THE BIOLOGY OF HYPERTHERMIA
#. Definition of Hyperthermia
HT means elevation of temperature to a supraphysiologic level. When cells or tumor tissues are subjected to elevated temperatures, a number of events follow that have important biologic consequences for cancer therapy. HT can kill cells in its own right, but perhaps more important, it can sensitize tumor cells to other forms of therpay, including RT and Chemotherapy(CT).
Some of the physiologic consequences of HT have implications for radiotherapy as well, such as thermally induced reoxygenation. Changes induced in microvessel pore size can lead to increased delivery of nanoparticle drugs as well as macromolecular therapeutic agents, such as monoclonal antibodies or drug-carrying polymers. The adaptive response to hyperthermia exposure(thermotolerance) may augment host immune reponse against tumor cells.
#. 온열치료의 정의
온열치료는 온도를 생리학적 수준 이상으로 상승시키는 것을 의미한다. 온도가 상승하면 세포나 종양 조직에서는 암 치료에 중요한 생물학적 영향을 미치는 많은 사건들이 일어난다. 온열치료 자체로도 세포를 죽일 수 있지만, 더 중요한 것은, 방사선치료와 항암치료를 포함한 다른 형태의 치료에 대한 종양 세포의 민감도를 올릴 수 있다는 점이다.
온열치료의 열 유도 재산소화 같은 생리학적 변화는 방사선치료에도 영향을 미친다. 미세혈관의 소공 크기를 변화시켜 나노입자 약물뿐만 아니라 단일 클론 항체 또는 약물 운반 중합체와 같은 고분자 치료제의 전달 증가시킬 수도 있다. 고열 노출에 대한 적응 반응(열내성)은 종양 세포에 대한 숙주의 면역 반응을 증가시킬 수도 있다.
#. Effects of Hyperthermia Along on Cell Survival
HT kills cells in a log-linear fashion, depending on the time at a defined temperature.
Resulting survival curves typically have an initial shoulder region, followed by an exponential portion. The initial shoulder region indicates that damage has to accumulate to a certain level before cells begin to die, analogous to the sublethal damage that is seen with ionizing radiation.
At lower temperatures, a resistant tail may appear at the end of the heating period. This resistant tail is not a resistant subpopulation, as might be seen for RT when there is a hypoxic subfraction, but rather is due to the induction of thermotolerance, which develops during the heating period.
At temperatures >43℃ the tail does not develop because thermotolerance is not observed at temperatures greater than this.
#.세포 생존에 따른 온열의 영향
온열치료의 세포 사망 효과는 온도와 시간의 로그-선형 함수를 그린다. 생존 곡선은 초기의 어깨 부분과 지수 부분으로 이루어져 있다. 초기 어깨 부분은 전리방사선에서 나타나는 치사상 손상과 유사한데, 세포 사멸이 일어나기 위해서는 일정 수준까지 손상이 축적되어야 한다는 것을 나타낸다.
가열이 끝나가는 낮은 온도에서는 저항성 꼬리가 나타날 수 있습니다. 이 저항성 꼬리가 나타나는 이유는, 방사선치료에서 볼 수 있는 저산소 분획에 의한 저항성 때문이 아니라, 가열 기간 동안 발생하는 열내성(thermotolerance)에 기인한다.
43℃ 이상에서는 열내성이 관찰되지 않기 때문에 이러한 꼬리가 나타나지 않는다.
#. Thermal Isoeffecct Dose: The Arrhenius Relationship
The temperature dependence of the rate of cell killing by heat is referred to as the Arrhenius relationship. Typically, one plots the log of the slope(1/D0) of cell survival curves as a function of temperature.
Characteristically, Arrhenius plots have a biphasic curve; the point at which the slope changes is referred to as a breakpoint. Above the breakpoint for nearly all cell types, a change in temperature of 1℃ will double the rate of cell killing. Below the breakpoint, the rate of cell killing drops by a factor of 4 to 8 for every drop in temperature of 1℃. The change in slope below the breakpoint is due to the development of thermotolerance during heating.
