MEDICAL INTELUGENCE UNIT
Hyperthermia in Cancer Treatment: A Primer Gian Franco Baronzio, M.D. Radiotherapy Unit Policlinico di Monza Monza, Italy
E. Dieter Hager, M.D., Ph.D., D.Sc. Department of Hyperthermia BioMed-Klinik GmbH Bad Bergzabern, Germany
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HYPERTHERMIA IN CANCER TREATMENT: A PRIMER Medical Intelligence Unit Landes Bioscience / Eurekah.com Springer Science+Business Media, LLC
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This Book Is Dedicated to: My tender wife, Anna, to Attilio, Paul Junior and Miriam, my dear children who unselfishly endured my work. My father, Paul Senior, and my mother, Louise, who inspired my life and without whom I would never have achieved what I have. Peter M. GuUino, Gian Luigi Monticelli and Fulvio Pezza, my teachers who helped me to mature from a naiVe scientist to a more mature physician and who share their knowledge, experience and friendship. Gian Franco Baronzio My wife Claudia, to my dear children Marsha, Jonas and Simon, to my teachers in hyperthermia, and to all the scientists doing tough research in the emerging field of hyperthermia. Erich Dieter Hager
CONTENTS Preface Introduction Section I: Physical Aspects of Hyperthermia 1. Hyperthermia, Physics, Vector Potential, Electromagnetic Heating: A Primer Ugo Cerchiari Direct Heating Heating by Ultrasounds Heating by Electromagnetic Methods 2. Thermometry: Clinical Aspects and Perspectives Barbara Baiotto and Piergiorgio Marini Calculation of the Produced Power Deposition Calculation of the Resulting Temperature Profile: The "Bio-Heat Equation" Experimental Measurement of Temperature Invasive Methods Non-Invasive Methods 3. Physical Background and Technical Realizations of Hyperthermia Andras SzasZy Oliver Szasz and Nora Szasz Characterization Demand Control-Parameters Technical Challenges Electromagnetic Heating Processes Comparison of the Methods New Directions in Electromagnetic Oncologic Hyperthermia 4. Thermotherapy and Nanomedicine: Between Vision and Reality Andreas Jordan Section II: Biological Aspects of Hyperthermia 5. Influence of Tumor Microenvironment on Thermoresponse: Biologic and CHnical ImpUcations Gian Franco Baronzio, Alberto Gramaglia, Attilio Baronzio and Isabel Freitas Hypoxia, HIF-1 and Angiogenesis Tumor Bioenergetic Status, Hypoxia, pH Hyperthermia Effects on Tumor Blood Flow and Endothelium Clinical Methods for Improving Thermoresponse Tumor Blood Flow (TBF) Modulation Microenvironment Modification Heat Delivery Methods
xv xvii 1 3 5 5 12 19 19 21 23 23 24 27 29 34 37 40 50 52 60
65
67
(cH 73 79 81 82 83 85
6. Hyperthermia and Angiogenesis: Results and Perspectives Cristina Roca andLuca Primo Bloodvessels, Blood Flow and Microenvironment Tumor Angiogenesis Molecular Mechanisms of Angiogenesis Inhibition by H T Perspectives 7. Vascular Effects of Localized Hyperthermia Debra K. Kelleher and Peter Vaupel Vascular Effects of Localized Hyperthermia Vascular Effects of Combined Modalities 8. On the Biochemical Basis of Tumour Damage by Hyperthermia Paola Pietrangeli and Bruno Mondovl Glycolysis and Respiration Hyperthermia and DNA, RNA and Protein Synthesis Tumour Membranes and Hyperthermia Hyperthermia and Liposomes Hyperthermia and Immune Response Heat Shock Proteins Hyperthermia and Oxygen Free Radicals Hyperthermia and Amine Oxidases 9. Results of Hyperthermia Alone or with Radiation Therapy and/or Chemotherapy Pietro Gahriele and Cristina Roca Hyperthermia Alone Hyperthermia and Radiotherapy Interstitial Thermo-Brachytherapy Intracavitary Hyperthermia Hyperthermia Radiation and Chemotherapy Prognostic Factors Affecting the Response to Hyperthermia Perspectives
92 93 94 95 96 99 100 102 110 Ill Ill 112 112 113 114 115 115
119 120 121 122 123 123 123 124
10. Thermo-Chemo-Radiotherapy Association: Biological Rationale, Preliminary Observations on Its Use on Malignant Brain Tumors 128 Gian Franco Baronzio, Vincenzo Cerreta, Attilio Baronzio, Isabel Freitas, Marco Mapelli and Alberto Gramaglia Radiotherapy and Hyperthermia Interaction 128 Chemotherapy and Hyperthermia Interaction 135 Effects of Hyperthermia on Drugs Uptake and Targeting 137 Methods for Enhancing Thermal Sensitivity 138 Thermo-Chemo-Radiotherapy for Malignant Brain Tumors 143 Experimental Studies 145 Results 148
11. A Step Deep on Hyperthermia, Hypoxia and Chemotherapy Interactions Giammaria Fiorentiniy Ugo De Giorgiy Maurizio CantorCy Andrea Mamhrini andStefano Guadagni The Problem of Tumor Hypoxia in Anticancer Therapy Hypoxia and Chemotherapy Hyperthermia and Chemotherapy Hyperthermia and Antineoplastic Drugs Selection
156 157 159 159
Section III: Clinical Aspects of Hyperthermia
165
12. Locoregional Hyperthermia E. Dieter Hager Clinical Trials on Hyperthermia Non-Thermal Effects
167
13. Hyperthermia and Radiotherapy in the Management of Prostate Cancer Sergio Villa Hyperthermia and Radiotherapy: Toxicity
156
170 176
183 185
14. Tumor Ablation Using Radiofrequency Energy: Technical Methods and Application on Liver Tumors 190 Johannes-Marcus Hdnslery Luigi Solhiatiy E. Dieter Hagery Tiziana leracey Luca Cova and Gian Franco Baronzio Technical Development 191 Treatment 194 15. Cytoreduction, Peritonectomy and Hyperthermic Antiblastic Peritoneal Perfusion for the Treatment of Peritoneal Carcinomatosis Michele De Simone and Marco Vaira Generalities and Indications Surgical Techniques Semiclosed HAPP Technique Clinical Experience and Results 16. Hyperthermic Isolated Limb Perfusion Michele De Simone and Marco Vaira Introduction and Indications of Limb Perfusion Surgical Procedure Clinical Experiences and Results
199 199 201 205 206 208 208 209 215
17. Intracavitary Hyperthermic Perfusion E. Dieter Hager Clinical Experience 18. Whole Body Hyperthermia at 43.5-44**C: Dream or Reality? Alexey V. Suvemev, Georgy V. Ivanov, Anatoly V. Efremov and Roman Tchervov Necessity of High-Level Whole Body Hyperthermia Risk Factors of Whole Body Hyperthermia Over 43 °C and Pathogenetic Substantiation of Their Overcoming What Are Possible Ways to Suppress the Activity of Trypsin during Hyperthermia? Technological and Anaesthetic Features of Whole Body Severe Hyperthermia 19. Extreme Whole-Body Hyperthermia with Water-Filtered Infirared-A Radiation Alexander von Ardenne andHolger Wehner Technical Realisation of Water-Filtered Infrared-A Radiation Tolerance of Extreme Whole-Body Hyperthermia w^ith Water-Filtered Infrared-A Radiation First Clinical Results
218 220 227
227 228 230 232
237 239 240 242
20. Effects of Local and Whole Body Hyperthermia on Immunity 247 Gian Franco Baronzio, Roberta Delia Seta, Mario DAmico, Attilio Baronzioy Isabel FreitaSy Giorgio Forzenigo, Alberto Gramaglia andE. Dieter Hager Tumor Immunity 247 Therapeutic Modalities 250 Effects of Thermal Component of Fever on Immune System 254 Effects of Induced Thermal Elevation (Hyperthermia) on Immunity 256 Specific Effects of Hyperthermia on Immune Therapeutic Modalities 259 Effects of Hyperthermia on Lymphocyte Homing 261 The Danger Model and Hyperthermia-Effects on Dendritic Cell Maturation and Stimulation of Innate-Adaptive Immunity 261 Effects of Hyperthermia on Metastatic Process 264
21.
Fever, Pyrogens and Cancer Ralf Kleefand E. Dieter Hager History and Background Rationale Epidemiology The Immunological Basis of Endo- and Exotoxin-Induced Tumor Regression Proposed Mechanism of Action Toxicity
22. Future Perspectives of Interstitial and Perfusional Hyperthermia Gian Franco Baronzioy Michele De Simone, Gianmaria Fiorentiniy Salvatore D*Angelay Giovanni Visconti andE. Dieter Hager Radiofrequency Ablation (RFA) Liver Cancer Renal Cell Carcinoma Breast Cancer Lung Tumors Bone Tumors Miscellaneous Tumors Treated with RFA Perfusional Treatment Addendum: Radiofrequency Thermal Ablation in the Treatment of Lung Malignancies Cosimo Gadaletay Anna Catino and Vittorio Mattioli Patients and Methods Results Discussion Index
276 276 277 279 286 299 308 338
340 342 342 343 343 344 344 344 352 353 354 356 361
EDITORS Gian Franco Baronzio Radiotherapy Unit Policlinico di Monza Monza, Italy Email:
[email protected] Chapters 5, 10, 14, 20, 22
E. Dieter Hager Department of Hyperthermia BioMed-KlinikGmbH Bad Bergzabern, Germany Email:
[email protected] Chapters 12, 14, 17. 20, 21, 22
CONTRIBUTORS Barbara Baiotto Medical Physics Unit Institute for Cancer Research and Treatment (IRCC) Turin, Italy Chapter 2
Ugo Cerchiari National Cancer Institute, Milan Milano, Italy Email:
[email protected] Chapter 1
Attilio Baronzio University of Novara Pharmacology DPT Novara, Italy Chapters 5. 10,20
Vincenzo Cerreta Radiotherapy Unit Policlinico Di Monza Monza, Italy Chapter 10
Maurizio Cantore Department of Oncology City Hospital Carrara Massa-Carrara, Italy Chapter 11
Luca Cova Interventional Radiology Unit Busto Arsizio Hospital Busto A (VA), Italy Chapter 14
Anna Catino Interventional Radiology Operative Unit Critical Area Department Oncology Institute Bari, Italy Chapter 22
Mario D'Amico Hematology and Oncology Unit Carrara Hospital Carrara, Italy Chapter 20
Salvatore D'Angelo Maffucci Liver Unit Moscati Hospital Avellino, Italy Chapter 22 Ugo De Giorgi Medical Oncology Unit "S. Giuseppe" Hospital Empoli, Florence, Italy Chapter 11 Michele De Simone Department of Surgical Oncology "S. Giuseppe" Hospital Empoli, Florence, Italy Email: m.desimone@usll 1 .tos.it Chapters 15> 16, 22 Roberta Delia Seta Hematology and Oncology Unit Carrara Hospital Carrara, Italy Chapter 20 Anatoly V. Efremov International Health Center Novosibirsk, Russia Chapter 18 Gianmaria Fiorentini Department of Oncology "S. Giuseppe" Hospital Empoli, Florence, Italy Email: oncologiaempoli@usl 11. toscana. it Chapters 11, 22 Giorgio Forzenigo Gynecologic Oncology Unit Gallarate Hospital Gallarate (VA), Italy Chapter 20
Isabel Freitas Department of Animal Biology CNR Institute of Molecular Genetics Section of Histochemistry and Cytometry University of Pavia Pavia, Italy Chapters 5, 10, 20 Cosimo Gadaleta Interventional Radiology Operative Unit Critical Area Department Oncology Institute Bari, Italy Email:
[email protected] Chapter 22 Pietro Gabriele Radiotherapy Unit Institute for Cancer Research and Treatment Candiolo, Turin, Italy Email:
[email protected] Chapter 9 Alberto Gramaglia Radiotherapy Unit Policlinico di Monza Monza, Italy Chapters 5, 10, 20 Stefano Guadagni Department of Surgical Sciences University of L'Aquila L^Aquila, Italy Chapter 11 Johannes-Marcus Hansler Department of Internal Medicine I University of Erlangen-Nuremberg Erlangen, Germany Email: j ohannes. haensler@ med 1 .imed. uni-erlangen.de Chapter 14
Tiziana lerace Interventional Radiology Unit Busto Arsizio Hospital Busto A (VA), Italy Chapter 14 Georgy V. Ivanov International Health Center Novosibirsk, Russia Chapter 18 Andreas Jordan MagForce Nanotechnologies GmbH and Center of Biomedical Nanotechnology (CBN) Department of Radiology Charit^ - University Medicine Berlin Berlin, Germany Email:
[email protected] Chapter 4 Debra K. Kelleher Institute of Physiology and Pathophysiology University of Mainz Mainz, Germany Email:
[email protected] Chapter 7
RalfKleef Institute for Hyperthermia and Immunotherapy Vienna, Austria Email:
[email protected] Chapter 21 Andrea Mambrini Department of Oncology City Hospital Carrara Massa-Carrara, Italy Chapter 11
Marco Mapelli Radiotherapy Unit Physics Department Policlinico di Monza Monza, Italy Chapter 10 Piergiorgio Marini Medical Physics Unit Institute for Cancer Research and Treatment (IRCC) Turin, Italy Email:
[email protected] Chapter 2 Vittorio Mattioli Interventional Radiology Operative Unit Critical Area Department Oncology Institute Bari, Italy Chapter 22 Bruno Mondovl Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and C.N.R. Centre of Molecular Biology University "La Sapienza" Roma, Italia Email:
[email protected] Chapter 8 Paola Pietrangeli Department of Biochemical Sciences "A. Rossi Fanelli" and C.N.R. Centre of Molecular Biology "La Sapienza" University Rome, Italy Chapter 8 Luca Primo Division of Molecular Angiogenesis Institute for Cancer Research and Treatment Candiolo, Italy Email:
[email protected] Chapter 6
Cristina Roca Division of Molecular Angiogenesis Institute for Cancer Research and Treatment (IRCC) Candiolo, Turin, Italy Email:
[email protected] Chapters 6, 9 Luigi Solbiati Interventional Radiology Unit Busto Arsizio Hospital BustoA(VA),Italy Chapter 14 Alexey V. Suvernev International Health Center Novosibirsk, Russia Chapter 18 Andras Szasz Biotechnics Department Faculty of Engineering Szent Istvan University Budapest, Hungary Email:
[email protected] Chapter 3 Nora Szasz Biotechnics Department Faculty of Engineering Szent Istvan University Budapest, Hungary Chapter 3 Oliver Szasz Biotechnics Department Faculty of Engineering Szent Istvan University Budapest, Hungary Chapter 3
Roman Tchervov Siberian Institute of Hyperthermia Iskitim, Russia Email:
[email protected] Chapter 18 Marco Vaira Department of Surgical Oncology "S. Giuseppe" Hospital Empoli, Florence, Italy Chapters 15, 16 Peter Vaupel Institute of Physiology and Pathophysiology University of Mainz Mainz, Germany Chapter 7 Sergio Villa National Cancer Institute Milan Radiotherapy Service Milano, Italy Email:
[email protected] Chapter 13 Giovanni Visconti Endocrinologist Ferno (VA), Italy Chapter 22 Alexander von Ardenne Von Ardenne Institute Dresden, Germany Email:
[email protected] Chapter 19 Holger Wehner Wilhelmshaven, Germany Chapter 19
PREFACE Although remarkable progress has been made in cancer therapy, many cancers, particularly solid cancers, are still untreatable by conventional therapies such as radiation, immunotherapy, surgery or chemotherapy. This creates the need to improve cancer treatment. Hyperthermia for its synergistic action with the aforementioned modalities may be considered the fifth modality of treatment. Hyperthermia is defined as a therapy in which tumor temperature is raised to values between 4 r C and 45**C by external means. It can be applied locally/ regionally or to the whole body depending from the stage of the cancer patients. For decades hyperthermia has been an area of laboratory investigation with moments of enthusiasm and disappointment, but now there is renewed interest. Its effectiveness as a cancer treatment has been demonstrated by many trials in Europe. These trials have highlighted that hyperthermia improves cancer treatment results while decreasing the side effects of conventional therapies. Following overviews on hyperthermia physics, this book comprehensively describes the biological rationale for associating hyperthermia with radiation and chemotherapy and the biological and clinical effects of heat on cancerous and normal tissues. Chapters are arranged in three main sections (physical and methodological studies, biologic principles, clinical studies). The first part devoted to the physical principles underlying heat generation in tumor tissue has been kept to a minimum, so as not to put off clinicians or students. An entire chapter regards thermometry since temperature measurements, or better, thermal dose calculations are clinically critical. Currendy no simple methods of temperature measurement inside tumor mass are available. The advent of noninvasive thermometry is warranted; some attempts have been made using ultrasound and magnetic resonance. Unfortunately, these measurements call for skilled teams composed of medical physicists and clinicians. Nanotherapy application with heat is reviewed by an expert in the field. The interactions of hyperthermia with tumor metabolism and its environment, particularly the effects on angiogenesis and vasculature, are discussed broadly and in depth in the second section. A chapter describes the tumor microenvironment and its manipulation in order to increase thermoresponse. A specific chapter is devoted to clinical trials with chemotherapy and radiotherapy, offering the opportunity to understand the therapeutic gain of heat. Aspects of tumor biology relevant to this kind of treatment and especially to brain tumors are described with particular attention to their clinical relevance. The third section of the book deals with the clinical applications of radiofrequency and perfusion hyperthermia; other methods for generating heat such as microwaves or ultrasound have been avoided. Interstitial hyperthermia applications on the liver and antiblastic limb perfiision applications are described by experts in the field.
Whole body hyperthermia treatment is described by two groups of authors that use different modalities of heating. Taking into account the side effects of various cancer therapies on immunity, we thought it appropriate to evaluate the various therapeutic approaches and interactions of this kind of therapy with immunity. A chapter on fever therapy has been also added, and the reader can understand the specific benefits of the thermal component of fever on the immune response. The main purpose of the book is clinical, and it must be considered a primer or an update for experts on the matter. One of our purposes is to provide physicists and engineers with information on the biological effects of heat on tumor tissue, aspects not deeply discussed in bioengineering curricula. We hope this book will be of interest to internists, oncologists and to all physicians involved in the management of cancer patients. Gian Franco Baronzio Erich Dieter Hager
= = =
INTRODUCTION
= = =
Introduction and Brief Historical Notes on Hyperthermia Ifknowledge can create problems, it is not through ignorance that we can solve them. —Isaac Asimov The art of medicine consists in amusing the patient while nature cures the disease. —Voltaire (1694-1778)
According to the National Institutes of Health (NIH) hyperthermia (also called thermal therapy or thermotherapy) is defined as a type of cancer treatment in which body tissue is exposed to high temperatures (up to 106*'F), to damage and kill cancer cells, or to make cancer cells more sensitive to the effects of radiation and certain anticancer drugs. Hyperthermia (HT) is used as an adjunct therapy to radiotherapy and/or chemotherapy to increase their effectiveness,^'^ but hyperthermia alone exhibits both antineoblastic and immunological effects. For decades hyperthermia has been an area of investigation with moments of excitement and disappointment, but now encouraging clinical results have renewed interest in its clinical application.^ Historically, hyperthermia was used many centuries ago by Romans, Greeks and Egyptians to cure breast masses.^ Indian ayurvedic physicians practiced local and whole body hyperthermia in 3000 BC. The method consisted of at least five stages of oleation, dietary regimens and purgation with locally or whole body heat application, e.g., a poultice of cotton wool or heated stones employed to treat the liver. Whole body hyperthermia (WBHT) was obtained using vapor produced by sprinkling liquids over heated stones, bricks or metal blocks. Many recommendations and simple methods for estimating the quantity of heat delivered have been described.^ In 1868 Busch in Germany concluded that fever induced by certain bacteria as erysipelas can cause tumor regression or cure cancer. This was concluded after the observations of a patient with a soft tissue sarcoma of the neck infected by erysipelas.^ The causative agent (streptococcus) at that time was not identified. Subsequently, in 1891, a young American surgeon named Coley, unaware of Buschs observation, observed a regression of a soft tissue sarcoma in a patient infected by erysipelas. Stunned by the finding, he searched the medical literature and found many publications confirming the same observation.^'^ Coley initially prepared a culture of streptococci injected at the tumor site with encouraging results. He even noted that the presence of Serratia marcenscens could enhance the virulence of streptococci and that a remote injection from the tumor could result equally in tumor regression. After these observations Coley incorporated Serratia marcenscens
into the streptococcal vaccine, forming the "Coley s toxin"/ The intravenous route was the most effective, and a dose of the toxin was considered sufficient only if accompanied by fever (39-40**C). Sustained pyrexia was considered the critical point in tumor regression.^'^ It was also observed that those who developed the highest fever were most often the ones with the longest survival. Other antitumoral effects not linked to fever are now recognized.^ The fever and pyrexia inducers are now recognized to be caused by tumor necrosis factor-alpha (TNF-a) and other cytokines.^ In the last decade numerous randomized or nonrandomized clinical trials have been conducted.^'^ Most studies were done in combination with radiotherapy (RT) and chemotherapy, in different order, to obtain a better loco-regional control of superficial tumors. Among these, recurrent and primary breast lesions, head and neck neoplasms and melanoma have been treated with radiotherapy in association with hyperthermia. The thermal enhancement ratio was increased for all cases from 1.4 to about 2. The most important prognostic factors for a complete response were radiation dose, tumor size, minimum thermal dose and temperature. The total number of heat fractionations delivered do not appear to be important, provided that adequate heat is delivered in at least one or two sessions. ^° Although there are positive clinical trials, oncologists are skeptical about hyperthermia even if hyperthermia is the only therapy able to exploit the unfavorable tumor microenvironment. In fact, within tumors, regions with reduced blood supply, with active anaerobic metabolism and low pH, are the most sensitive to the cytotoxic effects of hyperthermia as compared to radiotherapy or chemotherapy. For larger tumors, acidic-hypoxic environments are the rule. Furthermore, local hyperthermia (LHT) has been demonstrated to induce radiosensitization at temperatures < 42**C, and to increase oxygenation, an issue which would partially explain its radiosensitization effect.^ Some trials on esophageal cancers have been done using (triple modality) radiotherapy, hyperthermia, chemotherapy [RT-HT-CH]). The results are encouraging, depending on the disease stage, as two-year survival rates have been observed in the range of 20-30%. Despite this, progression of the disease and relapse are common. In the case of stomach and pancreatic cancers, combined therapy HT + chemotherapy (mitomycin-C, 5-fluorouracil) have been performed with positive effects on survival and on objective complaints. ^^ Interstitial hyperthermia (thermal ablation with RF or laser), has reached good therapeutic targets in terms of clinical results, side effects, limitations and costs. On the wave of these positive results, interstitial hyperthermia is now gaining new fields of applications, e.g., in liver, breast, kidney, bone and lung tumors.
Patients with peritoneal carcinomatosis or sarcomatosis have a poor prognosis even when the disease is confined within the abdominal cavity.^^'^^ Different therapies have been proposed, rangingfi*omsurgery to intracavitary chemotherapy. Regional perfiision chemotherapy achieves a high intraperitoneal concentration, minimizing systemic toxicity. However, an intraperitoneal route cannot guarantee adequate drug penetration into larger tumors. To overcome this problem, debulking surgery combined with chemotherapy has been proposed. ^"^ Despite this procedure, the combined treatment has not improved the clinical outcome markedly. Heat has been demonstrated to boost the activity of some antineoblastic drugs (cisplatinum, doxorubicin, mitomycin-C) suggesting that the combination of debulking surgery + chemo-hyperthermia (HIIC) can maximize the antitumor cytotoxic effect. ^^ Phase III studies are still in progress. Phase II studies were positive for overall and disease-free survival rates; however randomized clinical trials are necessary to provide a definitive response.^^'^^ On the basis of peritoneal chemo-hyperthermia experience, other perfiisional hyperthermia techniques have been developed for treating life threatening tumors confined to liver, lung, pleura and limbs. Isolated organ perfusion systems were developed by Creech. Subsequently, the method was adapted for treating various organs with hyperthermia by different authors including Cavaliere in Italy.^^ Perfusional techniques have become standardized and complications have been reduced to a minimum. Approximately 8 to 10% of primary melanomas involve extremities. In this case all the extremities are at risk, but amputation does not cure the disease. To try a definitive cure isolated limb perfusion (ILP) has become the choice. Initially melphalan was identified as the best agent to use with ILP, subsequently it was demonstrated that increased activity was achieved in combination with hyperthermia. Cytokines have also been approved for combination. Regional hyperthermia + ILP resulted in a response rate between 78 to 8 3 % for melanomas. Based on these results the method was applied to soft tissue sarcomas; melphalan or doxorubicin were used, reaching response rates between 4 5 % and 60%.^^ Recendy European investigators have shown that it is possible to reach a complete response rate near the 90% and a longer duration response by adding to melphalan tumor necrosis factor-alpha (TNF-a).^^' ^^ Different trials in Europe and overseas are in progress with TNF-a, and other biological response modifiers (BRMs) such as interleukin-2 (IL-2) and interferon gamma (INF-y). Notwithstanding these positive results, Takahashi clearly illustrated the problems that must be resolved to consider hyperthermia clinically relevant. They are: specific apparatus to maintain a stable temperature distribution, cost of therapy, long hospital stay, and clinical know-how to avoid complications. Furthermore the author concluded that "For clinicians to accept hyperthermia as conventional
therapy, more evidence must be drawn from prospective studies, and a definitive evaluation of the prognostic factors is needed". ^^ Insight from earUer cHnical trials with hyperthermia indicates: 1. For establishing a hyperthermia clinical trial, quahty criteria are needed; these have been collected separately by Overgaard and Nielsen.^^'^^ 2. Temperature measurements, or better, thermal dose calculations are critical. This confirms the in vitro studies that have established that thermal cytotoxicity is a function of both temperature and time. The measurement of the thermal dose is the great challenge for the future. Actually no simple cUnical methods of temperature acquisition inside tumor mass are available. The advent of noninvasive thermometry is warranted; some attempts have been made using ultrasound and magnetic resonance. Unfortunately, these measurements require skilled teams, composed of physician and clinicians. 3. How many hyperthermic applications are necessary to obtain good clinical results? Animal studies indicate that sometimes only one or two sessions of HT are sufficient. Clinical trials indicate that a single treatment once a week for at least five weeks is necessary but any definitive response has not yet been obtained. The role of the biological effects of hyperthermia like immunotoxicity, antiangiogenesis, and proteasome inhibition have to be further elucidated. Synergistic effects with antibody targeted therapy are also new, promising aspects in hyperthermia treatment. In conclusion, better use of the biological basis of hyperthermia, associated with better thermal dosimetry, will permit hyperthermia to become more than an unfulfilled promise. References 1. Van der Zee J. Heating the patient: A promising approach? Ann Oncol 2002; 13:1173-1184. 2. Urano M, Kuroda M, Nishimura Y. For the clinical application of thermochemotherapy given at mild temperature. Int J Hyperthermia 1999; 15:79-102. 3. Falk MH, Issels RD. Hyperthermia in oncology. Invited review. Int J Hyperthermia 2001; 17:1-18 4. Singh BB. Hyperthermia: an ancient science in India. Int J Hyperthermia 1991; 7:1-6. 5. Busch W.Verhandlungen artzlicher gesellschaften. Berl Klein Wochenschr 1868; 5:137-138. 6. Coley WB. The treatment of malignant tumors by repeated inoculations of erysipelas with a report on ten original cases: Am J Med Sci 1893; 105:487-511.
7. Bickels I, Kollender Y, Merinsky O et al. Coley's toxin: Historical perspective 2002; IMAJ; 4:471-472. 8. Hobohm U. Fever and cancer. Cancer Immunol Immunother 2001; 50:391-396. 9. Dinarello CA. Cytokines as Endogenous pyrogens. In: Macckowiak PA, ed. Fever: Basic mechanisms and management. 2 ed. Lippincott-Raven Publishers, Philadelphia. 1997: 7:87-117. 10. Engin K, Leeper DB, Tupchong L et al: Thermoradiotherapy in the management of superficial malignant tumors. Clin Cancer Res 1995; 1:139-145. 11. Takahashi I, Emi Y, Hasuda S et al. Clinical application of hyperthermia combined with anticancer drugs for the treatment of solid tumors. Surgery 2002; 131:578-84. 12. Rossi C, Foletto M, Pilati P et al. Hyperthermic intraoperative intraperitoneal chemotherapy with cisplatin and doxorubicin in patients who undergo cytoreductive surgery for peritoneal Carcinomatosis and Sarcomatosis. Phase 1 Study. Cancer 2002; 94:492-499. 13. Bozzetti F, Vaglini M, Deraco M. Intraperitoneal hyperthermic chemotherapy in gastric cancer: Rationale for a new approach. Tumori 1998; 84:483-488. 14. Begossi G, Gonzales-Moreno, Ortega-Perez G et al. Cytoreduction and intraperitoneal chemotherapy for the management of peritoneal Carcinomatosis, Sarcomatosis and mesothelioma. E JSO2002; 28:80-87. 15. Speyer JL, Collins JM, Dederick RL et al. Phase 1 and pharmacological studies of 5-Fluouracil administered intraperitoneally. Cancer Res 1980; 40:567-572. 16. Elias DM, Ouellet JF. Intraperitoneal chemohyperthermia: Surgical Oncology Clinics of North America 2001; 10:915-933. 17. Omlor GH. Introduction historical review. In: Omlor,Vaupel, Alexander, eds. Isolated hyperthermic limb perfusion. Austin: R.G. Landes; 1995:1-9. 18. Van der Zee J, Kroon BBR, Nieweg OE et al. Rationale for different approaches to combined melphalan and hyperthermia in regional isolated perfusion. Eur J Cancer 1997; 33:1546-1550. 19. Fraker DL. Hyperthermic regional perfusion for melanoma and sarcoma of the limbs. Curr Prob Surg 1999; 36:844-901. 20. Hokenberger P, Kettelhack C. Clinical management and current research in isolated limb perfusion for sarcoma and melanoma. Oncology 1998; 55:89-102. 21. Overgaard J. The rationale for clinical trials in hyperthermia. In: Field SB, Hand JW, eds. An introduction to the practical aspects of clinical hyperthermia. New York: Taylor&Francis Publishers; 1990:213-242. 22. Nielsen OS, Munro AJ, Warde PR. Assessment of palliative response in hyperthermia. Int J Hyperthermia 1992; 8:11-21.
Acknowledgements I would like to express my sincere gratitude to everyone involved in writing this book. To Francesco Miramonti for his help in realizing many of the figures present in this book, and to Isabel Freitas for her care in ameliorating my limited knowledge. Gian Franco Baronzio In this book Gian Franco Baronzio and I have acknowledged some of the researchers who have contributed to hyperthermia research. This should be a primer with the most relevant research and clinical applications in hyperthermia to date. I want to give a special thanks to all the authors who contributed to this book as a milestone in hyperthermia. Especially I want to thank Gian Franco who initiated this book and who has done so much work and spent so much time preparing this book over the last three years. I think he must have been living with this task day and night all this time. In addition, I would like to thank all colleagues and friends who have contributed to this book and given ideas about interesting, relevant aspects of hyperthermia. Many thanks to my teachers and pioneers in hyperthermia: Josef Issels, Paolo Pontiggia, Harry Le Veen, Manfred von Ardenne, Peter Vaupel, Sergej Osinsky, Sam P. Yarmonenko, Shigeru Fujimoto and Francois Gilly. Erich Dieter Hager Finally, we would like to thank Sara Lord from Landes Bioscience for her qualified assistance in revising this book.
SECTION I
Physical Aspects of Hyperthermia
CHAPTER 1
Hyperthermia, Physics, Vector Potential, Electromagnetic Heating: A Primer Ugo Cerchiari* Abstract
H
eating methodologies of restricted and specific body volumes as means to treat cancer are critically examined from the physical point of view. Difficulties in the application of heating methodologies are considered in relation to the different means giving more space to the means more suitable for modelling. Since the usual approach of electromagnetic heating and of its modelling is difficult, the use of vector potential is suggested and some simple calculations and considerations are presented for electromagnetic field modelling.
Introduction Heat is a mechanical energy of incoherent nature of small volumes of matter typically of atomic or molecular dimensions. Incoherent here means that velocities of near atoms are randomly directed and in solids represent vibration of those small portions of matter with random amplitudes and random phases. "Random" means that all possible movements (degrees of freedom) are present and share almost same energy. This energy is continuously exchanged among them. Temperature T is a parameter that is linked to the energy Q by a coefficient C called specific heat Q = CT. Thus in a homogeneous sample of matter temperature is a "measure" of heat. If the temperature of a volume of biological content increases, as a consequence biochemical reactions increase their speed. Initially this speeds up "life," as can be observed in reptiles or also in many species of mammalians, considering temperature and activity. Yet all functions must increase proportionately. In case of an insufficient supply, substrates are quickly consumed, and essential reactions enter in shortage of biological energy and substances and reject metabolites accumulate. These consequences can lead to essential impairment of functions including repair and transport.Thus, in case of stressed biology, heat may be sufficient to increase death rate in cells. These effects become apparent as temperature goes over a definite threshold (42.5°C). Increasing temperature (beyond 45°C) biological molecules, due to the vibrations of their atoms start to suffer new chemical reactions leading to unwanted products. Those modifications in nature of biological molecules (denaturation) further impair the ability of cells and tissues in doing what they normally do performing their functions. Finally a necrosis is induced. These last thermal effects damage both normal tissues as well as cancer. Thus heat may be used to treat cancer if this energy in mainly distributed to tumours, possibly in *Ugo Cerchiari—National Cancer Institute, Milan, Via G. Venezian 1, 20133 Milano, Italy. Email:
[email protected] Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.
Hyperthermia in Cancer Treatment: A Primer conjunction with drugs and /or radiotherapy and, if raise in temperature is in a limited interval, to avoid death of normal tissue. Cells killing by hyperthermia have to be evaluated by a special kind of dosimetry. Heat deposition measured by power per gram (SAR) has no significance since only thermal damaging effects due to temperature exposition should be taken into consideration. Dose as "the measure of thermal effects" is a function of temperature, of time and of thermal sensitivity of target tissue that may depend on the presence of substrates and of drugs or previous damage. Since even thermometry is not easy at the moment, only simple practical rules can be given, derived by very rough considerations. Chemical reactions starts effectively as thermal energy reaches an activation threshold and increase their velocity as temperature rises (roughly doubling their rate every 10°C) but biological chemistry requires that only a class of reactions should be allowed by limiting the temperature of activation of unwanted reactions. Thus useful reactions are supported by complex mechanisms of transport and catalysis as well as feedback regulation to allow working in a limited temperature range. Equally effective as temperature is shortage of substrates, oxygen especially, as it is well known. So it must be expected that this could be the primary parameter to control beyond temperature. Yet usually temperature is "the" parameter which is controlled and clinical practice su^ests that a temperature of 42.5°C should be kept for 4 hours to obtain the same effect of 45°C for ten minutes. In between of the two a working temperature and a practical time of treatment has to be clinically decided mainly in relation of the weight of the region to be heated and blood perfusion. Various methods have been developed to release heat in tumours having different features: 1. Direct heating by conduction by contact with a heat source: a. localized transcutaneous (superficial) b. invasive, interstitial and intracavitary c. extracorporeal circulation of hot blood in an organ d. heating generalized to whole body by conduction from a thermostatic bath 2. Indirect heating by deposition of coherent energy relaxing locally to incoherent energy (heat): a. mechanical vibration (ultrasound waves) b. low or high frequency electromagnetic fields All these methods face great difficulties deriving mainly from the complex nature of organs and tissues as regards their not uniform physical properties, geometry and blood flow. Since the temperature threshold of biological damage and the increase of the damage with temperature are critical it is immediately obvious that to predict or control the temperature distribution in a body region is mandatory. Unfortunately this is a difficult task. Accepted thermometry requirements are: 1. Overall accuracy < 0.2°C 2. Response time < 4 seconds 3. Sensor size < 2 mm 4. Possibly immunity or no substantial interference with the heating technique Since energy deposition in tissues as well as cooling by blood flow are difficult to model, a good thermometry control with few exceptions is always needed in clinical practice. Unfortunately not invasive thermometry, that in principle coiJd be attained by microwave radiometry, MRI temperature-dependent signal or electrical impedance tomography, still do not meet the requirements for clinical application. Thermometry will be treated in more details elsewhere in this book. As starting sources of Hyperthermia physics we suggest the articles in references 1-4.
HyperthermiUy Physics, Vector PotentiaU Electromagnetic Heating: A Primer
Direct Heating Direct heating is efficiently attained in a range of 10 mm in depth from a heat source covering the lesion with a border in excess of 20 mm. This condition can be met for cutaneous or intracavitary lesions. Interstitial treatments can be planned with linear heat sources spaced no more than 15 mm apart. Sources are tubes 1.6-2 mm in diameter with turbulent hot water flow or electrically heated probes. Normal tissue can be partly preserved by thermally insulating probes with low heat conducting coating in case of tubes or by electrical heated probes of suitable length. Temperature control poses no problem with any kind of invasive thermometry combined with sources. Unfortunately these devices are not commercially available as dedicated systems. Direct heating as a whole body technique as been used in the past by pyrogenic toxins or heat bath (water and/or air) but it is obviously limited in temperature and was usefully used in combination with other agents. Now could be used as a background temperature control in local heating. Yet the experience suggest that it is a risky treatment. Regional perfusion by external circulation of hot blood has also been attempted and also this technique has to be considered of difficult execution.
Heating by Ultrasounds Mechanical oscillations propagate in media and vibration, in contiguous portions of matter, induce a stress due to a difference in phase of local oscillations. During the cycle of stress and relaxation the medium transforms part of the mechanical coherent energy of oscillation (sound) into random movement thus absorbing "sound energy " and transforming it into heat. The portion of coherent energy transformed into heat is called absorbed energy and the loss in coherent energy is called attenuation.The velocity of sound in soft tissues is around 1500 m/s while in lung is significantly less (1000-600 m/s strongly dependent on inflation) and in bone considerably higher (2000-3500 m/s) .Temperature does not affect velocities to much. Mechanical waves are commonly induced in tissues by discs of piezoelectric materials. Piezoelectric discs have two electrodes on each face and if a voltage is applied across the crystals then mechanical compression or dilatation occurs. A piezoelectric disc (transducer) in contact with a medium acts as an oscillating piston if an oscillating voltage is applied. Thus a compression wave with oscillation in forward and backward direction (longitudinal in respect to the thickness of the disc) is generated. This in principle, what really happens is different, since piezoelectric crystals are sufficiently rigid to have their own oscillating frequencies and modes that are excited by mechanical stress making front face oscillate with not uniform amplitude. These not uniform amplitudes could generate small hot spots in front of crystals. To mitigate this problem the exciting frequency is modulated to mix secondary excitement modes and a bolus of water is placed between the disc and the skin to attenuate secondary (higher) frequencies. Transducers have dimensions between 3 and 12 cm in diameters driven by a frequency (v) in the range from 0.5 to 3 MHz (ultrasound US). At these frequencies the wavelength (k) in soft tissues is between 0.5 to 5 mm generating almost plane waves with litde diffiaction. Power densities in front of transducers is in mean 0.5 to 2 W/cm^. At higher power densities oscillation amplitudes can induce "boiling" (cavitation) in the water bolus and bubbles can induce scattering of waves. To avoid cavitation it is necessary to degas the water. Water in front of the disc firsdy heated by US can circulate in a cooler and thus provide cooling in the first centimetre of skin. Let us examine ultrasound waves in a uniform medium to understand some basic properties. A volume, of uniform medium, is excited by a plane transducer T at the left (Fig. 1). In front of T pressure waves are generated in the medium with fronts parallel to the surface of T. A small volume element of cylindrical shape is displaced by the wave from its equilibrium position X of a quantity A.
5
Hyperthermia in Cancer Treatment: A Primer
1r
A* •
A1 da
"
1
W
!
; ^
' • w
^
=;
;
1
dA/dx dx
A _w
^
J
^ 'W"
Figure 1. A cylindrical microscopic volume whose base at equilibrium has position x is displaced by pressure of an amount A. The opposite base is displaced ofA'. Cylinder lenght dx is reduced by an amount dA/dx dx. Displacement is not uniform along x in the volume since pressure wave strains the medium. As a consequence the volume element changes in length of an amount which is: dl = dA/dx dx This changes the original volume dK= dx da by the quantity:
8K=dlda = aA/axdxda Change in volume modifies internal pressure by an amount dp depending on a coefficient K, characteristic of the meditun, called bulk modulus, representing the change of pressure due to the fractional change in volume 8V/dV: dp = K 8V/dV= K aA/ax dx da/(dx da) = K dA/dx Since the strains before and after the volume element are not equal, pressures as well are not equal, thus moving the volume element. This difference in pressure, indicated by d p, depending on the second derivative of A is: d^p = Ka^A/ax2dx The force acting on the volume element, and effectively moving the element, is the pressure across the area da:
df = d^p da = K a^A/ax^ da dx The mass dm of the volume element is related to density p of the medium by: dm = p d a dx Acceleration of the volume element is dl^A/d^ i.e., the second time derivative of the displacement.
HyperthermiUy Physics, Vector PotentiaU Electromagnetic Heating: A Primer
7
Neglecting at the moment the attenuation of the motion due to the frictional force we may write the equation of motion (f = ma) for the volume element:
K a^A/ax^ da dx = p da dx a^A/at^ or a^A/ax^ - p/K a^A/at^ = o
(i)
This equation has the following solution : A = Aocos(27i;(vt-x/X))
(2)
where V is the frequency i.e., the number of oscillation made in one seconds at a fixed point and X is the wave length i.e., the space spanned by an oscillation at a fixed time and AQ represents the maximum displacement due to the vibration of the transducer. It is easily seen, substituting expression (2) into eq. (1), that A-V must be equal to (K/p)^^"^. The solution of eq. (1) represent a simple harmonic wave whose phases (i.e., the argument of the cosine) are constant along planes orthogonal to the direction of propagation x. The crests, where cos(27C(vt -x/A ) = 1 i.e., where the phase is equal to zero, are located where Vt -x/X=0 and thus move at velocity c = x/t = Xv = (K/p)^^^. Sound is only slighdy attenuated in tissues since it travels many X, s. Disc diameter is considerably larger than \ thus oscillations in the medium can be modelled by a simple exponentially attenuated plane wave whose amplitude of oscillation A is given by A=Ao e^-^^ ^ cos(27C(vt -x/X))
(3)
This expression is a solution of an equation slightly more complex than (1), taking in to account that, besides the elastic force K a^A/ax^,during the movement the volume element is also subject to a frictional force proportional to the velocity dA/dx and opposite to it i.e., -T|aA/ at. The coefficient T| is called viscosity. The new attenuated wave equation is :
Ka^A/ax^ - ^^A/^t - p a^A/at^ = o
(4)
The meaning of equation (3) is simply that the amplitude of oscillation decreases exponentially A(x)=Ao e^'^^ giving at a depth x a reduced oscillating amplitude. Substituting expression (3) into eq. (4) it is easily found that the conditions that allow to use (3) as a solution of eq. (4) are: K(|Li2 - {2%IXf) + p(27CV)2 = 0
and
\i = TivWK = r\dK
(5)
Since the frictional coefficient T| is very little, in relation to K, it may be considered zero in the second condition (5). This gives |X = 0, i.e., the exponential factor e^'^^^ is constant and equal to 1 as in eq. (2). The first condition (5) becomes K(27c/A,)^ = p(27CV)'^ corresponding to the previous condition Xv = (K/p) "^ for the velocity. For this reason, and for the great approximations implied in treating biological samples, the velocity c is considered c = {KJp) . Parameters in relations (5) depend on physics and technology: V is chosen by technology, K is very high and T| is experimentally found dependent on V as follows r| = r|tV" with n in the range 1 to 2. The exponent n and the coefficient T|t depend on tissue. Usually |X is of greater practical use then viscosity T|, thus the previous discussion leads to the consideration that: |I = |Xt v"
where |Xt = Tjtc/K.
