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Injury to DNA is the primary mechanism by which ionizing radiation kills cells (4,83). Most DNA damage is repaired, but lethal double-strand breaks are thought to persist in the form of locally multiply damaged sites (300) of about 15 to 20 nucleotides in size that cause micronuclei formation, chromosome aberrations, and cell death through loss of the reproductive integrity of the cell's genome (38,66,112,158,235,236). However, many biologic factors affect the relationship between the amount of physical energy deposited, the extent of DNA damage that is caused, the number of cells that are killed, and the severity of the tissue response.
The energy initially deposited by ionizing radiation is largely converted into the generation of free radicals. Since cells are made up largely of water (about 85%), most of the damage to biologic molecules caused by x-rays (perhaps 65% or more) is mediated through free radicals formed by activated water, and in particular, hydroxyl radicals, and most DNA damage is therefore indirect. A chain of physicochemical reactions is initiated that is heavily influenced by the intracellular milieu, which influences the persistence of free radicals and the other chemical species that are formed, and the damage that results. Perhaps the most important molecular presence is that of oxygen (5), although other electron-affinic molecules will also play a role. Oxygen can participate in the free radical-generation chain, fix free radical damage, and limit chemical repair. Conversely, sulfhydryl molecules, which vary in natural abundance, scavenge free radicals and may limit the extent of damage.
In contrast to sparsely ionizing x-rays, densely ionizing high linear energy transfer (LET) radiations (e.g., neutrons, α-particles) deposit their energy so intensely along their tracks that lethality relies more on direct ionization to cause damage in DNA and other molecules than on indirect action through ionization of water. The outcome of the interaction of the physicochemical events initiated by ionizing radiation with the biologic system therefore varies with the nature of the radiation and the intracellular milieu, in addition to obvious factors such as dose and dose rate.
Further complexities are that cells sense and respond to radiation damage and this mechanism varies, depending on their biochemical and genetic make-up. Several molecules have been identified by which cells sense damage to DNA (the DNA damage response), and to other intracellular structures and molecules, including mitochondria, membrane lipids, and certain growth factor receptors. Recognition of damage leads to activation of signal transduction pathways aimed at making a coherent and appropriate response to injury. The internal molecular signaling network that exists within a cell as well as the external signals they are receiving (e.g., from hypoxia, cytokines, cell-cell contact, and the extracellular matrix) influence the nature of the response. The resultant radiation-induced pathways can promote cell death or survival, cell cycle arrest or progression, and DNA repair or instability. In other words, the way the cell “perceives” radiation damage plays an important role in determining the final response. Since tumorigenesis requires mutations in molecular pathways that govern cell death, cell cycle, and DNA repair, it follows that genetic alterations associated with cancer frequently affect the response to radiation therapy. It should be noted that similar pathways are often activated in response to stresses other than radiation, including chemotherapy, hyperthermia, oxidative agents, and inflammation. However, radiation differs from these in that it causes a relatively large number of large lesions in DNA that are not only frequently lethal but drive a predominance of pathways triggered by DNA double-strand break formation.
Finally, in addition to molecular and cellular factors that determine intrinsic cellular radiosensitivity, tissue-related and clinical features of radiation exposure add several additional layers of complexity. For example, the number of cells in a tissue capable of regenerating function will be important, as will the way the regenerative potential is distributed as functional subunits within the tissue. Tissue responses may not relate directly to radiation's cytotoxic effects. For example, in tumors, although local control requires elimination of tumor clonogens, in some circumstances vascular damage could be important, especially when irradiation is combined with biologics or chemotherapeutic drugs. Also, irradiation modifies the tumor-host relationship, including interactions with infiltrating cells, such as macrophages and lymphocytes, which have been shown to be able to both promote and inhibit tumor growth. Effects have been described, mainly in vitro, in which the irradiated cell affects the viability or mutability of surrounding “bystander” cells. Such bystander effects may involve more than one mechanism but are presumably, in vivo in normal tissues, a “danger” signaling mechanism for responses to irradiation aimed at tissue healing. Further, some side effects of radiation therapy on
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normal tissues (13) may result from the release of cytokines and other biologic factors or may be associated with remodeling of the normal tissues, rather than cell death per se. For example, late radiation-induced normal tissue fibrosis depends to an extent on cell depletion by radiation, but is more an attempt at a healing response that can be modified by numerous factors, including health status, presence of infection, surgery, injury, and so on.
So, while the initial deposition of energy and subsequent radiochemical events are complete within thousandths of a second following irradiation, a chain of biologic events is initiated that induce programmed cell death or survival, tissue repair and remodeling, all of which depend on the intercellular signaling network, all of which are influenced by systemic and local physiologic conditions. Given the complexity of the biologic condition, it is impossible to predict biologic or clinical outcomes from the amount of physical energy deposited. In other words, biological dose differs from physical dose.
Remarkably, while cells and tissues may respond differently to the same physical dose of radiation, any given tissue appears to respond in a fairly predictable way. The reason for this apparent constancy is that tissue responses are governed largely by cell turnover and the regenerative reserve in the tissue that is generally similar between individuals. Therefore, normal mucosal reactions occur at the same time interval after the start of irradiation and have similar dose-response relationships in most patients. While tumor responses may be more variable than those in normal tissues, certain histologic types of tumors are regarded as more curable by irradiation, and others are not. Reproducible differences in biologic response between tissues are, in fact, exploited for therapeutic benefit. A good example is the use of standard dose fractionation in conventional radiation therapy. This protocol was derived empirically but actually exploits differences in the biologic response between tumor and normal tissues to the same physical dose of radiation. The radiobiologic rationale for the use of dose fractionation in standard radiation therapy has been encapsulated in the 4 “Rs” (reoxygenation, redistribution, repair, and repopulation) (311). This chapter aims to explain how radiobiologic concepts derived from studies dealing with responses within and between normal tissues and tumors are relevant in clinical radiotherapy.
Normal Tissue Radiobiology
Modes of Cell Death after Irradiation
Mitotic Death
Pioneers in radiobiology recognized that cells lethally injured by clinically relevant doses of radiation typically execute one or more divisions before undergoing “mitotic death,” the number depending on the size of the radiation dose (273,274). After 2 Gy, two to three attempts may be made. The progeny of these cells may all die or a proportion may survive to contribute to the reproductive pool, and in the case of tumor recurrence.
Interphase Death
The early pioneers also realized that, in contrast to mitotic death, certain cell types, including many lymphocytes, and some oligodendrocytes and serous cells in the salivary gland, thyroid, intestinal crypt, and hair follicles may undergo relatively rapid “interphase death” within about 2 to 6 hours after irradiation. Typically, only low doses of radiation are required; that is, interphase death is a characteristic of “radiosensitive” cells. Importantly, cells that die during interphase cannot contribute to the reproductive pool. While interphase death was a phenomenon recognized by radiobiologists for decades, recognition of the fact that cells could die by more than one pathway has only recently received wider credence as different pathways leading to cell death have become delineated (67,147,191,256). Interphase death is now acknowledged to represent death by rapid apoptosis.
Apoptotic Death
The morphologic characteristics of apoptosis are nuclear condensation, cell shrinkage, membrane blebbing, and nuclear fragmentation with formation of apoptotic bodies. At the biochemical level, endonucleases are activated that fragment DNA into nucleosomal-sized pieces that are multiples of 180 to 200 base pairs (the size of a nucleosome) and that produce a characteristic “ladder” pattern on agarose gel electrophoresis. In tissue sections, apoptosis can be recognized morphologically. Alternatively, fluorescein or other labels can be attached to the 5′-ends of apoptotic DNA strand breaks using terminal deoxynucleotidyl transferase (TUNEL technique) to allow visualization. In apoptosis, neighboring cells phagocytose cell remnants, and inflammation is not induced. Within a few hours, the whole process is complete, leaving no trace, which makes it easy to underestimate the role of apoptosis in cell loss. Radiation-induced apoptosis in normal tissues is often, but not always, dependent on activation of the tumor suppressor gene p53 (144). Importantly, apoptosis is an active form of cell “suicide” since it requires active metabolic processes. Apoptosis is the primary mechanism by which the body creates organ structures during morphogenesis and tissue sculpting. This is why apoptosis is often called programmed cell death type I. In the adult, apoptosis is required for normal tissue homeostasis; for example, removal of excess cells at sites of proliferation, self-reactive lymphocytes, some cell types as they age or lose the influence of survival signals, virally infected cells, hypoxic cells, or cells damaged by irradiation. It recycles many cell types and removes potentially harmful cells.
The ability to undergo rapid apoptosis is restricted to particular cell types, and even then only at certain positions within tissues that relate to their developmental stage (144). Only cells that have their internal molecular “rheostat” on a proapoptosis setting appear to undergo rapid apoptosis following irradiation. In other words, irradiation tends to increase the frequency of apoptosis only in tissues and tumors in which the cells already have a proapoptotic tendency. Thus, lymphomas generally have proapoptotic tendencies, whereas glioblastoma multiforme cells do not, with or without irradiation.
Necrotic Death
In contrast to apoptosis, necrosis is, for the most part, a pathologic, rather than a physiologic, process that does not require active metabolic cellular processes. It is involved in tissue healing in response to injury or invasion by pathogens. Necrosis can also be a pathologic response to vascular damage, as well as a “default” death pathway for cells that lack an effective apoptotic apparatus. Membrane integrity is lost, cells increase in size, lysosomal enzymes are released, and inflammatory responses are generated with release of cytokines that link cell death to the development of specific immunity. DNA from cells undergoing necrosis forms a “smear” on agarose gel electrophoresis.
Another alternative death style following irradiation is autophagy, where cells internalize cellular organelles within vacuoles and digest them. This is most often seen in nutrient deprivation but is also another form of “programmed” cell death (type II) involved in morphogenesis and tissue sculpting. Other outcomes may be important in specific cell types.
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Radiation-induced differentiation, senescence, or quiescence are possible outcomes that may achieve the same objective as death in removing damaged tumor cells from the reproductive pool and limiting the chances of carcinogenesis. A difference is that in these cases some function is retained for some time. For example, terminal differentiation of fibrocytes gives a cell phenotype that can contribute to radiation-induced fibrosis through enhanced collagen production (121).
While radiation-induced interphase death can easily be ascribed to apoptosis, mitotic death may involve several mechanisms; for example, failure of spindle formation in M phase, loss of the G2 checkpoint leading to “mitotic catastrophe,” or improper chromosome segregation due to damage and loss of genetic material, which may be manifested microscopically as chromosome alterations. Furthermore, mechanistically, the molecular machinery that is employed can be apoptotic, necrotic, or any other. Mitotic death, by its very nature, is normally delayed, occurring over a period of days. Unlike “rapid” (interphase) apoptosis, there is no evidence to suggest that cells that die by “delayed” (mitotic) apoptosis are particularly radiosensitive.