The recognitions that there is definable relationship between the rate of cell killing and temperature led Sapareto and Dewey to propose using this relationship to normalize thermal data from HT. HT results in temperatures within tumors that are almost always nonuniform, with variable time-temperature history. The formulation for this relationship is as follows:
CEM 43℃ = tR^(43-T)
where CEM 43℃ is the cumulative equivalent minutes at 43℃(the temperature suggested for normalization), t is the time of treatment, T is the average temperature during the interval of heating, and R is a constant. When above the breakpoint, which is usually assumed to be 43℃, R = 0.5. When below the breakpoint, R = 0.25.
For a complex time-temperature history, the heating profile is broken into intervals of time(t) where the temperature remains relatively constant. CEM 43℃ is calculated using the average T(Tavg) for each interval, and the resultant data are summed to give a final CEM 43℃ for the entire heating regimen:
CEM 43℃ = ∑tR^(43-Tavg)
The CEM 43℃(thermal isoeffect dose) formulation has been used extensively and successfully in clinical trials to describe thermal dose despite its derivation from rodent studies.
#. Mechanisms of Hyperthermic Cytotoxicity
> Celluar and Tissue Response to Hyperthermia: Targets for Hyperthermic Cytotoxicity
The predominant molecular target for hyperthermic cell killing appears to be protein. For many cells and tissues(both tumor and normal), the heat of inactivation for cell killing is in the range of that necessary for protein denaturation(130 to 170 kcal/mole). Additional evidence for proteins being the primary target is the importance of heat shock proteins in protecting cells from thermal damage. When cells are exposed to heat, the synthesis of nearly all proteins is stopped, with the exception of heat shock protiens, the synthesis of which is upregulated. One of the primary functions of heat shock proteins is to refold other proteins that have been denatured or damaged.
Some celluar organelles are especially important in controlling the thermal reponse. For example, modification of cellular 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. The cytoskeleton of cells is particularly heat sensitive. Cytoskeletal collapse also disrupts cytoskeletal-dependent signal transduction pathways. The heat sensitivity of the centriole leads to chromosomal aberrations following thermal injury. Finally, many DNA-repair proteins are heat sensitive. This may be one of the mechnisms that lead to heat-induced radiosensitization and chemosensitization.
Little information is available from human tumors to know what proportion of cells is killed with heat alone or what the underlying mechanisms of cell death might be. Such infromation may be important with respect to reoxygenation, which has been seen in rodent, canine, and human tumors after HT. Induction of apoptosis could lead to reoxygenation following heating as a result of reduced oxygen consumption, which could in turn increase RT sensitivity. Alternatively, induction of necrosis is not likely to affect hypoxia since both vessels and tumor cells would be killed in the process.
#. Thermotolerance
Thermotolerance is defined as a transient adaptation to thermal stress that renders surviving heated cells more resistant to additional heat stress. Whether or not a cell dies as a result of thermal insult is dependent on the net balance between how much protein is damaged and how much is protected and repaired via thermotolerance. Thermotolerance can develop either during or after heat stress and can persist for several days. If cells are not exposed to thermal stress again, thermotolerance will decay. The time of peak thermal resistance and the time of decay are related to the severity of the heat shock.
Concerns over the persistence of thermotolerance after HT has affected the design of many clinical trials of thermoradiotherapy. In most trials, a minimum of 48 hours has been suggested between HT fractions in order to avoid re-treatment during thermotolerance. This concern has proscribed the use of daily HT in conjunction with daily radiotherapy in most clinical trials or alternatively led to the use of hypofractionated RT, for example, large fractions twice weekly with concurrent heat.
Some investigators have suggested that one should take advantage of heat radiosensitization rather than hyperthermic cytotoxicity and ignore the issue of thermotolerance. The degree HT-mediated cytotoxicity may be low with HT as it is currently practiced because temperatures achieved are largely below that needed for direct cell killing. Furthermore, heat radiosensitization is relatively unaffected by thermotolerance.
