Contents

Editors: Skeel, Roland T.

Title: Handbook of Cancer Chemotherapy, 7th Edition

Copyright 2007 Lippincott Williams & Wilkins

> Table of Contents > Section I - Basic Principles and Considerations of Rational Chemotherapy > Chapter 1 - Biologic and Pharmacologic Basis of Cancer Chemotherapy and Biotherapy

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Chapter 1

Biologic and Pharmacologic Basis of Cancer Chemotherapy and Biotherapy

Roland T. Skeel

Samir N. Khleif

I. General mechanisms by which chemotherapeutic agents control cancer

The purpose of treating cancer with chemotherapeutic agents is to prevent cancer cells from multiplying, invading, metastasizing, and ultimately killing the host (patient). Most traditional chemotherapeutic agents currently in use appear to exert their effect primarily on cell proliferation. Because cell multiplication is a characteristic of many normal cells as well as cancer cells, most cancer chemotherapeutic agents also have toxic effects on normal cells, particularly those with a rapid rate of turnover, such as bone marrow and mucous membrane cells. The goal in selecting an effective drug, therefore, is to find an agent that has a marked growth-inhibitory or controlling effect on the cancer cell and a minimal toxic effect on the host. In the most effective chemotherapeutic regimens, the drugs are capable not only of inhibiting but also of completely eradicating all neoplastic cells while sufficiently preserving normal marrow and other target organs to permit the patient to return to normal, or at least satisfactory, function and quality of life.

Ideally, the cell biologist, pharmacologist, and medicinal chemist would like to look at the cancer cell, discover how it differs from the normal host cell, and then design a chemotherapeutic agent to capitalize on that difference. Until recently less rational means were used for most of the chemotherapeutic agents that are now in use. The effectiveness of agents was discovered by treating either animal or human neoplasms, after which the pharmacologist attempted to discover why the agent worked as well as it did. With few exceptions, the reasons why chemotherapeutic agents are more effective against cancer cells

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than against normal cells have been poorly understood. With the rapid expansion of information about cell biology and the factors within the neoplastic cell that control cell growth, the strictly empiric method of discovering effective new agents has changed. For example, antibodies against the protein product of the overexpressed HER2/ neu oncogene have been demonstrated to be effective in controlling metastatic breast cancer and reducing recurrences after primary therapy in patients whose tumors overexpress this gene. Discovery of the constitutively activated Bcr-Abl tyrosine kinase created as a consequence of the chromosomal translocation in Chronic Myelogenous Leukemia (CML) has led to an exciting new era of orally administered small molecular inhibitors of critical molecular changes in cancer cells and their environment. These sentinel events have presaged the development of a host of new therapeutic agents that are directed at known specific targets within and around the cancer cell. These targets have been selected, because they are altered in the cancer cell and are critical for cancer cell growth, invasion, and metastasis. This increased understanding of cancer cell biology has already provided more specific and selective ways of controlling cancer cell growth in several human cancers and will continue to dominate drug development for systemic therapy in the decade to come.

Inhibition of cell multiplication and tumor growth can take place at several levels within the cell and its environment:

• Macromolecular synthesis and function

• Cytoplasmic organization and signal transduction

• Cell membrane and associated cell surface receptor synthesis, expression, and function

• Environment of cancer cell growth

A. Classic chemotherapy agents

Most agents currently in use, with the exception of immunotherapeutic agents and other biologic response modifiers, appear to have their primary effect on either macromolecular synthesis or function. This effect means that they interfere with the synthesis of Deoxyribonucleic acid (DNA) Ribonucleic Acid (RNA) or proteins or with the appropriate functioning of the preformed molecule. When interference in macromolecular synthesis or function in the neoplastic cell population is sufficiently great, a proportion of the cells die. Some cells die because of the direct effect of the chemotherapeutic agent. In other instances, the chemotherapy may trigger differentiation, senescence, or apoptosis, the cell's own mechanism of programmed death.

Cell death may or may not take place at the time of exposure to the drug. Often, a cell must undergo several divisions before the lethal event that took place earlier finally results in the death of the cell. Because only a proportion of the cells die as a result of a given treatment, repeated doses of chemotherapy must be used to continue to reduce the cell number (Fig. 1.1). In an ideal system, each time the dose is repeated, the same proportion of cells not the same absolute number is killed. In the example shown in Fig. 1.1, 99.9% (3 logs) of the cancer cells are killed with each treatment, and there is a 10-fold (1-log) growth between treatments, for a net reduction of 2 logs with each treatment. Starting at 10¹⁰ cells (approximately

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10 g or 10 cm³ leukemia cells), it would take five treatments to reach fewer than 10 , or 1, cell. Such a model makes certain assumptions that rarely are strictly true in clinical practice:

• All cells in a tumor population are equally sensitive to a drug.

• Drug accessibility and cell sensitivity are independent of the location of the cells within the host and of local host factors such as blood supply and surrounding fibrosis.

• Cell sensitivity does not change during the course of therapy.

The lack of curability of most initially sensitive tumors is probably a reflection of the degree to which these assumptions do not hold true.

Figure 1.1. The effect of chemotherapy on cancer cell numbers. In an ideal system, chemotherapy kills a constant proportion of the remaining cancer cells with each dose. Between doses, cell regrowth occurs. When therapy is successful, cell killing is greater than cell growth.

B. Biologic response modifiers and molecular targeted therapy Molecular Targeted Therapy (MTT)

Within individual cells and cell populations are intricate interrelated mechanisms that promote or suppress cell proliferation, facilitate invasion or metastasis when the cell is malignant, lead to cell differentiation, promote (relative) cell immortality, or set the cell on the path to inevitable death (apoptosis). These activities are controlled in large part

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by normal genes and, in the case of cancer, by mutated cancer promoter genes, tumor suppressor genes, and their products. Included in these products are a host of cell growth factors that control the machinery of the cell. Some of these factors that affect normal cell growth have been biosynthesized and are now used to enhance the production of normal cells (e.g., epoetin and filgrastim) and to treat cancer (e.g., interferon [IFN]).

The recent expansion of our understanding of the biologic control of normal cells and tumor growth at the molecular level has only begun to offer improved therapy for cancer, although it has helped explain differences in response among populations of patients. New discoveries in cancer cell biology have provided insights into apoptosis, cell cycling control, angiogenesis, metastasis, cell signal transduction, cell surface receptors, differentiation, and growth factor modulation. New drugs in clinical trials have been designed to block growth factor receptors, prevent oncogene activity, block the cell cycle, restore apoptosis, inhibit angiogenesis, restore lost function of tumor suppressor genes, and selectively kill tumors containing abnormal genes. Further understanding of each of these holds a great potential for providing powerful and more selective means to control neoplastic cell growth and may lead to effective cancer treatments in the next decade.

II. Tumor cell kinetics and chemotherapy

Cancer cells, unlike other body cells, are characterized by a growth process whereby their sensitivity to normal controlling factors has been partially or completely lost. As a result of this uncontrolled growth, it was once thought that cancer cells grew or multiplied faster than normal cells and that this growth rate was responsible for the sensitivity of cancer cells to chemotherapy. Now it is known that most cancer cells grow less rapidly than the more active normal cells as in the bone marrow. Therefore, although the growth rate of many cancers is faster than that of normal surrounding tissues, growth rate alone cannot explain the greater sensitivity of cancer cells to chemotherapy.

A. Tumor growth

The growth of a tumor depends on several interrelated factors.

• Cell cycle time, or the average time for a cell that has just completed mitosis to grow, redivide, and again pass through mitosis, determines the maximum growth rate of a tumor but probably does not determine drug sensitivity. The relative proportion of cell cycle time taken up by the DNA synthesis phase may relate to the drug sensitivity of some types (S phase specific) of chemotherapeutic agents.

• Growth fraction, or the fraction of cells undergoing cell division, contains the portion of cells that are sensitive to drugs whose major effect is exerted on cells that are dividing actively. If the growth fraction approaches 1 and the cell death rate is low, the tumor-doubling time approximates the cell cycle time.

• Total number of cells in the population (determined at some arbitrary time at which the growth measurement is started) is clinically important because it is an index of how advanced the cancer is; it frequently correlates with normal organ dysfunction. As the total number of cells increases, so does the number of resistant cells, which in

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  turn leads to decreased curability. Large tumors may also have greater compromise of blood supply and oxygenation, which can impair drug delivery to the tumor cells as well as the sensitivity to both chemotherapy and radiotherapy.

• Intrinsic cell death rate of tumors is difficult to measure in patients but probably makes a major and positive contribution by slowing the growth rate of many solid tumors.

