Breast Cancer Research and Treatment

, Volume 161, Issue 1, pp 83–94 | Cite as

Longitudinally collected CTCs and CTC-clusters and clinical outcomes of metastatic breast cancer

  • Chun Wang
  • Zhaomei Mu
  • Inna Chervoneva
  • Laura Austin
  • Zhong Ye
  • Giovanna Rossi
  • Juan P. Palazzo
  • Carl Sun
  • Maysa Abu-Khalaf
  • Ronald E. Myers
  • Zhu Zhu
  • Yanna Ba
  • Bingshan Li
  • Lifang Hou
  • Massimo CristofanilliEmail author
  • Hushan YangEmail author



Circulating tumor cell (CTC) is a well-established prognosis predictor for metastatic breast cancer (MBC), and CTC-cluster exhibits significantly higher metastasis-promoting capability than individual CTCs. Because measurement of CTCs and CTC-clusters at a single time point may underestimate their prognostic values, we aimed to analyze longitudinally collected CTCs and CTC-clusters in MBC prognostication.


CTCs and CTC-clusters were enumerated in 370 longitudinally collected blood samples from 128 MBC patients. The associations between baseline, first follow-up, and longitudinal enumerations of CTCs and CTC-clusters with patient progression-free survival (PFS) and overall survival (OS) were analyzed using Cox proportional hazards models.


CTC and CTC-cluster counts at both baseline and first follow-up were significantly associated with patient PFS and OS. Time-dependent analysis of longitudinally collected samples confirmed the significantly unfavorable PFS and OS in patients with ≥5 CTCs, and further demonstrated the independent prognostic values by CTC-clusters compared to CTC-enumeration alone. Longitudinal analyses also identified a link between the size of CTC-clusters and patient OS: compared to the patients without any CTC, those with 2-cell CTC-clusters and ≥3-cell CTC-clusters had a hazard ratio (HR) of 7.96 [95 % confidence level (CI) 2.00–31.61, P = 0.003] and 14.50 (3.98–52.80, P < 0.001), respectively.


In this novel time-dependent analysis of longitudinally collected CTCs and CTC-clusters, we showed that CTC-clusters added additional prognostic values to CTC enumeration alone, and a larger-size CTC-cluster conferred a higher risk of death in MBC patients.


Circulating tumor cells (CTCs) Circulating tumor cell clusters (CTC-clusters) Metastatic breast cancer (MBC) Prognosis Time-dependent analysis 



This study is supported by Thomas Jefferson University Cancer Center Support Grant (5P30CA056036-17) for the CTC Core Facility, Pennsylvania Department of Health Grant (SAP# 4100062221), The Inflammatory Breast Cancer Network Foundation, The Jamie Lieberman Memorial Endowment Fund, and American Cancer Society Research Scholar Grant (123741-RSG-13-003-01-CCE).

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflict of interest.

Supplementary material

10549_2016_4026_MOESM1_ESM.docx (28 kb)
Supplementary material 1 (DOCX 27 kb)
10549_2016_4026_MOESM2_ESM.pptx (65 kb)
Supplementary material 2 (PPTX 65 kb)


