Steele Lab

Blood circulating endothelial cells and circulating progenitor cells as biomarkers of antiangiogenic therapy in cancer patients

Due to spectacular successes in clinical trials, the use of antiangiogenic agents in cancer patients is rapidly becoming part of clinical practice in the USA and elsewhere. But targeting angiogenic vessels requires new and adequate methods for the assessment of the biologic effect of various agents developed to control cancer progression. Traditionally, tumor angiogenesis has been evaluated by measuring microvessel density in biopsy specimens using immunohistochemistry.

Predicting and/or assessing accurately the efficacy of antiangiogneic therapies by this method is hampered by the heterogeneity of tumors, and by the difficulty to obtain specimens at multiple time points during treatments. Moreover, microvessel density, as well as the level of VEGF, Ras, P53, and thrombospondin 2 expression have all failed to predict the efficacy of bevacizumab with chemotherapy in the pivotal trial in colorectal cancer patients. Validating biomarkers for antiangiogenic agents is critically important, since traditional methods of evaluation of tumor response (e.g., RECIST) may not be appropriate for these targeted agents. On the other hand, obtaining peripheral blood at multiple time-points during treatment is a minimally invasive, routine procedure.

Because of the immediate exposure, blood circulating endothelial cells (CECs) and circulating progenitor cells (CPCS) may be directly and immediately affected by therapy with antiangiogenic agents. In 2001, CEC enumeration was proposed by the Bertolini group from Italy as a biomarker of response in mouse models. In patients, evaluation of CECs kinetics and viability predicted the clinical benefit of “metronomic” chemotherapy in metastatic breast cancer patients. Our group is evaluating in multiple trials of antiangiogenic agents targeting the VEGF pathway the changes in viable CECs (defined as CD31brightCD34+CD45–CD133– cells) and CPCs (identified as CD31+CD34brightCD45dimCD133+ cells) (Willett et al., Nature Medicine 2004; Willett et al., Journal of Clinical Oncology 2005; Duda et al., Journal of Clinical Oncology 2006; Hagendoorn et al., Nature Clinical Practice Oncology 2006; Duda et al., Journal of Clinical Oncology 2007; Duda et al., Nature Protocols 2007; Willett et al., Nature Clinical Practice Oncology 2007). This type of analysis is a departure from the techniques that used single markers for CEC identification, and relies on specific cell surface phenotypes using a combination of only four markers to identify both viable CECs and CPCs. We have shown that in rectal cancer patients the number of viable CECs and CPCs is decreased by bevacizumab (a VEGF-specific antibody, Genentech Inc., South San Francisco, CA) administration alone (Willett et al., Nature Medicine 2004). In recurrent glioblastoma patients, we have demonstrated that the number of viable CECs correlated with radiographic (MRI) progression through therapy with a pan-VEGF receptor tyrosine kinase inhibitor– AZD2171 (AstraZeneca Pharmaceuticals, Cheshire, UK) (Batchelor et al., Cancer Cell 2007). In the same study, we have also shown that CPCs had differential biomarker value, and predicted relapse of glioblastomas during drug interruptions. Given the variety of surface markers, methodologies and protocols used by different groups to identify CECs, the different potential origins of these endothelial cells (tumor, systemic, bone marrow), and the currently unresolved issue of circulating endothelial progenitor cell (referred to as CEP or EPC) phenotype, standardization is needed to allow cross-trial comparisons. We proposed a standard protocol for evaluation of viable CECs and of CPCs in cancer patients during treatment (Duda et al., Nature Protocols 2007). This protocol is currently employed by us for CEC/CPC enumeration in 7 ongoing clinical phase 2 studies of anti-VEGF agents at Massachusetts General Hospital, Boston and Duke University Medical Center, Durham, and a similar one is used by other groups at different institutions in the USA.

Figure 1: Multi-color flow cytometric analyses of human mononuclear cells in whole blood samples. a-d, Staining for CD31, CD34, CD133 and CD45 of a whole blood sample and gating on mononuclear cellular events on the forward-side scatter plot (in red in a) allow identification of two distinct populations of interest: CD31brightCD45–CD34+CD133– (green rectangles) and CD133+CD34brightCD31+CD45dim progenitor cells (blue, in yellow rectangles in b-d). (From Duda et al., Nature Protocols 2007.)

Figure 2: Multi-color flow cytometric analyses of human mononuclear cells separated from whole blood samples. a-d, Staining for CD31, CD34, CD133 and CD45 of a whole blood sample and gating on mononuclear cellular events on the forward-side scatter plot (in red in a) allow identification of two distinct populations of interest: CD31brightCD45–CD34+CD133– (green rectangles) and CD133+CD34brightCD31+CD45dim progenitor cells (blue, in yellow rectangles in b-d). (From Duda et al., Nature Protocols 2007.)

