The cellular and molecular basis of hyperthermia · Critical Reviews in Oncology/Hematology 43...

Critical Reviews in Oncology/Hematology 43 (2002) 33–56

The cellular and molecular basis of hyperthermia

Bert Hildebrandt a,*, Peter Wust b, Olaf Ahlers c, Annette Dieing a, Geetha Sreenivasa b,Thoralf Kerner c, Roland Felix b, Hanno Riess a

a Medical Clinic, Department of Hematology and Oncology, Charite Medical School, Humboldt-Uni!ersity, Campus Virchow Clinic,D-13344 Berlin, Germany

b Department of Radiation Oncology, Charite Medical School, Humboldt-Uni!ersity, Campus Virchow Clinic, D-13344 Berlin, Germanyc Department of Anesthesiology and Intensi!e Care Medicine, Charite Medical School, Humboldt-Uni!ersity, Campus Virchow Clinic,

D-13344 Berlin, Germany

Accepted 3 July 2001

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

2. Basic features of hyperthermic cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372.1. Cytotoxic effect of hyperthermia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372.2. Thermal isoeffect dose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382.3. Hyperthermic cell death in different phases of the cell cycle. . . . . . . . . . . . . . . . . . . . 382.4. Thermotolerance as an antagonist of hyperthermic cell death . . . . . . . . . . . . . . . . . . . 38

3. Special features of in vivo hyperthermia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393.1. Hyperthermia !42°C induces alterations of tumor blood flow and microenvironment . . . . 393.2. Whole-body hyperthermia and von Ardenne’s ‘systemic Cancer Multistep Therapy’ . . . . . . 403.3. Does ‘moderate’ hyperthermia increase tumor blood flow? . . . . . . . . . . . . . . . . . . . . 41

4. Synergistic effect of hyperthermia and radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414.1. Thermal radiosensitization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414.2. Radiation-heat sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

5. Interactions between hyperthermia and drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425.1. Thermal chemosensitization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425.2. Different modes of drug–heat interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435.3. Drug-heat sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435.4. Pharmacokinetics of drugs applied synchronously with hyperthermia . . . . . . . . . . . . . . 43

6. Cellular effectors of hyperthermia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446.1. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446.2. Cell membrane and cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446.3. Cellular proteins and nucleic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446.4. Heat-shock proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

7. Characteristics of hyperthermic cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467.1. Different modes of cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467.2. Hyperthermia-induced apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

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B. Hildebrandt et al. / Critical Re!iews in Oncology/Hematology 43 (2002) 33–5634

7.3. Immunogenic effects of heat-shock proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477.4. Signal transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

8. Hyperthermia-induced changes in cellular immune response . . . . . . . . . . . . . . . . . . . . . . 488.1. Pre-clinical effects of heat on lymphocytes and experimental tumors . . . . . . . . . . . . . . . 488.2. Immunologic changes in cancer patients treated with whole-body hyperthermia . . . . . . . . 48

9. Modulation of drug resistance by hyperthermia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499.1. Reversal of drug resistance induced by hyperthermia. . . . . . . . . . . . . . . . . . . . . . . . 499.2. Thermotolerance is often associated with drug resistance . . . . . . . . . . . . . . . . . . . . . 499.3. Does hyperthermia overcome or induce drug resistance? . . . . . . . . . . . . . . . . . . . . . . 49

10. Summary and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Abstract

In oncology, the term ‘hyperthermia’ refers to the treatment of malignant diseases by administering heat in various ways.Hyperthermia is usually applied as an adjunct to an already established treatment modality (especially radiotherapy andchemotherapy), where tumor temperatures in the range of 40–43 °C are aspired. In several clinical phase-III trials, animprovement of both local control and survival rates have been demonstrated by adding local/regional hyperthermia toradiotherapy in patients with locally advanced or recurrent superficial and pelvic tumors. In addition, interstitial hyperthermia,hyperthermic chemoperfusion, and whole-body hyperthermia (WBH) are under clinical investigation, and some positive compar-ative trials have already been completed. In parallel to clinical research, several aspects of heat action have been examined innumerous pre-clinical studies since the 1970s. However, an unequivocal identification of the mechanisms leading to favorableclinical results of hyperthermia have not yet been identified for various reasons. This manuscript deals with discussions concerningthe direct cytotoxic effect of heat, heat-induced alterations of the tumor microenvironment, synergism of heat in conjunction withradiation and drugs, as well as, the presumed cellular effects of hyperthermia including the expression of heat-shock proteins(HSP), induction and regulation of apoptosis, signal transduction, and modulation of drug resistance by hyperthermia. © 2002Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Hyperthermia-induced; Clinical trials; Radiotherapy; Cytostatic therapy; Thermal radiosensitization; Thermal chemosensitization;Tumor vascularization; Heat-shock proteins; Apoptosis; Drug resistance

1. Introduction

The term ‘hyperthermia’ refers to various tech-niques of heat application administered as an adjunctto already established strategies (especially radiother-apy and chemotherapy) in the treatment of cancerpatients. Regarding the different hyperthermia ap-proaches, therapeutical potentials, expenditure oftreatment, technical problems and evidence of effec-tiveness are diverse. Local/interstitial, and regional hy-perthermia are distinguished from whole-bodyhyperthermia (WBH), and hyperthermic perfusiontechniques (e.g. hyperthermic isolated limb perfusion(HILP), hyperthermic peritoneal perfusion (HPP)). Allhyperthermia modalities have in common that theirefficacy is not enough to replace any one of the estab-lished therapy modalities when applied alone, but, un-doubtedly, they are suitable enough to enhance the

cell-killing effect of cytotoxic drugs and/or radiation(‘thermal chemosensitization’, ‘thermal radiosensitiza-tion’). Therefore, hyperthermia aims at improving theresults of the conventional treatment strategies withinthe framework of multimodal treatment concepts.

One major argument for utilizing local and regionalhyperthermia, results from the convincing clinicalresults obtained from trials on patients with locallyad-vanced malignancies. Improved response and sur-vival rates were observed in patients treated with hy-perthermia and radiotherapy compared withradiotherapy alone in several comparative phase-IIItrials, with a very distinct response benefit in the hy-perthermia group in some of these studies (Table 1,[1–19]).

In spite of the inhomogeneous temperatures ac-quired (due to inhomogenous temperature depositionand physiological reasons), locoregional techniques of

B. Hildebrandt et al. / Critical Re!iews in Oncology/Hematology 43 (2002) 33–56 35

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Table 2Interactions between heat and drugs

PharmocodynamicsAcceleration of primary mode of action (alkylating reaction,

protein damage, oxygen-radicals; DNA-strand breaks)Increased intracellular drug concentration (drug uptake,

membrane damage, protein damage, pH changes)

PharmacokineticsDrug uptake (decreased gastrointestinal or transdermal

absorption)Distribution

(pH changes, fluid sequestration, increased tumor blood flow)Metabolism and excretion

(changes in hepatic and renal blood flow)

Table 4Molecular effectors of hyperthermia

Cell membrane, cytoskeletonChanges in fluidity/stability of cell membraneChanges in cell shapeImpaired transmembranal transportChanges in membrane potentialModulation of transmembranal efflux pumps (MDR)Apoptosis induction

Intracellular proteinsImpairment of protein synthesisProtein denaturationAggregation of proteins at the nuclear matrixInduction of HSP-synthesis

Nucleic acidsImpairment of RNA/DNA synthesisInhibition of repair enzymesAltered DNA conformation

Other alterations of cell functionIntracellular metabolism of other substratesGene expression, signal transduction

hyperthermia revealed a significant correlation betweenthermal dose and clinical outcome in these trials. Thisalso holds true for further investigations, where localand regional hyperthermia were combined withchemotherapy and radio-chemotherapy in the scope ofphase-II trials. These findings strongly suggest, on prin-ciple, the clinical effectiveness of hyperthermia [20–25].

Contrarily to hyperthermic radiotherapy, only fewcomparative trials have been completed to date wherehyperthermia was applied as an adjunct to chemother-apy [16–19]. All those trials refer to the comparison ofsurgery alone with surgery followed by adjuvant hyper-thermic chemoperfusion, applied either as HILP withmelphalan in patients with melanomas, or as HPP withmitomycin in patients with gastric cancer. A benefit forhyperthermic chemoperfusion was observed in two ofthese ‘adjuvant’ trials (Table 1). Regarding HILP in-duction therapy in patients with melanomas or sarco-mas limited to one limb, it is remarkable that nophase-III trials have been performed yet, due to thevery high response rates have been observed in some

non-randomized series (reviewed in [26,27]; Tables 2–4).

Regarding regional hyperthermia of the pelvis andextremities in conjunction with chemotherapy, encour-aging results of phase-II trials gave raise to the initia-tion of various comparative trials during the last years.Special attention is attracted to one multicenterEORTC/ESHO-trial subjecting the evaluation ofneoadjuvant chemotherapy #RHT in high-risk softtissue sarcomas of the limb or pelvis followed bysurgery, adjuvant radiotherapy, and subsequentchemotherapy again (#RHT). Further ongoing phase-III trials are addressing the effect of chemotherapy withCisplatin #RHT in pre-irradiated patients with recur-rent cervical cancer, as well as, preoperative neoadju-

Table 3Thermal enhancement ratios (TER) of selected drugs in various animal tumors

TER atTER at Tumor entity AuthorsDrug42.5–44 °C40–42 °C

(range) (range)

Honess, 1982; Monge, 1988; Hazen, 1981;1.52–2.28 1.27–2.74 RIF-1, Mammary-Ca, Fsa-II,CyclophosphamideUrano, 1985Lewis lung-Ca

1.5–2.96 2.71 RIF-1, KHT, Fsa-II Honess, 1982; Honess, 1985; Urano, 1991BCNUHoness, 1985; Urano, 1995RIF-1, KHT, Fsa-IIMelphalan 1.5–3.9 n.d.Mella, 1985; Douple, 1982; Herman, 1988;Cisplatin BT4A, Mammary-Ca, Lewis lung-Ca,1.39–4.961.48–3.9Nishimura, 1990; Lindegaard, 1992; vanSCC VII, R1-RMSBree, 1996

Bleomycin 1.24 1.65–2.90 Adeno-Ca 284, SCC, Fsa-II von Sazazepauski, 1981; Hassanzadeh, 1982;Urano, 1990

1.0 2.8Mitomycin C Mammary-Ca, Fsa-I1 Monge, 1989; Urano, 19945-Fluorouracil 1.0 1.0 Human leukemia, Colon-Ca, Fsa-II Mini, 1986; Rose, 1979; Urano, 1991

1.0 Mammary-Ca, Fsa-IIDoxorubicin 1.0 Monge, 1988; Urano, 1994

Data given as calculated by Urano et al., 1999; [27].


vant radiochemotherapy #RHT in patients with pri-mary non-metastatic, locally advanced rectal cancer[28–30].

Available data on WBH in conjunction withchemotherapy merely demonstrate feasibility and ac-ceptable toxicity of this approach when radiant heatapplicators are employed to induce homogenous body-care temperatures up to 42 °C for 1 h (Fig. 1). Theapplication of WBH is relatively invasive and accompa-nied with a broader spectrum of toxic effects than localand regional hyperthermia. On the other hand, WBH ispart of a systemic therapy with the potential to treatmetastatic disease. This is the reason for the commence-ment of the first phase-III trials in Germany on WBHwith adjunctive chemotherapy in patients withmetastatic cancer [31–34].

Recent reviews on clinical hyperthermia are given in[27,28,35].

Parallel to these encouraging clinical results, a largenumber of pre-clinical investigations have been per-formed on various aspects of heat effects since theearly-70s. In vitro and animal hyperthermic experi-ments exhibited a direct cell killing effect at tempera-tures ranging from 41 to 47 °C [27,36,37]. Furtherstudies have revealed a large variability of hyperthermiaeffects regarding cell death (especially with respect totumor entity, cell line, growth fraction). In general,hyperthermic cell death has been shown to be markedlyenhanced at temperatures above 43 °C, as well as, incombination with radiation and various cytostaticdrugs by sensitization. More recent publications havefocused on the effect of hyperthermia on distinct cellu-lar signalling pathways, particularly of those involvedin ‘heat shock response’, cell cycle regulation, and

apoptosis. Furthermore, hyperthermia influences tumorblood flow, oxygen and nutrient supply, as well as, thecellular immune response under in vivo conditions [38–46].

Despite a large number of publications on the pre-clinical aspects of hyperthermia, little certainty existsabout the extent of transferring these results into clini-cal practice. This is mostly due to difficulties in achiev-ing a direct correlation between clinical and moleculareffects for practical and ethical reasons (as discussed in[47,48]). Here, we outline the major topics on thecellular and molecular targets of hyperthermia. How-ever, since a medline-search for the term ‘hyperthermia,induced’ produces a total number of 11 233 citations(March 2001), we cannot give a complete summary ofthe facts here.

2. Basic features of hyperthermic cell death

2.1. Cytotoxic effect of hyperthermia

When exponentially growing cultured cells (e.g. Chi-nese hamster ovarian (CHO) cells) are exposed to adefined temperature between 41 and 47 °C, a dose–ef-fect curve can be defined by plotting the rate of celldeath against the duration of hyperthermia. The corre-sponding survival curves show a typical ‘shoulder’ thatreflects a two-step process of cell killing. This is markedby a linear growth arrest in the beginning of heatexposure (reflecting a reversible, non-lethal heat dam-age), that is followed by exponential cell death (Fig. 2).One fundamental observation is, that the capability toinduce cell death at lower temperatures $42–43 °C

Fig. 1. Examples for commercially available hyperthermia devices. ‘IRATHERM®-2000’ radiant heat applicator (WBH) and ‘BSD®-2000’ regionalhyperthermia system.


