Combination Therapy Is Required for Cure

The goals of combination therapy are numerous. First, because it is difficult to predict which single agent will be most effective against an individual patient’s tumor, the use of multiple drugs increases the potential for an initial complete response. Second, combination therapy is likely critical for preventing the development of therapeutic resistance. Third, drug combinations may have additive or synergistic interactions, increasing the total activity of the drugs and potentially enabling lower dosing and reduced toxicity. One example is the successful dose reduction of cytotoxic chemotherapy with the addition of all- trans -retinoic acid (ATRA) for the treatment of patients with acute promyelocytic leukemia (APL). With few exceptions, curative systemic therapy has required the combination of highly active agents rather than combinations of active and inactive agents.

The need for multiagent therapy was first appreciated in the treatment of acute lymphoblastic leukemia (ALL). At best, 60% of patients are estimated to achieve complete remission with single-agent chemotherapy. Despite continued use, almost all patients will relapse within 6 to 9 months with single-agent therapy. However, complete remission rates exceed 95% in patients treated with combination chemotherapy, and cure rates today for children with ALL exceed 80% with multiagent treatment regimens. Similarly, patients with APL had very poor remission and cure rates until the discovery of ATRA differentiation therapy for this disease. ATRA achieved a high complete remission rate in use as a single agent, but virtually all patients ultimately relapsed. It was not until the combination of cytotoxic chemotherapy with ATRA and the recent combination of ATRA with arsenic trioxide that these patients obtained the best long-term survival rates of any acute myeloblastic leukemia (AML) subtype.

Appropriate Dose Intensity Is Required for Improved Outcome

A guiding principle in the delivery of cytotoxic chemotherapeutic agents is the concept of the maximum tolerated dose (MTD): the simple idea that more is better. Drugs are delivered at the maximum dose possible, a dose greater than which unacceptable toxicity is encountered. Here, without a clear cellular biomarker of cytotoxic activity, it is not possible to relate the maximum molecular effect to maximum achievable efficacy. Thus cytotoxic drugs are first evaluated in phase I studies with the primary study goal of establishing the MTD. Subsequent phase II and III trials are then based on this dosing schedule. MTD assumes that some toxicity is acceptable and, in fact, expected in the pursuit of cure. The most extreme example of this concept has been the use of autologous stem cell transplantation to deliver what otherwise would be lethal doses of chemotherapy. In this case the MTD is surpassed by replacing the dose-limiting factor, the hematopoietic system, with stem cells harvested from the patient before delivery of the conditioning chemotherapy. While clearly a brute force approach, the MTD paradigm also established an important corollary: significant side effects can be tolerated when therapy is administered with curative intent (discussed later).

Many retrospective studies of chemotherapy dose intensity and outcome support this premise, particularly in the pediatric setting. In high-stage neuroblastoma the addition of dose intensification with stem cell transplantation has improved the outcome for patients with metastatic disease. In pediatric ALL several studies suggest that dose intensification is important for long-term outcome, including one randomized study com­paring standard versus dose-reduced methotrexate with mercaptopurine. Moreover, in Ewing sarcoma chemotherapy intensification through interval compression in a randomized trial was also associated with an improved outcome in patients with localized disease. However, fewer prospective studies than retrospective studies have been performed to address the issue of dose intensity and outcome for pediatric tumors. Significantly, while these studies begin to emerge, some previous assumptions about MTD have come into question. For example, retrospective studies support the notion that doxorubicin or methotrexate dose intensity is important for outcome in patients with localized high-grade osteosarcoma. However, prospective studies involving large patient numbers have called into question the role of further dose intensification for this disease. Furthermore, randomized trials for malignancies such as breast cancer have demonstrated that massively increasing dose with stem cell transplantion does not always improve long-term survival. Well-controlled prospective trials are critical to address the issue of dose intensification, even for cytotoxic agents.

Toxicity Is Acceptable When Therapy Is Curative

The oncology community has accepted toxicity as inherent in the mission of cure. With the implementation of dose-intensive regimens, however, comes additional toxicity. Because of the relatively nonspecific nature of traditional chemotherapy, morbidity from these treatment regimens is significant. Acute toxicity takes the form of infection, transfusion dependence, renal injury, and hepatic injury. There are also potential long-term consequences such as cardiac injury, infertility, chronic renal insufficiency, hearing loss, skeletal injury, and secondary malignancies. Ongoing efforts are focused on decreasing these short- and long-term side effects. For example, to minimize acute toxicity, chemotherapy dosing is usually individualized on the basis of body surface area (mg/m ² ). For some cytotoxic agents, such as methotrexate and busulfan, drug levels can be monitored to minimize toxicity. Dose intensity can also be achieved with regional tumor delivery, growth factor support, or rescue with stem cells. Furthermore, better supportive care, such as infection prophylaxis, has enabled tolerance of prolonged immunosuppression. To decrease long-term morbidity, “safe” maximum cumulative doses are being established, such as maximum cumulative doxorubicin to minimize late cardiac effects. Although toxicity can be tolerated, it is essential that the level of toxicity be commensurate with the level of benefit. Marginally effective therapeutic regimens that result in substantial toxicity should not become accepted standards.

Establish Curative Regimens and Attempt To Reduce Toxicity

Treatment strategies for most of the pediatric malignancies, including ALL and the solid tumors, have used disease stratification to decrease dose in treatment regimens. A dose-intensive regimen is initially used to establish efficacy. Then, patients with a good prognosis are treated with less intensive regimens, with the goal of com­parable disease-free outcomes but decreased treatment-related morbidity. This is particularly important for pediatric disease, in which the long-term toxicity of treatment is experienced over a lifetime. Historically, these stratifications were based on factors such as age, stage, and histology, but now they include molecular genetic markers, such as MYCN amplification in neuroblastoma, and MLL rearrangement in pediatric ALL. For example, the National Wilms’Tumor Study evaluated a half-dose chemotherapy regimen for infants with low-risk disease in an attempt to decrease the incidence of toxic deaths without compromising long-term survival. These infants had acceptable toxicity with no toxic deaths, and long-term survival was not compromised. A similar approach has been taken with infants and young children with low-stage neuroblastoma, and therapy for children with ALL has been dose-reduced based on rapid clearance of minimal residual disease.

While our understanding of the molecular basis of malignancy improves, so will our ability to develop fine-grained prognostic categories and patient-tailored treatment regimens, perhaps even for traditional chemotherapy agents. While we implement targeted therapy, it is expected that disease stratification and patient selection will be based on molecular genetic findings related to the credentialed target. In the clinical development of targeted therapies, we must continue to consider these key concepts: combination therapy, appropriate dose intensification, tolerance of toxicity for substantial efficacy and cure, and treatment adjustment within established favorable prognostic groups.

