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The emergence of targeted therapies has transformed ovarian cancer treatment. However, biomarker profiling for precision medicine is limited by access to quality, tumour-enriched tissue samples. The use of cell-free DNA (cfDNA) in ascites presents a potential solution to this challenge. In this study, next-generation sequencing was performed on ascites-derived cfDNA samples (26 samples from 15 human participants with ovarian cancer), with matched DNA from ascites-derived tumour cells (n = 5) and archived formalin-fixed paraffin-embedded (FFPE) tissue (n = 5). Similar tumour purity and variant detection were achieved with cfDNA compared to FFPE and ascites cell DNA. Analysis of large-scale genomic alterations, loss of heterozygosity and tumour mutation burden identified six cases of high genomic instability (including four with pathogenic BRCA1 and BRCA2 mutations). Copy number profiles and subclone prevalence changed between sequential ascites samples, particularly in a case where deletions and chromothripsis in Chr17p13.1 and Chr8q resulted in changes in clinically relevant TP53 and MYC variants over time. Ascites cfDNA identified clinically actionable information, concordant to tissue biopsies, enabling opportunistic molecular profiling. This advocates for analysis of ascites cfDNA in lieu of accessing tumour tissue via biopsy., (© 2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

3D co-cultures are key tools for in vitro biomedical research as they recapitulate more closely the in vivo environment while allowing a tighter control on the culture's composition and experimental conditions. The limited technologies available for the analysis of these models, however, hamper their widespread application. The separation of the contribution of the different cell types, in particular, is a fundamental challenge. In this work, ORACLE (OvaRiAn Cancer ceLl rEcognition) is presented, a deep neural network trained to distinguish between ovarian cancer and healthy cells based on the shape of their nucleus. The extensive validation that are conducted includes multiple cell lines and patient-derived cultures to characterize the effect of all the major potential confounding factors. High accuracy and reliability are maintained throughout the analysis (F1 score > 0.9 and Area under the ROC curve -ROC-AUC- score = 0.99) demonstrating ORACLE's effectiveness with this detection and classification task. ORACLE is freely available (https://github.com/MarilisaCortesi/ORACLE/tree/main) and can be used to recognize both ovarian cancer cell lines and primary patient-derived cells. This feature is unique to ORACLE and thus enables for the first time the analysis of in vitro co-cultures comprised solely of patient-derived cells., (© 2024 The Author(s). Advanced Biology published by Wiley‐VCH GmbH.)

3D cell cultures are a fundamental tool in ovarian cancer research that can enable more effective study of the main features of this lethal disease, including the high rates of recurrence and chemoresistance. A clearer, more comprehensive understanding of the biological underpinnings of these phenomena could aid the development of more effective treatments thus improving patient outcomes. Selecting the most appropriate model to investigate the different aspects of cell biology that are relevant to cancer is challenging, especially since the assays available for the study of 3D cultures are not fully established yet. To maximise the usefulness of 3D cell cultures of ovarian cancer, we undertook an in-depth review of the currently available models, taking into consideration the strengths and limitations of each approach and of the assay techniques used to evaluate the results. This integrated analysis provides insight into which model-assay pair is best suited to study different parameters of ovarian cancer biology such as cell proliferation, gene expression or treatment response. We also describe how the combined use of multiple models is likely to be the most effective strategy for the in vitro characterisation of complex behaviours., Competing Interests: The authors declare there are no competing interests., (©2024 Cortesi et al.)

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., Competing Interests: Declaration of competing interest KW declares potential financial conflict of interest due to stock ownership in the following companies that are developing cell-free DNA based clinical assays: Guardant Heath; Exact Sciences; EpiGenomics AG. All other authors declare no competing financial interests., (Copyright © 2024. Published by Elsevier Inc.)

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., Competing Interests: Financial disclosure/conflicts of interest: Kristina Warton owns stocks in Guardant Health, Epigenomics AG, and Exact Sciences and has an ongoing funding from World Scientific Publishing. The other authors have no conflict of interest or financial disclosures to disclose., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The Menopause Society.)

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.