The usefulness of equation (3) depends only on the fact that it gives the evaluation of attenuation in the medium but is not useful in reasoning locally where attenuation may be neglected and local values of energy, amplitudes and power may be considered to depend on local amplitude of oscillation given by A(x)=Ao e^'^^
Hyperthermia in Cancer Treatment: A Primer For instance local energy may be easily calculated from the fact that the oscillation energy of a volume element is all in kinetic form when velocity is at maximum and since, from eq. (2), velocity is: dkldi = -27CV A(x)sin (27c(vt -x/X)) the maximum volume velocity is 27CV A(x). Kinetic energy of the unit volume element (energy density) is one half of the product of the mass by the square of velocity. Thus local energy density is: E = p/2 (27CV A(x))2=2p7C^r2 Ao^ e^'^M^^
(6)
From eq. (6) it easily seen that energy density decreases with depth by a factor of e^'^^^ For this reason the energy absorbed in the volume in the unit time (Absorbed Power Density) is the difference of energy between two near points i.e., (the x differential of (6) divided by propagation time dt = dx/c (the time that energy takes to travel dx.) i.e.,: APD = dE/dx c/dx = -4 c^p7c\^ Ao^ e^'^M^^ = A c^ipTC^^ A(x)2
(7)
Firsdy equations (6) and (7) show that local energy and APD are proportional to the square of local amplitude. APD is the power density absorbed in the unit volume, that is only a fraction of the power (which is called intensity) transmitted to the unit volume and there present. Another quantity, often used, related to the APD is the Specific Absorption Rate (SAR) that refers the absorbed power to the mass of the unit volume and thus is related to APD by: SAR = APD/p
(8)
Considering eq. (3) and eq. (7) it is evident that what firsdy matters in relation to energy deposition are the exponential factors e^'^^ and e^'^^^ When the depth x equals the length l/|l, the wave has an ampUtude A reduced of a factor 1/ e (i.e., one third or 36.8%) of the input amplitude AQ. Usually p, is quoted in Np (Nepers) by metre yet to be intuitive it is interesting to consider that the value of l/p, ranges between 12 cm and 1.2 cm in soft tissues if frequency varies between 0.5 to 5 MHz. Attenuation in bone is much higher i.e., 3 - 0.1 cm for V in the same range between 0.5 to 5 MHz. As may be suspected by the reduction by a factor of 10 of 1/(1 related to a rise in frequency of factor of 10, attenuation increases roughly linearly with frequency in soft tissues and with the square of frequency in bone. Passing from a medium to another waves change velocity, direction and amplitude and can be pardy reflected at the interface. To understand what happens it is easier not to consider the interaction at the interface but to examine the behaviour of waves few wave lengths from it. Frequency of oscillation can not change but wave velocity is different in the two media. To statt and to clarify some ideas it is useful to do some simple considerations regarding wave fronts. A preview of wave evolution may be attained according to Huygens principle: considering each point of the medium as a source of a spherical wave inflating at the velocity of the perturbation, the envelope of the spherical waves gives the new wave front with the appropriate phase. By this principle it is possible to model the transmitted and reflected wave fronts considering every point at the interface as generating a spherical wave at the instant that it is interested by the impinging wave front The velocities vi of incoming and reflected waves are equal since the medium is the same. The transmitted wave in medium 2 has a velocity V2. For this reason the transmitted wave front changes direction (V2 > vi in Fig. 2). In Figure 2 it is represented the evolution of a wave front at time ti and at time t2 > ti.
Hyperthermia, Physics, Vector Potential, Electromagnetic Heating: A Primer
Otr =
«!
sin(a,) / sin(a,) ^ VjM (Snell's law)
o
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«r
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reil. wave front at tj t2 residual incident wave front
/ • ^ ^V?v
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0 Figure 2. The incident wave front at time ti with incidence angle ttj travels against the interface I between two media. At time t2 wave front is partly reflected with reflection angle CLj and partly transmitted with transmission angle (Xj. Transmission angle and incidence angle are related by Snell's law since wave fronts move with different velocities in the two media. Reflection angle (Xr is equal to ttj since in the same medium velocity is the same.
At time t2 the wave front is represented with a reflected and a transmitted front at an interface I and a residual incident front yet to be split. As the intersection of the front of the wave with the interface I moves from A to B, the wave front splits into one reflected and one transmitted wave front. If the intersection takes a unit of time to travel the length AB then vi = AB sin((Xi) and V2 = AB sin(at). Thus sin(ai)/sin(at) = vi/v2 If at= nil then sin(at)=l and sin((Xi) = vi/v2, thus if the angle OA > arcsin(vi/v2) there is no transmitted wave since "also" the transmitted wave is reflected back. Changing medium, waves change velocity, amplitude, direction and, keeping frequency obviously unaltered, change wavelength. The easiest way to understand these changes is to consider those parameters that remain unchanged i.e., momentum and energy. Mechanical momentum of impinging wave is conserved and thus must be equal to the sum of the two momenta of reflected and transmitted waves. Momentum is the product of mass by velocity of a small volume. This quantity is partly transmitted and partly reflected. The velocity of the volume element is given by the time derivative 3A/3t in each medium. Neglecting attenuation, as not interesting in the short range, and thus using solution (2) we have:
10
Hyperthermia in Cancer Treatment: A Primer dAJdt = -27CV A sin (27c(vt -x/Xi)
where the index i has value 1 or 2 according to the medium. This velocity, that incidentally is not the phase velocity of the waves but is the velocity of the volume element of the oscillating material, is different in each medium. The velocity of the volume changes also in ampUtude and direction (sign) during time since the factor sin (27C(vt - x/Xi) changes as time elapses. Yet the balance of the momentum conservation must hold instantaneously during the oscillation. This fact has two important consequences. The first is that oscillation at the interface in the two media must be in phase and the second is that the only thing that matters is the instantaneous relation among Ai^ific> Ai^refj A2,trasm i«c, the amplitudes of oscillation in media 1 and 2 for the incident, reflected and transmitted waves. The factor sin (27C(vt - x/X,) may be neglected since all waves are in phase. The mass to be taken into account during the exchange of momentum between the two media is obviously the mass of a volume of length proportional to the wavelength. In fact, during a period, a volume of length Xi is interested by the exchange of momentum at the interface. Thus we may write the following momentum conservation equation: vA^ipi d o dt Ai,inc = vA^ipi d o dt Ai,ref + VX2P2 dO dt A2,trasm
where vXiPi do dt = vi Pi do dt is the mass of a volume of density pi of length vi dt and section do.The As are the amplitudes that we have seen are proportional to the velocities. Simplifying and taking into account that the product Vj Pi is called acoustic impedance and it is indicated by Zj we have: ^ 1 Ai,inc = Zi Ai,ref + ^2 ^2,tnsm
(9)
(incidentally note that Z = v p do dt is also the mass of the interested volumes) This is a vector equation since the displacement A is a vector. Thus considering a wave impinging orthogonally on the interface line (wave front parallel to it) we obtain for the amplitudes: Zl Alpine = - Zi Aj^ref + Z2 A2,trasm
Now to conclude the evaluation of transmitted and reflected amplitudes we may use energy conservation. The kinetic energy of the same volume element is conserved as kinetic energy of the two volume element receiving reflected and transmitted energy. Since kinetic energy is mv^/2 and Zi,Z2 are the masses of these volumes and amplitudes are proportional to velocities we may write: Zl A\i,J2=
Zl A^Lref/2 + Z2
A\„^J2
these relations after some simple calculations lead to the following relations for the absolute amplitudes : Al,ref=(Z2-Zi)/(Z2+Z,)Ai.i„c
(10')
A2,trasm = 2 Z l / (Z2+ Zi) Ai,i„c
(10")
Or calling F the reflection coefBcient ((Z2- Zi)/ (Z2+ Zi))'^ Aucl=r"^Aunc
(10')
A2.,ra.m=(l-r^")Ai.i„c
(10")
Hyperthermia, Physics, Vector Potential, Electromagnetic Heating: A Primer
Figure 3. A) A spherical transducer can focus waves reducing problems related to scattering and diffusion. B) An array of transducers, acted with suitable phases, can do the same and can also slightly scan the treatment volume Since intensities are proportional to the square of the amplitudes, reflected energy is proportional to r . If we consider the following values of density and velocity we can evaluate the percent of power reflected for waves at the interface from soft tissues to bone or lung: Density of soft tissue will be considered 1 g/cm^ and sound velocity 1.5 10^ cm/s thus we have: Density Sound Velocity r % Cortical bone 2 g/cm^ 3 10^ cm/s 30% Lung 0.3g/cm^ 0.5 10^ cm/s 80% From these evaluations follows that lung is practically not penetrable as well as gas bubbles in the bowel bone also presents great difFiculties. Ultrasomid waves can be focused if a spherical wave front is created as can be easily understood applying repeatedly the Huygens principle. By this means an imploding wave can be focused nearly in a volume of dimension of a wavelength if the medium is homogeneous (Fig. 3A). The trick is based on the arrival in phase of all portions of the wave front to the same point F. An imploding spherical wave may be obtained distributing a set of transducers on a spherical surface and acting them in phase. This is a very effective mean to obtain a disruptive force at F as it is used to destroy renal calculi. Fortunately this approach is not necessary for hyperthermia since is not easy to drive transducers to obtain an arrival in phase at F through different paths in different media. To obtain hyperthermia at the point F, leaving surrounding tissues relatively cold, it is sufficient to send waves through different paths controlling that F is in the path for each beam and, preferably to avoid interference, acting each transducer during different time intervals. A spherical imploding wave front may be useful to reduce attenuation due to diffraction and scattering. Since direction and penetration of energy depends on the shape of wave fronts is useful to produce wave fronts with suitable shapes. The most effective way to obtain a wave front with a variable shape is to assemble a set of transducers acting them with designed phases. This possibility is illustrated in Figure 3B where a plane array is excited adding a suitable time delay to the central elements in respect to the
11
12
Hyperthermia in Cancer Treatment: A Primer
peripheral ones. By this trick according to Huygens principle the wave front detaches firsdy from the external set of arrays producing a spherical wave. By the same mean the focal spot of the wave can be driven to scan a volume and in principle also the different path length along each ray from the transducer to the focus may be corrected to take into account physical properties of different tissues and at least reduce diffraction. Since temperature control requires the insertion of probes in the volume to be treated, echoes from these probes could be used to assure that the volume of interest is in the ultrasound beam.
Heating by Electromagnetic Methods Heating by electromagnetic fields is mainly attained using time varying fields in a practical range of frequencies between 10 MHz and 2500 Mhz. Reaction of biological tissue to electromagnetic fields depends strongly on frequencies since at low frequency the wave behaviour of EM field may be completely n^lected. The wavelength of an EM field in tissues at a frequency between 10 and lOOMHz is in fact in the order of several decimetres. In this case the field may be considered quasi static and heating effects derive mainly by Ohmic considerations. At higher frequencies wavelength are shorter (few centimetres) and propagation behaviour as diffraction and interference cannot be neglected.
Heating by Low Frequencies There are two essentially different way to use low frequency fields to heat: capacitive heating and magnetic induction heating. Capacitive Heating If we put tissues between two plates belonging to a oscillating circuit, closing by this interposition an electric circuit, the charges contained in the tissues are put in motion. Charges in motion react electrically with the others of the same or opposite sign and put in motion atoms and molecules that are essentially charged entities. This effect (Joule effect) is easily locally modelled by the Ohm law: J = Eo
(11)
where E is the electricfieldJ is the local current density (the current in the unit volume) and a is the conductivity (the inverse of specific resistance) and by the Joule law: A P D = W = E2O.
(12)
From these two relations it follows that the power released to the unit volume is proportional to the square of the field. This result is very general and similar to the one already seen for ultrasound. The electric field may be modelled by line of force along which charges are put in motion according to the local difference of potential. Obviously conductivities of various tissues and their geometrical distributions are very influential in determining both electric field distribution and final currents. To understand the effects of currents on tissues let us consider the simple case of two layers of fat and muscle interposed between two plates at a difference of potential AV (Fig. 4). We must consider the value of conductivities for muscle and fat tissue at low frequency that are respectively of about Om = 0.6 Ohm'Vm and Of = 0.01 to 0.05 Ohm'Vm for fat tissue. The field is orthogonal to the plates and thus currents flow in parallel channels from one plate to the other. Current in the channel of section s is constant along the channel and is proportional to AV and to the global conductivity of the channel. Global conductivity is the inverse of resistance x^ of the channel i.e., the sum of the two resistances tf and rmof fat portion and muscle portion of the channel.
Hyperthermia, Physics, Vector Potential, Electromagnetic Heating: A Primer
13
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Hyperthermia in Cancer Treatment: A Primer
40
Tables. Selectivity of hyperthermia methods Conduction methods
Selective by the placement of the heatsource. In many applications the selectivity is not the case (e.g., systemic heating)
Convective methods
Selective by the localizability of the fluid convection. In most of the applications it is used non-selectively.
Radiation methods
Most of them (except systemic applications) are selective. Two main categories: artificial selectivity (focus arrangement) our self-selectivity (e.g., impedance selection).
Bioactive methods
Non-selective in most of the cases. Some stimulations (e.g., galvanic) could be used locally, and offers selectivity.
Table 6. Applied frequency ranges for hyperthermia
Frequency Range [Hz] From
To
From
To
0 10,000 1,000
100 500,000 1,000,000 45,000,000 200,000,000 2,400,000,000
1 5 50 50 150 50
5 50 500 800 2000 2000
Electromagnetic Method Galvano- treatment (Hz region) Impedance heating (kHz region) Inductive heating (kHz region) Capacitive heating (MHz region) Antenna (phase) array (100 MHz region) Microwave radiation (over 70 MHz)
Typical Forwarded Energy* [W]
1,000,000 60,000,000 70,000,000
•The absorbed energy could be substantially smaller. Thi;> depends on the arrangement of the applicators and the system tuning.
Electromagnetic Heating Processes Basic Effects The electromagnetic fields (magnetic induction B and electric field strength E) interact with the material and change its state. The interacting electromagnetic field is described by the Maxwellian equations adapted in the actual medium: roxB = floEo^—'^Jtffy ^^^ at
' dt
, divE = pff,
divB = 0, (11)
jco £[(36;r)"' lO"'], n„ =[(4;r)l0-^] where the notations rot and div are the derivatives by the space variables (field changes in the space), as well as e^ and ^o are the absolute permittivity and magnetic permeability, respectively, p^and/^are the material-dependent sources of fields, that is, the effective charge and current
Physical Background and Technical Realizations ofHyperthermia
41
density, respectively. Their values depend on the free charge density (p) and their current densities (/), as well as the polarization {P) and magnetization {M) vectors of the material: Peff =—-
div/'
(12)
jeff = jLLoj + " ^ + ^OXM.
Assuming that the polarization depends only on the external electric field and the magnetization by the external magnetic field: P^EoacE
M =
oc„ -B l + a„
(13)
where OCe and am are the electric and magnetic susceptibilities, respectively, creating the relative electric permittivity and relative magnetic permeability £r= I + ag and jU^= 1 + a^, respectively. The energy density (pw) of the electromagnetic radiation^^ is: P ^ = £ £ £ L £ 2 ^ _ ^ ^ 2
(14)
In the case of radiofrequency currents the energy source (10) of bio-heat equation (8) mainly derives from the dielectric loss, so: qc = iTzfEo \m{er )EI^
= PSARRF .
(15)
The temperature gain by the absorbed energy (if the physiologic modifications are neglected) is shown in Figure 3. The complex amplitude of the energy delivery (energy radiation, e.g., Poynting vector (5) expressed in W/m ) can be calculated as (16)
S= —ExB. ^0
/Zy^^yy^
^^/^2?
^ -^ - 1 0 ^g^.
,^^^^y
^ ^ ^ ^ ^
i^>S^2^^5^VW^^^^^
w^^^^^^^^^^g
^^!^x''^x\S?C\.A ^^^^^^^^*AX3a
4Cwii
time [JtJiB]
^^l\r"
r-15
Figure 3. Result of a model calculation of the temperature gain in homogeneous media with stabilized surface temperature (constant environment).
Hyperthermia in Cancer Treatment: A Primer
42
Transmissbn direction
\7 Transmitter material Zj8^, ^ , a^, (ct^, % y^, 5^, XJ)] Layer-boundaries
Zl[si,^ Pji, o J, (a^, p^, Y^^, 8^^ X^)] ^ ^^^P H^ ^2> (^2> P2> ^2> ^2> ^ ) ]
da
^
Layers
Zj[83, ji^, 03, (a3, P3, Y3, 83, X3)]
^i+ll%l' M-fl-ls ^'i+l? (^H-P Hi+1» Yt+l'^+P V l ) j Nf
^ - l t % - p >%•.!> %-1> (%-!> PN-1> YN-P ^N-l^ ^ - 1 ) ] ZITI%> %> ^ & * ' ' . ' - . ' . ' ; " :--/'--'->"-.-'"^^i
..-
-"''>>"' Figure 6. Penetration depth of planar waves into the tissue versus the frequency and the conductivity. The depth is approximated from simplified conditions of the imperfect conductor. (The shaded area represents the most common values for muscle tissue.)
Physical Background and Technical Realizations of
Hyperthermia
45
Figure 7. T h e possible risk of surface burn originates from the perpendicular gradient of the field absorption, the low dielectric constant of the skin and subcutan fatty tissues, as well as from the possible lateral (surface) currents. All of these problems can be eliminated by technical improvements.
The dangerous energy absorption and the consequent hot-spot on the surface are created not only by the maximal incident energy but by the low relative dielectric constant (large voltage drop) and the possible lateral currents (Fig. 7).
Technical Solutions The electromagnetic, "deep-thermal" (not convective and not conductive) heating can be considered as significantly better than the convective and conductive methods. While the convective and conductive methods are limited by the thermodynamic heat conduction, the efficacy of the electromagnetic methods is mainly determined by the extensive interactions determined microscopically. The utilized energy can be uncoupled at any essential position of oscillatory circuit producing the given frequency. Therefore, this fact determines the radiation (Poyntings vector), the magnetic (inductive) and electric (capacitive) treatment techniques (Fig. 8). The techniques are basically different in their dominating electromagnetic effects, and also differ in their actual technical solution (Fig. 9). Main characteristics of the methods are collected in Figure 10.
Electromagnetic radiation equal for the tissues, uses simple absorption
Antenna Magnetic field
Electric field
Magnetic field
Electric field
Selective by niagrietic permeability
selective by the polarisation (rcpolaiisiition tieiit)
(rein«gnetis»tfoii-i»««it)
.jCondensor (capacity)
Coil ^ , / (inductivity)
"X" Figure 8. Schematic representation of the basic electromagnetic methods for an oscillating circuit. T h e three basic effects (electric field, magnetic field and electromagnetic radiation) are interconnected, and their actual domination in one of the circuit elements decides about the effect.
46
Hyperthermia in Cancer Treatment: A Primer
Electric field
•
(capacldve coupling)
Patient is in the ^^^cond
y •
B
R a d i a t i v e (antenna array) Patient is in the antenna array
Magnetic field i(lnductive coupling)
^
Figure 9. Basic arrangements ofthe electromagnetic methods: A) Capacitive coupling, usingthe electric field in a capacitor, B) Magnetic (inductive) coupling, using the magnetic field of a coil, C) Radiative coupling, using the radiated field of an antenna-array. Generally, the transmitting medium is water in the capacitive coupling and antenna array, while in the case of magnetic coupling it is simple the ambient air.
RF supply
A
Magnetic coil
Figure 10. Basic electromagnetic methods: A) inductive (magnetic), B) capacitive (elearic), C) antenna-array (radiative).
Radiative Coupling T h e radiative coupling by antenna array has two distinguishing categories in accordance with the relation of the wavelength (frequency) and the source-target distance (Fig. 11). If the target distance measured from the source is smaller than the applied wavelength, then the wave can not be considered as radiaton, it acts by the change of the fields (E and B), and the Poynting vector (S) has no definite role. T h e two radiative methods are mainly different in their penetration depth (the far -field is more shallow) and the focusing ability (the interference can be modified by the phase-frequency characteristics, the attenuation only by the orientation of the beam). Basic disadvantage of the microwave treatments of higher frequency and shorter wavelength lies in its low penetration depth. (From this respect it slightly differs from the diffusion heating.) Other disadvantage is the possibility of destniaion during the heating process, namely, similarly to the ionization radiation this method has harmful influence on the healthy tissues because of the high specific energy input and the selective energy absorption regarding the hydrogen oxide molecides. As the usage of kitchen microwave oven has also demonstrated, the open microwave source may entrain serious health problems. A part of the near-field treatments uses significantly higher frequencies (up to the limit, which determines the wavelength and the target-distance), and uses interference focusing. T h e applied antennas (dipoles) work in the capacitive regime, however their optimal tuning is far away from the direct capacitive coupling; an array is harmonized to reach the interferences in the desired depth and place. D u e to its higher frequency the penetration depth significantly decreases, which is partly compensated by the additional effects of the multi-antenna array.
Physical Background and Technical Realizations ofHyperthermia
Radiative
47
(antenna array)
Figure 11. The radiative coupling is well described by the radiation alone (Poynting veaor) when the wavelength is small (frequency is high). In this case only the energy attenuation acts. If the wavelength is longer than the source-target distance, interference occurs. T h e SAR values depend on the situation (far- or near-field), and are reduced considerably in near cases. T h e empirical ratio^^ observed: 7SAR =
SARn SARf^
1+
c.
U^/2)
1+
a (^/2)
(20)
where f^ and C,h are frequency-dependent parameters, while Xy and A/, are the wavelength in air at the target place; the indexes denote the vertical and horizontal wave-positions. T h e ^^ and C^h parameters were measured^ in the 10-915 M H z region. Using these data, the actual values of )^AR are always smaller than 1, measured also by others.^^ For 13.56 MFiz and 135.6 MFiz the values are 0.95 and 0.22, respectively; so the low frequencies in fact have no differences between the near- and far-field applications, while the SAR decreases in near fields at high frequency by more than 4-times. T h e inductive (magnetic) and capacitive (electric) couplings are always near field applications due to their low frequency and the field effects.
Radio-Frequency Waves in Near-Field
Radiation
T h e radio-wave treatments belong to the lower frequency group of nonionizing radiations, covering the electromagnetic range approximately from 1 M H z to 30 M H z . W i t h the reduction of frequency the risky and uncertain effects typical of microwave decrease, while the penetration depth significantly increases, and the specific delivered energy shows more homogenous distribution. Magnetic Field A possibility of energy decoupling induced fundamentally by magnetic field may signify deep energy absorption. T h e penetration depth of magnetic field is extremely high in bio-systems, because of the very week interaction between the magnetic field and bio-material. For this reason, the energy absorbed by the tissues—from the energy transported by the magnetic field— manifests itself in the Eddy current or induced current part, namely, it is proportional to the
48
Hyperthermia in Cancer Treatment: A Primer
conductivity of material. In this manner the decoupling with magnetic field is weak, its control is difficult and its selectivity can be assured only as a function of conductivity. The magnetic coupling can be enhanced significandy, if a magnetic material is placed into the targeted area. Generally, nano- or microparticle suspensions, seed, grain or rod magnetic pieces are inserted invasively. In more sophisticated cases the magnetic material s Curie-temperature (ferro-/paramagnetic phase transition) is set to a desired temperature in order to control the heat-delivery and fix the maximal temperature of the magnetic material (the ferromagnetic material absorbs, the paramagnetic does not absorb the magnetic field). Electric Field The energy depletion is effectuated dominandy by electric field anyway. Its advantage, namely, the strong interaction with the highly organized bio-matter (which is typically good dielectrics) also could be a disadvantage, though the penetration depth decreases as compared to the magnetic field. Consequently, the capacitive arrangement offers other advantages (the absorbed energy can be significandy increased and extra selectivity factors can be included in the system) and sets other problems (low penetration and possible surface or subcutaneous burn). Next, we are going to discuss the case when the treated, layered part (for example a kernel and its pellicle and surface interface) is between the electrodes of the condenser supplied by radio-frequency power supply (Fig. 12, a simplified model of Fig. 4). Now, we are going to determine the electric power transformed into heat in the treated area with some simplifying assumptions. (1) The dielectric materials pursuant to Figure 12 are homogenous, namely, the Er relative permeability and the (T electric conductivity are constant by layer (2). The problem is symmetric from geometric and electric point of view, namely, the thickness of the appropriate layers and their electric parameters are identical; the treated area is spherical and is to be found in the geometrical centre of the arrangement (3). The extension of the area where the treated part can be found is large as compared to the extension of the treated part. This later assumption assures that a homogenous field is produced even in the tissue part including the treated area. Its advantage is that the field to be matched in accordance with the boundary
Figure 12. Principle of dielectric heating (Layers represent the actual structure).
Physical Background and Technical Realizations ofHyperthermia
49
electrodes
eotgaRe fr) = —j|/o(/>r)| [c + 6>eotga Re e^)
2d
(22)
where we utilized that in the case of sinusoidal feeding \] = U exp(/a>f) and Jo(pr) is the 0th order Bessel function.^"^ The SAR can then be approximately calculated in the form of -\2 SAR = —Rel
pR
1
-^-du ^ . F ^ - J^zr - ^» J *" ip^)J' (p^)
cmo
(23)
Hyperthermia in Cancer Treatment: A Primer
50
Figure 14. Focusing arrangement by artificial focusing (radiative and magnetic approaches).
Radiative approach
f
ji MMA^ f^^^K^^^^^^KB^^^' 1
' i^ii^^WHBiM^^plF:-'/"
^VHIA /
o^-^ o Figure 15. Focusing arrangement by capacitive arrangement (self-selective technics).
Comparison of the Methods The typical characteristics of these methods are summarized in Table 7. Note that these values are only orienting, covering the mainstream of the electromagnetic heating; however, the actual applications could be rather different. In the foUowings, due to its dominant presence and practical importance, only the near-field applications are discussed. A significant difference in application of the various methods lies in the focusing mechanisms. The artificial focusing (radiative and magnetic approaches) directs the energy absorption into the area where the malignancy is located, and tries to heat up the area homogeneously, which is in most of the cases not homogeneous at all. In the case of multi-centered liaisons the covered area includes both the healthy and malignant areas (Fig. 14). In the capacitive case the actual differences in complex impedance and conductivity determine the area of the heat absorption irrespective of its size or multiplicity (Fig. 15). The advantage is the self-supporting nature, and it has a positive feedback by the growing temperature when the selective differences between the malignant and healthy areas become even more pronounced.
Physical Background and Technical Realizations of Hyperthermia
51 (U
0
y C
O
0 _C
•^ ro 9 T3
CU
>-
• c Jo o .to "O
P Q- . u GU
e-2 .^ 3
OS
2
E-g
^
P
.y -t:
~ ~ V ^T + ^« ^
^ '
where 0 is the electric potential, and the Lik values are the transport coefficients. The observed values are one order of magnitude larger than the coupled one,^^ so we use the LqJLqq «10'^ approximation. Hence:y^«/^ {LqJLq^ « 1,510'^ {Alnt^. The usual data^^ ^^JNa = 1.26-10''^ (A/nr) 2jLidjjc= 4.53-10''^ {AJm^ for Na^ and K^ ions, respectively. In consequence, the forced current density is significandy larger than the natural one. This forced current is mainly Na^ influx. It depolarizes and therefore destabilizes the membrane, and stimulates the Na^/K^ pump activity which results in ATP transformations and further heating at the membrane. Membrane Damage by Increasing Pressure According to the Onsager's theorem, the heat flow is coupled to the volume (mass) transport as well. (The entire complex phenomenon is simplified on the intensive pairs only.) The relevant Onsager-relations are: JV = LwAp + Lvq — ,
Jq = LqvAp + Lqq —
.
(27)
The membrane permeability is much higher for water than for ions, so the main transported component in this coupling is water. The dynamic equilibrium hydrostatic pressure can be determined by the pressure equality on both sides of the membrane,^^ so:
^
=-£1
(28)
AT VTi where Q* is the transport heat and V is the molecular volume of the water. The transport-heat value for 1 liter water at normal conditions (room temperature, normal pressure) equals to Q^20 = 7\,2kJ, consequendy: ^ = -1.32.10^^ = - 1 3 2 ^ (29) AT K K This means that the 217"= 0.01K temperature difference generates 4 ^ = 1.32 bar =1.32-1 Or Pa pressure, which is a large value. Consequendy, the lateral tensile stress rises due to the lateral
Physical Background and Technical Realizations ofHyperthermia
55
dilatation generated by the radial mechanical pressure from the electric field, and the hydrostatic pressure difference tries to balance it. The lateral tensile stress calculated from the above nonequilibrium transports using Z) = 10 jim cell-diameter and B, = lOnm membrane thickness results in G„y = (DAp/ix) = 1.32(10 ^m/4l0 nm) « 3-lO^P^ = 30 MPa, which is a huge value! Comparing this value to the regular conditions: the induced scalar pressure (from the Maxwell stress-tensor ) is 0> = ££" « 3-10 Pay which is too small to be relevant and negligible in relation to the transport pressure. The maximal tolerable lateral tensile stress amounts to CTmax = (2 20)'10^Pa,^ which is smaller by two order of magnitudes than the temperature induced stress. The increase of the osmotic pressure also contributes as an additional factor to the damage of cellular membrane. The increase of the ECM temperature boosts the electro-chemical potentials in the ECM electrolyte. The chemical potentials are: Ji = IJ^io + V ip + RTlnyci + ZiFU, where V\ is the molar-volume, ^lo is the initial chemical potential, p is the pressure, / i s the activity coefficient, ci is the concentration and Zi is the ionizing state of the /-th component. F is the Faraday number and U\s the membrane potential. With the increasingy^j,, both^j^and jci decrease, therefore, in stationary membrane state the intracellular concentrations Ck (k = Na^, K^, CI') also grow. This process increases the osmotic pressure in the intracellular liquid, while decreases it in ECM. The above theoretical considerations can be used in many applications. Unfortunately, the complications in the analytical way force us to use different numerical calculations. The numerically calculated results for kernels are in good correspondence with the observations, and allow explaining some seed stimulation effects. Also the nondirected effect of the power lines in the plough-lands could be studied in this frame.
Summaiy In our opinion, the hyperthermia (definitely, it is a heat-dose treatment) is a temperaturedependent but not temperature-determined process. The temperature concept is not bad as long as the physiological factors (blood flow/vascularization, metabolism, chaperone-protein production, dissemination, apoptotic action, etc.) are included, and the tissue can be regarded as homogenic, semi-isolated from the surroundings. Unfortunately, these conditions are not common, so sometimes the temperature gives statistically significant, sometimes random results. This is the reason why some trials check the patients before, and divide them on the "beatable*' and "not-heatable" groups,^^ and randomize for trial only the previous group. For this group the anyway scientifically incorrect and assumed equivalence of the heat dose to the temperature is an acceptable approach. On the other hand, this preselection excludes a large number of patients receiving hyperthermia; however, this treatment could be a help for them as well. The exclusion was made on an insufficient characterization of the method. A relevant characterization of oncological hyperthermia for quality guidelines has to be started from the aims: it is to destroy the malignant cells! This demand contains some more precise requests: selectivity and blockage of fiirther proliferation and dissemination. Distortion could be promptly direct (the cells become necrotic during the treatment) or indirect (tune killing conditions; the cells become necrotic or apoptotic after the treatment). The demands actually do not contain any temperature request. Hyperthermia is an emerging effective treatment method in oncology. It has shown significant improvements in tumor response rates and patient morbidity in combination with other treatment methods, such as surgery, chemotherapy, radiation therapy and gene therapy or applied as a monotherapy. Nevertheless, hyperthermia is still in its infancy. The thermal dose quantification is likely to remain of practical importance. We have to characterize the hyperthermia by thermal dose and not by temperature. Thermal dose changes many energetic processes in the tissue, and in their physiology. Most of the changes (structural and chemical changes) are out of modification of the temperature, only the entropy changes. The nonequilibrium thermodynamics describes how the absorbed heat could excite various (e.g., diffiisional, electric, chemical, etc.) processes. The dynamism of the absorption determines the
56
Hyperthermia in Cancer Treatment: A Primer
dynamism of the processes (e.g., reaction rates, diffusion dynamism, etc.) and, by these, drives the efBcacy of the processes as well. These phenomena (which are anyway in the focus of the tumor destruction) are completely out of the possibility of temperature characterization. Hyperthermia suffers from a lack of standards and a lack of scientific consensus about its effects on malignant and healthy tissues. In order that hyperthermia shall gain widespread approval and clinical use, the technique requires extensive further research and standardization. For this we need an open mind and have to outgrow the dogmatic habits. The hyperthermia is an interdisciplinary approach. We have to use the historic roots, the scientific achievements on other areas. The hyperthermic oncology is in the wave of the demands: nontoxic, excellent in any combinations with other treatments, with minor contraindications. Hyperthermia has been considered a remarkably developing form of tumorous tissue overheating and tumour treatment. This method is based on the higher heat sensitivity of tumorous tissues and the totality of physiological processes resulting from the effect of heat.
Conclusions and Perspectives What / who is against the oncologic hyperthermia? Nothing / nobody from outside. We are against ourselves by constraining the temperature concept over all. The physics, biology and physiology are with us if we make a correct approach. We are convinced that the perspectives of hyperthermia in oncology are very bright and promising. What we have in hand is a practically non toxic effect with huge potential and advantages. However, we have to clarify the technical issues to make this capable method technically comparable, and provide for a control which is safe enough in terms of modern medical demands.
References I.Jones S, Henry W. Hippocrates. Cambridge: Harvard University Press, 1959. 2. Vesalius A. Dehumani corporis fabrica iibri septem, 1543. 3. Warren JC. Surgical observations on Tumors with cases and operations, 1837, (Ref: Pollay M: The first American book on tumors. Thesis, University of Madison, Wisconsin, 1955). 4. Seegenschmiedt MH, Vernon CC. A Historical perspective on hyperthermia in oncology. In: Seegenschmiedt MH, Fessenden P, Vernon CC, eds. Thermoradiotherapy and Thermochemotherapy. Vol 1. Berlin Heidelberg: Springer/Verlag, 1995. 5. Smith E. Egyptian surgical papyrus dated around 3000 B.C. (cited by: van der Zee J: Heating the patient: a promising approach? Ann Oncol 2002; 13:1173-1184). 6. Medieval literature—1. Medieval Turkish Surgical manuscript firom Charaf ed-Din, 1465 (Paris, BibHotheque Nationaie), 2. Armamentarium chirurgicum of Johann Schultes, Amsterdam 1672 (Paris, Bibliotheque de Faculte Medicine), cited by: Seegenschmiedt MH, Vernon CC. A historical perspective on hyperthermia in oncology. In: Seegenschmiedt MH, Fessenden P, Vernon CC, eds. Thermoradiotherapy and Thermochemotherapy. Vol 1. BerUn Heidelberg: Springer/Verlag, 1995. 7. Busch W. Uber den Einfluss welche heftigere Erysipeln zuweilig auf organisierte Neubildungenausuben, Vrh. Naturhist. Preuss Rhein Westphal 1866; 23:28-30. 8. Coley WB. The treatment of malignant tumors by repeated inoculationsof erysipelas, with a report often original cases. Am J Med Sci 1893; 105:488-511. 9. Westermark F. Uber die Behandlung des ulcerierenden Cervixcarcinoms mittels konstanter Warme. Zentralbl Gynaekol 1898; 22:1335-1337. 10. Westermark N. The effect of heat on rat tumors. Skand Arch Physiol 1927; 52:257-322. ll.Overgaard K. Uber Warmeterapie bosartiger Tumoren. Acta Radiol [Ther.] (Stockholm) 1934; 15:89-99. 12. MuUer C. Therapeutische Erfahrungen an 100 mit kombination von Rontgenstrahlen un Hochfrequenz, resp. Diathermic behandeleten bosartigen Neubildungen. Munchener Medizinische Wochenschrift 1912; 28:1546-1549. 13. Nielsen OS, Horsman M, Overgaard J. A future of hyperthermia in cancer treatment? Eur J Cane 2001; 37:1587-1589, (Editorial comment). 14. Osinsky S, Ganul V, Protsyk V et al. Local and regional hyperthermia in combined treatment of malignant tumors: 20 years experience in Ukraine. Awaji Japan: The Kadota Fund International Forum 2004.
Physical Background and Technical Realizations of Hyperthermia
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15. Dewey W C , Hopwood LE, Sapareto SA et al. Cellular response to combination of hyperthermia and radiation. Radiology 1977; 123:463-474. 16. Lindholm C-E. Hyperthermia and Radiotherapy. Ph.D. Sweden: Thesis, Lund University, Malmo, 1992. 17. Hafstrom L, Rudenstam C M , Blomquist E et al. Regional hyperthermic perfusion with melphalan after surgery for recurrent malignant melanoma of the extremities. J Clin Oncol 1991; 9:2091-2094. 18. Urano M. Thermochemotherapy: From in vitro and in vivo experiments to potential clinical application. In: U r a n o M , D o u p l e E, eds. H y p e r t h e r m i a and Oncology. U t r e c h t - T o k y o : VSP 1994:4:169-204. 19. Hasegawa T, Gu Y-H, Takahashi T et al. Enhancement of hyperthermic effects using rapid heatin. In: Kosaka M, Sugahara T, Schmidt KL et al, eds. Thermotherapy for Neoplasia, Inflammation, and Pain. Tokyo-Berlin: Springer Verlag, 2001:439-444. 20. Lindegaard JC. Thermosensitization induced by step-down heating. Int J Hyperthermia 1992; 8:561-582. 2 1 . Wust P, Hildebrandt B, Sreenivasa G et al. Hyperthermia in combined treatment of cancer. T h e Lancet Oncol 2002; 3:487-497. 22. Hayashi S, Kano E, Hatashita M et al. Fundamental aspects of hyperthermia on cellular and molecular level. In: Kosaka M, Sugahara T, Schmidt KL et al, eds. Thermotherapy for Neoplasia, Inflammation, and Pain. Tokyo-BerUn: Springer Verlag, 2001:335-345. 2 3 . Karasawa K, Muta N , Nakagawa K et al. Thermoradiotherapy in the treatment of locally advanced Nonsmall cell lung cancer. Int J Rad Oncol Biol Phys 1994; 30:1171-1177. 24. Kraybill W , Olenki T. A phase I study of fever-range whole body hyperthermia (FR-WBH) in patients with advanced solid tumors: Correlation with mouse models. Int J Hyperthermia 2002; 18:3, (253-266 and Burd R, Dziedzic ST. Tumor cell apoptosis, lymphocyte recruitment and tumor vascular changes are induced by low temperature, long duration (feverlike) whole body hyperthermiia. J Cellular Physiology 1998; 177:137-147). 25. Field SB. Biological aspects of hyperthermia. Physics and Technology of Hyperthermia. In: Field SB, Franconi C, eds. N A T O ASI Series, E: Applied Sciences, N o . 127. Dordrecht/Boston: Martinus Nijhoff Publ, 1987:19-53. 26. Szasz A, Szasz O, Szasz N . Electrohyperthermia: A new paradigm in cancer therapy. Wissenschaft and Forschung, Deutsche Zeitschrift fiir Onkologie 2001; 33:91-99. 27. de Pomarai D, Daniels C, David H et al. Nonthermal heat-shock response to microwaves. Nature 2000; 405:417-418. 28. Bukau B, Horwich AL. The HSP70 and HSP60 chaperone machines. Cell 1998; 92:351-366. 29. Feynman PR, Leighton RB, Sands M. The feynman lectures on physics. Reading and Caltech, MA and CA, USA: Addison-Wesley Publ Co.. 1963. 30. Katchalsky A, Curran PF. Nonequilibrium thermodynamics in biophysics. Cambridge: Harvard University Press, 1967. 3 1 . Lupis C H P . Chemical Thermodynamics of Materials. NewYork, Amsterdam, Oxford, North Holland: 1983. 32. Pennes H H . J AppUed Physiology 1948; 1:93-122. 33. Matay G, Zombory L. Physiological effects of radiofrequency radiation and their application for medical biology, [in Hungarian], Muegyetemi Kiado, Budapest, 2000:80. 34. Gautherie M. Temperature and blood-flow patterns in breast cancer during natural evolution and following radiotherapy. In: Alan R Liss, ed. Biomedical Thermology. New York, 1982:21-24. 35. ANSI C95.1-1966 (H. Schwan, Pennsylvania USA). 36. Lin H , Head M, Blank M et al. Myc-Mediated transactivation of HSP70 expression following explosure to magnetic fields. J Cell Biochem 1998; 69:181-188. 37. Goodman R, Blank M. Insights into electromagnetic interaction mechanisms. J Cellular Physiology 192:16-22. 38. Lin H , Blank M, Goodman R. A magnetic field-responsive domain in the human HSP70 promoter. J Cell Biochem 1999; 75:170-176. 39. Scholz B, Anderson R. O n Electrical impedance scanning—principles and simulations. Electromedica 68 - Onco 2000:35-44. 40. Barnett A. Electrical method for studying water metabolism and transaction in body segments. Proc Soc Exp Biol Med 1940; 44:142-147. 4 1 . Nyboer J, Bango S, Barnett A et al. Radiocardiograms - the electrical impedance changes of the heart in relation to electrocardiorganms and heart sounds. J Clin Invest 1940; 19:963-966. 42. McRae DA, Esrick MA, Mueller SC. Noninvasive, in-vivo electrical impedance of E M T - 6 tumors during hyperthermia: Correlation with morphology and tumour-growth delay. Int J Hyperthermia 1997; 13:1-20.
