Journal of Surgical Research 127, 208 223 (2005) doi:10.1016/j.jss.2005.02.

009

RESEARCH REVIEW Mechanisms of Focal Heat Destruction of Liver Tumors

Mehrdad Nikfarjam, M.B.B.S., Vijayaragavan Muralidharan, Ph.D., F.R.A.C.S., and Christopher Christophi, M.D., F.R.A.C.S., F.A.C.S., F.R.C.S.1
Department of Surgery, University of Melbourne, Austin Hospital, Melbourne, Victoria, Australia Submitted for publication October 26, 2004

Background. Focal heat destruction has emerged as an effective treatment strategy in selected patients with malignant liver tumors. Radiofrequency ablation, interstitial laser thermotherapy, and microwave treatment are currently the most widely applied thermal ablative techniques. A major limitation of these therapies is incomplete tumor destruction and overall high recurrences. An understanding of the mechanisms of tissue injury induced by focal hyperthermia is essential to ensure more complete tumor destruction. Here, the currently available scientic literature concerning the underlying mechanisms involved in the destruction of liver tumors by focal hyperthermia is reviewed. Methods. Medline was searched from 1960 to 2004 for literature regarding the use of focal hyperthermia for the treatment of liver tumors. All relevant literature was searched for further references. Results. Experimental evidence suggests that focal hyperthermic injury occurs in two distinct phases. The rst phase results in direct heat injury that is determined by the total thermal energy applied, tumor biology, and the tumor microenvironment. Tumors are more susceptible to heat injury than normal cells as the result of specic biological features, reduced heat dissipating ability, and lower interstitial pH. The second phase of hyperthermic injury is indirect tissue damage that produces a progression of tissue injury after the cessation of the initial heat stimulus. This progressive injury may involve a balance of several factors, including apoptosis, microvascular damage, ischemiareperfusion injury, Kupffer cell ac1 To whom correspondence and reprint requests should be addressed at Department of Surgery, University of Melbourne, Austin Hospital, Lance Townsend Building Level 8, Studley Rd, Heidelberg, Melbourne, Victoria 3084, Australia. Phone: 61 3 9496 5468. Fax: 61 3 9458 1650. E-mail: surgery-armc@unimelb.edu.au

tivation, altered cytokine expression, and alterations in the immune response. Blood ow modulation and administration of thermosensitizing agents are two methods currently used to increase the extent of direct thermal injury. The processes involved in the progression of thermal injury and therapies that may potentially modulate them remain poorly understood. Conclusion. Focal hyperthermia for the treatment of liver tumors involves complex mechanisms. Evidence suggests that focal hyperthermia produces both direct and indirect tissue injury by differing underlying processes. Methods to enhance the effects of treatment to achieve complete tumor destruction should focus on manipulating these processes. 2005 Elsevier Inc. All rights
reserved.

Key Words: focal hyperthermia; direct injury; thermal ablation; indirect injury; progressive injury; mechanisms; apoptosis; cytokines; Kupffer cells; growth factors; heat shock protein.

INTRODUCTION

Focal hyperthermia may be induced by several methods and is at present the preferred local ablative technique for primary hepatocellular cancer (HCC) and colorectal liver metastases [1]. Energy sources available for achieving tumor destruction by focal hyperthermia include radiofrequency (RF), laser, microwave, and high-intensity focused ultrasound (HIFU). Treatment can be delivered to liver tumors in a minimally invasive fashion to achieve tumor destruction without signicant damage to normal liver parenchyma. It is particularly applicable for treatment of patients with nonresectable disease and those with general contraindications to surgery [1, 2]. Long-term

0022-4804/05 $30.00 2005 Elsevier Inc. All rights reserved.

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survival has been achieved by thermal ablation in patients with HCC and colorectal cancer liver metastases [1 4]. In selected patients with HCC less than 5 cm in diameter, treatment by RF ablation or interstitial laser thermotherapy (ILT) achieves 5-year survival that ranges from 15% to 33% [5, 6]. In a recent thermal ablative series of 97 patients with HCC who were treated by predominately microwave therapy, the 3-year cumulative survival was 50% [7]. In the largest reported series of patients with colorectal liver metastases (n 603) treated by thermal ablation, the 5-year survival after magnetic resonance-guided ILT was 37% [2]. This series consisted of patients with both resectable and nonresectable liver metastases. It excluded patients with extrahepatic disease, tumors greater than 5 cm diameter, and those with more than ve lesions. Despite some encouraging survival results that rival surgical resection, disease recurrences occur in the majority of patients after thermal ablation [1, 2, 8]. Local recurrence appears to be related to tumor size and may represent incomplete tumor destruction [2, 8]. Local recurrences range from 0% to 21% in RF ablation series for treatment of HCC [4]. The recurrence rate after focal hyperthermia ablation is generally less than 10% when HCC does not exceed 4 cm maximum in diameter [6, 9]. Regional recurrences, however, occur in as many as 80% of patients at 5 years follow-up, which may reect the natural history of hepatocarcinogenesis [10, 11]. Similarly, the local recurrence rate of colorectal liver metastases that does not exceed 4 cm in maximum in diameter is generally less than 10% in large thermal ablative series [2, 4, 12]. Local recurrences in some series, however, occur in as many as 68% of cases for lesions greater than 4 cm in maximum in diameter [8]. Overall regional and extra-hepatic recurrences after complete ablation of colorectal liver metastases ranges from 60% to 90% at 5 years follow-up [2, 13, 14]. The exact underlying mechanisms of tumor destruction by focal hyperthermia and its inuence on tumor growth are unknown. A greater understanding of these underlying mechanisms and their manipulation may lead to increased tumor destruction, resulting in better local control, the reduction of overall recurrences, and the improvement of survival. This article reviews the underlying mechanisms of injury induced by focal hyperthermia in the treatment of liver tumors and outlines potential strategies to maximize tumor destruction.
METHODS OF HEAT GENERATION

tion. However, they vary according to the processes involved in heat generation and its delivery.
RF Ablation

