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Targeted Therapy of Colorectal Cancer: Clinical Experience with Bevacizumab

Department of Medical Oncology and Transplantation, Duke University Medical Center, Durham, North Carolina, USA

Correspondence: Herbert I. Hurwitz, M.D., Department of Medical Oncology and Transplantation, Box 3052, Duke University Medical Center, Durham, North Carolina 27710, USA. Telephone: 919-681-5257; Fax: 919-684-9712; e-mail: hurwi004{at}mc.duke.edu

	LEARNING OBJECTIVES

Top Learning Objectives Abstract Introduction VEGF in Colorectal Cancer Bevacizumab in Colorectal Cancer Preclinical Studies Clinical Studies Future Directions Conclusions References

 
After completing this course, the reader will be able to:

1. Describe the clinical experience of bevacizumab in colorectal cancer.
   
2. Explain the relevant biology and preclinical drug development of bevacizumab in colorectal cancer.
   
3. Discuss future directions of research.
   

	ABSTRACT

Top Learning Objectives Abstract Introduction VEGF in Colorectal Cancer Bevacizumab in Colorectal Cancer Preclinical Studies Clinical Studies Future Directions Conclusions References

 
Advanced colorectal cancer remains an urgent health concern, despite improvements in systemic chemotherapy. Targeted therapeutics promise effective tumor therapy with minimal side effects. Angiogenesis (the formation of new blood vessels) is essential for tumor growth and metastasis and may be an ideal target in the search for new antineoplastic agents. Vascular endothelial growth factor is one of the best characterized of the proangiogenic growth factors that regulate angiogenesis and is a logical target in colorectal cancer therapy. Bevacizumab (Avastin^TM; Genentech Inc.; South San Fransisco, CA), a humanized murine monoclonal antibody directed at vascular endothelial growth factor, is being evaluated in the treatment of various types of cancer. It has shown promising efficacy in phase II clinical trials in patients with metastatic colorectal cancer. Addition of bevacizumab at a dose of 5 mg/kg to chemotherapy (5-fluorouracil plus leucovorin) resulted in a higher objective response rate (40% versus 17%), longer time to disease progression (9.0 versus 5.2 months), and longer median survival time (21.5 versus 13.8 months). Hypertension and thrombosis were the principal safety concerns, but were manageable. Further phase II/III studies of bevacizumab, administered with 5-fluorouracil plus leucovorin, with or without irinotecan and/or oxaliplatin, in colorectal cancer, are under way.

Key Words. Colorectal cancer • Clinical trial • Bevacizumab • Vascular endothelial growth factor • Monoclonal antibodies

	INTRODUCTION

Top Learning Objectives Abstract Introduction VEGF in Colorectal Cancer Bevacizumab in Colorectal Cancer Preclinical Studies Clinical Studies Future Directions Conclusions References

 
Colorectal cancer remains a national health problem, with approximately 300,000 new cases and 55,000 deaths each year in the U.S. alone [1, 2]. Overall survival is highly dependent on the stage of disease at diagnosis, with estimated 5-year survival rates ranging from 85%-90% for patients with stage I disease to <5% for patients with stage IV disease. Adjuvant chemotherapy has been proven to increase disease-free and overall survival in patients with Duke’s stage C cancers, and greater survival is strongly suggested in Duke’s stage B2 cancers [3]. However, over 50% of patients with colorectal cancer presenting with metastatic or locally advanced disease experience local recurrence or develop distant metastases after potentially curative surgical excisions. The major current treatment for patients with stage IV disease is systemic chemotherapy. Although there have been recent advances in the field, with randomized trials confirming the activity of irinotecan and oxaliplatin, median survival remains at 15–18 months [4, 5].

There is, therefore, a strong medical need for more effective and better-tolerated therapies. One direction taken in research is to assess novel combinations of existing compounds. Combination regimens currently being assessed in colorectal cancer include capecitabine plus oxaliplatin (XELOX); irinotecan, 5-fluorouracil plus leucovorin (5-FU/LV) (modified de Gramont regimen); and oxaliplatin plus 5-FU/LV (FOLFOX-4) [6–10]. The other direction taken in research is to assess therapies able to selectively target pathways that are critical for tumor growth and development while sparing normal tissues. In this respect, angiogenesis is an obvious target in the treatment of cancer since neovascularization is essential for tumor growth [11].

