cfDNA Content Research Articles

IMMU-20. CIRCULATING CELL-FREE DNA DIRECTLY INHIBITS T CELL RESPONSES AND CONTRIBUTES TO SYSTEMIC IMMUNOSUPPRESSION IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is an aggressive form of brain cancer with poor survival despite standard of care. Checkpoint inhibitors aimed to revive dysfunctional T cells have significantly improved outcomes in other cancers, but have failed GBM. GBM patients have severe peripheral immunosuppression, including severe lymphopenia, immune organ atrophy, and defective T cell responses. This immunosuppression is a critical barrier to patient survival and the success of immunotherapies. Using parabiosis, we demonstrated that serum-derived factors were sufficient to drive hallmark features of immunosuppression, including inhibition of ex vivo T cell proliferation. This factor was nonsteroidal in nature and had molecular weights greater than 100kDa. Mass-spectrometry and pathway analysis of the suppressive serum implicated proteins involved in DNA response elements, cell death, and DNA-histone complexes. We hypothesized that the factor is cell-free DNA (cfDNA). Confirming this, serum levels of cfDNA are increased in glioma-bearing mice. Moreover, exposure to cfDNA from serum of glioma-bearing mice was sufficient to directly and potently inhibit T cell proliferation ex vivo. Interestingly, exposure to genomic DNA was not sufficient to induce proliferation defects in T cells, highlighting the unique features of cfDNA. Importantly, reducing cfDNA content using DNaseI partially restored proliferation capacity. We determined that the T cell proliferation defects were independent of DNA sensing mechanisms through AIM2, implicating a novel pathway of DNA sensing in T cells. Finally, to determine the origin of the cfDNA, we employed methyl-seq. The chromatin signature most closely represented neutrophils. This highlights a potential role for neutrophils-derived cfDNA as a targetable strategy in GBM. Together, we contend that cfDNA induces immunosuppressive effects in GBM and devising strategies to reduce circulating cfDNA could be a therapeutic approach to improve T cell functions and outcomes in GBM patients.

Quantitative Detection and Integrality Analysis of Plasma Circulating Cell-Free DNA in Multiple Myeloma

To investigate the role of plasma circulating cell-free DNA (cf-DNA) in the screening and diagnosis of patients with newly diagnosed multiple myeloma (MM) and explore the changes of cf-DNA in terms of content and integrality in the assessment of disease in patients treated with chemotherapy. Peripheral blood specimens were collected from 35 newly diagnosed MM patients and 18 healthy volunteers, and 13 of the 35 patients who had finished 3 courses of standard induction chemotherapy were selected as follow-up group. The ALU247 and ALU115 fragments were used as the target genes, and the cf-DNA content in the plasma of patients and healthy controls was measured by quantitative polymerase chain reaction (qPCR). The integrality of cf-DNA was calculated by the ratio of ALU247 to ALU115. Both the concentration of ALU247 and ALU115 fragments and the integrality of cf-DNA in patients were significantly higher than those in healthy controls (all P < 0.05). Patients who had underwent 3 courses of induction chemotherapy had significantly decreased concentration of ALU247 and ALU115 fragments and integrality of cf-DNA after treatment (all P < 0.05), and strong positive correlations were manifested between cf-DNA integrality and serum M protein content, as well as proportion of abnormal plasma cells in bone marrow before and after treatment (r =0.703, 0.705). Cf-DNA has certain positive significance for the screening and diagnosis of MM. Furthermore, cf-DNA may be a synergism or alternative to serum M-protein content and proportion of abnormal plasma cells in bone marrow in assessing the condition, curative effect and prognosis of patients.

Optimization and verification of conditions for induction of neutrophil extracellular traps in vitro

This study aims to optimize the conditions for the formation of neutrophil extracellular traps(NETs) in vitro, so as to establish a relatively stable experimental research platform. Different conditions were compared, including commonly used laboratory animals(rats and mice) and a variety of cell sources(bone marrow neutrophils and peripheral blood neutrophils separated by percoll density gradient centrifugation). Different inducers like lipopolysaccharide(LPS) and phorbol 12-myristate 13-acetate(PMA) were used for induction in vitro. Myeloperoxidase(MPO)/citrullinated histone H3(CitH3)/DAPI immunofluorescence and cell free DNA(cf-DNA) content determination were used for comprehensive evaluation to screen the optimal conditions for the formation of NETs induced in vitro. Furthermore, the stability of the selected conditions for inducing the formation of NETs in vitro was evaluated by tetramethylpyrazine(TMP), an active component in Chinese herbal medicines. The results showed that coated poly-D-lysine(PDL) induced the formation of NETs in bone marrow neutrophils of mice to a certain extent. Both LPS and PMA significantly up-regulated the protein levels of MPO and CitH3 in mouse bone marrow neutrophils and elevated the cfDNA level in the supernatant of rat peripheral blood neutrophils. The cfDNA level in the PMA-induced group increased more significantly than that in the LPS-induced group(P&lt;0.05). The results of immunofluorescence staining showed that the expression of MPO and CitH3 in mouse bone marrow neutrophils, rat bone marrow neutrophils, and rat peripheral blood neutrophils were significantly increased after PMA induction, especially in rat peripheral blood neutrophils. TMP significantly down-regulated the protein levels of MPO, CitH3, and neutrophil elastase(NE) in rat peripheral blood neutrophils induced by PMA. In conclusion, treating the peripheral blood neutrophils of rats with PMA is the optimal condition for inducing the formation of NETs in vitro. This study provides an optimal platform for in vitro studies based on NETs and a basis for studying the effects of traditional Chinese medicines targeting NETs.