RF ablation applies to methods that use electromagnetic energy sources with frequencies less than 900 kHz to generate heat [15]. Most devices function in the range of 375500 kHz. Electrode probes are placed within tumors percutaneously or at open operation. The passage of alternating high-frequency current through the probe tip displaces molecules within the tissue rst in one direction and then the opposite direction. Changes in the energy state of molecules results in a localized rise in temperature, reaching approximately 90C in the clinical setting.
Interstitial Laser Thermotherapy

Laser units consist of a power source, a lasing medium, and reecting mirrors. An infrared light wavelength of between 800 and 1100 nm achieves maximal tissue penetration and homogenous spread when delivered into tumors by an optical ber [14, 16, 17]. Neodyium Yttrium-Aluminum-Garnet (Nd-YAGwavelength of 1,064 nm) and diode (wavelength of 800 980 nm) lasers are most commonly used. Laser light delivered into tissue is absorbed by tissue-specic chromophores and photon energy is transferred into heat to produce thermal injury [18, 19]. ILT generally is performed at power and energy settings designed to achieve temperatures of 50C to 100C.
Microwave Ablation

Microwave ablation refers to methods that generate heat using devises with frequencies greater than or equal to 900 kHz. Microwave generators used for tumor ablation in the liver generally operate at 2000 3000 MHz. The electromagnetic radiation that is produces is delivered into tumors by an antenna (needle) and produce rapid agitation of water molecules within cells that causes ctional heating. The chosen power settings generally produce tissue temperatures ranging from 60C to 100C [20, 21].
Ultrasound Ablation

Local tumor destruction by RF, ILT, microwave, and HIFU rely on heat as the major mode of tumor abla-

Ultrasound energy can be applied by extracorporeal or direct needle application to achieve thermal tissue ablation. HIFU is an extracorporeal technique that is most commonly used for ultrasound ablation of liver tumors [22]. At frequencies typically between 0.8 and 1.6 MHz, ultrasound waves can be harmlessly propagated through soft tissue and brought into a tight focus. Sound energy is absorbed by tissue and deposited as heat. During treatment, temperatures at the point of focus rapidly rise greater than 80C. Although thermal ablation is the main mechanism of injury by

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TABLE 1 Effect of Temperature on Tissues

Temperature range (C) 4260 60140 100300 300 Minimum exposure time requirements 3060 minutes Seconds Seconds Fraction of a second Physical effects No physical changes Coagulation, ablation Vaporization Carbonization, smoke generation Biological effects Enzyme inactivation and mitochondrial injury Protein denaturation, membrane rupture, cell shrinkage, pyknosis, hyperchromasia Cell shrinkage and extracellular steam vacuole Carbonization

Note. Shown are the physical and biologic effects of heat application at various temperatures and exposures on tissue.

HIFU, ultrasound also causes tissues to vibrate, and can produce mechanical cell injury by the process of acoustic cavitation [22]. The mode of heat production varies with these different treatment methods; however, the mechanisms of resulting thermal injuries are essentially equivalent.
MECHANISMS OF HEAT INJURY

Tissue injury caused by focal hyperthermia occurs in two distinct phases. The initial phase is direct heat injury that is predominately determined by the total energy applied to the tumor, tumor biology, and tumor microenvironment [23]. The second phase is indirect injury after focal hyperthermia application that produces a progression in tissue damage. This progressive injury may involve a balance of several factors including microvascular damage, ischemia-reperfusion injury, induction of apoptosis, Kupffer cell activation, altered cytokine expression, and modulation of the immune response. Many of the concepts regarding the mechanisms of focal hyperthermic injury are derived from in vitro and whole-body hyperthermia studies. Direct injury is generally better dened than the secondary indirect effects that produce progressive tissue damage.
Direct Thermal Injury

The concept of treating tumors with hyperthermia is based on observations that cancer cells exhibit increased sensitivity to heat than normal cells. In vitro [24, 25] and in vivo [26] studies demonstrate that tumor cells are destroyed at lower temperatures than normal cells. The mechanisms of direct thermal injury and thermosensitivity involve complex interactions within tumor tissue at cellular and subcellular levels. These are inuenced by tumor biology and its microenvironment.
Tissue Effects

The effects of focal hyperthermia depend on tissue temperatures attained, determined by the total ther-

mal energy applied, rate of removal of heat, and the specic thermal sensitivity of the tissue (Table 1). Classical hyperthermia relies on a temperature of 42C to 45C for periods of 30 to 60 min to cause irreversible cellular damage [2730]. The inactivation of vital enzymes is a key feature of tissue injury at these temperatures [31]. As the tissue temperature rises to 60C, the time required to achieve irreversible cellular damage decreases exponentially. Protein denaturation occurs between 60C and 140C and leads to immediate cell death. An area of coagulative necrosis develops in the liver at these temperatures [32]. Vaporization of tissue water is superimposed on this process between 100C and 300C. Additionally, carbonization, charring and smoke generation occurs at 300C to 1000C [33]. Once carbonization occurs, there is a rapid increase in temperature, perpetuating the entire process. Paradoxically, carbonization limits the extent of tissue injury by creating a heat trap [34]. This carbonization also increases tissue interstitial pressures and may result in dissemination of cancer cells deep into the liver and blood vessels [31]. Distinct changes are evident in liver and tumor tissue after injury by focal hyperthermia. The changes are generally better visualized in liver than tumor tissue [35]. Four zones of cellular changes are described in the liver following treatment: application, central, transition, and reference tissue zones. The application zone is where the heat source contacts the tissue. The central zone immediately surrounds the application zone and consists of damaged tissue. The transition zone contains apparently undamaged tissue but exhibits signs of subacute hemorrhage. The reference zone refers to normal tissue surrounding the transition zone [36 38]. Cells adjacent to the application zone often are morphologically normal with the connective tissue scaffolding left intact when assessed immediately following injury [39, 40]. On examination with electron microscopy, features of irreversible injury are noted [41]. In a study examining the effects of microwave ablation in liver, cells within this zone were morphologically and

