Abeloff's Clinical Oncology, 4th Edition

Part I – Science of Clinical Oncology

Section D – Preventing and Treating Cancer

Chapter 31 – Principles of Molecularly Targeted Therapy: Present and Future

Anthony J. Murgo,Shivaani Kummar,
Martin Gutierrez,
Joseph E. Tomaszewski,
James H. Doroshow




Molecularly targeted anticancer agents (MTAs) are those that selectively target specific molecular features of cancer cells such as aberrations in genes, proteins, or pathways that regulate tumor growth, progression, and survival.



Molecular targets include the following: products of activating mutations and translocations, growth factors and receptors, aberrant signal transduction and apoptotic pathways, factors that control tumor angiogenesis and microenvironment, dysregulated proteins, DNA repair machinery, and aberrant epigenetic mechanisms.



The development of MTAs requires innovative strategies that differ from those traditionally applied to nontargeted conventional chemotherapy.



Successful development of an MTA depends largely on the importance of the target in controlling tumor cell proliferation and survival, a qualified assay to measure target modulation, and careful attention to patient selection.



Primary objectives of clinical trials of MTAs differ from those used in trials of conventional chemotherapy. An important objective in phase I trials of MTAs is determination of a phase II dose based on optimal target modulation (i.e., a biologically effective dose) rather than on maximum tolerated dose. In addition, objective tumor response may not be an adequate endpoint for the efficacy evaluation of MTAs, because they may have antitumor effects that are more cytostatic than cytotoxic in nature. Progression-free survival may be a more appropriate endpoint.



MTAs may be more effective when used in combination.



Functional and molecular imaging plays an increasingly important role in the development of MTAs.


Recent advances in cancer biology and synthetic chemistry have generated extraordinary opportunities for the development of molecularly targeted cancer therapeutics. Molecularly targeted anticancer agents (MTAs) are defined here as those that selectively target specific molecular features of cancer cells such as aberrations in genes, proteins, or pathways that regulate tumor growth, progression, and survival. In contrast, nontargeted therapeutics such as standard chemotherapeutic agents tend to be nonselective and thus produce considerable toxicity against normal cells. By identifying ways that cancer cells differ from normal healthy cells at the molecular level, scientists are now in a better position to exploit these differences to develop drugs that more effectively attack cancer cells while sparing normal cells. Consequently, an increasing number of MTAs are being developed with the goal of producing more effective and minimally toxic anticancer therapeutics. Furthermore, progress in the development of MTAs can shape cancer therapeutics into a more personalized form of cancer medicine. This chapter will review the principles of molecularly targeted therapy including strategies for preclinical and clinical development.


There is an increasing number and assortment of molecular targets, broadly categorized according to genetic or functional properties including the following: products of activating gene mutations and translocations; growth factors and receptors; aberrant signal transduction and apoptotic pathways; factors that control tumor angiogenesis and the tumor microenvironment; dysregulated proteins; DNA repair machinery and aberrant epigenetic mechanisms ( Table 31-1 ). Understandably, any such categorization of targets contains considerable overlap. For example, activation of the growth factor receptors epidermal growth factor receptor (EGFR) and HER2 can occur by a variety of mechanisms including altered transcriptional control, gene amplification, and activating mutations. [1] [2] Similarly, platelet-derived growth factor receptor (PDGFR)-α and PDGFR-β are receptors for growth factors in many types of tumors but are also expressed in stromal tissue and play a role in angiogenesis.[3] In addition, abnormal expression of PDGFR-α and PDGFR-β can result from genetic abnormalities; activating mutations of PDGFR-α in some cases of gastrointestinal stromal tumor (GIST)[4] and chromosomal translocations resulting in the TEL/PDGFR-β fusion protein in chronic myelomonocytic leukemia.[5]

Table 31-1   -- Tumor Targets




Disease Indication

FDA-Approved Agents(s)

Agent Type




Imatinib mesylate, dasatinib



Acute promyelocytic leukemia

All-trans retinoic acid




Imatinib mesylate





Imatinib mesylate, sunitinib malate




Imatinib mesylate, sunitinib malate




Non-small cell lung cancer




Colorectal cancer

Cetuximab, panitumumab



Head and neck cancer




HER-2 overexpressing breast cancer









Colorectal cancer, non-small cell lung cancer




Renal cell carcinoma

Sunitinib malate, sorafenib



Renal cell carcinoma


Rapamycin analogue


DNA methyltransferase

Myelodysplastic syndrome

Azacitidine, decitabine

Pyrimidine analogue

Histone deacetylase

Cutaneous T-cell lymphoma


Hydroxamic acid



Multiple myeloma


26S proteasome inhibitor




Role of Target

Disease Indication

Examples of Agents in Clinical Development



FLT-3 (FMS-like tyrosine kinase-3)

Regulates cell cycle progression, proliferation, and survival

Acute myeloid leukemia













bRAF mutation

Effects cell proliferation and angiogenesis via amplification of signal transduction in RAS/MAPK pathway

Papillary thyroid carcinoma










Single-strand DNA break repair

Various solid tumors





BRCA-positive breast and ovarian cancer


















O6-Alkylguanine DNA alkyltransferase

Prevents intrastrand DNA crosslinks






Regulates cell proliferation, differentiation and survival

Various adult and pediatric tumors

IMC-A12 (monoclonal antibody)





CP-751,871 (monoclonal antibody)





NVP-AEW541 (IGF-1R kinase inhibitor)





IGF-1R/AS ODN (antisense oligodeoxynucleotide)



Regulates cell proliferation, differentiation and survival

Various adult and pediatric tumors

INSM-120-101 (recombinant IGFBP3)



Hypoxia-inducible factor-1α

Regulates tumor cell response to oxygen deprivation

HIF-1α expressing solid tumors



αvβ3-integrin receptor

Involved in cell adhesion

Glioma and various solid tumors

Cilengitide (cyclic Arg-Gly-Asp peptide; EMD 121974)




Inhibits differentiation and induces growth factor–independent proliferation of pre–B cells

B-cell lymphomas




Regulate cell growth, antiapoptosis, altered cytoskeletal function

Solid tumors














Regulates tumor cell proliferation and survival







AZD6244 (ARRY-142886)



Promote cell proliferation and survival










Regulates tumor cell proliferation and survival

Melanoma and other solid tumors




Regulates tumor cell proliferation and survival

Medullary thyroid cancer













Akt kinase

Regulator of cell cycle and apoptotic pathway

Variety of solid and hematologic cancer




Promotes cell survival

Variety of solid tumors





Chaperone for several oncogenic proteins and growth factors

Myeloid leukemia and solid tumors






Alvespimycin (17-DMAG)










Lymphomas, solid tumors











Obatoclax mesylate (GX15-070MS)



Apoptotic mechanism

Various types of cancer








ALL, acute lymphoblastic leukemia; CMML, chronic monomyelocytic leukemia; DFSP, dermatofibrosarcoma protuberans; HIF-1α, hypoxia-inducible factor-1α; IGF-1R, insulin-like growth factor-1 receptor; IGFBP3, insulin-like growth factor binding protein-3; Mo-Ab, monoclonal antibody; mTOR, mammalian target of rapamycin; PARP, poly (ADP-ribose) polymerase; Ph + CML, Philadelphia chromosome positive myelogenous leukemia; TKI, tyrosine kinase inhibitor. TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.



