Characterization Of Cell-free DNA Research Articles

Isolation and Characterization of Cell-Free DNA from Cerebral Organoids.

Early detection of neurological conditions is critical for timely diagnosis and treatment. Identifying cellular-level changes is essential for implementing therapeutic interventions prior to symptomatic disease onset. However, monitoring brain tissue directly through biopsies is invasive and poses a high risk. Bodily fluids such as blood or cerebrospinal fluid contain information in many forms, including proteins and nucleic acids. In particular, cell-free DNA (cfDNA) has potential as a versatile neurological biomarker. Yet, our knowledge of cfDNA released by brain tissue and how cfDNA changes in response to deleterious events within the brain is incomplete. Mapping changes in cfDNA to specific cellular events is difficult in vivo, wherein many tissues contribute to circulating cfDNA. Organoids are tractable systems for examining specific changes consistently in a human background. However, few studies have investigated cfDNA released from organoids. Here, we examined cfDNA isolated from cerebral organoids. We found that cerebral organoids release quantities of cfDNA sufficient for downstream analysis with droplet-digital PCR and whole-genome sequencing. Further, gene ontology analysis of genes aligning with sequenced cfDNA fragments revealed associations with terms related to neurodevelopment and autism spectrum disorder. We conclude that cerebral organoids hold promise as tools for the discovery of cfDNA biomarkers related to neurodevelopmental and neurological disorders.

Abstract 786: Single molecule methylation sequencing of circulating DNA identifies individuals with breast cancer

Abstract Liquid biopsies are being investigated for the detection of cancer from blood. Epigenetic characterization of cell free DNA (cfDNA), specifically DNA methylation, is an emerging approach for sensitive detection and quantification of tumor burden. For breast cancer detection from blood, existing approaches for testing cfDNA have demonstrated a low performance in identifying affected patients. For this study, we developed a new approach that overcame this issue. Addressing the challenge of detecting breast cancer from cfDNA, this new approach relied on single-molecule methylation profiling of cfDNA. We used nanopore single molecule sequencing to provide quantitative characterization of cfDNA methylomes. This study determined if this approach provided the methylation features required for detecting the presence of breast cancer. The study population consisted of an extended cohort (~200 with untreated breast cancer and ~400 healthy controls). We identified 5mC-specific profiles from the nanopore sequencing signals of cfDNA. Importantly, this approach did not require any biosulfite or biochemical processing steps for determining methylation. Thus, we determined methylation profiles that were directly extrapolated from native cfDNA at single molecule resolution. Our results from this study demonstrated higher sensitivity of detecting breast cancer compared to other cfDNA methylation tests. We developed a new analytical framework for characterizing the methylation profiles of individuals with breast cancer compared to healthy individuals. Using a supervised bootstrapped algorithm, we identified CpG sites that were differentially methylated between the case and control groups. Afterwards, we calculated the similarity of any sample’s methylation profile to a training data. Our method also accommodates sparse and missing data. Based on separate validation datasets, we accurately identified those individuals with breast cancer. We validated our study with a different, independent cohort of healthy controls and individuals with breast cancer. Overall, this study demonstrates that a nanopore-based cfDNA sequencing and methylation profiling detects breast cancer from blood with higher performance then has been previously reported. Citation Format: Billy T. Lau, Xiangqi Bai, Alison Notestine, Brett Parkinson, Hanlee P. Ji, Lincoln Nadauld. Single molecule methylation sequencing of circulating DNA identifies individuals with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 786.

Multiplexed Quantification of Four Neuroblastoma DNA Targets in a Single Droplet Digital PCR Reaction

