Combining TNFR2-Expressing Tregs and IL-6 as Superior Diagnostic Biomarkers for High-Grade Serous Ovarian Cancer Masses.

We hypothesised that the inclusion of immunosuppressive and inflammatory biomarkers in HGSOC patients would improve the sensitivity and specificity of the preoperative marker prediction of malignancy in patients with ovarian masses. We tested a panel of 29 soluble immune factors by multiplex bead immunoassay and 16 phenotypic T cell markers by flow cytometry in pre-treatment blood samples from 66 patients undergoing surgery for suspected ovarian cancer or ovarian cancer risk reduction. The potential diagnostic utility of all parameters was explored using Volcano plots, principal component analysis (PCA) and receiver operator characteristic (ROC) analysis. We also assessed the effect of culturing PBMCs from 20 healthy donors in the presence of malignant ascites fluid. The combination of TNFR2+ Tregs and IL-6 in the pre-treatment blood of patients with advanced HGSOC effectively discriminated patients with benign or malignant ovarian masses. In vitro culturing of the PBMCs of healthy donors in malignant ascites promoted an increase in TNFR2-expressing Tregs, which were decreased following blockade with IL-6 or STAT3 activity. Pre-treatment serum IL-6 and peripheral blood TNFR2+ Tregs may be potential clinical biomarkers that can discriminate patients with malignant compared to benign ovarian cancer masses, and the relationship between IL-6 and TNFR2+ Treg is likely to be mediated via the STAT3 signalling pathway.

Pre-operative sera interleukin-6 in the diagnosis of high-grade serous ovarian cancer.

Pre-operative discrimination of malignant masses is crucial for accurate diagnosis and prompt referral to a gynae oncology centre for optimal surgical intervention. HGSOC progression is correlated with local and systemic inflammation. We hypothesised that inclusion of inflammatory biomarkers in sera may improve diagnostic tests. In the training cohort, we tested four existing clinical tests (RMI score and ROMA, CA125 and HE4) and a panel of 28 immune soluble biomarkers in sera from 66 patients undergoing surgery for suspected ovarian cancer. Six promising immune biomarkers alone, or in combination with conventional tests, were subsequently analysed in an independent validation cohort (n = 69). IL-6 was identified as the main driver of variability followed closely by conventional diagnostic tests. Median sera IL-6 was higher in HGSOC patients compared to those with a benign mass or controls with normal ovaries (28.3 vs 7.3 vs 1.2 pg/ml, p < 0.0001). The combination of IL-6 further improved the overall predictive probability of the conventional tests. Modelling a two-step triage of women with a suspicious ovarian mass, with IL-6 > 3.75 pg/ml as primary triage followed by conventional tests (CA125 or RMI score) identified ovarian cancer in patients with a misclassification rate of 4.54-3.03%, superior to the use of CA125 or RMI alone (9.09 to 10.60). The validation cohort demonstrated a similar improvement in the diagnostic sensitivity following addition of IL-6. IL-6 in combination with conventional tests may be a useful clinical biomarker for triage of patients with a suspected malignant ovarian mass.

Interleukin 6 Present in Inflammatory Ascites from Advanced Epithelial Ovarian Cancer Patients Promotes Tumor Necrosis Factor Receptor 2-Expressing Regulatory T Cells.

BACKGROUND: Epithelial ovarian cancer (EOC) remains a highly lethal gynecological malignancy. Ascites, an accumulation of peritoneal fluid present in one-third of patients at presentation, is linked to poor prognosis. High levels of regulatory T cells (Tregs) in ascites are correlated with tumor progression and reduced survival. Malignant ascites harbors high levels of Tregs expressing the tumor necrosis factor receptor 2 (TNFR2), as well as pro-inflammatory factors such as interleukin 6 (IL-6) and tumor necrosis factor (TNF). IL-6 is also associated with poor prognosis. Herein, we study the effect of IL-6 and TNF present in ascites on the modulation of TNFR2 expression on T cells, and specifically Tregs. METHODS: Ascites and respective peripheral blood sera were collected from 18 patients with advanced EOC and soluble biomarkers, including IL-6, sTNFR2, IL-10, TGF-ß, and TNF, were quantified using multiplexed bead-based immunoassay. Peripheral blood mononuclear cells (PBMC) from healthy donors were incubated with cell-free ascites for 48 h (or media as a negative control). In some experiments, IL-6 or TNF within the ascites were neutralized by using monoclonal antibodies. The phenotype of TNFR2+ Tregs and TNFR2- Tregs were characterized post incubation in ascites. In some experiments, cell sorted Tregs were utilized instead of PBMC. RESULTS: High levels of immunosuppressive (sTNFR2, IL-10, and TGF-ß) and pro-inflammatory cytokines (IL-6 and TNF) were present in malignant ascites. TNFR2 expression on all T cell subsets was higher in post culture in ascites and highest on CD4+CD25hiFoxP3+ Tregs, resulting in an increased TNFR2+ Treg/effector T cell ratio. Furthermore, TNFR2+ Tregs conditioned in ascites expressed higher levels of the functional immunosuppressive molecules programmed cell death ligand-1, CTLA-4, and GARP. Functionally, TNFR2+ Treg frequency was inversely correlated with interferon-gamma (IFN-γ) production by effector T cells, and was uniquely able to suppress TNFR2+ T effectors. Blockade of IL-6, but not TNF, within ascites decreased TNFR2+ Treg frequency. Results indicating malignant ascites promotes TNFR2 expression, and increased suppressive Treg activity using PBMC were confirmed using purified Treg subsets. CONCLUSION: IL-6 present in malignant ovarian cancer ascites promotes increased TNFR2 expression and frequency of highly suppressive Tregs.

Low dose cyclophosphamide: Mechanisms of T cell modulation.

Cyclophosphamide is considered one of the most successful chemotherapy drugs and is listed on the World Health Organisations List of Essential Medicines. Since its initial synthesis in 1958, it has been widely used to treat a range of cancers but its use has been declining due to the advent of platinum based and other chemotherapy agents. However, cyclophosphamide is still used either as a single agent or as adjuvant therapy to treat lymphomas, and breast and ovarian cancers at much lower doses. The efficacy of low dose cyclophosphamide is primarily due to its ability to promote anti-tumour immunity, by selectively depleting regulatory T cells and enhancing effector T cell function. Compared to effecter T cells, regulatory T cells have metabolic adaptations that make them more susceptible to cyclophosphamide-mediated cytotoxicity. In this review, we highlight the potential for improving the efficacy of low dose cyclophosphamide by combining insights on the mechanisms of cyclophosphamide-mediated cytotoxicity, and how these cytotoxic effects of cyclophosphamide influence T cell function, thereby contributing to anti-tumour immunity.

Paclitaxel and Its Evolving Role in the Management of Ovarian Cancer.

Paclitaxel, a class of taxane with microtubule stabilising ability, has remained with platinum based therapy, the standard care for primary ovarian cancer management. A deeper understanding of the immunological basis and other potential mechanisms of action together with new dosing schedules and/or routes of administration may potentiate its clinical benefit. Newer forms of taxanes, with better safety profiles and higher intratumoural cytotoxicity, have yet to demonstrate clinical superiority over the parent compound.