Valid to the degree that agents directed against them have proven effective in the treatment of one or more disease indications bearing the target.


The most promising molecular targets are those solely responsible for sustaining tumor growth and survival. Agents that potently and selectively inhibit these critical targets are likely to have a major clinical impact. Probably the best example of a critical target is BCR-ABL in chronic myelogenous leukemia (CML). BCR-ABL is a fusion protein formed by the reciprocal translocation of chromosomes 9 and 22. Knowledge that this dysregulated tyrosine kinase played a causal role in the pathogenesis of essentially all cases of CML spurred preclinical studies that led to the development of a potent and selective ABL tyrosine kinase inhibitor, imatinib mesylate.[6] Subsequent clinical trials established imatinib mesylate as the first highly effective molecularly targeted therapy for CML and a prototype for the development of others in the class. [6] [7] [8] Imatinib mesylate is also a potent inhibitor of other tyrosine kinases including PDGFR and KIT, and it is highly effective in the treatment of GIST bearing activating c-KIT mutations, and in some GIST bearing activating PDGFR mutations.[4]

Unfortunately, the majority of human tumors, including the most common types, are genetically complex and do not have a single critical target. In addition, what may be a critical target in one type of tumor may be expressed but not be as relevant in another. Most tumor types have various genetic and molecular abnormalities driving their growth and survival. The existence of multiple abnormalities is one mechanism to explain resistance to molecularly targeted therapy and provides a rationale for treatment strategies combining two or more targeted agents.[9] However, recent information suggests cancer cells may become “addicted” or physiologically dependent on the sustained activity of specific oncogenes for maintenance of a malignant phenotype and for survival. This dependence mechanism, termed oncogene addiction [10] [11] ( Fig. 31-1A ), is associated with differential attenuation rates of prosurvival and proapoptotic signals stemming from the oncoprotein, with predominant apoptotic signals resulting in cell killing. The latter process, termed “oncogenic shock,”[12] could explain the remarkably rapid clinical responses to tyrosine kinase inhibitors in some patients with solid tumors,[13] including those typically having complex molecular abnormalities. Other possible factors controlling sensitivity or resistance to molecularly targeted therapy include increased expression of the target due to gene amplification or transcription, emergence of resistant target gene mutations, and overexpression of multidrug transporter membrane proteins. [8] [14]


Figure 31-1  Proposed model of oncogene addiction. A, Proposed role of differential signal attenuation in creating a temporal window during which apoptotic signals persist in the absence of prosurvival signals following acute oncogene inactivation to promote cell death. B,Proposed role of differential signal attenuation in the cell death response to acute inactivation of oncogenic EGFR (EGFR*) upon which a tumor cell has become dependent. The upper panel illustrates the oncogene “addicted” state in which cells survive, and the lower panel illustrates the shift in the balance of prosurvival and proapoptotic signaling shortly after EGFR inactivation. Red, “off”; green, “on.”  (From Sharma SV, Gajowniczek P, Way IP, et al: A common signaling cascade may underlie “addiction” to the Src, BCR-ABL, and EGF receptor oncogenes. Cancer Cell 2006;10:425–435.)




There has been increased attention to the development of molecular therapy directed at factors controlling angiogenesis since approval by the United States Food and Drug Administration (FDA) of several agents that target vascular endothelial growth factor (VEGF) and its receptor (VEGFR). The VEGF pathway, involving the VEGF family of proteins and their receptors, is an important regulator of both physiologic and pathologic angiogenesis. Through its signaling pathways, VEGF/VEGFR activation contributes to increased vascular permeability, mobilization of bone marrow-derived endothelial cell precursors, degradation of the extracellular matrix, and endothelial cell division, differentiation, migration, and survival. [15] [16] VEGF overexpression occurs in most types of cancers, including colorectal, gastric, pancreatic, liver, lung, breast, thyroid, genitourinary, and in glioma and other intracranial tumors, as well as hematologic malignancies. [17] [18] In addition, VEGF overexpression is associated with tumor growth and clinical outcome in several of these tumor types.[17]

Potential therapeutic strategies to inhibit signaling through VEGF and VEGFR pathway activation include monoclonal antibodies directed against VEGF or VEGFR, tyrosine kinase inhibitors, and antisense strategies (antisense oligodeoxynucleotides, antisense RNA, small interfering RNAs). In 2004, the FDA approved bevacizumab, a humanized murine monoclonal antibody directed against VEGF, for treatment of metastatic colorectal cancer when used in combination with fluorouracil-based chemotherapy.[19] Subsequently, two small molecule tyrosine kinase inhibitors with activity against VEGFR, sunitinib and sorafenib, became FDA approved for the treatment of advanced renal cancer. [20] [21] In addition, sorafenib is effective in the treatment of hepatocellular carcinoma,[22] a tumor against which standard cytotoxic chemotherapy has little or no activity. The experience with these agents established the VEGF/VEGFR pathway as a valid target for cancer therapeutics.

Mammalian target of rapamycin (mTOR) has also emerged as a validated target with the demonstration that the small molecule mTOR inhibitor, temsirolimus, is effective in the treatment of renal cell carcinoma.[23] This activity of temsirolimus is attributed to the downregulation of factors that control cell growth and angiogenesis such as hypoxia inducible factor-1 (HIF-1).[24] Several other strategies to inhibit HIF-1 are under development including the use of the chemotherapeutic agent topotecan, which at low doses administered for relatively long periods has been shown in in vitro and animal models to reduce HIF-1 protein production thus inhibiting angiogenesis.[25]


The discovery and development of MTAs requires closely aligned laboratory and clinical research, integrating drug discovery, development, and clinical investigation. In such a cooperative setting, researchers can effectively take rational and iterative steps from target identification to clinical evaluation ( Fig. 31-2 ; Table 31-2 ). A crucial early step in developing an MTA is target validation, defined as experimental evaluation of the role of a given gene or protein in cancer. [26] [27] The process of target validation involves a variety of preclinical approaches, including genetic, cell-based, and animal models. [26] [28] [29] Validation and prioritization of molecular targets for therapeutic development depends on a variety of criteria taking into consideration chemical, biologic, clinical and practical factors (Table 31-3 ).[27] The fundamental goal is to provide evidence that the target is valid (affecting the target inhibits tumor growth, progression, or survival) and that making drugs that hit the target is feasible. The next major step in the development of MTAs is finding compounds directed against the target.


Figure 31-2  Central role of high-throughput screening (HTS) in the mechanism-based drug discovery process.  (With permission from Aherne GW, McDonald E, Workman P: Finding the needle in the haystack: why high-throughput screening is good for your health. Breast Cancer Res 2002;4:148–154.)




