Comparison of total and endometrial circulating cell-free DNA in women with and without endometriosis.

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.

Circulating cell-free DNA undergoes significant decline in yield after prolonged storage time in both plasma and purified form.

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.

Cell-free DNA is abundant in ascites and represents a liquid biopsy of ovarian cancer.

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.

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.

The influence of biological and lifestyle factors on circulating cell-free DNA in blood plasma.

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.

Total and endothelial cell-derived cell-free DNA in blood plasma does not change during menstruation.

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.

Circulating cell-free DNA from plasma undergoes less fragmentation during bisulfite treatment than genomic DNA due to low molecular weight.

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.

Comparison of 4 commercial kits for the extraction of circulating DNA from plasma.

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.

Evaluation of Streck BCT and PAXgene Stabilised Blood Collection Tubes for Cell-Free Circulating DNA Studies in Plasma.

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.