Skip to main content
SpringerLink
Log in

Personalized Treatment Through Detection and Monitoring of Genetic Aberrations in Single Circulating Tumor Cells

  • Chapter
  • First Online:
Isolation and Molecular Characterization of Circulating Tumor Cells

Part of the book series: Advances in Experimental Medicine and Biology ((AEMB,volume 994))

Abstract

Circulating tumor cells (CTCs) present a viable alternative to access tumor materials other than primary biopsies in cancer. This disease is among the most widespread in the world and is difficult to target because of its complex nature, challenges in getting quality samples and dynamic temporal changes in response to treatment. Conventional methods of detection and monitoring the disease profile do not suffice to be able to target the heterogeneity that exists at the cellular level. CTCs have been identified as a possible substitute for tumor tissue samples, and can be used to complement current disease management. Challenges in CTCs molecular analysis lie in the purity of the sample, which is masked by the presence of large quantities of white blood cells (WBCs) . In this chapter, we present a microfluidic biochip platform that performs secondary purification to isolate single CTCs efficiently. Studying single CTCs will allow for sensitive detection of critical mutations and addressing intercellular variances that will be otherwise missed easily due to low mutation frequencies when evaluating bulk cell retrieval. Using the biochip, we isolated single CTCs, and conducted personalized integrated EGFR mutational analysis using conventional polymerase chain reaction (PCR) and Sanger sequencing. We also demonstrated that high quality next generation sequencing (NGS) libraries can be readily generated from these samples. In our initial study, we revealed that the dominant EGFR mutations such as L858R and T790M could be detected in Non Small Cell Lung Cancer (NSCLC) patients with low CTC counts. We envision the biochip will enable efficient isolation of rare single cells from samples. This technology coupled with downstream molecular characterization of CTCs will aid in realizing the personalized medicine for cancer patients.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 199.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  • Aceto N, Bardia A, Miyamoto DT et al (2014) Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis. Cell 158(5):1110–1122

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Alix-Panabières C, Pantel K (2013) Circulating tumor cells: liquid biopsy of cancer. Clin Chem 59(1):110–118

    Article  PubMed  Google Scholar 

  • Allard WJ, Matera J, Miller MC et al (2004) Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases. Clin Cancer Res 10(20):6897–6904

    Article  PubMed  Google Scholar 

  • Andrykowski MA, Munn RK, Studts JL (1996) Interest in learning of personal genetic risk for cancer: a general population survey. Prev Med 25(5):527–536

    Article  CAS  PubMed  Google Scholar 

  • Ashworth TR (1869) A case of cancer in which cells similar to those in the tumours were seen in the blood after death. Aust Med J 14(3):146–149

    Google Scholar 

  • Autebert J, Coudert B, Bidard FC et al (2012) Microfluidic: an innovative tool for efficient cell sorting. Methods 57(3):297–307

    Article  CAS  PubMed  Google Scholar 

  • Bell DW, Gore I, Okimoto RA et al (2005) Inherited susceptibility to lung cancer may be associated with the T790M drug resistance mutation in EGFR. Nat Genet 37(12):1315–1316

    Article  CAS  PubMed  Google Scholar 

  • Bendall SC, Nolan GP (2012) From single cells to deep phenotypes in cancer. Nat Biotechnol 30(7):639–647

    Article  CAS  PubMed  Google Scholar 

  • Blainey PC, Quake SR (2013) Dissecting genomic diversity, one cell at a time. Nat Methods 11(1):19–21

    Article  Google Scholar 

  • Boshuizen R, Kuhn P, van den Heuvel M (2012) Circulating tumor cells in non-small cell lung carcinoma. J Thorac Dis 4(5):456–458

    PubMed  PubMed Central  Google Scholar 

  • Budd GT (2009) Let me do more than count the ways: what circulating tumor cells can tell us about the biology of cancer. Mol Pharm 6(5):1307–1310

    Article  CAS  PubMed  Google Scholar 

  • Chin CD, Linder V, Sia SK (2007) Lab-on-a-chip devices for global health: past studies and future opportunities. Lab Chip 7(1):41–57

    Article  CAS  PubMed  Google Scholar 

  • Cohen SJ, Punt CJ, Iannotti N et al (2008) Relationship of circulating tumor cells to tumor response, progression-free survival, and overall survival in patients with metastatic colorectal cancer. J Clin Oncol 26(19):3213–3221

