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16.5.1 Cellular and Tissue Responses
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Radiation treatment can cause loss of function in normal tissues. In renewal tissues, such as skin, bone marrow, and the gastrointestinal mucosa, loss of function may be correlated with loss of proliferative activity of stem cells. In these and other tissues, loss of function may also occur through damage to more mature cells and/or through damage to supporting stroma and vasculature, the influx of immune cells, and the induction of inflammatory responses (Stewart and Dorr, 2009). Traditionally, the effects of radiation treatment on normal tissues has been divided, based largely on directly observable functional and histopathological end points, into early (or acute) responses, which occur within 3 months of radiation treatment, and late responses that may take many months or years to develop. It should be noted that such endpoints do not assess early changes in gene expression associated with irradiation that occur in all tissues (see below). Acute responses occur primarily in tissues where rapid cell renewal is required to maintain the function of the organ. Because many cells express radiation damage during mitosis, there is early death and loss of cells killed by the radiation treatment. Late responses tend to occur in organs whose parenchymal cells divide infrequently (eg, liver or kidney) or rarely (eg, central nervous system or muscle) under normal conditions. Depletion of the parenchymal cell population as a result of entry of cells into mitosis, with the resulting expression of radiation damage and cell death, will thus be slow. Damage to the connective tissue and vasculature of the organ (which also proliferates slowly under normal conditions) may lead to progressive impairment of its circulation and secondary parenchymal cell death may occur as a consequence of nutrient deprivation. The loss of functional cells may induce other parenchymal cells to divide, causing further cell death as they express their radiation damage, leading eventually to functional failure of the organ. Consequential late effects may also occur where severe early reactions have led to impaired tissue recovery and/or development of infection. Several systems for documenting normal tissue responses (side effects) to irradiation in patients have been developed to facilitate cross-comparisons between investigators and institutions. These include the Radiation Therapy Oncology Group (RTOG)/European Organization for Research and Treatment of Cancer (EORTC) classification, the Common Terminology Criteria for Adverse Events (CTCAE v4) scale devised by the National Cancer Institute (NIH/NCI, 2009) and the Late Effects Normal Tissue Task Force Subjective, Objective, Management, and Analytic (LENT/SOMA) system, specifically designed to score late reactions (Hoeller et al, 2003).
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The radiosensitivity of the cells of some normal tissues can be determined directly using in situ assays that allow the observation of proliferation from single surviving cells in vivo. One such assay determines the fraction of regenerating crypts in the small intestine following radiation doses sufficient to reduce the number of surviving stem cells per crypt to 1 or less, and analysis of the results allows the generation of a survival curve (Tucker et al, 1991). Survival curves obtained for the cells of different normal tissues in mice and rats are shown in Figure 16–13. Considerable variability in sensitivity is apparent, and as with tumor cells, most of the difference appears to be in the shoulder region of the survival curve, suggesting differences in repair capacity.
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Alternative experimental analyses of normal tissue radiation damage most often use functional assays. The crudest functional assay is the determination of the dose of radiation given either to the whole body or to a specific organ that will cause lethality in 50% of the treated animals within a specified time (LD50). The relationship between lethality and single radiation dose is usually sigmoidal in shape, and some experimentally derived relationships for different normal tissues in mice are shown in Figure 16–14.
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For individual organs, a level of functional deficit is defined and the percentage of irradiated subjects that express at least this level of damage following different radiation doses is plotted as a function of dose. A tolerance dose for a specific organ can be defined as the dose above which more than 5% of patients express that level of functional deficit (TD5). In animal models, complete dose-response curves have usually been obtained and an example for the rat spinal cord using forelimb paralysis as the end point is shown in Figure 16–15. These curves are sigmoidal in shape and generally quite steep. Similar results have been reported for specific functional deficits in many other tissues (eg, increased breathing rate in lung, reduced flexibility as a result of increased fibrosis in subcutaneous tissue, elevated clearance rates in kidney).