Abstract 5040: Evaluation of commercially available cell free DNA extraction kits using biosynthetic reference materials

Abstract Extraction of cell free DNA (cfDNA) from a patient’s blood sample is a crucial preanalytic step of the liquid biopsy process. Whether testing is being performed for early disease diagnosis, therapy selection, or disease and treatment surveillance, the input material must be of sufficient quantity as well as high quality to return informative data. In addition, accurate capture of the full cfDNA content from a sample is necessary to evaluate tumor growth or recurrence. Fragmentomics is an emerging field that can indicate tumor origin among other information, and again, accurate and precise cfDNA content is critical. The cfDNA content in patient plasma is highly variable, and sample mass is quite limited compared to that of resected tumors. Healthy donor plasma, though abundant, varies in cfDNA levels batch to batch and should not contain any somatic variants relevant to liquid biopsy assay development or validation. Despite the challenges associated with cfDNA isolation and ctDNA analysis there are few reference materials available to optimize laboratory process for these preanalytic challenges. Here we evaluate various commercially available extraction kits using novel Seraseq® cfDNA Extraction Reference Materials. Genomic DNA from the GM24385 cell line was blended with 4 biosynthetic DNA EGFR variants targeted at 1% allele frequency and verified using the Bio-Rad QX-200 Droplet Digital PCR (ddPCR) System. The mixed DNA was fragmented and size selected to create ctDNA-like size profiles. The ctDNA was mixed into a synthetic plasma matrix to simulate native cfDNA samples and diluted to three distinct concentrations (20, 50, and 80 ng/mL). Verification of concentration was performed using the QIAGEN QIAamp Circulating Nucleic Acid Kit for extraction. The final materials were also extracted with other kits: the Maxwell RSC ccfDNA Plasma Kit, the MagMAX Cell-Free DNA Isolation kit, the Zymo MAGicBead™ cfDNA Isolation Kit, and the Takara NucleoSnap cfDNA Kit. Yields were measured with Qubit dsDNA BR assay and EGFR variants were measured using Bio-Rad ddPCR. Other sites evaluated the materials as well, using various kits. The kits showed variable yields, but there was a clear difference between the cfDNA extracted from each reference material correlating with the intended increasing concentration. The EGFR variants were all present at ~1% regardless of the cfDNA concentration and the kit used for extraction. Seraseq ctDNA Plasma Reference Materials serve to evaluate extraction of cfDNA in liquid biopsy testing workflows without having to source donor plasma, which is limited and variable in cfDNA content. The consistency and availability of these biosynthetic materials allow for optimization without risking patient samples. Citation Format: Dana Ruminski Lowe, Sanchita Jamindar, Andrew Anfora, David Merriam, Catherine Huang, Russell Garlick, Bharathi Anekella. Evaluation of commercially available cell free DNA extraction kits using biosynthetic reference materials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5040.

Abstract 5042: Urine as a non-invasive proxy for plasma in prostate cancer-related liquid biopsy applications