Does thermotolerance occur in humans after heating? Heat shock protein synthesis was evaluated in a small group of human patients(n = 23) with chest wall recurrences of breast cancer who underwent RT with or without HT. Elevated levels of heat shock proteins in biopsy specimens after treatment correlated with lower probability of attaining a complete response. In another study of patients treated with fluorouracil(5-FU) plus thermoradiotherapy for colorectal cancer, no correlation between HSP27 or HSP70 levels, either before or after treatment, and outcome was seen. Interpretation of clinical studies is complicated by the fact that heat shock protein expression is frequently upregulated in tumors in the absence of heat stress. Stresses other that HT, such as hypoxia and hypoxia-reoxygenation injury, can cause elevations in heat shock protein levels. Thus the question of the relevance of thermotolerance to the clinic and the best HT/RT fractionation scheme remains unsettled.
#. Immunologic Implications of the Heat Shock Response
There is emerging evidence that HT can augment the immunologic response toward tumors. Examples of effects that are known to occur after heating include (a) increaed immunogenicity, (b) increased T-cell, NK-cell, and dendritic cell maturation and activity, and (c) enhanced trafficking of immune effector cells into tumors and lymphatic organs. The trafficking is likely mediated by cytokines such as interleukin 6.
#. Hyperthermia and Physiology
In this section, we discuss what is known about the physiologic consequences of HT and then discuss how physiology can be manipulated to enhance the efficacy of HT.
As temperatures are elevated, tissue perfusion increases. In muscle, cyclic variations in temperature have been observed when delivered power is kept constant, demonstrating that thermoregulation is controlled by a threshold temperature. The temperature threshold for this change is 41℃ to 41.5℃ in skin. Changes in vascular permeability also occur, leading to edema formation in the heated volume. As temperature or time-at-temperature is increased, vascular stasis and hemorrhage develop.
The change in normal tissue perfusion upon heating is typically much greater than what one sees in tumors. Muscle and skin perfusion increase by about 10-fold, whereas tumor perfusion is not fully determined, but potential sources include arteriovenous shunting, thrombus formation, and leukocyte plugging. Hemorrhage probably occurs as a result of enlarged endothelial cell gaps or loos of endothelial cell and basement membrane integrity along the vessel wall.
These vascular effects may be exploited clinically. HT causes extravasation of nanoparticles into tumor parenchyma. Heating to 40℃ to 42℃ results in a marked increased in extravasation of liposomes in tumor but not in normal tissue vasculature. Above 42℃ vascular statsis and hemorrhage occur with reduced liposomal extravasation. These results are consistent with the hypothesis that the increase in extravasation is due to cytoskeletal collapse in the vessel wall(endothelial cell). The increase in liposomal extravasation can be exlpoited as a drug delivery vehicle, particulary since the effect appears to be preferential to tumors. Many investigators have shown that HT increase liposomal drug accumulation in tumorss, leading to enhanced antitumor efficacy of a variety of drugs compared with liposome administration alone or free drug administered with HT. When temperature-sensitive liposomes are used, even better antitumor effects can be achieved, particularly using low-temperature-sensitive liposomes that have entered into human clinical trials. The improved effectiveness of these drugs when combined with HT is directly related to the increase in drug delivery.
Although the changes in perfusion in tumors are relatively small in comparison with normal tissues, there have been a number of efforts to exploit them as a means to augment drug delivery to tomors. For relatively small agents, such as most chemotherapeutic drugs, there is no real advantage to using heat to augment delivery, although increaed cellular uptake has been seen with a number of drugs. For drugs with molecular weight <1,000, the primary mechanism that governs drug transport is diffusion, which is controlled by the concentration gradient across the vascular wall. The temperature dependence of diffusion is not large, so HT has relatively little effect.
However, convection is the primary driving force for transvascular transport for molecules >1,000 molecular weight. This is controlled by the pressure gradient across the blood vessel wall. HT increase transvascular delivery of monoclonal antibodies and polymeric peptides that can carry drugs or radioisotopes.
> Effects of Hyperthermia on Tumor Metabolism and Oxygenation
Enzymes for aerobic metabolism are more heat sensitive than those involved in anaerobic metabolism. The net result of this difference in heat sensitivity is that nutrient stores are more rapidly depleted during heat shock, leading to a reduction in adenosine triphosphate(ATP) and buildup of lactic acid. A number of rodent studies have demonstrated changes in energy balance and pH after heating. Kelleher et al. reported decrease in ATP and increases in lactate concentration occurring concomitantly with reduction in tumor blood flow after heating. In a series of human patients with soft tissue sarcomas who were treated preoperatively with HT and RT, a reduces oxygen consumption rates in murine and human tumor lines. These results are consistent with the theory that a reduction in tumor respiration occurs after HT.
