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OBJECTIVE: Adult granulosa cell tumors (aGCTs) represent a rare, hormonally active subtype of ovarian cancer that has a tendency to relapse late and repeatedly. Current serum hormone markers are inaccurate in reflecting tumor burden in a subset of aGCT patients, indicating the need for a novel biomarker. We investigated the presence of circulating tumor DNA (ctDNA) harboring a FOXL2 or TERT promoter mutation in serial plasma samples of aGCT patients to determine its clinical value for monitoring disease.METHODS: In a national multicenter study, plasma samples (n = 110) were prospectively collected from 21 patients with primary (n = 3) or recurrent (n = 18) aGCT harboring a FOXL2 402C > G and/or TERT (C228T or C250T) promoter mutation. Circulating cell-free DNA was extracted and assessed for ctDNA containing one of either mutations using droplet digital PCR (ddPCR). Fractional abundance of FOXL2 mutant and TERT mutant ctDNA was correlated with clinical parameters.RESULTS: FOXL2 mutant ctDNA was found in plasma of 11 out of 14 patients (78.6%) with aGCT with a confirmed FOXL2 mutation. TERT C228T or TERT C250T mutant ctDNA was detected in plasma of 4 of 10 (40%) and 1 of 2 patients, respectively. Both FOXL2 mutant ctDNA and TERT promoter mutant ctDNA levels correlated with disease progression and treatment response in the majority of patients.CONCLUSIONS: FOXL2 mutant ctDNA was present in the majority of aGCT patients and TERT promoter mutant ctDNA has been identified in a smaller subset of patients. Both FOXL2 and TERT mutant ctDNA detection may have clinical value in disease monitoring.

Purpose Adult granulosa cell tumors (AGCTs) of the ovary represent a rare malignancy in which timing and choice of treatment is a clinical challenge. This study investigates the value of FDG-PET/CT and FES-PET/CT in monitoring recurrent AGCTs and assessing eligibility for anti-hormonal treatment. Materials and methods We evaluated 22 PET/CTs from recurrent AGCT patients to determine tumor FDG (n = 16) and FES (n = 6) uptake by qualitative and quantitative analysis. We included all consecutive patients from two tertiary hospitals between 2003-2020. Expression of ERα and ERβ and mitoses per 2 mm2 were determined by immunohistochemistry and compared to FES and FDG uptake, respectively. Results Qualitative assessment showed low-to-moderate FDG uptake in most patients (14/16), and intense uptake in 2/16. One patient with intense tumor FDG uptake had a high mitotic rate (18 per 2 mm2) Two out of six patients showed FES uptake on PET/CT at qualitative analysis. Lesion-based quantitative assessment showed a mean SUVmax of 2.4 (± 0.9) on FDG-PET/CT and mean SUVmax of 1.7 (± 0.5) on FES-PET/CT. Within patients, expression of ERα and ERβ varied and did not seem to correspond with FES uptake. In one FES positive patient, tumor locations with FES uptake remained stable or decreased in size during anti-hormonal treatment, while all FES negative locations progressed. Conclusions This study shows that in AGCTs, FDG uptake is limited and therefore FDG-PET/CT is not advised. FES-PET/CT may be useful to non-invasively capture the estrogen receptor expression of separate tumor lesions and thus assess the potential eligibility for hormone treatment in AGCT patients.

Although patient engagement has become increasingly relevant, researchers find it difficult to involve patients, specifically in translational research. Particularly in rare disease research, patient engagement is crucial. First, since grant donors focus on common diseases, patients can support

Adult granulosa cell tumors (AGCTs) arise from the estrogen-producing granulosa cells. Treatment of recurrence remains a clinical challenge, as systemic anti-hormonal treatment or chemotherapy is only effective in selected patients. We established a method to rapidly screen for drug responses in vitro using direct patient-derived cell lines in order to optimize treatment selection. The response to 11 monotherapies and 12 combination therapies, including chemotherapeutic, anti-hormonal, and targeted agents, were tested in 12 AGCT-patient-derived cell lines and an AGCT cell line (KGN). Drug screens were performed within 3 weeks after tissue collection by measurement of cell viability 72 h after drug application. The potential synergy of drug combinations was assessed. The human maximum drug plasma concentration (Cmax) and steady state (Css) thresholds obtained from available phase I/II clinical trials were used to predict potential toxicity in patients. Patient-derived AGCT cell lines demonstrated resistance to all monotherapies. All cell lines showed synergistic growth inhibition by combination treatment with carboplatin, paclitaxel, and alpelisib at a concentration needed to obtain 50% cell death (IC50) that are below the maximum achievable concentration in patients (IC50 &lt, Cmax). We show that AGCT cell lines can be rapidly established and used for patient-specific in vitro drug testing, which may guide treatment decisions. Combination treatment with carboplatin, paclitaxel, and alpelisib was consistently effective in AGCT cell lines and should be further studied as a potential effective combination for AGCT treatment in patients.