Genetics Track

The genetics track relies on the idea that a cancer develops from a finite number of genetic alterations, that these alterations give the malignant cell a selective advantage, and that these genetic changes are selected for during malignant cell replication. Hence the malignant cell becomes dependent on a set of these changes for continued survival, a process sometimes referred to as oncogene addiction. This cancer cell dependence or addiction should provide a window for drug specificity for the malignant cell compared with normal cells. Targeted therapeutics taking advantage of genetic abnormalities should, in theory, be more injurious to the malignant cell than to the normal cellular counterpart. Advances in cytogenetic studies, high-throughput gene sequencing, detection of copy number alteration, whole genome expression profiling, and proteomic approaches have revolutionized candidate genetic target discovery. Now, tens of thousands of genes in hundreds of tumor samples can be analyzed simultaneously. These candidate targets must then be carefully credentialed for their suitability as a drug target. Is there cancer cell dependence on the genetic abnormality, and can the target be pharmacologically manipulated? Imatinib is one of a number of examples of clinically validated targeted therapeutics exploiting a genetic dependence, in this case the dependence of CML on the activity of the Abelson kinase (ABL). The very existence of the cancer-dependent state is dramatically illustrated by the emergence of resistance mechanisms that largely appear to restore Abelson activity (see further discussion later in this chapter).

Lineage Track

Cancer cells often maintain developmental features of the lineage from which they were derived. This is well supported with gene expression studies that report tumor cells more closely resembling the normal lineage counterpart than tumors of a different cell type. The notion that lineage or “legacy” features of a tumor cell may be exploited with targeted therapy is known as lineage addiction . One of the earliest examples of the success of this approach has been the antagonism of ER and its signaling pathway in patients with ER-positive breast cancer. Similarly, the vast majority of patients with prostate cancer derive significant benefit from antagonism of the androgen receptor (AR). In both ER-positive breast cancer and in prostate cancer (virtually all forms of which express AR), the emergence of hormone-refractory disease largely appears to rely on mechanisms that restore steroid receptor signaling. Thus as in the case of imatinib resistance, the dependent state is illustrated by these resistance mechanisms. In many cases lineage dependence may also derive from the normal hierarchy of differentiation beginning with the stem cell.

Host Track

The importance of tumor cell environment to the development and maintenance of the malignant state has become increasingly recognized and represents another potential inroad to targeted therapy. It is possible that tumors, by evolving in a particular environmental context, may become dependent on certain growth factors or neighboring cells for survival in that niche. Preclinical evidence suggests that many tumors require de novo blood vessel development for their growth and maintenance. The identification of these angiogenesis factors has enabled the development of modifiers of the angiogenesis process, leading to the successful clinical application of bevacizumab, an anti–vascular endothelial growth factor (VEGF) antibody, in patients with colorectal cancer and the approval of kinase insert dependent receptor inhibitors (sunitinib and sorafenib) in renal cell carcinoma (see further discussion later in this chapter). The interaction of tumors with the bone microenvironment has also been targeted using inhibitors of osteoclastogenesis. Here, multiple bisphosphonate agents are approved for the prevention of cancer-related fractures.

Synthetic Lethal or Empiric Track

A number of highly efficacious anticancer agents do not conveniently map to the first three categories. Admittedly, we can say that the understanding of the mechanisms of efficacy of such agents remains opaque. Nonetheless, it is quite likely that such agents, including many of the cytotoxic agents described previously, take advantage of aspects of cancer cell biology that remain unknown but may be broadly termed as synthetic lethal interactions. Synthetic lethal genetic interactions are defined as mutations that are lethal in combination but that do not produce a lethal phenotype as a single event. In a similar way, gain-of-fitness alterations in a malignant cell, which give the cell a survival advantage, can sensitize the cell to other stresses that have no consequence in a normal state but may be lethal with the malignancy promoting alteration. Thus cancer-promoting mutations give the malignant cell a survival advantage, but they may also incur a distinct liability that can be exploited therapeutically. Rather than two synthetic lethal genetic events, the clinician can also apply such synthetic lethal concepts to a preceding cancer-related molecular alteration followed by the application of a therapeutic. For example, a striking synthetic lethal relationship was discovered between loss of BRCA1 or BRCA2 function and inhibition of the poly(ADP-ribose) polymerase I enzyme (PARP). Here, loss of PARP1 was found to induce double-strand DNA breaks that required the recruitment of the RAD51 homologous recombination repair complex to the site of these breaks for effective repair. Because it was known that BRCA1 and 2 were required for RAD51 recruitment to such complexes, it was hypothesized that loss of BRCA1 or BRCA2 would create sensitivity to PARP inhibition. This was demonstrated both with siRNA and small-molecule inhibitors of PARP. Moreover, clinical activity of PARP inhibitors has now been seen in patients with BRCA1 and BRCA2 mutations with ovarian or breast cancer.

Target Epidemiology

Preclinical models of cancer are limited in both number and in their qualitative relationship to human cancers. Comprehensive collection of human tumor specimens could, in theory, be used to study a given target in a large array of samples broadly covering tissue distribution and stage distribution (primary versus metastatic sites). In practice, however, there are limited sets of comprehensive tumor collections, and generally the largest tumor-type specific collections are limited to fixed, paraffin-embedded specimens. The availability of tissue samples paired with robust clinical data remains limited. Furthermore, the vast majority of human cancer collections come from surgical resection samples, which do not necessarily reflect the disease state in which a therapeutic will be initially tested. Despite these limitations, molecular epidemiology studies in tissue samples, enabled by tissue microarrays, single nucleotide polymorphism (SNP) arrays, expression microarrays, reverse protein arrays, and next-generation sequencing technologies remain critical to building a case for the cancer relevance of a given target.

Genetic Alterations.

As illustrated by the success of ATRA, trastuzumab, and imatinib, direct genetic alteration leading to gene activation of a given drug target remains perhaps the best evidence for the causal role of a given gene in carcinogenesis. Genetic alterations that are highly frequent in a given tumor type and are present in the majority of cells are a strong indicator of causality. In the era of large-scale resequencing projects, it is critical that one distinguishes “driver” mutations (causal mutations) from so-called passenger mutations. In addition, driver mutations that arise late in the course of disease, are present in only a minor fraction of the tumors, or are present in only a fraction of tumor cells in a given single tumor must be given less weight. These findings would suggest a reduced likelihood of this lesion possessing a strong causal link to the initiation of the disease state, although they may pose an eventual mechanism of drug resistance. The types of genetic alterations (e.g., point mutations, focal amplifications, broad amplifications, and translocations) are also distinguished with respect to the extent to which they can be used to establish causality or to infer dependence.

Gain-of-Function Genetic Alterations.

Gain-of-function genetic alterations include translocations, activating point mutations, and increases in gene copy number. Translocations were among the first discovered genetic alterations initially described using chromosome spreads. Approaches to the discovery of chromosomal rearrangements include exon microarrays, which can potentially detect imbalanced or amplified translocations, cancer outlier profile analysis (COPA), paired-end resequencing of BAC clones from a genomic library (giving a structural map of a cancer genome), and newer methods of single-molecule sequencing (discussed later). Translocations are highly informative with respect to causality because they typically alter only two genes, and because background or random translocation events are relatively rare, with chromothripsis (massive DNA rearrangements affecting one or a few chromosomes that occur as a result of one catastrophic event) as a notable exception.

