Author: Ken Scholz, PhD Contributing Editor: Ricker Polsdorfer, MD

Author: Ken Scholz, PhD Contributing Editor: Ricker Polsdorfer, MD This continuing education program is sponsored and managed by Professional Education Services Group This continuing education program is supported through an independent educational grant from Wyeth Pharmaceuticals

Signal Transduction Inhibitors in Cancer Therapy Overview/Statement of Need The advent of molecular biology and its application to medicine have opened a new era in cancer treatment. Already multiple new agents have been developed, tested and released to the market. Many more are still in the development process. The future seems unlimited for identifying oncogenic molecular pathways; elucidating their role in the genesis, growth and spread of cancer; and developing effective agents to target those pathways. These agents are collectively known as signal transduction inhibitors since their function is to impede the transmission of molecular signaling down a particular metabolic pathway. The targeted pathways each contribute to oncogenesis by different mechanisms inhibited apoptosis, dedifferentiation, growth, spread, stimulation of angiogenesis and many others. The progress in this area is rapid and voluminous. Concerted efforts in continuing education are required to keep busy clinicians up-to-date on the rapid advances being made so that patients may receive the most recent information and best treatment options from the growing list of choices. Learning Objectives At the conclusion of this continuing medical education activity, participants should be able to: Interpret the molecular biology of cancer in the context of utilizing newer targeted therapies Develop effective treatment strategies employing traditional chemotherapy and newer molecular targeted therapies Identify ways to improve patient health care outcomes in terms of the therapeutic options that newer targeted therapies offer Target Audience This continuing medical education activity is intended for oncologists, surgical oncologists, researchers, and other health care professionals with an interest in the use of newer molecular targeted therapies in solid organ tumors. Accreditation Statement Professional Education Services Group is accredited by the Accreditation Council for Continuing Medical Education (AC- CME) to provide continuing medical education for physicians. This activity has been planned and implemented in accordance with the Essential Areas and policies of ACCME. Professional Education Services Group designates this educational activity for a maximum of 2.0 AMA PRA Category 1 Credits TM. Physicians should only claim credit commensurate with the extent of their participation in the activity. Acknowledgement of Commercial Support This continuing education activity is supported through an independent educational grant from Wyeth Pharmaceuticals. Statement of Sponsorship This continuing education activity is sponsored by Professional Education Services Group. Contributors Ken Scholz, PhD Ricker Polsdorfer, MD Disclosure Statement It is the policy of Professional Education Services Group to ensure balance, independence, objectivity, and scientific rigor in all CME activities. Faculty participating in this activity are required to disclose to the audience any relationship that they may have with the commercial supporters of this activity or with any other commercial organizations whose products or devices may be mentioned in their presentations. Ken Scholz, PhD Dr. Scholz has no significant financial relationships to disclose. Ricker Polsdorfer, MD Dr. Polsdorfer is an employee of Professional Education Services Group. PESG has received an independent educational grant from Wyeth Pharmaceuticals. Unlabeled/Unapproved Uses The authors acknowledge that much of the material reviewed in the manuscript constitutes either an unlabeled/unapproved use of a potential new drug or device. Please use your professional discretion when making treatment decisions and refer to product prescribing information.

Advanced Primer Introduction The receptor-tyrosine kinases (RTKs) are often the first step in a complex signal transduction cascade that commonly results in the regulation of gene transcription. Along these signal transduction pathways are numerous opportunities for deployment of pharmaceutical signal transduction inhibitors (STIs) that influence cell growth and survival. For example, an STI could inhibit the RTK (at the ligand-binding site or the kinase catalytic site), accessory proteins, or downstream transcription factors. In addition, inhibitors may be directed against the products of the newly stimulated gene transcription products which may include secreted growth factors, oncogene products, and other proteins that regulate cell proliferation. In recent years, several STIs have been developed as anticancer therapies and tested in clinical cancer trials. This monograph will describe the rationale for development of some prominent STIs currently available for the treatment of solid tumors, and the clinical evidence for their efficacy and safety. Renal Cell Carcinoma Until recently, renal-cell carcinoma (RCC) has been one of the most intractable forms of cancer, with a high proportion of patients presenting with metastatic disease, and with high rates of recurrence after treatment for localized disease. 1 RCC is highly resistant to treatment using conventional chemotherapy agents, 2 and median survival for patients with metastatic disease is only 13-18 months even with the most effective conventional treatments (high-dose interleukin-2). 3 In recent years, however, prospects for treating RCC have improved owing to a greater understanding of signal transduction pathways and their role in tumorigenesis. Several histologic forms of RCC have been identified including papillary, oncocytoma, chromophobe, and collecting duct forms but the most common and most aggressive form of RCC is clear-cell RCC. 1,4 Subsequent discussion will focus on clear-cell RCC (ccrcc) and the signal transduction mechanisms involved in its pathogenesis and treatment. Molecular Pathways Although familial cases of ccrcc account for only a small proportion of total cases, studies of susceptible families have been crucial for the identification of susceptibility genes and subsequent understanding of the molecular biology of ccrcc, including sporadic cases. 5,6 In particular, ccrcc occurs at unusually high frequency in persons with von Hippel-Lindau (VHL) disease, 7 a genetic disorder involving mutations of a tumor suppressor gene (VHL gene) on the short arm of chromosome 3. 8,9 Individuals with VHL disease are at increased risk of developing various tumors, including ccrcc. 7 Furthermore, a large proportion of sporadic cases of ccrcc are associated with deletion or loss-of-function mutations in both alleles of the VHL tumor-suppressor gene. 10,11 Together, these observations provided early evidence that the VHL gene played a crucial role in the development of many instances of ccrcc, in both sporadic and familial cases. The VHL gene encodes two protein isoforms (collectively known as pvhl) that each exhibit tumor-suppressing properties. 12-14 pvhl forms a complex with several other proteins in the cytoplasm. As a crucial component of this complex, pvhl binds to other cytoplasmic proteins and mediates the process of ubiquitylation, in which proteins are marked for subsequent destruction by proteosomes. 15 In this manner, pvhl controls the fate of a number of substrate proteins, including the alpha subunit of hypoxia-inducible factor-1 (HIFα). Under normal conditions, HIFα is continuously produced but rapidly ubiquitylated by pvhl, permitting its degradation by proteosomes (Fig. 1). 16-18 Under hypoxic conditions, however, HIFα is protected from ubiquitylation and accumulates. 16,19,20 Similarly, when pvhl is deficient or defective, as in many cases of ccrcc, HIFα also accumulates (Fig 2). 18 Excess HIFα is a feature common to a number of human cancers in addition to ccrcc. 21-23 Fig 1. Copied from Fig 1 of Potti and George. 24 Fig 2. First from Motzer review; second from Potti et al.

Signal Transduction Inhibitors in Cancer Therapy The significance of HIFα accumulation to ccrcc can be recognized by examining the protein s function. Viable HIFα binds to the HIFb subunit, translocates to the cell nucleus, and directs the enhanced expression of several gene products linked to both hypoxic stress and tumorigenesis. Some of these genes and their functions are listed in Table 1. The various products of HIF-directed gene transcription account for many of the invasive properties of ccrcc cells. Furthermore, VHL has been called a gatekeeper for the expression of the ccrcc phenotype. 6 Indeed, dysfunctional pvhl has been shown to be both necessary and sufficient for the proliferation of ccrcc tumor cell xenografts. Hence, a peptide that blocks the HIF-binding site of pvhl induces a cellular phenotype that mimics that seen in pvhl-deficient ccrcc cells. 25 A similar result is observed by expression of a HIF variant that is resistant to VHL-directed destruction. 26 Finally, inhibition of HIF or reintroduction of functional pvhl reverses the growthpromoting properties of pvhl-deficient tumor cells. 27-29 Identification of Potential Therapeutic Targets Elucidation of the molecular pathways underlying a majority of cases of ccrcc has allowed researchers to identify a number of steps along the pathway that may serve as targets for antitumor therapy. Figure 3 depicts several of the key targets, each described in detail below. Also shown in the figure are additional signal transduction pathways impinging upon the HIF/VHL system, some of which also serve as potential therapeutic targets. Strategy 1: Block Activity of HIF Gene Products Key products of HIF-mediated gene transcription are VEGF, PDGFβ, and TGFα. These secreted growth factors act separately and in concert to promote and stabilize angiogenesis and to promote tumor cell proliferation and survival (Table 1). Agents that prevent the actions of these factors have been proposed to reverse many of the tumor-supportive actions of HIF and reverse the effects of pvhl deficiency. This approach may require the use of combination therapies to target the Figure 3. Copied from Iliopoulos. 6 various gene products. Alternatively, multi-targeted agents may be useful in this setting. Within this strategy are embedded several potential substrategies, some of which have been utilized in the development of viable anticancer therapies. For example, an agent could be directed against any of the growth factors themselves (e.g., an antibody to VEGF, PDGFβ, or TGFα). Alternatively, because the receptors for these agents are closely related RTKs, smallmolecule inhibitors have been developed that inhibit the kinase activity of both the VEGF receptor (VEGFR) and the PDGF receptor (PDGFR). Inhibitors of TGFα or its receptor are a special case that will be addressed under Strategy 5. Strategy 2: Inhibit HIF-Directed Transcription In theory, it may be possible to develop an agent that specifically inhibits HIF-directed gene transcription. Currently, no such agents are in advanced clinical development. Strategy 3: Directly Inhibit HIF Expression The translation of HIFα mrna into protein is regulated by a protein known as mtor (mammalian target of rapamycin, relating to its inhibition by the drug rapamycin). 42 Thus, levels of HIFα are controlled not only by hypoxia and pvhl but also Table 1. Gene products whose expression is increased in the presence of elevated HIFα

