scholarly journals Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions

Diagnostics ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 234 ◽  
Author(s):  
Eun Young Lee ◽  
Eun-Ju Lee ◽  
Hana Yoon ◽  
Dong Hyeon Lee ◽  
Kwang Hyun Kim
Keyword(s):  
Cost Efficiency ◽  
High Sensitivity ◽  
Storage Conditions ◽  
Sample Storage ◽  
Sample Collection ◽  
Cell Free Dna ◽  
Free Dna ◽  
Zymo Research ◽  
Free Circulating Dna ◽  
Commercial Kits

Urinary cell-free DNA (cfDNA) is an attractive body fluid for liquid biopsy. In this study, we compared the efficiencies of four commercial kits for urinary cell-free DNA (cfDNA) isolation and of various sample storage conditions. Urinary cfDNA was isolated from 10 healthy individuals using four commercial kits: QIAamp Circulating Nucleic Acid Kit (QC; Qiagen), MagMAX™ Cell-Free DNA Isolation Kit (MM; Applied Biosystems), Urine Cell-Free Circulating DNA Purification Midi Kit (NU; Norgen Biotek), and Quick-DNA™ Urine Kit (ZQ; Zymo Research). To assess the isolation efficiency, an Agilent 2100 Bioanalyzer with High Sensitivity DNA chips was used, and cfDNA yield was defined as the amount of cfDNA obtained from 1 mL of urine. MM and QC provided the highest cfDNA yield in the 50–300 bp range, and MM and NU gave the highest cfDNA yield in the 50–100 bp range. In particular, the NU kit was efficient for isolation of more fragmented cfDNA in the range of 50–100 bp with the lowest cellular genomic DNA contamination. ZQ had the best cost-efficiency for isolating the same amount of urinary cfDNA. Samples stored at −70 °C with the addition of 10 mM EDTA resulted in the highest cfDNA yield 3 months after sample collection.

scholarly journals Enhanced specificity of clinical high-sensitivity tumor mutation profiling in cell-free DNA via paired normal sequencing using MSK-ACCESS

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
A. Rose Brannon ◽  
Gowtham Jayakumaran ◽  
Monica Diosdado ◽  
Juber Patel ◽  
Anna Razumova ◽  
...  
Keyword(s):  
Cancer Patients ◽  
De Novo ◽  
Low Frequency ◽  
High Sensitivity ◽  
Clinical Analysis ◽  
De Novo Mutation ◽  
Cell Free Dna ◽  
Free Dna ◽  
Somatic Alterations ◽  
Mutation Profiling

AbstractCirculating cell-free DNA from blood plasma of cancer patients can be used to non-invasively interrogate somatic tumor alterations. Here we develop MSK-ACCESS (Memorial Sloan Kettering - Analysis of Circulating cfDNA to Examine Somatic Status), an NGS assay for detection of very low frequency somatic alterations in 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance of MSK-ACCESS, we report results from 681 prospective blood samples that underwent clinical analysis to guide patient management. Somatic alterations are detected in 73% of the samples, 56% of which have clinically actionable alterations. The utilization of matched normal sequencing allows retention of somatic alterations while removing over 10,000 germline and clonal hematopoiesis variants. Our experience illustrates the importance of analyzing matched normal samples when interpreting cfDNA results and highlights the importance of cfDNA as a genomic profiling source for cancer patients.

Longitudinal monitoring of cell-free DNA to predict for transarterial chemo-embolization failure in hepatocellular carcinoma.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14544-e14544
Author(s):  
Joanne Evans ◽  
Caroline Ward ◽  
Madhava Pai ◽  
Rohini Sharma
Keyword(s):  
Hepatocellular Carcinoma ◽  
Early Stage ◽  
High Sensitivity ◽  
Cost Effective ◽  
Standard Of Care ◽  
Intermediate Stage ◽  
Cell Free Dna ◽  
Early Stage Disease ◽  
Free Dna ◽  
Synthetic Function

e14544 Background: Transarterial chemo-embolization (TACE) is the standard of care for patients with intermediate stage hepatocellular carcinoma (HCC) with adequate synthetic function, and as bridging treatment in early stage disease. Survival outcomes are heterogeneous, and improved biomarkers are needed for detection of early relapse. Current practice relies on monitoring of alpha-foetoprotein (AFP) and serial imaging. Circulating cell-free DNA (cfDNA) is a compelling emergent biomarker. Here, we describe the first study of cfDNA as prognostic biomarker in HCC following TACE. Methods: 34 patients treated with TACE (2012-2018) who had full demographic information and longitudinal stored plasma available were identified. Samples were retrieved and cfDNA extracted using the QIAamp Circulating Nucleic Acid Kit. cfDNA yields were quantified by high-sensitivity Qubit analysis. Results: 74% of patients were male, 66% had Childs Pugh A disease, and 56% of patients secreted AFP ( > 10ng/mL). Aetiologies of liver diseases were varied: viral hepatitis (44%), alcoholic or non-alcoholic steatohepatitis/cirrhosis (29%), hereditary or auto-immune (12%), and unexplained (15%). 79% of patients saw a fall in cfDNA titre following TACE. Where no reduction was seen, patients had a poorer median overall survival than those who did: 20 months (range 5.13 – 66.7 months) compared to 37 months (9.9 – 79.3 months). Of note, 44% of patients were AFP non-secretors. Here, cfDNA had a clear advantage in disease monitoring, with mean cfDNA titres in this group falling post treatment: from 1605.1 (range 185.9 – 6830) ng/mL to 693.7 (range undetectable - 2300) ng/mL and rising again with subsequent progression (mean of 1329.2 - range 616 – 3341 - ng/mL). Conclusions: These findings support a cost-effective monitoring role for plasma cfDNA analysis following treatment with TACE, with an added advantage in AFP non-secretors. Prospective validation is suggested to fully assess clinical utility.

