Cell-Free Nucleic Acids—New Insights into Physico-Chemical Properties, Analytical Considerations, and Clinical Applications

A special issue of Diagnostics (ISSN 2075-4418). This special issue belongs to the section "Pathology and Molecular Diagnostics".

Deadline for manuscript submissions: closed (10 June 2022) | Viewed by 90266

Special Issue Editors


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Guest Editor
1. Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, 53127 Bonn, Germany
2. Institute of Laboratory Medicine, German Heart Centre Munich, Technical University Munich, 80636 Munich, Germany
Interests: lab diagnostics in cardiology, oncology, immunology, neurology, pediatrics; pharmacogenomics, therapeutic drug monitoring; development and evaluation of new biomarkers: circulating nucleic acids, histone modifications miRNA, exosomes, immunogenic cell death markers, immunotoxicity, oligonucleotides; new technologies: liquid profiling, NGS, BEAMing, SPR, mass spectrometry, multiplex assays; establishment and coordination of the biofluid biobanks
Special Issues, Collections and Topics in MDPI journals

E-Mail Website
Guest Editor
Institute for Laboratory Medicine, German Heart Centre, Technical University Munich, Lazarettstraße 36, 80636 Munich, Germany
Interests: biology; molecular and cell biology; genetics; epigenetics; oncology; liquid biopsy; history of cell-free DNA and circulating tumor DNA (ctDNA) research; biological functions of cfDNA and potential roles of cfDNA in evolution; physico-chemical properties of cfDNA; histone modifications in cfDNA; next-generation sequenincg of cfDNA; bioinformatics approaches in cfDNA analysis; characterization of cfDNA using in vitro cell culture models; preanalytical standardization and optimization of cfDNA measurements; nomenclature of cfDNA
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Through programmed or accidental cell degradation, as well as purported extrusion from live cells, genetic material is continuously dispelled from different tissues and cells into body fluids. The capture and analysis of these cell-free DNA (cfDNA) molecules open up an unprecedented window of access for the minimally invasive characterization of inherited genetic codes, as well as static and temporal genomic changes that result from disease, environmental insults, and other means.

Until recently, the profiling of classical cfDNA mutations towards the characterization of solid tumors and fetal genetic abnormalities has attracted the most attention. However, from the rapidly growing body of evidence, the picture emerges that, not only is the scope of potential clinical applications of cfDNA analysis much wider than initially thought, but there is still much to be learned about the characteristics of cfDNA both as a molecular entity and as a biological phenomenon in general.  Unsurprisingly, studies into the complete physico-chemical features of cfDNA molecules (e.g., DNA methylation, sequence motifs, histone modifications, nucleosome density and spacing, fragment end-point patterns) as it relates to a variety of contexts has come rapidly to the front and will, in the next couple of years, likely become the principle centre of interest in the research field.

Within this context, the goal of this Special Issue is to advance the emerging perspective on the importance of a holistic knowledge on cfDNA.  This is crucial for accelerating our understanding of this intriguing biological phenomenon, and to fully harness its potential in both basic research and clinical settings.  Therefore, authors are invited to submit original research and review papers that are focused on, but not limited to, the following topics:

  • Nomenclature
  • The current status of cfDNA research:
    • The many different domains of research;
    • Important and emerging concepts in the cfDNA research field;
    • New technologies and opportunities;
    • Defining the edges of knowledge in the research field;
    • Highlighting neglected topics and areas of cfDNA research.
  • The clinical scope of cfDNA as a biomarker:
    • Assessments in different diseases and conditions (e.g., exercise, cancer, fetal genetic abnormalities, sepsis, clonal hematopoiesis, cardiovascular metabolic, inflammatory, autoimmune, hypoxic and ischemic diseases);
    • Assessment of different diseases in animals.
  • Preanalytical optimization, standardization, and quality control:
    • Comparative analysis of methods and technologies;
    • Development and testing of tailored methods and bioinformatics approaches;
    • Enrichment strategies for circulating tumor DNA.
  • New insights into the biology and structure of cfDNA:
    • Biological and physico-chemical properties of cfDNA;
    • cfDNA size analysis (fragmentomics);
    • Possible origins of cfDNA (consideration of apoptosis, necrosis, active release);
    • Possible biological functions of cfDNA;
    • Possible detrimental effects of cfDNA.
  • Analysis and management of high-throughput datasets.
  • Exciting applications of cfDNA analysis in oncology:
    • Epigenetic characterization of cfDNA and tissue specific signatures;
    • Gauging the use of cfDNA as a tool for early cancer detection;
    • Stratification and monitoring targeted therapies in early and advanced cancers;
    • Clonal evolution of cancer in response to microenvironment and immune response;
    • Plasma TMB and beyond for stratification and monitoring in immune therapies;
    • Liquid biopsy approaches for cell-based immune therapies;
    • Monitoring minimal residual disease in cancer;
    • Combinatorial diagnostic power of multimarker or multi-parameter assessments (inclusion of proteins, extracellular vesicles, and other nucleic acid species);
    • Longitudinal assessments of cfDNA.

Prof. Dr. Stefan Holdenrieder
Dr. Abel Bronkhorst
Guest Editors

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Keywords

  • cell-free DNA
  • circulating tumor DNA
  • liquid biopsy
  • cancer management
  • clinical oncology

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Published Papers (27 papers)

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Editorial

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7 pages, 212 KiB  
Editorial
Cell-Free Nucleic Acids: Physico-Chemical Properties, Analytical Considerations, and Clinical Applications
by Abel J. Bronkhorst and Stefan Holdenrieder
Diagnostics 2023, 13(13), 2312; https://doi.org/10.3390/diagnostics13132312 - 7 Jul 2023
Cited by 1 | Viewed by 787
Abstract
Human body fluids are rich sources of cell-free nuclear material, which exhibits unique characteristics [...] Full article

