Combining Chemotherapy With Molecularly Targeted Agents

Cytotoxic chemotherapy is likely to remain a key component of the oncologist's arsenal for some time. Nevertheless, the future of the medical treatment for cancer is likely to lie with the continued introduction of targeted molecular therapeutics that acts on the genetic and molecular abnormalities that drive the malignant phenotype. In the immediate future, treatment is likely to involve the combination of the new molecular therapeutics with conventional agents. There are considerable...

Introduction

A fundamental premise in the scientific enterprise is that technology has a powerful impact on research, allowing scientists to ask old questions in new and exciting ways. The recent sequencing of the complete human genome represented a fundamental shift in how biomedical research is performed such that biological phenomena can now be addressed with more global, comprehensive sets of tools. In parallel, advances in automation, high-throughput screening, gene-based libraries, combinatorial...

Imatinib Mesylate Gleevec

Imatinib is a 2-phenylaminopyrimidine derivative that functions as a potent inhibitor of a number of tyrosine kinase enzymes, with particularly high affinity for kinases involved in leukemia. Chronic myeloid leukemia (CML) has recently been successfully treated in the early stages of the disease (Druker et al., 2001), but the blastic phase (BP), which is characterized by rapid expansion of therapy-refractory and differentiation-arrested blast cells, still remains a challenge therapeutically. In...

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SiRNAs also suppressed T47D soft-agar growth (Fig. 8C), while no effect on cell proliferation (anchorage-dependent growth) was observed (data not shown). Therefore, knockdown of EphB4 in multiple transformed cell lines leads to reduced transformed phenotype. EphB4 is a member of erythropoietin-producing hepatocellular (Eph) receptor tyrosine kinases (RTKs), the largest subfamily of RTKs (Andres et al., 1994 Bennett et al., 1994 Stapleton et al., 1999). Like other Ephs, EphB4 is a large...

Genomics In Cancer Drug Discovery And Development

Perhaps the single-most important driver of current oncology drug discovery is our knowledge of the cancer genome, particularly at the level of expression of encoded genes, proteins, and regulatory elements. The coalescence of genome sequencing with laboratory automation and miniaturization now enables a truly global view of the state of a cancer cell's genes and their expression. The translation of this vast genomic resource into useful insights and tools for the detection and treatment of...

The Need For New Diagnostic Strategies

Refinements in more conventional diagnostic strategies, such as imaging, have had a substantial benefit to patients over the last 25 years. The potential to detect early breast cancer by mammography or the ability of computed tomography, ultrasonography, and magnetic resonance imaging to reveal small masses or tumor metastasis are but a few examples. However, hybrid strategies, combining imaging with other modalities should work better. Novel biomarkers, as additional tools to detect...

Identification of Patients for Adjuvant Therapy

After several years of profiling breast cancer gene expression at the Netherlands Cancer Institute using a microarray platform containing 25,000 genes, a prognostic classifier was identified that consists of 70 genes. This information can help distinguish those patients who are either at a high or low risk for developing distant metastases, and could indicate who might require adjuvant therapy and their response to it. Furthermore, this analysis has identified the estrogen receptor, HER-2...

Genetically Defined Tumor Models

In recent years, one major approach to improving tumor models has been to develop genetically engineered mouse (GEM) models in which tumor formation is driven by clinically relevant oncogenes, or loss of tumor suppressors. The use of GEM cancer models for elucidating cancer biology and drug discovery has been previously reviewed (Van Dyke and Jacks, 2002). The major advantages of GEM models include the ability to drive tumorigenesis with defined genetic changes and syngeneic tumor-host...

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Fig. 6 PK-PD clinical relationship for 17-AAG in a malignant melanoma patient with prolonged stable disease. This figure demonstrates the PK-PD clinical relationships in this patient. Pharmacokinetics The presence of active concentrations of 17-AAG, with peak plasma concentrations of > 10 mmol liter and concentrations of approximately 100 nmol liter achieved at 24 h. Pharmacodynamics Demonstration of the pharmacodynamic signature of HSP90 inhibition at 24-h postdose in tumor tissue with...

