Prevalence of predictive biomarkers in a large cohort of molecularly defined adult-type ovarian granulosa cell tumors.

OBJECTIVE: The objective of this study was to determine the prevalence of predictive biomarkers associated with FDA-approved therapies in molecularly defined adult-type ovarian granulosa cell tumors (aGCTs). METHODS: We performed a retrospective cross-sectional cohort study of tumor profiles using the inclusion criteria of molecularly defined (FOXL2 c.C402G positive) aGCTs previously sequenced at Foundation Medicine, Inc. The dataset included coding variants for up to 406 genes, microsatellite instability, tumor mutational burden, and genomic loss of heterozygosity (gLOH). PD-L1 expression was determined using the tumor proportion score, as measured using the DAKO 22C3 immunohistochemistry assay. RESULTS: 423 tumor profiles met inclusion criteria. The median age at the time of sample submission was 57 years (interquartile range 48-65). The mean tumor mutational burden was 1.8 mutations per megabase (range 0-8.8). No tumors exhibited microsatellite instability, and none were gLOH-High (≥16%). Sixty-seven tumors had PD-L1 expression measurement, and 94% were negative. Potentially actionable variants including MTAP deletion (12/173, 5.8%) and activating PIK3CA mutations (23/423, 5.4%) were identified. TP53-mutated aGCT had a higher tumor mutational burden (mean 2.4 mut/Mb, 95% CI 1.7-3.0 mut/Mb vs mean 1.7 mut/Mb, 95% CI 1.5-1.9 mut/Mb; P = .02) and higher gLOH score (mean 4.4%, 95% CI 2.7-6.1% vs mean 1.4%, 95% CI 1.2-1.6%; P = .002) than TP53 non-mutated tumors. CONCLUSIONS: No women with molecularly defined aGCT in this large cohort would be eligible for FDA-approved pembrolizumab based on either microsatellite instability or high tumor mutational burden. TP53 mutation identified a subset of this tumor type with distinct molecular features. The development of precision treatment options remains a critical unmet need for this rare disease.

The clinical efficacy and safety of single-agent pembrolizumab in patients with recurrent granulosa cell tumors of the ovary: a case series from a phase II basket trial.

Background Treatment of recurrent, unresectable granulosa cell tumor (GCT) of the ovary can be challenging. Given the rarity of the tumor, alternative therapies have been difficult to evaluate in large prospective clinical trials. Currently, to our knowledge, there are no reports of the use of immune checkpoint inhibitors in GCT patients. Here, we present a case series of GCT patients treated with pembrolizumab who were enrolled in a phase II basket trial in advanced, rare solid tumors (ClinicalTrials.gov: NCT02721732). Cases We identified 5 patients with recurrent GCT (4 adult and 1 juvenile type); they had an extensive history of systemic therapy at study enrollment (range, 3-10), with most regimens resulting in less than 12 months of disease control. Pembrolizumab was administered in these patients, as per trial protocol. Although there were no objective responses according to the irRECIST guidelines, 2 patients with adult-type GCT experienced disease control for ≥ 12 months (565 and 453 days). In one, pembrolizumab represented the longest duration of disease control compared to prior lines of systemic therapy (565 days vs. 13 months). In the other, pembrolizumab was the second longest systemic therapy associated with disease control (453 days vs. 22 months) compared to prior lines of therapy. In this patient, pembrolizumab was discontinued following withdrawal of consent. PD-L1 expression was not observed in any baseline tumor samples. Pembrolizumab was well tolerated, with no grade 3 or 4 treatment-related adverse events. Conclusions Although our results do not support the routine use of pembrolizumab monotherapy in unselected GCT patients, some patients with adult-type GCT may derive a clinical benefit, with a low risk of toxicity. Future studies should investigate the role of immunotherapy and predictors of clinical benefit in this patient population.

Ovarian granulosa cell tumor characterization identifies FOXL2 as an immunotherapeutic target.

