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.

Sperm Protein 17 Expression by Murine Epithelial Ovarian Cancer Cells and Its Impact on Tumor Progression.

The cancer testis antigen sperm protein 17 (Sp17) is a promising antigenic target in epithelial ovarian cancer (EOC) vaccine development. However, its role in ovarian cancer is unclear. We isolated and expanded Sp17⁺ and Sp17- clones from the murine EOC cell line ID8, and compared their in-vitro cell growth characteristics and in-vivo tumorigenicity. We also examined the potential co-expression of molecules that may influence cancer cell survival and interaction with immune cells. These include stimulatory and immunosuppressive molecules, such as major histocompatibility class I molecules (MHC I), MHC II, cytotoxic T lymphocyte associated antigen-4 (CTLA-4), CD73, CD39, tumor necrosis factor receptor II (TNFRII), signal transducer and activator of transcription 3 (STAT3) and programmed death-ligand 1 (PD-L1). Whilst the presence of Sp17 was not correlated with the ID8 cell proliferation/growth capacity in vitro, it was critical to enable progressive tumor formation in vivo. Flow cytometry revealed that Sp17⁺ ID8 cells displayed higher expression of both STAT3 and PD-L1, whilst MHC II expression was lower. Moreover, Sp17high (PD-L1⁺MHCII-) cell populations showed significantly enhanced resistance to Paclitaxel-induced cell death in vitro compared to Sp17low (PD-L1-MHCII⁺) cells, which was associated in turn with increased STAT3 expression. Together, the data support Sp17 as a factor associated with in-vivo tumor progression and chemo-resistance, validating it as a suitable target for vaccine development.

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.

Enterococcus hirae and Barnesiella intestinihominis Facilitate Cyclophosphamide-Induced Therapeutic Immunomodulatory Effects.

The efficacy of the anti-cancer immunomodulatory agent cyclophosphamide (CTX) relies on intestinal bacteria. How and which relevant bacterial species are involved in tumor immunosurveillance, and their mechanism of action are unclear. Here, we identified two bacterial species, Enterococcus hirae and Barnesiella intestinihominis that are involved during CTX therapy. Whereas E. hirae translocated from the small intestine to secondary lymphoid organs and increased the intratumoral CD8/Treg ratio, B. intestinihominis accumulated in the colon and promoted the infiltration of IFN-γ-producing γδT cells in cancer lesions. The immune sensor, NOD2, limited CTX-induced cancer immunosurveillance and the bioactivity of these microbes. Finally, E. hirae and B. intestinihominis specific-memory Th1 cell immune responses selectively predicted longer progression-free survival in advanced lung and ovarian cancer patients treated with chemo-immunotherapy. Altogether, E. hirae and B. intestinihominis represent valuable "oncomicrobiotics" ameliorating the efficacy of the most common alkylating immunomodulatory compound.

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.

Variability in CRP, regulatory T cells and effector T cells over time in gynaecological cancer patients: a study of potential oscillatory behaviour and correlations.

BACKGROUND: The inflammatory marker, C reactive protein has been proposed to also be a biomarker for adaptive immune responses in cancer patients with a possible application in time based chemotherapy. Fluxes in serum CRP levels were suggested to be indicative of a cyclical process in which, immune activation is followed by auto-regulating immune suppression. The applicability of CRP as a biomarker for regulatory or effector T cells was therefore investigated in a cohort of patients with gynaecological malignancies. METHODS: Peripheral blood samples were obtained from a cohort of patients at 7 time points over a period of 12 days. Serum and mononuclear cells were isolated and CRP levels in serum were detected using ELISA while regulatory and effector T cell frequencies were assessed using flow cytometry. To test periodicity, periodogram analysis of data was employed while Pearson correlation and the Wilcoxon signed rank test were used to determine correlations. RESULTS: The statistical analysis used showed no evidence of periodic oscillation in either serum CRP concentrations or Teff and Treg frequencies. Furthermore, there was no apparent correlation between serum CRP concentrations and the corresponding frequencies of Tregs or Teffs. Relative to healthy individuals, the disease state in the patients neither significantly affected the mean frequency of Tregs nor the mean coefficient of variation within the Treg population over time. However, both Teff mean frequency and mean coefficient of variation were significantly reduced in patients. CONCLUSION: Using our methods we were unable to detect CRP oscillations that could be used as a consistent serial biomarker for time based chemotherapy.