Adult granulosa cell tumors (AGCTs) harbor a somatic FOXL2 c.402C&gt, G mutation in ~95% of cases and are mainly surgically removed due to limited systemic treatment effect. In this study, potentially targetable genomic alterations in AGCTs were investigated by whole genome sequencing on 46 tumor samples and matched normal DNA. Copy number variant (CNV) analysis confirmed gain of chromosome 12 and 14, and loss of 22. Pathogenic TP53 mutations were identified in three patients with highest tumor mutational burden and mitotic activity, defining a high-grade AGCT subgroup. Within-patient tumor comparisons showed 29&ndash, 80% unique somatic mutations per sample, suggesting tumor heterogeneity. A higher mutational burden was found in recurrent tumors, as compared to primary AGCTs. FOXL2-wildtype AGCTs harbored DICER1, TERT(C228T) and TP53 mutations and similar CNV profiles as FOXL2-mutant tumors. Our study confirms that absence of the FOXL2 c.402C&gt, G mutation does not exclude AGCT diagnosis. The lack of overlapping variants in targetable cancer genes indicates the need for personalized treatment for AGCT patients.

Adult granulosa cell tumors (AGCTs) harbor a somatic FOXL2 c.402C>G mutation in ∼95% of cases and are mainly surgically removed due to limited systemic treatment effect. In this study, potentially targetable genomic alterations in AGCTs were investigated by whole genome sequencing on 46 tumor samples and matched normal DNA. Copy number variant (CNV) analysis confirmed gain of chromosome 12 and 14, and loss of 22. Pathogenic TP53 mutations were identified in three patients with highest tumor mutational burden and mitotic activity, defining a high-grade AGCT subgroup. Within-patient tumor comparisons showed 29-80% unique somatic mutations per sample, suggesting tumor heterogeneity. A higher mutational burden was found in recurrent tumors, as compared to primary AGCTs. FOXL2-wildtype AGCTs harbored DICER1, TERT(C228T) and TP53 mutations and similar CNV profiles as FOXL2-mutant tumors. Our study confirms that absence of the FOXL2 c.402C>G mutation does not exclude AGCT diagnosis. The lack of overlapping variants in targetable cancer genes indicates the need for personalized treatment for AGCT patients.

Introduction/Background Currently, researchers seem to aim for high impact scientific articles rather than research that is directly beneficial to patients. Patient engagement is therefore an increasingly relevant topic. Grant donors also focus on the translation of a scientific benchside discovery to the patient bedside. However, patients are mostly involved solely at study initiation but ideally should participate throughout the research process. Also, investigators often do not report back the results to the patients. Here, we aim to illustrate successful patient engagement in a Dutch multi-center study on a rare disease: granulosa cell tumours (GCTs) of the ovary. Methodology GCTs are neglected and often not included in ovarian cancer research due to their rarity and distinct behaviour. A patient‘s family initiated a fund to increase awareness and ultimately improve treatment, as it can be challenging to receive regular funding for rare diseases. Her particular experiences helped identifying the challenges that GCT patients face and determined the knowledge gaps. This resulted in patient engagement from the beginning. Additionally, we joined group meetings and informed patients through newsletters, explaining the current state of the research and giving them the possibility to directly ask the research team questions and brainstorm on future directions. Results A national, multi-center study was conducted and included 51 patients within a year. This comprises three times the yearly incidence (17–20 cases) of this rare disease in our country. This success is due to successful engagement of patients and their gynaecological-oncologists. Contact with the patient‘s organization guided future research directions and raised novel research questions. For example, two members who appeared to be distant relatives initiated a project on hereditary factors in these patients. Conclusion Patient engagement is crucial in research as it benefits both patients and researchers. Involvement facilitates patient enrollment and study publicity while also focusing on research that directly benefits patients. Disclosure No competing interests. Applicable funding sources: Granulosafonds Philine van Esch.