Animal Modeling In The Postgenomics

The dichotomization of therapies as either cytotoxic or targeted therapies is misnomered. In fact, the molecular targets for most cytotoxics are known (e.g., microtubules, nucleotide pools, topoisomerases, and so on). Conversely, many therapies developed to inhibit specific molecular targets may have widespread effects in both malignant and nonmalignant cells. In either case, animal models serve the same purposes in the drug discovery flow. Fundamentally, we want animal models to tell us (1) is...

Clinical and Molecular Prognostic Markers for Ovarian Cancer

Several clinical features have been shown to have significant impact on ovarian cancer patient survival. As discussed in Section I.A, patients with early stage diseases have significantly better survival rate than those with late-stage diseases. Optimally debulking of patient surgically (ability to remove residual tumor nodules which are greater than 2 cm) is associated with improved outcomes. In addition, patients with poorly differentiated tumors and clear cell lesions have a poorer prognosis...

Orthotopic Models

One of the primary rationales for the use of in vivo models is to replicate aspects of tumorigenesis that are not reflected in tissue culture. For example, nonmalignant stromal cells play an important role in promoting tumorigen-esis, including providing critical growth factors, nutrients, and angiogenesis (Condon, 2005 De Wever and Mareel, 2003 Kim et al., 2005). Although SQ xenografts may recapitulate certain aspects of the tumor-host microenvironment, it is clear that there are other aspects...

Chapters 16 Discovery Of Cancer Diagnostics And New Therapeutic Targets

By examining the signatures of gene or protein expression, cancer cells can now be readily differentiated from their normal counterparts at a molecular level, thus enabling the possibility of cancer diagnostics (Chapters 1 and 2). Expression analysis has also found significant use in aiding the selection of patients who may respond to therapy as well as delineating new therapeutic strategies for specific subtypes of cancer (Chapter 3). With improved diagnosis comes the need for new points of...

Pi3k Inhibitors

The PI3K pathway is frequently deregulated by various means in a wide range of malignancies, and is a key effector of several important cellular processes such as proliferation, growth, apoptosis, and cytoskeletal rearrangement (Bader et al., 2005 Vivanco and Sawyers, 2002). PI3K catalyzes the conversion of phosphatidylinositol 4,5-biphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3). PIK3CA, which encodes the p110a catalytic subunit of PI3K, has recently been found to exhibit...

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(TCR) by binding to the peptide-MHC complex, and the second involves so-called costimulatory signaling, such as the interaction between CD80 CD86 (B7.1 B7.2) on antigen-presenting cells (APCs) with CD28 on T-cells. For the generation of signal 1 there must be interaction between MHC-peptide and a specific TCR. The functional outcome of TCR-peptide-MHC interaction depends on costimulation (Fig. 1). Dendritic cells (DCs) are the key players in providing costimulation, and thereby provide the link...

Ribozymebased Genomic Technology In Cancer Gene Target Discovery And Validation

Cancers are genetic diseases caused by multiple alterations of three types of genes oncogenes, tumor suppressors, and DNA stability genes (Vogelstein and Kinzler, 2004). The cancer-related alterations include mutations leading to constitutive activation of oncogenes, inactivation of tumor suppressor and DNA repair genes, and aberrant expression of these gene products. Identification of these etiological genetic factors and understanding their functions are the foundation for future cancer...

Conclusions And Future Perspective

PD studies have the potential to have a dramatic impact on the drug development process. When used in conjunction with biomarkers for patient selection and with the concept of the pharmacological audit trail, these provide an intellectual and practical framework for the design of clinical studies and interpretation of data, as well as for critical decision making. To date, the uptake and use of PD biomarkers in early clinical trials has been disappointing, and their overall impact has been...

