Analysis of Non-Relapsed and Relapsed Adult Type Granulosa Cell Tumors Suggests Stable Transcriptomes during Tumor Progression.

Adult-type granulosa cell tumor (AGCT) is a rare ovarian malignancy characterized by slow growth and hormonal activity. The prognosis of AGCT is generally favorable, but one-third of patients with low-stage disease experience a late relapse, and over half of them die of AGCT. To identify markers that would distinguish patients at risk for relapse, we performed Lexogen QuantSeq 3' mRNA sequencing on formalin-fixed paraffin-embedded, archival AGCT tissue samples tested positive for the pathognomonic Forkhead Box L2 (FOXL2) mutation. We compared the transcriptomic profiles of 14 non-relapsed archival primary AGCTs (follow-up time 17-26 years after diagnosis) with 13 relapsed primary AGCTs (follow-up time 1.7-18 years) and eight relapsed tumors (follow-up time 2.8-18.9 years). Non-relapsed and relapsed primary AGCTs had similar transcriptomic profiles. In relapsed tumors three genes were differentially expressed: plasmalemma vesicle associated protein (PLVAP) was upregulated (p = 0.01), whereas argininosuccinate synthase 1 (ASS1) (p = 0.01) and perilipin 4 (PLIN4) (p = 0.02) were downregulated. PLVAP upregulation was validated using tissue microarray RNA in situ hybridization. In our patient cohort with extremely long follow-up, we observed similar gene expression patterns in both primary AGCT groups, suggesting that relapse is not driven by transcriptomic changes. These results reinforce earlier findings that molecular markers do not predict AGCT behavior or risk of relapse.

Real-life data on treatment and outcomes in advanced ovarian cancer: An observational, multinational cohort study (RESPONSE trial).

BACKGROUND: This study aimed to describe the treatment strategies and outcomes for women with newly diagnosed advanced high-grade serous or endometrioid ovarian cancer (OC). METHODS: This observational study collected real-world medical record data from eight Western countries on the diagnostic workup, clinical outcomes, and treatment of adult women with newly diagnosed advanced (Stage III-IV) high-grade serous or endometrioid OC. Patients were selected backward in time from April 1, 2018 (the index date), with a target of 120 patients set per country, followed for ≥20 months. RESULTS: Of the 1119 women included, 66.9% had Stage III disease, 11.7% had a deleterious BRCA mutation, and 26.6% received bevacizumab; 40.8% and 39.3% underwent primary debulking surgery (PDS) and interval debulking surgery (IDS), respectively. Of the patients who underwent PDS, 55.5% had no visible residual disease (VRD); 63.9% of the IDS patients had no VRD. According to physician-assessed responses (at the first assessment after diagnosis and treatment), 53.2% of the total population had a complete response and 25.7% had a partial response to first-line chemotherapy after surgery. After ≥20 months of follow-up, 32.9% of the patients were disease-free, 46.4% had progressive disease, and 20.6% had died. Bevacizumab use had a significant positive effect on overall survival (hazard ratio [HR], 0.62; 95% CI, 0.42-0.91; p = .01). A deleterious BRCA status had a significant positive effect on progression-free survival (HR, 0.60; 95% CI, 0.41-0.84; p < .01). CONCLUSIONS: Women with advanced high-grade serous or endometrioid OC have a poor prognosis. Bevacizumab use and a deleterious BRCA status were found to improve survival in this real-world population. LAY SUMMARY: Patients with advanced (Stage III or IV) ovarian cancer (OC) have a poor prognosis. The standard treatment options of surgery and chemotherapy extend life beyond diagnosis for 5 years or more in only approximately 45% of patients. This study was aimed at describing the standard of care in eight Western countries and estimating how many patients who are diagnosed with high-grade serous or endometrioid OC could potentially be eligible for first-line poly(adenosine diphosphate ribose) polymerase inhibitor (PARPi) maintenance therapy. The results highlight the poor prognosis for these patients and suggest that a significant proportion (79%) would potentially be eligible for first-line PARPi maintenance treatment.

