Implementing circulating tumor DNA analysis in a clinical laboratory: A user manual

Koessler, Thibaud; Addeo, Alfredo; Nouspikel, Thierry

doi:10.1016/bs.acc.2018.12.004

Cited by 11 publications

(8 citation statements)

References 135 publications

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“…The ccf-DNA is under extensive investigation as a biomarker for liquid biopsy aiming at early cancer detection, monitoring of disease progression, and therapeutic response [3][4][5][6][7][8][9][10]. In clinical studies, plasma DNA levels in lung cancer patients have been shown to correlate with disease stage, cancer histopathology, disease progression rate, and response to therapy [11].…”

Section: Introductionmentioning

confidence: 99%

Quantification of Circulating Cell Free Mitochondrial DNA in Extracellular Vesicles with PicoGreen™ in Liquid Biopsies: Fast Assessment of Disease/Trauma Severity

Marcatti

Saada

Okereke

et al. 2021

Cells

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The analysis of circulating cell free DNA (ccf-DNA) is an emerging diagnostic tool for the detection and monitoring of tissue injury, disease progression, and potential treatment effects. Currently, most of ccf-DNA in tissue and liquid biopsies is analysed with real-time quantitative PCR (qPCR) that is primer- and template-specific, labour intensive and cost-inefficient. In this report we directly compare the amounts of ccf-DNA in serum of healthy volunteers, and subjects presenting with various stages of lung adenocarcinoma, and survivors of traumatic brain injury using qPCR and quantitative PicoGreen™ fluorescence assay. A significant increase of ccf-DNA in lung adenocarcinoma and traumatic brain injury patients, in comparison to the group of healthy human subjects, was found using both analytical methods. However, the direct correlation between PicoGreen™ fluorescence and qPCR was found only when mitochondrial DNA (mtDNA)-specific primers were used. Further analysis of the location of ccf-DNA indicated that the majority of DNA is located within lumen of extracellular vesicles (EVs) and is easily detected with mtDNA-specific primers. We have concluded that due to the presence of active DNases in the blood, the analysis of DNA within EVs has the potential of providing rapid diagnostic outcomes. Moreover, we speculate that accurate and rapid quantification of ccf-DNA with PicoGreen™ fluorescent probe used as a point of care approach could facilitate immediate assessment and treatment of critically ill patients.

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Section: Introductionmentioning

confidence: 99%

Quantification of Circulating Cell Free Mitochondrial DNA in Extracellular Vesicles with PicoGreen™ in Liquid Biopsies: Fast Assessment of Disease/Trauma Severity

Marcatti

Saada

Okereke

et al. 2021

Cells

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“…To our knowledge, there is no such comparative study for the extraction of ctDNA in lymphoma patients, but there is no obvious reason to assume that lymphoma ctDNA extraction should be different from other cancer ctDNA. For a detailed review about this topic, the reader is referred to a recently published review [ 45 ].…”

Section: The Right Cfdna Extraction Methodsmentioning

confidence: 99%

How to Obtain a High Quality ctDNA in Lymphoma Patients: Preanalytical Tips and Tricks

et al. 2021

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The analysis of circulating tumor DNA (ctDNA) released by tumor cells holds great promise for patients with lymphoma, to refine the diagnostic procedure, clarify the prognosis, monitor the response to treatment, and detect relapses earlier. One of the main challenges of the coming years is to adapt techniques from highly specialized translational teams to routine laboratories as this requires a careful technical and clinical validation, and we have to achieve this as fast as possible to transform a promising biomarker into a routine analysis to have a direct consequence on patient care. Whatever the analytical technology used, the prerequisite is to obtain high yields of ctDNA of optimal quality. In this review, we propose a step-by-step description of the preanalytical process to obtain high-quality ctDNA, emphasizing the technical choices that need to be made and the experimental data that can support these choices.

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“…During the initial, straightforward step, individual sequence reads are aligned to the human reference genome, and divergences from the reference (variants) are collated. If molecular barcodes are used, reads can be aggregated based on the original DNA molecule they were amplified from, allowing for some level of error polishing (26). The second and most critical step is to classify variants: some will be germline polymorphisms that are easy to recognize as variant frequencies will approach 50 or 100%, depending on whether the patient is heterozygous or homozygous.…”

Section: Correlation Between Mutations Found In Tumor and Plasmamentioning

confidence: 99%

Tissue-Plasma TMB Comparison and Plasma TMB Monitoring in Patients With Metastatic Non-small Cell Lung Cancer Receiving Immune Checkpoint Inhibitors

et al. 2020

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Immuno-oncology is an ever growing field that has seen important progress across the spectrum of cancers. Responses can be deep and durable. However, as only a minority of patients respond to checkpoint inhibition, predictive biomarkers are needed. Cancer is a genetic disease arising from the accumulation of somatic mutations in the DNA of affected cells. Tumor mutational burden (TMB), represents the number of somatic mutations in a tumor that form neoantigens, responsible for the immunogenicity of tumors. Randomized controlled trials have so far failed to show a survival benefit when stratifying patients by tissue TMB. TMB has also been evaluated in plasma (PTMB). PTMB is anticipated to represent the biology of the entire cancer, whereas obtaining tissue of an amenable primary or a metastatic lesion may be prone to sampling bias because of tumor heterogeneity. For this reason, we are evaluating the correlation between TMB and PTMB, and prospectively evaluating the impact of these biomarkers on clinical outcomes. We also discuss the technical difficulties inherent to performing and comparing these analyses. Furthermore, we evaluate the correlation between the evolution of PTMB during an immunotherapy treatment and response at 3 and 6 months, as we believe PTMB may be a dynamic biomarker. In this paper, we present results from the first 4 patients in this project.

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Implementing circulating tumor DNA analysis in a clinical laboratory: A user manual

Cited by 11 publications

References 135 publications

Quantification of Circulating Cell Free Mitochondrial DNA in Extracellular Vesicles with PicoGreen™ in Liquid Biopsies: Fast Assessment of Disease/Trauma Severity

Quantification of Circulating Cell Free Mitochondrial DNA in Extracellular Vesicles with PicoGreen™ in Liquid Biopsies: Fast Assessment of Disease/Trauma Severity

How to Obtain a High Quality ctDNA in Lymphoma Patients: Preanalytical Tips and Tricks

Tissue-Plasma TMB Comparison and Plasma TMB Monitoring in Patients With Metastatic Non-small Cell Lung Cancer Receiving Immune Checkpoint Inhibitors

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