Circulating cell-free DNA is elevated in postmenopausal compared with pre- and perimenopausal women.

OBJECTIVE: With the rising use of circulating cell-free DNA (cirDNA) liquid biopsies for disease screening, it is important to understand biological differences that may impact the accuracy of cirDNA-based clinical tests. Although a number of biological factors have been researched, the relationship between menopause and cirDNA has not been thoroughly investigated. We aimed to compare plasma cirDNA concentration and DNA fragment integrity in healthy women pre- and postmenopause. METHODS: Blood was collected from healthy female volunteers 40 years and older. cirDNA was extracted from plasma (n = 52) and quantified by quantitative polymerase chain reaction (n = 47; 26 premenopause, mean age-46 y; 21 postmenopause, mean age-59 y). cirDNA concentration was quantitated using an ALU repetitive sequence with a 115-base-pair (bp) product (ALU-115), and long cirDNA fragments were quantitated using an ALU repetitive sequence with a 247-bp product (ALU-247). cirDNA integrity was expressed as a ratio of ALU-247 over ALU-115. Mann-Whitney U test was used to compare pre- and postmenopause qPCR results, and a two-tailed, unpaired t test was undertaken to compare the integrity ratio between the two groups. RESULTS: Postmenopause plasma samples were found to have a significantly higher cirDNA concentration (P < 0.0001, premenopause: mean, 3.10 ± 1.84 ng/mL; median, 2.90 ng/mL; postmenopause: mean, 5.28 ± 2.76 ng/mL; median, 4.56 ng/mL) and significantly higher concentration of long-stranded cirDNA fragments (P = 0.0033, premenopause: mean, 1.06 ± 0.48 ng/mL; median, 0.96 ng/mL; postmenopause: mean, 1.69 ± 0.89 ng/mL; median, 1.48 ng/mL). There was no significant difference in the integrity ratio between the groups (P = 0.1788). CONCLUSIONS: Plasma cirDNA concentrations are higher in postmenopausal women. This has important implications in cirDNA liquid biopsy development and screening, especially for diseases such as cancer where the majority of cases are diagnosed postmenopause.

Cell-free DNA in plasma and ascites as a biomarker of bevacizumab response- a translational research sub-study of the REZOLVE (ANZGOG-1101) clinical trial.

OBJECTIVE: To investigate cell-free DNA (cfDNA) in plasma and ascites and its association with clinical outcomes (paracentesis-free interval, overall survival) and CA125 level in participants with advanced ovarian cancer, treated with palliative intraperitoneal bevacizumab to delay re-accumulation of ascites. METHODS: cfDNA was extracted from 0.3 to 1 mL samples from 20/24 participants of the REZOLVE trial. Standard and methylation-specific PCRs were performed to measure 3 biomarkers: total cfDNA (Alu), tumour-derived cfDNA (ctDNA, methylated IFFO1 promoter) and endothelium-derived cfDNA (ec-cfDNA, unmethylated CDH5 promoter). Values were correlated to clinical outcomes. RESULTS: cfDNA was detected in all samples, with higher yield in ascites (mean 669 ng/mL) than plasma (mean 75 ng/mL, p < 0.0001). Ascites had a higher ctDNA proportion than plasma (74 % vs. 20 %, p < 0.0001) and plasma had a higher ec-cfDNA proportion than ascites (24 % vs. 16 %, p < 0.002). High ctDNA proportion (>75 %) in ascites was associated with a significantly shorter paracentesis-free interval (median interval 47.5 versus 84 days, hazard ratio (HR) 2.21, 95 % confidence interval (CI) 0.85 to 5.73, p = 0.039) and ctDNA presence in plasma was unfavourable for survival (median survival 56 versus 242 days, HR 3.21, 95 % CI 1.15 to 9.00, p = 0.008). A significant positive correlation was observed between ctDNA proportion in plasma and CA125 level (p = 0.012). No significant difference in total cfDNA, ctDNA nor ec-cfDNA was observed between participants who were responders versus non-responders. CONCLUSION: Sufficient cfDNA was detected in both plasma and ascites to study three biomarkers. These samples can provide useful information and should be considered in the design of future ovarian cancer trials.

