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Tomaskovic A, Weber V, Ochmann DT, Neuberger EW, Lachtermann E, Brahmer A, Haller N, Hillen B, Enders K, Eggert V, Zeier P, Lieb K, Simon P. Multimodal Web-Based Telerehabilitation for Patients With Post-COVID-19 Condition: Protocol for a Randomized Controlled Trial. JMIR Res Protoc 2025; 14:e65044. [PMID: 40397936 DOI: 10.2196/65044] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2024] [Revised: 03/22/2025] [Accepted: 03/26/2025] [Indexed: 05/23/2025] Open
Abstract
BACKGROUND Patients with post-COVID-19 condition (PCC) experience persistent, long-term health consequences following SARS-CoV-2 infection, including fatigue, hyperventilation, cognitive impairment, and limitations in daily activities. There is emerging evidence suggesting that exercise and respiratory therapy-based telerehabilitation is safe and could potentially improve physical capacity while reducing health care costs. OBJECTIVE This study aims to evaluate the superiority of a multimodal, symptom-titrated telerehabilitation program over standard care in patients with PCC who are severely affected, using the highest oxygen uptake rate (VO2peak [mL/min/kg]) achieved during the cardiopulmonary exercise test (CPET) and minute ventilation/carbon dioxide production slope (VE/VCO2 [full slope]) as primary outcomes. In addition, this study seeks to provide novel insights into the clinical and physiological adaptations associated with PCC, informing future rehabilitation strategies. METHODS This prospective, randomized, waitlist-controlled trial was approved by the Rhineland-Palatinate Medical Association ethics committee. All procedures comply with the Declaration of Helsinki. This study comprises 3 examination time points, which include patient-reported outcomes, clinical assessments, and a CPET. It is structured into an 8-week intervention phase followed by an 8-week follow-up phase. Following baseline assessment, patients will be randomly assigned to either the intervention group (IG) or the control group (CG). During the intervention phase, IG participants will receive a web-based, multimodal, symptom-titrated telerehabilitation program consisting of sports medicine consultations, weekly teleconsultations, a structured pacing approach, and exercise and respiratory therapy. In contrast, CG participants will receive treatment as usual, which includes a single sports medicine consultation on healthy habits and a self-directed pacing approach for managing symptoms and daily activities. During the follow-up phase, IG participants will continue training independently without teleconsultations, whereas CG participants will undergo the same telerehabilitation intervention as the IG. A follow-up assessment will be conducted for both groups to evaluate long-term effects. This study adheres to the SPIRIT (Standard Protocol Items: Recommendations for Interventional Trials) guidelines and follows the Consensus on Exercise Reporting Template. RESULTS Recruitment began in August 2023 and was extended until March 2025. As of March 2025, 80 participants have been recruited, and data analysis is ongoing. Final results are expected by December 2025, with a cross-sectional analysis of baseline data anticipated by July 2025. CONCLUSIONS This study is the first randomized controlled trial investigating the effectiveness of multimodal and symptom-titrated telerehabilitation in patients with PCC who are severely affected. The integration of various objective diagnostic systems will provide valuable insights into emerging postviral fatigue syndromes, supporting the development of CPET-based diagnostics, personalized rehabilitation strategies, and future research on long-term telerehabilitation effectiveness. The findings will be disseminated through peer-reviewed publications, professional networks, and patient advocacy groups to ensure scientific, clinical, and public impact. TRIAL REGISTRATION German Clinical Trials Register (DRKS) DRKS00032394; https://drks.de/search/de/trial/DRKS00032394. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID) DERR1-10.2196/65044.
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Affiliation(s)
- Aleksandar Tomaskovic
- Department of Sports Medicine, Prevention and Rehabilitation, Institute of Sport Science, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Vincent Weber
- Department of Sports Medicine, Prevention and Rehabilitation, Institute of Sport Science, Johannes Gutenberg University Mainz, Mainz, Germany
| | - David T Ochmann
- Department of Sports Medicine, Prevention and Rehabilitation, Institute of Sport Science, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Elmo Wanja Neuberger
- Department of Sports Medicine, Prevention and Rehabilitation, Institute of Sport Science, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Ella Lachtermann
- Department of Sports Medicine, Prevention and Rehabilitation, Institute of Sport Science, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Alexandra Brahmer
- Department of Sports Medicine, Prevention and Rehabilitation, Institute of Sport Science, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Nils Haller
- Department of Sports Medicine, Prevention and Rehabilitation, Institute of Sport Science, Johannes Gutenberg University Mainz, Mainz, Germany
- Department of Sport and Exercise Science, University of Salzburg, Salzburg, Austria
- Division of Exercise and Movement Science, Institute for Sport Science, University of Göttingen, Göttingen, Germany
| | - Barlo Hillen
- Department of Sports Medicine, Prevention and Rehabilitation, Institute of Sport Science, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Kira Enders
- Department of Sports Medicine, Prevention and Rehabilitation, Institute of Sport Science, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Viktoria Eggert
- Department of Sports Medicine, Prevention and Rehabilitation, Institute of Sport Science, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Peter Zeier
- Department of Clinical Psychology and Neuropsychology, Institute of Psychology, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Klaus Lieb
- Department of Psychiatry and Psychotherapy, Leibniz Institute for Resilience Research, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - Perikles Simon
- Department of Sports Medicine, Prevention and Rehabilitation, Institute of Sport Science, Johannes Gutenberg University Mainz, Mainz, Germany
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Macías M, Alba-Linares JJ, Acha B, Blanco-Luquin I, Fernández AF, Álvarez-Jiménez J, Urdánoz-Casado A, Roldan M, Robles M, Cabezon-Arteta E, Alcolea D, de Gordoa JSR, Corroza J, Cabello C, Erro ME, Jericó I, Fraga MF, Mendioroz M. Advancing Personalized Medicine in Alzheimer's Disease: Liquid Biopsy Epigenomics Unveil APOE ε4-Linked Methylation Signatures. Int J Mol Sci 2025; 26:3419. [PMID: 40244264 PMCID: PMC11989983 DOI: 10.3390/ijms26073419] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2025] [Revised: 03/31/2025] [Accepted: 04/03/2025] [Indexed: 04/18/2025] Open
Abstract
Recent studies show that patients with Alzheimer's disease (AD) harbor specific methylation marks in the brain that, if accessible, could be used as epigenetic biomarkers. Liquid biopsy enables the study of circulating cell-free DNA (cfDNA) fragments originated from dead cells, including neurons affected by neurodegenerative processes. Here, we isolated and epigenetically characterized plasma cfDNA from 35 patients with AD and 35 cognitively healthy controls by using the Infinium® MethylationEPIC BeadChip array. Bioinformatics analysis was performed to identify differential methylation positions (DMPs) and regions (DMRs), including APOE ε4 genotype stratified analysis. Plasma pTau181 (Simoa) and cerebrospinal fluid (CSF) core biomarkers (Fujirebio) were also measured and correlated with differential methylation marks. Validation was performed with bisulfite pyrosequencing and bisulfite cloning sequencing. Epigenome-wide cfDNA analysis identified 102 DMPs associated with AD status. Most DMPs correlated with clinical cognitive and functional tests including 60% for Mini-Mental State Examination (MMSE) and 80% for Global Deterioration Scale (GDS), and with AD blood and CSF biomarkers. In silico functional analysis connected 30 DMPs to neurological processes, identifying key regulators such as SPTBN4 and APOE genes. Several DMRs were annotated to genes previously reported to harbor epigenetic brain changes in AD (HKR1, ZNF154, HOXA5, TRIM40, ATG16L2, ADAMST2) and were linked to APOE ε4 genotypes. Notably, a DMR in the HKR1 gene, previously shown to be hypermethylated in the AD hippocampus, was validated in cfDNA from an orthogonal perspective. These results support the feasibility of studying cfDNA to identify potential epigenetic biomarkers in AD. Thus, liquid biopsy could improve non-invasive AD diagnosis and aid personalized medicine by detecting epigenetic brain markers in blood.
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Affiliation(s)
- Mónica Macías
- Neuroepigenetics Unit, Navarrabiomed, Hospital Universitario de Navarra, Universidad Pública de Navarra, Navarra Institute for Health Research (IdiSNA), 31008 Pamplona, Spain
| | - Juan José Alba-Linares
- Cancer Epigenetics and Nanomedicine Laboratory, Nanomaterials and Nanotechnology Research Center (CINN CSIC), 33940 El Entrego, Spain
- Health Research Institute of Asturias (ISPA FINBA), University of Oviedo, 33011 Oviedo, Spain
- Institute of Oncology of Asturias (IUOPA), University of Oviedo, 33006 Oviedo, Spain
- Rare Diseases CIBER (CIBERER) of the Carlos III Health Institute (ISCIII), 28029 Madrid, Spain
| | - Blanca Acha
- Neuroepigenetics Unit, Navarrabiomed, Hospital Universitario de Navarra, Universidad Pública de Navarra, Navarra Institute for Health Research (IdiSNA), 31008 Pamplona, Spain
| | - Idoia Blanco-Luquin
- Neuroepigenetics Unit, Navarrabiomed, Hospital Universitario de Navarra, Universidad Pública de Navarra, Navarra Institute for Health Research (IdiSNA), 31008 Pamplona, Spain
| | - Agustín F. Fernández
- Cancer Epigenetics and Nanomedicine Laboratory, Nanomaterials and Nanotechnology Research Center (CINN CSIC), 33940 El Entrego, Spain
- Health Research Institute of Asturias (ISPA FINBA), University of Oviedo, 33011 Oviedo, Spain
- Institute of Oncology of Asturias (IUOPA), University of Oviedo, 33006 Oviedo, Spain
- Rare Diseases CIBER (CIBERER) of the Carlos III Health Institute (ISCIII), 28029 Madrid, Spain
| | - Johana Álvarez-Jiménez
- Neuroepigenetics Unit, Navarrabiomed, Hospital Universitario de Navarra, Universidad Pública de Navarra, Navarra Institute for Health Research (IdiSNA), 31008 Pamplona, Spain
| | - Amaya Urdánoz-Casado
- Neuroepigenetics Unit, Navarrabiomed, Hospital Universitario de Navarra, Universidad Pública de Navarra, Navarra Institute for Health Research (IdiSNA), 31008 Pamplona, Spain
| | - Miren Roldan
- Neuroepigenetics Unit, Navarrabiomed, Hospital Universitario de Navarra, Universidad Pública de Navarra, Navarra Institute for Health Research (IdiSNA), 31008 Pamplona, Spain
| | - Maitane Robles
- Neuroepigenetics Unit, Navarrabiomed, Hospital Universitario de Navarra, Universidad Pública de Navarra, Navarra Institute for Health Research (IdiSNA), 31008 Pamplona, Spain
| | - Eneko Cabezon-Arteta
- Neuroepigenetics Unit, Navarrabiomed, Hospital Universitario de Navarra, Universidad Pública de Navarra, Navarra Institute for Health Research (IdiSNA), 31008 Pamplona, Spain
| | - Daniel Alcolea
- Department of Neurology, Institut d’Investigacions Biomèdiques Sant Pau (IIB Sant Pau), Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, 08025 Barcelona, Spain
- Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas, CIBERNED, 28029 Madrid, Spain
| | - Javier Sánchez Ruiz de Gordoa
- Neuroepigenetics Unit, Navarrabiomed, Hospital Universitario de Navarra, Universidad Pública de Navarra, Navarra Institute for Health Research (IdiSNA), 31008 Pamplona, Spain
- Neurology Department, Hospital Universitario de Navarra, Universidad Pública de Navarra, Navarra Institute for Health Research (IdiSNA), 31008 Pamplona, Spain
| | - Jon Corroza
- Neurology Department, Hospital Universitario de Navarra, Universidad Pública de Navarra, Navarra Institute for Health Research (IdiSNA), 31008 Pamplona, Spain
| | - Carolina Cabello
- Neuroepigenetics Unit, Navarrabiomed, Hospital Universitario de Navarra, Universidad Pública de Navarra, Navarra Institute for Health Research (IdiSNA), 31008 Pamplona, Spain
- Neurology Department, Hospital Universitario de Navarra, Universidad Pública de Navarra, Navarra Institute for Health Research (IdiSNA), 31008 Pamplona, Spain
| | - María Elena Erro
- Neuroepigenetics Unit, Navarrabiomed, Hospital Universitario de Navarra, Universidad Pública de Navarra, Navarra Institute for Health Research (IdiSNA), 31008 Pamplona, Spain
- Neurology Department, Hospital Universitario de Navarra, Universidad Pública de Navarra, Navarra Institute for Health Research (IdiSNA), 31008 Pamplona, Spain
| | - Ivonne Jericó
- Neurology Department, Hospital Universitario de Navarra, Universidad Pública de Navarra, Navarra Institute for Health Research (IdiSNA), 31008 Pamplona, Spain
| | - Mario F. Fraga
- Cancer Epigenetics and Nanomedicine Laboratory, Nanomaterials and Nanotechnology Research Center (CINN CSIC), 33940 El Entrego, Spain
- Health Research Institute of Asturias (ISPA FINBA), University of Oviedo, 33011 Oviedo, Spain
- Institute of Oncology of Asturias (IUOPA), University of Oviedo, 33006 Oviedo, Spain
- Rare Diseases CIBER (CIBERER) of the Carlos III Health Institute (ISCIII), 28029 Madrid, Spain
- Department of Organisms and Systems Biology (B.O.S.), University of Oviedo, 33006 Oviedo, Spain
| | - Maite Mendioroz
- Neuroepigenetics Unit, Navarrabiomed, Hospital Universitario de Navarra, Universidad Pública de Navarra, Navarra Institute for Health Research (IdiSNA), 31008 Pamplona, Spain
- Neurology Department, Hospital Universitario de Navarra, Universidad Pública de Navarra, Navarra Institute for Health Research (IdiSNA), 31008 Pamplona, Spain
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Passemiers A, Tuveri S, Jatsenko T, Vanderstichele A, Busschaert P, Coosemans A, Timmerman D, Tejpar S, Vandenberghe P, Lambrechts D, Raimondi D, Vermeesch JR, Moreau Y. DAGIP: alleviating cell-free DNA sequencing biases with optimal transport. Genome Biol 2025; 26:49. [PMID: 40055826 PMCID: PMC11887355 DOI: 10.1186/s13059-025-03511-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2023] [Accepted: 02/21/2025] [Indexed: 05/13/2025] Open
Abstract
Cell-free DNA (cfDNA) is a rich source of biomarkers for various pathophysiological conditions. Preanalytical variables, such as the library preparation protocol or sequencing platform, are major confounders of cfDNA analysis. We present DAGIP, a novel data correction method that builds on optimal transport theory and deep learning, which explicitly corrects for the effect of such preanalytical variables and can infer technical biases. Our method improves cancer detection and copy number alteration analysis by alleviating the sources of variation that are not of biological origin. It also enhances fragmentomic analysis of cfDNA. DAGIP allows the integration of cohorts from different studies.