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Hyperthermia in Cancer Treatment: A Primer
43. Esrick MA, McRae DA. The effect of hyperthermia induced tissue conductivity changes on electrical impedance temperature mapping. Phys Med Biol 1994; 39:133-144. 44. McRae DA, Esrick MA. The dielectric parameters of excised EMT-6 tumours and their change during hyperthermia. Phys Med Biol 1992; 37:2045-2058. 45. McRae DA, Esrick MA, Mueller SC. Changes in the noninvasive, in vivo electrical impedance of the xenograpfts during the necrotic cell-response sequence. Int J Radiat Oncol Biol Phys 1999; 43:849-857. 46. Dissado LA, Alison JM, Hill RM et al. Dynamic scaling in the dielectric response of excised EMT-6 tumours undergoing hyperthermia. Phys Med Biol 1995; 40:1067-1084. 47. Szendro P, Vincze G, Szasz A. Bio-response on white-noise excitation. Electro-and Magnetobiology 2001; 20:215-229. 48. Gersing E. Monitoring temperature induced changes in tissue during hyperthermia by impedance methods. In: Riu P, Rosell J, Bragos R et al, eds. Electrical Bioimpedance Methods: Applications to Medicine and Biotechnology. Ann New York Acad Sci 1999:873:13-20. 49. Haemmerich D, Staelin ST, Tsai JZ et al. In vivo electrical conductivity of hepatic tumors. Physiol Meas 2003; 24:251-260. 50. Smith SR, Foster KR, Wolf GL. IEEE Trans Biomed Eng BME 1986; 33:522-525. 51. Jossinet J. The impedivity of freshly excised human breast tissue. Physiol Meas 1998; 19:61-75. 52. Jossinet J, Schmitt M. A review parameters for the bioelectrical characterization of breast tissue. In: Riu P, Rosell J, Bragos R et al, eds. Electrical Bioimpedance Methods: Applications to Medicine and Biotechnology. Ann New York Acad Sci 1999:873:30-41. 53. Chauveau N, Hamzaoui L, Rochaix P et al. Ex vivo discrimination between normal and pathological tissues in human breast surgical biopsies using bioimpedance spectroscopy. In: Riu P, Rosell J, Bragos R et al, eds. Electrical Bioimpedance Methods: AppHcations to Medicine and Biotechnology. Ann New York Acad Sci 1999:873:42-50. 54. Tosso S, Piccoli A, Gusella M et al. Nutrition. 2000; 16:120-124. 55. Glickman YA, Filo O, David M et al. Electrical impedance scanning: A new approach to skin cancer diagnosis. Skin Res Techn 2003; 9(3):262. 56. TransCan TS2000, Transcan Medical Ltd. Migdal Ha'Emek, Israel, distributed by Siemens AG, Germany. 57. Skourou C, Hoopes PJ, Strawbridge RR et al. Feasibility studies of electrical impedance spectroscopy for early tumour detection in rats. Physiol Meas 2004; 25:335-346. 58. Chillcott TC, Coster HGL. Electrical impedance tomography study of biological processes in a single cell. In: Riu P, Rosell J, Bragos R et al, eds. The data Electrical Bioimpedance Methods: Apphcations to Medicine and Biotechnology. Ann New York Acad Sci 1999:873:269-286. 59. McRae, Esrick MA. Deconvolved electrical impedance spectra track distick cell morphology changes. IEEE Trans Biomed Eng 1996; 43:607-618. 60. Bioelectric impedance analysis in body composition measurement. National Institute of Health, USA: Technology Assessment Conference Statement, 1994:12-14. 61. Bowen WD, Beck CA, Iverson SJ. Bioelectrical Impedance analysis as a means of estimating total body water in grey seals. Can J Zool 1999; 77:418-422. 62. Talluri T, Lietdke RJ, Evangelisti A et al. Fat-free mass qualitative assessment with bioelectric impedance analysis. In: Riu P, Rosell J, Bragos R et al, eds. Electrical Bioimpedance Methods: Apphcations to Medicine and Biotechnology. Ann New York Acad Sci 1999:873:94-98. 63. Goovaerts HG, Faes THJC, DeValk-DeRoo GW et al. Estimation of extracellular volume by two frequency measurement. In: Riu P, Rosell J, Bragos R et al, eds. Electrical Bioimpedance Methods: Applications to Medicine and Biotechnology. Ann New York Acad Sci 1999:873:99-104. 64. McRae DA, Esrick MA. Changes in electrical impedance of skeletal muscle measured during hyperthermia. Int J Hyperthermia 1993; 9:247-261. 65. Shchepotin IB, McRae DA, Shabahang M et al. Hyperthermia and verapamil inhibit the growth of human colon cancer xenografts in vivo through apoptosis. Anticancer Res 1997; 17:2213-2216. GG. Casas O, Bragos R, Riu PJ et al. In vivo and in situ ischemic tissue characterization using electrical impedance spectroscopy. In: Riu P, Rosell J, Bragos R et al, eds. Electrical Bioimpedance Methods: Apphcations to Medicine and Biotechnology. Ann New York Acad Sci 1999:873:51-58. G7. Schafer M, Kirlum H-J, Schlegel C et al. Dielectric properties of scaletal muscle during ischemia in the frequency-range from 50Hz to 200 MHz. In: Riu P, Rosell J, Bragos R et al, eds. Electrical Bioimpedance Methods: Applications to Medicine and Biotechnology. Ann New York Acad Sci 1999:873:59-64. 68. Gheorghiu M, Gersing E, Gheorghiu E. Quantitative analysis of impedance spectra of organs during ischemia. In: Riu P, Rosell J, Bragos R et al, eds. Electrical Bioimpedance Methods: Applications to Medicine and Biotechnology. Ann New York Acad Sci 1999:873:65-71.
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69. Osterman KS, Paulsen KD, Hoopes PJ. Application of linear circuit models to impedance spectra in irradiated muscle. In: Riu P, Rosell J, Bragos R et al, eds. Electrical Bioimpedance Methods: Applications to Medicine and Biotechnology. Ann New York Acad Sci 1999:873:21-29. 70. Santini M T , Cametti C, Zimatore G et al. A dielectric relaxation study on the effects of the antitumor drugs Lomidamineand Rhein on the membrane electrical properties of Erlich ascites tumour cells. Anticancer Res 1995; 15:29-36. 7 1 . Keese CR, Wegener J, Walker SR et al. Electrical wound-healing assay for cells in vitro, PNAS, Proceedings. Nat Acad Sci USA 2004; 101:1554-1559. 72. Avitall B, Mughal K, Hare J et al. The effects of electrode-tissue contact on radiofrequency lesion generation. Pacing Chn Electrophysiol 1997; 20:2899-2910. 73. Schmidt D , Trubenbach J, Konig C W et al. Radiofrequency ablation ex vivo: Comparison of the efficacy impedance control mode versus manual control mode by using internally cooled clustered electrode. Rofo Fortschr Geb Rontgenstr Neuen Bildgeb Verfahr 2003; 175:967-972. 74. Szasz A, Vincze Gy, Szasz O et al. An energy analysis of extracellular hyperthermia. Magneto- and electro-biology 2003; 22:103-115. 75. Seegenschmiedt M H , Vernon C C . A Historical perspective on hyperthermia in oncology. In: Seegenschmiedt M H , Fessenden P, Vernon C C , eds. Thermoradiotherapy and Thermochemotherapy. Berlin: Clinical Applications, Springer Verlag, 1995:2:3-46. 7Ct. Blank M. Coupling of AC electric fields to cellular processes. First International Symposium on Nonthermal Medical/Biological Treatments Using Electromagnetic Fields and Ionized Gases, ElectroMed'99, Norfolk VA, USA, Symposium Record Abstracts, 1999:23. 77. Young RA. Stress proteins and immunology. Ann Rev Immunology 1990; 8:401-420. 78. Jackson J D . Classical Electrodynamics. New York: John Wiley and Sons Inc., 1999. 79. Rao N N . Elements of engineering electromagnetics. London: Prentice Hall International, 1994. 80. Matay G, Zombory L. Physiological effects of radiofrequency radiation and their application for medical biology, [in Hungarian], Muegyetemi Kiado, Budapest, 2000:71. 8 1 . Polk C, Postow E. Handbook of biological effects of electromagnetic fields. New York, London, Tokyo: C R C Press, 1996:15. 82. Tremoliers J. Effects biologiques des champs electromametiques non ionisants. Electron Appl 1978; 7:71-77. 83. Chatterjee I, Hagmann M, Gandhi O . An empirical realtionship for electromagnetic energy absorption in man for near-field exposure condition. IEEE Trans O n Microwave Theory and Techniques, M T T - 2 9 , 1981; 11:1235-1238. 84. Chain C. A theoretical basis for microwave and RF field effects on excitable cellular membranes. IEEE Trans O n Microwave Theory and Techniques, M T T - 2 9 , 1980; 2:142-147. 85. Iskander M, Olson S, MacCalmont J. Near-field absorption characteristics of models in the resonance frequency range. IEEE Trans O n Microwave Theory and Techniques, M T T - 3 5 , 1987; 8:776-779. 86. Kotnik T, Miklavcic D . Theoretical evaluation of the distributed power dissipation in biological cells exposed to electric field. Bioelectromagnetics 2000; 21:385-394. 87. Galeotti T, Borrello S, Minotti G et al. Membrane alterations in cancer cells: The role of oxy radicals. In: Bianchi G, Carafoli E, Scarpa A, eds. Membrane Pathology. Ann New York Acad Sci 1986:488:468-480. 88. Hager ED, Dziambor H, Hohmann D et al. Deep hyperthermiawith radiofrequencies in patients with liver metastases from colorectal cancer. Anticancer Res 1999; 19:3403-3408. 89. Hager D, Dziambor H , App EM et al. ASCO 2003 Meeting, Chicago USA, 2003:470. 90. Dani A, Varkonyi A, Nyiro I et al. Clinical experience of elecctro-hyperthermia for advanced pancreatic tumors. E S H O 2003 Conference, Munich 2003:41. 9 1 . Hodgkin AL, Huxley A. A quantitative description of membrane current and its application to conduction and excitation of nerve. J Physiol 1952; 117:500-544. 92. Weiss TF. Cellular Biophysics. Cambridge, MA, USA: A Bradford Book, The M I T Press, 1996. 93. Spanner D C . Symp Soc Exptl Biol 1954; 8:76. 94. Sackmann E. Physical basis of self-organisation and function of membranes: Physics of vesicles. In: Lipowsky, Sackmann E, Elsevier Science BV. eds. Handbook of Biological Physics 1995:1. 95. Vujaskovic Z, Jones EL, Oleson JR et al. A Randomized trial of hyperthermia and radiation for superficial tumors. Awaji Yumebutai, Japan: Presentation and Abstracts for The Kadota Fund International Forum, 2004.
CHAPTER 4
Thermotherapy and Nanomedicine: Between Vision and Reality Andreas Jordan* Summary
A
lthough nanoparticles have been already applied on patients in clinical trials, generally nanotechnology in medicine is regarded rather a vision than a realistic option. Progress in this field arises particularly from the combination of molecular biology and nano(bio) technology. From the viewpoint of entrepreneurs nanotechnology is only a tool to develop new products, however nanotechnology itself is not a product. We developed a new cancer treatment platform technology termed MagForce Nanotherapy, in which nanotechnology has the potential to cause a revolution in tumor therapy.
Introduction The idea of traveling through the vessels of a body in a "nanobot"* to heal diseases from "the inside", as shown in the Oscar-awarded movie "Fantastic Voyage" (Stephen Boyd, 1966), is indeed quite attractive, but unfortunately it also inherits general difficulties, especially in combating cancer. Theoretically, an ingeniously built nano-vehicle, controlled from outside the body, coiJd move through vessels. Nevertheless, the fantasy story would end very fast, because the inmiune system of the human body would quickly destroy the submarine, as it does enduringly with bacteria, viruses and other foreign particles. But even if this problem could be solved, the submarine woidd still not know how to destroy cancer cells selectively while sparing normal cells. Hereto defined molecules on the surface of tumor and normal cells ("targets") have to be identified for distinguishing between these cells. A solid tumor consists of a number of different sub-populations, whose genomes are different from each other expressing those targets or not. Therefore not all cells of the tumor are recognized by their specific target molecules, which build the source of recurrent, often multi-resistant tumor growth. Even the mixture of different target recognizing molecules is not a guarantee that all tumor cells are affected. This general problem cannot be solved by any "nanobot" approach. How we get along with this new knowledge without "nanobots",fixturewill tell. Answers to these problems may rather derive from research in the fields of molecular biology, where certain success has already been obtained concerning different tumor entities. Boundaries between nanotechnology and molecular biology blur. It is of general acceptance, that in the future even single molecules and atoms are to be controlled, and then nanotechnology will probably gain the same importance that molecular biology has today. As one of the first applications of nanotechnology in medicine the group of the authors developed a worldwide new Nano-cancer-therapy in more than 15 years of fundamental •Andreas Jordan—MagForce Nanotechnologies GmbH and Center of Biomedical Nanotechnology (CBN), Department of Radiology, Charite - University Medicine Berlin, Spandauer Damm 130, 14050 Berlin, Germany. Email:
[email protected] Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.
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research at the Charitd - University Medicine Berlin. Via foundation of the MagForce® Nanotechnologies GmbH in Berlin, research lead to products, which have already been tested in clinical trials and which are already requested by numerous cancer clinics, even before their approval. The principle of the method termed MagForce Nanotherapy is simple: Iron-oxide nanoparticles are directly injected into the tumor and release heat after inductively induced activation by an alternating magnetic field. Despite this simple-sounding approach, it was nanotechnology to be the key for realizing this new cancer-therapy: • Only nanoparticles extract high energy per applied mass from a magnetic field • Due to their enormous surface only nanoparticles are able to carry a huge number of binding sites for cancer cells /target molecules • Only nanoparticles are able to intrude deeply into tumor tissue • Only nanoparticles with special coatings • are recognized delayed by the immune system and thus reach their targets • can be ingested in great quantities by tumor cells • form a homogeneous fluid of low viscosity in water • remain in the tumor tissue even after interstitial application for a long time and are not being washed out So far, the MagForce Nanotherapy is, in a first step, a new form of local thermotherapy of deep-seated tumors. Clinical trials in this field done so far demonstrated a good feasibility and tolerability of the new technique. Later the nanoparticles are supposed to function as transport vehicles for medical agents, isotopes or genes. MagForce is doing research in this field for years now, predominantly in cooperation with the Leibniz-Institute for New Materials (INM), Saarbriicken, Germany and different departments of the Charit^ - University Medicine, Berlin, Germany. The MagForce Nanotherapy offers the possibility of repeated heat treatments of basically every region of the body very precisely without repeated application of the particles. Intratumoral temperatures can be varied according to clinical requirements between hyperthermia (up to 45°C for supporting radiochemotherapy) and thermoablation with temperatures of up to 70°C. The method is based on the defined power transfer to biocompatible iron-oxide nanoparticles in an alternating magnetic field. The patented nanotechnological design of the MagForce nanoparticle shell leads to preferred intracellular absorption into proliferating cells like tumor cells (Fig. 1). The particles function as "Trojan horses", thus destroying tumor cells, whereas healthy tissue is spared. Particles generate heat by relaxation processes of the particle core and emit it into the surrounding tissue.^ Thermotherapy is performed in a specially designed magnetic field applicator (Fig. 2). Due to its construction and safety standards, the system can be applied on diff^erent tumor entities in every region of the human body. To date, the MagForce Nanotherapy is being investigated only at the Charit^ - University Medicine Berlin, Germany. Treatment modalities are shown in Figure 2. In early 2007 the new method is supposed to be available for all clinics in Europe. From March 2003 to July 2004, the worldwide first feasibility study on thermotherapy using magnetic nanoparticles was performed on 14 patients with glioblastoma multiforme. In Germany, more than 2000 patients (Incidence: 3/100,000) die on this aggressive tumor every year. Median overall survival after first-line therapy does not exceed 12-15 months and no significant increase has been achieved over the last decade, despite modern diagnostics and treatments with surgery, radiotherapy and chemotherapy. ' The therapy was tolerated well by all patients, and in all cases intratumoral temperatures of 42-45°C could be achieved, even in deep-seated tumors. Signs of local efficacy could be observed in all patients. Detailed results will be published soon.
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Hyperthermia in Cancer Treatment: A Primer
Figure 1. Intracellular uptake of MagForce Nanoparticles in a human mammary carcinoma cell-line. Tumor cells marked like this can later be identified by MRI scans^ (electron micrograph from ref. 2).
Figure 2. A patient suffering from glioblastoma multiform during treatment in the magnetic field applicator (MFH® 300F, MagForce Nanotechnologies, Berlin, Germany).
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Subsequent to this trial, an efficaq^ study with 65 patients, suffering from recurrences of glioblastoma multiforme or anaplastic astrocytoma started in January 2005. Another trial started in February 2004 with 25 patients suffering from local tumor recurrences without distant metastases (e.g., sarcoma, cancer of the rectum, prostate-, ovarian- and cervix carcinoma) and who had received all kinds of conventional therapies before. These patients received repeated thermotherapies in combination with radiotherapy (afterloading method). Also in this study, positive results were obtained concerning feasibility and tolerability of the technique. Desired intratumoral temperatures were obtained and clear signs of local efficacy could be observed. Beginning in May 2004 a feasibility study with patients suffering from local recurrences of prostate carcinoma is now nearing completion. Other trials are in preparation.
Further Information http: //www. magforce. de
Acknowledgements The Center of Biomedical Nanotechnology (CBN) at the Charit^ Berlin, Germany is cofinanced by the European Community, European Fund for Regional Development (EFRE)Project NanoMed. The Federal Ministry of Education and Science (BMBF), Projekttrager Jiilich (PTJ), and Verein deutscher Ingenieure (VDI) within the framework of the support program Nanobiotechnology (Project MNC, TAN) granted project support.
References 1. Jordan A, Wust P, Fahling H et al. Inductive heating of ferrimagnetic particles and magnetic fluids: Physical evaluation of their potential for hyperthermia. Int J Hyperthermia 1993; 9:51-68. 2. Davis FG, Freels S, Grutsch J et al. Survival rates in patients with primary malignant brain tumors stratified by patient age and tumor histological type: An analysis based on Surveillance, Epidemiology, and End Results (SEER) data, 1973-1991. J Neurosurg 1998; 88:1-10. 3. Stupp R, Mason W P , Van Den Bent MJ et al. Concomitant and adjuvant temozolomide (TMZ) and radiotherapy (RT) for newly diagnosed glioblastoma multiforme (GBM). Conclusive results of a randomized phase III trial by the E O R T C Brain & RT Groups and N C I C Clinical Trials Group. J CHn Oncol, 2004 A S C O Annual Meeting Proceedings (Post-Meeting Edition) 2004; 22:2. 4. Pinkernelle J, Teichgraeber U, Neumann F et al. Imaging of single human carcinoma cells in vitro using a clinical whole body mr scanner at 3.0T. Magn Reson Med. 2005 May;53(5):l 187-92. 5. Stupp R, Mason W P , van den Bent MJ et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005; 352:987-996.
SECTION II
Biological Aspects ofHyperthemiia
CHAPTER 5
Influence of Tumor Microenvironment on Thermoresponse: Biologic and Clinical Implications Gian Franco Baronzio,* Alberto Gramaglia, Attilio Baronzio and Isabel Freitas Abstract
S
olid tumours tend to have a more acidic and hypoxic microenvironment than normal tissue. This hostile microenvironment results from a disparity between oxygen supply and demand of the tumor tissue. Overcoming hypoxia tumor induces a new vascular supply. This new vasculature is however inefficient and chaotic. It perpetuates the factors that have stimulated its induction. This review focuses on these processes and peculiarly on angiogenesis, tumor vascular morphology, hypoxia, pH, and the metabolic-vascular events induced or following tumour tissue heating. The various mechanisms that either modulate tumor microenvironments or blood perfusion during hyperthermia are described, providing also the many clinical modalities that may enhance or sensitize cancer cells to heat.
Introduction: Tumor Microenvironment Human solid neoplasia should be regarded as an intricate, yet poorly organized "organoid", whose function is maintained by a dynamic interplay between neoplastic and host cells. ^'^ This interplay constitutes the tumour metabolic microenvironment, defined by Vaupel as a complex pathophysiological entity resulting by the interactions of self-influencing factors, which go hand in hand: tumor perfusion, tumor oxygenation status, pH distribution and metabolic bioenergetic status.
Hypoxia, HIF-1 and Angiogenesis Hypoxioy HIF'l The growth of tumours beyond a critical mass >l-2 mm^ (10^ cells) is dependent on adequate blood supply. '^ Up to a distance from host vessel of 100-200 |im the initial foci of neoplastic cells receive their nutrients and oxygen by diffiision. Beyond this distance, hypoxia occurs and the need of adequate blood supply is crucial. ^'^ However, the establishment of this neovascular supply in the attempt to overcome hypoxia is inefficient and irregular. It may not occur at the same rate as the proliferation of the tumour. The result is the persistence within the tumour mass of •Corresponding Author: Gian Franco Baronzio—Family Medicine Area, ASL-01 Legnano; Radiotherapy Unit, Policlinico di Monza, Via Amati 11, 20052 Monza (Mi), Italy. Office address: P.O.B. 5, 20029 Turbigo (Mi), Italy. Email:
[email protected] Hyperthermia in Cancer Treatment: A Primer^ edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.
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Figure 1. Photomicrograph of a section of mammary tumor, developed by a 5 month old female transgenic MMTV-neu (erbB2) mouse. T: tumor cord; N = necrosis; Hyp: hypoxic, perinecrotic, rim of tumor cells; C: capillary. Hematoxylin and Eosin. 40x objective. heterogeneous microregions of nonproliferating hypoxic cells, which are surrounded by vital well nourished and proliferating cells (Fig. \).^ Several methods to measure hypoxia are currendy available and have demonstrated the presence of hypoxic cells both in experimental and in human tumours.^ The hypoxic microenvironments are characterized by low oxygen tension, low extracellular pHe, high interstitial fluid pressure, glucose deficiency, multidrug resistance, increased extracellular lactate concentration and tendency to metastasization. The formine of new blood vessels depends on a balance between angiogenesis inhibitors and promoters. '^ Tumor regional hypoxia and hypoglycaemia are the principal stimulators for the expression of local proangiogenic cytokines, especially vascular endothelial growth factor (VEGF) (Fig. 2).^'^ ^ The early response gene that produces hypoxia inducible factor-1 (HIF-1) and its subunits (HIF-1 a and -Ip) regulate VEGF expression. HIF-1 is a protein of 120 KDa, member of the basic helix-loop-heltx superfamilv transcription factors, and its expression is very sensitive to oxygen concentration (1% 02). ^'^^ The adaptation to hypoxia by earlier proliferating neoplastic cells results in the induction of genes that regulate the anaerobic metabolism, nitric oxide synthase and the angiogenesis process.^'^^ Recent studies have shown that HIF-1 and VEGF transcripts are overexpressed by several human neoplastic cells including breast, prostate, gastric, colon, lung, bladder and endometrium and they are more active in hypoxic and necrotic areas. ^ ^^ VEGF is correlated to vascular density, especially in brain tmnors and it is associated with bad prognosis. ^^ VEGF or vascular permeabilin^ factor (VPF), is a 32 to 44-KDa multifunctional potent stimulator of endothelial cells. ^^' It becomes active by binding to three high afFmity tyrosine kinase receptors [VEGFR-l(flt-l), VEGFR-2(KDR/Fik-1) andVEGFR-3(Flt-4)], diat are highly expressed
Influence ofTumorMicroenvironmenton Thermoresponse
69
OROWTH FACTOR & CYTOKINES (FDGF, TGf^, iL1^,TNFa, iLe4t8,IL10) '"
l,weHE«2tori^(fMHi)}
^
TUMOR BLOOD FLOW
INFLAMMATORY REACTION
PERITUMORAL INFLAMMATORY REACTION
j BLOOD STASIS
Platelet aggregation leukocyte sticking
, , i::::: Figure 2. In this figure the structural and functional eflFects of Hypoxia, HIF-1 and VEGF on tumor microcirculation, cancer metabolism and therapies are illustrated. The vicious circles that occur are also shown. (Modified with permission fi-om: Baronzio et al. Anticancer Res 1994; 14:1145-1154.)
on endothelial tumour vessels but not on mature vessels. They exert different effects on endothelial cells (ECs). VEGFR-1 mediates cell motility of ECs, whereas VEGFR-2 regulates vascular permeability and VEGFR-3 lymphoangiogenesis.^^'^ '^^ Hypoxia appears to be the principal stimulus for the production and the stabilization of VEGF and VEGF mRNA, but recent evidences suggest that VEGF expression has increased in many tumours, as well in absence of hypoxia. This increase results from at least three factors: (A) loss of function of some tumour suppressor genes (such as the von Hippel-Lindau (VHL), p53, pl6); (B) activation of oncogenes (including raf, ras,FiER2/erb2 (neu) and src '^^"^'^) (C) excessive quantity of growth factors, produced by tumour cells and their supporting network
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Hyperthermia in Cancer Treatment: A Primer
Table 1. Structural and functional abnormalities of tumor vasculature Decreased vascular density Heterogeneous distribution of microvessels Increased presence of sinusoids, dead ends and arterio-venous anastomosis Lack of arteriolar vessels Decreased perfusional pressure and increased geometric resistance absence of lymphatics, increased TIF, hemoconcentration, increased viscosity Incomplete basement membrane and absence of smooth muscle Decreased stabilization for absence of pericytes and mural cells Decreased leukocyte-endothelium adhesion Absence of innervation and regulation according to metabolic demand
Tumor Neovascularization The acquisition of new in-growing vessels may occur by different mechanisms. ECs are normally quiescent and tightly regulated by a delicate balance between proangiogenic and antiangiogenic molecules. '^^ In presence of an excessive secretion of angiogenic molecules by tumours, ECs are stimulated and they organize themselves in vessel structure through multistep sequential and distinct processes, depending on tumour type and anatomic localization. These processes include vessel Cooption, vasculogenesis and angiogenesis. Vessel-Cooption
It is the process in which tumours take up preexisting normal blood vessels and use them for their initial growrth. As just described, this is a limiting process and irrelevant in the great majority of solid tumours; in fact, cancer cells grow until oxygen demand exceeds supply and the distance from host vessels is lower than 100-200 jlm. Vasculogenesis
It is the mechanism in which precursors endothelial ECs from bone marrow are recruited by the tumor and aggregate to form new blood vessels. Recent studies have demonstrated this process in experimental animal tumours, but its relevance in human neoplasia is not fully elucidated.2^-^5 Angiogenesis Upon adequate stimulus, endothelial cells begin to sprout from preexisting capillaries and after the degradation of the extracellular matrix (ECM) by matrix metalloproteases, and the expression of adhesion molecules such as avp3 integrin, migrate and organize themselves in capUlary tube formation and ultimately in a vascular network '^
Tumor Vascular Morphology-Perjusion and Hypoxia Blood vessels associated with the tumor tend to be significantly different in architecture from the surrounding normal tissue (see Table 1)^^'^^ and they show, in presence of VEGF and other cytokines, a decreased expression of leukocyte-endothelial cell adhesion molecules (ICAM-1,VCAM-1, E-selectin ) and an enhanced expression of CD 44?^'^^ The decreased expression of adhesion molecules reduce significantly leukocytes and natural killer cells (NKs) recruitment, partially contributing to the phenomenon of immune-evasion,^^'^^ whereas the enhanced expression of CD44 may confer a growth advantage on many neoplastic cells. Moreover, VEGF induces tumor neovessels to become leakier and to lose a large quantity of fluids (proteins and other circulating macromolecules) towards the interstitium. Fluid accumulated inside the tumor interstitium, knovm as tumor interstitial fluid (TIF), occupies from 30% to 60% of the tumour volume and compared to normal fluids it has a different biochemical nature.^ '^^ TIF retention causes an increase in tumor interstitial pressure (TIFP)
Influence of Tumor Microenvironment on Thermoresponse
71
(Fig. 2).^^"^^ TIFP increase goes from tumour centre towards periphery, reaching the value of 50 mm Hg, as Jain and coworkers measured in human and experimental tumours on colon, breast, head-neck carcinomas and metastases specimens from lung, liver and lymph nodes. °" "^ Tumor blood flow (TBF) is determined by arteriovenous pressure difference (A Pa-v) divided by a factor T| for Z, where Tj expresses the flow resistance or blood viscosity and Z the geometric resistance.^ TBF=APa-v/TlZ
(1)
Normally in tumors, A Pa-v is lower than in normal microcirculation. This is caused by the increased number of arteriovenous fistulae present at tumor periphery, that creates a low resistance pathway at tumour surface and diverts blood from entering the tumour mass. Indicator dilution methods, angiography and experimental studies have confirmed this anomalous behaviour.^"^'^^ Associated to low perfusional tumour supply, Sevick and Jain have found that viscosity parameters Z and T| were abnormally high on tissue isolated mammary Adenocarcinoma (R3230AC) and on 22 carcinosarcoma. The geometric resistance Z is a complex function of vascular morphology (i.e., number of blood vessels, branching pattern, diameter, length and volume), and increases according to tumor size, length and vessel tortuosity. ^ Tumor neovessels are tortuous, heterogeneous, inefficient and devoid of hierarchisation, as confirmed by different Authors using biochemical, corrosion cast and electronic microscopic studies.^^'"^^ Tumor blood viscosity T| has been found elevated and correlated with the shear rate, hematocrit and circulating blood cells deformability. ' Normally capillary diameter ranges from 3.5 to 20 |xm, so at some point along their travel in the capillaries, red blood cells (RBCs) as well as white cells (WBCs) have to undergo deformation to pass through. By contrast in the tumor microvasculature, various factors can modify RBCs and WBCs deformability. Particularly the low oxygen partial pressure, the acidic pH, and the increased concentration of fibrinogen tend to make red blood cells and leucocytes less deformable, and more sticky, thus easily trapped intravascularly and blocking TBF.^ A further modifier factor of viscosity is the local increase of the hematocrit. Several authors^ '^^''^' have demonstrated that this phenomenon is to be ascribed to the fluid loss operating in tumor capillaries with a consequent local hemoconcentration and further worsening of blood flow. Blood flow measurements in human cancer show heterogeneous values going from highly vascularized organs, such as brain, and poorly vascularized, such as adipose tissue. Perfusion flow at tumour level is higher or lower than in the tissue of origin, depending on the physiological state of the latter. ' ^ It is higher at the tumour periphery than in the central zone and generally primary tumours are better supplied than metastatic lesions. In most studies, perfusion on rodent tumours decreases with tumour size when compared to normal tissue. However, in the minority of experiments the decreased flow was not confirmed even in a similar tumor type. Several pathophysiological mechanisms have been proposed to explain this difference such as transplantation site, stage of tumor growth, flow registration and recording methods.^ TBF is not regulated according to metabolic demand as in the case of normal tissues. This decreased metabolic adaptability to cells associated to an irregular blood availability (perfusion) produce a clusters of cells, lacking nutrients and oxygen (hypoxic cells). Two kinds of hypoxic or clusters of cells in situation of low energy state have been recognized: (A) Diffusion-limited or chronic hypoxic cells; (B) Transient or acute hypoxic cells. The two types of hypoxia have different origin and coexist together in a well-perfused zone of the tumour mass too, causing a functional disturbance of macro and microflow. '^ A more realistic vision shows that these two situations change continuously, because tumour blood flow is time fluctuating."^^"^^ Chronic hypoxia is the result of availability of nutrients and oxygen towards the tumoral tissue, the diffusion in the extracellular space and the respiration rate of cancer cells. ' It has been calculated and observed that when cancer cells are > 100-200 jim away from functional blood supply become hypoxic and suffering (Figs. 1,3).
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Hyperthermia in Cancer Treatment: A Primer
Figure 3. Photomicrograph of a section of mammary tumor, developed by a 5 month old female transgenic MMTV-neu (erbB2) mouse. T: tumor cord; N: necrosis; L: lymphatic vessel; C: capillary. Hematoxylin and Eosin. 20x objective. Cells adjacent to capillaries displayed a mean oxygen concentration of 2%, located at 200 P-m displayed a mean oxygen concentration of 0.2%.^ The distance from the nutritive vessels, the haemoglobin concentration and the blood flow crossing that tumour area are the only parameters responsible for chronic hypoxia. In fact, the removal of O2 by tumour cells, has been calculated to be efficient or better than the one of normal tissues, revealing that neoplastic cells in vivo do not have impaired ability to utilize oxygen as proposed in the past. Acute hypoxia is the restdt of intermittent opening and reopening of tumor blood vessels. Among the various factors responsible for this temporarily blood flow stop, two of them seem the most plausible: A. TIFP combined with the irregular expansion of tumour mass, whose three dimensional growth is subjected to a continuous remodelling in a confined space and it causes a temporary compression or occlusion of some tumour capillaries. ^'^^'^^ B. transient stop of tumor bloodflowor supply by platelets plug (see Fig. 4).5''^7 In our opinion, this intravascular thrombosis deserves to be taken into much higher account than usually done. ^ In fact, the majority of cancer patients have coagidation abnormalities associated to hypoxia. ^ Recently, it has been demonstrated that hypoxia not only induces VEGF but also stimulates endothelial cells to over express tissue factor (TF) and Plasminogen activator inhibitor (PAI-I). These factors induce endothelium to become prothrombotic and causefibrinformation and platelet activation.^^ Furthermore, VEGF binds tofibrinogenand fibrin by stimidating endothelial cell proliferation.^^ Fibrin has been demonstrated to be essential for supporting endothelial cells spreading and migration.^ The haemostatic system, in a certain sense, becomes a regulator of angiogenesis and it can partially explain acute hypoxia and its regional appearance and disappearance.^ Concluding hypoxia becomes a
Influence ofTumorMicroenvironmenton Thermoresponse
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Figure 4. Photomicrograph of a platelet thrombus in a peripheral blood smear of a mouse bearing an Ehrlich carcinoma implanted on the hind leg. MGG staining. X 63 objective. Differential interference contrast.
self-perpetuating mechanism able to trigger angiogenesis, intratumoral fluid accumulation and thrombosis (Figs. 2, 4)."^^
Tumor Bioenergetic Status, Hypoxia, pH Normally cancer cells display many altered metabolic abnormalities including an increased capacity to metabolise carbohydrates mainly by anaerobic glycolysis even under aerobic conditions. This metabolic behaviour results from the induction of many enzymes* involved in the intermediate reactions of glycolysis by HIF-1 genes. ^"^'^^ The relevance of these alterations is that oxidation of glucose stops at the stage of pyruvic acid and proceeds anaerobically producing for the same ATP amount six times more lactic acid and H^ than normal cells. The excessive lactic acid and H^ accumulation in tumour milieu, matched with the compromised interstitial fluid transport, causes the decrease of the external tumor pH (pHe).55-5^ Recently studies indicate that both local glucose-glutamine and oxygen availabilities affect tumour acidity independently. In the authors' opinion these findings have significant implications for cancer treatment.^^ Most pH estimations in tumour tissues were obtained by the insertion of microelectrodes.^^' They demonstrated the value of pHe in a range between 5.6 to 7.G (normal tissue pHe is 6.8) and they pointed out that tumors grow in an acidic environment. However for many years microelectrodes measurements were inaccurate and the calculated pHi was thought to be acidic. Recently the advent of ^^ P magnetic resonance spectroscopy, noninvasive method
*Enzymes HIP induced: [GLUT-1, type I exokinase, Aldolase A, Lactate dehydrogenase, Phosphofructokinase L, Posphoglycerate kinase 1 and pyruvate kinase M].
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Hyperthermia in Cancer Treatment: A Primer
Table 2. pH regulatorsand inhibitors pH Regulators
pH Inhibitors
1) V-ATPase (Vacuolar proton pump)
1A) IB) 2A) 2B) 2C) 3A) 3B) 4A) 4B)
2) Lactate/H'^Symport [MCT]
3) NAVH-'exchanger [NHE] 4) CL/HCOa-[BCT]
Bafilomycin Oximidine a-cyano-4-hydroxylcinnamic acid [CNCN] Lonidamine Quercetin Amiloride Cariporide Cariporide 4,4'-diisocyanatostil-bene-2, 2'-disulfonic acid [DIDS]
Mitochondrial Metabolic Inhibitors 1) Lonidamine 2) Metaiodobenzylguanidine [MIBG] of measurement, has permitted to measure pHi and p H e simultaneously more accurately. Associated to these measures other parameters of interest can be obtained and are: tissue perfusion and vessel permeability. These studies have shown that in a majority of animal and human tumors p H e is lower than pHi. In fact, the tumor pHi resulted, similar to that normal tissue or near neutrality (i.e., ± 0 . 1 to 0.2 p H units) whereas p H e obtained from different human tumors were 0.41 ± 0.27 units lower than the normal tissue one.^^'^^ T h e neutral p H i and p H gradient with the acidification of tumor milieu can be explained by the following factors: A. In normal tissue the lactic acid, accumulated in the interstitial fluid, is rapidly removed through lymphatic drainage, whereas in TIF is not so easily removed (due to a compromised vasculature and a absent lymphatic drainage).^^ B. Cancer cells, as normal cells, express a number of pH regulatory mechanisms to maintain a cytosol pH near neutrality (pHi = 7 A). The mechanisms underlying regulation of intracellular pH have been identified as their inhibitors and are illustrated and listed in Table 2 and Figure 5.^^ A brief description of the most important exchanger mechanisms active in cancer cells and their inhibitors is reported: 1. Vacuolar-type H* ATP-ase is a ion transporter regulated by ATP-dependent mechanism. 2. H+-lactate cotransport Na ^ / H * exchanger [MCT],is an exchanger mechanism revers ibly blocked by Amiloride and quercetin. 3. Na^dependent C 1 7 H C 0 3 ' exchanger [BCT], is blocked by disulfonic stilbene derivative 4,4'-diisothiocyanostilbene -2,2' disulfonic acid [DIDS] 4. Sodium-proton exchanger [NHE]. The N H E family of ion exchangers includes six isoforms (NHE1-NHE6) that function in an electroneutral exchange of intracellular H+ for extracellular Na^ They are blocked by Amiloride and its derivative Cariporide. 5,6. electrogenic N a ^ - H C 0 3 ' cotransport T h e lactate efflux and formation can be also blocked by metabolic inhibitors such as: lonidamine or by meta-Iodio-benzylguanidine (MIBG) or alpha-cyano-4-hydroxy-cinnamic acid ( C N C n ) . These drugs are active on mitochondrial generation of lactic acid by blocking Krebs cycle) (Table 2 and Fig. 5).
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GLUCOSE 10% Lonidaminie Quercetin CNCn
KREBS CYCLE
AMILORIDE CARIPORIDE
/5^
4i3 DiDS
fv-ATPaseJ
pHi>7
ypHe0.08). On these grounds, they suggest that extracellular pHg measurement may be a useful prognostic indicator of tumor response to thermotherapy. Although, a pHg reduction of «0.2 units is easily obtainable by glucose load, greater reduction >0.5 units are necessary for inducing acute intracellular acidosis. ^^^ The degree of reduction of pH; that accompanies acute extracellular acidification is the critical factor for sensitizing cells to hyperthermia and for abrogating the heat shock proteins induction. Recently various Authors have demonstrated that for obtaining such pHedrop in melanoma, metabolic inhibitors such as meta-Iodio-benzylguanidine (MIBG) or alpha-cyano-4-hydroxy-cinnamic acid (CNCn) must be added.^'^^ For understanding, the biochemical points of action of these inhibitors (see Fig. 5). In conclusion, the concomitant administration of glucose together with MIBG increases the tumour magnitude and duration of acidification and the oxygen tension. ^^^'^^^ This association has the potential to improve response to radiation therapy and to hyperthermia itself The protocol used in our laboratory was similar to that used by Nagata (Table 4).^^^ Following these studies, we used 500 cc of Glucose at 10% obtaining blood glucose value of 300-400 mg/dl without side effects (unpublished observations). Recently, following the suggestions of Leeper group we have added to the hyperglycemia two metabolic inhibitors such as quercetin and Amiloride (Moduretic®) (Table 4).
Modifiers of Thermal Sensitivily Lidocaine and anesthetics,^^^'^ Calcium antagonists,^^^ polyunsaturated fatty acids.^^^ cycloxygenase inhibitors,^^^ betulinic acid,^^^ aldehydes,^^^ vitamins and bioflavonoids.^^^'^ "^
Heat Delivery Methods Tumor cell killing curves by heat show a shape that it is both time and temperature-dependent and not dissimilar from those obtained for X-rays. The data in vitro are consistent with results in vivo and show that relatively small changes in temperature can have a large effect on cell killing^ ^'^ The critical temperature has been demonstrated to be between 42.5°C and 43°C.
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Hyperthermia in Cancer Treatment: A Primer
Unfortunately the hyperthermia devices now in use are not able to keep this range of temperature for enough time uniformly. This justifies the attempt done by different authors to change or to modify the treatment application.
Rapid Heating Rapid heating is a method developed by Hasegawa group^^^ in the attempt to shorten hyperthermic treatment still reaching temperature sufficient to kill tumour cells and to change tumor blood perfusion. The experiments have been made on C3H mice inoculated with SCC-VII tumor in the thigh, heated with warm water bath and RF heating devices. C3H mice were divided in two treatment groups and compared, the former in which the heating temperature was increased to the target temperature in 1 min, and the latter group in which the heating temperature was gradually increased. The following parameters were studied: changes in blood flow in tumour and normal tissue, tumour growth rate, cancer cells apoptosis. Changes in blood flow were not observed in the slow heating group before or after the hyperthermic treatment, whereas in the rapid heating group a significant increase in blood flow was observed in the normal tissue followed by a significant decrease after heat treatment in the tumour tissue. Tumor growth delay was more evident in the RF rapid heating group compared with warm water heating group. Apoptosis and cytokinetic activity modifications were favorable to the rapid heating group, revealing that a vascular injury was effectively obtained with a shortage in treatment time in this group. ^ Clinical studies on this methodology are warranted.
Mild Hyperthermia and Oxygenation As previous described, solid tumors contain regions of low extracellular pH and oxygen that may affect treatment outcome. Laboratory and clinical data confirm that hyperthermia may enhance the therapeutic index of ionizing radiation.^ ^ Several mechanisms have been found and are summarized in the recent reviews of Kampinga and Vujaskovic.^ Among these mechanisms, tumor oxygenation improvement after mild hyperthermia (HT with temperature between 39-42.5°C) is now considered to be of the utmost importance. As determined by Song et al,^^ normal and tumor tissue show a different behavior following heat deposition. Blood tumor vessels respond markedly different to a second heat application showing a greater vulnerability and vasodilatation to heat than normal surrounding blood vessels. This phenomena, referred by Song as vascular thermotolerance (VT), appears to account for the improvement in the tumour blood flow observed after the reheating at 42.5"C. As the blood flow increase, an improvement in tumor oxygenation follows which may last for as long as 24-48 j^ 94,95 Tumor oxygenation by mild HT has been found to be more effective than carbogen breathing in increasing the radiation response of experimental tumors.^ '^ Clinical studies on 18 patients with locally advanced breast cancer treated with thermo-chemo-radiotherapy have confirmed these experimental results. Tumour oxygenation improvement appeared to be temperature-dependent and associated with the lower thermal doses.
Sununaiy and Conclusions Tumour hypoxia is a problem that makes tumors more resistant to ionizing radiation and chemotherapeutic drugs. Hyperthermia represents a possibility in its overcoming; overall in association with other therapies such as Radiotherapy and chemo-immunotherapy.^ Moreover the effect of mild HT on oxygenation is of great relevance in fact, temperature of 39-39.5''C is more easily obtainable in clinic than killing temperature of 42.5°C.
