Therapeutic Strategies in Veterinary Oncology

By Nicole Amato, Jacob Cawley, Steven Dow and 12 more

This book is a comprehensive resource for veterinary oncologists and trainees, covering therapeutic strategies used in the treatment of veterinary patients. In the setting of a rapidly changing field like oncology, this timely text focuses on mechanisms of action and biological rationale rather than current specific clinical recommendations, allowing current and future clinicians to adapt treatment approaches as our understanding of the biology of cancer evolves.

With each chapter written by experts in their field, this book provides informative figures that convey this biological understanding and rationale of therapy. It starts from the mechanisms of treatment as we currently understand them, covering radiation therapy, chemotherapy, immunotherapy, targeted therapy, and many more. Dispensing vital, detailed and practical information about the different therapeutic strategies available, this book is a vital resource for practicing veterinarians, while also providing students of veterinary oncology with a better understanding of the key differences between different treatment strategies.

• Language: English
• Publisher: CAB International
• Release date: Aug 23, 2023
• ISBN: 9781789245820

Book preview

1 Radiation Therapy

Ira Gordon*

The Oncology Service, Springfield, Virginia, USA

Introduction: Types of Radiation

What radiation is and why it matters to clinicians

The term radiation refers to the emission of electromagnetic waves (photons) or subatomic particles (i.e., electrons, protons). When radiation is absorbed by matter, this can lead to excitation, which is the raising of the energy of an electron of an atom. Excitation can create heat, which is useful for devices like radios or microwaves (including for microwave or radiofrequency ablation medically) but does not otherwise have major biologic effects. Common types of nonionizing radiation include radiofrequency waves, microwaves, infrared light, and ultraviolet radiation. If the amount of energy in a discrete photon or particle is high enough, this can lead to ionization, which is the ejection of an electron from an atom. The term ionizing radiation refers to forms of radiation that have sufficient energy to cause ionization (Hall and Giaccia, 2011).

The distinction between ionizing and nonionizing radiation is of biologic importance due to the markedly greater biologic effects of ionizing radiation that occur from the localized release of large amounts of energy, capable of breaking chemical bonds. Breaking of chemical bonds can lead to alterations in molecules, potentially creating free electrons, reactive free radicals, or directly damaging or breaking molecules that are critical to the cell including DNA. For the purposes of this chapter, when the term radiation is used subsequently, this will always be in reference to ionizing radiation.

Medical uses of radiation

There are many types of ionizing radiation that can be used medically. The most common form of medical radiation is photons (usually X-rays). Electrons and protons are charged particles that are used therapeutically. In veterinary medicine, electron therapy is common because many clinical linear accelerators have both photon and electron therapy capability. Protons are another charged particle with therapeutic utility; they have significant dosimetric benefits compared to photons and electrons (Gibbons, 2019). Proton therapy equipment has not yet become commonly available in veterinary medicine outside of research facilities. Neutrons, positrons, and heavier ions (such as carbon ions) remain largely in experimental use only. This chapter focuses primarily on the effects of photons as these remain the predominant form of radiation used in radiotherapy in animals although the biologic effects of electrons are similar.

Fundamental Concepts in Radiation Oncology

Several related but distinct concepts in radiation are used to describe how radiation is delivered (Fig. 1). It is important to distinguish the differences between radiation dose, radiation technique, radiation course, and radiation intent.

Radiation dose

The concept of radiation dose is different from drug dosing in that radiation is typically dosed to a defined volume of tissue, rather than the entire body. Radiation effects differ when radiation is absorbed by different tissues. Absorbed radiation dose is not typically uniform throughout the target or body. As defined above, the unit Gy defines absorbed radiation dose. Although it would be lethal for the entire body to receive a uniform dose of 4–8 Gy, it is quite common for a portion of the body to receive doses far in excess of 4–8 Gy during cancer treatment. For example, a palliative course of radiation for canine osteosarcoma will frequently consist of two to four doses of 8–9 Gy each with a very low incidence of significant side effects (Vail et al., 2019).

Radiation dose is typically prescribed to a point or to a volume. For example, particularly when performing manual dose calculations, it is common to prescribe and calculate dose to a point within the center of the tumor or treatment field (International Commission of Radiation Units and Measurements, 1999). Since dose is typically heterogeneous, it is the responsibility of the radiation oncologist to understand how dose is distributed in a given treatment technique as they determine where to prescribe the dose to. When treatment plans are performed using cross-sectional imaging and computerized treatment planning software, dose can be calculated to every pixel within a treated volume. There are several ways to prescribe dose to a volume. Dose is often displayed with isodose lines, which are lines connecting all points receiving a given dose (or dose percentage). One can prescribe or normalize radiation dose to an isodose line such that the prescribed dose (i.e., 50 Gy) is received by that isodose line. Alternatively, it is common to prescribe dose based on a treated volume. One common convention is to prescribe dose such that 95% of that planned target volume receives the prescribed dose. One should note that such a dose prescription results in 5% of the volume receiving a dose that is less than prescribed and it is likely that within the treated volume, there will be areas that receive significantly greater than the prescribed dose. For some treatment plans, it is ideal to minimize dose heterogeneity such that there are no hot spots in excess of 10–15% greater than the prescribed dose. In other treatment techniques, significant areas of high dose are permissible within the target provided that those high doses do not reach sensitive normal tissue structures. As discussed later in this chapter, while it would be ideal to dose radiation based on the dose required to control a tumor, this is typically not known and instead, radiation is dosed based on expected or acceptable levels of predicted toxicity.