Amplifications are now routinely detected by methods of DNA copy number analyses: comparative genomic hybridization (CGH) array, high-density SNP arrays, and whole exome or whole genome sequencing. There are two general types of increases in gene copy: focal amplifications and broad copy number increases covering an entire chromosome or chromosome arm. With sufficient sample numbers it is possible to discern one to several genes contained within a focal amplicon. Typically, the minimal region of amplification is a guide. However, methods for detecting copy number alterations (e.g., genomic identification of significant targets in cancer [GISTIC]) also use the amplitude of amplification as an important parameter. Although it is not definitive proof, amplification lends evidence to a causal relationship between a given gene within an amplicon and a cancer state. In some amplicons, multiple genes remain strong candidates for oncogenes targeted by the amplification. One notable example is the 11q13 amplification with genes including Cyclin D1 , FGF3 , and FGF4 .

Larger-scale chromosomal alterations leading to trisomy or gain of an entire chromosome arm are frequent yet are far more difficult to study. As such, identifying single genes (or drug targets) within such broad regions remains a challenge. In this case the number of genes implicated in such large-scale alterations is so large that almost no importance can be ascribed to the cancer relevance of a gene contained within such broad chromosomal-level alterations. Outlier analysis may begin to allow the elucidation of multigene targeting by such broad regions and is illustrated by the analysis of trisomy 7 in glioma revealing a dependence on cMET and its ligand HGF, both located on chromosome 7.

Subtle nucleic acid level alterations, including point mutations and small insertions and deletions, are an important mechanism for the activation of oncogenes. Point mutations leading to the constitutive guanosine triphosphate (GTP)–bound and hence activated form of N-RAS and K-RAS, as well as genetic events leading to activation of the BRAF kinase in melanoma, thyroid cancer, colorectal cancer, pediatric pilocytic astrocytoma, and Langerhans cell histiocytosis are exemplars of this mechanism. Large-scale sequencing projects that unveiled new mutations activating oncogenes such as EGFR and PIK3CA have led to national and international cancer genome sequencing projects. From these studies it has become apparent that the detection of passenger mutations, constituting the background rate of mutation, can pose a significant challenge. These passenger mutations must be separated from the more prevalent and recurrent driver mutations. Such passenger mutations may be exceedingly high in certain types of tumors, particularly those bearing mutations in mismatch repair genes. Indeed, in one case inactivating mutations in the mismatch repair gene MSH6 were discovered in glioma tumors recurring after treatment with temozolomide. Here the sequencing of MSH6 was triggered by the very high rate of observed passenger mutations in treated versus untreated tumors. These data suggest that cautious interpretation of low-frequency events is warranted when considering mutation analysis from larger-scale modalities. A framework for context-specific mutation analysis has been provided by a systematic study of mutation data across many cancer types in The Cancer Genome Atlas (TCGA) project and others, and these studies are revealing remarkably simple genomes in many pediatric cancers ( Fig. 44-1 ).

Somatic mutation frequencies observed in exomes from 3083 tumor-normal pairs. Each dot corresponds to a tumor-normal pair, with vertical position indicating the total frequency of somatic mutations in the exome. Tumor types are ordered by their median somatic mutation frequency, with the lowest frequencies (left) found in pediatric tumors and hematologic malignancies, and the highest (right) in tumors induced by carcinogens such as tobacco smoke and ultraviolet light. Mutation frequencies vary more than 1000-fold between lowest and highest across different cancers and also within several tumor types. The bottom panel shows the relative proportions of the six different possible base-pair substitutions, as indicated in the label on the left. AML, Acute myeloblastic leukemia; CLL, chronic lymphocytic leukemia; DLBCL, diffuse large B-cell lymphyoma.

(Modified from Lawrence, et al: Mutational heterogeneity in cancer and the search for new cancer-associated genes. Nature 499:214–218, 2013.)

Loss-of-Function Genetic Alterations.

Loss-of-function alterations do not typically directly indicate or point to a specific gene as a candidate drug target. However, such alterations may predict sensitivity to agents acting against protein targets contained within downstream or parallel pathways. For example, clinical trials are under way testing inhibitors of mammalian target of rapamycin (mTOR) function against sporadic tumors or hereditable cancer predisposition syndromes resulting from loss of the tumor suppressors PTEN and TSC1 or TSC2. In both cases loss-of-function mutations in these tumor-suppressor genes are thought to lead to constitutive activation and hence constitutive dependence on mTOR (discussed later in this chapter).

Similarly, in the hedgehog pathway, a key development pathway regulating cell patterning, deletions or mutations in the gene encoding the Patched receptor (PTCH) are associated with hereditary and sporadic basal cell carcinomas and sporadic medulloblastoma. Loss of PTCH, a receptor for the ligands in the hedgehog family, leads to constitutive activation of the atypical G protein–coupled receptor (GPCR)–like protein Smoothened (Smo). Cyclopamine, a naturally derived inhibitor of Smo, and other, newer small-molecule inhibitors, have had significant activity in preclinical models lacking PTCH gene function and in clinical trials. These examples illustrate that identification of loss-of-function mutations can reveal downstream target molecules in critical cancer pathways.

Translocations are typically thought of as gain-of-function genetic events in that they act dominantly. However, they can functionally inactivate endogenous genes through the creation of dominant-negative protein fusions. For example, the PML-RARα fusion protein is believed to act, at least in part, as a dominant-negative inhibitor of endogenous RARα function. This type of molecular action can be difficult to elucidate in functional experiments. Nonetheless, it is worth remembering this possibility when evaluating proteins or genes as drug targets when they are involved in new or existing translocations.

Chromosomal-level alterations leading to loss-of-function events include homozygous deletions, heterozygous deletions, and loss of heterozygosity. Homozygous deletions are typically highly focal in their nature. When the boundaries are defined with large sample sets, in many cases, only a few genes are found in the overlapping region. For example, in the case of focal deletions on 9p, the typical minimal deleted regions in tumors such as melanoma contain only CDKN2A and CDKN2B . Heterozygous deletions are typically larger and usually involve entire chromosomes or chromosome arms. As in the case of amplifications, such large-scale broad genetic alterations make it difficult to ascertain the cancer relevance of any single gene contained within a larger region. Loss of heterozygosity (LOH) can occur as an event associated with heterozygous deletion leaving only one parental chromosome behind, which is thus homozygous. LOH can also occur when duplication of the remaining parental chromosome creates a situation known as uniparental disomy. This is significant because it may indicate preservation of a mutated allele, leading to two mutant alleles of a given gene. In the case of the tumor suppressor gene p53 , copy-neutral LOH is common.

Inactivating point mutations are a common means of gene inactivation. In this case stop codons are the most efficient mechanism for disrupting gene function. As a result, tumor suppressor gene mutations often are enriched for stop or truncating mutations. This creates certain technical and cost difficulties in sequencing genes for inactivating mutations because it is necessary to sequence all coding exons, consider sequencing of the regulatory elements governing splicing, and consider the detection of promoter mutations. As a result of these difficulties, precise clinical analysis of tumor suppressor gene mutations remains challenging even with the application of next-generation sequencing technologies.