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immunologically normal, but were non-viable based on assessment of metabolic activity [38]. The transition zone contains a peripheral hemorrhagic or hyperemic rim which surrounds the central area. It contains cells in the earliest stages of cellular death that demonstrate a loss of enzymatic activity [28, 42]. Histological techniques to access cell viability immediately after heat application are generally inaccurate because morphological changes are a poor indicator of cell vitality at this stage. Distinct changes indicative of lethal injury on histology develop well after the completion of therapy. Changes also occur in tumor vasculature after focal hyperthermia. However, these changes are not as well described as the tissue effects. Tumor blood vessels are usually at near maximal dilation, and their response to heat is limited [43, 44]. At temperatures between 40C and 42C and exposure times of 30 to 60 min, there is no signicant change in tumor blood ow [45]. In normal tissue, an inammatory response results in a mild increase in blood ow, which returns to baseline level after therapy [46]. Beyond 42C to 44C, there is an irreversible decrease in tumor blood ow, with vascular stasis and thrombosis resulting in heat trapping [45, 46]. Xenon-33 clearance studies in rats demonstrate that tumor vessels are more sensitive to thermal injury than those in normal liver and more likely to undergo irreversible injury [47]. At temperatures exceeding 60C, there is complete destruction of tumor microvasculature [48]. Muralidharan et al. [48] demonstrated that thermal ablation by laser results in marked disruption of blood vessels when applied to colorectal liver metastases in a murine model. At temperatures exceeding 60C, immediate cellular destruction occurs irrespective of the tissue histology. Tissue thermosensitivity, therefore, predominantly impacts tissue destruction in the transitional zone, exposed to lower temperatures than the application and central zones. Mechanical effects caused by abrupt changes in tissue temperature also contribute to the direct tissue injury produced by focal hyperthermia. Shock waves are created by phase changes or thermoelastic expansion of tissues in the treated regions. Secondary waves are created at tissue interfaces and may cause tissue disruption distal to the application site [19, 28, 49].
Cellular and Subcellular Effects

The direct effects of heat on tumors at a cellular and subcellular level appear complex, with all aspects of cellular mechanism disrupted to varying degrees. Cellular survival after thermal application is assessed in vitro by the arrhenius plot of survival versus the reciprocal of absolute temperature [50]. The fraction of tumor cells killed by heating can be increased by 100fold with minimal temperature elevations of 2C [51].

At temperatures between 50C to 55C, cellular death occurs instantaneously in cell culture [52]. Subcellular disruption exerts its effects through changes in nucleic acid, cell membrane, cytoskeleton, and mitochondrial function [53]. Changes in membrane function were traditionally considered the main cause of cell death. Observations of altered membrane uidity, permeability and surface blebbing with rising temperatures supported this concept [54, 55]. Actin laments become insoluble during hyperthermia and are inactivated in a temperature dependant manner. Cytoplasmic streaming decreases and depolymerization and inactivation of microtubules contributes to the overall membrane damage [56, 57]. This leads to impairment of facilitated diffusion, while preserving passive diffusion. The resultant accumulation of metabolites within cells, with inux of water, causes eventual cell death [58]. Some of these changes are related to free calcium elevation as the result of a heat-induced rise in inositol triphosphate, that causes protein and cytoskeleton malfunction [59]. Membrane damage and loss of integrity may not become evident during the heating process per se but occur during the recovery phase of the cells returning to baseline temperature [52, 60]. Despite changes in cellular membranes after hyperthermia, current evidence suggests that membrane destabilization is not the main cause of heat induced cell death but the end result of other process [59, 61]. There is no direct correlation between heat applied and membrane damage in the hyperthermic temperature range [52, 62, 63]. Mitochondrial dysfunction appears to be a more important determinant of irreversible heatinduced injury. Ultrastructural changes in the mitochondria accurately correlate with viability and metabolic functioning [30, 53, 64]. Changes that are observed include the formation of intramitochondrial dense granules, vesicularization of the cristi, intracristal space swelling, myelin degeneration, and formation of dense bodies [52]. Major ultrastructural changes in mitochondrial activity are observed within 15 min of heat injury [52]. Hyperthermia promotes, proton leaking across the inner mitochondrial membrane and impairs oxidative phosphorylation [65]. Mitochondrial vitality can be assessed histochemically in heat treated tissue. This involves staining for one of several mitochondrial enzymes, including nicotinamide adenine dinucleotide diaphorase (NADH-diaphorase). NADH-diaphorase activity ceases immediately after irreversible cellular injury [39, 66]. Staining for NADH-diaphorase activity clearly identies viable from nonviable tissue well before morphological changes of necrosis [23]. Heating also produces changes in the nucleolus polysome assembly, Golgi apparatus, and cytoskeleton components, such as intermediate laments [67]. Some of the injury may be attributable to the release of lysosomal

