Cellular and Tissue Responses to Hyperthermia

Protein is the primary target for hyperthermic cytotoxicity. When cells are exposed to elevated temperatures (≥41° C), damage is inflicted in multiple sites, but the predominant molecular target appears to be protein. The heat of inactivation for cell killing and thermal damage to tissues is in the range of that necessary for protein denaturation (130 to 170 kcal/mol). Additional evidence for protein being the primary target for cell killing is the importance of heat shock proteins in protecting thermotolerant cells from thermal damage. One of the primary functions of heat shock proteins is to refold other proteins that have been damaged.⁴

Some organelles are especially important in controlling thermal response. For example, modification of membrane lipid content or use of membrane active agents, such as alcohols, can sensitize cells to heat killing, but the sensitization is probably related to destabilization of the membrane as it relates to lipid-protein interactions.⁵ The cytoskeleton of cells is particularly heat sensitive.⁶ When it is collapsed by heat, there is disruption of cytoskeletal-dependent signal transduction pathways as well as inhibition of cell motility.^7,8 Enzymes in the respiratory chain are more heat sensitive than enzymes in the glycolytic pathway.⁹ The heat sensitivity of the centriole leads to chromosomal aberrations following thermal injury.¹⁰ Finally, the DNA repair process is heat sensitive, and this may be one of the mechanisms that leads to heat-induced radiosensitization and chemosensitization.¹¹

Physiologic Response to Hyperthermia

Tumor blood flow and metabolism have important influences on the efficacy of HT, and, conversely, the physiologic consequences of HT can influence the efficacy of other treatments.

As temperatures increase, there is an increase in blood flow. The temperature threshold for this change is 41° to 41.5° C in skin.¹² Changes in vascular permeability also occur, leading to edema formation in the heated volume. At higher thermal doses, vascular stasis and hemorrhage develop. The change in normal tissue perfusion upon heating is much greater (10 times) than one sees in tumors (1.5 to 2 times).¹³ Mechanisms underlying vascular stasis may include arteriovenous shunting, thrombus formation, and leukocyte plugging.¹⁴ Importantly, physiologic changes occur in tumors at lower temperatures that are potentially beneficial (Fig. 21-2).

Figure 21-2 Overview of the beneficial physiologic and metabolic effects of HT.

Taking Advantage of the Physiologic Response to Hyperthermia

Improvement in Macromolecular and Liposomal Drug Delivery

A liposome is a small lipid vesicle (≈100 nm in diameter) that contains water or saline in the center. Drugs can be loaded into liposomes at high concentration. It has been recently shown that HT increases microvascular pore sizes preferentially in tumor microvessels, which leads to enhanced liposomal accumulation in tumor.¹⁵ The threshold for increased liposomal extravasation is 40° C, and it increases by a factor of 2 for every degree of temperature rise until vascular damage occurs.¹⁶ The effects of HT on liposomal extravasation have been studied extensively in preclinical models, but a twofold to thirteenfold enhancement in accumulation occurs in spontaneous soft tissue sarcomas of pet cats, an encouraging result that may translate to human tumors.¹⁷ HT also increases the available volume fraction (fraction of tissue not occupied by cells or stromal fibers) that may develop as cells undergo either necrosis or apoptosis following heat treatment.¹⁸ This increases the tissue space available for nanoparticles to be deposited. The increase in liposomal drug delivery achieved with HT has been shown to result in increased antitumor effect in many preclinical studies.¹⁹ HT has been used in conjunction with liposomal doxorubicin and irradiation in one small clinical series of patients with chest wall recurrences of breast cancer. Encouraging results were obtained, although drug concentrations were not measured in this series.²⁰