Functional Studies to Address Cancer Dependence

Ideally, the functional relevance of a putative cancer target should be elucidated before embarking on the lengthy process of drug discovery. The concept is simple: It is preferable to “fail” or invalidate a putative target in preclinical studies than to fail later in the clinic. Although there are multiple reasons for the failure of a given therapeutic in clinical development, ideally failure should be avoided because the drug is made against an irrelevant target. Hence one should develop a robust understanding of the science surrounding a given drug target and tie this scientific understanding to cancer-relevant processes. It is useful to consider two questions in the context of designing experiments for functional validation. First, is the putative cancer target necessary for the maintenance of a cancer-related phenotype? Second, is the putative cancer target sufficient for inducing a cancer or transformation-related phenotype? These questions are generally answered by experiments directed at inducing loss of function of the target (addressing necessity) and by experiments directed at inducing gain of function (addressing sufficiency).

Loss-of-Function Experiments.

For many years there were no feasible methods for readily inducing loss-of-function alterations in mammalian systems. Consequently, the elaboration of a cell-penetrant small-molecule inhibitor or tool compound was one of the only ways to achieve the desired inhibitory effect. The development of robust small interfering RNA (siRNA) methods, taking advantage of the endogenous siRNA processing systems, now allows for transient, stable, or regulated knockdown of nearly any mammalian gene product. Genome-editing technologies, such as transcription activator-like effector nucleases (TALEN) and the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system have also been developed to enable targeted gene-disruption events. These powerful new tools are complemented by existing methods for loss-of-function experiments, including the use of inhibitory antibodies for extracellular targets, soluble protein traps for extracellular ligands and receptors, dominant-negative proteins expressed from cDNAs, murine germline knockouts, and inducible knockouts. Generally, loss-of-function experiments would be attempted first in human cancer cell line models. Increasingly, cell line models are being characterized in greater genetic detail, enabling more rational selection of models for functional inactivation experiments by either small molecules or genetic perturbations. Additionally, shRNAs have been merged with the bone marrow transplantation and the hepatoblast tissue reconstruction models described previously ( Fig. 44-2 ). Finally, regulated germline shRNAs can now be engineered in the mouse as a rapid method for generating tissue-specific and temporally regulated gene knockdowns.

Reactivation of p53 results in liver tumor regression. A, Embryonic liver progenitor cells were transduced with a tetracycline-regulatable p53 shRNA (TRE.shp53), tTA, and H-rasV12. After onset of liver tumors, p53 expression could be restored by doxycycline (Dox) treatment. B, Reactivation of p53 leads to rapid tumor regression. Tumor-bearing mice were treated with Dox starting at day 0 and imaged at the indicated time points ( n = 9). C, Subcutaneous tumors derived from ras-transformed liver progenitor cells with tet-off shRNA (TRE.shp53) or a nonregulatable shRNA (MLS.shp53) were grown in nude mice. Values represent mean s.d. ( n = 4). D, p53 reactivation is reversed by Dox withdrawal. Protein lysates from liver progenitor cells pulse-treated with Dox for 4 days were immunoblotted for p53. E, Representative mice ( n = 6) as in B were pulse-treated with Dox for 4 days and imaged at the indicated time.

(Modified from Xue, et al: Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas. Nature 445:656–60, 2007.)

Genetic Studies in Lower Organisms

The topic of genetic studies in lower organisms is too broad for a complete discussion in this chapter. However, it is worth noting the utility of defining genetic dependence in the context of highly conserved pathways that are shared between mammals, flies (Drosophila melanogaster) and worms (Caenorhabditis elegans). In some cases lower organisms have a reduced set of homologues for any given pathway member, and thus the necessity of a single gene can be tested directly. Although establishing the relationship between a gene and a cancer phenotype in these organisms is not usually possible, the genetic dependence or epistatic relationships elucidated in such model systems have proved highly informative in humans. A notable example is the discovery of the relationship between loss-of-function mutations in the tuberous sclerosis genes and activation of the mTOR and S6K (ribosomal S6 kinase) pathway (discussed later).

Target Credentialing Summary

The credentialing of drug targets ultimately must be conceived as a set of hypotheses to be tested experimentally. In each case these hypotheses must be built around the relevant biology. It is important to distinguish targets with cell-autonomous effects versus those in which the interaction between cancer cell and host factors is critical. In the case of drug targets that are encoded by bona fide human oncogenes with definitive genetic alterations, one can have a high degree of confidence in the likely therapeutic effect of inhibitory molecules. Unfortunately, this group is the minority of the drug targets for consideration. Thus the intent of credentialing is to enable a rational stratification of potential drug targets and a rational application of resources to specific drug discovery efforts.

Drugs

Small molecules are defined as carbon-containing compounds that usually have a molecular weight of less than 2000 g/mol. These small organic compounds have the advantage of purity, high permeability into target cells, ease of manufacturing, and stability, and they remain the primary class amenable to oral delivery. However, disadvantages include insufficient selectivity resulting in off-target toxicity, challenges with poor bioavailability, and challenges with metabolism of the molecule. Over the last decade synthetic methods have been developed for producing a diversity of chemical structures, either by using directed medicinal chemistry approaches or by using combinatorial chemistry, the synthesis of numerous organic compounds by combining variations of each of the building blocks that make up the compounds. Similarly, there has been the elaboration of a diverse set of methods for identifying the initial small-molecule hits against a desired target. High-throughput screening involves the in vitro assay of target activity against chemical libraries. In silico screening can be used to dock millions of compounds into known or modeled three-dimensional structures of a given target. Fragment-based screening uses either radiographic or nuclear magnetic resonance–based structural approaches to identify very-low-molecular-weight fragments that can interrogate the binding surface of a new target. Finally, target-specific cell-based screens can be used to identify cell-penetrant small molecules perturbing a target-specific reporter or phenotype.

Natural products have been important in drug development and have played a dominant role in cancer therapy. Approximately three fourths of the current anticancer drugs are natural products or derived from natural products. The chemical diversity of natural products complements that of synthetic libraries. As a result of a long evolutionary process, natural products tend to be sterically complex with greater diversity of ring systems compared with synthetic and combinatorial libraries.

Natural products are classified according to shared scaffolding elements and can be divided into several structural classes, such as terpenes, alkaloids, polyketides, and nonribosomal peptides. The building blocks for the natural products are most often monomeric components of primary metabolic pathways that are then shunted into secondary metabolic pathways. Plants and soil microbes are traditional sources of natural products with new sources, such as fungi and marine life (e.g., sponges and algae), under active exploration.

Drug development with natural products may have specific advantages to that of synthetic molecules. The complexity of natural products may lead to greater specificity. In addition, unanticipated activities may be discovered that would be difficult to engineer into a small molecule. For example, rapamycin (derived from a soil fungus from Easter Island [Rapa Nui]), creates a novel protein-drug-protein interaction between the protein FKBP12, rapamycin, and the mTOR kinase, resulting in an exquisitely selective inhibition of mTOR. However, although the natural products have been developed into very successful drugs, there are several disadvantages to these libraries. First, natural product extracts are generally impure, and hence identifying the active compound can be difficult. Second, the synthesis of these compounds in production level quantities for medicinal purposes can be challenging. Third, subsequent optimization of the lead natural product can be difficult in the absence of a complete in vitro synthesis of the relevant natural product. New high-throughput strategies are under development to tackle roadblocks to the use of natural products for medicinal purposes.

