The research, which led directly to initial clinical trials during the 1980s and 1990s that aimed to locally heat patients’ tumors to temperatures to approximately 43° C, focused on the observation that tumor cell damage from focused heat energy that could add to, or even synergize with, the antitumor effects of radiation without the systemic toxicity typical of chemical radiation sensitizers. When cells are heated in vitro to a sufficient temperature and for a long enough duration, they die in a predictable, exponential manner, and the rate of killing increases with temperature. Figure 28.1 shows a series of survival curves for cells exposed for various periods to a range of temperatures from 41.5° to 46.5° C. The cell survival curves for heat are similar in shape to those obtained for x-rays (i.e., an initial shoulder followed by an exponential region), except that the time of exposure to the elevated temperature replaces the absorbed dose of x-rays. For lower temperatures, the picture is complicated, because the survival curves flatten out after a protracted exposure to hyperthermia, possibly indicating the development of “thermotolerance” or an acquired resistance or tolerance to the elevated temperature. The similarity in the shape of the cell survival curves for heat energy and x-rays is misleading. It is important, therefore, not to draw conclusions for heat based on the interpretation of radiation dose-response curves, because the amount of energy involved in cell inactivation is a thousand times greater for heat than for x-rays. This reflects the different mechanisms involved in cell killing by heat and x-rays. Families of survival curves similar to those in Figure 28.1 have been obtained for many different cell types, and it is clear that cells differ somewhat in their sensitivity
to heat-induced cellular damage in this temperature range. However, as with ionizing radiation, there is no consistent difference between normal and malignant cells heated in vitro.

Survival data for Chinese hamster ovary cells exposed to various levels of hyperthermia (taken from Fig. 28.1) are replotted in Figure 28.2, with 1/D₀ on the ordinate and 1/T on the abscissa. T is the absolute temperature; D₀ is the reciprocal of the slope of the exponential region of the survival curve (i.e., the time at a given temperature that is necessary to reduce the fraction of surviving cells to 37% of their former value). This type of presentation is known as an Arrhenius plot; its slope gives the activation energy of the chemical process involved in the cell killing. The obvious change in slope, which is typical of Arrhenius plots, occurs at a temperature referred to as the “breakpoint” and is about 43° C for human cells. Above this temperature, an increase of 1° C doubles the rate of cell killing, and below the breakpoint, the rate of cell killing by heat drops by a factor of 2 to 4 for each drop of 1° C. The difference in activation energy above and below this temperature may reflect different mechanisms of cell killing (i.e., different targets for cytotoxicity above and below 43° C) or may reflect the development of thermotolerance within cells. Importantly, the slopes of Arrhenius plots derived from many in vitro and in vivo studies are nearly identical, and this consistency has provided long-standing usefulness of the Arrhenius analysis as a basis for assessing thermal dose in clinical hyperthermia applications up until the present time.

The similarity of the activation energy for protein denaturation to the activation energy for heat cytotoxicity, calculated from the Arrhenius analysis, led to the hypothesis that the target for heat cell killing in this temperature range resides in cellular proteins. Specifically, the heat of inactivation for cell killing and thermal damage is similar to the energy needed for protein denaturation (130-170 kcal/mol). Additional evidence for proteins as being the primary target for thermal cell killing is the importance of heat shock proteins (HSPs) in protecting thermotolerant cells from thermal damage. One of the primary functions of HSPs is to stabilize and refold other proteins that have been denatured or damaged, an event that signals their enhanced production. Structural chromosomal proteins, nuclear matrix, mitochondria and cytoskeleton, repair enzymes, transport proteins, and other membrane components are
all included among targets that have been shown to be denatured in a time-dependent manner by temperatures between 42° and 45° C. Enzymes in the respiratory chain are more heat sensitive than enzymes in the glycolytic pathway.

Protein damage represents an important mechanistic difference between the manner in which heat and ionizing radiation lead to cellular damage, and this suggested that the two therapies could result in at least an additive ability to kill tumor cells if used together. There are other differences between irradiation and heating, suggesting that they could have additive effects. After irradiation, cells die only in attempting the next or a subsequent mitosis (except in very unusual circumstances), whereas heat-induced damage is expressed early, causing cells to die by various necrotic or apoptotic mechanisms, depending on the temperature. In addition, heat affects differentiating as well as dividing cells. Heat-induced chromosomal aberrations can occur if cells are in S phase during the time of heating. Although there is a familiar delay in x-ray response related to the time it takes for stem cells to progress through the process of differentiation and become functional, this delay is absent in the case of heat-induced cell death because all cells are affected, differentiated, or dividing; the thermal damage to the tissue is expressed immediately if the temperature is high enough. The DNA repair process itself is heat sensitive and this may be one of the mechanisms that lead to heat-induced radiosensitization and chemosensitization. Finally, although hypoxia renders cells resistant to radiation, it does not appear to affect sensitivity to heat.

