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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.

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.