Therapeutic Ratio

The most fundamental of these concepts is what is termed the therapeutic ratio, in essence a risk-versus-benefit approach to planning a radiotherapy treatment regimen. Many of the radiobiologic phenomena to be discussed in this chapter are thought to play important roles in optimizing, or at least fine-tuning, the therapeutic ratio. In theory, it should be possible to eradicate any and all malignant tumors simply by delivering sufficiently high doses of radiation. Of course, in practice, the biologic consequences for normal tissues that are necessarily irradiated along with the tumor tissue limit the total dose that can be safely administered. As such, a balance must be struck between what is deemed an acceptable probability of a radiation-induced complication in a normal tissue and the probability of tumor control. Ideally, one would hope to achieve the maximum likelihood of tumor control that does not produce unacceptable normal tissue damage.

The concept of therapeutic ratio is best illustrated graphically, by making a direct comparison of dose-response curves for both tumor control and normal tissue complication rates plotted as a function of dose. Examples of this approach are shown in Figure 1-1, for cases in which the therapeutic ratio is either “unfavorable,” “favorable,” or “optimal,” bearing in mind that these are theoretical curves. Actual dose-response curves derived from experimental or clinical data are much more variable, particularly for tumors, which tend to show much shallower dose responses.¹ This serves to underscore how difficult it can be in practice to assign a single numerical value to the therapeutic ratio in any given situation.

Figure 1-1 The graphs illustrate the concept of therapeutic ratio under conditions in which the relationship between the normal tissue tolerance and tumor control dose-response curves is unfavorable (A), favorable (B), and optimal (C).

Many of the radiobiologic properties of cells (and, therefore, tissues) can have either a favorable or an adverse effect on the therapeutic ratio. Therefore, in planning a course of radiation therapy, the goal should be to optimize the therapeutic ratio as much as possible, in other words, using our graphical approach, increase the separation between the tumor control and the normal tissue complication curves. This can be accomplished either by shifting the tumor control curve to the left with respect to the dose axis (toward lower doses for the same level of tumor control, that is, tumor radiosensitization) or by shifting the normal tissue complication curve to the right (toward higher doses for the same level of normal tissue complications, that is, normal tissue radioprotection) or perhaps a combination of both. The key is to shift these curves differentially, not necessarily an easy task given that there are precious few exploitable differences in the radiobiology of cells derived from tumors and those derived from normal tissues.

Radiation Biology “Continuum”

There is a surprising continuity between the physical events that occur in the first picosecond (or less) after ionizing radiation interacts with biologic material and the ultimate consequences of that interaction on tissues. The consequences themselves may not become apparent until days, weeks, months, or even years after the radiation exposure. Some of the important steps in this radiobiology “continuum” are listed in Table 1-1. The orderly progression from one stage of the continuum to the next—from physical to physicochemical to biochemical to biologic—is particularly noteworthy not only because of the vastly different time scales over which the critical events occur, but also because of the increasing biologic complexity associated with each of the endpoints or outcomes. Each stage of the continuum also offers a unique radiobiologic window of opportunity: the potential to intervene in the process and thereby modify all the events and outcomes that follow.

TABLE 1-1 Stages in the Radiobiology Continuum

Levels of Complexity in Radiobiologic Systems

Another important consideration in all radiobiologic studies is the nature of the experimental system used to study a particular phenomenon, the assays used, and the endpoints assessed. For example, one investigator may be interested in studying DNA damage caused by ionizing radiation and, in particular, the frequency of DNA double-strand breaks (DSBs) produced per unit dose. As an experimental system, he or she might choose DNA extracted from mammalian cells. Then, using a DNA elution assay, the rate at which the irradiated DNA passes through a semipermeable membrane is measured as an endpoint and compared with the rate of elution of DNA extracted from cells that had not been previously irradiated. DNA containing more DSBs elutes faster than DNA containing fewer breaks, allowing a calibration curve to be generated that relates the dose received to the elution rate.

