Targeting Oncoproteins for Molecular Cancer Therapy

Turning Chemical Warfare into Cancer Therapy

Cancer and efforts to treat cancer are described in Ancient Egyptian documents dating back to 1600B.C. The first successful cancer treatments were arsenic-based therapies for leukemias, with the first reported application to cancer in the nineteenth century [ 1 ]. However, nitrogen mustards are often accredited as the first modern chemotherapy. Originally intended as a chemical warfare agent in World War I, nitrogen mustard was stockpiled by several countries. During World War II, Axis bombers sunk a ship containing large quantities of nitrogen mustard and killed numerous Allied sailors. Autopsies revealed that most of the victims’ white blood cells were depleted, suggesting that the nitrogen mustard destroyed these cells or inhibited cell division of these cells. This observation birthed the hypothesis that nitrogen mustards might prevent the rapid division of cancer cells, one of the few properties of cancer understood at that time. Today, the hallmarks of cancer as recently redefined by Hanahan and Weinberg [ 2 ] include several complex and connected cellular properties that allow for this phenotype: resistance to cell death, sustained angiogenesis, limitless ability to replicate, self-sufficiency in growth factor signaling, unresponsiveness to anti-growth factor signaling, genomic instability and mutation, deregulating cellular energetics, evading immune-mediated destruction, oncogenic inflammation, and invasiveness and metastasis [ 3 ]. The identification and understanding of these hallmarks is a direct result of our molecular understanding of cancer that has surfaced relatively recently. Each of these hallmarks is determined by a host of molecules which together represent distinct therapeutic opportunities to target molecules that give rise to these defining properties of cancer (Fig. 38.1 ). Fig. 38.1 Molecular therapies with targets associated with the . The inner yellow circle shows the hallmarks of cancer as defined by Hanahan and Weinberg. The middle purple ring contains proteins that govern these hallmarks. The outer green circle contains exemplary molecular therapies that explicitly such molecules.

The National Cancer Act of 1971, signed by President Richard Nixon, declared “… war on cancer …” and announced full Congressional and presidential support to eradicate the disease. As a result of increased funding and technological advancements, our understanding of cancer biology on the genetic and molecular level has exploded. Today, cancer phenotypes are associated with genetic and molecular culprits along with complex networks of various regulatory mechanisms that together cause and sustain cancer. Superficially, the genes capable of inducing carcinogenesis are divided into two categories. The first is a tumor suppressor, which is a gene that if inactivated, restricts cell division. Genes that confer pro-survival changes if activated are oncogenes. Intuitively, tumor suppressors such as p53 are commonly inactivated in cancer while oncogenes such as myc are activated and/or overexpressed. These carcinogenic changes manifest themselves by a number of avenues including overexpression, mutations, deletions, loss or gain of alleles, epigenetic modifications that alter genomic structure, alternative splicing, interference with the translation and transcription of the gene, chaperone-mediated protein folding, protein degradation processes, and posttranslational modifications that modulate localization, protein-protein interactions, and/or activity of the protein. Whether some of these changes occur simultaneously, sequentially, or otherwise is highly context-dependent and debated. Furthermore, the functional consequences of these alterations have a wide range of effects through crosstalk in cell signaling pathways. In the background of cancer-associated genomic instability, these wide-spread alterations and the functional redundancy of various genes provide a breeding ground for therapeutic resistance and make targeting cancer cells at the molecular level a challenging feat.

Radiation, surgical resection, and chemotherapy still comprise the vast majority of first-line cancer therapy today. While chemotherapy has yielded enormous patient benefit it is often accompanied by side effects that limit dose and therefore efficacy. Traditional chemotherapy is based on the notion that cancer cells divide more rapidly than normal cells and consequently will be differentially affected by an inhibitor of cell division. However, many types of normal cells need to divide for normal function. Our increased understanding of cancer has yielded numerous molecular targets for cancer therapy that may be less necessary for normal cell function and therefore may be less toxic. For instance, most normal cells divide a finite number of times, which is called the Hayflick limit. When cells divide, the ends of their chromosomes termed telomeres shorten as result of the DNA replication process, a phenomenon called the end replication problem. Upon reaching a critical telomere length, cells stop dividing and enter a dormant state referred to as cellular senescence. Cancer cells must evade senescence as they need to propagate indefinitely. Cancer cells escape this phenomenon by activating telomerase, an endogenous enzyme capable of elongating telomeres. As this oncogenic process is essential to cancer cells but not essential to most types of normal cells, telomerase is an attractive target for cancer therapy. Several types of inhibitors targeting various aspects of the telomerase molecular machinery and function are being investigated as a novel cancer therapy. There have been and continue to be efforts to discover therapies that alter the function of cancer-specific targets such as this. The rise of targeted therapies over the past two decades is a result of the rich marriage of our modern tools to understand cancer and our ancient desire to cure it. This chapter details the discovery, translation, development, and exemplified therapeutic concepts of several novel cancer therapies that specifically target oncoproteins and have molded how we discover and develop novel therapies today (Fig. 38.2 ). Fig. 38.2 Outline of exemplary molecular therapies targeting oncoproteins and their representative signaling networks.

Small Molecules with Big Consequences

A narrow library of atomic arrangements make up the relatively small number of molecular building blocks of cellular life such as nucleic acids that comprise DNA and RNA as well as amino acids that the end product, proteins, are made of. These building blocks are themselves synthesized from other molecules during cellular uptake and metabolism. Life has evolved to find chemical means for cells to convert small molecules to other molecules that are more useful for them. In a sense, we attempt to do the same for cancer therapy through medicinal chemistry to make more effective therapies from lead compounds. Small molecules offer a vast range of activity: sucrose sweetens foods and beverages, sodium pentothal is lethal, and amoxicillin can cure many bacterial infections. Our expanding knowledge in chemistry enables us to modify small molecules in nature or our synthetic libraries and create new molecules with altered functions, just as a cell does. Our understanding and ability to produce biological molecules on a therapeutic scale has been a relatively recent endeavor, therefore chemotherapies are almost entirely composed of small molecules. The availability, diversity, synthetic amenability, cost, and size of synthetic and natural compounds make small molecules an irreplaceable source of therapies.

A Rational Success Story: Imatinib

Chronic myelogenous leukemia (CML) is a cancer that causes increased amounts of white blood cells and is almost always associated with a specific translocation of chromosomes 9 and 22. The resulting shorter chromosome 22 is known as the Philadelphia chromosome, a tribute to the city housing the researchers who identified this in 1960s. The Philadelphia chromosome encodes a fusion of the two genes that results in the production Bcr-Abl. Abl is a tyrosine kinase that becomes constitutively active in the gene fusion product. This unregulated kinase activity causes oncogenic cell signaling shown to be sufficient to induce leukemia in mice. As a poster-child for translating modern cancer knowledge to clinical benefit, imatinib has significantly improved CML patient response rates. The FDA approved imatinib in 2001 for the treatment of CML as the first cancer therapy to target an intracellular molecule. Imatinib is a direct result of medicinal chemistry performed on a molecule identified as a protein kinase C (PKC) inhibitor [ 4 ]. Chemical modification of the PKC inhibitor altered the specificity of the molecule and rendered the derivative a potent inhibitor of v-abl [ 5 ], c-kit, and platelet-derived growth factor receptor (PDGFR) [ 6 ]. All of these proteins are receptor tyrosine kinases (RTKs) that bind extracellular factors and transduce the signal by phosphorylating specific protein substrates. The observation that imatinib inhibits Abl and BCR-Abl led to preclinical development and ultimately clinical trials of imatinib as a cancer therapy for CML. The concept of taking the currently available knowledge of a particular target and identifying a way to alter its function for therapeutic benefit is known as rational drug design.

Structural biology has played a key role in understanding how molecules look, how they move, how they interact with other molecules, and how all of these things change in different environments. Computational molecular docking in concert with an X-ray crystallographic structure of the catalytic domain of Abl bound to imatinib revealed that imatinib binds to the ATP-binding site of Abl preferentially when the protein is in the inactive form (Fig. 38.3 ) [ 7 , 8 ]. The crystal structure also showed that a chemical group added to increase solubility also forms hydrogens bonds with two residues of Abl. The insight gained from such structures provides an understanding of how and where a drug binds, what types of interactions are formed, and what role each residue or functional group plays. Due to the relatively facile excision and crystallization of the catalytic domains of many kinases, several atomic structures of these proteins are freely available in the Protein Data Bank (PDB). This provides fertile ground for numerous applications of computational chemistry to foster drug discovery and development. In addition to structural biology, medicinal chemistry also provides insight into the function of particular atoms of a molecule while searching for a more therapeutically potent derivative. The drug development process typically begins by identification of lead molecules using various screening techniques that search for a desired effect. Validation of these leads and optimization by medicinal chemistry ensues to identify the most promising compound to continue to develop. The process of lead optimization and elucidative structural biology intrinsically discerns the role of particular parts of drug and target molecules in the activity of the drug, giving rise to structure–activity relationships (SARs). Fig. 38.3 Crystal structure of bound to Abl kinase . The Abl kinase ( green ) is represented as a secondary structure cartoon with its activation loop highlighted in yellow . The confirmation is in an inactive kinase state. PBD accession 1IEP.

All of the kinases in the human genome, called the kinome, share a high degree of sequence and structural homology. This means that identifying inhibitors that are specific for a given kinase is challenging and often kinase inhibitors have multiple targets. Imatinib is no exception, as it inhibits autophosphorylation of c-KIT, platelet-derived growth factor (PDGF), and ARG [ 9 ] kinases in addition to BCR-ABL. c-KIT is a receptor tyrosine kinase that is almost ubiquitously mutated in gastrointestinal tumors (GISTs) during the transformation of interstitial cells of Cajal located in the gastrointestinal tract. This mutation typically occurs in exon 11 of the c-Kit gene, which results in constitutive autophosphorylation of the protein that continually activates downstream pro-survival signaling that is oncogenic. The observation that imatinib inhibits c-KIT lead to preclinical development and clinical trials with the treatment of GISTs. Seven years after its approval for treatment of advanced CML, the FDA approved imatinib for treatment of GISTs following surgical removal of the tumor.

This extension of clinical applications demonstrates a few key concepts in cancer therapy. Firstly, exclusive specificity of a cancer drug for a molecular target is a virtue rooted in our movement toward targeted therapy. We know that without specificity, effects on normal cells can yield side effects that are deleterious. However the distinction should be made that while cancer therapies should target cancer-specific properties, this does not have to be accomplished by targeting a single molecule. It is conceivable that evolving therapeutic resistance is easier against a single target rather than an array of molecules. Furthermore, different types of tumors seem to rely on alterations in multiple genes and so multiple targets may allow for broader spectrum and more potent antitumor activity. The clinical extension of imatinib to GISTs also underscores the importance of identifying and understanding the molecular targets of therapies and how they fit with our molecular understanding of cancer. The application of a therapy used in distantly related clinical settings is a concept that continues today. Such applications are often a direct result of rational drug design and typically have an expedited timeline for starting clinical trials as they have already been tested in humans. The time and cost associated with development of cancer therapies is astounding, taking an average of over 14 years and $2 billion to reach FDA approval of a cancer drug with a success rate of just over 7 % [ 10 ]. Clearly, reducing the time spent in early phase trials profiling safety of the drug would reduce cost and expedite evaluation and patient benefit.

Therapeutic resistance is frequent in cancer. Cancer is defined by its uncontrollable cellular division and therefore is a disease of evolution governed by natural selection. While our cells copy our genome during cell division with amazing fidelity, the molecular machinery that performs this task is not completely error free. This endogenous source of mutations, environmental mutagens such as UV radiation or tobacco, and the genomic instability associated with cancer provide a sufficient source of heritable variability. Cancer treatments serve as a selective force for the cancer cells. Imagine yourself looking at population of millions of CML cells circulating in the blood that have the oncogenic Bcr-Abl fusion gene. Now introduce imatinib into the blood stream, which inhibits Bcr-Abl, and watch as the CML cells begin to die due to their dependency on the function of this oncogenic protein. If any single cell out of these millions of CML cells evolved into cancer by a process not involving BCR-ABL or can continue to divide using other oncogenic alterations, the cell will survive and continue to divide. The entire offspring of such cells will not rely on BCR-ABL to propagate and thus the patient will now not respond to therapies that target BCR-ABL. The enhanced sources of genetic variability and the solitary goal to divide more rapidly give rise to heterogeneity of tumors. This heterogeneity has significant implications as to how cancer is diagnosed and managed.

There are several ways that cancer cells get around therapeutic road blocks in intracellular signaling to keep propagating. In the case of imatinib, mechanisms of resistance include increasing the amount of Bcr-Abl to saturate the drug, mutating Bcr-Abl to still be constitutively active in the presence of imatinib, bypassing BCr-Abl and activating its downstream targets to achieve the same end goal, or simply getting rid of the drug altogether. Following the first route, cell culture and patient data revealed that overexpression of Bcr-Abl by gene duplication occurred in refractory patients and that this was sufficient for imatinib resistance [ 11 – 14 ]. Additionally, several patients were found to have mutations in the BCR-ABL that allowed for sustained signaling in the presence of imatinib. Due to the availability of the drug-bound crystal structure of the Abl catalytic domain, the effects of these point mutations were rationalized at the molecular level. Another intriguing avenue of drug resistance is getting rid of drugs by upregulating molecular efflux pumps localized at the cell membrane. The most characterized member of this family of proteins that induce multidrug resistance is p-glycoprotein (PgP), exhibiting broad substrate specificity to include several chemotherapies such as vinblastine, doxorubicin, and paclitaxel. Upregulation of PgP was also found in imatinib-resistant clones and in advanced CML patients. Pharmacological inhibition of multidrug resistance proteins is being explored in clinical trials in combination with chemotherapy with mixed success [ 15 – 19 ]. Strategies to circumvent imatinib resistance in the clinic include increasing the dose, changing to other investigational therapies, and administering alternative Bcr-Abl inhibitors such as nilotinib and dasatinib. Nilotinib and particularly dasatinib also target Sarc-family kinase (Src) and have significantly improved patient outcome after imatinib failure [ 20 – 29 ]. Treating patients that are resistant to first-line therapies is a challenge in clinical oncology and is a major barrier in getting an investigational drug approved as these are often tested in these refractory patients.

Ten years after the discovery of imatinib, it was approved in the US, Japan, and Europe as the first-line therapy for CML. The story of imatinib is a testament to our modern molecular understanding of cancer. Computational biologists, chemists, cell biologists, translational oncology researchers, and clinical oncologists from various countries around the world collaborated to discover and translate this therapy. Such interdisciplinary integration is a recipe for success in modern drug discovery and development and is a theme found throughout molecular targeted cancer therapy and beyond.

Gefitinib

Growth factor signaling is intimately involved in tumorigenesis and propagation and is involved in two of the hallmarks of cancer. Growth factor signaling typically involves the binding of an extracellular factor by a transmembrane receptor on the cell surface, which triggers intracellular signaling events. These events such as substrate phosphorylation ultimately allow for cellular proliferation through various mechanisms such as turning on transcription factors that activate genes necessary for cell cycle progression. One group of receptors that mediate several of such signals is the epidermal growth factor receptor (EGFR) family and as a consequence, this family is commonly altered by mutations and/or overexpression in a wide range of solid tumors. These receptors form homo- or hetero-oligomers upon binding various ligands to trigger a range of intracellular signaling events via Ras/Raf/MAPK, PI3K/Akt, STAT, or Src kinase pathways. EGFR is one member of this family that homodimerizes upon binding and induces proliferation through ERK, PI3K/Akt, Ras, and STAT signaling pathways. Due to the frequency of their alteration in a variety of cancers and its plethora of potent downstream oncogenic targets, the EGFR family members have successfully targeted by a number of therapeutic approaches over the past two decades.

Gefitinib is an orally active EGFR inhibitor identified by Astra Zeneca and first reported in 1996. A quinazoline derivative was found to be an ATP-competitive inhibitor and highly specific to EGFR over its related family members. Cell-based events in accordance with the inhibition of EGFR activation were observed such as autophosphorylation, upregulation of the CDK inhibitor p27 Kip1 , and transcriptional inhibition of the transcription factor c-Fos [ 30 , 31 ]. Preclinical studies found that gefitinib had cooperative to synergistic combinations with several chemotherapies in EGFR-overexpressing cancer cell lines [ 32 ]. Interestingly, another group reported similar effects with gefitinib-chemotherapy combinations but in cancer cell lines with low EGFR expression [ 33 ]. Oral and intravenous administration of gefitinib in rats and dogs found the bioavailability of the drug to be ~50 % and that the drug was well distributed throughout the body [ 34 ]. Pharmacokinetic (PK) studies indicated that oral administration of gefitinib at 100–700 mg/day was well tolerated, had a terminal half-life between 1 and 2 days, and reached serum concentrations that inhibited 90 % of EGFR activity in vitro [ 35 – 39 ]. The first phase I trial of gefitinib was a dose escalation study in patients with various solid tumors that reported objective partial responses in NSCLC patients at oral doses ranging 300–700 mg per day on a 14 days-on, 14 days-off schedule [ 40 ]. The observed dose range with antitumor activity was below the dose-limiting toxicity (DLT) reported and was corroborated with another phase I trial [ 41 ]. These clinical trials used high-performance liquid chromatography (HPLC) with mass spectrometry (MS) to monitor serum concentrations of gefitinib [ 42 ].

HPLC along with complementary molecular identification techniques are often employed in clinical trials involving small molecules. HPLC allows for the separation and quantification of molecules based on their absorbance and interactions with a solid matrix. Molecular properties such as size and charge along with instrument and solvent parameters determine how the molecule interacts with this matrix. These interactions determine how long the molecule takes to migrate through the matrix column. HPLC conditions are optimized to allow for quantitative identification of a given molecule based on this empirically determined elution time, called retention time. The calculated serum concentrations can then be used to determine a plethora of pharmacokinetic parameters. These parameters are particularly important in guiding dosing schedules of new therapies and rationalizing patient responses. This technique is often coupled with mass spectrometry to allow for further verification that the molecule identified at a particular retention time is indeed the target molecule. Mass spectrometry is an electromagnetic separation method that distinguishes molecules based on their size to charge ratio of ionized forms of the molecule, which are generated by molecular collisions. Together, these ionized fragments yield a unique fingerprint for each molecule. Coupling HPLC and mass spectrometry has been instrumental in increasing the accuracy and reliability of pharmacokinetic data and identifying metabolites of drugs. Direct and indirect modifications of drugs often occur once delivered due to the staggeringly diverse mixture of molecules presence in the blood, digestive system, etc. As for gefitinib, HPLC-MS identified desmethyl-gefitinib as a metabolite of gefitinib that is inactive in vitro and in vivo [ 43 ]. Identification of metabolites is important for understanding and monitoring the various molecular species responsible for therapeutic activity as well as guiding chemical optimization.

Phase II trials with gefitinib again yielded some patient benefit in NSCLC along with low toxicity [ 44 , 45 ]. With data available from phase III clinical trials, the FDA granted accelerated approval for gefitinib as a third-line therapy in NSCLC in 2003. This type of approval is granted based on promising clinical evidence of efficacy when there is no current therapy for a particular clinical setting. However, this approval is temporary and full approval is contingent on a more complete clinical data set. In the few years following accelerated approval of gefitinib, several studies found responses in the overall population of NSCLC patients [ 44 – 46 ]. However, a small subset of responders was identified amongst these trials with the following characteristics: female, Asian, never-smokers, adenocarcinoma, and mutant EGFR [ 46 – 48 ]. Several other studies confirmed efficacy of gefitinib in NSCLC patients with EGFR mutations [ 49 – 52 ] though no benefit was found in NSCLC patients with EGFR gene amplification [ 53 ]. A breakthrough study published in 2004 found that the majority of gefitinib responders had EGFR deletions or point mutations clustered at the ATP-binding site of EGFR, which results in a ten-fold increase in sensitivity to gefitinib [ 54 ]. Others studies corroborated these EGFR alterations in gefitinib-responsive patients [ 55 , 56 ]. These genetic alterations were structurally modeled and rationalized using the crystal structure of the human EGFR kinase domain bound to gefitinib (Fig. 38.4 ) [ 57 ]. These response-determining mutations occur at the active site of the kinase domain in structural regions that are responsible for autoregulation of kinase activity. Further studies found that these particular mutations in EGFR stabilize the active form of the kinase and shift its affinity from ATP toward gefitinib [ 58 ]. Fig. 38.4 Molecular structure of gefitinib and bound to EGFR. ( a ) Molecular structure of gefitinib. ( b ) Crystal structure of gefitinib bound to EGFR kinase domain. Overlay of wild-type ( blue ), L858R ( green ), or G719S ( beige ) EGFR bound to gefitinib shown in gray , light green , and magenta , respectively. The L858R point mutation is shown in orange and its hydrogen as a black dashed line while the G719S point mutation is shown in red . PBD accessions 2ITY, 2ITZ, 2ITO.