Acknowledgements We thank for her secretarial assistance N. Tortolone and L. Scappini (Novara University Medical Library).
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References 1. Ruiter D, Bogenreider T, Herlyn M. Melanoma-stroma interactions: Structural and functional aspects: Lancet Oncol 2002; 3:35-42. 2. Delpech B. Le stroma des cancers. Bull Cancer 1991; 78:869-900. 3. Singh S, Ross SR, Acena M et al. Stroma is critical for preventing or permitting immunological destruction of antigenic cancer cells. J Exp Med 1992; 175:139-146. 4. Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply and metabolic microenvironment of human tumors, a review. Cancer Res 1989; 49:6449-6465. 5. Freitas I, Baronzio GF. Tumor hypoxia, reoxygenation and oxygenation strategies: Possible role in photodynamic therapy. J Photochem Photobiol B Biol 1991; 11:3-30. 6. Folkman J. T u m o r Angiogenesis: Therapeutic implications. N Engl J Med 1971; 285:1182-1186. 7. Berges G, Benjamin L. Tumorigenesis and the angiogenic switch. Nat Rev Cancer 2002; 3:401-410. 8. Mc Donald D M , Choyke PL. Imaging of angiogenesis: From microscope to clinic. N a t Med 2003; 9:713-725. 9. Pugh C W , Ratcliffe PJ. Regulation of angiogenesis by hypoxia role of the HIF system. Nat Med 2003; 9:677-684. 10. Semenza GL. HIF-1 and human disease: One highly involved factor. Gene Dev 2000; 14:1983-1991. 11. Semenza GL. Homeostatic regulation by Hypoxia -inducible factorl. Science and Medicine 2002; 8:338-347. 12. Dachs G U , Tozer G M . Hypoxia modulated gene expression: Angiogenesis, metastasis and therapeutic exploitation. Eur J Cancer 2000; 36:1649-1660. 13. Ferrara N , Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997; 18:4-25. 14. Papetti M, Herman I. Mechanisms of normal and tumor-derived angiogenesis. Am J Physiol Cell Physiol 2002; 282:c947-c970. 15. Griffioen AW, Molema G. Angiogenesis:Potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases, and chronic inflammation. Pharmacol Rev 2000; 52:238-268. 16. Kurebayashi J, Osuki T, Kunishe H et al. Expression of Vascular endothelial growth factor (VEGF) family members in breast cancer. Jpn J Cancer Res 1999; 90:977-981. 17. Balbay M D , Pettaway CA, Kuniyasu H et al. Highly metastatic human prostate cancer growing within prostate of athymic mice overexpresses vascular endothelial growth factor. Clin Cancer Res 1999; 5:783-789. 18. L Hlatky P, Hahnfeldt C, Tsionou C. Coleman: Vascular endothelial growth factor: Environmental controls and effects in angiogenesis. Br J Cancer 1996; (Suppl XII):sl51-sl56. 19. Pepper MS. Lymphangiogenesis and tumor metastasis: Myth or reality? Clin Cancer Res 2001; 7:462-468. 20. Polverini PJ. How the extracellular matrix and macrophages contribute to angiogenesis-dependent diseases. Eur J Cancer 32A; 14:2430-2437. 2 1 . Chiford SC, Maher ER. Von Hippel-Lindau disease. Adv Cancer Res 2001; 82:85-105. 22. Grugel S, Finkezeller G, Weindel K et al. Both v-Ha-ras and v-raf stimulate expression of Vascular endothelial growth factor in N I H 3T3 cells. J Biol Chem 1995; 270:25915-9. 23. Takakura N , Watanabe T, Suenobu S et al. A role for Hematopoietic stem cells in promoting angiogenesis. Cell 2000; 102:199-209. 24. Lyden D, Hattori K, Dias S et al. Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks t u m o r angiogenesis and growth. N a t u r e M e d 2 0 0 1 ; 7(11):1194-1201. 25. Abramsson A, Berlin O , Papayan H et al. Analysis of mural cell recruitment to tumor vessels. Circulation 2002; 105:112-117. 26. Dewirst M W , Tso CY, Oliver R et al. Morphologic and hemodynamics comparison of tumor and healing normal tissue microvasculature. It J Radiat Oncol Biol Phys 1989; 17:91-99. 27. Konerding MA, Malkusch W, Klaptor B et al. Evidence for characteristic vascular patterns in solid tumors: Quantitative studies using corrosion casts. Br J cancer 1999; 80:724-32. 28. Konerding MA, Steiberg F, van Ackern C et al. Vascular patterns of tumors: Scanning and trasmission electron microscopic studies on humam xenografts. Strahlentherapie un Onkologie 1992; 168:444-452. 29. Hirshi KK, D'Amore PA. Control of angiogenesis by pericytes: Molecular mechanism and significance. In: Goldberg ID, Rosen EM, eds. Regulation of Angiogenesis: Birkhauser Press, 1997:419-428. 30. Griffioen AW, Tromp SC, Hillen HFP. Angiogenesis modulates the tumor immune response. Int J Exp Path 1998; 79:363-368. 3 1 . Griffioen AW, Coenen M J H , Damen CA et al. C D 4 4 is involved in tumor angiogenesis; an activation antigen on human endothelial cells. Blood 1997; 90:1150-59.
88
Hyperthermia in Cancer Treatment: A Primer
32. Carlos TM. Leucocyte recruitment at sites of tumor: Dissonant orchestration. J Leucoc Biol 2001; 70:171-184. 33. Sevick EM, Jain RK. Measurement of capillary filtration coefficient in a solid tumor. Cancer Res 1991; 51:1352-1355. 34. Jain RK. Determinants of tumor blood flow: A review. Cancer Res 1988; 48:2641-2658. 35. GuUino PM. The internal Milieu of Tumors. Prog Exp Tum Res 1966; 8:1-25. 36. Gullino PM. Extracellular compartments of solid tumors. In: Beckert FB, ed. Cancer, a Comprehensive Treatise. Vol 3, Biology of Tumors: Cellular Biology and Growth. New York: Plenum, 1975:327-354. 37. Freitas I, Baronzio GF, Bono B et al. Tumor interstitial Fluid: Misconsidered component of internal milieu of a solid tumor. Anticancer Res 1997; 17:165-172. 38. Nagy JA, Herzberg KT, Dvorak JM et al. Pathogenesis of malignant ascites formation: Initiating events that lead to fluid accumulation. Cancer Res 1991; 53:2631-2643. 39. Steen RG. Oedema and tumor perfusion: Characterization by quantitative IH MR imaging. Am J Radiol 1992; 158:259-264. 40. Boucher Y, Less JR, Posner MC et al. Interstitial hypertension in human primary and metastatic tumors. In: Chapman JD, Dewey WC, Withmore GF, eds. Radiation Research: A Twentieth Century Perspective. Vol 1. San Diego: Academic Press, 1991:465. 41. Gutmann R, Leuning M, Feyh J et al Interstitial Hypertension in head and neck tumors in patients. Correlation with tumor size. Cancer Res 1992; 52:1993-1995. 42. Sevick EM, Jain RK. Geometric resistance to blood flow in solid tumours perfused ex vivo: Effects of tumor size and perfusion pressure. Cancer Res 1989; 49:3506-3512. 43. Sevick EM, Jain RK. Viscous resistance to blood flow in soHd tumors: Effect of hematocrit and intratumor viscosity. Cancer Res 1989; 49:3513-3519. 44. Peterson HI. Modification of tumor blood flow, a review. Int J Radiat Biol 1991; 60:201-210. 45. Vaupel P. Tumor blood flow. In: Molls M, Vaupel P, eds. Blood Perfusion and microenvironment of human tumors. Berlin Heidelberg, New York: SpringerVerlag, 2000:41-46. 46. Durand RE. Intermittent blood flow in soUd tumours an under appreciated source of "drug resistance". Cancer Metastasis Rev 2001; 20:57-61. 47. Baronzio GF, Freitas I, Kwann H. Tumor microenvironment (hypoxia-interstitial Fluid) and haemorheologic abnormalities. Semin Thromb Hemost 2003; 29:489-497. 48. Denko NC, Giaccia AJ. Tumor hypoxia, the physiological link between Trousseau's Syndrome (carcinoma -induced Coagulopaty) and metastasis. Cancer Res 2001; 61:795-798. 49. Verheul HMW, Hoekman JK, Broxterman HJ et al. Vascular endothelial factor-stimulated endothelial cells promote adhesion and activation of platelets. Blood 2000; 96:4216-4221. 50. Sahni A, Francis CW. Vascular endotheUal growth factor binds to fibrinogen and fibrin stimulates endotheUal cell proHferation. Blood 2000; 96:3772-3778. 51. Browder T, Folkman J, Pirie-Sheperd S. The hemostatic system as regulator of angiogenesis. J Biochem 2000; 275:1521-1524. 52. Dang CV, Semenza GL. Oncogenic alterations of metabolism. TIBS 1999; 24:68-72. 53. Shapot VS. Biochemical aspects of tumor growth. MIR Publishers, 1980. 54. Behrooz A, Ismail-Beigi F. Stimulation of glucose transport by Hypoxia: Signals and mechanisms. New Physiol Sci 1999; 14(6):105-110. 55. Younes M, Lechago LV, Somano JR et al. Wide expression of the human erythrocyte glucose transporter Glut 1 in human cancers. Cancer Res 1996; 56:1164-1167. 56. Gullino PM, Grantham FH, Smith SH et al. Modification of the acid base status of the internal miheu of tumors. J Nad Cancer Inst 1965; 34:857-869. 57. Asby BS, Cantab MB. pH studies in human malignant tumours. Lancet 1996; 2:312-315. 58. Griffiths JR, Mclntyre DJ, Howe FA et al. Why are cancers acidic? A carrier mediated diffusion model for H^transport in the interstitial fluid. In: Goodie JA, Chadwick DJ, eds. The Tumor Microenvironment: Causes and Consequences of Hypoxia and Acidity. Novartis Foundation Symposium. Chichester, New York: John Wiley, 2001:46-67. 59. Helmlinger G, Sckell A, Dellian M et al. Acid production in glycolysis-impaired tumors provides new insights into tumour metabolism. Clin Cancer Res 2002; 8:1284-1291. 60. Stubbs M, McSheehy PMJ, Griffiths JR et al. Causes and consequences of tumor acidity and implications for treatment. Mol Med Today 2000; 6:15-19. 61. Newell K, Tannock. Regulation of intracellular pH and viability of tumors cells. Funktionsanalyse Biologischer Systeme 1991; 20:219-234. 62. Izumi H, Torigoe T, Ishiguchi H et al. Cellular pH regulators: Potentially promising molecular targets for cancer chemotherapy. Cancer Treat Rev 2003; 29:541-549. 63. Vaupel P. Pathophysiological effects of hyperthermia in solid tumors and their clinical impHcation. In: Georg H Omlor, Peter Vaupel, Cristof Alexander RG, eds. Isolated Hyperthermic Limb Perfusion. Georgetown: Landes Bioscience, 1995:9-45.
Influence of Tumor Microenvironment
on Thermoresponse
89
64. Mueller-Klieser W, Walenta S, Paschen W et al. Metabolic imaging in microregions of tumors and normal tissues with bioluminescence and photon counting. J N C I 1988; 80:842-848. 65. Vaupel P. Pathophysiological mechanisms of hyperthermia in cancer therapy. In: Gautherie M, ed. Biological Basis of Oncologic Thermotherapy. Berlin, Heidelberg, New York: Springer-Verlag, 1990:73-134. GG. Gerweck LE. Tumor p H : Implication for treatment and novel drug design. Semin Radiat Oncol 1998; 8(3):176-182. G7. Hetzel FW. Biological rationale for hyperthermia. Radiol Clin North Am 1989; 27:499-508. 68. Dudar TE, Jain RK. Differential response of normal and tumor microenvironment to hyperthermia. Cancer Res 1984; 44:605-612. 69. Hall EJ. Hyperthermia. In: Hall EJ, ed. Radiobiology for the Radiologist. Lippincott: Wilkins Williams, 2000:495-520. 70. Suit H D , Gerweck LE. Potential for hyperthermia and radiation therapy. Cancer Res 1979; 39:2290-2298. 7 1 . Gerwek LE, Richards B. Influence of p H on thermal sensitivity of cultured human glioblastoma cells. Cancer Res 1981; 41:4019-4024. 72. Gerwek LE. Modifiers of Thermal effects. In: Urano M, Douple E, eds. Hyperthermia and Oncology. Vol 1. The Netherlands: VSP, 1988:83-98. . 73. Hahn G M , Shiu E. Adaptation to low p H modifies thermal and thermochemical response of mammalian cells. Int J Hyperthermia 1986; 2:379-387. 74. Gerwek LE, Richards B. Influence of p H on thermal sensitivity of cultured human gHoblastoma cells. Cancer Res 1981; 41:4019-4024. 75. Lyons JC, Kim G, Song CW. Modification of intracellular p H and Thermosensitivity. Radiation Research 1992; 129:79-87. 7G. Song CW, Park H, Griffin RJ. Theoretical and experimental basis of Hyperthermia. In: Kosaka M, Sugahara T, Schmidt KL et al, eds. Thermotherapy for neoplasia, inflammation, and pain. Tokyo: Springer Verlag, 2001:394-407. 77. Van der Berg A, Wike-Hooley JL, Broekmayer-Reurink et al. The relationship between the unmodified initial tissue p H of human tumors and the response to combined radiotherapy and local hyperthermia treatment. Eur J Cancer Clin Oncol 1989; 25:73-78. 78. Rhee JC, Eddy HA, Salazar O M et al. A differential low ph effect on tumour cells grown in vivo and in vitro when treated with hyperthermia. Int J Hyperthermia 1991; 7:75-84. 79. Hall EJ. Hyperthermia. In: Hall EJ, ed. Radiobiology for the radiologist. Lippincott: WilUams Wilkins, 2000:495-520. 80. Gerwek LE, Dahlberg WK, Greco B. Effect of p H on single or fractionated heat treatment at Al-AyC. Cancer Res 1983; 43:1163-1167. 8 1 . Nielsen O S , Overgaard J. Effect of extracellular p H on Thermotolerance and recovery of hyperthermic damage in vitro. Cancer Res 1979; 38:2772-2778. 82. Han JS, Storck C W , Wachsberger PR et al. Acute extracellular acidification increases nuclear associated protein levels in human melanoma cells during 42 degrees C hyperthermia and enhances cell killing. Int J Hyperthermia 2002; 18:404-415. 83. Field SB. In vivo aspects of hyperthermic oncology. In: Field SB, Hand JW, eds. An Introduction to the Practical Aspects of Clinical Hyperthermia. London, New York, Philadelphia: Taylor & Francis, 1990:55-68. 84. Van De Merwe SA, Van Den Berg Block E, Kroon BBR et al. Temporary vascular occlusion and glucose: Effects on tumour and normal tissue PH in animal experiments. Int J Hyperthermia 1995; 11:829-839. 85. Engin K, Leeper DB, Thistlethwaite AJ et al. Tumor extracellular p H as a prognostic factor in thermoradiotherapy. Int J Radiation Oncology Biol Phys 1994; 29:125-132. 86. Von Ardenne. Selective multiphase cancer therapy. Conceptual aspects and experimental basis. Adv Pharmacol Chemother 1972; 10:339-380. 87. Dixon JA, Calderwood SK. Effect of hyperglycaemia and hyperthermia on the, glycolsis p H and respiration of the Yoshida sarcoma in vivo. J Natl Cancer Inst 1979; 63:1371-1381. 88. MuUer-Klieser W , W a l e n t a S, Kellher D K et al. T u m o u r growth i n h i b i t i o n by i n d u c e d hyperglycaemia / hyperlactatacidaemia and localized hyperthermia. Int J Hyperthermia 1996; 12:501-11. 89. Lippmann H G , Graichen D. Glucose and K* balance during high dosage intravenous glucose infusion. Infusionsther Klin Ernahr 1977; 4:166-178. 90. van Den Berg AP, van Den Berg AE, Kal H B et al. A moderate elevation of blood glucose level increases the effectiveness of thermoradiotherapy in a rat tumor modelll. Improved tumor control at clinically achievable temperatures. Int J Radiat Oncol Biol Phys 2001; 50:793-801.
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91. Krag DN, Storm FK, Morton DL. Induction of transient hyperglycaemia in cancer patients. Int J Hyperthermia 1990; 6:741-744. 92. Ward KA, Jain RK. Response of tumours to hyperglycaemia: Characterization, significance and role in hyperthermia. Int J Hyperthermia 1988; 4:223-250. 93. Gullino PM, Grantham FH. Studies on the exchange of fluids between host and tumor,II. The blood flow of hepatomas and other tumors in rats and mice. J Nad Cancer Inst 1961; 27:1465-1491. 94. Song CW, Chelstrom LM, Sung JH. Effects of a second heating on tumor blood flow. Radiat Res 1990; 122:66-71. 95. Song CW. Effect of local hyperthermia on blood flow and microenvironment: A review. Cancer Res 1984; 44(suppl):4721-4730. 96. Li GC. Thermal biology and physiology in clinical hyperthermia: Current status and future needs. Cancer Res 1984; 44(suppl):4886s-4893s. 97. Vaupel P, KalUnowski F. Physiological effects of hyperthermia. Recent Results in Cancer 1987; 104:71-109. 98. Reinhold HS, Endrich B. Tumor microcirculation as a target for hyperthermia. Int J Hyperthermia 1986; 2:11-137. 99. LeVeen HH, Wapnick S, Piccione V et al. Tumor eradication by radiofrequency therapy. Response in 21 patients. J Am Med Assoc 1976; 235:2198-2200. 100. Kim JH, Hahn EW, Tokita N et al. Local tumor Hyperthermia in combination with radiation therapy. 1. Malignant cutaneous lesions. Cancer 1977; 40:161-69. 101. Hiraoka M, Shiken JO, Keizo A et al. Radiofrequency capacitive Hyperthermia for deep-seated tumors, 1. Studies on Thermometry Cancer 1987; 121-127. 102. Tanaka Y. Thermal response of microcirculation and modification of tumor blood flow in treating the tumors. In: Kosaka M, Sugahara T, Schmidt KL et al, eds. Theoretical and experimental basis of Hyperthermia. In Thermotherapy for neoplasia, inflammation, and pain. Tokyo: Springer Verlag, 2001:408-419. 103. Jain RK, Ward-Hardey K. Tumor bloodflow-Characterization,modifications and role in hyperthermia. IEEE Transactions on Sonics and Ultrasonic 1984; 31:504-526. 104. Fajardo LF, Prionas SD. EndotheUal cells and hyperthermia. Int J Hyperthermia 1994; 3:347-353. 105. Nishimura Y, Hiraoka M, Jo S et al. Microangiographic and histologic analysis of the effects of hyperthermia on murine tumor vasculature. Int J Radiat Oncol Biol Phys 1988; 15:411-420. 106. Evans SS, Frey M, Scheider DM et al. Regulation of leukocyte-endothelial cell interaction in tumor immunity. In: Mihich and Croce, eds. Biology of Tumors. Plenum Press, 1998:(Ch 20):273-286. 107. Roca C, Primo L, Valdembri D et al. Hyperthermia inhbits angiogenesis by a Plasminogen Activator Inhibitor -I dependent mechanism. Cancer Res 2003; 63:1500-1507. 108. Jirtle RL. Chemical modifications of tumor blood flow. Int J Hyperthermia 1988; 4:355-371. 109. Suit H, Shalek RJ. Response of spontaneous mammary carcinoma of the C3H mouse to X-irradiation given under conditions of local tissue anoxia. J Nat Cancer Inst 1963; 31:497-509. 110. Hill SA, Denekamp J. The effect of vascular occlusion on the thermal sensitisation of a mouse tumour. Br J Radiol 1978; 51:997-1002. 111. Stuart K- Chemoembolization in the management of liver tumors. The Oncologist 2003; 8:425-437. 112. Tanaka Y, Yamamoto K, Nagata K. Effects of multimodal treatment and hyperthermia on hepatic tumors. Cancer Chemother Pharmacol Suppl 1998; 1:111-114. 113. Hirst DG, Hirst VX, Shaffi KM et al. The influence of vasoactive agents on the perfusion of tumors growing in three sites in the mouse. Int J Radiat Oncol Biol Phys 1991; 60:211-218. 114. Baguley BC, Wilson WR. Potential of DMXAA combination therapy for solid tumors. Expert Rev Anticancer Ther 2002; 2:593-603. 115. Horsman MR, Murata R. Combination of vascular targeting agents with thermal or radiation therapy. Int J Radiat Oncol Biol Phys 2002; 54:1518-1523. 116. Calderwood SK, Dickson JA. Effect of hyperthermia on blood flow, pH and response to hyperthermia(42°C) of the Yoshida sarcoma in the rat. Anticancer Res 1980; 40:4728-4733. 117. Dickson JA, Calderwood SK. Thermosensitivity of neoplastic tissues in vivo. In: Storm FK, ed. Hyperthermia in Cancer Therapy. Boston, GK: Hall, 1983:63-140. 118. Nagata K, Tanaka Y, Akagi K et al. Enhancement of thermoradiotherapy by glucose administration for superficial malignant tumors. J Radiat Res 1998; 14:157-167. 119. Crandall E, Crtz A, Osher A et al. Influence of pH on elastic deformability of the human erythrocyte membrane. Am J Physiol 1978; 235:c269-c278. 120. Traykov TT, Jain RK. Effect of glucose and galactose on red blood cell membrane deformability. Int J Microcirc CUn Exp 1987; 6:35-44. 121. Hasegawa T, Gu Y-H, Takahashi T et al. Effects of hyperthermia-induced changes in pH value on tumor response and thermotolerance. In: Kosaka M, Sugahara T, Schmidt KL et al, eds. Thermotherapy for Neoplasia, Inflammation, and Pain.Tokyo: Springer Verlag, 2001:431-438.
Influence of Tumor Microenvironment
on Thermoresponse
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122. Shem BC, Dahl O . Thermal enhancement of A C N U and potentiation of thermochemotherapy with A C N U by hypertonic glucose in the BT4 A rat glioma. J Neuroncol 1991; 10:247-252. 123. Lippmann H G , Graichen D. Glucose and K* balance during high dosage intravenous glucose infusion. Infusionsther Klin Ernahr 1977; 4:166-178. 124. V o n A r d e n n e M . In vivo T h e o r i e zum glykolytishen Stoffwechsel der T u m o r e n ihrer Cbersauerbarkeit dutch Hyperglykamie. In: Hippokates Verlag Stuttgart, ed. Systemidche Krebs-Mehrschritt-Therapie. 1997:35-45. 125. Nagata K, Murata T, Shiga T et al. Enhancement of thermoradiotherapy by glucose administration for superficial malignant tumours. Int J Hyperthermia 1998; 14:157-167. 126. Leeper DB, Engin K, Wang J-H et al. Effect of I.V. glucose versus combined I.V. plus oral glucose on human tumour extracellular p H for potential sensitisation to Thermotherapy. Int J Hyperthermia 1998; 257-269. 127. Engin K, Leeper DB, Cater JR et al. Extracellular p H distribution in human tumors. Int J Hyperthermia 1995; 11:211-216. 128. Han JS, StorcK CW, Wachsberger PR et al. Acute extracellular acidification increases nuclear associated protein levels in human melanoma cells during 42°Chyperthermia and enhances cell killing. Int J Hyperthermia 2002; 18:404-415 129. Coss R, Storck CW, Daskalaqkis C et al. Intracellularacidification abrogates theheat shock response andcompromises survival of human melanoma cells. Mol Cancer Therapeut 2003; 383-388. 130. Burd R, Wachsberger PR, Biaglow JE et al. Absence of Crabtree effect in human melanoma cells adapted to growth at low p H : Reversal by respiratory inhibitors. Cancer Res 2001; 61:5630-5635. 131. Zhou R, Bansal N , Leeper D R et al. Enhancement of hyperglycemia-induced acidification of human melanoma xenografts with inhibitors of respiration and ion transport. Acad Radiol 2001; 8:571-582. 132. Zhou R, Bansal N , Leeper D R et al. Intracellular acidification of human melanoma xenografts by respiratory inhibitor m-Iodio-benzylguanidine plus hyperglycemia: A^^ P Magnetic resonance spectroscopy study. Cancer Res 2000; 61:3532-3536. 133. Hahn G M . Thermal Enhancement of the actions of anticancer agents. In: Hahn G M , ed. Hyperthermia and Cancer. New York, London: Plenum Press, 1982:55-85. 134. Sensiterra GA, Lepock JR. Thermal destabilization of transmembrane proteins by local anesthetics. Int J Hyperthermia 2000; 16:1-17. 135. Kameda K, Kondo T, Tanabe K et al. The role of intracellular Ca 2+ in apoptosis induced hyperthermia and ist enhancement by verapamil in U937 cells. Int J Radiat Oncol Biol Phys 2001; 49:1369-1379. 136. Kokura S, Yoshikawa T, Kaneko T et al. Efficacy of hyperthermia and polyunsaturated fatty acids on experimental carcinoma. Cancer Res 1997; 57:2200-2202. 137. Asea A, Mallick R, Lechpammer S et al. Cycloxygenase inhibitors are potent sensitizers of prostate tumours to hyperthermia and radiation. Int J Hyperthermia 2001; 17:401-414. 138. Wachsberger PR, Burd R, Wahl ML et al. Betulinic acid sensitization of low p H adapted human melanoma cells to hyperthermia. Int J Hyperthermia 2002; 18:153-164. 139. Kim J H . Modification of thermal effects: Chemical modifiers. In: Urano M, Douple E, eds. Hyperthermia and Oncology. Vol 1. The Netherlands: VSP, 1988:83-119. 140. Prasad K, Kumar B, Yan X-D et al. a-tocopheryl succinate, the most effective form of Vit. E for adjuvant cancer treatment: A review. J Am Coll N u t r 2003; 22:108-117. 141. Callari D , Sinatra F, Paravizzini GL et al. All trans retionic acid sensitizes colon cancer cells to hyperthermia cytotoxic effects. Int J Oncol 2003; 23:181-188. 142. Wachsberger PR, Burd R, Bhala SB et al. Quercetin sensitizes cells to hyperthermia. Int J Hyperthermia 2003; 19:507-519. 143. Van der Zee J. Heating the patient: A promising approach? Annals of Oncology 2002; 13:1173-1184. 144. Kampinga KH, Dikomey E. Hyperthermia radiosensitization: Mode of action and clinical relevance. Int J Radiat Biol 2001; 77:399-408. 145. Hasegawa T, Gu YH, Takahashi T et al. Enhancement of hyperthermic effects using rapid heating. In: Kosaka M, Sugahara T, Schmidt KL et al, eds. Thermotherapy for neoplasia, inflammation, and pain. Tokyo: Springer Verlag, 2001:439-444. 146. Vujaskovic Z, Song CW. Physiological mechanisms underlying heat-induced radiosensitization. Int J Hyperthermia 2004; 20:163-174. 147. Song CW, Park H, Griffin RJ. Improvement of tumour oxygenation by mild hyperthermia. Radiat Res 2001; 155:515-528. 148. Jones EL, Prosnitz LR, Dewhirst M W et al. Thermochemoradiotherapy improves oxygenation in locally advanced breast cancer. Clin Cancer Res 2004; 10:4287-4293. 149. Pontiggia P, Mc Laren JR, Baronzio GF et al. T h e biological responses to heat. Adv Exp Med Biol 1990; 267:271-291.
CHAPTER 6
Hyperthertnia and Angiogenesis: Results and Perspectives Cristina Roca and Luca Primo* Abstract
H
yperthermia (HT) is a promising method for cancer treatment when combined with radiotherapy or chemotherapy. The molecular mechanisms of anti-tumoral efficacy of HT are not well imderstood. Besides its direct cytotoxic effect on tumor cells, HT injures the normal microvasculature and, in particular, tumor vessels. This effect on microvasculature represents an important mechanism of tumor growth inhibition exerted by HT. Recendy, many studies have been made to understand the effects of HT on tumor vessels and, in particular, on new vessel formation that occurs during tumor progression. The tumor vasculature develops in a process known as angiogenesis that consists of the formation of new blood vessels from preexisting ones. Angiogenesis is essential for tumor progression and, without blood vessels, tumors can not grow beyond a critical size or metastatize to another organ. HT above 42°C inhibits endothelial cell (EC) differentiation on capillary-like structures both in vitro and in vivo. At least three distinct mechanisms have been described to be involved in angiogenesis inhibition by HT: direct cytotoxicity on proliferating ECs, down-modulation of vascular endothelial growth factor (VEGF) production by tumor cells and induction of the plasminogen activator inhibitor-1 (PAI-1) expression. These data indicate that inhibition of angiogenesis exerted by heat shock could represent an important mechanism of tumor control in clinical HT and coidd suggest a new rationale for a combined cancer therapy based on HT associated with anti-angiogenic molecules.
Introduction The scientific discipline of HT biology emerged in the 1970s largely from laboratories engaged in research in radiobiology. However, unequivocal identification of the mechanisms leading to favourable clinical research of HT have not yet identified for various reasons. ^"^ Although a large number of preclinical studies are available on different aspeas of HT action, the cellular and molecular pathways underlying this beneficial outcome of patients are still poorly understood. In in vitro studies and in animal experiments, HT exhibited a direct cell killing effect at temperatures ranging from 42°C to 45°C. Furthermore, acute and chronic heating of cells and tissue induces a variety of changes, including alterations of nuclear and cytoskeletal struaures, metabolic pathways and intracellular signals? HT affects fluidity and stability of cellular membranes and impedes the function of transmembrane transport proteins and cell surface receptors. Increasedfluidityof cell membranes was observed in thermosensitive, but not thermotolerant
•Corresponding Author: Luca Primo—Division of Molecular Angiogenesis, Institute for Cancer Research and Treatment, Str. Prov. 142 Km.3.95, Candiolo, TO 10100, Italy. Email:
[email protected] Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.
Hyperthermia and Angiogenesis: Results and Perspectives
cells. As a consequence of these effects, the heated cells undergo changes in membrane potential, elevated intracellular sodium and calcium content, as well as an elevation of potassium efflux. Besides, HT has been demonstrated to induce various changes of cytoskeletal organization (cell shape, mitotic apparatus, intracytoplasmatic membranes such as endoplasmatic reticulum and lysosomes), but there was no clear correlation found between these phenomenological changes and thermosensitivity of various cell lines. '^ Studies of the influence of HT on nucleic acid synthesis indicate that intracellular de novo synthesis and polymerization of both RNA and DNA molecules is rapidly and markedly inhibited after exposure to heat.^ Whereas RNA synthesis recovers rapidly after termination of heat exposure, DNA synthesis is inhibited for a longer period. ^^'^ Heat shock also induces a^regation of denaturated proteins at the nuclear matrix. This is mainly due to insolubility of cellular proteins after heat-induced protein unfolding, entailing enhancement of the nuclear protein concentration. On the trascriptional and translational level, heat shock induces the expression of a unique set of genes, the heat shock genes, that encode for of a number of proteins, called the heat shock proteins (HSP). HSP represent a heterogeneous group of molecular chaperones consisting of at least five subgroups with different molecular mass and partially varying biologic function. HSP are usually divided into small HSP (molecular mass < 40 KDa), and the HSP 60, HSP 70, HSP 90 and HSP 100 proteins families. All HSP families share their chaperoning function, i.e., they unselectively bind to hydrophobic protein sequences liberated by denaturation. In HT, HSPs are thought to be involved in the protection of cells against heat damage. One of the most interesting aspects of thermal biology is the response of heated cells to a heat challenge. Mammalian cells, when exposed to a nonlethal heat shock, have the ability to acquire a transient resistance to one or more subsequent exposures to elevated temperatures. This phenomenon has been termed thermotolerance and it has been suggested that HSPs are involved in the development of transient thermotolerance, in the acquisition of the permanent heat resistance and in the protection of cells from thermal stress. Moreover, HT may be able to cause damage preferentially in tumors relative to normal tissues because the structure and function of the vasculature and related microenvironment in tumors are rather different from those in normal tissues. In this chapter we will face several aspects of the effects of HT on the angiogenic process in light of the knowledge of some molecular aspects involved in angiogenesis inhibition by HT.
Blood Vessels, Blood Flow and Microenvironment One of the most important effects of HT on normal and tumor tissue is the modification of blood flow, and, as a consequence, the regulation of oxygen and nutrient supply. Moderate HT (< 42 °C) has been demonstrated to improve blood flow especially when applied on tumor tissue, which are normally characterized by reduced blood flow and hypoxic microenvironment. The increase of tumor oxygenation following HT improves the effectiveness of radiotherapy.^^ In addition, the delivery of anti-neoplastic drugs can be favored by increased tumor blood flow. Opposite effects on tissue microenvironment have been observed when temperatures were above 42 °C. HT at these temperatures has been shown to decrease blood flow and an excess exposure of tissue to heat results in a breakdown of vasculature followed by necrosis of the tissue.^ The microenvironment of malignant tumors is characterized by a reduction of blood flow and blood vessels density that favors hypoxia, acidosis and energy deprivation.^'^ Tumor vessels often show increased permeability and may have gaps between adjacent Ecs.^^ Unlike vessels of normal tissue, which respond to heat by dilation and increase in blood flow (and thus increase in heat loss by convention), tumor vessels, which lack appropriate innervation, do not dilate and heat diffusion is poor. Therefore, the heat dissipation by blood flow in tumor is slower than in normal tissue and the acidic and hypoxic environment increases the thermosensitivity of tumor cells. Moreover the tumor vasculature can be also severelv damaged at temperatures which may alter but not damage the vasculature of normal tissue.^ The alteration in tumor blood flow and microvasculature induced by HT can partially explain the increased thermal sensitivity of tumor compared to normal tissues. On the other
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hand, the preferential damage of tumor vessels as opposed to quiescent vessels is difficult to explain. Although some evidences support the hypothesis that ECs are more thermosensitive than other stromal cells, these data do not clarify whether HT is more cytotoxic on tumor ECs than on normal Ecs.^^'^^
Tumor Angiogenesis Angiogenesis, the formation of new blood vessels from preexisting ones, occurs primarily during embryonic development, although it also participates in many adult physiological processes such as the female reproductive cycle and wound healing. In adults, the vascular network is quiescent and angiogenesis is normally tri^ered only locally and transiendy. Angiogenesis is essential for tumor progression. During the premalignant stages of tumor development, cancer cells activate the quiescent vascidature to produce new blood vessels through an angiogenic switch. ^^ Without blood vessels, tumors can not grow beyond a critical size or metastatize to another organ. The control of tumor angiogenesis is separate from that of cancer cell proliferation. This raises the possibility that anti-angiogenic drugs can offer a treatment that is complementary to traditional chemotherapy and radiotherapy that directly target tumor cells. A balance between pro- and anti-angiogenic molecules rebates the process of angiogenesis. It is now widely accepted that the "angiogenic switch" is "off" when the effect of pro-angiogenic molecules is balanced by the one exerted by anti-anriogenic molecules, whereas is "on" when the net balance is tipped in favor of angiogenesis. Pro- and anti-angiogenic molecules can emanate from cancer cells, ECs, stromal cells, blood and the extracellidar matrix. Among such molecides, there are various soluble factors inducing angiogenesis and some endogenous inhibitors. The members of both families of VEGFs and angiopoietins have a predominant role in the new vessel formation, in the stimulation of proliferation, migration and differentiation of Ecs.^^ Tumor vessels develop with two distinct mechanisms: by sprouting or by inmssusception. The first type of angiogenesis involves sprouting of capillaries from preexisting blood vessels. In this case ECs degrade the extracellular matrix, migrate and proliferate, allowing the formation of coherent extension from the primary vessel.^^ The mechanism of intussusception involves the splitting of preexisting vessels by proliferation of ECs within a vessel.^^ This proliferation results in the formation of a large lumen that is then split by insertion of tissue colunms. Moreover, tumor cells can also grow around an existing vessel to form a perivascidar cuff or recruit circulating endothelial precursors.^^ VEGFs and angiopoietins are activators of all these angiogenesis mechanisms but their excessive tumor production causes the formation of vessels struaurally and frmctionally abnormal. Tumor vasculature is highly disorganized compared to normal vessels, with tortuous and dilated vessels, and excessive branching and shunts. Consequendy, blood flow is chaotic and vessels are leaky.^ Indeed, angiogenesis enhances the entry of tumor cells into the circulation by providing an increased density of immature, highly permeable blood vessels that have litde basement membrane and fewer intercellular junctional complexes than normal mature vessels. Various anti-angiogenic approaches to treat tumors are already in clinical trials, alone or in combination with conventional therapies.^^"^^ Strategies to inhibit angiogenesis includes the use of molecules that: (i) interfere with angiogenic ligands or their receptors (neutralizing antibodies to VEGF, synthetic inhibitors of VEGF receptor signaling), (ii) increase the level of endogenous inhibitors (angiostatin, endostatin), (iii) target tumor vasculature with different mechanism (TNP-470, Thalidomide, Combrestatin A-4) (iiii) inhibit matrix metalloproteinases (Marimastat, AG3340).^^ Endothelial cell-specific endogenous inhibitors, such as endostatin and angiostatin, appear particularly promising in preclinical studies and are actually in phase I of clinical trials.^ However, the most effective strategy will most likely be to combine angiogenesis inhibitors with traditional cancer therapies. Recent reports testify the power of such combination approaches: the tumor treatment with radiation combined with angiostatin or antibody anti-VEGF greatiy enhances the effects of radiation alone.^^'^^
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Figure 1. Effects of different temperatures of heating (37, 39, 41, 43°C) on in vitro angiogenesis. A) ECs plated on three-dimensional extracellular matrix preparation at the temperature of 43''C, spontaneously differentiate into multicellular capillary-like structures. B-D) a significant inhibition of morphogenesis is evident at 4 r C (C) and 43°C (D), while at 39''C there was only a slight reduction of tube formation (B).
Molecular Mechanisms of Angiogenesis Inhibition by HT Inhibition of angiogenesis has been suggested to play a role in tumor regression activity exerted by HT.^^ Since the 1960s a numbers of investigators have described and categorized the effects of H T on microvessels in vivo and on cultured ECs in vitro. Both ECs and microvessels can be lethally damaged by the H T doses used in antineoplastic therapy. Some studies have shown that the thermal sensitivity of ECs is dose-dependent within the therapeutic range of 42-45°C/30 min.^ Furthermore, proliferating capillary ECs are clearly more thermosensitive than nonproliferating cells. Since neoplasms contain a larger proportion of proliferating EC than normal tissues do,^ H T might damage preferentially the neoplastic microvasculature over the adjacent normal vasculature. Even though Fajardo et al demonstrated that H T inhibits angiogenesis in a dose-related manner,^^ the exact mechanisms by which H T exerts its anti-angiogenic activity are not clearly understood. Obviously it must be due to interference with one or more of the midtiple steps in the process of angiogenesis, from endothelial cell migration through EC replication to remodelling.^ '^^ Recently, some studies have been made to better address the molecidar aspects of this biological effect.^^'^^ H T at 42°C suppresses the gene expression and thus the production of VEGF in human fibrosarcoma HT-1080 cells and inhibits VEGF in vitro angiogenic action on human umbilical vein endothelial cells (HUVEC). These results suggest that H T acts as an anti-angiogenic tool by suppressing the expression of tumor-derived VEGF production thereby inhibiting endothelial-cell proliferation and extracellular matrix remodelling in blood vessels. Moreover, H T at 43°C for 1 hour is able to completely block the in vitro differentiation of ECs into capillary-like structures, without affecting EC survival and proliferation rate (Fig. 1).^^ This effect is not caused by direct cytotoxicity, but is dependent on modulation of
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20 18 16
I• I I II Vehicle
VEGF-A,65
VEGF.A,65 WW
VEGF-A,65 fAbaPAI-1
VEGF.A,65 fHlf AbaPAI-1
Figure 2. Effects of FiT on angiogenic response to VEGF-A165 of heat-treated CAM. In this assay gelatin sponges, adsorbed with VEGF-A165 or vehicle alone, are implanted on the top of growing chorioallantoic membranes (CAMs). After two days of stimulation, VEGF-A165 induces a strong angiogenic response in the CAM tissue, as shown in thefigureabove by the number of new vessels formed after stimulation with the angiogenic factor. Two hours of heat treatment at 43°C is sufficient to inhibit the VEGF-Ai65-induced angiogenesis in CAM, while the addition of a neutralizing antibody against PAI-1 is able to abrogate the inhibitory effect of FIT on angiogenesis. angiogenesis-involved genes. The gene profile analysis performed on heated ECs clearly shows that FiT activates a specific gene response that involves the transcription of the human plasminogen activator inhibitor-1, PAI-1, the key regulator of the plasminogen activation pathway. This is a proteolytic cascade implicated in many physiological and pathological processes, including vascular thrombosis, metastasis diffusion, inflammation and angiogenesis. During angiogenesis ECs secrete many extracellular matrix proteases, such as human urokinase plasminogen activator (u-PA) and matrix metalloproteases, (MMPs) that allow EC extravasation, invasion of the stromal space and basement membrane remodelling. After FiT, elevated levels of endogenous PAI-1 (the inhibitor of the above-mentioned proteases) in ECs, are sufficient to inhibit angiogenesis in vitro and in vivo. The neutralization of PAI-1 activity (with neutralizing anti PAI-1 antibody or in PAI-1 knock -out mice) is sufficient to partially block the anti-angiogenic effect of FIT both in vitro and in vivo angiogenic models (Fig. 2). These results indicate that the heat-mediated PAI-1 induction is an important pathway by which FIT exerts its anti-tumor activity thereby representing a rationale for a combined cancer therapy based on FiT associated with anti-angiogenic molecules.
Perspectives Clinically, FiT alone has no role to play in the curative treatment of tumors, but significant benefit has been reported in a number of clinical studies when FiT and radiation are combined."^^ Unfortunately, temperatures of around 42.5-43°C are required to efficiently enhance radiation damage, and these temperatures are difficult to obtain clinically. The failure to heat tumors to effective temperatures reduces the benefits of combined treatment. Growing evidence shows that the combination of cytotoxic drugs targeting vasctdature with FiT elicits a significant tumor response. In particidar, combretastatin A-4 and vinblastine enhance the antivascular activity of local FiT and induce a prolonged tumor growth delay. ' Moreover, the pretreatment with vascular target agents enhances the efficacy of FiT, performed at 40.5-41.5°C, alone or combined with radiation therapy."^ The combined antitumor activity of
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vinblastin, combretastatin and HT may be related to both their antiangiogenic activity and to direct disruption of tumor blood vessels. In a similar manner, HT, above 42 °C exhibits antivascular and antiangiogenic activity in in vitro experiments and in murine tumor models. The recent evidence that angiogenic vasculature is a target of HT tumor treatment, opens new approaches for combined therapies with angiogenesis inhibitors and HT. Blocking tumor neovascularization is a promising strategy to inhibit tumor growth, and various antiangiogenic drugs are actually in clinical trials. In spite of this, the failure of some angiogenesis inhibitors in advanced clinical trials suggests that they might be combined with other conventional drugs to be effective.^^ Likewise, recent studies have demonstrated that combination of radiation with anti-angiogenic drugs increases the anti-tumor effects of radiation and that VEGF-A expression is induced in tumor cells in vitro and in vivo after exposure to ionizing radiation. These last observations suggest that radiation-induced VEGF-A specifically protects tumor capillaries from the toxic effects of radiation.^^' ' ^ Thus, it is tempting to speculate that a treatment schedide including HT, radiation therapy and anti-angiogenic compounds could overcome the problem of ineffective heating of tumor, improve the efficacy of radiotherapy in tumor killing and enhance the anti-tumor activity of angiogenesis inhibitors.