For relatively small agents, such as most chemotherapeutic drugs, there is no advantage gained via increased vascular permeability to macromolecules or nanoparticles unless the drugs are protein-bound.²¹ For drugs with a molecular weight of less than 1000 MW, the primary mechanism that governs drug transport is diffusion,²² which is not highly temperature dependent. However, for molecules larger than 1000 MW, the primary driving force for transport is convection, which is controlled by the pressure gradient across the vessel wall. Accordingly, HT can augment the transvascular delivery of agents such as monoclonal antibodies²³ and polymeric peptides that carry drugs or radioisotopes.²⁴ Dreher and colleagues used a novel peptide polymer that undergoes an inverse-phase transition at 41° C to “pump” polymer into tumors. The inverse-phase transition refers to a physical feature of the polymer that renders it to be water soluble below 41° C; above this temperature, the polymer comes out of solution in aggregates. The aggregation occurs primarily intravascularly but is reversible. Therefore, when the tissue is cooled down, the particles rapidly disaggregate and diffuse into the tissue down their concentration gradient²⁵ (Fig. 21-3). This same type of peptide polymer could be used for noninvasive thermometry with magnetic resonance imaging (MRI) or electron paramagnetic resonance (EPR) imaging because the interaction of the polymer with water changes when it aggregates. Proof of principle for this method was shown using EPR imaging.²⁶

Figure 21-3 Images of thermally sensitive peptide polymer (green) and thermally insensitive polymer (red) in a solid tumor before, during, and after HT. The peptides were fluorescently labeled and injected into mice with a skin-fold window chamber, which permits direct visualization of the tumor microvessels using fluorescence microscopy. Upon heating to 41° C, the temperature-sensitive liposome begins to aggregate (green dots), whereas the red nonthermally sensitive polymer remains dissolved in the blood plasma, highlighting the location of the blood vessels. When the tissue is cooled down, the aggregates disappear, leaving the now-soluble polymer in the extravascular space (green background increases with accumulation). This method could be used to literally pump drug into tumors from the vascular space. Bar = 100 μm for all images.

Data reproduced from Dreher MR, Liu W, Michelich CR, et al: Thermal cycling enhances the accumulation of a temperature-sensitive biopolymer in solid tumors, Cancer Res 67(9):4418-4424, 2007.

Effects of HT on Tumor Metabolism and Oxygenation.

Enzymes for aerobic metabolism are more heat sensitive than those involved in anaerobic metabolism.⁹ Decreases in adenosine triphosphate (ATP) concentration after HT are inversely related to the temperatures achieved during HT in preclinical models and canine sarcomas^27,28 and are related to the pathologic complete response (pCR) rate in human sarcomas.²⁷ Decreases in ATP and increases in lactate concentration occur following tumor blood flow reduction after HT.²⁹ In human patients with soft tissue sarcomas who were treated preoperatively with HT and radiation therapy, reduction in the ratio of ATP to inorganic phosphate (Pi) (measured using phosphorus-31 [³¹P] magnetic resonance spectroscopy) was significantly correlated with a higher probability for pCR.²⁷ These results are consistent with the theory that tumor respiration can decrease after HT treatment.

A shift toward anaerobic metabolism would decrease oxygen consumption rates, which could lead to improvement in tumor oxygenation. Some of the benefits of HT may therefore result from improvements in oxygenation.³⁰ Several preclinical studies have shown that mild temperature heating (between 40° and 42° C for an hour) can lead to improvements in the oxygen partial pressure (pO₂) in the tumor up to 24 hours after heating.³¹ However, in some models, the extent of reoxygenation is quite heterogeneous between and even within individual tumors.³² It has been reported that HT improves tumor oxygenation in canine and human soft tissue sarcomas, with reoxygenation being associated with a greater probability for pCR in human tumors.^33,34 In a canine study, median temperatures of less than 44° C improved oxygenation, whereas median temperatures higher than 44° C for 60 minutes led to decreased oxygenation.³³ In patients with locally advanced breast cancer who were treated in a phase II study with preoperative HT plus radiotherapy and taxol, there was a significantly increased likelihood for achieving pCR if the tumors reoxygenated 24 hours after the first HT treatment. The probability for achieving a pCR was greater when the median temperatures were below 41.5° C.³⁵ Improvement in oxygenation does not occur in all tumors, however, and currently there is no method for predicting which tumors will show this effect.³⁶ In summary, mild temperature elevations during HT will benefit some patients if HT induces reoxygenation.³¹ To take full advantage of this effect, it will be necessary to use methods to enhance the reoxygenation effect, such that it occurs in the majority of patients. Song and Griffin have suggested that the addition of hyperoxic gas breathing after HT but during radiotherapy may achieve this goal.^37,38