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enzymes [68, 69]. Initiation of replication may be inhibited by abnormal condensations of nonhistone proteins around DNA, preventing key enzymes from becoming involved in DNA synthesis [70]. Heat injury also disrupts RNA synthesis and transport mechanisms [71, 72]. In addition, there is a disruption of glucose energy-dependent pathways [7274].
Inuence of Tumor Biology on Direct Injury

Inuence of Tumor Microenvironment on Direct Thermal Injury

The tissue changes that occur after focal hyperthermia also are inuenced by tumor biology. This is particularly relevant at the transition zone, where the tissue temperature rise is generally signicantly lower than the application and central zones. The heat sensitivity of tumors appears to be a type-specic feature of the cells and independent of the proliferation rate, absorbance or benign or malignant nature of the cells [75, 76]. It may reect differences in the structure and size of cellular components including the distribution of intermediate laments that alter susceptibility to heat injury [75, 77]. Tumor thermosensitivity is indirectly related to increased baseline metabolic stresses compared with normal cells [78, 79]. Tumors have a higher fraction of cells in the mitotic or S-phase of replication, have lower mitochondrial levels, reduced intracellular adenosine triphosphate (ATP) and lower overall oxygen intake compared to normal cells [78 81]. When tumor oxygen consumption requirement are substantially increased, with temperatures approaching 45C, the susceptibility of tumors to injury further increases [82, 83]. Intracellular levels of heat shock proteins (HSPs) appear to be important in the regulation of tumor cell thermosensisitivity [77]. HSP is the major cause of the phenomenon of acquired thermotolerance [84 86], produced as a result of a stress response, and may regulate the expression of certain genes [87]. HSP may prevent the aggregation of some cellular proteins and support refolding of others, leading to increased resistance to fragmentation [88, 89]. Induction of HSP 70 also increases cellular resistance to apoptosis [89 91]. The baseline levels of HSP and their expression following heat exposure varies with different cell lines. Increased expression of HSP is associated with a worse prognosis in some cancer cell lines [92]. The overall ability of cells to survive application of focal hyperthermia depends on the extent of protein loss and degradation. Cells with increased baseline HSP levels are afforded added protection from protein loss and exhibit greater thermoresistance [93]. Increased expression of these proteins in surviving tumor cells following focal hyperthermia may lead to the progressive development of more malignant and heat resistant tumor strains [94].

In addition to tumor biology, microenvironmental differences between tumor and normal liver tissue is likely to contribute to tumor thermosensitivity [95]. Tumor blood ow is a major determinant of direct heat-induced injury [95]. The relationship of major vessels to tumor necrosis in the liver is well established. In theoretical calculations of temperatures and thermal damage, blood ow is the most important variable in determining the extent of damage [95]. Heat is absorbed by hemoglobin in erythrocytes and transferred away from the region of focal hyperthermia limiting temperature build-up and necrosis [34]. Thermal injury is generally least pronounced in tumor adjacent to large intrahepatic vessels due to the heat dissipating effects described [96 98]. The same phenomenon is observed around major bile ducts, where the ow of bile may serve as a heat sink. The effect of blood ow on heat dissipation is greater in liver than tumor tissue, due to the presence of larger vessels and increased ability of normal liver to augment blood ow by vasodilatation. It is not surprising that the main region of treatment failure in local ablative techniques is at the host-tumor interface, where blood ow is greatest [99]. Tissue hypoxia and low pH also are implicated in the increased thermal sensitivity displayed by tumor tissue. Application of hyperthermia exacerbates tissue hypoxia, increases metabolism and shifts the oxyhemoglobin dissociation curve leading to reduced oxygen delivery [100 102]. Increases in the production of metabolites, inefcient vascular drainage, and heatinduced cellular injury cause a subsequent increase in intracellular and extracellular tumor acidity [103, 104]. A decrease in tumor pH is shown to increase thermosensitivity, possibly through processes of direct enhancement of thermal killing, inhibition of thermotolerance, impairment of DNA synthesis and reduction of cellular proliferation [100, 105111]. A decrease in intratumoral pH also may lead to further blood ow inhibition, through its effects on the microcirculation, thereby promote heat trapping [112].
Indirect Thermal Effects

Direct heat injury that occurs with focal hyperthermia, is followed secondary indirect injury. Clinical and experimental data indicate that tissue injury progresses after the cessation of the focal hyperthermic stimulus [30, 64, 98, 113]. The exact mechanism of this process is undetermined. The full extent of tissue damage becomes evident 1 to 7 days after focal hyperthermia application, depending on the model used [30, 37, 52, 64, 98, 113]. The lesion size then decreases slightly

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during the course of several weeks as a result of the breakdown of and removal of necrotic tissue by inammatory cells [114, 115]. Reports of progressive injury after focal hyperthermia are based on histological evaluation of tissue damage at various time points after thermal application [30, 37, 64, 98, 113]. However, routine histology by hematoxylin and eosin staining is unable to provide an accurate assessment of the extent of immediate focal hyperthermic injury. A progressive expansion of injury may reect several underlying mechanisms. The increase may purely represent a delay in identication of lethally damaged cells. Alternatively, it may present a true expansion in injury resulting from cellular mechanisms triggered by the initial heat stimulus. In our experience, with a model of colorectal liver metastases, tumor injury peaks at 4.5 days after application of focal hyperthermia. This progression of injury appears to be independent of the initial thermal effect, which is clearly demonstrated by NADH-diaphorase tumor staining [23]. Studies by Wiersinga and colleagues [113] support this nding. They demonstrated a continuous deterioration in tissue viability during the course of 24 h after RF ablation of porcine liver, using mitochondrial and cell membrane staining techniques. Experiments involving focal hyperthermia application to ank tumors in mice also produce progressive tissue injury for up to 48 h after the initial heat stimulus [53, 103, 116]. A true progression in injury represents a balance of several promoting and inhibiting mechanisms. These may include induction of apoptosis, Kupffer cell activation, cytokine release, ischemia-reperfusion injury and stimulation of the immune response.
Altered Apoptosis