The molecular pathways that participate in cell death are described in another chapter, but it is appropriate here to consider specific phenomena that might relate to in vivo responses. The presence of “survival” signals appropriate for the cell's environment, such as those provided by growth factors, cell-cell contact, and extracellular matrix, will be important in determining response to irradiation. Their loss leads to a phenomenon known as “anoikis” (97) or homelessness, which is a form of “death by neglect.” This is why lymphocytes activated by growth factors are more radioresistant than resting lymphocytes and endothelial cells are more radioresistant in the presence than in the absence of mitogenic basic fibroblast growth factor (98). This may be a broadly applicable concept and explain why blocking receptor tyrosine signaling with cetuximab, Iressa, or similar agents, or NF-κB activation with bBortezomib, may radiosensitize tumor cells.
Survival pathway signaling may also underlie some of the phenomena ascribed to potentially lethal damage repair (PLDR) following irradiation. In PLDR, the cellular microenvironment determines the likelihood of cell death. Classically, PLDR occurs when cells are irradiated and maintained in a contact-inhibited, plateau-phase culture (111,229). If such contact-inhibited cells are trypsinized immediately, or soon after irradiation, survival is compromised, as demonstrated, for example, by lowered clonogenicity. The cells are rendered “homeless” by trypsinization and are more likely to die. It must be noted, however, that PLDR has been invoked as a mechanism that increases survival under diverse sets of experimental conditions and that multiple mechanisms may contribute to the final outcome (282).
As opposed to “death by neglect,” certain positive signals can cause death of susceptible cells. Classically, such signals involve members of the tumor necrosis factor (TNF) family of cytokines (e.g. TNF-α, fasL, Trail). Activation of TNF receptor (TNFR) family members that contain a death domain in their cytoplasmic tail can trigger apoptosis. Other members of the same family of receptors that do not have a death domain, as well as soluble receptors and receptor antagonists, can counteract these death threats. Therefore, the type of receptor and related molecules that a cell expresses may determine its response to TNF. TNF-α causes proliferation of some cell types, such as fibroblasts or their CNS equivalent, astrocytes. In contrast, TNF-α can induce growth arrest or apoptosis in many tumors and in some normal cells, such as oligodendrocytes. Since radiation can induce expression of both TNF and TNFR family, these pathways can contribute indirectly to death or survival of some cell types following irradiation (114,115,125). For example, mice lacking TNFR2, which does not have a death domain and drives cell survival, are particularly sensitive to late effects of irradiation to the brain (61). The cytotoxicity of certain chemotherapeutic agents, such as 5-fluorouacil and cisplatin, can also involve TNF-α (206). Because TNF-α is a primary mediator of inflammation, a proinflammatory cytokine cascade is activated in tissues following irradiation that may also activate bystander cells.
Cell Death in Irradiated Normal Tissues
Radiation-induced apoptosis varies with location within a tissue, reflecting the fact that apoptosis is inherently programmed in a position-dependent manner. For understandable reasons, most of the information on radiation-induced apoptosis in normal tissues comes from studies in mice. Radiation-induced rapid apoptosis in the mouse small intestine is maximal around position 4 from the base of the crypt (232), which is the site of the proliferative compartment and of spontaneous apoptosis. In contrast, apoptosis is not so marked in the colon and is not seen in the proliferative region. Indeed, the antiapoptotic molecule Bcl-2 is expressed by cells in this region (184). In lymphoid tissues, small intestine, hair follicles, and ependyma, there is some concordance between the position of apoptotic cells and radiation-induced up-regulated expression of p53 (54,183,184,232). In subpopulations of cells in other tissues, p53 can be up-regulated with little evidence of rapid apoptosis, while most cells in liver, skeletal muscle, and brain show neither p53 nor much apoptosis (144,169,177,206). In the thymus, developing T lymphocytes with the potential to respond to foreign antigens are selected by apoptotic elimination of both self-reacting and nonreacting T cells in the cortex (positive and negative selection). About 98% of the cells that are generated by mitotic division die (about 5 × 107 cells per day in a young adult mouse). After irradiation, massive rapid, p53-dependent apoptosis of T cells is seen in the cortex, but less apoptosis is evident in the medulla, which contains more mature cells.
The role of apoptosis in normal tissue responses to irradiation has yet to be fully evaluated, but inevitably it will depend on the physiologic role of the proapoptotic cells. If the proapoptotic cells are superfluous to needs, radiation-induced cell death in this compartment may have little impact, but if they are critical to tissue function the opposite will be true. For example, in the mouse small intestine, the cells that die by rapid apoptosis may contribute little to the clonogenic crypt stem cell population. Thus, there is little difference between p53 wild type and p53-null mice in their clonogenic responses following gut irradiation (118), although variation with dose and dose rate is evident (117). In the mouse brain, radiation-induced apoptosis is seen in endothelial cells, oligodendrocytes, and the neuroepithelial subventricular zone (122) where it depends on the protein that is mutated in ataxia telengiectasia and p53, both of which are phosphorylated by radiation and act as sensors of DNA damage, as well as TNFR expression. The extreme radiosensitivity of the developing brain is probably due to its high apoptotic index. Although little information is available in humans, acute parotitis can develop in the first 24 hours of treatment of patients receiving head and neck irradiation, and this reflects apoptotic death of serous cells (251). There is no such acute death in the mucous cells; hence, the mouth is dry and the saliva more viscous.
Pathobiology and Kinetics of Radiation Injury in Normal Tissues
The time to development of most normal tissue injury depends critically on the turnover time of the tissue, that is, on the kinetics of cell differentiation, loss, and renewal. The terms acute,
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subacute, or late are commonly used to describe the time to occurrence of functional inadequacy after irradiation and reflect kinetic differences between tissues while saying little about the underlying pathogenesis of the response. The terms are also often loosely used to describe the tissues in which such effects are seen, as in “acute effects tissue,” but this can be misleading since tissues and organs comprise more than one cell type, each with its own turnover rate characteristics. Any one tissue can therefore express both acute and late symptoms of radiation damage, depending on the cell type that is limiting function at that time. Also, a severe acute injury from irradiation can lead to nonspecific late (consequential) changes such as fibrosis, atrophy, or ulceration (35,223,329) (e.g., stenosis consequent to mucosal ulceration of the bowel, or fibrosis or necrosis of skin or oropharyngeal tissues consequent to desquamation and acute ulceration).
Acute Responses
Acute responses to radiation therapy are defined as occurring during a standard 6- to 8-week course of therapy and are seen in tissues with large populations of cells that turn over rapidly (gastrointestinal mucosa, bone marrow, skin, and oropharyngeal and esophageal mucosa). Hierarchical organization exists in such tissues (187,188,189,303) with a small number of stem cells that proliferate slowly to produce a highly proliferative compartment of progenitor cells that differentiate into mature, nonproliferative, functional cells. Irradiation may deplete the stem and progenitor cell pools, but nonproliferating, differentiated cells maintain tissue function until they are lost through continuing normal cell turnover.
After irradiation, depleted stem and progenitor cell pools may first reconstitute their own numbers before differentiating to restore function, although the extent to which this occurs varies with the tissue. A useful model to consider is that under normal steady-state circumstances (i.e., not growing or involuting), tissues have, by definition, a cell-loss factor (φ) of 1. The only requirement for growth of a tissue is a decrease in φ to less than 1, which is characteristic of the embryo and fetus, tissue regeneration, and malignancy. After irradiation, some tissues (e.g., jejunal crypts) reduce φ to 0 and regenerate quickly; others (e.g., skin) may reduce it to about 0.5 and regenerate less quickly, but continuously produce some functional cells; others (e.g., seminiferous epithelium) show little change in φ and mostly continue in steady state, producing sperm in numbers that are reduced for months or years in direct proportion to the extent of stem cell depletion.
Because acute-responding tissues are organized in a hierarchical fashion, the severity of radiation injury depends on both the extent of stem/progenitor cell depletion and the length of the delay before new functional cells are released into the differentiated compartment. Severity of injury naturally increases with dose, but providing the proliferative pool does not fall below a critical value symptoms are transient and recovery is complete. Dose fractionation can lessen the severity of acute effects by allowing regeneration from the stem/progenitor cell compartment during the course of therapy. Unlike the extent of injury, the rate at which acute injury develops and the latent time to the appearance of symptoms is relatively (187), although not completely (175), independent of dose. This is because latency is mainly determined by the rate of loss of differentiated cells.
Because cell turnover kinetics determines the time to a normal tissue effect, latency is not an indicator of radiosensitivity. For example, in hematopoiesis, leukocyte and platelet numbers drop quickly after bone marrow irradiation because they have a fast turnover rate, whereas anemia is not an obvious acute effect because red cells turn over slowly. Similarly, in the testis, each spermatogenic stem cell division ultimately produces more than 1,000 sperm through successive divisions of spermatogonia and spermatocytes—a process that in humans takes more than 60 days. Early differentiating spermatogonia are few in number and are selectively depleted by doses that have little effect on cells in the more mature stages of spermatogenesis. This is why sperm counts remain normal for several weeks after exposure, falling steeply only at the time when the progeny of the irradiated spermatogonia would normally have reached the seminal vesicles. In the mucosa of the small bowel, mitotic activity is confined to the crypts; the cells lining the villus are nonproliferative. Because crypt cells divide rapidly (an average of more than once daily in humans), they are lost within days if sterilized by radiation. The villus shows no immediate effect of irradiation, with shortening becoming evident only as programmed shedding of differentiated cells into the lumen continues in the absence of renewal from the crypts. This is why symptoms take about 2 weeks to appear in patients undergoing standard daily doses of abdominal irradiation.
Subacute Responses
Certain tissues may display subacute reactions several months after irradiation, reflecting failure of a cell population with a longer turnover time. Symptoms are generally reversible, although in some instances they may be associated with severe damage and even death. Examples of transient effects are Lhermitte's syndrome after spinal cord irradiation, somnolence after brain irradiation, and subacute pneumonitis 2 to 3 months after the start of lung irradiation. Subacute effects occur most often during the remodeling phase in irradiated tissues and prior to the onset of late effects that are associated with slowly progressing damage.
Late Responses
Late reactions to radiation therapy in normal tissues can be severe, and recovery is often limited. They are generally considered to be the result of the depletion of slowly proliferating “target” cells that are lost from the tissue at a slow rate, for example, from central (oligodendroglia) or peripheral (Schwann cells) nervous tissue, kidney (tubule epithelium), blood vessels (endothelium), dermis (fibroblasts), and bones (osteoblasts and chondroblasts). However, abortive rounds of attempts at healing may involve different cell types in distinct cellular compartments, and lesions can appear to evolve with time in a dose-dependent manner (278). Some lesions, such as those associated with artherosclerosis and heart disease (238), can take decades to occur after irradiation and are an increasing problem as patients live longer following cancer therapy. Pathologic findings following collapse of late effects tissues can be very variable. For example, late demyelination after brain irradiation may be ascribed to loss of oligodendrocytes and subsequently of neurons (50), but coincident with and preceding any neurologic changes, proliferation of astrocytes and microglial cells can be observed (51), as can vascular lesions with edema, hemorrhage, or inflammatory infiltrates (45). Infiltrating cells may contribute to the pathogenesis of radiation injury, as is illustrated by involvement of infiltrating cells in the pathogenesis of radiation pneumonitis, or recovery from injury as in the slower healing of skin wounds in mice receiving total body irradiation compared with those irradiated only at the local site (293).
