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© 2003 American Society for Clinical Oncology Challenges of Developing Therapeutics That Target Signal Transduction in Patients With Gynecologic and Other MalignanciesFrom the Institute for Drug Development, Cancer Therapy and Research Center, San Antonio, TX. Address reprint requests to Eric K. Rowinsky, Institute for Drug Development, Cancer Therapy and Research Center, 7979 Wurzbach Rd, Fourth Floor Zeller Building, San Antonio, TX 78229; email: erowinsk{at}saci.org.
A greater understanding of cancer biology and major advances in biotechnology have resulted in the identification of a plethora of rationally designed, target-based anticancer therapeutics, particularly those that inhibit malignant-cell signal transduction, ushering in new therapeutic opportunities and extraordinary developmental challenges. Because these agents seem to principally target malignant cells, it is expected that they will produce less toxicity at clinically effective doses than nonspecific cytotoxic agents. The innate complexity of signaling networks, which have redundant relay systems that confer robustness, adaptability, and signaling diversity, also decreases the probability that any single therapeutic manipulation against any specific signaling element will be highly successful when used alone, particularly in patients with solid malignancies that have multiple relevant signaling aberrations. In addition, because the predominant therapeutic effect of inhibitors of signal transduction processes in preclinical studies is a decreased rate of tumor growth, it is anticipated that the predominant therapeutic outcome in the clinic will be similar; however, this end point is not readily detectable or quantifiable using traditional clinical evaluation methods. Furthermore, the results of preclinical and early clinical studies indicate that dose-toxicity relationships are not likely to be as steep as with nonspecific cytotoxic agents. Therefore, both regulatory and clinical practice end points, such as time to disease progression, disease-related symptoms, and quality of life, which are generally considered secondary for cytotoxic agents, may evolve into primary end points. The cumulative results of developmental evaluations to date indicate that the development, evaluation, and general clinical use of rationally designed, target-based anticancer therapeutics will require a radical departure from traditional paradigms to exploit the full potential of these new therapies.
The convergence of a plethora of recently acquired information about specific molecular abnormalities that drive the malignant phenotype with profound advances in biotechnology has given rise to an era of abundant novel therapeutic options for patients with gynecologic and other malignancies.1–10 It has also initiated many unique challenges for clinical investigators. As anticancer therapeutics with distinct targeting capabilities against malignant cells are developed, prioritization of these therapies for efficient allotment of clinical trial resources, identification of patients whose malignancies most likely express the molecular constituents resembling the true target, and derivation of relevant end points for both screening and assessment of clinical relevance will be critical to their successful development and optimization. The aforementioned projections are largely based on the results of a wide array of preclinical and early clinical evaluations of rationally designed, target-based agents in which rates of tumor regression are low, but dose-response and dose-toxicity relationships seem to be less steep than those of nonspecific cytotoxic agents. Thus, clinical and regulatory end points that are generally considered secondary for cytotoxic agents, such as improvements in disease-related symptoms and quality of life, may evolve into primary end points similar to the situations typically encountered in the assessment of therapeutic benefit in other medical disciplines. The number of rationally designed, target-based therapeutics undergoing development and evaluation is unprecedented.1–10 Whereas most anticancer compounds currently used in the clinic are nonselective in their actions against malignant and normal proliferative cells, it is presumed that rationally designed, target-based therapeutics will be far more selective against malignant cells, resulting in substantially less toxicity at effective doses. These agents can be classified by their specific target, such as farnesyltransferase (FTase) and ErbB receptor tyrosine kinase (RTK) inhibitors, or by their general mechanism of action, such as inhibitors of angiogenesis or cell-cycle modulators. They include natural products, small molecules, antibodies, ribozymes, gene therapy vectors, oligonucleotides, and peptides directed against critical elements of aberrant signal transduction pathways, aberrant cell survival pathways, survival genes and proteins, malignant angiogenesis, aberrant suppressor oncogenes, and aberrant epigenetic mechanisms. On the basis of the results of preclinical studies and early clinical trials to date, therapeutics directed against these targets are likely to result in less toxicity to normal bystander tissues and, hence, more breathing room to maximize the therapeutic indices of combination regimens.1–10 Because most of the new targets for antiproliferative therapies have not yet been validated in clinical practice, prioritizing the long list of rationally designed, target-based therapeutics entering clinical evaluations so that those with a high potential for improving clinical outcome are accurately identified for further study is a formidable challenge.
The broad term signal transduction refers to the means by which regulatory molecules that govern the fundamental processes of cell growth, differentiation, and survival communicate within the cell, resulting in the tight coordination of proliferative and other essential processes among various tissues. Signaling is astonishingly complex, involving a wide array of elements that interact through cascades of chemical signals arranged in intersecting and overlapping networks.4,11–13 Networks, consisting of redundant tracks, complex intersections, and intricate interconnections, enhance the robustness and diversity of signaling and permit fine-tuning and amplification and diminution of output, which may not be accomplished as efficiently by linear signaling cascades.11–14 The inherent redundancy of tracks within networks also confer protection against toxins, as well as decrease the likelihood that targeting any one specific signaling element will be highly successful in treating malignancies with many aberrations that contribute to the proliferative advantage. Targeting aberrant cell signaling is perhaps the most logical of ongoing developmental therapeutic endeavors against cancer because aberrations in signal transduction elements have been consistently demonstrated to enhance proliferation, invasiveness, metastasis, and angiogenesis and confer shortened survival and poor response to nonspecific cytotoxic modalities.15 The development of therapeutics against such processes is projected to yield broadly generalizable results because most malignancies possess at least one aberrant signaling element that confers a proliferative advantage.15 The most common aberrations result in unchecked or constitutive signaling activity or short-circuiting of elements that normally regulate cell signaling.11–14,16,17 In contrast to the situations represented by chronic myelogenous leukemia and gastrointestinal stromal cell tumors, in which a single aberration is the sole driver of tumor proliferation and its successful targeting results in dramatic antitumor effects, most malignancies possess multiple aberrations, many of which likely confer proliferative advantages.15,17–19 However, targeting one specific driver in a tumor that has multiple aberrations can hypothetically still produce efficacy, the magnitude of which relates to the relative importance of the driver itself. Nevertheless, even if the overall efficacy achieved by targeting only one of many drivers may be somewhat limited, the innate selectivity of the agent for the malignant cell over the normal cell may impart a favorable therapeutic index.