References 1. Hildebrandt B, Wust P, Ahlers O et al. The cellular and molecular basis of H T . Crit Rev Oncol Hematol 2002; 43(l):33-56. 2. Gerweck LE. H T in cancer therapy: The biological basis and unresolved questions. Cancer Res 1985; 45:3408-14. 3. Li GC, Mivechi N F , Weitzel G. Heat shock proteins, thermotolerance, and their relevance to clinical H T . Int J H T 1995; ll(4):459-88. 4. Coss R, Linnemanns W . The effects of H T on the cytoskeleton, a review. IntJ H T 1996; 12(2): 173-96. 5. Hahn G. H T and Cancer. NewYork: Plenum, 1982. 6. Overgaard J, Poulsen HS. Effect of H T and environmental acidity on the proteolytic activity in murine ascites tumor cells. J Natl Cancer Inst 1977; 58(4):1159-6l. 7. Streffer C. Aspects of metabolic change after H T . Recent Results Cancer Res 1988; 107:7-16. 8. Henle KJ, Leeper DB. Effects of H T (45 degrees) on macromolecular synthesis in Chinese hamster ovary cells. Cancer Res 1979; 39(7 Pt l):2665-74. 9. Song CW. Effect of local H T on blood flow and microenvironment: A review. Cancer Res 1984; 44(10 Suppl):4721s-30s. 10. Song CW, Park H, Griffin RJ. Improvement of tumor oxygenation by mild H T . Radiat Res 2001; 155(4):515-28. 11. Griffin RJ, Okajima K, Barrios B et al. Mild temperature H T combined with carbogen breathing increases t u m o r partial pressure of oxygen ( p 0 2 ) and radiosensitivity. Cancer Res 1996; 56(24):5590-3. 12. Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: A review. Cancer Res 1989; 49(23):6449-65. 13. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000; 407(6801):249-57. 14. Brown SL, H u n t JW, Hill RP. Differential thermal sensitivity of tumour and normal tissue microvascular response during H T . Int J H T 1992; 8(4):501-14. 15. Fajardo LF, Prionas SD. Endothelial cells and H T . Int J H T 1994; 10(3):347-53. 16. Fajardo LF, Schreiber AB, Kelly NI et al. Thermal sensitivity of endothelial cells. Radiat Res 1985; 103(2):276-85. 17. Hanahan D , Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996; 86:353-64. 18. Folkman J. Clinical applications of research on angiogenesis. N Engl J Med 1995; 333:1757-63. 19. Hanahan D , Weinberg RA. The hallmarks of cancer. Cell 2000; 100(l):57-70. 20. Yancopoulos G D , Klagsbrun M, Folkman J. Vasculogenesis, angiogenesis, and growth factors: Ephrins enter the fray at the border. Cell 1998; 93(5) :661-4. 2 1 . Bussolino F, Mantovani A, Persico G. Molecular mechanisms of blood vessel formation. TiBS 1997; 22:251-56. 22. Patan S, M u n n LL, Jain RK. Intussusceptive microvascular growth in a human colon adenocarcinoma xenograft: A novel mechanism of tumor angiogenesis. Microvasc Res 1996; 51(2):260-72. 23. Holash J, Maisonpierre PC, Compton D et al. Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF. Science 1999; 284(5422): 1994-8.
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24. Jain RK. Determinants of tumor blood flow: A review. Cancer Res 1988; 48(10):264l-58. 25. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995; 1:27-31. 26. Ferrara N, Alitalo K. Clinical applications of angiogenic growth factors and their inhibitors. Nat Med 1999; 5(12):1359-64. 27. Kerbel R, Folkman J. Clinical translation of angiogenesis inhibitors. Nat Rev Cancer 2002; 2(10):727-39. 28. O'Reilly MS, Holmgren L, Shing Y et al. Angiostatin: A novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 1994; 79:315-28. 29. Eder Jr JP, Supko JG, Clark JW et al. Phase I clinical trial of recombinant human endostatin administered as a short intravenous infusion repeated daily. J Clin Oncol 2002; 20(18):3772-84. 30. O'Reilly MS, Boehm T, Shing Y et al. Endostatin: An endogenous inhibitor of angiogenesis and tumor growth. Cell 1997; 88(2):277-85. 31. Mauceri HJ, Hanna NN, Beckett MA et al. Combined effects of angiostatin and ionizing radiation in antitumour therapy. Nature 1998; 394(6690):287-91. 32. Gorski DH, Mauceri HJ, Salloum RM et al. Potentiation of the antitumor effect of ionizing radiation by brief concomitant exposures to angiostatin. Cancer Res 1998; 58(24):5686-9. 33. Hobson B, Denekamp J. Endothelial proliferation in tumours and normal tissues: Continuous labeUing studies. Br J Cancer 1984; 49(4):405-13. 34. Denekamp J, Hobson B. Endothelial-cell proliferation in experimental tumors. British J Cancer 1982; 46:711-20. 35. Fajardo LF, Prionas SD, Kowalski J et al. HT inhibits angiogenesis. Radiat Res 1988; ll4(2):297-306. 36. Furcht LT. Critical factors controUing angiogenesis: Cell products, cell matrix, and growth factors. Lab Invest 1986; 55(5):505-9. 37. Folkman J. Angiogenesis: Initiation and control. Ann NY Acad Sci 1982; 401:212-27. 38. Roca C, Primo L, Valdembri D et al. HT inhibits angiogenesis by a plasminogen activator inhibitor-1 dependent mechanism. Cancer Res 2003; 0:0. 39. Sawaji Y, Sato T, Takeuchi A et al. Anti-angiogenic action of HT by suppressing gene expression and production of tumour-derived vascular endothelial growth factor in vivo and in vitro. Br J Cancer 2002; 86(10): 1597-603. 40. Falk MH, Issels RD. HT in oncology. Int J HT 2001; 17(1):1-18. 41. Nielsen OS, Horsman M, Overgaard J. A future for HT in cancer treatment? Eur J Cancer 2001; 37(13):1587-9. 42. Horsman MR, Murata R. Combination of vascular targeting agents with thermal or radiation therapy. Int J Radiat Oncol Biol Phys 2002; 54(5):1518-23. 43. Wust P, Hildebrandt B, Sreenivasa G et al. HT in combined treatment of cancer. Lancet Oncol 2002; 3(8):487-97. 44. Eikesdal HP, Bjerkvig R, Dahl O. Vinblastine and HT target the neovasculature in BT(4)AN rat gUomas: Therapeutic implications of the vascular phenotype. Int J Radiat Oncol Biol Phys 2001; 51(2):535-44. 45. Murata R, Overgaard J, Horsman MR. Combretastatin A-4 disodium phosphate: A vascular targeting agent that improves that improves the anti-tumor effects of HT, radiation, and mild thermoradiotherapy. Int J Radiat Oncol Biol Phys 2001; 51(4):1018-24. 46. Gorski DH, Beckett MA, Jaskowiak NT et al. Blockage of the vascular endothelial growth factor stress response increases the antitumor effects of ionizing radiation. Cancer Res 1999; 59(l4):3374-8. 47. Geng L, Donnelly E, McMahon G et al. Inhibition of vascular endothelial growth factor receptor signaling leads to reversal of tumor resistance to radiotherapy. Cancer Res 2001; 6l(6):24l3-9.
CHAPTER 7
Vascular Effects of Localized Hyperthermia Debra K. Kelleher* and Peter Vaupel Abstract
W
hen hyperthermia is applied in vitro, no fundamental differences can be seen between the response to normal and tumor cells. In vivo however, selective damage of tumor cells can be achieved and this phenomenon can be largely attributed to a number of characteristic properties of the blood vessels within solid tumors. Changes in blood flow induced by hyperthermia can influence the response of a tumor to heat either by affecting the delivery of heat through changes in heat dissipation or by a modulation of the tumor microenvironment which may in turn affect the thermosensitivity of tumor cells. Studies in experimental and human tumors suggest however that an accurate prediction of changes in blood flow during heating is not possible so that such changes cannot be used as a basis for the combination of hyperthermia with other therapy modalities. Even so, when the underlying mechanisms responsible for the antitumor effects of a combination of hyperthermia with either radiotherapy or chemotherapy are considered, it becomes evident that either an increase or a decrease in blood flow could potentially contribute to the cytotoxic effect. A further interesting approach is in the use of antivascular drugs or vascular-targeted photodynamic therapy in order to specifically reduce tumor blood flow prior to or during hyperthermia treatment. Experimental data suggest that a considerable enhancement of the antitumor effect can be achieved with this approach. By reducing heat dissipation, such an approach may in future also be of use in overcoming problems related to insufficient temperature increases frequently seen in the clinical setting.
Introduction The impact of tumor blood flow on the success of different forms of cancer therapy is already well established.^ When hyperthermia is considered, blood flow changes induced by this therapy form can influence the tumor response to heat in two ways. Firstly, changes in tumor blood flow will affect the delivery of heat to the tumor mass by causing changes in heat dissipation away from the tumor. Secondly, changes in blood flow will affect the metabolic microenvironment and are thus capable of modulating the thermosensitivity of tumor cells. With this in mind, numerous investigations have been undertaken to assess changes in blood flow in tumor and normal tissues during hyperthermia. These are summarized below. At the same time, blood flow changes occurring when hyperthermia is combined with other therapy modalities are examined.
*Corresponding Author: Debra K. Kelleher—Institute of Physiology and Pathophysiology, University of Mainz, Duesbergweg 6, 55128 Mainz, Germany. Email:
[email protected] Hyperthermia in Cancer Treatment: A Primer^ edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.
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Vascular ££Fects of Localized Hyperthermia When the effects of hyperthermia on normal and tumor cells are assessed under in vitro conditions, no fundamental differences are found with respect to thermosensitivity, The therapeutic benefits of hyperthermia cannot therefore be attributed to a greater susceptibility of tumor cells to heat. In vivo however, a quasi-selective damage of tumor cells can be achieved when the tissue is heated to temperatures between 40 and 44°C. A number of characteristic properties of blood vessels in solid tumors appear to be responsible for this "selectivity". Microscopic examination reveals a number of structural and functional features of tumor blood vessels including tortuosity, excessive branching, blind endings, lack of smooth muscle in the vessel walls together with a lack of pericytes, interrupted endothelial linings and basement membranes. Additionally, a hierarchical organization is missing and significant arterio-venous shimt perfusion and temporal variations in blood flow (including temporary stasis) have been shown to occur.^' The competence of the vasculature to regulate flow in response to changes in a tissues demands is limited in tumor tissue compared to normal tissue. Subsequendy, an insufficient nutrient and oxygen supply in tumor tissue results in the development of hypoxic tissue areas, acidosis and energy depletion.^ When cells in vitro are artificially exposed to hypoxic conditions, low pH or low intracellular ATP levels, they are found to be more susceptible to hyperthermia, so that the development of such conditions within tumors as found in vivo can at least partially explain the enhanced sensitivity of tumors to hyperthermia. The relative importance and the individual role of hypoxia, low pH and depleted energy levels is difficult to decipher since they are closely interrelated, so that alterations in any one of these parameters may affect the others. When the physiological response of nonnal tissue to being heated up to temperatures between 4l°C and 45°C is investigated, an increase in perfusion is typically seen since this is the major route by which heat is normally dissipated away from tissues such that a deleterious heat load can be avoided. Numerous investigators have attempted to quantify this response in terms of the increase in blood flow seen, and these investigations have been summarized in reviews by Vaupel and Song et al,^ where increases in skin perfusion and skeletal muscle perfusion of up to a factor of 15 and 10 respectively were found, although the magnitude of flow increase appears to vary considerably depending on the species and tissue investigated, the technique used for heating and the heating protocol. Comparable measurements in tumor tissue (both in experimental and human tumors^'^) have revealed pronounced tumor-to-mmor variation, with increases, decreases and lack of change in tumor perfusion having been reported. While some of this variability may again be explained by factors such as different heating-up rates, heating duration, thermal doses, temperature monitoring and heating systems, the use of tumors with different histology, implantation or growth sites and different tumor volumes together with the variability in the response to hyperthermia of different areas within the same tumor need to additionally be considered. When the available data are taken together, it appears that the change in blood flow upon heating is generally much greater in normal tissue than in tumors. Where increases in tumor perfusion occur, these are usually no greater than 1.5-2.0 fold.^'^ Within a single tumor entity, the changes seen depend upon the degree of heating applied, as exemplified by Song and colleagues who carried out investigations in SCK tumors in mice.^ This study showed a significant increase in blood flow following heating to 42.5°C for 1 h followed by a decline thereafter with recovery to the control level within 5 h. Heating to 43.5°C induced an initial increase in perfusion over the first 30 min followed by a pronounced reduction from which the tumor had not completely recovered even 24 h after heating. Upon 44.5°C hyperthermia, only a decrease in blood flow occurred, which became even more pronounced after completion of heating. Similar temperature-related effects were also seen in the R3230 adenocarcinoma growing in rats.^^ The variability in the response to heating within a single tumor model has also been assessed in a study of the micro regional physiology of rat DS-sarcomas during hyperthermia.^^ Here, tumors of different sizes were heated to 44°C for 60 min and micro regional perfusion was assessed
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Figure 1. Relative red blood cell flux (rel. LDF) during hyperdiermia in DS-sarcomas. Measurements were made simultaneously from two different sites within each tumor. The upper panel shows results from a tumor with a volume of 0.9 ml and the lower panel from a tumor with a volume of 2 ml. The shaded area indicates the time over which heating was applied. Adapted from reference 11.
continuously and simultaneously at multiple sites within the tumor using the laser Doppler technique. The data obtained indicated substantial inter-site variability which occurred in all tumor size ranges studied and was independent of the measurement site and of the actual temperature reached at individual measurement sites. Examples of measurements performed in individual tumors are shown in Figure 1. In light of these findings, it seems probable that the variations attributed to inter-tumor differences in other studies are partly caused by intra-tumor variations in the response to hyperthermia. At the same time, it would appear that measurements made at individual sites within a given tumor may not necessarily be representative of changes taking place at other sites within the same tumor. It is therefore questionable whether
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Hyperthermia in Cancer Treatment: A Primer
Table 1. Major mechanisms involved in the shutdown of tumor blood flow upon hyperthermia Intravascular events "sludging" erythrocyte aggregation platelet aggregation thrombus formation leukocyte adhesion high blood viscosity/viscous resistance to flow erythrocyte crenation intensified acidosis leading to erythrocyte rigidity formation of fibrinogen gel Vessel wall alterations endothelial swelling endothelial degeneration vessel wall rupture vasoconstriction in larger pre-existing arterioles at tumor periphery increased geometric resistance to flow Extravascular events interstitial edema hemorrhage into interstitial space Opposing host tissue/tumor mechanisms reactive hyperemia in adjacent normal tissue "steal" phenomenon (diversion of blood flow from tumor to normal tissue)
such measurements can provide information which is of relevance for an assessment of average changes in perfusion v^dthin a tumor, and highlights the difficulties encountered in accurately predicting the biological behavior of a tumor undergoing hyperthermia on the basis of such single site measurements. Nevertheless, when mean values from several sites within individual tumors were calculated in the latter study, the extent of the decrease in perfusion at the end of the measurement period was found to increase with enlarging tumor size. Since, in the clinical setting, hyperthermia is usually applied during a number of sessions, the effects of multiple heatings on tumor blood flow are of particular significance. Here again however, no clear-cut effects can be elucidated from the available literature, with contradictory results being obtained. Whereas Nah and colleagues^^ showed that heating the rat R3230 tumor to 42.5°C for 1 h induced vascular thermal adaptation so that when a second heating was applied, much greater increases in blood flow were seen, others saw an enhanced shutdown of the tumor microcirculation following sequential heat treatment. ^^ When a shutdown of tumor blood flow occurs upon hyperthermia, a wide range of mechanisms appear to be involved. These include both intra- and extravascular events, effects on the vessel wall and mechanisms in which opposing actions between host tissue and tumor tissue play a role. Table 1 shows a summary of such mechanisms which have been discussed in detail by Vaupel.
Vascular E£Fects of Combined Modalities Combined Irradiation and Hyperthermia Hyperthermia appears to be one of the most potent radiosensitizers known. ^ The complementary effect seen when radiotherapy and hyperthermia are combined occurs for a number of reasons. Firsdy, cells whose radiosensitivity is compromised by the presence of hypoxia and low pH areas are patticularly sensitive to heat. Secondly, cells in the late S-phase are resistant to
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irradiation but show an increased susceptibility to hyperthermia.^^ Furthermore, although studies in which blood flow changes during hyperthermic treatment of human tumors have been assessed have delivered inconclusive results with both increases and decreases being reported, hyperthermia-related changes in tumor blood flow, whether increases or decreases, may have an impact on the therapeutic efficacy. When tumor blood flow increases, an accompanying increase in oxygen delivery will occur and this should in turn result in increased radiosensitivity. Decreases in tumor blood flow on the other hand will reduce the tumor's oxygen supply, leading to hypoxia, making the tumor more resistant to standard radiotherapy, but at the same time rendering it highly susceptible to hyperthermia. A further important mechanism is that hyperthermia inhibits—probably via an effect on cellular proteins—the cellular repair of radiation-induced DNA damage. A number of animal studies have attempted to examine blood flow changes when irradiation and hyperthermia are applied. These investigations have generally shown that the greatest anti-tumor effect could be found when the two treatment components were applied simultaneously or when irradiation was followed closely by hyperthermia treatment. With increasing intervals between the application of irradiation and hyperthermia, the antitumor effect was seen to be reduced. In the latter of these studies. Song and colleagues attempted to assess whether vascular changes might be involved in this effect and found that when tumors were first irradiated (20 Gy, single dose) and subsequendy heated (43"C, 1 h), an increase in blood flow occurred which was generally greater than that seen upon irradiation or heat alone and persisted for approximately 4 weeks post treatment. These combined effects on blood flow diminished with increasing inter-treatment intervals. When these experimental data are extrapolated to the clinical setting it should however be remembered that although combination treatments involving irradiation and hyperthermia may show maximal cytotoxicitv when the two components are applied simultaneously, this will rarely be logistically possible. ^
Combined Chemotherapy and
Hyperthermia
The capability of hyperthermia to enhance the effects of chemotherapy in vitro or in vivo has been extensively reviewed by Dahl. The effects of a number of drugs, most prominendy the alkylating agents, can be potentiated by combination with hyperthermia. The main mechanisms involved appear to be increased drug uptake into the tumor cells, enhanced DNA damage, and—^where an increase in blood flow concurrently occurs—increased drug delivery (i.e., improved pharmacokinetics) to the tumor. Additionally, an impact on the pharmocodynamics of anticancer drugs in tumor cells undergoing heating needs to be considered. As with combined radiotherapy and hyperthermia, maximal effects were found when chemotherapy and hyperthermia were applied simultaneously.
Combined Photodynamic
Therapy and
Hyperthermia
Whereas the enhancement of tumor response in vivo is now clinically well-established for the combination of hyperthermia with radiotherapy or chemotherapy, the possibility of combining photodynamic therapy and hyperthermia has received less attention, despite the fact that there is considerable evidence from in vitro studies that such an application may result in a synergistic effect. ^^'^^ Investigations in vivo also showed promising effects. Henderson et al used a combination of Photofrin-based photodynamic therapy and microwave-induced hyperthermia for treating experimental fibrosarcomas in mice and showed that the combination of these modalities led to a potentiated cytotoxic effect with 45% of animals showing long-term tumor control in comparison to less than 10% with either hyperthermia or photodynamic therapy alone. In a similar study of rats bearing rhabdomyosarcomas treated with photofrin-based photodynamic therapy and a radiofrequency heating method for interstitial hyperthermia, Levendag et al obtained a 41 % cure rate, whereas no animals were cured when either of the treatment components were applied alone. Likewise, synergistic effects were reported for this therapy combination in the chick chorioallantoic membrane model.'^ Some
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studies, while not providing a thorough assessment of the optimum sequencing of the therapy components in vivo, did at least attempt to address this question. For example, in two studies,^ '^^ the sequence of application of the therapy components was found to have a dramatic effect on treatment outcome, with potentiation occurring when photodynamic therapy was applied before hyperthermia, whereas a reversal of the sequence produced only an additive effect. This dependency of treatment order on the extent of tumor response was also found in the in vitro studies discussed above. In general, the efficacy of a simultaneous application of the two components was however not investigated in any of these studies, primarily due to the technical difficulties of simultaneously inducing tissue heating and of delivering light for activation of the photosensitizer, despite the fact that a therapy combination which could be easily applied in a single session, would nevertheless, from a clinical point of view, be favorable. As far as mechanisms are concerned, traditionally in photodynamic therapy, it was thought that a photosensitizer—in order to be effective—^should primarily target the tumor cell. Effects on the vasculature, although considered to be important in the tumor eradication process, were believed to be detrimental to the photodynamic effect if they occurred during treatment, since even partial vascidar shutdown might lead to a decrease in oxygen delivery.^ As is the case with radiotherapy and some forms of chemotherapy, the photodynamic effect is inherently oxygen-dependent. ^Thus, an induction of oxygen deprivation was thought to be unfavorable since it might result in a "self-limitation" of the treatment effect. More recendy, several investigators have nevertheless examined a number of antitumor approaches targeted primarily at the tumor vasculature. These have included photodynamic therapy protocols aimed at inducing primarily a vascular effect rather than a direct cytotoxic effect.^ '^^ Such an approach is also now being clinically applied in verteporfin-based photodynamic treatment of age-related macular degeneration, where the blood vessels are the sole treatment target.^^ The residts of studies in tumors using second generation photosensitizers such as bacteriochlorophyll-serine or palladium-pheophorbide as photosensitizers suggest that these agents have a primarily anti-vascular action. Zilberstein et al, for example, found that the maximum antitumor effect was achieved when illumination coincided with the highest concentration of bacteriochlorophyll-serine in blood (i.e., direcdy after drug injection), and that this treatment resulted in extensive vascidar damage.^ ^ Palladium-pheophorbide appears to have a similar mode of action with extensive vascular damage having been found in rat C6 glioma xenografts'^ and in human prostatic small cell carcinoma xenografts,^' and changes suggestive of a primarily anti-vascular effect being seen during laser Doppler studies in rat tumors' and with blood oxygenation level-dependent (BOLD) contrast magnetic resonance imaging (MRI) in experimental melanoma.'^ On the basis of these effects the possibility of combining an antivascular photodynamic treatment approach with hyperthermia has now also been considered.' ''^ In these studies, bacteriochlorophyll-serine was used as the photosensitizer and an important aspect of the methodology employed was a simultaneous application of photodynamic therapy and heat using a single irradiator. Pronounced inhibitions of tumor growth were seen with this simultaneous combined treatment, with the effects being considerably greater than with either photodynamic therapy or hyperthermia alone. The probability of the tumors not reaching the target volume within 90 days following combined treatment was 78%, whereas with photodynamic therapy alone it was 36% and with hyperthermia alone 15%. The effects of the combination therapy using bacteriochlorophyll serine on tumor perfusion and oxygenation were subsequendy also evaluated. When hyperthermia alone was applied, the tumor perfusion was found to steadily increase reaching levels 80% greater than initial values, with this effect remaining for the duration of the treatment period. Corresponding to this, an increase (approximately 50%) in oxygenation also occurred. In contrast, under combined hyperthermia and photodynamic therapy, a dramatic decrease in tumor perfusion of approximately 90% was seen, with tumor oxygenation concurrendy reaching levels of anoxia (Fig. 2). In light of these effects, it is a paradox that the issue of hyperthermia was seen as a "problem** in the application of photodynamic therapy in the past. Early photodynamic studies using high fluence rates
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Hyperthermia in Cancer Treatment: A Primer
unavoidably resulted in uncontrolled (and generally unmonitored) tumor heating. As pointed out by Kinsey and coUeagues,^^ initial reports of cell killing solely due to a photodynamic process were presumably only partially correct since tissue heating will most certainly have contributed to the antitumor effect. A retrospective evaluation of the role of hyperthermia in these early studies on photodynamic therapy is not possible without detailed information on the tissue temperatures achieved due to the fact that the extent of tissue heating occurring during photodynamic therapy is dependent on a range of variables including light absorption and scattering, exposure time and tissue properties such as thermal conduction and perfusion, and is therefore difficult to predict.^^ At the same time, uncontrolled and immonitored tissue heating appears not to be a reasonable option for enhancing the photodynamic effect since the temperature window for this enhancement is apparendy narrow, lying between approximately 40 and 44.5°C. ^' The simultaneous application of hyperthermia and photodynamic therapy as described by Kelleher et al^ '^^ and outlined above involved a controlled hyperthermia treatment. It is also of interest that a potentiation of the effects of hyperthermia and photodynamic therapy could additionally be achieved using 5-aminolaevulinic acid, a photosensitizer requiring cellular uptake and conversion to a photoactive substance, which appears to rely predominandy on a direct cytotoxic effect on tumor cells rather than on vascular effects. The underlying mechanisms of the effects seen when hyperthermia is combined with either a "vascular'* or "nonvascular** targeted photosensitizer are of considerable interest and appear to be numerous. Since the photodynamic process has been shown to be inherendy temperature-dependent, ^ photodynamic therapy should show an increased cytotoxic effect whenever it is performed in heated tissues, irrespective of which photosensitizer is used. Additionally, in vitro studies have indicated that photodynamic therapy and hyperthermia can have a concerted effect on certain molecules or supramolecidar structiyes^^ and that photodynamic therapy-induced repairable lesions may be converted to irreparable ones when hyperthermia is additionally applied. Modification of the metabolic microenvironment upon therapy may also influence the efficacy of the treatment components since increased lactic acid formation was seen upon treatment which may result in a lower pH in tumor tissue.^^'"^^ When bacteriochlorophyll-serine-based photodynamic therapy was applied in combination with hyperthermia, injection of the photosensitizer close to the time of illumination was necessary due to the short biological half-life of this substance. ^ In the protocol developed for the combined therapy, bacteriochlorophyll-serine was injeaed 10 minutes after conmiencement of hyperthermia, at a time when the tumor temperature had reached approximately 40 °C and was still rising. The rationale for the choice of this timing was based on the findings of the laser Doppler studies which indicated that there was an approximately 10% increase in tumor perftision at this time, which could therefore be exploited to enhance the delivery of the photosensitizer to the tumor tissue. The selection of a vascular-targeted photodynamic therapy for combination with hyperthermia was also considered to be of interest in terms of achieving adequate tissue heating since, in the clinical setting, a major obstacle to an effective hyperthermia treatment is the failure of the protocol used to attain a therapeutically relevant temperature elevation in the tumor tissue. While this may pardy be due to limitations of the heating equipment employed, the relatively high perftision rates which have been found in human tumors may also lead to an undesirable dissipation of heat away from the malignancy.^ If the tumor perftision, and thus heat dissipation, is restricted by a vascular-based photodynamic therapy, then a more effective heating should be possible, an aspect which could be of particular relevance in the clinical setting.
Combination ofHyperthermia tuith Antivascular Drugs A further approach to enhancing the antitumor effects of hyperthermia is the combination with agents able to preferentially destroy an established tumor vessel network (vascular targeted therapy). This approach differs to antiangiogenic therapies since it attempts to exploit the differences between the vessels found in normal and tumor tissues in order to disrupt the
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existing tumor vasculature, rather than inhibiting the growth of new vessels. Experimental studies have shown that the use of vascular disrupting agents such as combretastatin, flavone acetic acid and DMXAA to reduce tumor blood flow can improve the response of tumors to hyperthermia. '^^ Where combretastatin was applied, the greatest response enhancement was seen when this agent was administered 2 to 6 hours prior to hyperthermia, corresponding to the time when the greatest reduction of tumor perfusion was seen. An attempt to assess the specificity of these effects of combretastatin showed that some enhancement of the susceptibility to heat occurred in normal tissues but this was generally not as pronounced as the effects occurring in tumor tissue. ^
Conclusions Despite many years of basic research on the physiological and pathophysiological actions of hyperthermia and the slowly increasing acceptance of hyperthermia as a clinical therapy option, an accurate prediction of the changes occurring in tumor blood flow during heating to therapeutically relevant temperatures is not possible. This is due to the large number of confounding variables (related both to treatment protocol and inherent tumor properties) which can influence tumor perfusion during hyperthermia. The assumption of reproducible and predictable heating-induced changes in tumor blood flow can therefore not form the premise for a combination of hyperthermia with other tumor modalities. However, conditions enhancing the effects of ionizing radiation typically reduce the effects of hyperthermia. Thus, low blood flow tumor areas will be more resistant to ionizing radiation but more susceptible to hyperthermia, whereas high blood flow areas will prove more difficult to heat but show higher radiosensitivity. In light of this complementary action, effects of hyperthermia on blood flow—in the form of either an increase or a decrease—should not prove deleterious for the effects of thermoradiotherapy. This should also be the case in instances where hyperthermia is combined with chemotherapy. A further approach, which has also been presented here is the use of photodynamic therapy or antivascular drugs to reduce tumor blood flow during or prior to hyperthermia treatment. Experimental data have confirmed that such methods can considerably enhance the antitumor effect so that clinical data will hopefully also show promise when they become available.
References 1. In: Vaupel P, Jain R, eds. Tumor Blood Supply and Metabolic Microenvironment. Stuttgart: Gustav Fischer, 1991. 2. StrefFer C. Molecular and cellular mechanisms of hyperthermia. In: Seegenschmiedt M H , Fessenden P, Vernon C C , eds. Thermoradiotherapy and Thermochemotherapy. Biology, Physiology, and Physics. Vol 1. Berlin: Springer, 1995:47-74. 3. Konerding MA, Malkusch W, Klapthor B et al. Evidence for characteristic vascular patterns in solid tumours: Quantitative studies using corrosion casts. Br J Cancer 1999; 80:724-732. 4. Vaupel P. Tumor microenvironmental physiology and its implications for radiation oncology. Semin Radiat Oncol 2004; 14:198-206. 5. Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: A review. Cancer Res 1989; 49:6449-6465. 6. Vaupel P. Pathophysiological mechanisms of hyperthermia in cancer therapy. In: Gautherie M, ed. Biological Basis of Oncologic Thermotherapy. Berlin: Springer, 1990:73-134. 7. Song CW, Choi IB, Nah BS et al. Microvasculature and perfusion in normal tissues and tumors. In: Seegenschmiedt M H , Fessenden P, Vernon C C , eds. Thermoradiotherapy and Thermochemotherapy. Biology, Physiology, and Physics. Vol 1. Berlin: Springer, 1995:139-156. 8. Vaupel PW, Kelleher DK. Metabolic status and reaction to heat of normal and tumor tissue. In: Seegenschmiedt M H , Fessenden P, Vernon C C , eds. Thermoradiotherapy and Thermochemotherapy. Biology, Physiology, and Physics. Vol 1. BerUn: Springer, 1995:157-176. 9. Song C W , Lin JC, Chelstrom LM et al. T h e kinetics of vascular thermotolerance in SCK tumors of A/J mice. Int J Radiat Oncol Biol Phys 1989; 17:799-802. 10. Shakil A, Osborn JL, Song CW. Changes in oxygenation status and blood flow in a rat tumor model by mild temperature hyperthermia. Int J Radiat Oncol Biol Phys 1999; 43:859-865.
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11. Kelleher DK, Engel T, Vaupel PW. Changes in microregional perfusion, oxygenation, ATP and lactate distribution in subcutaneous rat tumours upon water-filtered IR-A hyperthermia. Int J Hyperthermia 1995; 11:241-255. 12. Nah BS, Choi IB, Oh WY et al. Vascular thermal adaptation in tumors and normal tissue in rats. Int J Radiat Oncol Biol Phys 1996; 35:95-101. 13. Eddy HA, Chmielewski G. Effect of hyperthermia, radiation and adriamycin combinations on tumor vascular function. Int J Radiat Oncol Biol Phys 1982; 8:1167-1175. 14. Kampinga HH, Dikomey E. Hyperthermic radiosensitization: Mode of action and cUnical relevance. Int J Radiat Biol 2001; 77:399-408. 15. Streffer C. Biological basis of thermotherapy. In: Gautherie M, ed. Biological Basis of Oncologic Thermotherapy. BerUn: Springer, 1990:1-71. 16. Song CW, Kim JH, Rhee JG et al. Effect of X irradiation and hyperthermia on vascular function in skin and muscle. Radiat Res 1983; 94:404-415. 17. Wust P, Hildebrandt B, Sreenivasa G et al. Hyperthermia in combined treatment of cancer. Lancet Oncol 2002; 3:487-497. 18. Dahl O. Interaction of heat and drugs in vitro and in vivo. In: Seegenschmiedt MH, Fessenden P, Vernon CC, eds. Thermoradiotherapy and Thermochemotherapy. Biology, Physiology, and Physics. Vol 1. Berlin: Springer, 1995:103-121. 19. Christensen T, Wahl A, Smedshammer L. Effects of haematoporphyrin derivative and light in combination with hyperthermia on cells in culture. Br J Cancer 1984; 50:85-89. 20. Mang TS, Dougherty TJ. Time and sequence-dependent influence of in vitro photodynamic therapy (PDT) survival by hyperthermia. Photochem Photobiol 1985; 42:533-540. 21. Rasch MH, Tijssen K, Vansteveninck J et al. Synergistic interaction of photodynamic therapy with the sensitizer aluminum phthalocyanine and hyperthermia on loss of clonogenicity of CHO cells. Photochem Photobiol 1996; 64:586-593. 22. Henderson BW, Waldow SM, Potter WR et al. Interaction of photodynamic therapy and hyperthermia: Tumor response and cell survival studies after treatment of mice in vivo. Cancer Res 1985; 45:6071-6077. 23. Levendag PC, Marijnissen HPA, De Ru VJ et al. Interaction of interstitial photodynamic therapy and interstitial hyperthermia in a rat rhabdomyosarcoma—a pilot study. Int J Radiat Oncol Biol Phys 1988; 14:139-145. 24. Kimel S, Svaasand LO, Hammer-Wilson M et al. Demonstration of synergistic effects of hyperthermia and photodynamic therapy using the chick chorioallantoic membrane model. Laser Surg Med 1992; 12:432-440. 25. Chen Q, Chen H, Shapiro H et al. Sequencing of combined hyperthermia and photodynamic therapy. Radiat Res 1996; 146:293-297. 26. Henderson BW, Fingar VH. Oxygen limitation of direct tumor cell kill during photodynamic treatment of a murine tumor model. Photochem Photobiol 1989; 49:299-304. 27. Hockel M, Vaupel P. Tumor hypoxia: Definitions and current clinical, biologic, and molecular aspects. J Nad Cancer Inst 2001; 93:266-276. 28. Fingar VH. Vascular effects of photodynamic therapy. J CHn Laser Med Surg 1996; 14:323-328. 29. Fingar VH, Kik PK, Haydon PS et al. Analysis of acute vascular damage after photodynamic therapy using benzoporphyrin derivative (BPD). Br J Cancer 1999; 79:1702-1708. 30. Dougherty TJ. An update on photodynamic therapy applications. J Clin Laser Med Surg 2002; 20:3-7. 31. Zilberstein J, Schreiber S, Bloemers MC et al. Antivascular treatment of soHd melanoma tumors with bacteriochlorophyll-serine-based photodynamic therapy. Photochem Photobiol 2001; 73:257-266. 32. Schreiber S, Gross S, Brandis A et al. Local photodynamic therapy (PDT) of rat C6 glioma xenografts with Pd-bacteriopheophorbide leads to decreased metastases and increase of animal cure compared with surgery. Int J Cancer 2002; 99:279-285. 33. Koudinova NV, Pinthus JH, Brandis A et al. Photodynamic therapy with Pd-bacteriopheophorbide (TOOKAD): Successful in vivo treatment of human prostatic small cell carcinoma xenografts. Int J Cancer 2003; 104:782-789. 34. Kelleher DK, Thews O, Scherz A et al. Perfusion, oxygenation status and growth of experimental tumors upon photodynamic therapy with Pd-bacteriopheophorbide. Int J Oncol 2004; 24:1505-1511. 35. Gross S, Gilead A, Scherz A et al. Monitoring photodynamic therapy of solid tumors online by bold-contrast MRI. Nat Med 2003; 9:1327-1331. 36. Kelleher DK, Thews O, Rzeznik J et al. Water-filtered infrared-A radiation: A novel technique for localized hyperthermia in combination with bacteriochlorophyll-based photodynamic therapy. Int J Hyperthermia 1999; 15:467-474.
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37. Kelleher DK, Thews O , Scherz A et al. Combined hyperthermia and chlorophyll-based photodynamic therapy: Tumour growth and metabolic microenvironment. Br J Cancer 2003; 89:2333-2339. 38. Kinsey J H , Cortese DA, Neel H B . Thermal considerations in murine tumor killing using hematoporphyrin derivative phototherapy. Cancer Res 1983; 43:1562-1567. 39. Svaasand L O , Doiron DR, Dougherty TJ. Temperature rise during photoradiation therapy of malignant tumors. Med Phys 1983; 10:10-17. 40. Waldow SM, Henderson BW, Dougherty TJ. Enhanced tumor control following sequential treatments of photodynamic therapy (PDT) and localized microwave hyperthermia in vivo. Laser Surg Med 1984; 4:79-85. 4 1 . Kelleher DK, Bastian J, Thews O et al. Enhanced effects of aminolaevulinic acid-based photodynamic therapy through local hyperthermia in rat tumours. Br J Cancer 2003; 89:405-411. 42. Gottfried V, Kimel S. Temperature effects on photosensitized processes. J Photochem Photobiol B 1991; 8:419-430. 43. Prinsze C, D u b b e l m a n T M , Van Steveninck J. P o t e n t i a t i o n of thermal inactivation of glyceraldehyde-3-phosphate dehydrogenase by photodynamic treatment. A possible model for the synergistic interaction between photodynamic therapy and hyperthermia. Biochem J 1 9 9 1 ; 276:357-362. 44. Chen B, Xu Y, Agostinis P et al. Synergistic effect of photodynamic therapy with hypericin in combination with hyperthermia on loss of clonogenicity of RIF-1 cells. Int J Oncol 2 0 0 1 ; 18:1279-1285. 45. Rosenbach-Belkin V, Chen L, Fiedor L et al. Serine conjugates of chlorophyll and bacteriochlorophyll: Photocytotoxicity in vitro and tissue distribution in mice bearing melanoma tumors. Photochem Photobiol 1996; 64:174-181. 46. Siemann D W , Bibby M C , Dark GG et al. Differentiation and definition of vascular-targeted therapies. Clin Cancer Res 2005; 11:416-420. 47. Murata R, Overgaard J, Horsman MR. Combretastatin A-4 disodium phosphate: A vascular targeting agent that improves the anti-tumor effects of hyperthermia, radiation, and mild thermoradiotherapy. Int J Radiat Oncol Biol Phys 2001; 51:1018-1024. 48. Horsman MR, Murata R. Combination of vascular targeting agents with thermal or radiation therapy. Int J Radiat Oncol Biol Phys 2002; 54:1518-1523. 49. Eikesdal H P , Schem B-C, Mella O et al. The new tubulin-inhibitor combretastatin A-4 enhances thermal damage in the BT4An rat glioma. Int J Radiat Oncol Biol Phys 2000; 46:645-652. 50. Eikesdal H P , Bjerkvig R, Dahl O . Vinblastine and hyperthermia target the neovasculature in BT(4)AN rat gliomas: Therapeutic implications of the vascular phenotype. Int J Radiat Oncol Biol Phys 2001; 51:535-544. 51. Eikesdal H P , Bjerkvig R, Mella O et al. Combretastatin A-4 and hyperthermia; A potent combination for the treatment of solid tumors. Radiother Oncol 2001; 60:147-154.
CHAPTER 8
On the Biochemical Basis of Tumour Damage by Hyperthermia Paola Pietrangeli and Bruno Mondovi* Abstract
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umour cells are selectively inhibited by hyperthermia (41-42.5°C) in the same conditions where normal cells are not damaged. At higher temperature, also normal cells are injured. In spite of the large number of reports on the cytotoxic effect of hyperthermia the mechanisms of heat cytotoxicity are yet unclear. It appears plausible that concomitant phenomena, triggered by heat and related each other, may be involved. The major points on this subject are the following: i. DNA, RNA synthesis, DNA repair mechanism and cell respiration are affected; ii. Tumour cell membranes are damaged as it is demonstrated by alteration of their permeability and the effect of empty liposomes; iii. DNA polymerase-P, a key enzyme in muld-step repair system, should be involved; iv. Dilation of mitochondria cristae and dissociation of poliribosomes were observed; V. Heat shock proteins should be involved; vi. Heat appears to increase the flux of oxygen free radicals mediating in part the cytotoxicity.
Introduction First of all hyperthermia refers to temperatures above about 5 centigrades the normal body temperature of the animal being studied. In vitro experiments have demonstrated that neoplastic cells are more sensitive to heat than normal cells of the some histological type, even if the latter divide at a faster rate than the tmnour cells. ^ For man, the range is between 40 and 43 °C, where only tumour cells are damaged while at higher temperatiue also normal cells are injured. Therefore we can consider a selective heat sensitivity of tiunour cells for man in this range of temperature. Neoplastic cells acquired the thermosensitivity together with the malignant transformation. About one and half century ago Bush.^ observed that temperature above the physiological value appears to damage cancer cells. For over 200 years dramatic "spontaneous" regression of various types of cancer were observed and in last century a large number of authors reported their individual observations of the beneficial effects of infections, bacterial vaccines, inflammation, fever or incomplete surgery. It was found in all types of neoplastic disease the majority of spontaneous regression occurred following streptococcal infections (like erysipelas). The complete regression of melanomas was described in a patient affected by erysipelas after several days of fever over 40°C.5 Coley, in die end of 19th century suggested to induce fever by inoculating bacterial toxins for the treatment of tumour. Regression of sarcomas in patients receiving erysipelas infections was reported.^'^ •Corresponding Author: Bruno Mondovl—Dipartimento di Scienze Biochimiche "A. Rossi Panel I i", and C.N.R. Centre of Molecular Biology, Universita "La Sapienza", P.Le Aldo Mro, 5-00185 Roma, Italia. Email:
[email protected] Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.
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Systematic biochemical and clinical studies were performed by Rome and Madison groups'''^ in order to use hyperthermia for cancer therapy demonstrating that the treatment of cancer patients with hypertermia as such or together with chemical and/or radiation can give encouraging results in tumour cancer therapy. In this chapter a briefly summary of these researches, started on 1960 and some major biochemical questions of the thermosensitivity of tumour cells will be discussed.