Research

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11 pages, 2568 KiB  
Communication
Cell-Free Methylated PTGER4 and SHOX2 Plasma DNA as a Biomarker for Therapy Monitoring and Prognosis in Advanced Stage NSCLC Patients
by Michael Fleischhacker, Erkan Arslan, Dana Reinicke, Stefan Eisenmann, Gerit Theil, Jens Kollmeier, Christoph Schäper, Christian Grah, Frank Klawonn, Stefan Holdenrieder and Bernd Schmidt
Diagnostics 2023, 13(13), 2131; https://doi.org/10.3390/diagnostics13132131 - 21 Jun 2023
Cited by 1 | Viewed by 962
Abstract
Notwithstanding some improvement in the earlier detection of patients with lung cancer, most of them still present with a late-stage disease at the time of diagnosis. Next to the most frequently utilized factors affecting the prognosis of lung cancer patients (stage, performance, and [...] Read more.
Notwithstanding some improvement in the earlier detection of patients with lung cancer, most of them still present with a late-stage disease at the time of diagnosis. Next to the most frequently utilized factors affecting the prognosis of lung cancer patients (stage, performance, and age), the recent application of biomarkers obtained by liquid profiling has gained more acceptance. In our study, we aimed to answer these questions: (i) Is the quantification of free-circulating methylated PTGER4 and SHOX2 plasma DNA a useful method for therapy monitoring, and is this also possible for patients treated with different therapy regimens? (ii) Is this approach possible when blood-drawing tubes, which allow for a delayed processing of blood samples, are utilized? Baseline values for mPTGER4 and mSHOX2 do not allow for clear discrimination between different response groups. In contrast, the combination of the methylation values for both genes shows a clear difference between responders vs. non-responders at the time of re-staging. Furthermore, blood drawing into tubes stabilizing the sample allows researchers more flexibility. Full article
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13 pages, 1745 KiB  
Article
Pre-Analytical Evaluation of Streck Cell-Free DNA Blood Collection Tubes for Liquid Profiling in Oncology
by Inga Medina Diaz, Annette Nocon, Stefanie A. E. Held, Makbule Kobilay, Dirk Skowasch, Abel J. Bronkhorst, Vida Ungerer, Johannes Fredebohm, Frank Diehl, Stefan Holdenrieder and Frank Holtrup
Diagnostics 2023, 13(7), 1288; https://doi.org/10.3390/diagnostics13071288 - 29 Mar 2023
Cited by 5 | Viewed by 2579
Abstract
Excellent pre-analytical stability is an essential precondition for reliable molecular profiling of circulating tumor DNA (ctDNA) in oncological diagnostics. Therefore, in vitro degradation of ctDNA and the additional release of contaminating genomic DNA from lysed blood cells must be prevented. Streck Cell-Free DNA [...] Read more.
Excellent pre-analytical stability is an essential precondition for reliable molecular profiling of circulating tumor DNA (ctDNA) in oncological diagnostics. Therefore, in vitro degradation of ctDNA and the additional release of contaminating genomic DNA from lysed blood cells must be prevented. Streck Cell-Free DNA blood collection tubes (cfDNA BCTs) have proposed advantages over standard K2EDTA tubes, but mainly have been tested in healthy individuals. Blood was collected from cancer patients (n = 53) suffering from colorectal (n = 21), pancreatic (n = 11), and non-small-cell lung cancer (n = 21) using cfDNA BCT tubes and K2EDTA tubes that were processed immediately or after 3 days (BCTs) or 6 hours (K2EDTA) at room temperature. The cfDNA isolated from these samples was characterized in terms of yield using LINE-1 qPCR; the level of gDNA contamination; and the mutation status of KRAS, NRAS, and EGFR genes using BEAMing ddPCR. CfDNA yield and gDNA levels were comparable in both tube types and were not affected by prolonged storage of blood samples for at least 3 days in cfDNA BCTs or 6 hours in K2EDTA tubes. In addition, biospecimens collected in K2EDTA tubes and cfDNA BCTs stored for up to 3 days demonstrated highly comparable levels of mutational load across all respective cancer patient cohorts and a wide range of concentrations. Our data support the applicability of clinical oncology specimens collected and stored in cfDNA BCTs for up to 3 days for reliable cfDNA and mutation analyses. Full article
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17 pages, 2937 KiB  
Article
Cell-Free DNA in Plasma and Serum Indicates Disease Severity and Prognosis in Blunt Trauma Patients
by Inga Trulson, Juliane Stahl, Stefan Margraf, Martin Scholz, Eduard Hoecherl, Konrad Wolf, Juergen Durner, Frank Klawonn and Stefan Holdenrieder
Diagnostics 2023, 13(6), 1150; https://doi.org/10.3390/diagnostics13061150 - 17 Mar 2023
Cited by 6 | Viewed by 1408
Abstract
Background: Trauma is still a major cause of mortality in people < 50 years of age. Biomarkers are needed to estimate the severity of the condition and the patient outcome. Methods: Cell-free DNA (cfDNA) and further laboratory markers were determined in plasma [...] Read more.
Background: Trauma is still a major cause of mortality in people < 50 years of age. Biomarkers are needed to estimate the severity of the condition and the patient outcome. Methods: Cell-free DNA (cfDNA) and further laboratory markers were determined in plasma and serum of 164 patients at time of admission to the emergency room. Among them were 64 patients with severe trauma (Injury Severity Score (ISS) ≥ 16), 51 patients with moderate trauma (ISS < 16) and 49 patients with single fractures (24 femur neck and 25 ankle fractures). Disease severity was objectified by ISS and Glasgow Coma Scale (GCS). Results: cfDNA levels in plasma and serum were significantly higher in patients with severe multiple trauma (SMT) than in those with moderate trauma (p = 0.002, p = 0.003, respectively) or with single fractures (each p < 0.001). CfDNA in plasma and serum correlated very strongly with each other (R = 0.91; p < 0.001). The AUC in ROC curves for identification of SMT patients was 0.76 and 0.74 for cfDNA in plasma and serum, respectively—this was further increased to 0.84 by the combination of cfDNA and hemoglobin. Within the group of multiple trauma patients, cfDNA levels were significantly higher in more severely injured patients and patients with severe traumatic brain injury (GCS ≤ 8 versus GCS > 8). Thirteen (20.3%) of the multiple trauma patients died during the first week after trauma. Levels of cfDNA were significantly higher in non-surviving patients than in survivors (p < 0.001), reaching an AUC of 0.81 for cfDNA in both, plasma and serum, which was further increased by the combination with hemoglobin and leukocytes. Conclusions: cfDNA is valuable for estimation of trauma severity and prognosis of trauma patients. Full article
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8 pages, 1507 KiB  
