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Journal of Clinical Oncology, Vol 25, No 5 (February 10), 2007: pp. e3-e5 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2006.08.5100
In ReplySteele Laboratory for Tumor Biology, Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA
Center for Regenerative Medicine, Massachusetts General Hospital and Harvard Stem Cell Institute, Boston, MA
Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA
Center for Regenerative Medicine, Massachusetts General Hospital and Harvard Stem Cell Institute, Boston, MA
Department of Radiation Oncology, Duke University Medical Center, Durham, NC
Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA We read with interest the correspondence by Dignat-George et al regarding our article on the differential expression between tissue endothelial cells and viable blood circulating endothelial cells (CECs) in rectal cancer patients, published in the March 20 issue of the Journal of Clinical Oncology.1 We had considered and addressed most of the issues raised by these authors in the Discussion section of our article. For the most part, we subscribe to their conclusions and consider our findings and conclusions to be in good agreement. We commend the authors and their collaborators for establishing a unified immunoseparation protocol with CD146 magnetic beads. Using the criteria of positive Europaeus Lectin-1 (UEA-1) expression, number of immunomagnetic beads attached, and cell size larger than 10 µm, they have cultured, identified, and counted using fluorescence microscopy an extremely rare population of viable CD146+lectin+ CECs. As we discussed in our article, culture-based assay may enrich for viable CD146+ cells with the loss of leukocytes and nonviable CECs, and may induce CD146 upregulation in CECs, particularly in the CECs with progenitor capacity.2 Interestingly, the authors detected CD34+CD146+CD45– CECs using flow cytometry, and their data were in good agreement with the immunoseparation protocol.3 We believe that these results are in line with our overall conclusion in our paper that—regardless of the method used—multiple endothelial markers need to be used when evaluating CEC populations for at least two reasons: no one cell surface marker nor lectin binding is endothelial cell specific; and multiple cell populations with putative endothelial phenotype can de detected by flow cytometry in human blood (discussed later in this letter). Our findings in cancer patients,1 as well as of others in healthy individuals4,5 did not reveal in flow cytometric analyses the difference between the CD146 expression levels in T lymphocytes and on other blood cells described by Dignat-George et al when using bead separation. We found that the even the brightest CD146+ cells were distributed in both CD45dim/– and CD45+ cell populations (Fig 1A). The very rare CD146+CD45– mononuclear events occasionally detected in rectal cancer patients were largely CD31– (> 90%). Thus, the high and homogeneous level of CD146 expression observed by the authors and by us in human umbilicial vein endothelial cells may not be predictive of the CD146 level in the rare CD146+ CEC subpopulation.4,5 This is important because CD146 has been used both as a single marker and as an endothelial-specific marker. Some of these references were provided in our article.
We would also like to clarify some issues of apparent disagreement. Our studies were not intended to address the current debate on the ideal CEC marker. The authors imply that the CD146+ CECs are the true endothelial population in the human blood, and that the population of viable CECs reported by us may represent either circulating endothelial progenitor cells (referred to as EPCs or CEPs) or nonendothelial cells. In fact, by using a published flow cytometry protocol,6 we and others discovered the existence of several subpopulations of mononuclear cells that display an endothelial phenotype in blood of rectal cancer patients.1,7-9 This finding was confirmed now in blood analyses in patients suffering of ovarian cancer, glioblastoma, sarcoma, or pediatric tumors10 (and unpublished results). The two populations that we studied in depth as biomarkers of antiangiogenic treatment were CD31brightCD34dimCD133–CD45– cells (referred to as viable CECs in our reports) and CD31+CD133+CD34brightCD45dim circulating progenitor cells (See Figs 1A and 1B and supplemental Fig 1 in our article1). Our reasons for focusing on these two populations were threefold: these cells have a typical endothelial