BIOM-18. METHYLATION ANALYSIS TO REVEAL THE CELLULAR ORIGIN OF PROGNOSTIC CIRCULATING CELL-FREE DNA IN GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi14-vi14
Author(s):  
Jacob Till ◽  
Aseel Abdalla ◽  
Zhuoyang Wang ◽  
Wanding Zhou ◽  
S Ali Nabavizadeh ◽  
...  
Keyword(s):  
Fragment Size ◽  
Progression Free Survival ◽  
Gene Set Enrichment Analysis ◽  
Newly Diagnosed ◽  
Methylation Analysis ◽  
Cell Free Dna ◽  
Free Dna ◽  
A Genome ◽  
Newly Diagnosed Glioblastoma ◽  
Circulating Cell Free Dna

Abstract We have previously demonstrated an independent association between high levels of plasma circulating cell-free DNA (ccfDNA) concentration and poor survival outcomes in patients with newly diagnosed glioblastoma. Given that somatic mutations are rarely detected in glioblastoma patient plasma, we reasoned that DNA shed by tumor (circulating tumor DNA, ctDNA) was not likely to be a driver of prognostic increases in ccfDNA. To investigate the tissue of origin for this prognostic ccfDNA, we analyzed the plasma ccfDNA methylation signatures of 23 patients with newly diagnosed glioblastoma using the Illumina Infinium EPIC array, a genome-wide DNA methylation technique covering over 850,000 CpG sites. Deconvolution of the ccfDNA methylation signatures based on published reference methylomes revealed an increased proportion of ccfDNA derived from granulocytes in glioblastoma specimens compared to healthy controls (p < 0.0001). Further, this granulocyte proportion was increased in patients with high ccfDNA levels (above median value) compared to patients with low ccfDNA (p = 0.0001). Granulocyte proportion correlated with ccfDNA concentration (Spearman r = 0.64, p = 0.001). The top two gene sets identified by differential methylation analysis followed by gene set enrichment analysis (methylGSA) between high- and low-ccfDNA specimens were “neutrophil activation involved in immune response” (GO:0002283, p = 3.4E-23) and “neutrophil degranulation” (GO:0043312, p = 3.4E-23). Additional analysis of ccfDNA fragment size identified larger fragments ( > 700 bp) as the major source of the prognostic signal (log-rank p = 0.002 for progression-free survival) compared to traditionally sized ccfDNA fragments (50-700 bp, log-rank p = 0.12). Taken together, these data suggest that granulocytes are the primary contributing source of prognostic ccfDNA in glioblastoma. Future studies are needed to determine the mechanism through which granulocyte-derived ccfDNA is associated with clinical outcomes in glioblastoma and to explore related therapeutic opportunities.

scholarly journals Lengthening and shortening of plasma DNA in hepatocellular carcinoma patients

2015 ◽  
Vol 112 (11) ◽  
pp. E1317-E1325 ◽  
Author(s):  
Peiyong Jiang ◽  
Carol W. M. Chan ◽  
K. C. Allen Chan ◽  
Suk Hang Cheng ◽  
John Wong ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Hepatitis B ◽  
Molecular Diagnostic ◽  
Dna Molecules ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Free Dna ◽  
Size Profile ◽  
A Genome ◽  
Circulating Cell Free Dna

The analysis of tumor-derived circulating cell-free DNA opens up new possibilities for performing liquid biopsies for the assessment of solid tumors. Although its clinical potential has been increasingly recognized, many aspects of the biological characteristics of tumor-derived cell-free DNA remain unclear. With respect to the size profile of such plasma DNA molecules, a number of studies reported the finding of increased integrity of tumor-derived plasma DNA, whereas others found evidence to suggest that plasma DNA molecules released by tumors might be shorter. Here, we performed a detailed analysis of the size profiles of plasma DNA in 90 patients with hepatocellular carcinoma, 67 with chronic hepatitis B, 36 with hepatitis B-associated cirrhosis, and 32 healthy controls. We used massively parallel sequencing to achieve plasma DNA size measurement at single-base resolution and in a genome-wide manner. Tumor-derived plasma DNA molecules were further identified with the use of chromosome arm-levelz-score analysis (CAZA), which facilitated the studying of their specific size profiles. We showed that populations of aberrantly short and long DNA molecules existed in the plasma of patients with hepatocellular carcinoma. The short ones preferentially carried the tumor-associated copy number aberrations. We further showed that there were elevated amounts of plasma mitochondrial DNA in the plasma of hepatocellular carcinoma patients. Such molecules were much shorter than the nuclear DNA in plasma. These results have improved our understanding of the size profile of tumor-derived circulating cell-free DNA and might further enhance our ability to use plasma DNA as a molecular diagnostic tool.

scholarly journals Detection of Colorectal Cancer in Circulating Cell-Free DNA by Methylated CpG Tandem Amplification and Sequencing