Vascular Injury

It is well established that apoptosis increases in a temperature-dependent manner. Temperatures between 40C and 45C cause inactivation of vital enzymes and may initiate apoptosis [89, 117]. Focal hyperthermia application to liver or tumor tissue creates a temperature gradient that progressively decreases away from the site of probe insertion. The induction of apoptosis at a distance from the heat source may potentially contribute to the progression of injury. In a study of microwave ablation in liver, increased rate of apoptosis was observed for 24 h following treatment [37]. Based on caspase 3 activity, apoptosis peaked at 2 h after therapy, whereas progressive injury continued for a total of 12 h. The stimulation of apoptosis may be directly induced by temperature elevations, alterations in tissue microenvironment or triggered by various cytokines. Other factors may modulate apoptosis, including HSP 70 levels. The expression of HSP 70 has been shown to prevent cytochrome c/dATP-mediated caspase activation in vitro and inhibit apoptosis [118, 119].

Little evidence exists on the effects of focal hyperthermia on liver or tumor vasculature after the initial injury. Most reports are based on studies of focal hyperthermia in models of implanted tumors in extrahepatic locations [120 122]. In these models, focal hyperthermia produces endothelial cell damage. This increases vessel wall adhesiveness, vascular leakage and blood viscosity. This results in small vessel thrombosis after the cessation heat stimulus, leading to progressive tissue damage [53, 120, 123, 124]. A continuous decrease in blood ow for up to 4 to 6 h in the liver is demonstrated in several studies after focal hyperthermia treatment [124 127]. Unexpected areas of infarction observed at the periphery of focal hyperthermia treated regions may be explained by ischemia caused by vascular occlusion [64]. These effects may play a role in the therapeutics of hyperthermia [126]. In addition, reduced blood ow lowers tissue pH, which itself induces progressive cell death in some in vitro studies [45, 128]. A study at our institution on the effect of laser induced focal hyperthermia on vascular morphology was performed in a murine model of colorectal liver metastases (unpublished results). After the initial laser induced injury, there was a progressive increase over the course of 24 to 48 h, in the extent of the microvascular damage on scanning electron microscopy of vascular resin casts. The vascular damage preceded tissue injury, based on NADH-diaphorase staining at some time points. In other animals models progressive heatinduced vascular injury also has been demonstrated [103, 120]. In a ank implantation tumor model, radiofrequency hyperthermia ablation resulted in delayed tissue death, with morphological features suggestive of ischemia. These included cytoplasmic endothelial damage and capillary thrombosis at approximately 30 min after heat application [53].
Lysosomal Activity

It has been proposed that increased lysosomal activity, augmented by intratumor acidity may be responsible for progressive cell death. Studies of focal hyperthermia applied to implantable murine mammary tumors demonstrate a progression of cell death 12 h and 24 h after treatment directly related to increased lysosomal activity [129].
Kupffer Cell Activity

There is extensive evidence from in vitro and wholebody hyperthermia experiments that heat-induced inammatory reaction contributes to cell death [53]. However, there is a paucity of data on the effects of such inammatory reaction specically after focal hyperthermia.

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Kupffer cell activity may be the major factor involved in progressive injury after focal hyperthermia destruction of liver tumors through the production of tumoricidal cytokines [130]. Hyperthermia induces the production of IL-1 [131] and tumor necrosis factor- [132] by Kupffer cells, both of which are known to have in vivo antitumor activity [133, 134]. Hyperthermia induced release of these cytokines also is associated with an increase in apoptosis in some cancer cells [117]. Kupffer cell activation also is considered an important regulator of metastases in experimental models [135]. It is shown that Kupffer cells actively phagocytose cancer cells in vivo and impede the progression of metastases [136 140]. They also may have a role in controlling the growth of tumor in the liver after colonization. Inactivation of Kupffer cells is shown to increase tumor growth [135, 141, 142]. Kupffer cells require activation to acquire their tumoricidal functions [137]. There is a slow enhancement of macrophage cytotoxicity and more effective response to lymphokine activation with heating. However, beyond 43C, prolonged hyperthermia may damage macrophages and thus stimulate tumor growth [143146]. Kupffer cells also induce the production of interferon that augments the liver-associated natural killer cell activity [147]. All these described features suggest that Kupffer cells have a major role in focal hyperthermia induced tissue injury [148].
Cytokine Release and Adhesion Molecules

cytoprotective effects [165]. The role of epidermal growth factor and hepatocyte growth factor also has long been recognized to be a major factor in liver regeneration and potential tumor growth [166, 167]. The inuence of focal hyperthermia on the expression of these regenerative growth factors is however limited [164]. Ohono et al. [164] in experiments involving microwave coagulation of liver showed increased proliferation of tumor away from the region of heat application. This was associated with an increase in liver FGF and transforming growth factor levels. In a prospective study of 96 patients treated by ILT for the treatment of unresectable colorectal liver metastases, Christophi et al. [168] reported 4 patients with steady disease who developed rapid acceleration of residual tumor growth and multiple extrahepatic metastases shortly following treatment.
Immune Response