DNA repair biomarkers to guide usage of combined PARP inhibitors and chemotherapy: A meta-analysis and systematic review.

PURPOSE: The addition of PARP inhibitors to chemotherapy has been assessed in > 80 clinical trials across multiple malignancies, on the premise that PARP inhibitors will increase chemotherapy effectiveness regardless of whether cancers have underlying disruption of DNA repair pathways. Consequently, the majority of combination therapy trials have been performed on patients without biomarker selection, despite the use of homologous recombination deficiency to dictate use of PARP inhibitors in the maintenance setting. An unresolved question is whether biomarkers are needed to identify patients who respond to combination PARP inhibitors and chemotherapy. METHODS: A systematic literature review identified studies using PARP inhibitors in combination with chemotherapy versus chemotherapy alone, where the study included a biomarker of DNA repair function (BRCA1, BRCA2, homologous recombination deficiency test, ATM, ERCC1, SLFN11). Hazard ratios (HR) were pooled in a meta-analysis using generic inverse-variance, and fixed or random effects modelling. Subgroup analyses were conducted on biomarker selection and type of malignancy. RESULTS: Nine studies comprising 2547 patients met the inclusion criteria. Progression-free survival (PFS) was significantly better in patients with a DNA repair biomarker (HR: 0.57, 95% CI: 0.48-0.68, p < 0.00001), but there was no benefit in patients who lacked a biomarker (HR: 0.94, 95% CI: 0.82-1.08, p = 0.38). Subgroup analysis showed that BRCA status and SLFN11 biomarkers could predict benefit, and biomarker-driven benefit occurred in ovarian, breast and small cell lung cancers. The addition of PARP inhibitors to chemotherapy was associated with increased grade 3/4 side effects, and particularly neutropenia. CONCLUSIONS: Combination therapy only improves PFS in patients with identifiable DNA repair biomarkers. This indicates that PARP inhibitors do not sensitise patients to chemotherapy treatment, except where their cancer has a homologous recombination defect, or an alternative biomarker of altered DNA repair. While effective in patients with DNA repair biomarkers, there is a risk of high-grade haematological side-effects with the use of combination therapy. Thus, the benefit in PFS from combination therapy must be weighed against potential adverse effects, as individual arms of treatment can also confer benefit.

A comparative analysis of 2D and 3D experimental data for the identification of the parameters of computational models.

Computational models are becoming an increasingly valuable tool in biomedical research. Their accuracy and effectiveness, however, rely on the identification of suitable parameters and on appropriate validation of the in-silico framework. Both these steps are highly dependent on the experimental model used as a reference to acquire the data. Selecting the most appropriate experimental framework thus becomes key, together with the analysis of the effect of combining results from different experimental models, a common practice often necessary due to limited data availability. In this work, the same in-silico model of ovarian cancer cell growth and metastasis, was calibrated with datasets acquired from traditional 2D monolayers, 3D cell culture models or a combination of the two. The comparison between the parameters sets obtained in the different conditions, together with the corresponding simulated behaviours, is presented. It provides a framework for the study of the effect of the different experimental models on the development of computational systems. This work also provides a set of general guidelines for the comparative testing and selection of experimental models and protocols to be used for parameter optimization in computational models.

Endogenous cell-free DNA in fetal bovine serum introduces artifacts to in vitro cell-free DNA models.

Cell-free DNA (cfDNA) is of growing clinical and research significance. In vitro cfDNA models are a useful tool in cfDNA research; however, artifacts in these models may have implications for the interpretation of new and published data. This report aimed to establish how endogenous cfDNA in fetal bovine serum (FBS) may influence in vitro cfDNA measurements. Three commercial cell culture media, supplemented with 10% FBS, were analyzed for the presence of cfDNA, with and without culture with ovarian cancer cell lines. cfDNA from FBS was identified with all three commercial media and contributed a major portion of 167-bp cfDNA. Future studies should account for bovine cfDNA in FBS-supplemented media when conducting in vitro cfDNA research.

Targeting Homologous Recombination Deficiency in Ovarian Cancer with PARP Inhibitors: Synthetic Lethal Strategies That Impact Overall Survival.

