AHRR and SFRP2 in primary versus recurrent high-grade serous ovarian carcinoma and their prognostic implication.

BACKGROUND: The aim of this study was to analyse transcriptomic differences between primary and recurrent high-grade serous ovarian carcinoma (HGSOC) to identify prognostic biomarkers. METHODS: We analysed 19 paired primary and recurrent HGSOC samples using targeted RNA sequencing. We selected the best candidates using in silico survival and pathway analysis and validated the biomarkers using immunohistochemistry on a cohort of 44 paired samples, an additional cohort of 504 primary HGSOCs and explored their function. RESULTS: We identified 233 differential expressed genes. Twenty-three showed a significant prognostic value for PFS and OS in silico. Seven markers (AHRR, COL5A2, FABP4, HMGCS2, ITGA5, SFRP2 and WNT9B) were chosen for validation at the protein level. AHRR expression was higher in primary tumours (p < 0.0001) and correlated with better patient survival (p < 0.05). Stromal SFRP2 expression was higher in recurrent samples (p = 0.009) and protein expression in primary tumours was associated with worse patient survival (p = 0.022). In multivariate analysis, tumour AHRR and SFRP2 remained independent prognostic markers. In vitro studies supported the anti-tumorigenic role of AHRR and the oncogenic function of SFRP2. CONCLUSIONS: Our results underline the relevance of AHRR and SFRP2 proteins in aryl-hydrocarbon receptor and Wnt-signalling, respectively, and might lead to establishing them as biomarkers in HGSOC.

Stable expansion of high-grade serous ovarian cancer organoids requires a low-Wnt environment.

High-grade serous ovarian cancer (HGSOC) likely originates from the fallopian tube (FT) epithelium. Here, we established 15 organoid lines from HGSOC primary tumor deposits that closely match the mutational profile and phenotype of the parental tumor. We found that Wnt pathway activation leads to growth arrest of these cancer organoids. Moreover, active BMP signaling is almost always required for the generation of HGSOC organoids, while healthy fallopian tube organoids depend on BMP suppression by Noggin. Fallopian tube organoids modified by stable shRNA knockdown of p53, PTEN, and retinoblastoma protein (RB) also require a low-Wnt environment for long-term growth, while fallopian tube organoid medium triggers growth arrest. Thus, early changes in the stem cell niche environment are needed to support outgrowth of these genetically altered cells. Indeed, comparative analysis of gene expression pattern and phenotypes of normal vs. loss-of-function organoids confirmed that depletion of tumor suppressors triggers changes in the regulation of stemness and differentiation.

Impact of BRCA Mutation Status on Tumor Dissemination Pattern, Surgical Outcome and Patient Survival in Primary and Recurrent High-Grade Serous Ovarian Cancer: A Multicenter Retrospective Study by the Ovarian Cancer Therapy-Innovative Models Prolong Survival (OCTIPS) Consortium.

BACKGROUND: This study seeks to evaluate the impact of breast cancer (BRCA) gene status on tumor dissemination pattern, surgical outcome and survival in a multicenter cohort of paired primary ovarian cancer (pOC) and recurrent ovarian cancer (rOC). PATIENTS AND METHODS: Medical records and follow-up data from 190 patients were gathered retrospectively. All patients had surgery at pOC and at least one further rOC surgery at four European high-volume centers. Patients were divided into one cohort with confirmed mutation for BRCA1 and/or BRCA2 (BRCAmut) and a second cohort with BRCA wild type or unknown (BRCAwt). Patterns of tumor presentation, surgical outcome and survival data were analyzed between the two groups. RESULTS: Patients with BRCAmut disease were on average 4 years younger and had significantly more tumor involvement upon diagnosis. Patients with BRCAmut disease showed higher debulking rates at all stages. Multivariate analysis showed that only patient age had significant predictive value for complete tumor resection in pOC. At rOC, however, only BRCAmut status significantly correlated with optimal debulking. Patients with BRCAmut disease showed significantly prolonged overall survival (OS) by 24.3 months. Progression-free survival (PFS) was prolonged in the BRCAmut group at all stages as well, reaching statistical significance during recurrence. CONCLUSIONS: Patients with BRCAmut disease showed a more aggressive course of disease with earlier onset and more extensive tumor dissemination at pOC. However, surgical outcome and OS were significantly better in patients with BRCAmut disease compared with patients with BRCAwt disease. We therefore propose to consider BRCAmut status in regard to patient selection for cytoreductive surgery, especially in rOC.