Focal hyperthermia may induce both regional and systemic production of cytokines through activation of inammatory cells. The release of IL-1 and TNF- is well described after whole body hyperthermia [130, 149, 150]. Increased levels of TNF- have direct cytotoxic effects, can induce tumor endothelial injury and sensitize tumor cells to heat induced damage [151 156]. Alterations in expression of adhesion molecules also may inuence the extent of injury achieved by focal hyperthermia and the patterns of tumor spread [151]. Increased expression of intracellular adhesion molecule 1 on vascular endothelium occurs after hyperthermia [157]. Increased ingress of leukocytes to the site of thermal injury and subsequent activation promotes lysis of viable tumor cells [123, 158]. Focal hyperthermia in one study increased the rate of liver regeneration after partial hepatectomy [159]. This may be the result of increased expression of various growth factors [160, 161]. Golovneva et al. [162] identied an increased expression of broblast growth factor (FGF) in the liver peaking at 5 to 10 days after laser-induced focal hyperthermia application. The angiogenic response caused by increased expression of FGF and other growth factors may favor the proliferation of residual tumor cells [162164] and also have

Immunosuppression as the result of direct damage to lymphoid tissue is described with whole-body hyperthermia. When temperatures exceed 42C, there is T-cell and macrophage inhibition, which may stimulate tumor growth [143]. In contrast, whole-body hyperthermia appears to enhance the synergistic and antiproliferative activities of gamma-interferon, leading to an upgrading of immune surveillance [169 174]. However, this effect is reversed at temperatures greater than 42C [175]. Whether some of the changes described in whole-body hyperthermia occur with focal hyperthermia remains unknown. However, there is no conclusive evidence to suggest that focal hyperthermia applied to tumors increase the risk of dissemination [176]. Hyperthermia may modify tumor antigens to produce a host immune response [156]. This immune response may be one mechanism involved in progressive tissue injury after focal hyperthermia application and potentially inuence patterns of tumor recurrences [143, 177]. The effect of immunity on tissue destruction following focal hyperthermia largely depends on the antigenicity of cells in the experimental design. Human liver tumors are generally of low immunogenic potential [178]. Experimental results with frankly allogenic or strongly immunogenic tumors must be interpreted in this context when considering its relevance to the clinical situation [177 183]. Synergistic antitumor effects of immunotherapy combined with hyperthermia are demonstrated by some investigators [184]. It is highly likely that tumor destruction by focal hyperthermia increases expression of cancer antigens and stimulates the immune response. Further studies are needed to investigate the enhancement of such effects. Several studies demonstrate a dual role of thermotolerance and immune

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stimulation of HSPs [90, 185]. Ivarsson et al. [186] used an implantation model of colorectal liver metastases to identify increased expression and change in the localization of HSP 70 at 10 to 15 h after laser ablation. There was also an increase in peri-tumor Kupffer cell numbers, with positive immunostaining for cytoplasmic HSP 70. It was postulated that increased HSPtumor peptide complexes following focal hyperthermia are involved in tumor antigen presentation to macrophages and other antigen presenting cells [186 191]. In some studies T-lymphocyte and macrophage activity is rapidly stimulated by focal hyperthermia [192], followed by a peak in immunoblast and mast cell levels by 20 days after the heat application [193]. The heat evoked antitumor immune response may contribute to the mechanisms of heat induced tissue injury in all tumors to varying extents [143]. Moller et al. [194] using an implantation model of hepatic malignancy in rats found a reduction in intraperitoneal spread of tumors after focal hyperthermia by laser when compared with surgical resection. Isbert et al. [156, 195] showed in a rat model of implantable colorectal adenocarcinoma cells that laser ablation of a tumor in one lobe reduced the growth of an untreated treated tumor in the same animal compared with control and liver resection groups. Tumor volumes in control and surgical resection animals were 4- to 5-fold greater than tumor volumes in laser-treated animals. Peritoneal tumor dissemination was noted in 20% (4/20) of lasertreated animals compared with 65% (4/20) and 85% (17/20) in the control and resection group respectively. In the laser-treated animals, there was signicantly higher expression of costimulatory molecules (B7-2, CD8, MCH-II, LFA-1, ICAM-I) involved in T-cell activation and proliferation 24 to 96 h after therapy compared with control and resection groups. This was indicative of the generation of a cytotoxic immune response. However, the comparability of their ndings to the human situation requires further investigation.
THERAPEUTIC IMPLICATIONS

micro-environment and factors involved in indirect thermal injury are in their infancy.
Thermal Applicators

The type of applicator used to achieve thermal ablation is a major determinant of extent of direct thermal injury [1, 4, 14, 196]. The maximum diameter of necrosis with monopolar radiofrequency electrodes, bare ber laser probes and single microwave antennas does not generally exceed 3 cm in diameter. The use of multiple applicators or multitined expandable electrodes in the case of RF devices allows greater volumes of tissue to be heated and achieves synergistic heating between applicators or tines. Diameters of ablation approaching 6 cm have been described by such methods [1, 4, 14, 196]. Laser application by a single quartz diffusing cylindrical allows greater dispersion of laser light and tissue heating, producing an ablation diameter of approximately 5 cm [197, 198]. Microwave applicators using variable power output have been used to ablate up to 6.5 cm of liver in experimental settings [199]. Watercooled applicators that decrease temperature gradients around the applicator have been adapted to laser and RF systems to minimize tissue charring and maximize tissue necrosis. These systems achieve an ablation diameter of up to 6 cm [1, 4, 14, 196]. Concurrent intratumor deposition of sodium chloride solutions to enhance tissue electrical conductivity has also shown to increase tissue necrosis in experimental RF ablation studies [200, 201].
Blood Flow Manipulation

