Effect of short-term storage of blood samples on gene expression in lung cancer patients
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Eva Obermayr
,
Nina Koppensteiner
Abstract
Objectives
The stability of gene transcripts associated with the presence of circulating tumor cells (CTCs) has been predominantly studied in cultured cancer cell lines added to blood samples under artificial conditions. In the present study the effect of storage on CTC-related transcripts was assessed in blood samples taken from patients with non-small lung cancer (n=58).
Methods
The blood samples were split in two equal parts to compare the gene expression with and without storage for 24 h at ambient temperature without preservative added. After enrichment using the microfluidic Parsortix® technology, the expression levels of selected genes were assessed using quantitative PCR following a gene-specific pre-amplification. The prognostic relevance of each gene in fresh and stored blood samples was evaluated using the R-package Survminer.
Results
Some genes were either not affected (TWIST1, CDH5, CK19) or upregulated upon storage (NANOG, MET, UCHL1) but still associated with poor prognosis. In contrast, ERBB3, PTHLH, EpCAM, and TERT were no longer associated with the overall survival of the patients.
Conclusions
The study demonstrates the surprising stability of CTC-related transcripts, which makes overnight shipping of native blood samples possible. Careful verification is required when using model systems – such as normal blood spiked with tumor cells – or other CTC-related markers, as individual transcripts may respond differently to storage.
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Research funding: This study received support from Angle plc. in the form of an in-kind contribution of Parsortix® PR1 devices and microfluidic separation cassettes. ANGLE plc. had no influence on the design of the study, in the collection, analysis, and interpretation of data.
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Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
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Competing interests: All authors declare no conflict of interest.
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Informed consent: Informed consent was obtained from all individuals included in this study.
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Ethical approval: The study was approved by the Ethic Committee of the Medical University of Vienna, Austria (EK366/2003 and EK2266/2018).
References
1. https://clinicaltrials.gov/ct2/results?term=circulating+tumor+cells&Search=Apply&recrs=a.Suche in Google Scholar
2. Zavridou, M, Mastoraki, S, Strati, A, Tzanikou, E, Chimonidou, M, Lianidou, E. Evaluation of preanalytical conditions and implementation of quality control steps for reliable gene expression and DNA methylation analyses in liquid biopsies. Clin Chem 2018;64:1522–33. https://doi.org/10.1373/clinchem.2018.292318.Suche in Google Scholar PubMed
3. Obermayr, E, Agreiter, C, Schuster, E, Fabikan, H, Weinlinger, C, Baluchova, K, et al.. Molecular characterization of circulating tumor cells enriched by A microfluidic platform in patients with small-cell lung cancer. Cells 2019;8. https://doi.org/10.3390/cells8080880.Suche in Google Scholar PubMed PubMed Central
4. Obermayr, E, Maritschnegg, E, Agreiter, C, Pecha, N, Speiser, P, Helmy-Bader, S, et al.. Efficient leukocyte depletion by a novel microfluidic platform enables the molecular detection and characterization of circulating tumor cells. Oncotarget 2018;9:812–23. https://doi.org/10.18632/oncotarget.22549.Suche in Google Scholar PubMed PubMed Central
5. Stathopoulou, A, Ntoulia, M, Perraki, M, Apostolaki, S, Mavroudis, D, Malamos, N, et al.. A highly specific real-time RT-PCR method for the quantitative determination of CK-19 mRNA positive cells in peripheral blood of patients with operable breast cancer. Int J Cancer 2006;119:1654–9. https://doi.org/10.1002/ijc.22017.Suche in Google Scholar PubMed
6. Kassambara, A, Kosinski, M, Przemyslaw, B, Scheipl, F. survminer: drawing survival curves using “ggplot2”; 2020. Available from: https://cran.r-project.org/package=survminer.Suche in Google Scholar
7. Obermayr, E, Koppensteiner, N, Heinzl, N, Schuster, E, Holzer, B, Fabikan, H, et al.. Cancer stem cell-like circulating tumor cells are prognostic in non-small cell lung cancer. J Personalized Med 2021;11. https://doi.org/10.3390/jpm11111225.Suche in Google Scholar PubMed PubMed Central
