scholarly journals Application and optimization of minimally invasive cell-free DNA techniques in oncogenomics

Tumor Biology ◽  
2018 ◽  
Vol 40 (2) ◽  
pp. 101042831876034 ◽  
Author(s):  
Manish Kumar ◽  
Yashmin Choudhury ◽  
Sankar Kumar Ghosh ◽  
Rosy Mondal
Keyword(s):  
Dna Analysis ◽  
Storage Condition ◽  
Preoperative Chemoradiotherapy ◽  
Blood Collection ◽  
Limiting Factor ◽  
Cell Free Dna ◽  
Tissue Samples ◽  
Tissue Biopsy ◽  
Free Dna ◽  
Novel Biomarkers

The conventional method of measuring biomarkers in malignant tissue samples has already given subversive growth in cancer diagnosis, prognosis, and therapy selection. However, the regression and heterogeneity associated with tumor tissue biopsy have urged for the development of an alternative approach. Considering the limitations, cell-free DNA has emerged as a surrogate alternative, facilitating preoperative chemoradiotherapy (p < 0.0001) treatment response in rectal cancer and detection of biomarker in lung cancer. This potential of cell-free DNA in several other cancers has yet to be explored based on clinical relevance by optimizing the preanalytical factors. This review has highlighted the crucial parameters from blood collection to cell-free DNA analysis that has a significant impact on the accuracy and reliability of clinical data. The quantity of cell-free DNA is also a limiting factor. Therefore, a proper preanalytical factor for blood collection, its stability, centrifugation speed, and plasma storage condition are to be optimized for developing cancer-specific biomarkers useful for clinical purpose. Liquid biopsy–based origin of cell-free DNA has revolutionized the area of cancer research. Lack of preanalytical and analytical procedures may be considered for identification of novel biomarkers through next-generation sequencing of tumor-originated cell-free DNA in contradiction to tissue biopsy for cancer-specific biomarkers.

scholarly journals Bile-Based Cell-Free DNA Analysis Is a Reliable Diagnostic Tool in Pancreatobiliary Cancer

Cancers ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 39
Author(s):  
Caroline Driescher ◽  
Katharina Fuchs ◽  
Lena Haeberle ◽  
Wolfgang Goering ◽  
Lisa Frohn ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Invasive Disease ◽  
Primary Diagnosis ◽  
Ductal Adenocarcinoma ◽  
Extrahepatic Cholangiocarcinoma ◽  
Cell Free Dna ◽  
Tissue Samples ◽  
Invasive Approach ◽  
Less Invasive ◽  
Free Dna

Currently available serum biomarkers for pancreatobiliary cancers lack sensitivity and specificity and ultimate diagnosis still requires invasive procedures for histological confirmation. The detection of tumor-specific genetic aberrations with utilization of cell free DNA (cfDNA) is a less invasive approach than traditional tissue biopsies; however, it has not been implemented into clinical routine. In this study, we investigated bile as a liquid biopsy source in pancreatobiliary cancers and compared its potential as cell-free DNA source to plasma. Blood (n = 37) and bile (n = 21) samples were collected from patients affected by pancreatic ductal adenocarcinoma (PDAC) and extrahepatic cholangiocarcinoma (CCA) or with non-malignant biliary obstructions (blood n = 16; bile n = 21). Panel-based next generation sequencing (NGS) and digital droplet PCR (ddPCR) were applied for tumor mutation profiling. NGS results from matched tumor tissues (n = 29) served as comparison. Sequencing of cfDNA from bile resulted in detection of 96.2% of the pathogenic tumor mutations found in matched tissue samples. On the other hand, only 31.6% of pathogenic tumor mutations found in tissue could be detected in plasma. In a direct comparison, only half of the mutations detected in bile cfDNA were concordantly detected in plasma from the same patients. Panel NGS and ddPCR displayed comparable sensitivity. In conclusion, bile is a suitable source of cfDNA for the diagnosis of pancreatobiliary cancer and performs more reliably than plasma. Although primary diagnosis still requires histologic confirmation, bile-derived cfDNA could offer an alternative if tissue sampling is not feasible and might allow less invasive disease monitoring.

scholarly journals Evaluation of pre-analytical factors affecting plasma DNA analysis

2017 ◽  
Author(s):  
Havell Markus ◽  
Tania Contente-Cuomo ◽  
Winnie S. Liang ◽  
Mitesh J. Borad ◽  
Shivan Sivakumar ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Fragment Size ◽  
Digital Pcr ◽  
Clinical Samples ◽  
Blood Collection ◽  
Plasma Samples ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Free Dna ◽  
The Impact