Antisense oligonucleotides are single strands of short deoxynucleotide sequences (18-21 oligomers) that bind to target mRNA sequence by Watson-Crick hybridization and induce transcript destruction. They interact with the target transcript with sequence specificity and inhibit production of the target protein by several potential mechanisms. First, they activate endogenous nucleases, such as RNAse H, which then cleave the RNA strand of an RNA-DNA heteroduplex. A second potential mechanism of antisense activity is a noncatalytic effect with steric inhibition of the translational machinery with induction of translational arrest and inhibition of protein synthesis. A third mechanism is interference with normal RNA splicing, resulting in the inhibition of specific splice variants. Antisense therapeutics held the promise of pharmacologically inhibiting targets that had been intractable with small molecule approaches. In theory, virtually any gene can be targeted with antisense approaches with an expectation of high target selectivity. However, the development of antisense therapy has faced many challenges, including poor solubility, limited intracellular uptake, and rapid degradation. To overcome these issues, subsequent antisense therapy focused on chemical modification of the backbone to which the nucleoside bases are attached.

Despite these efforts, the utility of antisense therapy for cancer has been disappointing. However, there have been some clinical responses seen in trials. Oblimersen (Genasense), a phosphorothioate antisense therapy targeting BCL-2, has shown some activity in patients with heavily pretreated chronic lymphocytic leukemia (CLL). Furthermore, recent data from a randomized phase III trial for patients with relapsed or refractory CLL comparing fludarabine plus cyclophosphamide with or without oblimersen demonstrated an increase in complete response and nodular partial response in patients in the oblimersen arm, particularly for those patients with fludarabine-sensitive disease.

RNA interference (RNAi) is a more recent approach to nucleic acid–based therapy. Similar to antisense therapy, RNAi may have therapeutic application, particularly for targets that have been considered intractable by traditional drug discovery approaches. RNAi is a double-stranded RNA (dsRNA)–induced mechanism of gene silencing naturally occurring in plants and animals. Over the last few years, it has become possible to target virtually any gene in the genome for knockdown with small, dsRNA gene-silencing by using siRNAs or expressed shRNAs. The RNAi pathway is hypothesized to have evolved early in eukaryotes as a protection against viral and genetic pathogens. Double-stranded RNA viruses and genetic elements are subject to RNAi-dependent gene silencing as a means of cell-based immunity. Furthermore, endogenously expressed shRNAs may regulate gene expression during development through the RNAi pathway. If exploited as a potential cancer therapy, RNAi has the potential advantages of (1) relative speci­ficity for a particular gene target, (2) efficiency with the potential to achieve more than 90% knockdown, and (3) widespread applicability with the potential of targeting any gene in the human genome. Unlike antisense oligonucleotides, RNAi takes advantage of a naturally occurring cellular mechanism for gene silencing.

As in the case of oligonucleotides, there are impasses to the development of RNAi-based therapies, including the difficulty of delivering large nucleic acid molecules to intracellular targets in humans. Unmodified siRNAs have a short half-life in human plasma and are rapidly excreted by the kidneys. Furthermore, to have activity the siRNA or shRNA must reach the cytoplasm of the targeted cell, and naked siRNAs are not efficiently taken up by mammalian cells unaided. One method of delivering shRNA that is commonly used in the laboratory to develop stable knockdown is the use of viral vectors. However, with viral vector delivery, there remains significant concern regarding insertional mutagenesis, carcinogenesis, direct cellular toxicity and short-lived on-target effects. Other nonviral methods of delivery are actively under exploration. One possibility is to chemically modify the siRNA to increase its stability with modifications such as locked nucleic acid (LNA) residues or phosphorothioates. A second approach to increase the stability of the siRNA is to package it in liposomes or other nanoparticles such as the cationic polymer polyethylenimine or to use cholesterol modifications. These packaging systems can also be modified to contain tissue-specific homing signals, such as the attachment of specific antibodies. Despite the problems with delivery and stability of RNAi, novel therapeutics using RNAi have entered clinical trials for multiple diseases.

Biologicals

Antibody therapy with monoclonal antibodies (Mabs) is an increasingly important mode of targeted therapy for cancer. Early attempts at antibody-based therapy with murine Mabs were limited by immunogenicity, short half-life in humans, and poor ability to activate human immune effector mechanisms. With the evolution of recombinant DNA technology, it is now routine to create humanized Mabs. Recombinant antibodies with human crystallizable fragments (Fc) regions are much less immunogenic in humans than murine Mabs and can facilitate activation of immune effector mechanisms. Thus recombinant Mabs may have a dual mechanism of anticancer activity secondary to the specificity of epitope targeting with the antigen-binding fragment (Fab) and the recruitment of immune effectors with the Fc region. As a direct effect of the Mab variable domain binding to the target, they can have antagonist effects on receptor-ligand interactions and receptor-receptor interactions or agonistic effects on the target’s biological activity. Second, if engineered to be of the immunoglobulin G1 (IgG1) subclass and to contain an Fc region, they induce immune effector function against I the target cell after their interactions with complement or with receptors for the Fc region, such as FcγR, a class of surface glycoproteins expressed predominantly on white blood cells. The Fc region interactions with FcγR can induce antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, endocytosis of immune complexes followed by antigen presentation, and release of inflammatory mediators. Antibodies can be linked to chemical moieties with additional antitumor activity, such as in gemtuzumab ozogamicin, where an antibody to CD33 was conjugated to the antitumor antibiotic calicheamicin.

Bispecific antibodies: Whereas MAbs that are clinically effective usually recruit immune cells expressing an Fc receptor, T cells are notably FcR negative. To engage T cells in the antitumor response, bispecific antibodies (bsAbs) can be deployed. These are artificially designed molecules that bind simultaneously to two different antigens, one on the tumor cell, the other one on an immune cell, such as T cells, cross-linking tumor cells and T cells. For example, blinatumomab, a bsAb that directs T cells to CD19-expressing tumor cells, such as B-ALL and non-Hodgkin lymphoma (NHL) cells, has been demonstrated to have compelling activity in clinical trials, including complete responses.

Antibody-based therapy has several advantages to small molecule–based therapy ( Box 44-7 ). First, there is a high degree of specificity for the target of interest, resulting in reduced off-target effects. Second, pharmacokinetic properties are very similar from one antibody product to another, with the half-life of an infused antibody generally lasting 2 weeks. Unlike the case of small molecules, there is a consistent path for metabolism, thus eliminating a highly variable aspect of low-molecular-weight drug development. Third, the spectrum of targets amenable to antibody-based therapeutics is largely distinct from small molecules and includes large extracellular domains and secreted ligands that are otherwise “undruggable.”