Further reading

1. Duda DG, Batchelor TT, Willett CG, Jain RK. VEGF-targeted cancer therapy strategies: current progress, hurdles and future prospects. Trends Mol Med. 2007 Apr 24; [Epub ahead of print] PMID: 17462954 [PubMed - as supplied by publisher]
2. Duda DG, Cohen KS, Scadden DT, Jain RK. A protocol for phenotypic detection and enumeration of circulating endothelial cells and circulating progenitor cells in human blood. Nat Protoc. 2007;2(4):805-10. PMID: 17446880 [PubMed - in process]
3. Willett CG, Duda DG, di Tomaso E, Boucher Y, Czito BG, Vujaskovic Z, Vlahovic G, Bendell J, Cohen KS, Hurwitz HI, Bentley R, Lauwers GY, Poleski M, Wong TZ, Paulson E, Ludwig KA, Jain RK. Complete pathological response to bevacizumab and chemoradiation in advanced rectal cancer. Nat Clin Pract Oncol. 2007 May;4(5):316-21. PMID: 17464339 [PubMed - indexed for MEDLINE]
4. Duda DG, Cohen KS, Au P, Scadden DT, Willett CG, Jain RK. Detection of circulating endothelial cells: CD146- based magnetic separation enrichment or flow cytometric assay? J Clin Oncol. 2007; 25: e3-e5.
5. Batchelor TT, Sorensen AG, di Tomaso E, Zhang WT, Duda DG, Cohen KS, Kozak KR, Cahill DP, Chen PJ, Zhu M, Ancukiewicz M, Mrugala MM, Plotkin S, Drappatz J, Louis DN, Ivy P, Scadden DT, Benner T, Loeffler JS, Wen PY, Jain RK. AZD2171, a pan-VEGF receptor tyrosine kinase inhibitor, normalizes tumor vasculature and alleviates edema in glioblastoma patients. Cancer Cell. 2007 Jan;11(1):83-95. PMID: 17222792 [PubMed - indexed for MEDLINE]
6. Hagendoorn J, Padera TP, Yock TI, Nielsen GP, di Tomaso E, Duda DG, Delaney TF, Gaissert HA, Pearce J, Rosenberg AE, Jain RK, Ebb DH. Platelet-derived growth factor receptor-beta in Gorham's disease. Nat Clin Pract Oncol. 2006 Dec;3(12):693-7. PMID: 17139320 [PubMed - indexed for MEDLINE]
7. Duda DG, Cohen KS, di Tomaso E, Au P, Klein RJ, Scadden DT, Willett CG, Jain RK. Differential CD146 expression on circulating versus tissue endothelial cells in rectal cancer patients: implications for circulating endothelial and progenitor cells as biomarkers for antiangiogenic therapy. J Clin Oncol. 2006 Mar 20;24(9):1449-53. PMID: 16549839 [PubMed - indexed for MEDLINE]
8. Jain RK, Duda DG, Clark JW, Loeffler JS. Lessons from phase III clinical trials on anti-VEGF therapy for cancer. Nat Clin Pract Oncol. 2006 Jan;3(1):24-40. PMID: 16407877 [PubMed - indexed for MEDLINE]
9. Willett CG, Boucher Y, Duda DG, di Tomaso E, Munn LL, Tong RT, Kozin SV, Petit L, Jain RK, Chung DC, Sahani DV, Kalva SP, Cohen KS, Scadden DT, Fischman AJ, Clark JW, Ryan DP, Zhu AX, Blaszkowsky LS, Shellito PC, Mino-Kenudson M, Lauwers GY. Surrogate markers for antiangiogenic therapy and dose-limiting toxicities for bevacizumab with radiation and chemotherapy: continued experience of a phase I trial in rectal cancer patients. J Clin Oncol. 2005 Nov 1;23(31):8136-9. PMID: 16258121 [PubMed - indexed for MEDLINE]
10. Willett CG, Boucher Y, di Tomaso E, Duda DG, Munn LL, Tong RT, Chung DC, Sahani DV, Kalva SP, Kozin SV, Mino M, Cohen KS, Scadden DT, Hartford AC, Fischman AJ, Clark JW, Ryan DP, Zhu AX, Blaszkowsky LS, Chen HX, Shellito PC, Lauwers GY, Jain RK. Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer. Nat Med. 2004 Feb;10(2):145-7. Epub 2004 Jan 25. Erratum in: Nat Med. 2004 Jun;10(6):649. PMID: 14745444 [PubMed - indexed for MEDLINE]
11. Jain RK, Duda DG. of bone marrow-derived cells in tumor angiogenesis and treatment. Cancer Cell. 2003 Jun;3(6):515-6. PMID: 12842078 [PubMed - indexed for MEDLINE]