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Affiliation(s)
- Antoine Passemiers
- Dynamical Systems, Signal Processing and Data Analytics (STADIUS), KU Leuven, Leuven, Belgium.
| | - Stefania Tuveri
- Laboratory for Cytogenetics and Genome Research, Department of Human Genetics, KU Leuven, Leuven, Belgium
| | - Tatjana Jatsenko
- Laboratory for Cytogenetics and Genome Research, Department of Human Genetics, KU Leuven, Leuven, Belgium
| | - Adriaan Vanderstichele
- Department of Gynaecology and Obstetrics, University Hospitals Leuven, Leuven, Belgium
- Division of Gynaecological Oncology, Leuven Cancer Institute, KU Leuven, Leuven, Belgium
| | - Pieter Busschaert
- Department of Gynaecology and Obstetrics, University Hospitals Leuven, Leuven, Belgium
- Division of Gynaecological Oncology, Leuven Cancer Institute, KU Leuven, Leuven, Belgium
| | - An Coosemans
- Department of Oncology, Laboratory of Tumor Immunology and Immunotherapy, Leuven Cancer Institute, Leuven, Belgium
| | - Dirk Timmerman
- Department of Gynaecology and Obstetrics, University Hospitals Leuven, Leuven, Belgium
| | - Sabine Tejpar
- Department of Oncology, Molecular Digestive Oncology, KU Leuven, Leuven, Belgium
| | - Peter Vandenberghe
- Department of Human Genetics, Laboratory of Genetics of Malignant Diseases, KU Leuven, Leuven, Belgium
- Department of Hematology, University Hospitals Leuven, Leuven, Belgium
| | - Diether Lambrechts
- Center for Cancer Biology, VIB, Leuven, Belgium
- Laboratory for Translational Genetics, Department of Human Genetics, KU Leuven, Leuven, Belgium
| | - Daniele Raimondi
- Dynamical Systems, Signal Processing and Data Analytics (STADIUS), KU Leuven, Leuven, Belgium
- Institut de Génétique Moléculaire de Montpellier (IGMM), Université de Montpellier, Montpellier, France
| | - Joris Robert Vermeesch
- Laboratory for Cytogenetics and Genome Research, Department of Human Genetics, KU Leuven, Leuven, Belgium
| | - Yves Moreau
- Dynamical Systems, Signal Processing and Data Analytics (STADIUS), KU Leuven, Leuven, Belgium
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Enders K, Hillen B, Haller N, Brahmer A, Weber V, Simon P, Neuberger EWI. Pre-analytical pitfalls: How blood collection tubes influence exercise-induced cell-free DNA concentrations. Exp Physiol 2025. [PMID: 40033650 DOI: 10.1113/ep092284] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2024] [Accepted: 02/12/2025] [Indexed: 03/05/2025]
Abstract
Circulating cell-free DNA (cfDNA) is a promising biomarker for physiological stress, including exercise-induced responses. However, the lack of standardization in blood collection tubes (BCTs) for quantification of cfDNA hampers inter-study comparisons. In this study, we assessed the impact of different BCTs on exercise-induced cfDNA dynamics. Eleven participants [25 (SD 2.3) years of age] performed three different treadmill exercise protocols, including an all-out test and combinations of constant and interval load. Blood samples were collected before, 5 min and 30 min post-exercise using EDTA, lithium-heparin (LH) and serum BCTs. Concentrations of cfDNA were quantified using quantitative PCR. The cfDNA increased significantly across all protocols and BCTs. A significant effect of BCT on cfDNA concentrations (P = 0.034) was found, with serum showing higher concentrations than EDTA and LH. Although absolute differences from pre- to post-exercise were comparable across BCTs (P = 0.476), fold changes differed significantly (P = 0.012), with the highest observed in EDTA and the lowest in serum. Bland-Altman analyses demonstrated better agreement between EDTA and LH compared with serum. Significant correlations of cfDNA with energy expenditure and peak oxygen uptake were found. These correlations were stronger in EDTA and LH than in serum. Our findings highlight the crucial influence of BCT choice on cfDNA measurements in exercise settings. Given that EDTA and LH reflected exercise load better, they could be preferred for exercise physiology research. This work underscores the need to account for the choice of BCT to improve data comparability across studies. Additionally, these findings might have broader implications for clinical settings where cfDNA is used as a biomarker.
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Affiliation(s)
- Kira Enders
- Department of Sports Medicine, Disease Prevention and Rehabilitation, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Barlo Hillen
- Department of Sports Medicine, Disease Prevention and Rehabilitation, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Nils Haller
- Department of Sports Medicine, Disease Prevention and Rehabilitation, Johannes Gutenberg University Mainz, Mainz, Germany
- Department of Sport and Exercise Science, University of Salzburg, Salzburg, Austria
| | - Alexandra Brahmer
- Department of Sports Medicine, Disease Prevention and Rehabilitation, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Vincent Weber
- Department of Sports Medicine, Disease Prevention and Rehabilitation, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Perikles Simon
- Department of Sports Medicine, Disease Prevention and Rehabilitation, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Elmo W I Neuberger
- Department of Sports Medicine, Disease Prevention and Rehabilitation, Johannes Gutenberg University Mainz, Mainz, Germany
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Osei-Poku P, Tritten L, Fordjour F, Kwarteng A. Cell-free DNA as a complementary diagnostic tool for neglected tropical diseases towards achieving the WHO NTDs elimination by 2030. THE JOURNAL OF LIQUID BIOPSY 2025; 7:100283. [PMID: 40027229 PMCID: PMC11863940 DOI: 10.1016/j.jlb.2024.100283] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/14/2024] [Revised: 12/09/2024] [Accepted: 12/11/2024] [Indexed: 03/05/2025]
Abstract
Neglected Tropical Diseases (NTDs) continue to ravage the poorest regions of the world, with over 600 million people being affected in Sub-Saharan Africa. The global burden of NTDs within these regions is staggering, particularly post-COVID-19 pandemic, where the emerging infection intercepted the existing eradication efforts and protocols such as the Mass Drug Administration (MDA). This further complicated the approaches laid down to achieve the endgame program of eliminating the neglect and transmission of NTDs. To compensate for the detriment of COVID-19's interruption, accurate and timely diagnoses play a vital role in attaining the objectives of the WHO's goal of NTD elimination by 2030. To this effect, alternative approaches in diagnostics are urgently needed, particularly with the inadequacy of current diagnostic strategies for NTDs. Cell-free DNA (cfDNA) has shown great promise in detecting NTDs. Several studies have demonstrated its potential for diagnosing diseases such as malaria, leishmaniasis, and schistosomiasis. However, the adoption of cfDNA in NTD research faces several challenges, including the cost of the procedure, standardization, and technical expertise. Proper capacity building and training can mitigate some of these challenges. However, despite these limitations, the affordability of cfDNA detection is improving due to increased awareness of the approach and researchers' integration considerations into current diagnostic routines. In conclusion, while there are challenges to adopting cfDNA in NTD research, it remains a promising alternative strategy to be considered in the fight against NTDs.
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Affiliation(s)
- Priscilla Osei-Poku
- Department of Biochemistry and Biotechnology, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana
- Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR), Kumasi, Ghana
| | - Lucienne Tritten
- Institute of Parasitology, McGill University, Sainte-Anne-de-Bellevue, Quebec, Canada
| | - Fatima Fordjour
- Department of Biochemistry and Biotechnology, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana
- Department of Microbiology, University for Development Studies, Ghana
| | - Alexander Kwarteng
- Department of Biochemistry and Biotechnology, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana
- Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR), Kumasi, Ghana
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Asante DB, Tierno D, Grassi G, Scaggiante B. Circulating Tumour DNA for Ovarian Cancer Diagnosis and Treatment Monitoring: What Perspectives for Clinical Use? Int J Mol Sci 2025; 26:1889. [PMID: 40076521 PMCID: PMC11900478 DOI: 10.3390/ijms26051889] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2024] [Revised: 02/04/2025] [Accepted: 02/19/2025] [Indexed: 03/14/2025] Open
Abstract
Globally, ovarian cancer (OC) is the eighth most common malignant tumour in women. Unfortunately, its symptoms-especially at the early stages-are vague and non-specific, and, thus, most patients are diagnosed at the advanced stages of the disease (stage III and IV) when treatment is not curative. The currently available approved biomarkers are not sufficient for effective screening, prognosis, or monitoring of OC. Liquid biopsy tests such as circulating tumour DNA (ctDNA) analysis has the advantage of monitoring response to treatment in real time and providing a comprehensive genotypic profile of primary, metastatic, and recurrent tumours. Thus, ctDNA analysis can be used as a complementary test for effective diagnosis and monitoring of OC. We comprehensively review current studies (2019-2024) on OC, critically highlighting recent developments and applications of ctDNA for the diagnosis and management of the disease.
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Affiliation(s)
- Du-Bois Asante
- Department of Biomedical and Forensic Sciences, University of Cape Coast, Cape Coast P.O. Box CCLN 33, Ghana;
| | - Domenico Tierno
- Department of Medicine, Surgery and Health Sciences, University of Trieste, Strada di Fiume 447, I-34149 Trieste, Italy; (D.T.); (G.G.)
| | - Gabriele Grassi
- Department of Medicine, Surgery and Health Sciences, University of Trieste, Strada di Fiume 447, I-34149 Trieste, Italy; (D.T.); (G.G.)
| | - Bruna Scaggiante
- Department of Life Sciences, University of Trieste, Via Valerio 28, I-34127 Trieste, Italy
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Soleymani S, Naghib SM, Mozafari MR. Circulating Tumor Cells in Cancer Diagnosis, Therapy, and Theranostics Applications: An Overview of Emerging Materials and Technologies. Curr Pharm Des 2025; 31:674-690. [PMID: 39473210 DOI: 10.2174/0113816128328459241009191933] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2024] [Accepted: 09/06/2024] [Indexed: 04/11/2025]
Abstract
In recent years, immunotherapy, namely immune checkpoint inhibitor therapy, has significantly transformed the approach to treating various forms of cancer. Simultaneously, the adoption of clinical oncology has been sluggish due to the exorbitant expense of therapy, the adverse effects experienced by patients, and the inconsistency in treatment response among individuals. As a reaction, individualized methods utilizing predictive biomarkers have arisen as novel strategies for categorizing patients to achieve successful immunotherapy. Recently, the identification and examination of circulating tumor cells (CTCs) have gained attention as predictive indicators for the treatment of cancer patients undergoing chemotherapy and for personalized targeted therapy. CTCs have been found to exhibit immunological checkpoints in several types of solid tumors, which has contributed to our understanding of managing cancer immunotherapy. Circulating tumor cells (CTCs) present in the bloodstream have a crucial function in the formation of metastases. Nevertheless, the practical usefulness of existing CTC tests is mostly restricted by methodological limitations.
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Affiliation(s)
- Sina Soleymani
- Nanotechnology Department, School of Advanced Technologies, Iran University of Science and Technology, Tehran, Iran
| | - Seyed Morteza Naghib
- Nanotechnology Department, School of Advanced Technologies, Iran University of Science and Technology, Tehran, Iran
| | - M R Mozafari
- Australasian Nanoscience and Nanotechnology Initiative (ANNI), Monash University LPO, Clayton, VIC 3168, Australia
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Sue T, Ichikawa T, Hattori S, Otani H, Fujimura S, Higuchi T, Okumura N, Higuchi Y. Quantitative evaluation of citrullinated fibrinogen for detection of neutrophil extracellular traps. Immunol Res 2024; 72:409-417. [PMID: 38087184 DOI: 10.1007/s12026-023-09446-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2023] [Accepted: 11/28/2023] [Indexed: 07/03/2024]
Abstract
Activated neutrophils release neutrophil extracellular traps (NETs) composed of chromatin filaments containing bactericidal proteins and enzymes. This process, known as NETosis, is an innate host defense mechanism. However, NET accumulation can lead to uncontrolled inflammation and organ damage. Therefore, NET detection provides clinically important information for the assessment of inflammatory conditions. We investigated whether quantification of citrullinated fibrinogen (C-Fbg), which is catalyzed by peptidylarginine deiminase (PAD) released during NETosis, can be used to detect NETs. Human neutrophils were stimulated with fibrinogen using phorbol 12-myristate 13-acetate (PMA). The myeloperoxidase (MPO)-DNA complex and C-Fbg concentrations in the culture supernatants were quantified using an enzyme-linked immunosorbent assay. The protein levels of peptidylarginine deiminase 2 and 4 in culture supernatants and mRNA levels in PMA-stimulated neutrophils were also assessed. The levels of the MPO-DNA complex in the supernatants of PMA-stimulated neutrophils increased, indicating NETosis. C-Fbg level also increased, which was suppressed by both NETosis and PAD inhibitors. PAD2 was detected in the culture supernatant; however, PAD4, but not PAD2, mRNA levels increased in PMA-stimulated neutrophils. This study quantitatively demonstrates that fibrinogen is citrullinated by PAD derived from PMA-stimulated neutrophils upon NETosis. Although further studies are needed for clinical application, quantification of C-Fbg in blood may help detect the presence of NETs.
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Affiliation(s)
- Tsubasa Sue
- Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto, 390-8621, Japan
| | - Tomoki Ichikawa
- Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto, 390-8621, Japan
- Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan
| | - Shu Hattori
- Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto, 390-8621, Japan
| | - Hikaru Otani
- Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto, 390-8621, Japan
| | - Satoshi Fujimura
- Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto, 390-8621, Japan
- Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan
| | - Tsukasa Higuchi
- Department of General Pediatrics, Nagano Children's Hospital, Azumino, Japan
- Life Science Research Center, Nagano Children's Hospital, Azumino, Japan
| | - Nobuo Okumura
- Department of Biomedical Laboratory Sciences, Shinshu University School of Medicine, Matsumoto, Japan
| | - Yumiko Higuchi
- Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto, 390-8621, Japan.
- Department of Biomedical Laboratory Sciences, Shinshu University School of Medicine, Matsumoto, Japan.
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9
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Peng H, Pan M, Zhou Z, Chen C, Xing X, Cheng S, Zhang S, Zheng H, Qian K. The impact of preanalytical variables on the analysis of cell-free DNA from blood and urine samples. Front Cell Dev Biol 2024; 12:1385041. [PMID: 38784382 PMCID: PMC11111958 DOI: 10.3389/fcell.2024.1385041] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2024] [Accepted: 04/22/2024] [Indexed: 05/25/2024] Open
Abstract
Cell-free DNA (cfDNA), a burgeoning class of molecular biomarkers, has been extensively studied across a variety of biomedical fields. As a key component of liquid biopsy, cfDNA testing is gaining prominence in disease detection and management due to the convenience of sample collection and the abundant wealth of genetic information it provides. However, the broader clinical application of cfDNA is currently impeded by a lack of standardization in the preanalytical procedures for cfDNA analysis. A number of fundamental challenges, including the selection of appropriate preanalytical procedures, prevention of short cfDNA fragment loss, and the validation of various cfDNA measurement methods, remain unaddressed. These existing hurdles lead to difficulties in comparing results and ensuring repeatability, thereby undermining the reliability of cfDNA analysis in clinical settings. This review discusses the crucial preanalytical factors that influence cfDNA analysis outcomes, including sample collection, transportation, temporary storage, processing, extraction, quality control, and long-term storage. The review provides clarification on achievable consensus and offers an analysis of the current issues with the goal of standardizing preanalytical procedures for cfDNA analysis.
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Affiliation(s)
- Hongwei Peng
- Department of Biological Repositories, Human Genetic Resources Preservation Center of Hubei Province, Hubei Key Laboratory of Urological Diseases, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Ming Pan
- Taihe Skills Training Center, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei, China
| | - Zongning Zhou
- Department of Biological Repositories, Human Genetic Resources Preservation Center of Hubei Province, Hubei Key Laboratory of Urological Diseases, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Congbo Chen
- Department of Urology, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei, China
| | - Xing Xing
- Department of Urology, The First Affiliated Hospital of Yangtze University, Jingzhou, Hubei, China
| | - Shaoping Cheng
- Department of Urology, The First Affiliated Hospital of Yangtze University, Jingzhou, Hubei, China
| | - Shanshan Zhang
- Department of Biological Repositories, Human Genetic Resources Preservation Center of Hubei Province, Hubei Key Laboratory of Urological Diseases, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Hang Zheng
- Department of Urology, Laboratory of Precision Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Kaiyu Qian
- Department of Biological Repositories, Human Genetic Resources Preservation Center of Hubei Province, Hubei Key Laboratory of Urological Diseases, Zhongnan Hospital of Wuhan University, Wuhan, China
- Department of Urology, Laboratory of Precision Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China
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10
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Song HH, Park H, Cho D, Bang HI, Oh HJ, Kim J. Optimization of a Protocol for Isolating Cell-free DNA From Cerebrospinal Fluid. Ann Lab Med 2024; 44:294-298. [PMID: 38151854 PMCID: PMC10813833 DOI: 10.3343/alm.2023.0267] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2023] [Revised: 11/01/2023] [Accepted: 12/08/2023] [Indexed: 12/29/2023] Open
Abstract
A standardized protocol for the isolation of cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) is lacking. Therefore, we established a cfDNA isolation protocol optimized for clinical CSF specimens, integrating acceptable modifications and using artificial CSF generated from remnant CSF spiked with reference cell-free tumor DNA (ctDNA). We compared the isolation yields of in vitro diagnostic (IVD)-certified column-based (CB) and magnetic bead-based (MB) isolation. Furthermore, we modified both methods, including pre- and post-elution steps. To confirm ctDNA integrity and quantify the variant allele frequency after isolation, we performed droplet digital PCR (ddPCR) targeting IDH1 R132C in the reference ctDNA. MB isolation had a higher yield than CB isolation (P<0.0001), and post-isolation vacuum increased the final concentration in both methods, with little effect on cfDNA integrity. Our study provides a protocol to maximize CSF-ctDNA concentrations in IVD testing and future studies.