AbstractPre-analytical factors can significantly affect circulating cell-free DNA (cfDNA) analysis. However, there are few robust methods to rapidly assess sample quality and the impact of pre-analytical processing. To address this gap and to evaluate effects of DNA extraction methods and blood collection tubes on cfDNA yield and fragment size, we developed a multiplexed droplet digital PCR (ddPCR) assay with 5 short and 4 long amplicons targeting single copy genomic loci (mean amplicon size: 71 bp and 471 bp respectively). Using this assay, we compared performance of 7 cfDNA extraction kits and found cfDNA yield and fragment size varies significantly between them. We also compared 3 blood collection protocols used to collect plasma samples from 23 healthy volunteers (EDTA tubes processed within 1 hour and Cell-free DNA BCT tubes at ambient temperature processed within 24 hours and 72 hours of collection). To assess whether cell-stabilizing preservative in BCT tubes introduced noise in cfDNA, we performed digital targeted sequencing. We found no significant differences in cfDNA yield, fragment size and background sequencing noise between these protocols. In 219 clinical samples tested for quality using the ddPCR assay, cfDNA fragment size was significantly shorter in plasma samples immediately processed for ctDNA analysis compared to archived samples, suggesting background DNA contributed by lysed peripheral blood cells. In summary, we describe a multiplexed ddPCR approach that enables cfDNA quality assessment and could inform the design of future circulating tumor DNA studies.Gene namesNone

scholarly journals Comparison of Blood Collection Tubes from Three Different Manufacturers for the Collection of Cell-Free DNA for Liquid Biopsy Mutation Testing

2017 ◽  
Vol 19 (5) ◽  
pp. 801-804 ◽  
Author(s):  
Christina Alidousty ◽  
Danielle Brandes ◽  
Carina Heydt ◽  
Svenja Wagener ◽  
Maike Wittersheim ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Mutation Testing ◽  
Blood Collection ◽  
Cell Free Dna ◽  
Free Dna ◽  
Blood Collection Tubes

A dual target sequencing solution to assess genomic and epigenomic alterations in cell-free DNA with no sample splitting.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 3043-3043
Author(s):  
Grace Q. Zhao ◽  
Yun Bao ◽  
Heng Wang ◽  
Wanping Hu ◽  
John Coller ◽  
...  
Keyword(s):  
Cancer Detection ◽  
Dna Analysis ◽  
Residual Disease ◽  
Early Stage ◽  
Cancer Cell Line ◽  
Assay Sensitivity ◽  
Cell Free Dna ◽  
Target Sequencing ◽  
Free Dna ◽  
Dual Analysis

3043 Background: Assessing the genomic and epigenomic changes on plasma cell-free DNA (cfDNA) using next-generation sequencing (NGS) has become increasingly important for cancer detection and treatment selection guidance. However, two major hurdles of existing targeted NGS methods make them impractical for the clinical setting. First, there is no comprehensive, end to end, kit solution available for targeted methylation sequencing (TMS), let alone one that analyzes both mutation and methylation information in one assay. Second, the low yield of cfDNA from clinical blood samples presents a major challenge for conducting multi-omic analysis. Thus, an assay that is capable of both genomic and epigenomic analysis would be advantageous for clinical research and future diagnostic assays. Methods: Here, we report the performance of Point-n-SeqTM dual analysis, a kit solution that can provide in-depth DNA analysis with highly flexible and customizable focused panels to enable both genomic and epigenomic analysis without sample splitting. With custom panels of tens to thousands of markers designed with > 99% first-pass success rate, we conducted both performance validation and multi-center, multi-operator, reproducibility studies. Using spike-in titration of cancer cell-line gDNA with known mutation and methylation profiles, Point-n-Seq assay achieved a reliable detection level down to 0.003% of tumor DNA with a linear relationship between the measured and expected fractions. Benchmarked with conventional targeted sequencing and methylation sequencing, Point-n-Seq solution also demonstrated improved performance, speed and shortened hands-on time. Results: In a pilot clinical study, a colorectal cancer (CRC) TMS panel covering 560 methylation markers and a mutation panel with > 350 hotspot mutations in 22 genes were used in the dual assay. Using 1ml of plasma from late-stage CRC patients, cancer-specific methylation signals were detected in all samples tested, and oncogenic mutations. In an early-stage cohort (33 stage I/II CRC patient ), comparison of the analysis between tumor-informed, personalized-mutation panels (̃100 private SNVs) for each patient and the tumor-independent CRC methylation panels were conducted. The initial results showed that tumor-independent TMS assay achieved a comparable detection compared to the personalized tumor-informed approach. Moreover, cfDNA size information (fragmentome) is also integrated into the analysis of the same Point-n-Seq workflow to improve the assay sensitivity. Conclusions: Point-n-Seq dual analysis is poised to advance both research and clinical applications of early cancer detection, minimal residual disease (MRD), and monitoring.