  1. 1.
    Foster TS, Miller JD, Boye ME, Blieden MB, Gidwani R, Russell MW (2011) The economic burden of metastatic breast cancer: a systematic review of literature from developed countries. Cancer Treat Rev 37:405–415PubMedGoogle Scholar
  2. 2.
    Massihnia D, Perez A, Bazan V, Bronte G, Castiglia M, Fanale D, Barraco N, Cangemi A, Di Piazza F, Calo V, Rizzo S, Cicero G, Pantuso G, Russo A (2016) A headlight on liquid biopsies: a challenging tool for breast cancer management. Tumour Biol 37:4263–4273CrossRefPubMedGoogle Scholar
  3. 3.
    Esposito A, Criscitiello C, Locatelli M, Milano M, Curigliano G (2016) Liquid biopsies for solid tumors: understanding tumor heterogeneity and real time monitoring of early resistance to targeted therapies. Pharmacol Ther 157:120–124CrossRefPubMedGoogle Scholar
  4. 4.
    Swanton C (2012) Intratumor heterogeneity: evolution through space and time. Cancer Res 72:4875–4882CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Heitzer E, Auer M, Ulz P, Geigl JB, Speicher MR (2013) Circulating tumor cells and DNA as liquid biopsies. Genome Med 5:73CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Ignatiadis M, Lee M, Jeffrey SS (2015) Circulating tumor cells and circulating tumor DNA: challenges and opportunities on the path to clinical utility. Clin Cancer Res 21:4786–4800CrossRefPubMedGoogle Scholar
  7. 7.
    Powell AA, Talasaz AH, Zhang H, Coram MA, Reddy A, Deng G, Telli ML, Advani RH, Carlson RW, Mollick JA, Sheth S, Kurian AW, Ford JM, Stockdale FE, Quake SR, Pease RF, Mindrinos MN, Bhanot G, Dairkee SH, Davis RW, Jeffrey SS (2012) Single cell profiling of circulating tumor cells: transcriptional heterogeneity and diversity from breast cancer cell lines. PLoS ONE 7:e33788CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Krebs MG, Metcalf RL, Carter L, Brady G, Blackhall FH, Dive C (2014) Molecular analysis of circulating tumour cells-biology and biomarkers. Nat Rev Clin Oncol 11:129–144CrossRefPubMedGoogle Scholar
  9. 9.
    Aceto N, Bardia A, Miyamoto DT, Donaldson MC, Wittner BS, Spencer JA, Yu M, Pely A, Engstrom A, Zhu H, Brannigan BW, Kapur R, Stott SL, Shioda T, Ramaswamy S, Ting DT, Lin CP, Toner M, Haber DA, Maheswaran S (2014) Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis. Cell 158:1110–1122CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Hou JM, Krebs M, Ward T, Sloane R, Priest L, Hughes A, Clack G, Ranson M, Blackhall F, Dive C (2011) Circulating tumor cells as a window on metastasis biology in lung cancer. Am J Pathol 178:989–996CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Matera J, Miller MC, Reuben JM, Doyle GV, Allard WJ, Terstappen LW, Hayes DF (2004) Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 351:781–791CrossRefPubMedGoogle Scholar
  12. 12.
    Hayes DF, Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Miller MC, Matera J, Allard WJ, Doyle GV, Terstappen LW (2006) Circulating tumor cells at each follow-up time point during therapy of metastatic breast cancer patients predict progression-free and overall survival. Clin Cancer Res 12:4218–4224CrossRefPubMedGoogle Scholar
  13. 13.
    Bidard FC, Peeters DJ, Fehm T, Nole F, Gisbert-Criado R, Mavroudis D, Grisanti S, Generali D, Garcia-Saenz JA, Stebbing J, Caldas C, Gazzaniga P, Manso L, Zamarchi R, de Lascoiti AF, De Mattos-Arruda L, Ignatiadis M, Lebofsky R, van Laere SJ, Meier-Stiegen F, Sandri MT, Vidal-Martinez J, Politaki E, Consoli F, Bottini A, Diaz-Rubio E, Krell J, Dawson SJ, Raimondi C, Rutten A, Janni W, Munzone E, Caranana V, Agelaki S, Almici C, Dirix L, Solomayer EF, Zorzino L, Johannes H, Reis-Filho JS, Pantel K, Pierga JY, Michiels S (2014) Clinical validity of circulating tumour cells in patients with metastatic breast cancer: a pooled analysis of individual patient data. Lancet Oncol 15:406–414CrossRefPubMedGoogle Scholar