Granulosa cell tumors (GCT) are rare ovarian malignancies. Due to the lack of effective treatment in late relapse, there is a clear unmet need for novel therapies. Forkhead Box L2 (FOXL2) is a protein mainly expressed in granulosa cells (GC) and therefore is a rational therapeutic target. Since we identified tumor infiltrating lymphocytes (TILs) as the main immune population within GCT, TILs from 11 GCT patients were expanded, and their phenotypes were interrogated to determine that T cells acquired late antigen-experienced phenotypes and lower levels of PD1 expression. Importantly, TILs maintained their functionality after ex vivo expansion as they vigorously reacted against autologous tumors (100% of patients) and against FOXL2 peptides (57.1% of patients). To validate the relevance of FOXL2 as a target for immune therapy, we developed a plasmid DNA vaccine (FoxL2-tetanus toxin; FoxL2-TT) by fusing Foxl2 cDNA with the immune-enhancing domain of TT. Mice immunization with FoxL2-TT controlled growth of FOXL2-expressing ovarian (BR5) and breast (4T1) cancers in a T cell-mediated manner. Combination of anti-PD-L1 with FoxL2-TT vaccination further reduced tumor progression and improved mouse survival without affecting the female reproductive system and pregnancy. Together, our results suggest that FOXL2 immune targeting can produce substantial long-term clinical benefits. Our study can serve as a foundation for trials testing immunotherapeutic approaches in patients with ovarian GCT.

Serum IL-6 and IL-8 Correlate with Prognostic Factors in Ovarian Cancer.

The aim of the study was to correlate serum levels of IL-2, IL-5, IL-6, IL-8, IL-10, and TNF-α with clinical, laboratory, and pathological prognostic factors in patients with primary ovarian malignancy. Patients treated at the Pelvic Mass Ambulatory of the Discipline of Gynecology and Obstetrics/Oncology Research Institute (IPON) of the UFTM with confirmed diagnosis of malignant ovarian neoplasia (n = 26) were evaluated. Serum collection was performed preoperatively for the determination of tumor markers. The cytokines IL-2, IL-5, IL-6, IL-8, IL-10, and TNF-α were assayed by enzyme-linked immunosorbent assay (ELISA). The prognostic factors were compared using the Mann-Whitney test, with significance level lower than 0.05. When evaluating IL6, it was observed that higher serum levels were associated with overall survival less than 60 months (p = 0.0382). In the evaluation of IL8, higher serum levels were associated with neutrophil-to-lymphocyte ratio (NLR) ≥ 4 and platelet-to-lymphocyte ratio (PLR) ≥ 200 (p = 0.0198 and p = 0.0072, respectively), altered values of serum CA125 (p = 0.0457), and stage IIIC (p = 0.0486). Therefore, increased levels of IL-6 and IL-8 are associated with factors of worse prognosis in ovarian cancer. Additional studies with a larger sample of patients are needed to confirm the role of cytokines as prognostic factors, in the definition of treatment, and in the development of future target therapies.

The human Müllerian inhibiting substance type II receptor as immunotherapy target for ovarian cancer. Validation using the mAb 12G4.

Ovarian cancer has the highest mortality rate among gynecologic malignancies. The monoclonal antibody 12G4 specifically recognizes the human Müllerian inhibiting substance type II receptor (MISRII) that is strongly expressed in human granulosa cell tumors (GCT) and in the majority of human epithelial ovarian cancers (EOC). To determine whether MISRII represents an attractive target for antibody-based tumor therapy, we first confirmed by immunohistochemistry with 12G4 its expression in all tested GCT samples (4/4) and all, but one, EOC human tissue specimens (13/14). We then demonstrated in vitro the internalization of 12G4 in MISRII(high)COV434 cells after binding to MISRII and its ability to increase the apoptosis rate (FACS, DNA fragmentation) in MISRII(high)COV434 (GCT) and MISRII(medium)NIH-OVCAR-3 (EOC) cells that express different levels of MISRII. A standard (51)Cr release assay showed that 12G4 mediates antibody-dependent cell-meditated cytotoxicity. Finally, in vivo assessment of 12G4 anti-tumor effects showed a significant reduction of tumor growth and an increase of the median survival time in mice xenografted with MISRII(high)COV434 or MISRII(medium)NIH-OVCAR-3 cells and treated with 12G4 in comparison to controls treated with an irrelevant antibody. Altogether, our data indicate that MISRII is a new promising target for the control of ovarian GCTs and EOCs. A humanized version of the 12G4 antibody, named 3C23K, is in development for the targeted therapy of MISRII-positive gynecologic cancers.