Most current developmental efforts thwarting signal transduction processes are being directed against either membrane receptors or elements that comprise downstream signaling cascades. With regard to signal transduction receptors, developmental efforts are predominantly being directed against RTKs and G-protein receptors (GPCRs), which have secondary relay systems that permit signal amplification, diversification, and cross-talk.11,12,16 The complexity of signal transduction networks and the challenges related to the development and evaluation of therapeutics against these intricate systems are exemplified by the complex structural and functional aspects of the ErbB receptor family and related downstream processes, particularly with respect to the multifactorial determinants of output. When ErbB family receptors are overexpressed or constitutively overactivated, outputs include proliferation, invasiveness, angiogenesis, and resistance.13,16 Members of the ErbB receptor family include ErbB1 (also called EGFR), ErbB2 (Her2/neu), ErbB3, and ErbB4, which are often overexpressed in malignancies derived from epidermal tissues, including ovarian and other gynecologic malignancies.16 Ligand binding induces conformational changes in the receptor, which, in turn, facilitates dimerization with other ErbB receptors.13,16,20–25 Once ErbB receptors have dimerized, conformational changes favor phosphorylation or activation of specific tyrosine residues. Activated ErbB receptors, in turn, activate specific downstream signaling elements, thereby transducing chemical signals intracellularly.13,16,20–26 The specificity, potency, and diversity of intracellular signals are determined, in part, by positive and negative effectors of ErbB proteins by the identity of the ligand, dimer composition, and specific structural determinants of the receptors. However, the principal determinant is the vast array of phosphotyrosine-binding (PTB) proteins that associate with the C-terminal downstream docking tail of each ErbB molecule after engagement into dimeric complexes.13,22–26 The diversity of signals generated through the ErbB receptor family is largely determined by differences in the amino acid sequences of the C-terminal domains of the receptors.13,23,25,26 These critical sequences, which contain tyrosine residues that undergo phosphorylation on ligand binding and receptor dimerization, represent docking sites for various proteins involved in signal transduction.13,23,26 Docking sites are provided for proteins containing Src homology 2 or PTB domains, which recognize specific phosphotyrosine residues in the context of their surrounding amino acids. Each ErbB receptor displays a distinct pattern of C-terminal autophosphorylation sites. At least for ErbB2, which does not have a direct activating ligand, these PTB sites are essential for its transforming properties. It is now evident that there is a great deal of overlap in the signaling pathways activated by the four ErbB receptors. For example, the mitogen-activated protein kinase (MAPK) pathway, via Shc and Grb2, is an invariable target of all ErbB family members. However, there are also specific examples of preferential modulation of specific pathways. This is illustrated by the presence of multiple binding sites for the regulatory subunit of phosphatidylinositol 3-kinase (PI3K) on ErbB3 and ErbB4, which renders these receptors the most efficient activators of the PI3K pathway.19 Simultaneous activation of linear cascades, such as the MAPK pathway, the stress-activated protein kinase cascade, protein kinase C, and the PI3K pathway, translates in the nucleus into distinct transcriptional programs, the culmination of which is the net cellular response. The principal process by which ErbB signaling is turned off is ligand-mediated receptor endocytosis, and the kinetics of this process are often understated with regard to the overall magnitude of signaling.13,14,20,27–31 The kinetics of signal degradation are determined in part by the composition of the receptors. For ErbB, ligand stimulation results in rapid endocytosis and degradation of both the receptor and ligand. Ligand binding induces receptor clustering in clathrin-coated pits on the cell surface, followed by endocytosis, migration to multivesicular bodies, and eventual lysosomal degradation.11,13,14,20,27–31 Degradation of the ErbB receptor is dependent on RTK activity, and kinase-negative receptor mutants generally recycle to the cell surface for reuse. ErbB1 is more prone to degradation via endosome formation and hydrolysis, whereas the other ErbB receptors are relatively endocytosis impaired and tend to be recycled back to the cell surface.26,31 The rapid endocytosis and degradation of the activated ErbB receptor attenuate the signal generated at the cell surface in response to growth-factor stimulation. The particular mode and site of degradation are also determined in part by the composition of the dimer. For example, ErbB1 homodimers are processed primarily to the lysosome, ErbB3 molecules are constitutively recycled, and heterodimerization with ErbB2 decreases the rate of endocytosis and increases the recycling of its partners.14,28–30 ErbB2 homodimers, which are stable in endosomal vacuole, are rapidly tagged with ubiquitin and processed for digestion, resulting in weak signals, whereas ErbB2 heterodimers are relatively unstable in the endosome, resulting in a lower rate of degradation and a higher rate of receptor recirculation.32,33 To make matters even more complex, networks integrate heterologous signals from other networks. In the case of ErbB, heterologous signals, including those induced by hormones, neurotransmitters, lymphokines, and stress inducers, are integrated into downstream messengers.13 These interactions are mediated by protein kinases that directly phosphorylate the ErbB receptors, thereby affecting their kinase activity or endocytotic transport. One type of trans-regulatory mechanism involves the activation of GPCRs, such as those for lysophosphatidic acid, thrombin, and endothelin. Agonists of GPCRs may result in a net increase of tyrosine phosphorylation of ErbB1 and ErbB2. By a poorly defined mechanism, these agonists can also activate matrix metalloproteinases, which then cleave membrane-tethered ErbB ligands (eg, heparin-binding epidermal growth factor [EGF]), thereby freeing them to bind to ErbBs. Activation of GPCRs may also activate Src family kinases, leading to phosphorylation of tyrosine residues on the intracellular domains of ErbB. These activities can subsequently trigger events downstream of ErbB1, possibly contributing to the mitogenic potential of heterologous agonists. Furthermore, interconnections between other signaling pathways help to integrate and coordinate cellular