8. Obermayr, E, Castillo-Tong, DC, Pils, D, Speiser, P, Braicu, I, Van Gorp, T, et al.. Molecular characterization of circulating tumor cells in patients with ovarian cancer improves their prognostic significance – a study of the OVCAD consortium. Gynecol Oncol 2013;128:15–21. https://doi.org/10.1016/j.ygyno.2012.09.021.Suche in Google Scholar PubMed
9. Das, K, Dumais, J, Basiaga, S, Krzyzanowski, GD. Carbon-13 nuclear magnetic resonance analysis of formaldehyde free preservatives. Acta Histochem 2013;115:481–6. https://doi.org/10.1016/j.acthis.2012.11.004.Suche in Google Scholar PubMed
10. Qin, J, Alt, JR, Hunsley, BA, Williams, TL, Fernando, MR. Stabilization of circulating tumor cells in blood using a collection device with a preservative reagent. Cancer Cell Int 2014;14:23. https://doi.org/10.1186/1475-2867-14-23.Suche in Google Scholar PubMed PubMed Central
11. Das, K, Norton, SE, Alt, JR, Krzyzanowski, GD, Williams, TL, Fernando, MR. Stabilization of cellular RNA in blood during storage at room temperature: a comparison of cell-free RNA BCT((R)) with K3EDTA tubes. Mol Diagn Ther 2014;18:647–53. https://doi.org/10.1007/s40291-014-0118-z.Suche in Google Scholar PubMed PubMed Central
12. Rodriguez-Lee, M, Kolatkar, A, McCormick, M, Dago, AD, Kendall, J, Carlsson, NA, et al.. Effect of blood collection tube type and time to processing on the enumeration and high-content characterization of circulating tumor cells using the high-definition single-cell assay. Arch Pathol Lab Med 2018;142:198–207. https://doi.org/10.5858/arpa.2016-0483-oa.Suche in Google Scholar
13. Luk, AWS, Ma, Y, Ding, PN, Young, FP, Chua, W, Balakrishnar, B, et al.. CTC-mRNA (AR-V7) analysis from blood samples-impact of blood collection tube and storage time. Int J Mol Sci 2017;18. https://doi.org/10.3390/ijms18051047.Suche in Google Scholar PubMed PubMed Central
14. Koch, C, Joosse, SA, Schneegans, S, Wilken, OJW, Janning, M, Loreth, D, et al.. Pre-analytical and analytical variables of label-independent enrichment and automated detection of circulating tumor cells in cancer patients. Cancers (Basel) 2020;12. https://doi.org/10.3390/cancers12020442.Suche in Google Scholar PubMed PubMed Central
15. Hustler, A, Wishart, G, Templeman, A, Sales, EB, Torrecillas, SA, Mumford, K, et al.. Abstract 4880: Parsortix™ system: analytic performance evaluation using ovarian cancer cell lines. Cancer Res 2019;79:4880. https://doi.org/10.1158/1538-7445.am2019-4880.Suche in Google Scholar
16. D’Alessandro, A, Liumbruno, G, Grazzini, G, Zolla, L. Red blood cell storage: the story so far. Blood Transfus 2010;8:82–8.Suche in Google Scholar
17. Hashemi Tayer, A, Amirizadeh, N, Ahmadinejad, M, Nikougoftar, M, Deyhim, MR, Zolfaghari, S. Procoagulant activity of red blood cell-derived microvesicles during red cell storage. Transfus Med Hemotherapy 2019;46:224–30. https://doi.org/10.1159/000494367.Suche in Google Scholar PubMed PubMed Central
18. Matthews, K, Myrand-Lapierre, ME, Ang, RR, Duffy, SP, Scott, MD, Ma, H. Microfluidic deformability analysis of the red cell storage lesion. J Biomech 2015;48:4065–72. https://doi.org/10.1016/j.jbiomech.2015.10.002.Suche in Google Scholar PubMed
19. Sandlin, RD, Wong, KHK, Boneschansker, L, Carey, TR, Miller, KL, Rose, G, et al.. Preservative solution that stabilizes erythrocyte morphology and leukocyte viability under ambient conditions. Sci Rep 2017;7:5658. https://doi.org/10.1038/s41598-017-05978-7.Suche in Google Scholar PubMed PubMed Central
20. Aceto, N, Bardia, A, Miyamoto, DT, Donaldson, MC, Wittner, BS, Spencer, JA, et al.. Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis. Cell 2014;158:1110–22. https://doi.org/10.1016/j.cell.2014.07.013.Suche in Google Scholar PubMed PubMed Central
21. Wong, KHK, Tessier, SN, Miyamoto, DT, Miller, KL, Bookstaver, LD, Carey, TR, et al.. Whole blood stabilization for the microfluidic isolation and molecular characterization of circulating tumor cells. Nat Commun 2017;8:1733. https://doi.org/10.1038/s41467-017-01705-y.Suche in Google Scholar PubMed PubMed Central
22. Payne, K, Brooks, JM, Taylor, GS, Batis, N, Noyvert, B, Pan, Y, et al.. Immediate sample fixation increases circulating tumour cell (CTC) capture and preserves phenotype in head and neck squamous cell carcinoma: towards a standardised approach to microfluidic CTC biomarker discovery. Cancers (Basel) 2021;13. https://doi.org/10.3390/cancers13215519.Suche in Google Scholar PubMed PubMed Central