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An influx of immune cells (macrophages, lymphocytes, and neutrophils) into irradiated tissue and increased cytokine and chemokine expression have been observed within hours after irradiation when there are no apparent functional changes, and aspects of this inflammatory response may persist over months as the irradiated tissue transits to regeneration and repair (Schaue and McBride, 2010). Early increases in cytokine expression can occur after low doses of radiation (~1 Gy), but longer-term changes have been observed after larger doses (5 to 25 Gy). A wide range of cytokines is involved including pro- and anti-inflammatory factors, such as tumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1α and IL-1β), and TGF-β. In specific tissues, the response to radiation may include other growth factors that are associated with collagen deposition, fibrosis, inflammation, and aberrant vascular growth. These inflammatory factors may induce production of damaging radicals, such as reactive oxygen species, independently of those caused directly by the radiation treatment. The interplay between cell killing, cell repopulation, cytokine production, vascular damage, and immune cells infiltrates in producing the overall tissue damage remains poorly understood and is likely to vary from one organ to another (Stewart and Dorr, 2009).
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16.5.2 Acute Tissue Responses
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Acute radiation responses occur mainly in renewal tissues and have been related to death of critical cell populations such as the stem cells in the crypts of the small intestine, in the bone marrow, or in the basal layer of the skin. These responses occur within 3 months of the start of radiotherapy (in humans) but are not usually limiting for fractionated radiotherapy because of the ability of the stem cells in the tissue to undergo rapid repopulation to regenerate the transit and end cell populations. Radiation-induced cell death in normal tissues generally occurs when the cells attempt mitosis, thus the tissue tends to respond on a time scale similar to the normal rate of loss of functional cells in that tissue and the demand for proliferation of the supporting stem cells. Radiation-induced apoptosis can also be detected in many tissues, but is usually a minor factor in overall radiation-induced cell death, except in lymphoid and myeloid tissue.
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Endothelial cells in the vasculature supporting the crypts and villi of the small intestine of mice have been reported to be prone to radiation-induced apoptosis, and prevention of this effect by treatment with basic fibroblast growth factor can protect the animals against radiation-induced gastrointestinal injury, suggesting that dysfunction of the vasculature can reduce the ability of the crypts to regenerate (Paris et al, 2001). Contrary to most cell killing, which involves DNA damage, radiation-induced apoptosis of endothelial cells can occur via a cell membrane effect leading to activation of the ceramide pathway (see Chap. 15, Sec. 15.3.2) (Kolesnick and Fuks, 2003), and blocking this effect can protect the intestine from radiation damage (Rotolo et al, 2012). These effects appear to be more prominent following larger radiation doses (>10 Gy) such as those used in stereotactic body radiotherapy (SBRT) than at the doses used commonly for fractionated radiation therapy (~2 Gy).
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Following irradiation of mucosa (and skin), there is early erythema within a few days of irradiation as a result of increased vascular permeability related to the release of 5-hydroxytryptamine by mast cells. Similar mechanisms may lead to early nausea and vomiting observed following irradiation of the intestine. Expression of further acute mucositis (or moist desquamation in skin) and ulceration depends on the relative rates of cell loss and cell proliferation of the transit cells and the (basal) stem cells in the tissue. The time of expression for this damage depends on the time over which (intensity of) the dose is received (Fig. 16–16), and the extent of these reactions and the length of time for recovery is dependent on the total dose received and the volume (area) of mucosa (or skin) irradiated. Early recovery depends on the number of surviving basal stem cells that are needed to repopulate the tissue and these cells can migrate from undamaged areas into the irradiated area. Erythema occurs in humans at single doses greater than 24 Gy in 2 Gy fractions, whereas mucositis occurs after fractionated doses above approximately 50 Gy in 2 Gy fractions. Severe skin reactions in patients are relatively uncommon as high-energy radiation beams have a build-up region that results in a reduced dose at the skin surface (see Sec. 16.2.4 and Fig. 16–3A), but oral mucositis is prevalent during radiation treatment of head and neck cancers.
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16.5.3 Late Tissue Responses
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Late tissue responses occur in organs whose parenchymal cells normally divide infrequently and hence do not express mitosis-linked death until later times when called upon to divide. They also occur in tissues that manifest early reactions, such as skin/subcutaneous tissue and intestine, but these reactions (subcutaneous fibrosis, intestinal stenosis) are quite different from early reactions in these tissues. Late responses (usually regarded as those which occur more than 3 months after treatment) usually limit the dose of radiation that can be delivered to a patient during radiotherapy. Damage can be expressed as diminished organ function, such as radiation-induced nephropathy (symptoms of hypertension or increased serum creatinine) or myelopathy following spinal cord damage, as illustrated in Figure 16–15, and is usually progressive over time. The nature and timing of late reactions depends on the tissue involved. Damage to stromal and vascular elements of the tissue and the influx of inflammatory cells may cause secondary parenchymal cell death, resulting in increased cell proliferation and further death of parenchymal cells as they attempt mitosis. The latent period to manifestation of organ dysfunction depends on the dose received, because the higher the initial dose the smaller the fraction of surviving parenchymal cells that can repopulate the tissue.