Table 31-2   -- Steps in Discovery and Preclinical Development of Molecularly Targeted Therapy



Identify the molecular target against which an agent will be developed.



Validate the molecular target; confirm that affecting the target inhibits tumor growth/survival.



Screen for compounds that “hit” target (i.e., high-throughput screening).



Optimize compounds: select or modify structure to increase activity and selectivity while maintaining favorable pharmacologic drug properties.



Qualify assay methodology for drug-target effect, in in vitro and in animal models.



Evaluate lead drug(s) in vivo for efficacy and safety.



Prioritize and select drug candidate(s) for clinical testing.



Conduct necessary preclinical animal toxicology and pharmacokinetic studies to support investigational new drug status for first-in-human trial.



Table 31-3   -- Validation Criteria and Prioritization of New Targets for Drug Screening



High frequency of genetic or epigenetic deregulation of the molecular target or pathway in human cancer—indicates that the target or pathway is probably important in driving the disease.



Linkage of the deregulation to clinical outcome—strengthens case for causal involvement.



Evidence in a model system that the target pathway causes or contributes to the malignant phenotype—demonstrates a direct causal role in malignancy.



Demonstration of reversal of the malignant phenotype—provides greater confidence that modulation of the target by a drug will produce an anticancer effect.



Demonstration of “drugability” of the target (e.g., enzymes are generally much more druggable than are large-domain protein-protein interactions).



Availability of a robust, efficient biological test cascade to support the drug discovery program—to allow evaluation of lead compounds and to select a development candidate for preclinical toxicology testing and clinical trials.



Feasibility of establishing, validating, and running an affordable and robust high-throughput screen.



Potential for a drug design approach based on structural biology; such an approach, based on an x-ray crystallographic or nuclear magnetic resonance structure, can be highly complementary to a screening strategy.

Adapted from Aherne GW, McDonald E, Workman P: Finding the needle in the haystack: why high-throughput screening is good for your health. Breast Cancer Res 2002;4:148–154.




Empirical approaches traditionally used to screen for cytotoxic agents are not optimal for MTAs. Rather, screening for MTAs should be target based. The increasing number of potential targets and theavailability of sophisticated high-throughput screening technology provide tremendous opportunities to screen an enormous set of diverse small molecular compounds for promising therapeutics.[27]Furthermore, the availability of genetically engineered mouse models provides the opportunity to better screen compounds in vivo. [28] [29] A more in-depth discussion of these important drug discovery tools and their implications to the molecularly targeted drug development process is beyond the scope of this chapter.

Optimal development of an MTA requires careful assessment of pharmacokinetic (PK) and pharmacodynamic (PD) effects in relevant nonclinical models before initiating clinical trials. Preclinical in vivo pharmacologic and toxicologic testing is required to establish starting dose and schedule for clinical trials, evaluate effects on normal host tissue, and help make predictions about serum and tissue levels required for target modulation as well as effects on tumor growth. At present most in vivo efficacy studies involve human tumor cell murine xenograft models. [30] [31] However, xenograft models have limitations including the requirement for an immunocompromised host, and they are not an ideal method to simulate the complex relationship between tumor and microenvironment such as the angiogenesis process.[28] Moreover, xenograft models have not been very predictive of drug efficacy in cancer patients; activity in a particular histology in a xenograft tumor model does not closely correlate with activity in the same human cancer histology.[32] However, agents that have activity against a broad range of tumor types in xenografts have a better chance of clinical activity than those that do not. Major variables to consider in designing xenograft studies include the origin of the tumor, mutation status, site of implantation, size of the tumor at commencement of drug treatment, and dose and schedule of administration. Clearly, the future development of MTAs would benefit from more predictive in vivo models. Those based on target expression rather than histology promise better success, but this will require substantial additional testing.


Successful development of MTAs may require different strategies than those traditionally used for the development of conventional cytotoxic chemotherapeutic agents ( Table 31-4 ).[33] Consequently, the discovery of an increasing number of molecularly targeted compounds has created major challenges in drug development, both for dose determination and efficacy evaluation. In animal models, many of these newer agents may show marked growth-inhibitory and antimetastatic effects without impressive tumor regression, properties commonly referred to as cytostatic. Although the specific effects of each class are dependent on the target or targets inhibited, in patients many are more likely to result in tumor growth inhibition rather than tumor regression. Pivotal clinical trials of sorafenib in renal cell and hepatocellular carcinoma, for example, substantiate the cytostatic properties of this small molecule VEGFR inhibitor, with results showing marked improvement in progression-free survival with minimal objective tumor response by RECIST criteria. [21] [22]

Table 31-4   -- Properties and Development of Nontargeted versus Molecularly Targeted Anticancer Agents


Nontargeted Chemotherapy

Molecularly Targeted

Drug discovery

Empirical approaches; screening for compounds that inhibit tumor growth/survival

Target-based approaches; screening for compounds that hit tumor target

Antitumor effects

Cytotoxic; tumor shrinkage

May be cytostatic; growth inhibition

Host toxicity

Nonselective, toxic to many normal organ systems

Selective; depends on target(s) specificity; “off-target” effects

Therapeutic window

Usually narrow

Usually wider

Phase I primary endpoints


Target inhibition and OBD; PK



Intermittent or continuous

Phase II efficacy trial endpoints

Objective tumor response (tumor shrinkage)

Tumor response or progression-free survival

Phase II dose

Based on MTD


Patient selection


Target expression, if possible

Interval to clinical response

Relatively short (e.g., 4–8 weeks)


DLT, dose-limiting toxicity; MTD, maximum tolerated dose; OBD, optimal biologic dose; PK, pharmacokinetics.




Dose Determination

The primary objective of phase I trials of conventional cytotoxic agents is ordinarily to define maximum tolerated dose (MTD), under the assumption that the higher the dose the greater the antitumor activity ( Fig. 31-3A ). Thus, the dose recommended for phase II efficacy trials is typically the MTD. However, MTAs are generally less toxic and, because of their selectivity, may be effective at doses much lower than the MTD ( Fig. 31-3B ). MTAs act on highly specific targets differentially expressed or activated in cancer cells. Thus, they tend to spare normal tissue except at higher doses, resulting in a wide therapeutic index. Therefore, increasing the dose in a traditional phase I trial to the point of normal tissue tolerance (MTD) may be an irrelevant or nonachievable endpoint with an MTA. Rather, target inhibition may be a better endpoint, with the primary objective of determining the “optimal biologic dose,” that is, the lowest dose that maximally inhibits the relevant target or pathway. [33] [34] [35]


Figure 31-3  A, Hypothetical dose-effect curves for the antitumor and toxic effects of a conventional nontargeted cytotoxic agent. The therapeutic index is narrow, with the maximum antitumor effect close to the maximal tolerable dose (MTD). B, Hypothetical dose-effect curves for the antitumor and toxic effects of an MTA. The therapeutic index is relatively wide, with the optimal biologic dose (OBD) antitumor dose occurring at a dose considerably lower than the MTD.