responses to extracellular stimuli.34 The ErbB receptor family and related signaling network provide enormous signaling diversity at many levels, including ligand specificity, receptor partnering, providing scaffolding sites for effector signaling proteins and substrate specificity for their kinase activities, receptor degradation, and integration of heterologous signals.13,16,21–24,26 In addition, diversity between different types of cells and tissues can exist, depending on the expression levels and the preferred stoichiometry for interactions of the receptors and ligands. Taken together, it is clear that ErbB receptors couple to specific downstream pathways with differing efficiencies, thus affording an astonishing range of signaling possibilities. The particular cellular response to ErbB stimulation is a function of the cellular context, as well as the specific ligand and ErbB dimer. This has been shown best for mitogenic and transforming responses; homodimeric receptor combinations are less mitogenic and transforming than the corresponding heterodimeric combinations, and ErbB2-containing heterodimers are the most potent complexes. One of the best examples of the ability of the ErbB receptor family to generate a specific response is provided by the ErbB2-ErbB3 heterodimer. Although neither ErbB2 nor ErbB3 alone can be activated by ligand, the heterodimer is the most transforming and mitogenic receptor complex. The ErbB2-ErbB3 heterodimer also increases cell motility on stimulation with a ligand, but the other neuregulin receptor, ErbB4, which exists in several isoforms, has been associated with processes varying from cellular chemotaxis to proliferation and differentiation.
Many signaling processes are currently being targeted with various types of therapeutics. Nevertheless, there is concern that targeting any single aberrant signaling element of a complex network may be insufficient to produce a relevant degree of tumor growth inhibition, except in situations in which the aberration itself is the principal driver of tumor growth. Conversely, a relevant degree of tumor growth inhibition may result from targeting any one of many aberrant signaling elements, provided the element is a major driver of tumor growth and that normal cells are relatively unaffected. Furthermore, because the magnitude of tumor growth inhibition may be substantially less than that incurred in situations in which the aberration is the sole, albeit clinically relevant, driver of tumor proliferation, an optimal therapeutic paradigm may be one in which several different therapeutics are administered in tandem.18,19 An alternative approach to treating malignant neoplasms, in which the target is likely to be one of many drivers of proliferation, is the administration of rational combinations of different signal transduction inhibitors.
Targeting ErbB2 (HER2/neu) with monoclonal antibodies (trastuzumab; Genentech Inc, South San Francisco, CA) has resulted in impressive clinical activity, albeit limited to patients with breast cancer whose tumors have amplified HER-2/neu (ErbB2).14,35,36 Many other therapeutics that specifically target ErbB2, such as small-molecule RTK inhibitors (TAK-165; Takeda, Osaka, Japan; and CP724,714; Pfizer Inc, Groton, CT) and monoclonal antibodies targeting ErbB-containing heterodimers (2C4; Genentech), are under development.37,38 Still other investigational agents bind to the extracellular domain of ErbB subfamily members (eg, cetuximab, IMC-225; Imclone Systems Inc, New York, NY; EMD72000; Merck KGaA, Darmstadt, Germany; and ABX-EGF; Abgenex Inc, Freemont, CA); h-R3 (National Institute of Oncology, Havana, Cuba) and MDX-447 (Medarex Inc, Annadale, NJ) bind specifically to the extracellular domain of ErbB1, whereas other agents reversibly inhibit the tyrosine kinase (TK) activity of ErbB1 (eg, gefitinib, ZD1839; AstraZeneca, London, United Kingdom; erlotinib, OSI-774; OSIP Inc, Mellville, NY; and PKI116; Novartis, Basel, Switzerland) or multiple ErbB receptor subfamilies (eg, GW572016; Glaxo SmithKline, Middlesex, United Kingdom, inhibits RTK of ErbB1 and ErbB2).9,10,16,39–44 Still other small molecules form irreversible covalent linkages with cysteine residues in the TK domains of several types of ErbB receptors. For example, CI-1033 is an irreversible inhibitor of all four ErbB family member TKs, whereas EKB-569 irreversibly inhibits the TK activity of both ErbB1 and ErbB2.45,46 The relative therapeutic merits of antibodies versus small molecules, inhibitors of a single RTK versus multiple RTKs, and reversible receptor binding versus irreversible receptor binding are not known at this juncture, and therefore, clinical benchmarking studies are required to address these issues. In clinical evaluations, largely in unscreened patients or patients whose tumors demonstrate any degree of ErbB1 expression by immunohistochemical analysis, low rates of objective tumor regression have been observed in non–small-cell lung, colorectal, renal, head and neck, and ovarian carcinomas.16 Few studies have been performed to date in patients with gynecologic malignancies. In a phase II study of OSI-774 in previously treated patients with ovarian carcinoma and any degree of ErbB1 expression, three (10%) of 30 heavily pretreated subjects had partial responses and 15 (50%) had stable disease as their best response.17,47 For the most part, nonrandomized clinical evaluations with signal transduction inhibitors to date have not been designed to robustly assess the degree to which these agents affect other clinically relevant end points, such as time to tumor progression, survival, and tumor-related symptoms. Other important challenges will be to discern which patients have the highest likelihood of responding to ErbB-targeted therapies based on the biologic features of their tumors and whether these therapeutics should be developed concomitant with nonspecific cytotoxic agents. The latter issue emanates from the results of two phase III studies (Iressa Non–Small-Cell Lung Cancer Trial Assessing Combination 1 and 2) in which untreated patients with advanced non–small-cell lung carcinoma were randomly assigned to treatment with either standard cytotoxic therapy alone or standard cytotoxic therapy plus one of two doses of gefitinib.48 Although the failure of the experimental arms to demonstrate superior survival, response, and time to tumor progression could have been a result of many factors, including the inherent difficulty of affecting this specific disease, the substantial molecular heterogeneity of the patients regarding the presence of the target, the possibility of antagonist interactions between ErbB-targeting therapies that induce prominent arrest in G1, and cell-cycle dependent cytotoxic agents must also be considered.