Unlike acute responding hierarchical tissues, slowly proliferating, late effects tissues contain cells that are usually both functional and able to proliferate on demand. In an operational sense, such tissues can be regarded as “flexible” (187,188,189). This does not deny the presence of stem cells with limited function or functional cells that do not proliferate, but the roles of such cells are probably of lesser importance than in hierarchical tissues. The relative inability of late effects tissues to be repopulated
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from a stem cell pool makes radiation reactions in these tissues more chronic and debilitating, diminishing the quality of life for those afflicted.
As in acute-responding tissues, the rate at which radiation injury develops in late effects tissues reflects the turnover rate of proliferative cells, which may be also functional cells in this case (e.g., liver, kidney). Therefore, dose has a greater apparent influence on latency, with late injury developing more quickly with increase in dose. This may be because the greater the dose, the fewer the number of division cycles the cells can successfully negotiate before death (166,177,273). Another reason may be that as cells die, residual (mostly lethally injured) target cells are increasingly recruited to the proliferative pool, causing a cascade or “avalanche” of cell death and functional tissue failure. A third explanation may be that because interactions between cell types tend to be involved in the causation of late effects, the nature of the lesion may vary with time, depending on which cell type is critically limiting at that time (45,284,285). The time course to development of injury can be accelerated and the severity increased by various insults such as surgery, chemotherapy, infection, or physical trauma (105,106). Indeed, such factors may play a major role in precipitating the onset of late effects in humans (e.g., necrotic, nonhealing ulcers after trauma). Conversely, slowing the proliferation process and decreasing stress may reduce their incidence and severity.
Whereas a severe early response in a rapidly proliferating tissue permits adjustment of the dose schedule during the course of standard radiation therapy, this is not the case for late injuries, since they occur after completion of therapy. Tolerance doses for individual patients are therefore based on past experience. Such tolerance doses have not been precisely defined, even though, generally accepted limits to doses considered tolerable by various organs do exist in practice (85,241).
An issue of growing clinical importance is the extent to which late radiation effects can be reversed. It has been shown recently that certain agents given late after radiation can modify injury in tissues. For example, captopril, an angiotensin-converting enzyme inhibitor, slows the development of radiation-induced nephritis (194,196), pneumopathy, and lung fibrosis (192) in rats. Steroids also can prevent death from radiation pneumonitis in animals, although their withdrawal before the end of the usual period of pneumonitis can result in accelerated mortality (109). Pentoxifylline, alone or in combination with vitamin E, protects against radiation-induced late effects in some experimental models (69,151) and in a clinical study, Delanian et al. (63) found that the combination, but neither agent alone, reversed chronic radiation-induced fibrosis. It is not clear how these agents act and whether they promote cellular recovery, but such studies point to ways to improve the future management of late complications of radiation therapy (189).
Functional Subunits
The tolerance of a tissue to irradiation is determined by the number of cells with regenerative potential and the way they are organized, in addition to their intrinsic radiosensitivity. Tissues can be thought of as being composed of functional subunits (FSUs), which is the minimum clonogenic entity required for regeneration of a structure. For example, epilation requires doses lower than those for desquamation. This is not because the cells in the hair follicle differ in their radiosensitivity from those in the basal epithelium, but because there is a smaller number of clonogenic cells in the FSU that produces a hair than in the sheet of basal cells that is capable of regenerating itself. Similarly, hair is depigmented by relatively low doses of radiation (294), but the epidermis loses pigmentation only after higher doses. This is because each hair follicle contains a small number of melanocytes, sometimes only one, whereas melanocytes are more numerous in the epidermis.
In the kidney, each nephron is an FSU (324). If a tubule is completely de-epithelialized, it is lost permanently because it is not repopulated from adjacent nephrons. Therefore, the tolerance dose for the kidney is determined more by the number of tubule cells per nephron than the number of nephrons. For example, if the kidney contained 1011 clonogenic tubule cells distributed as 104 cells in each of 107 nephrons and any one of these 104 cells were capable of regenerating the tubule, then most tubules should regenerate after a dose that reduced survival to 10-4 (or an average of one cell per tubule). Because of the random nature of events, some tubules would then contain more than one surviving cell and others would contain none. From Poisson distribution statistics, 37% of FSU nephrons would be eliminated. The “tolerance” dose would be different if the organ were composed of 104 nephrons, each containing 107 cells: The dose that would eliminate 37% of the nephrons would be that to reduce survival to 10-7. In a multifractionated dose regimen, during which a logarithmic decline in cell number occurs, this is 7/4 (1.75) times that required to reduce survival to 10-4. This is why tolerance doses can vary so much among tissues and organs, even if the target cells have the same intrinsic radiosensitivity.
It is easy to appreciate the structural organization of FSUs for hair and kidney, but not for some other tissues. For example, in mouse skin, the survival of about 10 cells per cm2, from what is normally approximately 106 basal stem cells per cm2, is required to maintain uninterrupted integrity and prevent overt desquamation (308). Therefore, the FSU would be about 1/10 cm2. Organs with acinar or alveolar architecture (e.g., salivary glands, pancreas, sweat glands, testis, mammary epithelium, lung, and perhaps liver) may resemble the kidney in having structurally defined FSUs, while the target cells in dermis, CNS, mucosae, gut, and epidermis are less restricted by physical barriers and cellular migration may influence regeneration.
Known tolerance doses are consistent with FSUs being relatively small in tissues such as kidney with structurally well-defined FSUs, are intermediate in the spinal cord, and large in the dermis. Obviously, a primary tumor is just one large FSU in which one surviving clonogen can lead to recurrence. Clonogenic cell number will play a big role in determining local tumor control. For metastatic deposits, regional control will depend not only on the number of clonogenic cells each contains but also the number of metastatic deposits. In general, a larger number of small metastatic deposits will be cured with lower radiation doses than a small number of larger metastases, even if the total cell number is the same in both cases.
Volume Effects
Traditionally, radiation oncologists have reduced the total dose when treating large volumes of normal tissue. In fact, the now widespread reduction in dose per fraction from 2 to 1.8 Gy had its origin in a volume effect; the longer treatment duration enhanced mucosal tolerance in large head and neck treatment fields (93). In the orthovoltage era, a reduction in dose with increase in treatment volume was generally recommended (218), but with the advent of skin-sparing megavoltage beams, the volume effect received less attention, especially as larger tumors (and larger treatment volumes) require higher doses for their control.
In fact, the concept of decreasing dose with increasing treatment volume has little radiobiologic basis, except in specific circumstances. For example, if FSUs are arranged in series, like links in a chain, as they are in nerve tracts, spinal cord, and the cylindrical sheath of peritoneum covering the small intestine,
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the loss of one subunit may result in an overt expression of injury, regardless of the state of the other subunits in the series. The probability of injury increases with volume (number of FSUs exposed) (Fig. 2.1). Such a volume effect has been demonstrated clinically for small bowel obstruction (160,231) and experimentally for myelitis (126,246,286).
View Figure FIGURE 2.1. Diagrammatic representation of the influence on the probability of a complication from increasing the treatment volume in a tissue where functional subunits (FSUs) are arranged serially. The average survival of FSUs was 1 in 16, sterilized FSUs being denoted by the black squares. With the small volumes (A), the probability of myelitis was 6% (1/16), whereas it would approach 100% if 16 FSUs in one patient were exposed (E). The actual probabilities can be calculated using the equation in the text. (From Withers HR, Taylor JMG, Maciejewski B. Treatment volume and tissue tolerance. Int J Radiat Oncol Biol Phys 1988;14:751,with permission.)
The relationship between the number of FSUs irradiated (n) and the probability of a complication (P) is described by the following formula:
P = 1 - (1 - p)n
where p is the probability of the loss of one FSU. This relationship is illustrated in Figure 2.2. Increasing the volume (number of FSUs exposed) reduces the dose necessary to produce a complication and increases the steepness of the dose-response curves. The effect would be predicted to occur when the average number of surviving cells per FSU is reduced to almost one and when the length of serial arrangement of FSUs is small. This may be less true for the small bowel (100,160,319) than for spinal cord.
View Figure FIGURE 2.2. Curves illustrating how the probability of producing a complication increases with increase in the number of serially arranged functional subunits (FSUs) included in the treatment volume. The curves were positioned by assuming that 58 Gy in 2-Gy fractions sterilized 10% of FSUs and that for a series of 2-Gy fractions the effective D0 for the target cells was 4 Gy. The curves are shifted to the left and are steeper with increase in number of FSUs exposed, but this effect becomes less obvious once large numbers of subunits are involved. (From Withers HR, Taylor JMG, Maciejewski B. Treatment volume and tissue tolerance. Int J Radiat Oncol Biol Phys 1988;14:751, 1988,with permission.)
Nonradiobiologic “volume effects” exist that can result from multiple mechanisms. There are some mechanisms that can be excluded. For example, there is no evidence that cellular radiosensitivity is affected by an increase in treatment volume (332). The radiosensitivity of skin epithelium is constant over a 5,000-fold range of treatment area (308,332). Also, no evidence exists for an increased role for vascular damage as volume increases (326). On the other hand, “volume effects” can be seen when (332):
A patient tolerates a small area of injury (such as ulceration) better than a large area of the same severity because pain, exudation, infection are worse, healing is slower, and consequential contraction and scarring are more of a problem. The effect of increasing volume is to make the injury more incapacitating, even though the severity of the radiation response is independent of volume treated.
Large gradients in dose distribution and heterogeneity develop as volume increases. Without prudent planning, a tumor dose may be prescribed at the 80% level, leading to a 25% higher dose in the region of Dmax. Also, with large fields, large variations in contour may exist. These variations could result in a high-dose region where tissue thickness is less than that measured at the midplane; as, for example, in the spinal cord at the thoracic inlet in thoracic irradiation and in tangential fields for treatment of the breast. If the threshold-sigmoid curve of the probability of normal tissue complications against dose is steep, as it is in experimental studies (266), a 25% increase in total dose could produce a marked change in the incidence of complications. This increase in physical dose is further compounded by the biologic effectiveness of each dose per fraction being increased by more than 25%, or the “double trouble” of increased physical and biological dose. The magnitude of the increase in “biological” dose will depend on the dose per fraction and the type of normal tissue, but will be greatest in late-responding normal tissues for reasons that will be explained later in terms of the nonlinear rate of increase in injury with increase in
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dose per fraction. Because this additional augmentation of biologic doses is not evident from physical isodose contours, an increased biologic effect may be erroneously attributed to the large volume being treated per se.
If organ “reserve” is obliterated as volume is increased (e.g., lung, salivary gland), this is not a true volume effect because sequelae are determined by the volume and functional status of the tissue excluded from the treatment volume, not the volume irradiated.
