genes included GALNT16, MACROD2, GPD1 and WNT10B, of which MACROD2 is implicated in therapy-resistant breast cancers. Comparative analysis of MCF7 (EAR2 overexpression) and T47D (EAR2 knockdown) cells identified 54 common inversely regulated genes. Among these, DUSP4 and RUNX1 are implicated in early oncogenic transformation in breast cancer, while KRT8, KRT18 and KRT19 are associated with keratinization and epithelial differentiation.Our findings demonstrate that EAR2 is a novel driver of breast cancer proliferation, and may contribute to cancer progression in both ER+ and ER- subtypes. Notably, the role for EAR2 in tamoxifen resistance and its association with highly invasive, dedifferentiated tumors with poor prognosis suggest it could represent a novel, unexplored therapeutic target. Future studies are warranted to further explore EAR2’s functional role in breast cancer and its potential as a target for overcoming endocrine resistance. Presentation: Saturday, July 12, 2025

Background Patients with indeterminate thyroid nodules continue to be a challenge to clinicians. Molecular testing on fine needle aspirates (FNA) is well established in the United States, but its access is limited elsewhere. We aimed to explore the utility of a pre-existing Next Generation Sequencing (NGS) platform, and the role of BRAF mutation in patient selection for preoperative molecular testing. Methods Patients were recruited from the Monash University Endocrine Surgery Database for BRAF immunohistochemistry status on histopathological samples; and prospectively for NGS testing of preoperative FNA samples, using the Archer DX VariantPlex (31 genes) and FusionPlex (40 genes) Comprehensive Thyroid and Lung panels. Results The mean age of 90 patients (103 nodules) for NGS testing was 53, with 58 % female. Most of the nodules (n = 72, 70 %) had indeterminate cytology. Histology showed malignancy in 52 (50 %) nodules; of these, 45 had pathogenic DNA mutations and 3 had pathogenic RNA fusions on NGS testing of needle aspirates. The NGS panels were able to detect malignancy in indeterminate nodules with 75 % sensitivity, 85 % specificity, 80 % positive predictive value, and 81 % negative predictive value. 74 % of the papillary thyroid cancer population had BRAF mutations on immunohistochemistry, which showed 88 % concordance with the NGS results. Conclusion This is the first Australian study to demonstrate that pre-existing, non-proprietary NGS mutation and fusion panels can achieve high diagnostic specificity and positive predictive value for malignancy in indeterminate thyroid nodules. Furthermore, we propose exploration of using BRAF V600E testing to select patients for full NGS analysis.

Disclosure: T.S. Hegarty: None. P.J. Fuller: None. S. Chu: None. Background: Adult granulosa cell tumors (aGCT) are rare endocrine-related ovarian cancers, comprising approximately 5% of all ovarian cancers. These tumors harbor a pathognomonic FOXL2 gene mutation, involving a cysteine-to-tryptophan substitution at amino acid 134 (C134W). FOXL2 is a key transcriptional regulator of granulosa cell identity and function, and the FOXL2C134W mutant has been shown to alter its DNA binding specificity and transcriptional activity, inducing genes associated with oncogenesis, stemness and epithelial-to-mesenchymal transition (EMT). This study investigates the transcriptional impact of the FOXL2C134W mutation in an immortalized non-luteinized granulosa (hGrC1) cell line model. Methods: CRISPR-Cas9 gene editing introduced a homozygous FOXL2C134W mutation into hGrC1 cells. Cell proliferation was assessed using the xCELLigence Real-Time Cell Analysis SP system. RNA sequencing (RNA-seq) was performed to identify differentially expressed genes (DEGs) in FOXL2C134W hGrC1 cells compared to parental controls, with DEGs defined by log2FC≥1 and Q value of <0.05. Published wildtype FOXL2 ChIP-sequencing data (GSE236785) and FOXL2C134W ChIP-sequencing data (GSE126171) were analyzed to delineate target genes. Gene set enrichment analysis (GSEA) was used to assess pathway-level changes in gene expression. Results: FOXL2C134W mutation did not alter cell proliferation compared to parental hGrC1 cells. RNA-seq identified 355 DEGs in FOXL2C134W hGrC1 cells with 101 upregulated and 254 downregulated genes. Among these, 47 DEGs overlapped with FOXL2WT target genes, and 12 DEGs with FOXL2C134W target genes. GSEA revealed significant enrichment (p<0.01) of EMT-associated genes in FOXL2C134W hGrC1 cells. Notably, Microfibril associated protein 5 (MFAP5), an EMT-associated gene implicated in tumor progression and metastasis, exhibited a marked upregulation (log2FC = 10) in FOXL2C134W hGrC1 cells. Discussion: Our findings demonstrate that the FOXL2C134W mutation drives transcriptional reprogramming in granulosa cells, specifically upregulating EMT-associated pathways. The overexpression of MFAP5, a gene correlated with advanced aGCT stages and cancer invasiveness, underscores its potential as a biomarker and therapeutic target. These insights advance our understanding of the molecular mechanisms underlying aGCT pathogenesis and paves the way for targeted therapeutic strategies for this rare and challenging cancer. Presentation: Monday, July 14, 2025