The detection and characterization of cell-free DNA (cfDNA) in peripheral blood from neuroblastoma patients may serve as a minimally invasive approach to liquid biopsy. Major challenges in the analysis of cfDNA purified from blood samples are small sample volumes and low cfDNA concentrations. Droplet digital PCR (ddPCR) is a technology suitable for analyzing low levels of cfDNA. Reported here are two quadruplexed ddPCR assay protocols that reliably quantify MYCN and ALK copy numbers in a single reaction together with the two reference genes, NAGK and AFF3, and accurately estimate ALKF1174L (exon 23 position 3522, C>A) and ALKR1275Q (exon 25 position 3824, G>A) mutant allele fractions using cfDNA as input. The separation of positive and negative droplets was optimized for detecting two targets in each ddPCR fluorescence channel by the adjustment of the probe and primer concentrations of each target molecule. The quadruplexed assays were validated using a panel of 10 neuroblastoma cell lines and paired blood plasma and primary neuroblastoma samples from nine patients. Accuracy and sensitivity thresholds in quadruplexed assays corresponded well with those from the respective duplexed assays. Presented are two robust quadruplexed ddPCR protocols applicable in the routine clinical setting and that require only minimal plasma volumes for the assessment of MYCN and ALK oncogene status.

Abstract 1386: Application of PAXgene® Blood ccfDNA Tubes* as a standardized approach for therapy monitoring in late-stage lung cancer

Abstract Introduction In June 2016, the FDA approved the first blood-based genetic test to detect gene mutations of epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC). With this move, the genetic characterization of cell-free DNA became a routine application for the care of NSCLC patients. Nevertheless, there are many unresolved preanalytical issues. Particularly important is defining the optimal method for blood draw and sample handling before plasma preparation. The aim of this research study is to answer two questions: - Would the PAXgene Blood ccfDNA Tube* (PAXgene Tubes) be a viable alternative to EDTA blood tubes as the current gold standard? - Could a longitudinal analysis of methylated cell-free plasma DNA allow predicting response to NSCLC treatment? Experimental procedures To date, 45 advanced-stage lung cancer patients were enrolled in this ongoing research study. Blood samples from 17 were used for the first study part to compare the quantity of plasma mPTGER4 and mSHOX2 DNA in both tubes types. At diagnosis, blood from these therapy-naive subjects was drawn into EDTA and PAXgene tubes. The EDTA tubes were processed immediately (within 4 h after blood draw) and spun twice to separate the plasma. The PAXgene tubes were stored for up to 7 days at room temperature (15-25°C) before processing to mimic transport between clinical and laboratory sites. All plasma samples were stored at –80°C before DNA isolation and quantification of the two methylated sequences by real-time PCR. For the longitudinal study, blood was drawn from 28 subjects into PAXgene tubes only in approximately one week intervals, starting before therapy until conventional re-staging. The quantity of mPTGER4 and mSHOX2 plasma DNA was measured as in the comparative research study. Summary of data The amount of plasma mPTGER4 and mSHOX2 DNA in EDTA and PAXgene tubes was completely congruent for both markers in all 17 samples of the first study part. The analysis of the two methylation markers in the second study part allowed predicting response/non-response to the given therapy in 25/28 subjects (88%), when compared to standard computer tomography-based re-staging. PCR results show that an assessment of therapy success could already be possible within two to three weeks after therapy start. Conclusions PAXgene tubes stabilize blood cells and thus prevent a dilution of cell-free DNA by genomic DNA from white blood cells. No change in the amount of tumor-derived cell-free DNA was seen. Therefore, PAXgene Blood ccfDNA Tubes* could be a good alternative to EDTA tubes. Therapy monitoring by longitudinal measurements of mPTGER4 and mSHOX2 plasma DNA could be several weeks faster than re-staging by the currently used digital imaging method. *The PAXgene Blood ccfDNA Tube is for Research Use Only in the US. Citation Format: Bernd Schmidt, Erkan Arslan, Dana Reinicke, Thorsten Voss, Andrea Ullius, Stephan Eisenmann, Michael Fleischhacker. Application of PAXgene® Blood ccfDNA Tubes* as a standardized approach for therapy monitoring in late-stage lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1386.

Current status of liquid biopsies for the detection and management of prostate cancer.

