Free DNA Research Articles

Joint Analyses of Serum Proteins and Circulating Tumor DNA Improves Risk Assessment and Subtype Stratification in Large B-Cell Lymphomas

Introduction Inadequate dismantling of clinical and biological heterogeneity is an obstacle for improving outcome in large B-cell lymphoma (LBCL). Circulating tumor DNA (ctDNA)-based stratification holds great potential in resolving these challenges. However, the assessment is limited to genetic content, and excludes host response from the estimates. Previously, we have shown that an inflammatory signature identified from the pretreatment serum proteins translates to poor survival, and multiple serum proteins correlate with LBCL genetic subtypes. To further improve risk stratification and molecular characterization of LBCL from the liquid biopsy, we co-analyzed serum protein and ctDNA levels in LBCL patients treated in two Nordic Lymphoma Group (NLG) trials. Materials and methods Our discovery cohort consisted of 122 patients less than 65 years of age with clinically high-risk (age adjusted IPI ≥ 2) LBCL, who were treated in NLG-LBC-06 trial with immunochemotherapy. Plasma and serum samples were collected prior to treatment. Cell free DNA was extracted from plasma and sequenced with in-house adapted duplex sequencing platform using custom targeted panel of 347 lymphoma driver genes and immunoglobulin loci. An inflammation score was composed as log transformed mean of the most pertinent inflammatory serum proteins detected in the inflammatory signature (IL10, IL2RA, CXCL9, IL18, GZMB, PD-L1 and IFNG), and their levels were measured using antibody-based Luminex assay (pg/ml, R&D Systems). In addition, CCL17 and TNFRSF13B levels were analyzed to uncover their associations with genetic subtypes. An independent cohort of 91 patients treated in our previous NLG-LBC-05 trial was used for validation. Results Combined analysis of serum proteins and ctDNA identified a group of patients (n=12, 10%) with high serum inflammation scores and ctDNA burden (≥ 3.75 hGE/ml), which translated to poor survival compared to the patients with low concentrations of either inflammatory proteins or ctDNA, or both (progression free survival, PFS, hazard ratio 7.33, 95%-CI 2.59-20.79, P < 0.001, Figure 1A). This finding was confirmed in the independent validation cohort. In both cohorts, the combined analysis showed higher specificity in survival prediction as compared to the ctDNA-based assessment alone, indicating that inflammatory serum markers alongside with ctDNA can identify those patients who, despite of having high ctDNA burden, have excellent survival. Beyond inflammation, the levels of serum TNFRSF13B and CCL17 correlated with mutations associating with non-germinal center diffuse large B-cell lymphoma (non-GCB DLBCL)/MCD subtype and primary mediastinal B-cell lymphoma/grey-zone lymphoma (PMBCL/GZL), respectively. For example, serum TNFRSF13B levels correlated positively with mutations in MCD genes, such as CD79B, SETD1B, PIM2 and PRDM1, and negatively with mutations in EZB genes EZH2 and CREBBP in both cohorts (P < 0.05, Figure 1B). Supporting our earlier findings, high CCL17 levels revealed a group of patients diagnosed with DLBCL not otherwise specified or high-grade lymphoma but with a mutational landscape resembling PMBCL/GZL, including mutations in TNFAIP3, ITPKB, B2M, BTG1 and STAT6 (P < 0.05). Conclusion Our findings demonstrate that co-analysis of inflammatory serum markers along with the ctDNA improves ctDNA-based survival prediction. Moreover, analysis of serum proteins refines molecular classification. Altogether, joint analysis of serum proteins and ctDNA provides new tools to dismantle LBCL heterogeneity.

A Novel Approach: Nanopore Sequencing of Native Cell-Free DNA in Diffuse-Large B-Cell Lymphoma Patients

Introduction: Nanopore sequencing (NS), a third generation long-read sequencing technology, is increasingly being used to study cancer genomes. NS has many advantages over next-generation sequencing, including the capability to simultaneously extract base sequence as well as base modifications in the same run without any further library modification. Notably, its portability and ability to provide real-time data make it a potentially powerful tool for clinical applications. This study explores the use of NS to directly sequence native cell free DNA (cfDNA) from patients with diffuse-large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin lymphoma. Methods: For this exploratory study, 20 patients with either a new diagnosis or relapsed DLBCL were enrolled. We adapted NS methods for cfDNA and sequenced cfDNA before each chemotherapy cycle using both an Illumina platform (as a control) and the Nanopore platform. For differential methylation, we also sequenced plasma cfDNA from five healthy donors on the Nanopore. Results: Our findings demonstrate that we can directly sequence cfDNA on the Nanopore platform, obtaining a whole genome coverage (average 1.2x) with deeper coverage in some regions and also observe ~60% global DNA methylation level in CG context. From this data we are now analyzing regions that are differentially methylated (DMRs) between normal and DLBCL, determining SNV/Indel profiles, and comparing copy number variants (CNVs). These results will be available and presented at the time of the meeting. Conclusions: NS is capable of detecting cfDNA and measuring methylation levels, offering a promising and potentially clinically applicable new approach for the study of cfDNA. Future directions: As we continue this research, our vision is to leverage the portability and real-time data capabilities of NS for clinical applications. By simultaneously combining multiple genomic features with machine learning, we aim to develop a method for measuring lymphoma cfDNA-based MRD in DLBCL patients to be used in clinical settings.