Molecular Responses in Normal Tissues
As discussed in the introductory section, radiation-induced signal transduction pathways can be important in determining the cellular response to damage. Cell- and tissue-specific patterns of molecular responses are detected minutes to hours after irradiation that can vary with radiation dose, dose rate, and quality, and with dose-response relationships that are not always linear. The most rapid response includes transcriptional and posttranscriptional activation of members of immediate early gene families, such as c-jun and c-fos, ATM (ataxia telangiectasia), p53, c-abl, EGFR (epidermal growth factor receptor), and other molecules. Phosphorylation/dephosphorylation reactions or other activating mechanisms are invoked. Importantly, these pathways couple molecular damage to DNA repair, cell cycle arrest, phenotypic changes, and cell death. Another part of the early response involves the induction of sets of secreted molecules such as proinflammatory cytokines, proteases and antiproteases, cell adhesion molecules, and extracellular matrix materials that together form a regulated acute tissue reaction (125) to trigger subsequent tissue remodeling. Such “danger” signaling can extend beyond the radiation field and may be responsible for some of the observed “abscopal” effects of irradiation. The overall function of radiation-induced molecular responses is to preserve cell and tissue integrity after irradiation by promoting cell death/survival and tissue recovery and remodeling.
Radiation-induced inflammatory responses, as well as initiating healing responses in tissues, may “prime” cells and tissues for adaptive responses to further radiation doses. For example, radiation-induced basic fibroblast growth factor may act through autocrine pathways to promote survival of endothelial cells (98). In vivo, antagonists of radiation-induced interleukin (IL)-1 and TNF-α increase the intrinsic sensitivity of mice to bone marrow death after irradiation (200,201), suggesting that these responses also have adaptive survival value. On the other hand, radiation-induced TNF-α can cause certain cells to apoptose (115) and may trigger clinical symptoms that can not be ascribed to cell death (16,130). Examples are nausea or vomiting that can occur within hours of irradiation of the upper abdomen, acute erythema and edema associated with vascular leakage, fatigue in patients receiving irradiation to a large volume, especially within the abdomen, and somnolence that can develop within a few hours of cranial irradiation. Radiation-induced proliferative responses such as gliosis (51) or certain forms of fibrosis could also cause symptoms unrelated to cell depletion.
Recently, it has been shown that during the latent period leading up to the expression of late effects, waves of molecular responses occur that may reflect repeated attempts at tissue recovery and remodeling (49). As a result, the concept that late effects represent dysregulation of an integrated injury and healing process that involves both parenchymal and vascular elements, as well as inflammatory cells, has gained in prominence. Failure of any of the required elements could give rise to a late effect.
Cytokines and growth factors are thought to play important roles in late effects. For example, signaling through the TNF receptor 2 protects mice from late effects of brain irradiation (61). Anscher et al. (14,15) have reported that lung cancer patients with elevated plasma levels of transforming growth factor-β (TGF-β) prior to radiation therapy are more likely to develop radiation pneumonitis, illuminating the importance of the interaction of systemic change with local radiation damage. Elevated TGF-β levels could derive from the tumor or the stromal cells that invade it, or may be radiation-induced; the outcome will be the same. Inhibiting TGF-β activation during radiotherapy is being investigated as a strategy to lower the risk of pneumonitis in patients with non–small cell lung cancer. In addition, dose escalation is being attempted in patients whose TGF-β levels normalize during a course of radiation therapy (16).
Regeneration (Repopulation) in Normal Tissues
The time to onset of repopulation after irradiation and the rate at which it proceeds vary with the tissue. Both can be measured experimentally by a split-dose technique in which the increase in the number of cells with time after the first dose is reflected by an increase in size of the second dose required to produce a certain constant level of effect (isoeffect).
In acute-responding tissues, the onset of repopulation is early because cell loss is rapid. In the jejunal mucosa, the lag time before the onset of radiation-induced proliferation may be <24 hours. In the colon and stomach, it is slightly longer. In contrast, in renal tubules, there is no histologic evidence of cell depletion for many months after irradiation, and there is a long lag period before the onset of repopulation. In the mouse, it takes more than 12 months to reconstitute a tubule (324). The rate of repopulation has, similarly, not been well quantified in acutely and, especially, in late-responding tissues. In mice, some approximate doubling times for clonogenic cells are 8, 12, and 22 hours for jejunum, colon (310), and skin (309), respectively.
In humans, tissue turnover kinetics are slower than in mice. They have been approximated for oropharyngeal mucosa from consideration of responses to various dose-fractionation regimens. Mucositis begins to appear 14 to 21 days after the start of a regimen of 2 Gy given five times per week, but repopulation begins at about 10 to 12 days (288). High initial doses may shorten the lag period, but only by 1 or 2 days. Repopulation can increase the tolerance of the mucosa to a conventional dose regimen by an average of at least 1 Gy/day, which is equivalent to approximately a doubling of clonogenic cell numbers every 2 days, and it may be significantly faster (321). If daily irradiation is suspended (e.g., during a 10- to 14-day break in a split-course accelerated regimen), clonogenic cells may repopulate at two or three times this rate (9,12,288,299,321).
These values for lengths of lag time and repopulation rates are, at best, estimates. Figure 2.3 shows that in some tissues, regeneration begins within 1 or 2 days of the initiation of radiation therapy, whereas in others there is no evidence for regeneration even after 2 months. The critical point is that there is a lag period followed by a phase of rapid exponential growth. In general, the lag period is shorter for chemotherapy, hyperthermia, and surgery because the cell depletion that stimulates regeneration occurs more rapidly than after irradiation.
The importance of repopulation is implicit in the history of radiation therapy. The current standard protracted overall treatment times confer a benefit by allowing regeneration of acute-responding tissues, which reduces toxicity. When attempts are made to deliver curative therapy more quickly, acute responses become more severe and dose-limiting.
Growth factors may be useful in protecting normal tissues from irradiation by shortening the apparent lag phase and accelerating recovery in irradiated tissues. Hematopoietic growth
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factors such as G-CSF, GM-CSF, erythropoietin, and IL-11 can accelerate proliferation of hematopoietic cells (199). In doing so, they minimize the danger of infection. In epithelial tissues keratinocyte growth factor, which is specific for epithelial cells, has similar potential. It protects the oral mucosa, small intestine, lung, and hair follicles against chemotherapy or radiation injury (73,87,88,339) in preclinical models and has shown efficacy in clinical bone marrow transplantation trials.
FIGURE 2.3. Representation of the approximate kinetics of regeneration of irradiated normal tissues (solid lines, solid symbols) and tumors (dashed lines, open symbols). Curves are based on measurements or estimates of regeneration; symbols denote times at which an effect of regeneration has already appearedor has not yet appeared. The logarithmic abscissa is for convenience of presentation only and has no biologic rationale. In general, the human data are displaced to the right of experimental animal data, reflecting a slower initiation of repopulation because human tissues proliferate more slowly than do their rodent counterparts, they were exposed to protracted dose regimens; and less-sensitive end points were used to detect onset of repopulation in humans. Numbers on the curve and symbols refer to the sources of data. 1, Withers (310); 2, Withers (309); 3, Withers and Mason (322); 4, McCulloch and Till (179); 5, Hermens and Barendsen (120); 6, Suit et al. (259); 7, Choi et al. (53); 8, Denekamp (64); 9, Fletcher (93), Horiot et al. (128), van der Schueren et al. (288), Wang (297,298), and Wang et al. (299); 10, Barendsen and Broerse (21); 11, Arcangeli et al. (17); 12, Withers et al. (320) and Meistrich et al. (180); 13, Maciejewski et al. (168,169,170,171,172); 14, Allen (2); 15, Maciejewski et al. (169); 16, Barker et al. (22); 17, Wang (297,298) and Wang et al. (299); 18, Parsons et al. (216); 19, Maciejewski et al. (168); 20, Pedrick and Hoppe (219), Maciejewski et al. (170), Fisher and Hendry (91), Withers and Mason (324), and van der Kogel (284,285); 21, White and Hornsey (304); and Withers et al. (337); 22, Xu et al. (338) and Ang et al. (12); 23, Ang et al. (9); 24, Kummermehr and Trott (154); 25, Chen and Withers (48); and 26, Turesson and Notter (279).
“Remembered” Dose: Tolerance to Retreatment
Conventional teaching in radiation oncology has been that a heavily irradiated tissue will not tolerate retreatment. The postulated reason was that the basis of late effects was vascular damage and was irreversible. While irradiation may limit the tolerance of a tissue to retreatment, in fact, retreatment is often possible and may be better tolerated than previously expected (156). Factors that determine the extent to which residual injury will limit retreatment tolerance include the amount of cell depletion caused by prior treatment, the time elapsed since that treatment and therefore the extent of regeneration, and the tissue at risk. High prior doses, short intervals between treatment courses, and slow regeneration of target cells will reduce retreatment tolerance.
Some data for experimental radiation myelitis are shown in Figure 2.4. The plot shows the effect of size of the first dose on the dose required to produce myelitis in a second regimen. Recovery is complete after low doses, but is progressively compromised as the initial dose approaches tissue tolerance (174). It should be remembered that clinical “tolerance” doses for the spinal cord of 45 to 50 Gy in 1.8- to 2-Gy fractions are low in terms of the injury evaluated in Fig. 2.4 (50% incidence of myelitis). The time to recovery for the spinal cord is not accurately known, but in rats at 100 days it is about half of what it reaches by 200 days (287). In monkeys, there was extensive recovery from 44 Gy in 2.2-Gy fractions by 2 years, but a detailed profile of the time course could not be established (7).
Not all tissues, or elements within tissues, recover at an equal rate or to an equal extent from the effects of irradiation. Acute-responding epithelial and hemopoietic tissues generally recover quickly and demonstrate a high tolerance to retreatment in terms of acute responses. However, the fibrovascular support in skin and mucosa and the stroma in bone marrow are less tolerant to retreatment because they respond more slowly. The kidney shows poor retreatment tolerance as assessed functionally in mice (253). Reirradiation tolerance in this organ is inversely related to the initial dose, but tolerance decreases significantly with increasing interval between treatments, suggesting progression rather than recovery from the initial damage.
Because different tissues show different levels of tolerance to retreatment, caution should be exercised in the application of these concepts to the clinic. Also, the experimental studies deal with well-defined end points within a limited time scale. If different end points in the same tissue are examined or the time is extended, the same guidelines may not apply. It should
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also be noted that if slowly proliferating cells involved in late responses are extensively depleted, recovery may be permanently incomplete, and the organ will be vulnerable to further injury whether it is from radiation, trauma, cytotoxic drugs, or any other insult. For example, hyperthermia can precipitate myelitis in a patient who has had high, but otherwise tolerable, doses of x-irradiation (161), and trauma from dental intervention frequently precipitates mandibular necrosis.
View Figure FIGURE 2.4. The dependence of remembered dose on size of priming dose (as a percentage of the ED50) is shown for a variety of animal species at long periods (6 months to 2 years) after the initial radiation treatment: ○, adult rhesus monkey (10); ▲, 12-week-old rat (130); □, 1-day-old guinea pig (149); ●, young adult mouse (165); ▲, 8-week-old guinea pig (174); ▼, 8-week-old guinea pig (198); ▪, 3-week-old weanling rat (243); ♦, young adult rat (287). (From Mason KA, Withers HR, Chiang CS. Late effects of radiation on the lumbar spinal cord of guinea pigs: Re-treatment tolerance. Int J Radiat Oncol Biol Phys 1993;26:643,with permission.)