Ras is an integral signaling element and has been characterized as the primary switch that transmits proliferative signals through numerous intracellular signaling pathways. Ras belongs to a superfamily of guanine nucleotide triphosphatases that regulate a variety of cellular processes.49–52 At least 20 members of this superfamily are known. Three genes encode Ras proteins (neuroblastoma [N]-ras, Harvey [H]-ras, and Kirsten [K]-ras); the genes are closely related and similar in their ability to interact with both regulators and effectors. Ras mutations, which induce downstream signaling along many pathways by structural modifications that favor the active functional guanine nucleotide triphosphatase–binding form, are found in approximately 33% of all malignancies and invariably confer proliferative advantages of varying magnitudes. Ras is synthesized as a soluble protein that becomes membrane bound and functional after a complex series of posttranslational protein modifications, the most important of which is the addition of a 15-carbon farnesyl isoprenyl tail by a process known as farnesylation. This process is mediated by the enzyme FTase, which has become a major target for therapeutic development against both hematologic and solid malignancies. At this juncture, various farnesyl transferase inhibitors ([FTIs]; lonafarnib, SCH6636; Schering-Plough, Kenilworth, NJ; tipifarnib, R115777; Ortho Biotech, Bridgewater, NJ; and BMS214662; Bristol-Myers Squibb, Princeton, NJ) are undergoing broad clinical evaluations.9,10,49,53 Nevertheless, the results of disease-directed phase II and III studies in neoplasms that have high incidences of Ras mutations, albeit mostly K-ras mutations (eg, colorectal and pancreatic carcinomas), have been disappointing. Although tumors with K-ras mutations have been demonstrated to be sensitive to the growth-inhibitory effects of the FTIs in preclinical studies, the preponderance of experimental data indicate that H-ras–dependent tumors are relatively sensitive and K-ras–dependent tumors are relatively resistant.49 These observations are most likely a result of the fact that K-ras can be alternatively prenylated. Interestingly, several FTIs have demonstrated relevant degrees of clinical activity against malignancies that generally have low incidences of ras mutations. For example, tipifarnib has demonstrated intriguing activity in patients with refractory breast carcinoma, high-grade astrocytoma, acute myelogenous leukemia, myelodysplastic syndromes, and myeloproliferative disorders.53–57 One possible explanation for these unexpected results is that FTase is critical to the functionality of many critical proteins that have proliferative advantages of varying degrees, in addition to processes related to mutated ras. For example, FTase is required for the functionality of essential mitotic kinesins, components of the PI3K/Akt pathway, RhoB, and others, all of which may contribute to the effects of the FTIs.49,58–60
The MAPK pathway serves as a point of convergence for a wide array of signals initiated by the cell membrane, and the network of phosphorylation-mediated signals emanated from MAPKs are equally expansive.61–68 In the MAPK pathway, the Raf-1–mitogen-activated ERK kinase (MEK)–extracellular signal regulated kinase (ERK) module is used ubiquitously in the transduction of cell type–specific growth and differentiation signals from RTKs and GPCRs. The MAPK pathway does not function in isolation but, instead, is integrated into various cellular signaling networks that affect and are affected by MAPK signaling. Corresponding to the prominence of MAPKs in numerous signaling events, perturbations in the MAPK signaling pathway can have profound pathologic consequences. Therapeutic efforts are being directed at various components of the MAPK pathway; however, the Raf-1-MEK-ERK module seems to be most relevant at this time.
Raf-1 (C-Raf)
MEK (also called MAPK kinase)
ERK
Akt kinase/protein kinase B, which is transduced by PI3K and PDK1 and negatively regulated by the lipid phosphatase PTEN, is the focal point for survival signals from growth and survival-factor receptors.15,73,74 Akt kinase, when phosphorylated and activated, inhibits apoptosis by phosphorylating a variety of substrates, including apoptosis effectors such as FKHR, GSK-3, and caspase-9. The dominant survival function of Akt is emphasized by the frequent causal role in many cancers of activating aberrations in the PI3K/Akt pathway. The most important of these aberrations involves deletions and other mutations of PTEN, which occur in many solid malignancies, particularly high-grade astrocytoma and breast and endometrial carcinomas.75–79 Such mutations allow genomically compromised cells to survive and accumulate further DNA damage, leading to neoplastic transformation. Their pivotal roles in cell survival have made both PI3K and Akt kinase important targets for therapeutic discovery. Many efforts are being pursued along these lines, but such early attempts have been associated with decreased specificity.