Fig. 2. Dose–response relationship of thermal cell killing. Schematicdrawing of the surviving fraction of asynchronous CHO cells heatedat different temperatures, emphasizing the typical ‘shoulder’ ([40,205],with permission).

Calculation of the TID is using the fact that—as itknown from cell death curves—a temperature declineof 1 °C in the temperature range between 42.5 and47 °C can be compensated by doubling the expositiontime (R= 2, see above), whereas below 42.5 °C theheat exposure has to be prolonged even more (R= 4,see above). Although the TID is influenced by a num-ber of environmental factors in clinical hyperthermia(such as the time-behavior of temperature during theheating phase, temperature distribution, thermal toler-ance, intracellular pH and other milieu factors), itseems to be a valuable approach to describe the effec-tiveness of a clinical heat treatment. This has beendemonstrated in various trials on local and regionalhyperthermia, where significant correlations betweenEM43 and response parameters have been observed[21,22,24,49,52,53].

2.3. Hyperthermic cell death in different phases of thecell cycle

Synchronized cell cultures exhibit variations in theirsusceptibility to heat in accordance to their phase in thecell cycle. In general, highest heat sensitivity can beobserved during the mitotic phase. Microscopic exami-nations of M-phase cells subjected to hyperthermiashow damage of their mitotic apparatus leading toinefficient mitosis and consecutive polyploidy. S-phasecells are also sensitive to hyperthermia, where chromo-somal damage is observed. Both S- and M-phase cellsundergo a ‘slow mode of cell death’ after hyperthermia,whereas those exposed to heat during G1-phase arerelatively heat resistant and do not show any micro-scopic damage. Cells during G1-phase may follow a‘rapid mode of death’ immediately after hyperthermia.These variations existing between the different cell cyclephases indicate the possible diversity of molecularmechanisms of cell death following hyperthermia[46,54–56].

2.4. Thermotolerance as an antagonist of hyperthermiccell death

Malignant cells exposed to temperatures $43 °C orcooled down to 37 °C between two heat shock treat-ments !43 °C, show an impairment in their suscepti-bity to heat-induced cytotoxicity that results in aflattened inactivation curve (Fig. 4). This phenomenon,the so called ‘thermotolerance’, is principally reversible.Thermotolerance of multifactorial origin as it is notinherited in cell cultures. It is at least partially based onthe induction of heat-shock proteins (HSP) and otherpost-transitional adaptation processes (e.g. cell cyclearrest in the G2-phase, changes in cell metabolism). Theability to express thermal tolerance might be attenuatedunder some environmental conditions such as (sud-

(below a certain ‘breakpoint’), is markedly lower thanabove 43 °C. Therefore, one common definition of thethermal dose (D) derived from exposure time (t) andgiven temperature (T), is D= tRT= 43 with R= 2 fortemperatures "43 °C, and R= 4 for temperatures$43 °C, or, in the case of varying T, a summation ofdose fractions and duration (t+T).

The thermal dose required to induce hyperthermiccell death varies with factor 10 between different celltypes (Fig. 3). However, there is a particular thermaldose variation in the survival curves where the transi-tion into the exponential phase starts (‘activation en-ergy’). The thermal energy dose required to induceexponential cell death is closely correlated to that re-quired for cellular protein denaturation and amounts to140 kcal/mol in vitro, as well as, in experimental tu-mors. This led to the hypothesis that the cytotoxiceffect of hyperthermia is mainly based on denaturationof cytoplasmatic and membrane proteins, although cor-responding relationships have not been elaborated forsensitizing phenomena with respect to radiation and/orcytostatic drugs [40,46,49–52].

2.2. Thermal isoeffect dose

Calculation of the thermal dose applied in hyperther-mia, as it can be derived from the above-mentionedformula, has also been integrated into the concept ofthe ‘thermal isoeffect dose’ (TID) that is commonlyused in clinical practice in order to compare differenthyperthermia exposures with each other. The TID wasintroduced to convert a given thermal dose into so-called ‘equivalent heating minutes at 43 °C’ (EM43).


denly) lowered intracellular pH, and may also occurtogether with some forms of acquired or inherited drugresistance (see below) [57–61].

3. Special features of in vivo hyperthermia

3.1. Hyperthermia !42 °C induces alterations of tumorblood flow and microen!ironment

The microenvironment of malignant tumors is char-acterized by a reduction of blood flow and blood vessel

density that favors hypoxia, acidosis and energy depri-vation. Hyperthermia at temperatures above 42 °C,besides its cytotoxic effect, has been shown to decreasetumor blood flow in a number of fundamental studiesin the 70s and 80s, thereby impairing oxygen andnutrient supply, and inducing acidosis (although someexceptions have been reported). The thermal dose re-quired to alter tumor blood supply varies betweenindividual tumors and different tumor types. It seemsto mostly depend on the percentage of responsive ves-sels that have maintained their ability of thermal regu-lation. In addition, heat induced damage of tumor

Fig. 3. Differences of the dose–response relationship in different cell lines. Survival curves of two different colon carcinoma cell lines indicatedifferences in susceptibility to heat ([206], with permission).


Fig. 4. Thermotolerance. Surviving fraction of CHO-cells at 43 °Cthat were preheated at 40 °C for various periods of time ([207], withpermission).

swelling, shift of plasma fluid into the interstitium,microthrombosis due to activation of hemostasis, andchanges of the viscosity of blood cell membranes. All ofthese factors also promote a reduction of oxygen andnutrient supply, as well as, intratumoral acidosis [62–68] (Fig. 5).

3.2. Whole-body hyperthermia and !on Ardenne’s‘systemic Cancer Multistep Therapy’

Decrease of tumor blood flow in experimental tumorsat temperatures !42 °C may be further enhanced byreactive hyperaemia of surrounding (healthy) tissuesthat have maintained their ability of thermoregulation.In animal models, this ‘steal-phenomena’ can be inten-sified by additional application of vasodilatating agents,or by inducing intratumoral lactic acidosis with highly-concentrated glucose-infusion [63,65,69–73].

The idea that hyperthermia might be able to starveout the tumor represents the basis for the concept ofsCMT. sCMT has been concipated and propagated as apre-clinical construct by M. von Ardenne in Germanysince the late 1960s, consisting of WBH, induced hyper-glycemia, and hyperoxaemia. Here, WBH is thought toinduce a decrease in tumor blood and nutrition supplythat is further enhanced by (hyperglycaemia-induced)intratumoral lactate acidosis and the above-mentionedvascular steal-effect. In parallel, hyperoxemia was in-duced to ensure oxygenation of healthy tissues. It hasalso been hypothesized that sCMT sensitizes tumorcells for cytostatic therapy and radiation. Even if thesCMT-theory has never been proven, it gained world-wide interest in the early-1970s.

vasculature may occur at temperatures which may alter,but not damage those of normal tissue. In some cases,hyperthermia-induced changes of microcirculation werereported to be irreversible, and in others a breakdownof circulation even occurred after termination of heatexposure. Remarkably, the considerable inhomogenic-ity of blood supply within the same tumor persists afterheat treatment !42 °C. The morphological changesassociated with hyperthermia include endothelial

Fig. 5. Possible mechanisms involved in hyperthermia-induced breakdown of blood flow ([65], with permission).


The lack of systematic clinical trials on sCMT orWBH alone is mostly due to the fact that no appropri-ate devices were available up to the mid-1980s. Allpreviously used WBH-applicators have been either noteffective enough to induce body core temperatures of41.8–42 °C, or treatment was too toxic. Today, at leasttwo applicators are available at which temperatures inthe range of 41.8–42.2 °C can be induced and main-tained for 1 h by using radiant heat with acceptabletoxicity. This enables the evaluation of WBH in con-junction with cytostatic therapy in larger clinical trialsin the future. Interestingly, all recent WBH-investiga-tors—most of them disapproving the concept ofsCMT—generally recommend the application of hyper-glycaemia and hyperoxaemia during therapy. Anyway,this recommendation is not based on the assumedantineoplastic properties of these applications, but onthe consideration that supportive oxygen and nutrientsupply may be helpful to prevent severe organ toxicity[34,74–79].

3.3. Does ‘moderate’ hyperthermia increase tumorblood flow?

Contrary to those earlier studies on microenviron-mental changes in tumors treated with hyperthermia!42.0 °C, ‘moderate’ hyperthermia ($42.0 °C)—that is much more easy to apply in vivo and offers abetter comparison to the clinical situation—has beendemonstrated to rather improve tumor blood flow andthereby oxygen content. It, thereby, may increase theeffectiveness of radiotherapy (which is more effective intumors with higher oxygen supply) and chemotherapy(the delivery of which may be favored by increasedtumor blood flow). Indeed, this hypothesis would give agood explanation for the correlation of applied thermaldose and clinical outcome, as was observed in clinicaltrials on local/regional hyperthermia where intratu-moral temperatures do not exceed 42 °C during mostof the treatment time. However, the situation in cancerpatients appears to be much more complex than inexperimental systems, and behavior of tumor vascular-ity under hyperthermic conditions may also depend onthe method of hyperthermia applied. Provided the clin-ical effectiveness of certain hyperthermia applications isactually based on the breakdown of tumor vasculariza-tion, it may be useful to combine this effect with otherantiangionetic agents in the future (some of which havebeen recently introduced into anticancer therapy). If,on the other hand, hyperthermia acts by increasingtumor blood supply, one possible consequence could beto combine hyperthermia with antineoplastic drugs (#radiation) in tumors with low blood supply that can notbe treated adequately by conventional modalities (e.g.local recurrences of pre-irradiated cervical, rectal, orbreast cancer). At least, there are a number uncertain-

Fig. 6. Thermal radiosensitization. The effect of heating at 42 °C onthe thermosensitivity of V 79 cells. Heating was completed 10 minbefore acute X-irradiation ([208], with permission).

ties concerning the effect on hyperthermia on bloodflow, and this is why further investigations are desirable[62,66,80,81].

4. Synergistic effect of hyperthermia and radiation

4.1. Thermal radiosensitization

One of the most important observations from in vitrostudies on heat action was that hyperthermia and radia-tion act in a synergistic way. This synergism induces anincrease in cell killing even at lower temperatures,which is not the case when hyperthermia is imple-mented alone. This so-called ‘thermal radiosensitiza-tion’ results in a reduction of the shoulder of thedose–effect curve (Fig. 6). It appears most pronouncedin S-phase cells that are usually resistant to radiationalone. The extent of thermal radiosensitization can bequantified by the quotient of survival fraction of cellstreated with radiation alone and those treated withradiation and heat (‘thermal enhancement ratio’, TER)[36,40,42,46,52,82–84].


4.2. Radiation-heat sequence

The extent of synergism between heat and radiationdepends on the temperature applied, time interval be-tween heat and radiation, and treatment sequence. It ismost distinct when both modalities are applied syn-chronously. In vitro, a supraadditive effect of heat andradiation may occur for 8 h or longer when CHO-cellsare heated at !43 °C before radiation. Using an in-verse sequence, a similar effect is often observed for ashorter period of time (2–4 h), but also appears attemperatures below 42 °C. A synergistic effect of heatand radiation can also be observed in thermotolerantcells when single radiation doses of 2–4 Gy are applied,but this effect may be altered depending upon the celltype and degree of tolerance. Hypoxic cells, as well as,those with impaired nutrient supply and/or acidic pHhave been shown to react very sensitively to the com-bined treatment of heat and radiation [36,41,46,82,83,85] (Fig. 7).

Synchronous application of heat and radiotherapy isnot possible in clinical practice yet, even if respectivedevices have been designed and are under evaluation.As clear instructions on the optimal radiation-heatsequence can not be derived from experimental data,the general recommendation and practice today is dueto this reason, where heat and radiation are to beapplied within a short period of time. Some investiga-tors prefer a time interval of 2–4 h between radiationand heat to increase the therapeutic ratio. Others applyheat prior to irradiation as it may be advantageous dueto the prevention of vascular stasis, which probably

promotes radioresistance. Another aspect is that someradiotherapy fractions before the first course of hyper-thermia may be beneficial for radiobiological reasons.However, all of these strategies are not proven byclinical data and, at least, the optimal radiation-heatsequence in clinical hyperthermia remains to beclarified.

5. Interactions between hyperthermia and drugs

5.1. Thermal chemosensitization

Analogous to thermal radiosensitization, hyperther-mia also enhances the cytotoxicity of various antineo-plastic agents (‘thermal chemosensitization’).Additional application of selected chemotherapeuticdrugs has been shown to enhance the inhibition ofclonogenic cell growth at elevated temperatures both invitro and in animal experiments. The extent of a drug’s‘thermal chemosensitization’ can also be expressed bythe TER that basically is the ratio of cell survival at theelevated temperature to normal temperature for a cer-tain drug level. It mainly represents the pharmacody-namic features of drug–heat interaction (e.g. changesof the kinetics of the primary mode of drug action), butfurther attention should be attracted to clinical phar-macokinetics and modulation of side effects. At hyper-thermia, additional interactions between drugs andhyperthermia have been described. For example assome drugs lose their chemical stability at higher tem-peratures, or get impaired at contact with glass or

Fig. 7. Radiation–heat sequence. Effect of treatment intervals and sequences for combined radiation and hyperthermia—hyperthermia at43 °C/60 min is applied before, respectively, after 10 Gy radiation in growing rat cartilage ([209,210], with permission).


