Vascular Endothelial Growth Factor and Epidermal Growth Factor Signaling Pathways as Therapeutic Targets for Colorectal Cancer

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1 GASTROENTEROLOGY 2010;138: Vascular Endothelial Growth Factor and Epidermal Growth Factor Signaling Pathways as Therapeutic Targets for Colorectal Cancer Thomas Winder* Heinz Josef Lenz*, *Division of Medical Oncology and Department of Preventive Medicine, University of Southern California/Norris Comprehensive Cancer Center, Keck School of Medicine, Los Angeles, California Treatment of colorectal cancer (CRC) has developed considerably over the past decade, especially in the areas of targeted therapeutics and biomarker development. Multiple cellular pathways influence the growth and metastatic potential of CRC. Targeted therapies have been designed to interfere with specific molecular events in pathways that mediate tumor growth and progression. Preclinical and clinical studies have shown that the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) are valid therapeutic targets for patients with CRC. Monoclonal antibodies and tyrosine kinase inhibitors have been developed to target EGFR, VEGF, and VEGF receptors (VEGFRs) and are important additions to CRC treatment options. We review the most recent data on the VEGF and EGFR signaling pathways and therapeutic reagents designed to target them, provide insights into their mechanisms, and describe results from recent clinical trials. Keywords: EGFR-Pathway; VEGF-Pathway; Targeted Treatment. The systemic treatment options for patients with colorectal cancer (CRC) have progressed significantly; in 1995, patients were given only 5-fluorouracil (5-FU), but now they also receive cytotoxic chemotherapeutic and biologic agents. Despite significant improvements in response rate and survival over a little more than a decade, the efficacy of chemotherapeutic agents appears to have reached a plateau. This might be due to severe adverse effects that limit treatment duration and dose as well as drug resistance. As a result, patients with advanced or recurrent CRC need novel therapeutic agents that target specific biological pathways involved in tumor growth and dissemination. Targeted therapeutics include monoclonal antibodies (mabs) and small molecules that interfere with factors expressed by the tumor cells and the tumor microenvironment. Reagents have been developed to target the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) signaling pathways, which mediate progression of CRC. We review the mechanisms of these pathways in tumor growth, the reagents that target them, and the safety and efficacy of inhibiting these pathways in patients with CRC. EGFR EGFR is a transmembrane receptor tyrosine kinase of the ErbB (also known as HER) family that is abnormally activated in many epithelial tumors. This receptor family comprises EGFR (ErbB1/EGFR/HER1), ErbB2 (Her2/neu), ErbB3 (HER3), and ErbB4 (Her4). 1,2 The EGFR is activated by ligand-dependent, ligand-independent, and overexpression mechanisms. EGFR ligands include epidermal growth factor, transforming growth factor, amphiregulin, betacellulin, and epiregulin; their binding induces a conformational change in EGFR that activates its tyrosine kinase activity. EGFR can be activated by urokinase plasminogen in a ligand-independent mechanism. 3 Overexpression of EGFR by cancer cells results in ligand-independent dimerization and activation. 1 Abbreviations used in this paper: CRC, colorectal cancer; EGFR, epidermal growth factor receptor; 5-FU, 5-fluorouracil; FOLFIRI, irinotecan plus bolus and infusion fluorouracil and leucovorin; FOLFOX, leucovorin, fluorouracil, and oxaliplatin; HR, hazard ratio; IFL, irinotecan, 5-FU, and leucovorin; IL, interleukin; mab, monoclonal antibody; MAPK, mitogen-activated protein kinase; mfolfox, modified leucovorin, fluorouracil, and oxaliplatin; PI3-K, phosphatidylinositol 3-kinase; TKI, tyrosine kinase inhibitor; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; XELOX, capecitabine and oxaliplatin by the AGA Institute /10/$36.00 doi: /j.gastro

2 2164 WINDER AND LENZ GASTROENTEROLOGY Vol. 138, No. 6 Activated EGFR activates several signaling pathways, including the mitogen-activated protein kinase (MAPK) pathway, which leads to cell proliferation, survival, and, in some cases, transformation. 4 EGFR also activates the phosphatidylinositol 3-kinase (PI3K)-AKT pathway, which regulates cell survival. This pathway is negatively regulated by the tumor suppressor phosphatase and tensin homologue gene (PTEN), a phosphatase whose major substrate is phosphatidylinositol-3,4,5- triphosphate, produced by the activation of PI3-K. Loss of PTEN function results in increased phosphatidylinositol-3,4,5-triphosphate concentration and subsequent AKT hyperactivation, which protects cancer cells from various apoptotic stimuli. 5,6 EGFR also signals through the stress-activated protein kinase pathway, which includes protein kinase C and Jak and the Stat transcription factors. Activation of each of these pathways results in distinct transcriptional profiles that mediate multiple cellular responses, including apoptosis, differentiation, cellular proliferation, invasion, and DNA repair and survival (Figure 1). 7 More than 20 years ago, Kawamoto et al proposed that the EGFR be targeted for cancer therapy. 8 Two classes of anti-egfr agents have been shown to have anti-cancer activities in clinical trials. mabs Against EGFR Cetuximab Cetuximab is a recombinant, chimeric, humanmurine immunoglobulin (Ig)G1 mab that binds specifically to the extracellular domain of EGFR in normal and tumor cells, promoting receptor internalization and degradation without receptor phosphorylation and activation. 9 Several studies have shown that cetuximab inhibits growth of EGFR-expressing tumor cells in vitro and in human tumor xenografts in mice. Cetuximab reversed resistance to irinotecan in human colorectal tumor xenografts in mice. 10 This finding was followed by phase 2 trials in patients with metastatic CRC that showed cetuximab monotherapy produced notable antitumor activity (response rates of 9% 11.6%) in patients whose disease was refractory to irinotecan, oxaliplatin, and fluoropyrimidine therapies. 11,12 The phase 2 pivotal study Bowel Oncology with Cetuximab Antibody (BOND) compared the effects of cetuximab plus irinotecan with those of cetuximab alone. 13 The combination therapy was the most effective, with a response rate of 22.9% (vs 10.8% with cetuximab alone) and a median time to progression of 4.1 months (vs 1.5 months with cetuximab alone). The main toxicities reported with cetuximab in monotherapy and combination therapy were skin reactions mainly an acneiform rash in approximately 88% of patients and hypersensitivity reactions in approximately 10%. The acne-like or maculopapular rash arose because EGFR signaling maintains the integrity of the skin. Response and survival were correlated with the severity of the rash in phase 2 studies These findings led to the approval of cetuximab for irinotecan-refractory CRC in the United States and Europe. The combination of cetuximab and standard chemotherapy was tested as a first-line therapy, as well as in Figure 1. VEGF family members and receptors. The VEGF family comprises 5 VEGF glycoproteins (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E) and placenta growth factors (PlGF) 1 and 2. The best characterized of the VEGF ligands, VEGF-A, has different isoforms generated by alternate splicing. The primary effects of these ligands are mediated through binding to the VEGFR tyrosine kinases (TK) (VEGF- R1, VEGF-R2, VEGF-R3) and neuropilins 1 and 2, which function as coreceptors for VEGF binding.