Objectives: Circulating DNA (cirDNA) is generally purified from plasma that has been biobanked for variable lengths of time. In long-term experiments or clinical trials, the plasma can be stored frozen for up to several years. Therefore, it is crucial to determine the stability of cirDNA to ensure confidence in sample quality upon analysis. Our main objective was to determine the effect of storage for up to 2 years on cirDNA yield and fragmentation., Methods: We stored frozen EDTA plasma and purified cirDNA from 10 healthy female donors, then quantified cirDNA yield at baseline, and at regular intervals for up to 2 years, by qPCR and Qubit. We also compared cirDNA levels in non-haemolysed and haemolysed blood samples after 16 months of storage and tested the effect of varying DNA extraction protocol parameters., Results: Storage up to two years caused an annual cirDNA yield decline of 25.5% when stored as plasma and 23% when stored as purified DNA, with short fragments lost more rapidly than long fragments. Additionally, cirDNA yield was impacted by plasma input and cirDNA elution volumes, but not by haemolysis., Conclusions: The design of long-term cirDNA-based studies and clinical trials should factor in the deterioration of cirDNA during storage., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

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.

Research Question: Do women with laparoscopically confirmed endometriosis have higher plasma concentrations of circulating cell-free DNA (cirDNA) than those without endometriosis?, Design: Prospective study of women aged 18-45 years undergoing benign gynaecological laparoscopy at two tertiary hospitals. Venous blood was collected immediately before surgery, and women were allocated to the endometriosis or control groups based on surgical findings. Total plasma cirDNA and cirDNA integrity were measured by quantitative polymerase chain reaction (qPCR) targeting short (115 bases) and long (247 bases) ALU segments. Endometrial-derived cirDNA was measured by qPCR of bisulfite-treated cirDNA using primers selective for a FAM101A sequence uniquely unmethylated in endometrial tissue. Five cirDNA parameters were compared between the control and endometriosis cohorts: total cirDNA concentration, long-stranded cirDNA concentration, integrity ratio, endometrial cirDNA concentration and endometrial cirDNA proportion., Results: Twenty-eight endometriosis and 15 control samples were included. Women with and without endometriosis had cirDNA concentrations of 2.24 ± 0.89 ng/ml and 2.56 ± 0.92 ng/ml, respectively. Analysis by phenotype of endometriosis revealed a significantly higher endometrial cirDNA concentration in women with superficial disease (n = 10) compared with deep endometriosis (n = 18) (mean difference 0.14 ng/ml; 95% CI 0.15 to 0.26; P = 0.025), but not with controls., Conclusions: No significant differences were found in any of the cirDNA parameters between women with and without endometriosis. The low statistical power and heterogenous pelvic pathology in the control group render it difficult to determine whether the negative results reflect a true lack of increase in cirDNA in endometriosis., (Copyright © 2021 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Research and clinical use of circulating cell-free DNA (cirDNA) is expanding rapidly; however, there remain large gaps in our understanding of the influence of lifestyle and biological factors on the amount of cirDNA present in blood. Here, we review 66 individual studies of cirDNA levels and lifestyle and biological factors, including exercise (acute and chronic), alcohol consumption, occupational hazard exposure, smoking, body mass index, menstruation, hypertension, circadian rhythm, stress, biological sex and age. Despite technical and methodological inconsistences across studies, we identify acute exercise as a significant influence on cirDNA levels. Given the large increase in cirDNA induced by acute exercise, we recommend that controlling for physical activity prior to blood collection is routinely incorporated into study design when total cirDNA levels are of interest. We also highlight appropriate selection and complete reporting of laboratory protocols as important for improving the reproducibility cirDNA studies and ability to critically evaluate the results., Competing Interests: NY, CF No competing interests declared, KW holds stock in Guardant Health, Exact Sciences and Epigenomics AG, (© 2021, Yuwono et al.)

