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Journal of Clinical Oncology, Vol 25, No 13 (May 1), 2007: pp. 1816-1818 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2006.10.3051
Vascular Endothelial Growth Factor Levels in Immunodepleted Plasma of Cancer Patients As a Possible Pharmacodynamic Marker for Bevacizumab ActivityDivision of Medical Oncology, General Hospital, Livorno, Italy
Division of Medical Oncology, General Hospital, Livorno; Department of Oncology, University of Pisa, Pisa, Italy
Division of Medical Oncology, General Hospital, Livorno, Italy
Division of Pharmacology and Chemotherapy, Department of Internal Medicine, University of Pisa, Pisa, Italy
Department of Molecular and Cellular Biology, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, Canada
Division of Pharmacology and Chemotherapy, Department of Internal Medicine, University of Pisa, Pisa, Italy To the Editor: Bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF),1 has demonstrated clinical activity in various solid tumors, with proven efficacy for the treatment of advanced colorectal cancer, breast cancer, and non–small-cell lung cancer when used in combination with chemotherapy.2,3 The approval of bevacizumab, and subsequently other antiangiogenic drugs (eg, sunitinib and sorafenib), has stimulated enormous interest in medical oncology with a large number of phase II/III clinical studies under way testing various antiangiogenic drugs on many different cancer types.4 Nevertheless, the clinical development of molecularly targeted antiangiogenic drugs, including bevacizumab, still suffers from several handicaps such as monitoring biologic activity and determining the optimal biologic dose. Among the proposed surrogate biomarkers that may be applicable to antiangiogenic agents, circulating endothelial progenitors have attracted considerable interest.5 However, the complexity of flow cytometry procedures and the rarity of circulating endothelial progenitors in human blood6 could provide an advantage to other molecular approaches that can be used as surrogates of angiogenesis and antiangiogenic drug activity.7 Cristofanilli and colleagues8 suggested that measurement of serum VEGF could serve as such a surrogate biomarker for antibodies targeting this growth factor. This opinion was based mainly on previously published data by Gordon et al9 describing a reduction of free serum VEGF in cancer patients treated with escalating doses of an anti-VEGF antibody when compared with basal serum concentrations. Interestingly, these authors also showed a parallel increase in serum total VEGF concentrations across all dose groups. Willet et al10 reported in a phase I clinical trial the results from two dose escalation levels of bevacizumab (5 and 10 mg/kg) and the data regarding plasma VEGF levels, before and 12 days after single-dose infusion of bevacizumab.11 Surprisingly, a significant increase in the levels of plasma VEGF was observed, and the authors commented that it will be critical to establish how much of the VEGF in the plasma represents free protein (unbound to bevacizumab) at day 12.11 Similarly, Yang and colleagues12 in a phase II randomized trial reported a progressive increase of VEGF levels from baseline to 5 and 13 weeks after initiation of treatment with bevacizumab alone at two dose levels (3 and 10 mg/kg). In this experimental circumstance, the plasma VEGF assay detected both free and bevacizumab-bound VEGF equally, with a lower limit of detection of 40 pg/mL. As part of an effort to investigate the concentration of (biologically active) free VEGF after bevacizumab administration, we decided to explore the feasibility of the immunodepletion of plasma samples, removing all immunoglobulins, and thus separate the VEGF bound to bevacizumab from free VEGF, and to test the changes of free VEGF levels in cancer patients after treatment with bevacizumab. The study was in accordance with the precepts established by the Helsinki Declaration and approved by the local Ethics Committee; patients and healthy volunteers were enrolled after giving written consent. Blood samples (5 mL) were collected from five metastatic cancer patients (Table 1) receiving bevacizumab treatment at the dose of 5 mg/kg at day 0 (prebolus) and at day 14 of the first cycle of treatment, and from four healthy volunteers (male:female, 2:2; age 27 to 32 years). Blood samples were immediately centrifuged at 4°C and plasma fractions were divided in two aliquots. The split samples were subjected simultaneously to the immunodepletion protocol using Protein G-Sepharose 4 Fast Flow beads (Pharmacia Biotech, Uppsala, Sweden), as previously described,13 and to the same experimental procedure but adding only phosphate-buffered saline. To deplete plasma samples of the bevacizumab immunoglobulin antibody and bevacizumab-bound VEGF, 100 µL of protein G slurry (50% v/v protein G-Sepharose in phosphate-buffered saline) were added to 200 µL of plasma samples and incubated at 4°C for 4 hours. After centrifugation, 200 µL of plasma supernatants were removed and the immunodepletion was repeated by the addition of 100 µL of protein G slurry and overnight incubation at 4°C. Each plasma sample was assayed twice for human VEGF concentration by Endogen Human VEGF enzyme-linked immunosorbent assay Kit (Pierce Biotechnology, Rockford, IL) either after immunodepletion or not. Results are reported as the mean ± standard deviation. Student's paired two-tailed t test (GraphPad Prism software 4.0, San Diego, CA) was performed to compare depleted versus undepleted plasma samples at baseline and at day 14 after bevacizumab administration. The level of significance was set at P < .05.
