key: cord-0035365-vt9onpb4
authors: Allen, Joshua E.; El-Deiry, Wafik S.
title: Targeting Oncoproteins for Molecular Cancer Therapy
date: 2016-11-13
journal: The Molecular Basis of Human Cancer
DOI: 10.1007/978-1-59745-458-2_38
sha: 4c3b1a325d8894fee4142e09e49f572377d6cddb
doc_id: 35365
cord_uid: vt9onpb4

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. 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. 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 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. 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 that together represent distinct therapeutic opportunities to target molecules that give rise to these defining properties of cancer.

Cancer and efforts to treat cancer are described in Ancient Egyptian documents dating back to 1600B.C. The fi rst successful cancer treatments were arsenic-based therapies for leukemias, with the fi rst reported application to cancer in the nineteenth century [ 1 ] . However, nitrogen mustards are often accredited as the fi rst 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 redefi ned 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-suffi ciency in growth factor signaling, unresponsiveness to anti-growth factor signaling, genomic instability and mutation, deregulating cellular energetics, evading immune-mediated destruction, oncogenic infl ammation, and invasiveness and metastasis [ 3 ] . The iden-tifi cation 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 defi ning properties of cancer ( Fig. 38.1 ). 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. Superfi cially, the genes capable of inducing carcinogenesis are divided into two categories. The fi rst 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 modifi cations that alter genomic structure, alternative splicing, interference with the translation and transcription of the gene, chaperone-mediated protein folding, protein degradation processes, and posttranslational modifi cations 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 fi rst-line cancer therapy today. While chemotherapy has yielded enormous patient benefi t it is often accompanied by side effects that limit dose and therefore effi cacy. 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 fi nite number of times, which is called the Hayfl ick 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 indefi nitely. 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-specifi c 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 exemplifi ed 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 ).

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 fi nd 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.

Chronic myelogenous leukemia (CML) is a cancer that causes increased amounts of white blood cells and is almost always associated with a specifi c 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 identifi ed 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 suffi cient to induce leukemia in mice. As a poster-child for translating modern cancer knowledge to clinical benefi t, imatinib has signifi cantly improved CML patient response rates. The FDA approved imatinib in 2001 for the treatment of CML as the fi rst cancer therapy to target an intracellular molecule. Imatinib is a direct result of medicinal chemistry performed on a molecule identifi ed as a protein kinase C (PKC) inhibitor [ 4 ] . Chemical modifi cation of the PKC inhibitor altered the specifi city 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 specifi c 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 benefi t 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 identifi cation 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 structureactivity relationships (SARs).

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 specifi c 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 specifi city of a cancer drug for a molecular target is a virtue rooted in our movement toward targeted therapy. We know that without specifi city, effects on normal cells can yield side effects that are deleterious. However the distinction should be made that while cancer therapies should target cancer-specifi c 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 fi t 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 profi ling safety of the drug would reduce cost and expedite evaluation and patient benefi t.

Therapeutic resistance is frequent in cancer. Cancer is defi ned 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 fi delity, 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 suffi cient 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 signifi cant 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 fi rst route, cell culture and patient data revealed that overexpression of Bcr-Abl by gene duplication occurred in refractory patients and that this was suffi cient 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 effl ux 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 specifi city 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 signifi cantly improved patient outcome after imatinib failure [ 20 -29 ] . Treating patients that are resistant to fi rst-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 fi rst-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.

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.

Gefi tinib is an orally active EGFR inhibitor identifi ed by Astra Zeneca and fi rst reported in 1996. A quinazoline derivative was found to be an ATP-competitive inhibitor and highly specifi c 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 gefi tinib had cooperative to synergistic combinations with several chemotherapies in EGFR-overexpressing cancer cell lines [ 32 ] . Interestingly, another group reported similar effects with gefi tinibchemotherapy combinations but in cancer cell lines with low EGFR expression [ 33 ] . Oral and intravenous administration of gefi tinib 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 gefi tinib 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 fi rst phase I trial of gefi tinib 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 doselimiting 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 identifi cation techniques are often employed in clinical trials involving small molecules. HPLC allows for the separation and quantifi cation 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 identifi cation 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 verifi cation that the molecule identifi ed 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 fi ngerprint 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 modifi cations of drugs often occur once delivered due to the staggeringly diverse mixture of molecules presence in the blood, digestive system, etc. As for gefi tinib, HPLC-MS identifi ed desmethyl-gefi tinib as a metabolite of gefi tinib that is inactive in vitro and in vivo [ 43 ] . Identifi cation 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 gefi tinib again yielded some patient benefi t in NSCLC along with low toxicity [ 44 , 45 ] . With data available from phase III clinical trials, the FDA granted accelerated approval for gefi tinib as a third-line therapy in NSCLC in 2003. This type of approval is granted based on promising clinical evidence of effi cacy 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 gefi tinib, several studies found responses in the overall population of NSCLC patients [ 44 -46 ] . However, a small subset of responders was identifi ed amongst these trials with the following characteristics: female, Asian, never-smokers, adenocarcinoma, and mutant EGFR [ 46 -48 ] . Several other studies confi rmed effi cacy of gefi tinib in NSCLC patients with EGFR mutations [ 49 -52 ] though no benefi t was found in NSCLC patients with EGFR gene amplifi cation [ 53 ] . A breakthrough study published in 2004 found that the majority of gefi tinib responders had EGFR deletions or point mutations clustered at the ATPbinding site of EGFR, which results in a ten-fold increase in sensitivity to gefi tinib [ 54 ] . Others studies corroborated these EGFR alterations in gefi tinib-responsive patients [ 55 , 56 ] . These genetic alterations were structurally modeled and rationalized using the crystal structure of the human EGFR kinase domain bound to gefi tinib ( 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 affi nity from ATP toward gefitinib [ 58 ] .

One study mandated by the accelerated FDA approval of gefi tinib and another phase II clinical trial found no benefi t with gefi tinib in NSCLC refractory to fi rst-line or second-line therapies. This led the FDA to restrict its usage to patients who have previously benefi ted or are currently benefi ting from gefi tinib in 2005. Subsequent clinical trials have supported gefi tinib following chemotherapy resistance [ 59 , 60 ] . Together, the clinical trials comparing gefi tinib and chemotherapy are confusing as they conclude signifi cant to no benefi t of gefitinib over chemotherapy. These confl icting 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 gefi tinib as a fi rst-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 gefi tinib to its use as a fi rst-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.