3 May 2010 VEGF AND EGF AS TARGETS FOR CRC 2165 patients who were refractory to previous therapies. The phase 3 Cetuximab Combined With Irinotecan in First- Line Therapy for Metastatic Colorectal Cancer (CRYSTAL) trial investigated the effectiveness of cetuximab in combination with standard irinotecan plus bolus and infusion fluorouracil and leucovorin (FOLFIRI), compared with FOLFIRI alone, as first-line therapy for patients with metastatic CRC. The study reported a significant improvement in median progression-free survival (8.9 months for the combination of FOLFIRI and cetuximab vs 8 months for FOLFIRI alone) and in response (46.9% for the combination of FOLFIRI and cetuximab vs 38.7% for FOLFIRI alone). 16 Cetuximab has also been evaluated as a first-line treatment in combination with oxaliplatinbased chemotherapy. The phase 2 OPUS trial compared the effects of the combination of cetuximab and leucovorin, fluorouracil, and oxaliplatin (FOLFOX-4) with those of FOLFOX-4 alone; the combination therapy met the primary end point of increased response rate (45.6%) compared with FOLFOX-4 alone (35.7%). 17 Activating mutations in K-Ras, a small G protein downstream of EGFR in the MAPK signaling cascade, can affect patients response to EGFR inhibitors. A series of nonrandomized studies reported that patients with activating mutations in K-Ras had little or no response to anti-egfr (cetuximab) therapy. Lievre et al then showed that in patients with metastatic CRC, the presence of K-ras mutations predicted resistance to cetuximab therapy and was associated with a worse prognosis. 18 Results from the CRYSTAL trial have recently shown statistically significant differences between patients with wild-type K-Ras and those with mutant K-Ras in response to FOLFIRI plus cetuximab in terms of progression-free survival (9.9 vs 7.6 months) and best overall response (59% vs 36%). 19 Moreover, Van Cutsem et al showed a significant difference in overall survival for patients with wild-type K-Ras who were treated with FOLFIRI plus cetuximab versus FOLFIRI alone (23.5 months vs 20 months; P.0094). 20 Data from the OPUS trial showed that the combination of cetuximab and FOLFOX-4 has an overall response rate of 61% in patients with wild-type K-Ras compared with 33% in those with mutant K-Ras. 21 Moreover, Karapetis et al showed that patients with colorectal tumors that express mutant K-Ras did not respond to cetuximab (overall response rate of 1.2%), whereas patients with tumors that expressed wild-type K-Ras did benefit from cetuximab (overall response rate of 12.8%); this study analyzed tumor samples from 394 patients who were randomly assigned to receive cetuximab plus best supportive care or best supportive care alone. 22 Mutations in B-Raf, another component of the EGFR signaling pathway, have similar effects to those of K-Ras in patients treated with cetuximab. 23 However, the latest data on the CRYSTAL trial, presented at the 2009 annual meeting of the American Society of Clinical Oncology, did not confirm that B-Raf mutations can predict resistance to cetuximab treatment because of the limited sample size, which is due to the low B-Raf mutation frequency. 19 A prospective randomized trial is required to fully evaluate this potential marker. Nonetheless, there is a large amount of clinical data showing that K-Ras mutations affect the response of patients with metastatic CRC to the combination of cetuximab and either irinotecan- or oxaliplatin-based chemotherapies (Table 1). More markers of cetuximab response could be identified by gaining a better understanding of its molecular mechanisms. Functional polymorphisms in the FC receptors FCGR2A-H131R and FCGR3A-V158F were associated with clinical outcome in a study of 39 patients with refractory metastatic CRC treated with single-agent cetuximab. 24 This observation indicates that cetuximab might induce antibody-dependent, cell-mediated cytotoxicity. Moreover, in a retrospective phase 2 trial, Lurje et al were the first to show that the polymorphism G-765C in the gene that encodes cyclooxygenase-2 might predict outcome in patients with metastatic CRC treated with cetuximab as a single agent. 25 Larger trials of anti-egfr therapies that include biomarker analyses are needed to validate these preliminary findings. The EPIC trial was conducted to determine whether the addition of cetuximab to irinotecan prolongs survival as a second-line therapy for patients with metastatic CRC who already received fluoropyrimidine and oxaliplatin. 26 This phase 3 study showed that the combination of cetuximab and irinotecan was significantly more effective than irinotecan alone (median survival of 4 months vs 2.6 months and response rate of 16.4% vs 4.2%, respectively). Moreover, the combination therapy resulted in significantly better quality-of-life scores. Panitumumab Panitumumab is a fully human, recombinant IgG2 mab that binds specifically and with high affinity to the extracellular domain of EGFR in normal and tumor cells. Through competitive binding to EGFR ligands, panitumumab prevents EGFR dimerization, autophosphorylation, and signaling, thereby inhibiting proliferation and promoting apoptosis. 27 Panitumumab showed preliminary activity in phase 1 studies, 28 so phase 2 trials were initiated in patients with CRC. In a phase 2 study of 148 patients with metastatic CRC that progressed despite chemotherapy (fluoropyrimidine and irinotecan or oxaliplatin), patients given panitumumab as a single agent (2.5 mg/kg weekly) had a response rate of 9% and a median progression-free survival of 2.5 months. 29 The effects of panitumumab plus best supportive care were compared with those of best supportive care alone in a phase 3 study of 463 patients with metastatic CRC that was resistant to fluoropyrimidines, oxaliplatin, and irinotecan. 30 In this study, panitumumab was administered at a dosage of 6 mg/kg every 2 weeks until patients progressed or until the level of toxicity was unacceptable.