mTOR, which plays a critical role in the transduction of proliferative signals mediated through the PI3K/Akt signal transduction pathway, regulates the initiation of protein translation by altering the phosphorylation states of the translational regulators eukaryotic initiation factor 4E-binding protein and of p70s6k, the 70-kDa S6 kinase. Inhibition of mTOR function abolishes the proliferative signal mediated through the PI3K/Akt signal transduction pathway and results in cell cycle arrest in cancer cell lines.15 Therefore, inhibition of mTOR has the potential to inhibit tumor growth, and it has become a target for the development of novel cancer therapeutics. One such agent, rapamycin, a macrolide fungicide from the bacterium Streptomyces hygroscopicus, is a specific inhibitor of mTOR that exerts potent antimicrobial, immunosuppressant, and antineoplastic properties.15 Because of its profound immunosuppressive actions, rapamycin was initially developed and received regulatory approval for prevention of allograft rejection after organ transplantation. However, impressive antiproliferative activity occurs after treatment of a diverse range of experimental tumors, particularly tumors with PTEN mutations and other aberrations that activate the PI3K pathway.15,75–79 The antiproliferative actions of rapamycin seem to be a result of its ability to bind to the intracellular immunophilin KFB506 Binding Protein-12, and the complex then binds to and inhibits the activity of mTOR.15,80 This action blocks the linkage of mTOR to critical signal transduction pathways and the synthesis of proteins required for cell cycle traverse from G1 to S.15 However, the poor solubility and chemical stability of rapamycin precluded its clinical development as an anticancer agent, and several more feasible rapamycin analogs, including CCI-779 (Wyeth Ayerst, Philadelphia, PA) RAD001 (Novartis), and AP23573 (Ariad Pharmaceuticals, Cambridge, MA), are under development.15
Phase I and Feasibility Studies In contrast to the development of cytotoxic agents, in which toxicity in rapidly growing tissues relates, albeit loosely, to anticancer activity and can be used as a rough measure of drug effect, selecting an optimal dose of signal transduction inhibitors for subsequent disease-directed studies is a formidable challenge. Toxic effects may not be evident at doses that inhibit the purported target or are even related to target inhibition. Although pharmacologic studies may be used to gauge whether parameters associated with maximal inhibition of the target and antitumor activity in preclinical studies are being achieved in patients, interspecies differences in drug distribution in tissues, protein binding, clearance, and metabolism may preclude extrapolating results from animals to humans, thereby limiting the usefulness of pharmacologic comparisons. The development and validation of assays that reflect relevant drug effects in accessible tissues will undoubtedly facilitate efforts to define the optimal doses of signal transduction inhibitors in phase I evaluations. These assays may be helpful in selecting doses of therapeutics that have the highest likelihood of achieving maximal inhibition of the target as long as they have been validated to reflect the desirable target effect. Another important issue is discerning the optimal means to administer signal transduction inhibitors, particularly in regard to optimal scheduling and dose. In general, the preponderance of preclinical results to date indicates that continuous long-term treatment may be a highly effective means to achieve maximal and sustained efficacy. However, protracted treatment raises concerns about both acquired drug resistance and toxicity. In addition, protracted exposure may predispose patients to several unique toxic effects, which must be factored into the general equation regarding optimal scheduling. Furthermore, any long-term toxic effects associated with protracted continuous treatment may not be appreciated with standard preclinical toxicologic methods that are typically used in preclinical studies of conventional cytotoxic agents and principally focus on highly proliferative tissues. For rationally designed, target-based therapeutics, it will be important to rigorously monitor organs in which the target is highly expressed or that plays a role in organ function.
Disease-Directed Screening Evaluations The challenges involved in designing disease-directed studies of signal transduction inhibitors and other rationally designed, target-based therapeutics arise from the difficulty in selecting end points to assess the relative merits of agents that portend relatively low rates of tumor regression or no regressive effects whatsoever. Although many types of signal transduction inhibitors have been demonstrated to induce regressions of some well-established experimental tumors,69–71,81,82 their predominant therapeutic effect in preclinical studies is tumor growth delay, often referred to somewhat inappropriately as cytostasis. Although the net growth of any particular tumor may not change, the individual cells comprising the tumor are in a state of dynamic flux. Thus, the net change in the size of a tumor reflects the sum of the rates of tumor cell death and tumor cell proliferation. Hypothetically, when the rate of tumor cell growth is decreased or the rate of cell death is increased, so that the rates of cell proliferation and cell death are equivalent, there should be no change in the tumor size (ie, stable disease). However, a more pronounced decrease in the rate of tumor cell proliferation or a more profound enhancement of tumor cell death may theoretically result in a scenario in which cell death exceeds cell proliferation, thereby producing tumor regression. In fact, the regression of well-established tumors has been documented after treatment with many types of signal transduction inhibitors in preclinical studies.69–71,81,82 Unfortunately, this scenario is not noted with all types of signal transduction inhibitors, which, nonetheless, may produce clinical efficacy by substantially inhibiting tumor growth, leading to increased time to progression and survival. On the basis of this logic and the results of preclinical studies to date, the most common favorable clinical outcome with antiproliferative agents is projected to be delayed tumor growth, which can be manifested in at least three distinct scenarios. In the first scenario, treatment does not completely arrest tumor growth but variably decreases the growth rate. In this situation, the magnitude of the antiproliferative effect may not be readily apparent to the clinician who cannot objectively quantify drug-induced effects on the rate of tumor growth short of overt regression. Instead, the clinician may interpret any tumor growth whatsoever as disease progression or therapeutic failure, although the decrement in the tumor growth rate may result in increased time to tumor progression or overall survival, as well as a global improvement in quality of life, for the individual patient. In the second scenario, a more profound antiproliferative effect results in a situation in which the rates of tumor cell proliferation and cell death are equivalent. This situation is often interpreted as stable disease or cytostasis, and the clinician is likely to continue treatment as long as the patient does not experience intolerable toxic effects. Although preclinical studies indicate that both situations are likely to be the most common favorable clinical scenarios after treatment with rationally designed, target-based antiproliferative agents, the beneficial effects may not be apparent or unequivocally attributed to the agent in nonrandomized phase II screening trials, which, in fact, is the real problem for clinical