Maternal and Fetal Factors Affecting Cell-Free Fetal DNA (cffDNA) Fraction: A Systematic Review.

Cell-free fetal DNA (cffDNA) is a novel screening method for fetal aneuploidy that facilitated non-invasive prenatal testing (NIPT) through analysis of cffDNA in maternal plasma. However, despite increased sensitivity, it has a number of limitations that may complicate of its results interpretation. Therefore, elucidating factors affecting fetal fraction, as a critical limitation, guides its clinical application. In this report, systematic search was carried out through PubMed, Web of Science, and Scopus databases until February 11, 2022 by using keywords consist of "noninvasive prenatal screening", "NIPT", "noninvasive prenatal", "cell free DNA" and "fetal fraction". The articles were screened for eligibility criteria before data extraction. A total of 39 eligible studies, most published between 2010 and 2020, were included. Based on the results of studies, a negative correlation between maternal age and BMI/body weight with fetal fraction was found. Furthermore, LDL, cholesterol, triglyceride level, metformin, heparin and enoxaparin therapy, hemoglobin-related hemoglobinopathies, and physical activity showed to have negative associations. Interestingly, it seems the ethnicity of patients from South and East Asia has a correlation with fetal fraction compared to Caucasians. Positive correlation was observed between gestational age, free β-hCG, PAPP-A, living in high altitude, and twin pregnancy. Considering each factor, there was significant inconsistency and controversy regarding their impact on outcomes. Indeed, multiple factors can influence the accuracy of NIPS results, and it is worth noting that the impact of these factors may vary depending on the individual's ethnic background. Therefore, it is important to recognize that NIPS remains a screening test, and comprehensive pre- and post-NIPS counseling should be conducted as part of standard clinical practice.

Plasma cell-free DNA in patients with acute promyelocytic leukaemia treated with arsenic trioxide.

Cell-free DNA (cfDNA) is free DNA found in circulating blood that originates from apoptosis or necrosis, and elevated cfDNA concentrations have been reported in cancers and other diseases. In this study, the concentrations and fragment distributions of plasma cfDNA were preliminary investigated in elderly (n = 1) and paediatric (n = 1) patients with acute promyelocytic leukaemia (APL) treated with arsenic trioxide (ATO). A slight increase in cfDNA concentrations was observed in the APL patients compared with healthy controls. The change in plasma cfDNA concentrations corresponded to the change in plasma arsenic concentrations during ATO treatment. The fragment distribution pattern did not differ before and during treatment. Three ladder fragments were observed in part of the cfDNA in the second consolidation therapy in an elderly APL patient and the first consolidation therapy of a paediatric APL patient, while two fragments were observed in all other treatment periods. Moreover, APL-related gene mutations were successfully genotyped from plasma cfDNA by using polymerase chain reaction-based methods and these results are consistent with those from leukocytes. This study is the first to report the concentrations and fragment patterns of cfDNA from APL patients treated with ATO. The results suggested that plasma cfDNA concentration in APL patients increased with ATO treatment and that cfDNA is released mainly via neutrophil extracellular traps (and/or necrosis) in addition to apoptosis. To confirm whether cfDNA concentrations and fragment patterns can be used as a biomarker for APL treated with ATO, further accumulative data are needed.

Redox-Based Defect Detection in Packed DNA: Insights from Hybrid Quantum Mechanical/Molecular Mechanics Molecular Dynamics Simulations.

The impact of an 8-oxoguanine (8oxoG) defect on the redox properties of DNA within the nucleosome core particle (NCP) was investigated employing hybrid quantum mechanical/molecular mechanics (QM/MM) molecular dynamics simulations of native and 8oxoG-containing NCP systems with an explicit representation of a biologically relevant environment. Two distinct NCP positions with varying solvent accessibility were considered for 8oxoG insertion. In both cases, it is found that the presence of 8oxoG drastically decreases the redox free energy of oxidation by roughly 1 eV, which is very similar to what was recently reported for free native and 8oxoG-containing DNA. In contrast, the effect of 8oxoG on the reorganization free energy is even smaller for packed DNA (decrease of 0.13 and 0.01 eV for defect-free and defect-containing systems, respectively) compared to the one for free DNA (0.25 eV), consistent with the increased rigidity of the NCP as compared to free DNA. Furthermore, the presence of an 8oxoG defect does not yield any significant changes in the packed DNA structure. Such a conclusion favors the idea that in the case of chromatin, defect-induced changes in DNA redox chemistry can also be exploited to detect damaged bases via DNA-mediated hole transfer.

