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 (next-generation 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. However, the choice of blood collection tubes is a critical pre-analytical variable.

Agena Bioscience GmbH Hamburg Germany

Analysis of Circulating Tumor Cells Lab of Analytical Chemistry Department of Chemistry University of Athens Greece

Centre of Dermatology Elbe Clinics Buxtehude Germany

Department of Dermatology and Venereology University Medical Center Hamburg Eppendorf Germany

Department of Gene Expression Institute of Biotechnology Czech Academy of Sciences Vestec Czech Republic

Department of Tumor Biology University Medical Center Hamburg Eppendorf Germany

Orion Pharma Orion Corporation Espoo Finland

QIAGEN Inc GmbH Frederick MD USA

QIAGEN Inc GmbH Hilden Germany

TATAA Biocenter AB Gothenburg Sweden

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Alidousty C, Brandes D, Heydt C, Wagener S, Wittersheim M, Schafer SC, Holz B, Merkelbach‐Bruse S, Buttner R, Fassunke J et al (2017) Comparison of blood collection tubes from three different manufacturers for the collection of cell‐free DNA for liquid biopsy mutation testing. J Mol Diagn 19, 801–804. PubMed

Alix‐Panabieres C and Pantel K (2016) Clinical applications of circulating tumor cells and circulating tumor DNA as liquid biopsy. Cancer Discov 6, 479–491. PubMed

Andersen CL, Jensen JL and Ørntoft TF (2004) Normalization of real‐time quantitative reverse transcription‐PCR data: a model‐based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64, 5245–5250. PubMed

Anfossi S, Babayan A, Pantel K and Calin GA (2018) Clinical utility of circulating non‐coding RNAs—an update. Nat Rev Clin Oncol 15, 541–563. PubMed

Aya‐Bonilla C, Gray ES, Manikandan J, Freeman JB, Zaenker P, Reid AL, Khattak MA, Frank MH, Millward M and Ziman M (2019) Immunomagnetic‐enriched subpopulations of melanoma circulating tumour cells (CTCs) exhibit distinct transcriptome profiles. Cancers 11, 157. PubMed PMC

Aya‐Bonilla CA, Marsavela G, Freeman JB, Lomma C, Frank MH, Khattak MA, Meniawy TM, Millward M, Warkiani ME, Gray ES et al (2017) Isolation and detection of circulating tumour cells from metastatic melanoma patients using a slanted spiral microfluidic device. Oncotarget 8, 67355–67368. PubMed PMC

Bardelli A and Pantel K (2017) Liquid biopsies, what we do not know (Yet). Cancer Cell 31, 172–179. PubMed

Bartak BK, Kalmar A, Galamb O, Wichmann B, Nagy ZB, Tulassay Z, Dank M, Igaz P and Molnar B (2018) Blood collection and cell‐free DNA isolation methods influence the sensitivity of liquid biopsy analysis for colorectal cancer detection. Pathol Oncol Res 25, 915–923. PubMed

Cheng L, Sharples RA, Scicluna BJ and Hill AF (2014) Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell‐free blood. J Extracell Vesicles 3, 23743. PubMed PMC

Diaz IM, Nocon A, Mehnert DH, Fredebohm J, Diehl F and Holtrup F (2016) Performance of Streck cfDNA blood collection tubes for liquid biopsy testing. PLoS ONE 11, e0166354. PubMed PMC

Diehl F, Schmidt K, Choti MA, Romans K, Goodman S, Li M, Thornton K, Agrawal N, Sokoll L, Szabo SA et al (2008) Circulating mutant DNA to assess tumor dynamics. Nat Med 14, 985–990. PubMed PMC

Dillies M‐A, Rau A, Aubert J, Hennequet‐Antier C, Jeanmougin M, Servant N, Keime C, Marot G, Castel D, Estelle J et al. (2013) A comprehensive evaluation of normalization methods for Illumina high‐throughput RNA sequencing data analysis. Brief Bioinform 14, 671–683. PubMed

Elazezy M and Joosse SA (2018) Techniques of using circulating tumor DNA as a liquid biopsy component in cancer management. Comput Struct Biotechnol J 16, 370–378. PubMed PMC

