Journal: bioRxiv

Article Title: Brain-derived circulating cell-free DNA defines the brain region and cell specific origins associated with neuronal atrophy

doi: 10.1101/538827

Figure Lengend Snippet: A. Validation of brain-specific DNA methylation is performed using genomic DNA (gDNA) from tissue/cells-of-interest (top), for diagnostic application, cell-free DNA is extracted from patient plasma/serum. B. Schematic of targeted next generation bisulfite sequencing (tNGBS) (i) multiplex amplification of bisulfite converted gDNA/ cfDNA produces amplicon pool of regions with brain-specific CpG/CpH methylation. (ii) Tagmentation (Tn5) of the amplicon pool appends 19bp adapters (grey) that are used to append sequencing adapters (purple). The product can then be sequenced using NGS and data analysed by C. methylK pipeline performs two functions; firstly to define DNA methylation k-mers (top) DNA methylation (%) of tissues/ cells-of-interest are binarized (< 50% = 0, > 50% = 1) and converted to FASTA format within the context of surrounding nucleotides (0=T, 1=C). K-mer indexing is then performed on the tissues/cells-of-interest FASTA sequences using Kallisto . Secondly to quantify DNA methylation k-mers (bottom) bisulfite sequencing reads (fastq) from cfDNA samples are used within k-mer lookup of k-mer indexes defined. Correlated-methylation (co-methylation) thresholds based on signal-to-noise ratios are applied to each sequencing read before defining the tissue/ cell-of-origin of the single-molecule. D-E. Unsupervised hierarchical clustering (samples) of tNGBS data shows distinct clustering of DLPFC-NeuN+ cells from PBMC’s and DLPFC-NeuN-cells for example CpG assay (D) and CpH assay (E). F. Unsupervised hierarchical clustering (samples) of tNGBS shows distinct clustering of cerebellum tissue from PBMC’s and other brain regions (VWM, Hippocampus and DLPFC).

Article Snippet: Notably, the lowest WRR were observed in samples collected in specialized cfDNA collection tubes (Qiagen, ccf-DNA).

Techniques: Biomarker Discovery, DNA Methylation Assay, Diagnostic Assay, Clinical Proteomics, Methylation Sequencing, Multiplex Assay, Amplification, Methylation, Sequencing, CpG Assay

Journal: bioRxiv

Article Title: Brain-derived circulating cell-free DNA defines the brain region and cell specific origins associated with neuronal atrophy

doi: 10.1101/538827

Figure Lengend Snippet: A. Schematic of study; blast wave exposure of explosive entry personnel was measured (psi-ms) and cfDNA was analysed (tNGBS-methylK) post exposure on days 1,7,8 and 9. B. Blast wave exposure (peak daily impulse) of explosive entry personnel across training in which significantly higher exposure was recorded on day 7. C. DLPFC-NeuN+ cfDNA was detected in the serums of explosive entry personnel and was positively associated with blast wave exposure (Lm) in all 9 assays detected, of which 3 were significant (P-value < 0.05). D. Results from the most significant assay from the Lm (C) targeting CpG methylation of DLPFC-NeuN+ cells in which three individual personnel had detectable DLPFC-NeuN+ cfDNA on day 7 following increased blast exposure. E. Example of an explosive personnel’s DLPFC-NeuN+ cfDNA profile (only assays detectable) over all training days in which a peak in DLPFC-NeuN+ cfDNA was observed following day 7 exposure in all 3 assays.

Article Snippet: Notably, the lowest WRR were observed in samples collected in specialized cfDNA collection tubes (Qiagen, ccf-DNA).

Techniques: CpG Methylation Assay

Journal: bioRxiv

Article Title: Brain-derived circulating cell-free DNA defines the brain region and cell specific origins associated with neuronal atrophy

doi: 10.1101/538827

Figure Lengend Snippet: A. Schematic of study design; Two independent cohorts of PD and non-neurodegenerative controls were compared for brain-derived cfDNA (tNGBS-methylK). B. Volcano plot of P-values and fold change of detected DLPFC-NeuN+ cfDNA between PD and controls in cohort 1 (Raj). C & D Boxplots of detected DLPFC-NeuN+ cfDNA within controls and PD patients for the two DLPFC-NeuN+ assays with highest significance (B). Volcano plot of P-values (Lm) and fold change of detected DLPFC-NeuN+ cfDNA between PD and controls in cohort 2 (Walker). F. Boxplot of aggregate of detected DLPFC-NeuN+ cfDNA within controls and PD patients. G. Boxplot of aggregate of detected DLPFC-NeuN+ cfDNA detected within cohort 2 cognitively impaired PD patients and cognitively intact PD patients.