  14. 14.
    Zhang L, Riethdorf S, Wu G, Wang T, Yang K, Peng G, Liu J, Pantel K (2012) Meta-analysis of the prognostic value of circulating tumor cells in breast cancer. Clin Cancer Res 18:5701–5710CrossRefPubMedGoogle Scholar
  15. 15.
    Lv Q, Gong L, Zhang T, Ye J, Chai L, Ni C, Mao Y (2015) Prognostic value of circulating tumor cells in metastatic breast cancer: a systemic review and meta-analysis. Clin Transl Oncol 18:322–330CrossRefPubMedGoogle Scholar
  16. 16.
    Giuliano M, Giordano A, Jackson S, De Giorgi U, Mego M, Cohen EN, Gao H, Anfossi S, Handy BC, Ueno NT, Alvarez RH, De Placido S, Valero V, Hortobagyi GN, Reuben JM, Cristofanilli M (2014) Circulating tumor cells as early predictors of metastatic spread in breast cancer patients with limited metastatic dissemination. Breast Cancer Res 16:440CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Paoletti C, Li Y, Muniz MC, Kidwell KM, Aung K, Thomas DG, Brown ME, Abramson VG, Irvin WJ Jr, Lin NU, Liu MC, Nanda R, Nangia JR, Storniolo AM, Traina TA, Vaklavas C, Van Poznak CH, Wolff AC, Forero-Torres A, Hayes DF, Translational Breast Cancer Research C (2015) Significance of circulating tumor cells in metastatic triple-negative breast cancer patients within a randomized, phase II trial: TBCRC 019. Clin Cancer Res 21:2771–2779CrossRefPubMedGoogle Scholar
  18. 18.
    Mu Z, Wang C, Ye Z, Austin L, Civan J, Hyslop T, Palazzo JP, Jaslow R, Li B, Myers RE, Jiang J, Xing J, Yang H, Cristofanilli M (2015) Prospective assessment of the prognostic value of circulating tumor cells and their clusters in patients with advanced-stage breast cancer. Breast Cancer Res Treat 154:563–571CrossRefPubMedGoogle Scholar
  19. 19.
    Hou JM, Krebs MG, Lancashire L, Sloane R, Backen A, Swain RK, Priest LJ, Greystoke A, Zhou C, Morris K, Ward T, Blackhall FH, Dive C (2012) Clinical significance and molecular characteristics of circulating tumor cells and circulating tumor microemboli in patients with small-cell lung cancer. J Clin Oncol 30:525–532CrossRefPubMedGoogle Scholar
  20. 20.
    Smerage JB, Barlow WE, Hortobagyi GN, Winer EP, Leyland-Jones B, Srkalovic G, Tejwani S, Schott AF, O’Rourke MA, Lew DL, Doyle GV, Gralow JR, Livingston RB, Hayes DF (2014) Circulating tumor cells and response to chemotherapy in metastatic breast cancer: SWOG S0500. J Clin Oncol 32:3483–3489CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Bardia A, Haber DA (2014) Solidifying liquid biopsies: can circulating tumor cell monitoring guide treatment selection in breast cancer? J Clin Oncol 32:3470–3471CrossRefPubMedGoogle Scholar
  22. 22.
    Alix-Panabieres C, Pantel K (2014) Challenges in circulating tumour cell research. Nat Rev Cancer 14:623–631CrossRefPubMedGoogle Scholar
  23. 23.
    Hoevenaar-Blom MP, Spijkerman AM, Boshuizen HC, Boer JM, Kromhout D, Verschuren WM (2014) Effect of using repeated measurements of a Mediterranean style diet on the strength of the association with cardiovascular disease during 12 years: the Doetinchem Cohort Study. Eur J Nutr 53:1209–1215CrossRefPubMedGoogle Scholar
  24. 24.
    Marshall JA, Scarbro S, Shetterly SM, Jones RH (1998) Improving power with repeated measures: diet and serum lipids. Am J Clin Nutr 67:934–939PubMedGoogle Scholar
  25. 25.
    Therneau TM, Grambsch PM (2000) Modeling survival data: extending the Cox model. Springer, New YorkCrossRefGoogle Scholar
