Evaluation of Preanalytical Conditions and Implementation of Quality Control Steps for Reliable Gene Expression and DNA Methylation Analyses in Liquid Biopsies

Zavridou, Martha; Mastoraki, Sophia; Strati, Αreti; Tzanikou, Eleni; Chimonidou, Maria; Lianidou, Evi

doi:10.1373/clinchem.2018.292318

Cited by 42 publications

(34 citation statements)

References 39 publications

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“…Capture beads, coated with the monoclonal antibody BerEP4 against the human epithelial antigen, EpCAM, were used for CTCs enrichment (Dynabeads ® Epithelial Enrich, Life Technologies, USA) as previously described [17,18]. Exosomes were isolated from 2mL of plasma by affinity-based binding to a spin column (exoRNeasy Maxi kit, QI-AGEN ® , Germany) as previously described [19].…”

Section: Isolation Of Epcam-positive Ctcs and Exosomesmentioning

confidence: 99%

Prognostic Significance of Gene Expression and DNA Methylation Markers in Circulating Tumor Cells and Paired Plasma Derived Exosomes in Metastatic Castration Resistant Prostate Cancer

Zavridou

Strati

Bournakis

et al. 2021

Cancers

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Liquid biopsy, based on the analysis of circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), provides non-invasive real-time monitoring of tumor evolution and therapeutic efficacy. We performed for the first time a direct comparison study on gene expression and DNA methylation markers in CTCs and paired plasma-derived exosomes and evaluated their prognostic significance in metastatic castration resistant prostate cancer. This prospective liquid biopsy (LB) study was based on a group of 62 metastatic castration resistant prostate cancer (mCRPC) patients and 10 healthy donors (HD) as controls. Identical blood draws were used to: a) enumerate CTC and tumor-derived extracellular vesicles (tdEVs) using CellSearch (CS) and b) analyze CTCs and paired plasma-derived exosomes at the gene expression and DNA methylation level. CTCs were enumerated using CellSearch in 57/62 patients, with values ranging from 5 to 854 cells/7.5mLPB. Our results revealed for the first time a significantly higher positivity of gene expression markers (CK-8, CK-18, TWIST1, PSMA, AR-FL, AR-V7, AR-567 and PD-L1 mRNA) in EpCAM-positive CTCs compared to plasma-derived exosomes. GSTP1, RASSF1A and SCHLAFEN were methylated both in CTC and exosomes. In CTCs, Kaplan–Meier analysis revealed that CK-19(p = 0.009), PSMA(p = 0.001), TWIST1(p = 0.001) expression and GSTP1(p = 0.001) methylation were correlated with OS, while in exosomes GSTP1 (p = 0.007) and RASSF1A (p = 0.001) methylation was correlated with OS. Our direct comparison study of CTCs and exosomes at gene expression and DNA methylation level, revealed for the first time a significantly higher positivity in EpCAM-positive CTCs compared to plasma-derived exosomes. Future perspective of this study should be the evaluation of clinical utility of molecular biomarkers in CTCs and exosomes on independent multicentric cohorts with mCRPC patients.

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Section: Isolation Of Epcam-positive Ctcs and Exosomesmentioning

confidence: 99%

Prognostic Significance of Gene Expression and DNA Methylation Markers in Circulating Tumor Cells and Paired Plasma Derived Exosomes in Metastatic Castration Resistant Prostate Cancer

Zavridou

Strati

Bournakis

et al. 2021

Cancers

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“…Studies have shown that RNA-based analysis in CTCs is severely impeded by the preservatives used in many BCTs, but maintained in CTCs collected in EDTA BCTs. 21,22 In addition, CTCs isolated from EDTA tubes can be cultured and xenografted and were shown to capture some of the same information and heterogeneity found in tumor biopsies. 23 Blood processing into PBMCs is commonly performed either by density gradient centrifugation (e.g., Ficoll) or red blood cell (RBC) lysis, with different purity and yield results.…”

Section: Circulating Tumor Dnamentioning

confidence: 99%

Roadmap to Liquid Biopsy Biobanking from Pediatric Cancers–Challenges and Opportunities

Kahana-Edwin

Cain

Karpelowsky

2021

Biopreservation and Biobanking

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Liquid biopsy is rapidly gaining traction for potentially revolutionizing cancer diagnosis and treatment through blood-based utilization of shed biomolecules. This approach can provide a global picture of the cancer in real time, at multiple time points, and with minimal invasiveness. In this review, we familiarize cancer biobanks with the principles used for liquid biopsy work and highlight unique aspects of applying liquid biopsy approaches to pediatric cancers to enable high-quality and efficient translational research.

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“…Liquid biopsy has become an important alternative tool for cancer diagnostics and includes the analysis of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), extracellular vesicles (EVs), and circulating cell‐free microRNA (miRNA) (Alix‐Panabieres and Pantel, 2016; Anfossi et al , 2018; Bardelli and Pantel, 2017; Girotti et al , 2015; Zavridou et al , 2018). The amounts of both CTCs and ctDNA are often very low, and the half‐life time of both biomarkers is short (1–2 h) (Diehl et al , 2008; Meng et al , 2004).…”

Section: Introductionmentioning

confidence: 99%

Pre‐analytical factors affecting the establishment of a single tube assay for multiparameter liquid biopsy detection in melanoma patients

et al. 2020

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The combination of liquid biomarkers from a single blood tube can provide more comprehensive information on tumor development and progression in cancer patients compared to single analysis. Here, we evaluated whether a combined analysis of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and circulating cell-free microRNA (miRNA) in total plasma and extracellular vesicles (EV) from the same blood sample is feasible and how the results are influenced by the choice of different blood tubes. Peripheral blood from 20 stage IV melanoma patients and five healthy donors (HD) was collected in EDTA, Streck, and Transfix tubes. Peripheral blood mononuclear cell fraction was used for CTC analysis, whereas plasma and EV fractions were used for ctDNA mutation and miRNA analysis. Mutations in cell-free circulating DNA were detected in 67% of patients, with no significant difference between the tubes. CTC was detected in only EDTA blood and only in 15% of patients. miRNA NGS (nextgeneration sequencing) results were highly influenced by the collection tubes and could only be performed from EDTA and Streck tubes due to hemolysis in Transfix tubes. No overlap of significantly differentially expressed miRNA (patients versus HD) could be found between the tubes in total plasma, whereas eight miRNA were commonly differentially regulated in the EV fraction. In summary, high-quality CTCs, ctDNA, and miRNA data from a single blood tube can be obtained.

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Evaluation of Preanalytical Conditions and Implementation of Quality Control Steps for Reliable Gene Expression and DNA Methylation Analyses in Liquid Biopsies

Abstract: Standardization of preanalytical conditions and implementation of quality control steps is extremely important for reliable liquid biopsy analysis, and a prerequisite for routine applications in the clinic.

Cited by 42 publications

References 39 publications

Prognostic Significance of Gene Expression and DNA Methylation Markers in Circulating Tumor Cells and Paired Plasma Derived Exosomes in Metastatic Castration Resistant Prostate Cancer

Prognostic Significance of Gene Expression and DNA Methylation Markers in Circulating Tumor Cells and Paired Plasma Derived Exosomes in Metastatic Castration Resistant Prostate Cancer

Roadmap to Liquid Biopsy Biobanking from Pediatric Cancers–Challenges and Opportunities

Pre‐analytical factors affecting the establishment of a single tube assay for multiparameter liquid biopsy detection in melanoma patients

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