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 signifi cant clinical effi cacy in cancer including the EGFR-targeted therapies gefi tinib, 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, specifi city, 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 signifi cantly increased bioavailability of the drug, highlighting yet another clinical variable impacting therapeutic response [ 86 -88 ] . EGFRoverexpressing and Her2-overexpressing metastatic carcinoma patients receiving lapatinib showed signifi cant clinical responses and tolerated oral daily doses up to 1600 mg [ 89 ] . Lapatinib was also safe and effective as a fi rst-line monotherapy in Her2-amplifi ed advanced or metastatic breast cancer [ 90 ] . The agent has shown preliminary effi cacy 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 effi cacy. Based on the clinically active combination of trastuzumab with capecitabine, studies with this combination were conducted on Her2overexpressing advanced or metastatic breast cancer patients [ 97 , 98 ] . The study found that the time to progression doubled when lapatinib was added to capecitabine without signifi cant additional toxicity. Effi cacy 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 + /Her2infl ammatory breast cancer patients [ 100 ] . This suggests that Her2 inhibition is a key mediator of antitumor effi cacy 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 effi cacy when combined with TRAIL and TRAILreceptor antibodies that bind to these receptors [ 101 ] . This rationalizes the clinical exploration of higher doses of lapatinib to gain increased effi cacy 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 fi rst-line therapy yielded a signifi cant 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 effi cacy of combination with weekly paclitaxel [ 104 ] . In agreement with lapatinib and trastuzumab combinatorial preclinical data, PFS was signifi cantly 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.

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 fi rst 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 scientifi c literature. Over a decade later, a highprofi le 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 signifi cant conformational shift itself (Fig. 38.5 ) [ 118 ] .

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 identifi ed 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 specifi city 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 fl uorouracil. This observation caught the eye of the National Cancer Institute (NCI). NCI experiments and an independent report found rapamycin to have antitumor activity compara-ble 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 profi le [ 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 fi rst rapalogues and is a water soluble, chemically stable derivative of rapamycin (also called sirolimus) developed by Wyeth Pharmaceuticals. Preclinical studies found temsirolimus to have PTENdependent 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 pharmacody- namic 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 fi rst 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 benefi t [ 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 effi cacy in Hodgkin's lymphoma [ 178 ] , metastatic gastric cancer [ 179 ] , refractory NSCLC in combination with docetaxel [ 180 ] or gefi tinib, 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 profi les 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 effi cacy. This is a diffi culty inherent in the drug development process as clinical trial design dictates combinations with approved therapies. Combined with confl icting private interests, discerning the effi cacy 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.

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 modifi cations were explored and ultimately yielded sorafenib with a Raf1 IC 50 of 6 nM (Fig. 38.6 ) [ 202 ] .

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), fi broblast 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 trifl uoromethyl 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 affi nities. While the biophysical details of ligand binding are beyond the scope of this chapter, the affi nity 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 refl ect the motions of the molecule. Therefore crystal structures cannot provide direct entropic information themselves and by extension do not fully represent binding affi nity.

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 profi les [ 209 -214 ] . Renal cell carcinoma (RCC) is particularly resistant to the majority of chemotherapies and until 1997 was treated with interferon that causes signifi cant toxicities and limited responses. Due to the poor clinical outcomes with standard of care therapies, preclinical effi cacy 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 benefi ts regardless of previous cytokine therapy [ 218 ] and a follow-up >1 year from treatment initiation found sustained effi cacy and a well-tolerated toxicity profi le [ 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 effi cacy 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 diffi cult to fi nd. 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 benefi t 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 effi cacy of sorafenib has been seen as a neoadjuvant in advanced RCC [ 227 ] , as a monotherapy [ 228 ] or with erlotinib [ 229 ] or gefi tinib [ 230 ] in NSCLC, metastatic RCC with gemcitabine and capectibaine [ 231 ] . Sorafenib has had limited to no clinical effi cacy 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 ] .

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 amplifi cation 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 ] .

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 specifi c for a protein target associated with the biological material known as the hypervariable region (Fv), which allows for a high degree of diversity and specifi city. 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 specifi c 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 specifi city. Oncogene pathways mediated by soluble or cell-surface proteins naturally lend themselves as cancer therapeutic targets within the reach of antibodies.

CD20 is a B-cell-specifi c 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 affi nity 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 antigenexpressing cell. This process is known as complimentdependent cytotoxicity (CDC). CDC appears to be a key aspect of the antitumor activity of rituximab as rituximabresistant patient samples were associated with CD59, which negatively regulates this process [ 263 , 264 ] . Restoration of compliment has been shown to reverse resistance in smallscale patient studies [ 265 , 266 ] though this has been challenged in preclinical models [ 267 , 268 ] and follicular NHL [ 269 ] .

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-specifi c [ 270 ] . FcγR bound to IgG present on the surface of a cell can result in phagocytosis by 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. effector cells but these events are determined by the affi nity 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 affi nity 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 signifi cantly 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 effi cacy 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 signifi cant conclusion [ 290 ] . Other studies corroborated the signifi cant monotherapy effi cacy of rituximab in follicular lymphoma [ 291 , 292 ] . Adding rituximab to chemotherapy yielded an additive benefi t 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 fi rst 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 signifi cant increase in OS at a 2 year followup with the addition of rituximab to CHOP or other anthracycline-based chemotherapies. Analysis of biopsies from E4494 patients found p21 expression as a rituximab-specifi c, independent predictor of clinical outcome [ 297 ] .

NHL patients who previously received at least a single 4-week course of rituximab therapy had equivalent effi cacy and toxicity after an additional course with a median internal of 14.5 months [ 298 ] . The combination of rituximab and interferon yielded no signifi cant additional benefi t in the short term in follicular NHL [ 299 ] . Today, rituximab is also approved as a fi rst-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 fl udarabine 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 signifi cant neurological toxicity [ 312 ] . Also targeting CD20, the monoclonal antibody ocrelizumab was approved in 2009 for CLL refractory to fl udarabine 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.

