Biomarkers and targeted therapies for ovarian granulosa cell tumors

Adult-type granulosa cell tumor (AGCT) is a unique subtype of ovarian cancer, accounting for 5% of all ovarian malignancies. The prognosis of AGCTs is often favorable, however these tumors have a tendency for late relapse. Eventually AGCT recurs in every third patient, and half of them succumb to the disease. Surgery is the mainstay of treatment. Platinum-based chemotherapy is used in advanced and inoperable AGCTs, although its efficacy is only modest. The objectives of this study were to improve the preoperative diagnostics of AGCTs and to uncover targeted treatments for AGCT. Serum markers HE4 and CA125 are commonly used in the preoperative evaluation of unknown ovarian masses, but their roles in AGCTs are unknown. Based on our results, HE4 levels are not elevated in AGCTs. We detected increased CA125 levels in a subset of AGCTs; however, the marker had no prognostic significance. These results underline the possibility of malignant ovarian tumor even in situations where CA125 and HE4 are both normal. Based on our and previous findings, inhibin B and AMH are the most specific markers in distinguishing AGCT from other malignant or benign ovarian tumors. The equilibrium between proliferation and apoptosis is crucial in the physiology of normal granulosa cells and it has been proposed to be disturbed in AGCTs. We evaluated the role of TNF-related apoptosis inducing ligand (TRAIL) in clinical AGCT samples and detected a significant correlation of TRAIL to its receptors (DR4 and DR5) in AGCT tissue samples, indicating active TRAIL signaling in AGCTs. Furthermore, both circulating and tissue TRAIL expressions were decreased in patients with large AGCTs, implying a tumor suppressive role for TRAIL in AGCTs. Our results suggest a potential role for TRAIL in AGCT treatment. We characterized the hormonal milieu of AGCTs in a vast collection of AGCT samples and studied the functional effects of follicle-stimulating hormone (FSH) and estradiol (E2) in tumor proliferation by using cell culture assays. FSH receptor and estrogen receptor beta were found to be expressed in the majority of tumors whereas the expression of aromatase, a crucial enzyme in estrogen synthesis, varied significantly among the studied tumors. In functional assays, FSH increased the viable cell number in subset of AGCTs, whereas E2 increased the cell number only at high concentrations. The aromatase inhibitor letrozole was able to block E2 synthesis in AGCTs but had no effect on cell viability. To systematically screen for new targeted therapies for AGCTs, we performed drug sensitivity and resistance testing and transcriptomic profiling. Seven AGCT primary cell cultures were exposed to a collection of 230 oncologic regimens. Among these compounds, a multi tyrosine kinase inhibitor dasatinib emerged as the most effective targeted treatment. Furthermore, dasatinib in combination with the traditional chemotherapeutic paclitaxel showed a synergistic effect, suggesting clinical testing of these regimens in patients with advanced AGCT. In conclusion, we verified that inhibin B and AMH are the most specific serum markers in AGCT preoperative diagnostics. Even though aromatase inhibitors showed no efficacy in inhibition of AGCT cell growth, further clinical studies are needed to verify the role of hormonal treatments in AGCTs. Importantly, our results suggest a role for TRAIL and dasatinib in AGCT treatment and encourage clinical testing of these regimens.Aikuistyypin granuloosasolukasvain (AGSK) on munasarjasyövän alatyyppi, joka käsittää 5% pahanlaatuisista munasarjakasvaimista. Kasvaimella on taipumus myöhäiseen uusiutumiseen, jopa vuosikymmenten kuluttua taudin ensidiagnoosista. Noin kolmasosalla potilaista AGSK uusiutuu, ja näistä noin puolet menehtyy tautiin. Leikkaus on hoidon kulmakivi. Levinnyttä tautia hoidetaan lääkkein, joskin käytössä olevien solunsalpaajien teho AGSK:een on hyvin rajoittunut. Tutkimme kahden munasarjasyöpämerkkiaineen, CA125 ja HE4:n pitoisuuksia