Objective: Malignant ascites is a common clinical feature of ovarian cancer and represents a readily accessible sample of tumour cells and tumour DNA. This study aimed to characterise the cell-free DNA (cfDNA) in ascites in terms of its size profile, stability and cell-free tumour DNA (cftDNA) content., Methods: Cell spheroids, loose cells and cell-free fluid was collected from ascites from 18 patients with ovarian cancer. cfDNA was isolated and assessed for size by electrophoresis, concentration by fluorometry,cftDNA content by methylation specific qPCR of HOXA9 and IFFO1 promoter regions and by targeted sequencing. Stability was assessed after ascites fluid was stored at 4 °C for 24 and 72 h before fractionating., Results: The concentration of cfDNA in ascites ranged from 6.6 to 300 ng/mL. cfDNA size distribution resembled blood plasma-derived cfDNA, with major peaks corresponding to mono- and di-nucleosome DNA fragments. High molecular weight cfDNA was observed in 7 of 18 patients and appeared to be associated with extracellular vesicles. IFFO1 and HOXA9 methylation was proportionately higher in cfDNA than spheroid- and loose-cell fractions and was not observed in healthy primary cells. Variant allele frequency was highest in cfDNA compared to single cells and spheroids from ascites. Though cancer cell numbers in ascites declined to near zero in recurrent ascites from one patient undertaking chemotherapy, cftDNA could still be sampled. cfDNA size, concentration and tumour content was stable over 72 h., Conclusion: cfDNA in ovarian cancer ascites demonstrates inter-patient variability, yet is consistently a rich source of cftDNA, which is a stable substrate. This supports the wider clinical use of ascites in the molecular analysis of ovarian cancer., Competing Interests: Declaration of Competing Interest KW declares potential financial conflict of interest due to stock ownership in the following companies that are developing cell-free DNA based clinical assays: Guardant Heath; Exact Sciences; EpiGenomics AG. ADeF has received research funding from AstraZeneca. All other authors declare no competing financial interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Assays measuring cell-free DNA (cfDNA) in blood have widespread potential in modern medicine. However, a comprehensive understanding of cfDNA dynamics in healthy individuals is required to assist in the design of assays that maximise the signal driven by pathological changes, while excluding fluctuations that are part of healthy physiological processes. The menstrual cycle involves major remodelling of endometrial tissue and associated apoptosis, yet there has been little investigation of the impact of the menstrual cycle on cfDNA levels. Paired plasma samples were collected from 40 healthy women on menstruating (M) and non-menstruating (NM) days of their cycle. We measured total cfDNA by targeting ALU repetitive sequences and measured endothelial-derived cfDNA by methylation-specific qPCR targeting an endothelium-unique unmethylated CDH5 DNA region. CfDNA integrity and endothelial cfDNA concentration, but not total cfDNA, are consistent across time between NM and M. No significant changes in total (ALU-115 p = 0.273; ALU-247 p = 0.385) or endothelial cell specific (p = 0.301) cfDNA were observed, leading to the conclusion that menstrual status at the time of diagnostic blood collection should not have a significant impact on the quantitation of total cfDNA and methylation-based cancer assays., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: KW holds stock in Guardant Health, Exact Sciences and Epigenomics AG. No other authors have competing interests. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no restrictions on sharing of data and/or materials from this publication.

The build-up of fluid in the peritoneal cavity-ascites-is a hallmark of ovarian cancer, the most lethal of all gynaecological malignancies. This remarkable fluid, which contains a variety of cellular and acellular components, is known to contribute to patient morbidity and mortality by facilitating metastasis and contributing to chemoresistance, but remains largely under-researched. In this review, we will critically analyse the evidence associating ascites with metastasis and chemoresistance in ovarian cancer and provide an update on research in the field. We will argue the case for ascites as a unique and accessible substrate for tracking tumour progression and for translational research that will enhance our understanding of this cancer and lead to improvements in patient outcomes.