B. Cell cycle

The cell cycle of cancer cells is qualitatively the same as that of normal cells (Fig. 1.2). Each cell begins its growth during a postmitotic period, a phase called G₁, during which enzymes necessary for DNA production, other proteins, and RNA are produced. G₁ is followed by a period of DNA synthesis (S), in which essentially all DNA synthesis for a given cycle takes place. When DNA synthesis is complete, the cell enters a premitotic period (G₂), during which further protein and RNA synthesis occurs. This gap is followed immediately by mitosis (M), at the end of which actual physical division takes place, two daughter cells are formed, and each cell again enters G₁. G₁ phase is in equilibrium with a resting state called G₀. Cells in G₀ are relatively inactive with respect to macromolecular synthesis and are consequently insensitive to many chemotherapeutic agents, particularly those that affect macromolecular synthesis.

C. Phase and cell cycle specificity

Most classic chemotherapeutic agents can be grouped according to whether they depend on cells being in cycle (i.e., not in G₀) and, if they depend on the cell being in cycle, whether their activity is greater when the cell is in a specific phase of the cycle. Most agents cannot be assigned to one category exclusively. Nonetheless, these classifications can be helpful in understanding drug activity.

• Phase-specific drugs. Agents that are most active against cells in a specific phase of the cell cycle are called cell cycle phase specific drugs. A partial list of these drugs is shown in Table 1.1.

  • Implications of phase-specific drugs. Phase specificity has important implications for cancer chemotherapy.

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    • Limitation to single-exposure cell kill. With a phase-specific agent, there is a limit to the number of cells that can be killed with a single instantaneous (or very short) drug exposure because only those cells in the sensitive phase are killed. A higher dose does not kill more cells.

      Table 1.1 Cell cycle phase specific chemotherapeutic agents

      Phase of Greatest Activity	Class	Type	Characteristic Agents
      Gap 1 (G1)	Natural product	Enzyme	Asparaginase
      	Hormone	Corticosteroid	Prednisone
      G1/S junction	Antimetabolite	Purine analog	Cladribine
      DNA synthesis (S)	Antimetabolite	Pyrimidine analog	Cytarabine, fluorouracil, gemcitabine
      	Antimetabolite	Folic acid analog	Methotrexate
      	Antimetabolite	Purine analog	Thioguanine, fludarabine
      	Natural product	Topoisomerase I inhibitor	Topotecan
      	Miscellaneous	Substituted urea	Hydroxyurea
      Gap 2 (G2)	Natural product	Antibiotic	Bleomycin
      	Natural product	Topoisomerase II inhibitor	Etoposide
      	Natural product	Microtubule polymerization and stabilization	Paclitaxel
      Mitosis (M)	Natural product	Mitotic inhibitor	Vinblastine, vincristine, vindesine, vinorelbine
      DNA, deoxyribonucleic acid.

    • Increasing cell kill by prolonged exposure. To kill more cells requires either prolonged exposure to, or repeated doses of, the drug to allow more cells to enter the sensitive phase of the cycle. Theoretically, all cells could be killed if the blood level or, more importantly, the intracellular concentration of the drug remained sufficiently high while all cells in the target population passed through one complete cell cycle. This theory assumes that the drug does not prevent the passage of cells from one (insensitive) phase to another (sensitive) phase.

    • Recruitment. A higher number of cells could be killed by a phase-specific drug if the proportion of cells in the sensitive phase could be increased (recruited).

      

      Figure 1.2. Cell cycle time for human tissues has a wide range (16 to 260 hours), with marked differences among normal and tumor tissues. Normal marrow and gastrointestinal lining cells have cell cycle times of 24 to 48 hours. Representative durations and the kinetic or synthetic activity are indicated for each phase. RNA, ribonucleic acid; DNA, deoxyribonucleic acid.

  • Cytarabine. One of the best examples of a phasespecific agent is cytarabine (ara-C), which is an inhibitor of DNA synthesis and is therefore active only in the S phase (at standard doses). When used in doses of 100 to 200 mg/m² daily (i.e., not high-dose ara-C ), ara-C is rapidly deaminated in vivo to an inactive compound, ara-U, and rapid injections result in very short effective levels of ara-C. As a result, single doses of ara-C are nontoxic to the normal hematopoietic system and are generally ineffective for treating leukemia. If the drug is given as a daily rapid injection, some patients with leukemia respond well but not nearly as well as when ara-C is given every 12 hours. The apparent reason for the greater effectiveness of the 12-hour schedule is that the S phase (DNA synthesis) of human acute nonlymphocytic leukemia cells lasts for approximately 18 to 20 hours. If the drug is given every 24 hours, some cells that have not entered the S phase when the drug is first administered will not be sensitive to its effect. Therefore, these cells can pass all the way through the S phase before the next dose is administered and will completely escape any cytotoxic effect. However, when the drug is given every 12 hours, no cell that is in cycle will be able to escape exposure to ara-C because none will be able to get through one complete S phase without the drug being present.

    If all cells were in active cycle, that is, if none were resting in a prolonged G1 or G0 phase, it would be theoretically possible to kill any cells in a population by a continuous or scheduled exposure equivalent to one complete cell cycle. Experiments with patients who have acute leukemia have shown that if tritiated thymidine is used to label cells as they enter DNA synthesis, it may be 7 to 10 days before the maximum number of leukemia cells have passed through the S phase. This means that, barring permutations caused by itself or

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    other drugs, for ara-C to have a maximum effect on the leukemia, the repeated exposure must be continued for a 7- to 10-day period. Clinically, continuous infusion or administration of ara-C every 12 hours for 5 days or longer appears to be most effective for treating patients with newly diagnosed acute nonlymphocytic leukemia. However, even with such prolonged exposure, it appears that a few of the cells do not pass through the S phase.

• Cell cycle specific drugs. Agents that are effective while cells are actively in cycle but are not dependent on the cell being in a particular phase are called cell cycle specific (or phase-nonspecific) drugs. This group includes most of the alkylating agents, the antitumor antibiotics, and some miscellaneous agents, examples of which are shown in Table 1.2. Some agents in this group are not totally phase-nonspecific; they may have greater activity in one phase than in another, but not to the degree of the phase-specific agents. Many agents also appear to have some activity in cells that are not in cycle, although not as much as when the cells are rapidly dividing.

• Cell cycle nonspecific drugs. A third group of drugs appears to be effective whether cancer cells are in cycle or are resting. In this respect, these agents are similar to photon irradiation; that is, both types of therapy are effective, irrespective of whether or not the cancer cell is in cycle. Drugs in this category are called cell cycle nonspecific drugs and include mechlorethamine (nitrogen mustard) and the nitrosoureas (see Table 1.2).

Table 1.2 Cell cycle specific and cell cycle nonspecific chemotherapeutic agents

Class	Type	Characteristic Agents
Cell cycle specific
Alkylating agent	Nitrogen mustard	Chlorambucil, cyclophosphamide, melphalan
	Alkyl sulfonate	Busulfan
	Triazene	Dacarbazine
	Metal salt	Cisplatin, carboplatin
Natural product	Antibiotic	Dactinomycin, daunorubicin, doxorubicin, idarubicin
Cell cycle nonspecific
Alkylating agent	Nitrogen mustard	Mechlorethamine
	Nitrosourea	Carmustine, lomustine

D. Changes in tumor cell kinetics and therapy implications

As cancer cells grow from a few cells to a lethal tumor

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burden, certain changes occur in the growth rate of the population and affect the strategies of chemotherapy. These changes have been determined by observing the characteristics of experimental tumors in animals and neoplastic cells growing in tissue culture. Such model systems readily permit accurate cell number determinations to be made and growth rates to be determined. (Because tumor cells cannot be injected or implanted into humans and permitted to grow, studies of growth rates of intact tumors in humans must be limited largely to observing the growth rate of macroscopic tumors.)

• Stages of tumor growth. Immediately after inoculation of a tissue culture or an experimental animal with tumor cells, there is a lag phase, during which there is little tumor growth; presumably, the cells in this phase are becoming accustomed to the new environment and are preparing to enter into cycle. The lag phase is followed by a period of rapid growth called the log phase, during which there are repeated doublings of the cell number. In populations in which the growth fraction approaches 100% and the cell death rate is low, the population doubles within a period approximating the cell cycle time. As the cell number or tumor size becomes macroscopic, the doubling time of the tumor cell population becomes prolonged and levels off (plateau phase). Most clinically measurable human cancers are probably in the plateau phase, which may account, in part, for the slow doubling time observed in many human cancers (30 to 300 days). Because the rate of change in the slope of the growth curve during the premeasurable period is unknown for most human cancers, extrapolation from two points when the mass is measurable to estimate the onset of the growth of the malignancy is subject to considerable error. The prolongation in tumordoubling time in the plateau phase may be due to a smaller growth fraction, a change in the cell cycle time, an increased intrinsic death rate (predominantly apoptosis, which is a programmed and highly orchestrated cell death that occurs both naturally and under the influence of many types of chemotherapy), or a combination of these factors. Factors responsible for these changes include decreased nutrients or growth promotion factors, increased inhibitory metabolites or inhibitory growth factors, and inhibition of growth by other cell cell interactions. In the intact host, new blood vessel formation is a critical determinant of these factors.