Parity, menopausal hormone therapy, and risk of ovarian granulosa cell tumor - A population-based case-control study.

OBJECTIVE: Adult-type ovarian granulosa cell tumors (AGCTs) are hormonally active neoplasms with limited epidemiological data available. We evaluated the effect of parity and postmenopausal hormone therapy (HT) use on the risk of AGCT in a population-based case-control setting. METHODS: We identified all women diagnosed with AGCT during 1994-2015 (n = 505) from the Finnish Cancer Registry. For each case, five controls matched for age were selected from the National Population Registry, which also provided data on parity and ages at deliveries. Information on postmenopausal HT by different regimens (estradiol-only, sequential estrogen-progestin and continuous estrogen-progestin) was obtained from nationwide Prescription Register. The association between parity, ages at deliveries, HT use, and AGCT incidence was evaluated by odds ratios (ORs) using a conditional logistic regression model and stratified by age at index date (<55 years or ≥ 55 years). RESULTS: Parity and age at first or last delivery had no significant effect on AGCT risk. Systemic postmenopausal HT had been used by 20.4% of women who were later diagnosed with AGCT. The risk for subsequent AGCT was significantly decreased among users of estradiol-only therapy for at least five years (OR 0.28; 95% confidence interval 0.08-0.94) and continuous estradiol-progestin therapy for 6 months to 5 years (0.23; 0.08-0.71). CONCLUSIONS: Unlike in epithelial ovarian cancer, AGCT development is not clearly associated with parity, and users of postmenopausal HT do not seem to carry an excess risk for AGCT formation.

Roles of human epididymis protein 4, carbohydrate antigen 125, inhibin B and anti-Müllerian hormone in the differential diagnosis and follow-up of ovarian granulosa cell tumors.

OBJECTIVE: Evaluation of circulating tumor markers in ovarian cancer is crucial for optimal patient care. The goal of this study was to verify the most accurate circulating tumor markers for the diagnosis and follow-up of adult-type granulosa cell tumors (AGCTs). METHODS: The levels of circulating human epididymis protein 4 (HE4) and carbohydrate antigen 125 (CA125), together with AGCT markers inhibin B and anti-Müllerian hormone (AMH), were measured in 135 samples from AGCT patients, 37 epithelial ovarian carcinoma (EOC) patients, and 40 endometrioma (ENDO) patients. The levels were plotted with receiver operating characteristic (ROC) graphs, and the area under the curves (AUC) of the different markers were calculated and compared. RESULTS: HE4 levels were significantly lower in AGCTs than in EOCs (p<0.0001). CA125 levels were above 35IU/l in 25% of AGCT patients and 47.5% of ENDO patients, whereas inhibin B and AMH levels were elevated only in patients with AGCTs. In the AUC comparison analyses, inhibin B alone was sufficient to differentiate AGCT from EOC. In differentiating AGCT from ENDO, inhibin B and AMH performed similarly, and the combination of inhibin B and AMH increased the accuracy compared to either marker alone (sensitivity, 100%; specificity, 93%). Among AGCT patients, inhibin B was the best marker for detecting the presence of AGCT. CONCLUSIONS: HE4 and CA125 levels were low in AGCTs, and inhibin B was the most accurate circulating biomarker in distinguishing AGCTs from EOCs and from ENDOs. Inhibin B was also the best single marker for AGCT follow-up.

Systematic drug sensitivity testing reveals synergistic growth inhibition by dasatinib or mTOR inhibitors with paclitaxel in ovarian granulosa cell tumor cells.