Communication
Isolation and Quantification of Plasma Cell-Free DNA Using Different Manual and Automated Methods
by Eleni Polatoglou, Zsuzsanna Mayer, Vida Ungerer, Abel J. Bronkhorst and Stefan Holdenrieder
Diagnostics 2022, 12(10), 2550; https://doi.org/10.3390/diagnostics12102550 - 20 Oct 2022
Cited by 10 | Viewed by 3284
Abstract
Plasma cell-free DNA (cfDNA) originates from various tissues and cell types and can enable minimally invasive diagnosis, treatment and monitoring of cancer and other diseases. Proper extraction of cfDNA is critical to obtain optimal yields and purity. The goal of this study was [...] Read more.
Plasma cell-free DNA (cfDNA) originates from various tissues and cell types and can enable minimally invasive diagnosis, treatment and monitoring of cancer and other diseases. Proper extraction of cfDNA is critical to obtain optimal yields and purity. The goal of this study was to compare the performance of six commercial cfDNA kits to extract pure, high-quality cfDNA from human plasma samples and evaluate the quantity and size profiles of cfDNA extracts—among them, two spin-column based, three magnetic bead-based and two automatic magnetic bead-based methods. Significant differences were observed in the yield of DNA among the different extraction kits (up to 4.3 times), as measured by the Qubit Fluorometer and Bioanalyzer. All kits isolated mostly small fragments corresponding to mono-nucleosomal sizes. The highest yield and reproducibility were obtained by the manual QIAamp Circulating Nucleic Acid Kit and automated MagNA Pure Total NA Isolation Kit. The results highlight the importance of standardizing preanalytical conditions depending on the requirements of the downstream applications. Full article
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10 pages, 630 KiB  
Article
Low Impact of Clonal Hematopoiesis on the Determination of RAS Mutations by Cell-Free DNA Testing in Routine Clinical Diagnostics
by Cristin Roma, Alessandra Sacco, Laura Forgione, Riziero Esposito Abate, Matilde Lambiase, Serena Dotolo, Monica Rosaria Maiello, Daniela Frezzetti, Guglielmo Nasti, Alessandro Morabito, Antonella De Luca and Nicola Normanno
Diagnostics 2022, 12(8), 1956; https://doi.org/10.3390/diagnostics12081956 - 12 Aug 2022
Cited by 2 | Viewed by 1436
Abstract
Targeted sequencing of circulating cell-free DNA (cfDNA) is used in routine clinical diagnostics for the identification of predictive biomarkers in cancer patients in an advanced stage. The presence of KRAS mutations associated with clonal hematopoiesis of indeterminate potential (CHIP) might represent a confounding [...] Read more.
Targeted sequencing of circulating cell-free DNA (cfDNA) is used in routine clinical diagnostics for the identification of predictive biomarkers in cancer patients in an advanced stage. The presence of KRAS mutations associated with clonal hematopoiesis of indeterminate potential (CHIP) might represent a confounding factor. We used an amplicon-based targeted sequencing panel, covering selected regions of 52 genes, for circulating cell-free total nucleic acid (cfTNA) analysis of 495 plasma samples from cancer patients. The cfDNA test failed in 4 cases, while circulating cell-free RNA (cfRNA) sequencing was invalid in 48 cases. In the 491 samples successfully tested on cfDNA, at least one genomic alteration was found in 222 cases (45.21%). We identified 316 single nucleotide variants (SNVs) in 21 genes. The most frequently mutated gene was TP53 (74 variants), followed by KRAS (71), EGFR (56), PIK3CA (33) and BRAF (19). Copy number variations (CNVs) were detected in 36 cases, while sequencing of cfRNA revealed 6 alterations. Analysis with droplet digital PCR (ddPCR) of peripheral blood leukocyte (PBL)-derived genomic DNA did not identify any KRAS mutations in 39 cases that showed KRAS mutations at cfDNA analysis. These findings suggest that the incidence of CHIP-associated KRAS mutations is relatively rare in routine clinical diagnostics. Full article
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15 pages, 2721 KiB  
Article
Preanalytical Variables in the Analysis of Mitochondrial DNA in Whole Blood and Plasma from Pancreatic Cancer Patients
by Hannah Randeu, Abel J. Bronkhorst, Zsuzsanna Mayer, Angela Oberhofer, Eleni Polatoglou, Volker Heinemann, Michael Haas, Stefan Boeck and Stefan Holdenrieder
Diagnostics 2022, 12(8), 1905; https://doi.org/10.3390/diagnostics12081905 - 6 Aug 2022
Cited by 6 | Viewed by 1947
Abstract
Given the crucial role of mitochondria as the main cellular energy provider and its contribution towards tumor growth, chemoresistance, and cancer cell plasticity, mitochondrial DNA (mtDNA) could serve as a relevant biomarker. Thus, the profiling of mtDNA mutations and copy number variations is [...] Read more.
Given the crucial role of mitochondria as the main cellular energy provider and its contribution towards tumor growth, chemoresistance, and cancer cell plasticity, mitochondrial DNA (mtDNA) could serve as a relevant biomarker. Thus, the profiling of mtDNA mutations and copy number variations is receiving increasing attention for its possible role in the early diagnosis and monitoring therapies of human cancers. This applies particularly to highly aggressive pancreatic cancer, which is often diagnosed late and is associated with poor prognosis. As current diagnostic procedures are based on imaging, tissue histology, and protein biomarkers with rather low specificity, tumor-derived mtDNA mutations detected from whole blood represents a potential significant leap forward towards early cancer diagnosis. However, for future routine use in clinical settings it is essential that preanalytics related to the characterization of mtDNA in whole blood are thoroughly standardized, controlled, and subject to proper quality assurance, yet this is largely lacking. Therefore, in this study we carried out a comprehensive preanalytical workup comparing different mtDNA extraction methods and testing important preanalytical steps, such as the use of different blood collection tubes, different storage temperatures, length of storage time, and yields in plasma vs. whole blood. To identify analytical and preanalytical differences, all variables were tested in both healthy subjects and pancreatic carcinoma patients. Our results demonstrated a significant difference between cancer patients and healthy subjects for some preanalytical workflows, while other workflows failed to yield statistically significant differences. This underscores the importance of controlling and standardizing preanalytical procedures in the development of clinical assays based on the measurement of mtDNA. Full article
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17 pages, 1898 KiB  
Article
Cell-Free DNA Fragmentation Patterns in a Cancer Cell Line
by Vida Ungerer, Abel J. Bronkhorst, Carsten Uhlig and Stefan Holdenrieder