The energy initially deposited by ionizing radiation is largely converted into the generation of free radicals. Since cells are made up largely of water (about 85%), most of the damage to biologic molecules caused by x-rays (perhaps 65% or more) is mediated through free radicals formed by activated water, and in particular, hydroxyl radicals, and most DNA damage is therefore indirect. A chain of physicochemical reactions is initiated that is heavily influenced by the intracellular milieu, which influences the persistence of free radicals and the other chemical species that are formed, and the damage that results. Perhaps the most important molecular presence is that of oxygen (5), although other electron-affinic molecules will also play a role. Oxygen can participate in the free radical-generation chain, fix free radical damage, and limit chemical repair. Conversely, sulfhydryl molecules, which vary in natural abundance, scavenge free radicals and may limit the extent of damage.
In contrast to sparsely ionizing x-rays, densely ionizing high linear energy transfer (LET) radiations (e.g., neutrons, α-particles) deposit their energy so intensely along their tracks that lethality relies more on direct ionization to cause damage in DNA and other molecules than on indirect action through ionization of water. The outcome of the interaction of the physicochemical events initiated by ionizing radiation with the biologic system therefore varies with the nature of the radiation and the intracellular milieu, in addition to obvious factors such as dose and dose rate.
Further complexities are that cells sense and respond to radiation damage and this mechanism varies, depending on their biochemical and genetic make-up. Several molecules have been identified by which cells sense damage to DNA (the DNA damage response), and to other intracellular structures and molecules, including mitochondria, membrane lipids, and certain growth factor receptors. Recognition of damage leads to activation of signal transduction pathways aimed at making a coherent and appropriate response to injury. The internal molecular signaling network that exists within a cell as well as the external signals they are receiving (e.g., from hypoxia, cytokines, cell-cell contact, and the extracellular matrix) influence the nature of the response. The resultant radiation-induced pathways can promote cell death or survival, cell cycle arrest or progression, and DNA repair or instability. In other words, the way the cell “perceives” radiation damage plays an important role in determining the final response. Since tumorigenesis requires mutations in molecular pathways that govern cell death, cell cycle, and DNA repair, it follows that genetic alterations associated with cancer frequently affect the response to radiation therapy. It should be noted that similar pathways are often activated in response to stresses other than radiation, including chemotherapy, hyperthermia, oxidative agents, and inflammation. However, radiation differs from these in that it causes a relatively large number of large lesions in DNA that are not only frequently lethal but drive a predominance of pathways triggered by DNA double-strand break formation.
Finally, in addition to molecular and cellular factors that determine intrinsic cellular radiosensitivity, tissue-related and clinical features of radiation exposure add several additional layers of complexity. For example, the number of cells in a tissue capable of regenerating function will be important, as will the way the regenerative potential is distributed as functional subunits within the tissue. Tissue responses may not relate directly to radiation's cytotoxic effects. For example, in tumors, although local control requires elimination of tumor clonogens, in some circumstances vascular damage could be important, especially when irradiation is combined with biologics or chemotherapeutic drugs. Also, irradiation modifies the tumor-host relationship, including interactions with infiltrating cells, such as macrophages and lymphocytes, which have been shown to be able to both promote and inhibit tumor growth. Effects have been described, mainly in vitro, in which the irradiated cell affects the viability or mutability of surrounding “bystander” cells. Such bystander effects may involve more than one mechanism but are presumably, in vivo in normal tissues, a “danger” signaling mechanism for responses to irradiation aimed at tissue healing. Further, some side effects of radiation therapy on
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normal tissues (13) may result from the release of cytokines and other biologic factors or may be associated with remodeling of the normal tissues, rather than cell death per se. For example, late radiation-induced normal tissue fibrosis depends to an extent on cell depletion by radiation, but is more an attempt at a healing response that can be modified by numerous factors, including health status, presence of infection, surgery, injury, and so on.
So, while the initial deposition of energy and subsequent radiochemical events are complete within thousandths of a second following irradiation, a chain of biologic events is initiated that induce programmed cell death or survival, tissue repair and remodeling, all of which depend on the intercellular signaling network, all of which are influenced by systemic and local physiologic conditions. Given the complexity of the biologic condition, it is impossible to predict biologic or clinical outcomes from the amount of physical energy deposited. In other words, biological dose differs from physical dose.
Remarkably, while cells and tissues may respond differently to the same physical dose of radiation, any given tissue appears to respond in a fairly predictable way. The reason for this apparent constancy is that tissue responses are governed largely by cell turnover and the regenerative reserve in the tissue that is generally similar between individuals. Therefore, normal mucosal reactions occur at the same time interval after the start of irradiation and have similar dose-response relationships in most patients. While tumor responses may be more variable than those in normal tissues, certain histologic types of tumors are regarded as more curable by irradiation, and others are not. Reproducible differences in biologic response between tissues are, in fact, exploited for therapeutic benefit. A good example is the use of standard dose fractionation in conventional radiation therapy. This protocol was derived empirically but actually exploits differences in the biologic response between tumor and normal tissues to the same physical dose of radiation. The radiobiologic rationale for the use of dose fractionation in standard radiation therapy has been encapsulated in the 4 “Rs” (reoxygenation, redistribution, repair, and repopulation) (311). This chapter aims to explain how radiobiologic concepts derived from studies dealing with responses within and between normal tissues and tumors are relevant in clinical radiotherapy.
Normal Tissue Radiobiology
Modes of Cell Death after Irradiation
Mitotic Death
Pioneers in radiobiology recognized that cells lethally injured by clinically relevant doses of radiation typically execute one or more divisions before undergoing “mitotic death,” the number depending on the size of the radiation dose (273,274). After 2 Gy, two to three attempts may be made. The progeny of these cells may all die or a proportion may survive to contribute to the reproductive pool, and in the case of tumor recurrence.
Interphase Death
The early pioneers also realized that, in contrast to mitotic death, certain cell types, including many lymphocytes, and some oligodendrocytes and serous cells in the salivary gland, thyroid, intestinal crypt, and hair follicles may undergo relatively rapid “interphase death” within about 2 to 6 hours after irradiation. Typically, only low doses of radiation are required; that is, interphase death is a characteristic of “radiosensitive” cells. Importantly, cells that die during interphase cannot contribute to the reproductive pool. While interphase death was a phenomenon recognized by radiobiologists for decades, recognition of the fact that cells could die by more than one pathway has only recently received wider credence as different pathways leading to cell death have become delineated (67,147,191,256). Interphase death is now acknowledged to represent death by rapid apoptosis.
Apoptotic Death
The morphologic characteristics of apoptosis are nuclear condensation, cell shrinkage, membrane blebbing, and nuclear fragmentation with formation of apoptotic bodies. At the biochemical level, endonucleases are activated that fragment DNA into nucleosomal-sized pieces that are multiples of 180 to 200 base pairs (the size of a nucleosome) and that produce a characteristic “ladder” pattern on agarose gel electrophoresis. In tissue sections, apoptosis can be recognized morphologically. Alternatively, fluorescein or other labels can be attached to the 5′-ends of apoptotic DNA strand breaks using terminal deoxynucleotidyl transferase (TUNEL technique) to allow visualization. In apoptosis, neighboring cells phagocytose cell remnants, and inflammation is not induced. Within a few hours, the whole process is complete, leaving no trace, which makes it easy to underestimate the role of apoptosis in cell loss. Radiation-induced apoptosis in normal tissues is often, but not always, dependent on activation of the tumor suppressor gene p53 (144). Importantly, apoptosis is an active form of cell “suicide” since it requires active metabolic processes. Apoptosis is the primary mechanism by which the body creates organ structures during morphogenesis and tissue sculpting. This is why apoptosis is often called programmed cell death type I. In the adult, apoptosis is required for normal tissue homeostasis; for example, removal of excess cells at sites of proliferation, self-reactive lymphocytes, some cell types as they age or lose the influence of survival signals, virally infected cells, hypoxic cells, or cells damaged by irradiation. It recycles many cell types and removes potentially harmful cells.
The ability to undergo rapid apoptosis is restricted to particular cell types, and even then only at certain positions within tissues that relate to their developmental stage (144). Only cells that have their internal molecular “rheostat” on a proapoptosis setting appear to undergo rapid apoptosis following irradiation. In other words, irradiation tends to increase the frequency of apoptosis only in tissues and tumors in which the cells already have a proapoptotic tendency. Thus, lymphomas generally have proapoptotic tendencies, whereas glioblastoma multiforme cells do not, with or without irradiation.
Necrotic Death
In contrast to apoptosis, necrosis is, for the most part, a pathologic, rather than a physiologic, process that does not require active metabolic cellular processes. It is involved in tissue healing in response to injury or invasion by pathogens. Necrosis can also be a pathologic response to vascular damage, as well as a “default” death pathway for cells that lack an effective apoptotic apparatus. Membrane integrity is lost, cells increase in size, lysosomal enzymes are released, and inflammatory responses are generated with release of cytokines that link cell death to the development of specific immunity. DNA from cells undergoing necrosis forms a “smear” on agarose gel electrophoresis.
Another alternative death style following irradiation is autophagy, where cells internalize cellular organelles within vacuoles and digest them. This is most often seen in nutrient deprivation but is also another form of “programmed” cell death (type II) involved in morphogenesis and tissue sculpting. Other outcomes may be important in specific cell types.
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Radiation-induced differentiation, senescence, or quiescence are possible outcomes that may achieve the same objective as death in removing damaged tumor cells from the reproductive pool and limiting the chances of carcinogenesis. A difference is that in these cases some function is retained for some time. For example, terminal differentiation of fibrocytes gives a cell phenotype that can contribute to radiation-induced fibrosis through enhanced collagen production (121).
While radiation-induced interphase death can easily be ascribed to apoptosis, mitotic death may involve several mechanisms; for example, failure of spindle formation in M phase, loss of the G2 checkpoint leading to “mitotic catastrophe,” or improper chromosome segregation due to damage and loss of genetic material, which may be manifested microscopically as chromosome alterations. Furthermore, mechanistically, the molecular machinery that is employed can be apoptotic, necrotic, or any other. Mitotic death, by its very nature, is normally delayed, occurring over a period of days. Unlike “rapid” (interphase) apoptosis, there is no evidence to suggest that cells that die by “delayed” (mitotic) apoptosis are particularly radiosensitive.
The molecular pathways that participate in cell death are described in another chapter, but it is appropriate here to consider specific phenomena that might relate to in vivo responses. The presence of “survival” signals appropriate for the cell's environment, such as those provided by growth factors, cell-cell contact, and extracellular matrix, will be important in determining response to irradiation. Their loss leads to a phenomenon known as “anoikis” (97) or homelessness, which is a form of “death by neglect.” This is why lymphocytes activated by growth factors are more radioresistant than resting lymphocytes and endothelial cells are more radioresistant in the presence than in the absence of mitogenic basic fibroblast growth factor (98). This may be a broadly applicable concept and explain why blocking receptor tyrosine signaling with cetuximab, Iressa, or similar agents, or NF-κB activation with bBortezomib, may radiosensitize tumor cells.