OBJECTIVE: Resistance to standard chemotherapy poses a major clinical problem in the treatment of ovarian cancer patients. Adult-type granulosa cell tumor (AGCT) is a unique ovarian cancer subtype for which efficient treatment options are lacking in advanced disease. To this end, systematic drug response and transcriptomics profiling were performed to uncover new therapy options for AGCTs. METHODS: The responses of three primary and four recurrent AGCTs to 230 anticancer compounds were screened in vitro using a systematic drug sensitivity and resistance testing (DSRT) platform, coupled with mRNA sequencing. The responses of the AGCTs were compared with those of human granulosa luteal cells and bone marrow mononuclear cells. RESULTS: Patient-derived AGCT cells showed selective sensitivity to the Src family tyrosine kinase inhibitor dasatinib. A combination of either dasatinib or an mTOR-inhibitor everolimus with paclitaxel resulted in synergistic inhibition of AGCT cell viability. The key kinase targets of dasatinib and members of the mTOR pathway were constantly expressed at mRNA and protein levels, indicating multikinase signal addictions in the AGCT cells. Transcriptomic characterization of the tumors revealed no known oncogenic mutations, suggesting that the drug sensitivity of AGCTs was rather conveyed by selective target expression. CONCLUSIONS: We used a systematic functional approach to reveal novel treatment options for a unique gynecological cancer. The selective synergy found between taxanes and dasatinib or mTOR inhibitors warrants further clinical investigations of these combinations in relapsed or aggressive AGCTs and demonstrate that high-throughput drug screening and molecular profiling can provide an effective approach to uncover new therapy options.

Circulating levels of TNF-related apoptosis inducing-ligand are decreased in patients with large adult-type granulosa cell tumors-implications for therapeutic potential.

Targeted treatments are needed for advanced adult-type granulosa cell tumors (AGCTs). We set out to assess tumor tissue and circulating levels of TNF-related apoptosis-inducing ligand (TRAIL), a promising anti-cancer cytokine, in patients affected by AGCT. We analyzed tissue expression of TRAIL in 127 AGCTs using immunohistochemistry or RT-PCR. Soluble TRAIL was measured by means of ELISA from 141 AGCT patient serum samples, as well as the conditioned media of 15 AGCT patient-derived primary cell cultures, and the KGN cell line. Tissue and serum TRAIL levels were analyzed in relationship with clinical parameters, and serum estradiol, FSH, and LH levels. We found that AGCT samples expressed TRAIL mRNA and protein at levels comparable to normal granulosa cells. AGCT cells did not release soluble TRAIL. TRAIL protein levels were decreased in tumors over 10 cm in diameter (p = 0.04). Consistently, circulating TRAIL levels correlated negatively to tumor dimension (p = 0.01). Circulating TRAIL levels negatively associated with serum estradiol levels. In multiple regression analysis, tumor size was an independent factor contributing to the decreased levels of soluble TRAIL in AGCT patients. AGCTs associate with significantly decreased tumor tissue and serum TRAIL levels in patients with a large tumor mass. These findings encourage further study of agonistic TRAIL treatments in patients with advanced or recurrent AGCT.

Chemotherapy induces myeloid-driven spatial T-cell exhaustion in ovarian cancer.

To uncover the intricate, chemotherapy-induced spatiotemporal remodeling of the tumor microenvironment, we conducted integrative spatial and molecular characterization of 97 high-grade serous ovarian cancer (HGSC) samples collected before and after chemotherapy. Using single-cell and spatial analyses, we identify increasingly versatile immune cell states, which form spatiotemporally dynamic microcommunities at the tumor-stroma interface. We demonstrate that chemotherapy triggers spatial redistribution and exhaustion of CD8+ T cells due to prolonged antigen presentation by macrophages, both within interconnected myeloid networks termed "Myelonets" and at the tumor stroma interface. Single-cell and spatial transcriptomics identifies prominent TIGIT-NECTIN2 ligand-receptor interactions induced by chemotherapy. Using a functional patient-derived immuno-oncology platform, we show that CD8+T-cell activity can be boosted by combining immune checkpoint blockade with chemotherapy. Our discovery of chemotherapy-induced myeloid-driven spatial T-cell exhaustion paves the way for novel immunotherapeutic strategies to unleash CD8+ T-cell-mediated anti-tumor immunity in HGSC.

Functional Homologous Recombination Assay on FFPE Specimens of Advanced High-Grade Serous Ovarian Cancer Predicts Clinical Outcomes.