Predictive biomarker for surgical outcome in patients with advanced primary high-grade serous ovarian cancer. Are we there yet? An analysis of the prospective biobank for ovarian cancer.

BACKGROUND: High-grade serous ovarian cancer (HGSOC) is the most common subtype of ovarian cancer and is associated with high mortality rates. Surgical outcome is one of the most important prognostic factors. There are no valid biomarkers to identify which patients may benefit from a primary debulking approach. OBJECTIVE: Our study aimed to discover and validate a predictive panel for surgical outcome of residual tumor mass after first-line debulking surgery. STUDY DESIGN: Firstly, "In silico" analysis of publicly available datasets identified 200 genes as predictors for surgical outcome. The top selected genes were then validated using the novel Nanostring method, which was applied for the first time for this particular research objective. 225 primary ovarian cancer patients with well annotated clinical data and a complete debulking rate of 60% were compiled for a clinical cohort. The 14 best rated genes were then validated through the cohort, using immunohistochemistry testing. Lastly, we used our biomarker expression data to predict the presence of miliary carcinomatosis patterns. RESULTS: The Nanostring analysis identified 37 genes differentially expressed between optimal and suboptimal debulked patients (p < 0.05). The immunohistochemistry validated the top 14 genes, reaching an AUC Ø0.650. The analysis for the prediction of miliary carcinomatosis patterns reached an AUC of Ø0.797. CONCLUSION: The tissue-based biomarkers in our analysis could not reliably predict post-operative residual tumor. Patient and non-patient-associated co-factors, surgical skills, and center experience remain the main determining factors when considering the surgical outcome at primary debulking in high-grade serous ovarian cancer patients.

Ovarian tumours of different histologic type and clinical stage induce similar changes in lipid metabolism.

BACKGROUND: Previous results obtained from serum samples of late-stage, high-grade serous ovarian carcinoma patients showed large alterations in lipid metabolism. To validate and extend the results, we studied lipidomic changes in early-stage ovarian tumours. In addition to serous ovarian cancer, we investigated whether these changes occur in mucinous and endometrioid histological subtypes as well. METHODS: Altogether, 354 serum or plasma samples were collected from three centres, one from Germany and two from Finland. We performed lipidomic analysis of samples from patients with malignant (N = 138) or borderline (N = 25) ovarian tumours, and 191 controls with benign pathology. These results were compared to previously published data. RESULTS: We found 39 lipids that showed consistent alteration both in early- and late-stage ovarian cancer patients as well as in pre- and postmenopausal women. Most of these changes were already significant at an early stage and progressed with increasing stage. Furthermore, 23 lipids showed similar alterations in all investigated histological subtypes. CONCLUSIONS: Changes in lipid metabolism due to ovarian cancer occur in early-stage disease but intensify with increasing stage. These changes occur also in other histological subtypes besides high-grade serous carcinoma. Understanding lipid metabolism in ovarian cancer may lead to new therapeutic and diagnostic alternatives.

Characterisation of tumour microvessel density during progression of high-grade serous ovarian cancer: clinico-pathological impact (an OCTIPS Consortium study).