Cell-free DNA and preoperative chemoradiotherapy for rectal cancer: a systematic review

2018 ◽  
Vol 21 (7) ◽  
pp. 874-880 ◽  
Author(s):  
Anders Kindberg Boysen ◽  
Jakob Vasehus Schou ◽  
Karen-Lise Garm Spindler
Keyword(s):  
Systematic Review ◽  
Rectal Cancer ◽  
Preoperative Chemoradiotherapy ◽  
Cell Free Dna ◽  
Free Dna

scholarly journals The influence of biological and lifestyle factors on circulating cell-free DNA in blood plasma

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Nicole Laurencia Yuwono ◽  
Kristina Warton ◽  
Caroline Elizabeth Ford
Keyword(s):  
Acute Exercise ◽  
Blood Collection ◽  
Occupational Hazard ◽  
Biological Factors ◽  
Cell Free Dna ◽  
Biological Sex ◽  
Hazard Exposure ◽  
Free Dna ◽  
Complete Reporting ◽  
Circulating Cell Free Dna

Research and clinical use of circulating cell-free DNA (cirDNA) is expanding rapidly; however, there remain large gaps in our understanding of the influence of lifestyle and biological factors on the amount of cirDNA present in blood. Here, we review 66 individual studies of cirDNA levels and lifestyle and biological factors, including exercise (acute and chronic), alcohol consumption, occupational hazard exposure, smoking, body mass index, menstruation, hypertension, circadian rhythm, stress, biological sex and age. Despite technical and methodological inconsistences across studies, we identify acute exercise as a significant influence on cirDNA levels. Given the large increase in cirDNA induced by acute exercise, we recommend that controlling for physical activity prior to blood collection is routinely incorporated into study design when total cirDNA levels are of interest. We also highlight appropriate selection and complete reporting of laboratory protocols as important for improving the reproducibility cirDNA studies and ability to critically evaluate the results.

Blood Collection and Cell-Free DNA Isolation Methods Influence the Sensitivity of Liquid Biopsy Analysis for Colorectal Cancer Detection

2018 ◽  
Vol 25 (3) ◽  
pp. 915-923 ◽  
Author(s):  
Barbara Kinga Barták ◽  
Alexandra Kalmár ◽  
Orsolya Galamb ◽  
Barnabás Wichmann ◽  
Zsófia Brigitta Nagy ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cancer Detection ◽  
Liquid Biopsy ◽  
Dna Isolation ◽  
Blood Collection ◽  
Cell Free Dna ◽  
Free Dna ◽  
Isolation Methods

Cell-Free DNA Analysis in Maternal Blood: Differences in Estimates between Laboratories with Different Methodologies Using a Propensity Score Approach

2018 ◽  
Vol 45 (5) ◽  
pp. 302-311 ◽  
Author(s):  
Elisa Bevilacqua ◽  
Jacques C. Jani ◽  
Alexandra Letourneau ◽  
Silvia F. Duiella ◽  
Pascale Kleinfinger ◽  
...  
Keyword(s):  
Propensity Score ◽  
Dna Analysis ◽  
Maternal Blood ◽  
Cell Free Dna ◽  
Free Dna

Cell-Free DNA Screening for Fetal Aneuploidy

2021 ◽  
pp. 115-120
Author(s):  
Ashley N. Battarbee ◽  
Neeta L. Vora
Keyword(s):  
Trisomy 21 ◽  
Dna Analysis ◽  
Chromosomal Abnormalities ◽  
Area Under The Curve ◽  
First Trimester ◽  
Outcome Data ◽  
Cell Free Dna ◽  
High Risk Women ◽  
Free Dna ◽  
Trimester Screening

In a prospective, multicenter blinded study at 35 international centers, the Noninvasive Examination of Trisomy (NEXT) study evaluated the performance of cell-free DNA screening for fetal trisomy compared to standard first trimester screening with nuchal translucency and serum analytes in a routine prenatal population. Among the 15,841 women who had standard screening and cell-free DNA analysis with neonatal outcome data, there were 68 chromosomal abnormalities (1 in 236). Of these, 38 were Trisomy 21 (1 in 417). Cell-free DNA analysis had a higher area under the curve (AUC) for trisomy 21, compared to standard screening (0.999 vs. 0.958, p = 0.001). Cell-free DNA analysis also had greater sensitivity, specificity, and positive predictive value compared to standard screening for trisomy 21, 18, and 13. While cell-free DNA analysis cannot detect all chromosome abnormalities, it performed better than standard screening for detection of trisomies 21, 18, and 13 in a routine population including low- and high-risk women.

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