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 FDAapproved for the fi rst-line treatment of metastatic colorectal cancer in combination with irinotecan in 2004, making it the fi rst 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 fi rst clinical trial of mab 225. This trial found mab 225 to be safe in squamous cell lung carcinoma patients and profi led 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 specifi c 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 fi rst-line therapy consisting of leucovorin, 5-fl uorouracil (5-FU), and irinotecan [ 323 ] . Combining cetuximab with this treatment regimen also proved effective in a fi rst-line therapy setting [ 324 -326 ] and moderate effi cacy as a monotherapy [ 327 -330 ] . Safety and effi cacy 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 specifi city 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 EGFRcolorectal cancer patients [ 336 ] . This unexpected fi nding underscores the complex nature of therapeutic responses in the clinic and the principle that while rational design has well-evidenced benefi ts, 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 effi cacy 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 benefi t 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 signifi cance. Redesigning the clinical trial to include patients that harbor this response-determining characteristic may now show a strong therapeutic benefi t otherwise concluded as ineffective. The obvious challenge in patient stratifi cation in clinical trial design is determining what characteristic(s) confer therapeutic response. Clinical trials are sometimes analyzed retrospectively to fi nd such biomarkers. Some of biomarkers have even been added as stipulations for FDAapproved 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 signifi cantly 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.

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 fi rst 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 fi ndings 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 infl uence 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 sitedirected mutagenesis of the variable region of a human antibody to that of the murine A4.6.1 while maintaining a similar binding affi nity for VEGF [ 345 ] . This humanized version of A4.6.1, known as bevacizumab, is specifi c 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-specifi c fragment of bevacizumab bound to VEGF identifi ed a critical residue for binding and specifi city (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 fi rst-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 ] .

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 signifi cant improvement in OS, bevacizumab in combination with carboplatin and paclitaxel was approved for fi rst-line therapy in unresectable, locally advanced, recurrent, or metastatic NSCLC. Bevacizumab has now been approved for Her2negative 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 signifi cant 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 lifethreatening 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 diffi cult questions. Will the therapy provide a net benefi t 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 benefi t is defi ned. Obviously, most patients want to live longer but quality of life is also a consideration, which raises yet another patient-specifi c question: what is quality of life? Clinically, fewer adverse events and prolonged PFS and OS are quantifi able parameters that may act as a partial surrogate defi nition but this consideration is more complex. For bevacizumab, even this incomplete surrogate defi nition 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 diffi cult 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 signifi cantly extends PFS to varying degrees in several solid cancers, may shrink tumors to allow for resection, does not increase OS, and signifi cantly 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 benefi ts 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 infl uence. 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 diffi cult 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 benefi t? How do you weigh concomitant risks and benefi ts?

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 specifi city 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 antigenpresenting 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 infi ltration 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 singlenucleotides 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 effi cacy 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 fi rst trial to ever show survival benefi t in metastatic melanoma. Another phase III study measured the effi cacy of adding ipilimumab (10 mg/kg) to dacarbazine as a fi rst-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 fi rst-in-class cancer therapy that reactivates the adaptive immune system to restore antitumor immunity and provides unprecedented patient benefi t in melanoma. The clinical effi cacy and toxicity of ipilimumab highlights the potential benefi ts 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 effi cacy 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.

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 nor-mal cells [ 378 ] . TRAIL binds four transmembrane receptors in humans at similar affi nities 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 TRAILmediated apoptosis. The two decoy receptors compete for TRAIL binding with the death receptors at similar binding affi nities.

TRAIL-induced trimerization of the death receptors colocalizes their intracellular death domains, which recruit Fasassociated 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 effi cacy 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 DR4agonist mapatumumab are also currently in clinical trials.

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 benefi ted 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 diffi cult 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 benefi t 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 identifi ed, future failures may be avoided, and other successful treatment options can be discovered. 

Zwei Falle von Leukamie

Hallmarks of cancer: the next generation

The hallmarks of cancer

Phenylamino-pyrimidine (PAP) derivatives: a new class of potent and selective inhibitors of protein kinase C (PKC)

Potent and selective inhibitors of the Abl-kinase: phenylamino-pyrimidine (PAP) derivatives

Phenylaminopyrimidine (PAP)-derivatives: a new class of potent and highly selective PDGF-receptor autophosphorylation inhibitors

Structural mechanism for STI-571 inhibition of Abelson tyrosine kinase

STI571: a new treatment modality for CML? In: Anticancer agents

ARG tyrosine kinase activity is inhibited by STI571

Estimating the cost of new drug development: is it really $802 million?

Mechanism of resistance to the ABL tyrosine kinase inhibitor STI571 in BCR/ABL-transformed hematopoietic cell lines

Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplifi cation

Selection and characterization of BCR-ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor STI571: diverse mechanisms of resistance

Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplifi cation

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

A phase II study of the safety and effi cacy of the multidrug resistance inhibitor VX-710 combined with doxorubicin and vincristine in patients with recurrent small cell lung cancer

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

Phase II prospective study of the effi cacy of gefi tinib for the treatment of stage III/IV non-small cell lung cancer with EGFR mutations, irrespective of previous chemotherapy

First-line gefi tinib in patients with advanced non-small-cell lung cancer harboring somatic EGFR mutations

Multicentre prospective phase II trial of gefi tinib for advanced non-small cell lung cancer with epidermal growth factor receptor mutations: results of the West Japan Thoracic Oncology Group trial (WJTOG0403)

Epidermal growth factor receptor activating mutations in Spanish gefi tinib-treated nonsmall-cell lung cancer patients

Gefi tinib versus docetaxel in previously treated non-small-cell lung cancer (INTEREST): a randomised phase III trial

Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefi tinib

EGFR mutations in lung cancer: correlation with clinical response to gefi tinib therapy

Mutations of the epidermal growth factor receptor gene in lung cancer

Structure of the epidermal growth factor receptor kinase domain alone and in complex with a 4-anilinoquinazoline inhibitor

Kinetic analysis of epidermal growth factor receptor somatic mutant proteins shows increased sensitivity to the epidermal growth factor receptor tyrosine kinase inhibitor

Randomized phase III trial of gefi tinib versus docetaxel in non-small cell lung cancer patients who have previously received platinum-based chemotherapy

Randomized phase III trial of platinum-doublet chemotherapy followed by gefi tinib 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)

Reasons for response differences seen in the V15-32. INTEREST and IPASS trials

Gefi tinib or chemotherapy for non-small-cell lung cancer with mutated EGFR

Effectiveness of gefitinib (Iressa) as fi rst-line therapy for inoperable non-small-cell lung cancer with mutated EGFR gene (phase II study)