Advanced Primer Selected agents in late-stage clinical development Selected agents with antitumor efficacy that have been studied in ccrcc Selected agents in late-stage clinical development by mtor and the signal transduction pathways that regulate mtor (see Fig 3 and Strategy 4). Although rapamycin does not bind to mtor directly, it binds to a protein (FK506) that complexes with mtor, thereby inhibiting the kinase activity of mtor 43 and the translation of HIFα. 42 mtor also controls the expression of several other genes involved in control of the cell cycle and tumorigenesis, such as the cyclins and cmyc. 44 Analogs of rapamycin are in late-stage clinical development for treatment of ccrcc, as well as several other hematologic and solid tumors. Strategy 4: Inhibit the Signal Transduction Pathway Upstream of mtor As diagrammed in Figure 3, mtor is under the control of another kinase signaling pathway, known as the PI3K/Akt (phosphatidylinositol 3-kinase/Akt) pathway. Activation of this pathway activates mtor and promotes the translation and stability of HIFα. 6,47 Other signaling pathways impinge upon the PI3K/Akt/mTOR pathway, most notably the tumorsuppressors PTEN (phosphatase and tensin homolog) and TSC1 and 2 (tuberous sclerosis genes 1 and 2). At the current time, there are no agents in late-stage clinical development that specifically target these signal transduction elements. However, some of the RTK inhibitors in development also inhibit other elements of this signaling cascade. Furthermore, this system constitutes a very active area of research that holds much promise for future discovery. Strategy 5: Inhibit EGFR Tyrosine Kinase The epidermal growth factor receptor (EGFR/ErbB1) is a member of a family of RTKs that are strongly implicated in a number of human cancers. Another member of the family is HER2/neu/erbB2, the overexpression of which is a marker for a more aggressive form of breast cancer. As Figure 3 depicts, EGFR (as well as other members of the EGFR receptor family) activates the PI3K/Akt pathway. 48 In addition, overexpression of the EGFR is common in ccrcc. 49,50 Finally, TGFα, which we have already seen is upregulated in ccrcc, is an activating ligand of the EGFR, providing a potential positive-feedback loop for proliferation and survival of ccrcc cells. 51 Thus, inhibitors of EGFR have been studied as therapeutic agents for ccrcc. Strategy 6: Parallel or Serial Combination Therapy As described by Kaelin, 5 combination therapies may prove to be the most efficacious approach for treatment of ccrcc. Such therapies could be directed at multiple targets along the signal transduction pathways, acting either in series or in parallel (Fig 4). 5 Figure 4. Copied from Kaelin. 5

Signal Transduction Inhibitors in Cancer Therapy Other Strategies STIs utilizing other signaling pathways have found success in the treatment of other solid tumors, as well as hematologic malignancies. A prominent example is imatinib, which inhibits the abnormal RTK product of the Philadelphia chromosome (bcr-abl) that is a key defect in chronic myeloid leukemia. Such inhibition induces apoptosis of bcr-abl positive cells. Imatinib also inhibits other RTKs, such the PDGFR and the stem-cell factor receptor(c-kit). 52 Clinical Trials Overview A number of trials have tested the efficacy of agents targeting the epidermal growth factor receptor (EGFR) as antitumor therapy for ccrcc. Antibody-based therapies directed at the EGFR (cetuximab and panitumumab) have yielded disappointing results in randomized trials. 53,54 Similarly, smallmolecule inhibitors of the EGFR tyrosine kinase activity (gefitinib, lapatinib, erlotinib) have shown little promise when used alone, 55-57 although a subanalysis suggested that lapatinib may be more effective in tumors overexpressing EGFR. 55 The reasons for these disappointing results are not entirely clear, but they are likely related to additional signal transduction pathways that increase HIFα expression, 58 such as the Ras/Raf pathway. 6,59 There is also evidence for parallel genetic pathways leading to ccrcc tumorigenesis. 6 Although the EGFR receptor-directed agents have shown disappointing results as monotherapy for ccrcc, they may become important components of combination therapy regimens. For example, a nonrandomized trial of the combination of erlotinib and bevacizumab (an example of a serial approach as per Strategy 6), found objective responses in 15 of 59 (25%) evaluable patients with metastatic ccrcc. 60 An additional 36 patients (61%) had stable disease during 8 weeks of treatment. Further studies of this approach, as well as other combination therapies for ccrcc, are anticipated. More promising results have been obtained in late-stage clinical trials of therapies targeting signal transduction elements downstream from the EGFR, particularly those utilizing Strategies 1 and 3. The results of late-stage clinical trials of the relevant agents will be discussed in turn below. Bevacizumab Bevacizumab is a humanized, recombinant antibody to VEGF that was the first approved anticancer therapy to specifically target angiogenesis. Its mechanism of action relies on its ability to bind to VEGF in the extracellular space, thereby preventing the growth factor from binding to its receptor and stimulating angiogenesis. Bevacizumab is approved by the FDA for use in metastatic colorectal cancer in combination with 5-fluorouracil (5-FU)-based chemotherapy and in metastatic non-small cell lung cancer in combination with carboplatin and paclitaxel. In addition, it is being studied in clinical trials as therapy for numerous other solid tumors. 61 Bevacizumab has been studied in a randomized, blinded, placebo-controlled phase II trial in patients with metastatic ccrcc. 62 Patients (n=116) with histologically confirmed ccrcc were randomized to receive either placebo injections or one of two dosages of bevacizumab (3 mg or 10 mg per kg body weight) administered every two weeks. The trial was stopped early after the second interim analysis found strong evidence of efficacy that met the criteria for early termination. At that analysis, patients in the high-dose bevacizumab arm had a significantly longer median time to progression (4.8 months) compared to patients in the placebo arm (2.5 months; p<0.001). Patients in the low-dose bevacizumab arm also had a median time to progression that was longer than the placebo arm (3.0 months), which was marginally significant (p=0.041). Overall, 64% of patients in the high-dose bevacizumab arm exhibited no tumor progression after 4 months of randomized treatment. The corresponding percentages for the low-dose bevacizumab and placebo arms were 39% and 20%, respectively. The hazard ratios for time to progression were 2.55 (p<0.001) and 1.26 (p=0.53) for the high-dose and low-dose bevacizumab arms, respectively, as compared to the placebo arm (longer times to progression in the bevacizumab arms). Four patients (10%; 95% CI 2.9-24.2%) in the high-dose bevacizumab arm exhibited partial responses. As of the last published analysis, there was no significant difference in overall survival. 62 Patients receiving bevacizumab, especially those in the high-dose arm, experienced substantially higher rates of hypertension and proteinuria, consistent with observations in previous trials of this agent for other tumor types. 63 The investigators of this phase II trial concluded that the effects of bevacizumab on ccrcc were modest, even though they reached significance at an interim analysis. 62 They emphasized that bevacizumab may have an important role in a combination therapy regimen with an inhibitor of the EGFR pathway. As already discussed, an open-label trial of such combination therapy in metastatic ccrcc has been published, showing some promise for the combination of bevacizumab and erlotinib. 60 Additional phase III trials of bevacizumab for ccrcc are underway. 59,64 Sorafenib Sorafenib is a small-molecule inhibitor of receptor tyrosine kinases. It inhibits several tyrosine kinases that are part of the signal transduction pathway involved in ccrcc tyrosine kinases such as VEGFR and PDGFR, as well as Raf tyrosine kinase, FMS-like tyrosine kinase (Flt-3), c-kit protein, and RET receptor tyrosine kinase. 37,38 As such, it is often referred to as a multikinase inhibitor. The efficacy and safety of sorafenib for treatment of ccrcc have been studied in a randomized, blinded, placebo-controlled phase III trial. 65 Enrolled patients (n=903) had metastatic ccrcc that had progressed on one systemic treatment within the preceding 8 months. They were randomized to receive either sorafenib (400 mg twice daily) or placebo in 6-week cycles for