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Affiliation(s)
- Ho Hyun Song
- Department of Interdisciplinary Program in Biomedical Science, Graduate School, Soonchunhyang University, Asan, Korea
| | - Hyeran Park
- Department of Neurosurgery, Soonchunhyang University Seoul Hospital, Seoul, Korea
| | - Doohwan Cho
- Department of Laboratory Medicine, Soonchunhyang University Seoul Hospital, Seoul, Korea
| | - Hae In Bang
- Department of Laboratory Medicine, Soonchunhyang University Seoul Hospital, Seoul, Korea
| | - Hyuk-Jin Oh
- Department of Neurosurgery, Soonchunhyang University Cheonan Hospital, Cheonan, Korea
| | - Jieun Kim
- Department of Laboratory Medicine, Soonchunhyang University Seoul Hospital, Seoul, Korea
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11
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Terp SK, Pedersen IS, Stoico MP. Extraction of Cell-Free DNA: Evaluation of Efficiency, Quantity, and Quality. J Mol Diagn 2024; 26:310-319. [PMID: 38336350 DOI: 10.1016/j.jmoldx.2024.01.008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2023] [Revised: 01/03/2024] [Accepted: 01/10/2024] [Indexed: 02/12/2024] Open
Abstract
Cell-free DNA (cfDNA) serves as a valuable biomarker for early disease detection and monitoring. However, the use of cfDNA for analysis faces challenges owing to general low but variable abundance and fragmentation. Preanalytical factors, including cfDNA extraction, impact cfDNA quality and quantity. Efficient and robust cfDNA extraction is essential for reliable results in downstream applications, and various commercial extraction methods exist, each with trade-offs. To aid researchers and clinicians in choosing the proper cfDNA extraction method, manual, semiautomated, and automated methods were evaluated, including the QIAamp Circulating Nucleic Acid Kit (manual and QIAcube), QIAamp MinElute ccfDNA Kit (QIAcube), and QIAsymphony DSP Circulating DNA Kit (QIAsymphony). For each extraction method, cfDNA was extracted on two separate days, using samples obtained from 18 healthy donors. This study assessed extraction efficiency, quantity, and quality using droplet digital PCR and TapeStation. The QIAamp Circulating Nucleic Acid Kit, both manual and semiautomated, outperformed the QIAamp MinElute ccfDNA Kit (QIAcube) and QIAsymphony DSP Circulating DNA Kit (QIAsymphony), showing higher recovery rates and cfDNA quantity. All methods were reproducible, with no day-to-day variability and no contamination by high-molecular-weight DNA. The QIAamp Circulating Nucleic Acid Kit offers high yield without compromising quality. Implementation of the method should consider specific study and clinical needs, taking into account each method's advantages and limitations for optimal outcomes.
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Affiliation(s)
- Simone K Terp
- Department of Molecular Diagnostics, Aalborg University Hospital, Aalborg, Denmark; Clinical Cancer Research Center, Aalborg University Hospital, Aalborg, Denmark; Department of Clinical Medicine, Aalborg University, Aalborg, Denmark.
| | - Inge S Pedersen
- Department of Molecular Diagnostics, Aalborg University Hospital, Aalborg, Denmark; Clinical Cancer Research Center, Aalborg University Hospital, Aalborg, Denmark; Department of Clinical Medicine, Aalborg University, Aalborg, Denmark
| | - Malene P Stoico
- Department of Molecular Diagnostics, Aalborg University Hospital, Aalborg, Denmark; Clinical Cancer Research Center, Aalborg University Hospital, Aalborg, Denmark; Department of Clinical Medicine, Aalborg University, Aalborg, Denmark
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12
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van der Leest P, Schuuring E. Critical Factors in the Analytical Work Flow of Circulating Tumor DNA-Based Molecular Profiling. Clin Chem 2024; 70:220-233. [PMID: 38175597 DOI: 10.1093/clinchem/hvad194] [Citation(s) in RCA: 11] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2023] [Accepted: 10/30/2023] [Indexed: 01/05/2024]
Abstract
BACKGROUND Liquid biopsy testing, especially molecular tumor profiling of circulating tumor DNA (ctDNA) in cell-free plasma, has received increasing interest in recent years as it serves as a reliable alternative for the detection of tumor-specific aberrations to guide treatment decision-making in oncology. Many (commercially available) applications have been developed, however, broad divergences in (pre)analytical work flows and lack of universally applied guidelines impede routine clinical implementation. In this review, critical factors in the blood-based ctDNA liquid biopsy work flow are evaluated. CONTENT In the preanalytical phase, several aspects (e.g., blood collection tubes [BCTs], plasma processing, and extraction method) affect the quantity and quality of the circulating cell-free DNA (ccfDNA) applicable for subsequent molecular analyses and should meet certain standards to be applied in diagnostic work flows. Analytical considerations, such as analytical input and choice of assay, might vary based on the clinical application (i.e., screening, primary diagnosis, minimal residual disease [MRD], response monitoring, and resistance identification). In addition to practical procedures, variant interpretation and reporting ctDNA results should be harmonized. Collaborative efforts in (inter)national consortia and societies are essential for the establishment of standard operating procedures (SOPs) in attempts to standardize the plasma-based ctDNA analysis work flow. SUMMARY Development of universally applicable guidelines regarding the critical factors in liquid biopsy testing are necessary to pave the way to clinical implementation for routine diagnostics.
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Affiliation(s)
- Paul van der Leest
- Department of Pathology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands
- Department of Laboratory Medicine, Netherlands Cancer Institute, Amsterdam, the Netherlands
| | - Ed Schuuring
- Department of Pathology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands
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13
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Abstract
The cumulative pool of cell-free DNA (cfDNA) molecules within bodily fluids represents a highly dense and multidimensional information repository. This "biological mirror" provides real-time insights into the composition, function, and dynamics of the diverse genomes within the body, enabling significant advancements in personalized molecular medicine. However, effective use of this information necessitates meticulous classification of distinct cfDNA subtypes with exceptional precision. While cfDNA molecules originating from different sources exhibit numerous genetic, epigenetic, and physico-chemical variations, they also share common features that complicate analyses. Considerable progress has been achieved in mapping the landscape of cfDNA features, their clinical correlations, and optimizing extraction procedures, analytical approaches, bioinformatics pipelines, and machine learning algorithms. Nevertheless, preanalytical workflows, despite their profound impact on cfDNA measurements, have not progressed at a corresponding pace. In this perspective article, we emphasize the pivotal role of robust preanalytical procedures in the development and clinical integration of cfDNA assays, highlighting persistent obstacles and emerging challenges.
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Affiliation(s)
- Abel J Bronkhorst
- Munich Biomarker Research Center, Institute of Laboratory Medicine, German Heart Center, Technical University Munich, Munich, Germany
| | - Stefan Holdenrieder
- Munich Biomarker Research Center, Institute of Laboratory Medicine, German Heart Center, Technical University Munich, Munich, Germany
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14
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Bronkhorst AJ, Holdenrieder S. The changing face of circulating tumor DNA (ctDNA) profiling: Factors that shape the landscape of methodologies, technologies, and commercialization. MED GENET-BERLIN 2023; 35:201-235. [PMID: 38835739 PMCID: PMC11006350 DOI: 10.1515/medgen-2023-2065] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/06/2024]
Abstract
Liquid biopsies, in particular the profiling of circulating tumor DNA (ctDNA), have long held promise as transformative tools in cancer precision medicine. Despite a prolonged incubation phase, ctDNA profiling has recently experienced a strong wave of development and innovation, indicating its imminent integration into the cancer management toolbox. Various advancements in mutation-based ctDNA analysis methodologies and technologies have greatly improved sensitivity and specificity of ctDNA assays, such as optimized preanalytics, size-based pre-enrichment strategies, targeted sequencing, enhanced library preparation methods, sequencing error suppression, integrated bioinformatics and machine learning. Moreover, research breakthroughs have expanded the scope of ctDNA analysis beyond hotspot mutational profiling of plasma-derived apoptotic, mono-nucleosomal ctDNA fragments. This broader perspective considers alternative genetic features of cancer, genome-wide characterization, classical and newly discovered epigenetic modifications, structural variations, diverse cellular and mechanistic ctDNA origins, and alternative biospecimen types. These developments have maximized the utility of ctDNA, facilitating landmark research, clinical trials, and the commercialization of ctDNA assays, technologies, and products. Consequently, ctDNA tests are increasingly recognized as an important part of patient guidance and are being implemented in clinical practice. Although reimbursement for ctDNA tests by healthcare providers still lags behind, it is gaining greater acceptance. In this work, we provide a comprehensive exploration of the extensive landscape of ctDNA profiling methodologies, considering the multitude of factors that influence its development and evolution. By illuminating the broader aspects of ctDNA profiling, the aim is to provide multiple entry points for understanding and navigating the vast and rapidly evolving landscape of ctDNA methodologies, applications, and technologies.
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Affiliation(s)
- Abel J Bronkhorst
- Technical University Munich Munich Biomarker Research Center, Institute of Laboratory Medicine, German Heart Center Lazarettstr. 36 80636 Munich Germany
| | - Stefan Holdenrieder
- Technical University Munich Munich Biomarker Research Center, Institute of Laboratory Medicine, German Heart Center Lazarettstr. 36 80636 Munich Germany
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15
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Keup C, Kimmig R, Kasimir-Bauer S. The Diversity of Liquid Biopsies and Their Potential in Breast Cancer Management. Cancers (Basel) 2023; 15:5463. [PMID: 38001722 PMCID: PMC10670968 DOI: 10.3390/cancers15225463] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2023] [Revised: 11/10/2023] [Accepted: 11/14/2023] [Indexed: 11/26/2023] Open
Abstract
Analyzing blood as a so-called liquid biopsy in breast cancer (BC) patients has the potential to adapt therapy management. Circulating tumor cells (CTCs), extracellular vesicles (EVs), cell-free DNA (cfDNA) and other blood components mirror the tumoral heterogeneity and could support a range of clinical decisions. Multi-cancer early detection tests utilizing blood are advancing but are not part of any clinical routine yet. Liquid biopsy analysis in the course of neoadjuvant therapy has potential for therapy (de)escalation.Minimal residual disease detection via serial cfDNA analysis is currently on its way. The prognostic value of blood analytes in early and metastatic BC is undisputable, but the value of these prognostic biomarkers for clinical management is controversial. An interventional trial confirmed a significant outcome benefit when therapy was changed in case of newly emerging cfDNA mutations under treatment and thus showed the clinical utility of cfDNA analysis for therapy monitoring. The analysis of PIK3CA or ESR1 variants in plasma of metastatic BC patients to prescribe targeted therapy with alpesilib or elacestrant has already arrived in clinical practice with FDA-approved tests available and is recommended by ASCO. The translation of more liquid biopsy applications into clinical practice is still pending due to a lack of knowledge of the analytes' biology, lack of standards and difficulties in proving clinical utility.
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Affiliation(s)
- Corinna Keup
- Department of Gynecology and Obstetrics, University Hospital of Essen, 45147 Essen, Germany
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16
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Lelliott PM, Hobro AJ, Pavillon N, Nishide M, Okita Y, Mizuno Y, Obata S, Nameki S, Yoshimura H, Kumanogoh A, Smith NI. Single-cell Raman microscopy with machine learning highlights distinct biochemical features of neutrophil extracellular traps and necrosis. Sci Rep 2023; 13:10093. [PMID: 37344494 DOI: 10.1038/s41598-023-36667-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2023] [Accepted: 06/07/2023] [Indexed: 06/23/2023] Open
Abstract
The defining biology that distinguishes neutrophil extracellular traps (NETs) from other forms of cell death is unresolved, and techniques which unambiguously identify NETs remain elusive. Raman scattering measurement provides a holistic overview of cell molecular composition based on characteristic bond vibrations in components such as lipids and proteins. We collected Raman spectra from NETs and freeze/thaw necrotic cells using a custom built high-throughput platform which is able to rapidly measure spectra from single cells. Principal component analysis of Raman spectra from NETs clearly distinguished them from necrotic cells despite their similar morphology, demonstrating their fundamental molecular differences. In contrast, classical techniques used for NET analysis, immunofluorescence microscopy, extracellular DNA, and ELISA, could not differentiate these cells. Additionally, machine learning analysis of Raman spectra indicated subtle differences in lipopolysaccharide (LPS)-induced as opposed to phorbol myristate acetate (PMA)-induced NETs, demonstrating the molecular composition of NETs varies depending on the stimulant used. This study demonstrates the benefits of Raman microscopy in discriminating NETs from other types of cell death and by their pathway of induction.
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Affiliation(s)
- Patrick Michael Lelliott
- Laboratory of Biophotonics, Immunology Frontier Research Center, Osaka University, Yamadaoka 3-1, Suita, Osaka, 565-0871, Japan.
| | - Alison Jane Hobro
- Laboratory of Biophotonics, Immunology Frontier Research Center, Osaka University, Yamadaoka 3-1, Suita, Osaka, 565-0871, Japan
| | - Nicolas Pavillon
- Laboratory of Biophotonics, Immunology Frontier Research Center, Osaka University, Yamadaoka 3-1, Suita, Osaka, 565-0871, Japan
| | - Masayuki Nishide
- Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Yasutaka Okita
- Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Yumiko Mizuno
- Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Sho Obata
- Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
- Department of Otorhinolaryngology-Head and Neck Surgery, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Shinichiro Nameki
- Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Hanako Yoshimura
- Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Atsushi Kumanogoh
- Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
- Laboratory of Immunopathology, Immunology Frontier Research Center, Osaka University, Osaka, Japan
- Open and Transdisciplinary Research Institute (OTRI), Osaka University, Osaka, Japan
| | - Nicholas Isaac Smith
- Laboratory of Biophotonics, Immunology Frontier Research Center, Osaka University, Yamadaoka 3-1, Suita, Osaka, 565-0871, Japan.
- Open and Transdisciplinary Research Institute (OTRI), Osaka University, Osaka, Japan.
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17
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Assi T, Khoury R, Ibrahim R, Baz M, Ibrahim T, LE Cesne A. Overview of the role of liquid biopsy in cancer management. Transl Oncol 2023; 34:101702. [PMID: 37267803 DOI: 10.1016/j.tranon.2023.101702] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2023] [Revised: 05/12/2023] [Accepted: 05/23/2023] [Indexed: 06/04/2023] Open
Abstract
With the emergence of novel targeted therapeutic options in early-stage and advanced-stage malignancies, researchers have shifted their focus on developing personalized treatment plans through molecular profiling. Circulating tumor DNA (ctDNA) is a cell-free DNA (ctDNA) fragment, originating from tumor cells, and circulating in the bloodstream as well as biological fluids. Over the past decade, many techniques were developed for liquid biopsies through next-generation sequencing. This alternative non-invasive biopsy offers several advantages in various types of tumors over traditional tissue biopsy. The process of liquid biopsy is considered minimally invasive and therefore easily repeatable when needed, providing a more dynamic analysis of the tumor cells. Moreover, it has an advantage in patients with tumors that are not candidates for tissue sampling. Besides, it offers a deeper understanding of tumor burden as well as treatment response, thereby enhancing the detection of minimal residual disease and therapeutic guidance for personalized medicine. Despite its many advantages, ctDNA and liquid biopsy do have some limitations. This paper discusses the basis of ctDNA and the current data available on the subject, as well as its clinical utility. We also reflect on the limitations of using ctDNA in addition to its future perspectives in clinical oncology and precision medicine.