A second investigator, on the other hand, may be interested in improving the control rate of head and neck cancers with radiation therapy by employing a nonstandard fractionation schedule. In this case, the type of experiment would be a clinical trial. The “experimental system” would be a cohort of patients, some of whom are randomized to receive nonstandard fractionation, and the rest, standard fractionation. The endpoints assessed could be one or more of the following: locoregional control, long-term survival, disease-free survival, or normal tissue complication frequency, evaluated at specific times after completion of the radiation therapy.

In considering both the strengths and weaknesses of these two investigators’ studies, any number of pertinent questions may be asked. Which is the more complex or heterogeneous system? Which is the more easily manipulated and controlled system? Which is more relevant for the day-to-day practice of radiation therapy? What kinds of results are gleaned from each, and can these results be obtained in a timely manner? In this example, it is clear that human patients with spontaneously arising tumors represent a far more heterogeneous and complex experimental system than an extract of mammalian DNA. On the other hand, the DNA system is much more easily manipulated, possible confounding factors can be more easily controlled, and the measurement of the desired endpoint (elution rate) plus the data analysis can be completed within a day or two. Obviously, this is not the case with the human studies, where numerous confounding factors can and do influence results, manipulation of the system can be difficult if not impossible, and the experimental results typically take years to obtain.

The issue of relevance is an even thornier one. Arguably, both studies are relevant to the practice of radiation therapy insofar as the killing of cells is at the root of normal tissue and tumor toxicity caused by radiation exposure, and as cell killing usually is, either directly or indirectly, a consequence of irreparable damage to DNA. As such, any laboratory findings that contribute to the knowledge base of radiation-induced DNA damage are relevant. Clearly, however, clinical trials with human patients are not only a more familiar experimental system to clinicians but, also, their efficacy in cancer patients is, ultimately, the “gold standard” against which all new therapeutic strategies are judged.

All things considered, then, there is a time and place for both relatively simple systems and more complex ones. The relatively simple, homogeneous, and easily manipulated systems are best suited for the study of the mechanisms of radiation action, such as measuring DNA or chromosomal damage, changes in gene expression, perturbations of the cell cycle, or the clonogenic survival of cells maintained in culture. The more complicated and heterogeneous systems, with their unique endpoints, are more clinically relevant, such as assays of tumor control or normal tissue complication rates. Both types of assay systems have inherent strengths and weaknesses, yet both are critically important for hoped-for improvement in the practice of radiation therapy based on sound biologic principles.

Tissue Heterogeneity

Why is radiation therapy successful at controlling one patient’s tumor but not another’s, even when the two tumors seem identical? Why are we generally more successful at controlling certain types of cancers than others? The short answer to such questions is that although the tumors may appear identical “macroscopically,” their component cells may be quite different phenotypically and/or genotypically. Also, there may be important differences between the two patients’ normal tissues.

Normal tissues, being composed of more than one type of cell, are somewhat heterogeneous, and tumors, owing both to the genetic instability of individual tumor cells and to microenvironmental differences, are very heterogeneous. Different subpopulations of cells have been isolated from many types of human and experimental cancers, and these may differ in antigenicity, metastatic potential, sensitivity to radiation therapy and chemotherapy, and so on.2,3 This heterogeneity is manifest within a particular patient, and to a much greater extent, between patients with otherwise similar tumors.

Both intrinsic and extrinsic factors contribute to this heterogeneity. Intrinsic factors may include the following: inherent radiosensitivity, gene expression, biochemical repair processes, modes of cell death (clonogenic versus apoptotic, for example), genomic instability, cell cycle kinetics, and the structural and functional arrangement of the tissue. Extrinsic factors, on the other hand, tend to be related to physiologic differences between tissues, such as the degree of vascularity, availability of oxygen and nutrients, pH level, energy charge, and proximity of, and degree of contact between, normal host tissue and the tumor.

What are the practical implications of normal tissue and tumor heterogeneity? First, if one assumes that normal tissues are the more uniform and predictable in behavior of the two, then tumor heterogeneity is responsible, either directly or indirectly, for most radiotherapy failures. If so, this suggests that a valid clinical strategy might be to identify the radioresistant subpopulation(s) of tumor cells and then tailor therapy specifically to cope with them. This approach is much easier said than done. Some prospective clinical studies now include one or more pretreatment determinations of, for example, the extent of tumor hypoxia⁴ or the potential doubling time of tumor clonogens⁵ as criteria for assigning patients to different treatment groups.