One study mandated by the accelerated FDA approval of gefitinib and another phase II clinical trial found no benefit with gefitinib in NSCLC refractory to first-line or second-line therapies. This led the FDA to restrict its usage to patients who have previously benefited or are currently benefiting from gefitinib in 2005. Subsequent clinical trials have supported gefitinib following chemotherapy resistance [ 59 , 60 ]. Together, the clinical trials comparing gefitinib and chemotherapy are confusing as they conclude significant to no benefit of gefitinib over chemotherapy. These conflicting results have been ascribed to fundamental differences in the therapeutic mechanisms of chemotherapy and targeted therapy, differences in patient populations, and biomarker selection and technique accuracy [ 61 ]. Recent studies have supported gefitinib as a first-line therapy due to demonstrated superiority over standard of care therapy in mutant EGFR NSCLC patients [ 62 – 64 ]. This data is anticipated to extend the restricted use label of gefitinib to its use as a first-line therapy in NSCLC patients with mutant EGFR. The use of targeted therapy based on molecular determinants surfaces later in the chapter and is increasingly integrated into FDA approval stipulations as personalized medicine emerges in practice.

Two Birds with One Stone: Iapatinib

As discussed elsewhere, the EGFR family is overexpressed in numerous cancers. One particular EGFR member, Her2, does not bind ligands but can form heterodimers with other ligand-bound family members to transduce oncogenic signaling through the Ras/Raf/MAPK cascade. By 2001, several recent EGFR family-targeted molecular therapies had demonstrated significant clinical efficacy in cancer including the EGFR-targeted therapies gefitinib, erlotinib, and cetuximab along with anti-Her2 monoclonal antibody herceptin. Her2 and EGFR are well-established therapeutic cancer targets based on these clinical successes, the prevalence of EGFR family alternations in cancer, and the highly homologous and druggable ATP-binding site shared by EGFR family members. A collaboration of private companies including GlaxoSmithKline launched a large synthetic effort to simultaneously target these two proteins [ 65 – 67 ]. Published results of these efforts detail the synthesis, specificity, and biological activity of several quinazoline or pyridopyrimidine compounds. One initial compound, GW2974, produced tumor stasis given orally at a dose of 30 mg/kg in squamous cell head and neck carcinoma and breast cancer xenografts. A later report found yet another compound, GW572016 , to be a potent, reversible inhibitor of EGFR and Her2 even in the presence of excess EGF [ 68 ].

The full length 185 kDa Her2 protein can be proteolytically cleaved to shed its extracellular domain and give rise to its truncated form, p95 Her2 . p95 Her2 is constitutively active which causes autophosphorylation, has a high oncogenic transformation ability, and correlates with lymph-node positive metastasis and poor therapeutic response [ 69 – 76 ]. p95 Her2 was found to be insensitive to trastuzumab, preferentially dimerize with ErbB3, and be regulated by the ErbB3 ligand heregulin [ 77 ]. Lapatinib blocked baseline autophosphorylation and downstream signaling events induced by p95 Her2 . Her2 was found to contribute to androgen receptor transcriptional activity [ 78 , 79 ]. Accordingly, lapatinib cooperated with the small molecule estrogen receptor-agonist tamoxifen to reduce estragon receptor-dependent transcriptional activity and inhibit the growth of a tamoxifen-resistant xenograft [ 80 ]. Lapatinib-resistance clones were generated by chronic exposure of cancer cell lines and were enriched in androgen receptor signaling events, suggesting the importance of this signaling in lapatinib response [ 81 ]. Combining lapatinib with anti-Her2 antibodies such as trastuzumab enhanced downregulation of the anti-apoptotic protein survivin and apoptosis in Her2-overexpressing breast cancer cells [ 82 , 83 ] and trastuzumab-resistant cells [ 84 ]. These preclinical observations served as the basis for combining lapatinib with hormone therapy and trastuzumab.

Phase I studies in healthy volunteers found orally administered lapatinib to reach peak serum concentrations at 3 h and achieve steady state concentrations after 1 week [ 85 ]. High fat content of the patient significantly increased bioavailability of the drug, highlighting yet another clinical variable impacting therapeutic response [ 86 – 88 ]. EGFR-overexpressing and Her2-overexpressing metastatic carcinoma patients receiving lapatinib showed significant clinical responses and tolerated oral daily doses up to 1600 mg [ 89 ]. Lapatinib was also safe and effective as a first-line monotherapy in Her2-amplified advanced or metastatic breast cancer [ 90 ]. The agent has shown preliminary efficacy in head and neck squamous cell carcinoma [ 91 , 92 ], but not in NSCLC [ 93 ] or prostate cancer [ 94 ].

Lapatinib has been combined with a variety of chemotherapies, hormone agonists, and trastuzumab. Addition of lapatinib to the FOLFOX4 or FOLFIRI chemotherapy regimen was safe [ 95 , 96 ] and is now being explored for efficacy. Based on the clinically active combination of trastuzumab with capecitabine, studies with this combination were conducted on Her2-overexpressing advanced or metastatic breast cancer patients [ 97 , 98 ]. The study found that the time to progression doubled when lapatinib was added to capecitabine without significant additional toxicity. Efficacy within these Her2 + patients was not limited to a subgroup [ 99 ], but a separate study found lapatinib to be effective in Her2 + but not EGFR + /Her2 - inflammatory breast cancer patients [ 100 ]. This suggests that Her2 inhibition is a key mediator of antitumor efficacy in this malignancy. However, a higher lapatinib was recently found in preclinical models to have EGFR-independent and Her2-independent effects on death receptor upregulation that enhances efficacy when combined with TRAIL and TRAIL-receptor antibodies that bind to these receptors [ 101 ]. This rationalizes the clinical exploration of higher doses of lapatinib to gain increased efficacy via off-target mechanisms.

In 2007, the FDA approved trastuzumab and chemotherapy-resistant, for the treatment of EGFR + advanced or metastatic breast cancer. An increase in progression-free survival (PFS) from 3 to 8.4 months was observed with the addition of lapatinib to letrozole in estrogen receptor (ER)-positive metastatic breast cancer relative to letrozole monotherapy [ 102 ]. Based on this data, the FDA extended the indication for lapatinib to its use with letrozole in Her2 + ER + metastatic breast cancer in 2010. Addition of lapatinib to tri-weekly paclitaxel as a first-line therapy yielded a significant increase in time to progression of metastatic breast cancer patients that was restricted to the Her2 + patients [ 103 ]. A recent study has also demonstrated the safety and potential efficacy of combination with weekly paclitaxel [ 104 ]. In agreement with lapatinib and trastuzumab combinatorial preclinical data, PFS was significantly prolonged in trastuzumab-resistant Her2 + metastatic breast cancer with the combination of trastuzumab and lapatinib [ 105 ]. Lapatinib demonstrates the power of multitargeted therapies over single-targeted therapies such as trastuzumab and continues to be explored with various therapeutic combinations as a breast cancer therapy.

Rapamycin : From the Ground Up

The PI3K/Akt/mTOR pathway is a complex signaling network that controls cell survival, death, and division in response to a variety of stimuli such as hypoxia and growth factor deprivation. The elucidation of this pathway started in 1975, when rapamycin was first isolated as an antifungal agent from bacteria in a soil sample from the Polynesian island of Rapa Nui, where the compound got its name [ 106 , 107 ]. Follow up studies in rats found that rapamycin was a potent immunosuppressant [ 108 ] but the molecule lost attention in the scientific literature. Over a decade later, a high-profile immunosuppressant macrolide, FK506, was found to inhibit the proliferation of activated T-cells [ 109 ]. The same investigators that reported this observation also noticed structural homology between FK506 and rapamycin, which prompted a comparison study of their effects. Interestingly, rapamycin and FK506 antagonized the biological effects of each other. Experiments with radiolabelled FK506 found that rapamycin could directly compete with FK506 in cells for its undescribed binding target [ 110 ], which turned out to be FK506 binding protein (FKBP) [ 111 – 114 ]. However, the two molecules had different effects on signaling events involved in T-cell activation such as the transcriptional activity of NFAT or induced IL-2 transcription [ 115 ]. The structure of FKBP alone was determined by NMR [ 116 ] and in complex with FK506 by X-ray crystallography [ 117 ] which together revealed a rather unique drug binding site and revealed a strong conformational shift in FK506 and to a lesser extent in FKBP. Rapamycin was later found to bind to the same site but did not demonstrate a significant conformational shift itself (Fig. 38.5 ) [ 118 ]. Fig. 38.5 and its association with FKBP . ( a ) Molecular structure of rapamycin. ( b ) Surface representation of rapamycin ( yellow ) bound to FKBP ( green ) at a hydrophobic pocket. ( c ) Key interacting residues of FKBP involved in hydrophobic interactions ( beige ) and hydrogen bonds ( blue ) indicated with black dashed lines . PBD accession 1FKB.

Genetic studies in yeast found two homologous genes to be determinants of rapamycin-toxicity and as such were called targets of rapamycin (TOR1 and TOR2) [ 119 ]. Soon after, several reports identified a mammalian homologue of the proteins present in complex with FKBP that was dependent on the presence of rapamycin [ 120 – 122 ]. Furthermore, this mammalian target of rapamycin (mTOR) was required for the G1-arrest induced by rapamycin, which had been widely reported [ 110 , 115 , 123 – 126 ]. Rapamycin inhibits the function of the Akt-substrate mTOR [ 127 – 129 ], a serine/threonine kinase that phosphorylates p70 S6K , which mediates growth factor signaling in response to cytokines such as interleukin-2 [ 125 , 130 – 132 ]. mTOR was also found to impact EIF4E, a protein that inhibits translation by binding to the 5′ end of mRNA [ 133 – 136 ]. In the absence of rapamycin, mTOR can associated with adaptor proteins rictor or raptor to form mTORC1 or mTORC2, respectively. These complexes have different substrate specificity and cause distinct downstream signaling events.

In 1999, the FDA approved rapamycin as an immunosuppressant to prevent graft rejection in combination with cyclosporine A and steroids. However, the properties of rapamycin extend beyond this applicaiton. Phenotypic evaluation of growth inhibition in a particularly rapamycin-sensitive fungus, Candida albican , found nucleotide degradation and inhibition of synthesis as a primary mechanism of action in 1979 [ 137 ]. Nucleotide synthesis is a target of several clinically effective chemotherapies such as methotrexate and fluorouracil. This observation caught the eye of the National Cancer Institute (NCI). NCI experiments and an independent report found rapamycin to have antitumor activity comparable to that of cyclophosphamide and 5-FU in several solid malignancies and leukemia [ 138 ]. Other studies supported anticancer activity of rapamycin B-cell lymphoma [ 139 ], small cell lung cancer (SCLC) [ 140 ], rhabdomyosarcoma [ 141 ], melanoma [ 142 ], and pancreatic cancer [ 143 ]. Rapamycin was also found to inhibit angiogenesis under hypoxia [ 144 ] by causing transcriptional inhibition of VEGF [ 145 ], a process detailed later in the discussion of bevacizumab. Furthermore, rapamycin sensitized promyelocytic leukemia [ 141 ] and ovarian cancer [ 146 ] cell lines to cisplatin-induced apoptosis and inhibited transformation by PI3K or AKT. However, the clinical trial data generated in early phase trials of rapamycin as an immunosuppressant uncovered a poor pharmacokinetic profile [ 147 , 148 ]. To overcome this problem, a large array of rapamycin analogues were created that are collectively called rapalogues.

Temsirolimus (CCI-779) is one of the first rapalogues and is a water soluble, chemically stable derivative of rapamycin (also called sirolimus) developed by Wyeth Pharmaceuticals. Preclinical studies found temsirolimus to have PTEN-dependent antitumor activity in a number of cancers [ 149 – 155 ] and like rapamycin, bound FK506bp and inhibited phosphorylation of S6K and 4EBP-1 [ 156 , 157 ]. Phase I evaluation of temsirolimus found reversible mucositis or skin-related toxicity, no immunosuppressive functions, and that the major metabolite of temsirolimus was rapamycin [ 158 ]. Another study reported a linear correlation of time to progression and p70 S6K kinase activity as measured in peripheral blood mononuclear cells, thus providing a pharmacodynamic marker [ 159 ]. Temsirolimus had similar toxicities and response rates at a dose of 25, 75, and 250 mg in renal cell carcinoma. A multicenter phase III study in renal cell carcinoma (RCC) demonstrated extended overall survival (OS) and PFS in patients relative to interferon A but found no support for the combination of these therapies [ 160 ]. In parallel with the publication of this study, temsirolimus became the first FDA-approved cancer therapy to explicitly target mTOR and joined sorafenib and sunitinib for the treatment of advanced RCC. A comparison of temsirolimus with other approved therapies for CML as chosen by the investigator found superiority of temsirolimus in terms of improving PFS and OS [ 161 ]. Combinations with anti-angiogenic therapies such as sorafenib, sunitinib, and bevacizumab have yielded additional toxicity and largely no benefit [ 162 – 164 ]. Temsirolimus anticancer activity is also being clinically explored in breast cancer [ 165 ], gynecological malignancies [ 166 ], multiple myeloma [ 167 ], glioma [ 168 – 170 ], small cell lung cancer [ 171 ], and neuroendocrine carcinomas.

Everolimus and ridaforolimus are two other rapalogues that are being investigated in clinical trials. Unlike temsirolimus, everolimus retains the immunosuppressive properties of rapamycin and was approved in 2010 for organ rejection prophylaxis. Clinical trials found oral everolimus to be safe at an oral dose of 10 mg/kg [ 172 – 175 ]. A phase III study in metastatic (RCC) patients who had failed sorafenib, sunitinib, or the combination demonstrated an increased PFS with everolimus [ 176 ]. This resulted in FDA-approval of everolimus in 2009 for this indication. Based on promising early clinical data, this agent further received accelerated approval in 2010 for subependymal giant-cell astrocytoma patients with tuberous sclerosis who are not eligible for surgery [ 177 ]. Everolimus has promising preliminary efficacy in Hodgkin’s lymphoma [ 178 ], metastatic gastric cancer [ 179 ], refractory NSCLC in combination with docetaxel [ 180 ] or gefitinib, and in breast cancer as a monotherapy [ 181 ] or in combination with letrozole [ 182 , 183 ] or trastuzumab [ 184 ]. Interestingly, a phase II study with everolimus in refractory CLL reported partial responses along with an unexpected increase in the absolute lymphocytic count [ 185 ]. This has important implications for therapeutic sensitization as these cancer cells are likely to be more sensitive intravenous therapies when in circulation rather than in situ, however there is no clinical data to support this. Everolimus along with best supportive care was recently found to double PFS in patients with pancreatic neuroendocrine tumors [ 186 , 187 ]. Ridaforolimus is in earlier clinical development but has demonstrated some partial responses and acceptable toxicity profiles in as a monotherapy [ 188 – 190 ] and in combination with capecitabine [ 191 ] and paclitaxel [ 192 ] in solid and hematological malignancies.

These three rapalogues work by virtually the same mechanism of action and have been developed separately by pharmaceutical companies. As the development of these rapalogues has happened in a relatively short time span, one is left wondering if these therapies have the same clinical efficacy. This is a difficulty inherent in the drug development process as clinical trial design dictates combinations with approved therapies. Combined with conflicting private interests, discerning the efficacy and unique roles of competing therapies with a similar mechanism of action is challenging. A recent phase II study in advanced pancreatic cancer attempted to compare temsirolimus and everolimus but toxicity and lack of objective response in any treatment group confounded this comparison [ 193 ]. Nevertheless, the discovery and development of rapamycin and rapalogues has extended the life of numerous cancer patients across several malignancies, prevent transplanted rejections, and elucidated a critical cell signaling pathway. At a conceptual level, the rapamycin story highlights the ability of existing therapies to be applied to other medicinal situations, the insight that can be gleaned from mechanistic studies of pharmaceuticals, and the power of medicinal chemistry.

Sorafenib

Raf is a serine/threonine kinase that is the apical member of the MAPK signaling cascade, which mediates a variety of cellular processes such as cell death, proliferation, and differentiation in response to extracellular stimuli. Aberrant Raf is observed in about 30 % of human cancers and correlates with the progression of prostate cancer to androgen insensitivity [ 194 ]. This increased signaling can result from alterations in upstream members such as Ras or in one of the three isoforms of Raf1 (A-Raf, B-Raf, and C-Raf). The V600E mutation in B-Raf is seen commonly in melanoma and NSCLC [ 195 ]. C-Raf overexpression has been noted in hepatocellular carcinoma [ 196 ] and validated in preclinical models as a potent drug target for ovarian cancer [ 197 ]. Earlier reports found direct evidence for Raf in determining tumorigenicity and sensitivity to radiation that posed Raf as a drug target [ 198 , 199 ]. Bayer Pharmaceutical and Onyx Pharmaceutical jointly performed a high-throughput screen for Raf1 inhibitors. The screen found 3-thienyl as a lead compound with a Raf1 IC 50 of 17 μM and that adding a methyl group at a particular position results in a tenfold decrease in IC 50 . A follow-up screen with a library of analogues yielded 3-amino-isoxazole with an IC 50 of 1.2 μM [ 200 ]. Substitution of the phenyl group of this compound for a 4-pyridyl moiety lowered the Raf1 IC 50 to 230 nM as well as increased aqueous solubility. This compound was found to be orally active, inhibit signaling downstream of Ras through MEK and ERK, and inhibit cancer cell growth in vitro and in xenografts [ 201 ]. Based on SARs found throughout this process, further chemical modifications were explored and ultimately yielded sorafenib with a Raf1 IC 50 of 6 nM (Fig. 38.6 ) [ 202 ]. Fig. 38.6 Structure–activity relationships of Raf1 inhibitors explored during hit to lead development of sorafenib.

Sorafenib reduces MAPK signaling by inhibiting numerous oncogenic kinases: wild-type and V600E B-Raf, the angiogenic VEGFR family, platelet-derived growth factor receptor-β (PDGFRβ), fibroblast growth factor receptor 1 (FGFR1), the neurotrophin receptor RET, and the cytokine receptors c-Kit and Flt-3 [ 203 , 204 ]. The growth of several xenografts were inhibited by sorafenib though in a few cases, no change in MAPK signaling was detected but was rationalized by its anti-angiogenic effects of VEGFR inhibition [ 203 ]. Crystal structures of wild-type and V600E B-Raf in complex with sorafenib revealed key interactions with residues conserved in c-Raf [ 205 ]. The pyridyl ring occupies the ATP-binding pocket while the trifluoromethyl phenyl ring occupies a proximal hydrophobic pocket. Interestingly, the nitrogen of the pyridyl group forms a hydrogen bond with B-Raf and rationalizes the potent increase in activity following substitution of the pyridyl group for the phenyl group. The urea group bridging the rings was found to form a hydrogen bond network with protein, explaining its conservation throughout the analogue search and development. In this case, the structural data rationalized the previously found SARs. However, structural data can conversely be used to guide the exploration of analogue and can also potentiate in silico screen that computationally models the binding of a virtual library of ligands to protein structure. It should be noted that crystal structures do not provide a complete platform for computing binding affinities. While the biophysical details of ligand binding are beyond the scope of this chapter, the affinity of two molecules is determined by the change in enthalpy and entropy. As crystal structures represent a single average molecular conformation across the crystal lattice, they do not reflect the motions of the molecule. Therefore crystal structures cannot provide direct entropic information themselves and by extension do not fully represent binding affinity.

Since sorafenib inhibits a multitude of kinases, preclinical and early clinical trials explored a variety of malignancies. Several phase I trials found dose-dependent responses with an optimal oral dose of 400 mg [ 206 – 208 ] and addition of sorafenib to a variety of standard of care chemotherapy regimens did not increase toxicity profiles [ 209 – 214 ]. Renal cell carcinoma (RCC) is particularly resistant to the majority of chemotherapies and until 1997 was treated with interferon that causes significant toxicities and limited responses. Due to the poor clinical outcomes with standard of care therapies, preclinical efficacy of sorafenib, and an encouraging phase I result in a metastatic RCC patient, phase II studies were enriched with RCC patients. This study found a strong response in RCC patients [ 215 , 216 ] and led to a large-scale phase III study that reported a doubled PFS and ~40 % increase in OS [ 216 , 217 ]. These results gained sorafenib FDA-approval at the end of 2005 for RCC. A subset analysis of this phase III trial found similar clinical benefits regardless of previous cytokine therapy [ 218 ] and a follow-up >1 year from treatment initiation found sustained efficacy and a well-tolerated toxicity profile [ 219 ]. Liver transplant has traditionally been the only treatment option available for hepatocellular carcinoma. However, many patients become ineligible while waiting for the transplant as a result of disease progression. Human xenografts of liver cancer cell lines showed partial tumor regression from sorafenib and inhibition of ERK and EIF4 [ 220 ]. A phase II trial found moderate efficacy and that time to progression correlated with phospho-ERK levels [ 221 ]. A multicenter phase III trial demonstrated an unprecedented increased OS in HCC and solicited the FDA to extend the indication of sorafenib to unresectable HCC [ 222 ].

Due to the multitargeted nature of sorafenib, it is being explored as a monotherapy and in combination with chemotherapies in a variety of malignancies, though biomarkers are difficult to find. A recent trial in metastatic melanoma found no correlation between clinical responses and BRAF V600E mutation status, cyclin D1, or the proliferation marker Ki-67 [ 223 ]. Early evidence was promising with sorafenib, carboplatin, and paclitaxel in melanoma [ 224 ] but a recent phase III study in advanced melanoma failed to demonstrate any benefit as second-line therapy [ 225 ]. A phase III trial in advanced HCC doubled PFS and OS with sorafenib plus doxorubicin compared to doxorubicin alone [ 226 ]. Other promising efficacy of sorafenib has been seen as a neoadjuvant in advanced RCC [ 227 ], as a monotherapy [ 228 ] or with erlotinib [ 229 ] or gefitinib [ 230 ] in NSCLC, metastatic RCC with gemcitabine and capectibaine [ 231 ]. Sorafenib has had limited to no clinical efficacy in malignant mesothelioma [ 232 ], prostate cancer [ 233 , 234 ], uterine cancer [ 235 , 236 ], sarcomas [ 237 ], advanced and metastatic squamous cell carcinoma [ 238 ], with paclitaxel and carboplatin in NSCLC [ 239 , 240 ], or as a neoadjuvant in advanced ovarian cancer [ 241 ], sunitinib-refractory metastatic RCC [ 242 ].

