Exploiting molecular targets in pancreatic cancer

Hematol Oncol Clin N Am 16 (2002) 139 – 157

Exploiting molecular targets in pancreatic cancer Robert A. Wolff, MD Department of Gastrointestinal Medical Oncology, University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 426, Houston, TX 77030-4095, USA

Although there have been some encouraging results with the use of gemcitabine and other conventional cytotoxic agents in pancreatic cancer, median survival for patients with metastatic disease remains dismal [19,68, 87,90]. For patients who obtain objective tumor regression, few enjoy a durable response to chemotherapy. Pancreatic cancer is a disease associated with significant morbidity. In this setting, the identification of therapies with potential for less toxicity is desirable. Because pancreatic cancer has shown exceptional resistance to conventional chemotherapy, which may be associated with significant toxicity, this disease readily lends itself to the investigation of novel biologic agents. These are drugs that have been designed intentionally to interrupt or modulate known signal transduction pathways and other events involved in cell growth, invasion, and angiogenesis. Several of these agents are being tested for therapeutic activity, and some have produced promising preliminary results. Molecular targeting no longer is theoretical. There are exciting reports of clinically meaningful activity with a drug designed specifically to inhibit a known molecular defect. In chronic myelogenous leukemia, a single chromosomal translocation seems sufficient for the development of the malignant phenotype. This translocation causes the transcription of the bcr/abl fusion protein with constitutive tyrosine kinase activity. Gleevec (imatinib mesylate), a newly approved tyrosine kinase inhibitor, has been proved to inhibit the kinase activity of this fusion protein and lead to complete hematologic remission in chronic myelogenous leukemia without the administration of other chemotherapy [31]. This example is compelling evidence that previously

E-mail address: [email protected] (R.A. Wolff ). 0889-8588/02/$ - see front matter D 2002, Elsevier Science (USA). All rights reserved. PII: S 0 8 8 9 - 8 5 8 8 ( 0 1 ) 0 0 0 1 2 - 0

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resistant malignancies may be controlled with designer drugs without reliance on conventional cytotoxic agents.

Clinical development of signal transduction inhibitors and other agents The molecular events underlying pancreatic carcinogenesis, invasion, and metastasis have been described elsewhere in this issue and are not discussed here. In general, these events are not unique to this disease. Many of the agents currently being tested in pancreatic cancer also have been tested in other tumor types, particularly breast, colon, and non-small cell lung cancers. As a result, the bulk of clinical data currently available is based on investigations in these tumor types. Although many novel compounds are being tested extensively in pancreatic cancer, most reports of their use are preliminary and published only in abstract form. With an emerging understanding of these important signal transduction cascades, cell cycle events, and factors conferring metastatic potential, rational targeted treatments are being designed and delivered (Table 1). These treatments may be directed specifically at the tumor cell or more broadly at the host environment to impede cancer cell invasion and dissemination. Tumorspecific targeted therapy may be directed at the cell surface to inhibit extracellular growth-stimulating signals, at activated signal transduction pathways within the tumor cells, or at nuclear events controlling the cell cycle and apoptosis. Table 1 Potential molecular targets and specific agents being clinically tested Molecular target

Location

Agent

Mechanism of agent

HER 2

Cell membrane

EGFR

Cell membrane

Receptor tyrosine kinase inhibitor Receptor tyrosine kinase inhibitors

ras oncoprotein

Cell membrane (cytoplasmic portion)

Trastuzumab (Herceptin) Cetuxumab (C225) ZD 1839 (Iressa) OSI-774 SCH-66336 R-115777 BMS-214662

p53

Nucleus

Ad-p53 Onyx-015

mTOR

Cytoplasm

CCI-779

Matrix metalloproteinase inhibitors

Extracellular matrix

Marimastat

VEG-F

Endothelium

SU5416 SU6668

Angiogenesis inhibitors

Endothelium

Endostatin Angostatin

Farnesyl transferase inhibitors (prevent membrane localization of ras oncoprotein) Apoptosis of cells with mutant p53 Viral lysis of cells with mutant p53 Translational inhibitor/cell cycle arrest Inhibit extracellular matrix, basement membrane degradation VEG-F receptor tyrosine kinase inhibitors Inhibit endothelial proliferation and migration