BIOM-41. CITRULLINATED HISTONE H3 ELISA REVEALS NETOSIS AS A SOURCE OF PROGNOSTIC CIRCULATING CELL-FREE DNA IN GLIOBLASTOMA

Abstract BACKGROUND We have previously shown that higher concentrations of plasma cell free DNA (cfDNA), both at baseline and following chemoradiotherapy, are associated with worse survival in GBM. Because only scant amounts of GBM-derived cfDNA shed into the peripheral circulation, we sought to determine the predominant source of prognostic plasma cfDNA in these patients and hypothesized that this may help unravel the mechanism underlying the association of high cfDNA and survival. METHODS Plasma was obtained for 126 patients prior to resection of newly diagnosed GBM. A subset of 46 plasma samples had ccfDNA extracted, enzymatically converted, whole genome amplified, and analyzed on the Illumina Epic array for deconvolution of cfDNA methylation patterns. Based on these results demonstrating neutrophils as the predominant cfDNA source, we performed ELISA for citrullinated-histone 3 (citH3) in plasma from 113 of the patients as a specific marker of NETosis, a pro-tumor process of programmed neutrophil cell death during which DNA is released in web-like structures called neutrophil extracellular traps (NETs). RESULTS Deconvolution of cfDNA methylation patterns revealed that neutrophils were the dominant source of cfDNA (median percentage of cfDNA derived from neutrophils = 46% , range 17-67%) and correlated with baseline plasma cfDNA concentration (n = 46, r = 0.35, p = 0.017). Additionally, cfDNA concentration was significantly correlated with the concentration of citH3 (n = 113, r = 0.60, p < 0.0001), which was higher in GBM patients compared to 26 healthy controls (p < 0.0001). CONCLUSION Our findings suggest that NETosis may be the dominant source of plasma cfDNA, which we have previously shown to be highly prognostic in patients with GBM. Additional studies interrogating the tumor microenvironment for NETosis in GBM tumors are ongoing. These results lay the foundation for further exploration of NETosis as a therapeutic target and underscore the emerging importance of neutrophil biology in GBM.

IMMU-44. CIRCULATING CELL-FREE DNA DIRECTLY INHIBITS T CELL FUNCTION AND CONTRIBUTES TO PERIPHERAL IMMUNOSUPPRESSION IN EXPERIMENTAL GBM

Abstract Glioblastoma (GBM) is aggressive form of brain cancer that is associated with severe peripheral immunosuppression. This immunosuppression is a critical barrier to patient survival and the success of immunotherapies. Immunocompetent mice harboring intracranial gliomas present with the hallmark features of peripheral immunosuppression observed in GBM patients, including T cell lymphopenia. Using parabiosis, we determined that T cell lymphopenia can be transferred from glioma-bearing to healthy mice via circulation This led to the identification of a high molecular weight immunosuppressive factor in sera of glioma-bearing mice that inhibits T cell proliferation ex vivo. Proteomics analysis identified 119 proteins uniquely upregulated in the sera of glioma-bearing mice compared to controls. These proteins are associated with pathways involved in DNA response elements, cell death, and DNA-histone complexes which imply DNA release. Additional analysis revealed serum isolated from glioma-bearing mice harbor significantly higher concentrations of double stranded cell free DNA (cfDNA) compared to controls. Reducing DNA content using DNase partially reverses the proliferation defects observed in T cells when exposed to serum of glioma-bearing mice. This is while, addition of cfDNA purified from serum of glioma-bearing mice, but not genomic DNA, potently inhibited T cell function. These results were AIM2 independent implicating a novel pathway of cfDNA sensing in T cells. We contend that cfDNA induces immunosuppressive effects in experimental GBM. Devising strategies to reduce circulating cfDNA is therefore a therapeutic approach that could improve outcomes in GBM patients.

Prognostic potential of circulating cell free mitochondrial DNA levels in COVID-19 patients.

In viral infections, mitochondria act as one of the main hubs of the pathogenesis. Recent findings present new insights into the potential role of circulating cell-free mitochondrial DNA (ccf-mtDNA) in COVID-19 pathogenesis by the induction of immune response and aggressive cytokine storm in SARS-CoV-2 infection. The levels of ccf-mtDNA were investigated in 102 hospitalized patients with COVID-19 using the quantitative PCR (q-PCR) method. Statistical analysis confirmed a strong association between the levels of ccf-mtDNA and and mortality, ICU admission, and intubation. Also, our findings highlighted the pivotal role of comorbidities as a risk factor for COVID-19 mortality and severity. Higher levels of ccf-mtDNA can serve as a potential early indicator for progress and poor prognosis of COVID-19.

Analytical modeling and numerical simulation of graded JAM Split Gate-All-Around (GJAM-SGAA) Bio-FET for label free Avian Influenza antibody and DNA detection

This manuscript presents the analytical model of a novel biosensor called Graded JAM Split Gate-All-Around (GJAM-SGAA) Bio-FET for the detection of Avian Influenza antibody and DNA. The GJAM-SGAA Bio-FET utilizes a silicon Gate-All-Around FET which operates in the Junctionless Accumulation Mode (JAM), with a graded doping in the channel. This Bio-FET also features a gate underlap double sided cavity that enables detection of biomolecules without the use of labels. Gate underlap cavities overcome the fabrication complexity of nanocavities and provides structural stability. A comparative analysis between the GJAM-SGAA Bio-FET and non-graded JAM Split Gate-All-Around FET (SGAA-FET) demonstrates that the proposed BioFET has 5.72 times higher ION current sensitivity, 5.3 times increase in threshold voltage sensitivity (SVth), and 2.13 × 102 times improvement in switching ratio sensitivity for avian influenza biomolecule. The SVth of proposed biosensor is compared with the existing biosensors and found that a triple-metal engineered gate and graded doped channel substantially boosts the sensitivity of proposed biosensor.