pH Modification

It is well established that acidification can enhance sensitivity to HT. Changes in intracellular pH are responsible for increased thermal sensitivity.^39,40 The most widely studied method to achieve acidification has been induction of hyperglycemia. The logic is that excess glucose load will push tumors toward glycolysis and lactic acid production.^41,42 However, hyperglycemia alone is insufficient to reliably achieve adequate acidification.^43,44 The addition of agents that can selectively acidify tumor intracellular pH, such as glucose combined with the respiratory inhibitor meta-iodobenzylguanadine (MIBG), have the potential to further enhance hyperthermic cytotoxicity selectively in tumor tissues^45,46 because the acidification appears to be selective for tumors. Quercetin, a naturally occurring flavinoid, inhibits thermotolerance induction, particularly at a low pH, and can accordingly increase the thermal sensitivity of cells.^47–49 An alternative approach is to block the extrusion of hydrogen ions from cells, which is normally accomplished via membrane-bound pumps. Use of such agents, in combination with acidification of the extracellular space, can lead to enhanced hyperthermic cell killing both in vitro and in vivo.⁵⁰

Rationale for Combining Hyperthermia with Radiotherapy

When irradiation is combined with HT, complementary effects occur. Cells in S phase are radioresistant but are sensitive to HT. Hypoxic cells are three times more resistant to radiation compared with aerobic cells, but there is no difference in thermal sensitivity between aerobic and hypoxic cells. As discussed above, HT can lead to reoxygenation, which will further improve radiotherapy response.31,33,34 Finally, HT inhibits the repair of both sublethal and potentially lethal damage by inactivating crucial DNA repair pathways.^62–64

Factors to Consider when Combining Hyperthermia with Radiotherapy

The interaction between radiation and HT is described by the thermal enhancement ratio (TER), which is defined as the ratio of doses of radiation to achieve an isoeffect for radiotherapy alone compared with radiotherapy plus HT. TERs for local tumor control have been estimated for a number of human tumors using historical control data for radiation alone.⁶⁵ In most tumor types examined, these ratios were higher than 1. Assessment of normal tissue TER has not been attempted except in a few cases. For those examples, TER values for normal tissue damage have been less than those for tumor tissue damage in the same patient population, suggesting that there is potential for therapeutic gain with the use of radiotherapy combined with HT compared with radiotherapy alone.⁶⁵ Prospective randomized trials in dogs with spontaneous tumors have also shown evidence for improved local tumor control with radiotherapy plus HT compared with radiotherapy alone,^58,66,67,68 with no clinically observable increase in the frequency of clinically relevant late normal tissue complications. In one canine trial, enhancement of late radiation damage (as assessed histologically) was reported, and the duration of acute radiation complications was prolonged.⁶⁸ There is clinical evidence, however, that excessively high intratumoral temperatures (i.e., >45° C for 60 min) can lead to damage to surrounding normal tissues, an effect that is often caused by rapid tumor regression.^66,69 Such damage is not easily repaired and can lead to chronic tissue consequences, such as fibrosis, fistula formation, and bone necrosis.

Two cases of fatal pelvic necrosis have been reported as a late complication in patients with locally advanced cervical cancer treated with thermoradiotherapy,⁷⁰ and there was speculation as to whether the complication was the result of HT. More recently, however, this rare complication has been reported following chemoradiotherapy treatment of cervical cancer in two small series.^71,72 Thermal doses achieved in the HT series were several degrees lower than the radiotherapy doses reported to be associated with enhancement of normal tissue complications. This finding strongly suggests that these two cases were not the result of HT but more likely resulted from the radiotherapy treatment; An additional complication may have been the heavy smoking history of both patients.⁷⁰

In summary, most available data from preclinical and clinical studies indicate that therapeutic gain is achievable for the combination of HT with radiotherapy. There is very little evidence to suggest that HT enhances the incidence or severity of late normal tissue complications from radiotherapy, particularly when excessively high intratumoral temperatures are avoided.