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Treatments directed at tumor cells Targeting receptor tyrosine kinases Pancreatic cancer cells have a variety of cellular defects promoting uncontrolled growth. Transduction of extracellular growth-simulating signals to the tumor cell’s nucleus is one. At present, two members of the HER family of receptor tyrosine kinases have been studied intensively: HER2 and the epidermal growth factor receptor (EGF-R) [77]. These receptors have considerable homology and transduce signals arriving at the cell surface. Binding of growthstimulatory ligands from the extracellular environment leads to receptor tyrosine kinase activation. The downstream signal is mediated through the Raf-kinase/ mitogen-activated protein (MAP) kinase pathway [59]. HER2 and EGF-R may be overexpressed in a variety of solid tumor types leading to an augmentation of proliferative signals. In pancreatic cancer, EGF-R has been found to be involved in autocrine and paracrine loops that promote tumor cell proliferation. Two notable ligands for this receptor are EGF, and transforming growth factor (TGF)-a. In patients with pancreatic carcinomas, overexpression of EGF-R and either TGF-a or EGF in tumors is associated with an aggressive phenotype and portends poor survival [114]. The ligand for HER2 has yet to be identified, but signaling through this receptor appears to have similarities to EGF-R. One approach taken to inhibit the stimulatory signals transduced through these receptors has been the development of monoclonal antibodies that bind to the extracellular component of the receptor, inhibiting receptor activation [39]. To date, these have been murine monoclonal antibodies humanized to prevent immune degradation. Binding of antibody to receptor has been specific but requires sufficient expression of the receptor to cause inhibition by circulating antibody. Two antibodies are being investigated for activity in pancreatic cancer, trastuzumab (Herceptin) and cetuxumab (IM-C225). Monoclonal antibody to HER2: trastuzumab Trastuzumab is a humanized monoclonal antibody that specifically binds HER2 [27]. Preclinical studies have shown that trastuzumab leads to growth inhibition in cell lines overexpressing this receptor. In human tumor specimens, HER2 is overexpressed in many solid tumors, including breast, ovarian, lung, papillary thyroid cancers, gastric, colonic, and pancreatic carcinomas [18,33, 54,74,96,102,109]. It also has been found in a subset of patients with poorly differentiated carcinomas and adenocarcinomas of unknown primary site [52]. The bulk of clinical investigation with this antibody has focused on the treatment of advanced breast cancer. In patients with metastatic breast cancer whose tumors overexpressed HER2, trastuzumab as a single agent led to objective response rates ranging from 11% to 23% [12,111]. When combined with other chemotherapeutic agents, trastuzumab seems to be synergistic. In a phase III trial, response rate and survival were improved in women receiving the combination of

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trastuzumab and paclitaxel compared with women receiving paclitaxel alone [101]; all patients had metastatic breast cancer with overexpression of HER2. Trastuzumab is delivered intravenously weekly, using an initial loading dose followed by a weekly maintenance dose. Toxicity generally has been minimal with the exception of cardiotoxicity, which is most likely to occur in patients treated previously with an anthracycline or who receive trastuzumab concomitantly with an anthracycline [38,104]. The drug has led to death in a subset of women with metastatic breast cancer who were pretreated heavily before exposure to trastuzumab, although the reasons for this are elusive. Trastuzumab has been evaluated in preclinical models of pancreatic cancer using in vitro and in vivo systems and has shown antitumor activity in this setting [18]. Trastuzumab now is being investigated in combination with gemcitabine in advanced pancreatic cancer [94]. This study is limited to patients whose tumors overexpress HER2 based on immunohistochemical staining techniques. In this trial, HER2 overexpression is present in approximately 20% of tumor specimens tested. The treatment regimen consists of gemcitabine given at a dose of 1000 mg/m2 weekly and trastuzumab given as a loading dose of 4 mg/kg and weekly thereafter at a maintenance dose of 2 mg/kg. In 18 evaluable patients, the response rate to treatment with gemcitabine and trastuzumab has been 22%. So far, toxicity has been acceptable; no cardiotoxicity has been observed in this group. Targeting epidermal growth factor receptor: cetuximab (C225) Antibodies to EGF-R have been shown to compete with the growthstimulatory ligands (EGF and tumor necrosis factor-a) for binding to this receptor [39]. The humanized monoclonal antibody to EGF-R, cetuximab (C225), has shown potent competitive binding to the receptor, leading to growth inhibition [89]. C225 inhibits the growth of human pancreatic cancer xenografts in nude mice [84]. In vitro studies suggest an additive effect of C225 with cytotoxic agents [76]. Data from laboratories at M. D. Anderson Cancer Center also suggest a synergistic interaction in animal models of metastasis when gemcitabine and C225 are combined, with evidence suggesting an antiangiogenic effect [17]. Early clinical trials of this drug were performed in patients with tumors of the head and neck and non-small cell lung cancers [11,99]. In one study, pretreatment and posttreatment biopsy specimens showed that administering C225 at a loading dose of 400 mg/m2 and a maintenance dose of 250 mg/m2/wk thereafter achieved saturation of EFG-R in tumor tissue [99]. As a single agent, C225 has shown the ability to stabilize disease in patients with previously progressing disease [11]. Although experience with C225 in gastrointestinal tumors is limited, it has shown activity in advanced colon cancer resistant to cytotoxic therapy. In a small study of patients who either were progressing or had stable disease while receiving irinotecan, the addition of C225 to irinotecan led to an objective response rate of 22.5% [95]. This result has led to further interest in C225 for other gastrointestinal tumors, including pancreatic cancer. A phase II trial