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Affiliation(s)
- Tarek Assi
- Division of International Patients Care, Gustave Roussy Cancer Campus, Villejuif, France.
| | - Rita Khoury
- Division of International Patients Care, Gustave Roussy Cancer Campus, Villejuif, France
| | - Rebecca Ibrahim
- Division of International Patients Care, Gustave Roussy Cancer Campus, Villejuif, France
| | - Maria Baz
- Division of International Patients Care, Gustave Roussy Cancer Campus, Villejuif, France
| | - Tony Ibrahim
- Division of International Patients Care, Gustave Roussy Cancer Campus, Villejuif, France
| | - Axel LE Cesne
- Division of International Patients Care, Gustave Roussy Cancer Campus, Villejuif, France
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18
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Dameri M, Cirmena G, Ravera F, Ferrando L, Cuccarolo P, Stabile M, Fanelli GN, Nuzzo PV, Calabrese M, Tagliafico A, Ballestrero A, Zoppoli G. Standard Operating Procedures (SOPs) for non-invasive multiple biomarkers detection in an academic setting: A critical review of the literature for the RENOVATE study protocol. Crit Rev Oncol Hematol 2023; 185:103963. [PMID: 36931614 DOI: 10.1016/j.critrevonc.2023.103963] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2022] [Revised: 02/06/2023] [Accepted: 02/13/2023] [Indexed: 03/17/2023] Open
Abstract
Liquid biopsy has the potential to drastically change clinical practice, paving the way to a novel non-invasive approach for cancer diagnosis and treatment. One of the limitations for the implementation of liquid biopsy in clinical practice is the lack of shared and reproducible standard operating procedures (SOPs) for sample collection, processing and storage. Here, we present a critical review of the literature focusing on the available SOPs to guide liquid biopsy management in research settings and describe SOPs that our laboratory developed and employed in the context of a prospective clinical-translational trial (RENOVATE, NCT04781062). The main aim of this manuscript is to address common issues, towards the implementation of interlaboratory shared protocols for optimized preanalytical handling of blood and urine samples. To our knowledge, this work is one of the few up-to-date, freely available comprehensive reports on trial-level procedures for the handling of liquid biopsy.
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Affiliation(s)
- Martina Dameri
- Department of Internal Medicine and Medical Specialties (DiMI), University of Genoa, 16132 Genoa, Italy
| | | | - Francesco Ravera
- Department of Internal Medicine and Medical Specialties (DiMI), University of Genoa, 16132 Genoa, Italy; Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, 10044 New York, NY, USA
| | | | - Paola Cuccarolo
- Department of Internal Medicine and Medical Specialties (DiMI), University of Genoa, 16132 Genoa, Italy
| | - Mario Stabile
- Department of Internal Medicine and Medical Specialties (DiMI), University of Genoa, 16132 Genoa, Italy
| | - Giuseppe Nicolò Fanelli
- Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, 10021 New York, NY, USA; First Division of Pathology, Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, 56126 Pisa, Italy
| | - Pier Vitale Nuzzo
- Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, 10044 New York, NY, USA
| | | | - Alberto Tagliafico
- IRCCS Ospedale Policlinico San Martino, 16132 Genoa, Italy; Department of Health Sciences (DISSAL), University of Genoa, 16132, Genoa, Italy
| | - Alberto Ballestrero
- Department of Internal Medicine and Medical Specialties (DiMI), University of Genoa, 16132 Genoa, Italy; IRCCS Ospedale Policlinico San Martino, 16132 Genoa, Italy
| | - Gabriele Zoppoli
- Department of Internal Medicine and Medical Specialties (DiMI), University of Genoa, 16132 Genoa, Italy; IRCCS Ospedale Policlinico San Martino, 16132 Genoa, Italy.
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19
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Diaz IM, Nocon A, Held SAE, Kobilay M, Skowasch D, Bronkhorst AJ, Ungerer V, Fredebohm J, Diehl F, Holdenrieder S, Holtrup F. Pre-Analytical Evaluation of Streck Cell-Free DNA Blood Collection Tubes for Liquid Profiling in Oncology. Diagnostics (Basel) 2023; 13:diagnostics13071288. [PMID: 37046506 PMCID: PMC10093569 DOI: 10.3390/diagnostics13071288] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2023] [Revised: 03/27/2023] [Accepted: 03/27/2023] [Indexed: 04/03/2023] Open
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 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.
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20
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Aucamp J, van der Zwan H, Geldenhuys Z, Abera A, Louw R, van der Sluis R. Diagnostic applications and limitations for the use of cell-free fetal DNA (cffDNA) in animal husbandry and wildlife management. Res Vet Sci 2023; 158:106-116. [PMID: 36989830 DOI: 10.1016/j.rvsc.2023.03.013] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2022] [Revised: 02/02/2023] [Accepted: 03/15/2023] [Indexed: 03/18/2023]
Abstract
In animal breeding, a species sex can influence the value of the animal. For example, in the horse breeding industry, mares are preferred as polo horses, while in wildlife breeding males with larger horns are more valuable. Therefore, the economic advantages of knowing the unborn fetus' sex are important to successful animal management. Ultrasonography is used to determine the sex of unborn fetuses, but this method places additional stress on the animal and require specialized equipment and expertise. Conversely, molecular-based sexing techniques require less invasive sampling and can determine sex more reliably. Although in humans, various studies have evaluated the use of cell-free fetal DNA (cffDNA) for prenatal sexing, very few animal studies have been published in this field. Several factors can affect the sensitivity of cffDNA-based sex determination, for example the gestational age. These factors are often not optimized and validated when establishing a protocol for prenatal sexing. In this review, we summarize the current literature on cffDNA in animals. We discuss the diagnostic applications and limitations in the use thereof in animal husbandry and wildlife management. Lastly, the feasibility of implementing diagnostic tests is evaluated and solutions are given to the current drawbacks of the technology.
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21
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Soscia R, Della Starza I, De Novi LA, Ilari C, Ansuinelli M, Cavalli M, Bellomarino V, Cafforio L, Di Trani M, Cazzaniga G, Fazio G, Santoro A, Salemi D, Spinelli O, Tosi M, Terragna C, Robustelli V, Bellissimo T, Colafigli G, Breccia M, Chiaretti S, Di Rocco A, Martelli M, Guarini A, Del Giudice I, Foà R. Circulating cell-free DNA for target quantification in hematologic malignancies: Validation of a protocol to overcome pre-analytical biases. Hematol Oncol 2023; 41:50-60. [PMID: 36251440 DOI: 10.1002/hon.3087] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2022] [Revised: 10/05/2022] [Accepted: 10/11/2022] [Indexed: 02/03/2023]
Abstract
Circulating tumor DNA (ctDNA) has become the most investigated analyte in blood. It is shed from the tumor into the circulation and represents a subset of the total cell-free DNA (cfDNA) pool released into the peripheral blood. In order to define if ctDNA could represent a useful tool to monitor hematologic malignancies, we analyzed 81 plasma samples from patients affected by different diseases. The results showed that: (i) the comparison between two different extraction methods Qiagen (Hilden, Germany) and Promega (Madison, WI) showed no significant differences in cfDNA yield, though the first recovered higher amounts of larger DNA fragments; (ii) cfDNA concentrations showed a notable inter-patient variability and differed among diseases: acute lymphoblastic leukemia and chronic myeloid leukemia released higher amounts of cfDNA than chronic lymphocytic leukemia, and diffuse large B-cell lymphoma released higher cfDNA quantities than localized and advanced follicular lymphoma; (iii) focusing on the tumor fraction of cfDNA, the quantity of ctDNA released was insufficient for an adequate target quantification for minimal residual disease monitoring; (iv) an amplification system proved to be free of analytical biases and efficient in increasing ctDNA amounts at diagnosis and in follow-up samples as shown by droplet digital PCR target quantification. The protocol has been validated by quality control rounds involving external laboratories. To conclusively document the feasibility of a ctDNA-based monitoring of patients with hematologic malignancies, more post-treatment samples need to be evaluated. This will open new possibilities for ctDNA use in the clinical practice.
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Affiliation(s)
- Roberta Soscia
- Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy
| | - Irene Della Starza
- Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy.,GIMEMA Foundation, Rome, Italy
| | - Lucia Anna De Novi
- Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy
| | - Caterina Ilari
- Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy
| | - Michela Ansuinelli
- Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy
| | - Marzia Cavalli
- Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy
| | - Vittorio Bellomarino
- Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy
| | - Luciana Cafforio
- Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy
| | - Mariangela Di Trani
- Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy
| | - Giovanni Cazzaniga
- Tettamanti Research Center, Department of Pediatrics, University of Milano-Bicocca/Fondazione MBBM, Monza, Italy
| | - Grazia Fazio
- Tettamanti Research Center, Department of Pediatrics, University of Milano-Bicocca/Fondazione MBBM, Monza, Italy
| | - Alessandra Santoro
- Division of Hematology and Bone Marrow Transplantation, Ospedali Riuniti Villa Sofia-Cervello, Palermo, Italy
| | - Domenico Salemi
- Division of Hematology and Bone Marrow Transplantation, Ospedali Riuniti Villa Sofia-Cervello, Palermo, Italy
| | - Orietta Spinelli
- Hematology and Bone Marrow Transplant Unit, Ospedale Papa Giovanni XXIII, Bergamo, Italy
| | - Manuela Tosi
- Hematology and Bone Marrow Transplant Unit, Ospedale Papa Giovanni XXIII, Bergamo, Italy
| | - Carolina Terragna
- Seràgnoli Institute of Hematology, Azienda Ospedaliero-Universitaria Sant'Orsola-Malpighi, Bologna, Italy
| | - Valentina Robustelli
- Dipartimento di Medicina Specialistica Diagnostica e Sperimentale, Università di Bologna, Bologna, Italy
| | - Teresa Bellissimo
- Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy
| | - Gioia Colafigli
- Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy
| | - Massimo Breccia
- Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy
| | - Sabina Chiaretti
- Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy
| | - Alice Di Rocco
- Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy
| | - Maurizio Martelli
- Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy
| | - Anna Guarini
- Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy.,Department of Molecular Medicine, Sapienza University, Rome, Italy
| | - Ilaria Del Giudice
- Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy
| | - Robin Foà
- Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy
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22
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Della Starza I, De Novi LA, Elia L, Bellomarino V, Beldinanzi M, Soscia R, Cardinali D, Chiaretti S, Guarini A, Foà R. Optimizing Molecular Minimal Residual Disease Analysis in Adult Acute Lymphoblastic Leukemia. Cancers (Basel) 2023; 15:374. [PMID: 36672325 PMCID: PMC9856386 DOI: 10.3390/cancers15020374] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2022] [Revised: 01/02/2023] [Accepted: 01/03/2023] [Indexed: 01/09/2023] Open
Abstract
Minimal/measurable residual disease (MRD) evaluation has resulted in a fundamental instrument to guide patient management in acute lymphoblastic leukemia (ALL). From a methodological standpoint, MRD is defined as any approach aimed at detecting and possibly quantifying residual neoplastic cells beyond the sensitivity level of cytomorphology. The molecular methods to study MRD in ALL are polymerase chain reaction (PCR) amplification-based approaches and are the most standardized techniques. However, there are some limitations, and emerging technologies, such as digital droplet PCR (ddPCR) and next-generation sequencing (NGS), seem to have advantages that could improve MRD analysis in ALL patients. Furthermore, other blood components, namely cell-free DNA (cfDNA), appear promising and are also being investigated for their potential role in monitoring tumor burden and response to treatment in hematologic malignancies. Based on the review of the literature and on our own data, we hereby discuss how emerging molecular technologies are helping to refine the molecular monitoring of MRD in ALL and may help to overcome some of the limitations of standard approaches, providing a benefit for the care of patients.
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Affiliation(s)
- Irene Della Starza
- Hematology, Department of Translational and Precision Medicine, “Sapienza” University, Via Benevento 6, 00161 Rome, Italy
- GIMEMA Foundation, 00182 Rome, Italy
| | - Lucia Anna De Novi
- Hematology, Department of Translational and Precision Medicine, “Sapienza” University, Via Benevento 6, 00161 Rome, Italy
| | - Loredana Elia
- Hematology, Department of Translational and Precision Medicine, “Sapienza” University, Via Benevento 6, 00161 Rome, Italy
| | - Vittorio Bellomarino
- Hematology, Department of Translational and Precision Medicine, “Sapienza” University, Via Benevento 6, 00161 Rome, Italy
| | - Marco Beldinanzi
- Hematology, Department of Translational and Precision Medicine, “Sapienza” University, Via Benevento 6, 00161 Rome, Italy
| | - Roberta Soscia
- Hematology, Department of Translational and Precision Medicine, “Sapienza” University, Via Benevento 6, 00161 Rome, Italy
| | - Deborah Cardinali
- Hematology, Department of Translational and Precision Medicine, “Sapienza” University, Via Benevento 6, 00161 Rome, Italy
| | - Sabina Chiaretti
- Hematology, Department of Translational and Precision Medicine, “Sapienza” University, Via Benevento 6, 00161 Rome, Italy
| | - Anna Guarini
- Hematology, Department of Translational and Precision Medicine, “Sapienza” University, Via Benevento 6, 00161 Rome, Italy
| | - Robin Foà
- Hematology, Department of Translational and Precision Medicine, “Sapienza” University, Via Benevento 6, 00161 Rome, Italy
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23
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Maurer K, Schandl CA. Liquid Biopsy for Advanced Cancer: An Amplicon-Based Massively Parallel Sequencing Panel Approach to Precision Oncology. Methods Mol Biol 2023; 2621:111-126. [PMID: 37041443 DOI: 10.1007/978-1-0716-2950-5_8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/13/2023]
Abstract
Although discovered in the 1940s (Mandel and Metais, C R Seances Soc Biol Fil 142:241-243, 1948), cell-free DNA has only recently become a tool practical for use in clinical settings. The challenges associated with detection of circulating tumor DNA (ctDNA) in patient plasma are many and exist in the pre-analytical, analytical, and post-analytical periods. Initiation of a ctDNA program in a small academic clinical laboratory setting can be challenging. Thus, cost-effective, fast methods should be leveraged to promote a self-supporting system. Any assay should be based on clinical utility and have the potential to adapt in order to maintain relevance in a rapidly developing genomic landscape. Herein is described one of many approaches to ctDNA mutation testing - a massively parallel sequencing (MPS) method that is widely applicable and relatively easy to perform. Sensitivity and specificity are enhanced by unique molecular identification tagging and deep sequencing.