PURPOSE: Deficiency in homologous recombination (HR) repair of DNA damage is characteristic of many high-grade serous ovarian cancers (HGSC). It is imperative to identify patients with homologous recombination-deficient (HRD) tumors as they are most likely to benefit from platinum-based chemotherapy and PARP inhibitors (PARPi). Existing methods measure historical, not necessarily current HRD and/or require high tumor cell content, which is not achievable for many patients. We set out to develop a clinically feasible assay for identifying functionally HRD tumors that can predict clinical outcomes. EXPERIMENTAL DESIGN: We quantified RAD51, a key HR protein, in immunostained formalin-fixed, paraffin-embedded (FFPE) tumor samples obtained from chemotherapy-naïve and neoadjuvant chemotherapy (NACT)-treated HGSC patients. We defined cutoffs for functional HRD separately for these sample types, classified the patients accordingly as HRD or HR-proficient, and analyzed correlations with clinical outcomes. From the same specimens, genomics-based HRD estimates (HR gene mutations, genomic signatures, and genomic scars) were also determined, and compared with functional HR (fHR) status. RESULTS: fHR status significantly predicted several clinical outcomes, including progression-free survival (PFS) and overall survival (OS), when determined from chemo-naïve (PFS, P < 0.0001; OS, P < 0.0001) as well as NACT-treated (PFS, P < 0.0001; OS, P = 0.0033) tumor specimens. The fHR test also identified as HRD those PARPi-at-recurrence-treated patients with longer OS (P = 0.0188). CONCLUSIONS: We developed an fHR assay performed on routine FFPE specimens, obtained from either chemo-naïve or NACT-treated HGSC patients, that can significantly predict real-world platinum-based chemotherapy and PARPi response. See related commentary by Garg and Oza, p. 2957.

Optimized detection of homologous recombination deficiency improves the prediction of clinical outcomes in cancer.

Homologous recombination DNA-repair deficiency (HRD) is a common driver of genomic instability and confers a therapeutic vulnerability in cancer. The accurate detection of somatic allelic imbalances (AIs) has been limited by methods focused on BRCA1/2 mutations and using mixtures of cancer types. Using pan-cancer data, we revealed distinct patterns of AIs in high-grade serous ovarian cancer (HGSC). We used machine learning and statistics to generate improved criteria to identify HRD in HGSC (ovaHRDscar). ovaHRDscar significantly predicted clinical outcomes in three independent patient cohorts with higher precision than previous methods. Characterization of 98 spatiotemporally distinct metastatic samples revealed low intra-patient variation and indicated the primary tumor as the preferred site for clinical sampling in HGSC. Further, our approach improved the prediction of clinical outcomes in triple-negative breast cancer (tnbcHRDscar), validated in two independent patient cohorts. In conclusion, our tumor-specific, systematic approach has the potential to improve patient selection for HR-targeted therapies.

Functional Profiling of FSH and Estradiol in Ovarian Granulosa Cell Tumors.

Adult-type granulosa cell tumors (AGCTs) are sex-cord derived neoplasms with a propensity for late relapse. Hormonal modulators have been used empirically in the treatment of recurrent AGCT, albeit with limited success. To provide a more rigorous foundation for hormonal therapy in AGCT, we used a multimodal approach to characterize the expressions of key hormone biomarkers in 175 tumor specimens and 51 serum samples using RNA sequencing, immunohistochemistry, RNA in situ hybridization, quantitative PCR, and circulating biomarker analysis, and correlated these results with clinical data. We show that FSH receptor and estrogen receptor beta (ERß) are highly expressed in the majority of AGCTs, whereas the expressions of estrogen receptor alpha (ERα) and G-protein coupled estrogen receptor 1 are less prominent. ERß protein expression is further increased in recurrent tumors. Aromatase expression levels show high variability between tumors. None of the markers examined served as prognostic biomarkers for progression-free or overall survival. In functional experiments, we assessed the effects of FSH, estradiol (E2), and the aromatase inhibitor letrozole on AGCT cell viability using 2 in vitro models: KGN cells and primary cultures of AGCT cells. FSH increased cell viability in a subset of primary AGCT cells, whereas E2 had no effect on cell viability at physiological concentrations. Letrozole suppressed E2 production in AGCTs; however, it did not impact cell viability. We did not find preclinical evidence to support the clinical use of aromatase inhibitors in AGCT treatment, and thus randomized, prospective clinical studies are needed to clarify the role of hormonal treatments in AGCTs.