References

B., Davis, R. E., Ma, C., Lossos, I. S., Rosenwald, A., Boldrick, J. C., Sabet, H., Tran, T., Yu, X., Powell, J. I., Yang, L., et al. (2000). Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 403, 503-511. Arnold, N., Hagele, L., Walz, L., Schempp, W., Pfisterer, J., Bauknecht, T., and Kiechle, M. (1996). Overrepresentation of 3q and 8q material and loss of 18q material are recurrent findings in advanced human ovarian...

E DNA Methylases Epigenetic Silencing and Tumor Suppressors

Epigenetic mechanisms silence many tumor-suppressor genes, and this realization has spurred the assessment of novel tumor-suppressor genes. In one example, microarray analysis, among other techniques, was used to search for genes that are epigenetically silenced in human endometrial cancers. The endometrial cancer cell line Ishikawa exhibited changes in global gene expression that suggested the tazarotene-induced gene-1 (Tig1) and CCAAT enhancer-binding protein-a (C ebpa) might function as...

By Synthetic siRNAs

High-throughput gene expression studies have been performed primarily by the use of cDNA or oligonucleotide microarrays in which the expression of almost the complete genome can be analyzed simultaneously. The information produced by microarray analyses focus on the transcriptome and therefore does not provide any insights into the expression of proteins or their posttranslational modifications, often importantly altering the functional effects of gene expression. In contrast, RNAi screens can...

Viral Vectors

Retroviral vectors have been used to introduce siRNA expression constructs into cells. The vectors are based, in general, on oncoretroviruses as Moloney murine stem cell virus (MoMuLV) (Paddison and Hannon, 2002) or the murine stem cell virus (MSCV) (Brummelkamp et al., 2002a). Also another group of retroviruses has been applied, namely lentiviruses. These lentiviral vectors have the advantage to infect both dividing and nondividing cells (Naldini et al., 1996 Qin et al., 2003). Unlike...

Microarrays to Identify New Therapeutic Strategies for Cancer

Armstrong *Division of Hematology Oncology, Children's Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 I. Introduction II. Microarray Technologies C. Microarray Comparative Genomic Hybridization D. Nonsense-Mediated mRNA Decay III. Microarrays in Drug Development B. Toxicity and Pharmacogenomic Evaluation...

How Tumor Antigens Are Recognized

In order to manipulate the immune system to attack tumor cells, it is first necessary to understand the mechanism of CTL activation. Antigen naive T-cells must be primed in an antigen-specific manner to become functional effector cells. In the current model of CTL priming, two signals are required. The first is antigen-specific triggering of the T-cell receptor Table I Tumor Antigens Major Classifications Table I Tumor Antigens Major Classifications EBV (Burkitt's and nasopharangeal)

Cancer Cell Based Genomic and Small Molecule Screens

Genomics Institute of the Novartis Research Foundation, San Diego, II. Drugs, Druggability, and Target Validation III. Post-genomic Discovery of Novel Targets A. Functional cDNA Screening and Oncology B. RNA Interference Screening and Oncology IV. Small Molecule Screens in Oncology C. Data Analysis of Multidimensional Cellular Datasets This chapter focuses on the promising post-genomic technologies being used for discovery of new, safer, and better cancer drugs and drug targets. Since cancer is...

Gene Expression Microarrays and Proteomics

The use of gene expression microarrays to generate molecular signatures and explore molecular mechanisms of drug action is having a significant impact in the drug discovery and development process. The reader is directed to comprehensive reviews by Clarke et al. (2001, 2004) for a full commentary on the current status of gene expression microarray technology. The main technology platforms are Affymetrix and spotted cDNA arrays. Gene expression profiling of cells in response to novel molecular...

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Fig. 2 Characteristics of efficient siRNAs. The thermodynamic stability of the first base pairs of either siRNA strand guides incorporation into RISC. The unwinding of the double strand starts at the end with low stability (light gray box). To ensure an efficient incorporation of the antisense strand into the RISC complex, the 5' end of the antisense strand should lay in this region. The 5' half of the siRNA has a greater importance for target recognition than the 3' half of the molecule (red...