2019 ◽  
Vol 65 (7) ◽  
pp. 916-926 ◽  
Author(s):  
Jingyi Li ◽  
Xin Zhou ◽  
Xiaomeng Liu ◽  
Jie Ren ◽  
Jilian Wang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Early Stage ◽  
Plasma Samples ◽  
Dna Hypermethylation ◽  
Cell Free Dna ◽  
Noncancerous Tissue ◽  
Clinical Sensitivity ◽  
Free Dna ◽  
A Genome ◽  
Circulating Cell Free Dna

Abstract BACKGROUND Aberrant DNA hypermethylation of CpG islands occurs frequently throughout the genome in human colorectal cancer (CRC). A genome-wide DNA hypermethylation analysis technique using circulating cell-free DNA (cfDNA) is attractive for the noninvasive early detection of CRC and discrimination between CRC and other cancer types. METHODS We applied the methylated CpG tandem amplification and sequencing (MCTA-Seq) method, with a fully methylated molecules algorithm, to plasma samples from patients with CRC (n = 147) and controls (n = 136), as well as cancer and adjacent noncancerous tissue samples (n = 66). We also comparatively analyzed plasma samples from patients with hepatocellular carcinoma (HCC; n = 36). RESULTS Dozens of DNA hypermethylation markers including known (e.g., SEPT9 and IKZF1) and novel (e.g., EMBP1, KCNQ5, CHST11, APBB1IP, and TJP2) genes were identified for effectively detecting CRC in cfDNA. A panel of 80 markers discriminated early-stage CRC patients and controls with a clinical sensitivity of 74% and clinical specificity of 90%. Patients with early-stage CRC and HCC could be discriminated at clinical sensitivities of approximately 70% by another panel of 128 markers. CONCLUSIONS MCTA-Seq is a promising method for the noninvasive detection of CRC.

Targeted deep DNA methylation analysis of circulating cell-free DNA in plasma using massively parallel semiconductor sequencing

Epigenomics ◽  
2015 ◽  
Vol 7 (3) ◽  
pp. 353-362 ◽  
Author(s):  
Felipe Vaca-Paniagua ◽  
Javier Oliver ◽  
Andre Nogueira da Costa ◽  
Philippe Merle ◽  
James McKay ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Massively Parallel ◽  
Methylation Analysis ◽  
Cell Free Dna ◽  
Free Dna ◽  
Semiconductor Sequencing ◽  
Dna Methylation Analysis ◽  
Circulating Cell Free Dna

scholarly journals Nuclease deficiencies alter plasma cell-free DNA methylation profiles

2021 ◽  
pp. gr.275426.121
Author(s):  
Diana Siao Cheng Han ◽  
Meng Ni ◽  
Rebecca Wing Yan Chan ◽  
Danny Ka Lok Wong ◽  
Linda T Hiraki ◽  
...  
Keyword(s):  
Fragment Size ◽  
Cpg Islands ◽  
Periodic Pattern ◽  
Open Chromatin ◽  
Cell Free Dna ◽  
Free Dna ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale ◽  
Deficient Mice

The effects of DNASE1L3 or DNASE1 deficiency on cell-free DNA (cfDNA) methylation was explored in plasma of mice deficient in these nucleases and in DNASE1L3-deficient humans. Compared to wild-type cfDNA, cfDNA in Dnase1l3-deficient mice was significantly hypomethylated, while cfDNA in Dnase1-deficient mice was hypermethylated. The cfDNA hypomethylation in Dnase1l3-deficient mice was due to increased fragmentation and representation from open chromatin regions (OCRs) and CpG islands (CGIs). These findings were absent in Dnase1-deficient mice, demonstrating the preference of DNASE1 to cleave in hypomethylated OCRs and CGIs. We also observed a substantial decrease of fragment ends and coverage at methylated CpGs in the absence of DNASE1L3, thereby demonstrating that DNASE1L3 prefers to cleave at methylated CpGs. Furthermore, we found that methylation levels of cfDNA varied by fragment size in a periodic pattern, with cfDNA of specific sizes being more hypomethylated and enriched for OCRs and CGIs. These findings were confirmed in DNASE1L3-deficient human cfDNA. Thus, we have found that nuclease-mediated cfDNA fragmentation markedly affected cfDNA methylation level on a genome-wide scale. This work provides a foundational understanding of the relationship between methylation, nuclease biology and cfDNA fragmentation.

Circulating cell-free DNA in patients with newly diagnosed and recurrent cervical cancer

2020 ◽  
Vol 159 ◽  
pp. 33-34
Author(s):  
S.H. Kim ◽  
M. Wu ◽  
A. Stylianou ◽  
S. Ghafoor ◽  
Y. Lakhman ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Newly Diagnosed ◽  
Cell Free Dna ◽  
Recurrent Cervical Cancer ◽  
Free Dna ◽  
Circulating Cell Free Dna

Automated large-volume extraction of circulating, cell-free DNA to improve the sensitivity of tumor biomarker detection.