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Affiliation(s)
- Kristen Maurer
- Medical University of South Carolina, Charleston, SC, USA
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24
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Zhao Z, Pan Z, Zhang S, Ma G, Zhang W, Song J, Wang Y, Kong L, Du G. Neutrophil extracellular traps: A novel target for the treatment of stroke. Pharmacol Ther 2023; 241:108328. [PMID: 36481433 DOI: 10.1016/j.pharmthera.2022.108328] [Citation(s) in RCA: 31] [Impact Index Per Article: 15.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2022] [Revised: 11/30/2022] [Accepted: 11/30/2022] [Indexed: 12/12/2022]
Abstract
Stroke is a threatening cerebrovascular disease caused by thrombus with high morbidity and mortality rates. Neutrophils are the first to be recruited in the brain after stroke, which aggravate brain injury through multiple mechanisms. Neutrophil extracellular traps (NETs), as a novel regulatory mechanism of neutrophils, can trap bacteria and secret antimicrobial molecules, thereby degrading pathogenic factors and killing bacteria. However, NETs also exacerbate certain non-infectious diseases by activating autoimmune or inflammatory responses. NETs have been found to play important roles in the pathological process of stroke in recent years. In this review, the mechanisms of NETs formation, the physiological roles of NETs, and the dynamic changes of NETs after stroke are summarized. NETs participate in stroke through various mechanisms. NETs promote the coagulation cascade and interact with platelets to induce thrombosis. tPA induces the degranulation of neutrophils to form NETs, leading to hemorrhagic transformation and thrombolytic resistance. NETs aggravate stroke by mediating inflammation, atherosclerosis and vascular injury. In addition, the regulation of NETs in stroke, the potential of NETs as biomarker and the treatment of stroke targeting NETs are discussed. The increasing evidences suggest that NETs may be a potential target for stroke treatment. Inhibition of NETs formation or promotion of NETs degradation plays protective effects in stroke. However, how to avoid the adverse effects of NETs-targeted therapy deserves further study. In summary, this review provides a reference for the pathogenesis, drug targets, biomarkers and drug development of NETs in stroke.
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Affiliation(s)
- Ziyuan Zhao
- Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100050, China
| | - Zirong Pan
- Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100050, China
| | - Sen Zhang
- Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100050, China
| | - Guodong Ma
- Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100050, China
| | - Wen Zhang
- Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100050, China
| | - Junke Song
- Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100050, China
| | - Yuehua Wang
- Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100050, China
| | - Linglei Kong
- Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100050, China.
| | - Guanhua Du
- Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100050, China.
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25
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Bronkhorst AJ, Ungerer V, Oberhofer A, Gabriel S, Polatoglou E, Randeu H, Uhlig C, Pfister H, Mayer Z, Holdenrieder S. New Perspectives on the Importance of Cell-Free DNA Biology. Diagnostics (Basel) 2022; 12:2147. [PMID: 36140548 PMCID: PMC9497998 DOI: 10.3390/diagnostics12092147] [Citation(s) in RCA: 38] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2022] [Revised: 08/24/2022] [Accepted: 08/31/2022] [Indexed: 11/28/2022] Open
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, 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.
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Affiliation(s)
- Abel J. Bronkhorst
- Munich Biomarker Research Center, Institute for Laboratory Medicine, German Heart Centre, Technical University Munich, Lazarettstraße 36, D-80636 Munich, Germany
| | | | | | | | | | | | | | | | | | - Stefan Holdenrieder
- Munich Biomarker Research Center, Institute for Laboratory Medicine, German Heart Centre, Technical University Munich, Lazarettstraße 36, D-80636 Munich, Germany
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26
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Fathima N, Manorenj S, Vishwakarma SK, Khan AA. Role of cell-free DNA for predicting incidence and outcome of patients with ischemic stroke. World J Neurol 2022; 8:1-9. [DOI: 10.5316/wjn.v8.i1.1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/11/2022] [Revised: 06/11/2022] [Accepted: 07/31/2022] [Indexed: 02/08/2023] Open
Affiliation(s)
- Nusrath Fathima
- Central Laboratory for Stem Cell Research and Translational Medicine, Centre for Liver Research and Diagnostics, Deccan College of Medical Sciences, Hyderabad 500058, Telangana, India
| | - Sandhya Manorenj
- Department of Neurology, Princess Esra Hospital, Deccan College of Medical Sciences, Hyderabad 500002, Telangana, India
| | - Sandeep Kumar Vishwakarma
- Central Laboratory for Stem Cell Research and Translational Medicine, Centre for Liver Research and Diagnostics, Deccan College of Medical Sciences, Hyderabad 500058, Telangana, India
| | - Aleem Ahmed Khan
- Central Laboratory for Stem Cell Research and Translational Medicine, Centre for Liver Research and Diagnostics, Deccan College of Medical Sciences, Hyderabad 500058, Telangana, India
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27
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Bronkhorst AJ, Ungerer V, Oberhofer A, Holdenrieder S. The rising tide of cell-free DNA profiling: from snapshot to temporal genome analysis. J LAB MED 2022; 46:207-224. [DOI: 10.1515/labmed-2022-0030] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2025] Open
Abstract
Abstract
Genomes of diverse origins are continuously shed into human body fluids in the form of fragmented cell-free DNA (cfDNA). These molecules maintain the genetic and epigenetic codes of their originating source, and often carry additional layers of unique information in newly discovered physico-chemical features. Characterization of cfDNA thus presents the opportunity to non-invasively reconstruct major parts of the host- and metagenome in silico. Data from a single specimen can be leveraged to detect a broad range of disease-specific signatures and has already enabled the development of many pioneering diagnostic tests. Moreover, data from serial sampling may allow unparalleled mapping of the scantily explored landscape of temporal genomic changes as it relates to various changes in different physiological and pathological states of individuals. In this review, we explore how this vast dimension of biological information accessible through cfDNA analysis is being tapped towards the development of increasingly powerful molecular assays and how it is shaping emerging technologies. We also discuss how this departure from traditional paradigms of snapshot genetic testing may pave the way for an onrush of new and exciting discoveries in human biology.
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Affiliation(s)
- Abel Jacobus Bronkhorst
- Munich Biomarker Research Center , Institute of Laboratory Medicine , German Heart Centre Munich , Technical University Munich , Munich , Germany
| | - Vida Ungerer
- Munich Biomarker Research Center , Institute of Laboratory Medicine , German Heart Centre Munich , Technical University Munich , Munich , Germany
| | - Angela Oberhofer
- Munich Biomarker Research Center , Institute of Laboratory Medicine , German Heart Centre Munich , Technical University Munich , Munich , Germany
| | - Stefan Holdenrieder
- Munich Biomarker Research Center , Institute of Laboratory Medicine , German Heart Centre Munich , Technical University Munich , Munich , Germany
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28
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Preanalytical Variables in the Analysis of Mitochondrial DNA in Whole Blood and Plasma from Pancreatic Cancer Patients. Diagnostics (Basel) 2022; 12:diagnostics12081905. [PMID: 36010255 PMCID: PMC9406772 DOI: 10.3390/diagnostics12081905] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2022] [Revised: 07/28/2022] [Accepted: 08/05/2022] [Indexed: 02/07/2023] Open
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 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.
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29
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Cell-Free DNA Fragmentation Patterns in a Cancer Cell Line. Diagnostics (Basel) 2022; 12:diagnostics12081896. [PMID: 36010246 PMCID: PMC9406536 DOI: 10.3390/diagnostics12081896] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2022] [Revised: 07/29/2022] [Accepted: 08/03/2022] [Indexed: 12/20/2022] Open
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 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.
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30
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Sánchez-Herrero E, Serna-Blasco R, Robado de Lope L, González-Rumayor V, Romero A, Provencio M. Circulating Tumor DNA as a Cancer Biomarker: An Overview of Biological Features and Factors That may Impact on ctDNA Analysis. Front Oncol 2022; 12:943253. [PMID: 35936733 PMCID: PMC9350013 DOI: 10.3389/fonc.2022.943253] [Citation(s) in RCA: 51] [Impact Index Per Article: 17.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2022] [Accepted: 06/16/2022] [Indexed: 11/13/2022] Open
Abstract
Cancer cells release nucleic acids, freely or associated with other structures such as vesicles into body fluids, including blood. Among these nucleic acids, circulating tumor DNA (ctDNA) has emerged as a minimally invasive biomarker for tumor molecular profiling. However, certain biological characteristics of ctDNA are still unknown. Here, we provide an overview of the current knowledge about ctDNA biological features, including size and structure as well as the mechanisms of ctDNA shedding and clearance, and the physio-pathological factors that determine ctDNA levels. A better understanding of ctDNA biology is essential for the development of new methods that enable the analysis of ctDNA.
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Affiliation(s)
- Estela Sánchez-Herrero
- Liquid Biopsy Laboratory. Biomedical Sciences Research Institute Puerta de Hierro-Majadahonda, Majadahonda, Spain
- +D Department, Atrys Health, Barcelona, Spain
| | - Roberto Serna-Blasco
- Liquid Biopsy Laboratory. Biomedical Sciences Research Institute Puerta de Hierro-Majadahonda, Majadahonda, Spain
| | - Lucia Robado de Lope
- Liquid Biopsy Laboratory. Biomedical Sciences Research Institute Puerta de Hierro-Majadahonda, Majadahonda, Spain
| | | | - Atocha Romero
- Liquid Biopsy Laboratory. Biomedical Sciences Research Institute Puerta de Hierro-Majadahonda, Majadahonda, Spain
- Medical Oncology Department, Hospital Universitario Puerta de Hierro-Majadahonda, Majadahonda, Spain
- *Correspondence: Atocha Romero, ; orcid.org/0000-0002-1634-7397
| | - Mariano Provencio
- Liquid Biopsy Laboratory. Biomedical Sciences Research Institute Puerta de Hierro-Majadahonda, Majadahonda, Spain
- Medical Oncology Department, Hospital Universitario Puerta de Hierro-Majadahonda, Majadahonda, Spain
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31
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Doculara L, Trahair TN, Bayat N, Lock RB. Circulating Tumor DNA in Pediatric Cancer. Front Mol Biosci 2022; 9:885597. [PMID: 35647029 PMCID: PMC9133724 DOI: 10.3389/fmolb.2022.885597] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2022] [Accepted: 04/25/2022] [Indexed: 11/13/2022] Open
Abstract
The measurement of circulating tumor DNA (ctDNA) has gained increasing prominence as a minimally invasive tool for the detection of cancer-specific markers in plasma. In adult cancers, ctDNA detection has shown value for disease-monitoring applications including tumor mutation profiling, risk stratification, relapse prediction, and treatment response evaluation. To date, there are ctDNA tests used as companion diagnostics for adult cancers and it is not understood why the same cannot be said about childhood cancer, despite the marked differences between adult and pediatric oncology. In this review, we discuss the current understanding of ctDNA as a disease monitoring biomarker in the context of pediatric malignancies, including the challenges associated with ctDNA detection in liquid biopsies. The data and conclusions from pediatric cancer studies of ctDNA are summarized, highlighting treatment response, disease monitoring and the detection of subclonal disease as applications of ctDNA. While the data from retrospective studies highlight the potential of ctDNA, large clinical trials are required for ctDNA analysis for routine clinical use in pediatric cancers. We outline the requirements for the standardization of ctDNA detection in pediatric cancers, including sample handling and reproducibility of results. With better understanding of the advantages and limitations of ctDNA and improved detection methods, ctDNA analysis may become the standard of care for patient monitoring in childhood cancers.
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Affiliation(s)
- Louise Doculara
- Children’s Cancer Institute, Lowy Cancer Centre, UNSW Sydney, Sydney, NSW, Australia
- School of Women’s and Children’s Health, UNSW Sydney, Sydney, NSW, Australia
- University of New South Wales Centre for Childhood Cancer Research, UNSW Sydney, Sydney, NSW, Australia
| | - Toby N. Trahair
- Children’s Cancer Institute, Lowy Cancer Centre, UNSW Sydney, Sydney, NSW, Australia
- School of Women’s and Children’s Health, UNSW Sydney, Sydney, NSW, Australia
- Kids Cancer Centre, Sydney Children’s Hospital, Randwick, NSW, Australia
| | - Narges Bayat
- Children’s Cancer Institute, Lowy Cancer Centre, UNSW Sydney, Sydney, NSW, Australia
- School of Women’s and Children’s Health, UNSW Sydney, Sydney, NSW, Australia
- University of New South Wales Centre for Childhood Cancer Research, UNSW Sydney, Sydney, NSW, Australia
| | - Richard B. Lock
- Children’s Cancer Institute, Lowy Cancer Centre, UNSW Sydney, Sydney, NSW, Australia
- School of Women’s and Children’s Health, UNSW Sydney, Sydney, NSW, Australia
- University of New South Wales Centre for Childhood Cancer Research, UNSW Sydney, Sydney, NSW, Australia
- *Correspondence: Richard B. Lock,
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Haselmann V, Hedtke M, Neumaier M. Liquid Profiling for Cancer Patient Stratification in Precision Medicine—Current Status and Challenges for Successful Implementation in Standard Care. Diagnostics (Basel) 2022; 12:diagnostics12030748. [PMID: 35328301 PMCID: PMC8947441 DOI: 10.3390/diagnostics12030748] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2022] [Revised: 03/09/2022] [Accepted: 03/11/2022] [Indexed: 12/13/2022] Open
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 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.
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Morita S, Nakamaru Y, Fukuda A, Fujiwara K, Suzuki M, Hoshino K, Honma A, Homma A. The Quantification of Extracellular Trap Cell Death-Derived Products as Diagnostic Biomarkers for Otitis Media With Antineutrophil Cytoplasmic Antibody-Associated Vasculitis and Eosinophilic Otitis Media. Otol Neurotol 2022; 43:e337-e343. [PMID: 34802016 DOI: 10.1097/mao.0000000000003431] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
OBJECTIVE This study aimed to quantify the cell-free deoxyribonucleic acid (DNA), citrullinated-histone H3 (cit-H3)-DNA complex, and myeloperoxidase (MPO)-DNA complex as extracellular trap cell death (ETosis)-derived products in the middle ear fluid, and to identify diagnostic biomarkers for the discrimination of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (OMAAV) from eosinophilic otitis media (EOM). STUDY DESIGN Prospective study. SETTING Tertiary referral center. PATIENTS OMAAV patients were eligible for inclusion in this analysis. Patients with EOM were examined as controls. INTERVENTION All samples were obtained from the middle ear fluid in patients with OMAAV or EOM. The fluid samples were aspirated from the middle ear through the anterior-inferior portion of the tympanic membrane using a 1-ml tuberculin syringe with a 24- or 26-gauge needle under a microscope. MAIN OUTCOME MEASURES The levels of cell-free DNA, cit-H3-DNA complex and MPO-DNA complex in the fluid samples were quantified using an enzyme-linked immunosorbent assay. RESULTS Patients with OMAAV showed significantly higher levels of MPO-DNA complex compared to patients with EOM, regardless of the serum ANCA status at the time of sampling (p < 0.001 and p < 0.001, respectively). Meanwhile, there were no significant differences in the values of cell-free DNA or cit-H3-DNA complex between the OMAAV and EOM patients. CONCLUSION The findings of this study suggest that the detection and quantification of MPO-DNA complex in the otitis media fluid can be utilized to discriminate OMAAV, especially in cases of eosinophilic granulomatosis with polyangiitis, from EOM regardless of the serum ANCA status. It should be noted that it is possible for cell-free DNA and cit-H3-DNA complex in fluid samples to be derived from dead cells other than neutrophils that undergo ETosis.
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Affiliation(s)
- Shinya Morita
- Department of Otolaryngology - Head and Neck Surgery, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
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Kondratskaya VA, Pokrovskaya MS, Doludin YV, Borisova AL, Limonova AS, Meshkov АN, Drapkina OM. Influence of preanalytical variables on the quality of cell-free DNA. Biobanking of cell-free DNA material. КАРДИОВАСКУЛЯРНАЯ ТЕРАПИЯ И ПРОФИЛАКТИКА 2022. [DOI: 10.15829/1728-8800-2021-3114] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022] Open
Abstract
The search for early disease markers and the development of diagnostic systems has recently been expanding within genomics. Genomic deoxyribonucleic acid (DNA), cell-free DNA (cfDNA) and microbiome DNA obtained from different types of samples (tissues, blood and its derivatives, feces, etc.) are used as objects of genetic research. It has been shown that cfDNA that enters the bloodstream, in particular, as a result of apoptosis, necrosis, active tumor secretion and metastasis, is of great importance for studying molecular mechanisms of the pathological process and application in clinical practice. Circulating nucleic acid analysis can be used to monitor response to treatment, assess drug resistance, and quantify minimal residual disease. The review article reflects the following information about the biomaterial: source of cfDNA, methods of cfDNA isolation, storage and use for the diagnosis of certain diseases. Cell-free DNA can be present in biological fluids such as blood, urine, saliva, synovial and cerebrospinal fluid. In most cases, cfDNA is isolated from blood derivatives (serum and plasma), while it is most correct to use blood plasma for cfDNA isolation. Optimal and economically justifiable is the use of ethylenediaminetetra-acetic acid tubes for taking blood and obtaining plasma with subsequent cfDNA isolation. There is evidence that the optimal shelf life in an ethylenediaminetetra-acetic acid tube from the moment of blood sampling to subsequent isolation is a 2-hour interval. After centrifugation, cfDNA in plasma (or serum) can be stored for a long time at a temperature of -80O C. Storage at -20O C is undesirable, since DNA fragmentation increases.