Abstract Introduction: The non-invasive collection of urine renders it an attractive and pragmatic substitute for plasma in liquid biopsy applications, fostering the creation of diagnostic methods that are more patient-friendly and less cumbersome for clinicians. This study investigates the feasibility of urine-derived cfDNA as a non-invasive option for acquiring vital diagnostic insights, further enhancing the liquid biopsy field. Methods: Paired male first void urine (FVU) and venous blood samples were collected from healthy donors (n=40) using Colli-Pee UAS devices (Novosanis) and BD K2EDTA Vacutainer tubes, respectively. Out of these, 20 paired urine and blood samples were spiked with 10 ng of cfDNA reference standard containing the KRAS p.G12V mutation. ~35 ml of urinary cell-free supernatant and ~3.5 ml of plasma were obtained after centrifugation of FVU and blood sample, respectively, and used as input material for cell-free nucleic acids extraction using the nRichDX Revolution Max20 cfDNA Isolation Kit. The extracted cfDNAs profile was assessed on 4200 TapeStation System, Agilent, using cfDNA ScreenTape. Extracted nucleic acids from urine samples were subjected to a qPCR assay to quantify endogenous human cfDNA content using 2X iTaq Universal SYBR Mastermix (Bio-Rad). Human cfDNA quantity was assessed using Ct values obtained from the qPCR assay. The actionable cfDNA molecules recovery was determined by qPCR mutation detection assay using TaqMan Genotyping. Results: Male FVU collected in Colli-Pee UAS device showed the presence of 100-250 bp cell-free DNA fragments similar to plasma obtained from whole blood collected in 10 ml vacutainer tubes as demonstrated by the Agilent cfDNA ScreenTape analysis. The cfDNA yield was similar when comparing one tube of blood (~3.5 mL of plasma) to one Colli-Pee of urine (~35 mL) shown by endogenous cfDNA qPCR assay. Additionally, the amplifiability of the KRAS G12V mutation was consistent in both plasma and urine samples, with comparable Ct values. Conclusion: The nRichDX Revolution Sample Prep System demonstrates the ability to extract cfDNA from both plasma and urine samples, with comparable extraction efficiency observed between matched samples from the same donor. This study provides evidence supporting the viability of first void urine samples as a non-invasive alternative for prostate cancer-related liquid biopsy applications. It indicates that cfDNA can be recovered from a sample collected with a single BCT or Colli-Pee UAS urine collection device. Future investigations will be required to assess clinical samples and biomarkers to establish a robust correlation between cfDNA levels and various cancer types and stages. This study reinforces the utility of first void urine samples as a reliable proxy for blood-based liquid biopsy applications. Citation Format: Nafiseh Jafari, Jason Saenz, Lauren Lee, Carlos Hernandez, Andrew Dunnigan, Amit Arora, Rafal Iwasiow, Mayer Saidian. Urine as a non-invasive proxy for plasma in prostate cancer-related liquid biopsy applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5042.

Survival outcomes correlate with the level of cell-free circulating DNA in ST-elevation myocardial infarction.

Myocardial infarction (MI) can lead to higher cellular damage, making cell-free DNA (cfDNA) a potential biomarker for assessing disease severity. The aim of this study is to evaluate survival predictions using cfDNA measurements and assess its correlation with MI. A direct fluorescence assay was employed to measure cfDNA content in the blood samples of participants. The inclusion criteria included patients who gave informed consent, suffering from ST-elevation myocardial infraction (STEMI) based on established diagnostic criteria (joint ESC/ACC guidelines), between the age of 18 and 80 years old, and had elevated troponin biomarker levels. The study included 150 patients diagnosed with STEMI and 50 healthy volunteers as controls. Serial monitoring of patients was conducted to track their postdisease status. The rate of change of cfDNA was calculated and daily measurements for 7 days were recorded. Mean levels of cfDNA were found to be 5.93 times higher in patients with STEMI compared to healthy controls, providing clear evidence of a clinical correlation between cfDNA and STEMI. Patients were further categorized based on their survival status within a 90-day period. The study observed a strong predictive relationship between the rate of change of cfDNA during daily measurements and survival outcomes. To assess its predictive capability, a receiver operating characteristics (ROC) curve analysis was performed. The ROC analysis identified an optimal cutoff value of 2.50 for cfDNA, with a sensitivity of 81.5% and specificity of 74.0% in predicting disease outcomes. This study demonstrates a robust association between cfDNA and STEMI, indicating that cfDNA levels can be a valuable early prognostic factor for patients. Serial measurements of cfDNA during early disease onset hold promise as an effective approach for predicting survival outcomes in MI patients.

Extracellular vesicles as a potential source of tumor-derived DNA in advanced pancreatic cancer.

Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated EVs as a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. evDNA from both DNase-treated and untreated EV samples was analyzed to determine whether the DNA was primarily located internally or outside (surface-bound) the EVs. To assess whether methodology affected the results, we isolated EVs using four different methods for small EV isolation and differential centrifugation for isolating large EVs. Our results indicated that the DNA content of EVs was significantly less than the cfDNA content isolated from the same plasma volume (p < 0.001). Most of the detected evDNA was also located on the outside of the vesicles. Furthermore, the fraction of tumor-derived DNA in EVs was similar to that found in cfDNA. In conclusion, our results suggest that quantification of evDNA, as a source of tumor-derived DNA, does not add information to that obtained with cfDNA, at least not in patients with advanced pancreatic cancer.