A shift toward anaerobic metabolism would decrease oxygen consumption rates, which could improve tumor oxygenation. Oleson suggested that some of the benefits of HT in the clinical setting may result from improvements in oxygenation. Results from several studies in rodent tumors and human tumor xenografts support the notion that an overall improvement in tumor oxygenation can result from time-temperature combinations below those that cause vascular damage(e.g., 41℃ to 41.5℃ for 60 minutes). Higher thermal doses that cause vascular damamge(e.g., >43℃, 60 minutes) may lead to decreases in tumor oxygenation.
HT improves tumor oxygenation in canine and human tumors. In a human study of soft tissue sarcomas, failure to reoxygenate after the first HT fraction led to a significantly lower probability of achieving pathologic complete response at the time of surgery. In women with locally advanced breast cancer treated with neadjuvant thermochmoradiotherapy, reoxygenation after the first heat treatment was associated with higher likelihood for achieving a clinical response. In a canine soft tissue sarcoma study involving thermoradiotherapy treatment, there was an overall improvement in partial pressure of oxygen at 24 hours after the first heat treatment, but for those tumors in which the median temperature was greater than 44℃, perfusion and tumor oxygenation decreased. This threshold temperature for vascular damage is about 44℃ for human and canine tumors. It is difficult, however, to attain this temperature in nonanesthetized human patients.
Zywietz et al. reported that twice-weekly heating to 43℃ for 60 minutes in combination with 60 Gy delivered in 20 fractions over 4 weeks resulted in a steady decline in partial pressure of oxgen of a rat rhabdomyosarcoma. However, Thrall et al. reported that there were long-term improvements in oxygenation of canine tumors treated with fractionated thermoradiotherapy. Thus, It is not known with certainly whether reoxygenation or deoxygenation predominates after fractionated thermoradiotherapy and how this relates to treatment outcome. The sarcoma and breast clinical studies cited previously argue in favor of a positive effect on oxygen status, but only a few HT treatments may be necessaray to achieve this.
It has been reported that fractionated HT(42.5℃ for 60-minute conditioning dose followed by 44.5℃ for up to 90 minutes) can lead to vascular thermotolerance. This means that the likelihood of vascular damage decrases if a second HT treatment is given within 1 to 2 days after the first treatment, at a time when thermotolerance may still be present. It was reported that vascular thermotolerance is associated with vessel normalization. Normalization is a phenomenon popularized by Jain to explain the beneficial effects of antiangiogenic treatments to improve transport properties of tumors. It is associated with a deccrease in microvessel density and an increase in pericyte coverage, conditions reflective of a more mature vasculature.
The process of vascular normalization after heating may be mediated in part by upregulation of the transcription factor hypoxia-inducible factor-1(HIF-1). This transcription factor is known to regulate angiogenesis by controlling levels of vascular endothelial growth factor in tissue. Moon et al. reported that HT increases HIF-1 in tumors by activating the enzyme NADPH oxidase. The reactive oxygen species produced by this enzyme inhibited degradation of the laibe subunit of HIF-1, HIF-1α. The upregulation of HIF-1 levels was accompanied by increases in levels of vascular endothelial growth factor and perfused vascular density in heated tumors.
#. Physiologic Approaches to Enhance Thermal Cytotoxicity: pH Modification
It is well established that an acute reduction in extracelluar pH can greatly enhance sensitivity to HT. Cells adapted to grow at low pH, as occurs in tumors, have little reserve to further increase proton pumping. It is the reduction in inctracellular pH that is actually responsible for the increase in cytotoxicity. The potential degree of enhancement in killing is substantial, and, as a result, considerable effort has been made to accomplish this feat in vivo. The most widely studied method has been induction of hyperglycemia. The rationale is that excess glucose load to the tumor will push it toward glycolysis and lactic acid production because most tumors have a limited oxygen supply and also have defects in respiratory pathways. Induction of a hyperglycemic state may also reduce blood flow by increasing blood viscosity. Reduced perfusion compromises heat exchange capacity, thereby increasing temperatures in tumor during heating. When glucose has been administered intravenously, there has been little effect on perfusion, but reduction in extracellular pH has been observed.
