Liquid biopsies of plasma exosomal nucleic acids, plasma cell-free DNA, and survival of patients with advanced cancers.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 11551-11551
Author(s):  
Lino Moehrmann ◽  
Helen J. Huang ◽  
David S. Hong ◽  
Apostolia Maria Tsimberidou ◽  
Siqing Fu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Prognostic Factor ◽  
Kras Mutation ◽  
Tumor Tissue ◽  
Digital Pcr ◽  
Mutation Testing ◽  
Clinical Testing ◽  
Liquid Biopsies ◽  
Cell Free Dna ◽  
Free Dna

11551 Background: Blood-based liquid biopsies offer easy accessible genomic material for molecular diagnostics in cancer. Commonly used cell-free DNA (cfDNA) originates from dying cells. In contrast exosomal nucleic acid (exoNA) originates from living cells, which can better reflect underlying cancer biology. Methods: We isolated exoNA (EXO52) and cfDNA (QIAamp Circulating Nucleic Acid kit) from plasma of patients with progressing advanced cancers and tested for BRAFV600, KRASG12/G13, and EGFRexon19del/L858R mutations using next-generation sequencing (EXO1000), droplet digital PCR (ddPCR, QX200) and BEAMing digital PCR. The results were compared to clinical testing of archival tumor tissue and correlated with survival. Results: Of the 43 patients (colorectal cancer, 20; melanoma, 8; non-small cell lung cancer, 6; ovarian cancer, 2; papillary thyroid cancer, 2; other cancers, 5) 41 had a mutation in the tumor tissue (20 [47%] BRAF mutation, 17 [40%] KRAS mutation and 4 [9%] EGFR mutation). Mutation testing of plasma exoNA from all 43 patients detected 39 (95%) of 41 mutations present in tumor tissue with 100% specificity. Mutation testing of plasma cfDNA from 39 patients using ddPCR detected 33 (89%) of 37 mutations present in tumor and testing of plasma cfDNA from 37 patients using BEAMing detected 34 (97%) of 35 mutations present in tumor tissue; however, both cfDNA methods reported an additional KRAS mutation not present in tumor tissue. Patients with high mutation allele frequency (MAF, > median) had shorter median survival compared to patients with low MAF ( < median) when using exoNA (5.9 vs. 11.8 months, P= 0.006), but not cfDNA ddPCR (6.0 vs. 7.4 months, P= 0.06) or cfDNA BEAMing (6.5 vs. 7.4 month, P= 0.07). High MAF in exoNA was an independent prognostic factor for survival in multicovariate analysis (HR 0.13, P= 0.017). Conclusions: Mutation testing of plasma exoNA for common BRAF, KRAS, and EGFR mutations has high sensitivity compared to clinical testing of archival tumor tissue and better specificity than PCR testing of plasma cfDNA. High MAF in exoNA is the independent prognostic factor for shorter survival.

scholarly journals Analysis of KRAS Mutation Subtype in Tissue DNA and Cell-Free DNA Using Droplet Digital PCR and the Function of Cell-Free DNA as a Recurrence Predictive Marker in Pancreatic Cancer

Biomedicines ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 1599
Author(s):  
Eunsung Jun ◽  
Bonhan Koo ◽  
Eo Jin Kim ◽  
Dae Wook Hwang ◽  
Jae Hoon Lee ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Clinical Significance ◽  
Kras Mutation ◽  
Predictive Marker ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Cell Free Dna ◽  
Cancer Subtypes ◽  
Free Dna ◽  
Mutation Burden