Another consequence of tissue heterogeneity is that any radiobiologic endpoint measured in an intact tissue is necessarily related to the radiosensitivities of all the subsets of cells, plus all the other intrinsic and extrinsic factors contributing to the overall response of the tissue. And, because data on normal tissue tolerances and tumor control probabilities are also averaged among a number of patients, heterogeneity is even more pronounced.

“Powers of Ten”

Tumor control is achieved only when all clonogenic cells are killed or otherwise rendered unable to sustain tumor growth indefinitely. To estimate the likelihood of cure, it is necessary to know—or at least have an appreciation for—approximately how many clonogenic cells the tumor contains, how radiosensitive these cells are (i.e., some measure of killing efficiency per unit radiation dose), and what the relationship is between the number of clonogenic cells remaining after treatment and the probability of recurrence. The latter is perhaps the easiest to ascertain, given our knowledge of both the random and discrete nature of radiation damage and the general shape of dose-response curves for mammalian cells and tissues (approximately exponential for multifraction irradiation).

For a given number of surviving cells per tumor, the probability of local control can be derived from Poisson statistics using the equation P = e⁻ⁿ, where P is the tumor control probability and n is the average number of surviving clonogenic tumor cells. For example, when, for a large number of tumors, an average of two clonogenic cells remain per tumor at the end of radiation therapy, the tumor control rate will be about 10%, that is, that nine out of ten tumors of the same size and relative radiosensitivity will recur. Should the treatment reduce clonogenic cell numbers to an average of 0.1 per tumor, the tumor control probability would increase to 90%; for 0.05 surviving cells per tumor, control would be 95%; and for 0.01 surviving cells per tumor, control would be 99%.

The tumor control probability for a given fraction of surviving cells is not particularly helpful if the total number of cells at risk is unknown, however, and this is where an understanding of logarithmic relationships and exponential cell killing is useful. Based on the resolution of existing tools and technology for cancer detection, let us assume that a 1-cm³ (1-g) tumor mass can be identified reliably. A tumor of this size has been estimated to contain approximately 10⁹ cells,⁶ admittedly a theoretical value that assumes all cells are perfectly “packed” and uniformly sized and that the tumor contains no stroma. A further assumption, that all such cells are clonogenic (rarely, if ever, the case), suggests that at least 9 logs of cell killing would be necessary before any appreciable tumor control (about 37%) would be achieved, and 10 logs of cell killing would be required for a high degree of tumor control (i.e., 90%).

After the first log or two of cell killing, however, some tumors respond by shrinking, a partial response. After two to three logs of cell killing, the tumor may shrink to a size below the current limits of clinical detection, that is, a complete response. Although partial and complete responses are valid clinical endpoints, a complete response does not necessarily mean tumor cure. At least six more logs of cell killing would be required before any significant probability of cure would be expected. This explains why radiation therapy is not halted if the tumor “disappears” during the course of treatment; this concept is illustrated graphically in Figure 1-2.

Figure 1-2 The relationship between radiation dose and tumor cell survival during fractionated radiotherapy of a hypothetical 1-g tumor containing 10⁹ clonogenic cells. Although a modest decrease in cell surviving fraction can cause the tumor to shrink (i.e., partial response) or disappear below the limits of clinical detection (i.e., complete response), few cures would be expected until at least 9 logs of clonogenic cells have been killed. In this example, a total dose of at least 60 Gy delivered as daily 2-Gy fractions is required to produce a tumor control probability of 0.37, assuming each dose reduced the surviving fraction to 0.5.

Adapted from Steel G, Adams G, Peckham M, editors: The Biological Basis of Radiotherapy, New York, 1983, Elsevier.