• Growth rate and effectiveness of chemotherapy. Chemotherapeutic agents are most effective during the period of logarithmic growth. As might be expected, this result is particularly true for the antimetabolites, which are largely S phase specific. As a result, when human tumors become macroscopic, the effectiveness of many chemotherapeutic agents is reduced because only part of the cell population is dividing actively. Theoretically, if the cell population could be reduced sufficiently by other means such as surgery or radiotherapy, chemotherapy would be more effective because a higher fraction of the remaining cells would be in logarithmic growth. The validity of this theoretical premise is supported by the varying degrees

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  of success of surgery plus chemotherapy or radiotherapy plus chemotherapy in the treatment of breast cancer, colon cancer, Wilms' tumor, ovarian cancer, small cell anaplastic cell carcinoma of the lung, non small cell carcinoma of the lung, head and neck cancers, and osteosarcomas.

III. Combination chemotherapy

Combinations of drugs are frequently more effective in producing responses and prolonging life than are the same drugs used sequentially. Combinations are likely to be more effective than single agents for several reasons.

A. Reasons for effectiveness of combinations

• Prevention of resistant clones. If 1 in 10⁵ cells is resistant to drug A and 1 in 10⁵ cells is resistant to drug B, it is likely that treating a macroscopic tumor (which generally would have more than 10⁹ cells) with either agent alone would result in several clones of cells that are resistant to that drug. If, after treatment with drug A, a resistant clone has grown to macroscopic size (if the same mutant frequency persists for drug B), resistance to agent B will also emerge. If both drugs are used at the outset of therapy or in close sequence, however, the likelihood of a cell being resistant to both drugs (excluding, for a moment, the situation of pleiotropic drug resistance) is only 1 in 10^10. Therefore, the combination confers considerable advantage against the emergence of resistant clones. Compounding the problem of pre-existing resistant clones is the resistance that develops through spontaneous mutation in the absence of drug exposure. The use of multiple drugs with independent mechanisms of action or alternating non cross-resistant combinations (as well as the use of surgery or radiotherapy to eliminate macroscopic tumor) theoretically minimizes the chances for outgrowth of resistant clones and increases the likelihood of remission or cure.

• Cytotoxicity to resting and dividing cells. The combination of a drug that is cell cycle specific (phase nonspecific) or cell cycle nonspecific with a drug that is cell cycle phase specific can kill cells that are dividing slowly as well as those that are dividing actively. The use of cell cycle nonspecific drugs can also help recruit cells into a more actively dividing state, which results in their being more sensitive to the cell cycle phase specific agents.

• Biochemical enhancement of effect

  • Combinations of individually effective drugs that affect different biochemical pathways or steps in a single pathway can enhance each other. This may apply to some newer agents whereby blocking more than one molecular target in the interacting signal transduction pathways may magnify the interference of cell proliferation compared with that seen with either agent alone.

  • Combinations of an active agent with an inactive agent can potentially result in beneficial effects by several mechanisms, but has limited clinical utility.

    • An intracellular increase in the drug or its active metabolites, by either increasing influx or decreasing efflux (e.g., calcium channel inhibitors

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      with multiple agents affected by multidrug resistance [MDR] due to P-glycoprotein overexpression)

    • Reduced metabolic inactivation of the drug (e.g., inhibition of cytidine deaminase inactivation of ara-C with tetrahydrouridine)

    • Cooperative inhibition of a single enzyme or reaction (e.g., leucovorin enhancement of fluorouracil inhibition of thymidylate synthetase)

    • Enhancement of drug action by inhibition of competing metabolites (e.g., N-phosphonoacetyl-Laspartic acid inhibition of de novo pyrimidine synthesis with resultant increased incorporation of 5-fluorouridine triphosphate into RNA)

• Sanctuary access. Combinations can be used to provide access to sanctuary sites for reasons such as drug solubility or affinity of specific tissues for a particular drug type.

• Rescue. Combinations can be used in which one agent rescues the host from the toxic effects of another drug (e.g., leucovorin administration after high-dose methotrexate).

B. Principles of agent selection

When selecting appropriate agents for use in a combination, the following principles should be observed:

• Choose individually active drugs. Do not use a combination in which one agent is inactive when used alone unless there is a clear, specific biochemical or pharmacologic reason to do so, for example, high-dose methotrexate followed by leucovorin rescue or leucovorin followed by fluorouracil. This principle may not be applicable to the combined use of chemotherapeutic agents with biologic response modifiers or molecular targeted agents because the cooperativity of chemotherapy and these drugs may not depend on the independent cytotoxic effects of these nonclassic agents.

• When possible, choose drugs in which the doselimiting toxicities differ qualitatively or in time of occurrence. Often, however, two or more agents that have marrow toxicity must be used, and the selection of a safe dose of each is critical. As a starting point, two cytotoxic drugs in combination can usually be given at two-thirds of the dose used when the drugs are given alone. Whenever a new drug combination is tried, a careful evaluation of both expected and unanticipated toxicities must be carried out. Unexpected results such as the increased cardiotoxicity of the combination of trastuzumab with doxorubicin may occur, and this latter case has precluded the use of these agents together.

• Select agents for a combination for which there is a biochemical or pharmacologic rationale. Preferably, this rationale has been tested in an animal tumor system and in the appropriate model system, and the combination has been found to be better than either agent alone.

• Be cautious when attempting to improve on a successful two-drug combination by adding a third, fourth, or fifth drug simultaneously. Although this approach may be beneficial, two undesirable results may be seen:

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  • An intolerable level of toxicity that leads to excessive morbidity and mortality

  • Unchanged or reduced antitumor effect because of the necessity to reduce the dose of the most effective drugs to a level below which antitumor responses are not seen, despite the theoretical advantages of the combination. Therefore, the addition of each new agent to a combination must be considered carefully, the principles of combination therapy closely followed, and controlled clinical trials carried out to compare the efficacy and toxicity of any new regimen with a more established (standard) treatment program.

C. Clinical effectiveness of combinations

Combinations of drugs have been clearly demonstrated to be better than single agents for treating many, but not all, human cancers. The survival benefit of combinations of drugs compared with that of the same drugs used sequentially has been marked in diseases such as acute lymphocytic and acute nonlymphocytic leukemia, Hodgkin's lymphoma, non Hodgkin's lymphomas with more aggressive behavior (intermediate and high grade), breast carcinoma, anaplastic small cell carcinoma of the lung, colorectal carcinomas, ovarian carcinoma, and testicular carcinoma. The benefit is less notable in cancers such as non small cell carcinoma of the lung, non Hodgkin's lymphomas with favorable prognoses, head and neck carcinomas, carcinoma of the pancreas, and melanoma, although reports exist for each of these tumors in which combinations are better in one respect or another than single agents.

IV. Resistance to antineoplastic agents

Resistance to antineoplastic chemotherapy is a combined characteristic of a specific drug, a specific tumor, and a specific host, whereby the drug is ineffective in controlling the tumor without excessive toxicity. Resistance of a tumor to a drug is the reciprocal of selectivity of that drug for that tumor. The problem for the medical oncologist or pharmacologist is not simply to find an agent that is cytotoxic but to find one that selectively kills neoplastic cells while preserving the essential host cells and their function. Were it not for the problem of resistance of human cancer to antineoplastic agents or, conversely, the lack of selectivity of those agents, cancer chemotherapy would be similar to antibacterial chemotherapy in which complete eradication of infection is regularly observed. Such a utopian state of cancer chemotherapy has not yet been achieved for most human cancers. The problem of resistance and ways to overcome or even exploit it remain an area of major interest for the oncologist, pharmacologist, and cell biologist. This reductionist description glosses over the fact that each of these factors is a consequence of the complex genetic characteristics and changes of the cancer cell as it evolves.

Resistance to antineoplastic chemotherapeutic agents may be either natural or acquired. Natural resistance refers to the initial unresponsiveness of a tumor to a given drug, and acquired resistance refers to the unresponsiveness that emerges after initially successful treatment. There are three basic categories of resistance to chemotherapy: kinetic, biochemical, and pharmacologic.