Fig. 8. Thermal chemosensitization. Survival fractions of FsaII cellstreated with a fixed dose of cisplatin, heated at various temperatures([211] with permission).

transferred to clinical hyperthermia. In addition, differ-ent animal tumors vary in their susceptibility to acertain cytostatic agent (at room temperature, respec-tively, 37 °C as well as at elevated temperatures).Moreover, it is known fact that drugs most effective atnormal temperature do not necessarily have a highersensitization capacity at higher temperatures. Further,the extent of thermal chemosensitization of a drug in agiven experimental system varies among differentmodes of drug administration and dosage. Please notethat certain modes of heating may promote thermotol-erance and/or drug resistance, and that most pharma-cokinetic aspects cannot be simulated in animal models.At least, the lack of demonstrating thermal chemosensi-tization may be due to an inadequate interval betweendrug administration and heat exposure for variousagents (see below) [27,86–88].

5.3. Drug-heat sequence

Available data suggest that thermal chemosensitiza-tion can be yielded best by synchronous application oradministration within a short interval for most drugs,but there might be some exceptions. For example, theoxacephasporines cyclophosphamide and ifosfamideundergo an extensive hepatic metabolism, and, there-fore, should best be applied some hours before hyper-thermia. In opposite, favorable clinical results have alsobeen achieved by applying regional hyperthermia inconjunction with ifosfamide within a shorter interval oftime [21,27,90]. Another example is the antimetabolitegemcitabine, where a time interval of ca. 24 h betweendrug application and heat was necessary to achieve asynergistic effect in vitro and in a rat model. A correla-tion with clinical data has not been drawn yet [91]. Onthe contrary, it was shown that simultaneous applica-tion of etoposide (VP-16) and heat leads to a decreaseof cytostatic activity in vitro, whereas application ofthis drug led to persuading results when applied incombination with other agents and regional as well asWBH [92–95]. As a conclusion, further pharmacologi-cal studies are necessary to optimize the application ofcytostatic agents in conjunction with different hyper-thermia approaches in clinical practice. Continuing pre-clinical research may have setbacks regardingimportant factors concerning the interaction of heatand drugs, and will be helpful in the evaluation of noveldrugs for hyperthermic chemotherapy.

5.4. Pharmacokinetics of drugs applied synchronouslywith hyperthermia

As already discussed above, ‘thermal chemosensitiza-tion’ preferentially reflects pharmakodynamic aspects ofdrug action during hyperthermia, but does not considerthe assumed complex changes in drug pharmakokinet-

plastic. It has also to be considered that solventsor additives may interact with heat and thus mayinduce either thermotolerance or thermosensitization[39,86–88] (Fig. 8).

5.2. Different modes of drug–heat interaction

The interaction of heat with chemotherapeutic drugshas been classified by terms like ‘additive’ or ‘superad-ditive’ (linear increase with increasing temperature),‘threshold-behavior’ (little or no increase of cytotoxicityat lower temperatures, marked increase above a distinctthreshold temperature), or ‘independent’ (no depen-dency at all) [39,88]. It is generally accepted that mostalkylating agents (e.g. cyclophosphamide and ifos-famide) and the platinum compounds, are linearly en-hanced in their cytotoxic effect when temperatures areraised from 37 to above 40.5 °C. Conversely, doxoru-bicin has a temperature threshold, whilst most an-timetabolites (e.g. 5-fluorouracil) as well as vincaalkaloids and taxanes show no dependency to hyper-thermia. A further group of drugs, the so called ‘ther-mosensitizers’, act in a cytotoxic way only at elevatedtemperatures. Some well-known drugs like the localanesthetic drug lidocaine or the antifungal ampho-tericin B have been demonstrated to act as thermosensi-tizers [39,86–89].

While reviewing the published data on heat–druginteractions, attention is attracted to the controversialdiscussions about the findings on the mode of interac-tion of various drugs and heat, mainly for the followingreasons. For example, data on thermal chemosensitiza-tion often refer to in vitro studies in which tempera-tures !43 °C were applied, and thus cannot be


ics under hyperthermic conditions. Unfortunately, dataavailable on this topic are not very detailed so that thebasis of cytostatic therapy under hyperthermic condi-tions is still poorly understood. In general, one wouldexpect that changes in tumor blood supply occur whichaffect the distribution of cytostatic drugs in the neoplastictissue. It is also presumable that changes in fluid andelectrolyte balance as well as pH-changes may result inchanges of solubility and volume distribution of drugs.By influencing the effective dosage of a drug this couldbe applied, especially, for WBH. Gastric hyperchlorhy-dria and/or gastrointestinal fluid sequestration may rep-resent further sources of distribution problems, but atleast antineoplastic drugs will be mostly administeredintravenously during hyperthermia. At least, hepatic andrenal metabolism and excretion may show relevantchanges under various hyperthermic treatment modali-ties [96].

In a clinical phase-I study on WBH, a slight decreasein the renal elimination of carboplatin (CBCDA) wasdetected, and it was argued that this might be the reasonfor an increase in nephrotoxicity of CBCDA whilecombined with hyperthermia [97,98]. In another investi-gation on CBCDA—pharmacology in WBH, occurrenceof excess nephrotoxicity was mainly due to the use of anextracorporeal hemodialysis—system to induce WBH, amethod that is thought to produce a relevant rate ofnephrotoxicity by itself [97,99]. In regional hyperthermiaof the pelvis, a trend towards a higher peritoneal clear-ance after interperitoneal carboplatin application wasdetected in patients with ovarian cancer [100].

Altogether, only very few studies on clinical drugpharmacology under hyperthermic conditions have beenpublished. Data suggest that at least long lasting systemicheat exposure of larger parts of the body, e.g. in WBH,may influence especially the pharmacokinetics of syn-chronously administered cytotoxic drugs, due to changesin the organ circulation (e.g. of the liver or kidney), totemperature-depended metabolism rates, or to fluidshifts. In general, drug–heat interactions in cancer pa-tients appear to be much more dependant on environ-mental factors (e.g. blood supply, fluid balance orpH-value) than those of radiation and heat. As thosefactors usually cannot be simulated by pre-clinical exper-iments in a convincing manner, corresponding data ondrug–heat interaction and thermal chemosensitizationshould be interpreted with great caution. Further con-comitant research in the scope of clinical trials is re-quired.

6. Cellular effectors of hyperthermia

6.1. History

A large number of investigations concerning the cellu-

lar effects of hyperthermia exist, dating back to the 1970sand early-80s. Here, different aspects of hyperthermicaction were described that were conducted with theresearch techniques available at that time. As the interestin basic research of hyperthermia decreased markedlysince the mid-80s, information is insufficient to translateall the described phenomena into our more recentunderstanding of biological changes, which are domi-nated by molecular biological and genetical aspects.

6.2. Cell membrane and cytoskeleton

Hyperthermia affects fluidity and stability of cellularmembranes and impedes the function of transmembranaltransport proteins and cell surface receptors in vitro. Anincreased fluidity of cell membranes was observed inthermosensitive, but not thermotolerant cells. Suggestingthat membrane alterations represent an important targetin hyperthermic cell death [101–105], these observationgave raise to numerous reports on changes in membranepotential, elevated intracellular sodium and calciumcontent, as well as an elevation of potassium-efflux underhyperthermia. However, none of these phenomenasseems to correlate with the rate of cell death in vitro, evenif one may assume an effect of hyperthermia ontransmembrane transport of ions (Na+/H+ —respec-tively, HCO3

−/Cl−ATP— transport) and intracellularpH from these data [60,101,106–111].

Besides, hyperthermia has been demonstrated to in-duce various changes of cytoskeletal organization (cellshape, mitotic apparatus, intracytoplasmatic membranessuch as endoplasmatic reticulum and lysosomes), butagain there was no clear correlation found between thesephenomenological changes and thermosensitivity of var-ious cell lines [54,88,112–114]. In this context, theappearance of membrane blebbing in cultured cellsexposed to heat was described first, and it was noted thatcells that exhibited this phenomenon underwent celldeath after a single heat dose [115]. From a more recentpoint of view, membrane blebbing does not represent aprimary damage of the cell membrane, but is a typicalfeature of programmed cell death (apoptosis). Untiltoday, various authors have demonstrated that hyper-thermia is able to induce apoptosis in in vitro and inanimal experiments (see below).

6.3. Cellular proteins and nucleic acids

Intracellular de novo synthesis and polymerization ofboth RNA- and DNA-molecules during protein synthe-sis is decreased in vitro at temperatures between 42 and45 °C in a dose-dependant manner. Whereas RNA- andprotein-synthesis recover rapidly after termination ofheat exposure, DNA-synthesis is inhibited for a longerperiod [64,88,116].

Heat shock induces an aggregation of denaturated


proteins at the nuclear matrix. This is mainly due toinsolubility of cellular proteins after heat-inducedprotein unfolding, entailing an enhancement of thenuclear protein concentration. Recently, elevated bind-ing affinity and redistribution towards nuclear struc-tures has been described for more than 100 differentcellular proteins, including the so called heat shockproteins (see below) [64,88,116].

Increasal of the nuclear protein content by heatconsecutively affects several molecular functions (in-cluding DNA-synthesis and -repair) when a certainthermal dose is exceeded. This threshold dose is diversebetween distinct cell lines. HeLa-cells that enter theS-phase of the cell cycle at 41.5 °C despite an impair-ment of nuclear enzymes, consecutively undergo M-phase death after completion of DNA-synthesis. On theother hand, CHO-cells that exhibit a markedly im-paired DNA-replication at the same conditions experi-ence G1-arrest, and become thermotolerant. Both thethreshold character and the distinct susceptibility toheat between different cell lines can be explained bestby differences in recovery from heat shock, and not bythe extend of heat-induced cell damage itself [117–121].

In the 1960s, hyperthermia was supposed to actsimilar as radiation by inducing direct DNA-damageand double-strand breaks. Later, it became evident thatheat is not able to cause severe DNA-damage by itself,but instead hinders the repair of radiation-induced,sublethal cell damage, and thus boosts radiation-in-duced DNA-fragmentation. From a recent point ofview, this may be caused by a temperature-dependantinhibition of DNA-repair enzymes. Indeed, an inhibi-tion of the DNA-polymerases-! and -" by hyperther-mia has been demonstrated. Similarly, it was assumedthat hyperthermia may specifically inhibit the enzymesof the ‘mismatch repair’ or the ‘nuclear excision repair’-system, but this has not been proven yet. However,even if a temperature-dependent inhibition of DNA-re-pair enzymes would be an intriguing explanation, animpairment of DNA-enzymes may be caused by unspe-cific protein inactivation or interaction at nuclear struc-tures as well [122–127].

6.4. Heat-shock proteins

Whereas synthesis of most cellular proteins is im-paired under hyperthermic conditions, this cannot be

applied for the so called heat shock proteins. Heatshock proteins represent a heterogeneous group ofmolecular chaperones consisting of at least five sub-groups with different molecular mass and partiallyvarying biologic function. HSP are usually divided intosmall HSP (molecular mass $40 kDa), and the HSP60, HSP 70, HSP 90 and HSP 100 proteins families. AllHSP families share their chaperoning function, i.e. theyunselectively bind to hydrophobic protein sequencesliberated by denaturation. Thus they prevent irre-versible interaction with neighbor proteins (e.g. at thenuclear matrix, see above) that would result in a loss offunction. However, elevated expression and chaperon-ing function of HSPs are not restricted to elevatedtemperatures. They can also be observed under variousother stress conditions, and some HSPs even carry outsimilar functions during regular protein synthesis aslong as some amino acids have not developed complexstructures. HSP-synthesis can be induced within min-utes by activation of so called ‘heat shock factors’(HSF). Those HSFs rapidly bind to and activate thepromoter regions of various heat shock genes aftertrimerization, in particular those involved in the synthe-sis of HSP 70. Today, at least HSP 27 and HSP 70 aresupposed to represent ‘general survival proteins’ thatare able to defend cells against a variety of potentiallylethal (proapoptotic) stimuli [128–131] (Figs. 9–11).

In hyperthermia, HSPs are thought to be involved inthe protection of cells against heat damage. The con-nections between HSPs and thermotolerance on theone, and the interesting immunogenic features of theHSP on the other hand are discussed in a separatechapters of this article.

Intracellular HSP-synthesis increases when culturedcells are exposed to moderate heat application. Athigher temperatures, inhibition of HSP-synthesis occursabove a distinct threshold temperature. In general, thetemperature, respectively, thermal dose at which HSP-synthesis is inhibited in a given experimental systemvaries between different cell types, but the respectivethreshold can be lowered when further (proapoptotic)stimuli are added. As lack of HSP-synthesis is associ-ated with exponential cell death, it is generally acceptedthat HSPs prevent cells from lethal thermal damage.Indeed, an increase of apoptosis can also be observedwhen HSPs are inactivated at normothermia (e.g. byantisense technology). Vice versa, induction of apopto-

Fig. 9. Schematic illustration of heat shock protein 70 and its different epitopes ([150] with permission).


Fig. 10. Different ways by which heat shock proteins may act asinhibitors of cell death ([130] with permission).