In humans, the induction of hyperglycemia has been accomplished by either oral or intravenous glucose administration. Results using this approach have been mixed. On average, the drop in extracellular pH is about 0.17 pH unit, which is near the goal of 0.2 pH unit. However, there is considerable variation from one patient to another in terms of how effective this approach is. Furthermore, in prediabetic patients, the trend is toward an increase in pH rather than a decrease. In canines with soft tissue sarcomas, induction of hyperglycemia via intravenous administration did not result in any significant change in either intracellular or extracellular pH.
The use of hyperglycemia resulted in improved response to thermochemotherapy and thermoradiotherapy in rodent. Data in human tumors are sparse. One study in a limited number of patients suggested improvement in response with the use of hyperglycemia combined with thermoradiotherapy. However, pH was not measured, and the study was not randomaized.
The addition of agents that can selectively drive down tumor intracellular pH, such as glucose combined with the respiratory inhibitor metaiodobenzylguanidine, has the potential to further enhance hyperthermic cytotoxicity selectively in tumor tissues. Some groups have also focused on the use of pharmacologic agents that 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.
#. Blood Flow Manipulation
Blood perfusion is a major impediment to effective heating. This is because perfusion is the primary mechanism for conducting heat and maintaining homeostasis. Thus, if tumor blood flow can be effectively reduced, tmperatures in the tumor will increase. A number of vasoactive agents have been shown to reduce tumor blood flow, including some that are normally considered to be vasodilators. Agents investigated indclude hydralazine, nitroprusside, and angiotensin II; the first two drugs are vasodilators and the last is a vasoconstrictor. Clinically, changes in blood pressure have limited doses that can be safely used, minimizing effects on tumor blood flow. New agents are needed. It also makes sense to consider combinations of approaches that might reduce both tumor blood flow and pH and lead to improved temperature distributions, as well as to heat sensitization.
#. Radiation and Hyperthermia
> Rationale for Combining Hyperthermia with Radiotherapy
When RT is combined with HT, complementary effects occur.
Cells in the S phase of the cell cycle relatively radioresistant, but when heated, theses cells are most sensitive.
Hypoxic cells are known to be three times more resistant to RT as compared with aerobic cells. With HT, there is no difference in sensitivity between aerobic and hypoxic cells. As discussed in detail earlier, there is good evidence that HT can lead to reoxygenation, which will further improve RT response.
Finally, HT inhibits the repair of both sublethal and potentially lethal damage via its effects in inactivating crucial DNA repair pathways.
> Factors to Consider When Combining Hyperthermia with Radiotherapy
The interaction between RT and HT is described by th "thermal enhancement ratio"(TER), defined as the ratio of doses of RT to achieve an isoeffect for RT/HT + HT. TERs for local control have been estimated for a number of human tumors using historical control data for RT alone. In most tumor types examined, there ratios were greater 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 in the same patient population, suggesting potential for therapeutic gain for RT + HT compared with RT alone. Prospective, randomized trials in dog with spontaneous tumors have also shown evidence for improved local tumor control with RT + HT compared with RT alone, with no observable increase in the frequency of clinically relevant late normal-tissue complications. In one canine trial, enhancement of late RT damage(as assessed histologically) was reported and the duration of acute RT complications was prolonged. There is evidence, however, that excessively high intratumoral temperature(i.e., >45℃ for 60 minutes) can lead to damage to surrounding normal tissues, an effect that is often caused by rapid tumor regression. Such damage is nor easily repaired and can lead to chronic tissue consequences, such as fibrosis, fistula formation, and bone necrosis.
In summary, most available data from preclinical and clinical studies inidcate that therapeutic gain is achievable for the combination of HT with RT. There is little evidence to suggest that HT enhances the incidence or severity of late normal-tissue complications from RT, particularly when excessively high intratumoral temperatures are avoided.