Mass Spectrometry as a Cancer Diagnostic Tool

The concept and utility of multivariate protein markers as opposed to a single indicator to diagnose disease has been established for some time. More than 20 years ago, it became clear that different tumor cell types could be distinguished based on patterns of metabolites analyzed by GC-MS (Jellum et al., 1981). Investigators are currently using two types of proteomic technologies to mine the proteomic signature in order to differentiate between normal and diseased states protein microarrays...

Types Of Biomarkers

With the growth of biomarkers in clinical trials, a lack of agreement has been apparent in the definition of the varying types of markers. Consensus in the use of terminology is important to help clarity of thinking. An expert working group was created by the US National Institutes of Health to propose terms, definitions, and a conceptual model (Biomarkers Definitions Working Group, 2001 Frank and Hargreaves, 2003). A summary of the recommended terminology is given in the following paragraph...

Cancer Antigens

The aim of targeted immunotherapy for cancer is to generate an immunemediated specific anti-TAA response resulting in the abrogation or elimination of tumor growth. The choice of antigen may be TAA derived from whole protein, truncated long peptide, short peptide, or DNA sequences encoding specific epitopes, delivered to APCs by viral or nonviral vectors. DCs can be grown and modified ex vivo and exposed to antigens in the form of peptides mRNA, proteins, and so on, and then injected back into...

Mass Spectrometry Uncovering the Cancer Proteome for Diagnostics

Da-Elene van der Merwe,* Katerina Oikonomopoulou,*' John Marshall,z and Eleftherios P. Diamandis*' *Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5G1X5, Canada Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G1L5, Canada ZDepartment of Chemistry and Biology, Ryerson University, Toronto, Ontario M5G1G3, Canada I. Current Cancer Biomarkers II. Early Detection A. When Is an Early Detection Program Warranted...

By siRNA Expression Vectors

As an alternative to siRNA, screens in array format pooled siRNA libraries can be used. In array screens, each siRNA sequence is tested individually whereas in pooled library screens, the effect of silencing multiple genes in one cell can be examined, reducing the number of assays which have to be performed. In this type of screen shRNA vectors have been applied for their ability to be identified by a unique DNA barcode provided by the siRNA sequence within the vector insert. The barcodes are...

Magnetic Resonance Spectroscopy

Magnetic resonance spectroscopy (MRS) has the ability to determine concentrations of endogenous compounds, tracers, or drugs and their metabolites in a way that is minimally invasive. Data are produced and visualized in the form of a spectrum in which peaks correspond to different chemicals. Endogenous metabolites that can be measured as PD bioamark-ers include phosphomonoesters (PMEs), phosphocholine, or inorganic phosphate using 31P -MRS, or lactate, choline, or inositol compounds using 1H...

And Vaccines

George's University of London, Cranmer, London SW17 0RE, I. Introduction The Immune System and the Concept of Tumor Antigens II. How Tumor Antigens Are Recognized IV. Novel TAA Identification Techniques V. Effective Targeting of TAA in the Clinic VI. TAAS as Therapeutic Targets VIII. Tumor Antigens and Cancer Vaccines IX. Tumor Antigens as Surrogate Markers and Targets for Therapy and Vaccines X. Summary and Future Directions References There are a large number of tumor antigens, which may...

Other Inverse Genomics Screenings for Cancer Targets

We have previously described a few other in vitro cancer cell model systems for identifying cancer genes, for example, NIH3T3 cell focus formation (Li et al., 2000b) and BRCA1 promoter up-regulation (Beger et al., 2001). Evidence also suggests that ribozyme-mediated gene inactivation could potentially be used to evaluate in vivo models such as the tumor xenograft (see preceding section) and transgenic models (L'Huillier et al., 1996 Luo et al., 2003). Therefore, Inverse Genomics can also be...