BACKGROUND: High-grade serous ovarian cancer (HGSOC) intratumoural vasculature evolution remains unknown. The study investigated changes in tumour microvessel density (MVD) in a large cohort of paired primary and recurrent HGSOC tissue samples and its impact on patients' clinico-pathological outcome. METHODS: A total of 222 primary (pOC) and recurrent (rOC) intra-patient paired HGSOC were assessed for immunohistochemical expression of angiogenesis-associated biomarkers (CD31, to evaluate MVD, and VEGF-A). Expression profiles were compared between pOCs and rOCs and correlated with patients' data. RESULTS: High intratumoural MVD and VEGF-A expression were observed in 75.7% (84/111) and 20.7% (23/111) pOCs, respectively. MVDhigh and VEGF(+) samples were detected in 51.4% (57/111) and 20.7% (23/111) rOCs, respectively. MVDhigh/VEGF(+) co-expression was found in 19.8% (22/111) and 8.1% (9/111) of pOCs and rOCs, respectively (p = 0.02). Pairwise analysis showed no significant change in MVD (p = 0.935) and VEGF-A (p = 0.121) levels from pOCs to rOCs. MVDhigh pOCs were associated with higher CD3(+) (p = 0.029) and CD8(+) (p = 0.013) intratumoural effector TILs, while VEGF(+) samples were most frequently encountered among BRCA-mutated tumours (p = 0.019). Multivariate analysis showed VEGF and MVD were not independent prognostic factors for OS. CONCLUSIONS: HGSOC intratumoural vasculature did not undergo significant changes during disease progression. High concentration of CD31(+) vessels seems to promote recruitment of effector TILs. The study also provides preliminary evidence of the correlation between VEGF-positivity and BRCA status.

Morphology and tumour-infiltrating lymphocytes in high-stage, high-grade serous ovarian carcinoma correlated with long-term survival.

AIMS: Advanced-stage ovarian high-grade serous carcinoma (HGSC) is a poor-prognosis cancer; however, a small and poorly characterised subset of patients shows long-term survival. We aimed to establish a cohort of HGSC long-term survivors for histopathological and molecular analysis. METHODS AND RESULTS: Paraffin blocks from 151 patients with primary FIGO III/IV HGSC and progression-free survival (PFS) >5 years were collected within the Tumorbank Ovarian Cancer (TOC) Network; 77 HGSC with a PFS <3 years were used as a control group. A standardised analysis of histological type and morphological features was performed. Ki67 index, tumour-infiltrating lymphocytes (TILs) and major histocompatibility complex expression (MHC1/2) were determined by immunohistochemistry. A total of 117 of 151 tumours (77.5%) in the long-term survivor group fulfilled the World Health Organisation (WHO) criteria of HGSC after review, and of these, 83 patients (70.9%) fulfilled all clinical criteria for inclusion into our cohort. Tumours of long-term survivors had significantly higher CD3+ and CD8+ TILs and were more frequently positive for MHC2 than controls (P = 0.004, P = 0.025, P = 0.048). However, there were also long-term survivors (up to 20%) with low TILs or low MHC expression. TILs and MHC had no impact on survival in long-term survivors. Morphological and Ki67 analysis revealed no differences between long-term survivors and controls. CONCLUSIONS: HGSC from long-term survivors have higher-level T cell infiltration and antigen-presentation capacity; however, this is not a prerequisite for an excellent prognosis. Histopathological criteria are not capable to identify these patients. Further extensive clinical and molecular characterisation of this enigmatic subgroup is ongoing to understand the reasons of long-term survival in HGSC.

The prognostic impact of cancer stem-like cell biomarker aldehyde dehydrogenase-1 (ALDH1) in ovarian cancer: A meta-analysis.