Cell Free MYC DNA Copy Number Identifies High-Risk Smouldering Myeloma and Newly Diagnosed Multiple Myeloma

Introduction Smouldering multiple myeloma (SMM) is a heterogenous condition, with some patients progressing rapidly (high-risk) while others have MGUS-like disease (low-risk). The International Myeloma Working Group's (IMWG) 2/20/20 risk score predicts 2-year progression, but despite these improvements in risk-stratification, identification of patients requiring early intervention remains elusive. For newly diagnosed multiple myeloma (NDMM), survival varies, and current risk-stratification falls short for poor responders to standard first-line therapy (high-risk). MYC is a potent oncogene implicated in the initiation, maintenance, and progression of various malignancies. MYC overexpression on immunohistochemistry predicts poor clinical outcome in MM 1, implicating MYC overexpression in aggressive disease kinetics in MM. We hypothesised that MYC copy number could improve identification of high-risk SMM and ND MM. In this study, we developed a minimally invasive liquid biopsy approach to characterise the expression of MYC copy number in circulating cell-free DNA (cfDNA) on a unique cohort of peripheral blood samples obtained from the national liquid biopsy biobank, the M1000 that is linked to our clinical registry, the Myeloma and Related Diseases Registry (MRDR). Methods We obtained plasma samples from 64 SMM and 149 ND MM patients from the M1000. cfDNA was extracted from 1mL of plasma collected in Streck™ tubes using QIAamp Circulating Nucleic Acid Kit (QIAGEN) according to manufacturer instructions. Equal amount of cfDNA (5 uL), run in duplicates, from each patient was utilised for copy number assessment developed using the Biorad QX200 Digital Droplet TM PCR. The copy number of MYC was calculated by comparing the number of molecules (using a ratio of positive to negative droplets) to the number of molecules arising from the reference (RPP30) genomic locus using the QuantaSoft TM Analysis Pro Software (Biorad). MYC copy number were correlated with clinical data from MRDR on progression in SMM and overall survival (OS) in NDMM. We use median copy number as cut off to categorise high and low MYC. Results We were able to extract cfDNA from 1 ml of plasma samples from both SMM and NDMM patients, with the median amount being 35.11 ng (11.9 ng - 107.78ng) and 50.26 ng (5.85 ng - 243.45 ng), respectively. The median MYC copy number levels were 1.883 in SMM and 1.860 for NDMM. Within 36 months follow up in the SMM cohort, 18 patients progressed to MM with median time to progression of 14 months (2- 36 months, Figure 1). High MYC copy number (>1.9) at diagnosis was predictive of disease progression with hazard ratio of 5.27 (95% CI 1.99 -14; P = 0.0015). When combined with the 20/20/20 risk stratification model 3, MYC copy number improved the reliability of this score. In the NDMM cohort, median OS for the cohort was not reached at 36 months follow up. Patients with high MYC copy number (>1.9) at diagnosis had shorter OS at 36 months compared to those with low MYC copy number at 36 months (66.05% vs 82.02%. P=0.0012, Figure 2). Conclusion Our study demonstrates a significant association between MYC copy number, assessed via plasma cfDNA, and the risk of progression in patients with SMM. Additionally, in NDMM, MYC copy number at diagnosis directly correlates with OS. These findings require further investigation and validation in a larger cohort of M1000 samples, as they have the potential to introduce a minimally-invasive risk stratification methodology to identify high-risk patients and the introduction of MYC targeted therapies in patients with high MYC copy number.

Platelets Sequester Extracellular DNA, Capturing Tumour-Derived and Free Fetal DNA