Gefi tinib 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

The characterization of novel, dual ErbB-2/EGFR, tyrosine kinase inhibitors

Indazolylamino quinazolines and pyridopyrimidines as inhibitors of the EGFr and c-erbB-2

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

Anti-tumor activity of GW572016: a dual tyrosine kinase inhibitor blocks EGF activation of EGFR/erbB2 and downstream Erk1/2 and AKT pathways

NH2-terminally truncated HER-2/neu protein: relationship with shedding of the extracellular domain and with prognostic factors in breast cancer

erbB-2 is a potent oncogene when overexpressed in NIH/3T3 cells

Oncogenic activation of the neuencoded receptor protein by point mutation and deletion

Different structural alterations upregulate in vitro tyrosine kinase activity and transforming potency of the erbB-2 gene

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

The c-erbB transmembrane growth factor receptors as serum biomarkers in human cancer studies

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

Circulating HER2 extracellular domain and resistance to chemotherapy in advanced breast cancer

Truncated ErbB2 receptor (p95ErbB2) is regulated by heregulin through heterodimer formation with ErbB3 yet remains sensitive to the dual EGFR//ErbB2 kinase inhibitor GW572016

Heregulin-induced activation of HER2 and HER3 increases androgen receptor transactivation and CWR-R1 human recurrent prostate cancer cell growth

Inhibition of HER-2/neu kinase impairs androgen receptor recruitment to the androgen responsive enhancer

Cooperates with tamoxifen to inhibit both cell proliferation-and estrogen-dependent gene expression in antiestrogen-resistant breast cancer

A model of acquired autoresistance to a potent ErbB2 tyrosine kinase inhibitor and a therapeutic strategy to prevent its onset in breast cancer

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

Activity of the dual kinase inhibitor lapatinib (GW572016) against HER-2-overexpressing and trastuzumab-treated breast cancer cells

Lapatinib induces apoptosis in trastuzumab-resistant breast cancer cells: effects on insulin-like growth factor I signaling

Phase I pharmacokinetic studies evaluating single and multiple doses of oral GW572016, a dual EGFR-ErbB2 inhibitor, in healthy subjects

Effect of food on lapatinib pharmacokinetics

The value meal: effect of food on lapatinib bioavailability

Controversies in using lapatinib at reduced dosage with food

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

Effi cacy and safety of lapatinib as fi rst-line therapy for ErbB2-amplifi ed locally advanced or metastatic breast cancer

Phase I study of lapatinib in combination with chemoradiation in patients with locally advanced squamous cell carcinoma of the head and neck

A phase I and pharmacokinetic study of oral lapatinib administered once or twice daily in patients with solid malignancies

Randomized phase II multicenter trial of two schedules of lapatinib as fi rst-or secondline monotherapy in patients with advanced or metastatic nonsmall cell lung cancer

A multicenter phase II clinical trial of lapatinib (GW572016) in hormonally untreated advanced prostate cancer

Phase I pharmacokinetic study of the safety and tolerability of lapatinib (GW572016) in combination with oxaliplatin/fl uorouracil/leucovorin (FOLFOX4) in patients with solid tumors

A phase I and pharmacokinetic study of lapatinib in combination with infusional 5-fl uorouracil, leucovorin and irinotecan

Lapatinib plus capecitabine for HER2-positive advanced breast cancer

Phase I and pharmacokinetic study of lapatinib in combination with capecitabine in patients with advanced solid malignancies

A phase III randomized comparison of lapatinib plus capecitabine versus capecitabine alone in women with advanced breast cancer that has progressed on trastuzumab: updated effi cacy and biomarker analyses

Phase II study of predictive biomarker profi les for response targeting human epidermal growth factor receptor 2 (HER-2) in advanced infl ammatory breast cancer with lapatinib monotherapy

Off-target lapatinib activity sensitizes colon cancer cells through TRAIL death receptor upregulation

Lapatinib combined with letrozole versus letrozole and placebo as fi rst-line therapy for postmenopausal hormone receptor-positive metastatic breast cancer

Phase III, double-blind. Randomized study comparing lapatinib plus paclitaxel with placebo plus paclitaxel as fi rst-line treatment for metastatic breast cancer

A single-arm phase II trial of fi rst-line paclitaxel in combination with lapatinib in HER2-overexpressing metastatic breast cancer

Randomized study of lapatinib alone or in combination with trastuzumab in women with ErbB2-positive. Trastuzumab-refractory metastatic breast cancer

Rapamycin (AY-22, 989), a new antifungal antibiotic

AY-22, 989), a new antifungal antibiotic. III. In vitro and in vivo evaluation

Inhibition of the immune response by rapamycin, a new antifungal antibiotic

Novel immunosuppressive agent, FK506. In vitro effects on the cloned T cell activation

The immunosuppressive macrolides FK-506 and rapamycin act as reciprocal antagonists in murine T cells

A receptor for the immuno-suppressant FK506 is a cis-trans peptidyl-prolyl isomerase

A cytosolic binding protein for the immunosuppressant FK506 has peptidylprolyl isomerase activity but is distinct from cyclophilin

Molecular cloning and overexpression of the human FK506-binding protein FKBP

Complementary DNA encoding the human T-cell FK506-binding protein, a peptidylprolyl cis-trans isomerase distinct from cyclophilin

Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FK506 or rapamycin

Solution structure of FKBP, a rotamase enzyme and receptor for FK506 and rapamycin

Atomic structure of FKBP-FK506, an immunophilinimmunosuppressant complex

Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin

Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression

RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs

RAPT1, a mammalian homolog of yeast Tor, interacts with the FKBP12/rapamycin complex

Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells

A mammalian protein targeted by G1-arresting rapamycin-receptor complex

Effects of rapamycin on cultured hepatocyte proliferation and gene expression

Rapamycininduced inhibition of the 70-kilodalton S6 protein kinase

Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast

A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells

Regulation of cellular growth by the Drosophila target of rapamycin dTOR

Genetic and biochemical characterization of dTOR, the Drosophila homolog of the target of rapamycin

Rapamycin-FKBP specifi cally blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases

Rapamycin selectively inhibits interleukin-2 activation of p70 S6 kinase

Rapamycin inhibits the phosphorylation of p70 S6 kinase in IL-2 and mitogen-activated human T cells