scholarly journals High Sensitivity of Plasma Cell-Free DNA Genotyping in Cases With Evidence of Adequate Tumor Content

2021 ◽  
pp. 921-930
Author(s):  
Catherine B. Meador ◽  
Marina S. D. Milan ◽  
Emmy Y. Hu ◽  
Mark M. Awad ◽  
Michael S. Rabin ◽  
...  
Keyword(s):  
Plasma Cell ◽  
Nsclc Patient ◽  
False Negative ◽  
High Sensitivity ◽  
Driver Mutation ◽  
Driver Mutations ◽  
Resistance Mutations ◽  
Assay Sensitivity ◽  
Cell Free Dna ◽  
Free Dna

PURPOSE Plasma cell-free DNA (cfDNA) sequencing is a compelling diagnostic tool in solid tumors and has been shown to have high positive predictive value. However, limited assay sensitivity means that negative plasma genotyping, or the absence of detection of mutation of interest, still requires reflex tumor biopsy. METHODS We analyzed two independent cohorts of patients with advanced non–small-cell lung cancer (NSCLC) with known canonical driver and resistance mutations who underwent plasma cfDNA genotyping. We measured quantitative features, such as maximum allelic frequency (mAF), as clinically available measures of cfDNA tumor content, and studied their relationship with assay sensitivity. RESULTS In patients with EGFR-mutant NSCLC harboring EGFR T790M, detection of driver mutation at > 1% AF conferred a sensitivity of 97% (368/380) for detection of T790M across three cfDNA genotyping platforms. Similarly, in a second cohort of patients with EGFR or KRAS driver mutations, when the mAF of nontarget mutations was > 1%, sensitivity for driver mutation detection was 100% (43/43). Combining the two NSCLC patient cohorts, the presence of nontarget mutations at mAF > 1% predicts for high sensitivity (> 95%) for identifying the presence of the known driver mutation, whereas mAF of ≤ 1% confers sensitivity of only 26%-54% across platforms. Focusing on 21 false-negative cases where the driver mutation was not detected on plasma next-generation sequencing, other mutations (presumably clonal hematopoiesis) were detected at ≤ 1% AF in 14 (67%). CONCLUSION Plasma cfDNA genotyping is highly sensitive when adequate tumor DNA content is present. The likelihood of a false-negative cfDNA genotyping result is low in a sample with evidence of > 1% tumor content. Bioinformatic approaches are needed to further optimize the assessment of cfDNA tumor content in plasma genotyping assays.

scholarly journals Post-biopsy cell-free DNA from blood: an open window on primary prostate cancer genetics and biology

2021 ◽  
Author(s):  
Marinella Corbetta ◽  
Chiara Chiereghin ◽  
Ilaria De simone ◽  
Giulia Maria Emilia Antonietta Soldà ◽  
Monica Zuradelli ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Detection ◽  
Prostate Biopsy ◽  
Somatic Mutations ◽  
Cancer Genetics ◽  
High Sensitivity ◽  
Patient Specific ◽  
Cell Free Dna ◽  
Primary Tumors ◽  
Free Dna

Abstract BackgroundCirculating cell-free DNA (ccfDNA), released from normal and cancerous cells, is a promising biomarker for cancer detection as in neoplastic patients it is enriched in tumor-derived DNA (ctDNA). ctDNA contains cancer-specific mutations and epigenetic modifications, which can have diagnostic/prognostic value. However, in primary tumors, and in particular in localized prostate cancer (PCa), the fraction of ctDNA is very low and conventional strategies to study ccfDNA are unsuccessful.MethodsccfDNA was isolated from plasma before and after prostate biopsy by using the Maxwell RSC ccfDNA Plasma Kit and quantitatively and qualitatively analyzed by a fluorometer and by the Agilent High Sensitivity D5000 ScreenTape System. Somatic mutations were searched for by targeted RNAseq on prostate biopsies, sequencing libraries were prepared using the TruSight RNA Pan-Cancer Panel from Illumina. Detection of selected somatic mutations in ccfDNA was performed by ultra-deep sequencing of amplicons, using the NEBNext® Ultra™ II DNA Library Prep Kit for library preparation. All libraries were sequenced on the NextSeq550 platform.ResultsHere we demonstrate that prostate biopsy, by causing multiple injuries to the organ, leads to a significant increase in plasma concentration of ccfDNA (P < 0.0024) in primary PCa patients. By calculating the minor allele fraction at patient-specific somatic mutations pre- and post-biopsy, we show that ctDNA is significantly enriched (from 3.9 to 164 fold) after biopsy, representing a transient “molecular window” to access and analyze ctDNA. Moreover, we show that newly released ccfDNA contains a larger fraction of di-, tri- and multi-nucleosome associated DNA fragments. This feature could be exploited to further enrich prostate-derived ccfDNA and to analyze epigenetic markers.ConclusionOur data represent a proof-of-concept that liquid tumor profiling from peripheral blood performed just after the biopsy procedure can open a “valuable molecular metastatic window” giving access to the tumor genetic asset, thus providing an opportunity for early cancer detection and individual genomic profiling in the view of PCa precision medicine.