Introduction & Hypothesis Platelets are small, multi-functional cells that lack a nucleus but contain RNA and translational machinery for protein synthesis. Platelet RNA mostly derives from parent megakaryocytes, but they also sense and sequester endogenous and pathogen-derived nucleic acids during circulation (D'Ambrosi, 2021; Koupenova, 2019). Nucleated cells release DNA after cell death or aberrant mitosis, resulting in ‘cell-free’ DNA in plasma (cfDNA). Excess cfDNA is deleterious. cfDNA isolated from platelet-poor plasma is emerging as a major liquid biopsy tool in cancer and antenatal screening, but a major limitation is its low abundance, especially in early-stage disease. Given their role in sensing pathogen-derived nucleic acids, we hypothesized that platelets may clear cfDNA from plasma, and that clinically-relevant insights may be derived from the analysis of DNA fragments contained in platelets. Methods Live/fixed imaging using specific DNA probes and fluorescence in-situ hybridization (FISH), droplet digital PCR (ddPCR), NGS and flow cytometry were applied to platelets isolated from Streck, EDTA tubes or apheresis bags from healthy donors, pregnant women, patients with cancer and murine platelets from cancer models. DNA was extracted from platelet pellets or platelet-depleted plasma (cfDNA) using the Qiagen QIAamp Circulating Nucleic Acid Kit. Results Using live cell imaging and flow cytometry, significant DNA content (Draq5+) was detected in ~8% of platelets from healthy donors, including in platelets with low RNA content (SYTO-13-negative). Overall Draq5+ platelets were larger and primed for agonist activation. FISH and ddPCR of platelets from pregnant women carrying male fetuses detected Y-chromosome fragments (n=10), confirming that platelet DNA is not solely derived from parent megakaryocytes but also sequestered during circulation (Fig. 1a). In contrast, Y-chromosome genes were not detected in DNA isolated from mononuclear cells or red blood cells pellets. Acute platelet depletion in an ITP mouse model (n=20) led to a >2-fold increase in cfDNA extracted from platelet-poor plasma, suggesting a role for platelets in plasma DNA clearance. Platelet uptake in vitro was rapid, visible using live cell microscopy within 2 minutes of co-culture with cancer cells labelled with a probe that irreversibly intercalates to nuclear DNA. ddPCR of platelet DNA following co-culture of healthy donor platelets with cancer cell lines detected a range of canonical cancer driver mutations, including in PI3K, BRAF and JAK2. DNase treatment of co-cultured platelets did not reduce mutant allele content, indicating that platelets encapsulate DNA and protect it from degradation. We explored the mechanism of platelet DNA uptake. Platelets internalized DNA contained in extracellular vesicles (EVs). Platelet uptake of “free” DNA was also confirmed, with uptake of synthetic DNA fragments of varying lengths (120 bp to 650 bp). Importantly, platelet DNA detection was enhanced following exocytosis inhibition, confirming that platelet DNA uptake and release is an active process. Whole genome sequencing revealed that platelets contain a repertoire of DNA fragments that map across the nuclear genome, similar to cfDNA, and that the majority of platelet DNA is of nuclear not mitochondrial origin. We also found tumor-derived DNA showing multiple copy number aberrations in a patient with pancreatic carcinoma. In a transgenic mouse model of colorectal cancer, mutant KRASG12D alleles were readily detectable in platelet DNA and, notably, in higher abundance in platelets than platelet-poor plasma (cfDNA) in 11/16 mice (Fig. 1b). Similarly, KRASG12D was detected in platelets of mice with orthotopic pancreatic adenocarcinoma (n=4). Finally, to explore utility in cancer screening, we analysed patients with high-risk, pre-malignant colonic lesions (serrated polyps). Remarkably, the driver mutation BRAFV600E was detected in platelets in 16% patients despite the small-size lesions (5/30, Fig.1b). Conclusions This study establishes a role for platelets in sequestration of cfDNA, an aspect of platelet biology that has not previously been highlighted and is of substantial clinical relevance. Their abundance, ease of isolation, and continuous tissue perfusion make platelets ideal ‘sentinels’ for genetic perturbations including in early stage/pre-malignant disease.