Phase I trials intended primarily to determine a dose that optimally inhibits a target in tumor tissue usually involve pre- and post-treatment biopsies. However, the requirement for tissue biopsies is costly and hampers enrollment, because only patients with tumors accessible to biopsy are eligible, and not all otherwise eligible patients consent to the procedure. Therefore, in lieu of tumor biopsies, one strategy to evaluate target modulation and determine a biologically active dose is to use less invasive procedures involving target or biomarker assessment in surrogate tissue expressing the target, such as skin or peripheral blood mononuclear cells (PBMCs). [35] [36] [37] [38] For example, demonstrating a “biologically effective dose” in PBMCs may serve as a guide in dose-finding trials. This approach was invaluable in the initial studies of the proteasome inhibitor bortezomib.[36] Because a sufficiently sensitive PK assay was not yet available, the study used PBMC proteasome levels as a bioassay surrogate. This turned out to be a successful method for dose determination pending a sensitive PK assay. Phase I trials could also use target modulation in surrogate tissue such as PBMCs to determine a dose level to start obtaining more invasive tumor biopsies, based on the premise that measurable effects in tumor tissue are not likely to occur at lower doses.[35]

However, this approach may not be adequate for dose determination with all types of molecularly targeted therapy. For example, a phase I trial of the alkylguanine DNA alkyltransferase depleting agentO[6]-benzylguanine determined that substantially higher doses of the drug were required to inhibit the enzyme in tumor tissue as compared with PBMCs.[37] Thus, before relying on PBMCs or other potential surrogates for target effect in tumor tissue, authenticating studies should be done to directly compare the effects in the potential surrogate versus tumor tissue, preferably first performed in animal models and then confirmed in pre–phase I or phase 0 clinical trials.[39]

Another strategy to help define the optimal biologically effective dose is to incorporate into dose-finding trials noninvasive molecular and functional imaging, such as innovative magnetic resonance imaging (MRI) and positron emission tomography (PET). [40] [41] Dynamic contrast enhanced MRI (DCE-MRI), which can measure tumor permeability and vascularity, may be a useful biomarker for dose determination in studies of angiogenesis inhibitors. For example, assessment of changes in tumor vascularity and permeability by DCE-MRI has been successfully used in phase I studies as a biomarker for defining pharmacologically active doses for small molecule tyrosine kinase angiogenesis inhibitors. [42] [43] DCE-MRI is also applicable to the evaluation of early evidence of tumor responsiveness, a topic addressed later in this chapter.

Given the growing number of targeted agents in development and the complexities of determining a dose that yields optimal biologic activity based on target or biomarker inhibition rather than on toxicity, there is an increasing need for novel adaptive phase I trial statistical designs. One such design recommended for phase I trials of MTAs is based on the assumption that the target response in individual patients is a binary value (positive or negative) determined in each individual patient.[44] The goal of the design is to study a minimum number of patients to find a biologically adequate (rather than optimal) dose, with the intent to obtain sufficient data to move as quickly as possible from dose-finding to evaluating efficacy. Based on limited simulations, this type of design seems to perform adequately with only three or four patients treated at each dose level. The efficacy of this approach for dose determination for targeted agents warrants prospective evaluation in future clinical trials. [33] [39] [44]

However, to define a biologically effective dose it is essential to have a validated target (as defined previously) and a reliable and reproducible assay to evaluate for target inhibition. This is best accomplished by having a clear understanding of the dynamics and consequences of target inhibition, the degree of target effect required for tumor growth inhibition, whether the target must be inhibited continuously or intermittently, and the function of the target in normal cells. Thus, optimal clinical development of MTAs requires extensive preclinical studies, including better animal models that are predictive of eventual clinical effect and are applicable to thorough assay interrogation.[45] Following this, the drug and its effect on tumor (as evaluated by the assay) should preferably then be evaluated in first-in-human prephase I or “phase 0” studies before definitive phase I trials. [33] [39]

In addition to the potential to start substantially earlier than traditional phase I trials, phase 0 trials provide a better opportunity to establish feasibility and qualify target assay methodology in limited numbers of human samples before embarking on studies that involve larger numbers of patients receiving higher and potentially toxic doses of the study agent ( Table 31-5 ). Phase 0 trials characteristically involve rigorous preclinical development of assays for target modulation and PK analysis in advance of clinical investigation, and real-time analysis of patient samples incorporated into the clinical trial.

Table 31-5   -- Phase I versus Phase “0” Clinical Trials


Phase I Trials

Phase “0” Trials

Primary endpoint

Establish maximum tolerated dose.

Target modulation or ability to image target of interest.

Dose escalation

Determine safety and toxicity.

Achieve desired systemic exposure or target modulation, allowing dose selection for future studies.

Preclinical biomarker studies

Not consistently performed before initiating the trial

Required to have plasma drug (pharmacokinetic) and preclinical biomarker (pharmacodynamic) assay development and assay qualification before the initiation of the clinical trial

Biomarker assays

Not performed consistently, most phase I trials do not emphasize pharmacodynamic markers

Biomarker assays and/or imaging studies are integrated to establish mechanism of action in actual patient samples.

Number of patients

Usually >20





Therapeutic benefit

None expected; however, tumor response is evaluated to permit continued dosing in case evidence of clinical benefit is found.


Tumor biopsies


Serial tumor biopsies required to evaluate drug effect on target(s)

Pharmacokinetic/pharmacodynamic analysis

Samples are usually batched and analyzed at a later time point.


Adapted from Kummar S, Kinders R, Rubinstein L, et al: Compressing drug development timelines in oncology using phase “0” trials. Nat Rev Cancer 2007;7:131–139.




Because these pilot prephase I studies involve a limited number of subjects and very limited drug exposure with no therapeutic intent, fewer preclinical toxicity data may be required for them to be initiated in accordance with the FDA guidance for Exploratory Investigational New Drug (IND) studies.[46] There are several types of studies that can potentially be conducted under the purview of an Exploratory IND. These include studies that provide important PK information (e.g., oral bioavailability), are designed to select the most promising lead agent from several candidates, determine whether a mechanism of action (e.g., target inhibition) observed in animal models can be observed in humans, and evaluate in vivo binding properties of novel imaging agents using extremely small doses of the test agent (“microdosing”).

Another approach to determine an optimal dose of an MTA is to conduct a randomized phase II trial with a clinical response endpoint comparing two or more different doses. This approach may be necessary if dose-selection cannot rely on a biologic marker. However, if this design is to be effective, the agent must have some degree of clinical activity, yielding objective responses or affecting progression-free survival. Under these circumstances, the randomized phase II design is useful in selecting a maximally efficacious dose with the least amount of toxicity that is applicable to subsequent phase III trials. [47] [48] [49]

Efficacy Evaluation

Because of the expanding number of anticancer agents under development with novel mechanisms of action, there is an increased need to develop novel paradigms for demonstrating efficacy. A primary endpoint of objective response rate, considered standard for most phase II trials of traditional cytotoxic agents, is not adequate for targeted agents that produce growth inhibition without tumor regression.