Advanced Primer Figure 5. Copied from Escudier et al. 65 the first 24 weeks, and then 8-week cycles until progression or termination. After an interim analysis found evidence of efficacy associated with sorafenib, patients in the placebo arm were allowed to crossover into the sorafenib arm. The first analysis of survival data from this trial was performed at a median follow-up of 6.6 months, before placebo patients were allowed to crossover into the sorafenib arm. 65 The probability of overall survival is shown in Figure 5a. Overall, 22% (97/451) of patients in the sorafenib arm and 27% (123/452) of patients in the placebo arm had died. The median actuarial survival for patients in the placebo arm was 14.7 months but had not yet been reached in the sorafenib arm (hazard ratio 0.72; 95% CI 0.54-0.94; p=0.02, considered not significant for an interim analysis). Progression-free survival had been assessed at an earlier timepoint, at which data were available for 769 patients. At that time, the median progressionfree survival was 5.5 months for patients in the sorafenib arm and 2.8 months for patients in the placebo arm (p<0.001; Fig 5B). The hazard ratio for progression was 0.44 (95% CI 0.35-0.55) in the sorafenib arm compared to the placebo arm. One patient in the sorafenib arm exhibited a complete response, and partial responses were observed in 43 (10%) and 8 (2%) patients in the sorafenib and placebo arms, respectively. Stable disease was observed in 333 (74%) and 239 (53%) patients, respectively. Although sorafenib treatment was associated with improvements in overall and progression-free survival in patients with metastatic ccrcc, it was also associated with significant toxicity. 65,66 Indeed, sorafenib was associated with significantly higher rates of dose reduction (13% vs. 3%; p<0.001), dose interruption (21% vs. 6%; p<0.001), cardiac ischemia or infarction (3% vs. <1%; p<0.01), and serious adverse events leading to hospitalization or death (34% vs. 24%; p<0.01). Other adverse events occurring at higher rates in the sorafenib arm (relative to the placebo arm) were hypertension, bleeding, dermatologic events and diarrhea. Although the toxicity profile of sorafenib is of concern, the investigators considered its toxicity to be moderate and manageable, especially in comparison to cytotoxic chemotherapy. Overall, the results of this trial support the efficacy of sorafenib, in terms of progression-free survival in a pretreated population of patients with metastatic ccrcc. Such patients have few options for anticancer therapy. Furthermore, the large percentage of patients exhibiting stable disease while receiving sorafenib is consistent with the putative mechanism of action of the drug, which is expected to have a cytostatic effect on tumor cells rather than a cytotoxic effect. However, the primary endpoint (overall survival) was not statistically significant for an interim analysis, and sorafenib was associated with significant toxicity. Additional studies of sorafenib are underway to better define its role in the treatment of metastatic ccrcc, including use in combination therapy regimens. 59 Sorafenib is now approved by the FDA for treatment of advanced ccrcc. Because patients in this setting have few treatment options, sorafenib (and other new agents described below) will likely have significant roles in therapy of such patients. Sunitinib Sunitinib is another multikinase inhibitor that targets many of the same kinases as sorafenib. 39-41 It has also been studied in a recent randomized, controlled, phase III trial. Unlike the sorafenib trial, however, patients enrolled into the sunitinib trial had metastatic ccrcc that was previously untreated. Furthermore, in the sunitinib trial patients in the control arm were treated with interferon alfa rather than placebo. Thus, enrolled patients (n=750) were randomized to receive oral sunitinib (50 mg once daily) in 6-week cycles (4 weeks, on 2 weeks off) or interferon alfa (three times per week) by subcutaneous injection. Interferon alfa was initiated at a dosage of 3 MU for the first week, 6 MU for the second week, and 9 MU for all subsequent dosages, unless dose reduction was required to alleviate adverse events. Because of the different routes of drug administration, patients and site staff were not blinded to study drug allocation. Assessment of responses, however, was performed by a blinded central review. After a median duration of therapy of 6 months in the sunitinib arm and 4 months in the interferon alfa arm, patients in the

Signal Transduction Inhibitors in Cancer Therapy of sunitinib vs. sorafenib is unclear at this time. A phase II trial of sorafenib vs. interferon alfa has provided preliminary evidence that sorafenib is also superior to interferon alfa in treatment-naïve patients. 68 Additional studies of sunitinib are also ongoing. 59 Figure 6. Copied from Motzer. sunitinib arm had a median progression-free survival of 11 months (95% CI 10-12 months) versus 5 months (95% CI 4-6) for patients in the interferon alfa arm (hazard ratio 0.42 95% CI 0.32-0.54; p<0.001) (Figure 6). 67 At the time of the analysis, overall survival data were not mature but showed a trend toward better survival in the sunitinib arm (hazard ratio for death, 0.65; 95% CI 0.45-0.94; p=0.02, considered not significant for an interim analysis). No complete responses were observed, but 31% (103/335) of patients in the sunitinib arm, versus 6% (20/327) of patients in the interferon alfa arm, achieved partial responses (p<0.001). Stable disease was observed in 48% and 49% of patients in the respective arms. As observed in previous studies, interferon alfa was associated with numerous adverse events such as fatigue, pyrexia, chills, myalgia, and influenza-like symptoms. Patients in the sunitinib arm had higher rates of grade 3 diarrhea, vomiting, hypertension, and the hand-foot syndrome as compared to patients in the interferon alfa arm (p<0.05). Sunitinib was also associated with significantly higher rates of grade 3 or 4 leukopenia (5% vs. 2%), neutropenia (12% vs. 7%), and thrombocytopenia (8% vs. <1%) (all p<0.05). Interferon alfa was associated with higher rates of lymphopenia (12% vs. 22%; p<0.05). More patients in the sunitinib arm (versus the interferon alfa arm) had dose reductions (32% vs. 21%) and dose interruptions (38% vs. 32%) because of adverse events. 67 Sorafenib and sunitinib have not been directly compared in a head-to-head trial, and indirect comparison of results from these two trials is fraught with numerous difficulties. These difficulties include the use of different comparator arms (placebo for the sorafenib trial and interferon alfa for the sunitinib trial), as well as differences in enrollment criteria, the most notable of which is that patients in the sorafenib trial had failed a previous therapy for metastatic disease whereas those in the sunitinib trial were untreated for metastatic disease. Considering these differences in trial design, the sunitinib trial would be expected to yield longer times to disease progression and better objective response rates compared to the sorafenib trial. These expectations have been borne out. Whether these differences are a consequence of trial design or superiority Temsirolimus Temsirolimus is the most clinically advanced agent representing Strategy 3, as outlined previously. Temsirolimus is a derivative of rapamycin that inhibits mtor-directed gene transcription. Preliminary results have been presented of a randomized, 3-arm, phase III, open-label trial of temsirolimus in patients with metastatic ccrcc determined to be poor-risk 69 on the basis of criteria defined by Motzer et al. 70 (with the added criterion of multiple organ sites of metastasis). The focus on patients with features of poor prognosis was based on a subgroup analysis of earlier clinical studies in which temsirolimus appeared to be of greater benefit in patients with such features. 71 Patients enrolled into the trial (n= 626) had histologically confirmed, treatment-naive ccrcc that exhibited at least 3 (of 6) prespecified features associated with a high risk of disease progression. 69 Patients were randomized into one of three arms: Interferon alfa: escalating dosage up to 18 MU thrice weekly administered subcutaneously Temsirolimus-alone: 25 mg once weekly administered intravenously Combination: temsirolimus (15 mg IV once weekly) plus interferon alfa (6 MU thrice weekly, subcutaneous) Results are available from an interim analysis conducted after 442 enrolled patients had died. 69 At that time, patients in the temsirolimus-alone arm had a median overall survival (10.9 months; 95% CI 8.6-12.7 months) that was significantly longer than both the interferon alfa arm (7.3 months; 6.1-8.9 months) and the combination arm (8.4 months; 6.6-10.2 months) (Fig 7A). The hazard ratio for death in the temsirolimus arm (relative to the interferon alfa arm) was 0.73; 95% CI 0.57-0.92; p=0.0069). The overall survival in the combination therapy arm was not significantly different from the interferon alfa arm. Median progression-free survival was significantly longer in both the temsirolimus-alone arm (3.7 months; p<0.0001) and the combination arm (3.7 months; p<0.0019) as compared to the interferon alfa arm (1.9 months; Figure 7B). Objective response rates (complete response + partial response) were 7%, 9%, and 11% in the interferon alfa, temsirolimus-alone, and combination therapy arms, respectively. The safety profile of temsirolimus compared favorably with that of interferon alfa. 69 The most common grade 3-4 adverse events were asthenia (27%, 12%, and 30% in the interferon alfa, temsirolimus and combination arms, respectively), anemia (24%, 21%, 39%), and dyspnea (8%, 9%, 11%). Overall, patients in the temsirolimus-alone arm experienced significantly fewer grade 3-4 adverse events (p<0.001 vs.