Introduction/Background Adult granulosa cell tumours (aGCT) constitute a rare subtype of ovarian cancer, with >90% of tumours characterized by the FOXL2 c.402C>G mutation. Recurrences occur in nearly 50% of patients and are associated with a poor prognosis. Surgery is the mainstay of treatment, since the effect of adjuvant therapies are limited. Chemotherapy and hormone therapy response is difficult to predict and patient numbers insufficient to conduct informative clinical trials. Patient-specific aGCT organoid and 2D culture establishment could evaluate the effect of novel therapeutic options on a relatively large scale and enable personalized treatment in this neglected patient group. Methodology Samples from 46 tumours (4 primary and 42 recurrences) from 18 patients were collected, mechanically homogenized to single cells and small tissue pieces, and seeded in 50% Cultrex Basement Membrane Extract on day of surgery. Basal medium (DMEM/F12) supplemented with various growth factor combinations promoted organoid formation. Medium was refreshed every 3–4 days and cells passaged every 3 weeks. In parallel, 2D monolayer cell cultures were established to perform drug screens for later validation in the more physiologically relevant organoids as they became available. Organoid and 2D tumour origin verification is ongoing by FOXL2 c.402C>G PCR. Results Tumour tissue cultivation resulted in 3D structures that remained viable for 3–4 passages, with an establishment rate of approximately 75% in primary and 26% in recurrences. Morphology of masses ranged from grape-like to cystic, with primary organoids demonstrating a more cystic morphology (figure 1) that could be robustly passaged. Monolayer cultures in DMEM/F12/FBS were successfully established for future drug screening with carboplatin, paclitaxel, tamoxifen and other first-line treatments. Conclusion Patient-derived aGCT organoids can be established. Organoid establishment is more successful for primary tumours than for metastases, and organoid establishment optimization is necessary. Tumour cell culture establishment and drug screens are likely to provide insight into patient-specific treatment response. Disclosure Nothing to disclose.

Introduction/Background Adult granulosa cell tumours (aGCT) are rare ovarian malignancies, molecularly characterized by a single somatic c.402C>G mutation in FOXL2. Recurrent disease after initial surgical treatment occurs in 30–50% of patients. Serum markers are used for diagnosis and follow-up, but are not always accurate for GCT monitoring. The use of circulating tumour DNA (ctDNA) to monitor disease has been suggested in many cancer types. We aimed to assess the presence of FOXL2 mutant ctDNA in aGCT patients and perform an initial evaluation of its potential as a marker for disease activity. Methodology In a national, multicenter GCT study, plasma samples (n=27) were prospectively collected from 7 patients with primary (n=1) or recurrent (n=6) aGCT harbouring the FOXL2 c.402C>G mutation. Circulating cell-free DNA was extracted and assessed for the FOXL2 mutation using droplet digital PCR. Identification of FOXL2 mutation positive ctDNA, defined as ≥ 3 mutant droplets, was correlated with clinical parameters. Results The FOXL2 c.402C>G mutation was found in the plasma of 5 out of 7 aGCT patients (71%), with mutant ctDNA fractions ranging between 0.4% and 47.8%. The tumour load in two patients without FOXL2 mutant ctDNA was limited, while all aGCT patients with FOXL2 mutant ctDNA had multifocal recurrent disease. In all ctDNA positive patients, increasing FOXL2 mutant ctDNA levels were associated with disease progression or relapse defined by a rise in serum markers and/or CT scan findings. In 3 out of 4 ctDNA positive patients with plasma taken before and after treatment, the observed clinical response to therapy was accompanied by a marked decrease in FOXL2 mutant ctDNA fractions. Conclusion FOXL2 c.402C>G mutant ctDNA can be detected in patients with aGCT. Our results suggest that this test may have clinical value in disease monitoring, particularly when standard biomarkers inaccurately reflect tumour burden. Further research in a larger cohort is currently ongoing. Disclosure Nothing to disclose

Introduction/Background Adult granulosa cell tumours (aGCTs) are thought to arise from the stromal cells of the ovary and are molecularly characterized by a specific FOXL2 c.402C>G mutation. Cytoreductive surgery remains the mainstay of treatment of both primary and recurrent disease, because the effect of chemotherapy is limited. Unravelling this tumour on a genomic level could possibly identify targets for systemic therapy. However, most genomic studies on ovarian cancer do not focus on aGCTs. We investigated the genomic landscape and tumour stability of aGCTs, a crucial step towards targeted treatment. Methodology We performed whole genome sequencing (WGS; 30–90X coverage) on 31 fresh frozen aGCT samples (5 primary aGCTs and 26 recurrences) to detect single nucleotide variants and copy number variants. From 23 samples matched germline DNA was available, enabling somatic variation identification. We compared multiple tumour samples taken from the same patient at different locations and time points. The cohort will be further extended to identify novel (targetable) cohort mutations and affected pathways. Results Cohort analysis detected the FOXL2 mutation in 90% (28/31 samples) and a TERT promoter mutation in 68% ( C228T in 14/31 and C250T in 7/31) of samples. Copy number analyses showed polyploidy in 70% of the matched samples (16/23) and confirmed previous reported gains in chromosome 14 and losses in 22. Intra-patient comparisons showed 40–72% unique somatic variants with 1562–1650 overlapping somatic variants detected in all samples. Somatic variant analysis in the matched samples showed 1431 to 8102 (median 4103) single nucleotide variants and 462 to 2963 (median 2065) insertions/deletions. Conclusion The view of aGCTs as monogenetic and genetically stable is no longer supported. The large number of unique somatic mutations in intra-patient comparisons reveals a high degree of tumour heterogeneity. Future targeted treatment should focus on consistent genomic changes shared both between and within patients. Disclosure No competing interests. Applicable funding sources: Granulosafonds Philine van Esch.