Performance of the OncomineTM Lung cfDNA Assay for Liquid Biopsy by NGS of NSCLC Patients in Routine Laboratory Practice

Targeted next-generation sequencing (NGS) based on molecular tagging technology allowed considerable improvement in the approaches of cell-free DNA (cfDNA) analysis. Previously, we demonstrated the feasibility of the OncomineTM Lung cell-free DNA Assay (OLcfA) NGS panel when applied on plasma samples of post-tyrosine kinase inhibitors (TKIs) non-small cell lung cancer (NSCLC) patients. Here, we explored in detail the coverage metrics and variant calling of the assay and highlighted strengths and challenges by analyzing 92 plasma samples collected from a routine cohort of 76 NSCLC patients. First, performance of OLcfA was assessed using Horizon HD780 reference standards and sensitivity and specificity of 92.5% and 100% reported, respectively. The OLcfA was consequently evaluated in our plasma cohort and NGS technically successful in all 92 sequenced libraries. We demonstrated that initial cfDNA amount correlated positively with library yields (p < 0.0001) and sequencing performance (p < 0.0001). In addition, 0.1% limit of detection could be achieved even when < 10 ng cfDNA was employed. In contrast, the cfDNA amount seems to not affect the EGFR mutational status (p = 0.16). This study demonstrated an optimal performance of the OLcfA on routine plasma samples from NSCLC patients and supports its application in the liquid biopsy practice for cfDNA investigation in precision medicine laboratories.

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Peptide-Affinity Precipitation of Extracellular Vesicles and Cell-Free DNA Improves Sequencing Performance for the Detection of Pathogenic Mutations in Lung Cancer Patient Plasma

International Journal of Molecular Sciences ◽

10.3390/ijms21239083 ◽

2020 ◽

Vol 21 (23) ◽

pp. 9083

Author(s):

Catherine Taylor ◽

Simi Chacko ◽

Michelle Davey ◽

Jacynthe Lacroix ◽

Alexander MacPherson ◽

...

Keyword(s):

Lung Cancer ◽

Extracellular Vesicles ◽

Liquid Biopsy ◽

Nsclc Patient ◽

Egfr Mutations ◽

Single Nucleotide Variants ◽

Cell Free Dna ◽

Free Dna ◽

Nsclc Patients ◽

Peptide Affinity

Liquid biopsy is a minimally-invasive diagnostic method that may improve access to molecular profiling for non-small cell lung cancer (NSCLC) patients. Although cell-free DNA (cf-DNA) isolation from plasma is the standard liquid biopsy method for detecting DNA mutations in cancer patients, the sensitivity can be highly variable. Vn96 is a peptide with an affinity for both extracellular vesicles (EVs) and circulating cf-DNA. In this study, we evaluated whether peptide-affinity (PA) precipitation of EVs and cf-DNA from NSCLC patient plasma improves the sensitivity of single nucleotide variants (SNVs) detection and compared observed SNVs with those reported in the matched tissue biopsy. NSCLC patient plasma was subjected to either PA precipitation or cell-free methods and total nucleic acid (TNA) was extracted; SNVs were then detected by next-generation sequencing (NGS). PA led to increased recovery of DNA as well as an improvement in NGS sequencing parameters when compared to cf-TNA. Reduced concordance with tissue was observed in PA-TNA (62%) compared to cf-TNA (81%), mainly due to identification of SNVs in PA-TNA that were not observed in tissue. EGFR mutations were detected in PA-TNA with 83% sensitivity and 100% specificity. In conclusion, PA-TNA may improve the detection limits of low-abundance alleles using NGS.

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Disease monitoring and TKI resistance mutations of EGFR mutation-positive NSCLC patients via circulating tumor DNA.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.e21627 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. e21627-e21627

Author(s):

Corinna Woestmann ◽

Christine Ju ◽

Bernd Hinzmann ◽

Stephanie J. Yaung ◽

Michael Thomas ◽

...

Keyword(s):

Disease Progression ◽

Liquid Biopsy ◽

Kinase Inhibitors ◽

Outcome Data ◽

Egfr Mutations ◽

Driver Mutations ◽

Plasma Samples ◽

Resistance Mutations ◽

T790m Mutation ◽

Nsclc Patients

e21627 Background: 15–40% of NSCLC adenocarcinoma patients harbor EGFR sensitizing mutations. Tyrosine kinase inhibitors (TKI) provide significant clinical benefit in this population, yet all patients will develop resistance. Liquid biopsy has been demonstrated to reliably identify tumor associated somatic EGFR mutations. Quantitative assessment of mutated EGFR driven tumors could potentially be used to monitor disease progression, to assess therapeutic response, and to identify resistance mechanisms. Methods: 106 longitudinal plasma samples from 16 NSCLC patients who were treated with osimertinib as either first line or second line therapy were collected. A series of plasma samples collected during treatment and at the time of disease progression were analyzed with the AVENIO ctDNA Surveillance kit*. Mutations at each time point were identified and reported by the AVENIO software v2.0*. The mutation profile of each patient at different timepoints along with the treatment journey was examined in combination with clinical outcome data. Results: EGFR sensitizing mutations were detected in all plasma samples by sequencing except in 3 cases. Patients responsive to anti-EGFR therapy showed a rapid decrease of EGFR driver mutations to non-detectable levels. Meanwhile, patients who had stable disease or rapid disease progression had stable or slightly decreasing ctDNA levels after receiving the treatment. One patient had a MET amplification, FBXL7 SNV, and EGFR T790M detected at the time of disease progression which were not detected at baseline. One patient had both EGFR L858R and T790M mutations. This patient progressed very quickly on erlotinib. Detection of the T790M mutation decreased upon osimertinib administration, however, the L858R mutation level stayed the same. TP53 mutations were elevated in 3 patients at the time of progression, and could potentially be related to anti-EGFR resistance. Conclusions: This study clearly demonstrated that liquid biopsy could identify resistance mutations beyond EGFR prior to clinical progression. Plasma samples collected prior to or at disease progression could facilitate identification of novel resistant mutations to TKI therapy. Further studies to demonstrate the clinical utility of serial blood EGFR testing in NSCLC management are necessary. *For Research Use Only. Not for use in diagnostic procedures.