A. Cell kinetics and resistance

Resistance based on cell population kinetics relates to cycle and phase specificity,

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growth fractions and the implications of these factors for responsiveness to specific agents, and schedules of drug administration. A particular problem with many human tumors is that they are in a plateau growth phase with a small growth fraction. This factor renders many of the cells insensitive to the antimetabolites and relatively unresponsive to many of the other chemotherapeutic agents. Strategies to overcome resistance due to cell kinetics include the following:

• Reducing tumor bulk with surgery or radiotherapy

• Using combinations to include drugs that affect resting populations (with many G₀ cells)

• Scheduling of drugs to prevent phase escape or to synchronize cell populations and increase cell kill

B. Biochemical causes of resistance

Resistance can occur for biochemical reasons including the inability of a tumor to convert a drug to its active form, the ability of a tumor to inactivate a drug, or the location of a tumor at a site where substrates are present that bypass an otherwise lethal blockade. How cells become resistant is only partially understood. There can be decreased drug uptake, increased efflux, changes in the levels or structure of the in tracellular target, reduced in tercellular activation or increased inactivation of the drug, or increased rate of repair of damaged DNA. In one pre B-cell leukemia cell line, bcl-2 overexpression or decreased expression of the homolog bax renders cells resistant to several chemotherapeutic agents. Because bcl-2 blocks apoptosis, it has been proposed that its overexpression blocks chemotherapy-induced apoptosis. The interrelationship between mutations of p⁵³, HER2, and a host of other oncogenes and tumor suppressor genes and resistance to the cytotoxic effects of radiotherapy, chemotherapeutic, hormonal, and biologic agents, when better understood, may further our understanding of resistance and provide new therapeutic strategies.

Multidrug resistance (MDR), also called pleiotropic drug resistance, is a phenomenon whereby treatment with one agent confers resistance not only to that drug and others of its class but also to several other unrelated agents. MDR is commonly mediated by an enhanced energy-dependent drug efflux mechanism that results in lower in tracellular drug concentrations. With this type of MDR, overexpression of a membrane transport protein called P-glycoprotein (P meaning pleiotropic or permeability) is observed commonly. Other MDR proteins are those found in human lung cancer lines and the lung resistance protein. These proteins appear to have differing expression in different sets of neoplasms. Drugs that are effective in reversing resistance to P-glycoprotein do not reverse other MDR proteins. Combination chemotherapy can overcome biochemical resistance by increasing the amount of active drug in tracellularly as a result of biochemical interactions or effects on drug transport across the cell membrane. Calcium channel blockers, antiarrhythmics, cyclosporin A analogs (e.g., PSC-833, a nonimmunosuppressive derivative of cyclosporin D), and other agents have been found to modulate the MDR effect in vitro, but limited beneficial effects have been observed clinically.

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The use of a second agent to rescue normal cells may also permit the use of high doses of the first agent, which can overcome the resistance caused by a low rate of conversion to the active metabolite or a high rate of inactivation. Another way to overcome resistance is to follow marrow-lethal doses of chemotherapy by post-therapy infusion of stem cells obtained from the peripheral blood or bone marrow. This technique is effective for the treatment of lymphomas, chronic granulocytic leukemia, multiple myeloma, and a few other cancers. A more widely applicable technique may be to combine high-dose chemotherapy with blood cell growth factors, for example, granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GMCSF) or oprelvekin (interleukin [IL]-11) to stimulate platelets. These and other marrow-protective and marrow-stimulating agents are being used increasingly and may enhance the effectiveness of chemotherapy in the treatment of several types of cancer. High-dose therapy is discussed more extensively in Chapter 5.

C. Pharmacologic causes of resistance

Apparent resistance to cancer chemotherapy can result from poor tumor blood supply, poor or erratic absorption, increased excretion or catabolism, and drug interactions, all leading to inadequate blood levels of the drug. Strictly speaking, this result is not true resistance; but to the degree that the insufficient blood levels are not appreciated by the clinician, resistance appears to be present. The variation from patient to patient at the highest tolerated dose has led to dose modification schemes that permit dose escalation when the toxicities of the chemotherapeutic regimen are minimal or nonexistent as well as dose reduction when toxicities are great. This regulation is particularly important for some chemotherapeutic agents for which the dose response curve is steep or for patients who have genetically altered drug metabolism, such as can occur with irinotecan. Selection of the appropriate dose on the basis of predicted pharmacologic behavior is essential for some agents, not only to avoid serious toxicity but also to optimize effectiveness. This has been applied successfully to dose selection of carboplatin by predicting the time x concentration product (area under the curve) based on the individual patient's creatinine clearance.

True pharmacologic resistance is caused by the poor transport of agents into certain body tissues and tumor cells. For example, the central nervous system (CNS) is a site that many drugs do not reach well. Several drug characteristics favor transport into the CNS, including high lipid solubility and low molecular weight. For tumors that originate in the CNS or metastasize there, the drugs of choice should be those that achieve effective antitumor concentration in the brain tissue and that are also effective against the tumor cell type being treated.

D. Nonselectivity and resistance

Nonselectivity is not a mechanism for resistance but rather an acknowledgment that for most cancers and most drugs, the reasons for resistance and selectivity are only partially understood. Given a limited understanding of the biochemical differences between normal

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and malignant cells prior to the last 10 years, it is gratifying that chemotherapy has been as successful as frequently as it has. With the burgeoning of knowledge about the cancer cell, there is reason to hope that in 20 years, we will view current chemotherapeutic regimens as a fledgling if not crude beginning and will have found many more tumor molecular target directed agents that have a high potential for curing the human cancers that now resist effective treatment.

V. Molecular targeted therapy (MTT) introduction

MTT is a new approach to cancer treatment, which resulted from the plethora of molecular and biologic discoveries into the etiology of cancer that took place over the last quarter of a century. Many agents are currently being tested in clinical trials, a few have already been approved by the U.S. Food and Drug Administration (FDA) for clinical use, and their wide integration into the mainstream of therapy for cancer is expected to increase at an accelerated pace during the next 10 years.

Agents in this type of therapy are vastly different from the traditional chemotherapeutic agents that constitute most therapy described throughout the chapters of this book, in that they are designed with the intention to specifically target molecules that are uniquely or abnormally expressed within cancer cells, thereby sparing normal cells. This is possible because agents that qualify as molecularly targeted therapeutics take advantage of the special molecular characteristics of cancer cells to exert their mechanism of action. Within the remainder of this section, we will discuss drugs that are already available for clinical use, provide a brief description of the mechanism of action of these agents and the pathways they target, and address promising agents that may be coming soon to the clinic.

A. Characteristics and classification for MTT

• An ideal molecule for targeted therapy should have the following characteristics:

  • The molecule is uniquely expressed in cancer cells; hence the therapeutic agent will specifically target the cancer and not the normal cells.

  • The molecule is important for the maintenance of the malignant phenotype; therefore, once the target has been effectively hit, the cancer cell will not be able to develop resistance against the therapeutic agent by suppressing the function of or expelling the molecule from the cell.

  The degree to which target molecules do not embody these characteristics coupled with nonspecificity of the therapeutic agent determines in part the limitations of current targets and agents.

• Classification and type of MTT. The classification of MTT is a moving target. These therapies can be classified on the basis of the type of treatment, whether it is antibody based, small molecules, gene therapy etc., or based on the molecular target strategy of these agents. In this chapter, we will be using a combination of both approaches to classify these therapies.

B. Strategies for MTT development

• Function-directed therapy. This is a strategy that is intended to restore the normal function or abrogate the

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  abnormal function of the defective molecule or a pathway in the tumor cell. This is accomplished by reconstituting the normal molecule, inhibiting the production of a defective molecule, or aborting, altering, or reversing a newly acquired function. Some of the agents that will be discussed here under this category would target specific cellular pathways, for example, signal transduction pathway; angiogenesis, protein degradation, and immune modulators.

• Phenotype-directed therapy. This is a therapeutic strategy that is intended to target the unique phenotype of the cancer cell where killing is more dependent on nonspecific mechanism rather than targeting a specific pathway as outlined in Section V.B.1. in the preceding text. Such agents include monoclonal antibodies (MoAbs), immunotoxins, immunoconjugates, and vaccine therapy.

  In this chapter we will classify the agents based on the pathway that they target, if the molecular outcome of the mechanism of action is known; otherwise, the agent will be classified on the basis of the type of therapy.

VI. MTT molecular and functional mechanisms

A. Cell signaling targeted therapy

Targeting signal transduction is an important approach for therapy against cancer because the signal transduction pathways are crucial for delivering messages from the outside environment into the nucleus to enable it to carry on the crucial processes of survival of the cell including cell proliferation and differentiation. Many pathways are involved in signal transduction in the cell. These signals are initiated from the cell surface by the interaction of molecules (ligands) such as hormones, cytokines, and growth factors with cell receptors. These cell receptors, in turn, transfer the signal through a network of molecules to the nucleus that will lead to the transcription of new molecules responsible for engineering the desired outcome.