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combining gemcitabine with C225 was initiated in 2000 and has been reported in abstract form [1]. The objective response rate among 44 enrolled patients was 12%, not particularly better than the response rate expected with gemcitabine alone, but occasionally these responses were impressive (Fig. 1). Many of the study participants enjoyed a relatively prolonged stable phase, with 32% of

Fig. 1. Pre- (A) and post- (B) therapy CT images of a 54-year-old man with metastatic pancreatic cancer. The patient received 7 weekly injections of gemcitabine and C225 with significant response to therapy.

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patients surviving more than 1 year. One patient was treated with the combination for 17 months (JL Abbruzzese: personal communication, 2001). The toxicity of this combination is not significantly worse than that reported for single-agent gemcitabine except for an acneiform follicular eruption that frequently is induced by C225. The rash appears to be a manifestation of a superficial dermal inflammatory cell infiltrate and a suppurative superficial folliculitis [20]. Small molecule inhibitors of receptor tyrosine kinase activity Another approach to inhibit the HER family members uses small molecules to bind the tyrosine kinase domain located on the cytoplasmic portion of the cell membrane. Two agents currently under investigation are ZD1839 (Iressa) and OSI-774 (Tarceva). Iressa is an oral anilinoquinazoline analogue that can inhibit EGF-R potently and selectively [9]. It has cytotoxic activity in vitro when used alone or in combination with conventional chemotherapy [25,26,100]. Iressa is proceeding through early clinical testing, with some promising preliminary results [10]. Tarceva is another small molecular inhibitor of EGF-R. This drug also is orally bioavailable and is entering clinical trials [57]. A phase III trial of this agent is being planned for chemotherapy-naive patients with advanced pancreatic cancer. Patients will be randomized to receive gemcitabine alone or gemcitabine in combination with Tarceva. Targeting intracellular signal transduction pathways: the ras oncoprotein Mutation of the K-ras oncogene is one of the most frequent and early events in pancreatic oncogenesis [5,24]. The Ras oncoprotein is one of several guanosine triphosphate (GTP)-binding proteins that are part of the intracellular machinery necessary to tranduce extracellular signals [22]. The wild-type protein normally cycles between an inactive guanosine diphosphate (GDP)-bound form and an active GTP-bound form. The mutant Ras protein binds GTP, which cannot be hydrolyzed to the inactive GDP-bound form, and the mutation maintains the protein in its activated form. Activation leads to downstream proliferative signals. In pancreatic adenocarcinoma, the frequency of mutation probably approaches 90% [72]. Inhibition of Ras signaling long has been proposed as a target for therapy given its high mutational frequency and activation in pancreatic cancer and other solid tumors [2]. A common strategy to abrogate Ras protein function involves interruption of the posttranslational farnesylation of the protein [93]. This interruption is required for proper localization of the protein to the cytoplasmic side of the plasma membrane. Several specific small molecules have been identified and synthesized that inhibit farnesyl transferase activity necessary for localization to the plasma membrane [47,48]. As a consequence, the Ras oncoprotein is rendered functionally inactive. Preclinical studies of these drugs have shown cytotoxic activity in a variety of cell lines [36]. Many farnesyl transferase inhibitors (FTIs) currently are being tested clinically, including SCH 66336, R115777, and BMS 214662. These