A novel treatment strategy for ovarian cancer based on immunization against zona pellucida protein (ZP) 3.

We tested the principle of treating malignant ovarian tumors by vaccination against their ectopically expressed protein, zona pellucida glycoprotein (ZP) 3, using as the experimental model the granulosa cell tumors that develop in transgenic mice expressing the simian virus 40 T-antigen under the inhibin-α promoter (inhα/Tag). We found high ZP3 expression in granulosa cell tumors of the transgenic mice, in human surface ovarian cancer and granulosa cell lines, and in human granulosa cell tumors and their metastases. Early preventive immunization (between 2 and 5.5 mo of age) of transgenic mice with recombinant human (rh) ZP3 prevented ovarian tumorigenesis, and delayed therapeutic immunization (between 4.5 and 7 mo) reduced weights of existing tumors by 86 and 75%, respectively (P<0.001), compared to vehicle-treated control mice. No objective side effects of the immunizations were observed. Liver metastases were found in nontreated/vehicle-treated controls (n=7/39), but none following active rhZP3 immunizations (n=0/36; P<0.05). Immunization with rhZP3 was highly effective, as demonstrated by the induction of anti-ZP3 antibodies, as well as proliferative responses to the ZP3 antigen. These results signal rhZP3 immunization as a novel strategy to be developed for the immunotherapy of ovarian granulosa cell tumors, as well as for that of other malignancies that may express ZP3.

Ovarian granulosa cell tumors frequently express EGFR (Her-1), Her-3, and Her-4: An immunohistochemical study.

OBJECTIVE: Up to 50% of patients with ovarian granulosa cell tumors (GCTs) will develop recurrences; some of these recurrences can be seen as late as 30 years following the initial surgical treatment. Combined chemotherapy and radiotherapy are currently used for patients with advanced or recurrent disease. The aim of this study was to investigate the possible eligibility of patients with GCTs for anti-Her therapy. METHODS: The immunohistochemical expression of EGFR (Her-1), Her-2, Her-3, and Her-4 was analyzed in a group of ovarian GCTs encompassing 38 adult type and 2 juvenile type. RESULTS: Thirty-one cases (77.5%) were positive for at least one of the receptors EGFR (Her-1), Her-3, and Her-4. Twenty-six out of 40 (65%) GCTs showed positive reaction for EGFR (Her-1). Eight tumors (20%) were exclusively positive for EGFR (Her-1). None of 40 cases showed a positive reaction for Her-2. Positive reactions for Her-3 and Her-4 were observed in 18 (45%) and 23 (57.5%) tumors. Only one case (2.5%) was exclusively positive for Her-4. Four tumors (10%) showed positivity for Her-3 and Her-4 but were negative for EGFR (HER-1). While one of the two JGCTs was negative for all members of the Her-family, one showed reactivity for EGFR (Her-1), Her-3, and Her-4. CONCLUSION: In this study, most of the ovarian GCTs express at least one of the receptors EGFR (Her-1), Her-3, and Her-4. These findings provide some evidence to further explore the potential use of agents targeting these receptors (particularly EGFR) in the treatment of ovarian GCTs.

[The role of Ki-67, mutant suppressor gene p53 and low mitotic levels in defining the prognosis for adult granulosa cell tumor of the ovary].

Clinico-morphological examination of adult granulosa cell tumors of the ovary was carried out in 17 patients (mean follow-up duration--146.2 +/- 22.16 months). Overall and relapse-free survival appeared significantly higher in low expression of Ki-67 (8.63 +/- 1.4%), mutant suppressor gene p53 (3.55 +/- 1.9%) and low mitotic levels in tumor (2.33 +/- 0.9 per 10 fields of microscope) (p < 0.05).

The value of cell proliferation and angiogenesis in the prognostic assessment of ovarian granulosa cell tumors.