In cancer cells, these pathways are found to be altered through the mutation of some of their components. This leads to the dysregulation of the function of the pathways leading to uncontrolled proliferation and inhibition of apoptosis. Accordingly, targeting the components of these pathways is a prime goal for the development of MTT. The components of these pathways include the following:

• The ligand

• The receptors for these ligands most of which are kinase receptors

• The cascade of proteins that form these pathways, which are mainly protein kinases; other proteins are also present

Two of these pathways the phosphoinositide 3-OH kinase (PI3K) and the RAS-Raf-MAP kinase pathways are the most critical for the malignant transformation and most therapeutic interventions are being developed to target these pathways as will be discussed in the subsequent text.

Strategies that are followed to target signal transduction pathways include the following:

• Blocking of the ligand receptor binding: this leads to the inhibition of the initiation of the signal. This can be done by

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  either blocking circulating ligands or blocking ligand binding to the extracellular domain of the receptor. Many antibodies have been developed for this purpose and will be discussed in the subsequent text

• Inhibition of receptor protein kinases. This leads to the prevention of phosphorylation of the intracellular kinase domain of the receptor, hence aborting the cascade of protein reaction in the cell signaling pathways. This can be accomplished by blocking adenosine triphosphate (ATP) binding to the receptor

• Inhibition of intracellular signaling proteins

• Blocking of the ligand receptor binding. Blocking receptors and ligand-receptor interaction is currently achieved by utilizing specific MoAbs. MoAbs are biologic agents that are designed with the intention to specifically target membrane proteins that carry an extracellular domain. The MoAbs can exert their antitumor effect through multiple potential mechanisms including blocking the targeted receptor and preventing its function in transmitting proliferative signals to the nucleus, activating antibodydependent cellular cytotoxicity, or helping in internalizing the receptor and hence delivering toxic agents into the cells. The MoAb technology has been very much improved, in the last decade, by humanizing these biologic agents to form chimeric antibodies or, in some cases, fully humanized antibodies. Substitution with the human Fc portion of the molecule for the murine equivalent leads to significant decrease in the ability to generate human antimouse antibody (HAMA), although human antichimera antibodies (HACAs) may still occur for those MoAbs that are not fully humanized. This has made these biologic agents more usable in the treatment of cancer, particularly when repetitive dosing is needed. Here, this section will discuss MoAbs that are generated for specific signal transduction receptors. In Section VI.E., we will discuss those MoAbs that are generated against membrane nonreceptor antigens.

  • Epidermal growth factor receptor (EGFR) family. The EGFR is a family of proteins that includes at least four receptors: EGFR1, Her-2-neu (erbB2), Her3 (erbB3), Her4 (erbB4). These receptors are glycoproteins that consist of three domains: an extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain that contains tyrosine kinase activity. Binding of the ligands to the receptor leads to the activation of the intracellular tyrosine kinase and phosphorylation of the receptor, which, in turn, lead to downstream signal transduction pathway activation. The activation of this pathway leads to biologic effects that promote cell activation and proliferation and enhance survival. Agents have been developed against the first two receptors, EGFR and Her-2-neu, to treat cancer through the inhibition of this pathway. Some of these agents are antibodies that bind the external binding domain and inhibit the receptors, and others are small molecules that inhibit the tyrosine kinase activity of the internal domain.

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    • EGFR1-targeted therapy. EGFR1 is the first member of the EGFR family. It is activated by binding to epidermal growth factor (EGF) and to transforming growth factor alpha (TGF- ). EGFR1 is found to be overexpressed in many cancers including 50% to 70% of colon, lung, and breast cancer. Several antibodies are currently being developed to target EGFR; two have already been approved by the FDA for clinical use in patients with cancer:

      • Cetuximab is a humanized IgG1 chimeric MoAb that binds to the external ligand-binding domain of EGFR1. It binds to the EGFR with a much higher affinity than does either EGF or TGF- . It has been found that cetuximab can enhance the effect of both chemotherapy and radiation therapy. It has been found that the combination of cetuximab and irinotecan to treat patients with advanced colorectal carcinoma who express EGFR in their tumors and failed irinotecan therapy can improve disease response from10.8% to 22.9% and the disease-free survival from 1.5 to 4.1 months over the use of cetuximab alone. Accordingly, cetuximab was initially approved by FDA in combination with irinotecan to treat patients with advanced colon cancer who failed irinotecan or as a single agent in patients who cannot tolerate irinotecan. Many clinical trials are currently being conducted to test cetuximab in first-line therapy in combination with other chemotherapeutic regimens including FOLFOX4 (see Chapter 8). Clinical trials are also being conducted to test the efficacy of cetuximab in other diseases, the most advanced of which is in head and neck cancers for which cetuximab has also received FDA approval. Significant survival advantage has been demonstrated in one phase III trial adding cetuximab to high-dose radiation therapy in patients with locally advanced disease. Median survival was found to be 54 months in the combination arm versus 28 months in the radiation therapy alone arm. Other trials are currently being conducted, evaluating the addition of cetuximab to cisplatin in the metastatic setting.

      • Panitumumab is the only fully humanized MoAb that has been developed against EGFR. This gives it the advantage of being less likely to stimulate the development of anti-antibodies. Panitumumab was found to bind to EGFR with higher affinity than cetuximab. Serious toxicity has been reported only rarely, and as a consequence it does not require premedication for human use. Phase III trials of panitumumab have shown efficacy when given alone or in combination with chemotherapy in colorectal cancer with significant improvement in diseasefree survival (DFS). Other diseases in which there are some promising results with this agent include non small cell lung and renal cancers.

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      • EMD27000 is another antibody that is currently being developed against EGFR. It is a humanized IgG1 MoAb that has been found to have some activities seen in colon cancer in clinical trials.

    • Her-2-neu (HER2, erbB2)-targeted therapy. HER2 is the second member of the EGFR family. It is a 185-kd protein with 632 amino acids. It has the same basic structure as the other family members. However, no conjugate ligand has been identified for HER2. There have been no mutations identified in the HER2 gene in human cancers; however, it is overexpressed in many epithelial cancers including colon, pancreas, genitourinary, and 30% of breast cancers. HER2 works through the PI3K/Akt and MAPK pathway and its overexpression leads to inhibition of apoptosis and increase in cell proliferation.

      • Trastuzumab was one of the first MTTs to be introduced in clinical use. It is a humanized (chimeric) MoAb that binds the HER2 receptor. The FDA approved it in 1998 for the use in patients with metastatic breast cancer with tumors that overexpress the HER2 protein. In a large, multicenter phase III study in patients with metastatic breast cancer that overexpressed HER2, it was demonstrated that trastuzumab, when used as first-line therapy in combination with chemotherapy (with either the combination of anthracyclines and cyclophosphamide or paclitaxel as a single agent), can significantly increase both the duration of response and the overall survival. Trastuzumab is used in patients with breast cancer in two settings: as a single agent for second-line therapy or in combination with paclitaxel as firstline therapy. Furthermore, it has been found that trastuzumab is effective in the adjuvant setting for patients whose tumors overexpress HER2 protein. The optimal duration of treatment, however, is still undefined.

        Whereas the mechanism of action of trastuzumab is not entirely clear, it is believed to act through one or more of the following mechanisms: binding to the receptor, thereby inhibiting the tyrosine kinase signaling pathway; activating antibody-dependent cellular cytotoxicity; induction of apoptosis; inducing G1 arrest by modulating the cyclin-dependent kinases; inhibition of angiogenesis; and enhancing chemotherapy-induced antibody dependent cellmediated cytotoxicity (ADCC).

  • Vascular endothelial growth factor (VEGF). The VEGF family of proteins is one of the specific positive regulators of angiogenesis. It is composed of at least six different growth factors. Of these, the VEGF-A is the one

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    member that exerts the most influence on the angiogenesis process. The VEGF proteins bind to three tyrosinekinase receptors, VEGFR1 (vascular endothelial growth factor receptor1/Flt-1), VEGFR2 (KDR/Fetal liver kinase1, Flk-1) and VEGFR3 (Flt-4). Neuropilin-1 is a fourth receptor that can bind specifically to one of the VEGF isoforms, namely, VEGF165, which enhances its binding to VEGFR1. VEGFR2, through its interaction with VEGF, is thought to be the main mediator in tumor associated angiogenesis and metastatic processes whereas VEGFR1 plays a role in hematopoiesis. VEGF is expressed or overexpressed in many tumors including lung, breast, gastrointestinal stromal tumors (GISTs), ovarian, and in particular renal cancers, where the expression has been found to be high. Accordingly, targeting these molecules to abrogate their ability to stimulate tumor-associated angiogenesis constitutes a logical therapeutic strategy to control cancer. Both antibodies and small molecules have been developed as targeted therapies utilizing this pathway. Here we will discuss the antibodies. The small molecules will be discussed in the following section.