PHAS-I as a link between mitogen-activated protein kinase and translation initiation

cAMP-and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells

Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5′-cap function

The insulininduced signalling pathway leading to S6 and initiation factor 4E binding protein 1 phosphorylation bifurcates at a rapamycinsensitive point immediately upstream of p70s6k

AY-22, 989), a new antifungal antibiotic. IV. Mechanism of action

Activity of rapamycin (AY-22, 989) against transplanted tumors

Rapamycin, a potent immunosuppressive drug, causes programmed cell death in B lymphoma cells

Rapamycin inhibits constitutive p70s6k phosphorylation, cell proliferation, and colony formation in small cell lung cancer cells

Rapamycin causes poorly reversible inhibition of mTOR and induces p53-independent apoptosis in human rhabdomyosarcoma cells

Inhibition of the phosphatidylinositol 3-kinase/p70S6-kinase pathway induces B16 melanoma cell differentiation

Regulation of cell growth and cyclin D1 expression by the constitutively active FRAP-p70s6K pathway in human pancreatic cancer cells

Hypoxia enhances vascular cell proliferation and angiogenesis in vitro via rapamycin (mTOR) -dependent signaling

Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor

Rapamycin enhances apoptosis and increases sensitivity to cisplatin in vitro

Rapamycin: distribution, pharmacokinetics and therapeutic range investigations: an update

Pharmacokinetics of rapamycin

mTOR, a novel target in breast cancer: the effect of CCI-779, an mTOR inhibitor, in preclinical models of breast cancer

Antitumor activity of the rapamycin analog CCI-779 in human primitive neuroectodermal tumor/medulloblastoma models as single agent and in combination chemotherapy

The rapamycin analog CCI-779 is a potent inhibitor of pancreatic cancer cell proliferation

In vivo antitumor effects of the mTOR inhibitor CCI-779 against human multiple myeloma cells in a xenograft model

mTOR, a novel target in pancreatic cancer: the effect of CCI-779 in preclinical models

Effects of the mammalian target of rapamycin inhibitor CCI-779 used alone or with chemotherapy on human prostate cancer cells and xenografts

In vivo and in vitro effect of CCI-779 a rapamycin analogue on HNSCC

An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten+/+ mice

Biochemical correlates of mTOR inhibition by the rapamycin ester CCI-779 and tumor growth inhibition

Safety and pharmacokinetics of escalated doses of weekly intravenous infusion of CCI-779, a novel mTOR inhibitor, in patients with cancer

Pharmacodynamic evaluation of CCI-779, an inhibitor of mTOR, in cancer patients

Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma

Phase III study to evaluate temsirolimus compared with investigator's choice therapy for the treatment of relapsed or refractory mantle cell lymphoma

Phase I study combining treatment with temsirolimus and sunitinib malate in patients with advanced renal cell carcinoma

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

Can the combination of temsirolimus and bevacizumab improve the treatment of metastatic renal cell carcinoma (mRCC)? Results of the randomized TORAVA phase II trial

Phase II study of temsirolimus (CCI-779), a novel inhibitor of mTOR, in heavily pretreated patients with locally advanced or metastatic breast cancer

A phase I study of weekly temsirolimus and topotecan in the treatment of advanced and/or recurrent gynecologic malignancies

Phase II trial of temsirolimus in patients with relapsed or refractory multiple myeloma

Pharmacokinetic and tumor distribution characteristics of temsirolimus in patients with recurrent malignant glioma

Phase II study of CCI-779 in patients with recurrent glioblastoma multiforme

Phase II trial of temsirolimus (CCI-779) in recurrent glioblastoma multiforme: a North Central Cancer Treatment Group Study

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)

Phase I/II study of the mammalian target of rapamycin inhibitor everolimus (RAD001) in patients with relapsed or refractory hematologic malignancies

Phase I study of everolimus in pediatric patients with refractory solid tumors

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

Phase I pharmacokinetic and pharmacodynamic study of the oral mammalian target of rapamycin inhibitor everolimus in patients with advanced solid tumors

Effi cacy of everolimus in advanced renal cell carcinoma: a double-blind, randomised, placebo-controlled phase III trial

A pilot study of everolimus and gefi tinib in the treatment of recurrent glioblastoma

A phase II trial of the oral mTOR inhibitor everolimus in relapsed Hodgkin lymphoma

Multicenter phase II study of everolimus in patients with previously treated metastatic gastric cancer

Phase 1 and pharmacokinetic study of everolimus, a mammalian target of rapamycin inhibitor, in combination with docetaxel for recurrent/refractory nonsmall cell lung cancer

Randomized phase II study comparing two schedules of everolimus in patients with recurrent/metastatic breast cancer: NCIC Clinical Trials Group IND.163

The oral mTOR inhibitor RAD001 (everolimus) in combination with letrozole in patients with advanced breast cancer: Results of a phase I study with pharmacokinetics

Phase II randomized study of neoadjuvant everolimus plus letrozole compared with placebo plus letrozole in patients with estrogen receptor-positive breast cancer

Phase I study of everolimus plus weekly paclitaxel and trastuzumab in patients with metastatic breast cancer pretreated with trastuzumab

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

Daily oral everolimus activity in patients with metastatic pancreatic neuroendocrine tumors after failure of cytotoxic chemotherapy: a phase II trial

Everolimus for advanced pancreatic neuroendocrine tumors

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

A phase I trial to determine the safety, tolerability, and maximum tolerated dose of deforolimus in patients with advanced malignancies

Phase I trial of the novel mammalian target of rapamycin inhibitor deforolimus

MK-8669) administered intravenously daily for 5 days every 2 weeks to patients with advanced malignancies

Phase IB study of the mTOR inhibitor ridaforolimus with capecitabine

Phase Ib study of weekly mammalian target of rapamycin inhibitor ridaforolimus

MK-8669) with weekly paclitaxel

Inhibition of the mammalian target of rapamycin (mTOR) in advanced pancreatic cancer: results of two phase II studies

Raf-1 expression may infl uence progression to androgen insensitive prostate cancer

BRAF and RAS mutations in human lung cancer and melanoma

Over-expression of c-raf-1 proto-oncogene in liver cirrhosis and hepatocellular carcinoma

Association of c-Raf expression with survival and its targeting with antisense oligonucleotides in ovarian cancer

Effect of antisense c-raf-1 on tumorigenicity and radiation sensitivity of a human squamous carcinoma