Advanced Primer A B Figure 7. Screen captures from ASCO Presentation of Hudes. 69 both other arms), fewer dose reductions or interruptions, and fewer discontinuations because of adverse events. In general, temsirolimus exhibited significantly better overall and progression-free survival, and a better safety profile in comparison to interferon alfa or combination therapy for firstline therapy in high-risk patients. A new drug application for temsirolimus is currently under priority review with the FDA. Furthermore, studies of temsirolimus are underway to examine its efficacy in a broader range of patients and as a component of other combination therapy regimens. Summary A greater understanding of the signal transduction pathways involved in the development and progression of clear-cell RCC has permitted the rational development of agents targeting specific components of that signal transduction system. These efforts have now come to fruition with the development and approval of signal transduction inhibitors with established clinical efficacy against ccrcc. Thus, patients with metastatic ccrcc now have valuable treatment options where few if any treatment options existed before. Further studies, in both the clinical and experimental realms, are expected to lead to further improvements in the treatment of ccrcc, possibly involving the use of selective combinations of signal transduction inhibitors. Head and Neck Cancer As alluded to in the previous section on renal cell carcinoma, the epidermal growth factor receptor (EGFR) plays an important role in a number of human cancers. The relative contribution of the EGFR to the malignant phenotype, and hence the antitumor efficacy of therapies directed against the EGFR, varies widely across tumor types. According to our current understanding, the EGFR plays a more central role in squamous cell carcinoma of the head and neck (schnc) than in ccrcc. However, there is considerable heterogeneity among schnc tumor types, and even among different patients with seemingly related tumors. As we improve our understanding of molecular pathways involved in carcinogenesis, it is likely that we will uncover even greater degrees of heterogeneity. Ultimately, however, it is hoped that this understanding will give us the ability to tailor therapies to highly defined groups of patients, or even individual patients, in order to optimize the efficacy and safety of therapy. In the meantime, this section will provide an overview of our current understanding of the signal transduction pathways serving as therapeutic targets for the treatment of schnc. Molecular Pathways The EGFR (erbb1) is a member of the erbb/her family of RTKs and is strongly implicated in numerous human cancers including schnc. 72,73 These RTKs consist of three functional domains: the extracellular domain that binds ligand, the membrane-spanning domain, and an intracellular domain that contains the tyrosine kinase catalytic activity. 72 Ligands of EGFR include epidermal growth factor (EGF), transforming growth factor-α (TGFα), amphiregulin, betacellulin, and heregulin, many of which are thought to be produced and secreted by schnc cells. 74 Upon ligand binding, an EGFR dimerizes with other EGFRs (homodimers) or other members of the erbb family (heterodimers). Such dimerization leads to autophosphorylation of the EGFR and other erbb RTKs, allowing for the docking and activation of adaptor proteins, in turn leading to activation of downstream signaling pathways. 72 The nature of the downstream signal is influenced by the activating ligand, the type of dimers formed, as well as other properties of the cell and its environment. 75,76 As discussed in the preceding section on renal cell carcinoma, binding of ligand to EGFR (or an activating mutation of the EGFR) activates the PI3K/Akt/mTOR pathway. In addition,

Signal Transduction Inhibitors in Cancer Therapy Figure 8. Copied from part of Figure 1 of Kalyankrishna 73 and figure 2 of Hynes and Lane 72 EGFR hetero- or homodimers can activate several other signal transduction pathways, including the so-called mitogenactivated protein kinase (MAPK) pathways such as the Ras/ MEK/ERK pathway, the signal transducers and activators of transcription (STAT) pathways, and the phospholipase C-γ (PLC- γ) pathway (Figure 8). Each of these pathways has the potential to influence oncogenesis, cell proliferation, and cell survival. 72 A good example has already been discussed, the PI3K/Akt pathway, which activates mtor and promotes protein translation. Another example is the Ras/MEK/ERK pathway, which activates mitogenic processes 77 and stimulates transcription of VEGF, 76 which is secreted and promotes local angiogenesis. Figure 8 also hints at some of the crosstalk among the different signaling pathways, which are now understood to form a highly complex signaling network. 72,78 In addition, EGFR both influences and is influenced by other receptors, including G-protein-coupled receptors. 79 These aspects of the erbb signaling systems will become important when we discuss the efficacy of anticancer therapies targeting the EGFR, variations in responses in different patients with seemingly related tumors, and the development of resistance to anti-egfr therapies. The central role of the EGFR and related erbb RTKs in signaling systems controlling cell proliferation, differentiation, and survival has made these RTKs important targets for development of anticancer therapies. Furthermore, many lines of evidence indicate that EGFR plays a crucial role in tumorigenesis. In schnc cells, for example, more than 90% of tumors are found to overexpress EGFR mrna, and about 40% overexpress the protein. 80-82 There is also evidence that overexpression of EGFR is associated with poor differentiation, metastasis, and poor prognosis. 82,83 Another important observation is that one of the important ligands for EGFR, TGFα, is also overexpressed in a larger percentage of schnc tumors. 80,83 The overproduction of TGFα along with overexpression of EGFR constitutes a powerful autocrine loop providing positive feedback in promoting cell proliferation. 72 Other mechanisms that may contribute to overstimulation of EGFR, and which have been observed in a subset of schnc tumors, include: Decreased downregulation or destruction of EGFR protein Genetic mutations leading to constitutive activation of the receptor, seen in a small number of schnc cases. 73 Transactivation of EGFR via mechanisms involving G-protein-coupled receptors or other tyrosine kinases. With regard to G-protein-receptors, several studies have revealed that some G-protein-coupled receptors activate MAPK pathways via indirect activation of the EGFR. 79 A model for a prominent mechanism of such transactivation is shown in Figure 9. 79 In Figure 9. Copied from Gschwind. 79 10