Finally, it should be noted that while the goal of curative radiation therapy is to reduce tumor cell survival by at least nine logs, even for the smallest tumor likely to be encountered, it is much less clear how many logs of cell killing a particular normal tissue can tolerate before it loses its structural and/or functional integrity. This would depend on how the tissue is organized structurally, functionally, and proliferatively; which constituent cells are the most and least radiosensitive; and which cells are the most important to the integrity of the tissue. It is unlikely, however, that many normal tissues could tolerate a depletion of two logs (99%) of their cells, let alone nine or more logs.

The Interaction of Ionizing Radiation with Biologic Materials

As mentioned in the introductory section of this chapter, ionizing radiation deposits energy as it traverses the absorbing medium through which it passes. The most important feature of the interaction of ionizing radiation with biologic materials is the random and discrete nature of the energy deposition. Energy is deposited in increasingly energetic packets referred to as “spurs” (100 eV or less deposited), “blobs” (100 to 500 eV), or “short tracks” (500 to 5000 eV), each of which can leave from approximately three to several dozen ionized atoms in its wake. This is illustrated in Figure 1-4, along with a segment of (interphase) chromatin shown to scale. The frequency distribution and density of the different types of energy deposition events along the track of the incident photon or particle are measures of the radiation’s linear energy transfer or LET (see also the “Relative Biologic Effectiveness” section, later). Because these energy deposition events are discrete, it follows that although the average amount of energy deposited in a macroscopic volume of biologic material may be rather modest, the distribution of this energy on a microscopic scale may be quite large. This explains why ionizing radiation is so efficient at producing biologic damage; the total amount of energy deposited in a 70-kg human that will result in a 50% probability of death is only about 70 calories, about as much energy as is absorbed by drinking one sip of hot coffee.²⁷ The key difference is that the energy contained in the sip of coffee is uniformly distributed, not random and discrete.

Figure 1-4 Hypothetical alpha particle track through an absorbing medium, illustrating the random and discrete energy-deposition events along the track. Each event can be classified according to the amount of energy deposited locally, which determines how many ionized atoms will be produced. A segment of a DNA double helix is shown approximately to scale.

Adapted from Goodhead D: Physics of radiation action: microscopic features that determine biological consequences. In Hagen U, Harder D, Jung H, et al, editors: Radiation research 1895-1995, proceedings of the 10th international congress of radiation research, vol. 2. congress lectures, Wurzburg, 1995, Universitatsdruckerei H Sturtz, p 43.

Those biomolecules receiving a direct hit from a spur or blob, receive, relatively speaking, a huge radiation dose, that is, a large deposition of energy in a very small volume. For photons and charged particles, this energy deposition results in the ejection of orbital electrons causing the target molecule to be converted first into an ion pair and then into a free radical. Further, the ejected electrons—themselves energetic charged particles—can go on to produce additional ionizations. For uncharged particles such as neutrons, the interaction is between the incident particles and the nuclei of the atoms in the absorbing medium, causing the ejection of recoil protons (charged) and lower-energy neutrons. The cycle of ionization, free radical production, and release of secondary charged particles continues until all the energy of the incident photon or particle is expended. These interactions are complete within a picosecond after the initial energy transfer. After that time, the chemical reactions of the resulting free radicals predominate the radiation response (discussed later).

Any and all cellular molecules are potential targets for the localized energy deposition events that occur in spurs, blobs, or short tracks. Whether the ionization of a particular biomolecule results in a measurable biologic effect depends on a number of factors, including how probable a target the molecule represents from the point of view of the ionizing particle, how important the molecule is to the continued health of the cell, how many copies of the molecule are normally present in the cell and to what extent the cell can react to the loss of “working copies,” and how important the cell is to the structure or function of its corresponding tissue or organ. DNA, for example, is obviously an important cellular macromolecule and one that is present only as a single double-stranded copy. On the other hand, other molecules in the cell may be less crucial to survival yet are much more abundant than DNA, and therefore have a much higher probability of being hit and ionized. The most abundant molecule in the cell by far is water, comprising some 80% to 90% of the cell on a per weight basis. The highly reactive free radicals formed by the radiolysis of water are capable of adding to the DNA damage resulting from direct energy absorption by migrating to the DNA and damaging it indirectly. This mechanism is referred to as “indirect radiation action” to distinguish it from the aforementioned “direct radiation action.”²⁸ The direct and indirect action pathways for ionizing radiation are illustrated below.