Survival pathway signaling may also underlie some of the phenomena ascribed to potentially lethal damage repair (PLDR) following irradiation. In PLDR, the cellular microenvironment determines the likelihood of cell death. Classically, PLDR occurs when cells are irradiated and maintained in a contact-inhibited, plateau-phase culture (111,229). If such contact-inhibited cells are trypsinized immediately, or soon after irradiation, survival is compromised, as demonstrated, for example, by lowered clonogenicity. The cells are rendered “homeless” by trypsinization and are more likely to die. It must be noted, however, that PLDR has been invoked as a mechanism that increases survival under diverse sets of experimental conditions and that multiple mechanisms may contribute to the final outcome (282).
As opposed to “death by neglect,” certain positive signals can cause death of susceptible cells. Classically, such signals involve members of the tumor necrosis factor (TNF) family of cytokines (e.g. TNF-α, fasL, Trail). Activation of TNF receptor (TNFR) family members that contain a death domain in their cytoplasmic tail can trigger apoptosis. Other members of the same family of receptors that do not have a death domain, as well as soluble receptors and receptor antagonists, can counteract these death threats. Therefore, the type of receptor and related molecules that a cell expresses may determine its response to TNF. TNF-α causes proliferation of some cell types, such as fibroblasts or their CNS equivalent, astrocytes. In contrast, TNF-α can induce growth arrest or apoptosis in many tumors and in some normal cells, such as oligodendrocytes. Since radiation can induce expression of both TNF and TNFR family, these pathways can contribute indirectly to death or survival of some cell types following irradiation (114,115,125). For example, mice lacking TNFR2, which does not have a death domain and drives cell survival, are particularly sensitive to late effects of irradiation to the brain (61). The cytotoxicity of certain chemotherapeutic agents, such as 5-fluorouacil and cisplatin, can also involve TNF-α (206). Because TNF-α is a primary mediator of inflammation, a proinflammatory cytokine cascade is activated in tissues following irradiation that may also activate bystander cells.
Cell Death in Irradiated Normal Tissues
Radiation-induced apoptosis varies with location within a tissue, reflecting the fact that apoptosis is inherently programmed in a position-dependent manner. For understandable reasons, most of the information on radiation-induced apoptosis in normal tissues comes from studies in mice. Radiation-induced rapid apoptosis in the mouse small intestine is maximal around position 4 from the base of the crypt (232), which is the site of the proliferative compartment and of spontaneous apoptosis. In contrast, apoptosis is not so marked in the colon and is not seen in the proliferative region. Indeed, the antiapoptotic molecule Bcl-2 is expressed by cells in this region (184). In lymphoid tissues, small intestine, hair follicles, and ependyma, there is some concordance between the position of apoptotic cells and radiation-induced up-regulated expression of p53 (54,183,184,232). In subpopulations of cells in other tissues, p53 can be up-regulated with little evidence of rapid apoptosis, while most cells in liver, skeletal muscle, and brain show neither p53 nor much apoptosis (144,169,177,206). In the thymus, developing T lymphocytes with the potential to respond to foreign antigens are selected by apoptotic elimination of both self-reacting and nonreacting T cells in the cortex (positive and negative selection). About 98% of the cells that are generated by mitotic division die (about 5 × 107 cells per day in a young adult mouse). After irradiation, massive rapid, p53-dependent apoptosis of T cells is seen in the cortex, but less apoptosis is evident in the medulla, which contains more mature cells.
The role of apoptosis in normal tissue responses to irradiation has yet to be fully evaluated, but inevitably it will depend on the physiologic role of the proapoptotic cells. If the proapoptotic cells are superfluous to needs, radiation-induced cell death in this compartment may have little impact, but if they are critical to tissue function the opposite will be true. For example, in the mouse small intestine, the cells that die by rapid apoptosis may contribute little to the clonogenic crypt stem cell population. Thus, there is little difference between p53 wild type and p53-null mice in their clonogenic responses following gut irradiation (118), although variation with dose and dose rate is evident (117). In the mouse brain, radiation-induced apoptosis is seen in endothelial cells, oligodendrocytes, and the neuroepithelial subventricular zone (122) where it depends on the protein that is mutated in ataxia telengiectasia and p53, both of which are phosphorylated by radiation and act as sensors of DNA damage, as well as TNFR expression. The extreme radiosensitivity of the developing brain is probably due to its high apoptotic index. Although little information is available in humans, acute parotitis can develop in the first 24 hours of treatment of patients receiving head and neck irradiation, and this reflects apoptotic death of serous cells (251). There is no such acute death in the mucous cells; hence, the mouth is dry and the saliva more viscous.
Pathobiology and Kinetics of Radiation Injury in Normal Tissues
The time to development of most normal tissue injury depends critically on the turnover time of the tissue, that is, on the kinetics of cell differentiation, loss, and renewal. The terms acute,
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subacute, or late are commonly used to describe the time to occurrence of functional inadequacy after irradiation and reflect kinetic differences between tissues while saying little about the underlying pathogenesis of the response. The terms are also often loosely used to describe the tissues in which such effects are seen, as in “acute effects tissue,” but this can be misleading since tissues and organs comprise more than one cell type, each with its own turnover rate characteristics. Any one tissue can therefore express both acute and late symptoms of radiation damage, depending on the cell type that is limiting function at that time. Also, a severe acute injury from irradiation can lead to nonspecific late (consequential) changes such as fibrosis, atrophy, or ulceration (35,223,329) (e.g., stenosis consequent to mucosal ulceration of the bowel, or fibrosis or necrosis of skin or oropharyngeal tissues consequent to desquamation and acute ulceration).
Acute Responses
Acute responses to radiation therapy are defined as occurring during a standard 6- to 8-week course of therapy and are seen in tissues with large populations of cells that turn over rapidly (gastrointestinal mucosa, bone marrow, skin, and oropharyngeal and esophageal mucosa). Hierarchical organization exists in such tissues (187,188,189,303) with a small number of stem cells that proliferate slowly to produce a highly proliferative compartment of progenitor cells that differentiate into mature, nonproliferative, functional cells. Irradiation may deplete the stem and progenitor cell pools, but nonproliferating, differentiated cells maintain tissue function until they are lost through continuing normal cell turnover.
After irradiation, depleted stem and progenitor cell pools may first reconstitute their own numbers before differentiating to restore function, although the extent to which this occurs varies with the tissue. A useful model to consider is that under normal steady-state circumstances (i.e., not growing or involuting), tissues have, by definition, a cell-loss factor (φ) of 1. The only requirement for growth of a tissue is a decrease in φ to less than 1, which is characteristic of the embryo and fetus, tissue regeneration, and malignancy. After irradiation, some tissues (e.g., jejunal crypts) reduce φ to 0 and regenerate quickly; others (e.g., skin) may reduce it to about 0.5 and regenerate less quickly, but continuously produce some functional cells; others (e.g., seminiferous epithelium) show little change in φ and mostly continue in steady state, producing sperm in numbers that are reduced for months or years in direct proportion to the extent of stem cell depletion.
Because acute-responding tissues are organized in a hierarchical fashion, the severity of radiation injury depends on both the extent of stem/progenitor cell depletion and the length of the delay before new functional cells are released into the differentiated compartment. Severity of injury naturally increases with dose, but providing the proliferative pool does not fall below a critical value symptoms are transient and recovery is complete. Dose fractionation can lessen the severity of acute effects by allowing regeneration from the stem/progenitor cell compartment during the course of therapy. Unlike the extent of injury, the rate at which acute injury develops and the latent time to the appearance of symptoms is relatively (187), although not completely (175), independent of dose. This is because latency is mainly determined by the rate of loss of differentiated cells.
Because cell turnover kinetics determines the time to a normal tissue effect, latency is not an indicator of radiosensitivity. For example, in hematopoiesis, leukocyte and platelet numbers drop quickly after bone marrow irradiation because they have a fast turnover rate, whereas anemia is not an obvious acute effect because red cells turn over slowly. Similarly, in the testis, each spermatogenic stem cell division ultimately produces more than 1,000 sperm through successive divisions of spermatogonia and spermatocytes—a process that in humans takes more than 60 days. Early differentiating spermatogonia are few in number and are selectively depleted by doses that have little effect on cells in the more mature stages of spermatogenesis. This is why sperm counts remain normal for several weeks after exposure, falling steeply only at the time when the progeny of the irradiated spermatogonia would normally have reached the seminal vesicles. In the mucosa of the small bowel, mitotic activity is confined to the crypts; the cells lining the villus are nonproliferative. Because crypt cells divide rapidly (an average of more than once daily in humans), they are lost within days if sterilized by radiation. The villus shows no immediate effect of irradiation, with shortening becoming evident only as programmed shedding of differentiated cells into the lumen continues in the absence of renewal from the crypts. This is why symptoms take about 2 weeks to appear in patients undergoing standard daily doses of abdominal irradiation.
Subacute Responses
Certain tissues may display subacute reactions several months after irradiation, reflecting failure of a cell population with a longer turnover time. Symptoms are generally reversible, although in some instances they may be associated with severe damage and even death. Examples of transient effects are Lhermitte's syndrome after spinal cord irradiation, somnolence after brain irradiation, and subacute pneumonitis 2 to 3 months after the start of lung irradiation. Subacute effects occur most often during the remodeling phase in irradiated tissues and prior to the onset of late effects that are associated with slowly progressing damage.
Late Responses
Late reactions to radiation therapy in normal tissues can be severe, and recovery is often limited. They are generally considered to be the result of the depletion of slowly proliferating “target” cells that are lost from the tissue at a slow rate, for example, from central (oligodendroglia) or peripheral (Schwann cells) nervous tissue, kidney (tubule epithelium), blood vessels (endothelium), dermis (fibroblasts), and bones (osteoblasts and chondroblasts). However, abortive rounds of attempts at healing may involve different cell types in distinct cellular compartments, and lesions can appear to evolve with time in a dose-dependent manner (278). Some lesions, such as those associated with artherosclerosis and heart disease (238), can take decades to occur after irradiation and are an increasing problem as patients live longer following cancer therapy. Pathologic findings following collapse of late effects tissues can be very variable. For example, late demyelination after brain irradiation may be ascribed to loss of oligodendrocytes and subsequently of neurons (50), but coincident with and preceding any neurologic changes, proliferation of astrocytes and microglial cells can be observed (51), as can vascular lesions with edema, hemorrhage, or inflammatory infiltrates (45). Infiltrating cells may contribute to the pathogenesis of radiation injury, as is illustrated by involvement of infiltrating cells in the pathogenesis of radiation pneumonitis, or recovery from injury as in the slower healing of skin wounds in mice receiving total body irradiation compared with those irradiated only at the local site (293).