    Article  PubMed  Google Scholar 

  • Cristofanilli M, Budd GT, Ellis MJ et al (2004a) Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 351(8):781–791

    Article  CAS  PubMed  Google Scholar 

  • Cristofanilli M, Budd GT, Ellis MJ et al (2004b) Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 351(8):781–791

    Article  CAS  PubMed  Google Scholar 

  • Cristofanilli M, Hayes DF, Budd GT et al (2005) Circulating tumor cells: a novel prognostic factor for newly diagnosed metastatic breast cancer. J Clin Oncol 23(7):1420–1430

    Article  PubMed  Google Scholar 

  • Dey SS, Kester L, Spanjaard B et al (2015) Integrated genome and transcriptome sequencing of the same cell. Nat Biotechnol 33(3):285–289

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dong Y, Skelley AM, Merdek KD et al (2013) Microfluidics and circulating tumor cells. J Mol Diagn 15(2):149–157

    Article  PubMed  Google Scholar 

  • Eteshola E, Balberg M (2004) Microfluidic ELISA: on-chip fluorescence imaging. Biomed Microdevices 6(1):7–9

    Article  CAS  PubMed  Google Scholar 

  • Ettinger DS, Wood DE, Akerley W et al (2015) Non-small cell lung cancer, version 6.2015. J Natl Compr Cancer Netw 13(5):515–524

    Article  Google Scholar 

  • Fan T, Zhao Q, Chen JJ et al (2009) Clinical significance of circulating tumor cells detected by an invasion assay in peripheral blood of patients with ovarian cancer. Gynecol Oncol 112(1):185–191

    Article  CAS  PubMed  Google Scholar 

  • Garnett MJ, Edelman EJ, Heidorn SJ et al (2012) Systematic identification of genomic markers of drug sensitivity in cancer cells. Nature 483(7391):570–575

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gazdar AF (2009) Activating and resistance mutations of EGFR in non-small-cell lung cancer: role in clinical response to EGFR tyrosine kinase inhibitors. Oncogene 28(Suppl 1):S24–S31

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hou HW, Warkiani ME, Khoo BL et al (2013) Isolation and retrieval of circulating tumor cells using centrifugal forces. Sci Report 3:1259

    Article  Google Scholar 

  • Hussain SA, Palmer DH, Stevens A et al (2005) Role of chemotherapy in breast cancer. Expert Rev Anticancer Ther 5(6):1095–1110

    Article  CAS  PubMed  Google Scholar 

  • Hyun K-A, Jung H-I (2014) Advances and critical concerns with the microfluidic enrichments of circulating tumor cells. Lab Chip 14(1):45–56

    Article  CAS  PubMed  Google Scholar 

  • Karabacak NM, Spuhler PS, Fachin F et al (2014) Microfluidic, marker-free isolation of circulating tumor cells from blood samples. Nat Protoc 9(3):694–710

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kartalov EP, Quake SR (2004) Microfluidic device reads up to four consecutive base pairs in DNA sequencing-by-synthesis. Nucleic Acids Res 32(9):2873–2879

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kessler DA, Austin RH, Levine H (2014) Resistance to chemotherapy: patient variability and cellular heterogeneity. Cancer Res 74(17):4663–4670

    Article  CAS  PubMed  Google Scholar 

  • Kling J (2012) Beyond counting tumor cells. Nat Biotechnol 30(7):578–580

    Article  CAS  PubMed  Google Scholar 

  • Lohr JG, Adalsteinsson VA, Cibulskis K et al (2014) Whole-exome sequencing of circulating tumor cells provides a window into metastatic prostate cancer. Nat Biotech 32(5):479–484

    Article  CAS  Google Scholar 

  • Mach AJ, Kim JH, Arshi A et al (2011) Automated cellular sample preparation using a centrifuge-on-a-chip. Lab Chip 11(17):2827–2834

    Article  CAS  PubMed  Google Scholar 

  • Maheswaran S, Haber DA (2010) Circulating tumor cells: a window into cancer biology and metastasis. Curr Opin Genet Dev 20(1):96–99

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mark D, Haeberle S, Roth G et al (2010) Microfluidic lab-on-a-chip platforms: requirements, characteristics and applications. Chem Soc Rev 39(3):1153–1182

    Article  CAS  PubMed  Google Scholar 

  • Muinelo-Romay L, Vieito M, Abalo A et al (2014) Evaluation of circulating tumor cells and related events as prognostic factors and surrogate biomarkers in advanced NSCLC patients receiving first-line systemic treatment. Cancer 6(1):153