On the other hand, there are several disadvantages of antibody-based therapy compared with small-molecule therapy. Currently antibody-based therapies are generally delivered intravenously. A second disadvantage is that preclinical in vivo studies can be difficult to conduct. The researcher must address whether the antibody in development will recognize the appropriate antigen in the animal model being tested. In some cases only primates conserve the relevant human epitope, limiting the selection of species available for toxicology studies. Efficacy studies, in particular against stromal or host factors, may require generation of a “murine equivalent” antibody, although equivalence is difficult to quantitate. The robust evaluation of host effector function elicited by an antibody and the role of this function in antitumor activity is also difficult to assess in the typical human tumor xenotransplant study in rodents. A third challenge with antibody-based therapy is that the patient can develop neutralizing antibodies to the therapy. This was particularly problematic with murine-based antibody therapy. Fourth, antibodies cannot easily gain access to intracellular antigens. Fifth, the volume of distribution of antibodies is limited by the large molecular weight, and tumor penetration is therefore a concern, at least theoretically. Finally, antibody-based treatment is generally more costly to produce compared with small-molecule therapy.

Cellular Adoptive Therapy

Most recently, cell-based biological products have also seen dramatic clinical responses. The transfer of autologous T cells modified to express chimeric antigen receptors (CARs) specific for the B-cell antigen CD19 has shown antitumor activity in low-grade B-cell malignancies and in refractory adult and pediatric B-cell ALL. These exciting advances are discussed in more detail in Chapter 46 .

Genetics Examples of Targeted Therapy

Imatinib in Chronic Myelogenous Leukemia.

The development of imatinib for the treatment of CML stands out as the first example of a designed small-molecule inhibitor developed for the purpose of targeting an activated oncogene. Moreover, its dramatic single-agent activity in its initial phase I and II trials, along with the rapid path to registration, has provided the impetus for attempting to identify highly responsive patient populations during preclinical and early clinical development. In 1960 Nowell and Hungerford first detected the presence of the then-named Philadelphia chromosome in nearly all patients with CML. This aberrant chromosome, resulting from the fusion of chromosomes 9 and 22, was subsequently shown to encode a fusion protein linking a gene of unknown function ( BCR, or breakpoint cluster region ) to the product of the Abelson gene—the cellular homologue of an oncoprotein activated by the Abelson leukemia virus. The discovery that the Abelson gene product (ABL) could function as a protein tyrosine kinase and that the normal autoregulatory domain of ABL was replaced with coding sequences from BCR led to the idea that constitutive activation of ABL kinase activity was likely a causal genetic event in CML. In animal models production of BCR-ABL was sufficient to induce both acute and chronic leukemia. These data, along with the presence of a catalytic domain, made BCR-ABL kinase an attractive drug target. Based on these elements, a drug-discovery program was launched, culminating in the discovery and clinical testing of imatinib (Gleevec), a small molecule inhibitor of BCR-ABL.

In the initial phase I trial by Druker and colleagues, 83 patients with interferon-refractory CML were enrolled and treated with doses of imatinib ranging from 25 to 1000 mg per day. No MTD was reached, and nausea, myalgias, edema, and diarrhea were the most frequent side effects. Remarkably, at a dose of 140 mg or higher, all patients had a hematologic response. At doses of 300 mg per day or greater, 53 of 54 patients had complete hematologic responses, 17 had major cytogenetic responses, and 7 had complete cytogenetic remissions. Numerous subsequent trials demonstrated remarkable activity in CML and CML in accelerated phase, lymphoid or myeloid blast crisis or with Ph+ ALL in adults, including complete molecular responses.

On the basis of these data, imatinib became the gold standard for first-line therapy of CML. Nonetheless, resistance to imatinib does occur. In vitro experiments conducted by using randomly mutagenized ABL cDNA libraries inserted into sensitive CML cells revealed that a defined spectrum of mutations in the ABL gene itself could preserve kinase activity but could disrupt binding to imatinib. More important, sequencing of the ABL kinase domain in patients relapsing on imatinib revealed a similar spectrum of mutations in patient samples. When these mutations were isolated and placed into ABL kinase constructs and re-inserted into sensitive CML cells in vitro, these mutant ABL constructs also rescued lethality induced by imatinib inhibition. These data, although initially concerning because of the potential for long-term control of the CML disease state, definitively demonstrated in humans the absolute requirement for ABL kinase activity in CML and closed the loop on the notion of a fully validated drug target. Moreover, the identification of such mutants provided the impetus for testing second-generation inhibitors that might overcome these resistance alleles. Here, two separate approaches were considered. First, although imatinib is a fairly selective inhibitor, it is not a particularly potent inhibitor. Because many mutations shifted the IC ₅₀ of the kinase activity, it remained possible that a more potent inhibitor might be able to overcome or prevent the emergence of these resistance alleles. Nilotinib is a second-generation inhibitor designed to target BCR-ABL and preserve the interaction with the inhibited form of the ABL kinase, the type II binding mode. Nilotinib is more potent in biochemical and cellular assays of ABL activity and in phase I and phase II trials had significant activity in imatinib-resistant CML. Nilotinib was later demonstrated to be superior to imatinib in patients with newly diagnosed chronic phase CML. Dasatinib, initially developed as a src kinase inhibitor, binds to the “active” form of its kinase targets. Src kinases are highly related to ABL kinase activity, and in vitro testing showed that dasatinib had activity against imatinib-resistance alleles. In phase I and II trials dasatanib has shown significant activity in imatinib-resistant patients. Both dasatinib and nilotinib have gained FDA approval in imatinib-resistant patients with CML and in CML-accelerated phase and blast crisis. Ultimately, both drugs were approved for upfront therapy of patients with CML.

Unfortunately, both nilotinib and dasatinib lack activity against a common but difficult to target ABL mutation, the T315I or “gatekeeper” mutation. This mutation creates a steric problem in the adenosine triphosphate–binding domain. This particular kinase domain conformation creates challenges in the drug design process. Notably, it has been difficult to make potent and selective inhibitors of this conformation inhibition. Recently, a drug with activity against the T3151 mutation, ponatinib, was granted accelerated approval by the FDA for the treatment of adult patients with chronic, accelerated, or blast-phase CML that is resistant or intolerant to prior tyrosine kinase inhibitor therapy or BCR-ABL–positive ALL that is resistant or intolerant to prior tyrosine kinase inhibitor therapy ( Fig. 44-3 ). However, the safety of this drug was later called into question secondary to concerns about drug-related arterial thrombotic events.

Abl inhibitors. Shown are representative structural images of the indicated small molecule inhibitors (colored) bound to the ABL kinase domain (gray). Imatinib, nilotinib, and ponatinib are type II inhibitors and contain molecular components that occupy the hydrophobic pocket that is exposed when the kinase is in an “inactive” conformation. Dasatinib is a type I inhibitor and does not have extended interactions with the hydrophobic pocket and instead binds to the “active” conformation of the kinase.

(Courtesy Sandra Jacobson, Novartis Institutes for BioMedical Research, Emeryville, Calif.)

Dramatic Responses in Select Genotypes.

With the success of imatinib for the treatment of patients with CML, a high bar was set for subsequent small-molecule kinase inhibitors. The possibilities that this targeted approach held for the poorly treated adult epithelial malignancies generated great excitement. The EGFR pathway is believed to be important in the development, maintenance, and proliferation of many malignancies, and EGFR is frequently overexpressed in epithelial solid tumors. For these reasons EGFR was an attractive target for small-molecule inhibition. Gefitinib (Iressa) and erlotinib (Tarceva), both orally administered tyrosine kinase inhibitors, were developed to bind competitively at the adenosine triphosphate pocket of EGFR, inhibiting autophosphorylation, and suppressing downstream EGFR signaling. Surprisingly, these EGFR inhibitors have been largely ineffective in the treatment of solid tumors with one exception: the treatment of a subset of patients with non–small cell lung cancer (NSCLC).