Because there has not been significant standardization of how dose is prescribed in veterinary radiation oncology, there may be significant differences between institutions. This complicates the reporting and understanding of radiation dose in veterinary literature. Recently, some veterinary journals have begun requiring authors to report radiation dose prescription in a more detailed and standardized manner (Keyerleber et al., 2012).

Radiation technique

Radiation treatment technique describes the way that radiation is planned or delivered. Treatment techniques performed at a radiation facility are generally dependent upon the type of radiation equipment installed. It is worth mentioning that despite the recent and rapid evolution in radiation capabilities over the last 30 to 40 years in veterinary medicine, tumor control and survival outcomes for most tumor types are similar in recent investigations compared to studies from the 1980s and 1990s (Evans et al., 1989; Evans et al., 1993; Théon et al., 1993; Adams et al., 1998; Hunley et al., 2010; Mariani et al., 2015; Kubicek et al., 2016; Mayer et al., 2019). This may be due, in part, to a reluctance or inability to significantly escalate radiation dose despite these advances. Potential techniques/technologies include:

Manually planned/calculated radiation involves measuring a tumor target and manually setting up radiation beams to incorporate an appropriate margin and then calculating dose based on measurable features including field size, treatment distance, target depth, and attenuation factors of any beam-shaping tools such as blocks, trays, and wedges. If multiple beams are used, the dose from each must be calculated and summed to determine the total dose delivered.

Three-dimensional conformal radiation therapy (3D-CRT) refers to one of several advances in computer-based planning. This has become possible due to advances in radiation and imaging technology. Conformal radiation refers to shaping of radiation dose to better match the contour of the treatment target. 3D-CRT involves the use of cross-sectional imaging (computed tomography (CT) scan +/– magnetic resonance imaging (MRI)) and computer treatment planning systems to develop radiation plans consisting of multiple beam angles, beam-shaping blocks, and sometimes beam-shaping wedges or compensators to create radiation dose distributions that better approximate the shape of the treatment target.

Intensity-modulated radiation therapy (IMRT) is similar to 3D-CRT but uses fields consisting of hundreds or thousands of beamlets, which are thin beams of radiation of differing intensities delivered from multiple angles into the target. With most radiation equipment, these beamlets are created by a multi-leaf collimator (MLC).

Stereotactic radiation therapy (SRT) also refers to using highly conformal planning and treatment delivery techniques but also relies on extreme accuracy to treat minimal margins around a well-defined tumor target. Stereotactic radiosurgery (SRS) refers to the specific instance of delivering SRT in a single fraction, typically in intracranial radiation. When SRT is used to treat areas outside of the head/neck, it is sometimes referred to as stereotactic body radiation therapy (SBRT) or stereotactic ablative radiation therapy (SABR).

Radiation course

Radiation courses typically entail repeated treatments, delivering the same dose multiple times. Each treatment within a radiation course is known as a fraction of radiation. The total dose received is the sum of the individual fractions. Radiation courses are best defined by the dose, time/schedule, and fractionation of the treatment protocol. As an example, the patient is receiving a course of radiation consisting of 18 daily weekday fractions over 3.5 weeks, delivering a fractional dose of 2.8 Gy to 95% of the target volume and a total dose of 50.4 Gy. For simplicity, radiation courses are often described relative to what is considered standard. Standard courses of radiation are typically performed on a schedule that consists of one treatment per day, every weekday (Monday–Friday), for about 16–20 treatments. Hypofractionated refers to a course of radiation that consists of fewer total treatments, typically at higher doses per fraction and often at a lesser frequency (i.e., one or two treatments per week). Hyperfractionated treatments consisting of significantly greater than 23 treatments are rare in veterinary oncology. There are a few courses that are defined as accelerated, which implies that the entire treatment is given over a shorter time period, typically due to concern about tumor cell repopulation during a longer treatment course (Evans et al., 1989; Fidel et al., 2011; Marconato et al., 2020). An accelerated course could consist of a standard number of fractions by increasing to two fractions per day (e.g., a protocol for feline oral squamous cell carcinoma consisting of 14 fractions of 3.5 Gy in a 9-day period) and/or may be hypofractionated (e.g., a protocol for canine nasal tumors consisting of ten weekday fractions of 4.2 Gy over 2 weeks). It is important to distinguish between the treatment course and treatment technique as any treatment course can be applied to any treatment technique.

Radiation intent

Radiation treatment intent is classically defined as definitive (also known as curative intent) or palliative, although this may be incomplete. The distinction between these is that a definitive course of radiation is primarily intended to maximize the duration of tumor control whereas a palliative intent treatment is primarily intended to provide alleviation of clinical signs and minimize toxicity. It is generally true that definitive protocols are more finely fractionated and palliative protocols are usually hypofractionated, although there are now many short protocols utilizing SRT techniques that are definitive in intent.