  26. 26.
    Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, Dancey J, Arbuck S, Gwyther S, Mooney M, Rubinstein L, Shankar L, Dodd L, Kaplan R, Lacombe D, Verweij J (2009) New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 45:228–247CrossRefPubMedGoogle Scholar
  27. 27.
    Au SH, Storey BD, Moore JC, Tang Q, Chen YL, Javaid S, Sarioglu AF, Sullivan R, Madden MW, O’Keefe R, Haber DA, Maheswaran S, Langenau DM, Stott SL, Toner M (2016) Clusters of circulating tumor cells traverse capillary-sized vessels. Proc Natl Acad Sci USA 113:4947–4952CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Goldhirsch A, Winer EP, Coates AS, Gelber RD, Piccart-Gebhart M, Thurlimann B, Senn HJ (2013) Personalizing the treatment of women with early breast cancer: highlights of the St Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2013. Ann Oncol 24:2206–2223CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Khan MS, Kirkwood AA, Tsigani T, Lowe H, Goldstein R, Hartley JA, Caplin ME, Meyer T (2016) Early changes in circulating tumor cells are associated with response and survival following treatment of metastatic neuroendocrine neoplasms. Clin Cancer Res 22:79–85CrossRefPubMedGoogle Scholar
  30. 30.
    Normanno N, Rossi A, Morabito A, Signoriello S, Bevilacqua S, Di Maio M, Costanzo R, De Luca A, Montanino A, Gridelli C, Rocco G, Perrone F, Gallo C (2014) Prognostic value of circulating tumor cells’ reduction in patients with extensive small-cell lung cancer. Lung Cancer 85:314–319CrossRefPubMedGoogle Scholar
  31. 31.
    Coumans FA, Ligthart ST, Terstappen LW (2012) Interpretation of changes in circulating tumor cell counts. Transl Oncol 5:486–491CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Goldkorn A, Ely B, Quinn DI, Tangen CM, Fink LM, Xu T, Twardowski P, Van Veldhuizen PJ, Agarwal N, Carducci MA, Monk JP 3rd, Datar RH, Garzotto M, Mack PC, Lara P Jr, Higano CS, Hussain M, Thompson IM Jr, Cote RJ, Vogelzang NJ (2014) Circulating tumor cell counts are prognostic of overall survival in SWOG S0421: a phase III trial of docetaxel with or without atrasentan for metastatic castration-resistant prostate cancer. J Clin Oncol 32:1136–1142CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Cheng Y, Liu XQ, Fan Y, Liu YP, Liu Y, Ma LX, Liu XH, Li H, Bao HZ, Liu JJ, Zhang S, Wu CJ (2016) Circulating tumor cell counts/change for outcome prediction in patients with extensive-stage small-cell lung cancer. Future Oncol 12:789–799CrossRefPubMedGoogle Scholar
  34. 34.
    Dotan E, Cohen SJ, Alpaugh KR, Meropol NJ (2009) Circulating tumor cells: evolving evidence and future challenges. Oncologist 14:1070–1082CrossRefPubMedGoogle Scholar
  35. 35.
    Cohen SJ, Punt CJ, Iannotti N, Saidman BH, Sabbath KD, Gabrail NY, Picus J, Morse M, Mitchell E, Miller MC, Doyle GV, Tissing H, Terstappen LW, Meropol NJ (2008) Relationship of circulating tumor cells to tumor response, progression-free survival, and overall survival in patients with metastatic colorectal cancer. J Clin Oncol 26:3213–3221CrossRefPubMedGoogle Scholar
  36. 36.
    Gazzaniga P, Raimondi C, Gradilone A, Biondi Zoccai G, Nicolazzo C, Gandini O, Longo F, Tomao S, Lo Russo G, Seminara P, Vincenzi B, Chimenti I, Cristofanilli M, Frati L, Cortesi E (2013) Circulating tumor cells in metastatic colorectal cancer: do we need an alternative cutoff? J Cancer Res Clin Oncol 139:1411–1416CrossRefPubMedGoogle Scholar
  37. 37.
    de Bono JS, Scher HI, Montgomery RB, Parker C, Miller MC, Tissing H, Doyle GV, Terstappen LW, Pienta KJ, Raghavan D (2008) Circulating tumor cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer. Clin Cancer Res 14:6302–6309CrossRefPubMedGoogle Scholar