The extent of the interaction of heat and radiation can be expressed in terms of the thermal enhancement ratio (TER), defined as the ratio of doses of x-rays required to produce a given level of biologic damage with and without the application of heat. The TER has been measured for various normal tissues, including skin, cartilage, and intestinal epithelium. The data form a consistent pattern of increasing TER with increasing temperature, up to a value of about 2 for a 1-hour heat treatment (HT) at 43° C. The TER is more difficult to measure in transplanted tumors in laboratory animals, because the direct cytotoxic effect of the heat tends to dominate. Heat often can control experimental tumors with acceptable damage to normal tissues, because cell killing by heat is strongly enhanced by nutritional deprivation and increased acidity, conditions that are typical of the poorly vascularized parts of solid tumors. Thus, a moderate HT, which can be tolerated by well-vascularized normal tissues, destroys a large proportion of the cells of many solid tumors in experimental animals. In those cases in which thermal radiosensitization has been studied, typical TER values are 1.4 at 41° C, 2.7 at 42.5° C, and 4.3 at 43° C, with heat applied for 1 hour. TERs have been developed for canine and human tumors however in studies by Gillette et al. and Overgaard et al. These estimates came from retrospective analyses of dose-effect relationships with and without hyperthermia. In canine oral squamous cell carcinomas, it was estimated to be approximately 1.15, when hyperthermia was administered twice a week during a course of fractionated radiotherapy. Importantly, the use of hyperthermia increased the slope of the TCD₅₀ curve, such that the doses required to achieve a high level of local tumor control were maintained well below that which caused normal tissue damage (defined here as bone necrosis). TERs have also been estimated to be in the range of 1.5 for several superficial human tumor types.

The therapeutic gain factor can be defined as the ratio of the TER in the tumor to the TER in normal tissues. There is no advantage to using heat plus lower doses of x-rays if there is no therapeutic gain compared with the use of higher doses of x-rays alone. The question of a therapeutic gain factor is complicated in the case of heat, because the tumor and normal tissues are not necessarily at the same temperature. If the statement is made that heat preferentially damages tumor cells compared with normal tissue, it is implied that they are both at the same temperature. In a practical situation, however, this is not always the case. For example, if a poorly vascularized tumor is treated with microwaves, it may reach a higher temperature than the surrounding normal tissue, because less heat is carried away by the flow of blood. In addition, the overlying skin can be cooled actively by a draft of air or even a coldwater pack. In these circumstances, the normal tissues may be at a significantly lower temperature than the tumor, which therefore exaggerates the differential response in a favorable direction.

Because the earliest rationale for using heat as a radiation sensitizer involved maximizing direct heat-induced protein denaturation and cellular damage, there has been considerable attention by researchers given to factors that could modify tumor cell thermal sensitivity. For example, modification of cell membrane lipid content or use of membrane active agents, such as alcohols, was found to sensitize cells to heat killing, and the sensitization is probably related to destabilization of the membrane as it relates to lipid-protein interactions. Cells in an acid pH environment appear to be more sensitive to killing by heat. The pH dependence of cytotoxicity at elevated temperatures, however, is affected by pH history. Cells can adapt to pH changes and avoid the heat sensitivity shown for low pH.

Cells deficient in nutrients are quite heat sensitive. This can be demonstrated with cells in culture in which sensitivity to heat increases progressively as cells have their energy supply compromised, either by depriving them of glucose or by the use of a drug that uncouples oxidative phosphorylation. These conclusions about pH and nutrients, obtained under controlled conditions with cells in culture, led to the speculation that cells in tumors that are nutritionally deprived and at acid pH because of their location remote from a blood capillary may be particularly sensitive to heat. Because of their environment, it is likely that these cells are out of cycle and possibly hypoxic also. In this context, heat and x-rays would be predicted to be complementary in their action, because the cells that are most resistant to x-rays (out of cycle and hypoxic because of their remoteness from a capillary) show enhanced sensitivity to heat. A further enhancing factor that contributes to the complementary interaction of hyperthermia and radiation is that regions of a tumor in which the vasculature is poorly developed and potentially hypoxic tend to be at an elevated temperature because the cooling effect of blood flow is reduced.

Some studies in mice had shown that local hyperthermia (using temperatures of 43° to 44° C) could naturally damage tumor vascular endothelium as well as tumor cells. This was predicted to lead to an increased accumulation of heat (because the damaged blood vessels would carry less heat away) while the pH, pO₂, and nutrient status of the cells would decline, leading to enhanced tumor cell sensitivity to heat-induced cell killing. As a result of these studies, it was suggested that care was needed in combining heat and radiation, since tumor perfusion would be compromised by heating. For these reasons, several protocols have used hyperthermia in clinical applications after the first dose of radiation.