In veterinary radiation oncology, it has become common to utilize hypofractionated protocols with the goal of providing temporary tumor control and minimal side effects in pets that may or may not require palliation of symptoms. An example is a hypofractionated course of radiation consisting of 6 Gy per fraction twice weekly for five total fractions for canine macroscopic/nonresectable soft tissue sarcoma (Cancedda et al., 2016). In one study, this course provided a median progression-free interval of 286 days when used alone and 757 days when used in addition to metronomic chemotherapy. Many dogs with nonresectable soft tissue sarcoma are not experiencing significant symptoms of disease at the time of diagnosis and therapy, but are good candidates for treatment with the intent of delaying tumor progression. Accordingly, it is inappropriate to characterize this treatment course as palliative, but it is also clearly not a definitive protocol. It is common jargon to describe veterinary radiation courses as either a definitive course of radiation or a palliative course of radiation. However, there are many instances where the course and intent of radiation are not clearly aligned or implied. Accordingly, distinguishing between the course of radiation as described above and the intent of radiation is increasingly important. It may be preferable to shift away from these simplified descriptors of radiation intent although it will certainly remain important to discuss treatment intent and goals with pet owners.

Clinical Radiation Equipment

In medicine, there are several ways that patients may receive radiation. Most of the focus in oncology and in this chapter is on teletherapy, which is radiation administered by an external source or machine. For radiation therapy this is most commonly performed with a linear accelerator, although other technologies including cobalt-60 teletherapy machines and lower-energy orthovoltage treatment equipment have been used.

There are also systemic forms of radiation such as iodine-131 or samarium-153, which are radioactive isotopes that are injected and absorbed systemically. These radioisotopes then concentrate in areas of biologic utilization (thyroid gland for iodine and areas of bone turnover for samarium), resulting in high delivered doses in focal areas.

Brachytherapy involves the placement of a radiation source directly in or near a target and allows for a more concentrated and localized type of radiation to be delivered. There are several types of equipment that can be used for brachytherapy including small X-ray sources and remote-after loading systems which utilize a computer-driven mechanism to move a radioactive source through catheters that are pre-placed in the target tissue.

Plesiotherapy is a specific form of radiation involving the direct application of a radioactive probe to the surface of a lesion. An example of plesiotherapy is the strontium-90 applicators which are used in veterinary oncology for the treatment of various small superficial tumors, including feline squamous cell carcinoma of the planum nasale and pinna, feline mast cell tumor, and ophthalmic tumors in dogs, cats, and horses (Goodfellow et al., 2006; Turrel et al., 2006; Hammond et al., 2007; Plummer et al., 2007; Ware and Gieger, 2011; Nevile et al., 2015; Berlato et al., 2019).

Linear accelerators have become the most widely used type of radiation equipment due to their safety, high dose rate, and ability to produce megavoltage photons (having an average energy of > 1 million electron volts) (McEntee, 2004; Farrelly and McEntee, 2014). The most important properties of megavoltage photons that make them suitable/preferable for therapeutic oncology are excellent penetration into tissue, predictable dosimetry, and relatively consistent dose absorption and penetration through varying tissue types (skin, fat, muscle, bone).

Due to the predictable interactions of megavoltage photons with matter, accurate and advanced computerized radiation treatment planning systems have been developed. These systems allow for detailed and precise dose calculations throughout large volumes of irradiated tissue. This allows for the rapid calculation and delivery of complex radiation beam arrangements and treatment plans with dose distributions that can conform to the shape of complex tumors/targets.

Modern linear accelerators are equipped with a variety of accessories to augment treatment techniques and/or to aid in the localization and positioning of a patient with accuracy. An MLC is a field-shaping device consisting of many small independently moving leaves of shielding material. This device is computer-driven to create complex field shapes. The leaves can also be programmed to move while a radiation beam is on to create a field comprising many different zones of intensity or fluence of radiation in a technique referred to as IMRT. An electronic portal imaging device (EPID) is a calibrated digital X-ray plate that can be used to create digital radiographs from the primary treatment beam. This is very useful for verifying and adjusting patient setup and in some cases is used to validate dose delivered. On-board imaging (OBI) involves a second X-ray source that is used for setup validation, often using lower-energy X-rays for improved image contrast and/or for rotational imaging known as cone-beam computed tomography (CBCT). While not yet widely utilized, some of the newest linear accelerators can also use fiducial markers, surface tracking devices, or even built-in MRI for increasingly accurate patient setup procedures.

Despite continued advances in treatment delivery equipment and patient setup techniques that improve our capabilities, there remain inherent limitations to the precise delivery of photon radiation. It is documented that with appropriate positioning and imaging equipment/expertise, we can obtain submillimeter accuracy in patient setup. However, imaging techniques and clinical assessments remain insensitive at assessing the true microscopic extent of cancers. Several studies show that different trained experts, even within the same institution, will take the same imaging studies and draw significantly different tumor contours. Even tightly acceptable tolerances with respect to variations between planned and delivered doses of radiation are typically in the 2–3% and 2–3 mm range.

The Veterinary Radiation Team

A veterinary radiation team is responsible for a variety of aspects of patient care. In some practices, a single individual may be responsible for multiple facets. A significant emphasis is often placed on the specific type of radiation equipment that is installed at a facility when veterinarians or clients consider the treatment capabilities or quality of a veterinary radiation facility. It should be kept in mind that the radiation equipment is just one part of a facility’s capabilities. In most cases, the most important factors that relate to clinical outcomes and client satisfaction are not related to the type of radiation equipment or linear accelerator at a facility. A metaphor that may be useful is to think of the linear accelerator like a complex racing car. The highest-performing race teams do not necessarily have the best car. It is the skills of the driver (radiation oncologist) and the entire crew (radiation staff) that play a substantial role in the outcomes.