KRAS mutation is a major regulator in the tumor progression of pancreatic cancer. Here, we compared the frequency and mutation burden of KRAS mutation subtypes with paired tumor tissue and blood in patients and examined their clinical significance. DNA from tumor tissues and cell-free DNA (cfDNA) from preoperative blood were obtained from 70 patients with pancreatic cancer. Subtypes and mutation burdens of KRAS G12D and G12V mutations were evaluated using droplet digital PCR. Comparing the presence of mutations in tissue, accumulative and simultaneous mutations of G12D or G12V were identified of 67 (95.7%), and 48 patients (68.6%). Conversely, in blood, they were only identified in 18 (25.7%) and four (5.7%) patients; respectively. Next, comparing the mutation burden in tissue, the mutation burden varied from less than 0.1 to more than five, whereas that of cfDNA in blood was mostly between one and five, as cases with a mutation burden lower than 0.1 and higher than five were rare. Finally, the presence of the G12V mutation alone in cfDNA and the combination of the G12V mutation with elevated CA 19-9 levels were associated with poor recurrence-free survival. These fundamental data on the KRAS mutation subtypes and their clinical significance could support their potential as predictive markers for postoperative recurrence.

Abstract 5607: BRAF and KRAS mutation testing in plasma cell-free DNA with ICE COLD-PCR in patients with advanced cancers

2014 ◽  
Author(s):  
Filip Janku ◽  
Ben Legendre ◽  
Katherine Richardson ◽  
Gerald S. Falchook ◽  
Aung Naing ◽  
...  
Keyword(s):  
Plasma Cell ◽  
Kras Mutation ◽  
Mutation Testing ◽  
Cell Free Dna ◽  
Free Dna

Abstract 2407: MultiplexKRASG12/G13 mutation testing of 16ng of unamplified cell-free DNA from plasma of patients with advanced cancers using Droplet Digital PCR

2015 ◽  
Author(s):  
Helen J. Huang ◽  
Dawne N. Shelton ◽  
Siqing Fu ◽  
Sarina A. Piha-Paul ◽  
Apostolia M. Tsimberidou ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Mutation Testing ◽  
Droplet Digital Pcr ◽  
Cell Free Dna ◽  
Free Dna

BRAF and KRAS mutation testing in plasma cell-free DNA with ICE COLD-PCR in patients with advanced cancers.

2014 ◽  
Vol 32 (15_suppl) ◽  
pp. e22147-e22147
Author(s):  
Benjamin L. Legendre ◽  
Katherine Richardson ◽  
Gerald Steven Falchook ◽  
Aung Naing ◽  
Veronica R. Holley ◽  
...  
Keyword(s):  
Plasma Cell ◽  
Kras Mutation ◽  
Mutation Testing ◽  
Cell Free Dna ◽  
Free Dna

Development and analytical validation of a 523-gene clinical assay for cell-free DNA.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 3039-3039
Author(s):  
Robin Harrington ◽  
Biswajit Das ◽  
Tingting Jiang ◽  
Jennifer S. LoCoco ◽  
Rajesh Patidar ◽  
...  
Keyword(s):  
Cancer Patients ◽  
Residual Disease ◽  
Digital Pcr ◽  
Coding Region ◽  
Single Nucleotide Variants ◽  
Liquid Biopsies ◽  
Driver Genes ◽  
Cell Free Dna ◽  
Initial Validation ◽  
Free Dna

3039 Background: Liquid biopsies are emerging as a powerful complement to tumor biopsies for the clinical management of cancer patients. A large gene panel with robust analytical performance that accurately assesses variants, tumor mutational burden (TMB), and microsatellite instability in plasma would be of high value for immunotherapy studies, monitoring minimal residual disease and early cancer detection. To this end, we have completed the initial validation for the cell-free DNA (cfDNA) assay, TruSight Oncology 500 (TSO500), which interrogates the full coding region of 523 genes plus selected intronic regions for fusion detection in 23 driver genes. Methods: Cell-free DNA was extracted from plasma collected from Streck or EDTA blood tubes and quantitated to achieve an assay input of ≥10 ng. Libraries were constructed using unique molecular identifiers (UMIs) and duplex barcodes for error correction, then enriched by target capture and sequenced on a NovaSeq 6000. Healthy donor (HD) specificity assessment used matched white blood cell results to filter germline and clonal hematopoiesis variants. Contrived specimens were used to evaluate sensitivity. Single nucleotide variants (SNVs) (n = 36), insertion/deletions (indels) (n = 19), copy number variants (CNVs) (n = 6), and fusions (n = 5) were tested in 2 multi-site replicates. Results: Sensitivity of detection at 0.5% variant allele fraction (VAF) was > 95% and > 97% for SNVs and indels, respectively. All expected CNVs were identified at the targeted threshold of ≥1.3X change and showed strong correlation with matched digital PCR results. All fusions were identified at ≥0.4% VAF. Specificity in HD was > 99.99%. In 22 temporally matched tumor and blood samples from late-stage patients, 58% of all reportable mutations in tumor were identified in cfDNA. Preliminary TMB analysis identified one TMB high case with POLE p.P286R observed in both tissue and cfDNA. Conclusions: In this initial validation study the TSO500 cfDNA assay exhibited high sensitivity and specificity consistent with requirements for clinical applications. Ongoing studies will further evaluate TSO500 as a complement or potential alternative to tissue biopsy for the genomic profiling of cancer patients.

