Repair of Radiation Damage

A critical determinant of a cell population’s sensitivity to radiation is the ability of cells to repair DNA damage caused by radiation. One Gy of radiation from photons causes approximately 2500 base damages, 1000 single-strand breaks, and 40 double-strand breaks in DNA in each cell.⁴ Most of this damage is repaired by cells within 6 to 24 hours; the double-strand breaks are the most lethal because they may lead to severe chromosomal aberrations. A given dose of radiation is preferentially cytotoxic to proliferating cells, including tumor cells and renewing cell populations (e.g., epithelial stem cells), although slowly dividing and nondividing cells (e.g., bone and cells of the nervous system) are also affected by radiation.

Cell Cycle Effects

The period of the cell cycle in which DNA undergoes synthesis is known as S-phase. Before and after S-phase are periods without overt activity by the DNA; these periods are called G₁ phase and G₂ phase. G₂ phase is followed by mitosis. Cells are distributed throughout the cell cycle in a tumor or tissue at a given time. The individual cell sensitivity to irradiation varies, depending on the phase of the cell cycle at the time of irradiation. Cells in late S-phase are most resistant to irradiation, and cells in late G₂ or mitosis are most sensitive.

Oxygen Effects

Because of their rapid growth and abnormal vasculature, tumors often become partially hypoxic. This results in upregulation of hypoxia-inducible proteins that may prepare the tumor cells to handle stresses. Oxygen is also a critical factor in the response to irradiation because reactive oxygen species (ROS) generate much of the damage from radiation. As a result, normoxic cells are up to threefold more sensitive to radiation than hypoxic cells.

Relative Biologic Effectiveness

Although this chapter primarily discusses the effects of photons (x-rays and gamma rays) and electrons, there are many other forms of radiation that can be used in oncology, including protons and neutrons. The biologic effects of 1 Gy of electrons are the same as 1 Gy of photons, but 1 Gy of protons or neutrons may cause substantially more damage than 1 Gy of photons or electrons. This difference is known as the relative biologic effectiveness (RBE) of the type of radiation.

Time, Dose, and Fractionation

Early radiation oncologists found that higher total doses could be given if the doses were divided into smaller fractions. They observed that tumor response was improved, and less injury of normal tissue occurred. In veterinary medicine, standard fractionation denotes a regimen delivering 2.7 to 4 Gy per fraction, 3 to 5 times per week to a total dose of 42 to 57 Gy, although several other regimens are currently being used or investigated. Hyperfractionation refers to schedules in which the dose per fraction is reduced and the total dose is increased. Accelerated fractionation describes a treatment regimen in which the overall time of treatment is reduced, but the dose per fraction and total dose are unchanged. Hypofractionation describes the administration of high doses per fraction given in a small number of fractions to a lower total dose. The response of tumor and normal tissues between fractions throughout the course of radiation therapy has been described by Withers⁵ as the “four Rs” of radiation therapy: repair of DNA damage, redistribution of cells in the cell cycle, reoxygenation of tumor cells, and repopulation of tumor and normal tissues.

Division of the total radiation dose into fractions is important for a number of reasons. The first reason is to exploit potential differences in repair capabilities between tumors and normal tissues. Slowly dividing cells are somewhat less sensitive to small doses of radiation than more rapidly dividing cells; however, they appear to become relatively more sensitive if radiation is delivered in larger doses per fraction. If smaller doses per fraction are used, normal tissues with slowly dividing cells can be spared relative to tumor tissues with rapidly dividing cells.

Other events that occur between radiation fractions are cell cycle redistribution and reoxygenation. When a fraction of radiation therapy is administered, many of the cells in the sensitive portions of the cell cycle are killed. During the interval between fractions, cells from the late S-phase, which are more likely to be alive than other cells, progress to more sensitive parts of the cell cycle. This is known as redistribution. Reoxygenation also occurs during the interval between radiation fractions when many of the hypoxic tumor cells become aerobic and thus more sensitive to irradiation.

The length of time over which radiation therapy is administered is important, primarily because of tumor repopulation but also because of rapidly proliferating normal tissues, such as mucosa and skin. Tumor cells that have not been destroyed by irradiation continue to replicate during the course of therapy. This process is exacerbated by a phenomenon known as accelerated repopulation. Some have suggested that after approximately 4 weeks of therapy, tumors repopulate more rapidly than initially.⁶ The reason for this is not clear, but the phenomenon could be related to (1) a reduction in the cell cycle time, (2) an increase in the number of tumor cells that are actively dividing, or (3) a reduction in the number of tumor cells that normally die (cell loss factor). Regardless of the cause, when treatment lasts longer than 4 weeks, repopulation may affect the outcome unless total dose is increased to account for this phenomenon. Repopulation may have a greater impact on rapidly dividing tumors than on slowly dividing tumors.