Background: Methylation patterns in circulating cell-free DNA are potential biomarkers for cancer and other pathologies. Currently, bisulfite treatment underpins most DNA methylation analysis methods, however, it is known to fragment DNA. Circulating DNA is already short, and further fragmentation during bisulfite treatment is of concern, as it would potentially reduce the sensitivity of downstream assays., Methods: We used high molecular weight genomic DNA to compare fragmentation and recovery following bisulfite treatment with 2 commercially available kits (Qiagen). The bisulfite treated DNA was visualised on an agarose gel and quantified by qPCR. We also bisulfite treated, visualised and quantitated circulating DNA from plasma., Results: There was no difference in DNA fragmentation between the two kits tested, however, the Epitect Fast kit gave better recovery than the standard Epitect kit, with the same conversion efficiency. We also found that bisulfite treated circulating DNA migrates as distinct bands on agarose gels, suggesting that, in contrast to genomic DNA, it remains largely intact following treatment. Bisulfite treatment of 129 and 234 base PCR products confirmed that this was due to the short length of the circulating DNA fragments. Compared to double stranded DNA, bisulfite treated single stranded DNA gives a very weak signal on gel electrophoresis., Conclusions: DNA fragmentation during bisulfite treatment does not contribute to loss of sensitivity in methylation analysis of circulating DNA. The absence of DNA fragments below approximately 170 bases from agarose gel images of purified circulating DNA raises the possibility that these fragments are single stranded following the DNA extraction step., Competing Interests: I have read the journals competing interests policy and the authors of this manuscript have the following competing interests: KW- stock ownership Guardant Health, Exact Sciences and Epigenomics AG. This does not alter our adherence to PLOS ONE policies of sharing data and materials.

The utility of circulating DNA as a source of clinical biomarkers in blood is limited by its low concentration and small fragment size. Effective purification methods can maximize circulating DNA yield and contribute to the success of downstream protocols. We describe the evaluation of 4 commercial DNA purification kits-QIAamp Circulating Nucleic Acids kit, QIAamp DNA Blood Mini kit, QIAamp Ultrasens Virus kit and the QIASymphony DSP Virus kit-for the extraction of high and low molecular weight DNA from blood plasma. Using qPCR to quantitate endogenous Alu sequences, as well as spiked exogenous high and low molecular weight zebrafish DNA, we found that the Circulating Nucleic Acids kit and the DSP kit were both efficient at purifying DNA from plasma regardless of fragment size, whereas the DNA Blood Mini kit was only able to effectively extract high molecular weight DNA. The Ultrasens Virus Kit produced the lowest yields for both large and small fragments. The use of carrier RNA with the Circulating Nucleic Acids and the DSP kits improved yields. Appropriate choice of kit can be an important factor in determining experiment outcome., (Published by Elsevier Inc.)

Introduction: Blood samples for studies of circulating DNA in disease are often collected in clinical settings where prompt processing of samples is not possible. In order to avoid problems associated with leukocyte lysis after prolonged blood storage, stabilised blood tubes have been developed containing preservatives that prevent cell lysis. We evaluated Streck BCT tubes and PAXgene ccfDNA tubes, as well as standard EDTA blood collection tubes, in terms of DNA yield and fragment size., Methods: Blood was collected in EDTA, Streck BCT or PAXgene ccfDNA tubes and stored for 1 h at 4 °C, or 4 days at room temperature. DNA was extracted using the QIAamp Circulating Nucleic Acids kit, and visualised on an agarose gel or quantitated by qPCR. Ratios of a 247-base and a 115-base amplicon of the Alu repetitive element were used to infer size distribution., Results: While plasma DNA in EDTA tube blood samples increased by ~10- to 20-fold after 4 days of storage at room temperature, both Streck BCT tubes and PAXgene ccfDNA tubes maintained stable plasma DNA concentrations. A slight decrease in DNA yield following 1 h of blood storage at 4 °C was observed in Streck BCT and PAXgene ccfDNA tubes relative to EDTA tubes. This decrease was reversed by increasing the proteinase digest step of the DNA extraction protocol to 60 min, as recommended by Streck tube product literature. Visualisation of the extracted DNA on an agarose gel showed that after 4 days of room temperature storage, samples collected in EDTA tubes contained abundant high-molecular weight DNA, which was partially fragmented in a ladder pattern. A slight increase in high-molecular weight DNA in samples stored for 4 days at room temperature in Streck BCT tubes was also observed, but this was not reflected in a change in large and small Alu fragment ratios as measured by qPCR., Conclusions: Tubes containing preservative to prevent cell lysis can extend the scope for blood collection in clinical settings; however, slight differences between samples collected in different tube types underscore the requirement for standardised protocols, as well as attention to sample handling.