The advent of molecular targeted therapies has made a significant impact on survival of women with ovarian cancer who have defects in homologous recombination repair (HRR). High-grade serous ovarian cancer (HGSOC) is the most common histological subtype of ovarian cancer, with over 50% displaying defective HRR. Poly ADP ribose polymerases (PARPs) are a family of enzymes that catalyse the transfer of ADP-ribose to target proteins, functioning in fundamental cellular processes including transcription, chromatin remodelling and DNA repair. In cells with deficient HRR, PARP inhibitors (PARPis) cause synthetic lethality leading to cell death. Despite the major advances that PARPis have heralded for women with ovarian cancer, questions and challenges remain, including: can the benefits of PARPis be brought to a wider range of women with ovarian cancer; can other drugs in clinical use function in a similar way or with greater efficacy than currently clinically approved PARPis; what can we learn from long-term responders to PARPis; can PARPis sensitise ovarian cancer cells to immunotherapy; and can synthetic lethal strategies be employed more broadly to develop new therapies for women with ovarian cancer. We examine these, and other, questions with focus on improving outcomes for women with ovarian cancer.

Pan-Tissue and -Cancer Analysis of ROR1 and ROR2 Transcript Variants Identify Novel Functional Significance for an Alternative Splice Variant of ROR1.

ROR1/2 are putative druggable targets increasing in significance in translational oncology. Expression of ROR1/2 mRNA and transcript variants has not been systematically examined thus far. ROR1/2 transcript variant sequences, signal peptides for cell surface localisation, and mRNA and transcript variant expression were examined in 34 transcriptomic datasets including 33 cancer types and 54 non-diseased human tissues. ROR1/2 have four and eight transcript variants, respectively. ROR1/2 mRNA and transcript variant expression was detected in various non-diseased tissues. Our analysis identifies predominant expression of ROR1 transcript variant ENST00000545203, which lacks a signal peptide for cell surface localisation, rather than the predicted principal variant ENST00000371079. ENST00000375708 is the predominantly expressed transcript variant of ROR2. ROR1/2 expression in healthy human tissues should be carefully considered for safety assessment of targeted therapy. Studies exploring the function and significance of the predominantly expressed ROR1 transcript variant ENST00000545203 are warranted.

The Anti-ROR1 Monoclonal Antibody Zilovertamab Inhibits the Proliferation of Ovarian and Endometrial Cancer Cells.

The non-canonical Wnt signalling receptor ROR1 is aberrantly expressed in numerous cancers, including ovarian and endometrial cancer. We previously reported that silencing ROR1 could inhibit the proliferation and metastatic potential of ovarian and endometrial cancer cells in vitro. Zilovertamab is an ROR1-targeting humanised monoclonal antibody, with demonstrated safety and efficacy in clinical trials of several ROR1-related malignancies. The aim of this study was to investigate the potential of zilovertamab alone, or in combination with commonly utilised gynaecological cancer therapies (cisplatin, paclitaxel and the PARP inhibitor-Olaparib) on high-grade serous ovarian cancer (HGSOC), including models of platinum resistance and homologous recombination deficiency (CaOV3, CaOV3CisR, PEO1 and PEO4) and endometrial cancer (EC) cell lines (Ishikawa and KLE). The effect of zilovertamab (at 25 µg/mL or 50 µg/mL) +/- agents was investigated using the IncuCyte S3 Live Cell imaging system. Zilovertamab alone inhibited the proliferation of HGSOC and EC cells in vitro, including in models of platinum resistance and homologous recombination deficiency. In general, the addition of commonly used chemotherapies to a fixed dose of zilovertamab did not enhance the observed anti-proliferative activity. This study supports the potential of zilovertamab, or other ROR1-targeting therapies, for treating women with HGSOC and EC.

Transcending Blood-Opportunities for Alternate Liquid Biopsies in Oncology.