BACKGROUND:Optimal treatment of ovarian Granulosa Cell Tumors (GCT) is uncertain due to a lack of evidence from randomized trials. The role of radiotherapy is unclear and recommendations differ between countries. Through an ongoing collaboration with the multinational closed GCT Survivor Sisters (GCT-SS) Facebook™ group (membership n = 1800) we developed a survey to understand members' experiences of radiotherapy. METHODS:GCT-SS members (≥18+ years) were invited to complete the survey assessing i) diagnosis and treatment, ii) radiotherapy experiences and iii) radiotherapy impact. Two questions allowed free-text responses about radiotherapy experiences. Country of residence was assessed (USA, Canada, United Kingdom (UK), European Union (EU), Australia/New Zealand (Aus/NZ); Other). Logistic regression examined factors associated with radiotherapy. RESULT:Surveys from 1017 members (96 % Adult-GCT) are analysed. Respondents were mostly from the USA (62 %), diagnosed post-2015 (67 %), and 44 % had recurrent disease. Overall, 12 % of respondents reported radiotherapy (9 % received, 3 % planned), mostly for recurrent (23 %) disease. In univariate analyses, radiotherapy for recurrent disease differed by country (p = .019) (Canada (12 %), UK (14 %), EU (14 %), Aus/NZ (36 %), USA (25 %)); age (p = .02) (≥50 (26 %) ≤50 (17 %)) having chemotherapy (p = .019) and hormonal therapy (p < .001). Analyses of open-ended questions found five themes: four positive (it worked; easier than other treatments, gave me back my life, gives hope) and one negative reflecting severe side-effects. CONCLUSIONS:Radiotherapy for GCT is not common and reflects treatment recommendations in different countries. As women mostly report positive experiences, studies are needed to develop an evidence-base regarding its optimal timing and sequencing in managing GCT.

Mitochondrial-associated granulocyte macrophage colony-stimulating factor (Magmas) is a unique protein located in the inner membrane of mitochondria, with an active role in scavenging reactive oxygen species (ROS) in cellular systems. Ovarian cancer (OC), one of the deadliest gynaecological cancers, is characterised by genomic instability, affected by ROS production in the tumour microenvironment. This manuscript discusses the role of Magmas and efficacy of its novel small molecule inhibitor BT#9 in OC progression, metastasis, and chemoresistance. Magmas expression levels were significantly elevated in high-grade human OC compared to benign tumours by immunohistochemistry. The inhibition of Magmas by BT#9 enhanced ROS production and reduced mitochondrial membrane permeability, basal respiration, mitochondrial ATP production, and cellular functions, such as the proliferation and migration of OC cell lines in vitro. Oral administration of BT#9 in vivo significantly reduced tumour growth and spread and enhanced the survival of mice without having any effect on the peritoneal organs. These data suggest that Magmas is functionally important for OC growth and spread by affecting ROS levels and that the inhibition of Magmas activity by BT#9 may provide novel clinical benefits for patients with this malignancy.

Patient-derived xenograft (PDX) models play a crucial role for in vivo research. They maintain the original molecular characteristics of the human tumor and provide a more accurate tumor microenvironment, which cannot be replicated by in vitro models. This chapter describes four different transplantation methods, namely, intra-bursal, intrarenal capsule, intraperitoneal, and subcutaneous, to develop PDX models for ovarian cancer research.

Figure S2. Effects of PPARg activation.

S1. Total number of mutated genes identified within this study that are also mutated in the studies of Caburet et al [21], Zehir et al [22], Kandoth et al [23], Lawrence et al [24] and Nik-Zainal et al [25]. Genes mutated in this current study are also presented as a percentage.

Figure S1. XIAP mRNA expression in GCT and GCT-derived cell line.

Figure S4. Effects of combined PPARg activation and XIAP inhibition in GCT explant.

Table S1. Gene expression analysis by TaqMan Low-Density Array.

