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 ).

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.

304-OR: 5-Hydroxymethylcytosines in Circulating Cell-Free DNA Reveal Diabetic Nephropathy

Diabetes ◽  
2020 ◽  
Vol 69 (Supplement 1) ◽  
pp. 304-OR
Author(s):  
CHANG ZENG ◽  
YING YANG ◽  
ZHOU ZHANG ◽  
CHUAN HE ◽  
WEI ZHANG ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
Cell Free Dna ◽  
Free Dna ◽  
Circulating Cell Free Dna

Cell free DNA as an evolving liquid biopsy biomarker for initial diagnosis and therapeutic nursing in Cancer- An evolving aspect in Medical Biotechnology

2020 ◽  
Vol 21 ◽  
Author(s):  
Suman Kumar Ray ◽  
Sukhes Mukherjee
Keyword(s):  
Tumor Cells ◽  
Liquid Biopsy ◽  
Cancer Metastasis ◽  
Nuclear Dna ◽  
Fragment Size ◽  
Body Fluids ◽  
Response To Therapy ◽  
Significant Information ◽  
Cell Free Dna ◽  
Free Dna

: Cell-free DNA (cfDNA) is present in numerous body fluids in addition to initiates generally from blood cells. It is undoubtedly the utmost promising tool among all components of liquid biopsy. Liquid biopsy is a specialized method investigating the nonsolid biological tissue by revealing of circulating cells, cell free DNA etc. that enter body fluids. Since, cancer cells disengage from compact tumors circulate in peripheral blood, evaluating blood of cancer patients holds the opportunities for capture and molecular level analysis of various tumor-derived constituents. Cell free DNA samples can deliver a significant perceptions into oncology, for instance tumor heterogeneity, instantaneous tumor development, response to therapy and treatment, comprising immunotherapy and mechanisms of cancer metastasis. Malignant growth at any phase can outhouse tumor cells in addition to fragments of neoplasticity causing DNA into circulatory system giving noble sign of mutation in the tumor at sampling time. Liquid biopsy distinguishes diverse blood based evolving biomarkers comprising circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) or cfDNA, circulating RNA (cfRNA) and exosomes. Cell free DNA are little DNA fragments found circulating in plasma or serum, just as other fluids present in our body. Cell free DNA involves primarily double stranded nuclear DNA and mitochondrial DNA, present both on a surface level and in the lumen of vesicles. The probable origins of the tumor-inferred portion of cfDNA are apoptosis or tumor necrosis, lysis of CTCs or release of DNA from the tumor cells into circulation. The evolution of innovations, refinement and improvement in therapeutics for determination of cfDNA fragment size and its distribution provide significant information related with pathological conditions of the cell, thus emerging as promising indicator for clinical output in medical biotechnology.

scholarly journals Circulating Cell-Free DNA in Breast Cancer: Searching for Hidden Information towards Precision Medicine

Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 728
Author(s):  
Maria Panagopoulou ◽  
Manel Esteller ◽  
Ekaterini Chatzaki
Keyword(s):  
Breast Cancer ◽  
Treatment Options ◽  
Treatment Monitoring ◽  
Circulating Biomarkers ◽  
Hidden Information ◽  
Cell Free Dna ◽  
Free Dna ◽  
Current Review ◽  
Circulating Cell Free Dna

Breast cancer (BC) is a leading cause of death between women. Mortality is significantly raised due to drug resistance and metastasis, while personalized treatment options are obstructed by the limitations of conventional biopsy follow-up. Lately, research is focusing on circulating biomarkers as minimally invasive choices for diagnosis, prognosis and treatment monitoring. Circulating cell-free DNA (ccfDNA) is a promising liquid biopsy biomaterial of great potential as it is thought to mirror the tumor’s lifespan; however, its clinical exploitation is burdened mainly by gaps in knowledge of its biology and specific characteristics. The current review aims to gather latest findings about the nature of ccfDNA and its multiple molecular and biological characteristics in breast cancer, covering basic and translational research and giving insights about its validity in a clinical setting.

Sign in / Sign up

Export Citation Format

Share Document