Vemurafenib

Melanoma is particularly dependent on signaling through the c-Kit/NRAS/BRAF/MEK/ERK signaling axis. This solicits the clinical application of imatinib to melanoma as it inhibits c-Kit among other targets. However, targeting c-kit has been clinically limited in melanoma as several patients have gene amplification or have oncogenic alterations downstream in this signaling axis. Targeting BRAF in melanoma was explored during the clinical development of sorafenib but ultimately proved ineffective as a monoagent and did not improve OS in combination with carboplatin and paclitaxel in a phase III placebo-controlled trial [ 225 ]. Several reasons have been proposed for this failure such as unsaturated MAPK inhibition at the MTD of sorafenib [ 243 ] and furthermore, the ability sorafenib to target BRAF in vivo has been challenged [ 244 ].

The V600E activating mutation in BRAF is commonly observed in melanoma and results in resistance to therapies targeting upstream molecules. Using structural biology, vemurafenib was developed as a selective inhibitor of BRAF V600E [ 245 , 246 ], though other targets have been uncovered such as CRAF, ACK1, SRMS, and MAP4K5 [ 247 ]. Phase I studies with Vemurafenib reported objective responses [ 248 ] that were corroborated in phase II studies and found pERK to be a valid correlative response marker [ 249 ]. A large phase III study in therapy-naïve patients ineligible for resection was recently reported an increased PFS and OS relative to dacarbazine [ 250 ]. In 2011, the FDA approved vemurafenib for the treatment of unresectable or metastatic melanoma with BRAFV600E.

While verumafenib is clearly a clinical oncology success, patients with wild-type BRAF still need better treatment options, biomarkers are needed to preemptively identify unresponsive patients with melanoma harboring BRAF V600E , and many patients relapse despite the improvements in OS [ 251 ]. Verumafenib-resistant melanoma cells appear to upregulate PDGFR, NRAS, or MAPK signaling in vitro and in tumors [ 252 , 253 ]. Preclinical evidence suggests that targeting MEK, PI3K, and mTORC in vemurafenib-refractory patients may be an effective combinatorial strategy [ 254 ]. Other preclinical reports have suggested the combination of vemurafenib with metformin [ 255 ], immunotherapy [ 256 ], or a monoclonal antibody targeting chondroitin sulfate proteoglycan 4 [ 257 ].

Antibodies

While evolution has allowed cells to use small molecules to remain viable, it has allowed organisms to protect themselves from disease through the immune system. Antibodies are large proteins used by the immune system to recognize, respond to, and remember foreign biological material. Antibodies contain a region that is highly specific for a protein target associated with the biological material known as the hypervariable region (Fv), which allows for a high degree of diversity and specificity. The constant region (Fc) of the antibody consists of a particular immunoglobulin that determines the class of antibody and ultimately the type of immune response mounted. Several factors including the complexity of these large proteins have curtailed our ability to synthesize these highly specific binding molecules. However in 1975, medicine was forever changed by discovery the ability to harness cells to make antibodies [ 258 ]. This process consists of immunizing a mouse with the desired antigen, fusing splenic cells from the immunized mice with mouse myeloma cells using Sendai virus, selecting and growing desired clones. This capability has revolutionized modern medicine and biomedical research by offering an unparalleled ability to recognize any given protein with unparalleled specificity. Oncogene pathways mediated by soluble or cell-surface proteins naturally lend themselves as cancer therapeutic targets within the reach of antibodies.

Rituximab

CD20 is a B-cell-specific cell surface protein and as such, is expressed in B-cell cancers such as Non-Hodgkin’s lymphoma (NHL). While the function of CD20 remains unclear, it is known that the protein is not secreted or cleaved from the cell surface [ 259 ] nor is it internalized following antibody binding [ 260 ]. An early study found that administration of murine anti-CD20 in malignant B-cell lymphomas produced a 90 % reduction of circulating malignant cells within four hours in humans [ 261 ]. Variable regions of the murine antibody were cloned in to an expression vector to allow for an antibody with human constant regions and would later gain the name rituximab [ 262 ]. Rituximab demonstrated a binding affinity for CD20 of 5 nM and resulted in a near complete depletion of peripheral blood cells and a 40–70 % depletion of B-cells in lymph nodes that began recovery around 2 weeks after administration. How does sticking an antibody to a surface molecule that does not have an obvious functional importance potently inhibit cancer? The currently understood answer is that rituximab induces three modes of cell death mediated by immune and cancer cells (Fig. 38.7 ). Unlike its murine counterpart, this hybridized antibody bound C1Q in vitro and furthermore induced cell lysis in the presence of serum as a source of complement. C1Q is part of a large protein complex that is found in serum and binds IgG or IgM to trigger a series of intra-complex cleavage events that ultimately form a transmembrane complex, called the membrane attack complex, to induce osmotic lysis of the antigen-expressing cell. This process is known as compliment-dependent cytotoxicity (CDC). CDC appears to be a key aspect of the antitumor activity of rituximab as rituximab-resistant patient samples were associated with CD59, which negatively regulates this process [ 263 , 264 ]. Restoration of compliment has been shown to reverse resistance in small-scale patient studies [ 265 , 266 ] though this has been challenged in preclinical models [ 267 , 268 ] and follicular NHL [ 269 ]. Fig. 38.7 Antitumor mechanisms of . Antibody-dependent cytotoxicity (ADCC) is mediated by natural killer cells (NK), dendritic cells (DC), macrophages (Mφ), and mast cells (M). Complement-dependent cytoxicity (CDC) involves a series conformational changes and cleavage events upon binding IgM or IgG that ultimately leads to the formation of the membrane attack (MAC) which induces osmotic lysis. Induction of apoptosis directly by CD20 also occurs but is less clear in mechanism.

In addition to CDC, effector cells such as macrophages and natural killer cells (NK) express a family of activating and inhibitory receptors called FcγR that bind the constant region (Fc) of IgG. A study in mice showed that blocking these various receptors mediated anti-CD20 mAb-induced B-cell depletion and was isotype-specific [ 270 ]. FcγR bound to IgG present on the surface of a cell can result in phagocytosis by effector cells but these events are determined by the affinity and balance of activating and inhibitory FcγR molecules [ 271 – 273 ]. Accordingly, a follicular NHL study found significantly higher rituximab responses in patients that harbor the FcγR 158V allotype compared to the 158F allotype, which has a relatively weaker affinity for IgG1 [ 274 ]. Ex vivo studies demonstrated that rituximab causes NK-mediated cell lysis [ 275 – 277 ] in a dose-dependent manner that was determined by the FcγR allotype [ 275 ]. There is also evidence that CD20 has direct effects. Early studies found that antibodies against CD20 significantly mediated RNA synthesis and cell cycle progression [ 278 – 280 ]. More recent evidence has found that cross-linking CD20 with antibodies in some, but not all, B-cell lines causes caspase-mediated, Bcl-2-independent apoptosis, and effects the tyrosine kinases Src, Jnk, and p38 [ 281 – 284 ]. The contribution of these different mechanisms appears to be highly context dependent and is likely to be a dynamic process varying between patients or even within a single patient.

Early clinical data found rituximab to have an average half-life of 18.5 days [ 285 ] and similar efficacy and favorable toxicity in relapsed NHL patients relative to CHOP chemotherapy (cyclophosphamide, doxorubicin, vincristine and prednisone) [ 285 – 289 ]. A small-scale study of rituximab found antitumor activity concentrated in the follicular subtype of NHL, though the population size prevented any significant conclusion [ 290 ]. Other studies corroborated the significant monotherapy efficacy of rituximab in follicular lymphoma [ 291 , 292 ]. Adding rituximab to chemotherapy yielded an additive benefit and did not augment the toxicity of standard chemotherapy for NHL [ 293 , 294 ]. Furthermore, polymerase chain reaction (PCR) of NHL patients with follicular histology treated with this combination showed a depletion of the chromosome 14 and 18 translocation often associated with follicular NHL [ 295 , 296 ]. Another study found the disappearance of this translocation a year after a 4-week course of rituximab [ 292 ]. This translocation induces a sustained transcriptional upregulation of Bcl-2, a protein that prevents mitochondria-mediated apoptosis carried out by many tumor suppressors and inhibited by oncogenes. In 1996, rituximab became the first antibody approved by the FDA as a cancer therapy and was indicated for relapsed or refractory low-grade or follicular, CD20 + , B-cell NHL. Three phase III studies (E4494, GELA, and MiNT) that were highly enriched in therapy-naïve, large diffuse cell NHL patients found a significant increase in OS at a 2 year follow-up with the addition of rituximab to CHOP or other anthracycline-based chemotherapies. Analysis of biopsies from E4494 patients found p21 expression as a rituximab-specific, independent predictor of clinical outcome [ 297 ].

NHL patients who previously received at least a single 4-week course of rituximab therapy had equivalent efficacy and toxicity after an additional course with a median internal of 14.5 months [ 298 ]. The combination of rituximab and interferon yielded no significant additional benefit in the short term in follicular NHL [ 299 ]. Today, rituximab is also approved as a first-line therapy for low-grade or follicular cell CD20 + NHL with CHOP and large diffuse B-cell CD20 + NHL with CVP (cyclophosphamide, vincristine, and prednisone). Initial studies of rituximab were conducted with intravenous administration for a 4-week cycle. However, exploration of alternative dosing schedules has provided efficacy in small lymphocytic lymphoma [ 300 ] (SLL) and chronic lymphocytic leukemia (CLL) patients [ 300 , 301 ].

The addition of rituximab to fludarabine and cyclophosphamide in CLL was found to be safe [ 302 – 307 ] and result in unprecedented clinical responses in CLL patients [ 305 ]. A phase III study found that this combination increased the amount of patients without disease progression (65 to 45 %) and overall survival (87 to 83 %) [ 308 ], resulting in the extension of its indication to this malignancy in 2010. Shortly after, it was also approved as a maintenance therapy following a response with chemotherapy and rituximab in CD20 + NHL based on phase III evidence of prolonged PFS with 1 weekly dose [ 309 ]. Rituximab and the proteasome inhibitor bortezomib have been shown to synergistically induce apoptosis in preclinical cancer models [ 310 , 311 ] and a phase II supported the combination but noted significant neurological toxicity [ 312 ]. Also targeting CD20, the monoclonal antibody ocrelizumab was approved in 2009 for CLL refractory to fludarabine and the anti-CD52 antibody alemtuzumab. Other anti-CD20 antibodies are in clinical development for lymphoma including ocrelizumab, veltuzumab, AME-133V, PRO131921, GA101. As seen with rapamycin, the ongoing elucidation of the mechanism of action of rituximab has strongly augmented our biological understanding within and beyond cancer.

Outcompeting Growth Factors with Cetuximab

A number of recent cancer therapies target the EGFR and its family members by inhibiting the intracellular tyrosine kinase domain. An alternative approach is to inhibit EGFR signaling by blocking the extracellular binding of the growth factor through use of antibodies. Cetuximab was FDA-approved for the first-line treatment of metastatic colorectal cancer in combination with irinotecan in 2004, making it the first monoclonal antibody approved by the FDA for this type of cancer. The discovery of cetuximab started with the observation that antibodies secreted from mouse hybridoma cells that target EGF receptors were able to block EGF-induced signaling events and proliferation [ 313 ]. A follow up study found that these antibodies potently inhibited the growth of human cancer cells in a murine xenograft [ 314 ]. Further studies of a particularly potent antibody against EGFR, mab 225, found that it competes directly with EGF-binding [ 315 ], is internalized after two hours in cells [ 316 ], blocks EGFR autophosphorylation, induces G1 cell cycle arrest by p27 KIP1 induction [ 317 – 319 ], induces caspase-8-mediated apoptosis [ 320 ], and preferentially accumulate in EGFR-expressing tumors [ 321 ]. The latter was found using a radiolabeled form of the antibody that was subsequently used for the first clinical trial of mab 225. This trial found mab 225 to be safe in squamous cell lung carcinoma patients and profiled the overall and liver drug uptake, serum clearance, and whole-body clearance as monitored radiographically [ 322 ]. Noninvasive molecular imaging of drugs and therapeutic targets remains an active area of research and informs on the distribution, concentration, and kinetics of therapies, their targets, and/or therapeutic response markers in their clinical setting.

As with rituximab, mab 225 was engineered into a chimeric human/mouse antibody. This process retains residues in the binding region (F v ) of the antibody from the mouse and replaces other residues not specific for the antigen (F c ) with related human residues. This substitution in the F c region reduces side effects resulting from immune responses to foreign, e.g. mouse, immunoglobulins. The mab 225 chimeric antibody, called cetuximab and marketed as Erbitux, was used for further studies and clinical trials. An early phase II trial with cetuximab in combination with irinotecan, a topoisomerase I inhibitor, demonstrated objective responses in EGFR-expressing solid cancers that had become resistant to first-line therapy consisting of leucovorin, 5-fluorouracil (5-FU), and irinotecan [ 323 ]. Combining cetuximab with this treatment regimen also proved effective in a first-line therapy setting [ 324 – 326 ] and moderate efficacy as a monotherapy [ 327 – 330 ]. Safety and efficacy was reported with cetuximab in FOLFOX6 [ 331 , 332 ] and FOLFOX4 [ 333 – 335 ] treatment regimens which consist of leucovorin, 5-FU, and oxaliplatin. A phase III trial also found cetuximab to be effective as a monotherapy and in combination with irinotecan in irinotecan-refractory, EGFR + colorectal cancer patients [ 329 ]. Based on the rational design and specificity of cetuximab, most of these clinical trials exclusively included patients that had EGFR-expressing tumors as determined by immunohistochemical assays. Interestingly, the intensity of EGFR express did not correlate to cetuximab clinical response and another trial has shown responses in EGFR - colorectal cancer patients [ 336 ]. This unexpected finding underscores the complex nature of therapeutic responses in the clinic and the principle that while rational design has well-evidenced benefits, it only allows for hypotheses that ultimately must be tested.

However, the notion that certain patients will respond better than other patients based on certain characteristics is gaining importance in clinical trial design and interpretation. As required by the drug development process, testing therapies in large-scale clinical trials results in considerable patient diversity. Imagine a clinical trial is conducted to examine the efficacy of a new cancer therapy in 1000 patients where 500 patients receive FOLFOX4 and 500 patients receive FOLFOX4 plus the new therapy. The results show no benefit by clinical response or overall survival. However, let us say that ten patients within this group showed a clinical response and improved overall survival due to a unique genetic characteristic. These patients comprise 2 % of the investigational therapy population and therefore will not impact response parameters with any level of statistical significance. Redesigning the clinical trial to include patients that harbor this response-determining characteristic may now show a strong therapeutic benefit otherwise concluded as ineffective. The obvious challenge in patient stratification in clinical trial design is determining what characteristic(s) confer therapeutic response. Clinical trials are sometimes analyzed retrospectively to find such biomarkers. Some of biomarkers have even been added as stipulations for FDA-approved cancer therapies, cetuximab being no exception.

Two years after FDA approval in 2004, a study found that among downstream targets of EGFR signaling, particular KRAS mutations were significantly associated with lack of response to cetuximab [ 337 ]. Other cetuximab trials also found KRAS to be a strong predictor of cetuximab response [ 330 , 338 , 339 ], which led ImClone, the company marketing cetuximab, to petition the FDA to add WT KRAS as a requisite for cetuximab treatment [ 340 ]. In 2009, the FDA updated the indications and usage label of cetuximab in colorectal cancer to include that cetuximab is not recommended for patients harboring KRAS mutations in codon 12 or 13.

Bevacizumab

Cell division is an active energy-dependent process and therefore cancer cells must gain alterations that provide more energy to allow for proliferation. While several mechanisms of harvesting intracellular energy are available, cellular respiration is a mainstay that relies primarily on oxygen. The oxygen source for cells is the blood stream and perhaps it is unsurprising that generation of new blood vessels to supply additional oxygen is seen in tumors. A number of pathways and proteins mediate this process of generating new blood vessels in for tumors, particularly the vascular endothelial growth factor (VEGF) family. VEGF is transcriptionally upregulated in a variety of solid tumors particularly in hypoxic regions due to stabilization of the transcription factor HIF1α that is stabilized under hypoxia. VEGF has four family members, VEGF-A being the prototypical member and itself has four isoforms as a result of alternative splicing which determines its localization and binding properties. Soluble VEGF can bind to VEGF receptors (VEGFR1, VEGF-R2) and its coreceptors (NRP-1,NRP-2) that are expressed on the surface of endothelial cells. Secretion of VEGF ultimately causes the formation of new blood vessels by endothelial cell recruitment and proliferation. Due to the involvement of VEGF in several disease and disorders such as cancer and macular degeneration, antibodies designed to bind VEGF and thereby prevent cognate receptor binding were generated and first described in 1992 [ 341 ]. A follow up study found that one of these mouse monoclonal antibodies, A4.6.1, had potent antitumor activity in vivo but not in vitro [ 342 ]. Interestingly, xenografts of human cancer cells in mice found that introducing soluble VEGF receptors that bind both human and mouse VEGF was superior to a receptor binding only the human or mouse VEGF [ 343 , 344 ]. Together, these findings indicate that both the tumor and other cells in the tumor microenvironment induce participate in generating a new vasculature system through VEGF. Over the last decade, the tumor microenvironment has increasingly garnered attention as a dynamic and strong influence on aspects of tumor biology including therapeutic response, growth rates, and metastasis through a variety of mechanisms.

Due to the potent antitumor activity of the A4.6.1 antibody, a humanized form of the antibody was generated using site-directed mutagenesis of the variable region of a human antibody to that of the murine A4.6.1 while maintaining a similar binding affinity for VEGF [ 345 ]. This humanized version of A4.6.1, known as bevacizumab, is specific for all isoforms of VEGF-A and its cleaved products that result from extracellular proteins such as matrix metalloprotease-9 (MMP9) present in the tumor microenvironment. A crystal structure of the antigen-specific fragment of bevacizumab bound to VEGF identified a critical residue for binding and specificity (Fig. 38.8 ) [ 346 ]. Bevacizumab was evaluated for safety in cynomolgus monkeys due to the complete conservation of VEGF isoforms between humans and monkeys [ 347 ]. After several weeks of administration at doses up to 50 mg/kg, adverse effects on ocular, ovarian, and uterine angiogenesis-dependent processes were evident but were dose-dependent and reversible. A phase I clinical trial found no additional toxicity associated with adding bevacizumab to various chemotherapies and a terminal half-life of 2–3 weeks [ 348 , 349 ]. The following year, several phase II trials were conducted in a variety of solid tumors [ 350 – 354 ]. Particularly encouraging results were found in RCC as a first-line-therapy and metastatic colorectal cancer in combination with standard chemotherapy. A phase III trial in metastatic colorectal cancer with bevacizumab plus standard chemotherapy increased overall survival, progression-free survival, and objective response rate [ 355 ]. Fig. 38.8 Binding interface of Fab and VEGF . ( a ) Symmetric assembly of VEGF dimmers and Fab fragments of bevacizumab. There are two monomers of VEGF in the center ( light and dark yellow ) along with four flanking monomers of bevacizumab. ( b ) Binding interfaces of bevacizumab ( light yellow ) and VEGF ( green ) with interacting residues displayed and hydrogen bonds represented as dashed lines . PDB accession 1BJ1.

The addition of bevacizumab to a paclitaxel-carboplatin treatment regimen increased median survival and PFS but also increased treatment-related deaths, including pulmonary hemorrhage [ 356 ]. Based on the significant improvement in OS, bevacizumab in combination with carboplatin and paclitaxel was approved for first-line therapy in unresectable, locally advanced, recurrent, or metastatic NSCLC. Bevacizumab has now been approved for Her2-negative breast cancer, metastatic RCC, NSCLC, glioblastoma, and metastatic colorectal cancer. However, bevacizumab seems continually surrounded by controversy from a variety of perspectives including clinical, economic, and ethical issues. A highly debated meta-analysis of 15 clinical trials with bevacizumab found a significant increase in venous thrombosis [ 357 – 361 ]. A separate analysis also found an increased risk of high-grade bleeding [ 362 ]. At the end of 2010, the FDA decided to revoke its approval for bevacizumab in metastatic breast cancer based on the results of three large clinical trials (E2100 [ 363 ], AVADO, and RIBBON1). These trials found that while PFS was prolonged, this magnitude was variable among trials and life-threatening adverse events were increased without any change in OS. On the same day, the European Medicines Association decided to maintain its approval in metastatic breast cancer but only in combination with paclitaxel based on the same clinical trials.