Advanced Primer brief, activation of the G-protein-coupled receptor by its ligand produces intracellular signals that activate a metalloproteinase in the cell membrane. The metalloproteinase in turn cleaves growth-factor precursors (such as TGFα and amphiregulin) from their membrane-bound tethers allowing them to bind and activate EGFR. Several ligands at G-protein-coupled receptors have been shown to activate the Ras/MEK/ERK pathway in this manner in various tissues, including schnc cells. 73,79 Clinical Trials Preclinical studies have found strong support for clinical trials of EGFR inhibitors as anticancer therapy for schnc. 84 These observations, together with observations that aberrations in EGFR pathways are associated with metastatic potential and poor prognosis (discussed above), have led to the development of EGFR inhibitors for treatment of schnc. Two general approaches have been pursued: (1) Antibodies to the extracellular domain of EGFR that block ligand binding (represented by cetuximab) (2) Small-molecule tyrosine kinase inhibitors of the EGFR, which act at the intracellular tyrosine kinase domain to block signal transduction (represented by gefitinib and erlotinib) Cetuximab Cetuximab is a human-mouse chimeric monoclonal antibody directed against the extracellular domain of the EGFR. It has received FDA approval for two indications related to squamous cell carcinoma of the head and neck: (1) in combination with radiotherapy for treatment of locally or regionally advanced disease, and (2) as a single agent for treatment of recurrent or metastatic disease after failure of platinum-based therapy. Bonner and colleagues compared high-dose, hyperfractionated radiotherapy alone (RT; n=213) versus RT plus cetuximab (n=211) for treatment of schnc in patients with locally or regionally advanced disease. 85 Cetuximab was administered intravenously using a loading dose of 400 mg/m 2 followed by weekly doses of 250 mg/m 2 for the duration of radiotherapy. Patients in the cetuximab-containing arm achieved significantly longer median locoregional control (the primary endpoint) of 24.4 months vs. 14.9 months in the RT alone arm (hazard ratio (HR) for locoregional progression or death, 0.68; p=0.001). Cetuximab was also associated with significantly longer median progression-free survival (HR,0.70; p=0.006) and overall survival (49.0 months versus 29.3 months; HR, 0.73; p=0.03). Although cetuximab was associated with acneiform rash and infusion reactions, rates of grade 3 or higher toxicities were comparable in the two arms. Several trials have studied cetuximab in the setting of recurrent or metastatic disease with somewhat mixed results. The Eastern Cooperative Oncology Group (ECOG) compared cisplatin alone to cisplatin with cetuximab in patients who had received no prior therapy for advanced disease. 86 Patients in the cetuximab arm had a longer progression-free survival (the primary endpoints) of 4.2 months (vs. 2.7 months), but the difference was not statistically significant. Nor was there a significant difference in overall survival. However, patients who exhibited an acneiform skin reaction in response to cetuximab therapy had a substantially better overall survival compared to patients not exhibiting the reaction (HR, 0.42; 95% CI 0.21-0.86). A similar association between response to cetuximab and the presence of a skin reaction has been observed in other studies. 87 Other studies have examined the activity of cetuximab in patients who have progressed on platinum-based therapies. These studies suggest that a small percentage of patients may benefit from cetuximab either in combination with platinum-containing regimens 88,89 or as monotherapy. 90 Tyrosine Kinase Inhibitors To date, no phase III trials of TKIs in schnc have been reported. However, several phase II trials have provided preliminary evidence of modest efficacy for gefitinib and erlotinib as monotherapy in patients with recurrent or metastatic disease, although these trials were all single-arm studies without comparator groups. The trials are summarized in Table 2. Overall, they suggest that the agents are active but are more likely to benefit patients in a first-line setting. Ongoing trials are studying other TKIs, such as lapatinib, as well as combinations of TKIs with chemotherapy agents or the anti-angiogenesis agent bevacizumab. 91 Until these trials are completed, the roles of TKIs in the treatment of schnc will remain promising but not yet established. Table 2. Summary of phase II trials of gefitinib and erlotinib in schnc 11

Signal Transduction Inhibitors in Cancer Therapy Efficacy, Resistance, and Future Directions With the EGFR playing such a central role in signal transduction underlying tumorigenesis of schnc, why are inhibitors of the EGFR or its tyrosine kinase only modestly effective as anticancer therapies? The answers to this question are likely to be as varied and complex as the disease itself, but several properties of the signal transduction system provide strong clues. First, as with most tumors, the clinical diagnosis of schnc encompasses a diverse array of tumors, some of which are not likely to rely on EGFR-dependent processes for proliferation and survival. Secondly, some forms of schnc may involve constitutive activation of the EGFR, in which case receptor blockers such as cetuximab may be predicted to be ineffective. Similarly, constitutive activation of specific components of the signal transduction pathway may render all EGFR inhibitors useless. For example, there is evidence for constitutive activity in the PI3K/Akt pathway, the STAT pathways, and the Ras/MEK/ ERK pathways in schnc cells, resulting from either activating mutations in the pathway itself, or via activation of other growth factor receptors, such as the receptor for insulin-like growth factor-1. 73 Thirdly, the numerous redundant signaling pathways, as well as crosstalk among different pathways, are likely to provide alternative mechanisms for cell survival and proliferation, even if the EGFR is inhibited. Other members of the erbb family are likely to play important roles in such a scenario. 73 But there is also evidence that certain G-protein-coupled receptors can activate the PI3K pathway or ERK pathway in schnc cells in a manner that is independent of the EGFR. 96 Thus, many researchers anticipate that inhibitors of multiple pathways may be required to achieve more effective anticancer activity. 72 Preliminary evidence provides strong support for this concept. 97 Many of these same themes may account for the development of resistance to therapies directed against the EGFR in patients who initially respond. Ongoing research is attempting to address these issues using various approaches, including combination therapies, agents directed at central components of the signal transduction pathways (such as mtor or MAPKs), and identification of tumor biomarkers that can predict which patients are more likely to respond to specific therapeutic agents. 73,91 Non-Small Cell Lung Cancer The EGFR has also been implicated as a key molecular player in the pathogenesis of non-small cell lung cancer (NSCLC). 72 In various studies, 40-80% of patients with NSCLC exhibit overexpression of EGFR in their tumors, 98 although these percentages vary among the different histologic forms of the disease. In general, tumors with a squamous cell phenotype are the most likely to overexpress EGFR; the lowest percentages of tumors with EGFR overexpression are among those with an adenocarcinoma phenotype, and intermediate percentages are found among those with a large-cell phenotype. 99,100 Some, but not all, clinical studies have found that higher levels of EGFR expression are associated with poor prognosis. 99,101-106 Importantly, EGFR overexpression in NSCLC is associated with gene amplification or polysomy in about 45-62% of cases. 104,107 An important difference between NSCLC and schnc is that NSCLC is much more likely to be associated with mutations in the EGFR gene that affect activity of the molecule. In a recent analysis of EGFR mutations, Tsao and colleagues found that the regions of the gene that encode the tyrosine kinase domain (exons 18-21) contained at least one mutation in 22% of patients with NSCLC tumors. 107 The potential prognostic significance of such observations will be discussed below (See Predicting Response). The three EGFR inhibitors that have been studied the most extensively for treatment of NSCLC are the small-molecule TKIs erlotinib and gefitinib, and the monoclonal antibody cetuximab. Unfortunately, none has been as successful as hoped, but intense efforts are directed at understanding the mechanisms of response or resistance, with the goal of predicting which patients are the most likely to respond. The anti-vegf monoclonal antibody bevacizumab has also been studied in clinical trials of NSCLC. Anti-EGFR Erlotinib To date, erlotinib has proven to be the most successful anti- EGFR treatment for NSCLC. It is approved as monotherapy for locally advanced or metastatic NSCLC in patients who failed at least one prior chemotherapy regimen (usually platinum-based doublet chemotherapy). The trial supporting this indication was the BR.21 trial conducted by the National Cancer Institute of Canada. 108 Patients (n=731) with locally advanced or metastatic NSCLC were eligible if their disease had progressed on one or two prior chemotherapy regimens; more than 90% had received platinum-based chemotherapy. They were randomized in a 2:1 ratio to receive either erlotinib (150 mg daily) or placebo. The use of a placebo arm in this situation was considered ethical because patients have few options after failure of platinum-based doublet-chemotherapy and docetaxel. 109,110 The primary endpoint, median overall survival (Figure 10A), was significantly better in the erlotinib arm (6.7 months) as compared to the placebo arm (4.7 months; HR, 0.70; 95% CI 0.58-0.85; p<0.001). Erlotinib was also associated with better progression-free survival (Figure 10B) compared to placebo (2.2 months, versus 1.8 months; HR, 0.61; 95% CI 0.51-0.74; p<0.001). Complete or partial responses were observed in 8.9% (38/427) of evaluable patients in the erlotinib arm versus <1% (2/211) in the placebo arm (p<0.001). 12