The raf oncogene is associated with a radiation-resistant human laryngeal cancer

Discovery of heterocyclic ureas as a new class of raf kinase inhibitors: identifi cation of a second generation lead by a combinatorial chemistry approach

Discovery of a novel Raf kinase inhibitor

Design and discovery of small molecules targeting Raf-1 kinase

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

BAY 43-9006 inhibition of oncogenic RET mutants

Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF

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

Phase I safety and pharmacokinetics of BAY 43-9006 administered for 21 days on/7 days off in patients with advanced, refractory solid tumours

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

Results of a phase I trial of sorafenib (BAY 43-9006) in combination with oxaliplatin in patients with refractory solid tumors, including colorectal cancer

Phase I trial of sorafenib and gemcitabine in advanced solid tumors with an expanded cohort in advanced pancreatic cancer

Results of a phase I trial of sorafenib (BAY 43-9006) in combination with doxorubicin in patients with refractory solid tumors

Phase I study of BAY 43-9006 (sorafenib), a Raf kinase and VEGFR inhibitor, combined with irinotecan (CPT-11) in advanced solid tumors

Phase I trial of BAY 43-9006 (sorafenib) combined with dacarbazine (DTIC) in metastatic melanoma patients

Phase II trial of sorafenib plus interferon-alpha 2b (IFN-α2b) as fi rst-or second-line therapy in patients (pts) with metastatic renal cell cancer (RCC)

Phase II placebo-controlled randomized discontinuation trial of sorafenib in patients with metastatic renal cell carcinoma

Randomized phase II trial of the multi-kinase inhibitor sorafenib versus interferon (IFN) in treatment-naïve patients with metastatic renal cell carcinoma (mRCC)

Prognostic factors for survival in previously treated patients with metastatic renal cell carcinoma

Effi cacy and safety of sorafenib in patients with advanced renal cell carcinoma with and without prior cytokine therapy, a subanalysis of TARGET

Long-term safety of sorafenib in advanced renal cell carcinoma: follow-up of patients from phase III TARGET

Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5

Phase II study of sorafenib in patients with advanced hepatocellular carcinoma

Sorafenib in advanced hepatocellular carcinoma

A phase II trial of sorafenib in metastatic melanoma with tissue correlates

A phase I trial of the oral. multikinase inhibitor sorafenib in combination with carboplatin and paclitaxel

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

Doxorubicin plus sorafenib vs doxorubicin alone in patients with advanced hepatocellular carcinoma

Neoadjuvant clinical trial with sorafenib for patients with stage II or higher renal cell carcinoma

Phase II, multicenter, uncontrolled trial of single-agent sorafenib in patients with relapsed or refractory. advanced non-small-cell lung cancer

A multicenter phase II study of erlotinib and sorafenib in chemotherapy-naïve patients with advanced non-small cell lung cancer

Phase I trial of sorafenib in combination with gefi tinib in patients with refractory or recurrent non-small cell lung cancer

Activity of a multitargeted chemo-switch regimen (sorafenib, gemcitabine, and metronomic capecitabine) in metastatic renal-cell carcinoma: a phase 2 study (SOGUG-02-06)

A phase II study of sorafenib in malignant mesothelioma: results of Cancer and Leukemia Group B 30307

A phase II study of sorafenib in patients with chemo-naive castration-resistant prostate cancer

A phase II clinical trial of sorafenib in androgen-independent prostate cancer

A phase II study of sorafenib in advanced uterine carcinoma/carcinosarcoma: A trial of the Chicago, PMH, and California Phase II Consortia

Phase 2 trial of sorafenib in patients with advanced urothelial cancer

Phase II study of sorafenib in patients with metastatic or recurrent sarcomas

Phase II evaluation of sorafenib in advanced and metastatic squamous cell carcinoma of the head and neck: Southwest Oncology Group Study S0420

Phase III study of carboplatin and paclitaxel alone or with sorafenib in advanced nonsmall-cell lung cancer

Phase I clinical and pharmacokinetic study of sorafenib in combination with carboplatin and paclitaxel in patients with advanced non-small cell lung cancer

Sorafenib in combination with carboplatin and paclitaxel as neoadjuvant chemotherapy in patients with advanced ovarian cancer

Phase II study of sorafenib in patients with sunitinib-refractory metastatic renal cell cancer

Phase I/II, pharmacokinetic and pharmacodynamic trial of BAY 43-9006 alone in patients with metastatic melanoma

Gatekeeper mutations mediate resistance to BRAF-targeted therapies

Clinical effi cacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma

The RAF inhibitor PLX4032 inhibits ERK signaling and tumor cell proliferation in a V600E BRAF-selective manner

PLX4032, a potent inhibitor of the B-Raf V600E oncogene, selectively inhibits V600E-positive melanomas

Phase I study of PLX4032: proof of concept for V600E BRAF mutation as a therapeutic target in human cancer

Inhibition of mutated. Activated BRAF in metastatic melanoma

Improved survival with vemurafenib in melanoma with BRAF V600E mutation

Targeting the RAS pathway in melanoma

Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation

Hyperactivation of MEK-ERK1/2 signaling and resistance to apoptosis induced by the oncogenic B-RAF inhibitor, PLX4720, in mutant N-RAS melanoma cells

Combinatorial treatments that overcome PDGFROE≤-driven resistance of melanoma cells to V600EB-RAF inhibition

Combination therapy with vemurafenib (PLX4032/RG7204) and metformin in melanoma cell lines with distinct driver mutations

The oncogenic BRAF kinase inhibitor PLX4032/RG7204 does not affect the viability or function of human lymphocytes across a wide range of concentrations

The CSPG4-specifi c monoclonal antibody enhances and prolongs the effects of the BRAF inhibitor in melanoma cells

Continuous cultures of fused cells secreting antibody of predefi ned specifi city

Molecular cloning of the human B cell CD20 receptor predicts a hydrophobic protein with multiple transmembrane domains

Production of a mousehuman chimeric monoclonal antibody to CD20 with potent Fc-dependent biologic activity

Monoclonal antibody 1F5 (anti-CD20) serotherapy of human B cell lymphomas

Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20

Apoptotic-regulatory and complement-protecting protein expression in chronic lymphocytic leukemia: relationship to in vivo rituximab resistance

Tumor cell expression of CD59 is associated with resistance to CD20 serotherapy in patients with B-cell malignancies