Bevacizumab was granted accelerated approval in metastatic breast cancer based on promising data indicating prolonged PFS but was never shown to increase OS. Traditionally, OS has been the key parameter used to decide whether or not a new therapy receives FDA approval. This brings up difficult questions. Will the therapy provide a net benefit to the patient? How do you quantify this? These answers will be unique for every patient as each has a unique situation that determines how benefit is defined. Obviously, most patients want to live longer but quality of life is also a consideration, which raises yet another patient-specific question: what is quality of life? Clinically, fewer adverse events and prolonged PFS and OS are quantifiable parameters that may act as a partial surrogate definition but this consideration is more complex. For bevacizumab, even this incomplete surrogate definition is unclear as both adverse events and prolonged PFS have been reported in multiple large-scale clinical trials and the magnitudes of these are highly variable among these trials. These discrepancies and lack of improvement in OS have led to a range of decisions from removal to full approval by medical agencies across the globe. The U.K. National Institute of Health and Clinical Excellence (NICE) denied the approval of bevacizumab for metastatic breast cancer, citing no evidence in improvement of survival or quality of life.

One less obvious consideration of a therapy like bevacizumab is the possibility of its use as a neoadjuvant, i.e. shrinking tumors to a size where resection is then possibility. This type of consideration is difficult to evaluate in early clinical trials typically compare an investigational therapy to other therapies in patients where such data would be excluded. Proper examination of this nuance and its effect on OS is warranted in this case. In summary, bevacizumab significantly extends PFS to varying degrees in several solid cancers, may shrink tumors to allow for resection, does not increase OS, and significantly increases adverse events. Cost is the elephant in the room. The cost of bevacizumab treatment varies by country, about $90,000 per year in United States [ 364 ]. In addition to rejecting bevacizumab for metastatic breast cancer, NICE has also denied its approval in metastatic colorectal cancer and RCC. Cost was clearly an issue in renal cell cancer, as the benefits of bevacizumab with interferon was similar to sunitinib, a multi-kinase inhibitor already approved for RCC and available at a much lower cost. The various agencies that make the approval decisions on these therapies explain their decisions on the grounds of clinical parameters but patient and social burdens of treatment cost is clearly an underlying influence. Off-label explorations, more clinical trials, and altered clinical trial design are likely to yield more information on the utility of bevacizumab. Nevertheless, this controversial therapy highlights difficult questions lurking in the background of the clinical management of cancer: How do we determine quality of life? Should we consider costs in approval decision? What price is too high for a given therapeutic benefit? How do you weigh concomitant risks and benefits?

Ipilimumab

The immune surveillance of cancer is an endogenous mechanism of tumor suppression that is lost during cancer progression. Inactivating the RAG-2 gene, which is intimately involved in recombination events required for the activation and specificity of immune responses, renders mice more susceptible to carcinogenesis [ 365 ]. T-cells are part of the adaptive immune system that modulates the immune response to antigen threat. The immune escape of cancer may occur by multiple mechanisms such as altering proteins involved in antigen presentation or enriching for regulatory T-cells that secrete immune-inhibitory cytokines such as IL-10 [ 366 ]. Regulatory T-cells have other mechanisms of immune suppression such as elevated expression of cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) [ 367 ]. CTLA4 is an inducible cell surface receptor that binds CD80 and CD86, which are expressed on the surface of antigen-presenting cells and function in concert to activate naive T-cells through CD28. CD28 is a costimulatory receptor expressed on naïve T-cells that cooperatively acts with CD4 as part of the major histocompatibility complex to activate T-cells in response to antigen. Thus, CTLA4 plays a key function in regulatory T-cells by inhibiting T-cell activation as an inhibitory receptor for ligands that are essential for T-cell activation. Accordingly, blocking CTLA4 with antibodies in mice reduces T-cell response and increased tolerability to immunogenic tumors [ 368 ].

Ipilimumab is a fully human antibody developed by Medarex Inc. that targets CTLA4. Cynomolgus monkey studies found that ipilimumab induces a humoral response without autoimmunity [ 369 ]. Early human studies with ipilimumab in melanoma and ovarian cancer reported tumor necrosis and lymphocyte infiltration in ipilimumab-responsive tumors, though several patients experienced grade III/IV adverse events and some developed T-cell reactivity to normal melanocytes [ 370 , 371 ]. Autoimmunity was noted in other clinical studies with ipilimumab but was found to correlative with response [ 372 ]. Enterocolitis was the most frequent adverse event, suggesting that CTLA-4 plays a critical role in protection from immune-mediated enterocolitis [ 373 ]. Similar efficacy and toxicity events were seen in metastatic RCC [ 374 ]. The investigation of single-nucleotide polymorphisms (SNPs) in the CTLA4 gene amongst ipilimumab-treated melanoma patients revealed a response-associated and nonresponse-associated haplotype comprised of seven single-nucleotides in the CTLA4 gene.

A phase II study of ipilimumab in previously treated melanoma reported that 30 % of patients were alive at 2 years in the highest dose cohort of 10 mg/kg [ 375 ]. Another phase II study in melanoma evaluated the safety and efficacy of ipilimumab at 3 mg/kg in combination with dacarbazine, noting a preliminary increase in objective response rate with the combination. The pivotal phase III study compared ipilimumab, the gp100 peptide vaccine, and the combination [ 376 ]. The overall survival as 10.1 months in the combination cohort, 10.0 months in the ipilimumab alone cohort, and 6.4 months in the gp100 alone cohort. This study corroborated survival adverse event observations noted in previous trials and was revered as the first trial to ever show survival benefit in metastatic melanoma. Another phase III study measured the efficacy of adding ipilimumab (10 mg/kg) to dacarbazine as a first-in-line therapy [ 377 ]. Adding ipilimumab to dacarbazine increased the overall survival from 36 to 47 %, though adverse events were increased by the combination.

Based on these phase III trials, the FDA recently approved ipilimumab for the treatment of unresectable or metastatic melanoma at 3 mg/kg every 3 weeks for four doses. Ipilimumab is a first-in-class cancer therapy that reactivates the adaptive immune system to restore antitumor immunity and provides unprecedented patient benefit in melanoma. The clinical efficacy and toxicity of ipilimumab highlights the potential benefits and dangers of immunotherapy as it clinically emerge as a valid new modality of cancer treatment. Despite frequent and severe adverse events associated with ipilimumab, it has been approved based on its unparalleled efficacy in a clinical situation that desperately needed better treatment options. Future trials with ipilimumab are exploring other malignancies and the combination of ipilimumab with other approved agents including chemotherapy and the recently approved vemurafenib.

Lexatumumab

Cell death is a very tightly controlled process that may be initiated by various inputs inside and outside of the cell. TNF-related apoptosis-inducing ligand (TRAIL) is an endogenous mammalian protein utilized by the immune system in the immune surveillance of cancer to potently induce apoptosis in cancer cells while exerting little toxicity to normal cells [ 378 ]. TRAIL binds four transmembrane receptors in humans at similar affinities that results in a homotrimeric receptor–ligand complex [ 379 ]. These receptors include two pro-apoptotic death receptors, DR4 and DR5, and two decoy receptors, DcR1 and DcR2. The ratio of decoy receptors to death receptors along with mediators of downstream signaling events are thought to determine TRAIL sensitivity and be utilized by normal cells to afford protection from TRAIL-mediated apoptosis. The two decoy receptors compete for TRAIL binding with the death receptors at similar binding affinities.

TRAIL-induced trimerization of the death receptors colocalizes their intracellular death domains, which recruit Fas-associated death domain (FADD) and procaspase-8, forming death inducing signaling complex (DISC). At the DISC, procaspase-8 is activated by autocatalytic cleavage to form caspase-8 which can cleave effector caspase-3, caspase-6, and caspase-7 to induce apoptosis by the extrinsic death pathway. Alternatively, caspase-8 can initiate the intrinsic death pathway by cleaving Bid to tBid, which primarily interacts with Bax and Bak at the mitochondrial membrane. This interaction induces oligomerization of Bax and Bak to promote cytochrome c release. In the cytosol, cytochrome c binds to apoptotic peptidase activating factor 1 (Apaf-1) and caspase-9 to form the apoptosome, which initiates the caspase cascade. While TRAIL directly targets death receptors that generally play a tumor suppressor role, several oncoproteins mediate this process and serve as potent resistance mechanisms. One of the most striking examples is Mcl-1, an anti-apoptotic member of the Bcl-2 family that interacts with pro-apoptotic family members such as Bax to inhibit mitochondrial-mediated apoptosis. Notably, this resistance mechanism can be overcome by combining TRAIL with sorafenib [ 380 ].

The ability of TRAIL to selectively induce apoptosis in tumors cell has led to the clinical trials with recombinant TRAIL. However, TRAIL has a short serum half-life and cytotoxic resistance can result from elevated decoy receptor expression. As a result, TRAIL-agonist antibodies targeting either of two pro-apoptotic death receptors were created and are currently in clinical trials [ 381 ]. Lexatumumab is one of the most developed DR5 agonist antibodies. A phase I study with lexatumumab in previously treated advanced solid tumors reported a MTD of 10 mg/kg when given once every 21 days, a second-phase serum half-life of ~16 days, and stable disease in approximately a third of the treated patients [ 382 ]. Another phase I study found lexatumumab to be safe at 10 mg/kg once every 14 days [ 383 ].

Several preclinical studies have suggested the use of lexatumumab in combination with other therapies to increase efficacy such as radiation [ 384 ], paclitaxel [ 385 ], bortezomib [ 386 – 388 ], HDAC inhibitors [ 389 ], doxorubicin [ 390 ], and cisplatin [ 391 ]. Phase Ib studies evaluated lexatumumab in combination with gemcitabine, pemetrexed, doxorubicin, or FOLFIRI [ 392 ]. Severe adverse events potentially attributable to lexatumumab included anemia, fatigue, and dehydration. Tumor regression was noted in patient cohorts receiving the combination of lexatumumab and doxorubicin or FOLFIRI. Numerous other DR5-agonst antibodies including TRA-8, LBY135, Apomab, and Conatumumab and the DR4-agonist mapatumumab are also currently in clinical trials.

Custom Cures

We still have not won the war on cancer. However, we have greatly expanded our understanding and gained some highly effective therapies in the course of battle. Cancer was once understood as a simple clonal expansion of a cell that gained a growth advantage by a stepwise loss or gain of function of a few critical genes. This previously linear pathway of carcinogenesis is now a multinodal, interconnected network of events and we have begun to appreciate the immense heterogeneity of tumors and the dynamic parameters that govern their biology and therapeutic response. So how much has all of this research investment helped cancer patients? Clearly some types of cancer have benefited from new targeted agents such as NHL and breast cancer. Chemotherapy, radiation, and surgery remain the cornerstones of treatment regimens in many malignancies and none of these were developed with a molecular understanding of their mechanism of action. Serendipity has played a key role in the discovery of many cancer therapies still used today. Nevertheless, it has been our hypotheses and observations built upon our prior knowledge base that has guided the application of these serendipitous discoveries. The discovery of nitrogen mustards was certainly not found in a quest to cure cancer but it was applied to cancer by a hypothesis generated from the current understanding of cancer. It is the application of our knowledge to what is right under our nose that can yield new solutions, quite literally in the case of rapamycin. The more we understand how cancer works, the better we understand what we are looking for and how we can use the tools we already have.

Oncogenes, tumor suppressors, epigenetics, immunology, the tumor microenvironment, and several other factors play critical roles in tumor biology. Considering this multifactorial and complex nature of cancer and the level of effort put forth throughout history to cure it, it is likely that there will be no magic bullet that cures cancer. The future of cancer therapy lies in continued drug discovery, explorations of multimodal and combinatorial therapy, improved agent targeting, and personalized medicine. Combinations of existing therapies can yield antagonistic to synergistic responses in the clinic but is often difficult to interpret due to the heterogeneity of patient responses. Interpretation of inter-patient and intra-patient responses will also play a critical role in the future of cancer therapy and could benefit from the use of biomarkers and molecular imaging. It is important that we understand why a therapy fails in a certain situation so that successful situations can be clearly identified, future failures may be avoided, and other successful treatment options can be discovered.

References

1. Lissauer Zwei Falle von Leukamie Berliner Klinische Wochenschrift 1865 2 403 4

2. Hanahan D Weinberg RA Hallmarks of cancer: the next generation Cell 2011 144 646 74 10.1016/j.cell.2011.02.013 21376230

3. Hanahan D Weinberg RA The hallmarks of cancer Cell 2000 100 57 70 10.1016/S0092-8674(00)81683-9 10647931

4. Zimmermann J Caravatti G Mett H Phenylamino-pyrimidine (PAP) derivatives: a new class of potent and selective inhibitors of protein kinase C (PKC) Arch Pharm 1996 329 371 6 10.1002/ardp.19963290707 8764886

5. Zimmermann J Buchdunger E Mett H Meyer T Lydon NB Potent and selective inhibitors of the Abl-kinase: phenylamino-pyrimidine (PAP) derivatives Bioorg Med Chem Lett 1997 7 187 92 10.1016/S0960-894X(96)00601-4

6. Zimmermann J Buchdunger E Mett H Phenylamino-pyrimidine (PAP)—derivatives: a new class of potent and highly selective PDGF-receptor autophosphorylation inhibitors Bioorg Med Chem Lett 1996 6 1221 6 10.1016/0960-894X(96)00197-7

7. Schindler T Bornmann W Pellicena P Structural mechanism for STI-571 inhibition of Abelson tyrosine kinase Science 2000 289 1938 42 10.1126/science.289.5486.1938 10988075

8. Zimmermann J Furet P Buchdunger E STI571: a new treatment modality for CML? Anticancer agents 2001 Washington, DC, USA American Chemical Society 245 59

9. Okuda K Weisberg E Gilliland DG Griffin JD ARG tyrosine kinase activity is inhibited by STI571 Blood 2001 97 2440 8 10.1182/blood.V97.8.2440 11290609

10. Adams CP Brantner VV Estimating the cost of new drug development: is it really $802 million? Health Aff 2006 25 420 8 10.1377/hlthaff.25.2.420 16522582

11. Weisberg E Griffin JD Mechanism of resistance to the ABL tyrosine kinase inhibitor STI571 in BCR/ABL-transformed hematopoietic cell lines Blood 2000 95 3498 505 10828035

12. le Coutre P Tassi E Varella-Garcia M Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplification Blood 2000 95 1758 66 10688835

13. Mahon FX Deininger MWN Schultheis B Selection and characterization of BCR-ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor STI571: diverse mechanisms of resistance Blood 2000 96 1070 9 10910924

14. Gorre ME Mohammed M Ellwood K Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification Science 2001 293 876 80 10.1126/science.1062538 11423618

15. Rubin EH de Alwis DP Pouliquen I A phase I trial of a potent P-glycoprotein inhibitor, Zosuquidar.3HCl trihydrochloride (LY335979), administered orally in combination with doxorubicin in patients with advanced malignancies Clin Cancer Res 2002 8 3710 7 12473580

16. Gandhi L Harding MW Neubauer M A phase II study of the safety and efficacy of the multidrug resistance inhibitor VX-710 combined with doxorubicin and vincristine in patients with recurrent small cell lung cancer Cancer 2007 109 924 32 10.1002/cncr.22492 17285598

17. Bauer KS Karp JE Garimella TS A phase I and pharmacologic study of idarubicin, cytarabine, etoposide, and the multidrug resistance protein (MDR1/Pgp) inhibitor PSC-833 in patients with refractory leukemia Leuk Res 2005 29 263 71 10.1016/j.leukres.2004.07.002 15661261

18. Kolitz JE George SL Dodge RK Dose escalation studies of cytarabine, daunorubicin, and etoposide with and without multidrug resistance modulation with PSC-833 in untreated adults with acute myeloid leukemia younger than 60 years: final induction results of Cancer and Leukemia Group B Study 9621 J Clin Oncol 2004 22 4290 301 10.1200/JCO.2004.11.106 15514371

19. Agrawal M Abraham J Balis FM Increased 99mTc-sestamibi accumulation in normal liver and drug-resistant tumors after the administration of the glycoprotein inhibitor, XR9576 Clin Cancer Res 2003 9 650 6 12576431

20. Kantarjian H Shah NP Hochhaus A Dasatinib versus imatinib in newly diagnosed chronic-phase chronic myeloid leukemia N Engl J Med 2010 362 2260 70 10.1056/NEJMoa1002315 20525995

21. Giles FJ Abruzzese E Rosti G Nilotinib is active in chronic and accelerated phase chronic myeloid leukemia following failure of imatinib and dasatinib therapy Leukemia 2010 24 1299 301 10.1038/leu.2010.110 20520639 PMC3078756

22. Cortes J Rousselot P Kim D-W Dasatinib induces complete hematologic and cytogenetic responses in patients with imatinib-resistant or -intolerant chronic myeloid leukemia in blast crisis Blood 2007 109 3207 13 10.1182/blood-2006-09-046888 17185463

23. Cortes J Kim DW Raffoux E Efficacy and safety of dasatinib in imatinib-resistant or -intolerant patients with chronic myeloid leukemia in blast phase Leukemia 2008 22 2176 83 10.1038/leu.2008.221 18754032

24. Apperley JF Cortes JE Kim D-W Dasatinib in the treatment of chronic myeloid leukemia in accelerated phase after imatinib failure: the START a trial J Clin Oncol 2009 27 3472 9 10.1200/JCO.2007.14.3339 19487385 PMC4979080

25. Kantarjian H Cortes J Kim D-W Phase 3 study of dasatinib 140 mg once daily versus 70 mg twice daily in patients with chronic myeloid leukemia in accelerated phase resistant or intolerant to imatinib: 15-month median follow-up Blood 2009 113 6322 9 10.1182/blood-2008-11-186817 19369231 PMC4916944

26. Shah NP Kantarjian HM Kim D-W Intermittent target inhibition with dasatinib 100 mg once daily preserves efficacy and improves tolerability in imatinib-resistant and -intolerant chronic-phase chronic myeloid leukemia J Clin Oncol 2008 26 3204 12 10.1200/JCO.2007.14.9260 18541900

27. Ottmann O Dombret H Martinelli G Dasatinib induces rapid hematologic and cytogenetic responses in adult patients with Philadelphia chromosome positive acute lymphoblastic leukemia with resistance or intolerance to imatinib: interim results of a phase 2 study Blood 2007 110 2309 15 10.1182/blood-2007-02-073528 17496201

28. Guilhot F Apperley J Kim D-W Dasatinib induces significant hematologic and cytogenetic responses in patients with imatinib-resistant or -intolerant chronic myeloid leukemia in accelerated phase Blood 2007 109 4143 50 10.1182/blood-2006-09-046839 17264298

29. Hochhaus A Kantarjian HM Baccarani M Dasatinib induces notable hematologic and cytogenetic responses in chronic-phase chronic myeloid leukemia after failure of imatinib therapy Blood 2007 109 2303 9 10.1182/blood-2006-09-047266 17138817

30. Woodburn J, Kendrew J, Fennell M. ZD1839 (“Iressa”) a selective epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI): inhibition of c-fos mRNA, an intermediate marker of EGFR activation, correlates with tumor growth inhibition [abstract no. 2552]. Proc Am Assoc Cancer Res. 2000.

31. Budillon A, Gennaro ED, Barbarino M. ZD1839, an epidermal growth factor receptor tyrosine kinase inhibitor, upregulates p27Kip1inducing G1 arrest and enhancing the antitumor effect of Interferon α [abstract no. 4910]. Proc Am Assoc Cancer Res. 2000;41.

32. Ciardiello F Caputo R Bianco R Antitumor effect and potentiation of cytotoxic drugs activity in human cancer cells by ZD-1839 (Iressa), an epidermal growth factor receptor-selective tyrosine kinase inhibitor Clin Cancer Res 2000 6 2053 63 10815932

33. Sirotnak FM Zakowski MF Miller VA Scher HI Kris MG Efficacy of cytotoxic agents against human tumor xenografts is markedly enhanced by coadministration of ZD1839 (Iressa), an inhibitor of EGFR tyrosine kinase Clin Cancer Res 2000 6 4885 92 11156248

34. Woodburn J, Wakeling A, Kelly H. Preclinical studies with the oral epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) ZD1839 (“Iressa”) demonstrate significant anti-tumor activity. Presented at the Signal Transduction Pathways and Regulation of Gene Expression as Therapeutic Targets meeting. 2000.

35. Kelly H, Laight A, Morris C. Phase I data of ZD1839—an oral epidermal growth factor receptor tyrosine kinase inhibitor [abstract no. 419]. Ann Oncol. 1998;2.

36. Kelly H, Ferry D, Hammond L. ZD1839 (“Iressa”), an oral EGFR-TKI (epidermal growth factor tyrosine kinase inhibitor): pharmacokinetics in a Phase I study of patients with advanced cancer [abstract no. 3896]. Proc Am Assoc Cancer Res. 2000;41.

37. Goss G, Hirte H, Batist G. NCIC CTG IND.122: a two-part phase I pharmacokinetic (PK) and pharmacodynamic (PD) study of the epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) ZD1839 [abstract no. 880]. Proc Am Soc Clin Oncol. 2000;19.

38. Baselga J, Herbst R, LoRusso P. Continuous administration of ZD1839 (Iressa), a novel oral epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), in patients with five selected tumor types: evidence of activity and good tolerability [abstract no. 686]. Proc Am Soc Clin Oncol. 2000;19.

39. Nakagawa K, Yamamoto N, Kudoh S. A phase I intermittent dose-escalation trial of ZD1839 (Iressa) in Japanese patients with solid malignant tumours [abstract no. 711]. Proc Am Soc Clin Oncol. 2000;19.