Cell-free DNA (cfDNA) is a useful molecular biomarker in oncology research and treatment, but while research into its properties in blood has flourished, there remains much to be discovered about cfDNA in other body fluids. The cfDNA from saliva, sputum, cerebrospinal fluid, urine, faeces, pleural effusions, and ascites has unique advantages over blood, and has potential as an alternative 'liquid biopsy' template. This review summarises the state of current knowledge and identifies the gaps in our understanding of non-blood liquid biopsies; where their advantages lie, where caution is needed, where they might fit clinically, and where research should focus in order to accelerate clinical implementation. An emphasis is placed on ascites and pleural effusions, being pathological fluids directly associated with cancer. We conclude that non-blood fluids are viable sources of cfDNA in situations where solid tissue biopsies are inaccessible, or only accessible from dated archived specimens. In addition, we show that due to the abundance of cfDNA in non-blood fluids, they can outperform blood in many circumstances. We demonstrate multiple instances in which DNA from various sources can provide additional information, and thus we advocate for analysing non-blood sources as a complement to blood and/or tissue. Further research into these fluids will highlight opportunities to improve patient outcomes across cancer types.

ROR1 and ROR2 expression in pancreatic cancer.

BACKGROUND: The Wnt receptors ROR1 and ROR2 are generating increased interest as cancer therapeutic targets but remain understudied in pancreatic ductal adenocarcinoma (PDAC). Compared to canonical Wnt/ ß-catenin signalling, the role of noncanonical Wnt signalling in PDAC remains largely unknown. Only one study has investigated the prognostic significance of the noncanonical Wnt signalling receptor, ROR2 in PDAC. No studies have investigated the prognostic role of ROR1 in PDAC. METHODS: Here, we performed analysis of ROR1 and ROR2 mRNA expression in three publicly available datasets ICGC-PACA-AU (n = 81), TCGA-PAAD (n = 150) and CPTAC-PDAC (n = 137). ROR1 and ROR2 protein expression from the CPTAC-PDAC discovery cohort were also analysed. Immunohistochemistry (IHC) using the validated anti ROR1 monoclonal antibody (4A5) was performed on the Australian Pancreatic Cancer Genome Initiative (APGI) cohort of PDAC samples (n = 152). Association between ROR1 cytoplasmic staining intensity and clinicopathological parameters including stage, grade and overall survival (OS) was investigated. RESULTS: High ROR1 mRNA expression levels correlated with a favourable OS outcome in all of the ICGC-PACA-AU, TCGA-PAAD and CPTAC-PDAC cohorts. ROR1 protein expression was not associated with stage, grade or OS in the APGI cohort. CONCLUSION: ROR1 and ROR2 have potential as prognostic markers when measured at the mRNA level in PDAC. Our IHC cohort did not support ROR1 protein expression in predicting OS, and highlighted the discrepancy of prognostic biomarkers when measured by MS, IHC and RNAseq.

An organotypic model of high-grade serous ovarian cancer to test the anti-metastatic potential of ROR2 targeted Polyion complex nanoparticles.

High-grade serous ovarian cancer (HGSOC) is the most lethal gynaecological malignancy. Most patients are diagnosed at late stages when the tumour has metastasised throughout the peritoneal cavity. The Wnt receptor ROR2 has been identified as a promising therapeutic target in HGSOC, with limited targeting therapeutic options currently available. Small interfering RNA (siRNA)-based therapeutics hold great potential for inhibiting the function of specific biomarkers, however major challenges remain in efficient delivery and stability. The aim of this study was to investigate the ability of nanoparticles to deliver ROR2 siRNA into HGSOC cells, including platinum resistant models, and estimate the anti-metastatic effect via a 3D organotypic model for ovarian cancer. The nanoparticles were generated by conjugating poly[2-(dimethylamino) ethyl methacrylate] (PDMAEMA) of various chain length to bovine serum albumin (BSA), followed by the condensation of ROR2 siRNA into polyplexes, also termed polyion complex (PIC) nanoparticles. The toxicity and uptake of ROR2 siRNA PIC nanoparticles in two HGSOC cell lines, CaOV3 as well as its cisplatin resistant pair (CaOV3CisR), in addition to primary cells used for the 3D organotypic model were investigated. ROR2 knockdown at both transcriptional and translational levels were evaluated via real-time PCR and western blot analysis, respectively. Following 24 h incubation with the nanoparticles, functional assays were performed including proliferation (IncuCyte S3), transwell migration and 3D co-cultured transwell invasion assays. The PICs nanoparticles exhibited negligible toxicity in the paired CaOV3 cell lines or primary cells. Treating CaOV3 and CaOV3CisR cells with ROR2 siRNA containing PICs nanoparticles significantly inhibited migration and invasion ability. The biocompatible ROR2 siRNA conjugated PICs nanoparticles provide an innovative therapeutic option. ROR2 targeting therapy shows potential in treating HGSOC including platinum resistant forms.