Erdmann F, Lortet‐Tieulent J, Schuz J, Zeeb H, Greinert R, Breitbart EW and Bray F (2013) International trends in the incidence of malignant melanoma 1953–2008–are recent generations at higher or lower risk? Int J Cancer 132, 385–400. PubMed

Feng X, Wang Z, Fillmore R and Xi Y (2014) MiR‐200, a new star miRNA in human cancer. Cancer Lett 344, 166–173. PubMed PMC

Freeman JB, Gray ES, Millward M, Pearce R and Ziman M (2012) Evaluation of a multi‐marker immunomagnetic enrichment assay for the quantification of circulating melanoma cells. J Transl Med 10, 192. PubMed PMC

Girotti MR, Gremel G, Lee R, Galvani E, Rothwell D, Viros A, Mandal AK, Lim KHJ, Saturno G, Furney SJ et al. (2015) Application of sequencing, liquid biopsies and patient‐derived xenografts for personalized medicine in melanoma. Cancer Discov 6, 286–299. PubMed

Gorges K, Wiltfang L, Gorges TM, Sartori A, Hildebrandt L, Keller L, Volkmer B, Peine S, Babayan A, Moll I et al. (2019) Intra‐patient heterogeneity of circulating tumor cells and circulating tumor DNA in blood of melanoma patients. Cancers 11, 1685. PubMed PMC

Gray ES, Reid AL, Bowyer S, Calapre L, Siew K, Pearce R, Cowell L, Frank MH, Millward M and Ziman M (2015) Circulating melanoma cell subpopulations: their heterogeneity and differential responses to treatment. J Invest Dermatol 135, 2040–2048. PubMed PMC

Hoshino A, Costa‐Silva B, Shen T‐L, Rodrigues G, Hashimoto A, Mark MT, Molina H, Kohsaka S, Di Giannatale A, Ceder S et al. (2015) Tumour exosome integrins determine organotropic metastasis. Nature 527, 329. PubMed PMC

Huang SK and Hoon DS (2016) Liquid biopsy utility for the surveillance of cutaneous malignant melanoma patients. Mol Oncol 10, 450–463. PubMed PMC

Koch C, Joosse S, Schneegans S, Wilken O, Janning M, Loreth D, Müller V, Prieske K, Banys‐Paluchowski M, Horst L et al (2020) Pre‐analytical and analytical variables of label‐independent enrichment and automated detection of circulating tumor Cells in cancer patients. Cancers 12, 442. PubMed PMC

Koyanagi K, O'Day SJ, Boasberg P, Atkins MB, Wang HJ, Gonzalez R, Lewis K, Thompson JA, Anderson CM, Lutzky J et al (2010) Serial monitoring of circulating tumor cells predicts outcome of induction biochemotherapy plus maintenance biotherapy for metastatic melanoma. Clin Cancer Res 16, 2402–2408. PubMed PMC

Lawrence MS, Stojanov P, Polak P, Kryukov GV, Cibulskis K, Sivachenko A, Carter SL, Stewart C, Mermel CH, Roberts SA et al (2013) Mutational heterogeneity in cancer and the search for new cancer‐associated genes. Nature 499, 214–218. PubMed PMC

Luo X, Mitra D, Sullivan RJ, Wittner BS, Kimura AM, Pan S, Hoang MP, Brannigan BW, Lawrence DP and Flaherty KT (2014) Isolation and molecular characterization of circulating melanoma cells. Cell Rep 7, 645–653. PubMed PMC

Madadi S, Schwarzenbach H, Lorenzen J and Soleimani M (2019) MicroRNA expression studies: challenge of selecting reliable reference controls for data normalization. Cell Mol Life Sci 76, 3497–3514. PubMed PMC

Marsavela G, Aya‐Bonilla CA, Warkiani M, Gray E and Ziman M (2018) Melanoma circulating tumor cells: benefits and challenges required for clinical application. Cancer Lett 424, 1–8. PubMed

Meng S, Tripathy D, Frenkel EP, Shete S, Naftalis EZ, Huth JF, Beitsch PD, Leitch M, Hoover S, Euhus D et al (2004) Circulating tumor cells in patients with breast cancer dormancy. Clin Cancer Res 10, 8152–8162. PubMed

Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova‐Agadjanyan EL, Peterson A, Noteboom J, O'Briant KC, Allen A et al. (2008) Circulating microRNAs as stable blood‐based markers for cancer detection. Proc Natl Acad Sci USA 105, 10513–10518. PubMed PMC