40. Ranson M Hammond LA Ferry D ZD1839, a selective oral epidermal growth factor receptor–tyrosine kinase inhibitor, is well tolerated and active in patients with solid. malignant tumors: results of a phase I trial J Clin Oncol 2002 20 2240 50 10.1200/JCO.2002.10.112 11980995

41. Baselga J Rischin D Ranson M Phase I safety, pharmacokinetic, and pharmacodynamic trial of ZD1839, a selective oral epidermal growth factor receptor tyrosine kinase inhibitor, in patients with five selected solid tumor types J Clin Oncol 2002 20 4292 302 10.1200/JCO.2002.03.100 12409327

42. Jones HK Stafford LE Swaisland HC Payne R A sensitive assay for ZD1839 (Iressa) in human plasma by liquid-liquid extraction and high performance liquid chromatography with mass spectrometric detection: validation and use in phase I clinical trials J Pharm Biomed Anal 2002 29 221 8 10.1016/S0731-7085(02)00014-6 12062681

43. McKillop D Guy SP Spence MP Minimal contribution of desmethyl-gefitinib, the major human plasma metabolite of gefitinib, to epidermal growth factor receptor (EGFR)-mediated tumour growth inhibition Xenobiotica 2006 36 29 39 10.1080/00498250500523253 16507511

44. Kris MG Natale RB Herbst RS Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer JAMA 2003 290 2149 58 10.1001/jama.290.16.2149 14570950

45. Fukuoka M Yano S Giaccone G Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer J Clin Oncol 2003 21 2237 46 10.1200/JCO.2003.10.038 12748244

46. Janne PA Gurubhagavatula S Yeap BY Outcomes of patients with advanced non-small cell lung cancer treated with gefitinib (ZD1839, Iressa™) on an expanded access study Lung Cancer 2004 44 221 30 10.1016/j.lungcan.2003.12.014 15084387

47. Thatcher N Chang A Parikh P Gefitinib plus best supportive care in previously treated patients with refractory advanced non-small-cell lung cancer: results from a randomised, placebo-controlled, multicentre study (Iressa Survival Evaluation in Lung Cancer) Lancet 2005 366 1527 37 10.1016/S0140-6736(05)67625-8 16257339

48. Chang A Parikh P Thongprasert S Gefitinib (IRESSA) in patients of Asian origin with refractory advanced non-small cell lung cancer: subset analysis from the ISEL study J Thorac Oncol 2006 1 847 55 10.1016/S1556-0864(15)30415-9 17409969

49. Sunaga N Tomizawa Y Yanagitani N Phase II prospective study of the efficacy of gefitinib for the treatment of stage III/IV non-small cell lung cancer with EGFR mutations, irrespective of previous chemotherapy Lung Cancer 2007 56 383 9 10.1016/j.lungcan.2007.01.025 17368623

50. Sequist LV Martins RG Spigel D First-line gefitinib in patients with advanced non-small-cell lung cancer harboring somatic EGFR mutations J Clin Oncol 2008 26 2442 9 10.1200/JCO.2007.14.8494 18458038

51. Tamura K Okamoto I Kashii T Multicentre prospective phase II trial of gefitinib for advanced non-small cell lung cancer with epidermal growth factor receptor mutations: results of the West Japan Thoracic Oncology Group trial (WJTOG0403) Br J Cancer 2008 98 907 14 10.1038/sj.bjc.6604249 18283321 PMC2266849

52. Cortes-Funes H Gomez C Rosell R Epidermal growth factor receptor activating mutations in Spanish gefitinib-treated non-small-cell lung cancer patients Ann Oncol 2005 16 1081 6 10.1093/annonc/mdi221 15851406

53. Kim ES Hirsh V Mok T Gefitinib versus docetaxel in previously treated non-small-cell lung cancer (INTEREST): a randomised phase III trial Lancet 2008 372 1809 18 10.1016/S0140-6736(08)61758-4 19027483

54. Lynch TJ Bell DW Sordella R Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib N Engl J Med 2004 350 2129 39 10.1056/NEJMoa040938 15118073

55. Paez JG Janne PA Lee JC EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy Science 2004 304 1497 500 10.1126/science.1099314 15118125

56. Kosaka T Yatabe Y Endoh H Mutations of the epidermal growth factor receptor gene in lung cancer Cancer Res 2004 64 8919 23 10.1158/0008-5472.CAN-04-2818 15604253

57. Stamos J Sliwkowski MX Eigenbrot C Structure of the epidermal growth factor receptor kinase domain alone and in complex with a 4-anilinoquinazoline inhibitor J Biol Chem 2002 277 46265 72 10.1074/jbc.M207135200 12196540

58. Carey KD Garton AJ Romero MS Kinetic analysis of epidermal growth factor receptor somatic mutant proteins shows increased sensitivity to the epidermal growth factor receptor tyrosine kinase inhibitor Erlotinib Cancer Res 2006 66 8163 71 10.1158/0008-5472.CAN-06-0453 16912195

59. Lee DH Park K Kim JH Randomized phase III trial of gefitinib versus docetaxel in non-small cell lung cancer patients who have previously received platinum-based chemotherapy Clin Cancer Res 2010 16 1307 14 10.1158/1078-0432.CCR-09-1903 20145166

60. Takeda K Hida T Sato T Randomized phase III trial of platinum-doublet chemotherapy followed by gefitinib compared with continued platinum-doublet chemotherapy in Japanese patients with advanced non-small-cell lung cancer: results of a West Japan Thoracic Oncology Group Trial (WJTOG0203) J Clin Oncol 2010 28 753 60 10.1200/JCO.2009.23.3445 20038730

61. Saijo N Takeuchi M Kunitoh H Reasons for response differences seen in the V15-32. INTEREST and IPASS trials Nat Rev Clin Oncol 2009 6 287 94 10.1038/nrclinonc.2009.37 19390555

62. Maemondo M Inoue A Kobayashi K Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR N Engl J Med 2010 362 2380 8 10.1056/NEJMoa0909530 20573926

63. Moiseenko V Protsenko S Semenov I Effectiveness of gefitinib (Iressa) as first-line therapy for inoperable non-small-cell lung cancer with mutated EGFR gene (phase II study) Vopr Onkol 2010 56 20 3 20361610

64. Mitsudomi T Morita S Yatabe Y Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial Lancet Oncol 2010 11 121 8 10.1016/S1470-2045(09)70364-X 20022809

65. Rusnak DW Affleck K Cockerill SG The characterization of novel, dual ErbB-2/EGFR, tyrosine kinase inhibitors Cancer Res 2001 61 7196 203 11585755

66. Cockerill S Stubberfield C Stables J Indazolylamino quinazolines and pyridopyrimidines as inhibitors of the EGFr and c-erbB-2 Bioorg Med Chem Lett 2001 11 1401 5 10.1016/S0960-894X(01)00219-0 11378364

67. Rusnak DW Lackey K Affleck K The effects of the novel, reversible epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor, GW2016, on the growth of human normal and tumor-derived cell lines in vitro and in vivo Mol Cancer Ther 2001 1 85 94 12467226

68. Xia W, Mullin RJ, Keith BR, et al. Anti-tumor activity of GW572016: a dual tyrosine kinase inhibitor blocks EGF activation of EGFR/erbB2 and downstream Erk1/2 and AKT pathways. Oncogene. 2002;21:6255–63. 10.1038/sj.onc.1205794 12214266

69. Christianson TA Doherty JK Lin YJ NH2-terminally truncated HER-2/neu protein: relationship with shedding of the extracellular domain and with prognostic factors in breast cancer Cancer Res 1998 58 5123 9 9823322

70. Di Fiore PP Pierce JH Kraus MH erbB-2 is a potent oncogene when overexpressed in NIH/3T3 cells Science 1987 237 178 82 10.1126/science.2885917 2885917

71. Bargmann C Weinberg R Oncogenic activation of the neu-encoded receptor protein by point mutation and deletion EMBO J 1988 7 2043 52 2901345 10.1002/j.1460-2075.1988.tb03044.x PMC454481

72. Segatto O King CR Pierce JH Di Fiore PP Aaronson SA Different structural alterations upregulate in vitro tyrosine kinase activity and transforming potency of the erbB-2 gene Mol Cell Biol 1988 8 5570 4 10.1128/MCB.8.12.5570 2907606 PMC365664

73. Kandl H Seymour L Bezwoda W Soluble c-erbB-2 fragment in serum correlates with disease stage and predicts for shortened survival in patients with early-stage and advanced breast cancer Br J Cancer 1994 70 739 42 10.1038/bjc.1994.387 7522511 PMC2033409

74. Brandt-Rauf P The c-erbB transmembrane growth factor receptors as serum biomarkers in human cancer studies Mutation Res 1995 333 203 8 10.1016/0027-5107(95)00146-8 8538628

75. Yamauchi H O'Neill A Gelman R Prediction of response to antiestrogen therapy in advanced breast cancer patients by pretreatment circulating levels of extracellular domain of the HER-2/c-neu protein J Clin Oncol 1997 15 2518 25 9215820 10.1200/JCO.1997.15.7.2518

76. Colomer R Montero S Lluch A Circulating HER2 extracellular domain and resistance to chemotherapy in advanced breast cancer Clin Cancer Res 2000 6 2356 62 10873087

77. Xia W Liu L-H Ho P Spector NL Truncated ErbB2 receptor (p95ErbB2) is regulated by heregulin through heterodimer formation with ErbB3 yet remains sensitive to the dual EGFR//ErbB2 kinase inhibitor GW572016 Oncogene 2004 23 646 53 10.1038/sj.onc.1207166 14737100

78. Gregory CW Whang YE McCall W Heregulin-induced activation of HER2 and HER3 increases androgen receptor transactivation and CWR-R1 human recurrent prostate cancer cell growth Clin Cancer Res 2005 11 1704 12 10.1158/1078-0432.CCR-04-1158 15755991

79. Liu Y Majumder S McCall W Inhibition of HER-2/neu kinase impairs androgen receptor recruitment to the androgen responsive enhancer Cancer Res 2005 65 3404 9 15833875 10.1158/0008-5472.CAN-04-4292

80. Chu I Blackwell K Chen S Slingerland J The dual ErbB1/ErbB2 inhibitor, lapatinib (GW572016). Cooperates with tamoxifen to inhibit both cell proliferation- and estrogen-dependent gene expression in antiestrogen-resistant breast cancer Cancer Res 2005 65 18 25 15665275

81. Xia W Bacus S Hegde P A model of acquired autoresistance to a potent ErbB2 tyrosine kinase inhibitor and a therapeutic strategy to prevent its onset in breast cancer Proc Natl Acad Sci 2006 103 7795 800 10.1073/pnas.0602468103 16682622 PMC1472524

82. Xia W Gerard CM Liu L Combining lapatinib (GW572016), a small molecule inhibitor of ErbB1 and ErbB2 tyrosine kinases, with therapeutic anti-ErbB2 antibodies enhances apoptosis of ErbB2-overexpressing breast cancer cells Oncogene 2005 24 6213 21 10.1038/sj.onc.1208774 16091755

83. Konecny GE Pegram MD Venkatesan N Activity of the dual kinase inhibitor lapatinib (GW572016) against HER-2-overexpressing and trastuzumab-treated breast cancer cells Cancer Res 2006 66 1630 9 10.1158/0008-5472.CAN-05-1182 16452222

84. Nahta R Yuan LXH Du Y Esteva FJ Lapatinib induces apoptosis in trastuzumab-resistant breast cancer cells: effects on insulin-like growth factor I signaling Mol Cancer Ther 2007 6 667 74 10.1158/1535-7163.MCT-06-0423 17308062

85. Bence AK Anderson EB Halepota MA Phase I pharmacokinetic studies evaluating single and multiple doses of oral GW572016, a dual EGFR-ErbB2 inhibitor, in healthy subjects Invest New Drugs 2005 23 39 49 10.1023/B:DRUG.0000047104.45929.ea 15528979

86. Messori A Effect of food on lapatinib pharmacokinetics J Clin Oncol 2007 25 5332 3 10.1200/JCO.2007.13.9345 18024884

87. Rahman A Pazdur R Wang Y Huang S-M Lesko L The value meal: effect of food on lapatinib bioavailability J Clin Oncol 2007 25 5333 4 10.1200/JCO.2007.14.6381 18024885

88. Trippoli S Controversies in using lapatinib at reduced dosage with food J Clin Oncol 2007 25 5333 10.1200/JCO.2007.13.9501 18024886

89. Burris HA Hurwitz HI Dees EC Phase I safety, pharmacokinetics, and clinical activity study of lapatinib (GW572016), a reversible dual inhibitor of epidermal growth factor receptor tyrosine kinases, in heavily pretreated patients with metastatic carcinomas J Clin Oncol 2005 23 5305 13 10.1200/JCO.2005.16.584 15955900

90. Gomez HL Doval DC Chavez MA Efficacy and safety of lapatinib as first-line therapy for ErbB2-amplified locally advanced or metastatic breast cancer J Clin Oncol 2008 26 2999 3005 10.1200/JCO.2007.14.0590 18458039

91. Harrington KJ El-Hariry IA Holford CS Phase I study of lapatinib in combination with chemoradiation in patients with locally advanced squamous cell carcinoma of the head and neck J Clin Oncol 2009 27 1100 7 10.1200/JCO.2008.17.5349 19171712

92. Burris HA Taylor CW Jones SF A phase I and pharmacokinetic study of oral lapatinib administered once or twice daily in patients with solid malignancies Clin Cancer Res 2009 15 6702 8 10.1158/1078-0432.CCR-09-0369 19825948 PMC3232441

93. Ross HJ Blumenschein GR Aisner J Randomized phase II multicenter trial of two schedules of lapatinib as first- or second-line monotherapy in patients with advanced or metastatic non-small cell lung cancer Clin Cancer Res 2010 16 1938 49 10.1158/1078-0432.CCR-08-3328 20215545

94. Sridhar SS Hotte SJ Chin JL A multicenter phase II clinical trial of lapatinib (GW572016) in hormonally untreated advanced prostate cancer Am J Clin Oncol 2010 33 609 13 10.1097/COC.0b013e3181beac33 20042973

95. Siegel-Lakhai WS Beijnen JH Vervenne WL Phase I pharmacokinetic study of the safety and tolerability of lapatinib (GW572016) in combination with oxaliplatin/fluorouracil/leucovorin (FOLFOX4) in patients with solid tumors Clin Cancer Res 2007 13 4495 502 10.1158/1078-0432.CCR-07-0004 17671135

96. Midgley RS Kerr DJ Flaherty KT A phase I and pharmacokinetic study of lapatinib in combination with infusional 5-fluorouracil, leucovorin and irinotecan Ann Oncol 2007 18 2025 9 10.1093/annonc/mdm366 17846021

97. Geyer CE Forster J Lindquist D Lapatinib plus capecitabine for HER2-positive advanced breast cancer N Engl J Med 2006 355 2733 43 10.1056/NEJMoa064320 17192538

98. Chu QSC Schwartz G de Bono J Phase I and pharmacokinetic study of lapatinib in combination with capecitabine in patients with advanced solid malignancies J Clin Oncol 2007 25 3753 8 10.1200/JCO.2007.11.1765 17704424

99. Cameron D Casey M Press M A phase III randomized comparison of lapatinib plus capecitabine versus capecitabine alone in women with advanced breast cancer that has progressed on trastuzumab: updated efficacy and biomarker analyses Breast Cancer Res Treat 2008 112 533 43 10.1007/s10549-007-9885-0 18188694

100. Johnston S Trudeau M Kaufman B Phase II study of predictive biomarker profiles for response targeting human epidermal growth factor receptor 2 (HER-2) in advanced inflammatory breast cancer with lapatinib monotherapy J Clin Oncol 2008 26 1066 72 10.1200/JCO.2007.13.9949 18212337

101. Dolloff NG Mayes PA Hart LS Off-target lapatinib activity sensitizes colon cancer cells through TRAIL death receptor up-regulation Sci Transl Med 2011 3 86ra50 10.1126/scitranslmed.3001384 21653830 PMC3769950

102. Johnston S Pippen J Pivot X Lapatinib combined with letrozole versus letrozole and placebo as first-line therapy for postmenopausal hormone receptor-positive metastatic breast cancer J Clin Oncol 2009 27 5538 46 10.1200/JCO.2009.23.3734 19786658

103. Di Leo A Gomez HL Aziz Z Phase III, double-blind. Randomized study comparing lapatinib plus paclitaxel with placebo plus paclitaxel as first-line treatment for metastatic breast cancer J Clin Oncol 2008 26 5544 52 10.1200/JCO.2008.16.2578 18955454 PMC2651098

104. Jagiello-Gruszfeld A Tjulandin S Dobrovolskaya N A single-arm phase II trial of first-line paclitaxel in combination with lapatinib in HER2-overexpressing metastatic breast cancer Oncology 2010 79 129 35 10.1159/000318043 21088439

105. Blackwell KL Burstein HJ Storniolo AM Randomized study of lapatinib alone or in combination with trastuzumab in women with ErbB2-positive. Trastuzumab-refractory metastatic breast cancer J Clin Oncol 2010 28 1124 30 10.1200/JCO.2008.21.4437 20124187

106. Vezina C, Kudelski A, Sehgal SN. Rapamycin (AY-22, 989), a new antifungal antibiotic. J Antibiot. 1975;28:721–6. 10.7164/antibiotics.28.721 1102508

107. Baker H Sidorowicz A Sehgal SN Vezina C Rapamycin (AY-22, 989), a new antifungal antibiotic. III. In vitro and in vivo evaluation J Antibiot 1978 31 539 45 10.7164/antibiotics.31.539 28309

108. Martel R Klicius J Galet S Inhibition of the immune response by rapamycin, a new antifungal antibiotic Can J Physiol Pharmacol 1977 55 48 51 10.1139/y77-007 843990

109. Sawada S Suzuki G Kawase Y Takaku F Novel immunosuppressive agent, FK506. In vitro effects on the cloned T cell activation J Immunol 1987 139 1797 803 2442255

110. Dumont FJ Melino MR Staruch MJ The immunosuppressive macrolides FK-506 and rapamycin act as reciprocal antagonists in murine T cells J Immunol 1990 144 1418 24 1689353

111. Harding MW Galat A Uehling DE Schreiber SL A receptor for the immuno-suppressant FK506 is a cis-trans peptidyl-prolyl isomerase Nature 1989 341 758 60 10.1038/341758a0 2477715

112. Siekierka JJ Hung SHY Poe M Lin CS Sigal NH A cytosolic binding protein for the immunosuppressant FK506 has peptidyl-prolyl isomerase activity but is distinct from cyclophilin Nature 1989 341 755 7 10.1038/341755a0 2477714

113. Standaert RF Galat A Verdine GL Schreiber SL Molecular cloning and overexpression of the human FK506-binding protein FKBP Nature 1990 346 671 4 10.1038/346671a0 1696686

114. Maki N Sekiguchi F Nishimaki J Complementary DNA encoding the human T-cell FK506-binding protein, a peptidylprolyl cis-trans isomerase distinct from cyclophilin Proc Natl Acad Sci U S A 1990 87 5440 3 10.1073/pnas.87.14.5440 1695378 PMC54340

115. Bierer BE Mattila PS Standaert RF Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FK506 or rapamycin Proc Natl Acad Sci U S A 1990 87 9231 5 10.1073/pnas.87.23.9231 2123553 PMC55138

116. Michnick SW Rosen MK Wandless TJ Karplus M Schreiber SL Solution structure of FKBP, a rotamase enzyme and receptor for FK506 and rapamycin Science 1991 252 836 9 10.1126/science.1709301 1709301

117. Van Duyne GD Standaert RF Karplus PA Schreiber SL Clardy J Atomic structure of FKBP-FK506, an immunophilin-immunosuppressant complex Science 1991 252 839 42 10.1126/science.1709302 1709302

118. Van Duyne GD Standaert RF Karplus PA Schreiber SL Clardy J Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin J Mol Biol 1993 229 105 24 10.1006/jmbi.1993.1012 7678431

119. Kunz J Henriquez R Schneider U Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression Cell 1993 73 585 96 10.1016/0092-8674(93)90144-F 8387896

120. Sabatini DM Erdjument-Bromage H Lui M Tempst P Snyder SH RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs Cell 1994 78 35 43 10.1016/0092-8674(94)90570-3 7518356

121. Chiu MI Katz H Berlin V RAPT1, a mammalian homolog of yeast Tor, interacts with the FKBP12/rapamycin complex Proc Natl Acad Sci U S A 1994 91 12574 8 10.1073/pnas.91.26.12574 7809080 PMC45481

122. Sabers CJ Martin MM Brunn GJ Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells J Biol Chem 1995 270 815 22 10.1074/jbc.270.2.815 7822316

123. Brown EJ Albers MW Bum Shin T A mammalian protein targeted by G1-arresting rapamycin-receptor complex Nature 1994 369 756 8 10.1038/369756a0 8008069

124. Francavilla A Carr BI Starzl TE Effects of rapamycin on cultured hepatocyte proliferation and gene expression Hepatology 1992 15 871 7 10.1002/hep.1840150520 1568729 PMC2963996

125. Price DJ Grove JR Calvo V Avruch J Bierer BE Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase Science 1992 257 973 7 10.1126/science.1380182 1380182

126. Heitman J Movva NR Hall MN Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast Science 1991 253 905 9 10.1126/science.1715094 1715094

127. Sekulik A Hudson CC Homme JL A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells Cancer Res 2000 60 3504 13 10910062

128. Zhang H Stallock JP Ng JC Reinhard C Neufeld TP Regulation of cellular growth by the Drosophila target of rapamycin dTOR Genes Dev 2000 14 2712 24 10.1101/gad.835000 11069888 PMC317034

