Circulating Cell-Free DNA and RNA Analysis as Liquid Biopsy: Optimal Centrifugation Protocol

Sorber, Laure; Zwaenepoel, Karen; Jacobs, Julie; Winne, Koen De; Goethals, Sofie; Reclusa, Pablo; Casteren, Kaat Van; Augustus, Elien; Lardon, Filip; Roeyen, Geert; Peeters, Marc; Meerbeeck, Jan P. van; Rolfo, Christian; Pauwels, Patrick

doi:10.3390/cancers11040458

Cited by 78 publications

(59 citation statements)

References 36 publications

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“…State‐of‐the‐art updates on technologies and clinical applications of liquid biopsy, including the abovementioned CTCs, ctDNA, ctRNA, microvesicles, and single‐cell sequencing, hold the prospects for anticancer drug development, individualized treatment on cancer patients, and tumor risk assessment in general population (Anfossi et al, 2018; Best et al, 2015; Osumi et al, 2019; Sorber et al, 2019; Wang & Chen, 2014, Xu et al, 2018). Of them, CTCs recognized as the precursors of metastasis and the dissemination in patients’ bloodstream are involved in metastatic and recurrent cancers (Keller & Pantel, 2019).…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, CTCs in combination with other liquid biopsy technologies such as circulating tumor DNAs, microRNAs and noncoding RNAs (ctDNA, ctRNA), tumor‐educated platelets, extracellular vesicles, or even with single‐cell technologies have contributed to uncover cancer heterogeneity and hold the promise for providing unique insights into genomic aberrations as well (Anfossi et al, 2018; Best et al, 2015; Osumi et al, 2019; Sorber et al, 2019; Wang & Chen, 2014; Xu et al, 2018). For instance, we recently reported the genome‑wide pattern differences in copy number variations (CNVs) of CTCs in breast cancer patients with liver metastasis (Zou et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

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Multifaceted modifications for a cell size‐based circulating tumor cell scope technique hold the prospect for large‐scale application in general populations

Zhang

Zou

et al. 2020

Cell Biology International

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Circulating tumor cells (CTCs) indicate the diagnosis and prognosis of cancer patients, together with benefiting individual treatment and anticancer drug development. However, their large‐scale application in general population still requires systematically multifaceted modifications for currently proprietary new technologies based on filtration. We primitively utilized a cell size‐based platform to evaluate the recovery efficiency of spiked abnormal cell lines and analyzed circulating abnormal cells (CACs). To dissect the subpopulations of CACs, we conducted immunofluorescent (IF) staining with a combination of unique biomarkers of CTCs and circulating endothelial cells (CECs). Furthermore, we improved the CTC screening system by assessing the feasibility of transferring CTCs for automatic IF analysis, together with simulating and optimizing the circumstances for long‐term CTC storage and transportation. We detected CACs in 15 HD candidates with CTC characteristics such as abnormally large cytomorphology, high nuclear−cytoplasmic ratio, and positive for panCK or VIM staining. Thereafter, we improved accuracy of the platform by distinguishing CTCs from CECs, which satisfied the elementary requirement for small‐scale CTC screening in HD candidates. Finally, large‐scale CTC screening in general population was available after multifaceted modifications including automatic analysis by transferring CTCs on slides, choosing the appropriate blood‐collecting tube, optimizing the conditions for long‐term CTC storage and transportation, and evaluating the potential effect on the CTC phenotype. Hence, we systematically modified the scope of technique parameters, improved the accuracy of early cancer detection, and made it realizable for large‐scale CTC or CEC screening in general population.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Multifaceted modifications for a cell size‐based circulating tumor cell scope technique hold the prospect for large‐scale application in general populations

Zhang

Zou

et al. 2020

Cell Biology International

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“…It has been suggested that interventional therapies may influence the expression levels of hsa_circ_0003056 in the plasma. circRNAs are relatively stable in plasma, however, the total free nucleic acid content of the peripheral blood is low (28). There are no stable, internal or precise quantification methods for the determination of these levels.…”