4 2166 WINDER AND LENZ GASTROENTEROLOGY Vol. 138, No. 6 Table 1. Effect of Cetuximab in Phase 2 and 3 Studies of Patients With Metastatic CRC Study Phase Drug Patients (n) Overall response, % (median duration of response, mo) Median time to progression (mo) Median overall survival (mo) Saltz et al, Cetuximab 57; refractory to 9 (4.2) irinotecan Cunningham et al, 13 3 Cetuximab 218; refractory to 22.9 (5.7) BOND study, 2004 irinotecan irinotecan Cetuximab 111; refractory to 10.8 (4.2) irinotecan Lenz et al, Cetuximab 346; refractory to irinotecan, oxaliplatin, and fluoropyrimidines 12.4 (4.2) NR 6.6 Cetuximab in combination with standard chemotherapy in first-line treatment Van Cutsem et al, 16 3 FOLFIRI NR NR CRYSTAL trial, 2007 FOLFIRI cetuximab NR NR Bokemeyer et al, 17 2 FOLFOX NR NR OPUS trial, 2007 FOLFOX-4 cetuximab 45.6 NR NR K-Ras status and efficacy of cetuximab with standard chemotherapy Karapetis et al, 22 Advanced CRC, 2008 Kohne et al, 19 first-line CRC treatment, CRYSTAL trial, 2009 Bokemeyer et al, 21 first-line CRC treatment, OPUS trial, Cetuximab BSC 81 mt K-Ras patients 1.2 HR 0.99 a wt K-Ras patients 12.8 HR 0.40 b 4.5 BSC alone 83 mt K-Ras patients wt K-Ras patients FOLFIRI cetuximab 172 K-Ras wt patients 59 HR 0.68 c 24.9 FOLFIRI cetuximab 105 K-Ras mt patients 36 HR 1.07 c FOLFOX cetuximab 61 K-Ras wt patients 61 HR 0.57 c NR FOLFOX cetuximab 52 K-Ras mt patients 33 HR 1.83 c NR NR, not reported; FOLFOX-4, oxaliplatin 85 mg/m 2 intravenously on day 1, leucovorin 200 mg/m 2 on days 1 2, 5-FU 400 mg/m 2 intravenous bolus 600 mg/m 2 infusion over 22 hours (days 1 2) every 14 days; BSC, best supportive care; mt, mutant; wt, wild-type. a HR for progression-free survival in the K-Ras mt cetuximab group as compared with the K-Ras mutant BSC group. b HR for progression-free survival in the K-Ras wt cetuximab group as compared with the K-Ras wild-type BSC group. c Progression-free survival with cetuximab was reported as an HR relative to standard chemotherapy (FOLFIRI or FOLFOX). After a medium follow-up period of at least 12 months, patients given panitumumab had a significantly higher response rate (10% vs 0% of those with best supportive care) and time of progression-free survival (8 weeks vs 7.3 weeks with best supportive care). The most common side effects were skin toxicity (90% of patients in the panitumumab group vs 9% in the best supportive care group), diarrhea (21% vs 11%), and hypomagnesemia (36% vs 1%). There were no infusion reactions. Panitumumab increases progression-free survival in patients with metastatic CRC that is resistant to chemotherapy compared with best supportive care; these results led to the approval of panitumumab for treatment of metastatic CRC in 2006 in the United States and Europe. A phase 2 study investigated the effects of panitumumab in combination with irinotecan-containing regimens as a first-line therapy for patients with metastatic CRC. In this trial, conducted by Berlin et al, the combination of panitumumab and FOLFIRI was well tolerated; the median time of progression-free survival was 10.9 months, and median overall survival time was 22.5 months. 31 As expected, given the biology of EGFR inhibitors, skin-related toxicities were common but mostly limited to grade 3. The most frequent noncutaneous toxicities were diarrhea, nail abnormalities, and hypomagnesemia, occurring in more than 20% of patients, whereas only 4% of the patients developed hypersensitivity reactions, which were serious in 1% of the patients. These findings are in contrast to those from studies of the combination of cetuximab with irinotecancontaining regimens, in which 19% of the patients developed hypersensitivity reactions, most likely because panitumumab is a humanized antibody and cetuximab is not. The phase 3 study PRIME evaluated the combination of panitumumab with FOLFOX-4 versus FOLFOX-4 alone as a first-line treatment of metastatic CRC; the combination therapy significantly improved progressionfree survival time, compared with that of FOLFOX-4 alone, in patients with wild-type K-Ras (9.6 vs 8.0 months, respectively; P.023). 32 The effects of a combination of panitumumab with FOLFIRI in second-line

5 May 2010 VEGF AND EGF AS TARGETS FOR CRC 2167 Table 2. Effect of Panitumumab in Phase 2 and 3 Studies of Patients With Metastatic CRC Study Phase Drug Patients (n) Hecht et al, Panitumumab 148; refractory to fluoropyrimidine and irinotecan or oxaliplatin Van Cutsem et al, Panitumumab BSC 463; refractory to 5-FU, irinotecan, and oxaliplatin Overall response, % (median duration of response, mo) Median time to progression (mo) Median overall survival (mo) 9 (5.2) (4.2) HR 0.54 a HR 1.0 a BSC alone 0 Panitumumab in combination with standard chemotherapy in first-line treatment Berlin et al, Panitumumab IFL b 17 Panitumumab FOLFIRI b 22.5 Douillard J et al, 32 PRIME 3 Panitumumab mt K-Ras NR 7.3 b NR trial, 2009 FOLFOX-4 wt K-Ras b A total of 1183 patients FOLFOX-4 mt K-Ras NR 8.8 b NR wt K-Ras b K-Ras status and efficacy of panitumumab with standard chemotherapy Amado et al, 34 advanced CRC, Panitumumab BSC 84 mt K-Ras 0 NR wt K-Ras 17 (5) 8.1 BSC alone 100 mt K-Ras 0 NR wt K-Ras BSC, best supportive care; FOLFOX-4 oxaliplatin 85 mg/m 2 intravenously on day 1, leucovorin 200 mg/m 2 on days 1 2, 5-FU 400 mg/m 2 intravenous bolus 600 mg/m 2 infusion over 22 hours (days 1 2) every 14 days; mt, mutant; wt, wild-type; NR, not reported. a Time to progression and overall survival with panitumumab were reported as an HR compared with BSC. b Progression-free survival. treatment of metastatic CRC is under investigation in the study (Table 2). 