129. Oldham S Montagne J Radimerski T Thomas G Hafen E Genetic and biochemical characterization of dTOR, the Drosophila homolog of the target of rapamycin Genes Dev 2000 14 2689 94 10.1101/gad.845700 11069885 PMC317036

130. Chung J Kuo CJ Crabtree GR Blenis J Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases Cell 1992 69 1227 36 10.1016/0092-8674(92)90643-Q 1377606

131. Kuo CJ Chung J Fiorentino DF Rapamycin selectively inhibits interleukin-2 activation of p70 S6 kinase Nature 1992 358 70 3 10.1038/358070a0 1614535

132. Terada N Lucas JJ Szepesi A Rapamycin inhibits the phosphorylation of p70 S6 kinase in IL-2 and mitogen-activated human T cells Biochem Biophys Res Commun 1992 186 1315 21 10.1016/S0006-291X(05)81549-9 1380801

133. Lin TA Kong X Haystead TA PHAS-I as a link between mitogen-activated protein kinase and translation initiation Science 1994 266 653 6 10.1126/science.7939721 7939721

134. Graves LM Bornfeldt KE Argast GM cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells Proc Natl Acad Sci U S A 1995 92 7222 6 10.1073/pnas.92.16.7222 7638171 PMC41311

135. Pause A Belsham GJ Gingras A-C Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5′-cap function Nature 1994 371 762 7 10.1038/371762a0 7935836

136. von Manteuffel SR Dennis PB Pullen N The insulin-induced signalling pathway leading to S6 and initiation factor 4E binding protein 1 phosphorylation bifurcates at a rapamycin-sensitive point immediately upstream of p70s6k Mol Cell Biol 1997 17 5426 36 10.1128/MCB.17.9.5426 9271419 PMC232392

137. Singh K Sun S Vezina C Rapamycin (AY-22, 989), a new antifungal antibiotic. IV. Mechanism of action J Antibiot 1979 32 630 45 10.7164/antibiotics.32.630 381274

138. Eng C Sehgal S Vezina C Activity of rapamycin (AY-22, 989) against transplanted tumors J Antibiot 1984 37 1231 7 10.7164/antibiotics.37.1231 6501094

139. Muthukkumar S Ramesh T Bondada S Rapamycin, a potent immunosuppressive drug, causes programmed cell death in B lymphoma cells Transplantation 1995 60 264 70 10.1097/00007890-199508000-00010 7544036

140. Seufferlein T Rozengurt E Rapamycin inhibits constitutive p70s6k phosphorylation, cell proliferation, and colony formation in small cell lung cancer cells Cancer Res 1996 56 3895 7 8752154

141. Hosoi H Dilling MB Shikata T Rapamycin causes poorly reversible inhibition of mTOR and induces p53-independent apoptosis in human rhabdomyosarcoma cells Cancer Res 1999 59 886 94 10029080

142. Busca R Bertolotto C Ortonne J-P Ballotti R Inhibition of the phosphatidylinositol 3-kinase/p70S6-kinase pathway induces B16 melanoma cell differentiation J Biol Chem 1996 271 31824 30 10.1074/jbc.271.50.31824 8943224

143. Grewe M Gansauge F Schmid RM Adler G Seufferlein T Regulation of cell growth and cyclin D1 expression by the constitutively active FRAP-p70s6K pathway in human pancreatic cancer cells Cancer Res 1999 59 3581 7 10446965

144. Humar ROK Kiefer FN Berns H Resink TJ Battegay EJ Hypoxia enhances vascular cell proliferation and angiogenesis in vitro via rapamycin (mTOR) -dependent signaling FASEB J 2002 16 771 80 10.1096/fj.01-0658com 12039858

145. Guba M von Breitenbuch P Steinbauer M Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor Nat Med 2002 8 128 35 10.1038/nm0202-128 11821896

146. Shi Y Frankel A Radvanyi LG Rapamycin enhances apoptosis and increases sensitivity to cisplatin in vitro Cancer Res 1995 55 1982 8 7728769

147. Trepanier DJ Gallant H Legatt DF Yatscoff RW Rapamycin: distribution, pharmacokinetics and therapeutic range investigations: an update Clin Biochem 1998 31 345 51 10.1016/S0009-9120(98)00048-4 9721433

148. Yatscoff R Pharmacokinetics of rapamycin Transplant Proc 1996 28 970 3 8623483

149. Yu K Toral-Barza L Discafani C mTOR, a novel target in breast cancer: the effect of CCI-779, an mTOR inhibitor, in preclinical models of breast cancer Endocr Relat Cancer 2001 8 249 58 10.1677/erc.0.0080249 11566616

150. Geoerger B Kerr K Tang C-B Antitumor activity of the rapamycin analog CCI-779 in human primitive neuroectodermal tumor/medulloblastoma models as single agent and in combination chemotherapy Cancer Res 2001 61 1527 32 11245461

151. Asano T Yao Y Zhu J The rapamycin analog CCI-779 is a potent inhibitor of pancreatic cancer cell proliferation Biochem Biophys Res Commun 2005 331 295 302 10.1016/j.bbrc.2005.03.166 15845392

152. Frost P Moatamed F Hoang B In vivo antitumor effects of the mTOR inhibitor CCI-779 against human multiple myeloma cells in a xenograft model Blood 2004 104 4181 7 10.1182/blood-2004-03-1153 15304393

153. Ito D Fujimoto K Doi R Shimada Y Imamura M mTOR, a novel target in pancreatic cancer: the effect of CCI-779 in preclinical models AACR Meeting Abstr 2004 2004 896

154. Wu L Birle DC Tannock IF Effects of the mammalian target of rapamycin inhibitor CCI-779 used alone or with chemotherapy on human prostate cancer cells and xenografts Cancer Res 2005 65 2825 31 10.1158/0008-5472.CAN-04-3137 15805283

155. Nathan C-AO Amirghahari N Sibley D In vivo and in vitro effect of CCI-779 a rapamycin analogue on HNSCC AACR Meeting Abstr 2004 2004 850 1

156. Podsypanina K Lee RT Politis C An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten+/+ mice Proc Natl Acad Sci U S A 2001 98 10320 5 10.1073/pnas.171060098 11504907 PMC56959

157. Dudkin L Dilling MB Cheshire PJ Biochemical correlates of mTOR inhibition by the rapamycin ester CCI-779 and tumor growth inhibition Clin Cancer Res 2001 7 1758 64 11410517

158. Raymond E Alexandre JRM Faivre S Safety and pharmacokinetics of escalated doses of weekly intravenous infusion of CCI-779, a novel mTOR inhibitor, in patients with cancer J Clin Oncol 2004 22 2336 47 10.1200/JCO.2004.08.116 15136596

159. Peralba JM deGraffenried L Friedrichs W Pharmacodynamic evaluation of CCI-779, an inhibitor of mTOR, in cancer patients Clin Cancer Res 2003 9 2887 92 12912932

160. Hudes G Carducci M Tomczak P Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma N Engl J Med 2007 356 2271 81 10.1056/NEJMoa066838 17538086

161. Hess G Herbrecht R Romaguera J Phase III study to evaluate temsirolimus compared with investigator’s choice therapy for the treatment of relapsed or refractory mantle cell lymphoma J Clin Oncol 2009 27 3822 9 10.1200/JCO.2008.20.7977 19581539

162. Patel PH Senico PL Curiel RE Motzer RJ Phase I study combining treatment with temsirolimus and sunitinib malate in patients with advanced renal cell carcinoma Clin Genitourin Cancer 2009 7 24 7 10.3816/CGC.2009.n.004 19213664 PMC3740755

163. Patnaik A, Ricart A, Cooper J, et al. A phase I, pharmacokinetic and pharmacodynamic study of sorafenib (S), a multi-targeted kinase inhibitor in combination with temsirolimus (T), an mTOR inhibitor in patients with advanced solid malignancies. J Clin Oncol. 2007;25. 2007 ASCO Annual Meeting Proceedings Part I.

164. Escudier BJ, Negrier S, Gravis G, et al. Can the combination of temsirolimus and bevacizumab improve the treatment of metastatic renal cell carcinoma (mRCC)? Results of the randomized TORAVA phase II trial. J Clin Oncol. 2010;28.

165. Chan S Scheulen ME Johnston S Phase II study of temsirolimus (CCI-779), a novel inhibitor of mTOR, in heavily pretreated patients with locally advanced or metastatic breast cancer J Clin Oncol 2005 23 5314 22 10.1200/JCO.2005.66.130 15955899

166. Temkin SM Yamada SD Fleming GF A phase I study of weekly temsirolimus and topotecan in the treatment of advanced and/or recurrent gynecologic malignancies Gynecol Oncol 2010 117 473 6 10.1016/j.ygyno.2010.02.022 20347480

167. Farag SS Zhang S Jansak BS Phase II trial of temsirolimus in patients with relapsed or refractory multiple myeloma Leuk Res 2009 33 1475 80 10.1016/j.leukres.2009.01.039 19261329 PMC3771631

168. Kuhn JG Chang SM Wen PY Pharmacokinetic and tumor distribution characteristics of temsirolimus in patients with recurrent malignant glioma Clin Cancer Res 2007 13 7401 6 10.1158/1078-0432.CCR-07-0781 18094423 PMC4918812

169. Chang SM Wen P Cloughesy T Phase II study of CCI-779 in patients with recurrent glioblastoma multiforme Invest New Drugs 2005 23 357 61 10.1007/s10637-005-1444-0 16012795

170. Galanis E Buckner JC Maurer MJ Phase II trial of temsirolimus (CCI-779) in recurrent glioblastoma multiforme: a North Central Cancer Treatment Group Study J Clin Oncol 2005 23 5294 304 10.1200/JCO.2005.23.622 15998902

171. Pandya KJ Dahlberg S Hidalgo M A randomized, phase II trial of two dose levels of temsirolimus (CCI-779) in patients with extensive-stage small-cell lung cancer who have responding or stable disease after induction chemotherapy: a Trial of the Eastern Cooperative Oncology Group (E1500) J Thorac Oncol 2007 2 1036 41 10.1097/JTO.0b013e318155a439 17975496

172. Yee KWL Zeng Z Konopleva M Phase I/II study of the mammalian target of rapamycin inhibitor everolimus (RAD001) in patients with relapsed or refractory hematologic malignancies Clin Cancer Res 2006 12 5165 73 10.1158/1078-0432.CCR-06-0764 16951235

173. Fouladi M Laningham F Wu J Phase I study of everolimus in pediatric patients with refractory solid tumors J Clin Oncol 2007 25 4806 12 10.1200/JCO.2007.11.4017 17947729

174. Tabernero J Rojo F Calvo E Dose- and schedule-dependent inhibition of the mammalian target of rapamycin pathway with everolimus: a phase I tumor pharmacodynamic study in patients with advanced solid tumors J Clin Oncol 2008 26 1603 10 10.1200/JCO.2007.14.5482 18332469

175. O'Donnell A Faivre S Burris HA Phase I pharmacokinetic and pharmacodynamic study of the oral mammalian target of rapamycin inhibitor everolimus in patients with advanced solid tumors J Clin Oncol 2008 26 1588 95 10.1200/JCO.2007.14.0988 18332470

176. Motzer RJ Escudier B Oudard S Efficacy of everolimus in advanced renal cell carcinoma: a double-blind, randomised, placebo-controlled phase III trial Lancet 2008 372 449 56 10.1016/S0140-6736(08)61039-9 18653228

177. Kreisl T Lassman A Mischel P A pilot study of everolimus and gefitinib in the treatment of recurrent glioblastoma (GBM) J Neurooncol 2009 92 99 105 10.1007/s11060-008-9741-z 19018475

178. Johnston PB Inwards DJ Colgan JP A phase II trial of the oral mTOR inhibitor everolimus in relapsed Hodgkin lymphoma Am J Hematol 2010 85 320 4 20229590 10.1002/ajh.21664 PMC4420736

179. Doi T Muro K Boku N Multicenter phase II study of everolimus in patients with previously treated metastatic gastric cancer J Clin Oncol 2010 28 1904 10 10.1200/JCO.2009.26.2923 20231677

180. Ramalingam SS Harvey RD Saba N Phase 1 and pharmacokinetic study of everolimus, a mammalian target of rapamycin inhibitor, in combination with docetaxel for recurrent/refractory nonsmall cell lung cancer Cancer 2010 116 3903 9 10.1002/cncr.25264 20564143 PMC2940062

181. Ellard SL Clemons M Gelmon KA Randomized phase II study comparing two schedules of everolimus in patients with recurrent/metastatic breast cancer: NCIC Clinical Trials Group IND.163. J Clin Oncol 2009 27 4536 41 10.1200/JCO.2008.21.3033 19687332

182. Awada A Cardoso F Fontaine C The oral mTOR inhibitor RAD001 (everolimus) in combination with letrozole in patients with advanced breast cancer: Results of a phase I study with pharmacokinetics Eur J Cancer 2008 44 84 91 10.1016/j.ejca.2007.10.003 18039566

183. Baselga J Semiglazov V van Dam P Phase II randomized study of neoadjuvant everolimus plus letrozole compared with placebo plus letrozole in patients with estrogen receptor-positive breast cancer J Clin Oncol 2009 27 2630 7 10.1200/JCO.2008.18.8391 19380449

184. Andre F Campone M O'Regan R Phase I study of everolimus plus weekly paclitaxel and trastuzumab in patients with metastatic breast cancer pretreated with trastuzumab J Clin Oncol 2010 28 5110 5 10.1200/JCO.2009.27.8549 20975068

185. Zent CS LaPlant BR Johnston PB The treatment of recurrent/refractory chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) with everolimus results in clinical responses and mobilization of CLL cells into the circulation Cancer 2010 116 2201 7 20166206 10.1002/cncr.25005 PMC2861142

186. Yao JC Lombard-Bohas C Baudin E Daily oral everolimus activity in patients with metastatic pancreatic neuroendocrine tumors after failure of cytotoxic chemotherapy: a phase II trial J Clin Oncol 2010 28 69 76 10.1200/JCO.2009.24.2669 19933912 PMC4295034

187. Yao JC Shah MH Ito T Everolimus for advanced pancreatic neuroendocrine tumors N Engl J Med 2011 364 514 23 10.1056/NEJMoa1009290 21306238 PMC4208619

188. Rizzieri DA Feldman E DiPersio JF A phase 2 clinical trial of deforolimus (AP23573, MK-8669), a novel mammalian target of rapamycin inhibitor, in patients with relapsed or refractory hematologic malignancies Clin Cancer Res 2008 14 2756 62 10.1158/1078-0432.CCR-07-1372 18451242

189. Hartford CM Desai AA Janisch L A phase I trial to determine the safety, tolerability, and maximum tolerated dose of deforolimus in patients with advanced malignancies Clin Cancer Res 2009 15 1428 34 10.1158/1078-0432.CCR-08-2076 19228743 PMC3147153

190. Mita MM Mita AC Chu QS Phase I trial of the novel mammalian target of rapamycin inhibitor deforolimus (AP23573; MK-8669) administered intravenously daily for 5 days every 2 weeks to patients with advanced malignancies J Clin Oncol 2008 26 361 7 10.1200/JCO.2007.12.0345 18202410

191. Perotti A Locatelli A Sessa C Phase IB study of the mTOR inhibitor ridaforolimus with capecitabine J Clin Oncol 2010 28 4554 61 10.1200/JCO.2009.27.5867 20855840

192. Sessa C Tosi D Vigano L Phase Ib study of weekly mammalian target of rapamycin inhibitor ridaforolimus (AP23573; MK-8669) with weekly paclitaxel Ann Oncol 2010 21 1315 22 10.1093/annonc/mdp504 19901013

193. Javle M Shroff R Xiong H Inhibition of the mammalian target of rapamycin (mTOR) in advanced pancreatic cancer: results of two phase II studies BMC Cancer 2010 10 368 10.1186/1471-2407-10-368 20630061 PMC2910694

194. Mukherjee R Bartlett JMS Krishna NS Underwood MA Edwards J Raf-1 expression may influence progression to androgen insensitive prostate cancer Prostate 2005 64 101 7 10.1002/pros.20211 15666389

195. Brose MS Volpe P Feldman M BRAF and RAS mutations in human lung cancer and melanoma Cancer Res 2002 62 6997 7000 12460918

196. Hwang YH Choi JY Kim S Over-expression of c-raf-1 proto-oncogene in liver cirrhosis and hepatocellular carcinoma Hepatol Res 2004 29 113 21 10.1016/j.hepres.2004.02.009 15163433

197. McPhillips F Mullen P Monia BP Association of c-Raf expression with survival and its targeting with antisense oligonucleotides in ovarian cancer Br J Cancer 2001 85 1753 8 10.1054/bjoc.2001.2139 11742498 PMC2363986

198. Kasid U Pfeifer A Brennan T Effect of antisense c-raf-1 on tumorigenicity and radiation sensitivity of a human squamous carcinoma Science 1989 243 1354 6 10.1126/science.2466340 2466340

199. Kasid U Pfeifer A Weichselbaum RR Dritschilo A Mark GE The raf oncogene is associated with a radiation-resistant human laryngeal cancer Science 1987 237 1039 41 10.1126/science.3616625 3616625

200. Smith RA Barbosa J Blum CL Discovery of heterocyclic ureas as a new class of raf kinase inhibitors: identification of a second generation lead by a combinatorial chemistry approach Bioorg Med Chem Lett 2001 11 2775 8 10.1016/S0960-894X(01)00571-6 11591521

201. Lyons JF Wilhelm S Hibner B Bollag G Discovery of a novel Raf kinase inhibitor Endocr Relat Cancer 2001 8 219 25 10.1677/erc.0.0080219 11566613

202. Lowinger TB Riedl B Dumas J Smith RA Design and discovery of small molecules targeting Raf-1 kinase Curr Pharm Des 2002 10 2269 78 10.2174/1381612023393125 12369855

203. Wilhelm SM Carter C Tang L BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis Cancer Res 2004 64 7099 109 10.1158/0008-5472.CAN-04-1443 15466206

204. Carlomagno F Anaganti S Guida T BAY 43-9006 inhibition of oncogenic RET mutants J Natl Cancer Inst 2006 98 326 34 10.1093/jnci/djj069 16507829

205. Wan PTC Garnett MJ Roe SM Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF Cell 2004 116 855 67 10.1016/S0092-8674(04)00215-6 15035987

206. Strumberg D Richly H Hilger RA Phase I clinical and pharmacokinetic study of the novel Raf kinase and vascular endothelial growth factor receptor inhibitor BAY 43-9006 in patients with advanced refractory solid tumors J Clin Oncol 2005 23 965 72 10.1200/JCO.2005.06.124 15613696

207. Awada A Hendlisz A Gil T Phase I safety and pharmacokinetics of BAY 43-9006 administered for 21 days on/7 days off in patients with advanced, refractory solid tumours Br J Cancer 2005 92 1855 61 10.1038/sj.bjc.6602584 15870716 PMC2361774

208. Moore M Hirte HW Siu L Phase I study to determine the safety and pharmacokinetics of the novel Raf kinase and VEGFR inhibitor BAY 43-9006, administered for 28 days on/7 days off in patients with advanced, refractory solid tumors Ann Oncol 2005 16 1688 94 10.1093/annonc/mdi310 16006586

209. Kupsch P Henning B Passarge K Results of a phase I trial of sorafenib (BAY 43-9006) in combination with oxaliplatin in patients with refractory solid tumors, including colorectal cancer Clin Colorectal Cancer 2005 5 188 96 10.3816/CCC.2005.n.030 16197622

210. Siu LL Awada A Takimoto CH Phase I trial of sorafenib and gemcitabine in advanced solid tumors with an expanded cohort in advanced pancreatic cancer Clin Cancer Res 2006 12 144 51 10.1158/1078-0432.CCR-05-1571 16397036

211. Richly H Henning BF Kupsch P Results of a phase I trial of sorafenib (BAY 43-9006) in combination with doxorubicin in patients with refractory solid tumors Ann Oncol 2006 17 866 73 10.1093/annonc/mdl017 16500908

212. Steinbild S, Baas F, Gmehling D, et al. Phase I study of BAY 43–9006 (sorafenib), a Raf kinase and VEGFR inhibitor, combined with irinotecan (CPT-11) in advanced solid tumors. J Clin Oncol. 2005;23.

213. Eisen T, Ahmad T, Gore ME, et al. Phase I trial of BAY 43-9006 (sorafenib) combined with dacarbazine (DTIC) in metastatic melanoma patients. J Clin Oncol. 2005;23.