Advanced Primer Fig 10. Copied from Shepherd. 108 Among patients receiving erlotinib, 19% required dose reduction, and 5% discontinued treatment because of adverse drug effects (vs. 2% and 2%, respectively, in the placebo arm). 108 The most frequent adverse effects associated with dose reduction in the erlotinib arm were rash (12%) and diarrhea (5%). Patients in the erlotinib arm also experienced a significantly higher rate of infections, although the authors suspected that this was due to longer follow up. However, analysis of quality of life suggested that patients receiving erlotinib fared better, especially in terms of time-to-deterioration of cough (p<0.04), dyspnea (p<0.03), and pain (p<0.04). Another phase III trial was conducted to examine the therapeutic value of adding erlotinib to platinum-containing regimens in the first-line setting for patients with advanced or metastatic NSCLC. 111 This trial concluded that there was no survival advantage to erlotinib in this setting and no apparent benefit in terms of response rate or time to progression. However, an exploratory analysis suggested that certain subgroups of patients may benefit, especially those with no history of smoking 111 and those with mutations in the EGFR gene (see Predicting Response below). 112 Considered together, the clinical trials of erlotinib indicate that it is a valuable option for second- or third-line therapy of NSCLC in patients who have progressed on platinum-based doublet chemotherapy and docetaxel or pemetrexed, or who are intolerant of chemotherapy. Currently, there is intense interest in defining the molecular characteristics of tumors that have a high probability of responding to erlotinib or another EGFR antagonist. Such knowledge may allow these agents to be used in highly selected patients most likely to benefit. Gefitinib Gefitinib was actually the first anti-egfr agent approved for use in patients with NSCLC, but its approval was based on uncontrolled phase II trials using surrogate endpoints instead of survival. However, a more recent phase III trial which enrolled patients with advanced disease that had progressed on 1 or 2 prior chemotherapy regimens failed to show that gefitinib therapy was associated with a survival advantage. Therefore, use of gefitinib has been restricted to patients who have benefited or are benefiting from its use. 113 Complete results of the phase III trial have not been published, but preliminary results indicate that gefitinib therapy was associated with median survival of 5.6 months (versus 5.1 months for placebo; HR 0.89; p=0.11). 113 Key results of the preceding phase II trials of gefitinib are presented in Table 3. 114,115 These trials compared 250 mg daily versus 500 mg daily gefitinib in patients with advanced NSCLC that had progressed on 1 or 2 prior chemotherapy regimens. The results suggest that gefitinib is active in this setting. Two phase III trials have studied the first-line use of gefitinib in combination with chemotherapy (paclitaxel and carboplatin, or, gemcitabine and cisplatin). 116,117 Both trials concluded that there was no benefit associated with adding gefitinib to chemotherapy in this setting. As with erlotinib, there is Table 3. Selected results of 2 phase II trials of gefitinib in advanced, chemotherapy-refractory NSCLC. 114,115 13

Signal Transduction Inhibitors in Cancer Therapy evidence that tumors with specific molecular characteristics may be more likely to respond to gefitinib (e.g., Lynch et al. 118 ), and intense research is being directed at identifying predictive markers, especially EGFR mutations predictive of response to gefitinib. 98 Cetuximab A phase III trial of cetuximab in NSCLC is ongoing but has yet to be published. Phase I/II studies suggest that the agent is active in patients with advanced NSCLC when used in combination with either first-line 119,120 or second-line chemotherapy. 121 Predicting Response In the heterogeneous populations of patients with NSCLC studied in clinical trials to date, the targeted EGFR antagonists particularly erlotinib have shown promise, if not overt efficacy. However, most clinicians and investigators would agree that the observed response rates and improvements in survival or progression-free survival have not met the expectations these drugs garnered during preclinical testing. This realization has led to intense interest and research into demographic or molecular predictors of response hoping to identify patients with a strong likelihood of benefiting from therapy. As discussed in the section on schnc, acneiform skin rash is commonly associated with oncologic response. 87 However, this predictive sign does not spare unresponsive patients from being needlessly exposed to the adverse effects of therapy. In some studies, EGFR overexpression, or EGFR gene polysomy or amplification, has been associated with higher response rates. 104,107,122,123 But this association is variable and may not be seen in multivariate analyses. 107,124 It is likely that such an association is real, but it is not reliable enough to guide selection of therapy. Several studies have also found that Asian patients, women, those with adenocarcinoma tumor histology, and those with no history of smoking are more likely to respond than patients without these demographic characteristics. 98,108,115,123 However, these characteristics are not strong enough predictors to justify excluding other patients as candidates for anti-egfr therapies. Interestingly, these same demographic characteristics are also associated with an increased probability that a patient s tumor is associated with a mutation in the EGFR gene. 98,107,124,125 This observation has led to a search for EGFR mutations that might prove to be strong predictors of response to anti-egfr therapies. A large number of studies have now provided evidence that mutations in the EGFR gene are associated with greater tumor response to gefitinib or erlotinib. 107,112,118,122,124,126-139 The mutations most commonly associated with enhanced responsiveness are in the tyrosine kinase domain of the EGFR molecule. As an example, consider the phase III trial of gefitinib discussed above. The available results from the entire trial indicated no survival benefit associated with gefitinib (vs. placebo). However, subsequent analyses indicated that EGFR mutations were associated with higher response rates. 122 An earlier study also found that specific EGFR mutations were associated with strong responses to gefitinib. 118 Indeed, the authors of this latter study concluded that screening for specific mutations may be a useful way to predict response. In a related analysis of erlotinib responsiveness as a function of EGFR mutation, Tsao and colleagues concluded that screening patients for EGFR mutations was not a useful predictor of response, even though response rates were higher in patients with EGFR mutations in the tyrosine kinase domain. 107 This conclusion is unlikely to be the last word on the issue. As more is learned about which specific mutations are associated with greater responsiveness, screening for specific mutations is likely to become of increasing predictive value. Research on this topic is rapidly evolving. Other research paths toward greater responsiveness point to the role of other members of the erbb family. For example, several studies have found that prognosis in NSCLC is correlated with overexpression of other members of the erbb family or co-overexpression of both EGFR and another member of the family, most notably HER2 and erbb3. 140,141 These observations suggest the potential utility of multiple kinase inhibitors or single inhibitors with broad specificity for members of the erbb family, such as CI-1033. 142 Anti-Angiogenesis Bevacizumab Bevacizumab in combination with chemotherapy as first-line therapy for NSCLC has been studied in phase II 143 and phase III trials. 144 In the phase III trial, 144 patients (n=878) with newly diagnosed advanced or treatment-naïve recurrent NSCLC were randomized to paclitaxel and carboplatin with or without bevacizumab. Patients with tumors of squamous cell histology were excluded. Chemotherapy was administered every 3 weeks for 6 cycles. Bevacizumab (15 mg/kg) was administered intravenously every 3 weeks until disease progression or unacceptable toxicity. The trial was halted early because the prespecified criteria for significance at the second interim analysis had been met. After a median follow-up of 19 months, median survival in the bevacizumab-containing arm was significantly longer than the chemotherapy-alone arm (12.3 months versus 10.3 months; HR 0.79; 95% CI 0.67-0.92; p=0.003; Fig 11A). 144 Progressionfree survival was also significantly better in the bevacizumabcontaining arm (6.2 months versus 4.5 months; p<0.001; Fig 11B). Among patients with measurable disease, objective responses were observed in 35% (133/381) of patients in the bevacizumab-containing arm, versus 15% (59/392) of patients in the chemotherapy-alone arm (p<0.001). Although addition of bevacizumab to paclitaxel and carboplatin was associated with significantly better overall survival, it was also associated with a significantly higher rate of death due to toxic effects of drug therapy. 144 Two such deaths occurred 14