33 Like cetuximab, the response to panitumumab can be affected by K-Ras mutations. A phase 3 trial that compared the effects of panitumumab monotherapy with those of best supportive care in patients with metastatic CRC showed that panitumumab was only effective in patients with tumors with wild-type K-ras; response rates were 17% versus 0% in patients with mutant versus wildtype K-Ras tumors. 34 Therefore, the European Medicines Agency has approved panitumumab only for patients with metastatic colorectal tumors that have wild-type K-Ras. EGFR-Targeted Tyrosine Kinase Inhibitors Gefitinib Gefitinib (ZD1839) is an orally administered, small-molecule anilinoquinazoline that reversibly inhibits EGFR tyrosine kinase autophosphorylation and inhibits downstream signaling. 35 In phase 1 trials of patients with CRC, gefitinib was associated with prolonged stable disease but no objective responses. 36,37 Kuo et al performed a phase 2 study to investigate the effects of a gefitinib (500 mg/day, administered orally throughout the 14-day cycle), FU, leucovorin, and oxaliplatin regimen in 27 patients with progressive CRC after at least 1 chemotherapeutic regimen. 38 Thirty-six percent of patients achieved a partial response, whereas 48% had stable disease. The median overall survival time was 12 months, and the median event-free survival time was 5.4 months. However, toxicity levels were worse in patients given gefitinib compared with FOLFOX-4 alone. Overall, 48% of patients given gefitinib had severe or life-threatening neutropenia and an equal number experienced grade 3 to 4 diarrhea. Because of the increasing use of oxaliplatin in first-line treatment, Fisher et al investigated the gefitinib, FU, leucovorin, and oxaliplatin regimen as first-line therapy in patients with metastatic CRC. 39 Seventy-two percent of 43 patients who were assessable for response had either a complete or partial response; the median overall survival time was 20.5 months, and median time to progression was 9.3 months. Commonly encountered grade 3 or 4 toxicities included diarrhea in 67% and neutropenia in 60% of patients. Gefitinib does not appear to add substantial efficacy to irinotecan-based chemotherapy and shows excessive toxicities (mainly neutropenia and diarrhea). 40,41 Erlotinib Erlotinib is a small-molecule quinazolinamine that reversibly inhibits the EGFR tyrosine kinase and prevents receptor autophosphorylation. 42 Erlotinib, given in continuous doses of 150 mg/day, had no mean-

6 2168 WINDER AND LENZ GASTROENTEROLOGY Vol. 138, No. 6 ingful activity as a single agent in patients with metastatic CRC. 43 However, the combination of erlotinib and oxaliplatin and fluoropyrimidine regimens seemed promising, with a response rate of 25%. 44 Notably, the combination of erlotinib with FOLFOX and bevacizumab resulted in high incidences of toxicities (mainly rash, neuropathy, and diarrhea). 45 The combination of erlotinib and reduced doses of FOLFIRI was also investigated and resulted in unexpected severe toxicities (rash, diarrhea, and neutropenia). However, a dose-finding study of erlotinib, combined with capecitabine and irinotecan, recently identified an acceptable safety profile that will be suitable for use in future phase 2 studies. 46 Angiogenesis Angiogenesis, the development of new blood vessels from the preexisting vascular bed, is regulated by a number of factors. Proangiogenic factors include VEGFs, fibroblast growth factors, platelet-derived growth factors, insulin-like growth factor, and transforming growth factors; antiangiogenic factors include thrombospondin-1, angiostatin, and endostatin. Physiologic angiogenesis is only observed transiently, during embryogenesis, wound healing, and reproductive functions in adults. 47 Normal and pathologic angiogenic processes differ in the tightly regulated balance of proangiogenic and antiangiogenic signals. During normal physiologic angiogenesis, new vessels rapidly mature and become stable. By contrast, tumors described as wounds that never heal 48 lose the appropriate balances between positive and negative controls. 49 The neovasculature of tumors is morphologically different from that of normal tissues; for example, tumor vessels are leaky, which influences angiogenesis, tumor growth, metastasis, and also drug delivery. 50 Tumor vessels have also been reported to have cancer cells integrated into the vessel wall 51 ; some tumors promote vasculogenesis, recruiting endothelial precursor cells from the bone marrow. 52 VEGF-Dependent Regulation VEGF regulates normal and pathologic angiogenesis, activating multiple signaling networks that promote endothelial cell growth, migration, differentiation, and vascular permeability. The VEGF family of angiogenic and lymphangiogenic growth factors comprises 5 VEGF glycoproteins (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E) and placental growth factors 1 and 2. These ligands bind and signal through the VEGFRs, which are receptor tyrosine kinases, and neuropilins 1 and 2, which function as coreceptors (Figure 2). VEGFR Ligands The best characterized of the VEGF family members is VEGF-A. VEGF-A and its receptor, VEGFR-2, are key targets of antiangiogenic agents. Alternative splicing of the VEGF-A gene was initially shown to yield 4 different mature isoforms (VEGF 121, VEGF 165, VEGF 189, and VEGF 206 ). In addition, some less commonly expressed variants have also been identified (VEGF 145 and Figure 2. EGFR signaling. EGFR binding to ligands and receptor dimerization activate the Ras-MAPK, PI3K-AKT, and STAT signal transduction pathways. These signaling pathways activate transcription factors that regulate genes whose products control cell functions such as survival, proliferation, angiogenesis, invasion, and metastasis.