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Validation of Cell-Free DNA Collection Tubes for Determination of EGFR Mutation Status in Liquid Biopsy from NSCLC Patients

Oncology and Therapy ◽

10.1007/s40487-019-00099-9 ◽

2019 ◽

Vol 7 (2) ◽

pp. 131-139

Author(s):

Marta Sesé ◽

Rosa Somoza ◽

Inmaculada Maestu ◽

Maria Martín Ureste ◽

Alfredo Sanchez ◽

...

Keyword(s):

Liquid Biopsy ◽

Egfr Mutation ◽

Cell Free Dna ◽

Mutation Status ◽

Free Dna ◽

Dna Collection ◽

Nsclc Patients

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Sensitivity assessment of workflows detecting rare circulating cell-free DNA targets: A study design proposal

PLoS ONE ◽

10.1371/journal.pone.0253401 ◽

2021 ◽

Vol 16 (7) ◽

pp. e0253401

Author(s):

Thorsten Voss ◽

Andrea Ullius ◽

Maike Schönborn ◽

Uwe Oelmüller

Keyword(s):

Study Design ◽

Liquid Biopsy ◽

Limit Of Detection ◽

Test Procedure ◽

Blood Collection ◽

Assay Sensitivity ◽

Cell Free Dna ◽

Free Dna ◽

Sensitivity Assessment ◽

Circulating Cell Free Dna

The field of liquid biopsy has seen extensive growth in recent decades, making it one of the most promising areas in molecular diagnostics. Circulating cell-free DNA (ccfDNA) especially is used as an analyte in a growing number of diagnostic assays. These assays require specified preanalytical workflows delivering ccfDNA in qualities and quantities that facilitate correct and reliable results. As each step and component used in the preanalytical process has the potential to influence the assay sensitivity and other performance characteristics, it is key to find an unbiased experimental setup to test these factors in diagnostic or research laboratories. We defined one such setup by using blood from healthy subjects and commercially available products for blood collection, spike-in material, ccfDNA isolation, and qPCR assays. As the primary read-out, we calculated the probit model-based LOD95 (limit of detection of the 95th percentile) from the qPCR assay results. In a proof of principle study we tested two different but widely used blood ccfDNA profile stabilization technologies in blood collection tubes, the Cell-Free DNA BCT and the PAXgene Blood ccfDNA Tube. We tested assays for three different EGFR gene mutations and one BRAF gene mutation. The study design revealed differences in performance between the two tested technologies for all four mutations. In conclusion, we successfully established a blueprint for a test procedure capable of verifying and validating a liquid biopsy workflow from blood collection to the analytical result.

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Liquid Biopsy Analysis of FGFR3, TERT Promoter and STAG2 Hotspot Mutations for Disease Surveillance in Bladder Cancer

Clinical Oncology and Research ◽

10.31487/j.cor.2020.02.11 ◽

2020 ◽

pp. 1-7

Author(s):

Victor Romanov ◽

Dimitri Gnatenko ◽

Edward Forsyth ◽

Liang Xiaohui ◽

Olga Povcher ◽

...

Keyword(s):

Bladder Cancer ◽

Sensitivity And Specificity ◽

Liquid Biopsy ◽

Disease Surveillance ◽

Digital Pcr ◽

Cell Free Dna ◽

Free Dna ◽

Tert Promoter ◽

Mutational Status ◽

Muscle Invasive

Patients with non-muscle invasive bladder cancer (NMIBC) are followed by frequent cystoscopies. Innovative approaches partly replacing cystoscopy (uncomfortable, expensive, low sensitive procedure) are demanded. The current study aims to establish a fast, reliable, non-invasive, and inexpensive procedure for NMIBC patient surveillance. Liquid biopsy is a reliable source of biomarkers for cancer patient monitoring. Urine is the most suitable biological liquid to search for bladder cancer biomarkers. Cell-free DNA in urine represents tumor-related mutations for several cancers, including the bladder. We investigated mutations in FGFR3, TERT promoter, and STAG2 as markers for diagnostics and follow-up in NMIBC. Digital PCR was used to detect mutations in urine-derived cell-free DNA. The sensitivity and specificity of the markers in relation to clinical outcomes served as criteria of the assay efficiency. The sensitivity with a single marker (TERT) reached 87%, with a specificity of 77%. Combining two biomarkers (TERT+FGFR3) increased the specificity of the assay to 100% with a sensitivity of 72%. Different mutational status of STAG2 can indicate NMIBC presence or recurrence. Therefore, applying the suggested combination of biomarkers with simple detection procedures to larger patient cohorts will allow developing procedures for BC detection and surveillance with optimal sensitivity and specificity. Based on the results of this proof-in-concept study, we conclude that this simple, fast and inexpensive assay can add diagnostic and prognostic value to cystoscopy/cytology analysis of NMIBC patients.

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

Current Pharmaceutical Biotechnology ◽

10.2174/1389201021666201211102710 ◽

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.

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