#. Hyperthermia and Chemotherapy
> Rationale for using Hyperthermia with Chemotherpay
Many chemotherpeutic agents have demonstrated synergism with HT, including cisplatin and related compounds, melphalan, cyclophosphamide, nitrogen mustards, anthracyclines, nitrosoureas, bleomycins, mitomycin C, and hypoxic cell sensitizers.
The mechanisms may include (a) incrased cellular uptake of drug, (b) increased oxygen free radical production, and (c) increased DNA damage and inhibition of repair. Hypoxia and pH appear to be important in the thermochemotherapeutic response.
An important factor in the potential use of HT with many drugs is its ability to reverse, at least partially, drug resistance. Examples of drugs for which this has been shown include cisplatin, melphalan, nitrosoureas, and doxorubicin, when combined with the MDR inhibitor verapamil.
In vitro and in vivo results may not correlate. Paclitaxel, for example, shows little in vitro activity, but clinical results in combination with HT and RT have been encouraging.
Most antimetabolites do not interact with HT synergistically when given concomitantly. However, it is important to consider issues such as time of drug exposure and temperature, both of which may be important in determining where and when to expect a positive interaction.
When 5-FU has been given simultaneously with HT, there have been only additive effects. However, 5-FU has been shown to interact supra-additively with HT under specific conditions. Heating to 39℃ to 41℃ can lead to enhanced conversion to active metabolites, thereby increasing drug cytotoxicity. In addition, continuous-infusion protocols with this drug may lead to cell cycle block in S pahse, a relatively sensitive part of the cell cycle to HT.
For most drug(excluding 5-FU and perhaps other antimetabolites), the optimal sequence between heat and drug is to administer them simultaneously or to give the drug immediately before the onset of heating. For platinum-containing drugs, the tissue extraction rate of drug may be increased with HT, further substatiating the rationale for use of this sequence. The degree of interaction between drugs and HT is temperature and cell line depedent.
There are also some classes of drugs for which there has been no demonstrated synergistic interaction. Interactions with etoposide have been unpredictable, and current recommendations are that one cannot expect synergistic interactions with it. There is also no eivdence for synergistic interaction between vinca alkkaloids and HT.
The term trimodality therapy has been used to describe combination therapy of HT, drugs, and RT. It has been studied in both preclinical and clinical models and will be discussed at lenght in the clinical section of this chapter.
> Hyperthermia and Thermosensitive Liposomally Encapsulated Drugs
In a classic paper, Yatvin et al. suggested that a temperature-sensitive liposome could be used to selectively deliver drug to tumors. Several papers have been published using formulations similar to that Yatvin et al. The combination of HT with such drug carriers increased drug delivery and efficacy as compared with using drug carrier along or free drug with HT. However, the thermal properties of the original Yatvin formulation were not amenable to clinical application. The release temperature was too high, and the rate of drug release was relatively slow. A breakthrough occurred when Needham et al. reported the formulation for a low-temperature-sensitive liposome that rapidly released drug at 41.4℃. A second formulation with similar drug release properties was reported by Lindner et al. This type of liposome is referred to as a low-temperature-sensitive liposome(LTSL).
Direct comparison of the relative efficacy of a doxorubicin-containing LTSL, the Yatvin formulation, a traditional non-thermally sensitive liposome formulation similar to Doxil, and free drug has been made. The LTSL was clearly more effective than any other formulation, as assessed using tumor growth delay as an endpoint. The difference in effectiveness was demonstrated to be related to a significant improvement in drug delivery to tumor, as well as to increased drug binding to DNA. Further preclinical work with this formulation demonstrated that it has broad activity when combined with local HT across a range of different tumor types. The formulation was tested in a phase I trial of canine tumors, where the maximum tolerated dose was associated with bone marrow toxicity. This has been the dose-limiting feature of the toxicity in phase I trials in humans as well. The drug was tested in a double-blind, phase III trial in combination with thermal ablation for hepatocellular carcinoma. The tiral is now completed, and the sponsor is waiting for follow-up before reporting the results. The formuulation has also been used as a MRI agent, where drug and contents have been loaded in the same liposome. Changes in MR signal intensity were used to determine drug concentration in preclinical models. Concentrations measured with MRI were associated with antitumor activity in individual animals.