7 May 2010 VEGF AND EGF AS TARGETS FOR CRC 2169 VEGF 183 ). 53 VEGF 165 is the predominant isoform and is commonly overexpressed in a variety of human solid tumors. 54 VEGF primarily targets endothelial cells and is secreted by cancer cells to mediate tumor angiogenesis. 55 VEGF is up-regulated by hypoxia-inducible factor 1 and platelet-derived growth factor B and can also be released from the extracellular matrix by matrix metallopeptidase 9 to initiate an angiogenic switch that promotes tumor growth. VEGFRs VEGFRs comprise an extracellular component and an intracellular domain with a consensus tyrosine kinase sequence. VEGFR-1 and VEGFR-2 were originally discovered on vascular endothelial cells, and VEGFR-3 is primarily associated with lymphangiogenesis. 56 The VEGF family members each bind these receptors with different affinities. VEGFR-1 is a receptor for VEGF-B and placenta growth factor; during pathologic conditions, such as tumorigenesis, it is a potent, positive regulator of angiogenesis. Moreover, VEGFR-1 has an important role in hematopoiesis, recruitment of endothelial progenitors, and induction of growth factors (eg, interleukin [IL]-6, hepatocyte growth factor) in liver sinusoidal endothelial cells. 54,57 VEGFR-2 mediates most of the cellular effects of VEGF-A during angiogenesis, including microvascular permeability, endothelial cell proliferation, migration, and invasion. 54 Specific activation of VEGFR-2 by VEGF-E activates endothelial cells in vitro and in vivo. 58 VEGFR-3 binds with the highest affinities to VEGF-C and VEGF-D and mediates embryonic cardiovascular development; during development and in adults, its expression is limited to the lymphatic endothelial cells. 58 Neuropilins 1 and 2 serve as coreceptors for VEGF by increasing the binding affinity of ligands to VEGFRs. 58 VEGF-Independent Regulation of Angiogenesis VEGF-independent regulation of angiogenesis stimulates vasculogenesis and angiogenesis through a complicated network. Interactions between ligands and their receptors such as angiopoietin and tie, EphrinB2 and EphB4, and Delta and Notch mediate the angiogenic process, including vascular remodeling, maturation, and directed growth. 59 Angiopoietins bind to Tie2 to mediate a range of effects, such as tightening cell junctions to inhibit cell permeability and inflammation and activating endothelial cell migration and survival through the PI3K-AKT signaling pathway. Ang1, the first and the best-characterized angiopoietin receptor, is secreted by periendothelial cells in quiescent vasculature. 60 Ang2 decreases angiogenesis by destabilizing vessels and promoting apoptosis of vascular endothelial cells. The angiogenic molecule ephrinb2 is specifically expressed in arterial angioblasts and endothelial and perivascular mesenchymal cells, whereas its receptor, EphB4, is expressed by endothelial cells of only the venous lineage. Interactions between EphrinB2 and EphB4 at the arterial-venous interface might restrict intermingling of endothelial cells or stimulate formation of new capillary sprouts. 61 Delta activates the Notch signaling pathway, which regulates differentiation of arteries into veins and stimulates blood vessel formation. Delta-like 4 ligand (Dll4) is secreted by endothelial cells and contributes to angiogenesis by suppressing nonfunctional sprouting of blood vessels. An inhibitor for Dll4, such as a neutralizing antibody, might have antiangiogenic effects by increasing nonfunctional endothelial sprouting and branching. 62 IL-8, a member of the CXC chemokine family, also mediates tumor angiogenesis. Induction of IL-8 preserved the angiogenic response in hypoxia-inducible factor 1 deficient colon cancer cells, indicating that IL-8 mediates angiogenesis independently of VEGF signaling pathways. 63 Patients with stage III CRC who have undergone surgery and carry the IL-8 polymorphism T-251A (A/A genotype), which causes IL-8 to be highly expressed, are at higher risk for recurrence of CRC than patients without the polymorphism. 64 VEGF-Targeted mabs Bevacizumab Bevacizumab, developed in the early 1990s, is a recombinant, humanized IgG1 mab against all isoforms of VEGF-A that disrupts their interactions with VEGFRs. 65 By limiting activation of VEGFRs, bevacizumab ultimately causes the regression of microvessels, inhibits the formation of new tumor blood vessels, and normalizes the tumor vasculature. Several agents have been developed to target VEGF signaling pathways, but only bevacizumab has been approved by the US Food and Drug Administration and the European Medicines Agency for use in combination with chemotherapy in patients with advanced CRC. Administration of bevacizumab inhibited growth of human xenograft tumors and reduced the number and size of liver metastases in preclinical studies. 66 Phase 1 clinical trials showed that free serum VEGF concentrations are below the detectable limit of the assay after the administration of bevacizumab at doses of 0.3 mg/kg; VEGF remained undetectable for the duration of the study. 67 The half-life of bevacizumab ranged from 11 to 50 days, with a mean half-life of approximately 21 days. Bevacizumab in Combination With Chemotherapy The activity of bevacizumab, in combination with conventional chemotherapy, was first shown in a phase 2

8 2170 WINDER AND LENZ GASTROENTEROLOGY Vol. 138, No. 6 trial by Kabbinavar et al. They evaluated the efficacy of 2 dose levels of bevacizumab (5 and 10 mg/kg, administered every 2 weeks), administered in combination with 5-FU (500 mg/m 2 ) and leucovorin (500 mg/m 2 ), compared with that of the combination of 5-FU and leucovorin in patients with metastatic CRC. 68 Treatment with 5-FU/leucovorin/bevacizumab (at both dose levels) improved patients response rates and time to progression compared with 5-FU/leucovorin. Interestingly, the greatest improvement in response occurred in patients treated with 5 mg/kg bevacizumab compared with 10 mg/kg bevacizumab (response rates of 40% vs 24%). The most common side effects were thrombosis, hypertension, epistaxis, headache, fever, and rash; most were either manageable (proteinuria and hypertension) or clinically insignificant (epistaxis, headache, fever, and rash). In a 2004 phase 3 trial of bevacizumab in patients with untreated metastatic CRC, called AVF2107 and performed by Hurwitz et al, 69 patients were given a combination of irinotecan, 