OBJECTIVES: Most cases of granulosa cell tumors (GCT) of the ovary are characterized by a relatively good outcome. However, some tumors behave aggressively and some tend to recur many years after the initial diagnosis. Tumor growth depends on cell proliferation and angiogenesis. Thus, proliferative indices and microvessel density were studied to determine possible valuable methods to assess the GCT patient's outcome. METHODS AND STUDY DESIGN: Paraffin-embedded tissue blocks were available for 60 patients with primary GCT and were investigated by immunostaining with monoclonal antibodies against PCNA, Ki-67 and factor VIII-related antigen. The follow-up was available for 51 patients and ranged from 25 to 206 months. A clinical follow-up distribution of patients was made: 8 patients with recurrence (group I); 6 patients who lived with no evidence of recurrence for 100 months or more (group II), and 37 patients alive with no evidence of recurrence in the follow-up period of less than 100 months (group III). RESULTS: There was a statistical correlation between PCNA and Ki-67 proliferative indices. A significant increase (P <0.05) of mean PCNA and Ki-67 proliferative indices and mean tumor size was seen in patients of Group I compared to those of Group II. The mean PCNA proliferative index positively correlated with the mean Ki-67 proliferative index for Groups I and II. Mean microvessel density showed a positive correlation with mean PCNA and Ki-67 proliferative indices and with mean tumor size for Group I, whereas it was negatively correlated with PCNA proliferative index and tumor size for Group II. A positive correlation was found between mean mitotic count and both proliferative indices only for Group II. The following features were indicative of a relatively poor prognosis: GCT measuring >9 cm in diameter, PCNA >4.0%, Ki-67 >1.2%, and diffuse, insular and sarcomatoid histologic patterns. CONCLUSIONS: The findings support the importance of proliferative factors, tumor size and histologic patterns as possible prognostic indicators for estimating the biologic behavior of patients with GCT. Unfortunately, angiogenesis did not seem to be a useful determinant parameter of a possible aggressive behavior. However, a longer follow-up period with larger series may be required to assess the value of the parameters in prediction of patient survival.

Monoclonal antibodies against inhibin represent key markers of adult granulosa cell tumors of the ovary even in their metastases. A report of three cases with late metastasis, being previously misinterpreted as hemangiopericytoma.

Antibodies against human inhibin, a peptide hormone produced by ovarian granulosa cells to inhibit FSH, are widely applied to determine serum inhibin levels. Recently, they were, however, proved also to stain follicle cells in ovarian tissue by immunoreactions in histological sections. The commercially available inhibin antibody produced by Serotec, applied to sections of paraffin blocks, stained follicle epithelia in 6/6 samples of ovarian tissue from females under the age of 40 recruited from the archives. Adult granulosa cell tumor tissue samples from primary tumors of the ovary showed positive reaction in 6/6 cases. No positive reaction was found in staining tissues from hemangiopericytomas from males (0/3), leiomyomas, leiomyosarcomas, and a malignant melanoma (0/5), serving as negative controls. No positive reactions could be observed in tumor cells of 10 ovarian carcinomas, whereas in two of these cases single cells of the specialized ovarian stroma stained positively with inhibin. Positive immunostainings were revealed in three late metastases (two within the liver) from granulosa cell tumors in females, primarily misinterpreted as hemangiopericytomas or leiomyosarcomas, because the previously resected primaries of the ovary were not known at the time of liver surgery. The recognition of granulosa cell tumors, especially the distinction of the sarcomatoid growth type from soft tissue tumors, may be difficult, even if immunostaining for intermediate filaments are applied. Immunostaining by antibodies against inhibin, which can be applied reliably in histopathology, may therefore provide a useful tool to distinguish between granulosa cell tumors and genuine soft tissue tumors. This is also of clinical importance, because treatment of the former by cisplatin-based polychemotherapy and antisex hormone therapy proved to be helpful. Furthermore, the inhibin antibody can be used as an early serum marker for detecting tumor recurrence months before clinical evidence.

Tumor-associated antigen Ca 125 before and during the treatment of ovarian carcinoma.

