Investigational New Drugs

, Volume 31, Issue 1, pp 1–13 | Cite as

Circulating vascular endothelial growth factor receptor 2/pAkt-positive cells as a functional pharmacodynamic marker in metastatic colorectal cancers treated with antiangiogenic agent

  • Sang Joon Shin
  • Jee Won Hwang
  • Joong Bae Ahn
  • Sun Young Rha
  • Jae Kyung Roh
  • Hyun Cheol ChungEmail author



The anti-vascular endothelial growth factor (VEGF) antibody bevacizumab has received considerable attention as a first-line treatment of advanced colorectal cancers. Difficulties associated with effectively monitoring the activity of this drug have prompted us to seek a pharmacodynamic marker suitable for defining the optimum biological dose and schedule of bevacizumab administration against colon cancer in early clinical trials.


We evaluated inhibitory effects of bevacizumab on VEGF signaling and tumor growth in vitro and in vivo, and assessed phosphorylation of VEGF receptor 2 (VEGFR2) and downstream signaling in endothelial cells as pharmacodynamic markers using phospho-flow cytometry. We also validated markers in patients with metastatic colorectal cancer (mCRC) treated with bevacizumab-based chemotherapy.


In in vitro studies, bevacizumab inhibited proliferation of human umbilical vein endothelial cells in association with reduced VEGF signaling. Notably, bevacizumab inhibited VEGF-induced phosphorylation of VEGFR-2, Akt, and extracellular signal-regulated kinase (ERK). In vivo, treatment with bevacizumab inhibited growth of xenografted tumors and attenuated VEGF-induced phosphorylation of Akt and ERK. The median percentages of VEGFR2 + pAkt + and VEGFR2 + pERK + cells, determined by phospho-flow cytometry, were approximately 3-fold higher in mCRC patients than in healthy controls. Bevacizumab treatment decreased VEGFR2 + pAkt + cells in 18 of 24 patients on day 3.


Bevacizumab combined with chemotherapy decreased the number of VEGFR2 + pAkt + cells, reflecting impaired VEGFR2 signaling. Together, these data suggest that changes in the proportion of circulating VEGFR2 + pAkt + cells may be a potential pharmacodynamic marker of the efficacy of antiangiogenic agents, and could prove valuable in determining drug dosage and administration schedule.


VEGFR2 Akt Bevacizumab Pharmacodynamic marker Phospho-flow cytometry 



This study was supported by a grant of the Korea Health technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A090660) and a faculty research grant of Department of Internal Medicine, Yonsei University College of Medicine for 2009.

Disclosure Statement

The authors indicate no potential conflicts of interest.