Clinical Value of cfDNA Content in Peripheral Blood of Patients with Triple-Negative Breast Cancer

Objective: To explore the value of circulating free (cfDNA) content in the clinical diagnosis and treatment of triple-negative breast cancer (TNBC). Methods: A total of 39 TNBC patients, 45 non-TNBC patients, and 50 healthy individuals admitted to the Baoding First Central Hospital during 2019–2022 were recruited. The clinical data, peripheral blood cfDNA concentration, and clinicopathological indicators of the patients were observed and analyzed. Results: The difference in clinical indicators such as age, age range, tumor size, clinical stage, and lymph node metastasis between patients with TNBC and non-TNBC was insignificant (P &gt; 0.05). The cfDNA concentrations (ng/mL) of the TNBC group, non-TNBC group, and healthy group were 24.12 ± 4.98, 15.36 ± 4.12, and 3.12 ± 1.02, respectively, and they are statistically different (P &lt; 0.05). The difference in cfDNA concentration was insignificant between TNBC patients with tumors ≤ 2 cm and &gt; 2 cm (P &gt; 0.05) but was significant between TNBC patients with clinical stages I+II and III+IV (P &lt; 0.05). The cfDNA concentration in TNBC patients with lymph node metastasis was significantly higher than those without lymph node metastasis (P &lt; 0.05). Conclusion: cfDNA has an important application value in the diagnosis and treatment of breast cancer. By detecting the cfDNA level and its gene variation, valuable information about the progress and treatment effects of breast cancer can be obtained. This non-invasive detection method has a wide range of applications and can be used for early screening, auxiliary diagnosis, efficacy evaluation, and recurrence monitoring of breast cancer.

Abstract A053: Epigenomic analyses of circulating tumor DNA in patients with metastatic castration resistant prostate cancer (mCRPC) treated with immune checkpoint blockade and PARP inhibition

Abstract Background: Molecular characterization of mCRPC has revealed disruption of DNA damage repair genes that may improve sensitivity to PARP inhibitors such as olaparib (O), which in turn may further complement the activity of the PD-L1 immune checkpoint inhibitor, durvalumab (D), through mechanisms such as activation of the STING pathway. We hypothesized that (epi)genomic analyses of circulating tumor DNA (ctDNA) would identify mechanisms of treatment response in patients with mCRPC treated with O plus D (NCT02484404). Methods: Eligible patients for this study had mCRPC with prior treatment by abiraterone and/or enzalutamide. Study participants were administered 300 mg of O twice daily and 1500 mg of D every 4 weeks. We obtained blood samples in Streck tubes from 38 (out of 60) study participants. Plasma samples were acquired at baseline and at progression. Buffy coat was isolated from each sample. Genomic DNA and cell-free DNA (cfDNA) were separately isolated and assembled into sequencing libraries. Low-pass whole-genome sequencing (3-4 × coverage) was performed for somatic copy number and fragmentomic analyses. In a subset of samples with the greatest cfDNA content, chromatin immunoprecipitation sequencing (ChIP-seq) with antibodies against H3K4me2 and H3K4me3 was performed directly in additional aliquots of plasma. Results: Amongst the 38 patients with at least one cfDNA sample, 5 patients had a radiographic partial response (PR) per RECIST v1.1, 28 patients had stable disease (SD), and 6 patients had progressive disease (PD) during the first two months of treatment. At baseline, ctDNA content in plasma was significantly lower in patients who went on to have PR or SD versus PD (p=0.02). However, upon progression, ctDNA content in plasma was not significantly different between groups. Losses to PTEN and NKX3-1 were significantly enriched in ctDNA from patients with PD (p=0.041 and p=0.047, respectively). By contrast, biallelic combined losses to BRCA2, RB1, BRCA1 and TP53 were observed in all PRs at baseline but not in any patient with PD (p=0.048). In patients with SD, longitudinal fragmentomic analysis inferred that the activity of the mitogenic KLF4 transcription factor (TF) was the most increased upon progression, while activity of the protective HIF2A TF was the most decreased. However, the TF binding motifs predicted by H3K4me2 and H3K4me3 cfDNA ChIP-seq did not significantly change in patients with PD. Conclusions: Distinctive genomic and epigenomic profiles were observed in plasma from patients with responsive vs. progressive disease, and the profiles may predict responses to combination D+O therapy. Deeper whole-genome sequencing of these samples currently in progress will reveal the contribution of germline and somatic point mutations to clinical response. Ongoing efforts to estimate the phylogenetic architecture of patients’ tumors from these plasma samples will enable the inference of drug response phenotypes as a function of evolutionary potential and potentially identify new opportunities to target treatment-resistant disease. Citation Format: Anna Baj, Clara C. Y. Seo, Nicholas T. Terrigino, John R. Bright, S. Thomas Hennigan, Isaiah M. King, Shana Y. Trostel, William D. Figg, William L. Dahut, Jung-Min Lee, David Y. Takeda, Fatima Karzai, Adam G. Sowalsky. Epigenomic analyses of circulating tumor DNA in patients with metastatic castration resistant prostate cancer (mCRPC) treated with immune checkpoint blockade and PARP inhibition [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A053.