However, temperatures that can be achieved experimentally in mouse tumors are significantly higher than those which typically occur in human tumors under clinical conditions using locally applied, external heat energy; whether tumor vasculature in human tumors is damaged by the more modest temperatures reached under clinical conditions is still open to question. Further, it is important to also note that in animal studies in which more moderate temperatures are used (41° and 41.5° C), hyperthermia has been shown to promote tumor reoxygenation, with the degree of reoxygenation correlating with the level of the radiosensitivity of the tumor. Importantly, these data have been confirmed in a clinical study of patients with soft tissue sarcomas and breast cancer. Brizel and colleagues showed that one HT led to reoxygenation of human soft tissue sarcomas within 24 to 48 hours, whereas there was no measurable reoxygenation during a prior week of standard radiation therapy (RT). Jones and colleagues reported that mild hyperthermia (41° and 41.5° C at 90% of the measured points for 1 hour) significantly increased the pO₂ in hypoxic, but not normoxic, human breast cancers. Such increases in tumor oxygenation could significantly improve tumor response to radiotherapy and is likely to be the primary important effect of local/regional or whole body forms of clinical hyperthermia. If this assumption is correct, then there should be much more attention devoted to optimal scheduling of hyperthermia in future trials to maximize oxygen-dependent radiosensitization. Indeed, instead of aiming to kill cells, mild hyperthermia may have the ability to rapidly stimulate global changes in the tumor microenvironment that collectively could enhance RT; this includes effects on pH, oxygen concentration, metabolism, protein/gene expression, and vascular perfusion.

It is well documented in human physiology that in normal tissues, a modest increase in temperature prompts highly sensitive thermoregulatory homeostatic mechanisms that increase heat dissipation in the vasculature. However, the ability to optimally thermoregulate is itself a highly temperature-dependent phenomenon. Optimal thermoregulation in terms of vascular effects occurs at mild to moderate hyperthermia and begins to fail above 41° and 42° C, resulting in heatstroke symptoms. While at temperatures of 43° C or higher, direct vascular and cellular damage can occur, and blood flow can be impaired. In cooler regions, the largest contributing factor to radiation sensitization by heating appears may be its ability to stimulate vascular activity, which, in turn, enhances delivery of additional oxygenated blood into tumors, sensitizing the tumor to radiation.

Despite an incomplete understanding of the actual basis of radiation sensitization by hyperthermia, an overestimation of the role of thermotolerance and suboptimal sequencing of hyperthermia and radiation fractions, multiple trials have been completed, revealing a critical role for hyperthermia in enhancing RT. A very active, preclinical, and clinical research effort is under way at the present time to develop the optimal dose and scheduling protocols and to design new clinical trials based on a more complete understanding of the physiologic effects of hyperthermia.

Exploration of the changing view of the role of “thermotolerance” in hyperthermia serves as a useful example demonstrating how our understanding of the radiosensitizing effects of hyperthermia have evolved over the past 2 to 3 decades. Thermotolerance, or induced thermal resistance, is usually described as the development of a transient and nonhereditary resistance to subsequent heating by an initial HT. Specifically, in vitro clonogenic assays of thermal cytotoxicity reveal that although one dose of heat kills a substantial fraction of cells, subsequent daily treatments are comparatively ineffective. Thermotolerance can begin to occur a few hours after the first treatment and may take as much as a week to decay. This would suggest that thermotolerance would be a significant problem clinically when heat is combined with fractionated radiotherapy, and most clinical trials were designed to minimize thermotolerance by keeping at least several days between hyperthermia fractions. However, it is now clear that in actual tumors in situ, an increase in blood flow and consequent increase in oxygenation by heating may significantly enhance the response of tumors to radiotherapy to a greater degree than any inhibition of thermal killing related to the occurrence of thermotolerance. Further, heat-induced radiosensitization is not subject to thermotolerance. Thus, although fears regarding thermotolerance had a historical importance regarding scheduling of hyperthermia for patients (schedules that may have turned out to be suboptimal in terms of achieving maximal benefits of hyperthermia), it is now clear that under normal heating conditions used in the clinic, thermotolerance may not be induced to any significant degree. Newer trials that are being designed to use more frequent applications of heating are likely to result in improved clinical benefit.

Another related area of research that has undergone considerable evolution in terms of understanding radiation sensitization by hyperthermia involves HSPs. It has been known for a long time that if cells are exposed to heat, proteins of a defined molecular weights or families are observed to be strongly induced above normal levels of expression. It was shown long ago that the appearance of these HSPs coincides with the development of thermotolerance, and their disappearance coincides with the decay of thermotolerance. Later molecular studies that specifically altered expression of certain HSPs demonstrated that they clearly exert a significant protective effect against potentially lethal heat exposures. Indeed, it has been long understood that HSP families are ubiquitous in evolution and are found in all living organisms. Similarly, a thermotolerance phenomenon also appears to be a conserved response in evolution resulting from their increased expression following exposure to certain stress conditions. However, although thermotolerance was one of the earliest functions attributed to HSPs, it is recognized today that these proteins play essential roles in numerous macromolecular processes under normal physiologic conditions as well as under stress conditions. In fact, there is now considerable research exploring induction of HSPs by hyperthermia treatments because of their likely role in promoting antitumor immune responses.