  1. 1.
    Folkman J (1971) Tumor angiogenesis: therapeutic implications. N Engl J Med 285(21):1182–1186. doi: 10.1056/NEJM197111182852108 PubMedCrossRefGoogle Scholar
  2. 2.
    Folkman J (1995) Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1(1):27–31PubMedCrossRefGoogle Scholar
  3. 3.
    Jain RK (2001) Normalizing tumor vasculature with anti-angiogenic therapy: a new paradigm for combination therapy. Nat Med 7(9):987–989. doi: 10.1038/nm0901-987 PubMedCrossRefGoogle Scholar
  4. 4.
    Kerbel RS (2000) Tumor angiogenesis: past, present and the near future. Carcinogenesis 21(3):505–515PubMedCrossRefGoogle Scholar
  5. 5.
    Saltz LB, Clarke S, Diaz-Rubio E, Scheithauer W, Figer A, Wong R, Koski S, Lichinitser M, Yang TS, Rivera F, Couture F, Sirzen F, Cassidy J (2008) Bevacizumab in combination with oxaliplatin-based chemotherapy as first-line therapy in metastatic colorectal cancer: a randomized phase III study. J Clin Oncol 26(12):2013–2019. doi: 10.1200/JCO.2007.14.9930 PubMedCrossRefGoogle Scholar
  6. 6.
    Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, Berlin J, Baron A, Griffing S, Holmgren E, Ferrara N, Fyfe G, Rogers B, Ross R, Kabbinavar F (2004) Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 350(23):2335–2342. doi: 10.1056/NEJMoa032691 PubMedCrossRefGoogle Scholar
  7. 7.
    Sessa C, Guibal A, Del Conte G, Ruegg C (2008) Biomarkers of angiogenesis for the development of antiangiogenic therapies in oncology: tools or decorations? Nat Clin Pract Oncol 5(7):378–391. doi: 10.1038/ncponc1150 PubMedCrossRefGoogle Scholar
  8. 8.
    Poon RT, Fan ST, Wong J (2001) Clinical implications of circulating angiogenic factors in cancer patients. J Clin Oncol 19(4):1207–1225PubMedGoogle Scholar
  9. 9.
    Jubb AM, Hurwitz HI, Bai W, Holmgren EB, Tobin P, Guerrero AS, Kabbinavar F, Holden SN, Novotny WF, Frantz GD, Hillan KJ, Koeppen H (2006) Impact of vascular endothelial growth factor-A expression, thrombospondin-2 expression, and microvessel density on the treatment effect of bevacizumab in metastatic colorectal cancer. J Clin Oncol 24(2):217–227. doi: 10.1200/JCO.2005.01.5388 PubMedCrossRefGoogle Scholar
  10. 10.
    Bertolini F, Shaked Y, Mancuso P, Kerbel RS (2006) The multifaceted circulating endothelial cell in cancer: towards marker and target identification. Nat Rev Cancer 6(11):835–845. doi: 10.1038/nrc1971 PubMedCrossRefGoogle Scholar
  11. 11.
    Mancuso P, Burlini A, Pruneri G, Goldhirsch A, Martinelli G, Bertolini F (2001) Resting and activated endothelial cells are increased in the peripheral blood of cancer patients. Blood 97(11):3658–3661PubMedCrossRefGoogle Scholar
  12. 12.
    Shaked Y, Emmenegger U, Man S, Cervi D, Bertolini F, Ben-David Y, Kerbel RS (2005) Optimal biologic dose of metronomic chemotherapy regimens is associated with maximum antiangiogenic activity. Blood 106(9):3058–3061. doi: 10.1182/blood-2005-04-1422 PubMedCrossRefGoogle Scholar
  13. 13.
    Mancuso P, Colleoni M, Calleri A, Orlando L, Maisonneuve P, Pruneri G, Agliano A, Goldhirsch A, Shaked Y, Kerbel RS, Bertolini F (2006) Circulating endothelial-cell kinetics and viability predict survival in breast cancer patients receiving metronomic chemotherapy. Blood 108(2):452–459. doi: 10.1182/blood-2005-11-4570 PubMedCrossRefGoogle Scholar
  14. 14.
    Hlatky L, Hahnfeldt P, Folkman J (2002) Clinical application of antiangiogenic therapy: microvessel density, what it does and doesn’t tell us. J Natl Cancer Inst 94(12):883–893PubMedCrossRefGoogle Scholar
  15. 15.
    Tofts PS (1997) Modeling tracer kinetics in dynamic Gd-DTPA MR imaging. J Magn Reson Imaging 7(1):91–101PubMedCrossRefGoogle Scholar