Diagnostics 2022, 12(8), 1896; https://doi.org/10.3390/diagnostics12081896 - 4 Aug 2022
Cited by 10 | Viewed by 2986
Abstract
Unique bits of genetic, biological and pathological information occur in differently sized cell-free DNA (cfDNA) populations. This is a significant discovery, but much of the phenomenon remains to be explored. We investigated cfDNA fragmentation patterns in cultured human bone cancer (143B) cells using [...] Read more.
Unique bits of genetic, biological and pathological information occur in differently sized cell-free DNA (cfDNA) populations. This is a significant discovery, but much of the phenomenon remains to be explored. We investigated cfDNA fragmentation patterns in cultured human bone cancer (143B) cells using increasingly sensitive electrophoresis assays, including four automated microfluidic capillary electrophoresis assays from Agilent, i.e., DNA 1000, High Sensitivity DNA, dsDNA 915 and dsDNA 930, and an optimized manual agarose gel electrophoresis protocol. This comparison showed that (i) as the sensitivity and resolution of the sizing methods increase incrementally, additional nucleosomal multiples are revealed (hepta-nucleosomes were detectable with manual agarose gel electrophoresis), while the estimated size range of high molecular weight (HMW) cfDNA fragments narrow correspondingly; (ii) the cfDNA laddering pattern extends well beyond the 1–3 nucleosomal multiples detected by commonly used methods; and (iii) the modal size of HMW cfDNA populations is exaggerated due to the limited resolving power of electrophoresis, and instead consists of several poly-nucleosomal subpopulations that continue the series of DNA laddering. Furthermore, the most sensitive automated assay used in this study (Agilent dsDNA 930) revealed an exponential decay in the relative contribution of increasingly longer cfDNA populations. This power-law distribution suggests the involvement of a stochastic inter-nucleosomal DNA cleavage process, wherein shorter populations accumulate rapidly as they are fed by the degradation of all larger populations. This may explain why similar size profiles have historically been reported for cfDNA populations originating from different processes, such as apoptosis, necrosis, accidental cell lysis and purported active release. These results not only demonstrate the diversity of size profiles generated by different methods, but also highlight the importance of caution when drawing conclusions on the mechanisms that generate different cfDNA size populations, especially when only a single method is used for sizing. Full article
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9 pages, 1934 KiB  
Article
Cell-Free DNA for Genomic Analysis in Primary Mediastinal Large B-Cell Lymphoma
by Alfredo Rivas-Delgado, Ferran Nadeu, Marcio Andrade-Campos, Cristina López, Anna Enjuanes, Pablo Mozas, Gerard Frigola, Luis Colomo, Blanca Sanchez-Gonzalez, Neus Villamor, Sílvia Beà, Elías Campo, Antonio Salar, Eva Giné, Armando López-Guillermo and Beatriz Bellosillo
Diagnostics 2022, 12(7), 1575; https://doi.org/10.3390/diagnostics12071575 - 28 Jun 2022
Cited by 7 | Viewed by 2276
Abstract
High-throughput sequencing of cell-free DNA (cfDNA) has emerged as a promising noninvasive approach in lymphomas, being particularly useful when a biopsy specimen is not available for molecular analysis, as it frequently occurs in primary mediastinal large B-cell lymphoma (PMBL). We used cfDNA for [...] Read more.
High-throughput sequencing of cell-free DNA (cfDNA) has emerged as a promising noninvasive approach in lymphomas, being particularly useful when a biopsy specimen is not available for molecular analysis, as it frequently occurs in primary mediastinal large B-cell lymphoma (PMBL). We used cfDNA for genomic characterization in 20 PMBL patients by means of a custom NGS panel for gene mutations and low-pass whole-genome sequencing (WGS) for copy number analysis (CNA) in a real-life setting. Appropriate cfDNA to perform the analyses was obtained in 18/20 cases. The sensitivity of cfDNA to detect the mutations present in paired FFPE samples was 69% (95% CI: 60–78%). The mutational landscape found in cfDNA samples was highly consistent with that of the tissue, with the most frequently mutated genes being B2M (61%), SOCS1 (61%), GNA13 (44%), STAT6 (44%), NFKBIA (39%), ITPKB (33%), and NFKBIE (33%). Overall, we observed a 75% concordance to detect CNA gains/losses between DNA microarray and low-pass WGS. The sensitivity of low-pass WGS was remarkably higher for clonal CNA (18/20, 90%) compared to subclonal alterations identified by DNA microarray. No significant associations between cfDNA amount and tumor burden or outcome were found. cfDNA is an excellent alternative source for the accurate genetic characterization of PMBL cases. Full article
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13 pages, 1440 KiB  
Article
Diagnostic Potential of Exosomal microRNAs in Colorectal Cancer
by Jonas Dohmen, Alexander Semaan, Makbule Kobilay, Martin Zaleski, Vittorio Branchi, Anja Schlierf, Karina Hettwer, Steffen Uhlig, Gunther Hartmann, Jörg C. Kalff, Hanno Matthaei, Philipp Lingohr and Stefan Holdenrieder
Diagnostics 2022, 12(6), 1413; https://doi.org/10.3390/diagnostics12061413 - 8 Jun 2022
Cited by 8 | Viewed by 2144
Abstract
Background: Despite the significance of colonoscopy for early diagnosis of colorectal adenocarcinoma (CRC), population-wide screening remains challenging, mainly because of low acceptance rates. Herein, exosomal (exo-miR) and free circulating microRNA (c-miR) may be used as liquid biopsies in CRC to identify individuals at [...] Read more.
Background: Despite the significance of colonoscopy for early diagnosis of colorectal adenocarcinoma (CRC), population-wide screening remains challenging, mainly because of low acceptance rates. Herein, exosomal (exo-miR) and free circulating microRNA (c-miR) may be used as liquid biopsies in CRC to identify individuals at risk. Direct comparison of both compartments has shown inconclusive results, which is why we directly compared a panel of 10 microRNAs in this entity. Methods: Exo-miR and c-miR levels were measured using real-time quantitative PCR after isolation from serum specimens in a cohort of 69 patients. Furthermore, results were compared to established tumor markers CEA and CA 19-9. Results: Direct comparison of exo- and c-miR biopsy results showed significantly higher microRNA levels in the exosomal compartment (p < 0.001). Exo-Let7, exo-miR-16 and exo-miR-23 significantly differed between CRC and healthy controls (all p < 0.05), while no c-miR showed this potential. Sensitivity and specificity can be further enhanced using combinations of multiple exosomal miRNAs. Conclusions: Exosomal microRNA should be considered as a promising biomarker in CRC for future studies. Nonetheless, results may show interference with common comorbidities, which must be taken into account in future studies. Full article