In recent years, new therapeutic options have become available for prostate cancer (PC) patients, generating an urgent need for better biomarkers to guide the choice of therapy and monitor treatment response. Liquid biopsies, including circulating tumor cells (CTCs), circulating nucleic acids, and exosomes, have been developed as minimally invasive assays allowing oncologists to monitor PC patients with real-time cellular or molecular information. While CTC counts remain the most extensively validated prognostic biomarker to monitor treatment response, recent advances demonstrate that CTC morphology and androgen receptor characterization can provide additional information to guide the choice of treatment. Characterization of cell-free DNA (cfDNA) is another rapidly emerging field with novel technologies capable of monitoring the evolution of treatment relevant alterations such as those in DNA damage repair genes for poly (ADP-ribose) polymerase (PARP) inhibition. In addition, several new liquid biopsy fields are emerging, including the characterization of heterogeneity, CTC RNA sequencing, the culture and xenografting of CTCs, and the characterization of extracellular vesicles (EVs) and circulating microRNAs. This review describes the clinical utilization of liquid biopsies in the management of PC patients and emerging liquid biopsy technologies with the potential to advance personalized cancer therapy.

Analysis of mutation detection in cell-free DNA in ascites using comprehensive NGS panel.

e13029 Background: Ascites is one of complications for patients with malignancies in advanced stage. Detection and characterization of cell-free DNA (cfDNA) from ascites effusions have not yet been studied. Herein, we analysis genomic alterations present in advanced cancer with ascites. Methods: We implemented targeted capture NGS with a panel of 1021 cancer-relevant genes on 85 ascites ctDNA, also on 46 matched plasma and 9 tissue from 85 patients. Results: Among 85 patients with advanced cancer, 42 digest system cancer (DC), 16 gynecologic cancer (GC), 14 non-small cell lung cancer(NSCLC) and 13 other solid tumors included. Overall, 90%(77/85) patients detected somatic mutations, which were identified predominantly in TP53 and KRAS for DC and TP53 for GC. Among NSCLC patients with ascites, nine were treated with EGFR TKI-targeted therapy previously. Mutations lead to the resistance of EGFR-TKI were identified in 55.6% (5/9) cases, including acquired EGFR T790M/C797S and primary BRAF V600E. These indicate ctDNA in ascites can used to guide tumor treatment and monitor of resistance. For the 46 pairs of ascites and plasma samples, actionable variations with on label, off-label, or experimental drugs available, were identified in 61% (n = 28) of ascites effusion samples, whereas plasma was 46% (n = 21). Median number of ascites somatic mutations was 4, while the median number was 3 in plasma samples. Of that, 85% and 61% of plasma ctDNA and ascites cfDNA samples were observed with the highest MAF above 1%. In addition, 63% and 35% of plasma ctDNA and pleural cfDNA samples were observed with the highest MAF above 10%. For the 9 groups of plasma, ascites and tumor tissue samples, at least 1 mutations within tissue was detected in 8 ascites samples and 6 in plasma samples. Conclusions: Our results suggest that cfDNA from ascites is more sensitive than plasma and had higher similarity with the tumor tissues for advanced cancer patients. With higher MAF and detection rate of actionable variations, cfDNA from ascites might provide additional information not detected with plasma cell-free DNA guide more precise cancer treatment.

Quantification of cell-free DNA in normal and complicated pregnancies: overcoming biological and technical issues.

The characterization of cell-free DNA (cfDNA) originating from placental trophoblast in maternal plasma provides a powerful tool for non-invasive diagnosis of fetal and obstetrical complications. Due to its placental origin, the specific epigenetic features of this DNA (commonly known as cell-free fetal DNA) can be utilized in creating universal ‘fetal’ markers in maternal plasma, thus overcoming the limitations of gender- or rhesus-specific ones. The goal of this study was to compare the performance of relevant approaches and assays evaluating the amount of cfDNA in maternal plasma throughout gestation (7.2–39.5 weeks). Two fetal- or placental- specific duplex assays (RPP30/SRY and RASSF1A/β-Actin) were applied using two technologies, real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR). Both methods revealed similar performance parameters within the studied dynamic range. Data obtained using qPCR and ddPCR for these assays were positively correlated (total cfDNA (RPP30): R = 0.57, p = 0.001/placental cfDNA (SRY): R = 0.85, p<0.0001; placental cfDNA (RASSF1A): R = 0.75, p<0.0001). There was a significant correlation in SRY and RASSF1A results measured with qPCR (R = 0.68, p = 0.013) and ddPCR (R = 0.56, p = 0.039). Different approaches also gave comparable results with regard to the correlation of the placental cfDNA concentration with gestational age and pathological outcome. We conclude that ddPCR is a practical approach, adaptable to existing qPCR assays and well suited for analysis of cell-free DNA in plasma. However, it may need further optimization to surpass the performance of qPCR.