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agents all are orally bioavailable. BMS 214662 also may be administered intravenously. Phase I clinical testing has been completed for these agents [3,21,35,65,117]. Gastrointestinal toxicity and fatigue are common; hematologic toxicity, particularly neutropenia, also has been observed. Some of these compounds are undergoing further evaluation in combination with conventional cytotoxic chemotherapy [4,8,86]. Early reports of activity in pancreatic cancer have started to appear in abstract form. In a randomized phase II trial, SCH 66336 was compared with gemcitabine in patients with advanced pancreatic cancer. Patients received either gemcitabine at a dose of 1000 mg/m2/wk for 7 weeks followed by infusions weekly for 3 weeks or SCH 66336 at a dose of 200 mg twice daily. Two of 33 patients treated with SCH 66336 had a partial response (6%), which compared favorably with the response to single-agent gemcitabine, which in this trial had a response rate of 3% [71]. The FTIs also prevent the modification of other proteins. The antiproliferative effects of the FTIs may not be explained by the presence of a constitutively active Ras oncoprotein but of other signaling proteins. This idea is supported by the finding that FTIs having growth-inhibitory effects in pancreatic cancer cell lines with wild-type Ras proteins [103]. Another potential approach to inhibit Ras activity relies on antisense technology. This strategy has been employed successfully in lung adenocarcinoma cells using retroviral constructs coding for K-ras antisense RNA [79]. Evolving ribozyme technologies offer the possibility of improved inhibition compared with traditional antisense approaches [40]. These approaches have not yet been tested in the clinical setting. Nuclear targets: cell cycle regulation and apoptosis The molecular machinery required for cell cycle regulation involves a multitude of tightly controlled nuclear transcription factors, enzymatic proteins involved in regulating cell cycle progression, and inhibitory factors acting at check points in the cell cycle. This complex system of checks and balances is intertwined with factors responding to cellular injury and DNA damage normally resulting in apoptosis. It now is evident that dysregulation of these apoptotic pathways and cell cycle check points is integral to uncontrolled cell growth and resistance to cytotoxic therapy. Central functions for p53, NF-kB, and cyclin D now are being implicated as important in resistance to chemotherapy and ionizing radiation [7,66,73,112]. Agents intended to target these three factors are being developed. To date, however, only the tumor-suppressor gene p53 has been exploited successfully in the clinic. Strategies to exploit p53 Mutation of p53 is common in pancreatic cancer, being detected in at least 50% of these tumors [28,30]. Two approaches have been developed that take advantage of the presence of mutant p53 within the tumor cells. One strategy is to restore the normal function of the gene, resulting in apoptosis, and the other

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strategy targets cells with mutated p53 for destruction through viral replication and lysis. Restoration of wild-type p53 function has been accomplished in tumor cells using an adenoviral vector with wild-type p53 complementary DNA (Ad-p53). In preclinical models, reintroduction of normal p53 function into cells has been shown to be an effective way to alter cellular growth, induce apoptosis, and sensitize cells to chemotherapy and radiation [13,61]. Phase I testing of Adp53 was carried out in 21 patients with non– small cell lung cancer by direct intratumoral injection [108]. Doses used ranged from 106 to 1011 plaque-forming units. Adenovirus vector DNA was detected in 86% of posttreatment biopsy specimens, and vector-specific p53 mRNA was found in 42%. Evidence for increased apoptosis was present in about half of the posttreatment biopsy specimens. Objective clinical response was shown in 2 patients (8%), with stable disease lasting 2 to 14 months in 64% of patients. An alternative strategy to exploit p53 uses adenoviruses genetically modified to replicate only in p53-deficient cells. This approach relies on replicating virus to lyse only cells deficient in wild-type p53 [75]. The attenuated adenovirus, ONXY-015, has cytotoxic activity in preclinical tumor models and has been used clinically in the treatment of recurrent tumors of the head and neck [45,64,81,116]. In pancreatic cancer, delivering adenovirus as an intratumoral injection has been undertaken using direct injection under visual guidance and by computed tomography and endoscopic ultrasound guidance [53,80]. This approach has been proved to be feasible, but objective tumor regression has not yet been established firmly in pancreatic cancer. It seems that the current technology has some ability to control disease in a locoregional fashion in non-small cell lung cancer and in cancers of the head and neck. p53-targeting strategies for pancreatic cancer likely will require development of novel delivery techniques, however, capable of targeting the local and metastatic components of this neoplasm. Mammalian target of rapamycin Another interesting and potentially relevant signal transduction cascade involves the mammalian target of rapamycin (mTOR). Rapamycin is a macrolide fungicide that has antimicrobial, antiproliferative, and immunosuppressant activity. When rapamycin binds to the immunophilin FK506 binding protein 12 (FKBP12), it forms a complex that inhibits the protein kinase activity of mTOR; this is downstream to signal transduction pathways through phosphoinositide-3 kinase (P3k) and protein kinase B (Akt). Inhibition of mTOR’s protein kinase activity in turn impedes the translation of specific mRNAs required for cell cycle progression from G1 to S phase [6,56]. This activity has generated interest in the use of rapamycin analogues to inhibit cell proliferation [32,88]. Such a strategy seems to be relevant to pancreatic cancer. In vitro, rapamycin induces a marked decrease in cyclin D1 levels in pancreatic cancer cell lines and an ester analogue of rapamycin [50]. The rapamycin analogue, CCI-779, has been evaluated in phase I studies, and efficacy trials of this agent are under way, including trials in pancreatic cancer [56].