Rituximab infusion promotes rapid complement depletion and acute CD20 loss in chronic lymphocytic leukemia

Addition of fresh frozen plasma as a source of complement to rituximab in advanced chronic lymphocytic leukaemia

Importance of cellular microenvironment and circulatory dynamics in B cell immunotherapy

Type II (tositumomab) anti-CD20 monoclonal antibody out performs type I (rituximab-like) reagents in B-cell depletion regardless of complement activation

Expression of complement inhibitors CD46, CD55, and CD59 on tumor cells does not predict clinical outcome after rituximab treatment in follicular non-Hodgkin lymphoma

Antibody isotype-specifi c engagement of Fcgamma receptors regulates B lymphocyte depletion during CD20 immunotherapy

IgG Fc receptors

Divergent immunoglobulin G subclass activity through selective Fc receptor binding

Fc[gamma] receptors: old friends and new family members

Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor Fcgamma RIIIa gene

Rituximab-dependent cytotoxicity by natural killer cells

CD16 polymorphisms and NK activation induced by monoclonal antibody-coated target cells

Ex vivoactivated human macrophages kill chronic lymphocytic leukemia cells in the presence of rituximab: mechanism of antibodydependent cellular cytotoxicity and impact of human serum

Antibodies reactive with the B1 molecule inhibit cell cycle progression but not activation of human B lymphocytes

The CD20 (Bp35) antigen is involved in activation of B cells from the G0 to the G1 phase of the cell cycle

Activation of human B cell proliferation through surface Bp35 (CD20) polypeptides or immunoglobulin receptors

Signaling events involved in anti-CD20-induced apoptosis of malignant human B cells

Clustered CD20 induced apoptosis: Src-family kinase, the proximal regulator of tyrosine phosphorylation, calcium infl ux, and caspase 3-dependent apoptosis

The chimeric anti-CD20 antibody rituximab induces apoptosis in B-cell chronic lymphocytic leukemia cells through a p38 mitogen activated protein-kinase-dependent mechanism

Binding to CD20 by anti-B1 antibody or F(ab′)2 is suffi cient for induction of apoptosis in B-cell lines

Feasibility and pharmacokinetic study of a chimeric anti-CD20 monoclonal antibody (IDEC-C2B8, rituximab) in relapsed B-cell lymphoma

IDEC-C2B8 (rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin's lymphoma

IDEC-C2B8: results of a phase I multiple-dose trial in patients with relapsed non-Hodgkin's lymphoma

Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program

Rituximab (anti-CD20 monoclonal antibody) for the treatment of patients with relapsing or refractory aggressive lymphoma: a multicenter phase II study

IDEC-C2B8 anti-CD20 (rituximab) immunotherapy in patients with low-grade non-Hodgkin's lymphoma and lymphoproliferative disorders: evaluation of response on 48 patients

IDEC-C2B8 (rituximab) anti-CD20 antibody treatment in relapsed advancedstage follicular lymphomas: results of a phase-II study of the German Low-Grade Lymphoma Study Group

Rituximab (anti-CD20 monoclonal antibody) as single fi rst-line therapy for patients with follicular lymphoma with a low tumor burden: clinical and molecular evaluation

Treatment of patients with low-grade B-cell lymphoma with the combination of chimeric anti-CD20 monoclonal antibody and CHOP chemotherapy

Phase II study of rituximab in combination with CHOP chemotherapy in patients with previously untreated. Aggressive Non-Hodgkin's Lymphoma

CHOP plus rituximab chemoimmunotherapy of indolent B-cell lymphoma

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

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

Rituximab anti-CD20 monoclonal antibody therapy in non-Hodgkin's lymphoma: safety and effi cacy of re-treatment

Combination immunotherapy of relapsed or refractory low-grade or follicular non-Hodgkin's lymphoma with rituximab and interferon-2a

Rituximab using A thrice weekly dosing schedule in B-cell chronic lymphocytic leukemia and small lymphocytic lymphoma demonstrates clinical activity and acceptable toxicity

Rituximab doseescalation trial in chronic lymphocytic leukemia

Early results of a chemoimmunotherapy regimen of fl udarabine, cyclophosphamide, and rituximab as initial therapy for chronic lymphocytic leukemia

Phase 2 study of a combined immunochemotherapy using rituximab and fl udarabine in patients with chronic lymphocytic leukemia

Addition of rituximab to fl udarabine 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

Chemoimmunotherapy with fl udarabine, cyclophosphamide, and rituximab for relapsed and refractory chronic lymphocytic leukemia

Long-term results of the fl udarabine, cyclophosphamide, and rituximab regimen as initial therapy of chronic lymphocytic leukemia

Rituximab plus fl udarabine and cyclophosphamide prolongs progression-free survival compared with fl udarabine and cyclophosphamide alone in previously treated chronic lymphocytic leukemia

Addition of rituximab to fl udarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: a randomised, open-label, phase 3 trial

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

Combination bortezomib and rituximab treatment affects multiple survival and death pathways to promote apoptosis in mantle cell lymphoma

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

Phase 2 trial of rituximab and bortezomib in patients with relapsed or refractory mantle cell and follicular lymphoma

Biological effects in vitro of monoclonal antibodies to human epidermal growth factor receptors

Growth inhibition of human tumor cells in athymic mice by anti-epidermal growth factor receptor monoclonal antibodies

Monoclonal antibody against epidermal growth factor receptor is internalized without stimulating receptor phosphorylation

Toxicity of ligand and antibody-directed ricin A-chain conjugates recognizing the epidermal growth factor receptor

Involvement of p27KIP1 in G1 arrest mediated by an anti-epidermal growth factor receptor monoclonal antibody

Anti-epidermal growth factor receptor monoclonal antibody 225 up-regulates p27KIP1 and induces G1 arrest in prostatic cancer cell line DU145

Anti-epidermal growth factor receptor monoclonal antibody 225 upregulates p27KIP1 and p15INK4B and induces G1 arrest in oral squamous carcinoma cell lines

The monoclonal antibody 225 activates caspase-8 and induces apoptosis through a tumor necrosis factor receptor familyindependent pathway

Imaging of human tumor xenografts with an indium-111-labeled anti-epidermal growth factor receptor monoclonal antibody

Phase I and imaging trial of indium 111-labeled anti-epidermal growth factor receptor monoclonal antibody 225 in patients with squamous cell lung carcinoma

