Phagocytosis of Extracellular Vesicles Extruded From the Placenta by Ovarian Cancer Cells Inhibits Growth of the Cancer Cells.

OBJECTIVE: Ovarian cancer is a common gynecological cancer, and parity is negatively associated with the incidence of this disease. This negative association is hypothesized to be due in part to shifting the balance of estrogen and progesterone toward more progesterone and reduced ovulation during pregnancy. However, studies suggested that parity is also associated with estrogen-independent gynecological cancers suggesting balance of hormones may not be the only protective factor. Extracellular vesicles (EVs) play an important role in cell-to-cell communication in physiological and pathological conditions. During pregnancy, large amounts of EVs are extruded from the placenta, and they seem to be involved in the remarkable adaptation of a woman's body to normal pregnancy. We hypothesized that EVs extruded from the placenta play a role in this protective effect. METHODS: Placental EVs were collected from first-trimester placentae, and cancer cell EVs were isolated from ovarian cancer cells. The EVs were exposed to ovarian cancer cells for 48 hours. The proliferation of cancer cells and the cell cycle were measured. In addition, phagocytosis of deported placental EVs by cancer cells was also measured. RESULTS: The proliferation of cancer cells was significantly reduced by treatment with placental EVs (P = 0.001, analysis of variance), but not EVs from monocytes (P = 0.195), compared with untreated cancer cells. Furthermore, placental EVs also prevented the proliferation of cancer cells induced by cancer cell-derived EVs (P = 0.001). This inhibition of proliferation of ovarian cancer cells was partially due to phagocytosis of placental EVs by cancer cells. Phagocytosis of placental EVs delayed progression through the cell cycle. Calreticulin, a phagocytic "eat me" signal carried by placental EVs significantly inhibited ovarian cancer growth (P = 0.001). CONCLUSIONS: Our data demonstrated that EVs extruded from the placenta prevented ovarian cancer cell growth by a mechanism that involved delaying progression of the cell cycle after phagocytosis of the EVs.

Adult granulosa cell tumours (GCT): clinicopathological outcomes including FOXL2 mutational status and expression.

OBJECTIVES: The aim of this research was to use nucleic acids isolated from formalin-fixed paraffin-embedded (FFPE) tissue to investigate the diagnostic potential and prognostic significance of FOXL2 in adult-type GCTs, particularly as a marker of identifying early stage patients that are likely to relapse. METHODS: We performed a retrospective review of GCT patients referred to the Auckland Gynae-Oncology Multidisciplinary Team from 1955 to 2012. Baseline characteristics, clinical course, histopathology and survival data was recorded. Using nucleic acids extracted from FFPE tumour blocks, FOXL2 mutation status and expression was determined by DNA sequencing and RT-qPCR, respectively, and correlated with clinical data. RESULTS: 57 adult GCT patients were identified, however FFPE tumour blocks were available for only 37 of these patients. Sequencing results confirmed the presence of the FOXL2 mutation in 70% of patients. FOXL2 mutation positive adult tumours showed a trend towards higher FOXL2 expression than wildtype adult tumours, particularly in stage I patients (p=0.051). In addition, patients with homozygous FOXL2 mutations had a significantly higher relapse rate (p=0.04). There was no significant correlation between FOXL2 mutation status or FOXL2 expression and any other clinical variables. CONCLUSIONS: FFPE tumour blocks are a valuable resource of molecular information, especially when studying rare tumours such as GCTs. The FOXL2 mutation appears to have some diagnostic potential, however additional work in a larger cohort needs to be completed to confirm the prognostic significance of this gene mutation, and its expression.

MicroRNA profiling of ovarian granulosa cell tumours reveals novel diagnostic and prognostic markers.

BACKGROUND: The aim of this study was to explore the clinical utility of microRNAs (miRNAs) as improved markers of ovarian granulosa cell tumours (GCTs) for cancer diagnosis and prognosis prediction. Current histopathological and genetic markers, such as the presence of a FOXL2 gene mutation to distinguish between the two major subtypes are not wholly accurate and as such novel biomarkers are warranted. METHODS: The miRNA expression profiles of five formalin-fixed, paraffin-embedded (FFPE) adult-GCTs and five juvenile-GCTs were assessed using Affymetrix miRNA 3.0 Arrays and compared for differential expression. Ten miRNAs were assessed in an additional 33 FFPE tumours and four normal granulosa cell samples by quantitative RT-PCR, and their expression correlated to clinical information. RESULTS: MicroRNA array found 37 miRNAs as differentially expressed between the two GCT subtypes (p < 0.05, fold change ≥2 and among these, miRs -138-5p, -184, -204-5p, -29c-3p, -328-3p and -501-3p were validated by RT-qPCR as differentially expressed between the two GCT subtypes (p < 0.05). In addition, the expression of miR-184 was predictive of tumour recurrence in adult-GCTs, specifically for patients diagnosed with stage I and II and stage I only disease (p < 0.001 and p < 0.05, respectively). CONCLUSIONS: This study is the first to report on global miRNA expression profiles of human ovarian GCTs using FFPE tumour samples. We have validated six miRNAs as novel markers for subtype classification in GCTs with low levels of miR-138-5p correlating with early tumour stage. Low miR-184 abundance was correlated with tumour recurrence in early stage adult-GCT patients as a candidate predictive biomarker. Further studies are now needed to confirm the clinical utility of these miRNAs as diagnostic and recurrence markers, and understand their possible roles in the pathogenesis of GCTs.