Comparison of cfDNA content in different cancer types with different extraction methods.

e15026 Background: ctDNA liquid biopsy technology has been widely used in the whole course of cancer management, from early screening and early diagnosis of tumors to tumor monitoring and medication guidance. However, the performances of ctDNA as a biomarker in these scenarios are different among different cancer types. One possible reason is that different types of cancer release different amounts of ctDNA or cfDNA, therefore, we explored the cfDNA levels in different cancer types in this study. Methods: We collected 15913 peripheral blood samples from 14686 cancer patients with different clinicopathologies, 256 patients with pulmonary nodules, and 971 healthy people. We isolated their plasma and extracted the cfDNA using QIAamp Circulating Nucleic Acid Kit, Apostle MiniMax cfDNA Extraction Kit or HaploX magnetic bead method cfDNA extraction kit. We calculated the average concentration of cfDNA (ng/ml) for different groups. Results: The number of samples of cervical cancer, liver cancer, glioma, colon cancer, prostate cancer, nasopharyngeal carcinoma, gallbladder carcinoma, melanoma, cholangiocarcinoma, colorectal cancer, breast cancer, gastric cancer, endometrial carcinoma, lung cancer, urothelial carcinoma, rectal cancer, ovarian cancer, healthy people, and pulmonary nodule patients are 93, 469, 113, 277, 139, 76, 89, 132, 171, 4010, 407, 547, 68, 7750, 46, 203, 96, 971 and 256, respectively. The cfDNA level of corresponding groups are 88.07, 79.51, 75.57, 63.58, 61.37, 59.09, 57.59, 57.16, 42.67, 34.73, 31.75, 31.37, 31.33, 29.84, 28.72, 28.69, 23.83, 17.82 and 12.54 ng/ml, respectively. The main groups are shown in the table below. Conclusions: The cfDNA concentrations of healthy people or pulmonary nodule patients were significantly lower than that of tumor patients, and there were also significant differences between different types of cancer. What's interesting, the cfDNA concentrations of lung cancer patients, pulmonary nodule patients and healthy people were quite different. What’s more, there were differences between Colon cancer and Rectal cancer patients. So, the cfDNA concentration maybe a good dimention to be used in early cancer screening and tracing of tumor types. [Table: see text]

Circulating cell free DNA and citrullinated histone H3 as useful biomarkers of NETosis in endometrial cancer

BackgroundCancer mortality is mainly caused by organ failure and thrombotic events. It has been demonstrated that NETosis, a chromatin release mechanism implemented by neutrophils, may contribute to these lethal systemic effects. Our aim was to investigate NETosis biomarkers in endometrial cancer (EC).MethodsThe experiments were conducted on 21 healthy subjects (HS) with no gynecological conditions, and on 63 EC patients. To assess the presence of NETosis features, IHC and IF was performed using antibodies against citrullinated histone H3 (citH3), neutrophil elastase (NE) and histone 2B. Serum levels of cell free DNA (cfDNA), cell free mitochondrial DNA (cfmtDNA) and citH3 were measured by qPCR using one microliter of deactivated serum, and by ELISA assay respectively. Fragmentation pattern of serum cfDNA was analyzed using the Agilent 2100 Bioanalyzer and High Sensitivity DNA Chips. Receiver operating characteristic (ROC) analysis was used to identify a cut off for cfDNA and cfmtDNA values able to discriminate between ECs and HSs. Correlation analysis and multiple correspondence analysis (MCA) between cfDNA, mtcfDNA, citH3 and blood parameters were used to identify the potential association among serum parameters in EC grades.ResultsWe demonstrated the presence of NETosis features in tissues from all EC grades. Serum cfDNA and cfmtDNA levels discriminate ECs from HSs and a direct correlation between citH3 and cfDNA content and an inverse correlation between cfmtDNA and citH3 in EC sera was observed, not detectable in HSs. MCA indicates cfDNA, cfmtDNA and citH3 as features associated to G1 and G2 grades. A correlation between increased levels of cfDNA, citH3 and inflammation features was found. Finally, serum nucleosomal cfDNA fragmentation pattern varies in EC sera and correlates with increased levels of cfDNA, citH3, lymphocytes and fibrinogen.ConclusionOur data highlight the occurrence of NETosis in EC and indicate serum cfDNA and citH3 as noninvasive biomarkers of tumor-induced systemic effects in endometrial cancer.