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10 pages, 1802 KiB  
Article
Feasibility of Cell-Free DNA Measurement from the Earlobe during Physiological Exercise Testing
by Nils Haller, Aleksandar Tomaskovic, Thomas Stöggl, Perikles Simon and Elmo Neuberger
Diagnostics 2022, 12(6), 1379; https://doi.org/10.3390/diagnostics12061379 - 2 Jun 2022
Cited by 3 | Viewed by 1760
Abstract
Circulating, cell-free DNA (cfDNA) has been discussed as an upcoming blood-based biomarker in exercise physiology, reflecting important aspects of exercise load. cfDNA blood sampling has evolved from elaborate venous to efficient capillary sampling from the fingertips. In this study, we aimed to evaluate [...] Read more.
Circulating, cell-free DNA (cfDNA) has been discussed as an upcoming blood-based biomarker in exercise physiology, reflecting important aspects of exercise load. cfDNA blood sampling has evolved from elaborate venous to efficient capillary sampling from the fingertips. In this study, we aimed to evaluate the principal feasibility of cfDNA blood sampling from the earlobe. Therefore, we obtained cfDNA concentrations from the fingertips, earlobe, and the antecubital vein during physiological exercise testing. Significantly higher concentrations were obtained from the earlobe compared to fingertip samples. All of the measurement methods showed good to excellent repeatability (ICCs of 0.85 to 0.93). In addition, the control experiments revealed that repeated sampling from the earlobe but not from the fingertips increased cfDNA at rest. In summary, cfDNA sampling is feasible for all sampling sources. However, at rest, cfDNA collected from the earlobe tend to increase over time in the absence of physical load, potentially limiting this sampling method. Full article
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9 pages, 418 KiB  
Article
Analysis of Septin 9 Gene Hypermethylation as Follow-Up Biomarker of Colorectal Cancer Patients after Curative Surgery
by Miguel Leon Arellano, Mariano García-Arranz, Héctor Guadalajara, Rocío Olivera-Salazar, Teresa Valdes-Sanchez and Damián García-Olmo
Diagnostics 2022, 12(4), 993; https://doi.org/10.3390/diagnostics12040993 - 15 Apr 2022
Cited by 6 | Viewed by 1996
Abstract
Background: The Septin 9 test analyzes the methylation status of the SEPT9 gene, which appears to be hypermethylated in patients with colorectal cancer (CRC). This has been validated as a colorectal cancer screening test. Due to the high sensitivity and specificity found, the [...] Read more.
Background: The Septin 9 test analyzes the methylation status of the SEPT9 gene, which appears to be hypermethylated in patients with colorectal cancer (CRC). This has been validated as a colorectal cancer screening test. Due to the high sensitivity and specificity found, the justification was to use it as a biomarker tool for monitoring minimal residual disease after radical surgery and recurrence. Methods: A prospective study was carried out at the Fundación Jiménez Díaz University Hospital extracting peripheral blood from 28 patients and 4 healthy donors. Free circulating DNA was obtained and subsequently a PCR reaction to quantify the number of methylated genes. Samples were obtained preoperatively and postoperatively at five to seven days, one and three months after surgery. Results: A total of 32 preoperative samples were analyzed. The sensitivity of the test to detect CRC was 55.6% and specificity was 100%. There were 22 postsurgical samples obtained at 5–7 days after surgery, the sensitivity to detect tumor recurrences was 100% and specificity was 75%. There were 21 samples analyzed 1 month after surgery exhibiting a sensitivity and specificity of 100% and 94.7%, respectively. At 3 months, 31 postsurgical samples were analyzed and the sensitivity and specificity were 66.7% and 80%. Conclusions: Detection of methylation of Septin 9 gene in circulating plasma DNA, obtained from a peripheral blood sample, may be a useful, non-invasive and effective method for detecting minimal residual disease and could therefore predict CRC tumor recurrences. The optimal time in our series to obtain the best prediction results based on Septin 9 methylation levels was one month after surgery. Despite these considerable findings, a study with more patients is necessary to obtain more robust conclusions. Full article
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14 pages, 2004 KiB  
Article
Blood Plasma Exosomes Contain Circulating DNA in Their Crown
by Oleg Tutanov, Tatiana Shtam, Alina Grigor’eva, Alexey Tupikin, Yuri Tsentalovich and Svetlana Tamkovich
Diagnostics 2022, 12(4), 854; https://doi.org/10.3390/diagnostics12040854 - 30 Mar 2022
Cited by 12 | Viewed by 2723
Abstract
It is known that circulating DNA (cirDNA) is protected from nuclease activity by proteins that form macromolecular complexes with DNA. In addition, it was previously shown that cirDNA can bind to the outer surface of exosomes. NTA analysis and real-time PCR show that [...] Read more.
It is known that circulating DNA (cirDNA) is protected from nuclease activity by proteins that form macromolecular complexes with DNA. In addition, it was previously shown that cirDNA can bind to the outer surface of exosomes. NTA analysis and real-time PCR show that exosomes from healthy females (HF) or breast cancer patients (BCP) plasma contain less than 1.4 × 10−8 pg of DNA. Thus, only a minor part of cirDNA is attached to the outer side of the exosome as part of the vesicle crown: the share of exosomal DNA does not exceed 0.025% HF plasma DNA and 0.004% BCP plasma DNA. Treatment of plasma exosomes with DNase I with subsequent dot immunoassay reveals that H2a, H2b, and H3 histones are not part of the exosomal membrane, but are part of the cirDNA–protein macromolecular complex associated with the surface of the exosome either through interaction with DNA-binding proteins or with histone-binding proteins. Using bioinformatics approaches after identification by MALDI-TOF mass spectrometry, 16 exosomal DNA-binding proteins were identified. It was shown that four proteins—AIFM1, IGHM, CHD5, and KCNIP3—are candidates for DNA binding on the outer membrane of exosomes; the crown of exosomes may include five DNA-binding proteins: H2a, H2b, H3, IGHM, and ALB. Of note, AIFM1, IGHM, and CHD5 proteins are found only in HF plasma exosomes; KCNIP3 protein is identified only in BCP plasma exosomes; and H2a, H2b, H3, and ALB are revealed in all samples of plasma exosomes. Two histone-binding proteins, CHD5 and KDM6B, have been found in exosomes from HF plasma. The data obtained indicate that cirDNA preferentially binds to the outer membrane of exosomes by association with DNA-binding proteins. Full article
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Review