CAR19 Therapy Drives Expansion of Clonal Hematopoiesis and Associated Cytopenias

Background: For patients with relapsed/refractory large B-cell lymphomas (rrLBCL), CD19-directed chimeric antigen receptor T cells (CAR19) improve survival compared to autologous hematopoietic cell transplantation (HCT). However, major toxicities of CAR19 therapy include prolonged cytopenias and associated infections. To better understand the impact of CAR19 on such toxicities, we studied a cohort of LBCL patients achieving durable remissions to assess immune recovery after CAR19 treatment. We (1) compared immune reconstitution in HCT vs CAR19 treated patients, (2) performed molecular analysis of post-CAR19 cytopenic marrow specimens, and (3) profiled emergent mutations in cell free DNA (cfDNA) before and after CAR19. Methods & Results: We first propensity matched rrLBCL patients treated at Stanford with either CAR19 (100% axi-cel) or HCT who achieved durable remissions. Patients were matched based on age, sex, prior systemic lines of therapy, and time from infusion. From 471 eligible cases, propensity score matching was successful in 138 (n=69 per arm). When compared to HCT-treated counterparts, CAR19-treated patients had reduced recovery of absolute lymphocyte counts (ALC) and absolute neutrophil counts (ANC; longitudinal regression day 28-800 ANOVA p<0.005), but similar recovery of hemoglobin and platelets. CAR19-treated patients experienced significantly more Grade ≥3 infections in competing risk analyses (HR 2.61, CI 1.26-5.42), and higher non-relapse mortality (log-rank p=0.027). Next, to characterize mechanisms underlying cytopenias after CAR19, we profiled marrow aspirates from cases requiring biopsy due to prolonged cytopenia or clinical concern for myeloid neoplasms. No patient had evidence of lymphoma relapse at the time of biopsy, and all were >60d from CAR19 infusion (median 377d, range 86-1561d). Among 21 patients profiled by DNA sequencing, 18 (86%) had evidence of ≥1 somatic mutation in genes canonically associated with clonal hematopoiesis (CH) in the marrow ( Fig. 1A), with the most recurrently mutated genes being DNMT3A (38%), TP53 (33%), PPM1D (24%), ASXL1 (14%), and TET2 (14%). Among 6 patients diagnosed with and requiring therapy for a myeloid malignancy, all (100%) had a canonical myeloid karyotypic aberration detected by FISH, as compared with 0 in the 14 other evaluable cases ( Fig. 1A). CAR19 cells were proportionately enriched in the marrow relative to paired peripheral blood by flow cytometry (n = 12, median 2.5-fold enrichment, Wilcoxon p<0.001). Using 10x scRNA-Seq, viable CAR19 was detectable in 7 of 8 marrows (88%) and were predominantly CD4+. We hypothesized that CH clones detectable after CAR19 would also be present in pre-CAR19 blood samples and detectable in cfDNA. To test this, we profiled 786 longitudinal samples from 141 patients using CAPP-seq and serially monitored CH clones in cfDNA. Among 141 eligible cases, 40 (28%) were evaluable for CH, in having both pre- and post-CAR19 infusion specimens, while in durable remission (median follow up 967d). Of 51 CH clones detected in post-infusion cfDNA, 46 (90.1%) were also detectable prior to CAR19, typically expanding post-infusion (median = 1.8-fold, Fig. 1B). We next tested whether expanding CH clones showed evidence for selection after CAR19. Strikingly, among 48 recurrently mutated genes tested, DNMT3A and PPM1D showed significant evidence for clonal selection and expansion after CAR19 (Q-value < 0.05). When compared to patients without emerging CH clones, patients with ≥5-fold clonal expansions of canonical CH genes (23% of cases) had reduced ANC, but not ALC, recovery (median ANC=1.43 vs 2.43 at 6-mo, Wilcoxon p<0.001). This association remained significant in multivariate longitudinal regression that included age, sex, prior lines of therapy, and pre-infusion ANC (ANOVA p = 0.007). Conclusion: CAR19 treated patients experience reduced immune reconstitution and higher infection rates than HCT treated counterparts. Bone marrow findings reveal prolonged CAR19 cytopenia to commonly be associated with clonal cytopenia of undetermined significance (CCUS). Despite durable lymphoma remissions, canonical myeloid clonal expansions are common post-CAR19, and associated with myelosuppression. Non-invasive genotyping could aid in early identification of patients at risk for prolonged cytopenias after CAR19 and inform interventions to decrease associated complications.

Non-invasive monitoring of neoadjuvant radiation therapy response in soft tissue sarcomas by multiparametric MRI and quantification of circulating tumor DNA-A study protocol.

Wide resection remains the cornerstone of localized soft-tissue sarcomas (STS) treatment. Neoadjuvant radiation therapy (NRT) may decrease the risk of local recurrences; however, its effectiveness for different histological STS subtypes has not been systematically investigated. The proposed prospective study evaluates the NRT response in STS using liquid biopsies and the correlation of multiparametric magnetic resonance imaging (mpMRI) with histopathology and immunohistochemistry. Patients with localized high-grade STS, who qualify for NRT, are included in this study. Quantification of circulating tumor DNA (ctDNA) in patient blood samples is performed by targeted next-generation sequencing. Soft-tissue sarcoma subtype-specific panel sequencing in combination with patient-specific exome sequencing allows the detection of individual structural variants and point mutations. Circulating free DNA is isolated from peritherapeutically collected patient plasma samples and ctDNA quantified therein. Identification of breakpoints is carried out using FACTERA. Bioinformatic analysis is performed using samtools, picard, fgbio, and the MIRACUM Pipeline. Combination of conventional MRI sequences with diffusion-weighted imaging, intravoxel-incoherent motion, and dynamic contrast enhancement. Multiparametric MRI is performed before, during, and after NRT. We aim to correlate mpMRI data with the resected specimen's macroscopical, histological, and immunohistochemical findings. Preliminary data support the notion that quantification of ctDNA in combination with tumor mass characterization through co-registration of mpMRI and histopathology can predict NRT response of STS. The methods presented in this prospective study are necessary to assess therapy response in heterogeneous tumors and lay the foundation of future patient- and tumor-specific therapy concepts. These methods can be applied to various tumor entities. Thus, the participation and support of a wider group of oncologic surgeons are needed to validate these findings on a larger patient cohort.

Detection of cell-free histones in the cerebrospinal fluid of pediatric central nervous system malignancies by imaging flow cytometry.