Tumor Antigens And Cancer Vaccines

Prior to the identification of specific TSA and TAA sequences, tumor antigens were provided from tumor cells, either as lysates or as irradiated cell lines derived from either autologous or allogeneic tumors. The perception is that good anecdotal and phase I II studies have never translated into positive randomized trials. However, there are two positive randomized vaccine trials, one for colorectal cancer and the other for renal, both published in the Lancet (Jocham et al., 2004 Vermorken et...

Methodological Issues

It will be clear from the above discussion that PD markers have the potential to play a key role in the rational and mechanistically informed contemporary drug development process. However, there are a number of important methodological issues that should be discussed at this stage, relating in particular to the need for these assays to be rigorously validated and carefully implemented (Frank and Hargreaves, 2003). In their review of this key area, Hidalgo and Messersmith (2004) outlined three...

Effective Targeting Of Taa In The Clinic

The idea of a magic bullet specifically aimed at a TSA was first suggested by Paul Erlich. This was first considered a realistic possibility with the development of monoclonal antibodies (MABs) by Milstein and Kohler in the mid-seventies. Unfortunately, early optimism was dashed by a strong immune response to the murine sequences. It has taken over two decades of molecular engineering (together with the discovery and defining of TAA as targets) to get to the position we are in today, whereby...

Mass Spectrometrybased Diagnostics

Mass spectrometry has been used in two different settings in the area of cancer diagnostics, first for the discovery of novel cancer biomarkers and second as a cancer diagnostic and imaging tool. The discovery of biomar-kers and their use as early detectors of cancer is based on the hypothesis that a complex interplay exists between a tumor and its host microenvironment (Liotta and Kohn, 2001). As blood perfuses through a diseased organ, the serum protein profile is altered as a result of...

B RNA Interference Screening and Oncology

Whereas cDNA screens are useful for identifying genes that cause cell transformation and possibly cancer, RNA interference (RNAi) inhibition screens may be used to identify which genes can be inhibited to reverse the disease phenotype. Since RNAi inhibition of gene expression is in some respects analogous to inhibition of the same gene product by a small molecule antagonist, the utility of RNAi for drug target identification and validation has caught the interest of the drug discovery...

Data Analysis of Multidimensional Cellular Datasets

Understanding appropriate statistical analysis to mine multidimensional profiling data is of paramount importance to derive the most useful, predictive hypotheses. Sophisticated methods to analyze multidimensional datasets have come to the fore including techniques of hierarchical clustering analysis (HCA), multidimensional scaling (MDS), self-organizing maps (SOMs), and methods based on neural networks. Each of these methods has been adapted to parallel cell-based screening to extract...

Current Cancer Biomarkers

Currently, hundreds of tumor markers exist, yet most of them fall short of expectation. Clinicians expect that a marker should be beneficial to their patients in terms of improved morbidity, mortality, and quality of life. To illustrate the point, even if a biomarker is able to detect relapse a few months prior to clinical symptoms, if effective treatment does not exist, this information does not necessarily translate into improved outcome. Moreover, knowledge of tumor marker elevation may be...

Microarrays To Direct The Use Of Cancer Therapeutics

Microarrays are being heavily utilized in cancer research and are proving to be useful in all aspects of study, including the classification of cancer, the study of biochemical pathways, and the identification of potential targets for novel therapeutics. Gene expression technologies are also being used to distinguish on-target versus off-target effects of cancer therapeutics, mechanisms of resistance to treatment, mechanisms of therapeutic function, and prediction of drug response. Resistance...

Practices and Pitfalls of Mouse Cancer Models in Drug Discovery

Department of Pediatric Oncology, Dana-Farber Cancer Institute and Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115 I. Why Are Animal Models Needed II. Model Types Tumor Locations A. Subcutaneous Xenograft Models D. Genetically Defined Tumor Models A. Efficacy Endpoints Is Tumor Growth Effected B. Functional and Molecular Endpoints Is the Target Modulated V. Animal Modeling in the Post-genomics Age VI. Conclusions Mouse models of cancer are critical tools for elucidating...