OBJECTIVE: To investigate the association of cancer stem cell biomarker aldehyde dehydrogenase-1 (ALDH1) with ovarian cancer patients' prognosis and clinico-pathological characteristics. METHODS: The electronic searches were performed in January 2018 through the databases PubMed, MEDLINE and Scopus by searching the terms: "ovarian cancer" AND "immunohistochemistry" AND ["aldehyde dehydrogenase-1" OR "ALDH1" OR "cancer stem cell"]. Studies evaluating the impact of ALDH1 expression on ovarian cancer survival and clinico-pathological variables were selected. RESULTS: 233 studies were retrieved. Thirteen studies including 1885 patients met all selection criteria. ALDH1-high expression was found to be significantly associated with poor 5-year OS (OR = 3.46; 95% CI: 1.61-7.42; P = 0.001, random effects model) and 5-year PFS (OR = 2.14; 95% CI: 1.11-4.13; P = 0.02, random effects model) in ovarian cancer patients. No correlation between ALDH1 expression and tumor histology (OR = 0.60; 95% CI: 0.36-1.02; P = 0.06, random effects model), FIGO Stage (OR = 0.65; 95% CI: 0.33-1.30; P = 0.22, random effects model), tumor grading (OR = 0.76; 95% CI: 0.40-1.45; P = 0.41, random effects model) lymph nodal status (OR = 2.05; 95% CI: 0.81-5.18; P = 0.13, random effects model) or patients' age at diagnosis (OR = 0.83; 95% CI: 0.54-1.29; P = 0.41, fixed effects model) was identified. CONCLUSIONS: Basing on the available evidence, this meta-analysis showed that high levels of ALDH1 expression correlate with worse OS and PFS in ovarian cancer patients.

Genetic Versus Epigenetic BRCA1 Silencing Pathways: Clinical Effects in Primary Ovarian Cancer Patients: A Study of the Tumor Bank Ovarian Cancer Consortium.

OBJECTIVES: The aim of the present study was to assess in a large cohort of primary epithelial ovarian cancer patients the incidence and the clinical effect of BRCA1 genetic and epigenetic silencing mechanisms. METHODS: A total of 188 primary epithelial ovarian cancer patients, treated between 2000 and 2011 at the Charité University Hospital of Berlin, were included. The patients' tumor and blood samples were obtained from the Tumor Bank Ovarian Cancer Network (www.toc-network.de). Direct sequencing of BRCA1 exon 11 was performed to detect germline mutations, whereas tumor samples were assessed for BRCA1 promoter hypermethylation by bisulphite-converted methylation-specific polymerase chain reaction. Basing on their BRCA1 status, patients were compared regarding clinicopathological variables and survival. RESULTS: Twenty-one patients (11.2%) showed hypermethylation in BRCA1 promoter (HMB), and 18 patients (9.6%) presented germline mutations in BRCA1 exon 11 (GMB). Patients with HMB showed a significantly younger age at diagnosis compared with BRCA1 wild type (BWT) patients (54 vs 61 years, P = 0.045), and both GMB and HMB patients were more likely to have high-grade serous ovarian cancer (76.2% and 77.8% vs 52.7%, P = 0.043 and P = 0.043). Positive family history of breast or ovarian cancer (OC) was more frequently reported among GMB patients with respect to BWT patients (44.4% vs 13.5%, P = 0.003); GMB, HMB, and BWT patients did not show significant differences in terms of tumor dissemination pattern, surgical outcomes, platinum response or survival; neither mutational nor hypermethylation BRCA1 status was found to be an independent prognostic factor for OC patients. CONCLUSIONS: Hypermethylation in BRCA1 is associated with earlier occurrence of OC. In addition, the coexistence of both GBM and HMB is an infrequent event, occurring in 0.5% of OC cases. Silencing of BRCA1 through mutation and hypermethylation confers to distinct clinical characteristics of OC patients but similar clinical outcome with respect to BWT patients.

Murine CD27(-) Vγ6(+) γδ T cells producing IL-17A promote ovarian cancer growth via mobilization of protumor small peritoneal macrophages.