Direct effect

Indirect effect

The most highly reactive and damaging species produced by the radiolysis of water is the hydroxyl radical (^•OH), although other free radical species are also produced in varying yields.^29,30 Ultimately, it has been determined that cell killing by indirect action constitutes some 70% of the total damage produced in DNA for low LET radiation.

How do the free radicals produced by the direct and indirect action of ionizing radiation go on to cause the myriad lesions that have been identified in irradiated DNA? Because they contain unpaired electrons, free radicals are highly reactive chemically and will undergo any number of reactions in an attempt to either acquire new electrons or rid themselves of remaining unpaired ones. These reactions are considered quite slow compared with the speed of initial ionization events but are still quite fast relative to normal enzymatic processes in a typical mammalian cell. For all intents and purposes, free radical reactions are complete in less than a second after irradiation.

The ^•OH radical is capable of both abstraction of hydrogen atoms from other molecules and addition across carbon-carbon or other double bonds. More complex macromolecules that have been converted to free radicals can undergo a series of transmutations in an attempt to rid themselves of unpaired electrons, many of which result in the breakage of nearby chemical bonds. In the case of DNA, these broken bonds may result in the loss of a base or an entire nucleotide or in frank scission of the sugar phosphate backbone, involving one or both DNA strands. In some cases, chemical bonds are first broken but then rearranged, exchanged, or rejoined in inappropriate ways. Bases in DNA may be modified by the addition of one or more hydroxyl groups (e.g., the base thymine converted to thymine glycol), pyrimidines may become dimerized, and/or the DNA may become cross-linked to itself or to associated protein components. And again, because the initial energy deposition events are discrete, the free radicals produced also are clustered and therefore undergo their multiple chemical reactions and produce multiple damages in a highly localized area. This has been termed the “multiply-damaged site”³¹ or “cluster”³² hypothesis. Examples of the types of damage found in irradiated DNA are shown in Figure 1-5.

Figure 1-5 Types of DNA damage produced by ionizing radiation. A, Segment of irradiated DNA containing single- and double-strand breaks, cross-links, and base damage. B, Two types of modified bases observed in irradiated DNA include thymine glycol, which results from the addition of two hydroxyl (OH) groups across the carbon-carbon double bond of thymine, and 8-hydroxyguanine, produced by ^•OH radical addition to guanine.

Biochemical Repair of DNA Damage

DNA is unique insofar as it is the only cellular macromolecule with its own repair system. Until relatively recently, little was known about DNA repair processes in mammalian cells, particularly because of the complexities involved and the relative lack of spontaneously occurring mutants defective in genes involved with DNA repair. As a consequence, most studies of DNA repair were carried out either in bacteria or yeasts and usually employed UV radiation as the tool for producing DNA damage. Although these were rather simple and relatively “clean” systems in which to study DNA repair, their relevance to mammalian repair systems and to the broader spectrum of DNA damage produced by ionizing radiation ultimately limited their usefulness.

The study of DNA repair in mammalian cells received a significant boost during the late 1960s with publications by Cleaver33,34 that identified the molecular defect responsible for the human disease xeroderma pigmentosum (XP). Patients with XP are exquisitely sensitive to sunlight and highly cancer-prone (particularly to skin cancers). Cleaver showed that cells derived from such patients were likewise sensitive to UV radiation and defective in the nucleotide excision repair pathway (discussed later). These cells were not especially sensitive to ionizing radiation, however. Several years later, Taylor and colleagues³⁵ reported that cells derived from patients with a second cancer-prone disorder called ataxia telangiectasia (AT) were extremely sensitive to ionizing radiation and radiation-mimetic drugs but not to UV radiation. In the years that followed, cell cultures derived from patients with these two conditions were used to help elucidate the complicated processes of DNA repair in mammalian cells.