Standard cytotoxic agents result in cell kill and eventual tumor shrinkage, whereas cytostatic agents inhibit tumor growth without direct cytotoxicity. MTAs tend to be more cytostatic than cytotoxic. As a result of their specificity and growth-inhibitory properties, the efficacy evaluation of these agents should apply different approaches than those commonly used for nontargeted cytotoxic chemotherapy (seeTable 31-4 ). [34] [50] [51]

Phase II trials of nontargeted cytotoxics commonly use objective response rate as a measure of activity warranting further clinical evaluation. Furthermore, evidence of a therapeutic response to cytotoxic drugs is expected to occur relatively early in the course of treatment (e.g., after one to two cycles). MTAs have a wider therapeutic window, may require dosing for longer periods at low doses to be efficacious, may have maximum clinical effects in combination rather than as single agents, and may be cytostatic instead of cytotoxic. Because cytostatic drugs may not necessarily cause tumor shrinkage, the traditional criteria of objective tumor response may not be optimal in assessing efficacy.[50] This is exemplified by the clinical development of sorafenib. The single-agent rate of objective response, using RECIST criteria, with this multitargeted tyrosine kinase inhibitor is minimal (2% to 3%) in patients with advanced renal cell carcinoma. Yet a phase III trial of the agent in renal cell carcinoma resulted in a marked improvement in progression-free survival, forming the basis for FDA approval.[21] Similarly, sorafenib has minimal objective response activity (response rate < 3%) in hepatocellular carcinoma, but a phase III trial resulted in marked improvement in time to tumor progression and a 44% increase in overall survival.[22] If the main decision factor for further development had been objective tumor response, the true effectiveness of sorafenib may not have been established. For this reason, exploratory efficacy trials of molecularly targeted cytostatic agents increasingly include progression-free survival as a primary endpoint.

However, molecular heterogeneity and differences in patient and tumor characteristics make evaluation of progression-free survival difficult to interpret, particularly in relatively small exploratory studies without concomitant comparator controls. One trial design proposed to overcome some of these concerns is the randomized discontinuation design, which is considered a type of enrichment trial. [51] [52]With one such design, all eligible patients receive a study agent for a defined period followed by restaging; patients with a predefined degree of tumor shrinkage remain on drug and those with progressive disease are taken off study; only those patients who show evidence of stable disease undergo randomization to receive either the study drug or placebo. Thus, the randomized population is enriched with sensitive patients, because only those patients without early drug failure continue on therapy. That all patients have the opportunity to receive the investigational drug is considered a major attraction of this study design.[52]

The application of the randomized discontinuation design or other types of novel screening designs in exploratory phase II trials may be particularly useful in the early development of a molecularly targeted cytostatic agent for which a dependable assay to select patients based on target expression is not available. [49] [53] Furthermore, randomized phase II designs, in general, are applicable to exploratory trials evaluating two or more doses or schedules. [47] [48] [54] [55]

Sometimes the approach taken to evaluate the efficacy (e.g., progression-free survival) of an MTA is to conduct a trial comparing the results of treatment with the targeted agent in combination with a standard agent or regimen to that with the standard regimen alone. The intent of such studies is to show superior efficacy with the combination. The “add-on” approach is useful for the evaluation of agents expected to have little efficacy when used alone, and is particularly attractive because all patients receive an active agent or regimen. For example, these designs were critical to the successful development of trastuzumab in breast cancer and bevacizumab in colorectal cancer.

The approach, however, is not without significant risk of failure, as was the case with the randomized trials of gefitinib and erlotinib combined with chemotherapy in non-small-cell lung cancer. [56] [57]Reasons for failure include suboptimal dose of the MTA, antagonism between the MTA and the chemotherapy, lack of synergistic or additive antitumor effects, or dilution of the study population with patients having insensitive tumors.[58] Such designs should only be considered if there is a reasonable estimate of the single-agent activity of the MTA in the specific patient population to be studied, the agent is administered at an optimally efficacious dose, and the combination is supported by a strong scientific rationale. Furthermore, for those agents with targets expressed in only a small proportion of patients, the study should be enriched with patients who have tumors that express the target or have another marker predictive of activity.

Use of Pharmacodynamic Markers

Trial designs for exploratory studies of MTAs may need to rely more on PD endpoints as markers or surrogates for antitumor effect than on toxicity or objective tumor response. This requires the development of PD assays that reliably measure drug-target effect. To do so, the assay must be accurate, precise, reproducible, sensitive, and robust, as well as having a dynamic range tight enough to detect differences between baseline and post-treatment values ( Table 31-6 ).[39] Furthermore, the assay methodology assessing drug effect on the target should be developed and authenticated first in in vivo animal models and then in prephase I or phase 0 clinical trials.

Table 31-6   -- Pharmacodynamic Assay Glossary

Accuracy: The ratio of the observed assay readout to the actual quantity of bona fide analyte present at any point within the dynamic range of the assay. Traditional methods of establishing accuracy include recovered fraction of a known mass of the analyte added to a clinical specimen (spike recovery), and measurement of interferences from materials likely to be found in a typical specimen.

Dynamic range: The range of concentrations in which the assay is capable of accurately measuring analyte; ideally concentrations present in both treated and untreated specimens without additional specimen dilutions or processing steps.

Precision: A measure of variability of results for a specimen around the determined value, performed in the range in which measured values approximate the true value; usually accomplished by repeated assays of a set of specimens by multiple technicians on multiple days.

Reproducibility: The closeness of agreement between independent results obtained with the same method on identical test material but under different conditions (different operators, different apparatus, different laboratories, and/or after different intervals of time). It is measured as the total imprecision of the assay from all sources, measured at several points within the dynamic range of the assay. This imprecision must be much less than the clinical endpoint selected: that is, if an undeveloped assay had a total imprecision of about 40%, this would make measurement of a 50% effect impossible.

Robustness: The assay must be transferable to other laboratories. The results obtained from the assay must be stable over time.

Sensitivity: Analytically, this is the slope of the standard curve. The assay should be sensitive enough to allow repeat determinations of the same specimen.

Adapted from Kummar S, Kinders R, Rubinstein L, et al: Compressing drug development timelines in oncology using phase “0” trials. Nat Rev Cancer 2007;7: 131–139.