214. Gollob J, Richmond T, Jones J, et al. Phase II trial of sorafenib plus interferon-alpha 2b (IFN-α2b) as first- or second-line therapy in patients (pts) with metastatic renal cell cancer (RCC). J Clin Oncol. 2006;24. 10.1200/JCO.2007.10.8613 17664476

215. Ratain MJ Eisen T Stadler WM Phase II placebo-controlled randomized discontinuation trial of sorafenib in patients with metastatic renal cell carcinoma J Clin Oncol 2006 24 2505 12 10.1200/JCO.2005.03.6723 16636341

216. Escudier B, Szczylik C, Demkow T, et al. Randomized phase II trial of the multi-kinase inhibitor sorafenib versus interferon (IFN) in treatment-naïve patients with metastatic renal cell carcinoma (mRCC). J Clin Oncol. 2006;24. 10.1200/JCO.2008.19.3342 19171708

217. Motzer RJ Bacik J Schwartz LH Prognostic factors for survival in previously treated patients with metastatic renal cell carcinoma J Clin Oncol 2004 22 454 63 10.1200/JCO.2004.06.132 14752067

218. Negrier S Jäger E Porta C Efficacy and safety of sorafenib in patients with advanced renal cell carcinoma with and without prior cytokine therapy, a subanalysis of TARGET Med Oncol 2010 27 899 906 10.1007/s12032-009-9303-z 19757215

219. Hutson TE Bellmunt J Porta C Long-term safety of sorafenib in advanced renal cell carcinoma: follow-up of patients from phase III TARGET Eur J Cancer 2010 46 2432 40 10.1016/j.ejca.2010.06.121 20656473

220. Liu L Cao Y Chen C Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5 Cancer Res 2006 66 11851 8 10.1158/0008-5472.CAN-06-1377 17178882

221. Abou-Alfa GK Schwartz L Ricci S Phase II study of sorafenib in patients with advanced hepatocellular carcinoma J Clin Oncol 2006 24 4293 300 10.1200/JCO.2005.01.3441 16908937

222. Llovet JM Ricci S Mazzaferro V Sorafenib in advanced hepatocellular carcinoma N Engl J Med 2008 359 378 90 10.1056/NEJMoa0708857 18650514

223. Ott PA, Hamilton A, Min C, et al. A phase II trial of sorafenib in metastatic melanoma with tissue correlates. PLoS One. 2010;5. 10.1371/journal.pone.0015588 PMC3012061 21206909

224. Flaherty KT Schiller J Schuchter LM A phase I trial of the oral. multikinase inhibitor sorafenib in combination with carboplatin and paclitaxel Clin Cancer Res 2008 14 4836 42 10.1158/1078-0432.CCR-07-4123 18676756

225. Hauschild A Agarwala SS Trefzer U Results of a phase III, randomized. placebo-controlled study of sorafenib in combination with carboplatin and paclitaxel as second-line treatment in patients with unresectable stage III or stage IV melanoma J Clin Oncol 2009 27 2823 30 10.1200/JCO.2007.15.7636 19349552

226. Abou-Alfa GK Johnson P Knox JJ Doxorubicin plus sorafenib vs doxorubicin alone in patients with advanced hepatocellular carcinoma JAMA 2010 304 2154 60 10.1001/jama.2010.1672 21081728

227. Cowey CL Amin C Pruthi RS Neoadjuvant clinical trial with sorafenib for patients with stage II or higher renal cell carcinoma J Clin Oncol 2010 28 1502 7 10.1200/JCO.2009.24.7759 20159822 PMC4525128

228. Blumenschein GR Gatzemeier U Fossella F Phase II, multicenter, uncontrolled trial of single-agent sorafenib in patients with relapsed or refractory. advanced non-small-cell lung cancer J Clin Oncol 2009 27 4274 80 10.1200/JCO.2009.22.0541 19652055

229. Lind JSW Dingemans A-MC Groen HJM A multicenter phase II study of erlotinib and sorafenib in chemotherapy-naïve patients with advanced non-small cell lung cancer Clin Cancer Res 2010 16 3078 87 10.1158/1078-0432.CCR-09-3033 20395213

230. Adjei AA Molina JR Mandrekar SJ Phase I trial of sorafenib in combination with gefitinib in patients with refractory or recurrent non-small cell lung cancer Clin Cancer Res 2007 13 2684 91 10.1158/1078-0432.CCR-06-2889 17473200

231. Bellmunt J Trigo JM Calvo E Activity of a multitargeted chemo-switch regimen (sorafenib, gemcitabine, and metronomic capecitabine) in metastatic renal-cell carcinoma: a phase 2 study (SOGUG-02-06) Lancet Oncol 2010 11 350 7 10.1016/S1470-2045(09)70383-3 20163987

232. Dubey S Janne PA Krug L A phase II study of sorafenib in malignant mesothelioma: results of Cancer and Leukemia Group B 30307 J Thorac Oncol 2010 5 1655 61 10.1097/JTO.0b013e3181ec18db 20736856 PMC3823555

233. Chi KN Ellard SL Hotte SJ A phase II study of sorafenib in patients with chemo-naive castration-resistant prostate cancer Ann Oncol 2008 19 746 51 10.1093/annonc/mdm554 18056648

234. Dahut WL Scripture C Posadas E A phase II clinical trial of sorafenib in androgen-independent prostate cancer Clin Cancer Res 2008 14 209 14 10.1158/1078-0432.CCR-07-1355 18172272

235. Nimeiri HS Oza AM Morgan RJ A phase II study of sorafenib in advanced uterine carcinoma/carcinosarcoma: A trial of the Chicago, PMH, and California Phase II Consortia Gynecol Oncol 2010 117 37 40 10.1016/j.ygyno.2010.01.013 20117828 PMC2842466

236. Dreicer R Li H Stein M Phase 2 trial of sorafenib in patients with advanced urothelial cancer Cancer 2009 115 4090 5 10.1002/cncr.24467 19536901 PMC2774800

237. Maki RG D'Adamo DR Keohan ML Phase II study of sorafenib in patients with metastatic or recurrent sarcomas J Clin Oncol 2009 27 3133 40 10.1200/JCO.2008.20.4495 19451436 PMC2716936

238. Williamson SK Moon J Huang CH Phase II evaluation of sorafenib in advanced and metastatic squamous cell carcinoma of the head and neck: Southwest Oncology Group Study S0420 J Clin Oncol 2010 28 3330 5 10.1200/JCO.2009.25.6834 20498388 PMC2903329

239. Scagliotti G Novello S von Pawel J Phase III study of carboplatin and paclitaxel alone or with sorafenib in advanced non-small-cell lung cancer J Clin Oncol 2010 28 1835 42 10.1200/JCO.2009.26.1321 20212250

240. Okamoto I Miyazaki M Morinaga R Phase I clinical and pharmacokinetic study of sorafenib in combination with carboplatin and paclitaxel in patients with advanced non-small cell lung cancer Invest New Drugs 2010 28 844 53 10.1007/s10637-009-9321-x 19760364 PMC2955234

241. Pölcher M Eckhardt M Coch C Sorafenib in combination with carboplatin and paclitaxel as neoadjuvant chemotherapy in patients with advanced ovarian cancer Cancer Chemother Pharmacol 2010 66 203 7 10.1007/s00280-010-1276-2 20204367

242. Di Lorenzo G Carteni G Autorino R Phase II study of sorafenib in patients with sunitinib-refractory metastatic renal cell cancer J Clin Oncol 2009 27 4469 74 10.1200/JCO.2009.22.6480 19652053

243. Flaherty K Phase I/II, pharmacokinetic and pharmacodynamic trial of BAY 43-9006 alone in patients with metastatic melanoma J Clin Oncol 2005 23 3037

244. Whittaker S Kirk R Hayward R Gatekeeper mutations mediate resistance to BRAF-targeted therapies Sci Transl Med 2010 2 35ra41 10.1126/scitranslmed.3000758 20538618

245. Bollag G Hirth P Tsai J Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma Nature 2010 467 596 9 10.1038/nature09454 20823850 PMC2948082

246. Joseph EW Pratilas CA Poulikakos PI The RAF inhibitor PLX4032 inhibits ERK signaling and tumor cell proliferation in a V600E BRAF-selective manner Proc Natl Acad Sci 2010 107 14903 8 10.1073/pnas.1008990107 20668238 PMC2930420

247. Lee JT Li L Brafford PA PLX4032, a potent inhibitor of the B-Raf V600E oncogene, selectively inhibits V600E-positive melanomas Pigment Cell Melanoma Res 2010 23 820 7 10.1111/j.1755-148X.2010.00763.x 20973932 PMC3623294

248. Flaherty K Phase I study of PLX4032: proof of concept for V600E BRAF mutation as a therapeutic target in human cancer J Clin Oncol 2009 27 9000

249. Flaherty KT Puzanov I Kim KB Inhibition of mutated. Activated BRAF in metastatic melanoma N Engl J Med 2010 363 809 19 10.1056/NEJMoa1002011 20818844 PMC3724529

250. Chapman PB Hauschild A Robert C Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 2011 364 2507 16 10.1056/NEJMoa1103782 21639808 PMC3549296

251. Ji Z, Flaherty KT, Tsao H. Targeting the RAS pathway in melanoma. Trends Mol Med. 2012;18:27–35. 10.1016/j.molmed.2011.08.001 PMC3759017 21962474

252. Nazarian R Shi H Wang Q Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation Nature 2010 468 973 7 10.1038/nature09626 21107323 PMC3143360

253. Kaplan FM Shao Y Mayberry MM Aplin AE Hyperactivation of MEK-ERK1/2 signaling and resistance to apoptosis induced by the oncogenic B-RAF inhibitor, PLX4720, in mutant N-RAS melanoma cells Oncogene 2011 30 366 71 10.1038/onc.2010.408 20818433 PMC6591715

254. Shi H Kong X Ribas A Lo RS Combinatorial treatments that overcome PDGFRβ-driven resistance of melanoma cells to V600EB-RAF inhibition Cancer Res 2011 71 5067 74 10.1158/0008-5472.CAN-11-0140 21803746 PMC3149831

255. Niehr F von Euw E Attar N Combination therapy with vemurafenib (PLX4032/RG7204) and metformin in melanoma cell lines with distinct driver mutations J Transl Med 2011 9 76 10.1186/1479-5876-9-76 21609436 PMC3152784

256. Comin-Anduix B Chodon T Sazegar H The oncogenic BRAF kinase inhibitor PLX4032/RG7204 does not affect the viability or function of human lymphocytes across a wide range of concentrations Clin Cancer Res 2010 16 6040 8 10.1158/1078-0432.CCR-10-1911 21169256 PMC3057460

257. Yu L Favoino E Wang Y The CSPG4-specific monoclonal antibody enhances and prolongs the effects of the BRAF inhibitor in melanoma cells Immunol Res 2011 50 294 302 10.1007/s12026-011-8232-z 21717063

258. Kohler G Milstein C Continuous cultures of fused cells secreting antibody of predefined specificity Nature 1975 256 495 7 10.1038/256495a0 1172191

259. Einfeld DA Brown JP Valentine MA Clark EA Ledbetter JA Molecular cloning of the human B cell CD20 receptor predicts a hydrophobic protein with multiple transmembrane domains EMBO J 1988 7 711 7 2456210 10.1002/j.1460-2075.1988.tb02867.x PMC454379

260. Liu AY Robinson RR Murray ED Production of a mouse-human chimeric monoclonal antibody to CD20 with potent Fc-dependent biologic activity J Immunol 1987 139 3521 6 3119711

261. Press OW Appelbaum F Ledbetter JA Monoclonal antibody 1F5 (anti-CD20) serotherapy of human B cell lymphomas Blood 1987 69 584 91 3492224

262. Reff ME Carner K Chambers KS Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20 Blood 1994 83 435 45 7506951

263. Bannerji R Kitada S Flinn IW Apoptotic-regulatory and complement-protecting protein expression in chronic lymphocytic leukemia: relationship to in vivo rituximab resistance J Clin Oncol 2003 21 1466 71 10.1200/JCO.2003.06.012 12697868

264. Treon SP Mitsiades C Mitsiades N Tumor cell expression of CD59 is associated with resistance to CD20 serotherapy in patients with B-cell malignancies J Immunother 2001 24 263 71 10.1097/00002371-200105000-00011 11394505

265. Kennedy AD Beum PV Solga MD Rituximab infusion promotes rapid complement depletion and acute CD20 loss in chronic lymphocytic leukemia J Immunol 2004 172 3280 8 10.4049/jimmunol.172.5.3280 14978136

266. Klepfish A Schattner A Ghoti H Rachmilewitz EA Addition of fresh frozen plasma as a source of complement to rituximab in advanced chronic lymphocytic leukaemia Lancet Oncol 2007 8 361 2 10.1016/S1470-2045(07)70106-7 17395110

267. Gong Q Ou Q Ye S Importance of cellular microenvironment and circulatory dynamics in B cell immunotherapy J Immunol 2005 174 817 26 10.4049/jimmunol.174.2.817 15634903

268. Beers SA Chan CHT James S Type II (tositumomab) anti-CD20 monoclonal antibody out performs type I (rituximab-like) reagents in B-cell depletion regardless of complement activation Blood 2008 112 4170 7 10.1182/blood-2008-04-149161 18583569 PMC2582008

269. Weng W-K Levy R Expression of complement inhibitors CD46, CD55, and CD59 on tumor cells does not predict clinical outcome after rituximab treatment in follicular non-Hodgkin lymphoma Blood 2001 98 1352 7 10.1182/blood.V98.5.1352 11520782

270. Hamaguchi Y Xiu Y Komura K Nimmerjahn F Tedder TF Antibody isotype-specific engagement of Fcgamma receptors regulates B lymphocyte depletion during CD20 immunotherapy J Exp Med 2006 203 743 53 10.1084/jem.20052283 16520392 PMC2118227

271. Ravetch JV Bolland S IgG Fc receptors Annu Rev Immunol 2001 19 275 90 10.1146/annurev.immunol.19.1.275 11244038

272. Nimmerjahn F Ravetch JV Divergent immunoglobulin G subclass activity through selective Fc receptor binding Science 2005 310 1510 2 10.1126/science.1118948 16322460

273. Nimmerjahn F Ravetch JV Fc[gamma] receptors: old friends and new family members Immunity 2006 24 19 28 10.1016/j.immuni.2005.11.010 16413920

274. Cartron G Dacheux L Salles G Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor Fcgamma RIIIa gene Blood 2002 99 754 8 10.1182/blood.V99.3.754 11806974

275. Dall'Ozzo S Tartas S Paintaud G Rituximab-dependent cytotoxicity by natural killer cells Cancer Res 2004 64 4664 9 10.1158/0008-5472.CAN-03-2862 15231679

276. Bowles JA Weiner GJ CD16 polymorphisms and NK activation induced by monoclonal antibody-coated target cells J Immunol Methods 2005 304 88 99 10.1016/j.jim.2005.06.018 16109421

277. Lefebvre M-L Krause SW Salcedo M Nardin A Ex vivo-activated human macrophages kill chronic lymphocytic leukemia cells in the presence of rituximab: mechanism of antibody-dependent cellular cytotoxicity and impact of human serum J Immunother 2006 29 388 97 10.1097/01.cji.0000203081.43235.d7 16799334

278. Tedder TF Forsgren A Boyd AW Nadler LM Schlossman SF Antibodies reactive with the B1 molecule inhibit cell cycle progression but not activation of human B lymphocytes Eur J Immunol 1986 16 881 7 10.1002/eji.1830160802 3091375

279. Golay JT Clark EA Beverley PC The CD20 (Bp35) antigen is involved in activation of B cells from the G0 to the G1 phase of the cell cycle J Immunol 1985 135 3795 801 2415587

280. Clark EA Shu G Activation of human B cell proliferation through surface Bp35 (CD20) polypeptides or immunoglobulin receptors J Immunol 1987 138 720 5 3492530

281. Shan D Ledbetter JA Press OW Signaling events involved in anti-CD20-induced apoptosis of malignant human B cells Cancer Immunol Immunother 2000 48 673 83 10.1007/s002620050016 10752475 PMC11037214

282. Hofmeister JK Cooney D Coggeshall KM Clustered CD20 induced apoptosis: Src-family kinase, the proximal regulator of tyrosine phosphorylation, calcium influx, and caspase 3-dependent apoptosis Blood Cells Mol Dis 2000 26 133 43 10.1006/bcmd.2000.0287 10753604

283. Pedersen IM Buhl AM Klausen P Geisler CH Jurlander J The chimeric anti-CD20 antibody rituximab induces apoptosis in B-cell chronic lymphocytic leukemia cells through a p38 mitogen activated protein-kinase-dependent mechanism Blood 2002 99 1314 9 10.1182/blood.V99.4.1314 11830481

284. Cardarelli PM Quinn M Buckman D Binding to CD20 by anti-B1 antibody or F(ab′)2 is sufficient for induction of apoptosis in B-cell lines Cancer Immunol Immunother 2002 51 15 24 10.1007/s00262-001-0247-1 11845256 PMC11032933

285. Tobinai K Kobayashi Y Narabayashi M Feasibility and pharmacokinetic study of a chimeric anti-CD20 monoclonal antibody (IDEC-C2B8, rituximab) in relapsed B-cell lymphoma Ann Oncol 1998 9 527 34 10.1023/A:1008265313133 9653494

286. Maloney DG Grillo-Lopez AJ White CA IDEC-C2B8 (rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin’s lymphoma Blood 1997 90 2188 95 9310469

287. Maloney DG Grillo-Lopez AJ Bodkin DJ IDEC-C2B8: results of a phase I multiple-dose trial in patients with relapsed non-Hodgkin’s lymphoma J Clin Oncol 1997 15 3266 74 9336364 10.1200/JCO.1997.15.10.3266

288. McLaughlin P Grillo-Lopez AJ Link BK Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program J Clin Oncol 1998 16 2825 33 9704735 10.1200/JCO.1998.16.8.2825

289. Coiffier B Haioun C Ketterer N Rituximab (anti-CD20 monoclonal antibody) for the treatment of patients with relapsing or refractory aggressive lymphoma: a multicenter phase II study Blood 1998 92 1927 32 9731049

290. Nguyen D Amess J Doughty H Hendry L Diamond L IDEC-C2B8 anti-CD20 (rituximab) immunotherapy in patients with low-grade non-Hodgkin’s lymphoma and lymphoproliferative disorders: evaluation of response on 48 patients Eur J Haematol 1999 62 76 82 10.1111/j.1600-0609.1999.tb01725.x 10052709

291. Feuring-Buske M Kneba M Unterhalt M IDEC-C2B8 (rituximab) anti-CD20 antibody treatment in relapsed advanced-stage follicular lymphomas: results of a phase-II study of the German Low-Grade Lymphoma Study Group Ann Hematol 2000 79 493 500 10.1007/s002770000163 11043420

292. Colombat P Salles G Brousse N Rituximab (anti-CD20 monoclonal antibody) as single first-line therapy for patients with follicular lymphoma with a low tumor burden: clinical and molecular evaluation Blood 2001 97 101 6 10.1182/blood.V97.1.101 11133748

293. Czuczman MS Grillo-LÃpez AJ White CA Treatment of patients with low-grade B-cell lymphoma with the combination of chimeric anti-CD20 monoclonal antibody and CHOP chemotherapy J Clin Oncol 1999 17 268 10458242 10.1200/JCO.1999.17.1.268

294. Vose JM Link BK Grossbard ML Phase II study of rituximab in combination with CHOP chemotherapy in patients with previously untreated. Aggressive Non-Hodgkin’s Lymphoma J Clin Oncol 2001 19 389 97 11208830 10.1200/JCO.2001.19.2.389

295. Czuczman M CHOP plus rituximab chemoimmunotherapy of indolent B-cell lymphoma Semin Oncol 1999 26 88 96 10561023

296. Czuczman MS Grillo-Lopez AJ McLaughlin P Clearing of cells bearing the bcl-2 [t(14;18)] translocation from blood and marrow of patients treated with rituximab alone or in combination with CHOP chemotherapy Ann Oncol 2001 12 109 14 10.1023/A:1008395214584 11249036

297. Winter JN, Li S, Aurora V, et al. Expression of p21 protein predicts clinical outcome in DLBCL patients older than 60 years treated with R-CHOP but not CHOP: a prospective ECOG and Southwest Oncology Group Correlative Study on E4494. Clinical Cancer Res. 2010;16:2435–42. 10.1158/1078-0432.CCR-09-1219 PMC2865202 20371683

298. Davis TA Grillo-Lopez AJ White CA Rituximab anti-CD20 monoclonal antibody therapy in non-Hodgkin’s lymphoma: safety and efficacy of re-treatment J Clin Oncol 2000 18 3135 43 10963642 10.1200/JCO.2000.18.17.3135

299. Davis TA Maloney DG Grillo-Lopez AJ Combination immunotherapy of relapsed or refractory low-grade or follicular non-Hodgkin’s lymphoma with rituximab and interferon-2a Clin Cancer Res 2000 6 2644 52 10914705

300. Byrd JC Murphy T Howard RS Rituximab using A thrice weekly dosing schedule in B-cell chronic lymphocytic leukemia and small lymphocytic lymphoma demonstrates clinical activity and acceptable toxicity J Clin Oncol 2001 19 2153 64 11304767 10.1200/JCO.2001.19.8.2153

301. O’Brien SM Kantarjian H Thomas DA Rituximab dose-escalation trial in chronic lymphocytic leukemia J Clin Oncol 2001 19 2165 70 11304768 10.1200/JCO.2001.19.8.2165

302. Keating MJ OÂ’Brien S Albitar M Early results of a chemoimmunotherapy regimen of fludarabine, cyclophosphamide, and rituximab as initial therapy for chronic lymphocytic leukemia J Clin Oncol 2005 23 4079 88 10.1200/JCO.2005.12.051 15767648

303. Schulz H Klein SK Rehwald U Phase 2 study of a combined immunochemotherapy using rituximab and fludarabine in patients with chronic lymphocytic leukemia Blood 2002 100 3115 20 10.1182/blood-2002-03-0972 12384407

304. Byrd JC Rai K Peterson BL Addition of rituximab to fludarabine may prolong progression-free survival and overall survival in patients with previously untreated chronic lymphocytic leukemia: an updated retrospective comparative analysis of CALGB 9712 and CALGB 9011 Blood 2005 105 49 53 10.1182/blood-2004-03-0796 15138165