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Affiliation(s)
| | - M. S. Pokrovskaya
- National Medical Research Center for Therapy and Preventive Medicine
| | - Yu. V. Doludin
- National Medical Research Center for Therapy and Preventive Medicine
| | - A. L. Borisova
- National Medical Research Center for Therapy and Preventive Medicine
| | - A. S. Limonova
- National Medical Research Center for Therapy and Preventive Medicine
| | - А. N. Meshkov
- National Medical Research Center for Therapy and Preventive Medicine
| | - O. M. Drapkina
- National Medical Research Center for Therapy and Preventive Medicine
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Van Paemel R, Vandeputte C, Raman L, Van Thorre J, Willems L, Van Dorpe J, Van Der Linden M, De Wilde J, De Koker A, Menten B, Devalck C, Vicha A, Grega M, Schleiermacher G, Iddir Y, Chicard M, van Zogchel L, Stutterheim J, Lak NSM, Tytgat GAM, Laureys G, Speleman F, De Wilde B, Lammens T, De Preter K, Van Roy N. The feasibility of using liquid biopsies as a complementary assay for copy number aberration profiling in routinely collected paediatric cancer patient samples. Eur J Cancer 2021; 160:12-23. [PMID: 34794856 DOI: 10.1016/j.ejca.2021.09.022] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2021] [Revised: 07/27/2021] [Accepted: 09/11/2021] [Indexed: 12/15/2022]
Abstract
BACKGROUND Paediatric tumours are often characterised by the presence of recurrent DNA copy number alterations (CNAs). These DNA copy number profiles, obtained from a tissue biopsy, can aid in the correct prognostic classification and therapeutic stratification of several paediatric cancer entities (e.g. MYCN amplification in neuroblastoma) and are part of the routine diagnostic practice. Liquid biopsies (LQBs) offer a potentially safer alternative for such invasive tumour tissue biopsies and can provide deeper insight into tumour heterogeneity. PROCEDURE The robustness and reliability of LQB CNA analyses was evaluated. We performed retrospective CNA profiling using shallow whole-genome sequencing (sWGS) on paired plasma circulating cell-free DNA (cfDNA) and tissue DNA samples from routinely collected samples from paediatric patients (n = 128) representing different tumour entities, including osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, Wilms tumour, brain tumours and neuroblastoma. RESULTS Overall, we observed a good concordance between CNAs in tissue DNA and cfDNA. The main cause of CNA discordance was found to be low cfDNA sample quality (i.e. the ratio of cfDNA (<700 bp) and high molecular weight DNA (>700 bp)). Furthermore, CNAs were observed that were present in cfDNA and not in tissue DNA, or vice-versa. In neuroblastoma samples, no false-positives or false-negatives were identified for the detection of the prognostic marker MYCN amplification. CONCLUSION In future prospective studies, CNA analysis on LQBs that are of sufficient quality can serve as a complementary assay for CNA analysis on tissue biopsies, as either cfDNA or tissue DNA can contain CNAs that cannot be identified in the other biomaterial.
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Affiliation(s)
- Ruben Van Paemel
- Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium; Cancer Research Institute Ghent (CRIG), Ghent, Belgium; Department of Pediatric Hematology, Oncology and Stem Cell Transplantation, Ghent University Hospital, Ghent, Belgium; Department of Biomolecular Medicine, Ghent University, Ghent, Belgium; Research Foundation Flanders, Belgium
| | - Charlotte Vandeputte
- Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium; Cancer Research Institute Ghent (CRIG), Ghent, Belgium; Department of Biomolecular Medicine, Ghent University, Ghent, Belgium
| | - Lennart Raman
- Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium; Cancer Research Institute Ghent (CRIG), Ghent, Belgium; Department of Pathology, Ghent University Hospital, Ghent, Belgium
| | - Jolien Van Thorre
- Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium
| | - Leen Willems
- Department of Pediatric Hematology, Oncology and Stem Cell Transplantation, Ghent University Hospital, Ghent, Belgium
| | - Jo Van Dorpe
- Cancer Research Institute Ghent (CRIG), Ghent, Belgium; Department of Pathology, Ghent University Hospital, Ghent, Belgium
| | - Malaïka Van Der Linden
- Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium; Cancer Research Institute Ghent (CRIG), Ghent, Belgium; Department of Pathology, Ghent University Hospital, Ghent, Belgium
| | - Jilke De Wilde
- Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium; Cancer Research Institute Ghent (CRIG), Ghent, Belgium; Department of Pathology, Ghent University Hospital, Ghent, Belgium
| | - Andries De Koker
- Center for Medical Biotechnology, Flemish Institute Biotechnology (VIB), Ghent, Belgium; Research Foundation Flanders, Belgium
| | - Björn Menten
- Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium; Cancer Research Institute Ghent (CRIG), Ghent, Belgium; Department of Biomolecular Medicine, Ghent University, Ghent, Belgium
| | | | - Ales Vicha
- Department of Pediatric Hematology and Oncology, Charles University, Second Faculty of Medicine and University Hospital Motol, Prague, Czech Republic
| | - Marek Grega
- Department of Pathology and Molecular Medicine, 2nd Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic
| | - Gudrun Schleiermacher
- Translational Pediatric Oncology, Centre de recherche de l'Institut Curie, Paris, France
| | - Yasmine Iddir
- Translational Pediatric Oncology, Centre de recherche de l'Institut Curie, Paris, France
| | - Mathieu Chicard
- Translational Pediatric Oncology, Centre de recherche de l'Institut Curie, Paris, France
| | - Lieke van Zogchel
- Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands; Department of Experimental Immunohematology, Sanquin Research and Landsteiner Laboratory, Amsterdam University Medical Center, Amsterdam, the Netherlands
| | | | - Nathalie S M Lak
- Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands; Department of Experimental Immunohematology, Sanquin Research and Landsteiner Laboratory, Amsterdam University Medical Center, Amsterdam, the Netherlands
| | - G A M Tytgat
- Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands
| | - Geneviève Laureys
- Cancer Research Institute Ghent (CRIG), Ghent, Belgium; Department of Pediatric Hematology, Oncology and Stem Cell Transplantation, Ghent University Hospital, Ghent, Belgium
| | - Frank Speleman
- Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium; Cancer Research Institute Ghent (CRIG), Ghent, Belgium; Department of Biomolecular Medicine, Ghent University, Ghent, Belgium
| | - Bram De Wilde
- Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium; Cancer Research Institute Ghent (CRIG), Ghent, Belgium; Department of Pediatric Hematology, Oncology and Stem Cell Transplantation, Ghent University Hospital, Ghent, Belgium
| | - Tim Lammens
- Cancer Research Institute Ghent (CRIG), Ghent, Belgium; Department of Pediatric Hematology, Oncology and Stem Cell Transplantation, Ghent University Hospital, Ghent, Belgium
| | - Katleen De Preter
- Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium; Cancer Research Institute Ghent (CRIG), Ghent, Belgium; Department of Biomolecular Medicine, Ghent University, Ghent, Belgium
| | - Nadine Van Roy
- Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium; Cancer Research Institute Ghent (CRIG), Ghent, Belgium; Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.
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Kerachian MA, Azghandi M, Mozaffari-Jovin S, Thierry AR. Guidelines for pre-analytical conditions for assessing the methylation of circulating cell-free DNA. Clin Epigenetics 2021; 13:193. [PMID: 34663458 PMCID: PMC8525023 DOI: 10.1186/s13148-021-01182-7] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2021] [Accepted: 10/04/2021] [Indexed: 02/06/2023] Open
Abstract
Methylation analysis of circulating cell-free DNA (cirDNA), as a liquid biopsy, has a significant potential to advance the detection, prognosis, and treatment of cancer, as well as many genetic disorders. The role of epigenetics in disease development has been reported in several hereditary disorders, and epigenetic modifications are regarded as one of the earliest and most significant genomic aberrations that arise during carcinogenesis. Liquid biopsy can be employed for the detection of these epigenetic biomarkers. It consists of isolation (pre-analytical) and detection (analytical) phases. The choice of pre-analytical variables comprising cirDNA extraction and bisulfite conversion methods can affect the identification of cirDNA methylation. Indeed, different techniques give a different return of cirDNA, which confirms the importance of pre-analytical procedures in clinical diagnostics. Although novel techniques have been developed for the simplification of methylation analysis, the process remains complex, as the steps of DNA extraction, bisulfite treatment, and methylation detection are each carried out separately. Recent studies have noted the absence of any standard method for the pre-analytical processing of methylated cirDNA. We have therefore conducted a comprehensive and systematic review of the important pre-analytical and analytical variables and the patient-related factors which form the basis of our guidelines for analyzing methylated cirDNA in liquid biopsy.
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Affiliation(s)
- Mohammad Amin Kerachian
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
- Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
- Cancer Genetics Research Unit, Reza Radiotherapy and Oncology Center, Mashhad, Iran.
| | - Marjan Azghandi
- Cancer Genetics Research Unit, Reza Radiotherapy and Oncology Center, Mashhad, Iran
- Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran
| | - Sina Mozaffari-Jovin
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Alain R Thierry
- IRCM, Institute of Research in Oncology of Montpellier, Montpellier, France.
- INSERM, U1194, Montpellier, France.
- University of Montpellier, Montpellier, France.
- ICM, Regional Institute of Cancer of Montpellier, Montpellier, France.
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Vogt SL, Patel M, Lakha A, Philip V, Omar T, Ashmore P, Pather S, Haley LM, Zheng G, Stone J, Mayne E, Stevens W, Wagner-Johnston N, Gocke CD, Martinson NA, Ambinder RF, Xian RR. Feasibility of Cell-Free DNA Collection and Clonal Immunoglobulin Sequencing in South African Patients With HIV-Associated Lymphoma. JCO Glob Oncol 2021; 7:611-621. [PMID: 33909482 PMCID: PMC8162966 DOI: 10.1200/go.20.00651] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
PURPOSE Diagnosis of AIDS lymphoma in low-resource settings, like South Africa, is often delayed, leaving patients with limited treatment options. In tuberculosis (TB) endemic regions, overlapping signs and symptoms often lead to diagnostic delays. Assessment of plasma cell-free DNA (cfDNA) by next-generation sequencing (NGS) may expedite the diagnosis of lymphoma but requires high-quality cfDNA. METHODS People living with HIV with newly diagnosed aggressive B-cell lymphoma and those with newly diagnosed TB seeking care at Chris Hani Baragwanath Academic Hospital and its surrounding clinics, in Soweto, South Africa, were enrolled in this study. Each participant provided a whole blood specimen collected in cell-stabilizing tubes. Quantity and quality of plasma cfDNA were assessed. NGS of the immunoglobulin heavy chain was performed. RESULTS Nine HIV+ patients with untreated lymphoma and eight HIV+ patients with TB, but without lymphoma, were enrolled. All cfDNA quantity and quality metrics were similar between the two groups, except that cfDNA accounted for a larger fraction of recovered plasma DNA in patients with lymphoma. The concentration of cfDNA in plasma also trended higher in patients with lymphoma. NGS of immunoglobulin heavy chain showed robust amplification of DNA, with large amplicons (> 250 bp) being more readily detected in patients with lymphoma. Clonal sequences were detected in five of nine patients with lymphoma, and none of the patients with TB. CONCLUSION This proof-of-principle study demonstrates that whole blood collected for cfDNA in a low-resource setting is suitable for sophisticated sequencing analyses, including clonal immunoglobulin NGS. The detection of clonal sequences in more than half of patients with lymphoma shows promise as a diagnostic marker that may be explored in future studies.
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Affiliation(s)
- Samantha L Vogt
- Department of Medicine, Johns Hopkins School of Medicine, Baltimore, MD
| | - Moosa Patel
- Clinical Haematology Unit, Department of Medicine, Chris Hani Baragwanath Academic Hospital and Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Atul Lakha
- Clinical Haematology Unit, Department of Medicine, Chris Hani Baragwanath Academic Hospital and Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Vinitha Philip
- Clinical Haematology Unit, Department of Medicine, Chris Hani Baragwanath Academic Hospital and Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Tanvier Omar
- Division of Anatomical Pathology, National Health Laboratory Service, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Philippa Ashmore
- Clinical Haematology, Netcare Olivedale Hospital, Johannesburg, South Africa
| | - Sugeshnee Pather
- Division of Anatomical Pathology, National Health Laboratory Service, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | - Lisa M Haley
- Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD
| | - Gang Zheng
- Department of Pathology, Mayo Clinic, Rochester, MN
| | - Jennifer Stone
- Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD
| | - Elizabeth Mayne
- Molecular Medicine and Haematology, University of the Witwatersrand, Johannesburg, South Africa
| | - Wendy Stevens
- Department of Immunology, Faculty of Health Sciences, University of Witwatersrand and National Health Laboratory Service, Johannesburg, South Africa
| | - Nina Wagner-Johnston
- Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD
| | - Christopher D Gocke
- Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD.,Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD
| | - Neil A Martinson
- Department of Medicine, Johns Hopkins School of Medicine, Baltimore, MD.,Perinatal HIV Research Unit (PHRU), University of the Witwatersrand, Johannesburg, South Africa
| | - Richard F Ambinder
- Department of Medicine, Johns Hopkins School of Medicine, Baltimore, MD.,Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD.,Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD
| | - Rena R Xian
- Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD.,Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD
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The relevance of liquid biopsy in surgical oncology: The application of perioperative circulating nucleic acid dynamics in improving patient outcomes. Surgeon 2021; 20:e163-e173. [PMID: 34362650 DOI: 10.1016/j.surge.2021.06.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2021] [Revised: 06/13/2021] [Accepted: 06/23/2021] [Indexed: 11/20/2022]
Abstract
BACKGROUND Liquid biopsy is gaining increasing clinical utility in the management of cancer patients. The main components of a liquid biopsy are circulating nucleic acids, circulating tumour cells and extracellular vesicles such as exosomes. Circulating nucleic acids including cell free DNA (cfDNA) and circulating tumour DNA (ctDNA) in particular have been the focus of recent attention as they have demonstrated excellent potential in cancer screening, provision of prognostic information and in genomic profiling of a tumour without the need for repeated tissue biopsies. The aim of this review was to explore the current evidence in relation to the use of liquid biopsy in the perioperative setting and identify ways in which liquid biopsy may be applied in the future. METHODS This narrative review is based on a comprehensive literature search up to the 1st of June 2020 for papers relevant to the application of liquid biopsy in surgical oncology, focusing particularly on the perioperative period. RESULTS Recent evidence has demonstrated that perioperative liquid biopsy can accurately stratify patients' risk of recurrence compared to conventional biomarkers. Attention to the perioperative dynamics of liquid biopsy components can potentially provide new understanding of the complex relationship between surgery and cancer outcome. In addition, careful evaluation of liquid biopsy components in the perioperative window may provide important diagnostic and therapeutic information for cancer patients. CONCLUSION The rapidly evolving concept of the liquid biopsy has the potential to become the cornerstone for decision making around surveillance and adjuvant therapies the era of personalised medicine.