The development of mechanism-based biomarker assays would significantly expedite drug development, because such assays aid in determining early on if a drug modulates the target. This approach could also help select the lead agent from a group of compounds, help determine dose, guide patient selection and assessment of response, and provide the basis for combination trials. [33] [59] [60] [61] However, this requires having a validated target, understanding the biology of target inhibition, and defining optimal target inhibition. The evaluation of target expression is demanding, because it requires the development of a reliable assay to measure the target, as exemplified by the comparative value of the measurement of Her2/neu by immunohistochemistry versus fluorescence in situ hybridization before selection for trastuzumab therapy. [62] [63]

Adequate clinical evaluation of PD endpoints as markers of drug-target effect in tumor tissue in exploratory trials of MTAs requires pre- and post-treatment biopsies. To do this effectively, such trials require careful attention to the use of standardized and validated procedures for tissue acquisition, handling, processing, and assay methodology. This is critical, because these factors could significantly influence biologic processes and significantly increase inter- and intrapatient variability, making the interpretation of the results problematic. In addition, different tissue handling procedures may change the expression or level of activity of targets, such as enzymes or protein substrates. If care is not taken to stabilize the analyte immediately, it may degrade completely and produce a false positive effect. The standard operating procedures from tissue acquisition to target assay methodology should be developed and qualified before initiating clinical trials.

As discussed previously, to minimize the need for invasive tumor biopsies in subsequent trials, early exploratory studies should evaluate effects in other tissues such as skin or PBMCs as potential surrogates. Exploratory efficacy trials should also utilize newer techniques like molecular profiling of the tumor and normal tissue, and there should be a better understanding of the effects of inhibiting the target both in tumor and host tissue. As such, these approaches depend heavily on the availability of clinical and laboratory based investigators and their willingness to collaborate.


There is an increasing interest in utilizing novel imaging technologies in the development of MTAs. [64] [65] Molecular imaging has the potential to monitor both normal and abnormal biochemical and physiologic parameters in individual patients. Unlike anatomic imaging, molecular imaging displays biochemical and physiologic abnormalities underlying disease, rather than the structural consequences of these abnormalities. Functional and molecular imaging can be helpful in phase I studies for dose determination, as discussed previously, and may be applicable as noninvasive tools to detect drug-target effect, as well as to provide early evidence of efficacy. It may also prove useful in patient selection.

Detecting and monitoring responsiveness to a cytostatic agent is more difficult than doing so with a cytotoxic. Clinical efficacy of cytotoxic agents is usually associated with relatively early evidence of tumor shrinkage that is easily discernable by anatomic imaging with computed tomography (CT) or MRI. Conversely, a molecularly targeted agent could halt tumor growth without demonstrable changes visible by CT or MRI; but evidence of response may be detectable with the use of functional imaging, such as [18F]fluorodeoxyglucose (FDG) positron emission tomography (PET). Uptake of the glucose analogue FDG by tumor cells is a marker of glucose metabolism, number of viable cells, and the level of cell proliferation in a tumor mass. Thus, treatment that causes metabolic changes associated with inhibition of tumor cell proliferation or that causes cell death should have a relatively rapid affect on the degree of FDG uptake. Decrease in the maximum standardized uptake value (SUVmax) on FDG-PET was found to be an early predictor of response of advanced GIST to imatinib therapy. [66] [67] These changes can occur as early as 24 to 48 hours after initiating imatinib and predict for objective response or stabilization of disease as determined subsequently by CT scanning. Conversely, failure to achieve a decrease in FDG-PET uptake following treatment with imatinib is predictive of a poor clinical response for patients with GIST.

DCE-MRI is another noninvasive modality for evaluating pharmacologic response to MTAs; it is particularly useful in the development of angiogenesis inhibitors. Measurement of DCE-MRI parameters pre- and post-treatment has proven helpful in evaluating changes in tumor blood flow and vascular permeability in early-phase trials of a variety of antiangiogenesis inhibitors including the anti-VEGF antibody bevacizumab[68] and small molecule VEGFR inhibitors. [42] [69] DCE-MRI provides the potential to evaluate not only treatment-related changes in tumor vasculature and determination of PK-PD relationships but may also be a relevant marker for predicting clinical outcome. [42] [70]

Another promising area of mounting interest is the use of PET and novel imaging probes to visualize cancer targets and processes in vivo.[60] These include a wide variety from small molecules, peptides, and antibodies ( Table 31-7 ). The processes that can be imaged include cell proliferation, angiogenesis, apoptosis, hypoxia, PK, and multidrug resistance. In addition, probes exist or are in development to image tumor cell receptors such as EGFR, HER2, and bombesin, to name a few. Future progress in the clinical application of molecular imaging depends on developing probes that have better sensitivity and specificity as well as probes that increase the signal-to-noise or spatial resolution of molecular imaging devices.

Table 31-7   -- Imaging Probes Used to Visualize Molecular Targets and Processes in Cancer

Molecular Target/Process

Imaging Probes



2-[11C]Thymidine, FLT, 1-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl) thymine, 2′-deoxy-2′-fluoro-5-fluoro-1-β-D-arabinofuranosyluracil, [124I]iododeoxyuridine


[99mTc]Annexin V, [18F]Annexin V


[18F]misonidazole, 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide, fluoroerythronitroimidazole, fluoroetanidazole, diacetyl-bis(N4-methylthiosemicarbazone) copper (II), [124I]iodo-azomycin-galactoside, fluoroazomycinarabinofuranoside


5-Fluorouracil, N-[2-(dimethylamino)ethyl]acridine-4-carboxamide,1,3-bis(chloroethyl)-1-nitrosourea, [11C]temozolomide, [13N]cisplatin

Multidrug resistance

[99mTc]sestamibi, [11C]verapamil, [11C]daunorubicin, [11C]colchicine, [99mTc]methoxyisobutylisonitrile

Breast cancer (ER)


Prostate cancer (androgen receptor)



Somatostatin/somatostatin receptor

[90Y]1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-Tyr3-octreotide, [111In]diethylenetriamine pentaacetic acid-D-Phe(1)-octreotide, [90Y]1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-lanreotide/vapreotide

Vasoactive intestinal peptide/vasoactive intestinal peptide receptor-1

123 I-labeled vasoactive intestinal peptide, [99mTc]TP3654

Bombesin, gastrin-releasing peptide/gastrin-releasing peptide receptor

99m Tc-labeled bombesin

Cholecystokinin, gastrin/cholecystokinin receptor

111 In-labeled diethylenetriamine pentaacetic acid-minigastrin


18F-labeled Arg-Gly-Asp peptide targeted to αVβ3 integrin

Cathepsin proteases

Prosense (VM102)



Paramagnetic nanoparticles using antibodies to integrin αVβ3, the integrin αVβ3 ligand, vascular cell adhesion molecule 1, E-selectin


Arcitumomab (CEAscan), satumomab

Prostate-specific membrane antigen

Capromab pendetide (ProstaScint)


131 I-labeled tositumomab (Bexxar), 90Y-labeled ibritumomab tiuxetan (Zevalin) (FDA approved)



Adapted from Kelloff GJ, Krohn KA, Larson SM, et al: The progress and promise of molecular imaging probes in oncologic drug development. Clin Cancer Res 2005;11:7967–7985.