Circulating tumor DNA (ctDNA) in the plasma or serum of cancer patients provides an opportunity for non-invasive sampling of tumor DNA. This 'liquid biopsy' allows for interrogations of DNA such as quantity, chromosomal alterations, sequence mutations and epigenetic changes, and can be used to guide and improve treatment throughout the course of the disease. This tremendous potential for real-time 'tracking' in a cancer patient has led to substantial research efforts in the ctDNA field. ctDNA can be distinguished from non-tumor DNA by the presence of tumor-specific mutations and copy number variations, and also by aberrant DNA methylation, with both DNA sequence and methylation changes corresponding to those found in the tumor. Aberrant methylation of specific promoter regions can be a very consistent feature of cancer, in contrast to mutations, which typically occur at a wide range of sites. This consistency makes ctDNA methylation amenable to the design of widely applicable clinical assays. In this review, we examine ctDNA methylation in the context of monitoring disease status, treatment response and determining the prognosis of cancer patients., (© 2016 The authors.)

A range of molecular alterations found in tumor cells, such as DNA mutations and DNA methylation, is reflected in cell-free circulating DNA (circDNA) released from the tumor into the blood, thereby making circDNA an ideal candidate for the basis of a blood-based cancer diagnosis test. In many cancer types, mutations driving tumor development and progression are present in a wide range of oncogenes and tumor suppressor genes. However, even when a gene is consistently mutated in a particular cancer, the mutations can be spread over very large regions of its sequence, making evaluation difficult. This diversity of sequence changes in tumor DNA presents a challenge for the development of blood tests based on DNA mutations for cancer diagnosis. Unlike mutations, DNA methylation that can be consistently measured, as it tends to occur in specific regions of the DNA called CpG islands. Since DNA methylation is reflected within circDNA, detection of tumor-specific DNA methylation in patient plasma is a feasible approach for the development of a blood-based test. Aberrant circDNA methylation has been described in most cancer types and is actively being investigated for clinical applications. A commercial blood test for colorectal cancer based on the methylation of the SEPT9 promoter region in circDNA is under review for approval by the Federal Drug Administration (FDA) for clinical use. In this paper, we review the state of research in circDNA methylation as an application for blood-based diagnostic tests in colorectal, breast, lung, pancreatic and ovarian cancers, and we consider some of the future directions and challenges in this field. There are a number of potential circDNA biomarkers currently under investigation, and experience with SEPT9 shows that the time to clinical translation can be relatively rapid, supporting the promise of circDNA as a biomarker.

Background: Free circulating DNA (fcDNA) has many potential clinical applications, due to the non-invasive way in which it is collected. However, because of the low concentration of fcDNA in blood, genome-wide analysis carries many technical challenges that must be overcome before fcDNA studies can reach their full potential. There are currently no definitive standards for fcDNA collection, processing and whole-genome sequencing. We report novel detailed methodology for the capture of high-quality methylated fcDNA, library preparation and downstream genome-wide Next-Generation Sequencing. We also describe the effects of sample storage, processing and scaling on fcDNA recovery and quality., Results: Use of serum versus plasma, and storage of blood prior to separation resulted in genomic DNA contamination, likely due to leukocyte lysis. Methylated fcDNA fragments were isolated from 5 donors using a methyl-binding protein-based protocol and appear as a discrete band of ~180 bases. This discrete band allows minimal sample loss at the size restriction step in library preparation for Next-Generation Sequencing, allowing for high-quality sequencing from minimal amounts of fcDNA. Following sequencing, we obtained 37 × 10(6)-86 × 10(6) unique mappable reads, representing more than 50% of total mappable reads. The methylation status of 9 genomic regions as determined by DNA capture and sequencing was independently validated by clonal bisulphite sequencing., Conclusions: Our optimized methods provide high-quality methylated fcDNA suitable for whole-genome sequencing, and allow good library complexity and accurate sequencing, despite using less than half of the recommended minimum input DNA.