Glycolysis and Respiration Westermark^ found that glycolysis, both aerobic and anaerobic, was rapidly inhibited in two experimental rat tumours while the Rome-Madison group ' published that glycolysis was unaffected by exposure up to 44°C in a large number of several animal and human malignant tumours. In some cases, where the rate of lactate production seemed to decline upon prolonged thermal exposure, it was subsequently ascertained that the inhibition observed could be attributed to an acidification of the medium. Von Ardenne et al^^ reported an inhibition of glycolysis in Ehrlich ascites mouse carcinoma cells only at very last event. A much more reliable parameter was oxygen consumption which underwent a severe inhibition upon thermal exposure of the neoplastic cells. The oxygen uptake of Novikoff hepatoma cells at 38 and 42°C were measured manometrically in the conventional way with air as the gas phase. After one hour the endogenous respiration at 42°C plateaued rapidly and was considerably less than that at 38 °C; similar effects were observed with the addition of glucose. ^° Whether the cells were incubated for 2 hours or more at 42°C and then returned at 38°C, oxygen uptake was irreversibly inhibited. Partial inhibition was observed when the cells were incubated at 42-44°C for 30 min then by measuring the respiration at 38°C. Also in the case of Ehrlich ascite carcinoma and in a human melanoma cells an inhibition of oxygen uptake in hyperthermic conditions was demonstrated. No effect of heat on respiration was found with normal and regenerating rat liver cells. When a minimal deviation hepatoma 5123 cell suspension was incubated at 38, 42 and 43°C the variation was irrelevant. However, whether hepatoma 5123 cells were incubated for 3.5 hours at 43°C and then returned at 38°C in the presence of glucose and succinate, oxygen uptake was lower. Conversely, no inhibition of oxygen uptake was observed in regenerating liver cells treated in identical conditions or for 3.5 hours also at 44°C.^^
Hyperthermia and DNA, RNA and Protein Synthesis A different set of parameters which are much more affected by thermal exposure concern the synthesis of macromolecules necessary to the cells: proteins and nucleic acids. In cells from Novikoff rat hepatoma, as well as from Morris 5123 rat hepatoma and from a human osteosarcoma, the DNA, RNA and protein synthesis were all strongly inhibited while regenerating liver cells were not affected. A prominent role of rRNA synthesis in the genesis of heat induced cell damage was found by investigations on the simultaneous action of heat and of some specific metabolic inhibitors of DNA and protein synthesis that decrease the thermal sensitivity of HeLa cells, while agents interfering with RNA synthesis act as enhancer of hyperthermia.^ The mechanism by which exposure of ascites tumour cells to supranormal temperatures causes an irreversible inhibition of uridine incorporation into RNA was investigated. Heat-treated cells were still able to incorporate labeled nucleotides, and even nucleosides, into RNA, if these precursors were added at sufficiently high concentrations. The passive permeability of the cell membrane increased exponentially with temperature, but this increase was fully reversible. At variance with the results obtained with agents which increase cell permeability, or which inhibit nucleoside permeation, heat treatment of Ehrlich ascites cells did not modify the ability of labeled uridine to be metabolised by these cells. The incubation of Chinese hamster cells, grown at confluent monolayer cultures, at 42.5°C, significantly inhibits the DNA repair synthesis which follows exposure of the cells to UV irradiation: 3 hours preincubation at 42.5°C prevents almost completely the repair of DNA damage produced to a single UV dose of 10 ]lrc^.
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Whether hyperthermia follows the radiative treatment, the decline to DNA repair synthesis appears after 60 min incubation. DNA polymerase-p,^^ a key enzyme in multistep repair system, could be considered as a possible target because of its well known heat sensitivity.
Tumour Membranes and Hyperthermia The hypothesis of a primary lesion of the plasma membrane of the tumour cell is worth special consideration. In this context, it should be pointed out that heat-induced cell damage, as measured by oxygen uptake and by thymidine incorporation into DNA, is abolished upon disruption of the neoplastic cells. ^^' Polyenic antibiotic filipine and ethanoP^'^^ showing a marked selectivity for the lipid components of cell membrane, show a marked synergism with thermal exposure, acting as sensitisers for subsequent action of heat and inhibit the incorporation of labeled nucleic acids precursors into tumour cells at concentrations which did not significandy affect regenerating liver cells. These results suggest that the inhibition of DNA, RNA and protein synthesis may be related to a primary alteration of cellular membranes of tumours which are probably different from that of normal cells. In this context, the effect of temperature in potassium-dependent stimulation transcellular migration in normal and neoplastic cells was studied. ^'^ A shift from 38°C and 42°C, or even at 40°C, irreversibly modified, in Novikoff hepatoma cells but not in their counterpart (i.e., in normal or regenerating liver cells), the potassium-dependent transcellular migration of glutamate was observed. Ultrastructural changes induced by hyperthermia in Chinese hamster V79fibroblastwas demonstrated by Arancia et al.^^ In particular, the plasma membrane, mitochondria, ribosomes and nuclear envelope were studied compared to control cells maintained at 37°C, the heated cells, processed for electron microscopy under identical conditions, showed remarkable ultrastructural changes, depending on both the temperature employed and duration of treatment. In the heat-treated cells the plasma membrane shows small interruption after 1 hour of treatment at 42"C. This alteration becomes progressively more evident with the increase of both length and temperature of treatment. After 3 hours at 43°C the discontinuity of the membrane is clearly evident. Finally, treatment at 45*'C produces loss of large segments of the plasma membrane. Very interesting results were obtained on mitochondria: at 37''C cells they display a cylindrical shape with well preserved and parallel cristae in a dense matrix. After 1 hour at 42°C the mitochondria still have a dense matrix but some cristae appear to be dilated or vesicular. These changes are even more evident after 1 hour of treatment at 43°C: the mitochondria appear to be swollen and the intracristal spaces enlarged. At 45°C for 1 hour all mitochondria are aggregated in a perinuclear area and exhibit very irregular and dilated cristae in a matrix with decreased density. These alterations probably reflea changes in their functions: verv similar alterations were observed after exposure to uncouplers of oxidative phosphorylation. In the heated samples the polyribosomes appear dissociated, and the single ribosomal unites are free in a cytoplasmic matrix increasingly extracted with longer periods of treatment and increasing temperature. After 6 hours of heating an irregular dilation of the nuclear envelope was observed. In cells treated at 43°C the nuclear membranes are not well defined and after 1 hour at 45°C they are not parallel, gready dilated and several foldings of the outer membrane are present. ^^
Hyperthermia and Liposomes The possible involvement of cell membranes target of hyperthermia is further confirmed by the effect of the enhancement of hyperthermic damage of tumour cells by liposome treatment. In this context it should be pointed out that cell membrane modifications have been obtained by means of liposome-cell interaction^^ which could be of particular interest because of the well characterised difference in membrane composition and fluidity of tumour cells with respect to normal ones.^^ Cell survival experiments performed on the relatively thermoresistant human melanoma cell line Ml4^^ showed that cell pretreatment with L-a-dipalmitoylphosphatidylcholine-containing liposomes enhances the cell killing induced by hyperthermia.
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although Uposomes per se did not appear toxic when their concentration was below 1000 nmol phospholipid/2.5 x 10 cells, while liposome treatment of cells before heat exposure determined a marked damaging effect at 100 nmol phospholipid/2.5 x 10 cells. The observation by electron microscopy seem to demonstrate that, multilamellar vesicles can either fuse with the plasma membrane or be taken up by the cell. In particular, the outer lipid bilayer of the liposomes appears to be involved in the fusion event leading to the release of the inner layers into the cell. Probably the internalised vesicles are capable of fusing with the intracellular membranes. Thus, the lipid components of the liposomes would be inserted in both plasma and intracellular membranes upon the interaction of multilamellar vesicles with M14 cells. For that reason, the cell membranes could be a site at which liposome pretreatment of cell enhance the hyperthermic damage. The proximity of multilamellar liposomes and nuclear envelope, observed by electron microscopy,^^ suggested a possible interaction between liposomes and nuclear membranes. Electron spin resonance experiments demonstrated a membrane fluidity decreasing after liposome treatment. It seem likely that the insertion of lipid domains of different organisation into plasma membrane following the incorporation of the liposomes, as evidenced by electron microscopy results, could lead to impaired ftinctioning of some membrane proteins. The interaction of multilamellar liposomes and cultured cells was also studied at mild hyperthermia, i.e., 37 and 4l.5°C for 2 hours. A dose-dependent impairment of cell survival was observed as a function of liposomes concentration. An enhancement of the cytotoxic effect was observed at 4l.5°C. This effect went on even after 24 hours from the end of treatment. Probably hyperthermia acts at the initial stage of treatment by accelerating the kinetics of the membrane-liposome interaction. Since in recovered cells numerous vesicles could be detected inside the cytoplasm, it may argued that the internalised liposomes continue to exert their effects on intracellular membranes, and that these membranes may be considered more critical than the plasma membrane for liposome cytotoxic action. Probably when the liposome treatment is carried out under mild hyperthermia, the ftision of the outer layers of multilamellar liposomes with the plasma membrane, and then the entry of the inner layers inside the cells are favoured and accelerated. We can therefore concluded that the liposome administration with mild hyperthermia appears to have a synergistic effect on cultured melanoma cells. Although the role of the cell membranes in the cytotoxic effect of hyperthermia has not yet been established, a key role of the membrane protein components was proposed.^ '^^ It has been suggested that lipid (both cholesterol and phospholipid):protein weight ratios correlate with increasing resistance of cells to an elevation in temperature. The major membrane components can influence and perhaps predict cellular survival to hyperthermia.^
Hyperthermia and Immune Response The possibility that plasma membranes should be the primary site damaged by exposure to supranormal temperatures appears to be also confirmed by the results obtained on the increased immunogenicity of Ehrlich ascites cells after heat treatment. ^ It was in fact demonstrated that inoculation of 10 viable, untreated cells to swiss mice resulted in 100% tumour take, the mean survival time of the animals being about 18-20 days. Exposure to 38**C for 2, 3 or 6 hours caused little modification of these parameters; after 1 hour at 42°C, tumour taken declined sensibly and, after 3 hours or more at this temperature, there was 100% survival for more than 3 months. Immunisation was performed by two intraperitoneal inoculation of heat heated 10'^ cells, at 20 day interval. Two schedules of heat treatment were used comparatively, with exposure time at 42°C being 3 or 6 hours. Only the group immunised with cells exposed for 3 hours at 42°C had more than 50% survival after 35 days when challenged with 10'^ viable cells. Non immunised animals had, after the same challenged dose, 100% of tumour take. A plausible hypothesis of these results seem to be the unmasking or modification of some antigenic determinant cell surface during the initial phase of exposure at high temperature leading to increased immunogenicity. Upon further heat treatment, some damage to these surface determinants may occur, so that the immunity evoked by the heat-treated cells is either lower
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or less effective toward viable cells. A protection against spontaneous mouse manunary adenocarcinoma by inoculation of heat-treated syngeneic manmiary tumour cells was also observed.^^ These results support the possibility of the inunune defence mechanism may play a role in contributing to the regression after hyperthermic perftision of otherwise untreatable tumours.
Heat Shock Proteins The problem of the relationship between enhancement of immunogenicity after hyperthermia appears to be related on the heat shock proteins (hsp).^^The induction of hsp is a general phenomenon observed in almost all organisms and cultured cell lines after exposure to subletal temperature, as well as to the stimidi which are injurious to the cells.^ In eukaryotic cells the hsp, identified by SDS polyacrilamide gel electrophoresis, may be classified in four major groups designed 16-28, 69-70, 80-90, 110, based on their respective molecular weights expressed in kilodalton. Hsps of the same site are so similar each other in different cell types and organisms that antibodies against a particular hsp from one specie usually cross-reacts with similar proteins from phylogenetically distant species. In particular, the hsp with molecular weight around 70 and 90 KD seem to be the most highly conserved in nature. Each specific temperature value reached during the heating process induces the expression of a specific hsp pattern. In vitro conditions, the pattern of hsps induced in tumour cells has been reported to change in dependence of the different cell lines, culture conditions, protocol of hyperthermic treatment. Hsps appear to be related to the heat effect on tumour cells essentially by different mechanisms: i. Correlation between induction of hsps and development of tolerance to subsequent thermal shock, ii. Correlation between cytotoxicity and heat-induced increase in nuclear matrix associated proteins, iii. Use of hsps in immunotherapy. These facts are apparendy in contrast each other, but, taking into account their different mechanisms of action, a possible concatenation can exist. Initially, the role of hsps appeared to involve a thermotolerance mechanism: brief sublethal heat exposure of cells induced a quick expression of hsps that conferred total protection against a subsequent but lethal exposure to hyperthermia. Then, it was shown that hsps were able to protect cells from different sources of stress such as oxidant radicals or endotoxin.^^'^^ In this context, Frossard ^^ demonstrated that hsp70 prolongs survival in rats exposed to hyperthermia. In addition, hsp25 overexpressed in L929 cells were shown have increased expression of the manganese superoxide dismutase gene and its enzyme activity.^^ It shoidd be taking into account that acute heat shock might residt in a redistribution of critical cell proteins and their absorption on the cell of nucleoskeleton.'^' The subsequent functional sequentiation of these proteins results in cytotoxicity. This speculation is supported by the observation that a specific set of phospholipids with molecular mass similar to the HeLa hsps have been observed to become tighdy associated with the cell s nucleoskeleton during acute thermal shock^^ To investigate the usefulness of hsp promoter for heat cancer gene therapy, hyperthermia and HSV thimidine kinase (tk) suicide gene combination therapy was checked with mouse mammalian cancer cell line FM3A. Hsp promoter activity was markedly increased after heat shock (4l-45°C) with maximum activation at 3 hr. The suppression of heat-induced accumulation of hsp72 by bleomicin appears to contribute to enhance cytotoxicity of the simultaneous treatment of 40'*C hyperthermia and bleomicin.^^ A direct relationship between hsp and cancer inmiunotherapy was in recent years developed. Tumour derived hsp-peptide complexes (particularly hsp70 and grp94/gp96) have been demonstrated to serve as effective vaccines producing antitumour inmiune responses in animals and man.^^ In this context, it should be pointed out that the realisation of significance of hsp come from the observation that tumour-cell derived hsps could immunise against tumours.
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although there were no structural difference between hsps, from normal cells and cancer cells. Studies of this puzzle have uncovered two unique immunological properties of hsps. One is their ability to associate and present antigenic fmgerprints/peptides of cells to MHC antigens. The other is their ability to activate dendritic cells, which are the most efficient antigen- presenting cells. These surprising immunological attributes of hsps are now the basis for a number of already completed chemical trials for cancer immunotherapy.^^
Hyperthermia and Oxygen Free Radicals Thermotolerance in same way could be correlated with other biochemical mechanisms causing the heat damage of cancer cells. In CHO and ovarian carcinoma cells, a rise in Cu,Zn-SOD activity was reported after heating to induce thermotolerance.^^ The increase of the steady state concentration of the superoxide radical O2', H2O2 and the more reactive hydroxyl radical O H (by H2O2 and O2' in Haber-Weiss reaction catalysed by transition metal ions), could be responsible for the cellular damage after heat treatment. It is well known that oxygen free radicals (ROS) are imputable for inactivation of enzymes, degradation of DNA and polysaccarides and induction of lipid peroxidation. Christiansen et al^^ observed that oxidative phosphorylation of mouse liver mitochondria is uncoupled after treatment at 4l-45°C. In cloned normal mouse embryo cells and their Simian virus 40-transferred derivatives, a correlation between low levels of antioxidant enzymes and thermosensitivity have been demonstrated. In this context, it is important to focus that transferred cells containing undetectable Mn-superoxide dismutase (Mn-SOD) and markedly low level of Cu,Zn-SOD, catalase and glutathione peroxidase activities, are selectively killed by exposure to hyperthermia, whereas the normal cells, having significantly higher enzyme activities, showed to be resistant to heat treatment. In addition, in both cell types the hyperthermic effect appears to be enhanced after a pretreatment with diethyldithiocarbamate, a well known inhibitor of Cu,Zn-SOD. The role of production of ROS can also be affected by heat at the level of oxidases. In this regard, a special emphasis should be given to the oxidative degradation of hypoxanthine to xanthine, and xanthine to urate by xanthine oxidase. In fact, as a consequence of insufficient ATP production, imputable to decreased respiration in hyperthermic conditions, an increase of cytosolic Ca^^ may occur, which in turn activates a protease capable of converting xanthine dehydrogenase to the oxidase. ^ A human melanoma cell line (Ml4) was enriched in superoxide dismutase (SOD) activity by treatment with enzyme-containing liposomes. The effect of hyperthermia on SOD-liposome enriched cells was tested by evaluating cell survival and measuring the incorporation of labelled L-leucine. A balance of some protective and lethal effect imputable to SOD and liposome respectively was su^ested. '^ An accelerated rate of production of ROS could also a consequence of the increased rate of enzymatic activity of amine oxidases on biogenic amines.
Hyperthermia and Amine Oxidases Immobilised pig kidney diamine oxidase (DAO) injected into the peritoneal cavity of swiss mice 24 hr after the viable intraperitoneal transplantation of Ehrlich ascite cells remarkably inhibited tumour growth. ^ These results suggest a possible use of AOs in cancer therapy. It is interesting to consider that an inflexion point on the dependence of swine kidney diamine oxidase activity upon the temperature was found at 40-43 °C, exacdy the temperature used for hyperthermic treatment of tumours. Probably, this result is imputable to a conformational transition of this enzyme as a function of temperature. The activation energies with putrescine as substrate calculated from the Arrhenius plot were 38,23 Kcal/mol for the temperature interval 25-40°C and only 15,14 Kcal/mol for the range 45-60°C. These values suggest two different conformations, one corresponding to the interval below 40°C and another one between 43-60 °C, with intermediate transitory form corresponding to the inflexion point at 40-43"C.
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In order to have more informations on the mechanism involved in effect of amine oxidases on tumours, and to study a possible use of amine oxidases as adjuvants in hyperthermia, the effect of bovine serum amine oxidase were studied in cidtured cells. It was demonstrated ' that bovine serum amine oxidase (BSAO), in the presence of exogenous spermine, caused cytotoxicity in Chinese hamster ovary cells (CHO). Cytotoxicity occurred when cells were exposed to BSAO (0.06-16 jlg/ml) in the presence of spermine. BSAO and spermine alone were not toxic at these concentrations. This cytotoxicity was accelerated at 42°C relative to 37°C. As reported above, BSAO seems to have more then one conformation as a function of temperature. Kinetic analysis of the enzymatic reaction, as a function of spermine concentration, show a Michaelis-Menten saturation kinetics. The apparent Vmax increased from 19.1 ± 0.4 nM min'^ at 37'C to 23.0 ± 0.3 ^iM min'^ at 42'C. The apparent K^ decreased from 25.5 ± 2.6 |xM at 37°C to 17.7 ±1.3 |xM at 42°C. It was observed that heat increased the cytotoxicity of both exogenous H2O2 and exogenous aldehyde acrolein, thus, both these species could contribute to the thermal enhancement of cytotoxicity caused by BSAO and spermine. ^ The effect of temperature was observed in the presence of exogenous catalase, therefore this cytotoxicity was attributed also to aldehydes. The involvement of aldehydes in cytotoxicity at 42°C was also confirmed by complete inhibition of cytotoxicity with both exogenous aldehyde dehydrogenase and exogenous catalase added in the incubation mixture. A particular interesting finding was that by incubating the cells in the presence of BSAO, 50 |lM spermine and exogenous catalase which were not toxic at 37°C, contributed to cytotoxicity at 42°C and therefore the oxidation products of amines resemble to be thermosensitiser."^^ The thermosensitizing activity of aldehyde(s) produced in the BSAO-catalyzed oxidation of spermine has potential value for improving the therapeutic effects of hyperthermia and could be considered for future application in cancer therapy. Amine oxidases, in turn, can be internalised into the cells. ' In fact, cultured hepatocytes express binding sites for BSAO on their membrane surfaces evaluated at the electron microscope level by using enzyme-gold complexes. Hepatocytes show binding sites as small clusters of gold granules, not bound in a specialised region of the plasma membrane. The binding competition of enzyme-gold ligand to cells was achieved by preincubation with uncoupled BSAO. In addition, enrichment of a human leukemia cell line (K562) with a plant diamine oxidase was recendy obtained. ^ These results suggest the possible use of amine oxidases in physiological and hyperthermic conditions as potential antineoplastic drugs^^ taking in account, also their involvement in apoptotic phenomena. In fact, as demonstrated by Malorni et al^^ both pargyline and clorgyline, classical inhibitors of mitochondrial mono amine oxidases (MAO) are capable of protecting cells from apoptosis induced by serum starvation. In addition, Marcocci et al demonstrated that the structure and function of the mitochondrial membrane is modulated by the activity of MAO A and MAO B at low concentrations of benzylamine and octopamine, probably through the enzymatic production of H2O2. As mentioned above in this chapter, mitochondria are particularly sensitive to hyperthermia. Therefore, by considering the importance of apoptotic phenomena, in the regulation of human cells growing the simultaneous use of hyperthermia and enhancement of toxic products for cells, like H2O2 and aldehydes as metabolic products of amine oxidases, could be helpful in cancer therapy. As general conclusion, more then one biochemical systems appears to be involved in the hyperthermic damage, but so far it is not possible to describe a specific biochemical target of the hyperthermic damage, occurring most probably a simultaneous concurrence of different causes.
Acknowledgement This paper was supported by C.N.R. grant n° G002FD1 Agenzia 2000 and MURST
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References 1. Giovanella BC, Morgan AC, Stehlin JS et al. Selective lethal effect of supranormal temperatures on mouse sarcoma cells. Cancer Res 1973; 33:2568-2578. 2. Chen T T , Heidelberger C. Quantitative studies on the malignant transformation of mouse prostate cells by carcinogenic hydrocarbons in vitro. Int J Cancer 1969; 4:166-178. 3. Bush W. Ober den Einfluss, welchen heftigere Erysipeln zuweilen auf organisierte Neubildungen ausuben: Verhandl. Naturhist Preuss Rhein Westphal 1866; 23:28-37. 4. Nauts H C . The beneficial effects of bacterial infections on host resistance to cancer end results in 449 cases. A Study and abstracts of reports in the world medical literature (1775-1980) and personal communications. Monograph n" 8. 2nd ed. 1980. 5. Burns P. Die Heilwirkung des Erysipels auf Geschwulste. Beitr Klin Chir 1887; 3:443-453. 6. Coley W B . The treatment of malignant tumors by repeated inoculations of erysipelas with a report of ten original cases. Am J Med Sci 1893; 105:487-495. 7. Marcocci L, Mondovl B. Biochemical and ultrastructural changes in the hyperthermic treatment of tumor cells: An outline. Consensus on hyperthermia for the 1990s. In: Bicher H I , McLaren JR, Pighucci C M , eds. CHnical pratice in cancer treatment. New York: Plenum Press, 1990:99-120. 8. Giovanella BC, Mondovl B. Recent results in cancer research. In: Rossi-Fanelli A, Cavaliere R, Mondovl B et al, eds. Selective Sensitivity of Cancer Cells. 1977. 9. Westermark N . The effect of heat upon rat tumors. Scand Arch Physiol 1927; 52:257-266. 10. Cavaliere R, Cioccatto EC, Giovannella BC et al. Selective heat sensitivity of cancer cells cancer. Biochemical and clinical studies. Cancer 1967; 20:1351-1381. 11. Mondovl B, Strom R, Rotilio G et al. The biochemical mechanism of selective heat sensitivity of cancer cells. I. Studies on cellular respiration. Europ J Cancer 1969; 5:129-136. 12. Von Ardenne M, Reitnauer PG, Reiger F. Zur Erkundung therapeutischer Angriffspunktenim intermediaren Stofifwechsel der Krebszelle. In: Von Ardenne M, ed. Theoretische und experimentelle Grundlagen der Krebs-Mehrschritt-Therapie. Berlin: VEB Verlag Volk u n d G e s u n d h e i t , 1967:267-277. 13. Mondovl B, Finazzi-Agr6 A, Rotilio G et al. The biochemical mechanism of selective heat sensitivity of cancer cells. II. Studies on nucleic acids and protein synthesis. Europ J Cancer 1969; 5:137-146. 14. Strom R, Santoro AS, Crifo' C et al. The biochemical mechanism of selective heat sensitivity of cancer cells. IV. Inhibition of RNA synthesis. Eur J Cancer 1973; 9:103-112. 15. Dube DK, Seal G, Loeb LA. Differential heat sensitivity of mammalian D N A polymerases. Biochem Biophys Res Commun 1976; 76:483-487. 16. Emmelot P, Bos CJ. Studies on plasma membranes. VI. Differences in the effect of temperature on the ATPase and (Na*-K*)-ATPase activities of plasma membranes isolated from rat liver and hepatoma. Biochim Biophys Acta 1968; 150:354-363. 17. Strom R, Caiafa P, Mondovl B et al. Effect of temperature on potassium-dependent stimulation of transcellular migration in normal and neoplastic cells. Febs Letters 1969; 3:343-350. 18.Arancia G, Crateri Trovalusci P, Mariutti G et al. Ultrastructural changes induced by hyperthermia in Chinese hamster V79 fibroblasts. Int J Hyperthermia 1989; 5:341-350. 19. Buffa P, Guarriera-Bobyleva V, Muscatello U et al. Conformational changes of mitochondria associated with uncoupling of oxidative phosphorylation in vivo and in vitro. Nature 1970; 226:272-274. 20. Margolis LB. Cell interaction with model membranes probing, modification and simulation of cell surface functions. Biochim Biophys Acta 1984; 779:161-189. 2 1 . Shinitzki M. Membrane fluidity in malignancy adversative and recuperative. Biochim Biophys Acta 1984; 738:251-261. 22. Laudonio N , Marcocci L, Arancia G et al. Enhancement of hyperthermic damage on M l 4 melanoma cells by Uposome pretreatment. Cancer Res 1990; 50:5119-5126. 23. Arancia G, Calcabrini A, Matarrese P et al. Effects of incubation with liposomes at different temperatures on cultured melanoma cells (M14). Int J Hyperthermia 1994; 10:101-114. 24. Lepock JR. Involvement of membranes in cellular responses to hyperthermia. Radiat Res 1982; 92:433-438. 25. Lepock JR, Cheng KH, Al-Qysi H et al. Thermotropic lipid and protein transitions in chinese hamster lung cell membranes: Relationship to hyperthermic cell killing. Can J Biochem Cell Biol 1983; 61:421-427. 26. Cress AE, Culver PS, Moon T E et al. Correlation between amounts of cellular membrane components and sensitivity to hyperthermia in a variety of mammalian cell lines in culture. Cancer Res 1982; 42:1716-1721. 27. Mondovi B, Santoro AS, Strom R et al. Increased immunogenicity of Ehrlich ascites cells after heat treatment. Cancer 1972; 30:885-888.
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28. Check JH, Childs TC, Brady LW et al. Protection against spontaneous mouse mammary adenocarcinoma by inoculation of heat-treated syngeneic mammary tumor cells. Int J Cancer 1971; 7:403-408. 29. Manjili MH, Wang XY, Park J et al. Immunotherapy of cancer using heat shock proteins. Front Biosci 2002; 7:d43-52. 30. Hard FU. Molecular chaperones in cellular protein folding. Nature 1996; 381:571-579. 31. Kiang JG, Tsokos GC. Heat shock protein 70 kDa: Molecular biology, biochemistry, and physiology. Pharmacol Ther 1998; 80:183-201. 32. Frossard JL. Heat shock protein 70 (HSP70) prolongs survival in rats exposed to hyperthermia. Eur J Clin Invest 1999; 29:561-562. 33. Yi MJ, Park SH, Cho HN et al. Heat-shock protein 25 (Hspbl) regulates manganese superoxide dismutase through activation of Nfkb (NF-kappaB). Radiat Res 2002; 158:641-649. 34. Warters RL, Brizgys LM, Sharma R et al. Heat shock (45 degrees C) results in an increase of nuclear matrix protein mass in HeLa cells. Int J Radiat Biol Relat Stud Phys Chem Med 1986; 50:253-268. 35. Welch WJ, Feramisco JR. Purification of the major mammalian heat shock proteins. J Biol Chem 1982; 257:14949-14959. 36. Jin ZH, Shioura H, Kano E et al. Efi^ects of combined treatment with 40°C hyperthermia and bleomycin on the accumulation of heat shock protein in murine L cells. Int J Oncology 2002; 20:137-142. 37. Liu B, De Filippo M, Zihal L. Overcoming immune tolerance to cancer by heat shock protein vaccines. Molecular Cancer Therapeutics 2002; 1:1147-1151. 38. Lx)ven DP, Leeper DL, Oberley LW. Superoxide dismutase levels in Chinese hamster ovary cells and ovarian carcinoma cells after hyperthermia or exposure to cycloheximide. Cancer Res 1985; 45:3029-3033. 39. Christiansen EN, Kvamme E. Effect of thermal treatment on mitochondria of brain, liver ascites cells. Acta Physiol Scand 1969; 76:472-484. 40. Omar RA, Yano S, Kikkawa Y. Antioxidant enzymes and survival of normal and Simian Virus 40-transformed mouse embryo cells after hyperthermia. Cancer Res 1987; 47:3473-3476. 41. McCord JM. Oxygen-derived free radicals in postischemic tissue injury. New Engl J Med 1985; 312:159-163. 42. Bozzi A, Laudonio N, Zupi G et al. Interaction of superoxide dismutase-containing liposomes with a human melanoma cell line in hyperthermic conditions. Intern J Hyperthermia 1987; 3:553. 43. Mondovl B, Gerosa P, Cavaliere R. Studies on the effect of polyamines and their products on EhrUch ascites tumours. Agents and Actions 1982; 12:450-451. 44. Mondovl B, Agostinelli E, Przybytkowski F et al. Amine oxidases as a possible antineoplastic drugs. In: Alberghina L, Frontali L, Sensi P, eds. Proceedings of the 6th European Congress on Biotechnology. Elsevier Sciences BV, 1994:775-778. 45. Mondovl B, Befani O, Gerosa P et al. Specific temperature dependence of diamine oxidase activity and its thermal stability in the presence of polyvinylalcohol. Agents Actions 1992; 37:220-226. 46. Averill-Bates D, Agostinelli, Przybytkowski E et al. Cytotoxicity and kinetic analysis of purified bovine serum amine oxidase in the presence of spermine in Chinese Hamster ovary cells. Arch Biochem Biophys 1993; 300:75-79. 47. Agostinelli E, Przybytkowski E, Mondovl B et al. Heat enhancement of cytotoxicity induced by oxidation products of spermine in Chinese Hamster ovary cells. Biochem Pharmacol 1994; 48:1181-1186. 48. Dini L, AgostineUi E, Mondovl B. Cultured hepatocytes bind and internalize bovine serum amine oxidase-gold complexes. Biochem Biophys Res Commun 1991; 179:1169-1174. 49. Marcocci L, Nocera S, Roig MB et al. Enrichment of a human leukemia cell line (K562) with a plant histaminase. Inflamm Res 2001; 50:S134-S135. 50. Pietrangeli P, Mondovl B. Amine oxidases and tumors. NeuroToxicology 2004; 25:317-324. 51. Malorni W, GiammarioH AM, Matarrese P et al. Protection againsts apoptosis by monoamine oxidase A inhibitors. Febs Letters 1998; 426:155-159. 52. Marcocci L, De Marchi U, Milella ZG et al. Role of monoamine oxidases on rat liver mitochondrial ftinction. Inflamm Res 2001; 50:S132-S133.
CHAPTER 9
Results of Hyperthermia Alone or with Radiation Therapy and/or Chemotherapy Pietro Gabriele and Cristina Roca* Abstract
T
he interest in clinical hyperthermia (HT) was maximum in eighties and decreases during nineties of the last century but now, thank to possibility of heat deeply and to measure the temperature not invasively the interest is growing another time. On the other hand the biology of HT, clearly established during seventies, is now discussed with particular attention to the molecular pattern. Heat alone can be used as a cytotoxic agent. Results from 14 studies about lesions treated with HT alone reported complete response (CR) rate of 13% and overall response (OR) rate of 51%; the response time was short. HT alone can obtain results similar to some drugs employed as monochemotherapy. The result depends strictly from the possibility to obtain a good quality geometrical heat and to prescribe a sufficient number of heat session. The recommendation of the International Consensus meeting on HT held in 1989 was that results of heat alone should be used as a reference for such combinations. We performed from 1983 to 1996 some studies concerning the association of HT and radiations (RT). The most important results were the 90% and 75% of OR and CR for chest wall recurrences (the majority of them pretreated with radiotherapy); the data obtained were similar to residts of multicentric Italian data obtained in 212 lesions treated in 10 radiotherapy centres. In the Overgaard, Myerson and van der Zee reviews about numerous studies the therapeutic enhancement ratio for patients treated with the association versus patients treated with radiotherapy alone is for the great majority of studies between 1.5 and 2.0 From 22 randomized studies we found in 15 a statistically significant advantage for patients treated with the association of HT and RT or radiochemotherapy versus patients treated with RT or chemotherapy alone. We would emphasise that the two American studies by RTOG (Radiation Therapy Oncology Group) are inconclusive because of sub-optimal technical way of HT. The use of interstitial HT permits heat delivery to a well-defined volume which is frequently inaccessible to external local or deep HT. Interstitial HT uses placement into the treatment-planned volume of multiple microwave or radiofrequency antennas. Intracavitary HT associated with radiation therapy and/or chemotherapy is under study from about 20 years, in particular for the carcinoma of the oesophagus. Several hundred patients have been treated in phase I-II studies in the far East: all reports showed good treatment *Corresponding Author: Cristina Roca—Division of Molecular Angiogenesis, Institute for Cancer Research and Treatment (IRCC), Strada Provinciale 142, Candiolo, Turin 10060, Italy. Email:
[email protected] Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.
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Tabic 1. Results of HT alone Author
Year
Pts. N.
HT Method
Prescribed T
Corry^^ Gabriele^'' Kim8 Luk^7 Marmor^^ Perez^^
1982 1990 1979 1981 1982 1983 1980
28 60 19 11 44 6 6
US MW RF inductive MW US MW MW
43-50 °C 42-44 °C 41-43.5°C 42.5 °C 43-45 °C 41-43 °C 42-44 °C
U20
N. Treatments 6-12 2-12 2-9 5-12 6 6 2-9
CR (%)
OR
18 15 21 18 11 16.5 0
39 32 8 32 33 50
tolerance and a lack of significant late complications but most of the reports are based on small number of patients and not provide sufficient information. A strong biological rationale exists for the use of local HT and systemic chemotherapy in patients with superficial tumors. Superficial metastases are oft:en associated with additional occult distant metastases that warrant systemic treatment. Preliminary results employing cisplatinum and bleomycin with local HT revealed high response rate even in tumors located in previously irradiated sites: the better results were obtained in the treatment of breast carcinoma, head and neck and malignant melanoma. The most important prognostic factors affecting the response to HT are RT or heat dose: some of them may be more important than others in the clinical application, e.g., the temperature and total heating time, and, when HT is done in association with RT, radiation dose. The great challenge for HT in the next future is to provide adequate heating to the full tumor volume, in particular for deep seated tumors. Radiation Therapy Oncology Group (RTOG) studies demonstrated that 42°C minimum temperature not were obtained for most tumors; now some devices will ultimately lead to better minimum temperature not only for superficial tumors but also for deep seated lesions. Another way to ameliorate HT in clinical setting will be the possibility to measure the temperature not invasively by means of magnetic resonance (MR) or ultrasound (US).
Introduction From the first International Congress on Clinical HT in Washington in 1975, HT has increased in clinical oncology. The interest in clinical HT was maximum in eighties {W International Congress in Aahrus in 1984) and decreases during nineties of the last century but now, thank to possibility of heat deeply and to measure the temperature not invasively, the interest is growing another time. On the other hand the biology of HT, clearly established during seventies, ' is now discussed and (reviewed refs. 3-6) with particular attention to the molecidar pattern'^'^ and vascular targets. ^°'^^
Hyperthermia Alone Heat alone can be used as a cytotoxic agent. Results from 14 studies about lesions treated with HT alone including a total of 343 patients, reported in the Consensus Conference of 1989,^^ the CR rate was 13% (0-40%) and the OR rate was 51%; the response time (time to recurrence) was very short. In a personal experience regarding 60 superficial lesions, recurrent to surgery, radiotherapy and/or chemotherapy, treated with microwave (915 and 434 MHz) in a mean of 6 heat sessions, the complete response rate was 15%.^"^'^^ In Table 1 are summarized the results of some studies of HT alone with the technical detailed note, temperature and treatments number.
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121
Table 2, Results of HT and radiotherapy (University of Turin, 1983-1996) Site/Tumor
Pts. N.
% CR
% O.R.
Head and neck recurrent (SCC)^^'^^ Head and neck advanced (SCC)^^'^^ Parotid region (Adenocarcinoma like)^^'^^ Chest wall recurrences^^'^^ Adenocarcinoma vaginal recurrences^^ Perineal recurrences^^'^"^ Malignant melanoma^^ Recurrent Sarcoma^"^ Lymphoma^^
38 33 20 62.5
75 80 80 92
30 50 100 58
7 48 (86) 33
28 42
—
% L.C.
% Survival
— 62 60
75
84 87
—
— —
—
—
62
CR: complete response; OR: overall responsei; LC: local control
In conclusion H T alone can obtain results similar to some drugs employed as monochemotherapy. The result depends strictly from the possibility to obtain a good quality geometrical heat and to prescribe a sufficient number of heat session (mean 6). The recommendation of the International Consensus Meeting on HT is that data on heat alone should be used as a reference for such combinations.^^
Hyperthermia and Radiotherapy Not Randomised Studies We performed in a period of 13 years (1983-1996) some studies concerning the association of HT and radiations. Generally HT was done immediately before radiotherapy for 6 (range: 2-10) heat sessions of half-one hour. The temperature, controlled by means of invasive thermometry with fibre optic thermometers, was between 42 and 44°C in the measured points.^^'^^ In Table 2 are reported the results obtained in the various sites treated in our personal experience; the results are reported in terms of CR, OR (WHO criteria), LC (2/3 years minimum follow-up) and 3/5 years survival. The most important results were the 90% and 75% of OR and CR for chest wall recurrences (the majority of them pretreated with radiotherapy). The data obtained were similar to results of multicentric Italian data obtained in 212 lesions treated in 10 radiotherapy centres (Aviano, Catania, Genoa, Messina, Padoa, Ravenna, Rome, San Giovanni Rotondo, Trento and Turin).^^ Radiotherapy dose rarely exceeded 40 Gy in the treatment of recurrences whereas the dose for untreated advanced cases (parotid, head and neck and intact breast) was 60 Gy; for malignant melanoma large fraction of 4-9 Gy were employed for a total dose of 27-40 Gy^^ The complications rate varied from 4% in recurrent head and neck to only 1% in the treatment of parotid adenocarcinoma like and malignant melanoma. ^^ The most frequent complications were local burns, soft tissue necrosis and general symptoms like fever. We performed also some studies about H T in not cancerous diseases, in association or not with radiation therapy.^^'^^
Randomized Studies In the Overgaard,^^ Myerson ^ and van der Zee '^ reviews about numerous studies the therapeutic enhancement ratio for patients treated with the association versus patients treated with radiotherapy alone is for the great majority of studies between 1.5 and 2.0 (Table 3). From 22 randomized studies we found in 15 a statistically significant advantage for patients treated with the association of HT done with MW, RF or US and /radio or radiochemotherapy
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Table 3. Clinical results of the randomised studies of HT and raidiotherapy Author
Year
Pts. Number
RT
RT+HT
TER
Arcangeli"*^
1984 1984 1990 1986 1989 1989 1987 1986 2001 1984 1986 1988 1982 1987 1987 1987 1983 1987 1986 1994 1998 1998 1998 1993
163 45 65 86 92 24 65 46 40 33 66 238 124 87 313 101 154 185 90 78 41 70 306 122
38 9 46 50 63 63 86 33 50 25 35 39 29 25 25 39 41 33 31 36 41 35 41 5
73 42 66 60 82 83 100 50 85 71 72 72 54 46 63 62 69 64 65 73 83 62 59 23
1.95 4.67 1.43 1.20 1.28 1.32 1.16 1.52 1.70 2.84 2.06 1.85 1.86 1.84 2.52 1.59 1.68 1.94 2.10 2.03 2.02 1.78 1.45 4.60
Bey Datta^^ Dunlop^^ Egawa Fuwa^^ Goldobenko'^^ Gonzales^° Harima^^ Hiraoka" Hornback^^ Kim«
Li Lindholm^'^ Murakthodzaev Overgaard^^ Perez^^ Shidnia Steeves Valdagni^^ Van der Zee^^ Van der Zee^'' Van der Zee ^^
You
versus patients treated with radio or chemotherapy alone. We would emphasise that the two American studies by RTOG are inconclusive because of sub-optimal technical way of HT. In Table 3 are reported the most important studies of HT and radiotherapy and the results in terms of response/control and the TER.
Interstitial Thermo-Brachytherapy The use of interstitial HT permits heat delivery to a well-defined volume, which is frequendy inaccessible to external local or deep HT. Interstitial HT uses placement into the treatment-planned volume of multiple microwave or radiofrequency antennas.^^
Table 4. Clinical
results of the studies of interstitial
thermobrachytherapy
Author
Year
Pts Number
Follow-up
CR
PR
NR
Oleson^^ Cosset^^ Puthawala Emami^^ Petrovich^"^
1984 1985 1985 1987 1989
52 29 43 44 44
3-18m
39% 80% 86% 60% 65%
42% 20% 14% 26% 32%
9%
2m 6m 6-60m 6-30m
CR: complete response; PR: partial response; NR: no response
14% 3%
Results ofHyperthermia Alone or with Radiation Therapy and/or Chemotherapy
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Table 5. Clinical results of the studies ofHT and chemotherapy (and radiotherapy) Author
Year
Pts N.
Chemo
Chemo+HT
RT+HT
Arcangell^° Emami^^ Gabriele''^ Kitamura^^ Orecchia''^ Steindorfer''^ Zhang
1983 1989 1991 1995 1991 1987 1988
43 21 10 66 20 22 28
95% 49% 10% 6% 10% 8% 41%
45% (67% OR) (50% OR) 25% (70%) 80% (85% OR)
p53, might predispose tumours to a more malignant phenotype. Clinical data support this conclusion. Studies on both soft tissue sarcomas and on carcinomas of the cervix have shown that hypoxic tumours are more likely to be metastatic. However, others have proposed that tumour hypoxia can occur in a second way, by temporary obstruction or cessation of tumour blood flow, the so called acute hypoxia model. Definitive evidence for this type of acute hypoxia arising from fluctuating blood flow, has come from elegant studies with transplanted tumours in mice using diffusion limited fluorescent dyes. Because fluctuating blood flow has also been demonstrated in human tumours, it is likely that this type of hypoxia is also present in human tumours. The consequences of acute hypoxia will be similar to those of the diffusion-limited hypoxia. Any cells surrounding a closed blood vessel will be resistant to radiation killing because of their lack of oxygen at the time of radiation and will be exposed to lower levels of anticancer drugs than those surrounding blood vessels with a normalflow.This would be expected to lead to differences in response to anticancer agents, as has been observed in experimental tumours. The low oxygen levels in tumours can be probably turned from a disadvantage to an advantage in cancer treatment. Such a possibility was proposed 20 years ago by Lin, who reasoned that compounds based on the quinone structure of mitomycin C might be more active in hypoxic tumours. It was known that mitomycin C required metabolic reduction of the benzoquinone ring to produce the cytotoxic bifunctional alkylating agent. Lin reasoned that a lower oxidation reduction (redox) potential for tumour tissue, relative to most normal tissues, could increase reductive activation of these quinone derivatives in tumours. Although this was not the correct mechanism for the increased cytotoxicity of mitomycin C and certain analogues toward hypoxic cells (much lower levels of hypoxia are needed to change cellular redox potential), these studies were important in su^esting the potential of hypoxia-activated drugs and led to the concept of selectively killing the hypoxic cells in solid tumours.