    Article  Google Scholar 

  • Nagrath S, Sequist LV, Maheswaran S et al (2007) Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature 450(7173):1235–1239

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nahta R, Yu D, Hung MC et al (2006) Mechanisms of disease: understanding resistance to HER2-targeted therapy in human breast cancer. Nat Clin Pract Oncol 3(5):269–280

    Article  CAS  PubMed  Google Scholar 

  • Neves RP, Raba K, Schmidt O et al (2014) Genomic high-resolution profiling of single CKpos/CD45neg flow-sorting purified circulating tumor cells from patients with metastatic breast cancer. Clin Chem 60(10):1290–1297

    CAS  PubMed  Google Scholar 

  • Ozkumur E, Shah AM, Ciciliano JC et al (2013) Inertial focusing for tumor antigen-dependent and -independent sorting of rare circulating tumor cells. Sci Transl Med 5(179):179ra147

    Article  Google Scholar 

  • Paguirigan AL, Beebe DJ (2008) Microfluidics meet cell biology: bridging the gap by validation and application of microscale techniques for cell biological assays. BioEssays 30(9):811–821

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Peeters DJE, De Laere B, Van den Eynden GG et al (2013) Semiautomated isolation and molecular characterisation of single or highly purified tumour cells from CellSearch enriched blood samples using dielectrophoretic cell sorting. Br J Cancer 108(6):1358–1367

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Peto R, Boreham J, Clarke M et al (2000) UK and USA breast cancer deaths down 25% in year 2000 at ages 20–69 years. Lancet 355(9217):1822

    Article  CAS  PubMed  Google Scholar 

  • Pierga JY, Bidard FC, Mathiot C et al (2008) Circulating tumor cell detection predicts early metastatic relapse after neoadjuvant chemotherapy in large operable and locally advanced breast cancer in a phase II randomized trial. Clin Cancer Res 14(21):7004–7010

    Article  CAS  PubMed  Google Scholar 

  • Powell AA, Talasaz AH, Zhang H et al (2012) Single cell profiling of circulating tumor cells: transcriptional heterogeneity and diversity from breast cancer cell lines. PLoS One 7(5):e33788

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Punnoose EA, Atwal SK, Spoerke JM et al (2010) Molecular biomarker analyses using circulating tumor cells. PLoS One 5(9):e12517

    Article  PubMed  PubMed Central  Google Scholar 

  • Punnoose EA, Atwal S, Liu W et al (2012) Evaluation of circulating tumor cells and circulating tumor DNA in non-small cell lung cancer: association with clinical endpoints in a phase II clinical trial of pertuzumab and erlotinib. Clin Cancer Res Off J Am Assoc Cancer Res 18(8):2391–2401

    Article  CAS  Google Scholar 

  • Rask-Andersen M, Almen MS, Schioth HB (2011) Trends in the exploitation of novel drug targets. Nat Rev Drug Discov 10(8):579–590

    Article  CAS  PubMed  Google Scholar 

  • Reategui E, Aceto N, Lim EJ et al (2015) Tunable nanostructured coating for the capture and selective release of viable circulating tumor cells. Adv Mater 27(9):1593–1599

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Robert D, Pamme N, Conjeaud H et al (2011) Cell sorting by endocytotic capacity in a microfluidic magnetophoresis device. Lab Chip 11(11):1902–1910

    Article  CAS  PubMed  Google Scholar 

  • Saunders NA, Simpson F, Thompson EW et al (2012) Role of intratumoural heterogeneity in cancer drug resistance: molecular and clinical perspectives. EMBO Mol Med 4(8):675–684

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schubert C (2011) Single-cell analysis: the deepest differences. Nature 480(7375):133–137

    Article  CAS  PubMed  Google Scholar 

  • Spencer SL, Sorger PK (2011) Measuring and modeling apoptosis in single cells. Cell 144(6):926–939

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Stoecklein NH, Hosch SB, Bezler M et al (2008) Direct genetic analysis of single disseminated cancer cells for prediction of outcome and therapy selection in esophageal cancer. Cancer Cell 13(5):441–453

    Article  CAS  PubMed  Google Scholar 

  • Swennenhuis JF, Reumers J, Thys K et al (2013) Efficiency of whole genome amplification of single circulating tumor cells enriched by CellSearch and sorted by FACS. Genitourin Med 5(11):106