Objective: Altered DNA methylation patterns hold promise as cancer biomarkers. In this study we selected a panel of genes which are commonly methylated in a variety of cancers to evaluate their potential application as biomarkers for prognosis and diagnosis in high grade serous ovarian carcinoma (HGSOC); the most common and lethal subtype of ovarian cancer., Methods: The methylation patterns of 10 genes (BRCA1, EN1, DLEC1, HOXA9, RASSF1A, GATA4, GATA5, HSULF1, CDH1, SFN) were examined and compared in a cohort of 80 primary HGSOC and 12 benign ovarian surface epithelium (OSE) samples using methylation-specific headloop suppression PCR., Results: The genes were variably methylated in primary HGSOC, with HOXA9 methylation observed in 95% of cases. Most genes were rarely methylated in benign OSE, with the exception of SFN which was methylated in all HGSOC and benign OSE samples examined. Methylation of DLEC1 was associated with disease recurrence, independent of tumor stage and suboptimal surgical debulking (HR 3.5 (95% CI:1.10-11.07), p=0.033). A combination of the methylation status of HOXA9 and EN1 could discriminate HGSOC from benign OSE with a sensitivity of 98.8% and a specificity of 91.7%, which increased to 100% sensitivity with no loss of specificity when pre-operative CA125 levels were also incorporated., Conclusions: This study provides further evidence to support the feasibility of detecting altered DNA methylation patterns as a potential diagnostic and prognostic approach for HGSOC., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Aim: To establish the suitability of multiplex tandem polymerase chain reaction (MT-PCR) for rapid identification of oestrogen receptor (ER) and Her-2 status using a single, formalin-fixed, paraffin-embedded (FFPE) breast tumour section., Methods: Tissue sections from 29 breast tumours were analysed by immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH). RNA extracted from 10 mum FFPE breast tumour sections from 24 of 29 tumours (14 ER positive and 5 Her-2 positive) was analysed by MT-PCR. After establishing a correlation between IHC and/or FISH and MT-PCR results, the ER/Her-2 status of a further 32 randomly selected, archival breast tumour specimens was established by MT-PCR in a blinded fashion, and compared to IHC/FISH results., Results: MT-PCR levels of ER and Her-2 showed good concordance with IHC and FISH results. Furthermore, among the ER positive tumours, MT-PCR provided a quantitative score with a high dynamic range. Threshold values obtained from this data set applied to 32 archival tumour specimens showed that tumours strongly positive for ER and/or Her-2 expression were easily identified by MT-PCR., Conclusion: MT-PCR can provide rapid, sensitive and cost-effective analysis of FFPE material and may prove useful as triage to identify patients suited to endocrine or trastuzumab (Herceptin) treatment.

Recent advances in the field of molecular clonotype analysis have enabled detailed repertoire characterization of viably isolated antigen-specific T cell populations directly ex vivo. However, in the absence of a biologically contained FACS facility, peripheral blood mononuclear cell (PBMC) preparations derived from patients infected with agents such as HIV must be formaldehyde fixed to inactivate the pathogen; this procedure adversely affects nucleic acid template quality. Here, we developed and validated a method to amplify and sequence mRNA species derived from formaldehyde fixed PBMC specimens. Antigen-specific CD8+ cytotoxic T-lymphocyte populations were identified with standard fluorochrome-conjugated peptide-major histocompatibility complex class I tetramers refolded around synthetic peptides representing immunodominant epitopes from HIV p24 Gag (KRWII[M/L]GLNK/HLA B*2705) and CMV pp65 (NLVPMVATV/HLA A*0201 and TPRVTGGGAM/HLA B*0702), and acquired in separate laboratories with or without fixation. In the presence of proteinase K pre-treatment, the observed antigen-specific CD8+ T-cell repertoire determined by molecular clonotype analysis was statistically no different whether derived from fixed or unfixed PBMC. However, oligo-dT recovery methods were not suitable for use with fixed tissue as significant skewing of clonotypic representation was observed. Thus, we have developed a reliable RNA-based method for molecular clonotype analysis that is compatible with formaldehyde fixation and therefore suitable for use with primary human samples isolated by FACS outside the context of a biological safety level 3 containment facility.