    • Bevacizumab is a humanized murine anti-VEGF MoAb. Its mechanism of action is by binding VEGF and blocking the binding to the VEGF receptors, and hence, inhibiting the tumor-induced angiogenesis process. Clinical trials have demonstrated that the addition of bevacizumab to standard chemotherapy can improve the outcome in advanced disease. In colon cancer, it has been shown that the addition of bevacizumab to fluorouracil and leucovorin improved time to progression from 5.2 to 9 months with an increase in response rate from 17% to 40%. Adding bevacizumab to irinotecan, fluorouracil and leucovorin (IFL) improved median survival from 15.6 to 20 months. The addition of bevacizumab to FOLFOX4 (see Chapter 8) also resulted in higher median survival using the combination. Bevacizumab as a single agent showed inferior results compared with standard chemotherapy. Currently the drug is approved by the FDA for use as first-line treatment for advanced colon cancer in combination with fluorouracil-based chemotherapy. The drug has also been shown to be effective in other tumors including non small cell lung cancer (NSCLC) where the addition of bevacizumab to the combination of paclitaxel and carboplatin showed higher response rate, longer disease-free survival, and median survival. Similar data was seen in advanced renal cell cancer. Trials are under way to test the effect of bevacizumab in many other tumors in both the advanced and the adjuvant setting. The use of bevacizumab has been shown to almost double the incidence of arterial thrombosis and to increase the incidence of hemorrhage and hypertension in certain cases. Hemoptysis seems to be a particular risk in squamous cell lung cancer.

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• Inhibition of receptor tyrosine kinases (RTKs). Kinases are enzymes that have the capability of attaching a phosphate moiety to another protein. This occurs on a side chain of a serine, threonine, or tyrosine moiety, by which these kinases are classified. The phosphorylation of proteins then regulates the behavior of these proteins whether with binding activity, enzymatic activity, or trafficking within the cell, or with their degradation. As a consequence, the phosphorylation process is a crucial biochemical reaction in controlling the behavior of a cell. Their critical role in cancer is shown by the observation that mutations in these kinases may lead to drastic outcomes including, in some instances, uncontrolled proliferation. Receptor serine/ threonine kinases will be discussed in another section; here we will discuss the RTKs.

  RTKs are a combination of families of receptors that share several structural and functional features. These kinases are glycoprotein receptors with extracellular, transmembrane, and intracellular domains. Whereas the transmembrane domain acts as an anchor for the receptor within the membrane of the cell, the extracellular domain contains a binding site for a specific multipeptide ligand, which, when it binds, initiates signaling events that are specific to the receptor. The cytoplasmic domain contains a catalytic protein tyrosine kinase region and a regulatory region, which are integral to the transmission of the signal downstream to the nucleus. The autophosphorylation of the receptor's kinase region initiates a cascade of signal transduction that leads to cell proliferation, survival/apoptosis, migration, adhesion, and promotion of angiogenesis. Some of the subfamilies in this group of receptors include the platelet-derived growth factor receptor (PDGFR), EGFR, VEGFR, fibroblast growth factor receptor (FGFR), and others. These RTKs are overexpressed or mutated in many human cancers. Therefore, the ability to target RTK activity is an attractive strategy for cancer therapy. A few small molecules have already been introduced into clinical practice and many others are currently in clinical trials. Here we will discuss some of these molecules:

  • Erlotinib is an orally available small molecule with the chemical structure of N-(-3-ethynylphenyl)-6,7-bis (2-methoxyethoxy)-4-quinazolinamine. This compound is a reversible inhibitor of EGFR and it exhibits its function through the inhibition of the intracellular phosphorylation of tyrosine kinase by competing with ATP in binding the intracellular domain of the tyrosine kinase region. Accordingly, it blocks signal transduction of the EGFR, leading to the inhibition of the downstream effect of the pathway including the inhibition of cell propagation and survival, and angiogenesis. Erlotinib is a highly selective inhibitor for EGFR tyrosine kinase region; concentrations that reach more than 1,000-fold higher are required for the inhibition of other tyrosine kinases. Erlotinib has been shown to be effective in few tumors. In phase III placebo-controlled clinical

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    trials in patients with locally advanced or metastatic NSCLC, erlotinib resulted in a median survival of 6.7 months versus 4.7 months when used as a secondline therapy. In pancreatic cancer, the addition of erlotinib to gemcitabine was found to improve median survival by 13.8 days over gemcitabine alone with increase in 1-year survival from 17% to 24%. Accordingly, erlotinib has been approved by the FDA in November 2005 for the treatment of patients with locally advanced or metastatic NSCLC as a second- or third-line therapy. It was also approved as a first-line therapy in combination with gemcitabine for locally advanced or metastatic pancreatic carcinoma. The most common toxicities included skin rash (12%) and diarrhea (5%). Erlotinib showed no major benefit when used as a first-line therapy in combination with platinum-based chemotherapy in advanced NSCLC. Clinical trials are currently being conducted in testing erlotinib in combination with other agents as first-line therapy for advanced NSCLC.

  • Gefitinib is another small molecule that is designed to effectively inhibit the tyrosine kinase domain of the EGFR. This compound initially showed an effect in randomized phase II trials with symptomatic improvement in advanced NSCLC. On the basis of these results, the drug was approved as a third-line therapy for this disease by the FDA in 2003. However, further placebo-controlled phase III studies as frontline showed no survival benefit. Accordingly, the drug is being re-evaluated by regulatory agencies. The drug had a new label approved by the FDA that states that the medicine can be used in patients with cancer who have already taken the medicine and whose doctor believes it is helping them.

  • Sunitinib is an ATP competitive inhibitor that leads to the inhibition of the phosphorylation of the kinase and further signal transduction in multiple RTKs. It functions as an inhibitor to a closely related family of RTK including PDGFR- and - , VEGFR, stem cell factor receptor KIT, Fms-like tyrosine kinase-3 receptor (FLT-3), and the RET oncoprotein. Accordingly, the antitumor effect of sunitinib is multifactorial. It inhibits cell proliferation and has an antiangiogenesis effect. The antiangiogenesis effect of sunitinib comes from the inhibition of both the VEGFR and PDGFR that is important for the recruitment of pericytes. Because of the inhibition of both these RTKs, this agent has a stronger inhibiting effect than those that target VEGF alone. Furthermore, KIT and PDGFR play an important role in the development of the stromal gastrointestinal tumors. Therefore, sunitinib will be expected to play a role in the inhibition of such tumors. Because angiogenesis is the hallmark of renal cell carcinoma that has been shown to have overexpression of VEGF and platelet-derived growth factor (PDGF), sunitinib would be expected to play a therapeutic role in this disease. A recent multinational phase III clinical trial comparing sunitinib to IFN- as a

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    first-line treatment in advanced renal cell carcinoma showed a major advantage in overall survival of 11 months as compared with 5 months for the IFN. Sunitinib has been approved by the FDA as first-line therapy for advanced renal cell cancers. Furthermore, more than 85% of GISTs possess activating mutations of the KIT kinase and another 5% are associated with mutation in the PDGFR. On the basis of its mechanism of action, sunitinib forms a natural candidate for the treatment of GIST. Clinical trials demonstrated efficacy in GIST patients who failed imatinib. An open-label multicenter phase I/II trial showed an overall survival of 19.8 months in patients who failed imatinib. It has been approved for GIST patients whose disease has progressed or who are unable to tolerate treatment with imatinib. Trials are being conducted to test sunitinib in breast and neuroendocrine tumors.

• Inhibition of intracellular signaling proteins and protein kinases. This mechanism is directed at a group of proteins that function in a network of communicating cascades to transfer the signal from the receptors into the nucleus to produce an intended biologic effect including, cell proliferation, apoptosis, angiogenesis, etc. When mutated, these proteins produce dysregulated pathways that cause the development of a transformation status of the cell. Most of these proteins are kinases nonreceptor tyrosine kinases or serine/threonine kinases. The non-receptor tyrosine kinases are cytoplasmic kinases, many of which are attached to and closely linked to membrane receptors, and they are usually activated by the binding of ligand to their associated receptors; some of these kinases include src, abl, and JAK. The serine/threonine tyrosine kinases are intracellular kinases, some of which play a crucial role in carcinogenesis. They include raf, Akt, and MEK. On the basis of this mechanism, small molecules designed to block or reverse the effect of these pathways have been developed, some of which are already in clinical use. In general, these targeted therapies that inhibit the intracellular signal proteins and protein kinases can act on multiple targets, some of which include receptor kinases; therefore, they can also be classified as receptor kinase inhibitors. For the sake of simplicity, this chapter will deal with those that primarily inhibit intracellular protein in this section and will allude to their other roles within the description of the drug.