Hypoxia and Chemotherapy There are presently three different classes of hypoxia-specific drugs that are in use clinically or are being developed for clinical use. They are the quinone antibiotics, the nitroimidazoles, and the benzotriazine di-N-oxides. In the quinone class, the three principal agents of current clinical interest are mitomycin C, porfiromycin and E09. All are structurally similar and require reductive metabolism for activity. Each of them is converted by reductive metabolism to a bifunctional alkylating agent and probably produces its major cytotoxic activity through the formation of DNA interstrand cross-links.
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Mitomycin C, considered to be the prototype bioreductive drug, was introduced into clinical use in 1958 and has demonstrated efficacy towards a number of different tumours, in combination with other selective drugs whose toxicity towards hypoxic cells is modest, with values for hypoxic cytotoxicity ratios (the ratio of drug concentration to produce equal cell kill for aerobic and hypoxic cells) of one (no preferential toxicity) to approximately £IYC. However, based on this activity, mitomycin C has been combined with radiotherapy in two randomized trials of head and neck cancer, the pooled restdts of which gave a statistically significant disease-free survival benefif^'^ The third drug in this series, E09, is a much more efficient substrate for DT-diaphorase than either mitomycin C or porfiromycin and shows high toxicity to both aerobic and hypoxic cells with high DT-diaphorase levels. Cells with low DT-diaphorase levels are much less susceptible to killing by E09 under aerobic conditions, but this drug shows a high, up to 50-fold, preferential toxicity toward hypoxic cells. However, the pharmacokinetics of this agent work against its clinical utility, and phase I clinical studies have shown litde activity of this drug. A second class of bioreductive agents is that of the nitroimidazoles, the first two of which, metronidazole and misonidazole, have been extensively tested as hypoxic radiosensitizing agents. Further drug development by Adams^ produced a compound, RSU1069, which has been shown to be a highly efficient cytotoxic agent with activity both in vitro and in vivo. RSU1069 has an hypoxic cytotoxicity ratio of some 10-100 for different cell lines in vitro, and it, or its prodrug, RB6145, has shown excellent activity with mouse tumour models when combined with irradiation or agents that induce hypoxia. Unfortunately, however, clinical testing of RB6145 has been aborted due to irreversible cytotoxicity toward retinal cells. Tirapazamine (TPZ) is the first, and thus far, only representative of the third class of hypoxia-selective cytotoxins. The mechanism for the preferential toxicity of TPZ towards hypoxic cells is the result of an enzymatic reduction that adds an electron to the TPZ molecide, forming a highly reactive radical. This radical is able to cause celi killing by producing DNA damage leading to chromosome aberrations. Moreover, DNA damage occurs only from TPZ metabolism within the nucleus. TPZ produces specific potentiation of celi kill by radiation and cisplatinum. Specifically, the synergistic cytotoxic interaction observed when TPZ and cisplatinum are given in sequence depends on the TPZ exposure being under hypoxic conditions. In fact, there is no interaction when TPZ is given under aerobic conditions. It has also been demonstrated that the cytotoxic activity of TPZ under hypoxia is independent of p53 gene status of tumour cells. This drug has 100-fold differential toxicity toward hypoxic vs. aerobic cells. Based on experimental studies that evaluated the responsiveness of tumour cells under aerobic and hypoxic conditions, Teicher^^ classified chemotherapeutic agents into three groups: (1) preferentially toxic in aerobic conditions (bleomycin, procarbazine, streptonigril, actinomycin D, vincristine and melphalan); (2) preferentially toxic under hypoxic conditions (mitomycin C and adriamycin); (3) no major preferential toxicity to oxygenation (cisplatinum, 5-fluorouracil and methotrexate).
Hypoxia and Gene Therapy The newest direction for exploiting tumour physiology is aimed toward the evolving field of gene therapy. In this novel approach to anticancer therapy, genetic material is transferred into cells with the idtimate goal of selectively killing cancer cells and sparing normal cells. Recent studies have regarded the possibility of using the hypoxia-signalling pathway to selectively activate gene expression.^ ^' Hypoxia induces the expression of a number of genes, principally via the stabilization of members of the bHLH/PAS family of transcription factors that bind to a consensus DNA sequence, the hypoxia response element (HRE). Physiologically regulated expression vector systems, containing HRE sequences, are now under development, to target therapeutic gene expression to tumour cells characterized by low oxygen tension.
A Step Deep on Hyperthermia, Hypoxia and Chemotherapy Interactions
159
From a clinical point of view the combination of hyperthermia and hypoxia seems to add activity to intra-arterial chemotherapy.^'^ At the same lime the exposure of body regions, such as pelvis or limbs, to a locally high dose of bioreductive agent such as mitomycin C, in hypoxic conditions shows activity in refractory cancers. ^^"^^ Following the study of genes we understood that there are other ways in which hypoxia might contribute to drug resistance. One is through the amplification of genes, such as dihydrofolate reductase, conferring various glucose-regulated proteins that appear to be responsible for resistance to doxorubicin, etoposide and camptothecin.
Hyperthermia and Chemotherapy Preclinical thermo-chemotherapy studies have given valuable information on the schedule of the cytotoxic interaction between the different agents and on the molecular mechanisms responsible for the potentiating effect. Several studies have demonstrated that the cytotoxic activity of various chemotherapeutic agents is enhanced by mild or moderate hyperthermia (40.5-43°C).^^ In these investigations the effect of scheduling on the cytotoxic interaction between hyperthermia and drugs has also been investigated in in vitro experimental systems. There are data regarding doxorubicin, the platinum compounds cisplatin and carboplatin, the bifunctional alkylating agent melphalan and the antimetabolite methotrexate which indicate that in each case the maximal cytotoxicity occurs when the drug is administered simultaneously with hyperthermia. ^ '^ The mechanisms responsible for the effect of hyperthermia on cell killing by anticancer drugs are not entirely understood. For example, for melphalan, which is widely used in experimental and clinical thermo-chemotherapy studies, different putative mechanisms of potentiation have been suggested including: an increase in melphalan influx leading to a higher intracellular drug accumulation.^° The alteration of the DNA quaternary structure, which favours alkylation; the interference with drug-DNA adduct metabolism and inhibition of repair^ ^ the stabilization of drug-induced G2 phase cell accumulation^^ through the inhibition of p32^ kinase activity.^'^'^^ As regards cisplatin, it has been demonstrated that the cytotoxic activity of this compound, as well as that of the platinum derivatives lobaplatin and oxaliplatin, is increased under hyperthermic conditions as the consequence of an enhanced formation of DNA-platinum adducts."^ Preclinical studies have also significantly contributed to the proposition of potential cellular determinants of response to individual and combined treatments. The relevance of cell kinetic and DNA ploidy characteristics as indicators of thermoresponsiveness has been determined in primary cultures of human melanoma. Results from this study showed that the median 3H-thymidine labelling index of sensitive tumours was four-fold that of resistant tumours. Moreover, thermosensitivity was found more frequently in tumours with a diploid nuclear DNA content than in those with DNA aneuploidy. Since heat and drug sensitivity may be related to the ability of tumour cells to mount a stress response, the relationship between constitutive (and inducible) levels of heat shock proteins (HSPs) and thermosensitivity has been evaluated in testes and bladder cancer cell lines. No correlation between constitutive levels of HSP90 or HSP72/73 and cellular thermoresponsiveness was found. However, results suggest that low HSP27 expression might contribute to heat sensitivity.
Hyperthermia and Antineoplastic Drugs Selection The most effective agent(s) at elevated temperatures have yet to be determined. Some studies suggest that the drug of choice at elevated temperatures may be different from that at the physiological temperature, and that the alkylating agents may be most effective at elevated temperatures. To further investigate these possibilities, the effect of chemotherapeutic agents were compared by Takemoto. He studied these agents: cyclophosphamide, ifosfamide, melphalan, cisplatin, 5-fluorouracil, mitomycin C and bleomycin. Three tumours (mammary
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Hyperthermia in Cancer Treatment: A Primer
carcinoma, osteosarcoma and squamous cell carcinoma) were used. They were transplanted into the feet of C3H/He mice. When tumours reached 65 mm, a test agent was injected intraperitoneally. Tumours were inmiediately heated at 41.5°C for 30 min, and the tumour growth (TG) time was studied for each tumour. Using theTG times, theTG-50 (the time required for one-half of the total number of the treated tumours to reach the volume of 800 mm from 65 mm was calculated. Subsequendy, the tumour growth delay time (GDT) and the thermal enhancement ratio (TER) were obtained. The GDT was the difference between theTG-50 of treated tumours and that of nontreated control tumours. The TER was the ratio of the GDT of a group treated with an agent at 4l.5*'C to that of a group treated with the agent at room temperature. Results showed that the top three effective agents tested at 41.5°C were solely alkylating agents cyclofosphamide, ifosfamide and melphalan for each kind of tumour. A GDT of cisplatin was smaller than those of the alkylating agents. The smallest TER, 1.1, was observed for 5-fluorouracil, which was given for manmiary carcinoma, and for mitomycin C, which was given for squamous cell carcinoma. It could be concluded that the alkylating agents at elevated temperatures might be the drugs of choice for many types of tumours.
Alkylating Agents and Oxaliplatin Urano^^ studied the effects of various agents on animal tumours with different histopathology at elevated temperatures. His studies indicated that alkylating agents were most effective to all tumours at a moderately elevated temperature. Cisplatin was also effective to all tumours, but its effectiveness at 41.5°C was less than that of alkylating agents. To quantitatively study these findings, the magnitude of thermal enhancement of melphalan, an alkylating agent, and that of oxaliplatin, a new platinum compound, were studied by this author, at 37-44.5°C by the colony formation assay. The dose of each agent was kept constant, and cell survival was determined as a function of treatment time. The cell survival curve was exponentially related with treatment time at all test temperatures, and theT(O) (the time to reduce survival from 1 to 0.37) decreased with an increasing temperature. These results suggested that the cytotoxic effect of these agents occurred with a constant rate at 37°C, and the rate was facilitated with an increasing temperature. This suggests that heat can accelerate the cytotoxic chemical reaction, leading to substantial thermal enhancement. The thermal enhancement ratio (TER, the ratio of the T(0) at 37°C to the T(0) at an elevated temperature) increased with an increase in the temperature. The activation energy for melphalan at moderately elevated temperatures was largest among the agents tested in the laboratory and that for oxaliplatin was approximately half of the melphalan activation energy. This suggests that the thermal enhancement for the cytotoxicity of melphalan or alkylating agents might be the greatest.
Docetaxel Recent studies suggest that docetaxel may show improved response at elevated temperatures. Factors that may modify the thermal enhancement of docetaxel were studied by Mohamed to optimize its clinical use with hyperthermia. The tumor studied was an early-generation isotransplant of a spontaneous C3Hf/Sed mouse fibrosarcoma, Fsa-II. Docetaxel was given as a single intraperitoneal injection. Hyperthermia was achieved by immersing the tumor-bearing foot into a constant temperature water bath. Four factors were studied: duration of hyperthermia, sequencing of hyperthermia with docetaxel, intensity of hyperthermia, and tumor size. To study duration of hyperthermia tmnors were treated at 41.5 °C for 30 or 90 min inunediately after intraperitoneal administration of docetaxel. For sequencing of hyperthermia and docetaxel, animals received hyperthermia treatment of tumors for 30 min at 41.5°C immediately after drug administration, hyperthermia both immediately and 3 hr after docetaxel administration and hyperthermia given only at 3 hr after administration of docetaxel. Intensity of hyperthermia was studied using heat treatment of tumors for 30 min at 41.5 or 43.5°C immediately following docetaxel administration. Effect of tumor size was studied by delaying
A Step Deep on Hyperthermia, Hypoxia and Chemotherapy Interactions
161
experiments until three times the tumor volume was observed. Treatment of tumors lasted for 30 min at 41.5°C immediately following drug administration. Tumor response was studied using the mean tumor growth time. Hyperthermia in the absence of docetaxel had a small but significant effect on tumor growth time at 43.5°C but not at 4l.5°C. Hyperthermia at 4l.5°C for 90 min immediately after docetaxel administration significantly increased mean tumor growth time (P = 0.0435) when compared to tumors treated with docetaxel at room temperature. Treatment for 30 min had no effect. Application of hyperthermia immediately and immediately plus 3 hr following docetaxel was effective in delaying tumor growth. Treatment at 3 hr only had no effect. No significant difference in mean tumor growth time was observed with docetaxel and one half hour of hyperthermia at 41.5 or 43.5°C. For larger tumors, hyperthermia alone caused a significant delay in tumor growth time. Docetaxel at 41.5°C for 30 min did not significantly increase mean tumor growth time compared to large tumors treated with docetaxel at room temperature. Docetaxel shows a moderate increase in anti-tumor activity with hyperthermia. At 41.5°C the thermal enhancement of docetaxel is time-dependent if hyperthermia is applied immediately following drug administration. With large tumors docetaxel alone or docetaxel plus hyperthemia showed the greatest delays in tumor growth time in the experiments.
Hyperthermia and Gene Therapy Li reported the activity of adenovirus-mediated heat-activeted antisense Ku70 expression radiosensitizers tumor cells in vitro and in vivo. Ku70 is one component of a protein complex, Ku70 and Ku80, that functions as a heterodimer to bind DNA double-strand breaks and activates DNA-dependent protein kinase. The previous study of this group with Ku70-/- and Ku80-/- mice, and cell lines has shown that Ku70- and Ku80-deficiency compromises the ability of cells to repair DNA double-strand breaks, increases radiosensitivity of cells, and enhances radiation-induced apoptosis. In this study, Li examined the feasibility of using adenovirus-mediated, heat-activated expression of antisense Ku70 RNA as a gene therapy paradigm to sensitize cells and tumors to ionizing radiation. First, they performed experiments to test the heat inducibility of heat shock protein (hsp) 70 promoter and the efficiency of adenovirus-mediated gene transfer in rodent and human cells. Replication-defective adenovirus vectors were used to introduce a recombinant DNA construct, containing the enhanced green fluorescent protein (EGFP) under the control of an inducible hsp70 promoter, into exponentially growing cells. At 24 h after infection, cells were exposed to heat treatment, and heat-induced EGFP expression at different times was determined by flow cytometry. The data by Li clearly show that heat shock at 42°C, 43"C, or 44°C appears to be equally effective in activating the hsp70 promoter-driven EGFP expression (>300-fold) in various tumor cells. Second, the authors have generated adenovirus vectors containing antisense Ku70 under the control of an inducible hsp70 promoter. Exponentially growing cells were infected with the adenovirus vector, heat shocked 24 h later, and the radiosensitivity determined 12 h after heat shock. Our data show that heat shock induces antisense Ku70 RNA, reduces the endogenous Ku70 level, and significantly increases the radiosensitivity of the cells. Third, the author has performed studies to test whether Ku70 protein level can be down-regulated in a solid mouse tumor (FSa-II), and whether this results in enhanced radiosensitivity in vivo, as assessed by in vivo/in vitro colony formation and by the tumor growth delay. Their data demonstrate that heat-shock-induced expression of antisense Ku70 RNA attenuates Ku70 protein expression in FSa-II tumors, and significantly sensitizes the FSa-II tumors to ionizing radiation. Taken together, these interesting results suggest that adenovirus-mediated, heat-activated antisense Ku70 expression may provide a novel approach to radiosensitize human tumors in combination with hyperthermia.
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Hyperthermia in Cancer Treatment: A Primer
Conclusions The comprehension, about 50 years ago, that hypoxic cells are resistant to X-rays led to the concept that cancers might be resistant to radiotherapy and chemotherapy because of their poor oxygen supply and subsequent hypoxia. Now tumour hypoxia is seen as a mechanism of resistance to many antineoplastic drugs, as well as a predisposing factor toward increased malignancy and metastases. However tumour hypoxia is a unique target for hyperthermia and cancer bioreductive therapy that could be exploited for therapeutic use. A hypoxic cell is unable to have a stable pH; this increases the permeability of the cell membrane so that antineoplastic agents can easily move through the membrane improving the global concentration of the drug both inside and outside the cell. Hyperthermia seems the best opportunity to enhance these phenomena.
References 1. Gray LH, Conger AD, Ebert M et al. Concentration oi oxygen dissolved in tissue at the time of irradiation as a factor in radiotherapy. Br J Radiol 1953; 26:638-48. 2. Thomlinson RH, Gray LH. The histological structure of some human lung cancers and the possible implications for radiotherapy. Br J Cancer 1955; 9:539-49. 3. Moulder JE, Rockwell S. Tumor hypoxia: Its impact on cancer therapy. Cancer Metastasis Rev 1987; 5:313-41. 4. Brown JM. The hypoxic cell: A target for selective cancer therapy. Cancer Res 1999; 59:5863-70. 5. Rauth AM, Melo T, Misra V. Bioreductive therapies: An overview of drugs and their mechanis m of action. Int J Radiat Oncol Biol Phys 1998; 42:755-62. 6. Wouters BG, Wang LH, Brown JM. Tirapazamine: A new drug producing tumor specific enhancement of platinum-based chemotherapy in non small cell lung cancer. Ann Oncol 1999; 10(suppl 5):S29-S33. 7. Graeber TG, Osmanian C, Jacks T. Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature 1996; 379:88-91. 8. Lin A, Cosby L, Shansky C et al. Potential bioreductive alkylating j^ents. 1. Benzoquinone derivates. J Med Chem 1972; 15:1247-52. 9. Adams GE, Stratford IJ. Bioreductive drugs for cancer therapy: The search for tumour specificity. Int J Radiat Oncol Biol Phys 1994; 29:231-8. 10. Teicher BA, Holden SA, Al-Achi A et al. Classification of antineoplastic treatments by their differential toxicity toward putative oxygenated and hypoxic tumor subpopulation in vivo in the FSallC murinefibrosarcoma.Cancer Res 1990; 50:3339-44. 11. Binley L, Iqball S, Kingsman S et al. An adenoviral vector regulated by hypoxia for the treatment of ischaemic disease and cancer. Gene Ther 1999; 6:1721-7. 12. Zaffaroni N, Fiorentini G, De Giorgi U. Hyperthermia and hypoxia: New developments in anticancer chemotherapy. Eur J Surg Oncol 2001; 27:340-2. 13. Guadagni S, Fiorentini G, Palumbo G et al. Hypoxic pelvic perfiision with mitomycin C using a simpHfied ballon-occlusion technique in the treatment of patients with unresecuble locally recurrent rectal cancer. Arch Surg 2001; 136(1): 105-12. 14. Guadagni S, Russo F, Rossi CR et al. Deliberate hypoxic pelvic and limb chemoperfiision in the treatment of recurrent melanoma. Am J Sur 2002; 183:28-36. 15. Fiorentini G, Poddie D, Graziani G et al. Hypoxic isolated limb perfiision with mitomycin C in locally recurrent melanoma and sarcoma: Results of a phase II study. Reg Cancer Treat 1995; 8:135-9. 16. Brown JM, Giacca AJ. The unique physiology of solid tumore: Opportunities (and problems) for cancer therapy. Cancer Res 1998; 58:1408-16. 17. Urano M, Kuroda M, Nishimura Y. For the clinical application of thermochemotherapy given at mild temperatures. Int J Hyperther 1999; 2:79-107. 18. Zaffaroni N, Villa R, Daidone MG et al. Antitumor activity of hyperthermia alone or in combination with cisplatin and melphalan in primary cultures of human malignant melanoma. Int J Cell Cloning 1989; 7:385-94. 19. Kusumoto T, Holden SA, Ara G et al. Hyperthermia and platinum complexes: Time between treatments and synergy in vitro and in vivo. Int J Hyperther 1995; 11:575-86. 20. Bates DA, Mackillop WJ. Effect of hyperthermia on the uptake and cytotoxicity of melphalan in Chinese hamster ovary cells. Int ] Radiat Onco/ Biol Phys 1998; 16:187-91.
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2 1 . Zaffaroni N , Villa R, Orlandi L et al. Effect of hyperthrmia on the formation and removal of DNA interstrand cross-links induced by melphalan in primary cultures of human malignant melanoma. Int J Hyperther 1992; 8:341-9. 22. Orlandi L, Zaffaroni N , Bearzatto A et al. Effect of melphalan and hyperthermia on cell cycle progression and cyclin Bl expression in human melanoma cells. Cell ProUf 1995; 28:617-30. 23. Orlandi L, Zaffaroni N , Bearzatto A et al. Effect of melphalan and hyperthermia on p34cdc2 kinase activity in human melanoma cells. Br J Cancer 1996; 74:1924-8. 24. Rietbroek RC, van de Vaart PJ, Haveman J et al. Hyperthermia enhances the cytotoxic activity and platinum-DNA adduct formation of lobaplatin and oxaliplatin in cultured SW 1573 cells. J Cancer Res Clin Oncol 1997; 123:6-12. 25. Orlandi L, Costa A, Zaffaroni N et al. Relevance of cell kinetic and ploidy characteristics for the thermal response of malignant melanoma primary cultures. Int J Oncol 1993; 2:523-6. 26. Richards E H , Hickman JA, Masters JR. Heat shock protein expression in testis and bladder cancer ceh lines exhibiting differential sensitivity to heat. Br J Cancer 1995; 72:620-6. 27. Takemoto M, Kuroda M, Urano M et al. Effect of various chemotherapeutic agents given with mild hyperthermia on different types of tumours. Int J Hyperthermia 2003; 19(2): 193-203. 28. Urano M , Ling C C . Thermal enhancement of melphalan and oxaliplatin cytotoxicity in vitro. Int J Hyperthermia 2002; 18(4):307-15. 29. Mohamed F, Stuart OA, Glehen O et al. Docetaxel and hyperthermia: Factors that modify thermal enhancement. J Surg Oncol 2004; 8 8 ( l ) : l 4 - 2 0 . 30. Li G C , H e F, Shao X et al. Adenovirus-mediated heat-activated antisense Ku70 expression radiosensitizes tumor cells in vitro and in vivo. Cancer Res 2003; 63(12):3268-74.
SECTION III
Clinical Aspects of Hyperthermia
CHAPTER 12
Locoregional Hyperthermia E. Dieter Hager* Abstract
L
ocoregional hyperthermia can be differentiated into external, interstitial and endocavitary hyperthermia. Different heat delivery systems are available: antennae array, capacitive coupled, and inductive devices. Depending on localization and size of the tumour different methods and techniques can be applied: superficial, intratumoral (thermoablation), deep hyperthermia, endocavitary, and part-body hyperthermia. Randomized clinical trials have been performed mosdy with electromagnetic applicators for superficial hyperthermia in combination with radiotherapy, deep hyperthermia with and without radiation, and endocavitary hyperthermia in combination with chemotherapy and radiotherapy. In randomized clinical trials it could be demonstrated, that loco-regional deep hyperthermia with antennae array or capacitive coupled hyperthermia devices may increase response rate, disease free survival and overall survival of patients with cancer in combination with radiotherapy or chemotherapy without increasing the toxicity of standard therapies.
Introduction Hyperthermia is one of the most promising new multidisciplinary approaches to cancer therapy. The rationale for raising temperature in tumor tissue is based on a direct cell-killing effect at temperatures above 4l-42°C and a synergistic interaction between heat and radiation as well as various antineoplastic agents. The thermal dose-response depends also on microenvironmental factors such as pH, and p02 in the tumor tissue. Depending on the physical characteristics of the energy field applied, also other mechanisms of tumor destruction or growth retardation may be relevant. Tissue-specific electromagnetic interactions may be possible, depending on frequency and applicator technique used, due to inhomogeneities in the relative dielectric permittivity, relative magnetic permeability, specific conductivity, and ion distribution in cancer tissue compared to normal tissue. The effects of hyperthermia on the host and cancer tissue are pleio tropic and depend mainly on the temperature and the physical techniques applied. The biological and molecular mechanisms of these effects are changes in the membrane, ^'^ the cytoskeleton, the ion-gradient and membrane potential, svnthesis of macromolecules and DNA-replication,^^ intra- and extra-cellular pH (acidosis ) and decrease in intracellular ATP. ^^ Genes can be up-regidated or down-regulated by heat, for example the heat-shock proteins (HSP).^^ Synergistic effects by interactions with antineoplastic agents, radiation and heat can be several powers of ten even at moderate temperatures. In addition, reduced chemotherapy resistancy, possibly due to increased tissue penetration, increased membrane permeability, and activated metabolism, has been observed. *E. Dieter Hager—Department of Hyperthermia, BioMed-Klinik GmbH, Tischberger Str. 5-8, D-76887 Bad Bergzabern, Germany. Email:
[email protected] Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.
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Hyperthermia in Cancer Treatment: A Primer
Immunological effects of hyperthermia may play an additional role in cancer therapy such as immunological effects on cellular effector cells (emigration, migration and activation), induction of cytokines, chemokines and heat shock proteins (chaperones), and modulation of cell adhesion molecules. The induction of heat-shock proteins might increase specific immune responses to cancer cells. Locoregional hyperthermia can be differentiated into A. External hyperthermia • Local hyperthermia (short waves/radiofrequencies (SW/RF), microwaves (MW)) • Regional deep hyperthermia (RF, MW, ultrasound (US)) • Part-body hyperthermia (RF, MW, infrared (IR), heat perfusion) B. Interstitial hyperthermia with • RF electrodes (i.e., needles) • HF or MW antennas • Laser fibres • Ultrasound transducers • Magnetic rods/seeds and fluid C. Endocavitary hyperthermia (sy: intraluminal) • RF electrodes (i.e., coils) • Radiative (IR, laser) • Heat sources (hot fluid perfusion, extracorporal perfiision) depending on the method of the external heating devices and the area treated with hyperthermia (Fig. 1). With RF capacitive heating devices delivering 8-27 MHz and annidar phased-array systems delivering 60-430 MHz electromagnetic waves local and regional deep hyperthermia (DHT) can be applied for superficial and deep seated tumors. As a general physical rule: the higher the frequency of the electromagnetic field the less deep the penetration depth will be. Therefore lower frequencies are used more frequendy for deep seated tumors and higher frequencies for superficial tumors. Molecules with dipoles, like water, can be excited in such alternating electromagnetic fields which will be measured as heat. With capacitively-coupled electrodes and perfusion with heated fluid larger anatomical areas like the peritoneum, the bladder, the pleural cavity and the whole liver and lung or extremities can be heated up, which is called part-body hyperthermia (PBHT). Depending on the frequencies applied and with new applicator techniques and with sufficient monitoring PBHT is also possible with dipole antennae devices. Interstitial hyperthermia delivers the heat direcdy at the site of the tumor. For interstitial hyperthermia high frequency needle electrodes at 375 kHz (i.e., high frequency-induced thermotherapy; HiTT), microwave antennas, ultrasound transducers, laser fiber optic conductors (laser-induced thermotherapy; LiTT), or ferromagnetic rods, seeds or fluids (magnetic fluid hyperthermia (i.e., with nanoparticles), MFH) are injected or implanted into the tumor. In some cases the interstitial hyperthermia is combined with a brachytherapy by an afterloading method. With these applicators a heat can be applied high enough to induce in tumors thermonecrosis at a distance of 1 to 2 cm around the hot source. This technique is suitable for 1-5 tumors less than 5 cm in diameter. Insertion of antennas or electrodes into lumens of the human body such as the oesophagus, rectum, bladder, urethra, vagina and the uterine cervix are used for endocavitary hyperthermia. With this technique larger applicators than for interstitial hyperthermia with higher penetration depth can be applied. Perfusional hyperthermia with fluids (water, blood) is used to deliver heat withfluidsinto cavities like the peritoneum, the pleural space, or the bkdder. The perfusate is combined with antineoplastic agents or cytokines, like TNF-a (see Chapter entided "Perfiisional Peritoneal Hyperthermia"). Extracorporal heat exchange is commonly used to heat up blood for the perfusion of extremities. Deep hyperthermia (DHT) is referred to the induction of heat in deep seated tumors, e.g., of the pelvis, abdomen, liver, lung, or brain-by external energy applicators. The
Locoregional Hyperthermia
169
Figure 1. Technical devices for deep hyperthermia: A) high frequency induced thermo-dierapy; B) RF capacitively-coupied electrodes; C) multi-antenna applicator (dipole pairs).
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Hyperthermia in Cancer Treatment: A Primer
Table /. Different heat delivery methods Heat Delivery Methods
Examples
Conductive Radiative Mechanical Antennas Capacitive Inductive Bioactive
Cavitational water-heating; extra-corporal blood heating; RF needles Infrared light (IR-A, -B, -C) Ultrasound Multi-antenna-dipole applicators Condenser Ferromagnetic rods/seeds/fluids Pyrogens, cytokines
technical features for the treatment of deep seated tumors are interstitial applicators (i.e., conductive), electromagnetic antenna-dipole arrangements, capacitive-coupled electrodes, ultrasound, and magnetic fields (see Table 1). The technique used, will restrict the application to certain body areas. The different electromagnetic techniques used for transferring energy in regional deep hyperthermia are: • radiofrequencies (RF-DHT) between 5-27 MHz • highfrequencies(HF-DHT) between 60-430 MHz (decimetre waves) and • microwaves (MW-DHT) atfrequencieslarger than or equal 1 GHz (centimetre waves). The absorption of the electromagnetic field (EMF) is depending from physical properties of the penetrated tissue, like conductivity and dielectricity which may cause focusing effects and electromagnetic coupling. The distribution of the temperature within tumor tissue is inhomogeneous due to intra- and extratumoral perfusion r^ulations, electric characteristics of the tissues and thermal conductivity, and ranges between 39 and 43°C. In addition to the thermal effects, frequency dependent non-thermal effects may play an essential role. Physical aspects (impedance and interaction with dipoles) let expect a special role for EMF in the radiofrequency range between 1-30 MHz. First experimental and clinical trials have been performed in the 1960s with radiofrequencies in the range between 8 and 27 MHz (LeVeen). This technique is now most frequently used in Japan and Russia. In Japan most clinical research has been performed with RF-technique at 8 MHz."^^ In Europe, especially the Netherlands and Germany, most frequently high frequency technique systems with dipole antennae operating at frequencies of 60 to 120 MHz (BSD-2000) are used in clinical research. Since the end of the eighties 13.56 MHz RF capacitive heating devices are available also for superficial and deep hyperthermia in Europe, especially in Germany and Italy.
Clinical Trials on Hyperthermia Superficial Hyperthermia Superficial tumors can be heated by (a) waveguide applicator, (b) spiral applicator, (c) current sheet applicator, (d) ultrasonic applicator, (e) RF-needles and (f) infrared sources. Electromagnetic applicators for superficial hyperthermia have a typical frequency of 150-430 MHz. Most convenient for local hyperthermia are water-filtered infrared sources. The therapeutic depths with these applicators is about 3 cm. By Medline database research up to October 2003, six randomized prospective phase III trials (RCT) on radiotherapy alone compared with radiotherapy combined with hyperthermia could be identified (Table 2). In all of these trials the combination radiotherapy plus hyperthermia showed better response rates. Overall survival benefit was only noted in one RCT trial.
Locoregtonal Hyperthermia
Table 2, Randomized
controlled
171
trials on superficial
hyperthermia
Tumor Site
Experimental
No. Control of Pts
Primary Endpoints
HT Better
Survival Benefit
Ref.
Head and neck (primary)
RT + sHT
RT
65
Response at 8 v^eeks
Yes
No
39
Melanoma (metastatic or recurrent)
RT + sHT
RT
68
Complete response
Yes
No
40
Superficial RT + sHT (head and neck, breast, miscellaneous)
RT
245
Initial response
possibly
No
41
Head and neck (N3 primary) (2-6 times)
RT + sHT
RT
44
Response
Yes
Yes
42
Breast (advanced primary or recurrent)
RT + sHT
RT
307
Initial response
Yes
No
43
Head and neck, breast, sarcoma melanoma
RT + 2X sHT
RT+ 1xsHT
173
Response
No
No
44
Abbreviations: RT: radiotherapy; sHT: superficial hyperthermia
Interstitial
Hyperthermia
For direct thermal ablation of tumors by interstitial hyperthermia most frequently ferromagnetic rods or seeds are implanted into the tumor and excited by an alternating external magnetic field. For the treatment of glioblastoma this treatment modality has been shown to improve overall survival (Table 3). The percutaneous, minimal invasive interstitial thermal ablation by means of laser or high frequency current (radiofrequency or microwave fields) which are introduced through a fibre optic conductor (LiTT) or special HF needle electrodes (HiTT), is a new therapeutic modality
Table 3. Randomized
controlled
trials with interstitial
hyperthermia
Control
No. of Pts
Primary Endpoints
HT Better
Survival Benefit
Ref.
Head and neck, iRF + iHT breast, melanoma, others
iRT
184
Response
No
No
45
Glioblastoma
RT+iRT
79
2-year survival
Yes
Yes
46
Tumor Site
Experimental
RT+iRT+iHT
Abbreviations: iRF: interstitial radiofrequency; RT: radiotherapy; iRT: interstitial radiotherapy; iHT: interstitial hyperthermy
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Hyperthermia in Cancer Treatment: A Primer
for palliative and potentially curative therapy of primary liver tumors and liver metastases, especially if surgery is not acceptable or the tumors are not resectable. For RF thermo ablation multiple array needle electrodes (LeVeen needle) or hollow needle electrodes which can be perfused with physiological saline solution (Bechtold) are used. T h e needles are heated up with high frequency alternating current. The laser-induced thermotherapy was applied for the first time by Hashimoto et al^^ for the treatment of hepatic tumors and in the last years further developed by Vogel et al.^^ In a non-randomized trial Vogel et al, could show that in a total of 646 patients with 1.829 liver metastases up to 5 cm in diameter, mainly from colorectal (n = 1.126 metastases) and breast (n = 294 metastases) carcinoma by LiTT a local timior control rate of 97.3% after six months follow-up could be achieved.^^ The median siu^val rate of 39.8 months for colorectal liver metastases and 55.4 months for liver metastases of the breast are comparable with data from literature on surgical tumor resection. First results of the RF needle technique are comparable with LiTT or tumor resection.^ '^^ These methods for the non-surgical treatment of tumor patients, preferably for inoperable malignant nodules of the liver (hepatocellular carcinoma and metastases) is highly promising. Also other tumors from the brain, breast, thyroid, parathyroid, lung and bone, and malignant lymphomas can be treated by this method. T h e advantages of these methods are that they can be applied: • if surgery is not acceptable or the tumors are not respectable, • with low risk compared to surgery, • at different times repeatedly, and • on an outpatient basis and at lower costs. T h e perfused needle electrodes have advantages compared to other techniques: • increased thermolesion up to 40 to 50 mm diameter compared to 10 to 15 mm by increased conductivity around the needle • single needle system instead of multi array antennae systems • thin needles with about 2 mm diameters • ultrasound-guided application and • lower costs for the needles. In the future, magnetic fluid (f e, ferromagnetic nanopanicles) will be added to the therapeutic arsenal, which can be heated up by an external alternating magnetic field (magnetic field hyperthermia, MFH).^^ Very promising phase I/II studies have been closed.
Endocavitary Hyperthermia Via intraluminal placed antennas heat can be applied in organs such as the oesophagus, rectum, urethra (prostate), vagina, and the uterine cervix. Radiofrequencies and microwaves are most frequendy used for the endocavitary hyperthermia (Table 4). A survival benefit could be shown in most clinical studies. Regional
Deep
Hyperthermia
D e e p Hyperthermia w i t h Multi-Anteima Applicator Systems Tumours in the abdominal area can be heated up by arrays of antennas, which are arranged as dipole antenna pairs in a ring around the patient. T h e Sigma-60 applicator of the BSD-2000 system is a widely used applicator, which consists of four dipole antenna pairs. T h e novel multi-antenna applicator Sigma-Eye consists of 12 dipole pairs. Each antenna pair can be controlled in phase, amplitude, frequency and electric field to focus the heat in the area of the tumor. Frequencies in the range of 60-150 M H z are used for this technique. Two randomized phase III trials with multi-antenna applicators have been published up to the end of 2 0 0 3 and two trials are ongoing (Table 5). In two of these trials external radiotherapy was compared with combined radiotherapy and regional deep hyperthermia in the treatment of patients with primary cervix uteri (stage III) and primary or recurrent pelvic
173
Locore^onal Hyperthermia
Table 4. Randomized controlled and observational trials with endocavitary hyperthermia
Tumor Site
Experimental
Control
No. of Pts
OR [%] Control
OR [%] with HT
Survival Benefit Remarks
Ref.
Oesophagus
CT+HT
CT
40
19
41
No
RCT
28
Oesophagus
RT+HT
53
8
70
RCT
29
Oesophagus
RT + CT + HT
53
8
27
RCT
30
Oesophagus
RT + CT + HT
Rt + CT
66
59
81,2
Yes
RCT
47
Oesophagus
Ext. RT + MW+HT
ext. RT
66
Yes
OT
32
Rectum
RT+HT
RT
115
Yes
RCT
48
Yes
OT
52
Yes
RCT
53
Rectum
RT + CT + HT
RT + CT 36
Bladder, neoadj.
M W + CT
CT
52
CR:22
Bladder, adj.
M W + CT
CT
58
Rec: 64
Bladder, recurrent
hyperthermic perfusion + CT
10
CR:66 Rec: 15
Yes
RCT
54
90
Yes
OT
55
Abbreviations: RT: radiotherapy; CT: chemotherapy; MW: microwaves; RCT: randomized controlled trial; OT: open-label observational study; CR: complete response; Rec: recurrence after adjuvant treatment; neoadj.: neoadjuvant; adj.: adjuvant.
Table 5. Randomized trials on regional deep hyperthermia with antenna applicator systems
Tumor Site
Experimental Control
No. of Pts
Primary Endpoints
HT Better
Survival Benefit
Ref.
Cervix uteri (primary, stage III)
RT + DHT
RT
40
CR
Yes
No
18
Primary or recurrent pelvic (cervix, rectum, bladder)
RT + DHT
RT
361
CR, survival
Yes
Yes
19
Rectum (uT3/4)
RT + CT + DHT
RT + CT
>150
Disease-free survival
ongoing
Soft-tissue sarcoma (high risk)
CT + DHT
CT
>150
Disease-free survival
ongoing
Abbreviations: RT: radiotherapy; CT: chemotherapy; [DHT: deep hyperthlermia; CR: complete response
tumors. The number of complete response rates could be improved in both clinical studies and a survival benefit was demonstrated in one trial.
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Hyperthermia in Cancer Treatment: A Primer
Table 6. Randomized trials with RF capacitive coupled heating devices
Tumor Site
Experimental
Control
No. of Pts
OR[%] Control
OR[%] with HT
Survival Benefit
Cervix Cervix Cervix Cervix Colorectal Gastric Colorectal Bladder
RT+HT RT+HT RT+HT RT+HT RT+HT RT+HT RT+HT RT+HT
RT RT RT RT RT RT HT HT
65 66 37 40 24 293 71 49
46 35 53 50 10 35,5 36 48
66 72 83 85 43 57,6 54 83
n.d. n.d. n.d. Yes n.d Yes Yes
Ref. 57 58 59 60 26 33 61 62
Abbreviations: RT: radiotherapy; CT: chemotherapy; HT: hyperthermia; OR: overall response; Obs: open-label observational study; RCT: randomized controlled trial; n.d.: not defined
Regional Deep Hyperthermia with Radiofrequency Capacitiye-Coupled Electrodes Deep seated tumors can be heated by RF capacitive-coupled electrodes. For these systems mostly radiofrequencies in the range between 8 and 27 MHz are used. In the 1960s Le Veen developed a machine for induction of hyperthermia in tissue with radiofrequencies by capacitively-coupling of electromagnetic fields (EMF) at 13.56 MHz. It has been shown that RF capacitive heating devices can effectively raise the temperature of lung and liver tumors in humans (see for review ref 49), though van Rhoon failed to raise the temperature with capacitive plate applicators at 13.56 MHz in tumors of the pelvic area of patients above 40.9°C.^ This technique can even be applied for the treatment of brain tumors^^ RF Hyperthermia without Combination with Radio- or Chemotherapy Clinical trials with hyperthermia mostly have been performed in combination with radiation or antineoplastic agents (Tables 6, 7). But some first results from hyperthermia trials with capacitive coupled radiowaves with 13.56 MHz in the treatment of patients with primary tumors or metastases in the liver, lung, pancreas and brain without combination with radio- or chemotherapy are promising and should be validated with randomized trials. Lung Cancer In a prospective open-label observational study 63 patients with histological proven small cell lung cancer (n = 10) and non-small cell Itmg cancer (n = 53) at far advanced stage of disease have been treated with regional deep hyperthermia (DHT) with RF capacitive coupled short waves of 13.56 MHz.^^M patients were inoperable, refractory or at stage of relapse after prior surgery (30%), chemotherapy (46%), and/or radiotherapy (46%). Eighty-six percent of the patients presented with restrictive disorder of pulmonary ventilation. The median time between first diagnosis of inoperabel cancer or relapse (local and distant progression) and beginning of DHT was 3.9 months. Only 2 patients were treated vnth palliative chemotherapy 8.4 and 28.5 months after the start of DHT due to tumor-associated symptoms (e.g., pain). The median overall survival time (MST) of all patients was 14.0 months from first diagnosis of advanced lung cancer. From relapse after surgery or first diagnosis of inoperable stage of disease the MST was 10.3 months. The one- and two-year survival rates from progression of disease were 37% and 18%, respectively.