7. Characteristics of hyperthermic cell death

7.1. Different modes of cell death

Today it seems to be proven that the antineoplasticproperties of drugs and radiation are mainly based ontheir ability to (directly or in an indirect way) induceeither apoptotic (synonym programmed) or necrotic celldeath. Whereas necrosis is marked by a passive patho-logical cell damage followed by an inflammatory re-sponse originating from the surrounding tissue,apoptosis represents a genetically controlled, activedeath program. It may be activated by cell damage orphysiologically, hereby contributing to maintain tissuehemostasis as well as preventing severe cell damage thatmay lead to sustained viral infection or cancer. On theother hand, excess of programmed cell death may leadto impaired development and degenerative disease in agiven tissue or organism. Interestingly, most of thestimuli activating apoptosis are also able to inducenecrosis at prolonged or intensified exposure [136].

In apoptosis, potential lethal stimuli like cytotoxicdrugs, radiation, viruses, or starvation, activate cas-cades of specific cysteine proteases, the so-called cas-pases, in different ways. Each of these signalingpathways can be boosted in the mitochondriae. Regula-tion of programmed cell death is controlled through theexpression of a number of genes with activating (e.g.bax gene family) or inhibiting (e.g. bcl gene-family,p53) properties. As genetic alteration of these geneswere found to contribute to malignant transformationand disease progression in various hematological andsolid malignancies, basic research on apoptosis waspropelled during the last decade [130,136–140].

7.2. Hyperthermia-induced apoptosis

Hyperthermia is suitable to induce both necrotic andprogrammed cell death in vitro in a temperature-de-pending manner [141]. The susceptibility of culturedcells to apoptosis could be demonstrated, in particular,in a number of experiments using hematological celllines [141–144]. On the other hand, Yonezawa andcoworkers explicitly reported that out of various softtissue and osteosarcoma cell lines apoptosis could onlybe induced by heating up to 43 °C in a single culturedcell-line of malignant fibrous histiocytoma. It seemsthat at least some cell types exhibit different susceptibil-ity to apoptotic cell death induced by heat. Above adistinct temperature, it is more likely to induce necrosisinstead.

Considering in vivo experiments, a significant tumorgrowth delay due to apoptosis was observed in a xeno-transplanted Ward colon carcinoma, but not fibrosar-coma, in rats exposed to long-term moderatehyperthermia. Analyses of the host tissues moreover

sis by direct stimulation of the fas-ligand or by naturalkiller (NK)-mediated lysis cells cannot be prevented byelevated HSP-expression in experimental systems.Therefore, induction of programmed cell death mayoccur at normal intracellular HSP-levels using thesepathways. Taken together, data suggest a close connec-tion between HSP-expression and inhibition of hyper-thermic cell death, especially apoptosis, under certainconditions, but further investigations are necessary tofurther understand the underlying mechanisms in thefuture [129,130,132–135].

Fig. 11. Course of NK-cells in patients treated with chemotherapyand WBH compared with patients treated with chemotherapy (CTX)alone (Ahlers et al., previously unpublished). y-Axis: NK-cells(CD3−/CD16+/CD56+) calculated as percent of total bloodlymphocytes; x-axis: ‘WBH’, measurements at 37, 40, 41.8 and 39 °Cbody-core temperature; ‘CTX’, measurements before, immediatelyafter, and 2 and 4 h after the application of chemotherapy.


revealed the occurrence of programmed cell death invarious lymphatic tissues (especially thymus). Moder-ately increased apoptosis rates were also establishedwithin the small intestine, but not in any of the remain-ing organs [45,145].

Taken together, programmed cell death seems torepresent an important effector of heat action. Anyway,it has to be taken into consideration that most of thepre-clinical data refer to higher temperatures than thosethat can be applied in the treatment of cancer patientswith hyperthermia. As hyperthermia is always appliedin combination with radiation and/or antineoplasticdrugs in clinical practice, it is conceivable that at leastan additive proapoptotic effect may become relevant inthe scope of multimodal strategies. This might furtherbe supported by additional vascular, nutritive, andimmunologic changes.

7.3. Immunogenic effects of heat-shock proteins

HSP isolated from cancer cells, but not those fromnormal tissues, are able to induce a cytotoxic T-cell-ac-tivation against the tumor derived from. Interestingly,it was found that this reaction is not caused by theHSP-molecule itself, but by complexes of HSP withtumor specific peptides. These HSP/peptide complexesare thought to be internalized into antigen presentingcells by specific receptors in order to get presentedtogether with major histocompatibility complex (MHC)class I molecules. This unique property makes the HSPan attractive target for anticancer vaccination strate-gies-regardless of an additional hyperthermia treatment[146–148].

A connection between HSP-expression and hyper-thermia that goes beyond the finding of elevated HSP-synthesis during heat exposure was recentlydemonstrated by the group of Multhoff and Issels[149–154]. They reported about a stress-inducible formof HSP-70/72 that is expressed on the surface of distinctcell cultures. It was also found that this presentation ofHSP 70/72 on the cell-surface may appear either consti-tutively or heat-induced, and, is able to induce a MHC-independent tumor cell-lysis, too. However, themechanism by which cytoplasmatic HSP reaches thecell surface and induces cell death have not beenclarified in detail yet. It was speculated that HSP 70/72cell surface expression modulates immune responseagainst certain tumor cells (e.g. if it functions as aforeign antigen by itself or when complexed with othermolecules). A further suggestion is that HSP mimikrisanother antigen when expressed on the cell surface thusinducing immunostimulation against specific tumorcells. Whereas, cell surface expression of HSP 70/72 ontumor cells was not confirmed by other groups yet,Roigas and coworkers recently detected a HSP cell

surface expression by FACS analysis using an antibodydiffering from the one used by Multhoff and cowork-ers. Work is going on to further confirm these resultsand to clear up how and why HSP 70/72 gets onto thecell surface of some tumor cells [149–154].

7.4. Signal transduction

The antitumor efficacy of antineoplastic drugs de-pends on dosage, as well as, on tissue-specific or/andcellular characteristics. The way a potential lethal stim-uli finally results in cell death, is mediated by a numberof specific genes, the gene products of which playimportant roles in different signal transduction path-ways (e.g. regulation of the cell cycle, programmed celldeath, and DNA-repair). With regard to hyperthermia,heat-shock response represents a typical, but by nomeans specific reaction. All together, there is only littleknowledge about the signaling pathways that mediatethe cytotoxic effects of heat, and it also is not knownwhether these possess any specificity [43,57,130,131,136,155–157].

In this context, the p53 gene product represents animportant cellular protein that exhibits the features of atranscription factor. p53 protein expression becomeselevated when cells are exposed to potentially lethalstimuli, and activation of the p53-pathway may resultin either G1-arrest or apoptosis. In case, this pathway isdisturbed, e.g. by genetic alterations, predominance ofproliferative signals may occur, leading to malignanttransformation under certain circumstances. Vice versa,p53-mutations resulting in an absent protein synthesisor synthesis of a mutant protein with oncogenic poten-tial represent the most common genetic alterations ob-served in human solid tumors [140,158–160].

First evidence for a connection between hyperther-mia and p53-function came from the finding that HSP70/72 interacts with certain mutants but not wild-type(wt)-p53 protein. Furthermore, a heat-induced accumu-lation of wt-p53 was reported to result in an increasedaffinity of p53 to HSP 70/72. Cultured fibroblast andcolon carcinoma cells containing wt-p53 exhibit ahigher thermosensitivity than those with p53 null-muta-tions, and the rate of cultured colon carcinoma cellsundergoing spontaneous apoptosis was shown to besignificantly lower in cells when either the p53- orretinoblastoma (Rb) protein was inactivated[44,91,161–163]. From these findings, it was proposedthat tumors containing distinct p53- or Rb-mutationsmight be less thermosensitive than those with wild-typealleles, and that thermosensitivity in wt-p53 cells mightbe due to a suppression of the cell saving properties ofHSP 70.

The few clinical data available on this topic did notconfirm correlations of p53-alterations, HSP-expression


and outcome of patients treated with hyperthermia.In particular, members of our group did not find acorrelation between HSP70 expression and outcomein patients with rectal cancer treated with eitherneoadjuvant radiochemotherapy or radiochemother-apy plus regional hyperthermia. Others described theoccurrence of tumor cell apoptosis in 28 patients withrectal cancer who received a multimodal therapy con-sisting of 5-fluorouracil, radiation, and intraluminalhyperthermia. Apoptosis rate was significantly corre-lated with therapeutic effect (but not p53 status),while the effect of hyperthermia could not be studiedbecause of the lack of an adequate control group[164,165].

8. Hyperthermia-induced changes in cellular immuneresponse

8.1. Pre-clinical effects of heat on lymphocytes andexperimental tumors

There is no doubt about a close connection be-tween cancer and the immune system today, and dif-ferent immunotherapeutic strategies are already underclinical evaluation. In addition, knowledge on mecha-nisms contributing to malignant transformation in pa-tients with comprised immune system has markedlyincreased during the past years [166–169]. The con-sideration that hyperthermia seems to imitate fever,an apparent immunologic reaction, prompted variousin vitro studies on the effect of heat on humanlymphocytes since the early 1980s, mostly focussingon immunologic functions of non-migratorylymphocytes [170–176].

In this context, most investigators have observedimpairments of lymphocyte function after applyingnon-physiologically high temperatures (!42 °C) invitro, and especially NK-lymphocytes have beenfound to react very sensitive to heat. From a recentpoint of view, it is very difficult to come to a conclu-sion by comparing these studies with each other, asdifferent tests and calculations were used to measurelytic NK-cell activity, sometimes without regard tothe total NK-lymphocyte count. However, Shen andcoworkers demonstrated in a persuading way thatNK-cell function was increased at temperatures ofabout 40 °C, but impaired at temperatures above42 °C. More recent findings referring to in vitro tem-peratures below 41 °C actually revealed an increasedNK-cell proliferation, that was accompanied withheat-shock response, as well as secretion of selectin.The clinical relevance of these findings remains to beclarified [171,173,177,178].

Additional clues on the influence of heat on cellularimmune response came from a recent animal study by

Burd and coworkers. Here, a growth delay of breastcancer xenografts in SCID- und Balb/c mice was ob-served after long-lasting moderate hyperthermia.Thermal doses applied were too low to inducechanges in any host tissue here, but at the tumor siteaccumulation of both host lymphocytes and adop-tively transferred NK-cells was found to be responsi-ble for the marked rate of tumor cell apoptosisobserved in both animal models after hyperthermia.This effect was inhibited by selectively blocking theNK-cell function. Therefore, NK-cell mediated cell ly-sis may represent an important cytotoxic mechanisminduced by (moderate) hyperthermia [38].

8.2. Immunologic changes in cancer patients treatedwith whole-body hyperthermia

Most data published on immunologic changes inhumans exposed to systemic heat, refer to investiga-tions of lymphocyte subpopulations and/or serum cy-tokines in healthy persons whose body coretemperature was moderately raised to temperatures of39–39.5 °C in a water bath [177,179,180]. Others re-ported about immunologic changes in patients after aheat-stroke [181]. In addition, few publications areavailable on short-term changes in serum cytokinelevels in patients treated with either radiant heat orextracorporal WBH at 42 °C [182].

Regarding lymphocyte subpopulations, a significantreduction of both T4-cell count and T4/T8 ratio wasobserved in both healthy persons that underwentmoderate water bath-hyperthermia, as well as patientssuffering from heat-stroke. On the other hand, T8-and NK-cells were significantly elevated, thus result-ing in a slight increase of the total lymphocyte countdespite the decrease of T4-cells mentioned [180,183].As we recently demonstrated, a decrease of T4-cellsalso takes place in patients treated with WBH, but areduction of T8-cells was not detected in this context[184–186].

Changes of cellular immune response observed insubjects exposed to systemic heat are relatively unspe-cific and may be interpreted as part of a general re-sponse to major physiological stress, the presence ofwhich is clearly reflected by significant elevations ofheart rate and cardiac output in WBH patients[187,188]. Similar changes, including an elevation ofNK-cells, respectively, activity, can also be inducedby adrenaline-infusion or moderate exercise, whereasheavy exercise may entail an impairment of NK-cell-activity [180,189]. Today, it is generally believed thatthe immunologic response to different kinds of stressis relatively uniform [180,189]. On the one hand,lymphocyte migration into the bone marrow has beendemonstrated to be adrenaline-dependant [180,189].


On the other hand, "-2-receptor-mediated mobilizationof endothelium-bound lymphocytes into the bloodwould give a favorable explanation for the differingcourse of lymphocyte subpopulations during WBH[180,189]. Madden and coworkers demonstrated a dif-ferential "-2-receptor expression on the cell surface ofdifferent lymphocyte subpopulations. "2-receptor ex-pression on NK-cells was higher than expression onT8- and on T4-lymphocytes. Moderate plasma cate-cholamine-levels resulted in a stimulation of NK-cellfunction, whereas impaired NK-cell activity was foundin the presence of high catecholamine levels. Cate-cholamines may exert their influence on bloodlymphocytes by direct stimulation on the one, andsympathetic innervation of lymphatic tissues on theother hand. [190–194].

Investigation of serum cytokine le!els in patients un-dergoing WBH has been performed by Robins andcoworkers. They reported about alterations in patientstreated with WBH, comprising an elevation of theantiinflammatory interleukins (IL) 6 and 10, whereasIL-2 and interferon (IFN)-# remained unchanged.These findings have recently been confirmed by others,including our group. Remarkably, both changes incytokine levels in WBH-patients as well as those inlymphocyte subpopulations were reversible sponta-neously. Moreover, an increase of antiinflammatorycytokines can also be induced by physiological stressin animal models, sometimes even inducing decreasingIL-2 and IFN-gamma serum levels [182,185,195].

