Barrett C. Lawson

Multiplexed Imaging Mass Cytometry Reveals Tumor-immune Microenvironment–dependent Hormone Receptor Expression in Adult-Type Ovarian Granulosa Cell Tumors

Abstract Adult-type granulosa cell tumors (AGCT) are rare ovarian tumors with few effective treatments for recurrent disease. To elucidate spatial features and cellular interactions within the AGCT tumor microenvironment, we applied imaging mass cytometry using a 34-marker panel on 130 regions from 24 AGCT samples, profiling more than 900,000 single cells. Analysis confirmed the immune “cold” phenotype of AGCTs and showed higher macrophage abundance in recurrent compared with primary tumors. We observed substantial heterogeneity in tissue architecture across samples, including variable presence of FOXL2+ cells embedded in collagen-rich regions (FOXL2+COL1A1+ cells). Based on tumor microenvironment composition, we defined two AGCT subtypes: AGCT-1 and AGCT-2 with distinct FOXL2+ cell distributions, differences in progesterone receptor expression, and unique transcriptomic profiles. Our findings highlight the role of macrophages, Foxl2+ subpopulations, and the extracellular matrix in AGCT progression and suggest AGCT subtype–specific vulnerabilities that could inform personalized therapies for this rare malignancy. Significance: We discovered two histologically and molecularly distinct forms of AGCTs that differ in cell composition, immune activity, and hormone signals. These findings point to new opportunities for more personalized treatment of this rare ovarian cancer.

Clinical and Genomic Landscape of RAS Mutations in Gynecologic Cancers

Abstract Purpose: We aimed to describe RAS mutations in gynecologic cancers as they relate to clinicopathologic and genomic features, survival, and therapeutic implications. Experimental Design: Gynecologic cancers with available somatic molecular profiling data at our institution between February 2010 and August 2022 were included and grouped by RAS mutation status. Overall survival was estimated by the Kaplan–Meier method, and multivariable analysis was performed using the Cox proportional hazard model. Results: Of 3,328 gynecologic cancers, 523 (15.7%) showed any RAS mutation. Patients with RAS-mutated tumors were younger (57 vs. 60 years nonmutated), had a higher prevalence of endometriosis (27.3% vs. 16.9%), and lower grades (grade 1/2, 43.2% vs. 8.1%, all P < 0.0001). The highest prevalence of KRAS mutation was in mesonephric-like endometrial (100%, n = 9/9), mesonephric-like ovarian (83.3%, n = 5/6), mucinous ovarian (60.4%), and low-grade serous ovarian (44.4%) cancers. After adjustment for age, cancer type, and grade, RAS mutation was associated with worse overall survival [hazard ratio (HR) = 1.3; P = 0.001]. Specific mutations were in KRAS (13.5%), NRAS (2.0%), and HRAS (0.51%), most commonly KRAS G12D (28.4%) and G12V (26.1%). Common co-mutations were PIK3CA (30.9%), PTEN (28.8%), ARID1A (28.0%), and TP53 (27.9%), of which 64.7% were actionable. RAS + MAPK pathway-targeted therapies were administered to 62 patients with RAS-mutated cancers. While overall survival was significantly higher with therapy [8.4 years [(95% confidence interval (CI), 5.5–12.0) vs. 5.5 years (95% CI, 4.6–6.6); HR = 0.67; P = 0.031], this effect did not persist in multivariable analysis. Conclusions: RAS mutations in gynecologic cancers have a distinct histopathologic distribution and may impact overall survival. PIK3CA, PTEN, and ARID1A are potentially actionable co-alterations. RAS pathway-targeted therapy should be considered.

Long-Term Follow-up of Levonorgestrel Intrauterine Device for Atypical Hyperplasia and Early Endometrial Cancer Reveals Relapse Characterized by Immune Exhaustion

Abstract Purpose: Nonsurgical treatment options are increasingly needed for endometrial atypical hyperplasia (AH) and endometrioid endometrial cancer (EEC). Despite promising initial response rates, prospective long-term data and determinants for relapse are limited. Materials and Methods: Follow-up data from patients in our prospective phase II trial of levonorgestrel intrauterine device (LIUD) for AH/G1EEC were collected from medical records. Spatial transcriptomics (Nanostring GeoMX digital spatial profiling) with in silico cell type deconvolution and pathway analyses were employed on longitudinal biopsy samples from five patients across pre-treatment, on-treatment, and relapse. Results: Of 43 participants exhibiting initial response to LIUD, 41 had follow-up data. Sixteen (39%) experienced relapse. Clinical factors associated with shorter response duration included younger age, initial diagnosis of G1EEC, lack of response at 6 months, premenopausal status, and Hispanic ethnicity (P < 0.05), but only 6-month response status remained a significant predictor in a multivariate model (P = 0.023). LIUD increased abundance of NK cells (ΔMCP-counter score = 46.13, FDR = 0.004) and cytotoxic lymphocytes (ΔMCP-counter score = 277.67, FDR = 0.004), as well as lymphocyte cytotoxicity markers PRF1 (log2FC = 1.62, FDR = 0.025) and GZMA (log2FC = 2.47, FDR = 0.008). NK cells were reduced at relapse (ΔMCP-counter score = −55.96, FDR = 0.02). Immune-related pathways (IFNα response and TGFβ signaling) were enriched at relapse (FDR < 0.05). IDO1 expression, reflecting immune exhaustion, was upregulated at relapse (FDR < 0.05). Conclusions: Upfront resistance and relapse after initial response to LIUD for AH/G1EEC impacts nearly half of patients, remaining a major hurdle for nonsurgical treatment of AH/G1EEC. Molecular studies evaluating longitudinal biopsies from a small cohort implicate immune mechanisms at relapse, including reversal of progestin-related immunomodulation and increased immune exhaustion. See related commentary by Johannet and Friedman, p. 5001