2012 ◽  
Vol 30 (30_suppl) ◽  
pp. 19-19
Author(s):  
Annette Nocon ◽  
Martin Horlitz ◽  
Markus Sprenger-Haussels
Keyword(s):  
Magnetic Particles ◽  
Fragment Size ◽  
Tumor Biomarker ◽  
Biomarker Detection ◽  
The European Union ◽  
Cell Free Dna ◽  
Extraction Protocol ◽  
Free Dna ◽  
The Mean ◽  
Circulating Cell Free Dna

19 Background: Because of its low concentration and high degree of fragmentation, the extraction and detection of tumor-derived circulating cell-free DNA (ccfDNA) is technically challenging. An optimized ccfDNA extraction method was developed and evaluated by comparison to an existing extraction protocol running on the QIAsymphony instrument and to a manual reference. Methods: 5mlEDTA plasma from healthy donors (with IRB approval) was processed. ccfDNA was bound to magnetic particles with novel surface chemistry and recovered in 150µl. As an alternative method, the “Virus cell-free 1000 protocol” using the QIAsymphony Virus/Pathogen Midi Kit was modified for the processing of higher sample volumes. ccfDNA was extracted from 4ml plasma and eluted in 90µl. The QIAamp Circulating Nucleic Acid (CNA) Kit served as a reference. ccfDNA yield was quantified by qPCR (66bp within the 18S rDNA). To determine the DNA fragment size-dependent recovery, another qPCR assay was run, quantifying four target sequences of different lengths within the APP gene. 5ml plasma was processed and eluted in 150µl. Results: The mean ccfDNA recovery (18S 66bp target; compared to the QIAamp CNA Kit) was 87% (N=6; +/-46%) for the newly developed automated extraction chemistry and 85% (N=6; +/-8%) for the modified extraction protocol. For the APP assay, ratios between the copy numbers of different target sizes were calculated. The mean ratios were: 67/476bp = 11 (N=12; +/-6.2), 180/476bp = 8.1 (N=12; +/-3.6) and 67/180bp = 1.4 (N=12; +/-0.3). Conclusions: The automated protocol versions led to an overall similar ccfDNA recovery compared to the QIAamp CNA Kit. Using the new extraction chemistry a generally improved recovery of tumor-derived circulating DNA is possible. This improvement leads to a higher sensitivity of tumor biomarker detection, which is, besides a high specificity, very important for the use of tumor biomarkers as non-invasive tool in cancer diagnosis and prognosis. The applications presented here are for research use only. Not for use in diagnostic procedures. This work has received funding from the European Union FP7 Programme under grant agreement no. 222916, SPIDIA project ( www.spidia.eu ).

scholarly journals Circulating Cell-Free DNA-Based Liquid Biopsy Markers for the Non-Invasive Prognosis and Monitoring of Metastatic Pancreatic Cancer

Cancers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1754 ◽  
Author(s):  
Marta Toledano-Fonseca ◽  
M. Teresa Cano ◽  
Elizabeth Inga ◽  
Rosa Rodríguez-Alonso ◽  
M. Auxiliadora Gómez-España ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Hepatic Metastases ◽  
Progression Free Survival ◽  
The Body ◽  
Ductal Adenocarcinoma ◽  
Cell Free Dna ◽  
Ras Mutation ◽  
Non Invasive ◽  
Free Dna ◽  
Circulating Cell Free Dna

Liquid biopsy may assist in the management of cancer patients, which can be particularly applicable in pancreatic ductal adenocarcinoma (PDAC). In this study, we investigated the utility of circulating cell-free DNA (cfDNA)-based markers as prognostic tools in metastatic PDAC. Plasma was obtained from 61 metastatic PDAC patients, and cfDNA levels and fragmentation were determined. BEAMing technique was used for quantitative determination of RAS mutation allele fraction (MAF) in cfDNA. We found that the prognosis was more accurately predicted by RAS mutation detection in plasma than in tissue. RAS mutation status in plasma was a strong independent prognostic factor for both overall survival (OS) and progression-free survival (PFS). Moreover, RAS MAF in cfDNA was also an independent risk factor for poor OS, and was strongly associated with primary tumours in the body/tail of the pancreas and liver metastases. Higher cfDNA levels and fragmentation were also associated with poorer OS and shorter PFS, body/tail tumors, and hepatic metastases, whereas cfDNA fragmentation positively correlated with RAS MAF. Remarkably, the combination of CA19-9 with MAF, cfDNA levels and fragmentation improved the prognostic stratification of patients. Furthermore, dynamics of RAS MAF better correlated with patients’ outcome than standard CA19-9 marker. In conclusion, our study supports the use of cfDNA-based liquid biopsy markers as clinical tools for the non-invasive prognosis and monitoring of metastatic PDAC patients.

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