Cancer-associated inflammation mobilizes a variety of leukocyte populations that can inhibit or enhance tumor cell growth in situ. These subsets include γδ T cells, which can infiltrate tumors and typically provide large amounts of antitumor cytokines, such as IFN-γ. By contrast, we report here that in a well-established transplantable (ID8 cell line) model of peritoneal/ovarian cancer, γδ T cells promote tumor cell growth. γδ T cells accumulated in the peritoneal cavity in response to tumor challenge and could be visualized within solid tumor foci. Functional characterization of tumor-associated γδ T cells revealed preferential production of interleukin-17A (IL-17), rather than IFN-γ. Consistent with this finding, both T cell receptor (TCR)δ-deficient and IL-17-deficient mice displayed reduced ID8 tumor growth compared with wild-type animals. IL-17 production by γδ T cells in the tumor environment was essentially restricted to a highly proliferative CD27((-)) subset that expressed Vγ6 instead of the more common Vγ1 and Vγ4 TCR chains. The preferential expansion of IL-17-secreting CD27((-)) Vγ6((+)) γδ T cells associated with the selective mobilization of unconventional small peritoneal macrophages (SPMs) that, in comparison with large peritoneal macrophages, were enriched for IL-17 receptor A, and for protumor and proangiogenic molecular mediators, which were up-regulated by IL-17. Importantly, SPMs were uniquely and directly capable of promoting ovarian cancer cell proliferation. Collectively, this work identifies an IL-17-dependent lymphoid/myeloid cross-talk involving γδ T cells and SPMs that promotes tumor cell growth and thus counteracts cancer immunosurveillance.

Cancer cell-derived lymphotoxin mediates reciprocal tumour-stromal interactions in human ovarian cancer by inducing CXCL11 in fibroblasts.

We have investigated the role of cytokine lymphotoxin in tumour-stromal interactions in human ovarian cancer. We found that lymphotoxin overexpression is commonly shared by the cancer cells of various ovarian cancer subtypes, and lymphotoxin-beta receptor (LTBR) is expressed ubiquitously in both the cancer cells and cancer-associated fibroblasts (CAFs). In monoculture, we showed that ovarian cancer cells are not the major lymphotoxin-responsive cells. On the other hand, our co-culture studies demonstrated that the cancer cell-derived lymphotoxin induces chemokine expression in stromal fibroblasts through LTBR-NF-κB signalling. Amongst the chemokines being produced, we found that fibroblast-secreted CXCL11 promotes proliferation and migration of ovarian cancer cells via the chemokine receptor CXCR3. CXCL11 is highly expressed in CAFs in ovarian cancer biopsies, while CXCR3 is found in malignant cells in primary ovarian tumours. Additionally, the overexpression of CXCR3 is significantly associated with the tumour grade and lymph node metastasis of ovarian cancer, further supporting the role of CXCR3, which interacts with CXCL11, in promoting growth and metastasis of human ovarian cancer. Taken together, these results demonstrated that cancer-cell-derived lymphotoxin mediates reciprocal tumour-stromal interactions in human ovarian cancer by inducing CXCL11 in fibroblasts. Our findings suggest that lymphotoxin-LTBR and CXCL11-CXCR3 signalling represent therapeutic targets in ovarian cancer.

Vaccinia virus induces programmed necrosis in ovarian cancer cells.

The mechanisms by which oncolytic vaccinia virus induces tumor cell death are poorly understood. We have evaluated cell death pathways following infection of ovarian cancer cells with both wild-type and thymidine kinase-deleted (dTK) Lister strain vaccinia. We show that death does not rely upon classical apoptosis despite the appearances of some limited apoptotic features, including phosphatidylserine externalization and appearance of sub-G1 DNA populations. Vaccinia infection induces marked lipidation of LC3 proteins, but there is no general activation of the autophagic process and cell death does not rely upon autophagy induction. We show that vaccinia induces necrotic morphology on transmission electron microscopy, accompanied by marked by reductions in intracellular adenosine triphosphate, altered mitochondrial metabolism, and release of high mobility group box 1 (HMGB1) protein. This necrotic cell death appears regulated, as infection induces formation of a receptor interacting protein (RIP1)/caspase-8 complex. In addition, pharmacological inhibition of both RIP1 and substrates downstream of RIP1, including MLKL, significantly attenuate cell death. Blockade of TNF-α, however, does not alter virus efficacy, suggesting that necrosis does not result from autocrine cytokine release. Overall, these results show that, in ovarian cancer cells, vaccinia virus causes necrotic cell death that is mediated through a programmed series of events.