305. Wierda W O’Brien S Wen S Chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab for relapsed and refractory chronic lymphocytic leukemia J Clin Oncol 2005 23 4070 8 10.1200/JCO.2005.12.516 15767647

306. Tam CS O’Brien S Wierda W Long-term results of the fludarabine, cyclophosphamide, and rituximab regimen as initial therapy of chronic lymphocytic leukemia Blood 2008 112 975 80 10.1182/blood-2008-02-140582 18411418 PMC3952498

307. Robak T Dmoszynska A Solal-Celigny P Rituximab plus fludarabine and cyclophosphamide prolongs progression-free survival compared with fludarabine and cyclophosphamide alone in previously treated chronic lymphocytic leukemia J Clin Oncol 2010 28 1756 65 10.1200/JCO.2009.26.4556 20194844

308. Hallek M Fischer K Fingerle-Rowson G Addition of rituximab to fludarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: a randomised, open-label, phase 3 trial Lancet 2010 376 1164 74 10.1016/S0140-6736(10)61381-5 20888994

309. Salles G Seymour JF Offner F Rituximab maintenance for 2 years in patients with high tumour burden follicular lymphoma responding to rituximab plus chemotherapy (PRIMA): a phase 3, randomised controlled trial Lancet 2010 377 42 51 10.1016/S0140-6736(10)62175-7 21176949

310. Alinari L White VL Earl CT Combination bortezomib and rituximab treatment affects multiple survival and death pathways to promote apoptosis in mantle cell lymphoma mAbs 2009 1 31 40 10.4161/mabs.1.1.7472 20046572 PMC2715189

311. Czuczman MS Olejniczak S Gowda A Acquirement of rituximab resistance in lymphoma cell lines is associated with both global CD20 gene and protein down-regulation regulated at the pretranscriptional and posttranscriptional levels Clin Cancer Res 2008 14 1561 70 10.1158/1078-0432.CCR-07-1254 18316581

312. Baiocchi RA, Alinari L, Lustberg ME, et al. Phase 2 trial of rituximab and bortezomib in patients with relapsed or refractory mantle cell and follicular lymphoma. Cancer. 2011;117:2442–51. 10.1002/cncr.25792 PMC3116936 24048792

313. Sato JD Kawamoto T Le AD Mendelsohn J Polikoff J Sato GH Biological effects in vitro of monoclonal antibodies to human epidermal growth factor receptors Mol Biol Med 1983 1 511 29 6094961

314. Masui H Kawamoto T Sato JD Growth inhibition of human tumor cells in athymic mice by anti-epidermal growth factor receptor monoclonal antibodies Cancer Res 1984 44 1002 7 6318979

315. Sunada H Magun BE Mendelsohn J MacLeod CL Monoclonal antibody against epidermal growth factor receptor is internalized without stimulating receptor phosphorylation Proc Natl Acad Sci U S A 1986 83 3825 9 10.1073/pnas.83.11.3825 2424012 PMC323616

316. Vollmar AM Banker DE Mendelsohn J Herschman HR Toxicity of ligand and antibody-directed ricin A-chain conjugates recognizing the epidermal growth factor receptor J Cell Physiol 1987 131 418 25 10.1002/jcp.1041310314 3496342

317. Wu X Rubin M Fan Z Involvement of p27KIP1 in G1 arrest mediated by an anti-epidermal growth factor receptor monoclonal antibody Oncogene 1996 12 1397 403 8622855

318. Peng D Fan Z Lu Y Anti-epidermal growth factor receptor monoclonal antibody 225 up-regulates p27KIP1 and induces G1 arrest in prostatic cancer cell line DU145 Cancer Res 1996 56 3666 9 8706005

319. Kiyota A Shintani S Mihara M Anti-epidermal growth factor receptor monoclonal antibody 225 upregulates p27KIP1 and p15INK4B and induces G1 arrest in oral squamous carcinoma cell lines Oncology 2002 63 92 8 10.1159/000065726 12187077

320. Fan BLAZ The monoclonal antibody 225 activates caspase-8 and induces apoptosis through a tumor necrosis factor receptor family-independent pathway Oncogene 2001 20 3726 34 10.1038/sj.onc.1204490 11439335

321. Goldenberg A Masui H Divgi C Imaging of human tumor xenografts with an indium-111-labeled anti-epidermal growth factor receptor monoclonal antibody J Natl Cancer Inst 1989 81 1616 25 10.1093/jnci/81.21.1616 2795690

322. Divgi CR Welt S Kris M Phase I and imaging trial of indium 111-labeled anti-epidermal growth factor receptor monoclonal antibody 225 in patients with squamous cell lung carcinoma J Natl Cancer Inst 1991 83 97 104 10.1093/jnci/83.2.97 1988695

323. Saltz L, Rubin M, Hochster H, et al. Cetuximab (IMC-C225) plus irinotecan (CPT-11) is active in CPT-11-refractory colorectal cancer (CRC) that expresses epidermal growth factor receptor (EGFR). Proc Am Soc Clin Oncol. 2001;2001.

324. Rosenberg AH, Loehrer PJ, Needle MN, et al. Erbitux (IMC-C225) plus weekly irinotecan (CPT-11), fluorouracil (5FU) and leucovorin (LV) in colorectal cancer (CRC) that expresses the epidermal growth factor receptor (EGFR). Proc Am Soc Clin Oncol. 2002;21.

325. Laethem J-LV, Raoul J-L, Mitry E, et al. Cetuximab (C225) in combination with bi-weekly irinotecan (CPT-11), infusional 5-fluorouracil (5-FU) and folinic acid (FA) in patients (pts) with metastatic colorectal cancer (CRC) expressing the epidermal growth factor receptor (EGFR). Preliminary safety and efficacy results. Proc Am Soc Clin Oncol. 2003;22.

326. Folprecht G Lutz MP Schoffski P Cetuximab and irinotecan/5-fluorouracil/folinic acid is a safe combination for the first-line treatment of patients with epidermal growth factor receptor expressing metastatic colorectal carcinoma Ann Oncol 2006 17 450 6 10.1093/annonc/mdj084 16303861

327. Saltz LB Meropol NJ Loehrer PJ Phase II trial of cetuximab in patients with refractory colorectal cancer that expresses the epidermal growth factor receptor J Clin Oncol 2004 22 1201 8 10.1200/JCO.2004.10.182 14993230

328. Lenz HJ Mayer RJ Mirtsching B Consistent response to treatment with cetuximab monotherapy in patients with metastatic colorectal cancer J Clin Oncol 2005 23 3536 10.1200/JCO.2005.09.100 15908664

329. Cunningham D Humblet Y Siena S Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer N Engl J Med 2004 351 337 45 10.1056/NEJMoa033025 15269313

330. Bokemeyer C Bondarenko I Makhson A Fluorouracil, leucovorin, and oxaliplatin with and without cetuximab in the first-line treatment of metastatic colorectal cancer J Clin Oncol 2009 27 663 71 10.1200/JCO.2008.20.8397 19114683

331. Scott J, Dakhil S, Cosgriff T, et al. Cetuximab + FOLFOX 6 as first line therapy for metastatic colorectal cancer. J Clin Oncol. 2005;23.

332. Dakhil S, Cosgriff T, Headley D, Boccia RV, Badarinath S. Cetuximab + FOLFOX6 as first line therapy for metastatic colorectal cancer (An International Oncology Network study, I-03-002). J Clin Oncol. 2006;24:3557.

333. Tabernero J Van Cutsem E Diaz-Rubio E Phase II trial of cetuximab in combination with fluorouracil, leucovorin, and oxaliplatin in the first-line treatment of metastatic colorectal cancer J Clin Oncol 2007 25 5225 32 10.1200/JCO.2007.13.2183 18024868

334. Tabernero JM, Cutsem EV, Sastre J, et al. An international phase II study of cetuximab in combination with oxaliplatin/5-fluorouracil (5-FU)/folinic acid (FA) (FOLFOX-4) in the first-line treatment of patients with metastatic colorectal cancer (CRC) expressing Epidermal Growth Factor Receptor (EGFR). J Clin Oncol. 2004;22.

335. Rubio ED, Tabernero J, Cutsem EV, et al. Cetuximab in combination with oxaliplatin/5-fluorouracil (5-FU)/folinic acid (FA) (FOLFOX-4) in the first-line treatment of patients with epidermal growth factor receptor (EGFR)-expressing metastatic colorectal cancer: an international phase II study. J Clin Oncol. 2005;23:3535.

336. Chung KY Shia J Kemeny NE Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry J Clin Oncol 2005 23 1803 10 10.1200/JCO.2005.08.037 15677699

337. Lievre A Bachet J-B Le Corre D KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer Cancer Res 2006 66 3992 5 10.1158/0008-5472.CAN-06-0191 16618717

338. Karapetis CS Khambata-Ford S Jonker DJ K-ras mutations and benefit from cetuximab in advanced colorectal cancer N Engl J Med 2008 359 1757 65 10.1056/NEJMoa0804385 18946061

339. Van Cutsem E Kohne C-H Hitre E Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer N Engl J Med 2009 360 1408 17 10.1056/NEJMoa0805019 19339720

340. Mack GS FDA holds court on post hoc data linking KRAS status to drug response Nat Biotech 2009 27 110 2 10.1038/nbt0209-110c 19204679

341. Kim KJ Li B Houck K Winer J Ferrara N The vascular endothelial growth factor proteins: identification of biologically relevant regions by neutralizing monoclonal antibodies Growth Factors 1992 7 53 64 10.3109/08977199209023937 1380254

342. Kim KJ Li B Winer J Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo Nature 1993 362 841 4 10.1038/362841a0 7683111

343. Gerber H-P Kowalski J Sherman D Eberhard DA Ferrara N Complete inhibition of rhabdomyosarcoma xenograft growth and neovascularization requires blockade of both tumor and host vascular endothelial growth factor Cancer Res 2000 60 6253 8 11103779

344. Holash J Davis S Papadopoulos N VEGF-trap: a VEGF blocker with potent antitumor effects Proc Natl Acad Sci U S A 2002 99 11393 8 10.1073/pnas.172398299 12177445 PMC123267

345. Presta LG Chen H O'Connor SJ Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders Cancer Res 1997 57 4593 9 9377574

346. Muller YA Chen Y Christinger HW VEGF and the Fab fragment of a humanized neutralizing antibody: crystal structure of the complex at 2.4 Å resolution and mutational analysis of the interface Structure 1998 6 1153 67 10.1016/S0969-2126(98)00116-6 9753694

347. Ryan AM Eppler DB Hagler KE Preclinical safety evaluation of rhuMAbVEGF, an antiangiogenic humanized monoclonal antibody Toxicol Pathol 1999 27 78 86 10.1177/019262339902700115 10367678

348. Margolin K Gordon MS Holmgren E Phase Ib trial of intravenous recombinant humanized monoclonal antibody to vascular endothelial growth factor in combination with chemotherapy in patients with advanced cancer: pharmacologic and long-term safety data J Clin Oncol 2001 19 851 6 11157039 10.1200/JCO.2001.19.3.851

349. Gordon MS Margolin K Talpaz M Phase I safety and pharmacokinetic study of recombinant human anti-vascular endothelial growth factor in patients with advanced cancer J Clin Oncol 2001 19 843 50 11157038 10.1200/JCO.2001.19.3.843

350. Reese DM Fratesi P Corry M A phase II trial of humanized anti-vascular endothelial growth factor antibody for the treatment of androgen-independent prostate cancer Prostate J 2001 3 65 70 10.1046/j.1525-1411.2001.32007.x

351. Cobleigh MA Langmuir VK Sledge GW A phase I/II dose-escalation trial of bevacizumab in previously treated metastatic breast cancer Semin Oncol 2003 30 117 24 10.1053/j.seminoncol.2003.08.013 14613032

352. Yang JC Haworth L Sherry RM A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer N Engl J Med 2003 349 427 34 10.1056/NEJMoa021491 12890841 PMC2275324

353. Kabbinavar F Hurwitz HI Fehrenbacher L Phase II, randomized trial comparing bevacizumab plus fluorouracil (FU)/leucovorin (LV) with FU/LV alone in patients with metastatic colorectal cancer J Clin Oncol 2003 21 60 5 10.1200/JCO.2003.10.066 12506171

354. DeVore R, Fehrenbacher L, Herbst R, et al. A randomized phase II trial comparing Rhumab VEGF (recombinant humanized monoclonal antibody to vascular endothelial cell growth factor) plus carboplatin/paclitaxel (CP) to CP alone in patients with stage IIIB/IV NSCLC. Proc Am Soc Clin Oncol. 2000;19.

355. Hurwitz H Fehrenbacher L Novotny W Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer N Engl J Med 2004 350 2335 42 10.1056/NEJMoa032691 15175435

356. Sandler A Gray R Perry MC Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer N Engl J Med 2006 355 2542 50 10.1056/NEJMoa061884 17167137

357. Nalluri SR Chu D Keresztes R Zhu X Wu S Risk of venous thromboembolism with the angiogenesis inhibitor bevacizumab in cancer patients JAMA 2008 300 2277 85 10.1001/jama.2008.656 19017914

358. Minor DR Risk of venous thromboembolism with bevacizumab in cancer patients JAMA 2009 301 1434 10.1001/jama.2009.439 19351937

359. Kilickap S Arslan C Risk of venous thromboembolism with bevacizumab in cancer patients JAMA 2009 301 1435 10.1001/jama.2009.441 19351938

360. Cortes J Saura C Atzori F Risk of venous thromboembolism with bevacizumab in cancer patients JAMA 2009 301 1434 5 10.1001/jama.2009.440 19351936

361. Chu D Wu S Risk of venous thromboembolism with bevacizumab in cancer patients-reply JAMA 2009 301 1435 6 10.1001/jama.2009.442 19351938

362. Hapania S Shera A Chub D Wu S Increased risk of serious hemorrhage with bevacizumab in cancer patients: a meta-analysis Oncology 2010 79 27 38 10.1159/000314980 21051914

363. Miller K Wang M Gralow J Paclitaxel plus bevacizumab versus paclitaxel alone for metastatic breast cancer N Engl J Med 2007 357 2666 76 10.1056/NEJMoa072113 18160686

364. Fojo T Grady C How much is life worth: cetuximab, non-small cell lung cancer, and the $440 billion question J Natl Cancer Inst 2009 101 1044 8 10.1093/jnci/djp177 19564563 PMC2724853

365. Koebel CM Vermi W Swann JB Adaptive immunity maintains occult cancer in an equilibrium state Nature 2007 450 903 7 10.1038/nature06309 18026089

366. Suzanne O-R Immune surveillance: a balance between protumor and antitumor immunity Curr Opin Genet Dev 2008 18 11 8 10.1016/j.gde.2007.12.007 18308558 PMC2699403

367. Read S Malmström V Powrie F Cytotoxic T lymphocyte‚associated antigen 4 plays an essential role in the function of Cd25 + Cd4+ regulatory cells that control intestinal inflammation J Exp Med 2000 192 295 302 10.1084/jem.192.2.295 10899916 PMC2193261

368. Leach DR Krummel MF Allison JP Enhancement of antitumor immunity by CTLA-4 blockade Science 1996 271 1734 6 10.1126/science.271.5256.1734 8596936

369. Keler T Halk E Vitale L Activity and safety of CTLA-4 blockade combined with vaccines in cynomolgus macaques J Immunol 2003 171 6251 9 10.4049/jimmunol.171.11.6251 14634142

370. Hodi FS Mihm MC Soiffer RJ Biologic activity of cytotoxic T lymphocyte-associated antigen 4 antibody blockade in previously vaccinated metastatic melanoma and ovarian carcinoma patients Proc Natl Acad Sci 2003 100 4712 7 10.1073/pnas.0830997100 12682289 PMC153621

371. Phan GQ Yang JC Sherry RM Cancer regression and autoimmunity induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma Proc Natl Acad Sci 2003 100 8372 7 10.1073/pnas.1533209100 12826605 PMC166236

372. Attia P Phan GQ Maker AV Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 2005 23 6043 53 10.1200/JCO.2005.06.205 16087944 PMC1473965

373. Beck KE Blansfield JA Tran KQ Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyten associated antigen 4 J Clin Oncol 2006 24 2283 9 10.1200/JCO.2005.04.5716 16710025 PMC2140223

374. Yang JC Hughes M Kammula U Royal R Sherry RM Topalian SL Suri KB Levy C Allen T Mavroukakis S Lowy I White DE Rosenberg SA Ipilimumab (anti-CTLA4 antibody) causes regression of metastatic renal cell cancer associated with enteritis and hypophysitis J Immunother 2007 30 825 30 10.1097/CJI.0b013e318156e47e 18049334 PMC2134980

375. Wolchok JD Neyns B Linette G Ipilimumab monotherapy in patients with pretreated advanced melanoma: a randomised, double-blind, multicentre, phase 2, dose-ranging study Lancet Oncol 2010 11 155 64 10.1016/S1470-2045(09)70334-1 20004617

376. Hersh E O’Day S Powderly J A phase II multicenter study of ipilimumab with or without dacarbazine in chemotherapy-naïve patients with advanced melanoma Invest New Drugs 2011 29 489 98 10.1007/s10637-009-9376-8 20082117

377. Robert C Thomas L Bondarenko I Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 2011 364 2517 26 10.1056/NEJMoa1104621 21639810

378. Falschlehner C Schaefer U Walczak H Following TRAIL’s path in the immune system Immunology 2009 127 145 54 10.1111/j.1365-2567.2009.03058.x 19476510 PMC2691779

379. Jin Z El-Deiry WS Overview of cell death signaling pathways Cancer Biol Ther 2005 4 139 63 10.4161/cbt.4.2.1508 15725726

380. Ricci MS Kim S-H Ogi K Reduction of TRAIL-induced Mcl-1 and cIAP2 by c-Myc or sorafenib sensitizes resistant human cancer cells to TRAIL-induced death Cancer Cell 2007 12 66 80 10.1016/j.ccr.2007.05.006 17613437

381. Abdulghani J El-Deiry WS TRAIL receptor signaling and therapeutics Expert Opin Ther Targets 2010 14 1091 108 10.1517/14728222.2010.519701 20819019

382. Plummer R Attard G Pacey S Phase 1 and pharmacokinetic study of lexatumumab in patients with advanced cancers Clin Cancer Res 2007 13 6187 94 10.1158/1078-0432.CCR-07-0950 17947486

383. Wakelee HA Patnaik A Sikic BI Phase I and pharmacokinetic study of lexatumumab (HGS-ETR2) given every 2 weeks in patients with advanced solid tumors Ann Oncol 2010 21 376 81 10.1093/annonc/mdp292 19633048 PMC2813303

384. Marini P Denzinger S Schiller D Combined treatment of colorectal tumours with agonistic TRAIL receptor antibodies HGS-ETR1 and HGS-ETR2 and radiotherapy: enhanced effects in vitro and dose-dependent growth delay in vivo Oncogene 2006 25 5145 54 16636678 10.1038/sj.onc.1209516

385. Gong J Yang D Kohanim S Novel in vivo imaging shows up-regulation of death receptors by paclitaxel and correlates with enhanced antitumor effects of receptor agonist antibodies Mol Cancer Ther 2006 5 2991 3000 10.1158/1535-7163.MCT-06-0188 17148761

386. Saulle E Petronelli A Pasquini L Proteasome inhibitors sensitize ovarian cancer cells to TRAIL induced apoptosis Apoptosis 2007 12 635 55 10.1007/s10495-006-0025-9 17252198

387. Smith MR Jin F Joshi I Bortezomib sensitizes non-Hodgkin’s lymphoma cells to apoptosis induced by antibodies to tumor necrosis factor‚ ÄìRelated apoptosis-inducing ligand (TRAIL) receptors TRAIL-R1 and TRAIL-R2 Clin Cancer Res 2007 13 5528s 34 10.1158/1078-0432.CCR-07-0982 17875785

388. Luster TA Carrell JA McCormick K Sun D Humphreys R Mapatumumab and lexatumumab induce apoptosis in TRAIL-R1 and TRAIL-R2 antibody-resistant NSCLC cell lines when treated in combination with bortezomib Mol Cancer Ther 2009 8 292 302 10.1158/1535-7163.MCT-08-0918 19174554

389. Nawrocki ST Carew JS Douglas L Histone deacetylase inhibitors enhance lexatumumab-induced apoptosis via a p21Cip1-dependent decrease in survivin levels Cancer Res 2007 67 6987 94 10.1158/0008-5472.CAN-07-0812 17638911

390. Wu X-X Jin X-H Zeng Y El Hamed AMA Kakehi Y Low concentrations of doxorubicin sensitizes human solid cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor (R) 2-mediated apoptosis by inducing TRAIL-R2 expression Cancer Sci 2007 98 1969 76 10.1111/j.1349-7006.2007.00632.x 17922852 PMC11160035

391. Wu X-X Kakehi Y Enhancement of lexatumumab-induced apoptosis in human solid cancer cells by cisplatin in caspase-dependent manner Clin Cancer Res 2009 15 2039 47 10.1158/1078-0432.CCR-08-2667 19276256

392. Sikic BI Wakelee HA von Mehren M Lewis N Calvert AH Plummer ER Fox NL Howard T Jones SF Burris HA A phase Ib study to assess the safety of lexatumumab, a human monoclonal antibody that activates TRAIL-R2, in combination with gemcitabine, pemetrexed, doxorubicin or FOLFIRI J Clin Oncol 2007 25 14006