5-FU, leucovorin (IFL), and placebo or a combination of IFL and bevacizumab (5 mg/kg biweekly). Patients who received IFL plus bevacizumab had a significant improvement in overall survival (20.3 vs 15.6 months) and progression-free survival (10.6 vs 6.2 months). Moreover, patients treated with IFL and bevacizumab showed a significant improvement in response rate (45% vs 35%), with a median duration of response of 10.4 vs 7.1 months, indicating that the primary mechanism of bevacizumab is inhibition of tumor growth rather than cytoreduction. This clinical benefit was associated with relatively modest increases in side effects; grade 3 hypertension was more common in patients who received IFL plus bevacizumab than IFL plus placebo (11% vs 2.3%) but was managed with standard oral antihypertensive drugs. One uncommon, potential adverse effect from bevacizumab was the appearance of gastrointestinal perforations (1.5%). Different toxicities observed in phase 2 and 3 trials of bevacizumab make it difficult to determine its role in these adverse events, especially the development of vascular events. Hurwitz et al 69 observed only an insignificant increase in thrombotic or bleeding events, whereas in the phase 2 trial by Kabbinavar et al, 68 thrombosis (26%) and bleeding episodes (46%), even if mild (grade 1 or 2), were common side effects of treatment with bevacizumab. Patients in these trials who received the combination of IFL and bevacizumab had a significantly higher incidence of impaired wound healing (15%) compared with patients who received only the FU regimens without bevacizumab. 68,69 Scappaticci et al 70 reported that in patients given a combination of bevacizumab, 5-FU, and leucovorin, wound healing did not increase if bevacizumab was administered 28 to 60 days after surgery. AVF2107 (Hurwitz et al) established bevacizumab, in combination with IFL, as the standard first-line therapy for patients with advanced or metastatic CRC. Despite the lack of evidence from phase 3 trials, the combination of FOLFOX and bevacizumab has consistently been used as first-line treatment for advanced or metastatic CRC. Another phase 3 trial, NO16966, by Saltz et al evaluated the effects of the combination of capecitabine and oxaliplatin (XELOX) with those of FOLFOX, with or without bevacizumab. 71 Although the addition of bevacizumab to oxaliplatin-based chemotherapy in the NO16966 trial by Saltz et al significantly improved length of progressionfree survival, the additional benefit was much smaller than achieved in the study of Hurwitz et al (compared IFL with or without bevacizumab; 9.4 months in the NO16966 trial vs 10.6 months in the AVF2107 trial). Subset analysis of the results of the individual chemotherapies, with or without bevacizumab, showed that the gain in progression-free survival time among patients given bevacizumab was limited to those who received XELOX (hazard ratio [HR], 0.77; P.0026). In contrast, the increase in progression-free survival time among patients treated with FOLFOX-4 was not statistically significant (HR, 0.89; P.187). 71 The reason for this difference remains unclear but might have occurred because many patients discontinued XELOX treatment because of adverse events, such as neurotoxicity from oxaliplatin exposure. Consequently, the median overall duration of treatment with bevacizumab in the NO16966 trial was much shorter than in the AVF2107 trial (approximately 6 months vs approximately 10 months). 47 The 3 Regimens of Eloxatin Evaluation (TREE) study, by Hochster et al, investigated the tolerability of oxaliplatin in combination with 3 different fluoropyrimidine regimens (continuous infusion, bolus, or oral) with (TREE-2 cohort) or without (TREE-1 cohort) bevacizumab as a first-line therapy for metastatic CRC. 72 The median overall survival time of patients given FOLFOX-6 plus bevacizumab was 26.1 months, compared with 19.2 months among patients given FOLFOX-6 without bevacizumab; this is a greater extension of overall survival time than any other prospective randomized trial performed with patients with metastatic CRC. The trial Bevacizumab Regimens: Investigation of Treatment Effects and Safety (BRiTE) was the first to show that continuation of bevacizumab therapy, beyond progression, benefited patients with metastatic CRC. The median overall survival time of patients who received bevacizumab beyond progression increased compared with those who received postprogression treatment without bevacizumab (median overall survival of 31.8 vs 19.9 months, respectively). 73 However, the patients were not randomly assigned to the treatment groups, so confounding factors could have contributed to the outcome. Therefore, the National Surgical Adjuvant Breast and Bowel Project C-08 trial was initiated to test the potential benefit and safety of 6 months of FOLFOX or 6 months of FOLFOX with 12 months of bevacizumab therapy

9 May 2010 VEGF AND EGF AS TARGETS FOR CRC 2171 Table 3. Effect of Bevacizumab in Phase 2 and 3 Studies of Patients With Metastatic CRC Study Treatment Phase Regimen Kabbinavar et al, Hurwitz et al, 69 AVF2107 trial, 2004 Saltz et al, 71 NO16966 trial, 2008 Hochster et al, 72 TREE-1 study, 2008 Patients (n) Overall response (%) Time to progression (mo) Overall survival (mo) First-line 2 5-FU leucovorin Bevacizumab 5 mg/kg Bevacizumab 10 mg/kg Placebo First-line 3 IFL bevacizumab 5 mg/kg Placebo a a 15.6 First-line 3 Placebo FOLFOX-4 or a 19.9 XELOX Bevacizumab FOLFOX-4 or a 21.3 XELOX First-line 3 mfolfox bfol XELOX mfolfox-6 bevacizumab b TREE-2 study First-line 3 bfol bevacizumab b XELOX bevacizumab c Giantonio et al, 76 ECOG E3200, Second-line 3 FOLFOX-4 Bevacizumab 10 mg/kg a Placebo a 10.8 Bevacizumab 10 mg/kg alone a 10.2 Bevacizumab beyond progression Grothey et al, 73 BRiTE study, 2008 Wolmark et al, 75 NSABP C-08 trial, 2009 No post-pd treatment 253 NR NR 12.6 No BBP treatment 531 NR NR 19.9 BBP treatment 642 NR NR 31.8 mfolfox-6 bevacizumab 1334 NR HR 0.89 d NR molfox-6 alone 1338 NR p.15 NR FOLFOX-4 oxaliplatin 85 mg/m 2 intravenously on day 1, leucovorin 200 mg/m 2 on days 1 2, 5-FU 400 mg/m 2 intravenous bolus 600 mg/m 2 infusion over 22 hours (days 1 2) every 14 days; mfolfox-6 oxaliplatin 85 mg/m 2 intravenously on day 1, leucovorin 400 mg/m 2 intravenously on day 1, 5-FU 400 mg/m 2 intravenous bolus on day 1, and 5-FU 2400 mg/m 2 CI over 46 hours (days 1 2) every 14 days 12 times; bfol bolus fluorouracil and low-dose leucovorin with oxaliplatin; BBP, bevacizumab beyond first progression; ECOG, Eastern Cooperative Oncology Group; NR not reported; NSABP, National Surgical Adjuvant Breast and Bowel Project; post-pd, post-disease progression. a Progression-free survival. b Bevacizumab 5 mg/kg every 2 weeks. c Bevacizumab 7.5 mg/kg every 3 weeks. d HR for disease-free survival in mfolfox-6 bevacizumab group as compared with mfolfox-6 alone group. (beginning concurrently) in patients with stages 2 and 3 CRC who were randomly assigned to treatment groups. Bevacizumab was well tolerated as a surgical adjuvant in a selected population of patients eligible for the National Surgical Adjuvant Breast and Bowel Project C-08 trial. 