  • Imatinib mesylate is one of the first targeted therapy small molecules to be used in clinical practice. It is primarily a protein kinase inhibitor that is designed to inhibit Bcr-Abl tyrosine kinase. The Bcr-Abl fusion protein is the product of the translocation between the Bcr and Abl 1 genes. The Abl 1 gene encodes a nonreceptor tyrosine kinase whereas the Bcr produces a serine/threonine kinase. The product of the translocation produces a phosphorylated protein that activates many pathways including the RAS, PI3K, and STAT, which leads to malignant transformation.

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    Through the inhibition of the Bcr-Abl tyrosine kinase, imatinib mesylate induces apoptosis in Bcr-Abl-positive cells. Imatinib mesylate binds Abl 1 and leads to inhibition of the active tyrosine kinase of the fusion protein. It has been found to be active against Bcr-Abl-positive CML and Bcr-Abl-positive acute lymphocytic leukemia (ALL). In early studies, it was found to produce superior results over the combination of IFN- and cytarabine in CML, and subsequent experience has shown that it produces both hematologic and cytogenetic remissions that are often long term and sustainable. Imatinib has also been found to inhibit the receptor kinases PDGF, stem cell factor, and cKIT. As noted in the preceding text, cKIT is mutated in 85% of GIST tumors. Imatinib was tested in and found effective in GIST. The current FDA-approved indications for imatinib include CML that is Philadelphia chromosome positive, whether newly diagnosed, in chronic phase, in accelerated phase or blast crisis, or after failure of IFN- therapy, or recurrence after stem cell transplant. It is also indicated in malignant cKITpositive GIST that is unresectable or metastatic. Studies are currently under way to evaluate the role of imatinib in the adjuvant setting in GIST patients; in addition, indications in other diseases are currently under testing. Clinically significant resistance to imatinib is being increasingly noted and has been found to occur in patients who develop mutations within the kinase domain in the Bcr-Abl proteins. Therefore, the need to develop alternatives is very important. Some of the alternative kinase inhibitors are discussed in the subsequent text.

  • Dasatinib is an oral inhibitor of multiple tyrosine kinases including Bcr-Abl. Other families of kinases that it inhibits include cKIT and PDGFR. Clinical data with dasatinib showed that 31% to 38% of imatinib-resistant and 75% of imatinib-intolerant patients with chronic phase CML reached major cytogenetic response. In addition, 30% to 59% of patients with advanced CML and Ph+ ALL showed major hematologic response. No clinical data are available yet to show increase in survival. It is indicated for patients with chronic, accelerated, and myeloid or lymphoid blast phase of CML who are intolerant or resistant to prior therapy with imatinib. It is also indicated in patients with Ph+ ALL.

  • Nilotinib is another Abl kinase inhibitor. Similar to imatinib, it acts by competing with the ATP-binding site of Bcr-Abl. Nilotinib differs from imatinib by having a higher binding activity to Abl kinase with higher inhibitory activity in imatinib-sensitive cell lines. In recently published studies, nilotinib was found to induce both hematologic and cytologic responses in Ph+ CML patients who are resistant to imatinib.

  • Sorafenib is a drug used for the treatment of advanced renal cell cancer. It is a small molecular inhibitor of C-Raf kinase that leads to the inhibition of the Raf/MEK/ERK signaling pathway. The Raf protein is a serine/threonine kinase, it is part of the Ras pathway, and it gets activated

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    when Ras, in response to the activation of an RTK, recruits and phosphorylates Raf kinase at the membrane site. Raf, in turn, phosphorylates the MEK that activates and phosphorylates ERK. ERK then enters the nucleus where it activates many other transcription factors leading to cellular proliferation. Aberration in this pathway leads to deregulation of proliferation, resulting in transformation of the cell. Raf has been found to be mutated in many tumors. Therefore, inhibition of this kinase is a reasonable target in cancer treatment. Sorafenib has also been found to be a strong inhibitor of both VEGFR-2 and PDGF kinase. In a large, phase III study, interim results showed that Sorafenib can reduce the risk of death by 23% compared with placebo in advanced renal cell carcinoma.

B. Angiogenesis-targeted therapy

Angiogenesis is a biologic process that is crucial for the development of tumors. Tumors have exploited this physiologic process to provide the milieu to permit the growth of both primary and metastatic cancers. Although the antineoplastic effect of antiangiogenesis therapy is mediated through its effect on the environment for the cancer cell growth, the initial mechanism of current therapies is based on molecular targeting, which is described in Sections VI.A.1.b and VI.A.2.

C. Protein degradation targeted therapy

Protein degradation is one of the mechanisms by which cell function is regulated. The ubiquitin proteosome pathway plays a very important role in this regard. The proteosome is a large complex of proteins that degrades other proteins after being tagged with a ubiquitin chain. It exerts its degradation capability through coordinated catalytic activities of its three proteolytic sites that lead to chymotryptic, tryptic, and post glutamyl peptide hydrolytic-like activities. Many key proteins in cell cycle, apoptosis, and angiogenesis pathways are regulated by degradation, including the p⁵³, p²¹, p²⁷ (important cell cycle) proteins; NF- B, a key transcription factor that is activated by the proteosomes, translocates to the nucleus and leads to the transcription of many crucial proteins including cytokines; and ICAM-1, VCAM, and E selectin (cell adhesion molecules).

• Bortezomib is a dipeptidyl boronic acid derivative that inhibits the 26S proteosome, the principal regulator of the intracellular protein degradation. It is the first of its class to be approved for clinical use. Bortezomib can selectively inhibit the chymotryptic site of the proteosome. This leads to a selective inhibition of the degradation of proteins involved in cell proliferation and survival regulation, and, as a consequence, apoptosis is induced. Bortezomib has been found to be effective, particularly in myeloma. A phase III trial that randomized patients with myeloma, who failed one to three previous therapies, to bortezomib versus high-dose dexamethasone has found that bortezomib resulted in a superior outcome with respect to response frequency, time to progression, and overall survival. Currently bortezomib is approved by the FDA for the treatment of patients with multiple myeloma who have received at least one prior therapy.

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  Active research to identify other active proteosome inhibiting agents is ongoing.

D. Immune modulation targeted therapy

Immune modulators (IMiDs) are a new family of medications that are derivatives of thalidomide and known to be immunomodulatory drugs. These compounds are generated by minor structural modifications on thalidomide that leads to enhancement of its efficacy and improvement in the side effect profile including the neurologic toxicity and prothrombotic effects of thalidomide. The mechanism of action of this group of compounds is not clearly defined. Many pathways have been shown to be triggered by these medications including caspase-8, proteosome, NF B, and the antiangiogenesis pathways.

• Lenolidomide is one of the new-generation immunomodulators. It has been found to be active in refractory multiple myeloma. In one phase II clinical trial, the response rate reached 24% with more than 50% reduction in monoclonal protein in patients with relapsed or refractory disease. When combined with dexamethasone in newly diagnosed multiple myeloma patients, 91% of patients achieved an objective response including 11% with complete response. Compared with thalidomide, it showed no significant somnolence, constipation, or neuropathy. It is approved for the use of patients with multiple myeloma in combination with dexamethasone in patients who received at least one prior therapy, and in patients with myelodysplastic syndrome with 5q deletion who are transfusion dependent.

E. Phenotype-directed targeted therapy

• Non receptor protein-directed MoAbs. These are a group of antibodies that are developed to recognize specific antigens on the surface of cancer cells, not for the purpose of blocking a specific pathway or receptor proteins, but rather to induce direct cytotoxic effect. These MoAbs may be used alone or as a delivery system for cellular toxins, radionuclides, or chemotherapy.

  • Unconjugated antibodies

    • Rituximab is an IgG1- murine human chimeric MoAb that is generated against the CD20 antigen. CD20 is expressed on the cell surface of the B cells and hence on the surface of B-cell lymphoma. Rituximab is indicated for the treatment of relapsed or refractory B-cell non Hodgkin's lymphoma and chronic lymphocytic leukemia that expresses CD20 marker. Rituximab is also being increasingly used in combination with chemotherapy (e.g., cyclophosphamide, vincristine, doxorubicin, and prednisone), particularly in the more aggressive non Hodgkin's lymphomas.

    • Alemtuzumab is a humanized IgG1- murine human chimeric MoAb that is directed against CD52 cell surface glycoprotein. CD52 is expressed on the surface of normal and malignant B and T cells, natural killer cells, monocytes, and macrophages. Alemtuzumab is indicated for the treatment of B-cell chronic lymphocytic leukemia in patients who have failed fludarabine. Patients who have recently been treated

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      with this MoAb should not receive any live viral vaccines because of the immune suppression effect of the medication.