After these initial clinical studies, numerous reports characterized the clinical predictors of tumor response (as opposed to survival) in patients. It soon became apparent that these patients frequently shared similar demographic features: a nonsmoking history, a well-differentiated adenocarcinoma histology, female sex, and Asian origin, although the underlying genetic reason for these response differences was not known. One hypothesis was that expression levels of EGFR correlated with these clinical characteristics and hence response. However, these initial clinical trials failed to identify a correlation between receptor expression and clinical outcome.

Later, in 2004, two research groups independently identified a correlation between mutations in the tyrosine kinase domain of the EGFR and patient responders. These EGFR mutations enhance ligand-dependent EGFR activation and sensitivity to gefitinib/erlotinib and are more common in nonsmokers, women, Asians, and patients with adenocarcinoma. Approximately 90% of these mutations occur in a few amino acids. Between 45% and 50% of mutations are in-frame deletions of exon 19 (codons 746 to 750), and 35% to 45% of mutations are missense mutation leucine to arginine at codon 858 (L858R) in exon 21. Several studies have also reported that patients with EGFR mutant tumors have a longer survival when treated with EGFR small- molecule inhibitors compared with patients with wild-type EGFR tumors.

This work highlights the importance of developing a molecular understanding of disease and response. The molecular characterization of EGFR abnormalities in NSCLC and their relationship to response to tyrosine kinase inhibitors have been illuminating for the clinical application of targeted therapy. More recently, the dramatic responses seen to B-RAF inhibition in patients with V600E B-RAF -mutant melanoma and to ALK inhibition in patients with EML4-ALK rearranged lung cancer underscores the critical role of matching tumor genotype with targeted drug therapy ( Fig. 44-4 ).

Response to ALK inhibition in patients with ALK-positive non–small cell lung cancer. Shown is the best response of patients with ALK -positive tumors who were treated with crizotinib, compared with pretreatment baseline. Numbers along the x axis indicate arbitrarily assigned patient numbers from 1 to 79. The bars indicate the percentage of change in tumor burden from baseline. Black bars indicate disease progression, blue stable disease, yellow partial response, and orange complete response. Four patients had complete resolution of their target lesions but were classified as having had a partial response on the basis of stability in nontarget lesions. Eight patients had tumor shrinkage of more than 30% but were classified as having stable disease either because confirmatory scans were not available by the data-cutoff point or early restaging was performed at 6 weeks after crizotinib initiation. The dashed line indicates a tumor reduction of 30% from baseline, the minimal percentage of decrease that constitutes a partial response, according to Response Evaluation Criteria in Solid Tumors.

(Modified from Kwak, et al: Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 363:1693–703, 2010.)

Lineage-Dependence Examples of Targeted Therapy

Several successful targeted cancer therapies capitalize on the lineage dependence of the tumor as exemplified by antibody-based therapy directed against cancers with hematologic lineage markers such as rituximab (Rituxan). As discussed in the subsequent sections, these therapies have the advantage of target specificity, with predictability of side effects based on the expression pattern of the lineage marker.

Empiric or Synthetic Lethal

As described previously, classical chemotherapy is likely to exploit cancer-dependent states that are not fully understood. For example, microtubule inhibitors, such as the taxanes, specifically interact with protein components of microtubules and are efficacious in a number of cancers, although there is no current scientific rationale for their selective efficacy. A number of emerging targeted therapeutics target highly selective protein targets involved in diverse basic cellular processes and were elaborated according to specific synthetic lethal hypotheses.

Majority of Children with Cancer Are Cured

There are numerous special considerations in the development of targeted therapy for pediatric cancer. In contrast to adults with cancer, the majority of children with cancer will be cured of the malignancy, making the selection of pediatric patients for testing of new targeted therapies more challenging and raising the required level of activity necessary for “success.” For example, in de novo pediatric pre–B-cell ALL, with cure rates greater than 80%, it is difficult to introduce a new targeted agent for fear of detracting from an already excellent cure rate by diluting curative agents. Although there are many interesting new targets emerging, such as PAX5 and other genes important in B-cell developmental pathways, it would be difficult to justify the inclusion of upfront novel targeted therapy in patients with this excellent outcome. Thus it is likely in the relapsed setting, or in de novo disease with poor outcome, that novel targeted therapies will undergo initial testing. If efficacy and safety are demonstrated, researchers may then consider incorporation into upfront clinical trials.

Pediatric Cancer Is Rare

A total of 1,660,290 new diagnoses of cancer and 580,350 cancer deaths are projected to occur in the United States in 2013. In contrast, it is estimated that only 14,023 children in the United States younger than age 20 years were diagnosed with cancer in 2009, and 1964 died of cancer in 2009. Hence pediatric cancers are quite rare compared with adult malignancies, and there is a reduced market incentive for pediatric-specific targeted drug development by the pharmaceutical industry. Furthermore, researchers have been understandably hesitant to test new drugs in children. For these reasons, there are virtually no drugs that have been developed specifically for pediatric malignancies. Even when a common target has been identified in adult and pediatric cancer, it may be years before a drug reaches a pediatric trial. Because pediatric cancers are rare, even with a drug in hand to test, clinical trials are difficult. A rare disease incidence means slow accrual of patients and necessitates large cooperative groups to power the trial with appropriate patient numbers. The limited number of patients also precludes testing of all possible new agents. New methods are needed to more rationally choose novel targeted agents for testing in children.

One national attempt to prioritize the selection of new agents for testing in children with cancer was the development of the Pediatric Preclinical Testing Program (PPTP) by the National Cancer Institute. This group is prospectively testing new agents against a panel of xenografts and genetic models of pediatric cancer, such as neuroblastoma, rhabdomyosarcoma, osteosarcoma, and ALL. The PPTP builds on earlier work demonstrating the ability of preclinical testing of new agents in rhabdomyosarcoma and neuroblastoma xenografts to predict in vivo activity in children with these malignancies. The development of new genetically engineered models of pediatric cancer should also facilitate preclinical evaluation of targeted therapies and better prioritization of new agents for clinical trial, particularly for diseases such as Ewing sarcoma, for which no such model yet exists.

Developmental Considerations in Targeted Therapy for Children

Children may tolerate targeted therapies differently from adults secondary to age-specific differences in physiology that result in altered pharmacokinetics or to drug interference with critical developmental pathways. In normal development, vascularization of cartilage is prominent during embryogenesis and during periods of growth when neoangiogenesis occurs at growth plates. Inhibition of VEGF signaling has been reported in animal models to result in thickening of the epiphyseal growth plates secondary to an expansion of the hypertrophic chondrocyte zone. These findings have been attributed to a delayed vascular invasion of the epiphyseal growth plate with a subsequent reduced rate of chondrocyte apoptosis. Inhibition of VEGF signaling has also been reported to impair trabecular bone formation, induce dental dysplasia, and cause ovarian atrophy. Although the animal data are concerning, the actual long-term effects of this class of drug on human children is unknown.