Functional cDNA Screening and Oncology

Genetic dissection of signaling pathways in human disease is the current challenge in drug target identification. The ability to generate libraries of cDNAs from human tissue sources and clone into mammalian expression vectors is enabling this field. Harnessing viruses to introduce libraries blan-ketly into cells, paired with advances in human tissue culture systems and phenotypic readouts have ushered in the age of human cell genetics. In the late 1980s, Brian Seed established the expression...

Pharmacodynamic Biomarkers for Molecular Cancer Therapeutics

Signal Transduction and Molecular Pharmacology Team, Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey SM2 5NG, United Kingdom I. Introduction II. Types of Biomarkers III. The Pharmacological Audit Trail V. Rationale for Use of PD Markers to Facilitate Drug Development A. PD Markers in Preclinical Drug Development VI. The Need for Multidisciplinary and Broad-Based Collaborative Research and Development VII. Biomarker...

Small Molecule Screens In Oncology

While it is appreciated that oncogenesis arises as a result of the accumulation of selectively advantageous somatic mutations, many of which can be mapped and sequenced, therapies that effectively reverse the unchecked tumor growth phenotype are sorely needed. Although cDNA screens can identify proto-oncogenes, siRNA libraries can identify potential targets for therapeutic intervention, small molecule screens in cellular cancer models to selectively impair tumor cell growth may identify both...

HeLaHeLaHF Cervical Cancer Cell System and Anchorage Independent Growth

Cancers of cervical origin are the second most common gynecological cancers and HeLa is the most well-characterized experimental cell line of cervical cancer (adenocarcinoma) origin (Masters, 2002). HeLa cells show a strong transformed phenotype including anchorage-independent growth in vitro and tumorigenesis in vivo. HeLaHF is a revertant variant isolated from HeLa cells following exposure to the mutagen ethylmethane sulpho-nate (EMS) (Boylan et al., 1996). HeLaHF demonstrates a...

Ca125 Cea Ca199

Choriocarcinoma and testicular cancer Hepatic and testicular cancer Ovarian cancer Colorectal and pancreatic cancer Pancreatic cancer Prostate cancer aThese are some of the markers currently available in widespread clinical use. Due to the heterogenicity of tumors, some tumors do not secrete these proteins and that targeting them will not be effective. Moreover, in mixed clonality, tumor targeting may leave negative clones to survive be selected for. possibilities. The examples mentioned here...

Activation of Apoptosis Induced by External Stimuli

Situ Cell Death Fluorescein

Another hallmark of cancer development is resistance to apoptosis. Apoptosis is normally triggered either intrinsically by stress conditions (DNA damage, protein misfolding, and so on) through the activation of caspase 9 or extrinsically by extracellular death ligands (FasL, TRAIL, TNF-a) through activation of caspase 8. These two pathways converge downstream leading to the activation of caspases 3 and 7 to irreversibly commit cells to apoptosis. These pathways are regulated at many steps by a...

Rationale For Use Of Pd Markers To Facilitate Drug Development

Early clinical trials are increasingly seen to represent a continuation of the preclinical and clinical discovery and development process. It is important to consider the role of PD markers in both preclinical drug development and also as part of the experimental medicine component of early clinical trials. A. PD Markers in Preclinical Drug Development In considering the role of PD endpoints in the preclinical phase, it is useful to consider the stages involved in the contemporary development...

Ribozyme Technology for Cancer Gene Target Identification and Validation

Qi-Xiang Li, Philip Tan, Ning Ke, and Flossie Wong-Staal Immusol, Inc., San Diego, California 92121 I. Brief Biology of Ribozymes II. Ribozymes as Tools for Gene Inactivation A. Design of Ribozymes for Targeting a Specific mRNA of Interest B. Delivery of Ribozymes into Cells C. Factors That Influence the Effectiveness of Ribozyme-Mediated Gene Inactivation III. Ribozymes as Tools in Gene Target Discovery and Validation A. Principles of Ribozyme-Based Gene Target Validation B. Drug Target...