Regarding the above mentioned facts, WBH andother forms of systemic heat exposure !41 °C havebeen found to induce severe alterations in circulatingblood lymphocytes, resulting in a suppression ratherthan a stimulation of the immune system. However,future research will reveal if these findings just repre-sent para-phenomenas or if there is a connection be-tween changes in blood lymphocytes and increasedlymphocyte migration and NK-cell activity at the tu-mor site as it was demonstrated in experimental tu-mors.

9. Modulation of drug resistance by hyperthermia

9.1. Re!ersal of drug resistance induced byhyperthermia

Drug resistance represents the major cause of treat-ment failure in human malignancies, and can be in-duced by different mechanisms, of which the pleitrope‘multidrug resistance (MDR)’, mediated by thetransmembranal ‘glycoprotein p170’ efflux pump, hasgained particular interest [196–198]. Pre-clinical datasuggest that hyperthermia is a good candidate to over-

come various modes of drug resistance, and this hasbeen demonstrated in particular for the platinumderivate cisplatin (DDP). Overcoming DDP-resistanceby hyperthermia is exemplary, because its causes aremultifactorial (e.g. changes in transmembrane conduc-tivity, activity of sodium/potassium-ATPase, glu-tathion metabolism, DNA repair). It, therefore, givesthe possibility to investigate the effect of hyperthermiaon drug resistance at different cellular levels (butplease note that the MDR-phenotype is not involvedin DDP-resistance). However, one has to keep in mindthat temperatures and thermal doses were significantlyhigher in these in vitro studies, than those usuallyachieved in clinical practice [199]. On the contrary, thecourse of individual patients previously refractory toplatinum compounds who responded to therapy afteraddition of hyperthermia are strongly suggestive of areversal of drug resistance induced by hyperthermia atlower temperatures [99,200].

9.2. Thermotolerance is often associated with drugresistance

On the other hand, moderate heat exposure hasbeen demonstrated to induce HSP-expression in cul-tured cells, and elevated levels of intracellular HSP-70have been shown to be associated with thermotoler-ance. Vice versa, transfection of HSP-70 into culturedfibroblasts resulted in a marked decrease of ther-mosensitivity, with similar findings obtained for othermembers of HSP-families. Thermotolerance may beassociated with different forms of drug resistance (e.g.MDR), and hyperthermia has been shown to inducevarious forms of drug resistance, too (including MDRor the heat-dependant inactivation of the enzyme to-poisomerase II). Occurrence of a heat-inducible,simultaneous resistance to heat and drugs (thermotol-erance+MDR) has been reported to occur under cer-tain conditions [59,93,132,157,201–203].

9.3. Does hyperthermia o!ercome or induce drugresistance?

It seems curious that hyperthermia has been shownto both overcome and induce drug resistance in vitro.Reversal of drug resistance has been particularlyshown for platinum compounds at temperatures!42 °C, whereas induction of drug resistance (aloneor in conjunction with thermotolerance and HSP-accu-mulation) may appear when lower temperatures areapplied. Despite of this pre-clinical observations, afavorable effect of different hyperthermia approacheson drug sensitivity has been reported in the scope ofclinical trials. Here, patients with principallychemosensitive, but individually refractory diseases(e.g. germ cell tumors and sarcomas), were successfully


treated with additional hyperthermia to a givenchemotherapeutic schedule [30,92,94,95]. In addition,there is one study available in which tumor specimensfrom patients participating in a clinical hyperthermiatrial were investigated with regard to the modulationof drug resistance by hyperthermia on a molecularlevel. Here, an induction of MDR-drug resistance byregional hyperthermia of the pelvis in conjunctionwith radiotherapy and chemotherapy was excluded[204]. As a conclusion, available data on hyperther-mia and drug resistance suggest that the positive ef-fect (reversal of drug resistance) overweighs thedisadvantages (induction of drug resistance) in clinicalpractice. However, one should keep in mind, thatdrug resistance could be induced by hyperthermia onprinciple, particularly at moderate temperatures.

10. Summary and discussion

Local and regional hyperthermia have been demon-strated to improve treatment results in conjunctionwith radio- and/or chemotherapy for several indica-tions until now, thus encouraging further evaluationof various hyperthermia approaches. However, thecellular and molecular pathways underlying thisbeneficial outcome of patients are still poorly under-stood, although a large number of pre-clinical studiesare available on different aspects of hyperthermia ac-tion. It is well-known from these data that heat de-velops an independent cytotoxic effect on culturedcells in vitro at temperatures "43 °C. In addition,hyperthermia acts in a synergistic way when com-bined with radiation or cytostatic drugs at lower tem-peratures (40.5–43 °C) in vitro and in vivo. In cancerpatients, complex changes were found during and af-ter the application of the various hyperthermiamodalities, concerning blood, nutrient and oxygensupply of the tumor, metabolic changes, signal trans-duction, immunology, as well as, pharmacological ef-fects. Reports on these pre-clinical effects ofhyperthermia rather raise a cascade of further ques-tions (for example, the details of the immunologicalprocesses caused by hyperthermia), but definitely donot answer one of the various questions on heat ac-tion in the treatment of cancer patients. Indeed, it isextremely difficult to extract reliable informationabout molecular and immunological effects of hyper-thermia from clinical investigations, since amongother reasons, the application of hyperthermia is usu-ally only a part of multimodal treatment schemes.

Many of the pre-clinical investigations concerningheat action have been performed in the seventies andearly-1980s, and were followed by a comparativelylow number of systematic molecular investigations in

the scope of concomitant research to clinical studies.Unfortunately, it has been an unproven dogma inhyperthermia research for many years, that antineo-plastic heat action requires temperatures !43 °C.This is why many of those pre-clinical studies refer tothermal doses above values that cannot be realisti-cally achieved in clinical approaches, such as 43.5–45 °C for 60 min or longer. Another point toconsider, hyperthermia under experimental conditionsensures a more homogeneous heating than undermost clinical conditions, especially than in local andregional hyperthermia. Therefore, in vitro hyperther-mia at !43 °C may lead to an overestimation of thecytotoxic potency of heat. This may, vice versa, leadto an underestimation of undesired effects that mayoccur in certain situations, e.g. an impairment in theavailability of drugs and drug action, induction ofdrug resistance, genetic alterations, clonal selection ofresistant tumor cell clones, or dissemination of tumorcells. However, clinical data strongly support thedominance of the beneficial effects of hyperthermiaover its possible negative features.

It is curious that one of the fundamental questionsin hyperthermia— the temperatures and thermal dosesapplied at the tumor site necessary to achieve a clini-cal benefit— is still unresolved and thus discussedcontroversially. Recent data, however, suggest thatoptimal thermal parameters may differ between thevarious modalities of heat application. Clinical trialson local and regional hyperthermia in combinationwith radiotherapy and chemotherapy have revealed aclinical benefit at temperatures significantly lowerthan postulated in former in vitro data. Remarkably,a dose–response relationship could be establishedeven at this comparatively low thermal dose level. Incontrast, a similar correlation cannot be drawn forpatients treated with WBH and chemotherapy, wherea body-core temperature of 41.8–42 °C can be main-tained for 60 min or even longer in almost everypatient, but a clear benefit is not evident (even if thedifferent treatment indication for both approaches aretaken into consideration). On the other hand, animalstudies on ‘mild’ WBH (e.g. 40.5–42 °C, for 60–360min) and some clinical phase-I and -II studies suggestan antineoplastic activity of clinical hyperthermiaeven at these low temperatures.

Regarding future research, efforts should be madeto conduct modern randomized phase-III trials to fur-ther establish local and regional hyperthermia, wherehyperthermia should be applied as an adjunct to agenerally accepted standard treatment (e.g. hyperther-mic vs. normothermic radiochemotherapy in primarycancer of the uterine cervix). In addition, ongoingcomparative trials on regional and WBH should becompleted as soon as possible in a multicenter setting,performing concomitant laboratory investigations on


different aspects of heat action. Regarding technicalaspects of hyperthermia, clinical experience withpresently existing loco-regional applicators demon-strate that improvements are still necessary. Tempera-ture distributions achieved to date are far fromsatisfactory, regarding both the absolute values andhomogeneity. A better temperature control may beprovided by introducing novel thermometry units withmagnetic resonance tomography (MRT) support, cou-pled with hyperthermia control systems. Developmentof adapted antenna arrays for the different anatomicallocalizations may further improve effectiveness of localand regional hyperthermia. In addition, basic researchproduced some very exciting novel applications forhyperthermia, e.g. in gene therapy, where the targetingof therapeutic genes was performed by heat via theHSP promoter, or in pharmacology, where tempera-ture-dependant liposomes may be triggered by a tem-perature increase.

According to our opinion, it would be beneficial inhyperthermia research, to abandon the rather mecha-nistic idea of a dose-dependent antineoplastic effect attemperatures with a breakpoint above 43 °C. As dis-cussed earlier, the antineoplastic effect of hyperthermiacannot be explained in clinical studies from thesedose–effect curves alone, because administered ther-mal doses were markedly lower than necessary for invitro ‘hyperthermic cell death’. This strongly supportsthe hypothesis that the mechanisms responsible forhyperthermia action in the clinical situation are multi-factorial and complex. Undoubtedly, extensive pre-clinical data on thermal effects are important tounderstand the basic mechanisms of hyperthermia andto highlight some of these heat effects in a very de-tailed way, but most of the available data cannot betransposed into clinical schemes. It is very probablefrom current clinical data that the benefit of hyper-thermia clearly overweighs its possible disadvantageswhen applied in conjunction with radiation or cyto-toxic drugs for certain neoplastic diseases. In this con-text, one convincing issue is a repeatedly demonstratedcorrelation between various thermal parameters andclinical outcome, and this also holds true for the tem-perature range below 43 °C. This is one of the majorargument justifying further evaluation of hyperthermiain the laboratory and clinic. In addition, clinical trialsof hyperthermia have to be accompanied by labora-tory investigations on different aspects of heat actionin order to further understand the cellular and molec-ular targets of hyperthermia.

Reviewers

James R. Oleson, Department of Radiation Oncol-ogy, The university of Arizona Health Sciences Center,

1501 North Campbell Ave., P.O. Box 245081, Tucson,AZ 85724-5081, USA.

Claire C. Vernon, Hammersmith Hospital, DuCaneRd, London W12 OHS, UK.

J. van der Zee, University Hospital—Daniel denHoed Cancer Center, Department of Radiotherapy,Subdivision Hyperthermia, Postbus 5201, NL-3008 AERotterdam, The Netherlands.

References

[1] Datta NR, Bose AK, Kapoor HK, Gupta S. Head and neckcancers: results of thermoradiotherapy versus radiotherapy. IntJ Hyperthermia 1990;6(3):479–86.

[2] Overgaard J, Gonzalez Gonzalez D, Huslhof MCCH, et al.Hyperthermia as an adjuvant to radiation therapy of recurrentor metastatic malignant melanoma. A multicentre randomizedtrial by the European Society for Hyperthermic Oncolgy. Int JHyperthermia 1996;12:3–20.

[3] Perez CA, Pajak T, Emami B, Hornback NB, Tupchong L,Rubin P. Randomized phase III study comparing irradiationand hyperthermia with irradiation alone in superficial measur-able tumors. Final report by the Radiation Therapy OncologyGroup. Am J Clin Oncol 1991;14(2):133–41.

[4] Valdagni R, Amichetti M. Report of a long-term follow-up ina randomized trial comparing radiation therapy and radiationplus hyperthermia to metastatic lymph nodes in stage IV headand neck cancer patients. Int J Radiat Oncol 1993;28:163–9.

[5] Vernon C, Hand JW, Field SB, et al. Radiotherapy with orwithout hypethermia in the treatment of superficial localizedbreast cancer: results from five randomized controlled trials. IntJ Radiat Oncol Biol Phys 1996;35:731–44.

[6] Emami B, Myerson RJ, Cardenes H, et al. Combined hyper-thermia and irradiation in the treatment of superficial tumors:results of a prospective randomized trial of hyperthermia frac-tionation (1 vs. 2 weeks). Int J Radiat Oncol Biol Phys1992;24:145–52.

[7] Engin K, Tupchong L, Moylan DJ, et al. Radomized trial ofone versus two adjuvant hyperthermia treatments per week inpatients with superficial tumors. Int J Hyperthermia1993;9(3):327–40.

[8] Kapp DS. A phase III study on hyperthermia in head and neckcanine tumours: not hot enough. Int J Hyperthermia1996;12(3):437–41.

[9] Emami B, Scott C, Perez CA, et al. Phase III study of intersti-tial thermoradiotherapy compared with interstitial radiotherapyalone in the treatment of recurrent or persistent human tumors.A prospectively controlled randomized study by the RadiationTherapy Group. Int J Radiat Oncol Biol Phys 1996;34(5):1097–104.

[10] Sneed PK, Stauffer PR, McDermott MW, et al. Survival benefitof hyperthermia in a prospective randomised trial ofbrachytherapy boost #hyperthermia for glioblastoma multi-forme. Int J Radiat Oncol Biol Phys 1998;40:287–95.

[11] Berdow BA, Menteshashvili GZ. Thermoradiotherapy of pa-tients with locally advanced carcinoma of the rectum. Int JHyperthermia 1990;6(5):881–90.

[12] Kitamura K, Kuwano H, Watanabe M, et al. Prospectiverandomized study of hyperthermia combines with chemoradio-therapy for esophageal carcinoma. J Surg Oncol 1995;60:55–8.