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One common late reaction is the slow development of tissue fibrosis that occurs in many tissues (eg, subcutaneous tissue, muscle, lung, gastrointestinal tract), often several years after radiation treatment. Radiation-induced fibrosis is associated with a chronic inflammatory response following irradiation, the aberrant and prolonged expression of the growth factor TGF-β and radiation-induced differentiation of fibroblasts into fibrocytes that produce collagen (Hakenjos et al, 2000; Martin et al, 2000). Transforming growth factor-β also plays a major role in wound healing and the development of radiation fibrosis has similarities to the healing of chronic wounds (Denham and Hauer-Jensen, 2002). Another common late reaction is progressive vascular damage, including telangiectasia that can be observed in skin and mucosa, and loss of microvasculature leading to atrophy (and fibrosis) that is manifest in skin and other tissues. Figure 16–17 shows the development of telangiectasia in patients following fractionated treatment and illustrates that heterogeneity in response between different patients is not limited to tumors but can also occur with normal tissue effects (Turesson et al, 1990).
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The lung is an important site of late radiation damage. There are 2 types of reactions: pneumonitis that occurs 2 to 6 months after irradiation, and fibrosis that usually occurs more than 1 year after irradiation. These reactions can cause increases in tissue density on CT scans (see Chap. 14, Sec. 14.3.1) and increases in breathing rate if severe. Measuring changes in breathing rate has been used extensively to assay the dose–response relationship for radiation-induced lung damage in rats and mice, particularly the development of pneumonitis. Studies in rodents have documented that inflammatory cells and inflammatory cytokines play a major role in lung response to irradiation injury (Fig. 16–18), but the relationship between this inflammatory response and the later development of functional symptoms is unclear. Studies in lung cancer patients suggest that increases in TGF-β levels in plasma following radiotherapy can contribute to the likelihood of developing lung complications (Anscher et al, 1998; Evans et al, 2006).
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The dose required to cause functional impairment in lung depends on the volume irradiated, with small volumes being able to tolerate quite large doses (Marks, Bentzen et al, 2010); this is a result of the functional reserve of the remaining lung because the irradiated region will develop fibrosis. Studies in rodents, using the dose required to cause an increased breathing frequency in 50% of animals (ED50) as an end point, have defined a relationship between ED50 and lung volume irradiated, which indicates that the base of the lung is more sensitive than the apex (Travis et al, 1997). The underlying mechanisms may relate to the functional reserve in different regions of the lung and/or to the extent of cytokine production following irradiation of different regions of the lung. There is also evidence for regional effects following irradiation of human lung (Marks, Bentzen et al, 2010).
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A theoretical framework introduced by Withers et al (1988b) suggests that late responding tissues can be considered as arrays of functional subunits (FSU) containing groups of cells that are critical for function (eg, bronchioli in lung, nephrons in the kidney). These FSU were postulated to be able to be regenerated from a single surviving tissue stem cell. Furthermore, tissues were considered to have these FSU operating in parallel to achieve overall tissue function (such as occurs in lung, kidney, liver) or in series (such as in spinal cord or intestine) in analogy with electrical circuits. Tissues with a parallel structure of FSU have substantial reserve capacity and, although damage to a small volume may completely inactivate this volume, the remaining regions can maintain function and/or may undergo hypertrophy to replace any loss of function (eg, kidney and liver). Tissues with a series structure of FSU may cease to function if even a small region of the tissue is irreparably damaged, such as may occur in the spinal cord where localized injury can cause complete tissue dysfunction and myelopathy, or in the intestine if severe stenosis causes obstruction. In practice, tissues do not fall neatly into these 2 categories for various reasons, including the common role of the vasculature, the development of inflammatory responses that may extend beyond the treatment field, because FSU may require more than one type of undamaged stem cells for repair and these stem cells may migrate into areas of damage either locally or via the circulation. However, the concept that the volume irradiated to high dose is critical to tissue response and that this varies for different organs is well established and used in mathematical models designed to predict normal tissue complication probabilities (NTCP) (Bentzen et al, 2010; Marks, Yorke et al, 2010).