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Samoila A, Sosa J, Padilla J, Wutkowski M, Vanness K, Viale A, Berger M, Houck-Loomis B, Pessin M, Peerschke EI. Developing Quality Programs for Cell-Free DNA (cfDNA) Extraction from Peripheral Blood. J Appl Lab Med 2021; 5:788-797. [PMID: 32603443 DOI: 10.1093/jalm/jfaa050] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2019] [Accepted: 01/09/2020] [Indexed: 12/26/2022]
Abstract
BACKGROUND Cell-free DNA (cfDNA) analysis using peripheral blood represents an exciting, minimally invasive technology for cancer diagnosis and monitoring. The reliability of testing is dependent on the accuracy and sensitivity of specific molecular analyses to detect tumor-associated genomic variants and on the quantity and quality of cfDNA available for testing. Specific guidelines for standardization and design of appropriate quality programs focused specifically on cfDNA isolation are lacking, as are standardized quality control reagents. CONTENT This report describes and illustrates quality control and quality assurance processes, supported by generation of in-house quality control material, to ensure the reliability of the preanalytical phase of cfDNA analysis. SUMMARY We have developed a robust quality program to support high-volume automated cfDNA extraction from peripheral blood by implementing processes and procedures designed to monitor the adequacy of specimen collection, specimen stability, efficiency of cfDNA extraction, and cfDNA quality.
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Affiliation(s)
- Aliaksandra Samoila
- Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY
| | - Jose Sosa
- Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY
| | - Jessica Padilla
- Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY
| | - Michael Wutkowski
- Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY
| | - Katelynd Vanness
- Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY
| | - Agnes Viale
- Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY
| | - Michael Berger
- Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY
| | - Brian Houck-Loomis
- Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY
| | - Melissa Pessin
- Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY
| | - Ellinor I Peerschke
- Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY
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Assessment of Circulating Nucleic Acids in Cancer: From Current Status to Future Perspectives and Potential Clinical Applications. Cancers (Basel) 2021; 13:cancers13143460. [PMID: 34298675 PMCID: PMC8307284 DOI: 10.3390/cancers13143460] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2021] [Revised: 07/01/2021] [Accepted: 07/06/2021] [Indexed: 02/06/2023] Open
Abstract
Current approaches for cancer detection and characterization are based on radiological procedures coupled with tissue biopsies, despite relevant limitations in terms of overall accuracy and feasibility, including relevant patients' discomfort. Liquid biopsies enable the minimally invasive collection and analysis of circulating biomarkers released from cancer cells and stroma, representing therefore a promising candidate for the substitution or integration in the current standard of care. Despite the potential, the current clinical applications of liquid biopsies are limited to a few specific purposes. The lack of standardized procedures for the pre-analytical management of body fluids samples and the detection of circulating biomarkers is one of the main factors impacting the effective advancement in the applicability of liquid biopsies to clinical practice. The aim of this work, besides depicting current methods for samples collection, storage, quality check and biomarker extraction, is to review the current techniques aimed at analyzing one of the main circulating biomarkers assessed through liquid biopsy, namely cell-free nucleic acids, with particular regard to circulating tumor DNA (ctDNA). ctDNA current and potential applications are reviewed as well.
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Voss T, Ullius A, Schönborn M, Oelmüller U. Sensitivity assessment of workflows detecting rare circulating cell-free DNA targets: A study design proposal. PLoS One 2021; 16:e0253401. [PMID: 34228726 PMCID: PMC8260181 DOI: 10.1371/journal.pone.0253401] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2021] [Accepted: 06/03/2021] [Indexed: 11/18/2022] Open
Abstract
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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Affiliation(s)
- Thorsten Voss
- R&D Department, QIAGEN GmbH, Hilden, Germany
- * E-mail:
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Ungerer V, Bronkhorst AJ, Van den Ackerveken P, Herzog M, Holdenrieder S. Serial profiling of cell-free DNA and nucleosome histone modifications in cell cultures. Sci Rep 2021; 11:9460. [PMID: 33947882 PMCID: PMC8096822 DOI: 10.1038/s41598-021-88866-5] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2020] [Accepted: 04/08/2021] [Indexed: 02/07/2023] Open
Abstract
Recent advances in basic research have unveiled several strategies for improving the sensitivity and specificity of cell-free DNA (cfDNA) based assays, which is a prerequisite for broadening its clinical use. Included among these strategies is leveraging knowledge of both the biogenesis and physico-chemical properties of cfDNA towards the identification of better disease-defining features and optimization of methods. While good progress has been made on this front, much of cfDNA biology remains uncharted. Here, we correlated serial measurements of cfDNA size, concentration and nucleosome histone modifications with various cellular parameters, including cell growth rate, viability, apoptosis, necrosis, and cell cycle phase in three different cell lines. Collectively, the picture emerged that temporal changes in cfDNA levels are rather irregular and not the result of constitutive release from live cells. Instead, changes in cfDNA levels correlated with intermittent cell death events, wherein apoptosis contributed more to cfDNA release in non-cancer cells and necrosis more in cancer cells. Interestingly, the presence of a ~ 3 kbp cfDNA population, which is often deemed to originate from accidental cell lysis or active release, was found to originate from necrosis. High-resolution analysis of this cfDNA population revealed an underlying DNA laddering pattern consisting of several oligo-nucleosomes, identical to those generated by apoptosis. This suggests that necrosis may contribute significantly to the pool of mono-nucleosomal cfDNA fragments that are generally interrogated for cancer mutational profiling. Furthermore, since active steps are often taken to exclude longer oligo-nucleosomes from clinical biospecimens and subsequent assays this raises the question of whether important pathological information is lost.
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Affiliation(s)
- Vida Ungerer
- Institute for Laboratory Medicine, German Heart Centre, Technical University of Munich, Lazarettstraße 36, 80636, Munich, Germany
| | - Abel J Bronkhorst
- Institute for Laboratory Medicine, German Heart Centre, Technical University of Munich, Lazarettstraße 36, 80636, Munich, Germany
| | | | - Marielle Herzog
- Belgian Volition SRL, 22 Rue Phocas Lejeune, Parc Scientifique Crealys, 5032, Isnes, Belgium
| | - Stefan Holdenrieder
- Institute for Laboratory Medicine, German Heart Centre, Technical University of Munich, Lazarettstraße 36, 80636, Munich, Germany.
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43
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González de Aledo-Castillo JM, Casanueva-Eliceiry S, Soler-Perromat A, Fuster D, Pastor V, Reguart N, Viñolas N, Reyes R, Vollmer I, Paredes P, Puig-Butillé JA. Cell-free DNA concentration and fragment size fraction correlate with FDG PET/CT-derived parameters in NSCLC patients. Eur J Nucl Med Mol Imaging 2021; 48:3631-3642. [PMID: 33797597 DOI: 10.1007/s00259-021-05306-2] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2020] [Accepted: 03/07/2021] [Indexed: 01/26/2023]
Abstract
PURPOSE The aim of our study was to investigate the correlation between cfDNA concentration and fragment size fraction with FDG PET/CT- and CT-derived parameters in untreated NSCLC patient. METHODS Fifty-three patients diagnosed of locally advanced or metastatic NSCLC who had undergone FDG PET/CT, CT and cfDNA analysis prior to any treatment were included in this retrospective study. CfDNA concentration was measured by fluorometry and fragment size fractions were determined by microchip electrophoresis. [18F]F-FDG PET/CT was performed and standardised uptake values (SUV), metabolic tumour volume (MTV) and total lesion glycolysis (TLG) were calculated for primary, extrapulmonary and total disease. CT scans were evaluated according to RECIST 1.1 criteria. RESULTS CfDNA concentration showed a positive correlation with extrapulmonary MTV (r2 = 0.36, P = 0.009), and extrapulmonary TLG (r2 = 0.35, P = 0.009) and their whole-body (wb) ratios. Higher concentrations of total cfDNA were found in patients with liver lesions. Short fragments of cfDNA (100-250 bp) showed a positive correlation with extrapulmonary MTV (r2 = 0.49, P = 0.0005) and extrapulmonary TLG (r2 = 0.39, P = 0.006) and their respective wb ratios, and a negative correlation with SUVmean (r2 = -0.31, P = 0.03) and SUVmean/SUVmax ratio (r2 = -0.34, P = 0.02). A higher fraction of short cfDNA fragments was found in patients with liver and pleural lesions. CONCLUSIONS This study supports the hypothesis that cfDNA concentration and short cfDNA fragment size fraction reflect the tumour burden as well as metabolic activity in advanced NSCLC patients. This suggests their suitability as complementary tests for a more accurate diagnosis of tumour metabolic behaviour and to allow personalised therapies.
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Affiliation(s)
| | | | | | - D Fuster
- Nuclear Medicine Department, Hospital Clínic, Barcelona, Spain.,Thoracic Oncology Unit, Hospital Clínic, Barcelona, Spain.,August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Barcelona, Spain.,Faculty of Medicine, University of Barcelona, Barcelona, Spain
| | - V Pastor
- Molecular Biology CORE, Hospital Clínic, Villarroel 170, 08036, Barcelona, Spain
| | - N Reguart
- Thoracic Oncology Unit, Hospital Clínic, Barcelona, Spain.,Medical Oncology Department, Hospital Clínic, Barcelona, Spain.,August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Barcelona, Spain
| | - N Viñolas
- Medical Oncology Department, Hospital Clínic, Barcelona, Spain
| | - R Reyes
- Medical Oncology Department, Hospital Clínic, Barcelona, Spain
| | - I Vollmer
- Radiology Department, Hospital Clínic, Barcelona, Spain.,Thoracic Oncology Unit, Hospital Clínic, Barcelona, Spain
| | - P Paredes
- Nuclear Medicine Department, Hospital Clínic, Barcelona, Spain.,Thoracic Oncology Unit, Hospital Clínic, Barcelona, Spain.,August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Barcelona, Spain.,Faculty of Medicine, University of Barcelona, Barcelona, Spain
| | - J A Puig-Butillé
- Thoracic Oncology Unit, Hospital Clínic, Barcelona, Spain. .,August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Barcelona, Spain. .,Molecular Biology CORE, Hospital Clínic, Villarroel 170, 08036, Barcelona, Spain.
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44
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González de Aledo-Castillo JM, Arcocha A, Victoria I, Martinez-Puchol AI, Sánchez C, Jares P, Rodríguez GF, Viñolas N, Reyes R, Reguart N, Puig-Butillé JA. Molecular characterization of advanced non-small cell lung cancer patients by cfDNA analysis: experience from routine laboratory practice. J Thorac Dis 2021; 13:1658-1670. [PMID: 33841957 PMCID: PMC8024825 DOI: 10.21037/jtd-20-3142] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2020] [Accepted: 01/20/2021] [Indexed: 12/13/2022]
Abstract
BACKGROUND Analysis of circulating free DNA (cfDNA) by the real-time PCR cobas® EGFR Mutation Test v2 (cobas® EGFR Test) is a diagnostic approach used in clinical practice for the characterization of advanced non-small cell lung cancer (NSCLC) patients. The test additionally outputs a semiquantitative index (SQI) which reflects the proportion of mutated versus wild-type copies of the EGFR gene in cfDNA with potential use as a biomarker. CfDNA concentration and cfDNA fragmentation pattern have also shown potential utility as biomarkers for cancer patients. We evaluated the implementation of EGFR testing and cfDNA related parameters in NSCLC patients in routine clinical setting as biomarkers for disease stage and diagnosis. METHODS A prospective cohort of 173 locally advanced or metastatic NSCLC TKI-naïve patients analyzed by the cobas® EGFR Test were included in the study. Reproducibility of the test was assessed in 56 patients. The concentration of cfDNA and fragment size pattern was measured using fluorometry and microchip electrophoresis respectively. RESULTS The test showed high diagnostic accuracy when compared to the gold standard of biopsy tumor tissue testing. The SQI value showed a moderate reproducibility (r2=0.70) and did not correlate with cfDNA concentration (r2=0.17, P=0.28) or disease stage (stage III patients SQI =9.1±3.1 and stage IV patients SQI =11.5±4.8, P=0.41). We found differences in SQI values according to the type of EGFR mutation (Ex19Del mutations, SQI =13.6; p.L858R, SQI =8.88; P=0.001). Stage IV patients had higher concentrations of cfDNA (P<0.0001) and higher fractions of cfDNA 100-250 base pairs (bp) fragments (P=0.01) compared to stage III patients. From the ROC curve analysis, cfDNA concentration showed higher AUC compared to cfDNA 100-250 bp fragments (0.86 vs. 0.71). We obtained a cut-off value for cfDNA concentration of 20.3 ng/mL with 72.3% sensitivity and 95% specificity for predicting disease stage in TKI-naïve advanced NSCLC patients. CONCLUSIONS The study indicates that cfDNA analysis in plasma for EGFR testing by RT-PCR is an accurate and fast method to initially stratify NSCLC patients in a real-world clinical setting. However, the SQI has limited clinical value. The cfDNA concentration and fragmentation pattern have clear potential clinical utility for tumor staging in NSCLC patients.
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Affiliation(s)
| | - Ainara Arcocha
- Thoracic Oncology Unit, Hospital Clínic, Barcelona, Spain
- Medical Oncology Department, Hospital Clínic, Barcelona, Spain
| | - Iván Victoria
- Thoracic Oncology Unit, Hospital Clínic, Barcelona, Spain
- Medical Oncology Department, Hospital Clínic, Barcelona, Spain
| | | | | | - Pedro Jares
- Thoracic Oncology Unit, Hospital Clínic, Barcelona, Spain
- Molecular Biology CORE, Hospital Clínic, Barcelona, Spain
- Pathology Department, Hospital Clínic, Barcelona, Spain
| | | | - Núria Viñolas
- Thoracic Oncology Unit, Hospital Clínic, Barcelona, Spain
- Medical Oncology Department, Hospital Clínic, Barcelona, Spain
| | - Roxana Reyes
- Thoracic Oncology Unit, Hospital Clínic, Barcelona, Spain
- Medical Oncology Department, Hospital Clínic, Barcelona, Spain
| | - Noemí Reguart
- Thoracic Oncology Unit, Hospital Clínic, Barcelona, Spain
- Medical Oncology Department, Hospital Clínic, Barcelona, Spain
- August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Barcelona, Spain
| | - Joan Antón Puig-Butillé
- Thoracic Oncology Unit, Hospital Clínic, Barcelona, Spain
- Molecular Biology CORE, Hospital Clínic, Barcelona, Spain
- August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Barcelona, Spain
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45
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Martins VF, Dobson CR, Begur M, Parekh J, Ball ST, Gonzalez F, Hughes-Austin JM, Schenk S. Surgical site peptidylarginine deaminase 4 (PAD4), a biomarker of NETosis, correlates with insulin resistance in total joint arthroplasty patients: A preliminary report. PLoS One 2021; 16:e0245594. [PMID: 33481860 PMCID: PMC7822240 DOI: 10.1371/journal.pone.0245594] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2020] [Accepted: 01/04/2021] [Indexed: 01/12/2023] Open
Abstract
While obesity and insulin resistance are known risk factors for wound complications after total joint arthroplasty (TJA), the biologic causes remain to be elucidated. Recently, neutrophil extracellular trap formation (NETosis) was identified as a mediator of delayed wound healing in insulin resistant states. Herein, we explored the relationship between obesity, insulin resistance and biomarkers of NET formation in TJA subjects. We enrolled 14 obese (body mass index [BMI]≥30 kg/m2), and 15 lean (BMI<30 kg/m2) subjects undergoing primary knee or hip TJA. On the day of surgery, skeletal muscle proximal to the operated joint and plasma were collected. Protein abundance of NETosis biomarkers, peptidylarginine deaminase 4 (PAD4) and neutrophil elastase (NE) were assessed in skeletal muscle by immunoblotting and metabolic parameters (glucose, insulin, triglycerides, free fatty acids) and cell-free double-stranded DNA (cf-dsDNA) were assessed in plasma and were correlated with obesity and insulin resistance (as measured by the homeostatic model assessment for insulin resistance). When comparing lean and obese subjects, there were no significant differences in plasma cf-dsDNA or skeletal muscle NE or PAD4 abundance. In contrast, skeletal muscle PAD4 abundance, but not NE or plasma cf-dsDNA, was positively correlated with insulin resistance. Compared to insulin sensitive subjects, insulin resistant TJA subjects have higher expression of PAD4 at the surgical site and therefore may have higher rates of NET formation, which may lead to delayed surgical site wound healing.