Although factors such as basic fibroblast growth factor, platelet-derived growth factor, and platelet-derived endothelial-cell growth factor, also known as thymidine phosphorylase, have been implicated in angiogenesis, vascular endothelial growth factor (VEGF) has emerged as the most potent and specific of the identified angiogenic factors [11]. It is also markedly upregulated in the vast majority of human cancers examined to date, including colorectal cancer [12].

	VEGF IN COLORECTAL CANCER

Top Learning Objectives Abstract Introduction VEGF in Colorectal Cancer Bevacizumab in Colorectal Cancer Preclinical Studies Clinical Studies Future Directions Conclusions References

 
VEGF is expressed in approximately 50% of colorectal cancers, with minimal to no expression in normal colonic mucosa or adenomas [13]. Vessel counts correlate with disease recurrence, metastasis, and survival [14, 15]. Also, increased VEGF expression is significantly correlated with advanced lymph node status and distant metastasis. Among patients with the highest levels of VEGF expression, survival was significantly worse than in patients with negative or lower levels of VEGF expression. On multivariate analysis, VEGF expression was no longer a significant variable, implying that VEGF expression is closely correlated with other prognostic indicators [13]. Others have also noted expression of VEGF to be higher in metastatic than in nonmetastatic cancers and to directly correlate with the extent of neovascularization and the degree of proliferation [16]. In a further study, VEGF receptors (VEGF-R-1 and -2) and rRNA for these receptors were found to be highly expressed in human liver metastases from primary colorectal carcinomas [17]. Direct evidence implicating VEGF in tumor growth was provided by preclinical data. Transfection of VEGF into a human colon cancer cell line was shown to enhance angiogenesis, tumor growth, and metastasis when the cancer cells were xenografted into nude mice [18].

Preoperative serum VEGF levels have also been shown to correlate with advanced tumor stage or nodal status at the time of surgery [19]. When measured prospectively in 81 patients undergoing curative resection for colorectal cancer, serum VEGF levels were significantly higher in patients who went on to develop metastases than in those who did not [20]. VEGF levels were predictive of future metastases independent of nodal status and adjuvant chemotherapy, with a positive predictive value of 73% and a negative predictive value of 85%. Whether baseline measurement of VEGF may prove useful for selecting patients who require adjuvant therapy or for selecting those patients most appropriate for anti-VEGF or other antiangiogenic therapies in other settings is an intriguing, but still unproven, concept.

From the above evidence, it is apparent that anti-VEGF therapy has great potential in the treatment of cancer. The advantages of this approach are reviewed in this issue [11]. In brief, targeting VEGF prevents angiogenesis induced by all activators upstream of this molecule, and also affects several downstream pathways that can lead to death of a large tumor mass [21]. In addition to avoiding the nonspecific toxicities of cytotoxic therapy, targeting endothelial cells (which are stable host cells) removes the need for the drug to penetrate the tumor and minimizes the risk of acquired resistance to therapy. The challenges involved in developing anti-VEGF agents are exemplified elsewhere in this issue [22].

	BEVACIZUMAB IN COLORECTAL CANCER

Top Learning Objectives Abstract Introduction VEGF in Colorectal Cancer Bevacizumab in Colorectal Cancer Preclinical Studies Clinical Studies Future Directions Conclusions References

 
Inhibition of the signaling pathway can be accomplished through several strategies, including monoclonal antibodies to VEGF, antibodies to the VEGF receptors, soluble extracellular domains of VEGF receptors that trap free VEGF, small molecules that interfere with VEGF binding or receptor signaling, and ribozymes that degrade VEGF receptor RNA [23–28]. Many of these strategies are in clinical development, primarily in phase I and phase II trials. Clinical development is most advanced for bevacizumab (Avastin^TM; Genentech, Inc.; South San Francisco, CA), a recombinant humanized monoclonal antibody to VEGF, which is in phase II/III development.

Bevacizumab, derived from the murine antibody A.4.6.1, comprises 93% human IgG frameworks and 7% murine-derived antigen-binding regions, the humanization providing a longer half-life and less immunogenicity. A study by Kim et al. demonstrated that the antibody was able to neutralize the biological properties of human VEGF, including endothelial cell mitogenic activity, vascular-permeability-enhancing activity, and angiogenic properties [23]. The antibody did not recognize the other growth factors tested, including fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor.