  • Cellular toxin conjugated antibodies

    • Gemtuzumab ozogamicin is a humanized IgG4- antibody against the CD33 antigen conjugated with calicheamicin. Calicheamicin is a cytotoxic agent that is isolated from fermentation of the bacterium Micromonospora echinospora ssp calichensis. The CD33 antigen is a sialic acid dependent adhesion protein that is expressed on the surface of immature cells of the myelomonocytic lineage and the surface of leukemic blast cells but not on the normal pluripotent hematopoietic stem cells. When this fusion antibody binds to the CD33 receptors, it gets internalized into the cell, after which the calicheamicin is cleaved and released. The calicheamicin in turn binds to the minor grooves of the DNA, leading to DNA breaks and apoptosis.

      Gemtuzumab is indicated for the treatment of the first relapse of myeloid leukemia that expresses CD33 in older patients (more than 60 years old) who are not candidates for chemotherapy. Clinical trials have shown that, when given as single agent, gemtuzumab may lead to 16% complete response and 30% overall response with a median time to remission of 60 days.

  • Radioimmunoconjugate antibodies

    • Ibritumomab tiuxetan (Zevalin, IDEC-Y2B8) is a murine monoclonal anti-CD20 antibody conjugated to tiuxetan that chelates to the pure -emitting yttrium 90 (⁹⁰Y). The mechanism of action includes antibodymediated cytotoxicity and cellularly targeted radiotherapy (radioimmunotherapy [RIT]). It is indicated for use in non Hodgkin's lymphoma that is follicular, B-cell CD20 positive, and rituximab refractory. Experimentally it is used in non Hodgkin's lymphoma that has relapsed or is refractory to other agents, but not refractory to rituximab. It should be used with caution in patients with 25% or more marrow involvement with lymphoma, prior external beam radiotherapy to 25% or more of the bone marrow, or a history of HAMAs or HACAs. Because the drug does not emit radiation, hospitalization is not required. Neutropenia and thrombocytopenia are common and are related to the radionuclide dose. At the higher end of the dosing, 25% of patients will develop nadir neutrophil counts of less than 500/ L. Low-grade nausea and vomiting are common. Infusion-related fever, chills, dizziness, asthenia, headache, back pain, arthralgia, and hypotension are occasional.

    • Iodine 131 (¹³¹I)-Tositumomab (Bexxar). ¹³¹I-Tositumomab is a murine IgG2a monoclonal anti-CD20 antibody radiolabeled with ¹³¹I, an emitter of both and radiation. The mechanism of action includes antibody-mediated cytotoxicity and cellularly targeted

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      RIT. It is indicated in non Hodgkin's lymphoma, which is chemotherapy refractory, CD20 positive, low grade, or transformed low grade. Before dosimetric and therapeutic doses, patients are premedicated with acetaminophen 650 mg and diphenhydramine 50 mg. A saturated solution of potassium iodide, two to three drops orally three times daily, is given beginning 24 hours before the dosimetric dose and continuing for 14 days after the therapeutic dose to prevent uptake of ¹³¹I by the thyroid. It must be used cautiously in patients with 25% marrow involvement with lymphoma, prior external beam radiotherapy to 25% of the bone marrow, or a history of HAMAs or HACAs.

• Immunotoxins

  • Denileukin diftitox (Ontak). Denileukin diftitox is a recombinant construct that includes a fragment of the IL-2 protein (Ala₁- Thr₁₃₃) linked to a fragment of the diphtheria toxin fragment A and B (Met₁-Thr₃₈₇). This construct is designed to bind to the CD25 component of the IL-2 receptor (IL-2R) on the surface of the targeted cells that express the receptor; in turn, the complex becomes internalized into the cytoplasm and releases the toxin to exhibit its damaging effect. The high-affinity IL-2R is normally present on the activated T and B lymphocytes and activated macrophages. However, cutaneous T-cell lymphoma (CTCL) also expresses high-affinity IL-2R, which forms an appropriate target.

In two different clinical studies, investigators have shown that 30% of patients with CTCL demonstrate clinical response, including approximately 10% complete response. Therefore, the indications for this agent include persistent or recurrent CTCL that expresses the IL-2R CD25.

VII. Other biologic therapies

A. Bone marrow supportive agents

• Erythrocyte growth factors

  • Epoetin is a recombinant growth factor, identical to endogenous human erythropoietin, which promotes the proliferation and differentiation of committed erythroid precursors. It is indicated to be used in patients with nonhematologic malignancies who have chemotherapyinduced anemia to minimize transfusion requirements during therapy. It is not indicated in these patients when they have anemia due to other causes such as iron deficiency, bleeding, or hemolysis. Erythropoietin takes time to work, and a decision on the effectiveness should not be made before 4 to 8 weeks. If transfusion requirement does not change after 8 weeks, the dose may be increased. The dose should be withheld if the hematocrit increases to more than 40% and resumed with 25% reduction when the hematocrit is reduced to 36%. If the patient fails to show improvement after increasing the dose to 60,000 U (900 U/kg)/week, it is unlikely that the patient will respond and other causes of the anemia should be looked for carefully. Rarely it may

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    induce pure red cell aplasia with associated neutralizing antierythropoietin antibodies.

  • Darbepoetin is an erythropoiesis-stimulating protein closely related to erythropoietin that has a threefold longer terminal half-life than epoetin. Its indications are similar to epoetin.

• Myeloid monocytic growth factors

  • G-CSF (filgrastim). G-CSF is a 175 amino acid growth factor with proliferative activity for bone marrow progenitors committed to the neutrophil line. As discussed elsewhere in this volume, G-CSF is widely used in the setting of cytotoxic chemotherapy for solid tumors and leukemia to accelerate recovery of neutrophils and lessen the risk of bacterial infection. Filgrastim is indicated under the following conditions:

    • The likelihood of first-cycle febrile neutropenia is 20% or higher. It should be considered when the risk is 10% to 20%, depending on patient and cancer-related factors.

    • Further cycles of chemotherapy are needed after an occurrence of febrile neutropenia, and maintenance of dose intensity rather than dose reduction is appropriate.

    • High-dose chemotherapy followed by peripheral blood stem cell or autologous bone marrow support has been used.

    • The patient has established febrile neutropenia, and when the infection is life threatening or is expected to require prolonged antibiotic or antifungal therapy.

    Granulocyte macrophage colony-stimulating factor (sargramostim). GM-CSF is a 127 amino acid growth factor that exhibits its predominant proliferative effects on multipotent stem cells, inhibits neutrophil migration, potentiates the functions of neutrophils and macrophages, and results in production of a spectrum of cytokines from these activated cells. GM-CSF is used mainly for the following indications: to shorten neutrophil recovery time after induction therapy in acute myelogenous leukemia and accelerate myeloid recovery after bone marrow transplantation.

• Megakaryocyte growth factor; IL-11 (oprelvekin). IL-11 is a 177 amino acid growth factor that is a member of the same family of growth factors as G-CSF. IL-11 is a thrombopoietic growth factor that stimulates the proliferation of megakaryocyte progenitor cells and induces megakaryocyte maturation, leading to increase in platelets. Therefore, it is indicated in the acceleration of the recovery of platelets after cytotoxic chemotherapy and has been approved for clinical use for that purpose.

B. Other biologic therapy

• IL-2 (Proleukin). IL-2 is a cytokine that is secreted by activated T cells. IL-2 binds to a specific cell surface receptor on activated T lymphocytes and leads to T-cell proliferation. In addition, IL-2 can also activate natural killer cells. Through these mechanisms and perhaps

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  others, IL-2 has been found to exhibit antitumor properties. In clinical trials, it has been found that IL-2 has antitumor activity when used alone in high doses in patients with renal cell carcinoma and malignant melanoma. High-dose IV therapy with IL-2 given as a single agent has received FDA approval for the treatment of patients with metastatic renal cell carcinoma and metastatic melanoma. Careful selection of patients for such an intensive therapy is mandatory, especially for the cardiopulmonary status. This therapy is associated with significant toxicity and should be administered only by physicians experienced in its use.

• IFN- (IFN- 2a, IFN- 2b). The IFNs are a family of small molecular weight proteins and glycoproteins that are secreted by activated T cells and other cells secondary to viral infection. There are three major types of IFNs: , and . IFN- is produced by T cells, B cells, and macrophages when exposed to the appropriate antigens, IFN- is produced by fibroblasts when exposed to viral infection, and IFN- is produced by T cells after stimulation with IL-2 or specific or nonspecific antigens. IFN- is the only one of the IFNs that is approved by the FDA for human cancer therapy. Approved indications include hairy cell leukemia, melanoma with high risk of recurrence after resection, follicular lymphoma as initial treatment for the aggressive types, in combination with anthracyclines, and acquired immune deficiency syndrome related Kaposi's sarcoma.

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