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Affiliation(s)
- Vitor F. Martins
- Department of Orthopaedic Surgery, University of California San Diego, La Jolla, CA, United States of America
- Biomedical Sciences Graduate Program, University of California San Diego, La Jolla, CA, United States of America
| | - Christopher R. Dobson
- Department of Orthopaedic Surgery, University of California San Diego, La Jolla, CA, United States of America
| | - Maedha Begur
- Department of Orthopaedic Surgery, University of California San Diego, La Jolla, CA, United States of America
| | - Jesal Parekh
- Department of Orthopaedic Surgery, University of California San Diego, La Jolla, CA, United States of America
| | - Scott T. Ball
- Department of Orthopaedic Surgery, University of California San Diego, La Jolla, CA, United States of America
| | - Francis Gonzalez
- Department of Orthopaedic Surgery, University of California San Diego, La Jolla, CA, United States of America
| | - Jan M. Hughes-Austin
- Department of Orthopaedic Surgery, University of California San Diego, La Jolla, CA, United States of America
| | - Simon Schenk
- Department of Orthopaedic Surgery, University of California San Diego, La Jolla, CA, United States of America
- Biomedical Sciences Graduate Program, University of California San Diego, La Jolla, CA, United States of America
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46
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Doludin YV, Limonova AS, Kozlova VA, Efimova AI, Borisova AL, Meshkov AN, Pokrovskaya MS, Drapkina OM. Collection and storage of DNA-containing biomaterial and isolated DNA. КАРДИОВАСКУЛЯРНАЯ ТЕРАПИЯ И ПРОФИЛАКТИКА 2020. [DOI: 10.15829/1728-8800-2020-2730] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
The advances of biomedicine include the new technologies, diagnosis and treatment techniques, as well as the practical use of new types of biological targets, in particular, nucleic acids. Genomic deoxyribonucleic acid (DNA), extracellular DNA (exDNA) and microbiome DNA obtained from different types of samples (tissues, blood and its derivatives, feces, etc.) are used as objects of genetic research. The use of new technologies for DNA analysis required the development of standardized methods for processing biological samples in order to obtain high-quality DNA samples. The research uses various methods for collecting, preparing samples and storing various DNA-containing biomaterials and isolated DNA, as well as methods for assessing the quality of samples and biobank standards. It is obvious that the use of uniform standards will allow large-scale genetic research on the basis of biobanks and research laboratories. Specialists from professional organizations such as International Society for Biological and Environmental Repositories (ISBER), Biobanking and BioMolecular Resources Research Infrastructure-European Research Infrastructure Consortium (BBMRI-ERIC), European, Middle Eastern & African Society for Biopreservationa and Biobanking (ESBB) and the Russian National Association of Biobanks and Biobanking Professionals.
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Affiliation(s)
- Yu. V. Doludin
- National Medical Research Center for Therapy and Preventive Medicine
| | - A. S. Limonova
- National Medical Research Center for Therapy and Preventive Medicine
| | - V. A. Kozlova
- National Medical Research Center for Therapy and Preventive Medicine
| | - A. I. Efimova
- National Medical Research Center for Therapy and Preventive Medicine
| | - A. L. Borisova
- National Medical Research Center for Therapy and Preventive Medicine
| | - A. N. Meshkov
- National Medical Research Center for Therapy and Preventive Medicine
| | - M. S. Pokrovskaya
- National Medical Research Center for Therapy and Preventive Medicine
| | - O. M. Drapkina
- National Medical Research Center for Therapy and Preventive Medicine
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47
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Yang WY, Feng LF, Meng X, Chen R, Xu WH, Hou J, Xu T, Zhang L. Liquid biopsy in head and neck squamous cell carcinoma: circulating tumor cells, circulating tumor DNA, and exosomes. Expert Rev Mol Diagn 2020; 20:1213-1227. [PMID: 33232189 DOI: 10.1080/14737159.2020.1855977] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
Introduction: Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers worldwide. Due to a lack of reliable markers, HNSCC patients are usually diagnosed at a late stage, which will lead to a worse outcome. Therefore, it is critical to improve the clinical management of cancer patients. Nowadays, the development of liquid biopsy enables a minimally invasive manner to extract molecular information from HNSCCs. Thus, this review aims to outline the clinical value of liquid biopsy in early detection, real-time monitoring, and prognostic evaluation of HNSCC. Areas covered: This comprehensive review focused on the characteristics as well as clinical applications of three liquid biopsy markers (CTCs, ctDNA, and exosomes) in HNSCC. What is more, it is promising to incorporate machine learning and 3D organoid models in the liquid biopsy of HNSCC. Expert opinion: Liquid biopsy provides a noninvasive technique to reflect the inter and intra-lesional heterogeneity through the detection of tumor cells or materials released from the primary and secondary tumors. Recently, some evolving technologies have the potential to combine with liquid biopsy to improve clinical management of HNSCC patients.
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Affiliation(s)
- Wen-Ying Yang
- College & Hospital of Stomatology, Anhui Medical University, Key Lab. Of Oral Diseases Research of Anhui Province , Hefei, 230032, China
| | - Lin-Fei Feng
- Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Anhui Medical University , Hefei, 230032, China
| | - Xiang Meng
- College & Hospital of Stomatology, Anhui Medical University, Key Lab. Of Oral Diseases Research of Anhui Province , Hefei, 230032, China
| | - Ran Chen
- School of Stomatology, Anhui Medical University , Hefei, 230032, China
| | - Wen-Hua Xu
- College & Hospital of Stomatology, Anhui Medical University, Key Lab. Of Oral Diseases Research of Anhui Province , Hefei, 230032, China
| | - Jun Hou
- Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Anhui Medical University , Hefei, 230032, China
| | - Tao Xu
- School of Pharmacy, Anhui Key Laboratory of Bioactivity of Natural Products, Anhui Medical University , Hefei, 230032, China.,Institute for Liver Diseases of Anhui Medical University, Anhui Medical University , Hefei, 230032, China
| | - Lei Zhang
- College & Hospital of Stomatology, Anhui Medical University, Key Lab. Of Oral Diseases Research of Anhui Province , Hefei, 230032, China.,Periodontal Department, Anhui Stomatology Hospital affiliated to Anhui Medical University , Hefei, 230032, China
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48
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Pös Z, Pös O, Styk J, Mocova A, Strieskova L, Budis J, Kadasi L, Radvanszky J, Szemes T. Technical and Methodological Aspects of Cell-Free Nucleic Acids Analyzes. Int J Mol Sci 2020; 21:ijms21228634. [PMID: 33207777 PMCID: PMC7697251 DOI: 10.3390/ijms21228634] [Citation(s) in RCA: 31] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2020] [Revised: 11/12/2020] [Accepted: 11/13/2020] [Indexed: 02/07/2023] Open
Abstract
Analyzes of cell-free nucleic acids (cfNAs) have shown huge potential in many biomedical applications, gradually entering several fields of research and everyday clinical care. Many biological properties of cfNAs can be informative to gain deeper insights into the function of the organism, such as their different types (DNA, RNAs) and subtypes (gDNA, mtDNA, bacterial DNA, miRNAs, etc.), forms (naked or vesicle bound NAs), fragmentation profiles, sequence composition, epigenetic modifications, and many others. On the other hand, the workflows of their analyzes comprise many important steps, from sample collection, storage and transportation, through extraction and laboratory analysis, up to bioinformatic analyzes and statistical evaluations, where each of these steps has the potential to affect the outcome and informational value of the performed analyzes. There are, however, no universal or standard protocols on how to exactly proceed when analyzing different cfNAs for different applications, at least according to our best knowledge. We decided therefore to prepare an overview of the available literature and products commercialized for cfNAs processing, in an attempt to summarize the benefits and limitations of the currently available approaches, devices, consumables, and protocols, together with various factors influencing the workflow, its processes, and outcomes.
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Affiliation(s)
- Zuzana Pös
- Institute of Clinical and Translational Research, Biomedical Research Center, Slovak Academy of Sciences, 845 05 Bratislava, Slovakia; (Z.P.); (A.M.); (L.K.)
- Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, 841 04 Bratislava, Slovakia;
- Geneton Ltd., 841 04 Bratislava, Slovakia; (L.S.); (J.B.)
| | - Ondrej Pös
- Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, 841 04 Bratislava, Slovakia;
- Geneton Ltd., 841 04 Bratislava, Slovakia; (L.S.); (J.B.)
- Comenius University Science Park, Comenius University, 841 04 Bratislava, Slovakia;
| | - Jakub Styk
- Comenius University Science Park, Comenius University, 841 04 Bratislava, Slovakia;
- Faculty of Medicine, Institute of Medical Biology, Genetics and Clinical Genetics, 811 08 Bratislava, Slovakia
| | - Angelika Mocova
- Institute of Clinical and Translational Research, Biomedical Research Center, Slovak Academy of Sciences, 845 05 Bratislava, Slovakia; (Z.P.); (A.M.); (L.K.)
- Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, 841 04 Bratislava, Slovakia;
| | | | - Jaroslav Budis
- Geneton Ltd., 841 04 Bratislava, Slovakia; (L.S.); (J.B.)
- Comenius University Science Park, Comenius University, 841 04 Bratislava, Slovakia;
- Slovak Center of Scientific and Technical Information, 811 04 Bratislava, Slovakia
| | - Ludevit Kadasi
- Institute of Clinical and Translational Research, Biomedical Research Center, Slovak Academy of Sciences, 845 05 Bratislava, Slovakia; (Z.P.); (A.M.); (L.K.)
- Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, 841 04 Bratislava, Slovakia;
| | - Jan Radvanszky
- Institute of Clinical and Translational Research, Biomedical Research Center, Slovak Academy of Sciences, 845 05 Bratislava, Slovakia; (Z.P.); (A.M.); (L.K.)
- Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, 841 04 Bratislava, Slovakia;
- Comenius University Science Park, Comenius University, 841 04 Bratislava, Slovakia;
- Correspondence: (J.R.); (T.S.); Tel.: +421-2-60296637 (J.R.); +421-2-9026-8807 (T.S.)
| | - Tomas Szemes
- Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, 841 04 Bratislava, Slovakia;
- Geneton Ltd., 841 04 Bratislava, Slovakia; (L.S.); (J.B.)
- Comenius University Science Park, Comenius University, 841 04 Bratislava, Slovakia;
- Correspondence: (J.R.); (T.S.); Tel.: +421-2-60296637 (J.R.); +421-2-9026-8807 (T.S.)
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Van Paemel R, De Koker A, Caggiano C, Morlion A, Mestdagh P, De Wilde B, Vandesompele J, De Preter K. Genome-wide study of the effect of blood collection tubes on the cell-free DNA methylome. Epigenetics 2020; 16:797-807. [PMID: 33074045 PMCID: PMC8216177 DOI: 10.1080/15592294.2020.1827714] [Citation(s) in RCA: 28] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
The methylation pattern of cfDNA, isolated from liquid biopsies, is gaining substantial interest for diagnosis and monitoring of diseases. We have evaluated the impact of type of blood collection tube and time delay between blood draw and plasma preparation on bisulphite-based cfDNA methylation profiling. Fifteen tubes of blood were drawn from three healthy volunteer subjects (BD Vacutainer K2E EDTA spray tubes, Streck Cell-Free DNA BCT tubes, PAXgene Blood ccfDNA tubes, Roche Cell-Free DNA Collection tubes and Biomatrica LBgard blood tubes in triplicate). Samples were either immediately processed or stored at room temperature for 24 or 72 hours before plasma preparation. DNA fragment size was evaluated by capillary electrophoresis. Reduced representation bisulphite sequencing was performed on the cell-free DNA isolated from these plasma samples. We evaluated the impact of blood tube and time delay on several quality control metrics. All preservation tubes performed similar on the quality metrics that were evaluated. Furthermore, a considerable increase in cfDNA concentration and the fraction of it derived from NK cells was observed after a 72-hour time delay in EDTA tubes. The methylation pattern of cfDNA is robust and reproducible in between the different preservation tubes. EDTA tubes processed as soon as possible, preferably within 24 hours, are the most cost effective. If immediate processing is not possible, preservation tubes are valid alternatives.
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Affiliation(s)
- Ruben Van Paemel
- Cancer Research Institute Ghent (CRIG), Ghent, Belgium.,Department of Internal Medicine and Pediatrics, Ghent University Hospital, Ghent, Belgium.,Department of Biomolecular Medicine, Ghent University, Ghent, Belgium
| | - Andries De Koker
- Cancer Research Institute Ghent (CRIG), Ghent, Belgium.,Center for Medical Biotechnology, Flemish Institute Biotechnology (VIB), Ghent, Belgium
| | - Christa Caggiano
- Departments of Neurology and Computational Medicine, University of California, Los Angeles, CA, USA
| | - Annelien Morlion
- Cancer Research Institute Ghent (CRIG), Ghent, Belgium.,Department of Biomolecular Medicine, Ghent University, Ghent, Belgium
| | - Pieter Mestdagh
- Cancer Research Institute Ghent (CRIG), Ghent, Belgium.,Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.,Biogazelle NV, Ghent, Belgium
| | - Bram De Wilde
- Cancer Research Institute Ghent (CRIG), Ghent, Belgium.,Department of Internal Medicine and Pediatrics, Ghent University Hospital, Ghent, Belgium.,Department of Biomolecular Medicine, Ghent University, Ghent, Belgium
| | - Jo Vandesompele
- Cancer Research Institute Ghent (CRIG), Ghent, Belgium.,Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.,Biogazelle NV, Ghent, Belgium
| | - Katleen De Preter
- Cancer Research Institute Ghent (CRIG), Ghent, Belgium.,Department of Biomolecular Medicine, Ghent University, Ghent, Belgium
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50
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Pellini B, Szymanski J, Chin RI, Jones PA, Chaudhuri AA. Liquid Biopsies Using Circulating Tumor DNA in Non-Small Cell Lung Cancer. Thorac Surg Clin 2020; 30:165-177. [PMID: 32327175 DOI: 10.1016/j.thorsurg.2020.01.005] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Liquid biopsies for the diagnosis and treatment of lung cancer have developed rapidly, driven primarily by technical advances in sensitivity to detect circulating tumor DNA (ctDNA). Still, technical limitations such as the challenge of detecting low-level ctDNA variants and distinguishing tumor-related variants from clonal hematopoiesis remain. With further technical advancements, new applications for ctDNA analysis are emerging including detection of post-treatment molecular residual disease (MRD), clinical trial selection, and early cancer detection. This chapter reviews the current state of ctDNA testing in NSCLC, the underlying technological advances enabling ctDNA detection, and the potential to expand ctDNA analysis to new applications.
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Affiliation(s)
- Bruna Pellini
- Department of Medicine, Division of Oncology, Washington University School of Medicine, Division of Oncology Campus Box 8056, 660 South Euclid Avenue, St Louis, MO 63110, USA
| | - Jeffrey Szymanski
- Department of Radiation Oncology, Division of Cancer Biology, Washington University School of Medicine, Radiation Oncology Campus Box 8224, 660 South Euclid Avenue, St Louis, MO 63110, USA
| | - Re-I Chin
- Department of Radiation Oncology, Division of Cancer Biology, Washington University School of Medicine, Radiation Oncology Campus Box 8224, 660 South Euclid Avenue, St Louis, MO 63110, USA
| | - Paul A Jones
- Department of Radiation Oncology, Division of Cancer Biology, Washington University School of Medicine, Radiation Oncology Campus Box 8224, 660 South Euclid Avenue, St Louis, MO 63110, USA
| | - Aadel A Chaudhuri
- Department of Radiation Oncology, Division of Cancer Biology, Washington University School of Medicine, Radiation Oncology Campus Box 8224, 660 South Euclid Avenue, St Louis, MO 63110, USA.
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