Introduction: Pediatric brain tumours (PBT) are one of the most common malignancies during childhood, with variable severity according to the location and histological type. Certain types of gliomas, such a glioblastoma and diffuse intrinsic pontine glioma (DIPG), have a much higher mortality than ependymoma and medulloblastoma. Early detection of PBT is essential for diagnosis and therapeutic interventions. Liquid biopsies have been demonstrated using cerebrospinal fluid (CSF), mostly restricted to cell free DNA, which display limitations of quantity and integrity. In this pilot study, we sought to demonstrate the detectability and robustness of cell free histones in the CSF. Methods: We collected CSF samples from a pilot cohort of 8 children with brain tumours including DIPG, medulloblastoma, glioblastoma, ependymoma and others. As controls, we collected CSF samples from nine children with unrelated blood malignancies and without brain tumours. We applied a multichannel flow imaging approach on ImageStream(X) to image indiviual histone or histone complexes on different channels. Results: Single histones (H2A, macroH2A1.1, macroH2A1.2 H2B, H3, H4 and histone H3 bearing the H3K27M mutation), and histone complexes are specifically detectable in the CSF of PBT patients. H2A and its variants macroH2A1.1/macroH2A1/2 displayed the strongest signal and abundance, together with disease associated H3K27M. In contrast, mostly H4 is detectable in the CSF of pediatric patients with blood malignancies. Discussion: In conclusion, free histones and histone complexes are detectable with a strong signal in the CSF of children affected by brain tumours, using ImageStream(X) technology and may provide additive diagnostic and predictive information.

Serum miRNA-21, miRNA-146a and plasma cell free DNA as novel biomarkers for assessing systemic lupus erythematosus activity

BackgroundMicroRNA and cell-free DNA have shown significant correlations with several autoimmune disorders including systemic lupus erythematosus (SLE). SLE has been associated with challenges in determining its activity, so that the need for biomarkers contributing to assessing its activity is emerging. The current study investigated miRNA-21, miRNA-146a and plasma cf-DNA in determination of SLE activity, in addition their association with clinical data including complement factor 3 (C3), complement factor(C4), anti-dsDNA, and other disease activity indices.Methods and resultsEighty subjects divided into; twenty active patients (with SLE-DAI2K score of 16–18) twenty inactive patients (with SLE-DAI2K score of 1–3), and forty healthy control participants) were included in this study. Serum miR-21, miR-146a, and plasma cf-DNA were quantified by real time PCR and their correlation with clinical data was statistically analyzed. The results demonstrated that active cases have significant upregulation of serum miRNA-21 and plasma cf-DNA. Moreover, miR-21 showed a negative, significant pertaining to C3, C4 and was positively related to Systemic Lupus Erythematosus Disease Activity Index 2 K score (SLE-DAI Index2K score) and Systemic-Lupus-Erythematosus-Disease Activity-Index 2 K activity (SLE-DAI 2 K activity). Also, Active group miRNA-146a was negatively, significantly correlated with C3, as well as a positive significant relationship with SLE-DAI2K score and SLEDAI 2 K activity, in addition to anti DNA Autoantibodies. Furthermore, miR-21 and cf-DNA demonstrated a differential value through Receiver Operating Characteristic (ROC) curve’s study.Conclusionsthe present study illustrated miR-21, miR-146a, and cf-DNA relationship with SLE clinical data. In addition to their potential value in SLE diagnosis, and activity determination.

Role of Histone Deacetylase Inhibitor in Diabetic Painful Neuropathy.

Diabetic painful neuropathy (DPN) is one of the most detrimental complications of diabetes. Alterations in neuroinflammatory mediators play significant roles in the development of DPN. Infiltration of the neutrophils and monocyte/macrophages contributes substantial role in the degenerative process of the distal sciatic nerve by forming neutrophil extracellular traps (NETs) under diabetic condition. Citrullination of histones due to increase in protein arginine deiminase (PAD) enzyme activity under hyperglycemia may promote NET formation, which can further increase the cytokine production by activating macrophages and proliferation of neutrophils. This study reveals that the increase in histone deacetylases (HDAC) is crucial in DPN and inhibition of HDAC using HDAC inhibitor (HDACi) FK228 would suppress NETosis and alleviate diabetic nerve degeneration and pain. FK228, also known as romidepsin, is FDA approved for the treatment of cutaneous T-cell lymphoma yet the molecular mechanisms of this drug are not completely understood in DPN. In this study, type 2 diabetic (T2D) mice with pain were treated with HDACi, FK228 1mg/kg; I.P. 2 × /week for 3weeks. The results demonstrate that FK228 treatment can alleviate thermal hyperalgesia and mechanical allodynia significantly along with changes in the expression of HDACs in the dorsal root ganglia (DRG) and spinal cord dorsal horn neurons of diabetic animals. The results also indicate that FK228 treatment can alter the expression of neutrophil elastase (NE), extracellular or cell free DNA (cfDNA), citrullinated histone-3 (CitH3), PADI4, growth-associated protein (GAP)-43, and glucose transporter (GLUT)-4. Overall, this study suggests that FK228 could amend the expression of nerve regeneration markers and inflammatory mediators in diabetic animals and may offer an alternative treatment approach for DPN.

GAS-STING: a classical DNA recognition pathways to tumor therapy.