74 However, the addition of bevacizumab to FOLFOX did not significantly prolong disease-free survival. 75 As of September 2009, bevacizumab should not be integrated into adjuvant therapy regimens outside of clinical trials. The addition of bevacizumab (10 mg/kg) to secondline chemotherapy with FOLFOX-4 after progression on a chemotherapy regimen without bevacizumab was evaluated in the Eastern Cooperative Oncology Group E3200 phase 3 trial. 76 A bevacizumab-only arm was originally included in the trial design, but an interim analysis determined that efficacy in this arm was less efficacious than the other 2 arms. Therefore, accrual was discontinued. The addition of bevacizumab to FOLFOX-4 improved progression-free and overall survival and response rates in patients whose CRC had already progressed on irinotecan-containing chemotherapy; these findings led to the approval of bevacizumab in combination with chemotherapy as second-line therapy for CRC. It is not clear why the same chemotherapy regimen (FOLFOX-4) did not show any benefit in first-line therapy; 71 it could have arisen from the doubled dose (10 mg/kg) of bevacizumab administered in the second-line (Eastern Cooperative Oncology Group 3200) trial (Table 3). VEGFR Tyrosine Kinase Inhibitors Vatalanib Vatalanib (PTK787/ZK222584) is a tyrosine kinase inhibitor (TKI) that blocks the function of VEGFR-1, VEGFR-2, and VEGFR-3, preventing VEGFmediated angiogenesis to reduce tumor growth and metastasis. 77 The combination of vatalanib (daily doses up to 1250 mg) and FOLFOX-4 was well tolerated as a

10 2172 WINDER AND LENZ GASTROENTEROLOGY Vol. 138, No. 6 Table 4. Ongoing Clinical Trials in Metastatic CRC Clinical trial Phase Randomization CALGB-C FOLFOX/FOLFIRI cetuximab and/or bevacizumab in wildtype K-Ras patients VELOUR 2 FOLFIRI aflibercept (AVE0005) or placebo AFFIRM 2 FOLFOX6 aflibercept (AVE0005) or placebo PETACC-8 3 FOLFOX cetuximab (adjuvant treatment of fully resected stage 3 CRC) first-line treatment for metastatic CRC in a phase 1b study. The regimen produced overall responses in 48.6% of patients, with a median progression-free survival time of 11.4 months. 78 The drug combination increased only slightly the incidence of diarrhea and the hematologic toxicities compared with FOLFOX-4 alone. Despite encouraging results in phase 1/2 trials, the results from the phase 3 study of colorectal oral novel therapy for the inhibition of angiogenesis and retarding of metastases (CONFIRM-1) showed that the addition of vatalanib to FOLFOX-4 did not improve progression-free survival, compared with FOLFOX-4 alone, as a first-line treatment for metastatic CRC (7.7 vs 7.6 months; P.118) or relative risk (42% vs 46%, respectively). 79 However, a preplanned meta-analysis from this study and a subsequent study (CONFIRM-2; similar design, but a second-line treatment from metastatic CRC) showed that vatalanib specifically benefits patients with high levels of lactate dehydrogenase. 80 These results are in contrast to the clinical benefit observed from bevacizumab in patients with the same tumor type. One possible explanation for these results is that vatalanib has a shorter half-life (4 6 hours) than bevacizumab; the once-daily schedule might have been insufficient to inhibit the enzyme for 24 hours. Aflibercept Aflibercept (VEGF-Trap) is a fully recombinant, decoy fusion protein of the second Ig domain of VEGFR-1 and the third Ig domain of VEGFR-2 fused to the Fc domain of human IgG1. VEGF-Trap binds all isoforms of VEGF-A, placenta growth factor, and VEGF-B with high affinities and prevents them from stimulating angiogenesis. 81 This agent has shown significant activity in preclinical models as well as in early-stage clinical trials. 82 Multiple clinical trials are currently under way to determine the clinical benefits of aflibercept in patients with prostate, non small cell lung, colorectal, ovarian, and pancreatic cancers (Table 4). Sunitinib Sunitinib is an oral, multi-target TKI of VEGFR, platelet-derived growth factor receptor, c-kit, RET, and flt3. Two dose finding, open-label, phase 1 trials were designed to determine the maximum tolerated dose, safety, and pharmacokinetic profiles of sunitinib, in combination with modified FOLFOX-6 (mfolfox-6) or FOLFIRI, in patients with advanced tumors. Preliminary results indicated that sunitinib (50 mg/day, given 2 weeks on and 2 weeks off) given in combination with mfolfox-6 (given every 2 weeks with 37.5 mg sunitinib per day, given on a 4 weeks on/2 weeks off schedule with FOLFIRI) is safe and well tolerated. 83,84 Combination Targeted Therapies The availability of a variety of different targeted therapies provides an opportunity to combine agents and improve anti-tumor activity. In solid tumors, the VEGF and EGFR pathways seem to be linked, particularly with respect to angiogenesis. Epidermal growth factor and transforming growth factor both induce VEGF expression in cancer cells via activation of EGFR, and tumorassociated endothelial cells express EGFR. 85 It is likely that the EGFR pathway modulates angiogenesis by upregulating VEGF or other key mediators in the angiogenic process. 86 Preclinical models showed that EGFR blockade with the mab cetuximab down-regulated proangiogenic factors, including VEGF and IL Once the therapeutic efficacies of cetuximab and bevacizumab were shown, the safety and efficacy of concurrent administration of these 2 agents, with or without irinotecan, were explored in a phase 2 study of patients with metastatic CRC (the BOND-2 study). 