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27 pages, 746 KiB  
Review
The Clinical Utility of Droplet Digital PCR for Profiling Circulating Tumor DNA in Breast Cancer Patients
by Ugur Gezer, Abel J. Bronkhorst and Stefan Holdenrieder
Diagnostics 2022, 12(12), 3042; https://doi.org/10.3390/diagnostics12123042 - 5 Dec 2022
Cited by 16 | Viewed by 7131
Abstract
Breast cancer is the most common cancer affecting women worldwide. It is a malignant and heterogeneous disease with distinct molecular subtypes, which has prognostic and predictive implications. Circulating tumor DNA (ctDNA), cell-free fragmented tumor-derived DNA in blood plasma, is an invaluable source of [...] Read more.
Breast cancer is the most common cancer affecting women worldwide. It is a malignant and heterogeneous disease with distinct molecular subtypes, which has prognostic and predictive implications. Circulating tumor DNA (ctDNA), cell-free fragmented tumor-derived DNA in blood plasma, is an invaluable source of specific cancer-associated mutations and holds great promise for the development of minimally invasive diagnostic tests. Furthermore, serial monitoring of ctDNA over the course of systemic and targeted therapies not only allows unparalleled efficacy assessments but also enables the identification of patients who are at risk of progression or recurrence. Droplet digital PCR (ddPCR) is a powerful technique for the detection and monitoring of ctDNA. Due to its relatively high accuracy, sensitivity, reproducibility, and capacity for absolute quantification, it is increasingly used as a tool for managing cancer patients through liquid biopsies. In this review paper, we gauge the clinical utility of ddPCR as a technique for mutational profiling in breast cancer patients and focus on HER2, PIK3CA, ESR1, and TP53, which represent the most frequently mutated genes in breast cancers. Full article
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51 pages, 39021 KiB  
Review
New Perspectives on the Importance of Cell-Free DNA Biology
by Abel J. Bronkhorst, Vida Ungerer, Angela Oberhofer, Sophie Gabriel, Eleni Polatoglou, Hannah Randeu, Carsten Uhlig, Heiko Pfister, Zsuzsanna Mayer and Stefan Holdenrieder
Diagnostics 2022, 12(9), 2147; https://doi.org/10.3390/diagnostics12092147 - 3 Sep 2022
Cited by 28 | Viewed by 6041
Abstract
Body fluids are constantly replenished with a population of genetically diverse cell-free DNA (cfDNA) fragments, representing a vast reservoir of information reflecting real-time changes in the host and metagenome. As many body fluids can be collected non-invasively in a one-off and serial fashion, [...] Read more.
Body fluids are constantly replenished with a population of genetically diverse cell-free DNA (cfDNA) fragments, representing a vast reservoir of information reflecting real-time changes in the host and metagenome. As many body fluids can be collected non-invasively in a one-off and serial fashion, this reservoir can be tapped to develop assays for the diagnosis, prognosis, and monitoring of wide-ranging pathologies, such as solid tumors, fetal genetic abnormalities, rejected organ transplants, infections, and potentially many others. The translation of cfDNA research into useful clinical tests is gaining momentum, with recent progress being driven by rapidly evolving preanalytical and analytical procedures, integrated bioinformatics, and machine learning algorithms. Yet, despite these spectacular advances, cfDNA remains a very challenging analyte due to its immense heterogeneity and fluctuation in vivo. It is increasingly recognized that high-fidelity reconstruction of the information stored in cfDNA, and in turn the development of tests that are fit for clinical roll-out, requires a much deeper understanding of both the physico-chemical features of cfDNA and the biological, physiological, lifestyle, and environmental factors that modulate it. This is a daunting task, but with significant upsides. In this review we showed how expanded knowledge on cfDNA biology and faithful reverse-engineering of cfDNA samples promises to (i) augment the sensitivity and specificity of existing cfDNA assays; (ii) expand the repertoire of disease-specific cfDNA markers, thereby leading to the development of increasingly powerful assays; (iii) reshape personal molecular medicine; and (iv) have an unprecedented impact on genetics research. Full article
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26 pages, 4480 KiB  
Review
Tracing the Origin of Cell-Free DNA Molecules through Tissue-Specific Epigenetic Signatures
by Angela Oberhofer, Abel J. Bronkhorst, Carsten Uhlig, Vida Ungerer and Stefan Holdenrieder
Diagnostics 2022, 12(8), 1834; https://doi.org/10.3390/diagnostics12081834 - 29 Jul 2022
Cited by 13 | Viewed by 5556
Abstract
All cell and tissue types constantly release DNA fragments into human body fluids by various mechanisms including programmed cell death, accidental cell degradation and active extrusion. Particularly, cell-free DNA (cfDNA) in plasma or serum has been utilized for minimally invasive molecular diagnostics. Disease [...] Read more.
All cell and tissue types constantly release DNA fragments into human body fluids by various mechanisms including programmed cell death, accidental cell degradation and active extrusion. Particularly, cell-free DNA (cfDNA) in plasma or serum has been utilized for minimally invasive molecular diagnostics. Disease onset or pathological conditions that lead to increased cell death alter the contribution of different tissues to the total pool of cfDNA. Because cfDNA molecules retain cell-type specific epigenetic features, it is possible to infer tissue-of-origin from epigenetic characteristics. Recent research efforts demonstrated that analysis of, e.g., methylation patterns, nucleosome occupancy, and fragmentomics determined the cell- or tissue-of-origin of individual cfDNA molecules. This novel tissue-of origin-analysis enables to estimate the contributions of different tissues to the total cfDNA pool in body fluids and find tissues with increased cell death (pathologic condition), expanding the portfolio of liquid biopsies towards a wide range of pathologies and early diagnosis. In this review, we summarize the currently available tissue-of-origin approaches and point out the next steps towards clinical implementation. Full article
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20 pages, 697 KiB  
Review
Applications of Liquid Biopsies in Non-Small-Cell Lung Cancer
by Martin Pesta, Dattatrya Shetti, Vlastimil Kulda, Tereza Knizkova, Katerina Houfkova, Mahyar Sharif Bagheri, Martin Svaton and Jiri Polivka
Diagnostics 2022, 12(8), 1799; https://doi.org/10.3390/diagnostics12081799 - 25 Jul 2022
Cited by 9 | Viewed by 3376
Abstract
The concept of liquid biopsy as an analysis tool for non-solid tissue carried out for the purpose of providing information about solid tumors was introduced approximately 20 years ago. Additional to the detection of circulating tumor cells (CTCs), the liquid biopsy approach quickly [...] Read more.
The concept of liquid biopsy as an analysis tool for non-solid tissue carried out for the purpose of providing information about solid tumors was introduced approximately 20 years ago. Additional to the detection of circulating tumor cells (CTCs), the liquid biopsy approach quickly included the analysis of circulating tumor DNA (ctDNA) and other tumor-derived markers such as circulating cell-free RNA or extracellular vesicles. Liquid biopsy is a non-invasive technique for detecting multiple cancer-associated biomarkers that is easy to obtain and can reflect the characteristics of the entire tumor mass. Currently, ctDNA is the key component of the liquid biopsy approach from the point of view of the prognosis assessment, prediction, and monitoring of the treatment of non-small-cell lung cancer (NSCLC) patients. ctDNA in NSCLC patients carries variants or rearrangements that drive carcinogenesis, such as those in EGFR, KRAS, ALK, or ROS1. Due to advances in pharmacology, these variants are the subject of targeted therapy. Therefore, the detection of these variants has gained attention in clinical medicine. Recently, methods based on qPCR (ddPCR, BEAMing) and next-generation sequencing (NGS) are the most effective approaches for ctDNA analysis. This review addresses various aspects of the use of liquid biopsy with an emphasis on ctDNA as a biomarker in NSCLC patients. Full article
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23 pages, 1309 KiB  
Review
Neutrophil Extracellular Traps and Neutrophil-Derived Extracellular Vesicles: Common Players in Neutrophil Effector Functions
by Heiko Pfister
Diagnostics 2022, 12(7), 1715; https://doi.org/10.3390/diagnostics12071715 - 14 Jul 2022
Cited by 10 | Viewed by 5527
Abstract