investigators. In the third scenario, which is projected to be less common than the aforementioned situations, the antiproliferative agent profoundly inhibits tumor cell proliferation and/or enhances tumor cell death, resulting in net tumor regression. This scenario may be expected to occur in situations in which the target is a major driver of tumor proliferation. Designing phase II and III disease-directed studies to gauge whether or not signal transduction inhibitors possess relevant antitumor activity is a formidable challenge. Although many types of therapeutics targeting signal transduction processes are capable of regressing some well-established tumors in animals, tumor growth inhibition may not be the principal therapeutic effect in situations in which the tumor is not driven by a solitary major aberration but rather multiple contributory aberrations of the specific target. Therefore, developmental plans that provide for clinical situations that are sufficiently sensitive to detect a relevant magnitude of tumor growth inhibition will need to be implemented in disease-directed clinical evaluations. Although many therapeutics targeting signal transduction processes are capable of inhibiting the growth of tumors with and without major functional aberrations of the target, nonrandomized screening evaluations may have the greatest likelihood of detecting meaningful clinical activity if performed in malignancies with inherently high rates of target aberrations. Therefore, nonrandomized studies are much more likely to be useful in screening rationally designed, target-based agents if patient eligibility is restricted to patients with malignancies that are likely driven by functional aberrations of the target. After restrictive and rigorous proof-of-principle trials, the scope of disease-directed evaluations can be broadened, and patient eligibility requirements can be less restrictive. Nonetheless, if there are compelling preclinical or early clinical data indicating that a particular rationally designed, target-based agent is capable of inducing tumor regressions, tumor regression may be a reasonable end point in phase II screening trials. However, such trials must be sized appropriately to provide sufficient statistical power to detect a low, albeit meaningful, level of major responses, so as not to reject a potentially useful therapeutic. Realistically, some indication that the therapeutic possesses relevant clinical activity and may be capable of modifying disease progression will ultimately be needed before resource-intensive phase III evaluations are begun. Although a compellingly high tumor regression rate will suffice as proof of principle, the potential of many therapeutics may not be appreciated if tumor regression is used as the sole end point in screening evaluations.83,84 Therefore, this traditional end point, which may be appropriate in nonrandomized phase II studies of signal transduction inhibitors that have a reasonable likelihood of inducing tumor regression, particularly in patients whose tumors are demonstrated to be driven by the target, may be suboptimal for agents in which tumor growth inhibition is expected to be the predominant therapeutic effect based on preclinical results. Alternate screening end points include survival, fractional survival at relevant intervals, time to tumor progression, proportion of patients without progressive disease as their best response, and quality of life.83–87 It may be reasonable to measure the relative time to tumor progression of patients receiving single-agent treatment with the therapeutic against the time to tumor progression of patients receiving treatment with a relevant standard therapy or supportive care, measured just before administration of the experimental agent. On the basis of experience with agents that were later shown to have relevant clinical activity in randomized trials, a 30% prolongation in the time to tumor progression may be a reasonable threshold to use before proceeding to phase III studies.88 As an alternative, exploratory single-arm or randomized phase II studies that are designed with sufficient power to detect and quantify tumor growth inhibition may provide meaningful leads about activity before randomized evaluations. For example, the percentage of patients surviving for at least 1 year in exploratory nonrandomized studies may be considered a reasonable end point to guide decisions about proceeding with randomized phase III evaluations.87,88 Considering the results of phase II and III studies of gemcitabine in patients with advanced pancreatic cancer, a novel therapeutic demonstrating a 1-year survival rate with a lower limit of a 95% confidence interval of at least 20% might be viewed encouragingly as a candidate for phase III development. Such thresholds would have to be established for other types of malignancies using retrospective data ascertained with therapeutics that have previously demonstrated therapeutic effect. On a similar note, the proportion of patients with progressive disease as their best response seems to inversely relate to the ultimate utility of any particular agent in a specific clinical setting, and a maximum acceptable threshold of patients with progressive disease as their best response may be used to forecast the usefulness of the agent.83–86 A retrospective analysis of the National Cancer Institute of Canada Clinical Trial Group’s phase II studies of new cytotoxic agents indicates that the rates of disease progression (ie, the proportion of patients whose disease progressed in the first 8 weeks of therapy) in patients treated with agents thought to be the most promising in breast carcinomas, lung carcinomas, and glioma seem to be less than 20%, 30%, and 40%, respectively.83,84 In this review of the group’s phase II experience, agents identified as interesting because they surpassed a minimum response rate (ie, the usual phase II decision threshold) were the same agents that would have been selected because they produced less than a maximum rate of progressive disease. Thus, ignoring the response rate would have identified the same active agents. The use of such threshold values, especially those that have been validated, may be useful in screening antiproliferative agents before undertaking randomized trials. However, historical data may not be appropriate for such comparisons in all instances because progressive improvement in supportive management during the last several years may have affected the natural progression of disease over time. In contrast to the traditional notion of randomized phase II studies, which lack the requisite robustness and statistical power to discern small-to-moderate, but relevant, differences between treatment groups, the randomized discontinuation design uses the process of natural enrichment to screen for relevant tumor growth–inhibitory activity before resource-intensive phase III evaluations.89,90 At the end of an initial evaluation period, the duration of which is somewhat dependent on the mechanism of action of the therapeutic, patients who experience significant tumor growth, toxic effects, or noncompliance are removed from the study. This initial weeding-out process enriches the randomized patient population with those individuals who may most likely benefit from the agent, thereby increasing the likelihood that the subsequent randomization step will be more efficient at detecting drug-induced effects on clinical end points that reflect the rate of tumor growth. The remaining patients are randomly assigned to either continue or discontinue treatment, ideally in a double-blind, placebo-controlled fashion. Because randomization does not occur until after the initial evaluation period, eligibility criteria can be relatively nonrestrictive. The