    Google Scholar 

  • Swennenhuis JF, Tibbe AG, Stevens M et al (2015) Self-seeding microwell chip for the isolation and characterization of single cells. Lab Chip 15(14):3039–3046

    Article  CAS  PubMed  Google Scholar 

  • Tan SJ, Yobas L, Lee GY et al (2009) Microdevice for the isolation and enumeration of cancer cells from blood. Biomed Microdevices 11(4):883–892

    Article  PubMed  Google Scholar 

  • Thorsen T, Maerkl SJ, Quake SR (2002) Microfluidic large-scale integration. Science 298(5593):580–584

    Article  CAS  PubMed  Google Scholar 

  • Tsiatis AC, Norris-Kirby A, Rich RG et al (2010) Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications. J Mol Diagn 12(4):425–432

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • VanDelinder V, Groisman A (2006) Separation of plasma from whole human blood in a continuous cross-flow in a molded microfluidic device. Anal Chem 78(11):3765–3771

    Article  CAS  PubMed  Google Scholar 

  • Wang D, Bodovitz S (2010) Single cell analysis: the new frontier in ‘omics’. Trends Biotechnol 28(6):281–290

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang J, Fan HC, Behr B et al (2012) Genome-wide single-cell analysis of recombination activity and de novo mutation rates in human sperm. Cell 150(2):402–412

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • White AK, VanInsberghe M, Petriv OI et al (2011) High-throughput microfluidic single-cell RT-qPCR. Proc Natl Acad Sci U S A 108(34):13999–14004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Whitesides GM (2006) The origins and the future of microfluidics. Nature 442(7101):368–373

    Article  CAS  PubMed  Google Scholar 

  • Williams SC (2013) Circulating tumor cells. Proc Natl Acad Sci U S A 110(13):4861

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xu X, Hou Y, Yin X et al (2012) Single-cell exome sequencing reveals single-nucleotide mutation characteristics of a kidney tumor. Cell 148(5):886–895

    Article  CAS  PubMed  Google Scholar 

  • Yang Y, Rho HS, Stevens M et al (2015) Microfluidic device for DNA amplification of single cancer cells isolated from whole blood by self-seeding microwells. Lab Chip 15(22):4331–4337

    Article  CAS  PubMed  Google Scholar 

  • Yeo T, Tan SJ, Lim CL et al (2016) Microfluidic enrichment for the single cell analysis of circulating tumor cells. Sci Report 6:22076

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Clearbridge Accelerator Pte Ltd, 81 Science Park Drive, The Chadwick, #02-03, Singapore Science Park 1, Singapore, 118257, Singapore

    Swee Jin Tan & Trifanny Yeo

  2. Mechanobiology Institute of Singapore, 5A Engineering Drive 1, Singapore, 117411, Singapore

    Sarvesh Abhay Sukhatme & Chwee Teck Lim

  3. Genome Institute of Singapore, 60, Biopolis Street, #02-01, Genome, Singapore, 138672, Singapore

    Say Li Kong

  4. Division of Medical Oncology, National Cancer Centre Singapore, 11 Hospital Drive, Singapore, 169610, Singapore

    Wan-Teck Lim

  5. Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore, 138673, Singapore

    Wan-Teck Lim

  6. Department of Biomedical Engineering, National University of Singapore, 9 Engineering Drive 1, Block EA, #03-12, Singapore, 117575, Singapore

    Chwee Teck Lim

Authors
  1. Swee Jin Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Trifanny Yeo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sarvesh Abhay Sukhatme
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Say Li Kong
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wan-Teck Lim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chwee Teck Lim
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Chwee Teck Lim .

Editor information

Editors and Affiliations

  1. University of California San Francisco, San Francisco, California, USA

    Mark Jesus M. Magbanua

  2. University of California San Francisco, San Francisco, California, USA

    John W. Park

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer International Publishing AG

About this chapter

Cite this chapter

Tan, S.J., Yeo, T., Sukhatme, S.A., Kong, S.L., Lim, WT., Lim, C.T. (2017). Personalized Treatment Through Detection and Monitoring of Genetic Aberrations in Single Circulating Tumor Cells. In: Magbanua, M., Park, J. (eds) Isolation and Molecular Characterization of Circulating Tumor Cells. Advances in Experimental Medicine and Biology, vol 994. Springer, Cham. https://doi.org/10.1007/978-3-319-55947-6_14

Download citation

Publish with us

Policies and ethics

Search

Navigation