Neutrophil granulocytes are a central component of the innate immune system. In recent years, they have gained considerable attention due to newly discovered biological effector functions and their involvement in various pathological conditions. They have been shown to trigger mechanisms that can either [...] Read more.
Neutrophil granulocytes are a central component of the innate immune system. In recent years, they have gained considerable attention due to newly discovered biological effector functions and their involvement in various pathological conditions. They have been shown to trigger mechanisms that can either promote or inhibit the development of autoimmunity, thrombosis, and cancer. One mechanism for their modulatory effect is the release of extracellular vesicles (EVs), that trigger appropriate signaling pathways in immune cells and other target cells. In addition, activated neutrophils can release bactericidal DNA fibers decorated with proteins from neutrophil granules (neutrophil extracellular traps, NETs). While NETs are very effective in limiting pathogens, they can also cause severe damage if released in excess or cleared inefficiently. Since NETs and EVs share a variety of neutrophil molecules and initially act in the same microenvironment, differential biochemical and functional analysis is particularly challenging. This review focuses on the biochemical and functional parallels and the extent to which the overlapping spectrum of effector molecules has an impact on biological and pathological effects. Full article
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13 pages, 314 KiB  
Review
Clinical Significance of Clonal Hematopoiesis of Indeterminate Potential in Hematology and Cardiovascular Disease
by Gregor Hoermann
Diagnostics 2022, 12(7), 1613; https://doi.org/10.3390/diagnostics12071613 - 2 Jul 2022
Cited by 6 | Viewed by 2844
Abstract
Liquid profiling uses circulating tumor DNA (ctDNA) for minimal invasive tumor mutational profiling from peripheral blood. The presence of somatic mutations in peripheral blood cells without further evidence of a hematologic neoplasm defines clonal hematopoiesis of indeterminate potential (CHIP). CHIP-mutations can be found [...] Read more.
Liquid profiling uses circulating tumor DNA (ctDNA) for minimal invasive tumor mutational profiling from peripheral blood. The presence of somatic mutations in peripheral blood cells without further evidence of a hematologic neoplasm defines clonal hematopoiesis of indeterminate potential (CHIP). CHIP-mutations can be found in the cell-free DNA (cfDNA) of plasma, are a potential cause of false positive results in liquid profiling, and thus limit its usage in screening settings. Various strategies are in place to mitigate the effect of CHIP on the performance of ctDNA assays, but the detection of CHIP also represents a clinically significant incidental finding. The sequelae of CHIP comprise the risk of progression to a hematologic neoplasm including therapy-related myeloid neoplasms. While the hematological risk increases with the co-occurrence of unexplained blood count abnormalities, a number of non-hematologic diseases have independently been associated with CHIP. In particular, CHIP represents a major risk factor for cardiovascular disease such as atherosclerosis or heart failure. The management of CHIP requires an interdisciplinary setting and represents a new topic in the field of cardio-oncology. In the future, the information on CHIP may be taken into account for personalized therapy of cancer patients. Full article
17 pages, 906 KiB  
Review
The Utility of Repetitive Cell-Free DNA in Cancer Liquid Biopsies
by Ugur Gezer, Abel J. Bronkhorst and Stefan Holdenrieder
Diagnostics 2022, 12(6), 1363; https://doi.org/10.3390/diagnostics12061363 - 1 Jun 2022
Cited by 9 | Viewed by 3094
Abstract
Liquid biopsy is a broad term that refers to the testing of body fluids for biomarkers that correlate with a pathological condition. While a variety of body-fluid components (e.g., circulating tumor cells, extracellular vesicles, RNA, proteins, and metabolites) are studied as potential liquid [...] Read more.
Liquid biopsy is a broad term that refers to the testing of body fluids for biomarkers that correlate with a pathological condition. While a variety of body-fluid components (e.g., circulating tumor cells, extracellular vesicles, RNA, proteins, and metabolites) are studied as potential liquid biopsy biomarkers, cell-free DNA (cfDNA) has attracted the most attention in recent years. The total cfDNA population in a typical biospecimen represents an immensely rich source of biological and pathological information and has demonstrated significant potential as a versatile biomarker in oncology, non-invasive prenatal testing, and transplant monitoring. As a significant portion of cfDNA is composed of repeat DNA sequences and some families (e.g., pericentric satellites) were recently shown to be overrepresented in cfDNA populations vs their genomic abundance, it holds great potential for developing liquid biopsy-based biomarkers for the early detection and management of patients with cancer. By outlining research that employed cell-free repeat DNA sequences, in particular the ALU and LINE-1 elements, we highlight the clinical potential of the repeat-element content of cfDNA as an underappreciated marker in the cancer liquid biopsy repertoire. Full article
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15 pages, 1418 KiB  
Review
The History and Future of Basic and Translational Cell-Free DNA Research at a Glance
by Peter B. Gahan, Heidi Schwarzenbach and Philippe Anker
Diagnostics 2022, 12(5), 1192; https://doi.org/10.3390/diagnostics12051192 - 10 May 2022
Cited by 4 | Viewed by 1824
Abstract
We discuss the early history of the structure of DNA and its involvement in gene structure as well as its mobility in and between cells and between tissues in the form of circulating cell-free DNA (cfDNA). This is followed by a view of [...] Read more.
We discuss the early history of the structure of DNA and its involvement in gene structure as well as its mobility in and between cells and between tissues in the form of circulating cell-free DNA (cfDNA). This is followed by a view of the present status of the studies on cfDNA and clinical applications of circulating cell-free tumor DNA (ctDNA). The future developments and roles of ctDNA are also considered. Full article
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12 pages, 1153 KiB  
Review
Cell-Free DNA Fragmentomics in Liquid Biopsy
by Spencer C. Ding and Y.M. Dennis Lo
Diagnostics 2022, 12(4), 978; https://doi.org/10.3390/diagnostics12040978 - 13 Apr 2022
Cited by 43 | Viewed by 7888
Abstract
Cell-free DNA (cfDNA) in bodily fluids has rapidly transformed the development of noninvasive prenatal testing, cancer liquid biopsy, and transplantation monitoring. Plasma cfDNA consists of a mixture of molecules originating from various bodily tissues. The study of the fragmentation patterns of cfDNA, also [...] Read more.
Cell-free DNA (cfDNA) in bodily fluids has rapidly transformed the development of noninvasive prenatal testing, cancer liquid biopsy, and transplantation monitoring. Plasma cfDNA consists of a mixture of molecules originating from various bodily tissues. The study of the fragmentation patterns of cfDNA, also referred to as ‘fragmentomics’, is now an actively pursued area of biomarker research. Clues that cfDNA fragmentation patterns might carry information concerning the tissue of origin of cfDNA molecules have come from works demonstrating that circulating fetal, tumor-derived, and transplanted liver-derived cfDNA molecules have a shorter size distribution than the background mainly of hematopoietic origin. More recently, an improved understanding of cfDNA fragmentation has provided many emerging fragmentomic markers, including fragment sizes, preferred ends, end motifs, single-stranded jagged ends, and nucleosomal footprints. The intrinsic biological link between activities of various DNA nucleases and characteristic fragmentations has been demonstrated. In this review, we focus on the biological properties of cell-free DNA unveiled recently and their potential clinical applications. Full article
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25 pages, 1846 KiB  
Review
Combinatorial Power of cfDNA, CTCs and EVs in Oncology
by Corinna Keup, Rainer Kimmig and Sabine Kasimir-Bauer
Diagnostics 2022, 12(4), 870; https://doi.org/10.3390/diagnostics12040870 - 31 Mar 2022
Cited by 17 | Viewed by 2739
Abstract
Liquid biopsy is a promising technique for clinical management of oncological patients. The diversity of analytes circulating in the blood useable for liquid biopsy testing is enormous. Circulating tumor cells (CTCs), cell-free DNA (cfDNA) and extracellular vesicles (EVs), as well as blood cells [...] Read more.