natural selection or enrichment of the population of patients who eventually undergo randomization increases the efficiency of the trial, with as little as 20% of the standard number of randomly assigned patients. Thus, this design may markedly shorten the phase II stage of therapeutic development and also yields information about the rate of tumor regression. Nonetheless, a potential shortcoming of the approach relates to an inability to precisely quantify the magnitude of antitumor activity. However, if there is a clear difference in disease progression between the patients who continue with treatment and those randomly assigned off-treatment, a general conclusion can still be made in regard to the activity of the agent. A principal concern regarding this design is whether the new agent had the potential to produce a prolonged effect after a short course. The randomized discontinuation design is being used by the Cancer and Leukemia Group B and Bayer Pharmaceuticals to screen for the utility of the signal transduction inhibitors carboxyamidotriazole and Bay 43–9006, respectively. Measurement of relevant target activity in accessible tissues may assist in ascertaining proof of principle in early screening studies, particularly if the end points have been validated to reflect tumor growth delay and/or regression in preclinical models. For example, the phosphorylation status of tyrosine residues on the ErbB receptor and indirect measurements of the phosphorylation status of signal transduction proteins downstream of EGFR are currently being assessed as indices of targeting activity in the course of developing inhibitors of ErbB.91,92 In addition to confirming that the agent is inducing relevant biologic effects in patients, such studies can potentially be integrated into decisions about whether to continue the development of rationally designed, target-based therapeutics. For example, in the presence of unacceptable toxicity plus evidence of relevant target inhibition or biologic activity, it may be logical to proceed with drug development efforts, perhaps using an alternate strategy. The same decision would likely be made if dose-related target inhibition were achieved without unacceptable toxicity. In contrast, neither toxic effects nor evidence of target inhibition in a validated assay would give rise to concerns about the continued evaluation of the compound. However, the scenario of unacceptable toxicity in the absence of evidence of target inhibition is the most difficult of all. If there is uncertainty about the validity of the assay as a surrogate for target inhibition and antitumor activity, the decision to proceed to phase II and III evaluations should depend on the strength of the preclinical and clinical efficacy data, as well as the uniqueness of the therapeutic. Other surrogate end points that may be considered for phase II screening evaluations include assessment of target inhibition and relevant changes on positron emission tomographic scanning using isotopes that reflect tumor cell proliferation.93,94 However, these imaging procedures must be validated as reliable predictors of delayed tumor growth, tumor regression, and clinical impact. Although all of these potential end points remain intriguing possibilities for future phase II trials, no proposed alternative end points have, in fact, been validated. Thus, the challenge will be to successfully integrate them as we search for new paradigms for evaluating these novel agents.
Phase III Evaluations Although the principal end points of phase III trials will continue to be based on survival end points, other therapeutic end points that may not be directly related to survival are likely to be used to achieve regulatory approval, provided that the safety profiles of novel, rationally designed, targeted-based therapeutics differ from those of traditional cytotoxic agents. Relatively flat dose-toxicity relationships permit greater emphasis on end points related to clinical benefit, such as performance status, weight loss, pain control, improvement in specific disease-related symptoms, and improvement in overall quality of life. To provide for these changes, greater emphasis will need to be placed on developing and validating scales and indices that enable reliable quantification of clinical benefit, such as the quality-adjusted time without symptoms or toxicity measure.95–97
Rationally designed, target-based antiproliferative agents will have the greatest likelihood of benefiting patients whose tumors possess the putative target. Thus, phase II and III evaluations will have the greatest probability of detecting meaningful clinical activity if studies are performed in patients with tumor types that have a high likelihood of possessing the target, especially if the target is a meaningful driver of proliferation. For such agents, the scope of disease-directed evaluations can be broadened and patient eligibility requirements made less restrictive after proof-of-principle trials in the most logical clinical situations. However, implementation of stringent eligibility restrictions requires confidence about the mechanism of action and precise target of the agent. The implementation of this strategy in the development of the FTIs, which were initially believed to target tumors with ras mutations, would have restricted study enrollment to patients with tumor types that have high rates of mutated ras. In retrospect, the adoption of this strategy would never have led to the appreciation that the FTIs possess antitumor activity in patients with advanced breast cancer and high-grade astrocytoma, in which ras is rarely mutated, and the subsequent realization that the antitumor activity of the FTIs may not be solely related to the mutational status of ras.49,53–56 Another example of the potential benefits and pitfalls of eligibility requirements to enrich clinical trials with patients who are most likely to benefit from treatment with therapeutics targeting signal transduction processes pertains to the development of inhibitors of the mTOR (ie, rapamycin analogs). Tumors that rely on paracrine or autocrine stimulation of receptors that constitutively stimulate the PI3K/Akt and mTOR-dependent pathways or tumors with mutations that activate the PI3K/Akt signal transduction pathway may depend on rapamycin-sensitive pathways for growth and, thus, may be especially sensitive to rapamycin analogs. For example, PTEN mutations occur commonly in a wide variety of tumor types, which results in constitutive activation of the PI3K pathway and resistance to apoptosis. As predicted, experimental tumors with PTEN mutations are extraordinarily sensitive to rapamycin analogs.15,76–80 In addition to the increased sensitivity to rapamycin conferred by PTEN mutations, abnormalities of regulators of the G1 checkpoint, such as pRB, p16, p27, and cyclin D1, may increase the sensitivity of tumors to rapamycin and may predict for drug efficacy.98 However, both preclinical and early clinical investigations indicate that patients with tumors that do not have, as yet, identifiable susceptibility factors may also derive benefit.15 Nonetheless, it is clear that defining the molecular characteristics of tumors of patients enrolled onto clinical trials of mTOR inhibitors may help identify which patients may benefit from treatment. Furthermore, although investigations with highly restrictive eligibility requirements, which ultimately enrich the study populated with patients who have tumors with highly relevant molecular aberrations, may lead to the most compelling results, other, as yet unknown, patient populations may also benefit, and eligibility should be less restrictive in situations in which there is insufficient information regarding the determinants of clinical benefit.