In a clinical setting, the aim focal hyperthermia therapy is to achieve complete tumor ablation, including a rim of normal tissue. The extent of initial tissue necrosis is predominately determined by the thermal power and energy applied to the tissue before charring. Most reported studies aim at enhancing direct thermal injury by methods that increase tissue temperatures while minimizing charring. The use of different thermal applicators, particularly cooled-tip applicators and blood ow manipulation are currently the most investigated techniques [1]. Thermosensitizing agents, chemotherapy, and radiotherapy in combination with hyperthermia to increase direct injury also have been investigated. At present, investigation and manipulation of cellular and sub-cellular events, the tissue

Blood ow occlusion is shown to increase the area of tissue injury caused by focal hyperthermia signicantly when measured immediately after the application of heat [30, 64, 202204]. The lesion size can be increased by as much as 20% under reduced blood ow conditions [204]. The effects of blood ow occlusion on the progression of tissue necrosis is however unknown. In tumors, the highest blood ow is at its periphery, near the tumorliver interface. The relative reduction in blood ow following portal clamping is greatest at the periphery of tumors [43, 99]. Therefore, the effects of heat-induced injury after blood ow occlusion may be greatest in this region. However, decreased blood ow in some cases leads to signicant temperature rises that can cause charring and limit further tissue destruction [99, 202]. This may be particularly significant when heat is applied directly to center of tumors where blood-ow is most sluggish [99, 205]. The reduction of blood ow can be achieved by clamping of the portal pedicle at laparoscopic or open surgery, selective trans-arterial embolization procedures, or via portal vein occlusion using a balloon catheter [206 209]. In patients with cirrhosis and portal

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hypertension, the magnitude of arterial ow as a potential cooling source may be more pronounced than the portal vein [204, 210]. Experimental studies also have shown that vascular targeting drugs and other pharmacological agents can signicantly reduce blood ow and also enhance the effects of thermotherapy [211214]. Combretastatin A-4 disodium phosphate and its analogues are vascular targeting agents that disrupt tubulin in tumor vessels, reduce blood ow and have been shown to augment focal hyperthermia injury in animal models [212]. The clinical utility of such interventions is however undetermined. The absolute benet of portal clamping for focal hyperthermia treatment of liver tumors is undetermined. A recent study in a porcine model identied increased rates of delayed portal vein, hepatic artery and biliary complications when RF ablation was combined with vascular inow occlusion [215]. There also was no increased cellular destruction. Most of the effects of blood ow occlusion may relate to promoting the rapid attainment of increased local tissue temperatures. There are no reports to date of the effects of blood ow occlusion on tissue necrosis in settings where the applied energy is such that tissue temperatures remain relatively constant. In most settings, blood ow occlusion results in increased tissue temperatures that can be achieved by altering power and exposure settings of the heat source, particularly with the advent of cooled tipped applicators [2].
Thermosensitizers

esthetics also have a potential role [221]. There is also experimental evidence of thermal sensitizing effects of bioavinoids and nonsteroidal anti-inammatory drugs on HSP expression [90]. The true clinical utility of these agents is undetermined [211213].
Chemotherapy and Radiotherapy

The manipulation of the microenvironment of tumors to increase its sensitivity to heat is another potential method for maximizing heat induced injury. Clinically, there is little information regarding the use of thermosenitizing agents to maximize tissue injury. Most of the data are derived from in vitro studies or in experimental animal models. Metronidazole used in vivo has been shown to cause a rise in temperature of 2.5C because of the ability of its nitro compounds to disrupt temperature regulatory mechanisms after whole-body hyperthermia [216]. Its effects in vivo immediately after focal hyperthermia was tested in a murine model of hepatic metastases without a signicant increase in injury [217]. In this study, the effects were determined by histological measurement of tumor necrosis immediately after injury. Some of the effects of metronidazole may be through stimulation of the indirect effects of focal hyperthermia injury, which become fully evident well after the initial injury. There are a number of other agents that have demonstrated increased thermal cytotoxicity [218 220]. Amphotericin B and mercaptoethylamine are two additional agents shown to increase cellular injury during hyperthermia [218]. Membrane targeting local an-

In the clinical situation, the combination of chemotherapy and radiotherapy with hyperthermia is well described. Hyperthermia produces direct injury by damaging the entire cellular machinery, including nucleic acids, cytoskeleton, and cell membranes. Radiotherapy and many chemotherapeutic agents have similar mechanisms of action. There are reports of synergistic effects of regional or whole-body hyperthermia for cancer treatment that include radiotherapy, bleomycin, mitomycin C, Adriamycin, 5-urouricil, cisplatin, and carboplatin [222]. Experimental and clinical evidence shows synergistic effects when RF ablation combined with doxorubicin [223, 224]. In mammary tumor rodent models, RF ablation combined with intratumoural injection of doxorubicin resulted in an almost 2-fold increase in the maximum diameter of injury, compared to RF ablation alone [225]. Similar encouraging results have been observed with intravenous liposomal doxorubicin administered before or within 72 h of RF ablation [223, 226]. In a pilot clinical study of liposomal doxorubicin and RF ablation in patients with liver tumors, there was a median 32% increase in the volume of ablation, compared to controls receiving RF ablation alone [227]. This was based on imaging studies 2 to 4 weeks after therapy. Laser ablation combined with doxorubicinbased transarterial chemoembolization for the treatment of HCC has shown similar improved results [224]. Reduction of thermosensitivity, augmentation of indirect thermal injury, and heat-induced increased in deposition of doxorubicin in RF treated tissue may be responsible for the observed synergistic effects [227].
Others

There is the potential to manipulate the extent of heat induced injury by other more novel means. This may involve the use of agents that augment the mechanisms involved in the progression of injury following focal hyperthermia therapy. The possible mechanisms of progressive indirect tissue injury described earlier in this report include alterations in apoptotic activity, Kupffer cell stimulation, cytokine release, and the immune response. Manipulation of these processes may allow more complete tumor destruction than current techniques. Particular interest may focus on the manufacture of drugs aimed at the molecular chaperone function of HSPs that is involved in tumor-derived antigenic immune response [90]. In addition, combination of hyperthermia with apoptosis inducing chemo-

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therapeutic agents is likely to have synergistic effects, as shown in pilot studies with doxorubicin.
10.