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Molecular strategies aimed at the host environment Historically, cancer therapies have focused on the direct killing of tumor cells. These efforts have been fruitful in many hematologic malignancies and, to a lesser extent, solid tumors. Treatment of advanced disease has been especially frustrating, however, in the common cancers, including breast, colorectal, lung, prostate, and pancreatic cancer. In high-risk and metastatic breast cancer, evidence suggests no advantage of dose-intense therapies using myeloablative regimens and hematologic rescue over lower dose therapies [85,105]. Although the ultimate goal of most therapies is eradication of malignant cells, strategies designed to impede cancer dissemination and metastatic growth may be targeted better at the relatively permissive host environment. Although the bulk of clinical research efforts focus on cytotoxic therapies, there is growing interest in modulating the host environment to prevent cancer cell invasion and to inhibit the angiogenic process. These events are complex, interdependent, and tightly regulated [41,63]. As a primary tumor grows, it begins to invade the surrounding normal tissues. This invasion usually is associated with neovascularization. As proliferating tumor cells contact lymphatic channels and capillaries, secreted degradative enzymes break down a variety of structural stromal proteins in the basement membrane and the extracellular matrix (ECM). Through this mechanism, tumor cells gain access to the systemic circulation. Subsequent adherence of tumor emboli to the walls of small vessels in distant sites is followed by invasion into the organ through the ECM. Bound within the ECM are many angiogenic factors, which are released and activated as the matrix is degraded. Invasion leads to release of angiogenic factors with resultant endothelial cell migration and proliferation. Just as angiogenic activators are sequestered in the ECM, however, it also is apparent that some of the products of degradation may act as angiogenic inhibitors, reflecting a crucial balance between activating and inactivating factors in host tissues. Several key factors necessary for these events have been identified and represent potential targets for exploitation. Efforts to inhibit tumor cell invasion have focused on preventing basement membrane and ECM degradation. Pancreatic cancer cells produce high levels of matrix metalloproteinases (MMPs), specifically metalloproteinase-2 (MMP-2) [67]. The MMPs are a large family of proteases involved in physiologic tissue remodeling required for wound healing and in pathogenic processes, such as arthritis and cancer invasion. Preclinical studies suggest a central role for the MMPs in the metastatic process [15]. These proteases represent a putative target to inhibit tumor cell invasion into surrounding stromal tissues and distant sites. Matrix metalloproteinase inhibitors Endogenous matrix metalloproteinase inhibitors (MMPIs) exist that tightly regulate the activity of proteases in the extracellular milieu. The two primary endogenous inhibitors are tissue metalloproteinase inhibitors 1 and 2. Both are