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16.5.4 Whole-Body Irradiation
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The response of animals to single-dose whole-body irradiation can be divided into 3 separate syndromes (hematological, gastrointestinal, and neurovascular) that manifest following different doses and at different times after irradiation (Mettler and Voelz, 2002; Dainiak et al, 2003). The neurovascular syndrome occurs following large doses of radiation (>20 Gy) and usually results in rapid death (hours to days) as a consequence of cardiovascular and neurological dysfunction. The gastrointestinal syndrome occurs after doses greater than approximately 5 to 15 Gy and, in rodents, doses at the upper end of this range usually result in death at about 1 week after irradiation as a consequence of severe damage to the mucosal lining of the gastrointestinal tract; this causes a loss of the protective barrier with consequent infection, loss of electrolytes, and fluid imbalance. Intensive nursing with antibiotics, fluid, and electrolyte replacement can prevent early death from this syndrome in human victims of radiation accidents, but these patients may die later as a result of damage to other organs, particularly skin, if large areas are exposed. The hematopoietic syndrome occurs at doses in the range of 2 to 8 Gy in humans (3 to 10 Gy in rodents) and is caused by severe depletion of blood elements as a result of killing of precursor cells in the bone marrow. This syndrome causes death in rodents (at the higher dose levels) between approximately 12 to 30 days after irradiation, and somewhat later in larger animals, including humans. Death can sometimes be prevented by bone marrow transplantation (BMT) and cytokine therapy (eg, GM-CSF [granulocyte-macrophage colony-stimulating factor], G-CSF [granulocyte colony-stimulating factor], stem cell factor) provided that the radiation exposure is not too high when damage to other organs may become lethal. Following the Chernobyl accident, 28 of the emergency workers (of 104 identified as showing symptoms of acute radiation syndrome) died within 4 months. Most of these workers received bone marrow doses greater than 4 Gy and much higher doses to the skin (10 to 30 times). Bone marrow failure was the primary cause of death, particularly for those dying within the first 2 months. Although 13 of these patients had BMT, most died, probably because of serious radiation damage to the skin (UNSCEAR, 2008). There are substantial differences in the doses required to induce death from the hematopoietic syndrome (ie, LD50 value) between different species of animals and even between different strains of the same species. The LD50 value for humans has been estimated at 4 to 7 Gy, depending on the available level of supportive care (excluding BMT). Following doses greater than approximately 2 Gy, humans will develop early nausea and vomiting within hours of irradiation (prodromal syndrome), which may be controlled with 5-hydroxytryptamine antagonists.
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16.5.5 Retreatment Tolerance
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Although tissues may repair damage and regenerate after irradiation, previously irradiated tissues may have a reduced tolerance for subsequent radiation treatments, indicating the presence of residual injury. For tissues that undergo only an early response to radiation, there is almost complete recovery in a few months, so that a second high dose of radiation can be tolerated. For late-responding tissue damage, the extent of the residual injury depends on the level of the initial damage and is tissue dependent. There is substantial recovery in skin, mucosa, spinal cord, and lung over a period of 3 to 6 months, but kidney and bladder show little evidence of recovery (Stewart and Dorr, 2009). Clinical studies have demonstrated that retreatment to high doses with curative intent is possible depending on the tissues involved but usually entails increased risk of normal tissue damage.
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16.5.6 Predicting Normal Tissue Response
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Patients receiving identical radiation treatments may experience differing levels of normal tissue injury (see, eg, Fig. 16–17). Thus predictive assays might be useful in identifying those patients who are at greater risk of experiencing the side effects of radiotherapy. The enhanced radiosensitivity of patients with ataxia telangiectasia (AT) and other DNA repair-deficiency syndromes (see Chap. 5, Sec. 5.5) supports a genetic contribution to individual variability in radiosensitivity, although other factors, such as diet or environment, could also play a role. Studies of women with breast cancer show individual correlation of acute and late skin reactions in one treatment field with those in a different treatment field (Bentzen et al, 1993). Several studies have quantified the in vitro radiosensitivity of fibroblasts and peripheral lymphocytes as a potential predictive assay for normal tissue damage. These studies show variations in the radiosensitivity of fibroblasts from individual patients, but are inconsistent in predicting late radiation fibrosis (Russell and Begg, 2002). Fibroblasts from people who are heterozygous for mutations in the ATM gene are reported to have increased radiosensitivity, but it has not been possible to directly link severity of normal tissue reactions to heterozygosity in this gene. Thus, although cellular sensitivity is an important contributor to normal tissue damage, other factors, such as cytokine induction and the response of the tissue stroma and vasculature, likely also play an important role in normal tissue injury. In rodents, genetic differences associated with different strains have been reported to influence the development of normal tissue damage, for example, pneumonitis and fibrosis following lung irradiation, although these factors do not affect the radiosensitivity of lung fibroblasts directly (Haston et al, 2002). Extensive genetic screening of large populations is underway to identify genetic alterations or gene signatures that may predict patients predisposed to differential responses to irradiation (West et al, 2010).