Unlike acute responding hierarchical tissues, slowly proliferating, late effects tissues contain cells that are usually both functional and able to proliferate on demand. In an operational sense, such tissues can be regarded as “flexible” (187,188,189). This does not deny the presence of stem cells with limited function or functional cells that do not proliferate, but the roles of such cells are probably of lesser importance than in hierarchical tissues. The relative inability of late effects tissues to be repopulated
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from a stem cell pool makes radiation reactions in these tissues more chronic and debilitating, diminishing the quality of life for those afflicted.
As in acute-responding tissues, the rate at which radiation injury develops in late effects tissues reflects the turnover rate of proliferative cells, which may be also functional cells in this case (e.g., liver, kidney). Therefore, dose has a greater apparent influence on latency, with late injury developing more quickly with increase in dose. This may be because the greater the dose, the fewer the number of division cycles the cells can successfully negotiate before death (166,177,273). Another reason may be that as cells die, residual (mostly lethally injured) target cells are increasingly recruited to the proliferative pool, causing a cascade or “avalanche” of cell death and functional tissue failure. A third explanation may be that because interactions between cell types tend to be involved in the causation of late effects, the nature of the lesion may vary with time, depending on which cell type is critically limiting at that time (45,284,285). The time course to development of injury can be accelerated and the severity increased by various insults such as surgery, chemotherapy, infection, or physical trauma (105,106). Indeed, such factors may play a major role in precipitating the onset of late effects in humans (e.g., necrotic, nonhealing ulcers after trauma). Conversely, slowing the proliferation process and decreasing stress may reduce their incidence and severity.
Whereas a severe early response in a rapidly proliferating tissue permits adjustment of the dose schedule during the course of standard radiation therapy, this is not the case for late injuries, since they occur after completion of therapy. Tolerance doses for individual patients are therefore based on past experience. Such tolerance doses have not been precisely defined, even though, generally accepted limits to doses considered tolerable by various organs do exist in practice (85,241).
An issue of growing clinical importance is the extent to which late radiation effects can be reversed. It has been shown recently that certain agents given late after radiation can modify injury in tissues. For example, captopril, an angiotensin-converting enzyme inhibitor, slows the development of radiation-induced nephritis (194,196), pneumopathy, and lung fibrosis (192) in rats. Steroids also can prevent death from radiation pneumonitis in animals, although their withdrawal before the end of the usual period of pneumonitis can result in accelerated mortality (109). Pentoxifylline, alone or in combination with vitamin E, protects against radiation-induced late effects in some experimental models (69,151) and in a clinical study, Delanian et al. (63) found that the combination, but neither agent alone, reversed chronic radiation-induced fibrosis. It is not clear how these agents act and whether they promote cellular recovery, but such studies point to ways to improve the future management of late complications of radiation therapy (189).
Functional Subunits
The tolerance of a tissue to irradiation is determined by the number of cells with regenerative potential and the way they are organized, in addition to their intrinsic radiosensitivity. Tissues can be thought of as being composed of functional subunits (FSUs), which is the minimum clonogenic entity required for regeneration of a structure. For example, epilation requires doses lower than those for desquamation. This is not because the cells in the hair follicle differ in their radiosensitivity from those in the basal epithelium, but because there is a smaller number of clonogenic cells in the FSU that produces a hair than in the sheet of basal cells that is capable of regenerating itself. Similarly, hair is depigmented by relatively low doses of radiation (294), but the epidermis loses pigmentation only after higher doses. This is because each hair follicle contains a small number of melanocytes, sometimes only one, whereas melanocytes are more numerous in the epidermis.
In the kidney, each nephron is an FSU (324). If a tubule is completely de-epithelialized, it is lost permanently because it is not repopulated from adjacent nephrons. Therefore, the tolerance dose for the kidney is determined more by the number of tubule cells per nephron than the number of nephrons. For example, if the kidney contained 1011 clonogenic tubule cells distributed as 104 cells in each of 107 nephrons and any one of these 104 cells were capable of regenerating the tubule, then most tubules should regenerate after a dose that reduced survival to 10-4 (or an average of one cell per tubule). Because of the random nature of events, some tubules would then contain more than one surviving cell and others would contain none. From Poisson distribution statistics, 37% of FSU nephrons would be eliminated. The “tolerance” dose would be different if the organ were composed of 104 nephrons, each containing 107 cells: The dose that would eliminate 37% of the nephrons would be that to reduce survival to 10-7. In a multifractionated dose regimen, during which a logarithmic decline in cell number occurs, this is 7/4 (1.75) times that required to reduce survival to 10-4. This is why tolerance doses can vary so much among tissues and organs, even if the target cells have the same intrinsic radiosensitivity.
It is easy to appreciate the structural organization of FSUs for hair and kidney, but not for some other tissues. For example, in mouse skin, the survival of about 10 cells per cm2, from what is normally approximately 106 basal stem cells per cm2, is required to maintain uninterrupted integrity and prevent overt desquamation (308). Therefore, the FSU would be about 1/10 cm2. Organs with acinar or alveolar architecture (e.g., salivary glands, pancreas, sweat glands, testis, mammary epithelium, lung, and perhaps liver) may resemble the kidney in having structurally defined FSUs, while the target cells in dermis, CNS, mucosae, gut, and epidermis are less restricted by physical barriers and cellular migration may influence regeneration.
Known tolerance doses are consistent with FSUs being relatively small in tissues such as kidney with structurally well-defined FSUs, are intermediate in the spinal cord, and large in the dermis. Obviously, a primary tumor is just one large FSU in which one surviving clonogen can lead to recurrence. Clonogenic cell number will play a big role in determining local tumor control. For metastatic deposits, regional control will depend not only on the number of clonogenic cells each contains but also the number of metastatic deposits. In general, a larger number of small metastatic deposits will be cured with lower radiation doses than a small number of larger metastases, even if the total cell number is the same in both cases.
Volume Effects
Traditionally, radiation oncologists have reduced the total dose when treating large volumes of normal tissue. In fact, the now widespread reduction in dose per fraction from 2 to 1.8 Gy had its origin in a volume effect; the longer treatment duration enhanced mucosal tolerance in large head and neck treatment fields (93). In the orthovoltage era, a reduction in dose with increase in treatment volume was generally recommended (218), but with the advent of skin-sparing megavoltage beams, the volume effect received less attention, especially as larger tumors (and larger treatment volumes) require higher doses for their control.
In fact, the concept of decreasing dose with increasing treatment volume has little radiobiologic basis, except in specific circumstances. For example, if FSUs are arranged in series, like links in a chain, as they are in nerve tracts, spinal cord, and the cylindrical sheath of peritoneum covering the small intestine,
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the loss of one subunit may result in an overt expression of injury, regardless of the state of the other subunits in the series. The probability of injury increases with volume (number of FSUs exposed) (Fig. 2.1). Such a volume effect has been demonstrated clinically for small bowel obstruction (160,231) and experimentally for myelitis (126,246,286).
View Figure FIGURE 2.1. Diagrammatic representation of the influence on the probability of a complication from increasing the treatment volume in a tissue where functional subunits (FSUs) are arranged serially. The average survival of FSUs was 1 in 16, sterilized FSUs being denoted by the black squares. With the small volumes (A), the probability of myelitis was 6% (1/16), whereas it would approach 100% if 16 FSUs in one patient were exposed (E). The actual probabilities can be calculated using the equation in the text. (From Withers HR, Taylor JMG, Maciejewski B. Treatment volume and tissue tolerance. Int J Radiat Oncol Biol Phys 1988;14:751,with permission.)
The relationship between the number of FSUs irradiated (n) and the probability of a complication (P) is described by the following formula:
P = 1 - (1 - p)n
where p is the probability of the loss of one FSU. This relationship is illustrated in Figure 2.2. Increasing the volume (number of FSUs exposed) reduces the dose necessary to produce a complication and increases the steepness of the dose-response curves. The effect would be predicted to occur when the average number of surviving cells per FSU is reduced to almost one and when the length of serial arrangement of FSUs is small. This may be less true for the small bowel (100,160,319) than for spinal cord.
View Figure FIGURE 2.2. Curves illustrating how the probability of producing a complication increases with increase in the number of serially arranged functional subunits (FSUs) included in the treatment volume. The curves were positioned by assuming that 58 Gy in 2-Gy fractions sterilized 10% of FSUs and that for a series of 2-Gy fractions the effective D0 for the target cells was 4 Gy. The curves are shifted to the left and are steeper with increase in number of FSUs exposed, but this effect becomes less obvious once large numbers of subunits are involved. (From Withers HR, Taylor JMG, Maciejewski B. Treatment volume and tissue tolerance. Int J Radiat Oncol Biol Phys 1988;14:751, 1988,with permission.)
Nonradiobiologic “volume effects” exist that can result from multiple mechanisms. There are some mechanisms that can be excluded. For example, there is no evidence that cellular radiosensitivity is affected by an increase in treatment volume (332). The radiosensitivity of skin epithelium is constant over a 5,000-fold range of treatment area (308,332). Also, no evidence exists for an increased role for vascular damage as volume increases (326). On the other hand, “volume effects” can be seen when (332):
A patient tolerates a small area of injury (such as ulceration) better than a large area of the same severity because pain, exudation, infection are worse, healing is slower, and consequential contraction and scarring are more of a problem. The effect of increasing volume is to make the injury more incapacitating, even though the severity of the radiation response is independent of volume treated.
Large gradients in dose distribution and heterogeneity develop as volume increases. Without prudent planning, a tumor dose may be prescribed at the 80% level, leading to a 25% higher dose in the region of Dmax. Also, with large fields, large variations in contour may exist. These variations could result in a high-dose region where tissue thickness is less than that measured at the midplane; as, for example, in the spinal cord at the thoracic inlet in thoracic irradiation and in tangential fields for treatment of the breast. If the threshold-sigmoid curve of the probability of normal tissue complications against dose is steep, as it is in experimental studies (266), a 25% increase in total dose could produce a marked change in the incidence of complications. This increase in physical dose is further compounded by the biologic effectiveness of each dose per fraction being increased by more than 25%, or the “double trouble” of increased physical and biological dose. The magnitude of the increase in “biological” dose will depend on the dose per fraction and the type of normal tissue, but will be greatest in late-responding normal tissues for reasons that will be explained later in terms of the nonlinear rate of increase in injury with increase in
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dose per fraction. Because this additional augmentation of biologic doses is not evident from physical isodose contours, an increased biologic effect may be erroneously attributed to the large volume being treated per se.
If organ “reserve” is obliterated as volume is increased (e.g., lung, salivary gland), this is not a true volume effect because sequelae are determined by the volume and functional status of the tissue excluded from the treatment volume, not the volume irradiated.