Cyclic GMP-AMP synthetase (cGAS), recognized as the primary DNA sensor within cells, possesses the capability to identify foreign DNA molecules along with free DNA fragments. This identification process facilitates the production of type I IFNs through the activator of the interferon gene (STING) which induces the phosphorylation of downstream transcription factors. This action characterizes the most archetypal biological functionality of the cGAS-STING pathway. When treated with anti-tumor agents, cells experience DNA damage that triggers activation of the cGAS-STING pathway, culminating in the expression of type I IFNs and associated downstream interferon-stimulated genes. cGAS-STING is one of the important innate immune pathways,the role of type I IFNs in the articulation between innate immunity and T-cell antitumour immunity.type I IFNs promote the recruitment and activation of inflammatory cells (including NK cells) at the tumor site.Type I IFNs also can promote the activation and maturation of dendritic cel(DC), improve the antigen presentation of CD4+T lymphocytes, and enhance the cross-presentation of CD8+T lymphocytes to upregulating anti-tumor responses. This review discussed the cGAS-STING signaling and its mechanism and biological function in traditional tumor therapy and immunotherapy.

The SAP domain of Ku facilitates its efficient loading onto DNA ends.

The evolutionarily conserved DNA repair complex Ku serves as the primary sensor of free DNA ends in eukaryotic cells. Its rapid association with DNA ends is crucial for several cellular processes, including non-homologous end joining (NHEJ) DNA repair and telomere protection. In this study, we conducted a transient kinetic analysis to investigate the impact of the SAP domain on individual phases of the Ku-DNA interaction. Specifically, we examined the initial binding, the subsequent docking of Ku onto DNA, and sliding of Ku along DNA. Our findings revealed that the C-terminal SAP domain of Ku70 facilitates the initial phases of the Ku-DNA interaction but does not affect the sliding process. This suggests that the SAP domain may either establish the first interactions with DNA, or stabilize these initial interactions during loading. To assess the biological role of the SAP domain, we generated Arabidopsis plants expressing Ku lacking the SAP domain. Intriguingly, despite the decreased efficiency of the ΔSAP Ku complex in loading onto DNA, the mutant plants exhibited full proficiency in classical NHEJ and telomere maintenance. This indicates that the speed with which Ku loads onto telomeres or DNA double-strand breaks is not the decisive factor in stabilizing these DNA structures.

Extracellular vesicles are the main contributor to the non-viral protected extracellular sequence space

Environmental virus metagenomes, commonly referred to as “viromes”, are typically generated by physically separating virus-like particles (VLPs) from the microbial fraction based on their size and mass. However, most methods used to purify VLPs, enrich extracellular vesicles (EVs) and gene transfer agents (GTAs) simultaneously. Consequently, the sequence space traditionally referred to as a “virome” contains host-associated sequences, transported via EVs or GTAs. We therefore propose to call the genetic material isolated from size-fractionated (0.22 µm) and DNase-treated samples protected environmental DNA (peDNA). This sequence space contains viral genomes, DNA transduced by viruses and DNA transported in EVs and GTAs. Since there is no genetic signature for peDNA transported in EVs, GTAs and virus particles, we rely on the successful removal of contaminating remaining cellular and free DNA when analyzing peDNA. Using marine samples collected from the North Sea, we generated a thoroughly purified peDNA dataset and developed a bioinformatic pipeline to determine the potential origin of the purified DNA. This pipeline was applied to our dataset as well as existing global marine “viromes”. Through this pipeline, we identified known GTA and EV producers, as well as organisms with actively transducing proviruses as the source of the peDNA, thus confirming the reliability of our approach. Additionally, we identified novel and widespread EV producers, and found quantitative evidence suggesting that EV-mediated gene transfer plays a significant role in driving horizontal gene transfer (HGT) in the world’s oceans.

Mitochondrial DNA is a key driver in cigarette smoke extract-induced IL-6 expression.

Smoking is an independent risk factor for atherosclerosis, the primary pathogenesis of which is inflammation. We recently reported that cigarette smoke extract (CSE) causes cytosolic and extracellular accumulation of both nuclear (n) and mitochondrial (mt) DNA, which leads to inflammation in human umbilical vein endothelial cells (HUVECs). In this study, we examined whether inflammation induction depends more on cytosolic nDNA or mtDNA, and which chemical constituents of CSE are involved. Acrolein (ACR), methyl vinyl ketone (MVK), and 2-cyclopenten-1-one (CPO) were used in the experiments, as these are the major cytotoxic factors in CSE in various cell types. Stimulation with ACR, MVK, or CPO alone resulted in the accumulation of DNA double-strand breaks (DSBs), but not oxidative DNA damage, accumulation of cytosolic DNA, or increased expression of inflammatory cytokines. Simultaneous administration of all three constituents (ALL) resulted in oxidative DNA damage in both the nucleus and mitochondria, accumulation of DSBs, reduced mitochondrial membrane potential, induction of minority mitochondrial outer membrane permeabilization, accumulation of cytosolic free DNA, and increased expression of inflammatory cytokines such as IL-6 and IL-1α. Treatment with N-acetyl-L-cysteine, a reactive oxygen species scavenger, suppressed oxidative DNA damage and the increased expression of IL-6 and IL-1α induced by ALL or CSE. The ALL- or CSE-induced increase in IL-6 expression, but not that of IL-1α, was suppressed by mtDNA depletion. In conclusion, ACR, MVK, and CPO may strongly contribute to CSE-induced inflammation. More importantly, cytosolic free mtDNA is thought to play an important role in IL-6 expression, a central mediator of inflammation.