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Adult-type granulosa cell tumors (aGCT) are rare ovarian sex cord tumors with few effective treatments for recurrent disease. The objective of this study was to characterize the tumor microenvironment (TME) of primary and recurrent aGCTs and to identify correlates of disease recurrence. Total RNA sequencing (RNA-seq) was performed on 24 pathologically confirmed, cryopreserved aGCT samples, including 8 primary and 16 recurrent tumors. After read alignment and quality-control filtering, DESeq2 was used to identify differentially expressed genes (DEG) between primary and recurrent tumors. Functional enrichment pathway analysis and gene set enrichment analysis was performed using “clusterProfiler” and “GSVA” R packages. TME composition was investigated through the analysis and integration of multiple published RNA-seq deconvolution algorithms. TME analysis results were externally validated using data from independent previously published RNA-seq datasets. A total of 31 DEGs were identified between primary and recurrent aGCTs. These included genes with known function in hormone signaling such as LHCGR and INSL3 (more abundant in primary tumors) and CYP19A1 (more abundant in recurrent tumors). Gene set enrichment analysis revealed that primarily immune-related and hormone-regulated gene sets expression was increased in recurrent tumors. Integrative TME analysis demonstrated statistically significant depletion of cancer-associated fibroblasts in recurrent tumors. This finding was confirmed in multiple independent datasets. Implications: Recurrent aGCTs exhibit alterations in hormone pathway gene expression as well as decreased infiltration of cancer-associated fibroblasts, suggesting dual roles for hormonal signaling and TME remodeling underpinning disease relapse.

RNA Sequencing QC Metrics

Adult-type granulosa cell tumors (aGCT) are rare ovarian sex cord tumors with few effective treatments for recurrent disease. The objective of this study was to characterize the tumor microenvironment (TME) of primary and recurrent aGCTs and to identify correlates of disease recurrence. Total RNA sequencing (RNA-seq) was performed on 24 pathologically confirmed, cryopreserved aGCT samples, including 8 primary and 16 recurrent tumors. After read alignment and quality-control filtering, DESeq2 was used to identify differentially expressed genes (DEG) between primary and recurrent tumors. Functional enrichment pathway analysis and gene set enrichment analysis was performed using “clusterProfiler” and “GSVA” R packages. TME composition was investigated through the analysis and integration of multiple published RNA-seq deconvolution algorithms. TME analysis results were externally validated using data from independent previously published RNA-seq datasets. A total of 31 DEGs were identified between primary and recurrent aGCTs. These included genes with known function in hormone signaling such as LHCGR and INSL3 (more abundant in primary tumors) and CYP19A1 (more abundant in recurrent tumors). Gene set enrichment analysis revealed that primarily immune-related and hormone-regulated gene sets expression was increased in recurrent tumors. Integrative TME analysis demonstrated statistically significant depletion of cancer-associated fibroblasts in recurrent tumors. This finding was confirmed in multiple independent datasets.Implications:Recurrent aGCTs exhibit alterations in hormone pathway gene expression as well as decreased infiltration of cancer-associated fibroblasts, suggesting dual roles for hormonal signaling and TME remodeling underpinning disease relapse.

Differentially Expressed Genes

Clinical Characteristics by Patient

S1. Heatmap of unsupervised hierarchical clustering of the top 1000 most variable genes.S2. Violin plots showing the expression levels of genes previously identified as differentially expressed genes between primary and recurrent tumors in Haltia et al., 2020.S3. Gene set enrichment analysis performed with GO terms and KEGG pathways showing that primarily immune-related and hormone-regulated gene sets expression are altered between primary and recurrent aGCTs.S4. Gene set enrichment analysis results showing that top enriched gene sets are significantly enriched in recurrent tumors in comparison with primary tumors.S5. Boxplot showing the fraction of each noncancerous cell type identified by CIBERSORTx (A) and xCell (B) in primary and recurrent tumor samples.S6. Heatmap showing the correlation between immune cells fractions.

A major challenge in diagnostic pathology is to identify sensitive and specific markers for tumor origin. Although PAX8 is the most widely used marker for gynecological tumors, it is not entirely specific, which makes it difficult to determine the origin of some tumors with high confidence. Through mining The Cancer Genome Atlas mRNA expression profile data, we identified SOX17 as a potential specific marker at the mRNA level for gynecological tumors. To test SOX17 expression at the protein level, we performed immunochemical staining in a large cohort of ovarian and endometrial cancer cases (n = 402) together with a large cohort of solid tumors from other organs (n = 1269). Similar to PAX8, SOX17 was highly expressed in all subtypes of ovarian carcinoma (97.5% for SOX17 vs. 97% for PAX8 in serous, 90% vs. 90% in endometrioid, and 100% vs. 100% in clear cell and transitional cell types) except for mucinous carcinoma (0% vs 27%), and also highly expressed in endometrial endometrioid carcinoma (88% vs 84%). However, SOX17 was completely negative in thyroid carcinoma and renal cell carcinoma, whereas PAX8 expression was high (86% and 85%, respectively). In addition, SOX17 had no expression in breast carcinoma, lung carcinoma, pancreatic adenocarcinoma, colonic and gastric adenocarcinomas, salivary ductal carcinoma, hepatocellular carcinoma, and cholangiocarcinoma. Our data indicate that SOX17 is not only a sensitive but also a specific marker for the origin of ovarian and endometrial carcinomas.