	PRECLINICAL STUDIES

Top Learning Objectives Abstract Introduction VEGF in Colorectal Cancer Bevacizumab in Colorectal Cancer Preclinical Studies Clinical Studies Future Directions Conclusions References

 
Extensive preclinical studies have demonstrated the efficacy of the parent A.4.6.1 antibody in several tumor models. Kim and coworkers injected three human tumor cell lines subcutaneously into nude mice: SK-LMS-1 leiomyosarcoma, G55 glioblastoma multiforme, and A673 rhabdomyosarcoma [29]. The antibody was then injected intraperitoneally twice weekly. Growth inhibition occurred, ranging from 70% to >95%. Examination of the posttreatment tumors revealed a significantly lower blood-vessel density compared with those from control animals. Studies with in vitro cell lines were also performed and did not reveal a direct effect of the antibody on the growth of tumor cells, a finding subsequently confirmed by others [17, 29]. Both these observations support the hypothesis that tumor suppression is mediated through inhibition of neovascularization.

Further investigations have confirmed these initial results in numerous tumor models. Warren and coworkers demonstrated that treatment with anti-VEGF antibodies was effective in suppressing primary tumor growth as well as liver metastasis growth in a murine model of colorectal carcinoma [17]. Mice who received active antibody demonstrated a dose-response reduction in primary tumor volume, with a 90% reduction in volume at the highest dose. Treated mice also had a marked reduction in the average number and weight of hepatic lesions. Perhaps more importantly, 91% of the liver metastases in treated mice were <1 mm and only one animal had a liver metastasis >3 mm. In contrast, only 38% of tumors in control mice were <1 mm and all mice had tumors >8 mm. These findings correspond well to the paradigm that tumors require neovascularization for growth beyond a diameter of 2–3 mm [30].

	CLINICAL STUDIES

Top Learning Objectives Abstract Introduction VEGF in Colorectal Cancer Bevacizumab in Colorectal Cancer Preclinical Studies Clinical Studies Future Directions Conclusions References

 
Based on preclinical data, a phase I/II program with bevacizumab was initiated. The initial phase I trial enrolled 25 patients in a dose-escalation fashion (0.1–10.0 mg/kg on days 0, 28, 35, and 42) [31]. No grade 3 or 4 toxicity was seen that was directly attributable to therapy. There were three episodes of tumor-related bleeding, including a hemorrhage in a previously undetected cerebral metastasis. Grade 1 and 2 toxicities, possibly or probably related to treatment, included asthenia, headache, and nausea. There were no complete or partial responses seen, although one patient with renal cell carcinoma achieved a mixed response. Disease stabilization was seen in 48% of patients evaluable at day 70. Importantly, no patient developed antibodies to bevacizumab, presumably due to the humanization. A subsequent phase Ib trial investigated the combination of bevacizumab with either doxorubicin, carboplatin/paclitaxel, or 5-FU/LV in 12 patients with advanced cancer [32]. The grade 3 toxicities seen were all attributable to the chemotherapy component. One patient in each combination regimen achieved a partial response and continued on therapy.

A randomized, open-label, phase II multicenter trial evaluated the efficacy, safety, pharmacokinetics, and pharmacodynamics of bevacizumab in combination with 5-FU/LV as first-line chemotherapy in patients with metastatic colorectal cancer [33]. The study enrolled 104 patients between June and November 1998. The study randomized patients to treatment with standard 5-FU (500 mg/m²)/LV (500 mg/m²) alone or in combination with a high (10 mg/kg) or low (5 mg/kg) dose of bevacizumab. Weekly doses of 5-FU/LV were given for the first 6 weeks of each 8-week cycle, and bevacizumab was administered every 2 weeks. Treatment with 5-FU/LV was continued for six cycles or until disease progression, and bevacizumab treatment was continued for up to 48 weeks or until disease progression, whichever occurred first. Patients in the control arm who developed progressive disease had the option to cross over and receive single-agent bevacizumab.

Table 1 presents the results for the end points of response, time to disease progression, and survival from that trial. Compared with 5-FU/LV alone, both combination regimens were associated with higher response rates (17% in the control arm versus 40% in the low-dose bevacizumab arm and 24% in the high-dose bevacizumab arm).