Mocellin S, Del Fiore P, Guarnieri L, Scalerta R, Foletto M, Chiarion V, Pilati P, Nitti D, Lise M and Rossi CR (2004) Molecular detection of circulating tumor cells is an independent prognostic factor in patients with high‐risk cutaneous melanoma. Int J Cancer 111, 741–745. PubMed

Mosko MJ, Nakorchevsky AA, Flores E, Metzler H, Ehrich M, van den Boom DJ, Sherwood JL and Nygren AO (2016) Ultrasensitive detection of multiplexed somatic mutations using MALDI‐TOF mass spectrometry. J Mol Diagn 18, 23–31. PubMed

Nikolaev S, Lemmens L, Koessler T, Blouin JL and Nouspikel T (2018) Circulating tumoral DNA: preanalytical validation and quality control in a diagnostic laboratory. Anal Biochem 542, 34–39. PubMed

Po JW, Ma Y, Balakrishna B, Brungs D, Azimi F, de Souza P and Becker TM (2019) Immunomagnetic isolation of circulating melanoma cells and detection of PD‐L1 status. PLoS ONE 14, e0211866. PubMed PMC

Puhka M, Nordberg M, Valkonen S, Rannikko A, Kallioniemi O, Siljander P and Af Hällström T (2017) KeepEX, a simple dilution protocol for improving extracellular vesicle yields from urine. Eur J Pharm Sci 98, 30–39. PubMed

Qin J, Alt JR, Hunsley BA, Williams TL and Fernando MR (2014) Stabilization of circulating tumor cells in blood using a collection device with a preservative reagent. Cancer Cell Int 14, 23. PubMed PMC

Schwarzenbach H, da Silva AM, Calin G and Pantel K (2015) Data Normalization Strategies for MicroRNA Quantification. Clin Chem 61, 1333–1342. PubMed PMC

Tan CL, Lim TH, Lim T, Tan DS, Chua YW, Ang MK, Pang B, Lim CT, Takano A, Lim AS et al (2016) Concordance of anaplastic lymphoma kinase (ALK) gene rearrangements between circulating tumor cells and tumor in non‐small cell lung cancer. Oncotarget 7, 23251–23262. PubMed PMC

van Kempen LC, van den Hurk K, Lazar V, Michiels S, Winnepenninckx V, Stas M, Spatz A and van den Oord JJ (2012) Loss of microRNA‐200a and c, and microRNA‐203 expression at the invasive front of primary cutaneous melanoma is associated with increased thickness and disease progression. Virchows Arch 461, 441–448. PubMed

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A and Speleman F (2002) Accurate normalization of real‐time quantitative RT‐PCR data by geometric averaging of multiple internal control genes. Genome Biol 3, RESEARCH0034. PubMed PMC

Verrando P, Capovilla M and Rahmani R (2016) Trans‐nonachlor decreases miR‐141‐3p levels in human melanocytes s promoting melanoma cell characteristics and shows a multigenerational impact on miR‐8 levels in Drosophila. Toxicology 368–369, 129–141. PubMed

Vickers KC, Palmisano BT, Shoucri BM, Shamburek RD and Remaley AT (2011) MicroRNAs are transported in plasma and delivered to recipient cells by high‐density lipoproteins. Nat Cell Biol 13, 423. PubMed PMC

Ward Gahlawat A, Lenhardt J, Witte T, Keitel D, Kaufhold A, Maass KK, Pajtler KW, Sohn C and Schott S (2019) Evaluation of storage tubes for combined analysis of circulating nucleic acids in liquid biopsies. Int J Mol Sci 20, 704. PubMed PMC

Xu Y, Brenn T, Brown ER, Doherty V and Melton DW (2012) Differential expression of microRNAs during melanoma progression: miR‐200c, miR‐205 and miR‐211 are downregulated in melanoma and act as tumour suppressors. Br J Cancer 106, 553–561. PubMed PMC

Zavridou M, Mastoraki S, Strati A, Tzanikou E, Chimonidou M and Lianidou E (2018) Evaluation of preanalytical conditions and implementation of quality control steps for reliable gene expression and DNA methylation analyses in liquid biopsies. Clin Chem 2018, 292318. PubMed