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16.5.7 Radioprotection
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Many agents can protect against radiation damage to cells in culture (Weiss and Landauer, 2009). These include agents that can scavenge radiation-produced radicals, such as dimethyl sulfoxide (DMSO), or the superoxide dismutase enzymes (SODs), or sulfhydryl-containing compounds, such as glutathione, and cysteine. These latter compounds can also donate a hydrogen atom back to a radical site created on a macromolecule such as DNA by hydroxyl radicals produced by water radiolysis. Because of the short lifetimes of radiation-induced radicals, exogenously added agents have to be present in the cell at the time of the irradiation. They are equally effective for tumor and normal cells in vitro; thus specificity for therapeutic application in vivo depends largely on preferential uptake of such agents into the normal tissue. One agent that appears to fulfill this criterion is amifostine, a prodrug that is converted into a sulfhydryl-containing compound in vivo by the action of alkaline phosphatases. The selective activity of this compound in normal tissue is believed to be a result of poor penetration from tumor blood vessels and reduced levels of alkaline phosphatase in tumors. Amifostine was shown to protect a variety of normal tissues with variable, mostly small, protection of tumors in animal models (for review, see Lindegaard and Grau, 2000). Studies in patients with head and neck and lung cancers show substantial protection of normal tissue, including salivary gland, lung, and mucosa, without detectable change in tumor response (Brizel et al, 2000; Antonadou et al, 2003), although the compound is not widely used clinically because of unrelated toxicities.
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Another strategy for protection of normal tissue is to block the development of late radiation effects with treatment given after the end of the radiation. The use of steroids after irradiation to prevent lung injury is an example, although this treatment appears to delay the development of symptoms rather than prevent them. Agents that block angiotensin-converting enzyme (ACE) activity (eg, captopril, enalapril) or that block directly the action of angiotensin II mitigate the development of radiation-induced pulmonary fibrosis and nephropathy, respectively (Cohen et al, 2011). Various strategies, including antiinflammatory agents and antioxidants, are being studied in patients to determine whether they can reverse the progressive nature of radiation-induced fibrosis and necrosis (Delanian and Lefaix, 2007; Delanian et al, 2011). Extensive efforts are underway to develop agents to mitigate multiple organ damage in cases of accidental radiation exposure (Williams and McBride, 2011).
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16.5.8 Therapeutic Ratio
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The concept of therapeutic ratio is illustrated in Figure 16–19, which shows theoretical dose–response curves for tumor control and normal tissue complications as described in Sections 16.3.1 and 16.5.1 (see also similar curves for systemic therapy in Chap. 17, Fig. 17–9). Tumor-control curves (red lines) tend to be shallower than those for normal tissue response (blue lines) because of the large heterogeneity among tumors as discussed in Section 16.3.1. The therapeutic ratio is often defined as the percentage of tumor cures that are obtained at a given level of normal tissue complications (ie, by taking a vertical cut through the 2 curves at a tolerance dose, eg, at 5% complications after 5 years). In experimental studies, an alternative approach is to define the therapeutic ratio as the ratio of radiation doses Dn/Dt required to produce a given percentage of complications and tumor control (usually 50%). It is then a measure of the horizontal displacement between the 2 curves. It remains imprecise, however, because it depends on the shape of the dose–response curves for tumor control and normal tissue complications. The curves shown in Figure 16–19A depict a situation in which the therapeutic ratio is favorable because the tumor-control curve is displaced to the left of that for normal tissue damage. The greater this displacement, the more radiocurable the tumor. Because the tumor-control curve is shallower than that for normal tissue damage, the therapeutic ratio tends to be favorable only for low and intermediate tumor-control levels. If the 2 curves are close together or the curve for tumor control is displaced to the right of that for complications (Fig. 16–19B), the therapeutic ratio is unfavorable because a high level of complications must be accepted to achieve even a minimal level of tumor control.
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