IMC-C225) plus irinotecan (CPT-11) is active in CPT-11-refractory colorectal cancer (CRC) that expresses epidermal growth factor receptor (EGFR)

IMC-C225) plus weekly irinotecan (CPT-11), fl uorouracil (5FU) and leucovorin (LV) in colorectal cancer (CRC) that expresses the epidermal growth factor receptor (EGFR)

Cetuximab (C225) in combination with bi-weekly irinotecan (CPT-11), infusional 5-fl uorouracil (5-FU) and folinic acid (FA) in patients (pts) with metastatic colorectal cancer (CRC) expressing the epidermal growth factor receptor (EGFR). Preliminary safety and effi cacy results

Cetuximab and irinotecan/5-fl uorouracil/folinic acid is a safe combination for the fi rst-line treatment of patients with epidermal growth factor receptor expressing metastatic colorectal carcinoma

Phase II trial of cetuximab in patients with refractory colorectal cancer that expresses the epidermal growth factor receptor

Consistent response to treatment with cetuximab monotherapy in patients with metastatic colorectal cancer

Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer

leucovorin, and oxaliplatin with and without cetuximab in the fi rstline treatment of metastatic colorectal cancer

Cetuximab + FOLFOX 6 as fi rst line therapy for metastatic colorectal cancer

Cetuximab + FOLFOX6 as fi rst line therapy for metastatic colorectal cancer (An International Oncology Network study, I-03-002)

Phase II trial of cetuximab in combination with fl uorouracil, leucovorin, and oxaliplatin in the fi rst-line treatment of metastatic colorectal cancer

An international phase II study of cetuximab in combination with oxaliplatin/5-fl uorouracil (5-FU)/folinic acid (FA) (FOLFOX-4) in the fi rst-line treatment of patients with metastatic colorectal cancer (CRC) expressing

Epidermal Growth Factor Receptor (EGFR)

Cetuximab in combination with oxaliplatin/5-fl uorouracil (5-FU)/folinic acid (FA) (FOLFOX-4) in the fi rst-line treatment of patients with epidermal growth factor receptor (EGFR)-expressing metastatic colorectal cancer: an international phase II study

Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry

KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer

K-ras mutations and benefi t from cetuximab in advanced colorectal cancer

Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer

FDA holds court on post hoc data linking KRAS status to drug response

The vascular endothelial growth factor proteins: identifi cation of biologically relevant regions by neutralizing monoclonal antibodies

Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo

Complete inhibition of rhabdomyosarcoma xenograft growth and neovascularization requires blockade of both tumor and host vascular endothelial growth factor

VEGF-trap: a VEGF blocker with potent antitumor effects

Humanization of an antivascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders

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

Preclinical safety evaluation of rhuMAbVEGF, an antiangiogenic humanized monoclonal antibody

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

Phase I safety and pharmacokinetic study of recombinant human anti-vascular endothelial growth factor in patients with advanced cancer

A phase II trial of humanized anti-vascular endothelial growth factor antibody for the treatment of androgen-independent prostate cancer

A phase I/II doseescalation trial of bevacizumab in previously treated metastatic breast cancer

A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer

Phase II, randomized trial comparing bevacizumab plus fl uorouracil (FU)/leucovorin (LV) with FU/LV alone in patients with metastatic colorectal cancer

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

Bevacizumab plus irinotecan, fl uorouracil, and leucovorin for metastatic colorectal cancer

Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer

Risk of venous thromboembolism with the angiogenesis inhibitor bevacizumab in cancer patients

Risk of venous thromboembolism with bevacizumab in cancer patients

Risk of venous thromboembolism with bevacizumab in cancer patients

Risk of venous thromboembolism with bevacizumab in cancer patients

Risk of venous thromboembolism with bevacizumab in cancer patients-reply

Increased risk of serious hemorrhage with bevacizumab in cancer patients: a meta-analysis

Paclitaxel plus bevacizumab versus paclitaxel alone for metastatic breast cancer

How much is life worth: cetuximab, non-small cell lung cancer, and the $440 billion question

Adaptive immunity maintains occult cancer in an equilibrium state

Immune surveillance: a balance between protumor and antitumor immunity

Cytotoxic T lymphocyte‚associated antigen 4 plays an essential role in the function of Cd25 + Cd4+ regulatory cells that control intestinal infl ammation

Enhancement of antitumor immunity by CTLA-4 blockade

Activity and safety of CTLA-4 blockade combined with vaccines in cynomolgus macaques

Biologic activity of cytotoxic T lymphocyte-associated antigen 4 antibody blockade in previously vaccinated metastatic melanoma and ovarian carcinoma patients

Cancer regression and autoimmunity induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma

Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4

Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyten associated antigen 4

Ipilimumab (anti-CTLA4 antibody) causes regression of metastatic renal cell cancer associated with enteritis and hypophysitis

Ipilimumab monotherapy in patients with pretreated advanced melanoma: a randomised, double-blind, multicentre, phase 2, dose-ranging study

A phase II multicenter study of ipilimumab with or without dacarbazine in chemotherapy-naïve patients with advanced melanoma

Ipilimumab plus dacarbazine for previously untreated metastatic melanoma

Following TRAIL's path in the immune system

Overview of cell death signaling pathways

Reduction of TRAIL-induced Mcl-1 and cIAP2 by c-Myc or sorafenib sensitizes resistant human cancer cells to TRAIL-induced death

TRAIL receptor signaling and therapeutics

Phase 1 and pharmacokinetic study of lexatumumab in patients with advanced cancers

Phase I and pharmacokinetic study of lexatumumab (HGS-ETR2) given every 2 weeks in patients with advanced solid tumors

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

Novel in vivo imaging shows up-regulation of death receptors by paclitaxel and correlates with enhanced antitumor effects of receptor agonist antibodies

Proteasome inhibitors sensitize ovarian cancer cells to TRAIL induced apoptosis

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

Mapatumumab and lexatumumab induce apoptosis in TRAIL-R1 and TRAIL-R2 antibody-resistant NSCLC cell lines when treated in combination with bortezomib

Histone deacetylase inhibitors enhance lexatumumab-induced apoptosis via a p21Cip1-dependent decrease in survivin levels

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

Enhancement of lexatumumab-induced apoptosis in human solid cancer cells by cisplatin in caspase-dependent manner

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