The veterinary radiation oncologist oversees and should be primarily responsible for all aspects of patient care including patient evaluation for radiation and consultation with pet owners about treatment options. In addition, the radiation oncologist should prescribe the radiation dose and course. In some practices, the radiation oncologist will maintain full responsibility for all aspects of patient care (including client communications, prescribing medications, and treatment follow-up). At other practices, those aspects of patient care are overseen by other veterinarians (often medical oncologists).

Radiation treatment planning is most commonly performed by radiation dosimetrists in human radiation oncology, although in veterinary medicine this is often performed by the attending radiation oncologist. A benefit of doing this is that veterinary radiation oncologists are specifically trained in veterinary treatment planning. In addition, treatment planning requires multiple trade-offs to be made regarding where dose is delivered to, which may have clinical consequences. When the attending radiation oncologist creates their own treatment plan, they can plan treatment while considering the goals and priorities that were discussed with the pet owner. It is generally viewed as acceptable for a radiation dosimetrist or a remote radiation oncologist to perform treatment planning provided that the plan is approved by the attending radiation oncologist.

The existence of veterinary radiation facilities without an on-site, attending radiation oncologist is controversial. There is clear benefit to having a veterinary radiation oncologist who will consult with pet owners, perform or approve treatment plans, and oversee the delivery of radiation at a facility. They should be intimately familiar with the members of the care team and all aspects of the equipment, software, as well as safety and quality assurance procedures. Miscommunications or insufficient training within a radiation team is likely to result in major errors impacting staff and patient safety, including significant potential for fatal complications. It is increasingly possible for many of these aspects of radiation to be performed remotely but care must be taken to ensure sufficient training, communication, and quality assurance. In addition, it is incumbent upon each facility to comply with state regulations regarding veterinary client–patient relationship requirements as relate to the practice of medicine.

Once a treatment plan is approved, it must be delivered to the patient. This is typically done by a radiation therapy technologist (RTT or radiation therapist). Some veterinary facilities will employ a human trained RTT although many will train veterinary staff members (most commonly, licensed veterinary technicians) to properly operate radiation equipment. The radiation oncologist is typically responsible for ensuring adequate training and skill of the acting radiation therapist. In many practices, the acting radiation therapist is also responsible for positioning radiation patients during simulation imaging (CT scan) and creating positioning/immobilization devices for those patients.

In veterinary radiation therapy, patients require anesthesia for treatment. This must be prescribed and overseen by a veterinarian who is responsible for anesthesia and has examined the patient. In most practices, the radiation oncologist oversees anesthesia for treatment, but this can also be performed by another veterinarian. Anesthesia is typically performed by a licensed veterinary technician with sufficient training and experience in anesthesia. Adapting to monitoring patients remotely from outside of the treatment vault and without touching/moving the patient is important in radiation therapy.

Finally, medical physicists play an important role in veterinary radiation oncology. Depending upon the radiation equipment at a veterinary facility and the types of treatments performed, an appropriate regimen for machine commissioning and quality assurance in collaboration with a medical physicist is critical. The Veterinary Radiation Therapy Oncology Group (VRTOG) has previously published recommendations on quality assurance of radiation equipment but now recommends following guidelines published in the American Association of Physicists in Medicine (AAPM) task group recommendations.

Mechanisms of Radiation Damage

The biologic basis of radiation damage to cells is the result of rapidly generated reactive electrons and free radicals that develop when radiation interacts with atoms in the cell. Much of the damage from radiation occurs due to indirect effects after radiation initially interacts with water in the cell. This interaction with water generates reactive intermediates, primarily hydroxyl radicals, that go on to cause oxidative damage to critical cellular macromolecules with DNA being the major target (Hall and Giaccia, 2011).

It is estimated that 1 Gy of ionizing photon radiation to a cell results in about 1000 DNA base damages, 500 single-strand DNA breaks, and 50 double-strand DNA breaks (Gibbons, 2019). Much of this damage is repaired by cells but cell death is often correlated with the number of DNA double-strand breaks, likely due to increased lethality when multiple double-strand breaks occur within a short distance on DNA (Mladenov et al., 2013; Nickoloff et al., 2020). Interestingly, radiation effects are considerably more complex than these effects on cells and DNA. Bystander effects of radiation, which are effects on cells that were not traversed by the radiation beam, have been well documented. This has been shown in microbeam experiments showing that radiation focused on a specific cell in a population of cells can cause damage and death of nontargeted cells and in experiments showing that transfer of the media from irradiated cell to unirradiated cells leads to cell death (Hall, 2003; Ponnaiya et al., 2007; Prise et al., 2009; Autsavapromporn et al., 2019).

The distribution of radiation damage within a treated area is random without preferential damage to tumor cells. Therapeutic benefit is derived by increased intrinsic radiosensitivity of tumor cells due to increased rates of cell division and/or decreased DNA repair capacity relative to many normal tissues (Alsbeih et al., 2000; Gerweck et al., 2006; Maeda et al., 2016). In addition, because radiation dose is targeted at a tumor site, surrounding normal tissues will often receive less radiation dose than the tumor.