  38. 38.
    Shiomi-Mouri Y, Kousaka J, Ando T, Tetsuka R, Nakano S, Yoshida M, Fujii K, Akizuki M, Imai T, Fukutomi T, Kobayashi K (2016) Clinical significance of circulating tumor cells (CTCs) with respect to optimal cut-off value and tumor markers in advanced/metastatic breast cancer. Breast Cancer 23:120–127CrossRefPubMedGoogle Scholar
  39. 39.
    Liotta LA, Saidel MG, Kleinerman J (1976) The significance of hematogenous tumor cell clumps in the metastatic process. Cancer Res 36:889–894PubMedGoogle Scholar
  40. 40.
    Glaves D (1983) Correlation between circulating cancer cells and incidence of metastases. Br J Cancer 48:665–673CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Fidler IJ (1973) The relationship of embolic homogeneity, number, size and viability to the incidence of experimental metastasis. Eur J Cancer 9:223–227CrossRefPubMedGoogle Scholar
  42. 42.
    Karabacak NM, Spuhler PS, Fachin F, Lim EJ, Pai V, Ozkumur E, Martel JM, Kojic N, Smith K, Chen PI, Yang J, Hwang H, Morgan B, Trautwein J, Barber TA, Stott SL, Maheswaran S, Kapur R, Haber DA, Toner M (2014) Microfluidic, marker-free isolation of circulating tumor cells from blood samples. Nat Protoc 9:694–710CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Harb W, Fan A, Tran T, Danila DC, Keys D, Schwartz M, Ionescu-Zanetti C (2013) Mutational analysis of circulating tumor cells using a novel microfluidic collection device and qPCR assay. Transl Oncol 6:528–538CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Gorges TM, Penkalla N, Schalk T, Joosse SA, Riethdorf S, Tucholski J, Lucke K, Wikman H, Jackson S, Brychta N, von Ahsen O, Schumann C, Krahn T, Pantel K (2016) Enumeration and molecular characterization of tumor cells in lung cancer patients using a novel in vivo device for capturing circulating tumor cells. Clin Cancer Res 22:2197–2206CrossRefPubMedGoogle Scholar
  45. 45.
    Sarioglu AF, Aceto N, Kojic N, Donaldson MC, Zeinali M, Hamza B, Engstrom A, Zhu H, Sundaresan TK, Miyamoto DT, Luo X, Bardia A, Wittner BS, Ramaswamy S, Shioda T, Ting DT, Stott SL, Kapur R, Maheswaran S, Haber DA, Toner M (2015) A microfluidic device for label-free, physical capture of circulating tumor cell clusters. Nat Methods 12:685–691CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Chun Wang
    • 1
  • Zhaomei Mu
    • 2
  • Inna Chervoneva
    • 3
  • Laura Austin
    • 1
  • Zhong Ye
    • 1
  • Giovanna Rossi
    • 2
  • Juan P. Palazzo
    • 4
  • Carl Sun
    • 1
  • Maysa Abu-Khalaf
    • 1
  • Ronald E. Myers
    • 1
  • Zhu Zhu
    • 1
  • Yanna Ba
    • 1
  • Bingshan Li
    • 5
  • Lifang Hou
    • 6
  • Massimo Cristofanilli
    • 2
    Email author
  • Hushan Yang
    • 1
    Email author
  1. 1.Department of Medical Oncology, Sidney Kimmel Cancer CenterThomas Jefferson UniversityPhiladelphiaUSA
  2. 2.Division of Hematology and Oncology, Robert H Lurie Comprehensive Cancer Center, Department of Medicine, Feinberg School of MedicineNorthwestern UniversityChicagoUSA
  3. 3.Department of Pharmacology and Experimental TherapeuticsThomas Jefferson UniversityPhiladelphiaUSA
  4. 4.Department of PathologyThomas Jefferson UniversityPhiladelphiaUSA
  5. 5.Center for Human Genetics Research, Department of Molecular Physiology and BiophysicsVanderbilt UniversityNashvilleUSA
  6. 6.Robert H Lurie Comprehensive Cancer Center, Division of Hematology & Oncology, Feinberg School of MedicineNorthwestern UniversityChicagoUSA

Personalised recommendations