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.

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 Undetectable Tumor Cell-Free DNA in a Patient With Metastatic Breast Cancer With Complete Response and Long-Term Remission

2020 ◽  
Vol 18 (4) ◽  
pp. 375-379
Author(s):  
Natasha Hunter ◽  
Sarah Croessmann ◽  
Karen Cravero ◽  
Daniel Shinn ◽  
Paula J. Hurley ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Metastatic Breast Cancer ◽  
Primary Tumor ◽  
Large Scale ◽  
Residual Disease ◽  
Metastatic Breast ◽  
Liquid Biopsies ◽  
Her2 Positive ◽  
Cell Free Dna ◽  
Free Dna

The ability to serially monitor tumor-derived cell-free DNA (cfDNA) brings with it the potential to measure response to anticancer therapies and detect minimal residual disease (MRD). This report describes a patient with HER2-positive metastatic breast cancer with an exceptional response to trastuzumab and nab-paclitaxel who remains in complete remission several years after cessation of therapy. Next-generation sequencing of the patient’s primary tumor tissue showed several mutations, including an oncogenic hotspot PIK3CA mutation. A sample of cfDNA was collected 6 years after her last therapy and then analyzed for mutant PIK3CA using digital PCR. No detectable mutations associated with the primary tumor were found despite assaying >10,000 genome equivalents, suggesting that the patient had achieved a molecular remission. Results of this case study suggest that serial monitoring of MRD using liquid biopsies could provide a useful method for individualizing treatment plans for patients with metastatic disease with extreme responses to therapy. However, large-scale clinical studies are needed to validate and implement these techniques for patient care.

scholarly journals The pitfalls and promise of liquid biopsies for diagnosing and treating solid tumors in children: a review

2020 ◽  
Vol 179 (2) ◽  
pp. 191-202 ◽  
Author(s):  
Ruben Van Paemel ◽  
Roos Vlug ◽  
Katleen De Preter ◽  
Nadine Van Roy ◽  
Frank Speleman ◽  
...  
Keyword(s):  
Solid Tumors ◽  
Liquid Biopsy ◽  
Residual Disease ◽  
Therapy Response ◽  
Response Monitoring ◽  
Liquid Biopsies ◽  
Cell Free Dna ◽  
Pediatric Solid Tumors ◽  
Free Dna ◽  
Tumor Types

AbstractCell-free DNA profiling using patient blood is emerging as a non-invasive complementary technique for cancer genomic characterization. Since these liquid biopsies will soon be integrated into clinical trial protocols for pediatric cancer treatment, clinicians should be informed about potential applications and advantages but also weaknesses and potential pitfalls. Small retrospective studies comparing genetic alterations detected in liquid biopsies with tumor biopsies for pediatric solid tumor types are encouraging. Molecular detection of tumor markers in cell-free DNA could be used for earlier therapy response monitoring and residual disease detection as well as enabling detection of pathognomonic and therapeutically relevant genomic alterations.Conclusion: Existing analyses of liquid biopsies from children with solid tumors increasingly suggest a potential relevance for molecular diagnostics, prognostic assessment, and therapeutic decision-making. Gaps remain in the types of tumors studied and value of detection methods applied. Here we review the current stand of liquid biopsy studies for pediatric solid tumors with a dedicated focus on cell-free DNA analysis. There is legitimate hope that integrating fully validated liquid biopsy–based innovations into the standard of care will advance patient monitoring and personalized treatment of children battling solid cancers. What is Known:• Liquid biopsies are finding their way into routine oncological screening, diagnosis, and disease monitoring in adult cancer types fast.• The most widely adopted source for liquid biopsies is blood although other easily accessible body fluids, such as saliva, pleural effusions, urine, or cerebrospinal fluid (CSF) can also serve as sources for liquid biopsies What is New:• Retrospective proof-of-concept studies in small cohorts illustrate that liquid biopsies in pediatric solid tumors yield tremendous potential to be used in diagnostics, for therapy response monitoring and in residual disease detection.• Liquid biopsy diagnostics could tackle some long-standing issues in the pediatric oncology field; they can enable accurate genetic diagnostics in previously unbiopsied tumor types like renal tumors or brain stem tumors leading to better treatment strategies

scholarly journals KRAS G12V Mutation Detection by Droplet Digital PCR in Circulating Cell-Free DNA of Colorectal Cancer Patients

2016 ◽  
Vol 17 (4) ◽  
pp. 484 ◽  
Author(s):  
Susana Olmedillas López ◽  
Dolores García-Olmo ◽  
Mariano García-Arranz ◽  
Héctor Guadalajara ◽  
Carlos Pastor ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cancer Patients ◽  
Mutation Detection ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Cell Free Dna ◽  
Free Dna ◽  
Colorectal Cancer Patients ◽  
Circulating Cell Free Dna

scholarly journals Liquid Biopsies beyond Mutation Calling: Genomic and Epigenomic Features of Cell-Free DNA in Cancer

Cancers ◽  
2021 ◽  
Vol 13 (22) ◽  
pp. 5615
Author(s):  
Arlou Kristina Angeles ◽  
Florian Janke ◽  
Simone Bauer ◽  
Petros Christopoulos ◽  
Anja Lisa Riediger ◽  
...  
Keyword(s):  
Residual Disease ◽  
Treatment Options ◽  
Acquired Resistance ◽  
Therapy Response ◽  
Driver Mutations ◽  
Clinical Value ◽  
Liquid Biopsies ◽  
Cell Free Dna ◽  
Free Dna ◽  
Invasive Method

Cell-free DNA (cfDNA) analysis using liquid biopsies is a non-invasive method to gain insights into the biology, therapy response, mechanisms of acquired resistance and therapy escape of various tumors. While it is well established that individual cancer treatment options can be adjusted by panel next-generation sequencing (NGS)-based evaluation of driver mutations in cfDNA, emerging research additionally explores the value of deep characterization of tumor cfDNA genomics and fragmentomics as well as nucleosome modifications (chromatin structure), and methylation patterns (epigenomics) for comprehensive and multi-modal assessment of cfDNA. These tools have the potential to improve disease monitoring, increase the sensitivity of minimal residual disease identification, and detection of cancers at earlier stages. Recent progress in emerging technologies of cfDNA analysis is summarized, the added potential clinical value is highlighted, strengths and limitations are identified and compared with conventional targeted NGS analysis, and current challenges and future directions are discussed.

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.

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