Today, many rodent and human genes involved in DNA repair have been cloned.³⁶ Nearly 20 distinct gene products participate in excision repair of base damage, and even more are involved in the repair of strand breaks. Many of these proteins function as component parts of larger repair complexes; some of these parts are interchangeable and participate in other DNA repair and replication pathways as well. It is also noteworthy that some are not involved with the repair process per se but rather link DNA repair to other cellular functions, including transcription, cell cycle arrest, chromatin remodeling, and apoptosis.³⁷ This attests to the fact that the maintenance of genomic integrity results from a complex interplay between not only the repair proteins themselves but between others that serve as damage sensors, signaling mediators and transducers, and effectors.

For example, the defect responsible for the disease AT is not in a gene that codes for a repair protein but rather in a gene that participates in damage recognition and in a related pathway that normally prevents cells from entering S phase of the cell cycle and beginning DNA synthesis while residual DNA damage is present. This was termed the G₁/S checkpoint response.³⁸ Because of this genetic defect, AT cells do not experience the normal G₁ arrest after irradiation and enter S phase with residual DNA damage. This accounts both for the exquisite radiosensitivity of AT cells and the resulting genomic instability that can lead to carcinogenesis.

What is known about the various types of DNA repair in mammalian cells is outlined below.

Cytogenetic Effects of Ionizing Radiation

When cells divide following radiation exposure, chromosomes frequently contain visible structural aberrations, most of which are lethal to the cell. In some cases, these aberrations physically interfere with the processes of mitosis and cytokinesis and result in prompt cell death. In other cases, cell division can occur, but the loss or uneven distribution of genetic material between the cell’s progeny is ultimately lethal as well, although the affected cells may linger for several days before they die (some may even be able to go through a few more cell divisions in the interim). It is clear that these aberrations are the result of any unrepaired or mis-rejoined DNA damage that persists from the time of irradiation until the time of the next cell division. What is not known, however, is how an unrepaired DSB in essentially “naked” DNA is transformed into such a large lesion—a broken arm of a chromosome, for example—given that the chromosome has undergone many levels of modification, folding and packaging, association with chromatin and other proteins, and condensation in preparation for mitosis.

Most chromosome aberrations result from an interaction between two (or more; discussed later) damage sites, and, therefore, can be grouped into three different types of “exchange” categories. A fourth category is reserved for chromosome aberrations thought to result from a single damage site.⁴⁹ These categories are described below, and representative types of aberrations from each category are shown in Figure 1-6:

Figure 1-6 Types of radiation-induced chromosome aberrations that are the result of unrepaired or mis-rejoined DNA damage. Aberrations are classified according to whether they involve a single chromosome or multiple chromosomes; whether the damage is thought to be caused by the passage of a single charged particle track (i.e., one-hit aberration) or by the interaction of damages produced by two different tracks (i.e., two-hit aberration); and whether the irradiation occurred before or after the chromosomes had replicated (i.e., chromosome-type vs. chromatid-type aberrations, respectively; only chromosome-type aberrations are shown). The aberrations can be further subdivided according to whether broken pieces of the chromosome rearrange themselves symmetrically (with no net loss of genetic material) or asymmetrically (acentric fragments produced).

These four categories can be further subdivided according to whether the initial radiation damage occurred before or after the DNA is replicated during S phase of the cell cycle (a chromosome-type vs. a chromatid-type of aberration, respectively) and, for the three exchange categories, whether the lesion interaction was symmetric or asymmetric. Asymmetric exchanges always lead to the formation of acentric fragments that are usually lost in subsequent cell divisions and therefore are nearly always fatal to the cell. These fragments may be retained transiently in the cell’s progeny as extranuclear chromatin bodies called micronuclei. Symmetric exchanges are more insidious in that they do not lead to the formation of acentric fragments and the accompanying loss of genetic material at the next cell division, they are sometimes difficult to detect cytologically, and they are not invariably lethal to the cell. As such, they will be transmitted to all the progeny of the original cell. Some types of symmetric exchanges (a reciprocal translocation, for example) have been implicated in radiation carcinogenesis, insofar as they have the net effect of either bringing new combinations of genes together or of separating preexisting groups of genes.²⁷ Depending on where in the genome the translocation takes place, genes normally on could be turned off, or vice versa, possibly with adverse consequences.

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