Advanced Primer in the chemotherapy arm versus 15 in the bevacizumabcontaining arm (p<0.001). The causes of death in the bevacizumab containing arm were pulmonary hemorrhage (5), complications of febrile neutropenia (5), cerebrovascular event (2), gastrointestinal hemorrhage (2), and probable pulmonary embolus (1). Three patients in the bevacizumab-containing arm also died of cardiac events, but these were deemed unrelated to the study drug. Patients in the bevacizumab-containing arm also experienced significantly higher rates of grade 3 or higher hypertension, proteinuria, bleeding, neutropenia, febrile neutropenia, thrombocytopenia, hyponatremia, rash, and headache (all p<0.05). Bevacizumab is now approved for use in combination with paclitaxel and carboplatin as first-line therapy for NSCLC. Patients should be selected carefully on the basis of non-squamous cell tumor histology, and with consideration given to the risk for severe or life-threatening toxicity. Colorectal Cancer Inhibitors of EGFR and VEGF have also found success in the treatment of advanced colorectal cancer (CRC). Cetuximab and bevacizumab were first approved for treatment of advanced CRC in 2004, albeit in different settings as dictated by the pivotal clinical trials that led to their approval. Additional clinical trials have expanded the roles of these two agents in the treatment of advanced CRC. Furthermore, a number of ongoing trials are addressing issues of their clinical roles in various treatment settings and as part of combination regimens. Another monoclonal antibody inhibitor of EGFR, panitumumab, was approved in 2006 for treatment of patients with metastatic CRC after progression of disease on chemotherapy. Anti-Angiogenesis Bevacizumab The initial approval of bevacizumab for treatment of metastatic colorectal cancer arose from a successful trial of its use as firstline therapy in combination with IFL (irinotecan, fluorouracil, and leucovorin), 63 which was the standard of care at the time the trial was initiated. Patients receiving bevacizumab in combination with IFL achieved significantly longer median overall survival (20.3 months versus 15.6 months; p<0.001) and progression-free survival, as well as significantly better Fig 11. Copied from Sandler. 144 response rates in comparison to patients receiving IFL alone. 63 As a result, bevacizumab was approved for firstline use in combination with 5-fluorouracil (5-FU)-containing chemotherapy. During this same period, oxaliplatin began to supplant irinotecan as part of 5-FU-containing regimens (cf. Colucci et al. 145 ), mostly because of a better toxicity profile. To examine the use of bevacizumab in combination with newer first-line regimens, several ongoing clinical trials were expanded to include bevacizumab-containing arms in combination with chemotherapy regimens. Recent results from two of these trials support the safety and efficacy of adding bevacizumab to oxaliplatin-containing regimens (such as FOLFOX4) for first-line therapy. 63,146,147 Bevacizumab also improves efficacy when added to first-line regimens including only 5-FU and leucovorin. 148 Bevacizumab has also been studied as second-line therapy in combination with FOLFOX4 in patients who progressed on 5-FU- or irinotecan-containing regimens and who did not receive bevacizumab as part of first-line therapy. 149 In this trial conducted by the Eastern Cooperative Oncology Group (ECOG), patients were randomized to one of three arms: FOLFOX4, FOLFOX4 + high-dose bevacizumab (10 mg/kg every 2 weeks), or high-dose bevacizumab alone. Enrollment in the bevacizumab-alone arm was halted after an interim analysis provided evidence of inferior efficacy. Results discussed here will include only the two remaining arms. As shown in Table 4, patients in the bevacizumab-containing arm had significantly longer median overall survival and progression-free survival, and significantly higher objective response rates as compared to patients in the FOLFOX4-alone arm. Patients in the bevacizumab-containing arm experienced higher rates of hypertension, bleeding, vomiting, and neuropathy as compared to the FOLFOX4-alone arm. The increased incidence of neuropathy in the FOLFOX + bevacizumab arm may be due to the fact that those patients were likely to be on treatment longer and were thus exposed to greater cumulative doses of oxaliplatin, which is known to be associated with risk of neuropathy at high cumulative doses. This trial established the efficacy of bevacizumab as second-line therapy in selected patients. At this time it is unclear if bevacizumab should be continued from first-line therapy to second-line therapy after 15

Signal Transduction Inhibitors in Cancer Therapy Table 4: Results of a phase III trial of FOLFOX4 vs. FOLFOX4 + bevacizumab for second-line therapy of metastatic CRC. 149 failure of first-line therapy. New approaches that blur the distinctions between first- and second-line patients may help address such questions. 150 Anti-EGFR Cetuximab Cetuximab was first approved for use in CRC as secondline therapy in patients who had progressed on first-line therapy with irinotecan. 151 This trial showed that both cetuximab monotherapy and cetuximab in combination with irinotecan were active in patients who had progressed on irinotecan. Interestingly, patients in the combination therapy arm had significantly higher response rates and significantly longer progression-free survival compared to cetuximab monotherapy, suggesting that cetuximab resensitized patients to irinotecan. 151 More recent trials have studied cetuximab in second- or third-line settings and in the first-line setting in combination with FOLFOX. One phase III trial was designed to assess cetuximab in combination with FOLFOX4 versus FOLFOX4 alone as second-line therapy in patients who had progressed on irinotecan-containing first-line therapy. 152 Because of the increasing use of oxaliplatin in the first-line setting, however, fewer patients were available for enrollment in this trial than anticipated. Nevertheless, with 102 EGFR-positive patients enrolled, 9 of 43 patients (21%) in the cetuximab plus FOLFOX4 arm achieved an objective response versus 4 of 42 patients (10%) in the FOLFOX4-alone arm. There was no significant difference in progression-free survival (4.4 and 4.1 months, respectively). Preliminary results are available from another trial, an uncontrolled phase II trial of cetuximab plus FOLFOX4 in the firstline setting. 153 In this study, complete responses were observed in 4 of 43 patients (9%), and partial responses were observed in 27 (63%). Ten patients (23%) exhibited stable disease, and the median progression-free survival was 10.2 months. In general, the adverse effects of cetuximab were manageable and often comparable to those observed in chemotherapyalone arms with the exception of skin toxicity. As in NSCLC, acneiform skin rash is commonly associated with an increased likelihood of response to cetuximab in patients with CRC. 154 Interestingly, however, studies to date have not found strong evidence that response to cetuximab is correlated with tumor expression of EGFR in patients with CRC. 155 Panitumumab Panitumumab is the most recently approved signal transduction inhibitor for advanced CRC. It is a fully human monoclonal antibody directed against the EGFR. Approval of panitumumab was based on an open-label, randomized, controlled trial in patients with heavily pretreated, metastatic CRC. 156,157 All patients were required to have disease that was positive for EGFR and that had progressed on prior chemotherapy regimens containing 5-FU, oxaliplatin, and/or irinotecan (the median number of prior chemotherapy regimens for metastatic disease was 2.4). Patients (n=463) were randomized to best supportive care or best supportive care plus panitumumab (6 mg/kg every 2 weeks). Patients in the panitumumab arm exhibited significantly longer median progression-free survival (96 days) as compared to patients receiving best supportive care alone (60 days; p<0.0001). Nineteen of 231 patients (8%) in the panitumumab arm had partial responses, versus no patients in the best supportive care arm. According to the available data, no significant survival difference has been observed between the two arms. As with cetuximab, panitumumab was associate with skin toxicity. 156,157 Combinations of Biologic/Targeted Therapies A small, exploratory trial has found preliminary evidence that the combination of bevacizumab and cetuximab is active in patients with advanced CRC who have progressed on irinotecan-containing first-line therapy. 158 In this trial, patients who had never received either therapy were randomized to receive bevacizumab and cetuximab with or without irinotecan. Among patients receiving bevacizumab plus cetuximab (without irinotecan) partial responses were observed in 8 of 35 (23%) patients, and the median time to progression was 4.0 months. Among patients in the irinotecan-containing arm 14 of 40 (35%) exhibited a partial response, and the median time to progression was 5.8 months. At this time, however, clinical use of bevacizumab plus cetuximab combination therapies must await the results of several ongoing trials. One such trial, the Phase III Intergroup/CALGB trial 80405, is randomizing treatment-naïve patients with metastatic CRC to either cetuximab, bevacizumab, or the combination. These randomized treatments will be administered in combination 16