The use of mobile phones is increasing, which also increases the population's exposure to global system of mobile communications (GSM) signals. Questions of safety and possible biological effects are of concern and to date, remain largely unanswered. In order to examine possible biological effects of a GSM-like signal at a cellular level, we exposed two human cell lines (one of neuronal (SK-N-SH) and the other of monocytoid (U937) origin) to a 900 MHz RF signal, pulsed at 217 Hz, producing a specific absorption rate (SAR) of 0.2 W/kg. Putative effects were assessed by comparing radiofrequency-exposed cells to sham-exposed cells using a variety of assay techniques. For the cell line SK-N-SH, effects were specifically assessed by gene microarray, followed by real-time PCR of the genes of interest, Western blot analysis was used to measure heat shock protein levels, and flow cytometry to measure cell cycle distributions and apoptosis. Effects of radiofrequency on the cell line U937 were assessed by cell viability and cell cycle analysis. From our study of these two cell lines, we found no significant difference between sham-exposed versus radiofrequency-exposed cells in any of the assays or conditions examined.

The aim of this study was to characterise a novel family of inflammatory genes induced by pro-inflammatory cytokines in primary human endothelial cells. Using a genome-wide array screen two previously uncharacterised genes, NLF1 and NLF2 were identified that were upregulated over 30 fold by treatment with interleukin 1beta for 2 h. They were also found to respond to tumour necrosis factor alpha, suggesting a general role in inflammation. Expression of both genes peaked 2 h after addition of interleukin 1beta, with similar kinetics to the fastest nuclear factor kappaB (NF-kappaB) induced genes. The activation of both genes by interleukin 1beta was abrogated by the proteasomal inhibitor, lactacystin which blocks activation of NF-kappaB by preventing IkappaB degradation. Furthermore, two sequences with homology to NF-kappaB binding sites in the promoter of NLF1 were found to be essential for rapid elevation in expression in response to interleukin 1beta. NLF1 and NLF2 transcripts were found predominantly in endothelial cells, and the encoded proteins were localised to the nuclear compartment suggesting a role in the regulation of transcription. Transfection of recombinant NLF into endothelial cells resulted in upregulation of the Rho kinases, Rnd1 and Gem GTPase. We propose that NLF1 and NLF2 belong to a novel gene family encoding nuclear factors with a role in regulating genes which control cellular architecture. This might increase vascular permeability in acute inflammation.

CLIC1 (NCC27) is an unusual, largely intracellular, ion channel that exists in both soluble and membrane-associated forms. The soluble recombinant protein can be expressed in Escherichia coli, a property that has made possible both detailed electrophysiological studies in lipid bilayers and an examination of the mechanism of membrane integration. Soluble E. coli-derived CLIC1 moves from solution into artificial bilayers and forms chloride-selective ion channels with essentially identical conductance, pharmacology, and opening and closing kinetics to those observed in CLIC1-transfected Chinese hamster ovary cells. The process of membrane integration of CLIC1 is pH-dependent. Following addition of protein to the trans solution, small conductance channels with slow kinetics (SCSK) appear in the bilayer. These SCSK modules then appear to undergo a transition to form a high conductance channel with fast kinetics. This has four times the conductance of the SCSK and fast kinetics that characterize the native channel. This suggests that the CLIC1 ion channel is likely to consist of a tetrameric assembly of subunits and indicates that despite its size and unusual properties, it is able to form a completely functional ion channel in the absence of any other ancillary proteins.

NCC27 is a nuclear chloride ion channel, identified in the PMA-activated U937 human monocyte cell line. NCC27 mRNA is expressed in virtually all cells and tissues and the gene encoding NCC27 is also highly conserved. Because of these factors, we have examined the hypothesis that NCC27 is involved in cell cycle regulation. Electrophysiological studies in Chinese hamster ovary (CHO-K1) cells indicated that NCC27 chloride conductance varied according to the stage of the cell cycle, being expressed only on the plasma membrane of cells in G2/M phase. We also demonstrate that Cl- ion channel blockers known to block NCC27 led to arrest of CHO-K1 cells in the G2/M stage of the cell cycle, the same stage at which this ion channel is selectively expressed on the plasma membrane. These data strongly support the hypothesis that NCC27 is involved, in some as yet undetermined manner, in regulation of the cell cycle.