B regulatory cells and the tumor-promoting actions of TNF-α during squamous carcinogenesis.

The inflammatory cytokine TNF-α has been recognized as a critical tumor promoter, but the effector cells that mediate its action have not been fully characterized. Because B cells regulate squamous and prostate carcinogenesis, and Tnf(-/-) mice harbor B-cell defects, we investigated the hypothesis that B cells are important effector cells for TNF-α-mediated promotion of cancer development. Using an adoptive transfer strategy and the 7,12-dimethylbenz[α]anthracene/terephthalic acid (DMBA/TPA) two-stage model of skin carcinogenesis, we found that both B cells and TNF-α are critical for the development of DMBA/TPA-induced papilloma. Transfer of B cells from DMBA/TPA-treated wild-type mice to Tnf(-/-) mice rescued papilloma development to a wild-type level, a result not observed when B cells from Tnf(-/-) mice were transferred to Rag2(-/-) mice or when TNF-α was eliminated selectively in B cells. Resistance to papilloma development in Tnf(-/-) mice was associated with increased IFN-γ and CD8(+) T cells in skin and a significant reduction in IL-10-producing B regulatory cells alongside an increase in IFN-γ-producing CD8(+) T cells in the spleen. These data indicate that during DMBA/TPA-induced squamous carcinogenesis TNF-α mediates tumor-promoting activity via regulatory B cells that repress antitumor immunity.

Pre-Operative Malnutrition in Patients with Ovarian Cancer: What Are the Clinical Implications? Results of a Prospective Study.

BACKGROUND: Malnutrition was associated with worse survival outcomes, impaired quality of life, and deteriorated performance status across various cancer types. We aimed to identify risk factors for malnutrition in patients with epithelial ovarian cancer (EOC) and impact on survival. METHODS: In our prospective observational monocentric study, we included the patients with primary and recurrent EOC, tubal or peritoneal cancer conducted. We assessed serum laboratory parameters, body mass index, nutritional risk index, nutritional risk screening score (NRS-2002), and bio-electrical impedance analysis. RESULTS: We recruited a total of 152 patients. Patients > 65 years-old, with ascites of >500 mL, or with platinum-resistant EOC showed statistically significant increased risk of malnutrition when evaluated using NRS-2002 (p-values= 0.014, 0.001, and 0.007, respectively). NRS-2002 < 3 was an independent predictive factor for complete tumor resectability (p = 0.009). The patients with NRS-2002 ≥ 3 had a median overall survival (OS) of seven months (95% CI = 0-24 months), as compared to the patients with NRS-2002 < 3, where median OS was forty-six months (p = 0.001). A phase angle (PhAα) ≤ 4.5 was the strongest predictor of OS. CONCLUSIONS: In our study, we found malnutrition to be an independent predictor of incomplete cytoreduction and independent prognostic factor for poor OS. Preoperative nutritional assessment is an effective tool in the identification of high-risk EOC groups characterized by poor clinical outcome.

Dynamics of clonal hematopoiesis under DNA-damaging treatment in patients with ovarian cancer.

Clonal hematopoiesis (CH) driven by mutations in the DNA damage response (DDR) pathway is frequent in patients with cancer and is associated with a higher risk of therapy-related myeloid neoplasms (t-MNs). Here, we analyzed 423 serial whole blood and plasma samples from 103 patients with relapsed high-grade ovarian cancer receiving carboplatin, poly(ADP-ribose) polymerase inhibitor (PARPi) and heat shock protein 90 inhibitor (HSP90i) treatment within the phase II EUDARIO trial using error-corrected sequencing of 72 genes. DDR-driven CH was detected in 35% of patients and was associated with longer duration of prior PARPi treatment. TP53- and PPM1D-mutated clones exhibited substantially higher clonal expansion rates than DNMT3A- or TET2-mutated clones during treatment. Expansion of DDR clones correlated with HSP90i exposure across the three study arms and was partially abrogated by the presence of germline mutations related to homologous recombination deficiency. Single-cell DNA sequencing of selected samples revealed clonal exclusivity of DDR mutations, and identified DDR-mutated clones as the origin of t-MN in two investigated cases. Together, these results provide unique insights into the architecture and the preferential selection of DDR-mutated hematopoietic clones under intense DNA-damaging treatment. Specifically, PARPi and HSP90i therapies pose an independent risk for the expansion of DDR-CH in a dose-dependent manner.