[13] Sugimachi K, Kuwano H, Ide T, Toge T, Saku M, Oshiumi Y.Chemotherapy combined with or without hyperthermia forpatients with oesophageal carcinoma: a prospective randomizedtrial. Int J Hyperthermia 1994;10(4):485–93.


[14] Harima Y, Nagata K, Harinma K, Ostapenko VV, TanakaY, Sawada S. A randomized clinical trial of radiation therapyversus thermoradiotherapy in stage III cervical carcinoma. IntJ Hyperthermia 2001;17(2):97–105.

[15] van der Zee J, Gonzalez Gonzales D, van Rhoon GC, et al.Comparison of radiotherapy alone with radiotherapy plus hy-perthermia in locally advanced pelvic tumors: a prospective,randomised, multicentre trial. Lancet 2000;355:1119–25.

[16] Hamazoe R, Maeta M, Kaibara N. Intraperitoneal ther-mochemotherapy for prevention of peritoneal recurrence ofgastric cancer. Cancer 1994;73:2048–52.

[17] Ghussen F, Kruger I, Smalley R, Groth W. Hyperthermicperfusion with chemotherapy for melanoma of the ex-tremeties. World J Surg 1989;13:598–602.

[18] Hafstrom L, Rudenstam CM, Blomquist E, et al. Regionalhyperthermic perfusion with melphalan after surgery for re-current melanoma of the exremities. Swedish MelanomaStudy Group. J Clin Oncol 1991;9(12):2085–7.

[19] Koops HS, Vaglini M, Suciu S, et al. Prophylactic isolatedlimb perfusion for localized, high-risk limb melanomas: re-sults of a multicenter randomized phase III trial. J ClinOncol 1998;16:2906–12.

[20] Hildebrandt B, Wust P, Rau B, Schlag PM, Riess H. Re-gional hyperthermia in rectal cancer. Lancet2000;356(9231):771–2.

[21] Issels R, Prenninger SW, Nagele A, et al. Ifosfamide plusetoposide combined with regional hyperthermia in patientswith locally advanced sarcomas. J Clin Oncol 1990;11:1818–29.

[22] Oleson JR, Samulski TV, Leopold KA, et al. Sensitivity ofhyperthermia trial outcomes to temperature and time: impli-cations for thermal goals of treatment. Int J Radiat OncolBio Phys 1993;25(2):289–97.

[23] Wust P, Gellermann J, Rau B, et al. Hyperthermia in themultimodal therapy of advanced rectal carcinomas. RecentRec Cancer Res 1996;142:281–309.

[24] Wust P, Stahl H, Dieckmann K, et al. Local hypert-hermiaof N2/N3 cervical lymphnode metastases: correlation of tech-nical/thermal parameters and response. Int J Radiat OncolBiol Phys 1996;34:635–46.

[25] Rau B, Wust P, Tilly W, et al. Preoperative radiochemother-apy in locally advanced or recurrent rectal cancer: regionalradiofrequency hyperthermia correlates with clinical parame-ters. Int J Radiat Oncol Biol Phys 2000;48(2):381–91.

[26] Hohenberger P, Kettekhack C. Clinical management and cur-rent research in isolated limb perfusion for sarcoma andmelanoma. Oncology 1998;55(2):89–102.

[27] Urano M, Kuroda M, Nishimura Y. For the clinical applica-tion of themochemotherapy given at mild temperatures. Int JHyperthermia 1999;15:79–107.

[28] Falk MH, Issels RD. Hyperthermia in oncology. Int J Hyper-thermia 2001;17(1):1–18.

[29] Rau B, Wust P, Hohenberger P, et al. Preoperative hyper-thermia combined with radiochemotherapy in locally ad-vanced rectal cancer: a phase II clinical trial. Ann Surg1998;227(3):380–9.

[30] Rietbroek RC, Schilthuis MSPJB, et al. Phase II trial ofweekly locoregional hyperthermia and cisplatin in patientswith a previously irradiated recurrent carcinoma of the uter-ine cervix. Cancer 1997;79(5):935–43.

[31] Bakshandeh A, Bruns I, Eberhardt K, et al. Ifosfamide, car-boplatin and etoposide combined with aquatherm-induced41.8 °C whole-body hyperthermia for adult patients with ma-lignant pleural mesothelioma. Ann Oncol 2000;11(Suppl.4):513P.

[32] Gruber Y, Hegewisch-Becker S, Bakshandeh-Bath A, SommerH, Hoffmann R, Hossfeld DK. Whole-body hyperthermia at41.8 °C combined with ifosfamide and carboplatin in re-lapsed ovarian carcinoma pretreated with a platin-containingregimen. Ann Oncol 2000;11(Suppl. 4):377P.

[33] Hegewisch-Becker S, Gruber Y, Panse J, Atanackivoc D,Nierhaus A, Hossfeld DK. Whole-body hyerthermia as anadjunct to oxaliplatin/5-FU/FA in high-dose 5-FU/FA or 5-FU/FA and CPT-11 refractory/relapsed advanced colorectalcancer. ESMO proceedings. Ann Oncol 2000;11(Suppl.4):A267.

[34] Wust P, Riess H, Hildebrandt B, et al. Utilizing the iratherm-2000 infrared system for whole-body hyperthermia at42 °C—a feasibility study. Int J Hyperthermia2000;16(4):325–39.

[35] Dahl O, Dalene R, Schem BC, Mella O. Status of clinicalhyperthermia. Acta Oncol 1999;38(7):863–73.

[36] Dewey WC. Arrhenius relationships from the molecule andcell to the clinic. Int J Hyperthermia 1994;10(4):457–83.

[37] Dewhirst MW, Prosnitz L, Thrall D, et al. Hyperthermictreatment of malignant disease: current status and a viewtoward the future. Sem Oncol 1997;24(6):616–25.

[38] Burd R, Dziedzic TS, Xu Y, Caliguiri MA, Subjeck JR, Re-pasky EA. Tumor cell apoptosis, lymphocyte recruitment andtumor vascular changes are induced by low temperature,long-duration (fever-like) whole-body hyperthermia. J CellPhysiol 1998;177:137–47.

[39] Bull JMC. An update on the anticancer effects of a combina-tion of chemotherapy and hyperthermia. Cancer Res1984;44(Suppl.):4853–6.

[40] Dewey WC, Thrall D, Gilette EL. Hyperthermia and radia-tion—a selective thermal effect on chronically hypoxic tumorcells in vivo. Int J Radiat Oncol Biol Phys 1977;2:99–103.

[41] Dewey WC, Hopwood LE, Sapareto SA, Gerweck LE. Cellu-lar responses to combinations of hyperthermia and radiation.Radiology 1977;123:463–74.

[42] Dewhirst MW, Ozimek EJ, Gross J, Cetas TC. Will hyper-thermia conquer the elusive hypoxic cell. Radiology1980;137:811–7.

[43] Fuller KJ, Issels RD, Slosmann DO, Guillet J-G, Soussi T,Polla BS. Cancer and the heat shock response. Eur J Cancer1994;30:1884–91.

[44] Matsumoto H, Takahashi A, Wang X. Transfection of p53-knockout mouse fibroblasts with wild-type p53 increases thethermosensitivity and stimulates apoptosis induced by heatstress. Int J Radiat Oncol Biol Phys 1997;39(1):197–203.

[45] Sakaguchi Y, Stephens LC, Makino M, Kaneko T, et al.Apoptosis in tumors and normal tissues induced by wholebody hyperthermia in rats. Cancer Res 1995;55:22.

[46] Westra A, Dewey WC. Variation in sensitivity to heat shockduring the cell-cycle of Chinese hamster cells in vitro. Int JRadiat Biol Relat Stud Phys Chem Med 1971;19(5):467–77.

[47] Feyerabend T, Wiedemann GJ, Richter E, Hegewisch-BeckerS. Hyperthermiea as an adjunct to the standard treatment ofneoplastic diseases: few cures but some advances. Onkologie1999;22:122–7.

[48] Hildebrandt B, Loffel J, Ahlers O, Wust P, Riess H. Hyper-thermia: different methods, different aims, and different prob-lems. Onkologie 1999;22:5.

[49] Gerner EW. Definition of a thermal dose. In: Overgaard J,editor. Hyperthermic Oncology. London: Taylor and Francis,1985:253–62.

[50] Henle KJ. Arrhenius analysis of thermal responses. In: StormFK, editor. Hyperthermia and Cancer Therapy. Boston: Hall,1983:47–53.

[51] Overgaard J, Suit HD. Time– temperature relationship th hy-perthermic treatment of malignant and normal tissue in vivo.Cancer Res 1979;39(8):3248–53.


[52] Sapareto SA. Thermal isoeffect dose: addressing the problem ofthermotolerance. Int J Hyperthermia 1987;3(4):297–305.

[53] Jordan A, Scholz R, Schuler J, Wust P, Felix R. Arrheniusanalysis of the thermal response of human colonic adenocar-cinoma cells in vitro using the multi-target, single-hit and thelinear quadratic model. Int J Hyperthermia 1997;13(1):83–8.

[54] Coss RA, Dewey WC, Bamburg JR. Effects of hyperthermia ondividing Chinese hamster ovary cells and on microtubules invitro. Cancer Res 1982;42(3):1059–71.

[55] Dewey WC. The search for critical cellular targets damaged byheat. Radiat Res 1989;20:191–204.

[56] Vidair C, Dewey WC. Two distinct modes of hyperthermic death.Radiat Res 1988;116:157–71.

[57] Chin KV, Tanaka S, Darlington G, et al. Heat shock and arseniteincrease expression of the multi-drug resistance (MDR 1) genein human carcinoma cells. J Biol Chem 1990;26:221–6.

[58] Jung H. A generalized concept for cell killing by heat. Radiat Res1986;106(1):56–72.

[59] Li GC, Mivechi NF, Weitzel G. Heat shock proteins, thermotol-erance, and their relevance for clinical hyperthermia. Int JHyperthermia 1995;11(4):459–88.

[60] Song CW, Lyons JC, Griffin RJ, Makepeace CM, Cragoe EJ.Increase in thermosensitivity of tumor cells by lowering intracel-lular pH. Cancer Res 1993;53:1599–601.

[61] Wachsberger PR, Landry J, Storck C, et al. Mammalian cellsadapted to growth at pH 6.7 have elevated HSP27 levels and areresistant to cisplatin. Int J Hyperthermia 1997;13(3):251–5.

[62] Folkman J. What is evidence that tumors are angiogenesisdependant. J Natl Cancer Inst 1990;82:4–6.

[63] Song CW. Effect of local hyperthermia on blood flow andmicroenvironment: a review. Cancer Res 1984;44:4721–30.

[64] Streffer C. Aspects of metabolic change in hyperthermia. Rec ResCancer Res 1988;197:7–43.

[65] Vaupel P, Kallinowski F, Kluge M, Egelhof E, Fortmeyer HP.Microcirculatory and pH alterations in isotransplanted rat andxenotransplanted human tumors associated with hyperthermia.Rec Res Cancer Res 1988;109:174–82.

[66] Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen andnutrient supply and microenvironment of human tumors: areview. Cancer Res 1989;49:6449–65.

[67] Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen andnutrient supply, and metabolic microenvironment of humantumors: a review. Cancer Res 1989;49(23):6449–65.

[68] von Ardenne M, Reitnauer PG. Manipulated selective inhibitionof microcirculation in cancer tissues, article in German. J CancerRes Clin Oncol 1982;103(3):269–79.

[69] Hiroaka M, Hahn GM. Comparison between tumor pH and cellsensitivity to heat in RIF-1 tumors. Cancer Res 1989;49(14):3734–6.

[70] Overgaard J. Effect of misodinazole and hyperthermia on theradiosensitivity of a C3H mouse mammary carcinoma and itssurrounding normal tissue. Br J Cancer 1980;41:10–21.

[71] Reinhold HS, Endrich B. Tumour microcirculation as a targetfor hyperthermia. Int J Hyperthermia 1986;2(2):111–37.

[72] Vaupel P, Kallinowski F. Physiological aspects of hyperthermia.Rec Res Cancer Res 1987;104:71–109.

[73] Vaupel P, Okunieff P, Kluge M. Response of tumor red bloodcell flux to hyperthermia and/or hyperglycaemia. Int J Hyperther-mia 1989;5(2):199–210.

[74] Kerner T, Deja M, Gerlach H, et al. Whole-body hyperther-mia—a secure procedure for patients with various malignancies.Intensive Care Med 1999;25:959–65.

[75] Robins HI, Dennis WH, Neville AJ, et al. A nontoxic system for41.8 °C whole body hyperthermia: results of a phase I studyusing a radiant heat device. Cancer Res 1985;45:3937–44.

[76] Robins HI, Rushing D, Kutz M, et al. Phase I clinical trial of

melphalan and 41.8 °C whole-body hyperthermia in cancerpatients. J Clin Oncol 1997;15:158–64.

[77] Steinhausen D, Mayer WK, Ardenne MV. Evaluation of Sys-temic Tolerance of 42.0 °C Infrared—A Whole-Body Hyper-thermia in Combination with Hyperglycemia and Hyperoxemia,1994.

[78] von Ardenne M. Principles and concept 1993 of the systemiccancer multistep therapy (sCMT). Strahlenther Onkol1993;170:581–9.

[79] von Ardenne M. Systemische Krebs-Mehrschritt-Therapie, Hy-perthermie und Hyperglykamie als Therapiebasis. Stuttgart:Hippokrates-Verlag, 1997.