Only a few years ago, it seemed that anticancer therapeutic development had come to a standstill with a paucity of new agents with potential for a major effect. The exponential rate of acquisition of information about the cancer cell during the last decade has led to the development of agents targeted against the inherent basis of cancer, especially aberrant growth signal transduction and the microenvironment. It is expected that such therapeutics will result in greater specificity, less toxicity, and higher therapeutic indices. However, it may be necessary to implement radically different therapeutic development, evaluation, and treatment paradigms to realize the full potential of these new therapies.
DR. CANNISTRA: Is there some merit to developing a consensus among clinical investigators regarding the proper approach to studying biological agents with novel mechanisms of action? For instance, you mentioned doing a randomized phase 2 trial, not using a clinical endpoint but using a biological endpoint, in order to determine a biologically active dose of your agent, before proceeding to a more standard trial that evaluates a clinical endpoint. The other issue is that in ovarian cancer and other diseases, achievement of stable disease may be a clinically important endpoint, and it may be the only effect that we see in the presence of bulky disease. Are we recognizing this endpoint in some of the studies that are currently being conducted? How is that endpoint, as opposed to our more standard CR/PR endpoints, being integrated into clinical trials? DR. ROWINSKY: By shot-gunning these agents into large patient populations, we’re seeing major dilutional effects of patients who don’t have the targets, and we’re also seeing a tremendous lack of interest in companies now because of the design of these initial trials with indiscriminate patient eligibility. Initial "go or no go trials" will have to provide principle. I think that will have to be possibly aimed at very small populations that are driven by aberrations in the target. Industry doesn’t want to hear about this approach, because they assume that the first trials will dictate the market, but the commercial ramifications will be extraordinary. In addition, I think we have to profile our cancers. There’s an extraordinary amount of information we’re not profiling. DR. CANNISTRA: Dr Bookman, can you share with us the experience you’ve had in the GOG trying to obtain interesting compounds from industry, and how you’ve dealt with this issue of stable disease as a potentially valid clinical endpoint? DR. BOOKMAN: There are two ways that we collaborate with the industry. One is to have the sponsor initiate a Cooperative Research Development Agreement (CRADA) with the NCI. The other way is to directly collaborate with industry independent of the NCI. There are pros and cons to both approaches in terms of the practicalities of running phase 2 trials in a cooperative setting. We tried to address the question of endpoints based on our historical database running trials using overall response rate (complete response and partial response) as the primary endpoint and setting a bar to see if the agent crosses the bar. That may not be optimal for many of these new biologic agents, and so we’ve tried to develop alternative phase 2 paradigms based on disease stabilization rates or progression-free intervals. However, we don’t have as much of a historical database, and those endpoints are more subject to selection bias and variability. The problem is that the industry trials, where they’ve used randomized phase 2 designs, have really been masquerading as underpowered phase 3 trials, where they’ve tried to obtain preliminary data on survival and other long-term clinical endpoints. DR. CANNISTRA: What I’m suggesting is that the randomized phase 2 design for biologic agents be primarily used for determining whether a drug is biologically active, and at what dose, using a biologic surrogate of activity. Once this is established, a proper phase 3 trial could follow, using more traditional clinical endpoints of response and benefit. DR. ROWINSKY: Certainly, ovarian cancer would be a set-up for that type of trial. DR. BEREK: The same paradigm applies to immunotherapies, because you can define the subset of patients who are unlikely to benefit. Clearly, some of these strategies are only going to work in patients who are not totally overwhelmed with tumor by the time you test them, and you may miss the potential benefit that could be done in sequence or combination with some other agent. DR. BAST: We need more studies of tumor biology, clinical tumor biology, and relevant targets. In ovarian cancers, you can show that the PI3 kinase pathway is aberrant in about 70% or more of cases, but with many different abnormalities [Semin Oncol 28:125–141, 2001]. Studies of tumor heterogeneity need to look not only upstream, where a lot of these inhibitors work, but downstream as well. It may be necessary, but not sufficient, that the tyrosine kinase inhibitors are present or that PI3 kinase is activated. If you’ve also got amplification of AKT downstream of that, inhibiting upstream targets won’t help. Consequently, it may not just be a single biological target but a combination of targets. You should be able to identify subsets of patients who are much more likely to respond. We’re also going to have to look at combination of agents that affect signaling with cytotoxin drugs. If we are fortunate, there will be single agents that stabilize tumor growth, but almost all of these agents will also be potentiators of more standard cytotoxic drugs. If we accept this possibility and move to clinical trials of combination therapy monitored with molecular diagnostics or functional imaging, progress should accelerate. Another issue is that endothelial cells are genetically stable in contrast to the genetic instability of cancers that evolve so rapidly. Different angiogenic factors will be important for the growth of different tumors. For ovarian cancer, VEGF, interleukin 8, and beta-FGF are important, whereas other factors are probably not so important. Ultimately, we are going to have to look for markers that will monitor disease stability. One solution to this problem is the more intelligent use of serum tumor markers, provided the inhibitors of signaling pathways used for therapy don’t affect the secretion or processing of those markers.
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ABSTRACT
CURRENT CHALLENGES WITH...