Pathogenesis and treatment of adult-type granulosa cell tumor of the ovary.

Adult-type granulosa cell tumor is a clinically and molecularly unique subtype of ovarian cancer. These tumors originate from the sex cord stromal cells of the ovary and represent 3-5% of all ovarian cancers. The majority of adult-type granulosa cell tumors are diagnosed at an early stage with an indolent prognosis. Surgery is the cornerstone for the treatment of both primary and relapsed tumor, while chemotherapy is applied only for advanced or non-resectable cases. Tumor stage is the only factor consistently associated with prognosis. However, every third of the patients relapse, typically in 4-7 years from diagnosis, leading to death in 50% of these patients. Anti-Müllerian Hormone and inhibin B are currently the most accurate circulating biomarkers. Adult-type granulosa cell tumors are molecularly characterized by a pathognomonic somatic missense point mutation 402C->G (C134W) in the transcription factor FOXL2. The FOXL2 402C->G mutation leads to increased proliferation and survival of granulosa cells, and promotes hormonal changes. Histological diagnosis of adult-type granulosa cell tumor is challenging, therefore testing for the FOXL2 mutation is crucial for differential diagnosis. Large international collaborations utilizing molecularly defined cohorts are essential to improve and validate new treatment strategies for patients with high-risk or relapsed adult-type granulosa cell tumor. Key Messages: Adult-type granulosa cell tumor is a unique ovarian cancer with an indolent, albeit unpredictable disease course. Adult-type granulosa cell tumors harbor a pathognomonic somatic missense mutation in transcription factor FOXL2. The key challenges in the treatment of patients with adult-type granulosa cell tumor lie in the identification and management of patients with high-risk or relapsed disease.

FIGO 1988 versus 2009 staging for endometrial carcinoma: a comparative study on prediction of survival and stage distribution according to histologic subtype.

OBJECTIVE: The surgical staging system for endometrial carcinoma developed by International Federation of Gynecology and Obstetrics (FIGO) in 1988 was revised in 2009. Given the importance of continuous validation of the prognostic performance of staging systems, we analyzed the disease specific survival for patients with endometrial carcinoma using FIGO 1988 and 2009 systems. Further, the stage distribution of endometrioid and nonendometrioid carcinomas was studied. METHODS: Eight hundred twenty-one women with endometrial carcinoma were retrospectively staged using FIGO 1988 and 2009 systems. RESULTS: FIGO 1988 IC was associated with an inferior survival compared with IA-IB. Survival overlapped for 1988 IA and IB, for 1988 IC and IIA, and for 2009 IB and II. FIGO 2009 IA-II patients with negative peritoneal cytology had a superior survival compared with 1988 IIIA patients with positive cytology only. The survival was similar for 1988 IIIA with positive cytology only and for 2009 IIIA. Cox proportional hazards model recognized grade 3 endometrioid and nonendometrioid histology, tumor spread beyond the uterine corpus and cervix, and positive peritoneal cytology as significant predictors of death. Among 2009 IIIC substages, the proportion of IIIC2 tumors was higher for nonendometrioid than for endometrioid carcinomas (p=0.003). CONCLUSION: Stage I with deep myometrial invasion and stage II endometrial carcinoma seem to have similar survival outcomes. Although positive peritoneal cytology does not alter the stage according to the FIGO 2009 system, it should be considered a poor prognostic sign. The high proportion of nonendometrioid carcinomas in the stage IIIC2 category may reflect different patterns of retroperitoneal spread among tumors with different histologic subtypes.