and progenitor phenotype, respectively, and thus, their blood concentration may be affected by antiangiogenic therapy; these cells were consistently detected in numbers that allowed kinetic analyses by flow at different time points during treatment with antiangiogenic agents in several hundred patient samples (after acquiring 50,000 to 150,000 gated cellular events and excluding red cells and fragments from each sample); and we and others have discovered promising clinical correlations between the concentration of these two populations and the effect of antiangiogenic therapy and/or tumor response in phase I-II clinical trials of antiangiogenic therapy7-9,11 (and unpublished results). These viable CEC/progenitors were CD34+ cells identified by gating on mononuclear events, which makes it highly unlikely to be contaminated by platelets. The CD31bright cellular events correlated with increasing forward scatter, and were negative for the monocyte marker CD14 and for the nonviability markers PI and TO-PRO-38 (and data not shown). Before analyses, we used Fc (the constant region on an immunoglobulin molecule)-receptor blocking, and then analyzed all data using isotype-matched immunoglobulin G controls. Collectively, these data show that the flow events defined by us as viable CECs were viable mononuclear cells with a mature endothelial surface marker phenotype.8 We have also discovered that when changing the flow cytometry gate from mononuclear cells to other blood cell populations, the sensitivity of CD31+/CD45– analyses decreases and the complexity of these analyses extends further. The processed blood may contain cell fragments, including endothelial microparticles12 as well as CD31+ platelets. Moreover, we detected CD34+ and CD146+ cells in the neutrophil gate (which contains approximately 70% of the nucleated cells in human blood; Fig 1A).7 These cells were distributed among both CD45dim/– and CD45+ populations, independent of CD146 staining intensity (Fig 1A). Additional staining for vascular endothelial growth factor receptor 2 (VEGFR2) showed that VEGFR2 was detectable—to a variable degree—on most of the subsets of hematopoietic and endothelial populations described herein, as well as on others (for example, a small subset of CD14+ monocytes, not shown). Collectively, these findings indicate that comparative evaluations between treatment time points in clinical studies, and equally important, comparative evaluations between different studies using distinct markers for CEC evaluation in cancer patients are impossible and misleading without a good understanding of these phenotypic characteristics and methodology issues. Finally, we do not consider the log difference in the number of CECs as measured by flow cytometry and CD146-magnetic beads to be controversial for two main reasons. First, while immunomagnetic bead separation has a high degree of specificity, the yield is known to be lower and variable. Second, as reported in our article, CD146 expression was virtually undetectable in mononuclear CD31brightCD34dimCD133–CD45– CECs, in contrast to cultured or tissue endothelial cells. Thus, we think that the CD146 staining/bead selection—while detecting CD146+ cells—is simply overlooking the much larger CD31brightCD34dimCD133–CD45– CECs, as well as the CD34brightCD133+ progenitor cell populations. In cancer patients, these viable cells could originate from vascular endothelium shed during antiangiogenic treatment or, more likely, be mobilized from niches such as the bone marrow by tumor-derived factors. We agree that functional studies of these putative endothelial cell populations are warranted, and we plan to perform them. But ultimately, the validity of any of these cell populations as a biomarker of antiangiogenic treatment of cancer remains to be proven in correlative studies in phase II-III trials of antiangiogenic agents. In addition to time- and cost-efficient analyses of fresh whole blood samples, flow cytometry has the added benefit of quantitatively and comparatively analyzing multiple blood cell populations, while dissecting their surface marker phenotype and viability. We think that these qualities make this method highly qualified for the type of comparative and kinetic viable CEC analyses described herein. With the development of flow cytometers able to simultaneously analyze up to 11 colors and of fluorescent probes with narrow emission spectra (for example, nanocrystals), we anticipate a rapid and significant improvement in the ability to quantitatively analyze blood cell