Serum concentrations of Ca 125, a tumor-associated antigen of epithelial ovarian cancer, were measured in 29 ovarian cancer patients before cytoreductive surgery and in 112 patients during and after treatment. Ca 125 levels were increased (greater than 30 IU/mL) in 89.8% of patients with clinically demonstrable ovarian tumors and were negative in 92.1% of clinically disease-free patients. Low levels of Ca 125 were associated with early clinical stages or a minimal tumor burden, and predicted a successful response to treatment and a low recurrence rate. High values indicated advanced disease and a poor response to cytotoxic chemotherapy. In 77% of patients the operation was explorative, with a preoperative Ca 125 level higher than 1000 IU/mL, whereas all the patients with values less than 100 IU/mL could be operated radically. Serum levels of Ca 125 were increased in similar frequency in epithelial, sex cord, and germ cell ovarian malignancies. The assay of Ca 125 seems to be a reliable noninvasive method for monitoring the presence and clinical behavior of ovarian cancer. Preoperative values have prognostic significance in predicting operability and response to chemotherapy.

Cellular differentiation in ovarian sex-cord-stromal and germ-cell tumors studied with antibodies to intermediate-filament proteins.

Seventy ovarian sex-cord-stromal and germ-cell tumors were immunohistochemically studied for the presence of intermediate-filament proteins of different types used as markers for cellular differentiation. Cells of ovarian granulosa-cell tumors constantly expressed vimentin and appeared to lack cytokeratin. Two tumors previously classified as granulosa-cell tumors were reclassified as poorly differentiated "common" epithelial tumors based on their cytokeratin positivity, vimentin negativity, and morphologic features. Dysgerminomas and Leydig-cell tumors showed only vimentin positivity. Tubular structures in androblastomas, which are considered to represent Sertoli-cell differentiation, were cytokeratin positive, and thus differed from the majority of normal Sertoli cells that are known to express vimentin and not cytokeratin. Embryonal carcinomas, choriocarcinomas, and endodermal sinus tumors showed cytokeratin positivity in the neoplastic cells whereas vimentin was observed in the stromal cells. In immature teratomas, epithelial differentiation was demonstrated with cytokeratin antibodies, and neural and glial differentiation was also frequently demonstrated by immunostaining with antibodies to neurofilaments and glial fibrillary acidic protein. The results show that antibodies to intermediate filaments can be used in the differential diagnosis between ovarian epithelial and nonepithelial tumors, and they provide a very accurate additional method to characterize the cellular differentiation of ovarian neoplasms.

[The clinical significance of CA125 in patients with gynecological tumors--a comparative study on CA125 and other tumor markers].

Serum CA125 levels were measured by radioimmunoassay patients with various gynecological tumors. Elevated levels of CA125 were detected in the serum of patients with malignant ovarian tumors. Among patients with uterine tumors, CA125 levels were elevated in those with malignant uterine tumors, but not with uterine myomas. The correlations between tumor markers (CA125, IAP, ferritin, PTA) and malignant ovarian tumors were measured, and only CA125 levels were found to correlate with progression or regression of the disease in patients with malignant ovarian tumors. These results suggest that CA125 may be a useful marker for monitoring the response to treatment in patients with malignant ovarian tumors.

Carcinoembryonic antigen (CEA) in ovarian cancer: factors influencing its incidence and changes which occur in response to cytotoxic drugs.

We report the serum levels of carcinoembryonic antigen (CEA) in 109 patients with ovarian cancer. Histology, degree of differentiation, and clinical stage influenced the incidence of positive CEA. Although CEA was significantly raised in patients with a variety of tumours, the highest incidence (77 per cent) was found in those with serious cystadenocarcinoma. Nearly all (94 per cent) of the poorly differentiated tumours were associated with a positive CEA result. Serial CEA levels provided a useful guide to management during cytotoxic chemotherapy, rapidly falling levels indicating a favourable tumour response which was reflected clinically. However, only two-thirds of tumours were associated with detectable CEA levels in serum, day-to-day variations in individual serum levels occurred, and CEA levels tended to fall paradoxically during terminal illness. The significance of persistently low levels in the apparent absence of disease was uncertain.