There are several described mechanisms of cell death after radiation. Importantly, many cells do not show visible evidence of damage or death until they attempt to divide. For the majority of cells, death from mitotic catastrophe is the largest mechanism of cell death. Apoptosis occurs more commonly in some cells such as lymphocytes. Cells may also undergo senescence or terminally arrest and stop proliferating while remaining metabolically active. Cell necrosis may occur as well, often after a period of senescence. These mechanisms explain why many tumors respond very slowly after radiation therapy (Roninson et al., 2001; Adjemian et al., 2020; Sia et al., 2020).

Radiation Biology and the Basic Science of Oncology

In many respects, radiation biology is merely an extension of the basic science of oncology. The drivers of oncogenesis play a major role in the response to radiation. The conceptual framework of the Hallmarks of Cancer, by Hanahan and Weinberg, described the fundamental features of cancer (Hanahan and Weinberg, 2000, 2011). These include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis. In a recent review, Boss, Bristow, and Dewhirst describe the many ways that radiation biology reflects these fundamentals (Boss et al., 2014). Radiation biology has both contributed to and benefitted from discoveries surrounding these hallmark features of cancer as described herein.

Sustaining proliferative signaling

The growth of cells in normal tissues is regulated by a variety of growth factors that signal cellular differentiation and proliferation. Normal cells typically do not proliferate in the absence of stimulatory or mitogenic signals (Musgrove, 2006). There are a variety of mechanisms by which neoplastic cells acquire the ability to maintain self-sufficiency from these growth factor requirements including autocrine stimulation, growth factor receptor overexpression or mutation, and downstream alterations in mitogenic signals (i.e., Ras pathway).

Radiation biology correlate

In order for a radiation treatment course to achieve tumor regression or control, aberrant cell proliferation must be inhibited or overcome. There is evidence that in some tumors, radiation exposure can lead to accelerated repopulation of tumor cells (Jones, 1991; Trott and Kummermehr, 1991; Schmidt-Ullrich et al., 1999). This is the rationale for accelerated courses of radiation therapy as described previously to overcome the consequence of increasing duration of a radiotherapy course resulting in decreasing local tumor control. This is likely one respect in which veterinary radiation protocols outperform human radiation protocols as veterinary full-course protocols are typically completed within 21–30 calendar days, which is accelerated compared to corresponding human treatment protocols that may extend over 6–8 weeks.

Another radiobiologic correlate is the concept of combining therapy that interferes with a tumor’s ability to sustain proliferative signaling over a course of radiation. One example of this approach is the use of growth pathway inhibitors as radiation sensitizers, such as the use of epidermal growth factor receptor (EGFR) inhibitors in head and neck carcinoma (Harari and Huang, 2006; Bernier et al., 2009). In veterinary medicine, this may provide a biologic rationale for the combination of Palladia with radiation therapy in the treatment of mast cell tumor.

Evading growth suppressors

Cells in normal tissues are maintained in a quiescent state due to antiproliferative signals including soluble growth inhibitors and inhibitors within the extracellular matrix (ECM) and nearby cells (Rowe and Weiss, 2009; Boss et al., 2014). These work to block cell proliferation by moving cells from the cell cycle to quiescence (G0) or by inducing the cell to differentiate into a postmitotic state. Cancer cells must continue to go through the cycle, bypassing growth checkpoints.

Radiation biology correlate

As cancer cells do not terminally differentiate or stay quiescent, they are typically moving through the cell cycle repeatedly. Radiation is selectively cytotoxic to cells in different phases of the cell cycle (cells in S phase are most resistant and cells in M phase are most radiosensitive) (Pawlik and Keyomarsi, 2004). In addition, sublethal radiation can result in temporary growth arrest (G1 arrest or G2 arrest). These delays are presumed to be due to DNA damage detection and repair prior to entry into the next phase of the cell cycle. Attempts to target this for therapeutic benefit include the use of DNA checkpoint inhibitors (Chk1/2) as radiation sensitizers (Fernet et al., 2010; Mitchell et al., 2010).

Resisting cell death

The ability of a tumor to grow is a function of both the rate of cell proliferation and the rate of cell attrition. Accordingly, the cells of a growing cancer must acquire mechanisms to resist cell death, including apoptosis. There are likely many mechanisms by which cancer cells can acquire resistance to cell death; one of the most common is through mutations of the p53 tumor suppressor gene. Functional inactivation of the p53 protein is suggested to be present in over 50% of human cancers (Hollstein et al., 1991). Not surprisingly, similar types of mutations/aberrations have been seen in canine osteosarcoma, intestinal carcinoma, brain tumors, and lymphoma (Setoguchi et al., 2001; York et al., 2012).

Radiation biology correlate

The effectiveness of radiation therapy is intimately correlated with its ability to induce cell death. DNA represents the critical target of radiation as unrepaired double-strand breaks and chromosomal damage lead to cell death. Apoptosis represents a major mode of cell death for lymphoid cells, but mitotic cell death is the major mechanism of cell death for most cancer cells after irradiation. Attempts to target this for therapeutic benefit include the use of DNA damage repair inhibitors as radiation sensitizers. The role of p53 in radiation response is complex but it has been shown to be important for induction of apoptosis after radiation. The role of p53 loss or mutation in conferring clinical radiation resistance is unclear (Fei and El-Deiry, 2003).