CEA, carcinoembryonic antigen; FDHT, 16-fluoro-5-dihydrotestosterone; FES, 16α-fluoroestnadiol-17β; FLT, 3’-[18F]fluoro-3’-deoxythymidine.





3′-[18F]Fluoro-3′-deoxythymidine PET is particularly useful as an in vivo marker of tumor cell proliferation and for monitoring response to antiproliferative therapy.[71] Finally, there is increasing interest in the use of optical imaging by spectral fluorescence enhancement because of its sensitivity and ability to detect very small clusters of cancer cells.[72] However, optical imaging has the disadvantage of being limited to settings where direct imaging is possible (e.g., intra-operative or endoscopic), because whole-body imaging is not yet feasible. Unquestionably, noninvasive imaging will have increasingimportance in the development and application of molecularly targeted therapy.


An important consideration in the enrollment of patients on exploratory efficacy trials of MTAs is the use of patient selection that optimizes the chance for a positive effect. This is not feasible unless patients enrolled have tumors that express or have a high probability of expressing the target. Until recently, essentially all phase II trials have evaluated a drug in tumors based on organ of origin with further categorization by histologic type (e.g., breast cancer, non-small-cell lung cancer). The presumption is that a given histologic type within an organ site represents a relatively uniform disease entity. However, there is increasing evidence that tumors are very heterogeneous at the molecular level within any given histologic type in a particular organ and that genetic variations in the tumor may significantly affect drug sensitivity. Clearly, patient selection beyond organ site and histologic type is becoming increasingly important in drug development.

Good examples of patient selection based on molecular phenotype include the use of the estrogen and progesterone receptor expression and HER2/neu expression to select breast cancer patients for treatment with selective estrogen receptor modulators (such as tamoxifen) and trastuzumab, respectively. Another example is the use of EGFR expression to identify patients with colorectal cancer amenable to treatment with cetuximab, an anti-EGFR monoclonal antibody FDA approved in this indication.

Further support for selection based on molecular phenotype is the exceptional responsiveness of non-small-cell lung cancer patients who have exon 19–21 EGFR gene mutations to the small molecule EGFR tyrosine kinase inhibitor gefitinib.[73] Because these mutations occur in only about 8% of non-small-cell lung cancer patients, the potential benefit of gefitinib in this small proportion of patients can be missed in a group of unselected patients, unless an extraordinary number of patients are studied ( Table 31-8 ).[58] Similarly, exon 11 mutations of c-kit in GIST[4] and T315I bcr/abl mutations in CML,[74] confer sensitivity and resistance to imatinib, respectively.

Table 31-8   -- Number of Patients Required for Randomization in Trial of a Target-Directed Agent[*]



Percent of Patients with Target in Study




















Data from Dancey JE, Freidlin B: Targeting epidermal growth factor receptor—are we missing the mark? Lancet 2003;362:62–64.


The model makes several assumptions: all patients are eligible regardless of target expression, only patients with the target benefit from treatment, target expression does not affect prognosis, lack of target does not result in a negative effect with treatment, median survival is about 10 months, about 100 patients are entered per month over 1–6 years and are followed up for 1.5–2 years, and the study has a power of 0.8 (a one-sided α of 0.05).


Thus, it is increasingly important when designing phase II clinical trials of MTAs to consider the effect of molecular heterogeneity on drug efficacy.[75] For example, to maximize the chance of success in non-small-cell lung cancer trials of EGFR tyrosine kinase inhibitors, enrollment should be restricted to patients with a reasonable probability of responding, for example those with tumors that are EGFR positive by immunohistochemistry, have increased numbers of copies of the EGFR gene, or where the presence of EGFR activating mutations can be demonstrated.[76] Another example is evaluating poly(ADP-ribose) polymerase inhibitors in breast and ovarian cancer patients with BRCA1BRCA2 mutations, who may be a uniquely sensitive patient population.[77]


An increasing number of MTAs in development have some clinical activity against selected types of cancer. However, with few exceptions, such as imatinib in the treatment of CML and GIST, MTAs have limited effectiveness as single agents and any clinical benefit is restricted to a relatively small proportion of patients. Consequently, there is a clear need to improve the effectiveness and utility of MTAs in the treatment of the vast majority of malignancies. One approach to improve the therapeutic potential of MTAs is to use them in combination with other agents or modalities ( Table 31-9 ).

Table 31-9   -- Strategies Combining Molecularly Targeted Anticancer Agents with Other Modalities

Combination Strategies

Potential Mechanism

MTAs plus cytotoxic therapy

Modulate targets involved in tumor sensitivity/resistance or repair mechanisms.

 Combination with chemotherapy


 Combination with radiotherapy


Two or more MTAs


 Antibody directed at ligand or receptor plus a small molecule TKI directed at the same receptor (e.g., anti-VEGF antibody plus VEGFR TKI)

Maximizes target inhibition.

 Agents that inhibit two or more signal transduction molecules in the same pathway (e.g., EGFR TKI plus RAF inhibitor)

Maximizes pathway inhibition.

 Agents that inhibit parallel pathways (EGFR inhibitor plus HER2 inhibitor)

Maximize tumor inhibition by affecting multiple cellular mechanisms.

 Agents that inhibit a target and a compensatory feedback loop (e.g., mTOR inhibitor plus an Akt inhibitor)

Maximize tumor response by inhibiting compensatory resistance mechanisms.

 Agents that target the tumor microenvironment/vasculature plus an agent directed against tumor cell proliferation

Maximize clinical benefit by inhibiting tumor cell proliferation, invasion and metastasis.

 Epigenetic remodeling agents (e.g., DNA methylation inhibitors; histone deacetylase inhibitors) used in combination or in concert with agents directed against a re-expressed target

Enhance restoration of expression of tumor suppressor genes, apoptotic mechanisms, or tumor antigens.

MTA plus surgery


 Administer MTA following complete anatomic resection

Maximize benefit by targeting minimal residual disease.

MTA, Molecularly targeted anticancer agents; mTOR, mammalian target of rapamycin; TKI, tyrosine kinase inhibitor.




As noted previously, the use of MTAs in combination with standard chemotherapy has met with mixed success. Among the successes are trials demonstrating improved survival with the addition of trastuzumab in breast cancer[63] and bevacizumab in colorectal [19] [78] and non-small-cell lung cancer.[79] Among the failures are randomized trials of gefitinib and erlotinib combined with chemotherapy in non-small-cell lung cancer. [56] [57] The reasons why these latter trials failed to show superiority of the combination over chemotherapy alone are not clear, but they include the possibility of suboptimal dosing (as may have been the case for gefitinib), antagonism with the chemotherapy agents, or dilution of the study population with patients having insensitive tumors.[58] Consequently, there is a need to better elucidate these variables in the future using reliable predictive preclinical models and appropriately designed early-phase clinical trials in advance of large phase II–III clinical trials.