IMGN853 Induces Autophagic Cell Death in Combination Therapy for Ovarian Cancer

Abstract Antibodies targeting folate receptor 1 (FOLR1) induce autophagic cell death in addition to antibody-dependent cytotoxicity, but the biological relevance of anti-FOLR1 antibody–induced autophagy for clinical applications remains unclear. In this study, we investigated the role of autophagic cell death triggered by IMGN853 (mirvetuximab soravtansine), an FOLR1-targeted antibody–drug conjugate, and explored potential combinations of IMGN853 with chemotherapeutic drugs used for ovarian cancer treatment. We discovered that FOLR1 was predominantly expressed in epithelial ovarian cancer cells, with similar expression levels observed in both primary ovarian tumors and metastatic omental tumors from patients with high-grade serous ovarian cancer (HGSC). Treatment with IMGN853 improved survival in mice bearing patient-derived xenografts of HGSC, enhanced autophagic flux, and induced cell death in HGSC cells. Additionally, it increased expression of the autophagy-related marker LC3B-II and cell death as marked by cleaved caspase-3, in a manner dependent on beclin-1, in HGSC models. Notably, combinations of IMGN853 with topotecan or the anti–VEGF-A antibody (B20) significantly reduced tumor growth compared with IMGN853 alone, whereas no significant effect was observed with olaparib. Our findings indicate that IMGN853 induces autophagic cell death, which contributes to its tumor-inhibiting effects. The identification of these effective combination therapies and the mechanisms behind FOLR1-mediated autophagic cell death could facilitate further clinical development of IMGN853. Significance: FOLR1 is heterogeneously overexpressed in epithelial ovarian cancer. We examined the combined effects of the anti-FOLR1 antibody–drug conjugate (IMGN853) with other drugs, including topotecan, anti–VEGF-A antibody, and olaparib. These findings could contribute to the continued development of IMGN853 in the treatment of ovarian cancer.

Surgical and Blood-Based Minimal Residual Disease in Patients with Ovarian Cancer after First-line Therapy: Clinical Outcomes and Translational Opportunities

Abstract Purpose: Minimal residual disease (MRD) after first-line treatment of advanced-stage ovarian cancer remains a long-standing barrier to cure. We investigated the prognostic and translational value of MRD detection by second-look laparoscopy (SLL) and ctDNA at the completion of first-line therapy. Experimental Design: Patients with high-grade epithelial ovarian cancer who had a complete clinical response to first-line therapy and underwent SLL and plasma collection for ctDNA were included. Progression-free survival (PFS) and overall survival (OS) were estimated based on MRD and clinicopathologic status. Spatial transcriptomics (GeoMx and Visium) and proteomics (CODEX) profiling were performed on serial samples from select patients. Results: Forty of 95 (42.1%) patients had surgically detected MRD, which was associated with worse PFS (median PFS 7.4 vs. 23.8 months; P < 0.001) and OS (median OS 33.9 vs. not reached; P < 0.001). SLL positivity was an independent negative prognostic factor for OS (HR, 4.40; 95% confidence interval, 1.37–14.21; P = 0.013) in multivariable analysis. Among 44 patients who underwent SLL and had ctDNA testing, 34% (15/44) were ctDNA-positive, which was associated with worse PFS (6.4 vs. 28.1 months; P < 0.001) and OS (32.4 months vs. not reached; P = 0.008). We demonstrated the feasibility of spatial multiomics in studying MRD and their ability to provide hypothesis-generating observations, implicating the upregulation of the hypoxia signaling pathway, expression of multiple druggable targets (CDK6, GLS, MSLN, ERBB2), and immune exclusion in MRD lesions. Conclusions: Approximately half of patients in clinical remission after first-line therapy have assessable MRD, which can inform prognosis, therapeutic target discovery, and clinical trials.

Comparative Tumor Microenvironment Analysis of Primary and Recurrent Ovarian Granulosa Cell Tumors

Abstract 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.

Gain-of-Function Chromatin Remodeling Activity of Oncogenic FOXL2C134W Reprograms Glucocorticoid Receptor Occupancy to Drive Granulosa Cell Tumors

Abstract Adult type ovarian granulosa cell tumors (AGCT) are rare malignancies with the near universal c.C402G (p.Cys134Trp) somatic mutation in FOXL2, a forkhead box family transcription factor important for ovarian function. Relapsed AGCT is incurable, but the mechanism of the unique FOXL2 mutation could confer therapeutic vulnerabilities. To identify FOXL2C134W-dependent pharmacologic synergies, we created and characterized endogenous FOXL2 isogenic AGCT cells and an AGCT tumoroid biobank. A drug screen identified that glucocorticoids promote FOXL2C134W-dependent AGCT growth. Epigenetic investigation revealed that the Cys134Trp mutation exposes latent DNA sequence–specific chromatin remodeling activity in FOXL2. FOXL2C134W-dependent chromatin remodeling activity redirected glucocorticoid receptor chromatin occupancy to drive hyaluronan synthase 2 gene expression and increase extracellular hyaluronan secretion. Treatment of AGCT models with hyaluronidase reduced viability, and dexamethasone rescued this effect. Combinatorial drug–drug interaction experiments demonstrated that dexamethasone antagonizes the potency of paclitaxel, a chemotherapy agent frequently used in the treatment of AGCT. Thus, gain-of-function pioneering activity contributes to the oncogenic mechanism of FOXL2C134W and creates a potentially targetable synergy with glucocorticoid signaling. Significance: Glucocorticoids promote granulosa cell tumor growth via epigenetic coregulation with the disease driver FOXL2C134W, providing mechanistic insight into disease oncogenesis and uncovering a potential treatment strategy.