Enabling replicative immortality

Normal cells typically have a finite potential to replicate and will eventually undergo senescence. A key limiting factor impacting a cell’s ability to replicate indefinitely is the loss of telomeric DNA after each trip through the cell cycle. Nearly all malignant cells are able to maintain telomere length, with most doing so by expressing high levels of the telomerase enzyme (Kim, 1997; Greider, 1998).

Radiation biology correlate

The importance of replicative immortality is central to how radiation sensitivity is assessed in cells. The gold standard test assessing cellular radiosensitivity is the clonogenic assay. The clonogenic assay is an in vitro assay that compares the number of colonies formed by tumor cells in tissue culture at varying doses of radiation to that of a control. The clonogenic survival curves generated from these assays are the basis for many key radiation experiments that characterize tumor response to radiation (Fiebig et al., 2004).

Inducing angiogenesis

In order for cells to survive, they need to be within 100 μm of a capillary due to limitations of diffusion of oxygen and/or nutrients. For a tumor to grow beyond a very small size, it must be able to induce and sustain angiogenesis. The importance of angiogenesis is underscored by experiments showing the impact of angiogenic inhibitors on the growth of tumors in vivo. Many tumors have been shown to increase expression of proangiogenic proteins such as vascular endothelial growth factor (VEGF) (McMahon, 2000; Carmeliet, 2005). However, it is also established that tumor vasculature is frequently abnormal.

Radiation biology correlate

Although angiogenesis is necessary for tumor growth, tumor hypoxia plays a significant role in the radiation response. Radiation biologists were among the first cancer researchers to recognize the importance of tumor hypoxia in treatment resistance. Hypoxic tumor cells may be up to three times more resistant than identical normoxic cells to radiation (Höckel et al., 1996). Attempts to target this for therapeutic benefit include the use of hyperbaric oxygen or carbogen to increase oxygen levels in tumors undergoing radiation (Hockel et al., 1996). In addition, there are hypoxic cell radiosensitizers and hypoxic cell cytotoxins that aim to overcome or target this mechanism of radiation resistance (Weinmann et al., 2003).

Activating invasion and metastasis

A unique and deadly feature of cancerous cells is the ability to invade normal tissues and metastasize to distant sites in the body. Metastasis is reportedly the cause of tumor-associated death in 90% of human cancers and a significant proportion of veterinary cancers (Bogenrieder and Herlyn, 2003; Dillekås et al., 2019).

Radiation biology correlate

Tumor invasion is a major reason why radiation is needed in many veterinary patients. Invasion of tumor cells into surrounding tissues may result in local tumor recurrence, particularly after marginal excision. In radiation, we consider the need for a treatment target that extends significantly beyond the gross tumor volume to account for subclinical microscopic disease from invasive tumor cells. The larger volume is referred to as the clinical tumor volume (CTV). Interestingly, abscopal effects of radiation have been described where irradiation of a primary tumor can result in the regression of distant metastatic lesions. The mechanism for this is unclear but it may be the result of adaptive immune system recognition, similar to a tumor vaccine (Demaria et al., 2004; Siva et al., 2015).

Acute versus Late Effects of Radiation

Normal tissue reactions to radiation can be characterized as acute, late, consequential, and early delayed. Acute effects are expected during or shortly after treatment. This occurs in tissues with rapidly proliferating cells such as the oral mucosa and skin. The severity of acute effects may vary between patients, but these effects generally are self-limiting. While potentially unpleasant and/or distressing, most acute effects will heal with modest intervention and time provided that self-trauma such as licking or chewing by the pet is prevented (Collen and Mayer, 2006).

Early delayed radiation effects are seen sporadically in neurologic tissues between 2 weeks and 4 months after treatment and may be due to transient demyelination or cytokine release and edema (Kim et al., 2008).

Late effects typically involve more slowly proliferating tissues, such as bone, lung, and nervous system, although late effects to skin are possible as well. The dose of radiation administered to the patient is frequently 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, and loss of function. Severe late effects threaten life or quality of life. Late effects occur from the loss of normal tissue stem cells with concurrent radiation-induced vascular changes and inflammation. A subtype of late effects is known as consequential late effects. These are late effects that result because of extensive damage to the proliferating cells of acutely responding tissues, often as a sequela of severe acute toxicity. Radiation carcinogenesis or secondary tumor formation is also frequently included under the category of severe late radiation effects. Severe late effects are quite rare (generally <5%) with most veterinary radiation protocols in clinical use but there is concern that the increasing use of hypofractionated radiation including with SRT may be associated with higher rates of severe late effects, particularly when used to treat larger targets (Hoban et al., 1999; Pettersson et al., 2009; Nahum, 2015).

Modeling Cell Survival and Tumor Response from Radiation

Radiation biologists have developed predictive models that describe how radiation time, dose, and fractionation impact expected tumor cell surviving fractions and correspond accordingly to clinical tumor control probabilities. The most widely used of these models is the linear–quadratic model, which can be used to describe the radiation survival curve for most types of cells. In this model, it is assumed that there are two components or mechanisms of cell death. There is a lethal event that may occur at any dose but increases in likelihood linearly with dose (i.e., twice as likely if dose is doubled). In addition, cell death can occur due to accumulation of multiple harmful but nonlethal events. The likelihood of cell death occurring due to this accumulation of multiple events increases exponentially with dose. This model has been used and manipulated mathematically to allow for the calculation of a biologically effective dose (BED). The BED is a standardized number that allows for a comparison of different courses of radiation containing different numbers of fractions and doses per fraction. The equation can be augmented to account for tumor cell proliferation over time in longer courses of radiation. BED calculations do require assumptions about the cellular characteristics, specifically the alpha/beta ratio of the target or organ of interest (Fowler, 2010).