Table S1: Somatic single nucleotide variants, indel and sequence alteration mutations identified across each sample Table S2: Mutated genes identified in samples with matched germline DNA: 1, 18 and 24 Table S3: Microarray analysis by -124C>T TERT promoter mutation status Table S4: Ingenuity Pathway Analysis on genes that differed by TERT promoter mutation status Table S5: Comparison with a series of large scale analyses looking at mutations in genes that overlap with data in this study Table S6: Mutations in genes identified by Zehir, A et al in 11 aGCT samples

Abstract Disclosure: S. Chu: None. V.M. White: None. M. Alexiadis: None. K. Eroh: None. K. Ackermann: None. S. Rodgers: None. L.M. Langdale: None. N.E. Armour: None. T.W. Jobling: None. P.J. Fuller: None. Background: Engaging with online social media consumer groups for rare cancers may help to develop collaborations between consumers and researchers. This study, a collaboration with the Granulosa Cell Tumour-Survivor Sisters (GCT-SS) Facebook® group, explores the results of their survey of member’s treatment and follow-up experiences. Method: Members of the closed multinational GCT-SS Facebook® group completed a 43-item survey covering symptoms, diagnosis, treatment, recurrence, follow-up, and possible risk factors for GCT. Group members had either adult (aGCT) or juvenile (jGCT) disease. Data was collected via an online survey between 2014 and 2019. Open-ended responses were reviewed by the study team and coded into agreed categorical responses or binary-type variables. Statistical analysis was conducted using SPSS V28. Descriptive statistics (e.g. frequencies) were used to describe the sample in terms of individual, disease and treatment characteristics. Pearson Chi-square or Fisher’s exact test were used to compare proportions between groups (e.g., recurrence or not). Logistic regression was used to examine factors associated with recurrent disease. Results: 744 members (average 4.4 years (SD=5.9) post-diagnosis) participated including 52 with jGCT. 67% had stage I disease and 8% stage III–V at diagnosis, although 30% of aGCT and 25% of jGCT reported recurrent disease at survey completion. 48% of aGCT had laparoscopic surgery, tumor encapsulation was reported by 49% and tumor bagging reported by 29% overall (37% laparoscopic; 8% open). Recurrence rates were higher when the tumor was cut or ruptured (ruptured: p<.001; cut: p=.01). 19% of aGCT had chemotherapy with this most common for stage II-III disease. BEP (bleomycin, etoposide and cisplatin) protocols became less common over time (diagnosed < 2015: 47% vs diagnosed post-2015: 21%). Conclusions: While there is a growing recognition of the need for involvement of patients and the public across the entire research process, to date consumer involvement has mainly focused on setting research questions/focus and recruitment. The development of information and support groups on social media platforms has provided a unique opportunity for greater collaboration between consumers, researchers and clinicians. This may be of particular benefit when the disease is rare as the global reach of these platforms allows relatively large numbers of people with the condition to connect. Our study suggests the feasibility of engaging with communities that have developed via social media platforms. Members of the GCT-SS group report treatment patterns generally in line with those found from clinical audits. Utilising naturally forming consumer groups may assist with developing the evidence base for care and supporting those living with GCT ovarian cancer. Presentation: Thursday, June 15, 2023

Figure S3. Effects of XIAP inhibition alone or in combination with PPARg activation.

Abstract Disclosure: S. Chu: None. A. Carolan: None. C. Giatrakis: None. T. Nguyen: None. M. Alexiadis: None. T. Yu: None. J. Silke: None. P.J. Fuller: None. Background: Granulosa cell tumours (GCT) are uncommon ovarian cancers characterised by an indolent clinical course and significant late recurrence rates. Aside from invasive surgery, there are limited therapeutic options, highlighting the need for targeted therapies. Our laboratory has previously shown that targeting X-linked Inhibitor of Apoptosis Protein (XIAP) using small molecule inhibitors known as Smac-mimetics (SM) in combination with other compounds, is a compelling therapeutic strategy in GCT. XIAP inhibition sensitises cancer cells to anti-cancer therapies through the regulation of key pro-survival pathways, namely NFκB. Methods: High-throughput screening (HTS) using established drug libraries was performed in a GCT-derived cell line (KGN) and a transformed non-luteinized granulosa cell line (hGrC1), following pre-treatment with the SM, Compound A (CmpdA; 500nM). Drug combination hits were defined as a greater than> 80% reduction in viability compared to CmpdA monotherapy alone. Subsequent validation studies were conducted to measure the effects on cell proliferation, apoptosis, and NFκB transactivation. The impact of the combination therapy was assessed by RNA-seq analysis to identify differentially expressed genes, significant pathways, and functional enrichment. Results: Here we report the use of a histone deacetylase inhibitor (HDACi), panobinostat, in combination with CmpdA, as a potential treatment for GCT. Using cell proliferation and viability assays, we demonstrated that 100nM panobinostat acts synergistically with 500nM CmpdA to significantly decrease cell proliferation and increase apoptosis, as demonstrated by increased caspase 3/7 activity. Apoptotic pathways were further assessed using flow cytometry. A potential mechanism of action for the compounds was tested using real-time PCR and luciferase reporter assays. In KGN cells, panobinostat demonstrated increased NFκB activity and TNFα expression, which decreased when combined with CmpdA, suggesting that there is enhanced activation of the extrinsic pathway of apoptosis. Transcriptomic analysis showed the top significantly enriched pathways related to mechanisms of cancer pathogenesis. In particular, we observed significant downregulation of SIRT1, a non-classical Class III HDAC that is potentially important in GCT tumorigenesis through its interactions with the FOXL2 transcription factor, affecting cancer pathways such as cell senescence, proliferation, and apoptosis. Conclusion: We present a promising combination therapeutic strategy for GCT that reduced cell proliferation, increased apoptosis, and reduced inflammatory gene expression. Further studies are now needed to confirm and translate these findings, and to identify the links between SIRT1, and the pathognomonic FOXL2C134W mutation of GCT. Presentation: Thursday, June 15, 2023