Rationale for Using Hyperthermia with Chemotherapy

Many chemotherapeutic agents have been shown to exhibit synergism with HT, including cisplatin and related compounds, melphalan, cyclophosphamide, nitrogen mustards, anthracyclines, nitrosoureas, bleomycin, mitomycin C, and hypoxic cell sensitizers.²¹ The mechanisms that underlie the synergy may include (1) increased cellular uptake of drug, (2) increased oxygen radical production, (3) increased DNA damage and inhibition of repair, and (4) reversal of drug resistance mechanisms.^73,74–76 Hypoxia and pH are also important in the thermochemotherapeutic response.^77–81 There are some classes of drugs, such as etoposide and vinca alkaloids, that do not synergistically interact with HT.²¹

Rationale for Using Hyperthermia with Targeted Agents

Very little is known about whether HT affects the cytotoxicity of newer targeted agents, such as those that block epidermal growth factor receptor (EGFR), mitogen-activated protein (MAP) kinase, mammalian target of rapamycin (mTOR), or other signal transduction pathways. Identification of targets can be accomplished by examining changes in gene expression brought on by HT treatment. Such studies have been conducted in vitro and in vivo. Genomic analysis of cells from U937, a human myelomonocytic tumor, exhibited differential expression in approximately 1000 genes, following a noncytotoxic treatment of 41° C for 30 minutes.⁸² A higher thermal exposure of 42° C for 90 minutes led to more significant change in proapoptotic signaling pathways,⁸³ in addition to the expected heat shock response in both cases. Genome-wide analysis has also been done on one rat tumor line in vivo, following local heating (43° C for 60 min).^84,85 The effects of HT were rather pleiotropic, involving over 1200 genes and a host of cellular responses, including apoptosis regulation, cell cycle control, MAP kinase- and calcium-regulated cell signaling, and genes involved in angiogenesis and metabolism regulation. There was also substantial down-regulation of a number of genes involved in immune function at 3 hours after treatment, which recovered to baseline by 24 hours. These results suggested that HT does not adversely affect immune function. The fact that HT appears to augment proapoptotic signaling pathways suggests that it could work additively with targeted agents that promote apoptosis. To date, such strategies have not been examined, however.

It has been reported recently that HT can induce autophagy via activation of nuclear factor kappa B (NFκB) as a protective mechanism against cell death.⁸⁶ Therefore it would make sense to consider combining agents that inhibit NFKB activation with HT as a means of enhancing thermal cell killing. Alternatively, the combination of HT at 43° C with increased oxidative stress can increase cell killing via inhibition of the Akt pathway and induction of irreversible autophagy.⁸⁷ Pajonk and coworkers⁸⁸ reported that inhibition of proteasome activity in combination with HT at 44° C for 60 minutes was particularly effective in augmenting apoptosis, and, in addition, caused substantial radiosensitization.

It is important to note that in most of the studies cited above, a single thermal exposure was used and the severity of the exposure ranged from sublethal to exposures that would be expected to kill logs of cells (i.e., 41° C for 30 min vs. 44° C for 60 min). It is not legitimate to make comparisons between studies with such divergent treatment conditions, let alone the variations in cell types and other methodologic differences. Nevertheless, it appears that there is an emerging rationale for combining HT with the targeting of various signal transduction pathways and other cellular adaptation processes as a means of enhancing cell killing.

There has been some effort, however, related to the idea of using HER2-targeted thermosensitive liposomes to enhance specificity of drug delivery to HER2-positive tumors.⁸⁹ Alternatively, magnetic nanoparticles have been coated with HER2 antibodies to enhance delivery to tumor cells, where they can induce HT when placed in a high-powered magnetic field.^90,91 Targeted nanorods have also been investigated for therapeutic potential with HT.⁹²

Factors to Consider when Combining Hyperthermia with Chemotherapy

Electromagnetic Heating Devices

Electromagnetic heating devices can be separated into two categories: superficial applicators with effective penetration into tissue of less than 4 cm, and deep heating devices that have effective penetration of more than 4 cm. Superficial devices include waveguides (Fig. 21-5) and microstrip or patch antennas (Fig. 21-6) operating at microwave frequencies of 433, 915, or 2450 MHz.^{111,112,113,114} Microwave energy is usually coupled into tissue through a temperature-controlled de-ionized water bolus to maintain the skin temperature below 44° C.