Vallone, P. Radiofrequency ablation of hepatocellular cancer in 110 patients with cirrhosis. Ann. Surg. 232: 381, 2000. Poon, R. T., Fan, S. T., Lo, C. M., Ng, I. O., Liu, C. L., Lam, C. M., and Wong, J. Improving survival results after resection of hepatocellular carcinoma: a prospective study of 377 patients over 10 years. Ann. Surg. 234: 63, 2001. Lau, W. Y., Leung, T. W., Yu, S. C., and Ho, S. K. Percutaneous local ablative therapy for hepatocellular carcinoma: a review and look into the future. Ann. Surg. 237: 171, 2003. Christophi, C., Nikfarjam, M., Malcontenti-Wilson, C., and Muralidharan, V. Long-term survival of patients with unresectable colorectal liver metastases treated by percutaneous interstitial laser thermotherapy. World J. Surg. 28: 987, 2004. Abdalla, E. K., Vauthey, J. N., Ellis, L. M., Ellis, V., Pollock, R., Broglio, K. R., Hess, K., and Curley, S. A. Recurrence and outcomes following hepatic resection, radiofrequency ablation, and combined resection/ablation for colorectal liver metastases. Ann. Surg. 239: 818; discussion 825, 2004. Nikfarjam, M., and Christophi, C. Interstitial laser thermotherapy for liver tumours. Br. J. Surg. 90: 1033, 2003. Siperstein, A. E., and Gitomirski, A. History and technological aspects of radiofrequency thermal ablation. Cancer J. 6: S293, 2000. Parrish, J. A. New concepts in therapeutic photomedicine: photochemistry, optical targeting and the therapeutic window. J. Invest. Dermatol. 77: 45, 1981. Ivarsson, K., Olsrud, J., Sturesson, C., Moller, P. H., Persson, B. R., and Tranberg, K. G. Feedback interstitial diode laser (805 nm) thermotherapy system: ex vivo evaluation and mathematical modeling with one and four-bers. Lasers Surg. Med. 22: 86, 1998. Muralidharan, V., and Christophi, C. Interstitial laser thermotherapy in the treatment of colorectal liver metastases. J. Surg. Oncol. 76: 73, 2001. Jacques, S. L. Laser-tissue interactions. Photochemical, photothermal, and photomechanical. Surg. Clin. North Am. 72: 531, 1992. Izzo, F. Other thermal ablation techniques: microwave and interstitial laser ablation of liver tumors. Ann. Surg. Oncol. 10: 491, 2003. Goldberg, S. N., Charboneau, J. W., Dodd, G. D., 3rd, Dupuy, D. E., Gervais, D. A., Gillams, A. R., Kane, R. A., Lee, F. T. Jr., Livraghi, T., McGahan, J. P., Rhim, H., Silverman, S. G., Solbiati, L., Vogl, T. J., and Wood, B. J. Image-guided tumor ablation: proposal for standardization of terms and reporting criteria. Radiology 228: 335, 2003. Kennedy, J. E., Ter Haar, G. R., and Cranston, D. High intensity focused ultrasound: surgery of the future? Br. J. Radiol. 76: 590, 2003. Nikfarjam, M., Malcontenti-Wilson, C., and Christophi, C. Focal hyperthermia produces progressive tumor necrosis independent of the initial thermal effects. J. Gastrointest. Surg. 9: 410, 2005. Dickson, J. A., and Calderwood, S. K. Temperature range and selective sensitivity of tumors to hyperthermia: a critical review. Ann. N Y Acad. Sci. 335: 180, 1980. Bender, E., and Schramm, T. [Further studies of the problem of different thermosensitivity of tumor and normal cells in vitro and in vivo]. Arch Geschwulstforsch. 32: 215, 1968. Overgaard, K., and Overgaard, J. Investigations on the possibility of a thermic tumour therapy. I. Short-wave treatment of a transplanted isologous mouse mammary carcinoma. Eur. J. Cancer 8: 65, 1972.

CONCLUSIONS

Focal hyperthermia is the most widely used local ablative technique for the treatment of liver tumors. The application of heat to tumor cells is based on the nding that tumor cells are generally more sensitive to a heat than normal tissue. Focal hyperthermia achieves tumor destruction by both direct and indirect thermal effects. The direct effects reect local temperatures increases that cause immediate cell death through interruption of cellular metabolism. There is also an indirect effect that results in an evolving area of necrosis that occurs following the cessation of the heat stimulus. The evolving area of necrosis at the tumor margins may relate to a balance of several interacting mechanisms that include HSP-induced thermotolerance, altered apoptosis, localized cytokine stimulation, growth factor release, and triggering of the immune response. Further elucidation and subsequent pharmacological manipulation of there process may allow more complete destruction of liver tumors, minimize recurrences and improve overall survival outcomes.
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