Bronchoalveolar Lavage Fluid (BALF) as a Compartment for Donor-Derived Cell-Free DNA (dd-cfDNA) Assessment in Lung Recipients

<h3>Purpose</h3> Circulating dd-cfDNA has been proposed as a biomarker to discriminate acute lung rejection. It is unknown whether BALF may represent a more sensitive compartment for dd-cfDNA analysis in lung recipients. Additionally, the relative contribution of the recipient or donor to the BALF cfDNA content remains uncertain. We sought to understand the feasibility of distinguishing dd-cfDNA in the BALF and determine the greatest contributor of BALF cfDNA. <h3>Methods</h3> The cohort was drawn from the Clinical Trials in Organ Transplantation-20 study and comprised 126 first adult lung recipients with paired BALF and plasma samples (n=320 each). Total cfDNA was isolated from ∼1mL of sample using automated nucleic acid purification. The % cfDNA contributed by the donor vs. recipient was distinguished in plasma and BALF samples using a next generation sequencing (NGS) assay that measures single nucleotide polymorphisms (SNPs) followed by bioinformatics algorithms. Because these algorithms were developed on plasma, for each BALF sample the SNP typing from the paired plasma sample was used to verify the accuracy of the donor vs. recipient cfDNA contribution in the BALF. <h3>Results</h3> Applying standard QC filters, 168 BALF samples "passed" and generated a dd-cfDNA result while 96 "failed" and did not generate a dd-cfDNA result. 45 samples yielded insufficient total cfDNA to support NGS while 11 generated a potentially invalid dd-cfDNA result. To understand reasons underlying inability to estimate BALF dd-cfDNA, we compared the volume of BALF used for total cfDNA isolation and the total cfDNA yield from the BALF in passed vs. failed samples. The BALF volume was similar between these groups, however the total cfDNA yield was notably less among samples that failed (median total cfDNA 0.38ng/uL in failed vs. 1.42ng/uL in passed samples). Considering only passed BALF samples, the median (25^th, 75^th percentile) fraction of cfDNA contributed by the donor was 6.95% (2.40%, 14.00%), with the remainder contributed by the recipient. <h3>Conclusion</h3> Our data point to challenges in consistently generating dd-cfDNA data from the BALF and suggest the dilute nature of BALF may be a contributing factor. Additionally, these results indicate that the recipient, rather than the donor, is the largest contributor to the BALF cfDNA content potentially reflecting intragraft recipient immune cells.

Single-step measurement of cell-free DNA for sepsis prognosis using a thread-based microfluidic device.

Cell-free DNA (cfDNA) content in plasma has been studied as a biomarker for sepsis. Recent publications show that the cfDNA content in sepsis patients entering intensive care unit who were likely to survive had a total cfDNA concentration of 1.16 ± 0.13μg/mL compared to 4.65 ± 0.48μg/mL of non-survivors. Current methods for measuring cfDNA content in plasma were designed to amplify and measure low concentrations of specific DNA, making them unsuitable for low-cost measurement of total cfDNA content in plasma. Here, we have developed a point of care (POC) device that uses a thread silicone device as a medium to store a fluorescent dye which eliminates the need for preparatory steps, external aliquoting and dispensing of reagents, preconcentration, and external mixing while reducing the detection cost. The device was paired with a portable imaging system with an excitation filter at 472 ± 10nm and an emission filter of 520 ± 10nm that can be operated with just 100mA current supply. The device was demonstrated for use in the quantification of buffered cfDNA samples in a range 1-6μg/mL with a sensitivity of 5.72 AU/μg/mL and with cfDNA spiked in plasma with a range of 1-3μg/mL and a sensitivity of 5.43 AU/μg/mL. The results showed that the device could be used as a low-cost, rapid, and portable POC device for differentiating between survivors and non-survivors of sepsis within 20min.

Quantitative analysis of plasma DNA in anal cancer patients.

IntroductionThe availability and non-invasiveness of circulating cell-free DNA (cfDNA) opens up new possibilities for real-time serial testing. The relationship between cfDNA concentration, clinical factors and suitability for monitoring was analyzed in patients with newly diagnosed anal squamous cell carcinoma (ASCC).Material and methodsBlood samples were collected at several points during and after treatment. Patients were homogeneously treated with chemoradiotherapy.ResultsThe concentration of cfDNA strongly correlated with the tumor volume (r = 0.9, p = 0.00006) and number of neutrophils (r = 0.706, p = 0.0069). Monitoring of cfDNA levels during treatment showed an increase after initiation of therapy, a peak at the end of treatment with significantly higher values for advanced than in T1/T2 tumors, and a decrease (T3/T4) during follow-up. However, neither the concentration of cfDNA before treatment nor its changes correlated with the response to chemoradiotherapy. There was no association between baseline cfDNA levels and T, N, age and gender.ConclusionsSubstantial changes in plasma cfDNA content can be observed after chemoradiotherapy for ASCC. However, the small number of cases studied makes it difficult to assess the usefulness of the cfDNA test.