AGSK-potilaiden verinäytteissä, ja arvioimme, voiko näitä merkkiaineita käyttää AGSK:n erotusdiagnostiikassa. AGSK-potilailla ei havaittu koholla olevia HE4-tasoja. CA125 oli koholla osalla AGSK-potilaista, mutta tällä ei ollut ennusteellista merkitystä. Normaalit CA125 ja HE4 -tasot eivät poissulje pahanlaatuisen munasarjakasvaimen mahdollisuutta. Tutkimuksemme vahvisti aiemmat havainnot siitä, että inhibiini B ja AMH ovat tarkkoja merkkiaineita erottamaan AGSK:t sekä pahanlaatuisista että hyvänlaatuisista munasarjamuutoksista. Normaalien granuloosasolujen elinkaarta säätelevät nopeaa kasvua edistävät tekijät, sekä ohjelmoidun solukuoleman mekanismit. Näiden tasapainon on ajateltu olevan AGSK:ssa häiriintynyt. Tutkimme TNF-related apoptosis inducing ligand (TRAIL) -proteiinin roolia AGSK-potilailla, ja osoitimme AGSK-kudoksen TRAIL-ligandin ilmentymisen korreloivan positiivisesti TRAIL-reseptoreiden ilmentymiseen, mikä viittaa aktiiviseen TRAIL-signalointiin AGSK:ssa. AGSK-potilailla, joilla oli keskimääräistä kookkaammat kasvaimet, TRAIL-proteiinin määrä sekä verenkierrossa että kasvaimessa oli vähentynyt. Tulokset viittaavat TRAIL:n säätelevän AGSK:n kasvua ja puoltavat jatkotutkimuksia TRAIL:ista AGSK-potilaiden hoidossa. Tässä väitöskirjatyössä tutkittiin myös hormonaalisten tekijöiden roolia AGSK:ssa. FSH-reseptori ja estrogeenireseptori beta ilmentyivät valtaosassa AGSK:ssa. Sen sijaan vain noin puolet kasvaimista ilmensi aromataasia, joka on välttämätön entsyymi kudoksen estradiolituoton kannalta. Solumalleilla tehdyissä tutkimuksissa FSH lisäsi AGSK-solujen määrää osassa kasvaimista, kun taas estradioli vaikutti solujen määrää lisäävästi vain hyvin suurilla annoksilla. Aromataasinestäjä letrotsoli esti kasvaimen estradiolituoton, mutta solujen elinvoimaisuuteen sillä ei ollut vaikutusta. Tutkimuksessa tehtiin myös laaja-alaista lääketestausta AGSK-soluille, tavoitteena löytää uusia täsmällisesti kasvaimeen kohdennettuja hoitoja. Seitsemästä AGSK:sta tehtiin soluviljelmä, ja solujen herkkyys testattiin 230 eri lääkeaineelle. Tyrosiinikinaasiestäjä dasatinibi osoittautui tehokkaaksi kaikissa testatuissa kasvaimissa. Kun dasatinibi yhdistettiin solunsalpaaja paklitakseliin, havaittiin näillä lääkkeillä synergiavaikutus, joten yhdistelmää kannattaa jatkossa kliinisesti testata AGSK-potilaiden hoidossa. Tässä tutkimuksessa vahvistettiin inhibiini B:n ja AMH:n rooli AGSK:n erotusdiagnostiikassa ennen leikkausta. Kohdennettujen hoitojen suhteen aromataasi-inhibiittoreilla ei havaittu olevan solujen kasvua hillitsevää vaikutusta. Osa potilaista saattaa kuitenkin hyötyä hormonaalisista hoidoista. Sekä TRAIL-proteiini että tyrosiinikinaasi-inhibiittori dasatinibi ovat yhdisteitä, joita on aiheellista jatkossa testata kliinisin tutkimuksin AGSK-potilaiden hoidossa

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.Peer reviewe

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.acceptedVersionPeer reviewe

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-Mullerian 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.Peer reviewe

Roles of human epididymis protein 4, carbohydrate antigen 125, inhibin B and anti-Mullerian 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 (AGCT5). Methods. The levels of circulating human epididymis protein 4 (HE4) and carbohydrate antigen 125 (CA125), together with AGCT markers inhibin B and anti-Mtillerian 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 AGCT5 than in EOCs (p <0.0001). CA125 levels were above 35 IU/1 in 25% of AGCT patients and 47.5% of ENDO patients, whereas inhibin B and AMH levels were elevated only in patients with AGCT5. 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. (C) 2016 Elsevier Inc. All rights reserved.Peer reviewe

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

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\u27 mRNA sequencing on formalin-fixed paraffin-embedded, archival AGCT tissue samples tested positive for the pathognomonic Forkhead Box L2

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