There is increasing interest in developing MTAs in combination with radiotherapy based on the premise that some may function as effective radiation sensitizers or modulators without increasing normal tissue toxicity. Radiation can activate multiple cellular processes and signaling pathways, which in turn can regulate radiation-induced cell death.[80] For example, radiation-induced signaling through growth factor receptors such as EGFR may activate multiple downstream compensatory signals that provide radioprotection.[81] In addition, radiation increases the expression of EGFR in cancer cells, overexpression of EGFR correlates with radiation resistance, and blocking EGFR signaling sensitizes cells to radiation effects. [82] [83] Furthermore, clinical data support the radiation-sensitizing effects of EGFR inhibition. For example, the administration of radiotherapy in combination with the anti-EGFR monoclonal antibody cetuximab in patients with head and neck cancer results in a significant improvement in local tumor control and reduced mortality without increased toxicity as compared with radiation alone.[84]

Although the results with cetuximab plus radiation in head and neck cancer are very encouraging, the use of an MTA directed at a single target may not be sufficient for optimal radiation sensitizer/modulator effects, because many pathways and processes can regulate radiation-induced cell death. A potentially more effective approach is to target many radiation response–regulatory molecules. One recent line of research focuses on the role of the molecular chaperone heat shock protein 90 (Hsp90), because Hsp90 has several client proteins, many of which are involved in signal transduction and critical to the regulation of radiosensitization. Hsp90 inhibition destabilizes client proteins, leading to their ubiquitination and proteasomal degradation. Preclinical in vitro and in vivo experiments demonstrate that the Hsp90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) sensitizes tumor cells to the effects of radiation therapy, possibly by abrogating radiation-induced G2 - and S-phase arrest.[85] The potential effectiveness of 17-DMAG and other agents that modulate multiple signal transduction pathways and processes is under clinical investigation.

Because molecular abnormalities in cancer are usually complex and involve multiple processes and pathways responsible for tumor cell proliferation and survival, the likelihood that hitting any one specific target will have a major antitumor impact is relatively low. Consequently, there is considerable interest in developing combinations of MTAs that strategically hit two or more critical targets with the expectation that such regimens will have more pronounced antitumor effects.[9] However, given the large number of targets and MTAs in development, the number of potential combinations is almost limitless. Thus, the design of clinical trials evaluating MTAs in combination should not be empiric but based on a sound scientific rationale. Examples of rational combinations are those that maximize inhibition of a single target or pathway, those that inhibit two or more parallel cellular processes or pathways, those that inhibit tumor cellular processes and the microenvironment in concert, and those that use epigenetic remodeling agents to increase the expression of a target ( Fig. 31-4 and see Table 31-9 ). In regard to the last example, inhibitors of DNA methyltransferase and histone deacetylase are being studied alone and in combination to restore the expression of tumor suppressor genes and other genes silenced by DNA methylation, including for example hormone receptors and tumor antigens, with the intent to make tumor cells more sensitive to targeted hormonal and vaccine therapy, respectively.[86]


Figure 31-4  A, Maximize inhibition of a target such as a growth factor (GF) receptor by inhibiting both receptor-ligand binding and tyrosine kinase activity. B, Maximize inhibition of a pathway by inhibiting a series of signaling components within the pathway. C, Inhibit parallel pathways by inhibiting two growth factor receptors or inhibiting downstream components in parallel pathways. D, Inhibit a target and the feedback loop that results in resistance. MEK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol 3-kinase; RTK, receptor tyrosine kinase.  (From Dancey JE, Chen HX: Strategies for optimizing combinations of molecularly targeted anticancer agents. Nat Rev Drug Discov 2006;5:649–659.)




An increasing number of clinical trials are evaluating combinations of two or more MTAs; these trials have recently demonstrated greater than expected level of responsiveness than treatment with either agent alone. Such is the case with the combination of the anti-VEGF antibody bevacizumab plus the VEGFR/RAF kinase inhibitor sorafenib in patients with advanced ovarian cancer.[87] Another promising combination is bevacizumab plus erlotinib in non-small-cell lung cancer.[88] Because this is a very promising area of research, many additional studies exploring rational combinations of MTAs are underway.

The potential advantages of using two or more MTAs in combination are clear. However, another approach to disrupting multiple molecular targets is to design a single agent with specificity against multiple targets of interest. One successful example is the small molecule HER2/EGFR inhibitor lapatinib, an agent that recently received FDA approval for the treatment of advanced breast cancer.[89]Incentives for pharmaceutical companies to take this latter approach are to avoid complex trial designs that may be necessary to isolate the effects of each agent, as well as complicated intellectual property negotiations with another company. Regardless of whether developing single agents with multiple targets or combinations of agents that focus on a limited number of targets, it is important to minimize the risk of toxicity if possible by avoiding overlapping or off-target toxicity.


Recent advances in cancer genetics, biology, and drug discovery, as well as an enhanced capacity to characterize tumors at the molecular level have created many opportunities for improved treatment of malignancy and accelerated development of new molecularly targeted therapeutics. The expansion of MTAs with more selectivity and less toxicity than conventional chemotherapy over the past decade has been remarkable. However, this paradigm shift has presented new challenges.

The development of MTAs requires different strategies than those traditionally applied to nontargeted conventional chemotherapy. Empirical methods used in the past for the development of standard agents are mostly inadequate for the development of MTAs. Rather, optimal development of MTAs from drug discovery to clinical testing requires the integration of innovative methods that are mainly target based. Furthermore, novel clinical trial designs have evolved with the introduction of MTAs. Most phase I trials of MTAs should have as a primary objective the determination of a recommended phase II dose based on optimal target modulation, because escalation to MTD may not be needed or feasible; however, accomplishing this successfully requires the development of reliable procedures for tissue acquisition and handling and reproducible authenticated assay methodologies to measure target modulation. These target assay procedures are best performed in preclinical in vitro and in vivo models and, if feasible, in phase 0 clinical studies in a limited number of patients in advance of large, more definitive trials.

Another challenge is designing clinical trials that sufficiently evaluate the efficacy of MTAs administered alone or in combination. Because many MTAs are more cytostatic than cytotoxic, objective tumor response defined by RECIST criteria may not be adequate. In addition, it is increasingly apparent that it may be better to base patient selection on target expression rather than on histology and organ site. There is also an increasing appreciation of the value of integrating innovative molecular and functional imaging technologies into early-phase clinical trials as noninvasive means for assessing target modulation, dose determination, patient selection, and early evidence of clinical response.

The development of MTAs has taken cancer therapeutics further on the road of personalized medicine. The major challenge in further advancing and refining these agents lies in understanding them within the context of networks in the cancer cell as well as in association with similar intracellular networks within the tumor microenvironment at both the primary and metastatic sites. The evaluation of the efficacy of MTAs must be based on this complex analysis of the target within the patient. Progressive advancements in molecularly targeted therapy required to meet this challenge are expected in the near future with the development of preclinical models more predictive of human outcome, applications of rationally designed MTA combination therapy, and the more effective integration of molecular imaging and profiling into clinical trials.


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