Background: A missense mutation in the Forkhead domain-containing FOXL2 (Foxl2 p.C134W) transcription factor is found in nearly all adult-type granulosa cell tumors, but the oncogenic mechanism of this mutation is not known. Other Forkhead family transcription factors have well-described “pioneer” activity, binding to compacted, nucleosome-bound DNA and increasing accessibility for other regulatory proteins. Objectives: To develop novel cell culture model systems and determine whether oncogenic Foxl2-C134W has pioneering activity. Methods: CRISPR/Cas9 editing was used to generate isogenic granulosa cell tumor cells lacking either the FOXL2 wild-type allele (single knock-out; SKO) or both the mutant and wild-type FOXL2 alleles (double knock-out; DKO). ATAC-Seq and endogenous Foxl2 ChIP-Seq were performed on these isogenic lines to determine the differential chromatin accessibility at Foxl2-bound regulatory regions across genotypes. ENCODE pipelines and data standards were used for analysis, and the irreproducible discovery rate was used to identify high-reliability ATAC-seq and ChIP-seq peaks. De novo motifs were identified with STREME. Promoter capture HiC was performed using a bespoke genome-wide hybrid capture probe set, and CHiCAGO was used to identify significant HiC interactions. Results: Endogenous ChIP-seq of Foxl2-C134W from the SKO cell line identified 1147 high-confidence peaks. De novo motif discovery identified the canonical Foxl2 binding motif (TGTTTACATT, P = 1.4 x 10-7) in 44.9% of peaks, as well as a novel variant motif (TGTTTTGTCT, P = 6.7 x 10-15) in 68.5% of peaks. Median chromatin accessibility at Foxl2-C134W peak regions, as measured by ATAC-seq, was significantly decreased in the DKO cells compared to either SKO or parental cell lines (P < 2 x 10-16 for both comparisons). No difference was observed between SKO and parental cell lines (P = 0.19). Decreased chromatin accessibility at Foxl2-C134W ChIP-seq peaks in the DKO cells was driven by peaks containing the novel variant Foxl2 binding motif. Promoter capture HiC identified promoter-enhancer interactions lost in the DKO cell line involving several genes, including HUNK, NOVA1, andSFRP2 (adjusted P < 0.05). Conclusion: Foxl2-C134W exhibits “pioneering” activity, increasing chromatin accessibility at key gene regulatory elements with associated re-programming of promoter-enhancer interactions. The oncogenic mechanism of Foxl2-C134W in granulosa cell tumors may involve changes in DNA binding specificity, re-directing this pioneering function to sites containing a novel variant Foxl2 binding motif. Citation Format: Veena Vuttaradhi, Eleonora Khlebus, Thomas Welte, Namrata Khurana, Barrett Lawson, Robert Tyler Hillman. Oncogenic Foxl2 is a chromatin-remodeling pioneer transcription factor in adult-type ovarian granulosa cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1462.