Liquid biopsy is a promising technique for clinical management of oncological patients. The diversity of analytes circulating in the blood useable for liquid biopsy testing is enormous. Circulating tumor cells (CTCs), cell-free DNA (cfDNA) and extracellular vesicles (EVs), as well as blood cells and other soluble components in the plasma, were shown as liquid biopsy analytes. A few studies directly comparing two liquid biopsy analytes showed a benefit of one analyte over the other, while most authors concluded the benefit of the additional analyte. Only three years ago, the first studies to examine the value of a characterization of more than two liquid biopsy analytes from the same sample were conducted. We attempt to reflect on the recent development of multimodal liquid biopsy testing in this review. Although the analytes and clinical purposes of the published multimodal studies differed significantly, the additive value of the analytes was concluded in almost all projects. Thus, the blood components, as liquid biopsy reservoirs, are complementary rather than competitive, and orthogonal data sets were even shown to harbor synergistic effects. The unmistakable potential of multimodal liquid biopsy testing, however, is dampened by its clinical utility, which is yet to be proven, the lack of methodical standardization and insufficiently mature reimbursement, logistics and data handling. Full article
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15 pages, 635 KiB  
Review
Liquid Profiling for Cancer Patient Stratification in Precision Medicine—Current Status and Challenges for Successful Implementation in Standard Care
by Verena Haselmann, Maren Hedtke and Michael Neumaier
Diagnostics 2022, 12(3), 748; https://doi.org/10.3390/diagnostics12030748 - 19 Mar 2022
Cited by 9 | Viewed by 2326
Abstract
Circulating tumor DNA (ctDNA), accurately described by the term liquid profiling (LP), enables real-time assessment of the tumor mutational profile as a minimally invasive test and has therefore rapidly gained traction, particular for the management of cancer patients. By LP, tumor-specific genetic alterations [...] Read more.
Circulating tumor DNA (ctDNA), accurately described by the term liquid profiling (LP), enables real-time assessment of the tumor mutational profile as a minimally invasive test and has therefore rapidly gained traction, particular for the management of cancer patients. By LP, tumor-specific genetic alterations can be determined as part of companion diagnostics to guide selection of appropriate targeted therapeutics. Because LP facilitates longitudinal monitoring of cancer patients, it can be used to detect acquired resistant mechanisms or as a personalized biomarker for earlier detection of disease recurrence, among other applications. However, LP is not yet integrated into routine care to the extent that might be expected. This is due to the lack of harmonization and standardization of preanalytical and analytical workflows, the lack of proper quality controls, limited evidence of its clinical utility, heterogeneous study results, the uncertainty of clinicians regarding the value and appropriate indications for LP and its interpretation, and finally, the lack of reimbursement for most LP tests. In this review, the value proposition of LP for cancer patient management and treatment optimization, the current status of implementation in standard care, and the main challenges that need to be overcome are discussed in detail. Full article
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4 pages, 211 KiB  
Reply
Multi Cancer Early Detection by Using Circulating Tumor DNA—The Galleri Test. Reply to Klein et al. The Promise of Multicancer Early Detection. Comment on “Pons-Belda et al. Can Circulating Tumor DNA Support a Successful Screening Test for Early Cancer Detection? The Grail Paradigm. Diagnostics 2021, 11, 2171”
by Oscar D. Pons-Belda, Amaia Fernandez-Uriarte and Eleftherios P. Diamandis
Diagnostics 2022, 12(5), 1244; https://doi.org/10.3390/diagnostics12051244 - 17 May 2022
Cited by 12 | Viewed by 3475
Abstract
We recently published some concerns with new technologies which are based on circulating tumor DNA (ctDNA) for early cancer detection. Most of our published criticism, including a commentary in this journal, has focused on tests developed by the biotechnology company GRAIL (their commercial [...] Read more.
We recently published some concerns with new technologies which are based on circulating tumor DNA (ctDNA) for early cancer detection. Most of our published criticism, including a commentary in this journal, has focused on tests developed by the biotechnology company GRAIL (their commercial product is also known as The Galleri Test). Scientists from GRAIL provided explanations and rebuttals to our criticism. They also posed some questions. Here, we reiterate our position and provide rebuttals, explanations and answers to these questions. We believe that constructive scientific debates, like this one, can profoundly contribute to advancements in scientific fields such as early cancer detection. Full article
5 pages, 1526 KiB  
Comment
The Promise of Multicancer Early Detection. Comment on Pons-Belda et al. Can Circulating Tumor DNA Support a Successful Screening Test for Early Cancer Detection? The Grail Paradigm. Diagnostics 2021, 11, 2171
by Eric A. Klein, Tomasz M. Beer and Michael Seiden
Diagnostics 2022, 12(5), 1243; https://doi.org/10.3390/diagnostics12051243 - 17 May 2022
Cited by 9 | Viewed by 3377
Abstract
Multicancer Early Detection (MCED) represents a new and exciting paradigm for the early detection of cancer, which is the leading cause of death worldwide. Current screening tests, recommended for only five cancer types (breast, lung, colon, cervical, and prostate), are limited by a [...] Read more.
Multicancer Early Detection (MCED) represents a new and exciting paradigm for the early detection of cancer, which is the leading cause of death worldwide. Current screening tests, recommended for only five cancer types (breast, lung, colon, cervical, and prostate), are limited by a lack of complete adherence to guideline-based use and by the fact that they have cumulative high false positive rates. MCED tests agnostically detect cancer signals in the blood with good sensitivity and low false positive rates, can predict the cancer site of origin with high accuracy, can detect highly lethal cancers that have no current screening tests, and promise to improve cancer screening by improving efficiency and reducing the overall number needed to screen. Herein we outline this promise and clarify several published misconceptions about this field. Full article
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8 pages, 1089 KiB  
Commentary
Can Circulating Tumor DNA Support a Successful Screening Test for Early Cancer Detection? The Grail Paradigm
by Oscar D. Pons-Belda, Amaia Fernandez-Uriarte and Eleftherios P. Diamandis
Diagnostics 2021, 11(12), 2171; https://doi.org/10.3390/diagnostics11122171 - 23 Nov 2021
Cited by 23 | Viewed by 3899
Abstract
Circulating tumor DNA (ctDNA) is a new pan-cancer tumor marker with important applications for patient prognosis, monitoring progression, and assessing the success of the therapeutic response. Another important goal is an early cancer diagnosis. There is currently a debate if ctDNA can be [...] Read more.
Circulating tumor DNA (ctDNA) is a new pan-cancer tumor marker with important applications for patient prognosis, monitoring progression, and assessing the success of the therapeutic response. Another important goal is an early cancer diagnosis. There is currently a debate if ctDNA can be used for early cancer detection due to the small tumor burden and low mutant allele fraction (MAF). We compare our previous calculations on the size of detectable cancers by ctDNA analysis with the latest experimental data from Grail’s clinical trial. Current ctDNA-based diagnostic methods could predictably detect tumors of sizes greater than 10–15 mm in diameter. When tumors are of this size or smaller, their MAF is about 0.01% (one tumor DNA molecule admixed with 10,000 normal DNA molecules). The use of 10 mL of blood (4 mL of plasma) will likely contain less than a complete cancer genome, thus rendering the diagnosis of cancer impossible. Grail’s new data confirm the low sensitivity for early cancer detection (<30% for Stage I–II tumors, <20% for Stage I tumors), but specificity was high at 99.5%. According to these latest data, the sensitivity of the Grail test is less than 20% in Stage I disease, casting doubt if this test could become a viable pan-cancer clinical screening tool. Full article
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