88 The combination of bevacizumab and cetuximab produced a partial response in 20% of patients and a median time to progression of 4.9 months. Toxicities observed were as would have been expected from the single agents. This trial provided further evidence that bevacizumab increases the therapeutic efficacy of other therapeutic regimens for metastatic CRC. The first phase 3 trial to evaluate the combination of mabs against EGFR and VEGF was the Panitumumab in Advanced Colorectal Cancer Evaluation (PACCE) trial. 89 Patients were treated with FOLFOX or FOLFIRI (physicians choice) plus bevacizumab and then randomly assigned to groups that were or were not given concurrent panitumumab. The combination therapies were not found to be more effective than chemotherapy alone, with no differences reported in response rates, progression-free survival times, or overall survival. The recently reported phase 3 trial CAIRO-2 reported similar results for the combination of capecitabine, oxaliplatin, and bevacizumab, with or without cetuximab. 90 Therefore, neither panitumumab nor cetuximab plus bevacizumab in combination with chemotherapy should be given to patients with CRC outside of clinical trials. Discussion In the past 10 years, we have witnessed a radical change in the use of systemic therapy for CRC, with the

11 May 2010 VEGF AND EGF AS TARGETS FOR CRC 2173 introduction of 3 cytotoxic therapies and 3 mabs. The availability of sequential therapy has created new challenges in determining an actual overall survival benefit for any new agents or regimens. Despite this exciting era, some questions associated with these new treatment options need to be resolved. For instance, why is EGFR mab therapy in patients with K-Ras wild-type CRC effective while EGFR-targeted TKIs as downstream inhibitors of the EGFR are ineffective? It is important to note that although EGFR mabs and TKIs intend to inhibit the same target, they mediate their effects by different mechanisms. EGFR mabs disrupt the interaction between ligands and receptors whereas TKIs are ligand independent. EGFR kinase domain mutations that constitutively activate the EGFR kinase (ligand independent) may confer sensitivity to EGFR TKIs and resistance to EGFR mabs. 91 The discovery and characterization of EGFR-activating mutations and their relationship to sensitivity to EGFR TKIs have increased response rates to EGFR TKI therapy from less than 10% to 60% 80% in patients with non small cell lung cancer. 92,93 Notably, these mutations exist in non small cell lung cancer but are absent in CRC. 94 On the contrary, most mabs are developed to inhibit ligand binding and receptor activation. In addition, some mabs (eg, cetuximab) can induce antibody-dependent cellmediated toxicity in preclinical models, which is difficult to prove clinically. Another controversial discussed question is why bevacizumab given as a single agent was inactive in protocol E3200, which was conducted by the Eastern Cooperative Oncology Group. Bevacizumab exerts its function through a number of potential mechanisms, including inhibition of new vessel growth, regression of newly formed tumor vasculature, alteration of vascular function and tumor blood flow, and direct effects on tumor cells. The proposed mechanisms of action suggest that bevacizumab has primarily antiangiogenic effects and not cytotoxic effects. The fact that bevacizumab, given as a single agent, in protocol E3200 was ineffective may support the hypothesis of normalization of tumor vasculature: specifically, vasoconstriction to transiently improve flow dynamics and pruning of immature, functionally abnormal vessel sprouts. Therefore, the delivery of cytotoxic agents might be improved, with subsequent increases in the efficacy of chemotherapy. 95 Moreover, anti-vegf therapy changes vascular function. Blocking VEGF signaling limits the secretion of vasodilators, which has been suggested to change tumor blood flow, but this is still controversial. 96 Finally, how can we explain the observation in the PACCE and CAIRO-2 studies that the use of chemotherapy plus bevacizumab with an anti-egfr antibody in first-line treatment of metastatic CRC resulted in an inferior outcome? An explanation could be signaling pathway interactions caused by the different drug combinations. This is supported by results from the CAIRO-2 study, in which the addition of cetuximab resulted in a significantly reduced incidence of severe hypertension (9.3% vs 14.8%). 16 Because hypertension is a known side effect of bevacizumab, which has been correlated with antitumor efficacy, 97 this suggests an inhibitory effect of cetuximab on the mechanism of action of bevacizumab. Another explanation why the combination of bevacizumab with an anti-egfr antibody is detrimental in first-line therapy but has shown activity in second- and third-line therapy might be a different genetic makeup in first-line tumors than in third-line tumors. The optimal use of targeted therapy will depend on recognizing the mechanism of action of each targeted agent. Moreover, we still have insufficient data on the interaction of targeted agents and chemotherapy. Understanding resistance mechanisms remains the major challenge in the treatment of patients with CRC. Future Directions With the development of new, effective anticancer drugs and the integration of novel, targeted therapies, it will be important to gain a better understanding of how these agents are metabolized and mechanisms of resistance. Better markers of efficacy are needed for anti- EGFR and anti-vegf agents, which would allow oncologists to individualize therapeutic strategies by maximizing efficacy and minimizing side effects. K-Ras mutation status is the only marker available to predict response to anti-egfr therapeutics; it is a challenge to identify and implement new biomarkers of prognosis or those that predict response to therapies into routine clinical practice. Future studies should seek to identify new markers of disease progression and response to therapy, optimize treatment durations for targeted therapeutics, and identify effective combination therapies. References 1. Mendelsohn J, Baselga J. Status of epidermal growth factor receptor antagonists in the biology and treatment of cancer. J Clin Oncol 2003;21: Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2001;2: Liu D, Aguirre Ghiso J, Estrada Y, et al. 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