Molecular Responses in Normal Tissues
As discussed in the introductory section, radiation-induced signal transduction pathways can be important in determining the cellular response to damage. Cell- and tissue-specific patterns of molecular responses are detected minutes to hours after irradiation that can vary with radiation dose, dose rate, and quality, and with dose-response relationships that are not always linear. The most rapid response includes transcriptional and posttranscriptional activation of members of immediate early gene families, such as c-jun and c-fos, ATM (ataxia telangiectasia), p53, c-abl, EGFR (epidermal growth factor receptor), and other molecules. Phosphorylation/dephosphorylation reactions or other activating mechanisms are invoked. Importantly, these pathways couple molecular damage to DNA repair, cell cycle arrest, phenotypic changes, and cell death. Another part of the early response involves the induction of sets of secreted molecules such as proinflammatory cytokines, proteases and antiproteases, cell adhesion molecules, and extracellular matrix materials that together form a regulated acute tissue reaction (125) to trigger subsequent tissue remodeling. Such “danger” signaling can extend beyond the radiation field and may be responsible for some of the observed “abscopal” effects of irradiation. The overall function of radiation-induced molecular responses is to preserve cell and tissue integrity after irradiation by promoting cell death/survival and tissue recovery and remodeling.
Radiation-induced inflammatory responses, as well as initiating healing responses in tissues, may “prime” cells and tissues for adaptive responses to further radiation doses. For example, radiation-induced basic fibroblast growth factor may act through autocrine pathways to promote survival of endothelial cells (98). In vivo, antagonists of radiation-induced interleukin (IL)-1 and TNF-α increase the intrinsic sensitivity of mice to bone marrow death after irradiation (200,201), suggesting that these responses also have adaptive survival value. On the other hand, radiation-induced TNF-α can cause certain cells to apoptose (115) and may trigger clinical symptoms that can not be ascribed to cell death (16,130). Examples are nausea or vomiting that can occur within hours of irradiation of the upper abdomen, acute erythema and edema associated with vascular leakage, fatigue in patients receiving irradiation to a large volume, especially within the abdomen, and somnolence that can develop within a few hours of cranial irradiation. Radiation-induced proliferative responses such as gliosis (51) or certain forms of fibrosis could also cause symptoms unrelated to cell depletion.
Recently, it has been shown that during the latent period leading up to the expression of late effects, waves of molecular responses occur that may reflect repeated attempts at tissue recovery and remodeling (49). As a result, the concept that late effects represent dysregulation of an integrated injury and healing process that involves both parenchymal and vascular elements, as well as inflammatory cells, has gained in prominence. Failure of any of the required elements could give rise to a late effect.
Cytokines and growth factors are thought to play important roles in late effects. For example, signaling through the TNF receptor 2 protects mice from late effects of brain irradiation (61). Anscher et al. (14,15) have reported that lung cancer patients with elevated plasma levels of transforming growth factor-β (TGF-β) prior to radiation therapy are more likely to develop radiation pneumonitis, illuminating the importance of the interaction of systemic change with local radiation damage. Elevated TGF-β levels could derive from the tumor or the stromal cells that invade it, or may be radiation-induced; the outcome will be the same. Inhibiting TGF-β activation during radiotherapy is being investigated as a strategy to lower the risk of pneumonitis in patients with non–small cell lung cancer. In addition, dose escalation is being attempted in patients whose TGF-β levels normalize during a course of radiation therapy (16).
Regeneration (Repopulation) in Normal Tissues
The time to onset of repopulation after irradiation and the rate at which it proceeds vary with the tissue. Both can be measured experimentally by a split-dose technique in which the increase in the number of cells with time after the first dose is reflected by an increase in size of the second dose required to produce a certain constant level of effect (isoeffect).
In acute-responding tissues, the onset of repopulation is early because cell loss is rapid. In the jejunal mucosa, the lag time before the onset of radiation-induced proliferation may be <24 hours. In the colon and stomach, it is slightly longer. In contrast, in renal tubules, there is no histologic evidence of cell depletion for many months after irradiation, and there is a long lag period before the onset of repopulation. In the mouse, it takes more than 12 months to reconstitute a tubule (324). The rate of repopulation has, similarly, not been well quantified in acutely and, especially, in late-responding tissues. In mice, some approximate doubling times for clonogenic cells are 8, 12, and 22 hours for jejunum, colon (310), and skin (309), respectively.
In humans, tissue turnover kinetics are slower than in mice. They have been approximated for oropharyngeal mucosa from consideration of responses to various dose-fractionation regimens. Mucositis begins to appear 14 to 21 days after the start of a regimen of 2 Gy given five times per week, but repopulation begins at about 10 to 12 days (288). High initial doses may shorten the lag period, but only by 1 or 2 days. Repopulation can increase the tolerance of the mucosa to a conventional dose regimen by an average of at least 1 Gy/day, which is equivalent to approximately a doubling of clonogenic cell numbers every 2 days, and it may be significantly faster (321). If daily irradiation is suspended (e.g., during a 10- to 14-day break in a split-course accelerated regimen), clonogenic cells may repopulate at two or three times this rate (9,12,288,299,321).
These values for lengths of lag time and repopulation rates are, at best, estimates. Figure 2.3 shows that in some tissues, regeneration begins within 1 or 2 days of the initiation of radiation therapy, whereas in others there is no evidence for regeneration even after 2 months. The critical point is that there is a lag period followed by a phase of rapid exponential growth. In general, the lag period is shorter for chemotherapy, hyperthermia, and surgery because the cell depletion that stimulates regeneration occurs more rapidly than after irradiation.
The importance of repopulation is implicit in the history of radiation therapy. The current standard protracted overall treatment times confer a benefit by allowing regeneration of acute-responding tissues, which reduces toxicity. When attempts are made to deliver curative therapy more quickly, acute responses become more severe and dose-limiting.
Growth factors may be useful in protecting normal tissues from irradiation by shortening the apparent lag phase and accelerating recovery in irradiated tissues. Hematopoietic growth
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factors such as G-CSF, GM-CSF, erythropoietin, and IL-11 can accelerate proliferation of hematopoietic cells (199). In doing so, they minimize the danger of infection. In epithelial tissues keratinocyte growth factor, which is specific for epithelial cells, has similar potential. It protects the oral mucosa, small intestine, lung, and hair follicles against chemotherapy or radiation injury (73,87,88,339) in preclinical models and has shown efficacy in clinical bone marrow transplantation trials.
FIGURE 2.3. Representation of the approximate kinetics of regeneration of irradiated normal tissues (solid lines, solid symbols) and tumors (dashed lines, open symbols). Curves are based on measurements or estimates of regeneration; symbols denote times at which an effect of regeneration has already appearedor has not yet appeared. The logarithmic abscissa is for convenience of presentation only and has no biologic rationale. In general, the human data are displaced to the right of experimental animal data, reflecting a slower initiation of repopulation because human tissues proliferate more slowly than do their rodent counterparts, they were exposed to protracted dose regimens; and less-sensitive end points were used to detect onset of repopulation in humans. Numbers on the curve and symbols refer to the sources of data. 1, Withers (310); 2, Withers (309); 3, Withers and Mason (322); 4, McCulloch and Till (179); 5, Hermens and Barendsen (120); 6, Suit et al. (259); 7, Choi et al. (53); 8, Denekamp (64); 9, Fletcher (93), Horiot et al. (128), van der Schueren et al. (288), Wang (297,298), and Wang et al. (299); 10, Barendsen and Broerse (21); 11, Arcangeli et al. (17); 12, Withers et al. (320) and Meistrich et al. (180); 13, Maciejewski et al. (168,169,170,171,172); 14, Allen (2); 15, Maciejewski et al. (169); 16, Barker et al. (22); 17, Wang (297,298) and Wang et al. (299); 18, Parsons et al. (216); 19, Maciejewski et al. (168); 20, Pedrick and Hoppe (219), Maciejewski et al. (170), Fisher and Hendry (91), Withers and Mason (324), and van der Kogel (284,285); 21, White and Hornsey (304); and Withers et al. (337); 22, Xu et al. (338) and Ang et al. (12); 23, Ang et al. (9); 24, Kummermehr and Trott (154); 25, Chen and Withers (48); and 26, Turesson and Notter (279).
“Remembered” Dose: Tolerance to Retreatment
Conventional teaching in radiation oncology has been that a heavily irradiated tissue will not tolerate retreatment. The postulated reason was that the basis of late effects was vascular damage and was irreversible. While irradiation may limit the tolerance of a tissue to retreatment, in fact, retreatment is often possible and may be better tolerated than previously expected (156). Factors that determine the extent to which residual injury will limit retreatment tolerance include the amount of cell depletion caused by prior treatment, the time elapsed since that treatment and therefore the extent of regeneration, and the tissue at risk. High prior doses, short intervals between treatment courses, and slow regeneration of target cells will reduce retreatment tolerance.
Some data for experimental radiation myelitis are shown in Figure 2.4. The plot shows the effect of size of the first dose on the dose required to produce myelitis in a second regimen. Recovery is complete after low doses, but is progressively compromised as the initial dose approaches tissue tolerance (174). It should be remembered that clinical “tolerance” doses for the spinal cord of 45 to 50 Gy in 1.8- to 2-Gy fractions are low in terms of the injury evaluated in Fig. 2.4 (50% incidence of myelitis). The time to recovery for the spinal cord is not accurately known, but in rats at 100 days it is about half of what it reaches by 200 days (287). In monkeys, there was extensive recovery from 44 Gy in 2.2-Gy fractions by 2 years, but a detailed profile of the time course could not be established (7).
Not all tissues, or elements within tissues, recover at an equal rate or to an equal extent from the effects of irradiation. Acute-responding epithelial and hemopoietic tissues generally recover quickly and demonstrate a high tolerance to retreatment in terms of acute responses. However, the fibrovascular support in skin and mucosa and the stroma in bone marrow are less tolerant to retreatment because they respond more slowly. The kidney shows poor retreatment tolerance as assessed functionally in mice (253). Reirradiation tolerance in this organ is inversely related to the initial dose, but tolerance decreases significantly with increasing interval between treatments, suggesting progression rather than recovery from the initial damage.
Because different tissues show different levels of tolerance to retreatment, caution should be exercised in the application of these concepts to the clinic. Also, the experimental studies deal with well-defined end points within a limited time scale. If different end points in the same tissue are examined or the time is extended, the same guidelines may not apply. It should
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also be noted that if slowly proliferating cells involved in late responses are extensively depleted, recovery may be permanently incomplete, and the organ will be vulnerable to further injury whether it is from radiation, trauma, cytotoxic drugs, or any other insult. For example, hyperthermia can precipitate myelitis in a patient who has had high, but otherwise tolerable, doses of x-irradiation (161), and trauma from dental intervention frequently precipitates mandibular necrosis.
View Figure FIGURE 2.4. The dependence of remembered dose on size of priming dose (as a percentage of the ED50) is shown for a variety of animal species at long periods (6 months to 2 years) after the initial radiation treatment: ○, adult rhesus monkey (10); ▲, 12-week-old rat (130); □, 1-day-old guinea pig (149); ●, young adult mouse (165); ▲, 8-week-old guinea pig (174); ▼, 8-week-old guinea pig (198); ▪, 3-week-old weanling rat (243); ♦, young adult rat (287). (From Mason KA, Withers HR, Chiang CS. Late effects of radiation on the lumbar spinal cord of guinea pigs: Re-treatment tolerance. Int J Radiat Oncol Biol Phys 1993;26:643,with permission.)
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