The peritoneal tumour microenvironment of high-grade serous ovarian cancer.

High-grade serous ovarian cancer (HGSC) disseminates early and extensively throughout the peritoneal space, causing multiple lesions that are a major clinical problem. The aim of this study was to investigate the cellular composition of peritoneal tumour deposits in patient biopsies and their evolution in mouse models using immunohistochemistry, intravital microscopy, confocal microscopy, and 3D modelling. Tumour deposits from the omentum of HGSC patients contained a prominent leukocyte infiltrate of CD3(+) T cells and CD68(+) macrophages, with occasional neutrophils. Alpha-smooth muscle actin(+) (α-SMA(+) ) pericytes and/or fibroblasts surrounded these well-vascularized tumour deposits. Using the murine bowel mesentery as an accessible mouse peritoneal tissue that could be easily imaged, and two different transplantable models, we found multiple microscopic tumour deposits after i.p. injection of malignant cells. Attachment to the peritoneal surface was rapid (6-48 h) with an extensive CD45(+) leukocyte infiltrate visible by 48 h. This infiltrate persisted until end point and in the syngeneic murine ID8 model, it primarily consisted of CD3(+) T lymphocytes and CD68(+) macrophages with α-SMA(+) cells also involved from the earliest stages. A majority of tumour deposits developed above existing mesenteric blood vessels, but in avascular spaces new blood vessels tracked towards the tumour deposits by 2-3 weeks in the IGROV-1 xenografts and 6 weeks in the ID8 syngeneic model; a vigorous convoluted blood supply was established by end point. Inhibition of tumour cell cytokine production by stable expression of shRNA to CXCR4 in IGROV-1 cells did not influence the attachment of cells to the mesentery but delayed neovascularization and reduced tumour deposit size. We conclude that the multiple peritoneal tumour deposits found in HGSC patients can be modelled in the mouse. The techniques described here may be useful for assessing treatments that target the disseminated stage of this disease.

In Silico Analysis Predicts Nuclear Factors NR2F6 and YAP1 as Mesenchymal Subtype-Specific Therapeutic Targets for Ovarian Cancer Patients.

BACKGROUND: Tumour heterogeneity in high-grade serous ovarian cancer (HGSOC) is a proposed cause of acquired resistance to treatment and high rates of relapse. Among the four distinct molecular subtypes of HGSOC, the mesenchymal subtype (MES) has been observed with high frequency in several study cohorts. Moreover, it exhibits aggressive characteristics with poor prognosis. The failure to adequately exploit such subtypes for treatment results in high mortality rates, highlighting the need for effective targeted therapeutic strategies that follow the idea of personalized medicine (PM). METHODS: As a proof-of-concept, bulk and single-cell RNA data were used to characterize the distinct composition of the tumour microenvironment (TME), as well as the cell-cell communication and its effects on downstream transcription of MES. Moreover, transcription factor activity contextualized with causal inference analysis identified novel therapeutic targets with potential causal impact on transcription factor dysregulation promoting the malignant phenotype. FINDINGS: Fibroblast and macrophage phenotypes are of utmost importance for the complex intercellular crosstalk of MES. Specifically, tumour-associated macrophages were identified as the source of interleukin 1 beta (IL1B), a signalling molecule with significant impact on downstream transcription in tumour cells. Likewise, signalling molecules tumour necrosis factor (TNF), transforming growth factor beta (TGFB1), and C-X-C motif chemokine 12 (CXCL12) were prominent drivers of downstream gene expression associated with multiple cancer hallmarks. Furthermore, several consistently hyperactivated transcription factors were identified as potential sources for treatment opportunities. Finally, causal inference analysis identified Yes-associated protein 1 (YAP1) and Nuclear Receptor Subfamily 2 Group F Member 6 (NR2F6) as novel therapeutic targets in MES, verified in an independent dataset. INTERPRETATION: By utilizing a sophisticated bioinformatics approach, several candidates for treatment opportunities, including YAP1 and NR2F6 were identified. These candidates represent signalling regulators within the cellular network of the MES. Hence, further studies to confirm these candidates as potential targeted therapies in PM are warranted.