Repopulation of proliferating (also known as acutely responding) normal tissues is also affected by time. The same total dose of radiation administered over a short period results in somewhat more severe acute effects than if administered over a longer course. Nonproliferating (late-responding) normal tissues are not significantly affected by the length of time over which therapy is administered. Fraction size and the interval between treatment fractions are more important in late-responding tissues. Fractions should be separated by at least 6 hours to allow repair of DNA damage to normal tissues. Cells of the brain and spinal cord may require additional time for complete repair, and the impact of multiple fractions per day on these late-responding tissues is not clearly understood.

The total dose administered to a patient should have a low probability for causing significant late normal tissue reactions in the region of therapy. However, the response of tissues also depends on the fraction size. For example, 48 Gy administered in 4 Gy fractions has a higher probability of causing late effects than 48 Gy administered in 3 Gy fractions (Figure 12-1). The probability of tumor control is not as affected because rapidly proliferating tissues, including tumors, are not as sensitive to the change in dose per fraction. The benefits of protocols that use small doses per fraction are clear: they allow a higher total dose to be administered without increasing the probability of damage to late-responding normal tissues.

The total dose tolerated depends on the specific normal tissues present in the irradiated field. For example, brain and spinal cord are less tolerant to the effects of irradiation than muscle or bone. Another factor that must be considered when selecting the appropriate dose is the volume of tissue in the field. Large volumes of normal tissues are more susceptible to damage from irradiation than smaller volumes.

Although time, dose and fractionation are still the underpinnings of SRT, the paradigm is different than for fractionated radiation therapy. SRT involves the use of high doses per fraction but overcomes the radiobiologic limitations with stereotactically verified positioning and treatment delivery techniques that leave a minimal volume of normal tissue in the high dose area. Stereotactic radiation therapy, by definition, requires (1) a tumor for targeting (not microscopic disease), (2) treatment planning and administration that will provide a dramatic dose drop-off between the tumor and the surrounding normal tissue structures, and (3) a method of stereotactically verifying patient positioning. The result is that normal late-responding tissue structures are spared through dose avoidance rather than by administering small doses per fraction. The normal tissue structures still receive dose, and the dose per fraction is higher than for traditional radiation therapy. However, the total dose to the normal structures is lower than what is typical for fractionated radiation therapy. Normal tissue tolerance data are just evolving for SRT, as is long-term follow-up. Estimates of tolerance have been based on limited clinical data, toxicity observation, and educated guessing.⁷ It is inappropriate to extrapolate dose constraints from fractionated protocols. An additional difference between SRT and fractionated radiation therapy is that with SRT, acutely responding normal tissues in the surrounding region such as skin, esophagus, and colon may be susceptible to consequential late effects. A consequential late effect is a late effect that develops from severe acute effects that may be associated with stem cell depletion. Dose constraints therefore must also be applied to these tissues. SRT treatment is generally delivered in 1 to 5 fractions over a period of 1 week or less for most tumors; therefore accelerated repopulation is unlikely to impact tumor control. Although historically stereotactic treatment was delivered in a single fraction (referred to as stereotactic radiosurgery), current technology allowing precise repositioning makes limited fractionation feasible. Even this minimal fractionation should allow higher total doses to be administered safely to late-responding tissues in the region and presumably take advantage of tumor reoxygenation and redistribution. However, biologic response following SRT has not been comprehensively evaluated. From a practical standpoint, SRT minimizes the number of anesthesia episodes to these older and sometimes debilitated patients and is also generally more convenient for the owner. Acute effects are minimal and tumor-associated signs such as discomfort or dysfunction often improve rapidly. Long-term tumor control and late effects need to be quantitated.