scholarly journals Circulating DNA, a Potentially Sensitive and Specific Diagnostic Tool for Future Medicine

Dose-Response ◽  
2019 ◽  
Vol 17 (4) ◽  
pp. 155932581989101
Author(s):  
Fan Jiang ◽  
Xiaoxiao Yang ◽  
Xiping He ◽  
Mingming Yang
Keyword(s):  
Early Stage ◽  
Point Of Care ◽  
Circulating Dna ◽  
Fetal Cell ◽  
Early Screening ◽  
Cell Free Dna ◽  
Promising Tool ◽  
Free Dna ◽  
Cancer Types ◽  
Commercial Kits

Liquid biopsy has the great potential of detecting early diseases before deterioration and is valued for screening abnormalities at early stage. In oncology, circulating DNA derived from shed cancer cells reflects the tissue of origin, so it could be used to locate tissue sites during early screening. However, the heterogenous parameters of different types limit the clinical application, making it inaccessible to encompass all the cancer types. Instead, for reproducible scenario as pregnancy, fetal cell-free DNA has been well utilized for screening aneuploidies. Noninvasive and convenient as is, it would be of great value in the next decades far more than early diagnosis. This review recapitulates the discovery and development of tumor and fetal cell-free DNA. The common factors are also present that could be taken into consideration when collecting, transporting, and preserving samples. Meanwhile, several protocols used for purifying cell-free DNA, either classic ones or through commercial kits, are compared carefully. In addition, the development of technologies for analyzing cell-free DNA have been summarized and discussed in detail, especially some up-to-date approaches. At the end, the potential prospect of circulating DNA is bravely depicted. In summary, although there would be a lot of efforts before it’s prevalent, cell-free DNA remains a promising tool in point-of-care diagnostic medicine.

scholarly journals PSV-36 Optimization of Collection, Storage, and Extraction of Samples for Large-Scale 16s Sequencing Project

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 338-338
Author(s):  
Robert D Jacobs ◽  
Mary Beth E Gordon ◽  
Morghan Bowman
Keyword(s):  
Large Scale ◽  
Storage Conditions ◽  
Primary Objective ◽  
Rna Transport ◽  
Sample Collection ◽  
16S Sequencing ◽  
Sequencing Project ◽  
Horse Owner ◽  
Zymo Research

Abstract Correlations between the microbiome of the horse and various physiological parameters continue to be determined. Most evaluations of the equine microbiome have been regionally focused with limited numbers or variability. In order to elucidate the role of the microbiome on equine health, a large-scale trial has been initiated with a primary objective of analyzing and characterizing the microbiome of the horse. A study of this scope relies on the horse owner to collect samples from various locations and ship them to a centralized lab. Therefore, collection protocols and extraction techniques need to be optimized to maintain sample integrity. A trial was conducted to evaluate the storage conditions and extraction methodologies necessary to develop an optimized method of sample collection, preparation, and extraction. Samples were collected from a single horse and subjected to one of four treatments in duplicate (1-fresh, 2-frozen, 3-stored in 99% EtOH, and 4-stored in a commercially available DNA/RNA transport medium; DNA/RNA Shield®, Zymo Research, Irvine, CA). Frozen samples were placed at -80◦ C immediately following collection and stored for 48-hr. Frozen samples were thawed immediately prior to extraction. All other treatments were stored as dictated by treatment at room temperature for 48-hr prior to extraction to mimic shipping. Samples were processed utilizing a commercially available DNA extraction kit (Quick-DNA™ Fecal/Soil Microbe Miniprep, Zymo Research). DNA extract was analyzed for quantity utilizing a Qubit Fluorometer (ThermoFisher, Waltham, MA), and quality via spectrophotometry (NanoDrop 2000; ThermoFisher, Waltham, MA). Swabs stored in DNA/RNA Shield® yielded higher concentrations of DNA with superior 260/280 values than other treatments. These data indicate that storage of fecal swabs in an appropriate buffer combined with optimized extraction protocols can deliver adequate extracts that can be utilized for 16s sequencing and validates the use of this method for large-scale microbiome analysis.

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