Although 70-80% of newly diagnosed ovarian cancer patients respond to first-line therapy, almost all relapse and five-year survival remains below 50%. One strategy to increase five-year survival is prolonging time to relapse by improving first-line therapy response. However, no biomarker today can accurately predict individual response to therapy. In this study, we present analytical and prospective clinical validation of a new test that utilizes primary patient tissue in 3D cell culture to make patient-specific response predictions prior to initiation of treatment in the clinic. Test results were generated within seven days of tissue receipt from newly diagnosed ovarian cancer patients obtained at standard surgical debulking or laparoscopic biopsy. Patients were followed for clinical response to chemotherapy. In a study population of 44, the 32 test-predicted Responders had a clinical response rate of 100% across both adjuvant and neoadjuvant treated populations with an overall prediction accuracy of 89% (39 of 44, p < 0.0001). The test also functioned as a prognostic readout with test-predicted Responders having a significantly increased progression-free survival compared to test-predicted Non-Responders, p = 0.01. This correlative accuracy establishes the test's potential to benefit ovarian cancer patients through accurate prediction of patient-specific response before treatment.

Endometriosis is a highly prevalent, chronic, heterogeneous, fibro-inflammatory disease that remains recalcitrant to conventional therapy. We previously showed that loss of KLF11, a transcription factor implicated in uterine disease, results in progression of endometriosis. Despite extensive homology, co-expression, and human disease association, loss of the paralog Klf10 causes a unique inflammatory, cystic endometriosis phenotype in contrast to fibrotic progression seen with loss of Klf11. We identify here for the first time a novel role for KLF10 in endometriosis. In an animal endometriosis model, unlike wild-type controls, Klf10(-/-) animals developed cystic lesions with massive immune infiltrate and minimal peri-lesional fibrosis. The Klf10(-/-) disease progression phenotype also contrasted with prolific fibrosis and minimal immune cell infiltration seen in Klf11(-/-) animals. We further found that lesion genotype rather than that of the host determined each unique disease progression phenotype. Mechanistically, KLF10 regulated CD40/CD154-mediated immune pathways. Both inflammatory as well as fibrotic phenotypes are the commonest clinical manifestations in chronic fibro-inflammatory diseases such as endometriosis. The complementary, paralogous Klf10 and Klf11 models therefore offer novel insights into the mechanisms of inflammation and fibrosis in a disease-relevant context. Our data suggests that divergence in underlying gene dysregulation critically determines disease-phenotype predominance rather than the conventional paradigm of inflammation being precedent to fibrotic scarring. Heterogeneity in clinical progression and treatment response are thus likely from disparate gene regulation profiles. Characterization of disease phenotype-associated gene dysregulation offers novel approaches for developing targeted, individualized therapy for recurrent and recalcitrant chronic disease.

Increased toxicant exposure and resultant environmentally induced diseases are a tradeoff of industrial productivity. Dioxin [2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD)], a ubiquitous byproduct, is associated with a spectrum of diseases including endometriosis, a common, chronic disease in women. TCDD activates cytochrome (CYP) p450 metabolic enzymes that alter organ function to cause disease. In contrast, the transcription factor, Krüppel-like factor (KLF) 11, represses these enzymes via epigenetic mechanisms. In this study, we characterized these opposing mechanisms in vitro and in vivo as well as determining potential translational implications of epigenetic inhibitor therapy. KLF11 antagonized TCDD-mediated activation of CYP3A4 gene expression and function in endometrial cells. The repression was pharmacologically replicated by selective use of an epigenetic histone acetyltransferase inhibitor (HATI). We further showed phenotypic relevance of this mechanism using an animal model for endometriosis. Fibrotic extent in TCDD-exposed wild-type animals was similar to that previously observed in Klf11-/- animals. When TCDD-exposed animals were treated with a HATI, Cyp3 messenger RNA levels and protein expression decreased along with disease progression. Fibrotic progression is ubiquitous in environmentally induced chronic, untreatable diseases; this report shows that relentless disease progression can be arrested through targeted epigenetic modulation of protective mechanisms.

We evaluated the association of disease outcome with T cell immune-related characteristics and T cell receptor (TCR) repertoire in malignant ascites from patients with high-grade epithelial ovarian cancer. Ascitic fluid samples were collected from 47 high-grade epithelial ovarian cancer patients and analyzed using flow cytometry and TCR sequencing to characterize the complementarity determining region 3 TCR β-chain. TCR functions were analyzed using the McPAS-TCR and VDJ databases. TCR clustering was implemented using Grouping of Lymphocyte Interactions by Paratope Hotspots software. Patients with poor prognosis had ascites characterized by an increased ratio of CD8+ T cells to regulatory T cells, which correlated with an increased productive frequency of the top 100 clones and decreased productive entropy. TCRs enriched in patients with an excellent or good prognosis were more likely to recognize cancer antigens and contained more TCR reads predicted to recognize epithelial ovarian cancer antigens. In addition, a TCR motif that is predicted to bind the TP53 neoantigen was identified, and this motif was enriched in patients with an excellent or good prognosis. Ascitic fluid in high-grade epithelial ovarian cancer patients with an excellent or good prognosis is enriched with TCRs that may recognize ovarian cancer-specific neoantigens, including mutated TP53 and TEAD1. These results suggest that an effective antigen-specific immune response in ascites is vital for a good outcome in high-grade epithelial ovarian cancer.

Ovarian cancer is associated with a high mortality rate due to diagnosis at advanced stages. Dissemination often occurs intraperitoneally within the ascites fluid. The microenvironment can support dissemination through several mechanisms. One potential ascites factor which may mediate dissemination are EVs or extracellular vesicles that can carry information in the form of miRNAs, proteins, lipids, and act as mediators of cellular communication. We present our observations on EVs isolated from ascitic supernatants from patients diagnosed with high grade serous ovarian carcinoma in augmenting motility, growth, and migration towards omental fat. MicroRNA profiling of EVs from malignant ascitic supernatant demonstrates high expression of miR 200c-3p, miR18a-5p, miR1246, and miR1290 and low expression of miR 100- 5p as compared to EVs isolated from benign ascitic supernatant. The migration of ovarian cancer spheroids towards omental fat is enhanced in the presence of malignant ascitic EVs. Gene expression of these cells showed increased expression of ZBED2, ZBTB20, ABCC3, UHMK1, and low expression of Transgelin and MARCKS. We present evidence that ovarian ascitic EVs increase the growth of ovarian cancer spheroids through miRNAs.