Article Snippet: Notably, the lowest WRR were observed in samples collected in specialized cfDNA collection tubes (Qiagen, ccf-DNA).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Brain-derived circulating cell-free DNA defines the brain region and cell specific origins associated with neuronal atrophy

doi: 10.1101/538827

Figure Lengend Snippet: A. Schematic of study design; cognitive assessments were performed in a cohort of type-2 diabetic patients 3 time-points over 36 months and cfDNA was analysed (tNGBS-methylK) at each follow-up. B. Results of linear modelling of DLPFC-NeuN+ cfDNA detected within type-2 diabetic serums and matched Clinical Dementia Rating (CDR) scores. All DLPFC-NeuN+ assays were negatively associated with CDR, an aggregate of all DLPFC-NeuN+ assays was marginally significant (P=0.05). C. CDR scores (top) and aggregate DLPFC-NeuN+ cfDNA results (bottom) from cognitively stable type-2 diabetics (n=12). D. CDR scores (top) and aggregate DLPFC-NeuN+ cfDNA results (bottom) from type-2 diabetics exhibiting cognitive decline over 36 months (n=10). E. Cerebellum cfDNA was detected within the type-2 diabetic cohort (n samples = 64) at significantly higher levels compared to explosive entry personnel and Parkinson’s Disease (n samples = 100) patients’ samples. F. Results of linear modelling of cerebellum cfDNA detected within type-2 diabetic serums and matched CDR scores. G. Cerebellum cfDNA detected within T2D-stable (top) and T2D-decliners (bottom) for the most significant assay detected (F). H. Examples of DLPFC-NeuN+ and Cerebellum cfDNA changes over 36-month follow-ups within T2D-stable (top) and T2D-decliners (bottom). *p-value = 0.03, students t-test.

Article Snippet: Notably, the lowest WRR were observed in samples collected in specialized cfDNA collection tubes (Qiagen, ccf-DNA).

Techniques:

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Quantification of cell-free DNAfor the analysis of CD19-CAR-T cells during lymphoma treatment

doi: 10.1016/j.omtm.2021.10.009

Figure Lengend Snippet: ddPCR assay and in vitro experiments (A) Two-dimensional plots of ddPCR analysis. Two cfDNA samples from patients were analyzed by CARTP-A1 primer-probe pair. Top: Peripheral Blood sample from a patient treated with axi-cel. Bottom: Sample from a patient not treated with axi-cel. black: negative droplets, green: Hex (reference) positive droplets, blue: FAM (CAR-DNA) positive droplets, orange: FAM + Hex double-positive droplets. (B) Spike-in experiments. gDNA of CAR-T cells (2 ng/μL, isolated from the leftovers of an infusion bag) was spiked into DNA collected from CM of Jurkat and Karpas cell lines. No false positive signals were observed. The amount of reference cfDNA was higher in CM obtained from Karpas cells, most likely due to higher rates of apoptosis in the cell culture. In spike-in experiments, reference DNA increased concomitant to CAR-DNA, as the spiked gDNA comprises both the CAR-DNA and reference DNA. Green: Hex-positive droplets ( TERT reference), blue: FAM-positive droplets (CAR-DNA). (C) Analysis of CM from increasing amounts of cultured CAR-T cells. Cells were seeded in 200 μL medium and cultured for 24 h. Numbers of CAR-T cells correlated with cfCAR-DNA ( r 2 = 0.99). Increasing amounts cfCAR-DNA occured concurrently with increasing amounts of reference DNA. Every CAR-T cell harbored the CAR-DNA and the reference DNA ( TERT ), which were both released from apoptotic CAR-T cells. Accordingly, the ratio of CAR-DNA and reference DNA was constant.

Article Snippet: Blood samples were collected in EDTA tubes and special cfDNA collection tubes (Cell-Free DNA BCT; Streck).