Cell-Free DNA Variant Sequencing Using Plasma and AR-V7 Testing of Circulating Tumor Cells in Prostate Cancer Patients

Prostate cancer (PCa) is the second most common malignant cancer and is a major cause of morbidity and mortality among men worldwide. There is still an urgent need for biomarkers applicable for diagnosis, prognosis, therapy prediction, or therapy monitoring in PCa. Liquid biopsies, including cell-free DNA (cfDNA) and circulating tumor cells (CTCs), are a valuable source for studying such biomarkers and are minimally invasive. In our study, we investigated the cfDNA of 34 progressive PCa patients, via targeted sequencing, for sequence variants and for the occurrence of CTCs, with a focus on androgen receptor splice variant 7 (AR-V7)-positive CTCs. The cfDNA content was associated with overall survival (OS; p = 0.014), disease-specific survival (DSS; p = 0.004), and time to treatment change (TTC; p = 0.001). Moreover, when considering all sequence variants grouped by their functional impact and allele frequency, a significant association with TTC (p = 0.017) was observed. When investigating only pathogenic or likely pathogenic gene variants, variants of the BRCA1 gene (p = 0.029) and the AR ligand-binding domain (p = 0.050) were associated with a shorter TTC. Likewise, the presence of CTCs was associated with a shorter TTC (p = 0.031). The presence of AR-V7-positive CTCs was associated with TTC (p < 0.001) in Kaplan–Meier analysis. Interestingly, all patients with AR-V7-positive CTCs also carried TP53 point mutations. Altogether, analysis of cfDNA and CTCs can provide complementary information that may support temporal and targeted treatment decisions and may elucidate the optimal choice within the variety of therapy options for advanced PCa patients.

THE EFFECT OF EMBRYO MORPHOLOGY AND CULTURING METHODS ON THE PERFORMANCE OF NON-INVASIVE PREIMPLANTATION GENETIC TESTING FOR ANEUPLOIDIES

The objective of this study was to evaluate the impact of common embryological culturing methods and blastocyst morphology on the cell-free DNA (cfDNA) content of spent media and the performance of non-invasive preimplantation genetic testing for aneuploidies (niPGT-A).

Comparative analysis of nuclear and mitochondrial DNA from tissue and liquid biopsies of colorectal cancer patients

The current standard for molecular profiling of colorectal cancer (CRC) is using resected or biopsied tissue specimens. However, they are limited regarding sampling frequency, representation of tumor heterogeneity, and sampling can expose patients to adverse side effects. The analysis of cell-free DNA (cfDNA) from blood plasma, which is part of a liquid biopsy, is minimally invasive and in principle enables detection of all tumor-specific mutations. Here, we analyzed cfDNA originating from nucleus and mitochondria and investigated their characteristics and mutation status in a cohort of 18 CRC patients and 10 healthy controls using targeted next-generation sequencing (NGS) and digital PCR. Longitudinal analyses of nuclear cfDNA level and size during chemotherapy revealed a decreasing cfDNA content and a shift from short to long fragments, indicating an appropriate therapy response, while shortened cfDNAs and increased cfDNA content corresponded with tumor recurrence. Comparative NGS analysis of nuclear tissue and plasma DNA demonstrated a good patient-level concordance and cfDNA revealed additional variants in three of the cases. Analysis of mitochondrial cfDNA surprisingly revealed a higher plasma copy number in healthy subjects than in CRC patients. These results highlight the potential clinical utility of liquid biopsies in routine diagnostics and surveillance of CRC patients as complementation to tissue biopsies or as an attractive alternative in cases where tissue biopsies are risky or the quantity/quality does not allow testing.

The Potential Use of Dynamics Changes of ctDNA and cfDNA in the Perioperative Period to Predict the Recurrence Risk in Early NSCLC.