Advanced Primer with FOLFOX or FOLFIRI according to each investigator s discretion. Another trial, the BOND3 trial, is studying the addition of cetuximab to therapy in patients who progressed on first-line bevacizumab-containing regimens. Finally, a phase III trial is studying the addition of panitumumab to a regimen of chemotherapy plus bevacizumab for first-line therapy. 159 Breast Cancer The prototypical signal transduction system implicated in the development and progression of breast cancer is the estrogen-progesterone system, in which the steroid estrogen binds to an intracellular receptor that also acts as a DNA transcription factor. 160 For women with hormone receptorpositive breast cancer, inhibitors of this system such as the aromatase inhibitors that block estrogen production or the selective estrogen receptor modulators that block the estrogen receptor are arguably the most thoroughly tested and proven therapies in all of oncology. 161 Unfortunately, a significant number of breast cancer tumors are hormone receptornegative, for which hormonal therapies have no value. In some cases of hormone receptor-negative disease, as well as some cases of hormone receptor-positive disease, researchers have demonstrated a crucial role for another member of the erbb family, erbb2/her2/neu (henceforth referred to as HER2). Like other members of the erbb family, HER2 is a tyrosine kinase that activates downstream signaling mechanisms. But unlike EGFR (erbb1) and other members of the erbb family, HER2 has no known ligand. 78 However, HER2 is able to dimerize with other members of the erbb family. 72 In this manner, HER2 acts as a coreceptor with other erbb receptor tyrosine kinase and is known as a particularly strong activator of downstream signal transduction pathways. 162,163 HER2 is normally expressed in many cells and, through dimerization with other erbb receptors, is involved in the regulation of cell proliferation, differentiation, and survival. 164-166 For unknown reasons, breast tumors are particularly susceptible to exhibiting HER2 overexpression. At the molecular and cellular level, overexpression of HER2 is associated with stimulation of cell proliferation, enhanced cell invasiveness, and stimulation of angiogenesis. 165 At the tissue level, tumors that overexpress HER2 are likely to have a number of distinguishing characteristics associated with poor prognosis. 167,168 These characteristics include poor differentiation, high proliferation rate, increased metastatic potential (as evidenced by nodal status), and increased risk of disease recurrence after primary treatment. 167,169 Overexpression of HER2 is observed in about 20-24% of breast tumors, and it is frequently, but not always, associated with amplification of the HER2 gene. 167,169 As discussed above, patients with HER2-overexpressing breast tumors have a worse prognosis compared to patients with normal tumor expression of HER2. 167 Breast tumors exhibiting overexpression of HER2 are also less likely to express hormone receptors (estrogen and/or progesterone receptors), 170,171 leaving many more patients without the valuable option of hormonal therapy. Some investigators have concluded that hormone receptor-positive tumors that also overexpress HER2 are less likely to respond to hormonal therapies, (e.g. Gago et al. 172 ), but others have found no evidence for such an association (e.g. Elledge et al. 173 ). However, HER2-overexpressing tumors are more likely to respond to certain chemotherapy regimens (specifically, anthracycline-containing regimens). 174 Anti-HER2 Therapy for Breast Cancer Because of the role of HER2 in signal transduction, the unique characteristics of HER2-overexpressing breast tumors, and the strong association of this molecular feature with disease aggressiveness and poor prognosis, HER2 is an attractive target for antitumor therapies for breast cancer. This observation has led to the development of specific agents targeting HER2, the most clinically advanced of which is trastuzumab, a humanized monoclonal antibody to HER2. Basic and clinical research on trastuzumab culminated in its approval, in 1998, for use in first-line treatment of metastatic, HER2-overexpressing breast cancer in combination with chemotherapy. 175 Recent trials have improved upon the success of trastuzumab plus paclitaxel regimens with the addition of a platinum-based agent. 176 In the meantime, other clinical trials have expanded the settings in which trastuzumab has proven efficacy in metastatic disease, including first-line monotherapy. 177,178 In general, the experience with trastuzumab in the setting of metastatic breast cancer has been consistent with observations from preclinical studies, in which the agent was most effective in tumor models that overexpressed HER2, and in which trastuzumab had additive or synergistic effects when used in combination with certain cytotoxic agents. 165 In 2005, results from 5 clinical trials provided remarkably strong and consistent evidence for overwhelming efficacy of trastuzumab in patients with HER2-overexpressing, early-stage breast cancer when used as part of adjuvant therapy regimens for operable disease. 179-182 One of the trials presented updated data from a second interim analysis in 2006. 183 Figure 12 shows trial schema for each of the 5 trials and Table 5 shows a summary of the treatment arms and disease-free survival for each trial (both from Gonzalez-Angulo et. al. 184 ). The key enrollment criteria were similar in all of the trials HER2-overexpressing early breast cancer treated with primary surgery. The FinHer trial 179 had additional enrollment criteria of node-positive or T2 (if node-negative) disease and negative assay for progesterone receptor (an indication that the estrogen receptor was either absent or dysfunctional). The HERA trial 180 allowed chemotherapy to be administered either after surgery (adjuvant) or before (neoadjuvant). The NSABP B-31 trial and the NCCTG N9831 trial were presented in a combined analysis. 181 17

Signal Transduction Inhibitors in Cancer Therapy Figure 12. Copied from Gonzalez-Angulo et al. 184 Several of the trials of adjuvant trastuzumab released results after the first interim analysis because their independent data monitoring committees concluded that there was overwhelming evidence of superior efficacy in the trastuzumab-containing arms compared to the control arms. 180-182 Considered collectively, the results of these 5 trials indicate that addition of trastuzumab to adjuvant chemotherapy reduces the risk of disease recurrence or death by about 50% in women with HER2-overexpressing early breast cancer. This remarkable degree of efficacy has led some to conclude that trastuzumab is the critical component of adjuvant therapy for this group of patients and that chemotherapy and hormonal therapy should be considered as adjuvant to trastuzumab. 184 Owing to the different treatment regimens used in the various trials, the regimen with the best balance of long-term efficacy and safety is still in question. It is hoped that additional follow up of these trials coupled with additional clinical trials will resolve these issues. Trastuzumab is not without drawbacks, the most serious of which is an increase in the rate of serious cardiac complications, particular congestive heart failure. This complication has been noted since the early clinical trials of trastuzumab in the metastatic disease setting 175 and is of particular concern when trastuzumab is combined with anthracycline-containing regimens or when trastuzumab is administered to older patients. 185 In a meta-analysis of seven trials of trastuzumab in the setting of metastatic breast cancer, cardiac dysfunction was found in 3-7% of patients receiving trastuzumab monotherapy, 13% of patients receiving trastuzumab in combination with paclitaxel, and 27% of patients receiving trastuzumab in combination with an anthracycline. 185 Most patients in this analysis (79%) showed improvement of CHF symptoms with standard treatment. 185 In the trials of adjuvant trastuzumab in early breast cancer, trastuzumab therapy was again associated with an increase in the rate of cardiac dysfunction in each of the trials. 178,180,181,183 In the four largest trials, rates of class III or IV heart failure ranged from 0.06-0.8% in the control arms 18

Advanced Primer Table 5. Copied from Gonzalez-Angulo et al. 184 versus 0.38-4.1% in the trastuzumab-containing arms. 178,180-182 The lowest rates in any trastuzumab-containing arm were observed in the BCIRG006 trial. 182,183 At the second interim analysis, patients receiving concurrent docetaxel, carboplatin, and trastuzumab (TCH) had significantly lower rates of class III/IV CHF (4 of 1075 patients; 0.38%) as compared to patients receiving 4 cycles of doxorubicin plus cyclophosphamide followed by docetaxel plus trastuzumab (AC TH; 20 of 1068 patients; 1.9%; p<0.0015). At that same analysis, the diseasefree survival rates were comparable in these two arms (both significantly better than non-trastuzumab-containing arm (AC T). It is hoped that additional follow-up of patients in these trials, coupled with additional studies, will clarify which regimen for use of trastuzumab in early breast cancer represents the best possible balance of efficacy and safety. Other Signal Transduction Inhibitors in Breast Cancer Patients with breast cancer receiving either hormonal therapies or trastuzumab are known to be at risk of developing resistance to these agents. Preclinical studies have identified several potential biological explanations for such resistance, some of which involve crosstalk with other signal transduction pathways, including the EGFR pathways. 186-188 Indeed, various preclinical studies have found evidence that inhibitors of EGFR and/or HER2 may improve or restore responsiveness of breast tumor cells to hormonal agents. 189-193 Similarly, dual inhibition of HER2 and EGRF has been proposed as a potentially valuable therapeutic strategy in patients who have developed resistance to the HER2 inhibitor trastuzumab. This latter concept has now led to the development and very recent approval of lapatinib for treatment of metastatic, HER2-overexpressing breast cancer in patients who have progressed on trastuzumab-containing therapy. Lapatinib is another small-molecule tyrosine kinase inhibitor that targets both EGFR and HER2. 194 A phase III trial of lapatinib enrolled women with HER2-overexpressing, metastatic breast cancer that had progressed on anthracycline-, taxane, and trastuzumab-containing regimens. 195 Patients were randomized to receive either capecitabine or capecitabine plus lapatinib. At the first interim analysis, the data monitoring committee determined that the criteria for release of data had been satisfied on the basis of overwhelming efficacy of the combination-therapy arm. The median time to progression among patients in the capecitabine plus lapatinib arm (8.4 months) was significantly longer than the capecitabine-alone arm (4.4 months; HR for time to progression, 0.48; 95% CI 0.34-0.71; p<0.001). Lapatinib was not associated with significant increases in serious adverse events. 195 Thus, lapatinib provides a dramatic improvement in treatment options for patients who have failed other therapies for HER2-overexpressing breast cancer. Such strong evidence for the efficacy of lapatinib in heavily pre-treated patients warrants studies of this TKI in earlier stages of therapy. Bevacizumab has also been studied in phase III trials in women with advanced breast cancer. One trial studied bevacizumab in combination with capecitabine in patients with metastatic breast cancer who had progressed on previous treatment using an anthracycline and a taxane. 196 A second trial studied the addition of bevacizumab to paclitaxel for firstline therapy in women with locally recurrent or metastatic breast cancer. 197 Both trials have found strong evidence for efficacy of bevacizumab in these settings. Thus, although it is not yet approved for use in breast cancer, bevacizumab is likely to become an important treatment option for women with advanced breast cancer. Conclusions As the science of cancer treatment has evolved into its current form, in which molecularly targeted agents play a central role, clinicians have witnessed both disappointments and startling successes. But even the disappointments have been accompanied by improvements in our understanding of disease processes and mechanisms. Although it is tempting to credit molecular techniques for many of the advances witnessed in recent decades, most advances have arisen from 19