scholarly journals From Sampling to Sequencing: A Liquid Biopsy Pre-Analytic Workflow to Maximize Multi-Layer Genomic Information from a Single Tube

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 3002
Author(s):  
Kendra K. Maass ◽  
Paulina S. Schad ◽  
Agnes M. E. Finster ◽  
Pitithat Puranachot ◽  
Fabian Rosing ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Pcr Amplification ◽  
Genomic Analysis ◽  
Digital Pcr ◽  
Great Promise ◽  
Sample Collection ◽  
Liquid Biopsies ◽  
Cell Free Dna ◽  
Dna And Rna ◽  
Free Dna

Liquid biopsies hold great promise for the management of cancer. Reliable liquid biopsy data depend on stable and reproducible pre-analytical protocols that comply with quality measures, irrespective of the sampling and processing site. We established a workflow for plasma preservation, followed by processing, cell-free nucleic acid isolation, quantification, and enrichment of potentially tumor-derived cell-free DNA and RNA. Employing the same input material for a direct comparison of different kits and protocols allowed us to formulate unbiased recommendations for sample collection, storage, and processing. The presented workflow integrates the stabilization in Norgen, PAX, or Streck tubes and subsequent parallel isolation of cell-free DNA and RNA with NucleoSnap and NucleoSpin. Qubit, Bioanalyzer, and TapeStation quantification and quality control steps were optimized for minimal sample use and high sensitivity and reproducibility. We show the efficiency of the proposed workflow by successful droplet digital PCR amplification of both cell-free DNA and RNA and by detection of tumor-specific alterations in low-coverage whole-genome sequencing and DNA methylation profiling of plasma-derived cell-free DNA. For the first time, we demonstrated successful parallel extraction of cell-free DNA and RNA from plasma samples. This workflow paves the road towards multi-layer genomic analysis from one single liquid biopsy sample.

scholarly journals Circulating Cell-Free DNA in Liquid Biopsies as Potential Biomarker for Bladder Cancer: A Review

Cancers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1448
Author(s):  
Raquel Herranz ◽  
Julia Oto ◽  
Emma Plana ◽  
Álvaro Fernández-Pardo ◽  
Fernando Cana ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Cancer Progression ◽  
Specific Treatment ◽  
Predictive Biomarkers ◽  
Liquid Biopsies ◽  
Cell Free Dna ◽  
Specific Patient ◽  
Free Dna ◽  
Potential Biomarker ◽  
Cancer Types

Bladder cancer (BC) is among the most frequent cancer types in the world and is the most lethal urological malignancy. Presently, diagnostic and follow-up methods for BC are expensive and invasive. Thus, the identification of novel predictive biomarkers for diagnosis, progression, and prognosis of BC is of paramount importance. To date, several studies have evidenced that cell-free DNA (cfDNA) found in liquid biopsies such as blood and urine may play a role in the particular scenario of urologic tumors, and its analysis may improve BC diagnosis report about cancer progression or even evaluate the effectiveness of a specific treatment or anticipate whether a treatment would be useful for a specific patient depending on the tumor characteristics. In the present review, we have summarized the up-to-date studies evaluating the value of cfDNA as potential diagnostic, prognostic, or monitoring biomarker for BC in several biofluids.

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
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