BackgroundPostoperative circulation tumor DNA (ctDNA) is a promising method to predict the risk of recurrence. However, the amount of ctDNA in patients with early NSCLC is too small. Cell damages caused during the intraoperative period leads to a significant increase in cell free DNA (cfDNA). Whether cfDNA content is restored to the preoperative level within a short time after surgery may indicate the degree of surgical trauma. In this study, dynamic changes of cfDNA combined with ctDNA in the perioperative period of NSCLC were used to explore the possibility of them as a biomarker to indicate the risk of recurrence.MethodsNSCLC patients who planned to undergo radical resection were investigated. 10ml of peripheral blood was collected before, during and 7 days after surgery. DNA concentration was measured, and a 23-gene NGS panel was performed to detect gene mutations. All the patients would be followed-up for at least 18 months.ResultsA total of 7 patients were sampled. The amount of cfDNA before surgery was 36.6 ± 14.7ng, and increased to 127.2 ± 52.2ng during surgery. 7 days after surgery, it dropped to 45.23 ± 9.41ng in 3 patients and rose to 173.7 ± 80.80ng in the remaining 4. Only 1 patient was ctDNA positive after surgery, with decreasing cfDNA, and he was the only one that relapsed and died within 18 months.ConclusionThe use of ctDNA to predict the risk of postoperative recurrence of NSCLC is a very valuable method, and it may be more reliable if combined with the dynamic changes of cfDNA. The amounts of cfDNA are raised by the operation, but will be polarized after surgery in 7 days. Postoperative NSCLC patients with positive ctDNA and reduced cfDNA have a higher risk of recurrence.

Circulating nuclear DNA structural features, origins, and complete size profile revealed by fragmentomics.

To unequivocally address their unresolved intimate structures in blood, we scrutinized the size distribution of circulating cell-free DNA (cfDNA) using whole-genome sequencing (WGS) from both double- and single-strand DNA library preparations (DSP and SSP, n = 7) and using quantitative PCR (Q-PCR, n = 116). The size profile in healthy individuals was remarkably homogenous when using DSP sequencing or SSP sequencing. CfDNA size profile had a characteristic nucleosome fragmentation pattern. Overall, our data indicate that the proportion of cfDNA inserted in mono-nucleosomes, di-nucleosomes, and chromatin of higher molecular size (>1000 bp) can be estimated as 67.5% to 80%, 9.4% to 11.5%, and 8.5% to 21.0%, respectively. Although DNA on single chromatosomes or mono-nucleosomes is detectable, our data revealed that cfDNA is highly nicked (97%–98%) on those structures, which appear to be subjected to continuous nuclease activity in the bloodstream. Fragments analysis allows the distinction of cfDNA of different origins: first, cfDNA size profile analysis may be useful in cfDNA extract quality control; second, subtle but reliable differences between metastatic colorectal cancer patients and healthy individuals vary with the proportion of malignant cell-derived cfDNA in plasma extracts, pointing to a higher degree of cfDNA fragmentation and nuclease activity in samples with high malignant cell cfDNA content.

The Combination of Neutrophil-Lymphocyte Ratio and Platelet-Lymphocyte Ratio with Liquid Biopsy Biomarkers Improves Prognosis Prediction in Metastatic Pancreatic Cancer.

Simple SummaryLiquid biopsy is a noninvasive approach that provides tumor molecular profiling. On the other hand, the vast majority of pancreatic tumors are pancreatic ductal adenocarcinomas (PDAC), which are characterized by pronounced inflammation. Therefore, we hypothesized that the combination of biomarkers of systemic inflammation, such as the neutrophil-to-lymphocyte-ratio (NLR) and platelet-to-lymphocyte ratio (PLR), with liquid biopsy-based biomarkers may increase their clinical usefulness. Our study shows that combining NLR, PLR, and the standard PDAC marker CA19-9 with circulating cell-free DNA and circulating RAS-mutated DNA outperforms traditional clinical tools for the clinical management of metastatic PDAC patients.Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a highly inflammatory microenvironment and liquid biopsy has emerged as a promising tool for the noninvasive analysis of this tumor. In this study, plasma was obtained from 58 metastatic PDAC patients, and neutrophil–lymphocyte ratio (NLR), platelet–lymphocyte ratio (PLR), circulating cell-free DNA (cfDNA) concentration, and circulating RAS mutation were determined. We found that NLR was significantly associated with both overall survival (OS) and progression-free survival. Remarkably, NLR was an independent risk factor for poor OS. Moreover, NLR and PLR positively correlated, and combination of both inflammatory markers significantly improved the prognostic stratification of metastatic PDAC patients. NLR also showed a positive correlation with cfDNA levels and RAS mutant allelic fraction (MAF). Besides, we found that neutrophil activation contributed to cfDNA content in the plasma of metastatic PDAC patients. Finally, a multi-parameter prognosis model was designed by combining NLR, PLR, cfDNA levels, RAS mutation, RAS MAF, and CA19-9, which performs as a promising tool to predict the prognosis of metastatic PDAC patients. In conclusion, our study supports the idea that the use of systemic inflammatory markers along with circulating tumor-specific markers may constitute a valuable tool for the clinical management of metastatic PDAC patients.