Alpha/beta ratio

Alpha/beta ratio (α/β) is a commonly referenced but often poorly understood metric that describes the biologic response of a tumor or tissue to radiation as the dose per fraction increases. A potentially useful metaphor is to consider that a specific type of cell is a boxer, and that radiation represents punches being thrown. The number of punches thrown is the dose and a round of boxing is analogous to a fraction of radiation. Finally, a boxer that is knocked out is the metaphor for a cell kill and if the boxer is not knocked out, this is equivalent to cell survival.

Any punch thrown may hit the boxer, may miss altogether, or may be blocked. There are two ways to knock out an opponent. Alpha is a single strong punch to the head that is a knockout event and beta is a strong punch to the body that requires the accumulation of multiple punches (for descriptive purposes, we will use the example of five) landed to the body to yield a knockout. If less than five beta punches to the body land successfully, whether zero, one, two, three, or four, the boxer will recover.

Certain boxers may be more susceptible to the single alpha punch head knockout (i.e., boxers with a glass jaw) and others more susceptible to knockout by accumulation of beta body punches. For the alpha punch to the head type of knockout, the likelihood of this happening is ten times more likely if ten times more punches are thrown, regardless of whether they are thrown in succession or thrown in different rounds. This is a linear relationship of knockouts to punching dose. However, the likelihood of landing all five beta body punches increases exponentially with dose since it is very unlikely to occur at a low punching dose and exponentially more likely at a higher punching dose. A boxer can only be knocked out once (either by an alpha punch to the head or by the accumulation of beta punches to the body).

If we now imagine that there are millions of identical boxers (corresponding to the millions of cells of a tissue or tumor that are interacting with radiation), the response to being punched could be described by the following relationship. At any given punching dose (i.e., 10 punches), some proportion of the boxers will be knocked out due to a single alpha head punch and an additional proportion of the boxers will be knocked out due to multiple beta body punches. The remaining boxers are surviving.

Mathematically, this could be described as follows. α represents a coefficient that can be multiplied by any dose (D) and linearly corresponds to the proportion of boxers knocked out by single head punches at that dose. β represents a similar coefficient that can be multiplied by exponential dose (dose squared). The probability that a boxer is NOT knocked out (or survives) at a given dose can be described by the following equation:

An equation reads Survival probability equals e x p, left bracket, minus, left parenthesis, alpha uppercase D plus beta uppercase D square, right parenthesis, right bracket.

(1.1)

There is a dose where the amount of knockouts from single head shots is equal to the amount of knockouts from multiple body blows. This dose is the alpha/beta ratio for the boxer. Boxers that have a high alpha/beta ratio (i.e., 20) are primarily knocked out by single alpha head punches. Accordingly, a single big dose in one round versus 1/12 of the dose in each of 12 rounds results in a similar number of total knockouts. However, another boxer has a very low alpha/beta ratio (i.e., 1.5), which means that they are much more susceptible to knockout by multiple body punches and not as susceptible to single head punches. These boxers would be much more likely to be knocked out by a high single dose in a round (because of the accumulation of multiple body blows) compared to 1/12 of the dose in each of 12 rounds if they can fully recover between rounds.Following the preceding boxer analogy conveying how tissues are variably impacted by fractionation, it would be useful to have a quantitative measure that conveys the relative effect of various dosing schedules. In the setting of radiation oncology, this is accomplished using the concept of Biologically Equivalent Dose (BED).

One of the commonly used formulas for BED is:

An equation reads B E D equals n multiplied by lowercase d, left bracket, 1 plus lowercase d over, left parenthesis, alpha over beta, right parenthesis, right bracket. (1.2)

where d is the dose per fraction in Gy, n is the number of treatment fractions, and α/β is the dose at which the linear and quadratic components of cell kill are equal for the cell/tissue type (the alpha/beta ratio). A related concept is the biologically equivalent dose, which is the dose delivered in a conventionally sized fraction that would have the same biologic effect as an alternative protocol for comparison. For humans, this is normally calculated as the equivalent dose in 2 Gy fractions but for veterinary medicine, it is more appropriate to use equivalent dose in 3 Gy fractions as this is a more common fractional dose. The equation is as follows:

An equation reads E Q D subscript 3 equals uppercase D multiplied by, left bracket, left parenthesis, lowercase d plus alpha over beta, right parenthesis, over, left parenthesis, 3 plus alpha over beta, right parenthesis, right bracket. (1.3)

where EQD3 is the equivalent dose in 3 Gy fractions and D is the total dose (or n × d).

To illustrate the concept of equivalent dose, compare several published treatment protocols for nasal tumors in dogs. We will look at course 1 consisting of 16 fractions of 3 Gy, course 2 consisting of ten fractions of 4.2 Gy, and course 3 consisting of three fractions of 10 Gy. We will look at the equivalent dose for tumor with an assumed alpha/beta ratio of 10 Gy and for brain with an assumed alpha/beta ratio of 2 Gy.