populations in patients during cancer therapy. Detection of very rare cell populations such as CD146+ CECs is within the sensitivity range of flow cytometry, but it may require the acquisition of a very large number of events (1 to 2 million4). We agree that the exceedingly low concentration of some of these cell populations may pose significant challenges for reliable quantitative comparisons in patients in large clinical trials regardless of the technique used. In summary, our study along with reports from other laboratories demonstrates that due to lack of specificity CD146 is inappropriate as single marker for CEC evaluation in primary blood samples of cancer patients as a biomarker of antiangiogenic therapy. Moreover, that flow cytometric analyses using four to six different endothelial/hematopoietic markers reveal the existence of multiple and distinct endothelial populations positive for each of the endothelial markers in human blood. These distinct populations may represent a different phenotype or stage of differentiation of CECs, or simply identify the surface marker overlap with different hematopoietic lineage-committed populations. Moving forward, we hope that our results on the phenotypic characterization of CECs by flow cytometry and our methodologic insights for this technique provide data to the research community that will impact the development, validation, and utility of both the biomarkers and the therapeutics whose target is the tumor vasculature. AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST Although all authors completed the disclosure declaration, the following authors or their immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors. Employment: N/A Leadership: N/A Consultant: Rakesh K. Jain, Pfizer, AstraZeneca, ThromboGenics, Nektar Therapeutics Stock: N/A Honoraria: Christopher G. Willett, Genentech; Rakesh K. Jain, Roche Research Funds: Rakesh K. Jain, AstraZeneca Testimony: N/A Other: N/A
ACKNOWLEDGMENTS This study was supported by two National Cancer Institute grants (PO1-CA80124 [R.K.J.] and R21 CA099237 [C.G.W.]) and by a grant from the National Foundation for Cancer Research (R.K.J.). Dan G. Duda's research is supported by an American Association for Cancer Research-Genentech BioOncology Award. REFERENCES
1. Duda DG, Cohen KS, di Tomaso E, et al: 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 24:1449-1453, 2006 2. Ingram DA, Mead LE, Tanaka H, et al: Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. Blood 104:2752-2760, 2004 3. Goon PK, Boos CJ, Stonelake PS, et al: Detection and quantification of mature circulating endothelial cells using flow cytometry and immunomagnetic beads: A methodological comparison. Thromb Haemost 96:45-52, 2006[Medline] 4. Khan SS, Solomon MA, McCoy JP Jr: Detection of circulating endothelial cells and endothelial progenitor cells by flow cytometry. Cytometry B Clin Cytom 64:1-8, 2005[Medline] 5. Elshal MF, Khan SS, Takahashi Y, et al: CD146 (Mel-CAM), an adhesion marker of endothelial cells, is a novel marker of lymphocyte subset activation in normal peripheral blood. Blood 106:2923-2924, 2005 6. Mancuso P, Burlini A, Pruneri G, et al: Resting and activated endothelial cells are increased in the peripheral blood of cancer patients. Blood 97:3658-3661, 2001 7. Duda DG, Cohen KS, di Tomaso E, et al: Diferential circulation kinetics during antiangiogenic therapy of four distinct blood cell populations expressing endothelial markers (ASCO 42). J Clin Oncol 24:130s, 2006 (abstr 3038) 8. Willett CG, Boucher Y, di Tomaso E, et al: Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer. Nat Med 10:145-147, 2004[CrossRef][Medline] 9. Rugo HS, Dickler MN, Traina TA, et al: Change in circulating endothelial cells (CEC) predicts progression free survival (PFS) in patients (pts) with hormone receptor positive metastatic breast cancer (MBC) receiving letrozole and bevacizumab (ASCO 42). J Clin Oncol 24:130s, 2006 (abstr 3039) 10. Hagendoorn J, Padera TP, Yock TI, et al: Platelet-derived growth factor receptor-beta in Gorham's disease. Nat Clin Pract Oncol 3:639-697, 2006 11. Willett CG, Boucher Y, Duda DG, et al: 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 23:8136-8139, 2005 12. Goon PK, Lip GY, Boos CJ, et al: Circulating endothelial cells, endothelial progenitor cells, and endothelial microparticles in cancer. Neoplasia 8:79-88, 2006[CrossRef][Medline]
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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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