23. Apostolou, P, Ntanovasilis, DA, Papasotiriou, I. Evaluation of a simple method for storage of blood samples that enables isolation of circulating tumor cells 96 h after sample collection. J Biol Res (Thessalon) 2017;24:11. https://doi.org/10.1186/s40709-017-0068-9.Suche in Google Scholar PubMed PubMed Central
24. Mendelaar, PAJ, Kraan, J, Van, M, Zeune, LL, Terstappen, L, Oomen-de Hoop, E, et al.. Defining the dimensions of circulating tumor cells in a large series of breast, prostate, colon, and bladder cancer patients. Mol Oncol 2021;15:116–25. https://doi.org/10.1002/1878-0261.12802.Suche in Google Scholar PubMed PubMed Central
25. Shaw Bagnall, J, Byun, S, Begum, S, Miyamoto, DT, Hecht, VC, Maheswaran, S, et al.. Deformability of tumor cells versus blood cells. Sci Rep 2015;5:18542. https://doi.org/10.1038/srep18542.Suche in Google Scholar PubMed PubMed Central
26. Hasmim, M, Noman, MZ, Messai, Y, Bordereaux, D, Gros, G, Baud, V, et al.. Cutting edge: hypoxia-induced Nanog favors the intratumoral infiltration of regulatory T cells and macrophages via direct regulation of TGF-beta1. J Immunol 2013;191:5802–6. https://doi.org/10.4049/jimmunol.1302140.Suche in Google Scholar PubMed
27. Nagata, A, Itoh, F, Sasho, A, Sugita, K, Suzuki, R, Hinata, H, et al.. The evolutionarily conserved deubiquitinase UBH1/UCH-L1 augments DAF7/TGF-beta signaling, inhibits dauer larva formation, and enhances lung tumorigenesis. J Biol Chem 2020;295:9105–20. https://doi.org/10.1074/jbc.ra119.011222.Suche in Google Scholar
28. Mekki, MS, Mougel, A, Vinchent, A, Paquet, C, Copin, MC, Leroy, C, et al.. Hypoxia leads to decreased autophosphorylation of the MET receptor but promotes its resistance to tyrosine kinase inhibitors. Oncotarget 2018;9:27039–58. https://doi.org/10.18632/oncotarget.25472.Suche in Google Scholar PubMed PubMed Central
Supplementary Material
The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2022-0738).
© 2022 Walter de Gruyter GmbH, Berlin/Boston
Artikel in diesem Heft
- Frontmatter
- Editorial
- Standardization and harmonization in laboratory medicine: not only for clinical chemistry measurands
- Mini Review
- The effect of the immunoassay curve fitting routine on bias in troponin
- Opinion Papers
- An overview of the most important preanalytical factors influencing the clinical performance of SARS-CoV-2 antigen rapid diagnostic tests (Ag-RDTs)
- Time to address quality control processes applied to antibody testing for infectious diseases
- Perspectives
- Definition and application of performance specifications for measurement uncertainty of 23 common laboratory tests: linking theory to daily practice
- The gaps between the new EU legislation on in vitro diagnostics and the on-the-ground reality
- General Clinical Chemistry and Laboratory Medicine
- Second-generation Elecsys cerebrospinal fluid immunoassays aid diagnosis of early Alzheimer’s disease
- Evaluation of the interchangeability between the new fully-automated affinity chrome-mediated immunoassay (ACMIA) and the Quantitative Microsphere System (QMS) with a CE-IVD-certified LC-MS/MS assay for therapeutic drug monitoring of everolimus after solid organ transplantation
- Quantitative detection of anti-PLA2R antibodies targeting different epitopes and its clinical application in primary membranous nephropathy
- Reference Values and Biological Variations
- A zlog-based algorithm and tool for plausibility checks of reference intervals
- Harmonization of indirect reference intervals calculation by the Bhattacharya method
- Red blood cell parameters in early childhood: a prospective cohort study
- Cancer Diagnostics
- Association of circulating free and total oxysterols in breast cancer patients
- Effect of short-term storage of blood samples on gene expression in lung cancer patients
- Infectious Diseases
- Operation Moonshot: rapid translation of a SARS-CoV-2 targeted peptide immunoaffinity liquid chromatography-tandem mass spectrometry test from research into routine clinical use
- Seroprevalence of SARS-CoV-2 antibodies in Italy in newborn dried blood spots
- Positivization time of a COVID-19 rapid antigen self-test predicts SARS-CoV-2 viral load: a proof of concept
- New insights into SARS-CoV-2 Lumipulse G salivary antigen testing: accuracy, safety and short TAT enhance surveillance
- Preanalytical stability of SARS-CoV-2 anti-nucleocapsid antibodies