  16. 16.
    Hale MB, Nolan GP (2006) Phospho-specific flow cytometry: intersection of immunology and biochemistry at the single-cell level. Curr Opin Mol Ther 8(3):215–224PubMedGoogle Scholar
  17. 17.
    Li Q, Yano S, Ogino H, Wang W, Uehara H, Nishioka Y, Sone S (2007) The therapeutic efficacy of anti vascular endothelial growth factor antibody, bevacizumab, and pemetrexed against orthotopically implanted human pleural mesothelioma cells in severe combined immunodeficient mice. Clin Cancer Res 13(19):5918–5925. doi: 10.1158/1078-0432.CCR-07-0501 PubMedCrossRefGoogle Scholar
  18. 18.
    Presta LG, Chen H, O’Connor SJ, Chisholm V, Meng YG, Krummen L, Winkler M, Ferrara N (1997) Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders. Cancer Res 57(20):4593–4599PubMedGoogle Scholar
  19. 19.
    Johnson DH, Fehrenbacher L, Novotny WF, Herbst RS, Nemunaitis JJ, Jablons DM, Langer CJ, DeVore RF 3rd, Gaudreault J, Damico LA, Holmgren E, Kabbinavar F (2004) Randomized phase II trial comparing bevacizumab plus carboplatin and paclitaxel with carboplatin and paclitaxel alone in previously untreated locally advanced or metastatic non-small-cell lung cancer. J Clin Oncol 22(11):2184–2191. doi: 10.1200/JCO.2004.11.022 PubMedCrossRefGoogle Scholar
  20. 20.
    Yang JC, Haworth L, Sherry RM, Hwu P, Schwartzentruber DJ, Topalian SL, Steinberg SM, Chen HX, Rosenberg SA (2003) A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. N Engl J Med 349(5):427–434. doi: 10.1056/NEJMoa021491 PubMedCrossRefGoogle Scholar
  21. 21.
    Fox WD, Higgins B, Maiese KM, Drobnjak M, Cordon-Cardo C, Scher HI, Agus DB (2002) Antibody to vascular endothelial growth factor slows growth of an androgen-independent xenograft model of prostate cancer. Clin Cancer Res 8(10):3226–3231PubMedGoogle Scholar
  22. 22.
    Inoue S, Hartman A, Branch CD, Bucana CD, Bekele BN, Stephens LC, Chada S, Ramesh R (2007) mda-7 In combination with bevacizumab treatment produces a synergistic and complete inhibitory effect on lung tumor xenograft. Mol Ther 15(2):287–294. doi: 10.1038/ PubMedCrossRefGoogle Scholar
  23. 23.
    Monestiroli S, Mancuso P, Burlini A, Pruneri G, Dell'Agnola C, Gobbi A, Martinelli G, Bertolini F (2001) Kinetics and viability of circulating endothelial cells as surrogate angiogenesis marker in an animal model of human lymphoma. Cancer Res 61(11):4341–4344PubMedGoogle Scholar
  24. 24.
    Strumberg D, Richly H, Hilger RA, Schleucher N, Korfee S, Tewes M, Faghih M, Brendel E, Voliotis D, Haase CG, Schwartz B, Awada A, Voigtmann R, Scheulen ME, Seeber S (2005) Phase I clinical and pharmacokinetic study of the Novel Raf kinase and vascular endothelial growth factor receptor inhibitor BAY 43-9006 in patients with advanced refractory solid tumors. J Clin Oncol 23(5):965–972. doi: 10.1200/JCO.2005.06.124 PubMedCrossRefGoogle Scholar
  25. 25.
    Ludwig JA, Weinstein JN (2005) Biomarkers in cancer staging, prognosis and treatment selection. Nat Rev Cancer 5(11):845–856. doi: 10.1038/nrc1739 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Sang Joon Shin
    • 1
    • 2
  • Jee Won Hwang
    • 2
  • Joong Bae Ahn
    • 1
    • 2
  • Sun Young Rha
    • 1
    • 2
  • Jae Kyung Roh
    • 1
    • 2
  • Hyun Cheol Chung
    • 1
    • 2
    Email author
  1. 1.Department of Internal Medicine, Yonsei Cancer CenterYonsei University College of MedicineSeoulSouth Korea
  2. 2.Cancer Metastasis Research Center, Yonsei Cancer CenterYonsei University College of MedicineSeoulSouth Korea

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