Prognostic value of regulatory T cells and T helper 17 cells in high grade serous ovarian carcinoma.

PURPOSE: In recent years the tumor microenvironment and its interaction with the tumor has emerged into research focus with increased attention to the composition of Tumor-infiltrating lymphocytes. We wanted to quantify the composition of Regulatory T cells (Tregs) and T helper 17 cells (Th17 cells) and their prognostic impact in high-grade serous tubo-ovarian carcinoma. METHODS: Tregs and Th17 cells were determined by immunohistochemical analysis of CD25 FoxP3 and RORγt, respectively on tissue microarrays of a cohort of 222 patients with reviewed histology and available clinical data. Expression was analyzed with Qupath for quantification and integration with clinical data enabled calculation of prognostic impact. For validation FOXP3 and RORC mRNA expression levels from 502 patients with HGSC in publicly available datasets were evaluated. RESULTS: An average percentage of 0.93 Tregs and of 0.06 Th17 cells was detected per cells in overall tissue. Optimal cut-offs were determined and higher Tregs were associated with a better overall survival in stroma (p = 0.006), tumor area (p = 0.0012) and overall tissue (p = 0.02). After accounting for well-known prognostic factors age at diagnosis, residual tumor and FIGO stage, this association remained significant for stromal Tregs with overall survival (p = 0.02). Survival analysis for Th17 cells revealed no significant association with survival rates. Moreover, lower Th17/Treg ratios had a positive impact on patient overall survival (p = 0.025 tumor, p = 0.049 stroma and p = 0.016 overall tissue). CONCLUSION: Our results outline a positive prognostic effect for higher Tregs but not for Th17 in high grade serous tubo-ovarian carcinoma.

High EVI1 and PARP1 expression as favourable prognostic markers in high-grade serous ovarian carcinoma.

BACKGROUND: Mechanisms of development and progression of high-grade serous ovarian cancer (HGSOC) are poorly understood. EVI1 and PARP1, part of TGF-ß pathway, are upregulated in cancers with DNA repair deficiencies with DNA repair deficiencies and may influce disease progression and survival. Therefore we questioned the prognostic significance of protein expression of EVI1 alone and in combination with PARP1 and analyzed them in a cohort of patients with HGSOC. METHODS: For 562 HGSOC patients, we evaluated EVI1 and PARP1 expression by immunohistochemical staining on tissue microarrays with QuPath digital semi-automatic positive cell detection. RESULTS: High EVI1 expressing (> 30% positive tumor cells) HGSOC were associated with improved progression-free survival (PFS) (HR = 0.66, 95% CI: 0.504-0.852, p = 0.002) and overall survival (OS) (HR = 0.45, 95% CI: 0.352-0.563, p < 0.001), including multivariate analysis. Most interestingly, mutual high expression of both proteins identifies a group with particularly good prognosis. Our findings were proven technically and clinically using bioinformatical data sets for single-cell sequencing, copy number variation and gene as well as protein expression. CONCLUSIONS: EVI1 and PARP1 are robust prognostic biomarkers for favorable prognosis in HGSOC and imply further research with respect to their reciprocity.