Figure 1 shows that free VEGF levels measured after immunodepletion of plasma samples significantly decrease from day 0 (prebolus) to day 14 of bevacizumab treatment (from 157 ± 92 pg/mL to 45 ± 27 pg/mL; P = .0283) suggesting that the anti-VEGF antibody truly and effectively reduced the plasma level of the biologically active growth factor. Moreover, a slight but not significant variation has been demonstrated between the prebolus VEGF levels to those detected at day 14 in undepleted plasma samples, confirming the necessity of the immunodepletion process in order to detect significative variations between samples before and after therapy. The differences of the human VEGF levels between the undepleted and depleted samples were not significant before the bevacizumab dose but significantly differed after antibody treatment (129 ± 53 pg/mL v 45 ± 27 pg/mL; P = .0211). Moreover, no differences in VEGF concentrations were found between undepleted and depleted samples (48 ± 18 pg/mL v 59 ± 20 pg/mL, respectively; P > .05) of the healthy volunteers. These results clearly suggest that the immunodepletion procedure did not significantly impact on the free plasma VEGF in the absence of bevacizumab, demonstrating the validity of measurement of the biologically active growth factor.
Surprisingly, the hypothesis of Cristofanilli and colleagues8 regarding using VEGF levels as a surrogate marker has never been tested with immunodepleted plasma samples, and until now, the decrease of free human VEGF levels during treatment with bevacizumab has been observed only in the study by Gordon et al.9 Others have reported increases in VEGF after bevacizumab treatment. These experimental findings could be related to assays that do not discriminate between free and bevacizumab-bound VEGF. Our assay was designed to eliminate this problem. In this regard, our results show that immunodepletion of plasma samples is feasible, easy, rapid, and able to eliminate, among the other immunoglobulins, bevacizumab and bevacizumab-bound VEGF, leading to the enzyme-linked immunosorbent assay determination of free plasma VEGF and allowing significant reduction of the biologic active growth factor after bevacizumab administration. This simple procedure could not only be relevant for obtaining correct measurements of free VEGF concentrations during bevacizumab treatment, but could also help to identify those patients in which the antibody seems to be acting as an antiangiogenic agent. Thus, assessing free VEGF could become a candidate among many proposed surrogate markers of bevacizumab antiangiogenic activity. By way of example, treatment of cancer patients with antiangiogenic small molecule receptor tyrosine kinase inhibitors, such as sunitinib, leads to increases in the levels of plasma VEGF, which are reversed when therapy is stopped.14 Such changes may be a reflection of the antiangiogenic activity of the drug. In conclusion, the immunodepletion of plasma samples from patients treated with bevacizumab represents a potentially promising procedure to discriminate and quantitate free plasma VEGF, a possible pivotal pharmacodynamic marker for the antiangiogenic activity of the antibody compared with other novel but more cumbersome and expensive strategies. Hopefully, our results will provide incentives to undertake randomized phase III trials testing free plasma VEGF after the immunodepletion procedure as a means of validating its efficacy and routine use in the clinic. 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: N/A Stock: N/A Honoraria: Robert S. Kerbel, Roche, Genentech Research Funds: N/A Testimony: N/A Other: N/A
ACKNOWLEDGMENTS Supported by a research grant from AIRC (A.F.) and from the Ministry of Education, University and Research (PRIN project 2004; M.D.T.). REFERENCES 1. Ferrara N, Hillan KJ, Novotny W: Bevacizumab (Avastin), a humanized anti-VEGF monoclonal antibody for cancer therapy. Biochem Biophys Res Commun 333:328-335, 2005[CrossRef][Medline] 2. Lyseng-Williamson KA, Robinson DM: Bevacizumab: A review of its use in advanced colorectal cancer, breast cancer, and NSCLC. Am J Cancer 5:43-60, 2006[CrossRef] 3. Hurwitz H, Fehrenbacher L, Novotny W, et al: Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 350:2335-2342, 2004 4. Jain RK, Duda DG, Clark JW, et al: Lessons from phase III clinical trials on anti-VEGF therapy for cancer. Nat Clin Pract Oncol 3:24-40, 2006[CrossRef][Medline] 5. Shaked Y, Bertolini F, Man S, et al: Genetic heterogeneity of the vasculogenic phenotype parallels angiogenesis: Implications for cellular surrogate marker analysis of antiangiogenesis. Cancer Cell 7:101-111, 2005[Medline] 6. Bertolini F, Shaked Y, Mancuso P, et al: The multifaceted circulating endothelial cell in cancer: Towards marker and target identification. Nat Rev Cancer 6:835-845, 2006[CrossRef][Medline] 7. Shaked Y, Bocci G, Munoz R, et al: Cellular and molecular surrogate markers to monitor targeted and non-targeted antiangiogenic drug activity and determine optimal biologic dose. Curr Cancer Drug Targets 5:551-559, 2005[CrossRef][Medline] 8. Cristofanilli M, Charnsangavej C, Hortobagyi GN: Angiogenesis modulation in cancer research: Novel clinical approaches. Nat Rev Drug Discov 1:415-426, 2002[CrossRef][Medline] 9. Gordon MS, Margolin K, Talpaz M, et al: Phase I safety and pharmacokinetic study of recombinant human anti-vascular endothelial growth factor in patients with advanced cancer. J Clin Oncol 19:843-850, 2001 10. 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] 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. Yang JC, Haworth L, Sherry RM, et al: A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. N Engl J Med 349:427-434, 2003 13. Bocci G, Man S, Green SK, et al: Increased plasma vascular endothelial growth factor (VEGF) as a surrogate marker for optimal therapeutic dosing of VEGF receptor-2 monoclonal antibodies. Cancer Res 64:6616-6625, 2004 14. Motzer RJ, Michaelson MD, Redman BG, et al: Activity of SU11248, a multitargeted inhibitor of vascular endothelial growth factor receptor and platelet-derived growth factor receptor, in patients with metastatic renal cell carcinoma. J Clin Oncol 24:16-24, 2006 This article has been cited by other articles:
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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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P < .05 immunodepleted samples after BV versus undepleted samples after BV administration.