Engaging with online social media consumer groups for rare cancers may help to develop collaborations between consumers and researchers. This study, a collaboration with the Granulosa Cell Tumor‐Survivor Sisters (GCT‐SS) Facebook group, explores the results of their survey of member's treatment and follow‐up experiences.

Recent studies have suggested that the unique FOXL2C134W mutation, which is pathognomonic for adult granulosa cell tumours of the ovary, is a tumour suppressor gene. In a recent issue of The Journal of Pathology, a detailed study by Pilsworth et al seeks to rebut the proposition that the FOXL2C134W mutation, which uniquely characterises adult granulosa cell tumours of the ovary, leads to reduced transcript levels with the implication that FOXL2 is a tumour suppressor gene. The study provides compelling evidence that both wild-type and mutant FOXL2 transcripts and protein are expressed at equivalent levels. In the context of other recent studies, one is drawn to the conclusion that FOXL2C134W is a gain-of-function mutation whose impact is mediated through enhanced interactions with the SMAD transcription factor complex. © 2021 The Pathological Society of Great Britain and Ireland.

BACKGROUND:The endogenous tissue inhibitor of metalloproteinase-2 (TIMP-2), through its homeostatic action on certain metalloproteinases, plays a vital role in remodelling extracellular matrix (ECM) to facilitate cancer progression. This study investigated the role of TIMP-2 in an ovarian cancer cell line in which the expression of TIMP-2 was reduced by either siRNA or CRISPR/Cas9.METHODS:OVCAR5 cells were transiently and stably transfected with either single or pooled TIMP-2 siRNAs (T2-KD cells) or by CRISPR/Cas9 under the influence of two distinct guide RNAs (gRNA1 and gRNA2 cell lines). The expression of different genes was analysed at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence (IF) and western blot. Proliferation of cells was investigated by 5-Ethynyl-2'-deoxyuridine (EdU) assay or staining with Ki67. Cell migration/invasion was determined by xCELLigence. Cell growth in vitro was determined by 3D spheroid cultures and in vivo by a mouse xenograft model.RESULTS:Approximately 70-90% knock down of TIMP-2 expression were confirmed in T2-KD, gRNA1 and gRNA2 OVCAR5 ovarian cancer cells at the protein level. T2-KD, gRNA1 and gRNA2 cells exhibited a significant downregulation of MMP-2 expression, but concurrently a significant upregulation in the expression of membrane bound MMP-14 compared to control and parental cells. Enhanced proliferation and invasion were exhibited in all TIMP-2 knocked down cells but differences in sensitivity to paclitaxel (PTX) treatment were observed, with T2-KD cells and gRNA2 cell line being sensitive, while the gRNA1 cell line was resistant to PTX treatment. In addition, significant differences in the growth of gRNA1 and gRNA2 cell lines were observed in in vitro 3D cultures as well as in an in vivo mouse xenograft model.CONCLUSIONS:Our results suggest that the inhibition of TIMP-2 by siRNA and CRISPR/Cas-9 modulate the expression of MMP-2 and MMP-14 and reprogram ovarian cancer cells to facilitate proliferation and invasion. Distinct disparities in in vitro chemosensitivity and growth in 3D culture, and differences in tumour burden and invasion to proximal organs in a mouse model imply that selective suppression of TIMP-2 expression by siRNA or CRISPR/Cas-9 alters important aspects of metastasis and chemosensitivity in ovarian cancer.