For tumor:

An equation reads Course 1 colon E Q D subscript 3 equals 48 multiplied by, left bracket, left parenthesis, 3 plus 10, right parenthesis, over, left parenthesis, 3 plus 10, right parenthesis, right bracket, equals 48 G y.

(1.4)

An equation reads Course 2 colon E Q D subscript 3 equals 42 multiplied by, left bracket, left parenthesis, 4 plus 10, right parenthesis, over, left parenthesis, 3 plus 10, right parenthesis, right bracket, equals 45.2 G y.

(1.5)

An equation reads Course 3 colon E Q D subscript 3 equals 30 multiplied by, left bracket, left parenthesis, 10 plus 10, right parenthesis, over, left parenthesis, 3 plus 10, right parenthesis, right bracket, equals 46.2 G y.

(1.6)

For brain:

An equation reads Course 1 colon E Q D subscript 3 equals 48 multiplied by, left bracket, left parenthesis, 3 plus 2, right parenthesis, over, left parenthesis, 3 plus 2, right parenthesis, right bracket, equals 48 G y. (1.7)

An equation reads Course 2 colon E Q D subscript 3 equals 42 multiplied by, left bracket, left parenthesis, 4 plus 2, right parenthesis, over, left parenthesis, 3 plus 2, right parenthesis, right bracket, equals 50.4 G y. (1.8)

An equation reads Course 3 colon E Q D subscript 3 equals 42 multiplied by, left bracket, left parenthesis, 10 plus 2, right parenthesis, over, left parenthesis, 3 plus 2, right parenthesis, right bracket, equals 72 G y.

(1.9)

This mathematical exercise shows that for these three published nasal tumor radiation protocols, the equivalent dose in 3 Gy fractions for tumor effects/response is predicted to be relatively similar, although slightly lower for courses 3 and 2 than for course 1. However, the predicted effect/response to brain is slightly higher for course 2 and much higher for course 3. This illustrates the rationale of fractionation, which is that for a given equivalent effect on a tumor, a more finely fractionated protocol has a lesser effect on late-responding normal tissues. This is largely the reason why to safely administer a treatment protocol such as course 3 requires a stereotactic technique that would minimize the volume of adjacent late-responding normal tissues that receive a dose approaching the prescribed dose to the tumor. It would also predict that there might be a higher risk of late tissue complications when a significant degree of sparing is not possible. For nasal tumors, this is likely to correspond to increased risks of bone necrosis (potentially leading to fistula development) due to the alpha/beta ratio of bone.

A related concept to the above topic is that of tumor control probability (TCP) and normal tissue complication probability (NTCP). For a given dose or course of radiation, there is some probability of tumor control and a corresponding probability of a severe normal tissue complication (Hall and Giaccia, 2011). At low doses, the probability of either is minimal and at high doses, both become increasingly likely. Ideally, we would select a radiation dose that maximizes TCP while maintaining a low and acceptable NTCP, thus maximizing the therapeutic index of radiation. Advanced radiation techniques may allow the radiation oncologist to escalate the effective dose to a tumor target (increasing TCP) while still sparing or minimizing dose to neighboring normal structures (thus decreasing or maintaining low NTCP). As discussed later in this chapter, the goal of using drugs that could act as radiation sensitizers or radiation protectors is to similarly widen the therapeutic index by improving TCP without a corresponding increase in NTCP (radiation sensitizer) or to decrease NTCP without a corresponding decrease in TCP (radiation protector).

Factors Influencing Radiation Response (The Rs of Radiation)

How any tumor responds to radiation is impacted by a variety of factors. There are factors associated with the tumor itself including its intrinsic radiosensitivity, the total tumor burden/volume, and the rate of cell proliferation and cell loss. There are also factors associated with the radiation course itself including total radiation dose, radiation dose per fraction, and the period of time the radiation course extends over, collectively referred to as time, dose, and fractionation. Finally, there are factors related to the patient that may include the patient’s normal tissue radiosensitivity, immune response to the tumor, and perfusion to the tumor.

Radiation courses are typically fractionated or divided into multiple doses in order to improve the rate of tumor control for a given level of toxicity. Classically, the four Rs of radiobiology were described by Withers and have been used to describe the factors or biologic events that occur between fractions of a radiation course that impact tumor response (Lett and Adler, 1975). These are Repair, Repopulation, Redistribution, and Reoxygenation. An expansion of this concept to include a fifth factor known as intrinsic Radiosensitivity was introduced by Steel (Steel et al., 1989). More recently, additional Rs describing tumor and host factors that influence the response to a course of radiation have been proposed including Remodeling of the tumor microenvironment, Rejection by the immune system, and Remote bystander effects (Brown et al., 2014; Golden and Formenti, 2014; Kim et al., 2015; Arnold et al., 2018).

Repair

Many experiments have demonstrated the importance of cellular repair and recovery in the response to fractionated doses of radiation. Specifically, repair of DNA that is damaged by radiation plays the largest role. Based on experiments looking at split doses of radiation or continuous irradiation at varying dose rates, radiation damage can be characterized as lethal damage or repairable/sublethal damage (Frankenberg-Schwager et al., 1985; Bedford, 1991). Sublethal damage can be repaired unless it compounded by additional