View larger version (13K): [in this window] [in a new window]   Figure 1. Time to disease progression in patients treated with 5-FU/LV alone or in combination with bevacizumab. Reproduced with permission from Kabbinavar et al. 2003 [33].

Therapy with bevacizumab was generally well tolerated, with the most common toxicities of diarrhea, leukopenia, fever, and stomatitis seen at incidences and severities that could be attributable to 5-FU/LV. Greater incidences of hemorrhagic complications were seen in the bevacizumab arms. Approximately 50% of patients reported transient minor epistaxis. An additional three patients had a grade 3 or 4 gastrointestinal hemorrhage. Thrombosis was the most significant adverse effect, with greater incidences seen in the bevacizumab arms, including one fatal pulmonary embolus (Table 2). Patients were able to restart bevacizumab after administration of an anticoagulant. Other potential safety concerns included proteinuria and epistaxis (as expected from phase I data) and hypertension; 16 patients required antihypertensive therapy.

Patients with various tumor types (including 17 with colorectal cancer) from phase I/II clinical trials have been treated with bevacizumab under an extension protocol [35]. Thirty-five patients received bevacizumab for 1 year and six patients received the drug for 2 years. At the time of writing of this paper, three complete and 15 partial responses had been reported, and 14 patients had stabilized disease. The median survival had not been reached, but was >27.5 months at the time of data capture, with 25 patients (71%) still alive at 2 years. Three of the patients with colorectal cancer received bevacizumab treatment for more than 3 years. The authors of the study concluded that there appeared to be a subset of patients who could achieve long-term disease stabilization with bevacizumab therapy. No unexpected adverse events were observed after 1 year of treatment. Deep venous thrombosis was observed, but patients were able to remain on bevacizumab with anticoagulant therapy.

The phase III study of standard bolus irinotecan/ 5-FU/LV (IFL) plus bevacizumab (5 mg/kg) was recently reported [36]. This randomized, double-blind, placebocontrolled study of 923 patients demonstrated that the addition of bevacizumab to bolus IFL improved overall survival, time to progression, and response rate. Toxicities were generally mild, consisting primarily of hypertension, which was readily manageable with standard blood pressure medications. Importantly, this was the first phase III validation of an antiangiogenic approach for the treatment of human cancer. Several further, large phase III trials of bevacizumab in metastatic colorectal cancer are under way. These include a first-line study of standard 5-FU/LV with or without bevacizumab in patients who are not appropriate for irinotecan therapy. A further phase III study will assess the FOLFOX regimen with or without bevacizumab in the second-line setting for patients who have failed previous irinotecan plus 5-FU treatment [37]. These results will help to define the extent to which anti-VEGF therapy can be used for the treatment of colorectal cancer.

	FUTURE DIRECTIONS

Top Learning Objectives Abstract Introduction VEGF in Colorectal Cancer Bevacizumab in Colorectal Cancer Preclinical Studies Clinical Studies Future Directions Conclusions References

 
The clinical data obtained to date indicate that bevacizumab monotherapy is an effective and well-tolerated treatment for patients with colorectal cancer. There are, however, issues that need to be addressed. For example, it remains to be determined which subset of patients benefits most from bevacizumab therapy. Importantly, phase III data suggest that all clinical subgroups benefit from therapy. Evaluation of molecular predictors is ongoing. The optimal dose of bevacizumab is also currently being explored. While the available phase II and phase III data for colorectal cancer have demonstrated a benefit for bevacizumab at a dose of 5 mg/kg every 2 weeks, it should be noted that the current ECOG second-line study of the FOLFOX regimen, alone or in combination with bevacizumab, is using a dose of 10 mg/kg. It is possible that both doses will be active, and further study may be needed to define the optimal dose for this agent in colorectal cancer.

From a safety point of view, the mechanism of bleeding and thrombosis associated with bevacizumab treatment remains to be clarified. VEGF appears to act as a critical survival factor for endothelial cells in newly formed vessels by inhibiting apoptosis [12]. Increased apoptosis of these cells through the blockade of VEGF receptors, leading to exposure of subendothelial cells, may trigger a coagulation cascade. Therefore, thrombosis has been speculated to be a class effect for all antiangiogenic agents. Importantly, however, the incidence of thrombosis and cardiovascular events was not elevated in the phase III study of bevacizumab plus IFL in metastatic colorectal cancer, suggesting these potential complications are more related to advanced colorectal cancer and systemic chemotherapy than to bevacizumab treatment. Modest elevations in blood pressure occurred occasionally and were easily managed with standard antihypertensive medications.