Lenalidomide-based maintenance therapy reduces TNF receptor 2 on CD4 T cells and enhances immune effector function in acute myeloid leukemia patients.

A major limitation to improved outcomes in acute myelogenous leukemia (AML) is relapse resulting from leukemic cells that persist at clinical remission. Regulatory T cells (Tregs), which are increased in AML patients, can contribute to immune evasion by residual leukemic cells. Tumor necrosis factor (TNF), a pro-inflammatory cytokine present at high levels within patients, can induce TNF receptor-2 (TNFR2) expression on Tregs. We hypothesized that since TNFR2 is required for Treg stabilization and TNFR2+ Tregs are potent suppressors, targeting TNFR2+ Tregs may restore the effectiveness of immune-surveillance mechanisms. In this pilot study, we report AML patients in clinical remission have substantially increased levels of TNFR2+ T cells, including TNFR2+ Tregs and impaired effector CD4 T cell function with reduced IL-2 and IFNγ production. The immunomodulatory drug, lenalidomide, and the demethylating agent, azacitidine have been moderately successful in treating AML patients, but their combined effects on TNFR2+ T cells, including Tregs are currently unknown. Our data indicates that although treatment with lenalidomide and azacitidine increased cytokine production by effector T cells in all patients, durable clinical remissions may be observed in patients with a concomitant reduction in TNFR2+ T cells and TNFR2+ Tregs. In vitro studies further demonstrated that lenalidomide can reduce TNFR2 expression and can augment effector cytokine production by T cells, which can be further enhanced by azacitidine. These results indicate that reduction of TNFR2+ T cells in AML postremission phase may result from combined azacitidine/lenalidomide therapy and may contribute to an improved clinical outcome.

Impaired Th1 immunity in ovarian cancer patients is mediated by TNFR2+ Tregs within the tumor microenvironment.

Ovarian cancer is a prevalent gynecological malignancy with potent immune-suppression capabilities; regulatory T cells (Tregs) are significant contributors to this immune-suppression. As ovarian cancer patients present with high levels of TNF and Tregs expressing TNFR2 are associated with maximal suppressive capacity, we investigated TNFR2+ Tregs within these patients. Indeed, TNFR2+ Tregs from tumor-associated ascites were the most potent suppressor T cell fraction. They were abundantly present within the ascites and more suppressive than peripheral blood TNFR2+ Tregs in patients. The increased suppressive capacity can be explained by a distinct cell surface expression profile, which includes high levels of CD39, CD73, TGF-ß and GARP. Additionally, CD73 expression level on TNFR2+ Tregs was inversely correlated with IFN-γ production by effector T cells. This Treg fraction can be selectively recruited into the ascites from the peripheral blood of patients. Targeting TNFR2+ Tregs may offer new approaches to enhance the poor survival rates of ovarian cancer.

Tumour eradication and induction of memory against murine mesothelioma by combined immunotherapy.

Numerous immunotherapy treatments for cancer are undergoing clinical trials or are already approved for use. One particular area of interest is targeting mechanisms of immune tolerance. Using a murine model of mesothelioma, we investigated the roles of regulatory T-cells, intratumoural transforming growth factor (TGF)-ß and the negative regulator molecule cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) in immune tolerance to tumours. It was found that treatments targeting a single negative regulator molecule mechanism were not as effective against tumours as targeting multiple mechanisms simultaneously. Most importantly, it was found that a combined triple treatment of anti-CD25 monoclonal antibody (mAb), anti-CTLA-4 mAb and TGF-ß soluble receptor resulted in long-term clearance of tumours and memory against tumour rechallenge. These data suggest that clinical application of immunotherapies against tumours may be improved by simultaneously targeting multiple mechanisms of immune suppression.