Figure 21-5 Example of a microwave waveguide applicator heating system for superficial HT treatments. Typical systems operate at 915 MHz or 430 MHz for heating a depth of 2 to 4 cm. Energy is usually coupled into tissue through a thin layer of deionized water that is temperature controlled to maintain the skin surface below 44° C. A single waveguide applicator provides no control of power distribution in the x-y plane, aside from what can be achieved by physically moving the applicator.

Figure 21-6 Two examples of thin, flexible microstrip antenna array applicators currently under development for superficial HT. A temperature-controlled, deionized water bolus less than 1 cm thick is used to couple microwaves into tissue and maintain the skin surface below 44° C. These conformal array applicators are driven with eight or more microwave power sources to provide adjustment of the power distribution across the tissue surface.

In order to heat at depths of more than 4 cm, radiofrequencies between 5 and 200 MHz are used. There are three basic techniques for electromagnetic deep heating: magnetic induction, capacitively coupled electric fields, and radiated fields from multiple antenna phased arrays.

Radiofrequency Phased Array

The third option for heating with electromagnetic fields is the radiofrequency phased array.119,120,121,122 These devices consist of an array of radiofrequency antennas arranged in a geometric pattern surrounding the target body region.¹²³ Antennas are driven with multiple independently controllable power amplifiers but use a common radiofrequency source, allowing the radiofrequency fields from all antennas to add together to form a focus in the center of the array. The focus can usually be shifted off center and into the tumor by variation of the relative phase of the signals driving each amplifier. For an array with multiple antennas in phase, one can achieve better and deeper power deposition into tissue than is possible with operation of the antennas independently (i.e., driving the antennas without phase addition). By varying the phase and amplitude of multiple antennas, the phased array technique has more flexibility for controlling power deposition than the magnetic induction and capacitive techniques, which depend heavily on tissue properties and have few power control variables. Figure 21-7 shows one example of a 12-antenna annular phased array applicator mounted on the patient table of an MRI system. This MR-compatible 100-MHz radiofrequency phased array applicator fits inside the bore of a 1.5-T magnet, allowing MR imaging of a temperature rise inside the body during HT treatment.

Figure 21-7 Example of a 12-antenna radiofrequency phased array applicator for deep regional HT treatments. This array is driven at 100 MHz and has appropriate filtering to enable use inside a magnetic resonance (MR) imaging system for monitoring deep tissue temperatures with proton resonance frequency shift-based MR thermal imaging. The large focus of heating inside the phased array applicator may be guided into the tumor by adjusting phase and amplitude of the 12 antennas, in response to real-time feedback from the MR thermal images.

Invasive Thermometry

Invasive thermometry, the current standard, involves physically placing thermometry probes into the tumor within implanted needles or catheters to read subsurface temperatures.129,130,131 Although the accuracy of invasive thermometers is sufficiently precise (typically, ±0.2° C) to resolve important differences in thermal dose (as described above), this type of thermometry has many disadvantages. Disadvantages include discomfort to the patient and risk of hemorrhage and/or infection, the expense of physician time required for catheter placement, and the necessity for imaging to verify placement of thermometers. In addition, the sparse nature of the data obtained makes it difficult to spatially control power deposition or to alter treatment to improve temperature distributions.^132,133 The most common method for doing invasive thermometry is to insert blind-ended catheters into a tumor, using either ultrasound or computed tomography (CT) guidance.¹³⁰ Alternatively, for deep-seated tumors, thermometers may be placed inside orifices that are surrounded by tumor, such as the rectum, urethra, or cervix.^134,135 Thermometers are then placed inside these catheters during treatment. The probes can be multipoint sensors that remain fixed in place, or the sensors can be moved cyclically during treatment to record temperatures at many points along the catheter. There are guidelines published for how these measurements should be taken for all HT devices.^{130,136–138} In spite of its sparse nature, invasive thermometry can provide valuable information about the quality of a treatment. A number of clinical reports have correlated temperature-related parameters to clinical response.^{66,139,140,141–144}

Noninvasive Thermometry

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