Section: Discussionmentioning

confidence: 99%

Selective downregulation of distinct circRNAs in the tissues and plasma of patients with primary hepatic carcinoma

et al. 2019

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Multiple studies have indicated that circular RNAs (circRNAs) are closely associated with malignant tumor development and metastasis. However, the significance of circRNAs in primary hepatic carcinoma (PHC), particularly in the plasma, remains largely undetermined. In the current study, circRNA expression profiles in three pairs of tumor and adjacent normal samples from patients with PHC, were examined using circRNA chip screening. A total of 80 circRNAs were upregulated, while 75 circRNAs were downregulated in PHC tissues, relative to para-tumor tissues (fold change, ≥1.5). A total of two upregulated circRNAs and three downregulated circRNAs were selected as candidates for further validation of their differential expression. This was performed using reverse transcription-quantitative PCR with 11 pairs of PHC tissues and para-tumor tissues. The results indicated that hsa_circ_0003056 exhibited reduced expression in PHC tissues. Moreover, hsa_circ_0003056 and hsa_circ_0067127 were quantified in the plasma samples of 35 PHC patients and 32 healthy donors. The results revealed that hsa_circ_0067127 was significantly downregulated in the patients' plasma. Finally, a competing endogenous RNA network was constructed, which consisted of one circRNA (hsa_circ_0003056 or has_circ_0067127), five miRNAs and miRNA-targeted genes (mRNAs). Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that differentially expressed (DE) genes were significantly enriched in the pathway associated with ‘regulation of the pluripotency of stem cells’ for hsa_circ_0003056, and ‘ubiquitin-mediated proteolysis’ and ‘prostate cancer’ for hsa_circ_0067127. Gene ontology analysis revealed that DE genes were primarily associated with the ‘modulation of kinase activity’ and ‘intracellular and transmembrane-ephrin receptor activity’ for hsa_circ_0003056, ‘artery morphogenesis activity’, ‘HOPS complex and transferase activity’ and in ‘transferring acyl groups’ for hsa_circ_0067127. This approach indicated that hsa_circ_0003056 in PHC tissue, and hsa_circ_0067127 in PHC plasma, are downregulated and may be implicated in the tumorigenesis of PHC.

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“…It is recommended to perform plasma isolation soon after blood withdrawal, while inappropriate handling and storage leads to non-informative liquid biopsy samples due to leukocyte lysis and therefore contamination with genomic DNA [ 13 , 14 , 15 ], thereby leading to non-suitable therapeutic options and treatment failure of those inappropriately handled samples [ 16 ]. Further, the usage of different blood collection tubes and centrifugation protocols for plasma isolation might cause a contamination of the plasma samples with high molecular genomic DNA to variable degrees [ 17 , 18 , 19 , 20 ]. However, how to use those non-appropriate handled liquid biopsy samples for further NGS approaches and how to rescue ctDNA from those samples remains a matter of debate.…”

Section: Introductionmentioning

confidence: 99%

Rescue of Non-Informative Circulating Tumor DNA to Monitor the Mutational Landscape in NSCLC

Mayer¹,

Schmidtke-Schrezenmeier

Buske

et al. 2020

Cancers

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In non-small cell lung cancer (NSCLC) the usage of plasma-derived circulating tumor DNA (ctDNA) have come into focus to obtain a comprehensive genetic profile of a given lung cancer. Despite the usage of specific sampling tubes, archived plasma samples as well as inappropriately treated blood samples still cause a loss of information due to cell lysis and contamination with cellular DNA. Our aim was to establish a reliable protocol to rescue ctDNA from such non-informative samples to monitor the mutational landscape in NSCLC. As a proof-of-concept study we used archived plasma samples derived from whole blood EDTA samples of 51 patients suffering from NSCLC. Analysis of the isolated plasma DNA determined only a small fraction of ctDNA in a range of 90–250 bp. By applying a specific purification procedure, we were able to increase the informative ctDNA content and improve in a cohort of 42 patients the detection of driver mutations from 32% to 79% of the mutations found in tissue biopsies. Thus, we present here an easy to perform, time and cost effective procedure to rescue non-informative ctDNA samples, which is sufficient to detect oncogenic mutations in NGS approaches and is therefore a valuable technical improvement for laboratories handling liquid biopsy samples.

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Circulating Cell-Free DNA and RNA Analysis as Liquid Biopsy: Optimal Centrifugation Protocol

Cited by 78 publications

References 36 publications

Multifaceted modifications for a cell size‐based circulating tumor cell scope technique hold the prospect for large‐scale application in general populations

Multifaceted modifications for a cell size‐based circulating tumor cell scope technique hold the prospect for large‐scale application in general populations

Selective downregulation of distinct circRNAs in the tissues and plasma of patients with primary hepatic carcinoma

Rescue of Non-Informative Circulating Tumor DNA to Monitor the Mutational Landscape in NSCLC

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