[80] Iwata K, Shakil A, Hur WJ, Makepeace CM, Griffin RJ, SongCW. Tumour pO2 can be increased markedly by mild hyperther-mia. Br J Cancer Suppl 1996;27(21):S217–21.

[81] Song CW, Shakil A, Griffin RJ, Okajima K. Improvement oftumor oxygenation status by mild temperature hyperthermiaalone or in combination with carbogen. Sem Oncol1997;24(6):626–32.

[82] Kim SH, Kim JH, Hahn EW. The enhanced killing of irradiatedHeLa cells in synchronous culture by hyperthermia. Radiat Res1976;66(2):337–45.

[83] Mills MD, Meyn RE. Hyperhtermic potentiation of unrejoinedDNA strand breaks following irradiation. Radiat Res1983;95:327–38.

[84] Schlag H, Lucke Huhle C. Cytokinetic studies on the effect ofhyperthermia on Chinese hamster lung cells. Eur J Cancer1976;12(10):827–31.

[85] Streffer C, van Beuningen D, Uma Devi P. Radiosenzitation byhyperthermia in human melanoma cells: single and fractionatedtreatments. Cancer Treat Rev 1984;11:179–85.

[86] Dahl O. Interaction of hyperthermia and chemotherapy. Rec ResCancer Res 1988;107:157–69.

[87] Engelhardt R. Hyperthermia and drugs. Rec Res Cancer Res1987;104:136–203.

[88] Hahn GM. Hyperthermia and Cancer. New York: Plenum Press,1982.

[89] Issels R. Hyperthermia combined with chemotherapy—biologi-cal rationale, clinical application, and treatment results. Onkolo-gie 1999;22:374–81.

[90] Wiedemann GJ, Siemens HJ, Mentzel M, et al. Effects oftemperature on the therapeutic efficacy and pharmacokinetics ofifosfamide. Cancer Res 1993;53:4268–72.

[91] van Bree C, van der Maat B, Ceha HM, Franken NAP, HavemanJ, Bakker PJM. Inactivation of p53 and of pRb protects humancolorectal carcinoma cells against hyperthermia-induced cytotox-icity and apoptosis. J Cancer Res Clin Oncol 1999;125:549–55.

[92] Hildebrandt B, Wust P, Rick O, et al. Whole body hyperthermiain germ cell tumors. Onkologe 2001;7(3):324–30.

[93] Kampinga HH. Hyperthermia, thermotolerance and topoiso-merase II inhibitors. Br J Cancer 1995;72(2):333–8.

[94] Wessalowksy R, Kruck H, Pape H, Kahn T, Willers R, GobelU. Hyperthermia for the treatment of patients with malignantgerm cell tumors: a phase I/II study in ten children or adolescentswith recurrent or refractory tumors. Cancer 1998;82(4):793–800.

[95] Wiedemann GJ, Katschinsky DM, Mentzel M, d’Oliere F,Wagner T, Robins HI. Ifosfamide, carboplatin and etoposidecombined with aquatherm induced 41.8 °C whole-body hyper-thermia (WBH) for refractory sarcoma. J Clin Oncol1996;14:1751.

[96] Vanakoski J, Seppala T. Heat exposure and drugs. Clin Pharma-cokinet 1998;34(4):311–22.

[97] Gerke P, Filejski W, Robins HI, Wiedemann GJ, Steinhoff J.Nephrotoxicity of ifosfamide, carboplatin and etoposide (ICE)alone or combined with extracorporal or radiant-heat-inducedwhole-body hyperthermia. J Cancer Res Clin Oncol2000;126(3):173–7.


[98] Robins HI, Cohen JD, Schmitt CL, et al. Phase I clinical trialof carboplatin and 41.8 °C whole-body hyperthermia in cancerpatients. J Clin Oncol 1993;9:1787–94.

[99] Wiedemann G, d’Oleire F, Knop E, et al. Ifosfamide andcarboplatin combined with 41.8 °C whole-body hyperthermia inpatients with refractory sarcoma and malignant teratoma. CancerRes 1994;54:5346–50.

[100] Formenti SC, Shrivastava PN, Sazozink M, et al. Ab-dominopelvic hyperthermia and intraperitoneal carboplatin inepithelial ovarian cancer: feasibility, tolerance, and pharmacol-ogy. Int J Radiat Oncol Biol Phys 1996;35(5):993–1001.

[101] Calderwood SK, Hahn GM. Thermal sensitivity and resistanceof insulin-receptor binding. Biochim Biophys Acta1983;756(1):1–8.

[102] Coss RA, Linnemanns WAM. The effects of hyperthermia on thecytoskeleton: a review. Int J Hyperthermia 1996;12(2):173–96.

[103] Konings AW, Ruifrok AC. Role of membrane lipids andmembrane fluidity in thermosensitivity and thermotolerance ofmammalian cells. Radiat Res 1985;102(1):86–98.

[104] Majda JA, Gerner EW, Vanlandingham B, Gehlsen KR, CressAE. Heat shock-induced shedding of cell surface integrins inA549 human lung tumor cells in culture. Exp Cell Res1994;210(1):46–51.

[105] Stevenson MA, Minton KW, Hahn GM. Survival and con-canavalin-A-induced capping in CHO fibroblasts after exposureto hyperthermia, ethanol, and X irradiation. Radiat Res1981;86(3):467–78.

[106] Liu F-F, Miller N, Levin W, et al. The potential role of hsp 70as an indicator of response to radiation and hyperthermiatreatments for recurrent breast cancer. Int J Hyperthermia1996;12(2):197–208.

[107] Malhotra A, Heynen MLP, Lepock JR. Role of extracellularcalcium in the hyperthermic killing of CHL V79 cells. Radiat Res1987;112:487–9.

[108] Nishida T, Akagi K, Tanaka Y. Correlation between cell killingeffect and cell membrane potential after heat treatment: analysisusing fluorescent dye and flow cytometry. Int J Hyperthermia1997;13(2):227–34.

[109] Ruifrok AC, Kanon B, Konings AW. Correlation betweencellular survival and potassium loss in mouse fibroblasts afterhyperthermia alone and after a combined treatment with X-rays.Radiat Res 1985;101(2):326–31.

[110] Stevenson MA, Calderwood SK, Hahn GM. Effect of hyperther-mia (45 °C) on calcium flux in Chinese hamster ovary HA-1fibroblasts and its potential role in cytotoxicity and heat resis-tance. Cancer Res 1987;47(14):3712–7.

[111] Vidair CA, Dewey WC. Evaluation of a role for intracellularNa+, K+, Ca2+ , and Mg2+ in hyperthermic cell killing. RadiatRes 1986;105(2):187–200.

[112] Overgaard J. Influence of extracellular pH on the viability andmorphology of tumor cells exposed to hyperthermia. J NatlCancer Inst 1976;56(6):1243–50.

[113] Overgaard J. Ultrastructure of a murine mammary carcinomaexposed to hyperthermia in vivo. Cancer Res 1976;36(3):983–95.

[114] von Ardenne M, Chaplain RA, Reitnauer PG. Selective injuryto cancer cells by a combined attack with acidification, heat,vitamin A, dimethyl sulfoxide and other agents facilitating therelease of lysosomal enzymes, article in German. Arch Geschwul-stforsch 1969;33(4):331–44.

[115] Borrelli MJ, Garlini WG, Ransom BR, Dewey WC. A directcorrelation between hyperhtermia induced membranne blebbingand survival in synchronous G1 CHO cells. J Cell Physiol1986;126:181–90.

[116] Henle KJ, Leeper DB. Effects of hyperthermia (45 °C) onmacromolecular synthesis in Chinese hemster ovary cells. CancerRes 1979;39(7 Pt. 1):2665–74.

[117] Higashikubo R, White RA, Roti Roti JL. Flow cytometricBrdUrd-pulse-chase study of heat-induced cell-cycle progressiondelays. Cell Prolif 1993;26(4):337–48.

[118] Kampinga HH. Thermotolerance in mammalian cells. Proteindenaturation and aggregation, and stress proteins. J Cell Sci1993;104(Pt. 1):11–7.

[119] Mackey MA, Anolik SL, Roti Roti JL. Changes in heat andradiation sensitivity during long duration, moderate hyperther-mia in HeLa S3 cells. Int J Radiat Oncol Biol Phys1992;24(3):543–50.

[120] Mackey MA, Roti Roti JL. A model of heat-induced clonogeniccell death. J Theor Biol 1992;156(2):133–46.

[121] Roti Roti JL, Kampinga HH, Malyapa RS, Wright WD,vanderWaal RP, Xu M. Nuclear matrix as a target for hyperther-mic killing of cancer cells. Cell Stress Chaperones 1998;3(4):245–55.

[122] Dahm-Daphi J, Brammer I, Dikomey E. Heat effects on therepair of DNA double-strand breaks in CHO cells. Int J RadiatBiol 1997;72(2):171–9.

[123] Dikomey E, Franske J. Effect of heat on induction and repairof DNA strand breaks in X-irradiated CHO cells. Int J RadiatBiol 1992;61:221–34.

[124] Dikomey E, Jung H. Correlation between thermal radiosenzita-tion and slowly-rejoining DNA strand breaks in CHO cells. IntJ Radiat Biol 1995;68:227–33.

[125] Jorritsma JBM, Kampinga HH, Scaf AHJ, Konings AWT.Strand break repair, DNA polymerase activity and heat radiosen-zitation in thermotolerant cells. Int J Hyperthermia 1985;1:131–45.

[126] Kampinga HH, Jorritsma JBM, Konings AWT. Heat-inducedalterations in DNA polymerase activity in HeLa cells and ofisolated nuclei. Int J Radiat Biol 1985;64:225–30.

[127] Spiro I-J, Denman DL, Dewey WC. Effect of hyperthermia onCHO DNA polymerase a and ß. Radiat Res 1982;89:134–49.

[128] Agashe VR, Hartl FU. Roles of molecular chaperones incytoplasmatic protein folding. Semin Cell Dev Biol2000;11(1):15–25.

[129] Buchner J. Supervising the fold: functional principles of molec-ular chaperones. FASEB J 1996;10:10–9.

[130] Jaattela M. Heat shock proteins as cellular lifeguards. Ann Med1999;31:261–71.

[131] Morimoto RI. Cells in stress: trancriptional activation of heatshock genes. Science 1993;259:1409–10.

[132] Ciocca DR, Oesterreich S, Chamness GC, McGuire WL, FuquaSA. Biological and clinical implications of heat shock protein27 000 (Hsp27): a review. J Natl Cancer Inst 1993;85(19):1558–70.

[133] Fuller KJ, Issels RD, Slosman DO, Guillet JG, Soussi T, PollaBS. Cancer and the heat shock response. Eur J Cancer1994;30A(12):1884–91.

[134] Kaur J, Kaur J, Ralhan R. Induction of apoptosis by abrogationof HSP70 expression in human oral cancer cells. Int J Cancer2000;85(1):1–5.

[135] Samali A, Holmberg CI, Sistonen L, Orrenius S. Thermotoler-ance and cell death are distinct cellular responses to stress:dependence on heat shock proteins. FEBS Lett 1999;461(3):306–10.

[136] White E. Life, death, and the pursuit of apoptosis. Genes Dev1996;10:1–15.

[137] Ashkenazi A, Dixit VM. Death receptors: signaling and modu-lation. Science 1998;281(5381):1305–8.

[138] Dewey WC, Ling CC, Meyn RE. Radiation-induced apoptosis:relevance to radiotherapy. Int J Radiat Oncol Biol Phys1995;4:781–96.

[139] Kaufmann SH, Earnshaw WC. Induction of apoptosis by cancerchemotherapy. Exp Cell Res 2000;256(1):42–9.


[140] Neubauer A, Thiede C, Huhn D, Wittig B. p53 and inductionof apoptosis as a target for anticancer therapy. Leukemia1996;10(Suppl. 3):S2–4.

[141] Harmon BV, Corder AM, Collins RJ, et al. Cell death inducedin a murine mastocytoma by 42–47 °C heating in vitro: evi-dence that the form of death changes from apoptosis to necrosisabove a critical heat load. Int J Radiat Biol 1990;58:845–58.

[142] Allan DJ, Gobe GC, Harmon BV. The morphological charac-terization of cell death induced by mild hyperthermia andcomparison with death induced by ionizing radiation and cato-toxic drugs. Scanning Electron Microsc 1988;3:1121–33.

[143] Baxter BD, Lavin MF. Specific protein dephosphorylation inapoptosis induced by ionizing radiation and heat shock inhuman lymphoid tumor lines. J Immunol 1992;148:1949–54.

[144] Gabai VL, Zamulaeva IV, Mosin AF, et al. Resistance ofEhrlich tumor cells to apotptosis can be due to the accumula-tion of heat shock proteins. FEBS Lett 1996;375:21–6.

[145] Yonezawa M, Otsuka T, Matsui N, Tsuji H, et al. Hyperther-mia induces apoptosis in malignant fibrous histiocytoma cells invitro. Int J Cancer 1996;66(3):347–51.

[146] Menoret A, Chandawarkar R. Heat-shock protein-based im-munotherapy: an idea whose itime has come. Sem Oncol1998;25(6):654–60.

[147] Suto R, Srivastatva PK. A mechanism for the specific immuno-genecity of heat-shock protein chaperoned peptides. Science1995;269:1585–8.

[148] Tamura Y, Peng P, Lui K, Daou M, Srivastatva P. Im-munotherapy of tumors with autologous tumor-derived heatshock protein prepations. Science 1997;278:117–20.