An important area of enquiry is the role of bevacizumab in combination therapy. Given the potentially cytostatic nature of anti-VEGF therapy, the successful use of bevacizumab may require combinations with cytotoxic therapy and/or radiotherapy. This rationale has been strengthened by preclinical evidence.

Kabbinavar et al. studied the combination of the anti-VEGF antibody A.4.6.1 with cisplatin in a subcutaneous mouse model of human lung cancer (Calu-6) [38]. The combination of the two therapies markedly enhanced biological activity against the tumor compared with either agent alone. Similar results were seen when A.4.6.1 and doxorubicin were combined in a human breast cancer cell line [39]. Anti-VEGF treatment resulted in significant reductions in angiogenesis as assessed by intravital videomicroscopy, but only modest reductions in tumor size. Tumor regression alone was seen after treatment with doxorubicin, without any significant effect on angiogenesis. When the two drugs were combined, tumor regression was even more profound, with complete responses seen in some cases. The mechanism by which anti-VEGF therapy enhances the activity of chemotherapy is not fully understood. Inhibition of vascular and endothelial cell function may reduce stromal-derived tumor growth and survival signals and may also increase tumor apoptotic rates. In addition, a reduction in tumor vascular permeability may reduce interstitial pressures, allowing enhanced drug delivery. Alternatively, by selectively targeting small aberrant tumor vessels, there may be a relative normalization of tumor blood flow, again enhancing drug delivery.

Similar positive interactions have been seen when anti-VEGF therapies were combined with radiation therapy. In general, hypoxia is thought to play a prominent role in radiation and chemotherapy resistance. In a murine tumor xenograft model, hypoxia reduced the tumor growth delay seen with radiation [40]. Treatment with a monoclonal antibody to VEGF decreased interstitial pressure, increased oxygenation, and reversed radiation resistance. These data suggest that anti-VEGF treatment is likely to aid delivery of large molecule therapeutic agents within tumors and reverse the resistance to radiotherapy conferred by hypoxia.

These experiments support the idea that tumor growth and tumor angiogenesis are intricately coordinated. Importantly, almost all classic antiproliferative agents will inhibit proliferating endothelial cells, resulting in a direct antiangiogenic effect. Most agents that inhibit tumor growth will also inhibit production of proangiogenic signals, either directly or indirectly. Furthermore, inhibition of tumor angiogenesis may lead to decreased tumor proliferation and increased tumor apoptosis. Thus, there are many potential mechanisms for synergy between conventional chemotherapies and novel antiangiogenic agents. Whether this potential is borne out and how a strategy could be optimized clinically are still to be determined. Combinations of bevacizumab with other agents, such as cyclooxygenase-2 inhibitors, thalidomide, and small molecule, tyrosine-kinase inhibitors also need to be assessed.

Lastly, evaluation of bevacizumab in the early stages of disease (adjuvant and neoadjuvant) is warranted. While bevacizumab has, thus far, been remarkably well tolerated, the effects of long-term therapy may uncover novel mechanisms of tumor resistance or toxicities in the late stages of disease that are not yet appreciated. There is a strong rationale for using antiangiogenic therapy in early disease when tumor neovascularization is particularly critical. In addition, such therapy may reduce metastasis.

	CONCLUSIONS

Top Learning Objectives Abstract Introduction VEGF in Colorectal Cancer Bevacizumab in Colorectal Cancer Preclinical Studies Clinical Studies Future Directions Conclusions References

 
Targeting of tumor-expressed VEGF is a highly attractive approach to the treatment of human cancer. Bevacizumab, a humanized murine monoclonal antibody directed at VEGF, has shown promising efficacy in the treatment of colorectal cancer. The results of ongoing phase III trials are eagerly awaited to help determine whether and when bevacizumab therapy will become a standard entry in the armamentarium against this disease.

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Top Learning Objectives Abstract Introduction VEGF in Colorectal Cancer Bevacizumab in Colorectal Cancer Preclinical Studies Clinical Studies Future Directions Conclusions References

 

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