No perfect radiation therapy protocol exists, and all protocols commonly used in veterinary and human medicine have advantages and disadvantages. It is not within the scope of this chapter to prescribe specific radiation doses or fractionation schedules because many factors must be considered. Rather, referring veterinarians must know what to expect when sending patients to a radiation oncology center, and they should be able to explain some of the fundamental principles to clients. The radiation oncologist should inform the referring veterinarian and owner of the probabilities of late effects and tumor control and estimate the degree of acute effects expected with a specific protocol. The goal of radiation therapy is to destroy the reproductive capacity of the tumor without excessive damage to surrounding normal tissues. This goal is best achieved by dividing the total dose into a number of smaller fractions (fractionation) that are administered over a period of time or by applying stereotactic technology. Regardless of the approach, the relationship of these three parameters (time-dose-fractionation) must be carefully considered in the development of radiation treatment plans for successful therapy.

Acute and Late Effects

Reactions from radiation therapy are classified as acute (also called early) or late. Acute effects occur during or shortly after radiation therapy. Acute effects involve rapidly proliferating tissues, such as the oral mucosa, intestinal epithelium, and epithelial structures of the eyes and skin. Concurrent chemotherapy can exacerbate acute effects from radiation. These effects generally are self-limiting, and recovery is rapid. However, acute effects can be unpleasant for the patient and distressing to the owner, and in rare instances they can be life-threatening if the proper care is not given. The referring veterinarian often is called on to treat recently irradiated patients. Acute effects will heal without medical intervention in the vast majority of cases over the course of weeks or occasionally months. In veterinary patients, the most important provision to allow healing during this time is prevention of self-trauma of the radiation site by the patient. Therefore pain management plays an important role and should be addressed. Pain management for cancer patients is discussed in Chapter 15, Section A, of this text, and specific protocols have been published.⁸ Additional treatment is based on common sense, supportive care, and the knowledge that the signs will resolve with time.

Late effects involve more slowly proliferating tissues, such as bone, lung, heart, kidneys, and nervous system. The dose of radiation administered is limited by the tolerance of these normal tissue structures in the field. Late reactions can be difficult to treat; it is the radiation oncologist’s obligation to minimize the incidence of late effects with appropriate dose prescriptions and careful radiation planning and treatment. When late effects occur, they may be quite severe, resulting in fibrosis, necrosis, loss of function, or even death.⁹ Late effects occur from the loss of normal tissue stem cells with concurrent radiation-induced vascular changes and inflammation. These changes are multifactorial, but the cytokine transforming growth factor-β (TGF-β) is believed to play a critical role in radiation fibrosis. Strategies attempted in human radiation oncology to mitigate late radiation effects include the use of antioxidants and free radical scavengers (superoxide dismutase, vitamin E, thiol radioprotectors), vascular-directed therapies (clopidogrel, statins, pentoxifylline), antiinflammatory agents (corticosteroids), inhibitors of the renin-angiotensin system (angiotensin-converting enzyme [ACE] inhibitors), and stem cell therapies.⁹ In veterinary patients, severe late reactions such as fibrosis and tissue necrosis should be managed under the guidance of or referred to a surgeon and/or radiation oncologist experienced in dealing with radiation injury.

Radiation-Induced Neoplasia

Ionizing radiation is a complete carcinogen, capable of initiating, promoting, and progressing cellular changes that lead to cancer. Therefore it is possible to see radiation-induced neoplasia develop in a radiation treatment field. It appears that orthovoltage radiation and radiation with high linear energy transfer (high-LET) such as neutrons result in carcinogenesis at a higher frequency than the megavoltage photons typically used in veterinary radiation therapy.¹⁰ Other factors that influence the risk of radiation carcinogenesis include the age of the patient (young patients are more likely to develop subsequent tumors) and the tissues irradiated. Certain tissues are also more prone to development of radiation-induced tumors, such as the thyroid gland. For a tumor to be considered radiation induced, the following criteria must be met^10,11:

The overall incidence of radiation-induced tumors in patients treated with radiotherapy is thought to be extremely low (<1% to 2% of patients treated).¹¹

Cell Survival After Irradiation

After a tissue or population of cells is exposed to any dose of radiation, a fraction of the cells will be killed. The proportion of remaining cells is known as the surviving fraction (S). The sensitivity of a tumor or tissue to radiation can be shown as a graph of the radiation dose (D) versus the surviving fraction (Figure 12-2).¹² The relationship between a dose of radiation and the surviving fraction of cells is commonly described by the linear quadratic equation:

where S is the surviving fraction at a dose (D).¹³ Alpha (α) and beta (β) are constants that vary according to the tissue with α corresponding to the cell death that increases linearly with dose and β corresponding to cell death that increases in proportion to the square of the dose (also known as the quadratic component). The α/β ratio is a useful number that is the dose in Gy when cell kill from the linear and quadratic components of the cell survival curve is equal. Cells with a higher α/β ratio have a more linear appearance when plotted on a log scale, and cells with a low α/β ratio have a parabolic shape. The α/β ratio is also an important description of the radiosensitivity of a cell. At low-dose fractions, tissues or cells with low α/β ratio are relatively radiation resistant compared to tissues or cells with high α/β ratio. It has been suggested that tissues and cells with low α/β ratios have a greater capacity for repair of sublethal radiation damage. Sublethal radiation damage is defined as damage that can become lethal if it interacts with additional damage. Sublethal damage repair is the reason that cell survival increases when a radiation dose is split into two fractions separated by a time interval.

Most early responding tissues and tumors have a high α/β ratio, whereas late-responding tissues have a low α/β ratio.¹³ There are some tumors that may have a low α/β ratio, which can influence the optimal radiation prescription in terms of total dose, time, and fractionation. Tumors that may have lower α/β ratios include melanoma, prostatic tumors, soft tissue sarcomas, transitional cell carcinoma, and osteosarcomas.^14–16

The concept of biologic effective dose (BED) is used to predict how changes in dose prescription may preferentially affect different cells or tissues based on their α/β ratio in the linear quadratic model of survival. The formula for BED is as follows:

where n is the number of fractions and d is the dose per fraction. If the α/β ratio of a tissue is known or can be estimated, one can calculate the BED for any dose prescription. It is possible to use this formula to assess how dosimetry changes or errors alter the effective dose of a protocol. It is important to note that there are several limitations to the use of this equation, including that it does not account for differences in the overall length of time of the radiation protocol or accelerated repopulation. There are other formulas that can be used to account for time. Also, the true α/β ratio of any cell or tissue is rarely known; therefore calculations made with this model involve making assumptions or predictions that may be incorrect. Nevertheless, this formula is a useful tool when considering hyperfractionating or hypofractionating a standard radiation protocol in order to create a new protocol that will have expected outcomes related to either tumor control or tissue complications. However, the validity of BED for SRT is unclear.

Alternative Mechanisms of Radiation Injury

In addition to direct radiation cytotoxicity, the extracellular matrix and local microenvironment also appear to play an important role in radiation response. Some evidence suggests that apoptosis of endothelial cells may precede damage to proliferating cells and cause some of the effects observed after radiation. Apoptosis of endothelium primarily occurs when high-dose fractions are administered and may play a more important role when hypofractionated and radiosurgical doses of radiation are delivered.

Chemical Modifiers of Radiation

Although rarely used clinically in veterinary medicine, many drugs can modify the cellular and tissue response to radiation. Radioprotectors (e.g., amifostine/WR-2721) are compounds that decrease the amount of radiation damage to targeted normal cells without providing similar protection to tumor cells. Radiosensitizers are chemicals that achieve greater tumor inactivation than would be expected from the additive effect of treatment with either radiation or the chemical alone. Mechanisms of action of radiation sensitizers include hypoxic cell sensitizers or cytotoxins and agents that damage or incorporate into DNA.

Palliative Radiation Therapy

Palliative radiation therapy is commonly used in human medicine, and its use in veterinary medicine has increased in recent years. Palliative radiation is generally hypofractionated compared to curative intent protocols, often administered in larger doses per fraction (6 to 10 Gy per fraction) in 1 to 4 total fractions once or twice weekly. Conversely, palliative therapy can be delivered in conventional doses or modestly hypofractionated daily or twice daily for a short but intense treatment regimen. The goal of palliative radiation therapy is not to provide long-term or definitive tumor control; rather, it is intended to relieve pain or improve function or quality of life in patients in which other factors (e.g., advanced metastatic disease) are likely to lead to early demise. Palliative radiation therapy has been used most often for metastatic or primary bone tumors, principally canine osteosarcoma (see Chapter 24). Palliative therapy is more convenient for the owner, and the cost is modest compared with curative radiation therapy because fewer fractions are administered. It is, however, important to remember that palliative radiation is not a substitute for curative-intent protocols despite the convenience. Some palliative therapy protocols may have an increased probability of causing late radiation effects but because they are prescribed to patients that have a poor long-term prognosis, these late effects may not have time to manifest. Curative-intent radiation protocols require strict adherence to radiation biologic principles; palliative radiation protocols, on the other hand, are far more flexible. As in human hospital settings, the protocols may vary dramatically from radiation center to radiation center.