[149] Botzler C, Schmidt J, Luz A, Jennen L, Issels R, Multhoff G.Differential Hsp70 plasma-membrane expression on primaryhuman tumors and metastases in mice with severe combinedimmunodeficiency. Int J Cancer 1998;77:942–8.

[150] Botzler C, Li G, Issels RD, Multhoff G. Definition of extracel-lular localized epitopes of Hsp70 involved in an NK immuneresponse. Cells Stress Chaperones 1998;3(1):6–11.

[151] Multhoff G, Botzler C, Wiesnet M, et al. A stress-inducible 72kDa heat shock protein (HSP72) is expressed on the surface ofhuman tumor cells, but not on normal cells. Int J Cancer1995;61:272–9.

[152] Multhoff G, Botzler C, Jennen L, Schmidt J, Ellwart J, IsselsR. Heat shock protein 72 on tumor cells: a recognition struc-ture for natural killer cells. J Immunol 1997;158(9):4341–50.

[153] Multhoff G, Mizzen L, Winchester CC, et al. Heat shockprotein 70 (Hsp70) stimulates proliferation and cytolytic activ-ity of natural killer cells. Exp Hematol 1999;27(11):1627–36.

[154] Roigas J, Wallwn ES, Loenings SA, Moseley PL. Heat shockprotein (HSP72) surface expression enhances the lysis of ahuman renal cell carcinoma by IL-2 stimulated NK-cells. AdvExp Med Biol 1998;451:225–9.

[155] Edwards MJ. Apoptosis, the heat shock response, birth defects,disease and cancer. Where are the common links. Cell StressChaperons 1998;3(4):213–20.

[156] Leppa S, Sistonen L. Heat-schock response-pathophysiologicalimplications. Ann Med 1997;29:73–8.

[157] Lindquist S, Craig EA. The heat shock proteins. Ann Rev Gen1988;22:631–77.

[158] Heide I, Thiede C, Sonntag T, et al. The status of p53 in themetastatic progression of colorectal cancer. Eur J Cancer1997;33(8):1314–22.

[159] Sturm I, Kohne C-H, Wolff G, et al. Analysis of the p53/BAXpathway in colorectal cancer: low BAX is a negative prognosticfactor in patients with resected liver metastases. J Clin Oncol1999;17:1364–74.

[160] Vogelstein B, Kinzler KW. p53 Function and dysfunction. Cell1993;70:523–6.

[161] Agoff SN, Hou J, Linzer DI, Wu B. Regulation of the human

hsp70 promoter by p53. Science 1993;259(5091):84–7.[162] Finlay CA, Hinds PW, Tan TH, Eliyahu D, et al. Activating

mutations for transformation by p53 produce a gene productthat forms an hsp-p53 complex with an altered half life. MolCell Biol 1988;8:531–9.

[163] Matsumoto H, Shimura M, Omatsu T, Okaichi K, Majima H,Ohnishi T. p53 Proteins accumulated by heat stress associatewith heat shock proteins HSP72/HSC73 in human glioblastomacell lines. Cancer Lett 1994;87:39–46.

[164] Rau B, Gaestel M, Wust P, et al. Preoperative radio-chemo-thermo-therapy of rectal cancer: analysis of treatment efficacyand heat shock response. Radiat Res 1999;151(4):479–88.

[165] Sakakura C, Koide K, Ichikawa D, et al. Analysis of histolog-ical therapeutic effect, apoptosis rate and p53 status aftercombined treatment with irradiation, hyperthermia and 5-flourouracil suppositories for advanced rectal cancers. Br JCancer 1998;77(1):159–66.

[166] Jonas S, Rayes N, Neumann U, et al. De novo malignanciesafter liver transplantation using tacrolimus-based protocols orcyclosporine-based quadruple immunosuppression with an in-terleukin-2 receptor antibody or antithymocyte globulin. Can-cer 1997;80(6):1141–50.

[167] Oertel S, Anagnostopoulos I, Bechstein WO, Liehr H, RiessHB. Treatment of posttransplant lymphoproliferative disorderwith the anti-CD20 monoclonal antibody rituximab alone in anadult after liver transplantation: a new drug in the treatment ofposttransplant lymphoproliferative disorder after solid organtransplantation. Transplantation 2000;2000(69):3.

[168] Shakhar G, Ben Eliyahu S. In vivo "-adrenergic stimulationsuppresses natural killer activity and compromises resistance totumor metastasis in rats. J Immunol 1998;160:3251–8.

[169] Wang CY, Snow JL, Su WP. Lymphoma associated withhuman immunodeficiency virus infection. Mayo Clin Proc1995;70(7):665–72.

[170] Amaning EP, Olszewski WL. Kinetics of distribution of recircu-lating lymphocytes during whole body hyperthermia. Arch Im-munol Ther Exp Warsz 1994;42:107–13.

[171] Azocar J, Yunis EJ, Essex M. Sensitivity of human naturalkiller cells to hyperthermia. Lancet 1982;1:16–7.

[172] Kearns RJ, Ringler S, Krakowka S, Tallman R, Sites J, Ogles-bee MJ. The effects of extracorporal whole body hyperthermiaon the functional and phenotypic features of canine peripheralblood mononuclear cells (PBMC). Clin Exp Immunol1999;116:188–92.

[173] Shen RN, Lu L, Young P, Shidnia H, Hornback NB, Brox-meyer HE. Influence of elevated temperature on natural killercell activity, lymphokine-activated killer cell activity and lectin-dependent cytotoxicity of human umbilical cord blood andadult blood cells. Int J Radiat Oncol Biol Phys 1994;29:821–6.

[174] Wang WC, Goldman LM, Schleider DM, et al. Fever-rangehyperthermia enhances L-selectin-dependent adhesion oflymphocytes to vascular endothelium. J Immunol1998;160(2):961–9.

[175] Yang H, Lauzon W, Lemaire I. Effects of hyperthermia onnatural killer cells: inhibition of lytic function and microtubuleorganization. Int J Hyperthermia 1992;8:87–97.

[176] Evans SS, Bain MD, Wang WC. Fever-range hyperthermiastimulates !4"7 integrin-dependent lymphocyte-endothelial ad-hesion. Int J Hyperthermia; 16(1):45–9.

[177] Kappel M, Stadeager C, Tvede N, Galbo H, Pedersen BK.Effects of in vivo hyperthermia on natural killer cell activity, invitro proliferative responses and blood mononuclear cell sub-populations. Clin Exp Immunol 1991;84:175–80.

[178] Di YP, Repasky EA, Subjeck JR. Distribution of HSP70,protein kinase C, and spectrin is altered in lymphocytes duringa fever-like hyperthermia exposure. J Cell Physiol1997;172(1):44–54.


[179] Downing JF, Martinez Valdez H, Elizondo RS, Walker EB,Taylor MW. Hyperthermia in humans enhances interferon-#synthesis and alters the peripheral lymphocyte population. JInterferon Res 1988;8:143–50.

[180] Kappel M, Poulsen TD, Galbo H, Pedersen BK. Influence ofminor increases in plasma catecholamines on natural killer cellactivity. Horm Res 1998;49(1):22–6.

[181] Hammami MM, Bouchama A, Shail E, Aboul Enein HY, alSedairy S. Lymphocyte subsets and adhesion molecules expres-sion in heatstroke and heat stress. J Appl Physiol1998;84:1615–21.

[182] Robins HI, Kutz M, Wiedemann GJ, et al. Cytokine inductionby 41.8 °C whole body hyperthermia. Cancer Lett1995;97:195–201.

[183] Downing JF, Taylor MW. The effect of in vivo hyperthermiaon selected cytokines in men. Lymphokine Res 1987;6:103–9.

[184] Ahlers O, Boehnke T, Kerner T, et al. Lymphocyte alterationsduring hyperthermia. Br J Anesthesia 1998;80(Suppl. 1):301.

[185] Ahlers O, Boehnke T, Kerner T, et al. Changes in serumcytokine levels during induced whole body hyperthermia. CritCare 1999;3(Suppl. 1):P082.

[186] Hegewisch-Becker S, Nierhaus A, Panse J, Wiedemann G,Hossfeld DK. Whole body hyperthermia has a stimulatoryeffect on the immune cell activity in cancer patients. Ann Oncol1998;9(Suppl. 4):136–7.

[187] Faithfull NS, Reinhold HS, Berg APVD, Rhoon GCV, ZeeJVD, Wike-Hooley JL. Cardiovascular changes during wholebody hyperthermia treatment of advanced malignancy. Eur JAppl Physiol 1984;53:274–81.

[188] Kerner T, Deja M, Ahlers O, et al. Whole body hyperthermia:a secure procedure for patients with various malignancies.Intensive Care Med 1999;25:959–65.

[189] Hoffman Goetz L, Pedersen BK. Exercise and the immunesystem: a model of the stress response. Immunol Today1994;15:382–7.

[190] Benschop RJ, Schedlowski M, Wienecke H, Jacobs R, SchmidtRE. Adrenergic control of natural killer cell circulation andadhesion. Brain Behav Immun 1997;11:321–32.

[191] Felten SY, Madden KS, Bellinger DL, Kruszewska B, Moyni-han JA, Felten DL. The role of the sympathetic nervous systemin the modulation of immune responses. Adv Pharmacol1998;42:583–7.

[192] Kerner T, Ahlers O, Spielmann S, et al. L-selectin in traumapatients: a marker for organ dysfunction and outcome. Eur JClin Invest 1999;29(12):1077–86.

[193] Madden KS, Sanders VM, Felten DL. Catecholamine influ-ences and sympathetic neural modulation of immune respon-siveness. Annu Rev Pharmacol Toxicol 1995;35:417–48.

[194] Maes M, Van Bockstaele DR, Gastel A, et al. The effects ofpsychological stress on leukocyte subset distribution in humans:evidence of immune activation. Neuropsychobiology1999;39:1–9.

[195] Iwakabe K, Shimada M, Ohta A, et al. The restraint stressdrives a shift in Th1/Th2 balance toward Th2-dominant immu-nity in mice. Immunol Lett 1998;62:39–43.

[196] Hegewisch-Becker S. MDR 1 reversal: criteria for clinical trialsdesigned to overcome the multidrug resistance phenotype.Leukemia 1996;10(Suppl. 3):S32–8.

[197] Goldstein LJ, Galski H, Fojo A, et al. Expression of a multi-drug resistance gene in human cancers. J Natl Cancer Inst1989;81:116–24.

[198] Filipits M, Suchomel RW, Zochbauer S, et al. Clinical rele-vance of drug resistance genes in malignant diseases. Leukemia1996;10(Suppl. 3):S10–7.

[199] Hettinga JV, Konings AW, Kampinga HH. Reduction of cellu-lar cisplatin resistance by hyperthermia—a review. Int J Hyper-thermia 1997;13(5):439–57.

[200] Hildebrandt B, Loeffel J, Deja M, et al. Whole body hyperther-mia induces a renewed remission in a patient with refractorygerm cell tumor after high-dose chemotherapy. Ann Hematol1998;77(Suppl. 2):222.

[201] Chin KV, Tanaka S, Darlington G, et al. Heat shock andarsenite increase expression of the multi-drug resistance (MDR1) gene in human carcinoma cells. J Biol Chem 1990;26(5):221–6.

[202] Fisher B, Kraft P, Hahn GM, Anderson RL. Thermotolerancein the absence of induced heat shock proteins in a murinelymphoma. Cancer Res 1992;52(10):2854–61.

[203] Oh HJ, Chen X, Subjeck JR. Hsp 110 protects heat-denatu-rated proteins and confers cellular thermoresistance. J BiolChem 1997;272(50):31636–40.

[204] Stein U, Rau B, Wust P, Walther W, Schlag PM. Hyperthermiafor treatment of rectal cancer. Evaluation for induction ofmultidrug resistance (mdr1) expression. Int J Cancer1999;80:5–12.

[205] Streffer C. Biological basis of thermotherapy (with specialreference to oncology). In: Gautherie M, editor. BiologicalBasis of Oncologic Thermotherapy. Berlin: Springer, 1990:1–72.

[206] Wust P, Rau B, Gellermann J, et al. Radiochemotherapy andhyperthermia in the treatment of rectal cancer. Rec Res CancerRes 1998;146:175–91.

[207] Jung H. Interaction of thermotolerance and thermosensitizationinduced in CHO-cells by combined hyperthermic treatments at40 and 43 °C. Radiat Res 1982;91:433–46.

[208] Dewey WC. Mechanism of thermal radiosensitization. In:Urano M, Douple E, editors. Biology of Thermal Potentiationof Radiotherapy, vol. 2. Utrecht, Tokyo: VSP, 1989:1–16.

[209] Myers R, Field RB. The response of the rate tail on tocombined heat and X-rays. Br J Radiol 1977;52:581–6.

[210] Urano M. Thermal effect on the radiation response of normaltissues. In: Urano M, Douple E, editors. Biology of ThermalPotentiation of Radiotherapy, vol. 2. Utrecht, Tokyo: VSP,1989:83–112.

[211] Urano M. Thermochemotherapy: from in vitro and in vivoexperiments to potential clinical application. In: Urano M,Douple E, editors. Chemopotentiation by Hyperthermia, vol. 4.Utrecht, Tokyo: VSP, 1994:169–204.

Biographies

The authors, members of the ‘Berlin HyperthermiaStudy Group’, are medical doctors. The work wasfinanced by grants of the German Research Ministry(SFB 273-Technical, clinical, and biological hyperther-mia research) and the Deutsche Krebshilfe/GermanCancer Aid (Whole-body hyperthermia).

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