Ethical Considerations in Targeted Therapy for Children

The testing of drugs in children raises issues universal to cytotoxic and targeted therapies, as well as challenges more specific to targeted treatments. Adults can make informed decisions about the risk and potential benefit of experimental therapies and biological studies and actively participate in treatment decisions. Particularly for young children, however, true informed consent from the patients for experimental therapies cannot be obtained. Here it is the parent or legal guardian making decisions about participation in a trial. In light of this potential conflict of interest, special regulations have been instituted to afford additional protection to children. These include the Code of Federal Regulations, Part 46; Protection of Human Subjects, Subpart D; and the related FDA regulation, Part 50, Protection of Human Subjects, Subpart D. These regulations outline allowable research in children based on the risk to the child, potential benefit to the child or other children, and alternative therapies. In the testing of targeted therapies, pharmacodynamic studies with sampling of tumor tissue before and after therapy initiation become more critical. For obvious reasons these studies can be difficult to perform with the current regulations, particularly for solid tumors or brain tumors. In general, tissue collection in children for the purpose of biological studies must be considered no greater than a minor increase in minimal risk. Innovative methods are needed to evaluate pharmacodynamic response, such as new imaging technologies, and better methods are needed to assess surrogate tissues.

FLT3 and the Acute Leukemias

Although much progress has been made in the treatment of childhood leukemias, patients with AML with mutations in the Fms-like tyrosine kinase 3 (FLT3) gene remain a high-risk group. FLT3 is a class III receptor tyrosine kinase that plays a critical role in normal hematopoiesis and shares structural homology with PDGFR, c-KIT, and CSF1R. Upon the binding of FLT3 ligand, wild-type receptors dimerize and become activated by phosphorylation, leading to activation of downstream signal transduction pathways. Mutations in FLT3 can be identified in blasts from 30% of adult patients with AML with constitutive activation of the kinase resulting from internal tandem duplications (ITD) in the juxtamembrane domain or point mutations in the activation loop. The FLT3 ITD is present in 15% and FLT3 point mutations in 7% of pediatric AML, and children with FLT3 ITD have a particularly poor prognosis.

FLT3 has also been implicated as a target in pediatric patients with MLL -rearranged ALL. Gene-expression profiling studies comparing MLL -rearranged ALL with all other ALL and AML have demonstrated a unique expression profile for MLL -rearranged leukemia. FLT3 is the gene most frequently overexpressed in MLL -rearranged leukemia compared with the other acute leukemias. An initial study reported mutations in FLT3 in approximately 15% of samples of MLL -rearranged ALL. All of these were point mutations in the activation loop, including mutations not previously reported in patients with AML (deletion of isoleucine 836). These mutations lead to constitutive activation of the kinase and increased sensitivity to small-molecule inhibitors of FLT3. Furthermore, MLL -rearranged lymphoblasts with high levels of wild-type FLT3 were sensitive to the FLT3 inhibitor PKC412. Subsequent studies have reported an incidence of FLT3 mutations in infant MLL samples ranging from 3% to 18%. These studies also confirmed increased expression of FLT3 in MLL -rearranged infant ALL compared with both infant and noninfant patients with germline MLL . Even in the absence of documented mutation, a high level of FLT3 expression was associated with FLT3 ligand independence and with increased sensitivity to the FLT3 inhibitor PKC412. In addition to its role as a target in MLL -rearranged ALL, FLT3 is a potential target in children with T-cell ALL (T-ALL), particularly with early precursor T-ALL, and in children with hyperdiploid ALL, wherein FLT3 mutations have been reported.

Several FLT3 inhibitors have been tested in clinical trials for adults with AML. Most of the early trials testing these compounds have had some transient responders with decreased peripheral blood or marrow blasts, and the compounds have generally been well tolerated. However, very few patients enter a complete remission, and the majority of patients eventually progress. The high rate of initial response failure, as well as the development of progressive disease despite an initial clinical response, has tempered enthusiasm for FLT3 inhibitors in AML. There are several possible explanations for the lack of dramatic clinical responses, including issues related to pharmacology and the failure to achieve sustained, complete inhibition of FLT3. Another explanation is the acquisition of resistance mutations in FLT3 itself. Indeed, the newer drug quizartinib (AC220), with greater potency and sustained inhibition of FLT3, has had promising initial results in clinical trials. Moreover, point mutations within the kinase domain of FLT3 -ITD, conferring quizartinib resistance, were recently reported. This finding is similar to the emergence of BCR-ABL –resistance mutations in patients with CML treated with imatinib. Significantly, this study provided gold-standard validation that FLT3-ITD is an oncogenic driver of AML.

The clinical study of FLT3 inhibitors in children with leukemia is ongoing. A phase I trial testing the multikinase inhibitor sorafenib, with activity against FLT3, in children with refractory solid tumors and leukemia, was reported by the Children’s Oncology Group (COG). This trial established a recommended phase II dose of sorafenib for children with leukemias. Rash, diarrhea, and liver enzyme elevation were the most common sorafenib-related toxicities. Two patients with AML with the FLT3-ITD achieved less than 5% bone marrow blasts with 4 or 5 cycles of sorafenib. A second phase I trial evaluated the effects of sorafenib in combination with clofarabine and cytarabine in children with relapsed or refractory leukemia. On day 8 of this trial, 10 of 12 patients demonstrated a decrease in blast percentage after sorafenib alone. After combination chemotherapy, six patients (three of these with FLT3-ITD) achieved a complete remission, two with FLT3-ITD had a complete remission with incomplete count recovery, and one patient with FLT3 wild-type had a partial remission. This trial also demonstrated downregulation of AKT, RPS6, and 4E-BP1 phosphorylation in leukemic blasts. Finally, there was another report of three patients with relapsed FLT3-ITD AML who achieved a sustained remission with sorafenib in combination with other cytotoxic chemotherapy. Based on these encouraging initial results, there are now multiple ongoing trials to test the efficacy of FLT3 inhibitors in pediatric patients with leukemia.

JAK Mutations in Pediatric Leukemia

With the marked success of imatinib in the treatment of BCR-ABL -rearranged ALL in children ( Fig. 44-5 ), there has been interest in the identification of other kinase abnormalities in childhood cancers, including leukemia. Recurrent mutations in Janus kinases (JAKs) have emerged as immediately targetable in a subset of pediatric patients with leukemia. JAKs phosphorylate substrates, including STAT proteins, on ligand binding to a type I cytokine receptor, leading to altered transcription of growth-enhancing and anti-apoptotic genes. Interest in targeting JAK2 was sparked by the finding of recurrent JAK2 mutations (most frequently involving valine 617) in patients with myeloproliferative disorders. Subsequently, activating mutations in JAK2 and JAK3 were reported in patients with AML and Down syndrome–associated acute megakaryoblastic leukemia and in 18% to 28% of patients with Down syndrome–associated ALL. In the case of Down syndrome–associated ALL, the mutations affect a highly conserved arginine residue (R683) in the pseudokinase domain of JAK2 and have been demonstrated to induce JAK/STAT activation and promote growth factor independence in BaF3 cells and response to JAK inhibitors.

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