Background: Adult-type granulosa cell tumors (AGCT) are rare ovarian sex cord tumors that exhibit near-universal FOXL2 c.C402G (p.Cys134Trp) hotspot mutations. AGCT recurrence is difficult to predict and is almost always incurable after relapse. Little is known about the relationship between intra-tumor immune and stromal composition and AGCT relapse. Objective: To compare global gene expression profiles between primary and recurrent AGCTs, characterize the tumor microenvironment (TME), and identify correlates of disease recurrence. Methods: Total RNA sequencing was performed on 24 pathologically confirmed, cryopreserved AGCT samples, including 8 primary and 16 recurrent tumors. Standard methods were applied for read alignment, quality control, and quantification of gene-specific read counts. DESeq2 was used to identify statistically significant (adjusted P-value < 0.05) differentially expressed genes between primary and recurrent tumors with fold change > 2. Gene set enrichment analysis was performed using clusterProfiler. Integrative TME composition de-convolution was performed using multiple published algorithms including CIBERSORTx, quanTIseq, xCell, MCP-counter, and EPIC. TME analysis results were externally validated using data from smaller, previously published RNA sequencing datasets. Results: Thirty-one genes were identified as differentially expressed between primary and recurrent AGCTs, including NELL2, GDF6, TUBB2B, AQP3. These included genes with known function in hormone signaling such as LHCGR (adjusted P-value = 0.002) and INSL3 (adjusted P-value = 0.017) which were highly expressed in primary tumors and CYP19A1 (adjusted P-value = 0.009) which was highly expressed in recurrent tumors. Gene set enrichment analysis revealed increased expression of hormone-regulated and immune-related gene sets in recurrent tumors. Integrative, multi-platform TME analysis showed recurrent AGCT to exhibit reduced fractions of cancer-associated fibroblasts and enrichment of myeloid lineages such as neutrophils and macrophages. Conclusions: Recurrent AGCTs exhibit alterations in hormone pathway gene expression as well as decreased infiltration of cancer-associated fibroblasts, suggesting dual roles for hormonal signaling and TME remodeling underpinning disease relapse. Citation Format: Eleonora Khlebus, Veena K. Vuttaradhi, Thomas Welte, Namrata Khurana, Joseph Celestino, Hannah C. Beird, Curtis Gumbs, Latasha Little, Alejandra Flores Legarreta, Tri Nguyen, Barrett Lawson, Russell R. Broaddus, David M. Gershenson, P. Andrew Futreal, R. Tyler Hillman. Tumor microenvironment composition correlates with relapse in ovarian granulosa cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2503.

Background: Nearly all adult-type granulosa cell tumors of the ovary (aGCTs) carry the same oncogenic mutation affecting the second winged helix domain of the Forkhead family transcription factor FOXL2 (c.C402G; p.C134W). There are no currently available therapies that exploit the high prevalence of FOXL2 mutations in this disease. Objectives: Exploit FOXL2-C402G oncogenic addiction in order to identify FDA-approved compounds with differential potency in KGN-FOXL2WT/C402G cells and isogenic KGN-FOXL2—/— cells. Methods: CRISPR/Cas9 was used to generate isogenic derivatives of KGN, a cell line derived from a recurrent aGCT. Parental cells (KGN-FOXL2WT/C402G) or isogenic cells lacking both FOXL2 alleles (KGN-FOXL2—/—) were functionally characterized and loss of Foxl2 protein was confirmed by immunoblot. Two drug libraries comprising a total of 2,416 compounds (majority FDA approved) were screened for effectiveness in KGN-FOXL2WT/C402G cells and KGN-FOXL2—/— cells. Our assay included staining with DAPI, followed by automated image analysis of each well to enumerate nuclei/cell numbers. Based on Fraction Affected (FA) statistics, compounds were ranked according to effectiveness in KGN-FOXL2WT/C402G cells. FA-area under the curve (FA-AUC) cutoff >0.1 yielded 152 drug candidates, categorized into 13 drug classes. 40 drug candidates, including the top 1-4 hits in each drug class were chosen for further validation. Validation rate in the confirmatory screen was at 95%. A novel primary tumor organoid culture system for aGCT was developed and validated using whole exome sequencing and immunophenotype. Selected candidate compounds were further investigated in aGCT organoid cultures from two relapsed tumors. Results: We identified classes of drugs such as the inhibitors of Bromodomain family proteins and Hydroxy-Camptothecin analogs, with enhanced activity in KGN-FOXL2WT/C402G cells relative to KGN-FOXL2—/— cells. Unexpectedly glucocorticoids as a class were found to stimulate proliferation of KGN-FOXL2WT/C402G cells whereas they lacked this activity in KGN-FOXL2—/— cells. For 36 glucocorticoids present in the screen, FA-AUC ranged from -0.3191 to -0.1342 (highest proliferation index among all test compounds) in KGN-FOXL2WT/C402G cells compared to -0.0353 to +0.1667 in KGN-FOXL2—/— cells. In recurrent aGCT organoids, the high-potency glucocorticoid dexamethasone induced cell proliferation and co-treatment with glucocorticoids increased the IC50 of paclitaxel in KGN-FOXL2WT/C402G cells from 1.43 nM to 4.04 nM. Conclusion: Mutant FOXL2 mediates a proliferative response to glucocorticoids in cultured cells and primary aGCT organoids. Paclitaxel is a standard of care chemotherapy in the treatment of aGCTs and is often administered with glucocorticoid pre-medication. The adverse effect of glucocorticoids on paclitaxel efficiency shown here should thus be taken into consideration in the clinic. Citation Format: Thomas Welte, Veena Vuttaradhi, Eleonora Khlebus, Barrett Lawson, Robert T. Hillman, Nghi Nguyen, Mary Sobieski, Reid T. Powell, Clifford Stephan. Targeting FOXL2C402Gvulnerabilities in adult type ovarian granulosa cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 453.