Prognostic utility and characterization of cell-free DNA in patients with severe sepsis

IntroductionAlthough sepsis is the leading cause of death in noncoronary critically ill patients, identification of patients at high risk of death remains a challenge. In this study, we examined the incremental usefulness of adding multiple biomarkers to clinical scoring systems for predicting intensive care unit (ICU) mortality in patients with severe sepsis.MethodsThis retrospective observational study used stored plasma samples obtained from 80 severe sepsis patients recruited at three tertiary hospital ICUs in Hamilton, Ontario, Canada. Clinical data and plasma samples were obtained at study inclusion for all 80 patients, and then daily for 1 week, and weekly thereafter for a subset of 50 patients. Plasma levels of cell-free DNA (cfDNA), interleukin 6 (IL-6), thrombin, and protein C were measured and compared with clinical characteristics, including the primary outcome of ICU mortality and morbidity measured with the Multiple Organ Dysfunction (MODS) score and Acute Physiology and Chronic Health Evaluation (APACHE) II scores.ResultsThe level of cfDNA in plasma at study inclusion had better prognostic utility than did MODS or APACHE II scores, or the biomarkers measured. The area under the receiver operating characteristic (ROC) curves for cfDNA to predict ICU mortality is 0.97 (95% CI, 0.93 to 1.00) and to predict hospital mortality is 0.84 (95% CI, 0.75 to 0.94). We found that a cfDNA cutoff value of 2.35 ng/μl had a sensitivity of 87.9% and specificity of 93.5% for predicting ICU mortality. Sequential measurements of cfDNA suggested that ICU mortality may be predicted within 24 hours of study inclusion, and that the predictive power of cfDNA may be enhanced by combining it with protein C levels or MODS scores. DNA-sequence analyses and studies with Toll-like receptor 9 (TLR9) reporter cells suggests that the cfDNA from sepsis patients is host derived.ConclusionsThese studies suggest that cfDNA provides high prognostic accuracy in patients with severe sepsis. The serial data suggest that the combination of cfDNA with protein C and MODS scores may yield even stronger predictive power. Incorporation of cfDNA in sepsis risk-stratification systems may be valuable for clinical decision making or for inclusion into sepsis trials.

Quick recovery and characterization of cell-free DNA in seminal plasma of normozoospermia and azoospermia: implications for non-invasive genetic utilities

We established a quick and reliable method for recovering cell-free seminal DNA (cfsDNA), by using the binding-washing-elution procedure on the DNA purification column. Low variations (below 15%) among the triplicate values of cfsDNA quantity verified the reproducibility of our cfsDNA recovery method. Similar cfsDNA yield and size distribution between seminal plasma acquired by filtration and centrifugation confirmed the presence of cfsDNA. To investigate the general characterization of cfsDNA, the quantitation and size distribution of cfsDNA from normozoospermic and azoospermic semen were analyzed by real-time PCR and electrophoresis, respectively. CfsDNA concentration in semen with normozoospermia (n = 11) was 1.34 +/- 0.65 microg mL(-1), whereas a higher cfsDNA concentration was observed in azoospermia (2.56 +/- 1.43 microg mL(-1), n = 9). The continuous distribution of DNA fragments ranging from approximately 1 kb to 15 kb and a spectrum of multiples of 180-bp fragments were observed in each normozoospermic and azoospermic sample. Distinct characteristic DNA ladder fragmentations in some azoospermic samples implicated that cfsDNA originate partly from apoptotic cells. CfsDNAs of 36 selected azoospermic patients with known information of Y chromosome microdeletion were subjected to the same microdeletion analysis by multiplex PCR and PCR amplification of sY114 (1450 bp). All multiplex PCR reactions with cfsDNA amplified successfully and provided the same result as leukocyte DNA. PCR amplification of sY114 gave a 1450-bp amplicon as expected. Our data suggested the potential use of cfsDNA in search of biomarker or diagnostic procedures.