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fairly large proteins and unlikely to be clinically relevant because of their poor pharmacologic properties [15]. Many synthetic MMPIs have been developed, however, including marimastat, BAY 12-9566, and BMS-275291 [14,46,51,107]. Of these three agents, marimastat has been studied most extensively. It is an orally bioavailable drug that inhibits MMP-2 and MMP-9, and it has been tested in patients with advanced prostate cancer, small cell lung cancer, colorectal cancer, ovarian cancer, and pancreatic cancer [106]. The drug has been given at doses ranging from 5 mg orally once daily to 75 mg orally twice daily. Observed doselimiting toxicity has been musculoskeletal pain with arthralgias and myalgias [91]. Phase III studies of marimastat have been undertaken in a variety of solid tumors. In a phase III trial, patients with limited or extensive small cell lung cancer were randomized to receive oral marimastat at 10 mg twice daily or a placebo, if they had achieved a complete or partial response to first-line therapy [98]. Median survival for the entire group was 9.5 months. During an integrated symposium held at the 2001 meeting of the American Society of Clinical Oncology, the principal investigator reported there was no added survival benefit based on randomization to marimastat. Marimastat may have clinical utility, however, for patients with pancreatic cancer without significant toxicity. Three phase III trials of marimastat have been conducted in patients with pancreatic cancer. In one study, patients with metastatic pancreatic cancer were assigned randomly to one of four treatment arms: one of three dose levels of marimastat (5, 10, or 25 mg) twice daily, or standard-dose gemcitabine [16]. The study enrolled 414 patients, and the median survival was 111, 105, or 125 days for patients receiving marimastat at 5, 10, or 25 mg twice a day. The authors reported a statistically significant difference in survival between patients receiving the lower marimastat doses (5 or 10 mg) compared with patients receiving gemcitabine ( P < 0.003) but no difference in survival between patients randomized to the highest doses of marimastat and patients treated with gemcitabine ( P = 0.46). This difference suggested a dose response to marimastat’s activity as a cytostatic agent. Grade 3 and 4 toxicity was more common using gemcitabine than marimastat (22% versus 15%). In another phase III trial, patients with advanced pancreatic cancer are being randomized to receive gemcitabine with marimastat or gemcitabine alone; the results of this trial have not been disclosed yet. Theoretically, marimastat would be expected to have greater impact when the primary tumor has been resected and the tumor burden is limited to microscopic metastatic disease. Marimastat also is being investigated in a randomized double-blind, placebo-controlled trial designed to evaluate whether it can prevent or delay significantly the onset of metastatic disease after pancreaticoduodenectomy in patients undergoing surgery with curative intent. The primary end point of this trial is survival; accrual has been completed, but the results have not been determined yet (PJW Pisters: personal communication, 2002). Another oral MMPI, BAY 12-9566, has been studied in a phase I trial. Common side effects included headache, nausea, and vomiting. Some abnormalities in hepatic function also were observed [92]. Another orally bioavailable

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metalloproteinase has entered clinical testing, BMS-275291. This agent is a nonhydroxamate inhibitor of MMP-2 and MMP-9, and to date, dose-limiting musculoskeletal toxicity has not been observed in early clinical testing [60]. Antiangiogenic agents for pancreatic cancer Angiogenesis has been proposed as a target for anticancer therapy for since the 1970s [43]. Until more recently, however, few endogenous angiogenic factors had been identified. Now, many factors have been isolated and shown to promote endothelial cell growth and migration [63]. These angiogenic factors provide a mechanism for tumor foci to expand beyond a few millimeters. One such factor is vascular endothelial growth factor (VEG-F), which is up-regulated in pancreatic cancer and associated with a poor prognosis [34,97]. Its regulation may be linked to Ras activation in these tumors [62]. Strategies have been developed to inhibit the effects of VEG-F, including monoclonal antibodies to VEG-F and receptor tyrosine kinase inhibitors to the receptor for VEG-F (Flk-1/KDR) [44,49]. One such agent is SU5416, a specific receptor tyrosine kinase inhibitor for VEG-F [44]. Clinical trials of SU5416, a drug administered intravenously, currently are being conducted in many solid tumors, including pancreatic cancer [110]. Another agent, SU6668, is a receptor tyrosine kinase inhibitor of Flk-1/KDR that is orally bioavailable and is a potent angiogenic inhibitor [70]. It is beginning to be tested in early clinical trials. Many other antiangiogenic compounds are under investigation, including TNP-470, an analogue of fumagillin. Fumagillin is derived from the fungus Aspergillus fumigatus fresenius and was found to be a potent angiogenesis inhibitor in an in vitro model. TNP-470 has a half-life of only 1 hour, suggesting prolonged or frequent infusions of the agent may be required for activity [42]. Nevertheless, as a single agent, TNP-470 has been reported to lead to a complete response in a patient with metastatic cervical cancer, with other study subjects having stable disease after previous known progression [69]. At M. D. Anderson Cancer Center, TNP-470 is being investigated in the setting of locally advanced pancreatic cancer after initial chemoradiotherapy. The objective is to determine if TNP-470 prevents or delays the onset of overt metastatic disease. More recently, two potent inhibitory molecules have been isolated from tumorbearing animals: endostatin, a fragment from collagenase XVIII, and angiostatin, which is a peptide fragment of plasminogen [82]. Angiostatin and endostatin have shown significant antitumor activity in animal models, and angiostatin has induced tumor dormancy in mice and tumor regression in an animal model of pancreatic cancer [23,83,115]. Endostatin and angiostatin have completed phase I testing, and further evaluations of these agents are under way [29,55].