- Prognostic value of procalcitonin in cancer patients with coronavirus disease 2019
- 24/7 workflow for bloodstream infection diagnostics in microbiology laboratories: the first step to improve clinical management
- Diagnostic performance of machine learning models using cell population data for the detection of sepsis: a comparative study
- Diagnostic value of procalcitonin, hypersensitive C-reactive protein and neutrophil-to-lymphocyte ratio for bloodstream infections in pediatric tumor patients
- Letters to the Editor
- Pseudohyponatremia: interference of hyperglycemia on indirect potentiometry
- Ligand binding assay-related underestimation of 25-hydroxyvitamin D in pregnant women exaggerates the prevalence of vitamin D insufficiency
- Urokinase-type plasminogen activator soluble receptor (suPAR) assay in clinical routine: evaluation one year after its introduction in the high automation corelab of the A. Gemelli hospital
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Artikel in diesem Heft
- Frontmatter
- Editorial
- Standardization and harmonization in laboratory medicine: not only for clinical chemistry measurands
- Mini Review
- The effect of the immunoassay curve fitting routine on bias in troponin
- Opinion Papers
- An overview of the most important preanalytical factors influencing the clinical performance of SARS-CoV-2 antigen rapid diagnostic tests (Ag-RDTs)
- Time to address quality control processes applied to antibody testing for infectious diseases
- Perspectives
- Definition and application of performance specifications for measurement uncertainty of 23 common laboratory tests: linking theory to daily practice
- The gaps between the new EU legislation on in vitro diagnostics and the on-the-ground reality
- General Clinical Chemistry and Laboratory Medicine
- Second-generation Elecsys cerebrospinal fluid immunoassays aid diagnosis of early Alzheimer’s disease
- Evaluation of the interchangeability between the new fully-automated affinity chrome-mediated immunoassay (ACMIA) and the Quantitative Microsphere System (QMS) with a CE-IVD-certified LC-MS/MS assay for therapeutic drug monitoring of everolimus after solid organ transplantation
- Quantitative detection of anti-PLA2R antibodies targeting different epitopes and its clinical application in primary membranous nephropathy
- Reference Values and Biological Variations
- A zlog-based algorithm and tool for plausibility checks of reference intervals
- Harmonization of indirect reference intervals calculation by the Bhattacharya method
- Red blood cell parameters in early childhood: a prospective cohort study
- Cancer Diagnostics
- Association of circulating free and total oxysterols in breast cancer patients
- Effect of short-term storage of blood samples on gene expression in lung cancer patients
- Infectious Diseases
- Operation Moonshot: rapid translation of a SARS-CoV-2 targeted peptide immunoaffinity liquid chromatography-tandem mass spectrometry test from research into routine clinical use
- Seroprevalence of SARS-CoV-2 antibodies in Italy in newborn dried blood spots
- Positivization time of a COVID-19 rapid antigen self-test predicts SARS-CoV-2 viral load: a proof of concept
- New insights into SARS-CoV-2 Lumipulse G salivary antigen testing: accuracy, safety and short TAT enhance surveillance
- Preanalytical stability of SARS-CoV-2 anti-nucleocapsid antibodies
- Prognostic value of procalcitonin in cancer patients with coronavirus disease 2019
- 24/7 workflow for bloodstream infection diagnostics in microbiology laboratories: the first step to improve clinical management
- Diagnostic performance of machine learning models using cell population data for the detection of sepsis: a comparative study
- Diagnostic value of procalcitonin, hypersensitive C-reactive protein and neutrophil-to-lymphocyte ratio for bloodstream infections in pediatric tumor patients
- Letters to the Editor
- Pseudohyponatremia: interference of hyperglycemia on indirect potentiometry
- Ligand binding assay-related underestimation of 25-hydroxyvitamin D in pregnant women exaggerates the prevalence of vitamin D insufficiency
- Urokinase-type plasminogen activator soluble receptor (suPAR) assay in clinical routine: evaluation one year after its introduction in the high automation corelab of the A. Gemelli hospital
- Is this a true lambda free light chain?
- Effect of various blood collection tubes on serum lithium and other electrolytes: our perspective from a tertiary healthcare institute