Targeting the actin/tropomyosin cytoskeleton in epithelial ovarian cancer reveals multiple mechanisms of synergy with anti-microtubule agents.

BACKGROUND: Anti-microtubule agents are widely used to treat ovarian cancers, but the efficacy is often compromised by drug resistance. We investigated co-targeting the actin/tropomyosin cytoskeleton and microtubules to increase treatment efficacy in ovarian cancers and potentially overcome resistance. METHODS: The presence of tropomyosin-3.1 (Tpm3.1) was examined in clinical specimens from ovarian cancer patients using immunohistochemistry. Combinatorial effects of an anti-Tpm3.1 compound, ATM-3507, with vinorelbine and paclitaxel were evaluated in ovarian cancer cells via MTS and apoptosis assays. The mechanisms of action were established using live- and fixed-cell imaging and protein analysis. RESULTS: Tpm3.1 is overexpressed in 97% of tumour tissues (558 of 577) representing all histotypes of epithelial ovarian cancer. ATM-3507 displayed synergy with both anti-microtubule agents to reduce cell viability. Only vinorelbine synergised with ATM-3507 in causing apoptosis. ATM-3507 significantly prolonged vinorelbine-induced mitotic arrest with elevated activity of the spindle assembly checkpoint and mitotic cell death; however, ATM-3507 showed minor impact on paclitaxel-induced mitotic defects. Both combinations substantially increased post-mitotic G1 arrest with cyclin D1 and E1 downregulation and an increase of p21Cip and p27Kip. CONCLUSION: Combined targeting of Tpm3.1/actin and microtubules is a promising treatment strategy for ovarian cancer that should be further tested in clinical settings.

ROR2 Is Epigenetically Regulated in Endometrial Cancer.

The Wnt signalling receptor ROR2 has been identified as a possible therapeutic target in numerous cancers; however, its exact role remains unclear. The aim of this study was to investigate the role of ROR2 in endometrial cancer (EC) and the potential mechanism associated with its altered expression. The association between ROR2 mRNA expression levels and clinicopathological parameters, including overall survival (OS), in EC was analysed in The Cancer Genome Atlas Uterine Corpus Endometrial Carcinoma (TCGA-UCEC) cohort and GEO dataset GSE17025. Four EC cell lines (KLE, MFE-296, Ishikawa and ARK-1) and eight clinical EC samples were analysed for ROR2 methylation via Combined Bisulphite Restriction Analysis (COBRA) and bisulphite genomic sequencing (BGS). In addition, the functional effects of ROR2 overexpression were investigated in Ishikawa and ARK-1 cells following ectopic ROR2 expression. ROR2 promoter methylation or reduced ROR2 expression were both found to correlate with shorter OS, high grade and serous subtype in the TCGA-UCEC and GEO datasets. ROR2 was epigenetically silenced by promoter methylation in both patient samples and cell lines. A significant correlation between ROR2 expression levels and promoter methylation was observed in patient samples (r = -0.797, p = 0.018). ROR2 restoration in ARK-1 significantly decreased invasion ability, with associated changes in epithelial-mesenchymal transition (EMT) markers. ROR2 plays a tumour-suppressor role in EC and is epigenetically suppressed with the development of disease. It may represent a diagnostic or therapeutic candidate for EC.

ROR1 is upregulated in endometrial cancer and represents a novel therapeutic target.