Clinical trial design in the era of molecular therapy Rational anticancer therapy for pancreatic cancer requires an accurate knowledge of the natural history of this disease [113]. Patient survival depends

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primarily on tumor burden and performance status at diagnosis. Variations in patient outcome using cytotoxic therapy often have failed to recognize this. For purposes of patient management and clinical trial design, patients should be defined as having resectable, locally advanced, or metastatic disease. Despite resection with curative intent, most patients undergoing surgery ultimately develop metastatic disease, signifying the presence of disseminated microscopic disease at presentation. Patients with locally advanced, nonmetastatic disease are certain to harbor subclinical metastatic deposits. In advanced pancreatic cancer, previously published therapeutic trials teach valuable lessons that should be acknowledged in clinical trial design. First, studies of investigational agents and approved drugs often enroll patients who are described as having advanced pancreatic cancer. This is a heterogeneous group composed of patients with locally advanced disease and patients with overt metastatic disease. Median survival in such studies may seem promising, but if a substantial proportion of the study group have locally advanced disease, median survival is more likely dependent on this variable than on the delivered therapy. Second, although high objective response rates have been reported in some phase II trials in pancreatic cancer, this has not usually translated into meaningful improvements in survival in phase III studies [37]. Clinical trials investigating the activity of agents with specific molecular targets need to be designed with these variables in mind and with the understanding that a sequence of trials may be necessary to show superior outcomes compared with standard therapy. Several of the treatment strategies described earlier may be described more aptly as potentially cytostatic, rather than cytotoxic. Tumor regression may not be the appropriate end point compared with tumor or symptom stabilization as evidence of biologic activity. In the phase II trial of gemcitabine and C225, the objective response rate to therapy was only 12% with a 1-year survival rate of 32%. All patients had measurable bidimensional disease, and this represents a substantial proportion of patients surviving greater than 1 year, suggesting a significant cytostatic effect. Antiangiogenic treatments may require several weeks to months to produce an observable biologic effect, and in the intervening time, some degree of asymptomatic tumor progression should be allowed. In this setting, measurements of metabolic activity or tumor perfusion have been proposed as surrogates for biologic activity and potentially more relevant than conventional anatomic imaging modalities. Despite these caveats, rational trial design for resectable, locally advanced, or metastatic disease is possible. Treatment of a homogeneous patient population based on clinical stage and performance status is encouraged, however. For patients with resectable disease, chemoradiotherapy often is delivered as adjuvant therapy. Subsequent single-agent targeted therapy with a FTI, a MMPI, or an antiangiogenic agent would be reasonable investigational approaches, with time to treatment failure and survival as primary end points. All patients should have uniform treatment before entry (surgery with curative intent and all or no patients receiving chemoradiotherapy) For patients with locally advanced disease, chemoradiotherapy followed by targeted therapy is appealing. Here the appropriate end

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points could include survival, time to symptomatic local disease progression, or time to onset of distant disease. Response to therapy is unlikely to be a relevant end point in this setting. For patients with metastatic disease, measurable disease is preferred if tumor regression is a valued goal. If not, time to objective or symptomatic disease progression, 1-year survival, and overall survival are reasonable end points. Whenever possible, careful assessment of treatment-related toxicity should be recorded and kept to a minimum. Serial measurements of tumor perfusion or metabolic activity may be reasonable tools to determine biologic activity, especially when complemented by conventional cross-sectional imaging [58,78].

Summary Oncology has entered an era of molecular therapy. Given the multitude of molecular defects involved with pancreatic carcinogenesis, invasion, and mestastasis, it is unlikely that single-agent targeted therapy will alter the course of this disease. Given the emergence of molecular targets and a growing number of agents available for clinical development, however, meaningful improvements in patient outcomes are expected, particularly if treatments are designed and delivered rationally.

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