Techniques: In Vitro, Isolation, Cell Culture

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Quantification of cell-free DNAfor the analysis of CD19-CAR-T cells during lymphoma treatment

doi: 10.1016/j.omtm.2021.10.009

Figure Lengend Snippet: The ratio (%) of cfCAR-DNA to reference cfDNA over time (A) Follow-up of patients with tumor response after treatment with axi-cel. In these patients, ratio of cfCAR-DNA to reference cfDNA increases within the first days after infusion of CAR-T cells. (B) Follow-up of patients with disease progression after axi-cel treatment. Development of the cfCAR-DNA ratio is heterogeneous. It tends to be lower compared with patients with tumor response in some patients (<1%, patients 3 and 9), but not uniformly (patients 7 and 12).

Article Snippet: Blood samples were collected in EDTA tubes and special cfDNA collection tubes (Cell-Free DNA BCT; Streck).

Techniques: Biomarker Discovery

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Quantification of cell-free DNAfor the analysis of CD19-CAR-T cells during lymphoma treatment

doi: 10.1016/j.omtm.2021.10.009

Figure Lengend Snippet: Absolute copies/μL of cfCAR-DNA and reference cfDNA ( TERT ) over time (A) Follow-up of patients with tumor response after treatment with axi-cel. In all patients, increases of cfCAR-DNA were associated with increasing amounts of reference DNA. (B) Follow-up of patients with disease progression within 6 months after axi-cel treatment. Absolute copies/μL of reference DNA showed an immediate decrease in 3 of 4 patients. An increase of cfCAR-DNA in patients 7 and 12 was not accompanied by a substantial increase of reference DNA. With disease progression, reference cfDNA increased in patients 3 and 12.

Article Snippet: Blood samples were collected in EDTA tubes and special cfDNA collection tubes (Cell-Free DNA BCT; Streck).

Techniques: Biomarker Discovery

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Quantification of cell-free DNAfor the analysis of CD19-CAR-T cells during lymphoma treatment

doi: 10.1016/j.omtm.2021.10.009

Figure Lengend Snippet: Co-culture experiments of CAR-T cells with Karpas422 and Jurkat cells (n = 4) Cells were seeded in an effector:target cell ratio of 1:1 (20,000 cells absolute) and 2:1 (30,000 cells absolute) and were cultured for 4 h and 24 h, respectively. (A) CAR-T cells and CD19 + Karpas422. Top: Comparison of cfCAR-DNA and reference cfDNA. Absolute amounts of cfCAR-DNA were higher if more CAR-T cells were seeded (left: 10,000, right: 20,000 CAR-T cells) or due to longer incubation time (CAR-T cell expansion). In both conditions, reference cfDNA (TERT) was lower compared with cfCAR-DNA after 4-h incubation time. After 24-h incubation time, more reference cfDNA was measured, compared with cfCAR-DNA. Bottom: Comparison of IgH-BCL2 cfDNA and reference cfDNA ( PPID ). Concurrently with the reference DNA, Karpas-specific IgH-BCL2 cfDNA increases during incubation of Karpas422 with CAR-T cells. (B) CAR-T cells and CD19 - Jurkat. As expected, the amounts of cfCAR-DNA were again higher if more CAR-T cells were seeded and due to longer incubation time. The amounts of reference cfDNA were not increasing as strong as in (A) and were lower compared with the cfCAR-DNA.

Article Snippet: Blood samples were collected in EDTA tubes and special cfDNA collection tubes (Cell-Free DNA BCT; Streck).

Techniques: Co-Culture Assay, Cell Culture, Comparison, Incubation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Quantification of cell-free DNAfor the analysis of CD19-CAR-T cells during lymphoma treatment

doi: 10.1016/j.omtm.2021.10.009

Figure Lengend Snippet: Ratio of cfCAR-DNA in co-culture experiments of CAR-T cells and target cells The ratio of cfCAR-DNA to reference cfDNA decreased over time due to overshooting increase of reference cfDNA in co-cultures with CD19 + cells (blue columns). In co-cultures with CD19 − Jurkat cells, the ratio was constant (red columns).

Article Snippet: Blood samples were collected in EDTA tubes and special cfDNA collection tubes (Cell-Free DNA BCT; Streck).

Techniques: Co-Culture Assay