ROR1 and ROR2 are receptor tyrosine kinases with altered expression in a range of cancers. Silencing ROR1 or ROR2 in different tumour types has been shown to inhibit proliferation and decrease metastatic potential. The aim of this study was to investigate the role of ROR1 and ROR2 in endometrial cancer via immunohistochemistry (IHC) in a large endometrial cancer patient cohort (n = 499) and through in vitro analysis in endometrial cancer cell lines. Correlation was assessed between ROR1/2 expression and clinicopathological parameters. Kaplan Meier curves were produced for 5-year progression free survival (PFS) and overall survival (OS) with low/moderate versus high ROR1/2 intensity. Cox multivariate regression was applied to analyse the effect of selected covariates on the PFS and OS. The effect of ROR1 and/or ROR2 modulation on cell proliferation, adhesion, migration and invasion was analysed in two endometrial cancer cell lines (KLE and MFE-296). We observed a significant decrease in OS and PFS in patients with high ROR1 expression. ROR1 silencing and ROR2 overexpression significantly inhibited proliferation of KLE endometrial cancer cells and decreased migration. This study supports the oncogenic role of ROR1 in endometrial cancer, and warrants investigation of future application of ROR1-targeting therapies in endometrial cancer patients.

A new 3D organotypic model of ovarian cancer to help evaluate the antimetastatic activity of RAPTA-C conjugated micelles.

INTRODUCTION: Ovarian cancer is often diagnosed at a late stage, when disease has spread to extra-pelvic regions such as the omentum. There are limited treatment options available for women with extensive disease and tumours often relapse after current chemotherapy regimens. Therefore, novel drugs should be investigated for the treatment of ovarian cancer. A 3D organotypic model of ovarian cancer can provide a specific platform for the evaluation of nano-drugs. Using patient derived primary cells, the 3D model mimics the ovarian metastatic microenvironment allowing efficient and reproducible testing of many nanoparticles. Dichlororuthenium(ii) (p-cymene) (1,3,5-triaza-7-phosphaadamantane) (RAPTA-C) conjugated fructose-micelles have been used as the promising nano-drug for the treatment of metastatic cancer. Therefore we aimed to investigate the anti-metastatic properties of RAPTA-C conjugated micelles in ovarian cancer metastasis. METHODS: Ovarian cancer cell adhesion and invasion into a model of omentum were analyzed with and without RAPTA-C conjugated micelles in a range of conditions. RESULTS: We observed that RAPTA-C showed low general toxicity to both primary healthy and cancer cell lines. RAPTA-C loaded micelles significantly enhance the internalization of ruthenium inside the cells compared to free drugs. RAPTA-C did not affect adhesion of OVCAR4 ovarian cancer cells; however, it significantly inhibited invasion of these cells within the omentum model, either in its free form or as cargos inside the micelles. However, when OVCAR4 were treated prior to implantation, invasion was not inhibited. CONCLUSION: A 3D organotypic model provides a clinically relevant and simple method to evaluate the efficiency of nano-drug treatment of ovarian cancer. The ability to inhibit metastasis of RAPTA-C delivered in fructose coated nanoparticles was investigated for the first time via this model. These results provide a good basis to continue the development of this nano-drug in vivo.

Selective modulation of Wnt-binding receptor tyrosine kinase ROR2 expression by human cytomegalovirus regulates trophoblast migration.

Cytomegalovirus (CMV) infection during pregnancy may lead to adverse pregnancy outcomes and permanent neurological disabilities in infants infected in utero. Congenital CMV disease of the foetus and neonate results from both direct viral cytopathic damage and indirect effects through placental dysfunction. Infection specifically alters Wnt signalling, an essential pathway involved in trophoblast migration and placental development. We examined CMV regulation of trophoblast migration. This virus controls expression of Wnt-binding receptor tyrosine kinase ROR2, but not alternate receptor tyrosine kinases ROR1 or RYK. Ectopic expression of ROR2 reduced Wnt5a-induced trophoblast migration, whilst overexpression of ROR1 or RYK did not affect trophoblast migration. CMV infection increased ROR2 protein expression in trophoblasts, with no effect on ROR1 and RYK expression. These data further support the proposal that specific inhibition of this mechanism may be a target for therapeutic intervention to reduce placental damage and consequent foetal disease due to congenital CMV infection.