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Table 7. Non-randomized clinical trials with RF capacitive coupled heating devices Tumor Site
Experimental
Cervix Cervix Breast Breast Breast Colorectal Colorectal Colorectal Colorectal Gastric Gastric, adv. Gliomas "III, IV Liver (HCC) Liver (Met) Liver (HCC) Liver (Met) Lung (SCLC, SCLC) Lung (NSCLC) Lung (SCLN, NSCLC) Oesophagus Pancreas Pancreas Pancreas Sarcoma
C T + HT RT+HT RT+HT RT+HT RT+ HT RT+HT RT+HT RT+HT RT+HT RT + CT + HT CT+HT HT CT+HT HT + CT HT HT HT RT+HT RT+HT RT+ HT CT+HT HT HT RT+HT
Control
_ RT RT RT RT RT RT RT
CT CT
RT
-
No. of Pts 23 40 9 24 13 48 117 101 14 21 33 36 48 80 73 45 63 20 25 313 22 20 46 31
OR [%] with HT
Survival Better
-
52 80 100 83 92 11 69 71 100 89 39
43
56
-
-
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
OR [%] Control
_ 50 63 84 0 33 55 20
25
-
31 (SD51) 31 (SD27)
75 80 63 36
74
Yes Yes Possibly
Ref. 63 64 20 21 65 22 23 24 25 35 25 38 27 37 66 67 36 67 90 31 25 68 69 70
Abbreviations: HCC: hepatocellular carcinoma; Met: metastases: SCLC: small cell lung cancer; NSCLC: non-small cell lung cancer; HT: hyperthermia; RT: radiotherapy; CT: chemotherapy; adv: advanced; n.d.: not defined
Liver Metastases from Colorectal Cancer Patients at advanced stage of colorectal cancer with liver metastases have been treated with deep hyperthermia alone or in combination with chemotherapy (5-FU + FA). RF capacitive coupled electrodes with a radiofrequency of 13.56 MHz (RF-DHT) was applied.^^ Median total survival time of all 80 patients from first diagnosis of disease was 34.4 months, and from first diagnosis of progression (metastases or relapse) 24.5 months, and from beginning of first RF-DHT alone (n = 50) 16 months. Patients who received RF-DHT followed by chemotherapy in combination with hyperthermia (n = 30) survived at a median of 11 months. Survival rates of all patients (n = 80) from first diagnosis of progression (metastases or relapse) were 91 ± 3 % , 51 ± 6% and 31 ± 6 % at 1, 2 and 3 years, respectively. Pancreas Cancer In a retrospective analysis of the treatment of 20 patients with inoperable or relapsed cancer of the pancreas the treatment with RF-DHT (13.56 MHz) resulted in a median survival time of 12months.^^ In a prospective open trial with AG patients with far advanced (non-resectable, relapsed or metastatized) pancreatic carcinoma were treated with RF capacitive heating at 13.56 MHz. Median age at study entry was 62 years (range 38-82), median Karnofskys index 50% (range 30-90). Six patients suffered from jaundice and 10 showed ascites at study entry. The
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multimodal non-toxic treatment consisted of regional RF-deep hyperthermia (13.56 MHz, Synchrotherm, Italy) combined with complementary therapies (prteolytic enzymes, antihormonal therapy, etc.). The median overall survival of the patients was 10.5 months (range 2-76, mean 18 months) fromfirstdiagnosis of disease and 5 months from the beginning of the multimodal treatment. Most patients experienced essential improvement in quality of life (68% freedom from pain, 24% marked pain relief); 64% improved appetite (thereof 24% normal appetite) over a relatively long period of time, and reduction of jaundice and ascites. Gliomas The primary aim of this study was to define the feasibility of RF-DHT deep hyperthermia (RF-DHT) in the treatment of patients with progressive gliomas afi:er standard therapy and to estimate the effect on survival.^^ Between 09/97 and 09/02, 36 patients with gliomas (9 patients with anaplastic astrocytome WHO grade III, and 27 patients with glioblastoma multiforme WHO grade IV were treated with RF-DHT and Boswellia caterii, an inhibitor of leukotriene synthesis for inhibition of peritoneal edema. DHT was performed with a 13.56 MHz capacitive coupled RF-device. Patients with inoperable or subtotally resected and recurrent gliomas (WHO grade III and IV) with progression after radio- and/or chemotherapy and a Karnofsky Performance Score of > 50% were included. The study was designed as a prospective open-label, single-arm, mono-centred observational phase II trial. Primary endpoints were median survival time and survival-rate (Kaplan-Meier estimation). The survival was calculated on the basis of an intention-to-treat-analysis. Deep hyperthermia of brain tumors with RF capacitive hyperthermia at 13.56 MHz is feasible and without severe side effects. The RF-DHT-treatment is well tolerated and even patients at far advanced stages of disease can be treated. Complete and partial remission or retardation of tumor growth could be observed (Fig. 2). Prolongation of MST compared to historical controls and improvement of quality of live (EORTC QLQ-C30 questionnaires) is clinically significant. The median overall survival time of patients with anaplastic astrocytoma (WHO grade III) was 106±47 months [95% conficence intervall 14 to 197 months] and for patients with glioblastoma multiforme (WHO grade IV) 20±5 months [95% confidence interval 10 to 31 months]. The survival rates are listed in Tables 8 and 9.
Non-Thermal Effects The differences in the relative dielectric permittivity and magnetic permeability, the electric conductivity and the different ion distribution between normal and malignant tissue may explain different physical and physiological behaviour of the cells in an electric or magnetic field. It is possible that especially electromagnetic fields in the range between 1 and 30 MHz exhibit non-thermal antineoplastic effects on cancer cells by direct electromagnetic coupling, i.e., with the cell membrane, receptors or ion channels. Tumour growth inhibition has been shown also for interactions with alternating magnetic fields.^^ The application of low power electric fields ( 3 months, the median survival time is 726 days. For the 25 patients with plurirelapsing disease, the median survival time is definitely shorter: 427 days. Mesothelioma: in the patients who underwent cytoreduction and HAPP morbidity was 21%; 3 patients presented ARDS. The median survival time is 40 months. Sarcoma: the residts are not statistically valuable. Gastric Carcinoma: results in our casuistry were very poor, with a median survival time of about 6 months. Those results, combined to aggressiveness of the treatment brought us to quit this technique for patients with peritoneal carcinomatosis from gastric cancer. Anyway there are some Authors reporting good residts in the treatment with neo-adjuvant intent of gastric cancer with serosal invasion, without evident peritoneal carcinomatosis at the moment of operation.
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References 1. Averbach A M , Sugarbaker PH. Methodologic considerations in treatment using intraperitoneal chemotherapy. Cancer Treat Res 1996; 82:289-309. 2. Cavaliere F, Perri P, Di Filippo F et al. Treatment of peritoneal carcinomatosis with intent to cure. J Surg Oncol 2000; 7 4 ( l ) : 4 l - 4 . 3. De Simone M, Barone R, Vaira M et al. Semi-closed hyperthermic-antiblastic peritoneal perfusion (HAPP) in the treatment of peritoneal carcinosis. J Surg Oncol 2003; 82(2): 138-40. 4. Elias D, Antoun S, Raynard B et al. Treatment of peritoneal carcinomatosis using complete excision and intraperitoneal chemohyperthermia. A phase I-II study defining the best technical procedures] Chirurgie 1999; 124(4):380-9. 5. Gilly FN, Carry PY, Sayag AC et al. Regional chemotherapy (with mitomycin C) and intra-operative hyperthermia for digestive cancers with peritoneal carcinomatosis. Hepatogastroenterology 1994; 4l(2):124-9. 6. Glehen O , Sugarbaker PH, Elias D et al. Cytoreductive surgery combined with perioperative intraperitoneal chemotherapy for the management of peritoneal carcinomatosis from colorectal cancer. A multi-institutional study for 506 patients. J Clin Oncol 2004; 22:3284 7. Loggie BW, Fleming RA, McQuellon RP et al. Cytoreductive surgery with intraperitoneal hyperthermic chemotherapy for disseminated peritoneal cancer of gastrointestinal origin. Am Surg 2000 Jun;66(6):561-8. 8. Sugarbaker PH. Peritonectomy procedures. Ann Surg 1995; 221(l):29-42. 9. Sugarbaker P H , Ronnett BM, Archer A et al. Pseudomyxoma peritonei syndrome. Adv Surg 1996; 30:233-80. 10. Sugarbaker P H , Chang D. Results of treatment of 385 patients with peritoneal surface spread of appendiceal malignancy. Ann Surg Oncol 1999; 6(8):727-31.
CHAPTER 16
Hyperthermic Isolated limb Perfusion Michele De Simone* and Marco Vaira Abstract
I
n this overview we describe surgical procedures and hyperdierniic-isolated limb perfusion techniques for the treatment of in transit metastases from melanoma and sarcoma of the Umbs. We also briefly analyze the rationale of limb perfusion. The procedures are divided, for teaching piuposes, in three phases (surgical procedure, perfiision time, reconstructive phase). Finally we present a brief summary of our results obtained in the treatment of sarcoma and melanoma. We have performed 91 limb perftisions on 86 patients (5 patients have been treated twice). We obtained an objective response on 93.6% of patients with in-transit metastases from melanoma (45.5% presented a complete response and 48.1% a partial response). About sarcoma of limbs, we reached an objective response on 80% of patients. Side effects have been mild and not life threatening (e.g., edema of the limb, leukopenia and a compartment syndrome)
Introduction and Indications of Limb Perfusion The principles underlying the synergistic effects of cytostatic drugs and hyperthermia have been extensively described in previous chapters; briefly, we must emphasize that isolated limb perfusion offers two main pharmacokinetic advantages compared to systemic neoplastic treatment: (a) high drug concentration in the tumor area and (b) low systemic toxicity. Those important eff^ects have been clinically applied initially for in-transit metastases from melanoma. In this condition, melanoma disseminates through the whole limb from the initial site to the regional lymph nodes. This situation is not easily managed with conventional surgical or chemotherapeutic treatments but is still a locally-advanced disease. Isolated limb perfusion achieves good survival and quality of life results without the toxic effects of systemic chemotherapy. Limb perfusion consists of three phases: 1. Surgical ablative phase (node dissection and vessel preparation) 2. Perfusion phase 3. Reconstructive phase Basically, the treatment consists in isolating the limb from systemic circulation and perform limb perfusion with cytostatic drugs for 60 minutes with extracorporeal circulation (ECC) (Fig. 1). At the beginning of the perfusion phase, blood in the limb is heated; when the tumor site and whole limb reach an homogeneous temperature of 41.3-41.5°C, the drugs are injected into the perfusion circuit at high concentration with low side effects for the rest of the body. The pharmacological benefit is linked not only to high concentration but also to a continuous circulation of drugs in the limb that increases the cytostatic uptake from tumor. As previously *Corresponding Author: Michele De Simone—Department of Surgical Oncology "S. Giuseppe" Hospital, Empoli, Florence, Italy. Email:
[email protected] /
[email protected] Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.
Hyperthermic Isolated Limb Perfusion
209
Esophageal temperaturej detector T* = 36*-37n
\
i I
J ' i
IVescical temperature detector T* = 36° 37/
-2.
Venous line
•
Anticancer Drug
A
Abductors J ' ' = m a x 41,8\
^
pump (40-80 mi/l Vol. arto)
Oxygen 95% Heat
^^Ox^Q^n^ior
exchanger T* » m a x '
mm Thermostatic Sink with pump
Figure 1. Circuit scheme of isolated limb perfusion. described, hyperdiermia interacts with anticancer drugs at multiple levels. O n e direct effect is selective damage of tumor cells due to the decreased adaptability of tumor vessels to elevated tempeerature. This effect manifests as increased heat-entrapment in tumor areas compared to normal tissue, rendering hyperthermia a selective therapy. Another effect is related to increased permeability of tumor-cell membrane, induced by heat, that allows accumulation of drugs inside cancer cells. T h e intracellular concentration of drugs combined with heat shock causes impairment of D N A repair and disrupts D N A duplication. This action is especcially marked in tumor cells which have a high proliferative index. Hyperthermic isolated limb perfusion is indicated in following disease stages: • treatment of in transit metastases from melanoma (Figs. 2, 3, 4) • palliative treatment for melanoma when residual life quality is the target. • curative treatment of soft tissue sarcomas • limb-saving procedure and • neoadiuvant treatment to reduce bulky tumors and allow successive conservative surgery.
Surgical Procedure Ablative Phase Lower Limbs Iliac access is preferred for lower limbs. This access allows both iliac lymph node dissection and inguinal lymph no perfusion. Iliac node dissection is mandatory because in a non-negligible percentage of patients (30% in some studies) iliac-obturator lymph node disease can be present although the inguinal lymph nodes appear uninvolved. In selected cases (second perfusion or previous iliac lymphadenectomy), femoral access has to be utilizedd. Iliac access requires external iliac and obturator lymph node dissection with isolation of internal and external iliac vessels. Illiac perfusion, when groin lymphatic metastases are present, requires inguinal-crural and iliac-obturator nodes en-bloc dissection. In this casee, wide incision must be d o n e with section and reconstruction of the inguinal ligament. This perfusion access is accompanied by high morbidity (diastasis of w o u n d margins, lymphorrhhea, w o u n d
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Hyperthermia in Cancer Treatment: A Primer
Figure 2. Lower limb after limb perfusion.
Figure 3. Same limb of Figure 2. Thirty days after perftision.
Figure 4. Same limb of Figure 2. Sixty days after perftision.
Hyperthermic Isolated Limb Perfusion
211
Figure 5. Iliac vessels after lymph node dissection.
infection). Recently, to reduce this morbidity, we combine laparoscopic iliac obturator node dissection with an "open" crural dissection. After this procedure we cannulate the femoral vessels for perfusion. Going back to iliac access, when iliac vessels are isolated, we proceed towards the femoral vessels, with isolation, ligature and division of circumflex, epigastric and other main collateral vessels. This manoeuvre reduces drug leakage into the systemic circulation. In fact leakage is responsible both for systemic toxicity and ineffective treatment due to systemic hemodilution of the drug. After the isolation of collateral branches of iliac vessels, radical iliac vessel node dissection is performed to the common iliac biftircation (Fig. 5). Then follows isolation on elastic drain of the obturator nerve to preserve it from iatrogenic injury, and obturator fossa dissection (Figs. 6,7,8). In our experience, lymphadenectomy is easier if performed with an electrosurgical knife with bipolar forceps; moreover, the application of metal clips assures hemostasis and postoperative control of lymphorrhea due to lymph node dissection. When iliac access is not feasable or indicated, femoral access is done. We perform a longitudinal elliptical incision 2 cm medial and 2 cm caudal to the iliac anterior superior spine, that extends caudally to the apex of Scarpa's triangle. This is followed by a subcutaneous flap 1 cm in thickness extending to the muscular sheath which is removed en bloc with subcutaneous flap, lymph nodes and perivascular tissue. During preparation of the sheath and the lymphadenectomy, the saphenous vein and the deep circumflex and epigastric vessels are tied and dissected. Upper Limbs The patient is positioned with limbs abducted; we incise from the sternoclavicular joint to the insertion of the large pectoral muscle on the humerus. Then we lance the large pectoral sheath and separate the muscle fibers to reach the cephalic vein, that is tied at its origin. The following step is division of the small pectoral muscle. After this we isolate and tie collateral vessels of the axillary artery and vein to reduce drug leakage. When vessels are isolated, the node dissection is performed.
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Hyperthermia in Cancer Treatment: A Primer
Figure 6. Obturator nerve isolated on elastic string (blue string).
Figure 7. Obturator fossa nodes dissection.
Figure 8. Obturator nodes dissection completed.
Hyperthermic Isolated Limb Perfusion
213
Figure 9. Operating room, limb heater linked to double-sheet drape.
Perfusion Time During the preparatory phase, different temperature probes are positioned on the limb (skin, tumor site, adductor muscles, pretibial muscles) for monitoring the temperature of whole limb during perfusion. To keep temperature uniformity along the treated region, the limb is covered with a double-sheet drape linked to an air-heater which is activated at the beginning of the operation (Fig. 9). When the iliac-obturator dissection has been completed, we isolate on a tourniquet the internal and external iliac vessels. If internal vein isolation is difficult or dangerous, we isolate the common iliac vein. When vessels are completely isolated and prepared for cannulation, we heparinize thee patient (300 i.u. of heparin/kg); then we clamp external and internal iliac vessels (or common iliac vein) with tourniquets. On external vessels we perform an incision and place vascular catheters into iliac vessels. The position of catheters is very important: the tips of the catheters must be placed before the bifurcation of common femoral vessels, just under the inguinal ligament (Fig. 10). In femoral access, the femoral vessels are cannulated with the most cranial incision that can be done. The catheters are linked to the extracorporeal circulation system, consisting of an oxygenator, a heat exchanger, and a peristaltic pump. The extracorporeal circulation starts at a minimum flow that gradually increases until it reaches flow appropriate to limb volume (usually a flow between 50 and 70 ml/liter of limb). After some minutes of circulation, the superficial blood circulation of the limb is blocked by a rubber drain twisted around the root of the limb and anchored to the operating table (Fig. 11). When circulation is started, an important target is a constant level of solution inside the oxygenator; this level is directly related to a balance between systemic and isolated limb circulation. This is accomplished by equilibrating arterial and venous flow. Venous flow is determined by gravity so the critical flow that we can regulate is arterialflow.All these contrivances, combined with ligation of collateral vessels and proper balance between systemic circulation and limb flow of perfusion, limits drug leakage. Fieat comes both from the heat exchanger and the double-sheet drape wrapped around the limb until an homogeneous temperature of 41.5°C is reached. When we achieve optimal temperature and perfusion flow balance, we
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Hyperthermia in Cancer Treatment: A Primer
Figure 10. Cannulation of iliac vessels.
Figure 11. During perfusion, rubber tourniquet on the root of the limb. inject the drug (melphalan 10 mg/liter tissues for lower limb and 12 mg/liter for upper limb) into the circuit. The drug dose is divided in three parts administered in successive periods of time with an interval of 5 minutes. Perfusion is maintained for 60 minutes after drug injection. Leakage Monitoring During hyperthermic isolated limb perftision, the control of drug leakage into the systemic circidation is of vital importance in order to reduce systemic toxicity and maintain maximum drug concentration in the tumor. We measure leakage infusing in the ECC circuit with human serum albumin labelled with ^^'"Tc. The ^^"^Tc-labelled albumin is traced on a Geiger-Midler counter placed over the liver. Systemic leakage is quantitatively expressed as a percentage, whereby a 100% leak is considered to give a homogeneous distribution of the tracer in the body (Fig. 12). We consider a value of leakage < 5% acceptable. It is important not to overcome a threshold value of 10%.
Hyperthermic Isolated Limb Perfusion
215
Figure 12. Geiger-Muller counter for leakage measurement. The detection probe is positioned on the projection of the liver on the abdominal wall.
Reconstruction Time Once the perfusion phase is finished, the vessels are uncannulated and sutured. Heparin is neutralized by protamine solfate. When hemostasis is achiieved and, two drains are placed (Fig. 13). In iliac access, one drain is positioned near the vessels that underwent lymphadenectomy and the other in the subcutaneous space. In femoral access, transposition of the sartorius muscle on the femoral vessels is performed.This procedure is accomplished by separating the cranial head of the muscle from its bone insertion and transposing it to adjacent vessels. The transposed muscle is fixed on the inguinal ligament with nonresorbable suture. This surgical procedure supports subcutaneous tissu. It prevents dehiscence of the femoral vessels due to infection of the surgical wound and, at the very least, prevents inguinal hernia that may follow this kind of operation.
Clinical Experiences and Results From 1995 to December 2004, 79 patients with metastases from melanoma and 12 with sarcoma were treated with 91 limb perfusions; in fact, 5 patients with melanoma have been treated twice. The main features of the patients were: • Median age 65 years (35-81 range) • 69 patients were at stage III (26 on stage Ilia; 43 on stage IIIAB) • 10 patients at stage rV • 76 lower limbs; 3 upper limbs • All patients, except 5, were previously treated with surgical removal of metastases and/or with lymph node dissection • In 22 cases, more than 10 in-transit metastases were present. The follow-up and statistical elaboration of results confirmed the eff^ectiveness of isolated limb perfusion. N o perioperative mortality was observed.
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Hyperthermia in Cancer Treatment: A Primer
Figure 13. At the end of perfusion: iliac vessels sutured. Major morbidity as follows: • Edema of perfused limb (not only a complication, but an indirect sign of well-done perfusion), more or less, in each patient treated • 8 cases of transitory leukopenia and platelet disorder • 1 case of retroperitoneal hemorrhage treated conservatively Late complications inccluded a wound diastasis and a chronic ischemia. Our results are summarized in Table 1 and are comparable to those reported in the literature. The median survival has been evaluated in 48 months for patients at stage III and in 43 months for stage III AB. It is remarkable that even for patients in stages III and IV that demonstrated a partial response, we obtained adequate control of bulky disease and good quality of life until the death which generally occurred from systemic spread. Of those patients with sarcomas, 12 patients have been treated with isolated limb perfusion. There was no perioperative mortality and the side effects were: • edema of the limb • leukopenia (1 case) • a compression syndrome treated with decompressive fasciotomy.
Table 1. Clinical results in melanoma A) Complete response B) Partial response (Lesions Necrosis > 50%) C) Objective response (A+B)
36 (45.5%) 38(48.1%) 74 (93.6%)
Hyperthermic Isolated Limb Perfusion
217
For this kind of disease we obtained an objective response in 80% of treated cases, and we must emphasize that for almost all of them, limb salvage was possible, with improvement of quality of life.
References 1. Cavaliere R, Di Filippo F, Cavaliere F et al. Medical radiology, thermoradiotherapy and thermochemotherapy. In: Seegenschmiedt M H , ed. Clinical practice of hyperthermic extremity perfusion in combination with radiotherapy and chemotherapy. Springer-Verlag 1996; 2:323-345. 2. Krementz ET. Regional perfusion. Current sophistication, what next? Cancer 1986; 57:416. 3. SchrafFordt H , Koops BBR, Kroon FJ et al. Management of local recurrence, satellites and in transit metastases of the limbs with isolation perfusion. In: Lejeune FJ, ed. Malignant Melanoma. Pub McGraw-Hill 1994; 221-231 4. Hahn G C . Hyperthermia and cancer. Plenum Press, New York: 1982 5. McBride M , M c M u r t r e y MJ, Copeland EM et al. Regional chemotherapy by isolation-perfusion. Int Adv Surg Oncol 1978; 1:1-9. 6. Autier P. Epidemiology of Melanoma. In: Lejeune FJ, ed. Malignant Melanoma. Pub McGraw-Hill 1994; 1-7 7. Fraker DL, Coit D G . Isolated perfusion of extremity tumor. In: Lotze M T , ed. Regional Therapy of Advanced Cancer. Philadelphia: Pub. Lippincott-Raven, 1997; 333-349 8. Cavaliere F, Di Filippo, CavaUere F et al. Clinical practice of hyperthermic extremity perfusion in combination with radiotherapy and chemotherapy. In: Seegenschmiedt M H , ed. Medical Radiology, Thermoradiotherapy and Thermochemotherapy. Springer-Verlag, 1996; 2:323-345. 9. Clark AJ, Grabs PG, Parsons et al. Melphalan uptake, hyperthermic synergism and drug resistance in a human cell culture model for the isolated limb perfusion of melanoma. Melanoma Res 1994; 4(6):365-370. 10. Di Fihppo, Calabro A, GiannareUi D et al. Prognostic variables in recurrent limb melanoma treated with hyperthermic antiblastic perfusion. Cancer 1989; 63:2551-2561. 11. Bowers J, Copeland EM. Surgical limb perfusion for extremity melanoma. Surg Oncol 1994; 3:91-102.
CHAPTER 17
Intracavitary Hyperthermic Perfiision E. Dieter Hager* Introduction
D
irect intraperitoneal (IP) installation of anticancer agents for the treatment of patients with peritoneal carcinomatosis or sarcomatosis has pharmacological advantages compared to intravenous systemic therapy in terms of local drug concentrations (Table 1). The ratio of antineoplastic agent in the dialysate compared to the levels in the blood is 18-1,000 times greater, depending on the drug (Table 2).^ In a pharmacological study comparing intravenous versus intraperitoneal infusion of carboplatin the 24 hr platinum AUG in the peritoneal cavity was 280 times higher when carboplatin was administered with IP route.^ In addition, pharmacological studies showed, that IP infusion is a pharmacologically more reasonable route for systemic chemotherapy of carboplatin. The peritoneal space/plasma barrier provides dose-intensive therapy. The increase of local concentrations of antineoplastic drugs leads to improved response rates and significant increased recurrence-free survival in patients with peritoneal carcinosis. Recent studies showed in woman with stage III epithelial ovarian cancer, that integrated intraperitoneal chemotherapy into front-line therapy reported promising results with median survival times of 49-63 months and 2-year survival rates of 70-80%,^ compared to median survival of 41-52 months and 2-year survival of 65-70% in women who undergone optimal debulking surgery (Abbreviations: NFD: no evidence of disease; RFC: recurrence; vs: versus; R: residual
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Hyperthermia in Cancer Treatment: A Primer
Ovarian Cancer Epithelial ovarian cancer spread primarily by continuous extension of exfoliating cells into the peritoneal cavity and rarely systemically. Therefore, regional intraperitoneal chemotherapy might benefit these patients. Because antineoplastic agents can penetrate into tumor tissue in combination with heat much deeper than with normothermic perfusion, hyperthermia may have additional advantages. Several phase I and II trials of intraperitoneal hyperthermic chemoperfusion with platinum compounds after cytoreductive surgery have been reported or initiated. Wether the pharmacokinetic advantage of intraperitoneal hyperthermic chemoperfiision will translate into a clinical benefit has yet to be confirmed in randomized phase III trials. Colorectal Cancer After curative colorectal cancer resection, peritoneal carcinomatosis is the second most common site for recurrence. Intraperitoneal hyperthermic perfusion in the treatment of patients with peritoneal carcinomatosis with mitomycin C or cisplatin has been reported in numerous phase I and II trials.^^'^^ A prospective phase III trial comparing intravenous chemotherapy plus cytoreduction plus intraperitoneal hyperthermic chemoperfusion with intravenous therapy alone, in patients with peritoneal carcinomatosis of colorectal cancer, was initiated at the Netherlands Cancer Institute. The preliminary residts are promising. ^^ Mesothelioma^ Pseudomyxoma and Sarcoma Mesothelioma, pseudomyxoma, and sarcoma are relatively unresponsive to systemic chemotherapy. Intraperitoneal normothermic chemotherapy increases response rate, but early relapse is conunon in most patients. With a two-year survival rate of 80% recent results of a trial with intraperitoneal hyperthermic chemoperfusion of patients with peritoneal mesothelioma reported from the National Cancer Institute in the USA demonstrate superior antineoplastic activity and gives hope for new treatment options.^^ Case reports from Gilly et al Sugarbaker and Hager about good response on IPHC-treatment of patients with locally aggressive pseudomyxoma peritonei are very promising. Percutaneous Intraperitoneal Hyperthermic Chemoperfusion This conservative technique can be performed without abdominal surgery and can be repeated oftenly. Long-term treatment is possible by this method which is a great advantage compared to the periopertive I PHC. Therefore, this technique can be applied not only in patients with peritoneal carcinomatosis inunediately after cytoreductive surgery but also in patients after relapse, in chemotherapy resistant or refractory cases, far advanced stages of peritoneal carcinomatosis or sarcomatosis and in patients with recurrent malignant ascites. Ovarian Cancer Hager et al demonstrated a 65%, 39% and 16% survival rate of patients with progredient far advanced and chemotherapy resistant or refractory peritoneal disseminated epithelial and stromal ovarian cancer with primary stage III and IV disease from first IPHC-treatment at one-year, two-years and five-years, respectively.^^ The patients have been treated before entry to the trial with platinum- and/or taxane-based regimens at an average of 12.5 cycles. The overall median survival of these patients from first diagnosis of disease was 49 months. Considering that the expected 1-year survival rate of these patients would be smaller than 2%, this prospective, open-label trial gives evidence for a new very effective treatment option for patients with peritoneal disseminated disease. The advantage of this technique is, that it can be repeated as long as perfusion of the abdomen is possible. Also patients with stromal cancer respond to this percutaneous IPHC.
Intracavitary Hyperthermic Perfusion
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Gastrointestinal Cancer Also patients with peritoneal disseminated colorectal and gastric carcinoma can be treated in combination with mitomycin C, 5-FU, and/or oxaliplatin repeatedly safe and effectively with this treatment modality. The response in the case of colorectal cancer is less than in patients with ovarian or gastric cancer. Conclusion: Further trials have to proof evidence of efficacy of IPHC with this indications. Cervical Uterine Cancer Patients with peritoneal disseminated cervical or uterus carcinoma can be treated with intraperitoneal hyperthermic perfusion in combination with platinum compounds (cisplatin, carboplatin, oxaliplatin) or mitomycin (personal experiences). Conclusion: Preliminary case reports of the treatment of patients with chemotherapy resistant peritoneal disseminated cervical cancer are indicating good responses. Bladder Cancer Depending on stage and grade of the tumors 40-80% of patients with superficial bladder cancer will have recurrent tumors after intravesicular chemo- or immunotherapy with MMC, ADM, BCG, or IFN-a. Repeated intravesicular hyperthermic chemotherapy with perfiision technique via a catheter or by microwaves can be used to treat patients with recurrent superficial or muscle invading bladder cancer. In a neoadjuvant intravesicular hyperthermic chemotherapy Colombo et al^^ demonstrated pathological complete responses in GG% of the cases compared with 22% of the cases in the conventional with a normothermic intravesicular chemotherapy treated control group using a 915 MHz microwave source that directly heats the bladder walls within a temperature range of 42.5 to 45.5°C. The expected high recurrence rate of superficial bladder cancer after TUR of the bladder and normothermic intravesical chemotherapy with MMC could be reduced from 64% in the control group to 15.5% in the adjuvant combined hyperthermia with MMC treated group, as could be shown by Colombo et al in a randomized, multicentric study. Intravesicular hyperthermic chemoperfusion with MMC at temperatures above 42°C may prevent cystectomy in advanced, inoperable patients with bladder cancer as has been shown by Hager et al.^^ The recurrence rate and -frequency could be reduced substantially. Conclusion: Intravesical hyperthermic chemotherapy is safe and widely more effective than chemotherapy alone in ablating superficial or recurrent bladder cancer. It can be used also as an organ-sparing treatment of muscle-invasive bladder cancer.
Toxicity Perioperative Intraperitoneal Hyperthermic Chemoperfusion Postoperartive morbidity and mortality after intraperitoneal hyperthermic chemoperfiision (IHCP) relate mainly to the extent and duration of surgery, and not to hyperthermic perfusion itself Extensive cytoreductive surgery followed by intraperitoneal hyperthermic chemoperfiision is associated with considerable rates of morbidity and mortality. In a study of morbidity and mortality rates following IPHC with MMC after intensive cytoreductive surgery of patients with colorectal cancer the postoperative mortality rate was 5% and complications were noted in 35% of patients.^ Mainly digestive fistula, prolonged ileus, and pleurisy have been reported."^^ In some studies comparable side effects have been described as with cytoreductive surgery alone. More experience may reduce the risks of treatment. Percutaneous Intraperitoneal Hyperthermic Chemoperfusion Percutaneous intraperitoneal or intravesicular hyperthermic chemotherapy is feasible, safe and exhibits much less toxicity than systemic chemotherapy.^"^ In 1.8% of the treatments, peritoneal dismrbances with symptoms of subileus were observed and in 5% peritoneal irritations.
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depending on dosage of antineoplastic agents. Most adverse effects were due to W H O grade 1 and 2 nausea and vomiting. This treatment can be repeated ofteniy without remarkable increased toxicity. Up to 30 treatments could have been performed in patients with peritoneal carcinomatosis and sarcomatosis.^^ Limitations for this method are extended adhesions or massive tumor burden.
Conclusions Excellent survival in patients with peritoneal carcinomatosis and sarcomatosis can be achieved by intraperitoneal hyperthermic chemoperfusion with or without optimal debulked cancer. Hyperthermia may increase response rates, reduce resistance to antineoplastic agents and increase quality of Ufe and survival. Most of the studies are phase I and II trials. Some of these studies suffer from methodological flaws with respect to sample size, inclusion of different tumor types, absence of clearly defined endpoints and statistical estimations. Some of the studies have been performed as randomized controlled phase III trials and some phase III trials are ongoing. IPHC is one of the most promising new cancer treatment modalities at this time. Well designed randomized studies should be performed to establish the role of this relatively well tolerated technique in thefixturetreatment of peritoneal disseminated malignant diseases.
References 1. Markmann M. The role of intraperitoneal chemotherapy in ovarian cancer. Cancer Treat Res 1994; 70:73-82. 2. Miyagi Y, Fujiwara K, Fujiwara M et al. Intraperitoneal (IP) infiision is a pharmacologically more reasonable route for systemic chemotherapy of carboplatin. A comparative pharmacolokinetic ananlysis of platinum using a new mathematical model after IP vs. IV infiision of carboplatin. A Sankai Gynecology Study Group (SGSG) study. Proc Am Soc CHn Oncol 2002; 21:2167. 3. Rothenberg ML, Liu PY, Wilcznyski S et al. Excellent 2-year survival in w o m e n with optimally-debulked ovarian cancer treated with intraperitoneal and intravenous chemotherapy: A S W O G - E C O G - N C I C study (S9619). Proc Am Soc CHn Oncol 2002; 21:809. 4. Hahn G M , Shiu EC. Effect of p H and elevated temperatures on the cytotoxicity of some chemotherapeutic agents on Chinese hamster cells in vitro. Cancer Res 1983; 43:5789-5791. 5. Hahn G M . Potential for therapy of drugs and hyperthermia. Cancer Res 1979; 39:2264-2268. 6. Teicher BA, Kowal C D , Kennedy KA et al. Enhancement by hyperthermia of the in vitro cytotoxicity of mitomycin C toward hypoxic tumor cells. Cancer Res 1981; 41:1096-1099. 7. O h n o S, Siddik Z H , Baba H et al. Effect of carboplatin combined with whole body hyperthermia on normal tissue and tumors in rats. Cancer Res 1991; 51:2994-3000. 8. Rietbroek RC, van de Vaart PJM, Haveman J et al. Hyperthermia enhances the cytotoxicity and platinum-DNA adduct formation of lobaplatin and oxaliplatin in cultured SW 1573 cells. J Cancer Res CHn Oncol 1997; 123:6-12. 9. Zakris EL, Dewhirst M W , Riviere JE et al. Pharmacokinetics and toxicity of intraperitoneal cisplatin combined with regional hyperthermia. J CHn Oncol 1987; 5:1613-1620. 10. Dogramatzis D, Nishikawa K, Newman RA. Interaction of hyperthermia with bleomycin and liblomycin: Effects on C H O cells in vitro. Anticancer Res 1991; 11:1359-1364. 11. Jacquet P, Averbach A, Stuart OA et al. Hyperthermic intraperitoneal doxorubicin: Pharmacokinetics, metabolism, and tissue distribution in a rat model. Cancer Chemother Pharmacol 1998; 41:147-154. 12. Katschinski D M , Robins HI. Hyperthermic modulation of SN-38-induced topoisomerase I D N A cross-linking and SN-38 cytotoxicity through altered topoisomerase I activity. Int J Cancer 1999; 80:104-109. 13. Kutz ME, Mulkerin DL, Wiedemann GJ et al. In vitro studies of the hyperthermic enhancement of activated ifosfamide (4-hydroperoxy-ifosfamide) and glucose isophosphoramide mustard. Cancer Chemother Pharmacol 1997; 40:167-171. 14. Haveman J, Rietbroek RC, Geerdink A et al. Effect of hyperthermia on the cytotoxicity of 2'-2'-difluorodeoxycytidine (gemicitabine) in cultured SW1573 cells. Int J Cancer 1995; 62:627-630. 15. Dumontet C, Bodin F, Michal Y. Potential interactions between antitubulin agents and temperature: Implications for modulation of multidrug resistance. Clin Cancer Res 1998; 4:1563-1566.
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16. Yamauchi N , Watanabe N , Maeda M et al. Mechanism of synergistic cytotoxic effect between tumor necrosis factor and hyperthermia. Jpn J Cancer Res 1992; 83:540-545. 17. Song C W , Lin JC, Lyions JC. Antitumor effect of interleukin l a in combination with hyperthermia. Cancer Res 1993; 4:34-39. 18. Hager E D , Dziambor H, Strama H et al. Intraperitoneal hyperthermic perfusion (IPHP) chemotherapy of patients with peritoneal disseminated drug resistano ovarian cancer. Southern Med J 1996; 89:143. 19. Spratt JS, Adcock RA, Muscovin M et al. Clinical delivery system for intraperitoneal hyperthermic chemotherapy. Cancer Res 1980; 40:256-260. 20. Koga S, Hamazoe R, Maeta M et al. Prophylactic therapy for peritoneal recurrence of gastric cancer by continuous hyperthermic peritoneal perfusion with mitmycin C. Cancer 1988; 61:232-237. 2 1 . Fujimura T , Yonemura Y, Fushida S et al. Coninuous hyperthermic peritoneal perfusion for the treatment of peritoneal dissemination in gastric cancers and subsequent second-look operation. Cancer 1990; 65:65-71. 22. Fujimoto S, Takahashi M, Okui K. A prospective study on combined treatment of intraperitoneal hyperthermic and surgery for patients with refractory gastric cancer. In: Taguchi T, Aigner KR, eds. Mitomycin C in Cancer Chemotherapy Today. Excerpta Medica, 1991. 23. Fujimoto S, Takahashi M, Mutou t et al. Successful intraperitoneal hyperthermic chemoperftision for the prevention of postoperative peritoneal recurrence in patients with advanced gastric carcinoma. Cancer 1999; 85:529-534. 24. Deraco M , Rossi CR, Pennacchioli E et al. Cytoreductive surgery followed by intraperitoneal hyperthermic perfusion in the treatment of recurrent epithelial ovarian cancer: A phase II clinical study. Tumori 2001; 87:120-126. 25. Sugarbaker P H , Gianola FJ, Speyer JL et al. Prospective randomized trial of intravenous vs. intraperitoneal 5-FU in patients with advanced primary colon or rectal cancer. Semin Oncol 1995; 12:101-111. 26. Sugarbaker P H . Treatment of peritoneal carcinomatosis from colon or appendiceal cancer with induction intraperitoneal chemotherapy. Cancer Treat Res 1996; 82:317-325. 27. Loggie BW, Fleming RA, McQuellon RP et al. Cytoreductive surgery with intraperitoneal hyperthermic chemotherapy for disseminated peritoneal cancer of gastrointestinal origin. Am Surg 2000; 66:561-568. 28. De Simone M, Aimone M, Izzo G et al. Peritonectomy and hyperthermic antiblastic peritoneal perfusion (HAT) for peritoneal carcinomatosis. J Exp Clin Cancer Res 1997; 16:356-357. 29. Glehen O , Mithieux F, Osinsky D et al. Surgery combined with peritonectomy procedures and intraperitoneal chemohpyerthermia in abdominal cancers with peritoneal carcinomatosis: A phase II study. J Clin Oncol 2003; 21:799-806. 30. Zoetmulder FA, van der Vange N , Witkamp AJ et al. Hyperthermia intra-peritoneal chemotherapy (HIPEC) in patients with peritoneal pseudomyxoma or peritoneal metastases of colorectal carcinoma; good preliminary results from the Netherlands Cancer Institute. Ned Tijdschr Geneesk 1999; 143:1863-1868. 3 1 . Park BJ, Alexander HR, Libutti SK et al. Treatment of primary peritoneal mesotherlioma by continuous hyperthermic peritoneal perfusion (CHPP). Ann Surg Oncol 1999; 6:582-590. 32. Hager ED, Dziambor H, Hohmann D et al. Intraperitoneal hyperthermic perfusion chemotherapy of patients with chemotherapy-reistant peritoneal disseminated ovarian cancer. Int J Gynecol Cancer 2001; l l ( S u p p l l):57-63. 33. Colombo R, Da Pozzo LF, Lev A et al. Neoadjuvant combined microwave induced local hyperthermia and topical chemotherapy versus chemotherapy alone for superficial bladder cancer. J Urol 1996; 155(4): 1227-3. 34. Colombo R, Da Pozzo LF, Lev a et al. Adjuvant microwave hyperthermia and mitomycin C versus mitomycin C alone for superficial bladder cancer. Eur Urol 1999; 35(Suppl 2). 35. Hager ED, Strama H , Hohmann D et al. Prevention of cystectomy of recurrent bladder cancer by intravesical hyperthermic perfusion chemotherapy (IVHP). Anticancer Res 1998; 18:4807-5006. 36. Jacquet P, Stephens A D , Averbach AM et al. Analysis of morbidity and mortality in 60 patients with peritoneal carcinomatosis treated by cytoreductive surgery and heated intraoperative intraperitoneal chemotherapy. Cancer 1996; 77:1037-1042. 37. McQuellon RP, Loggie BW, Fleming RA et al. Quality of life after intraperitoneal hyperthermic chemotherapy (IPHC) for peritoneal carcinomatosis. Eur J Surg Oncol 2001; 27:65-73. 38. Sugarbaker et al. Management of Peritoneal surface malignancy using intraperitoneal chemotherapy and cytoreductive surgery. Ludann Company, USA, 1998. 39. Fujimura T, Yonemura Y, Fujita H et al. Chemohyperthermic peritoneal perfusion for peritoneal dissemination in various intrabdominal malignancies. Int Surg 1999; 84:60-66.
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40. Beaujard AC, Glehen O, Caillot JL et al. Intraperitoneal chemohyperthermia with mitomycin C for digestive tract cancer patients with peritoneal carcinomatis. Cancer 2000; 88:2512-2519. 41. Cavaliere F, Di Filippo F, Botti C et al. Peritonectomy and hyperthermic antiblastic perfusion in the treatment of peritoneal carcinomatosis. Eur J Surg Oncol 2000; 26(5):486-491. 42. Pestieau SR, Sugarbaker PH. Treatment of primary colon cancer with peritoneal carcinomatosis: Comparison of concomitant vs. delayed management. Dis Colon Rectum 2000; 43:1341-1346. 43. Loggie BW, Fleming RA et al. Cytoreductive Surgery with Intraperitoneal Hyperthermic Chemotherapy for Disseminated Peritoneal Cancer of Gastrointestinal Origin. Am Surg 2000; 66:561-568. 44. Hager ED, Dziambor H, Hohmann et al. Intraperitoneal hyperthermic perfusion chemotherapy of patients with chemotherapy-resistant peritoneal disseminated ovarian cancer. Int J Gyn Cancer 2001; ll(Suppl l):57-63. 45. Elias D, Blot F, El Otmany A et al. Curative treatment of peritoneal carcinomatosis arising from colorectal cancer by complete resection and intraperitoneal chemotherapy. Cancer 2001; 92:71-76. 46. Witkamp AJ, de Bree E, Kaag MM et al. Extensive cytoreductive surgery followed by intra-operative hyperthermic intraperitoneal chemotherapy with Mitomycin-C in patients with peritoneal carcinomatosis of colorectal origin. Eur J Cancer 2001; 37:979-984. 47. CuUiford Att, Brooks Ad, Sharma S et al. Surgical debulking and intraperioneal chemotherapy for established peritoneal metastases from colon and appendix cancer. Ann Surg Oncol 2001; 8:787-795. 48. Verwaal V, Ruth S, Bree E et al. Randomized trial of cytoreduction and hyperthermic intraperitoneal chemotherapy versus systemic chemotherapy and palliative surgery in patients with peritoneal carcinomatosis of colorectal cancer. J Clin Oncol 2003; 21:3737-3743. 49. Shen P, Levine EA, Hall J et al. Factors predicting survival after intraperitoneal hyperthermic chemotherapy with Mitomycin C after cytoreductive surgery for patients with peritonad carcinomatosis. Arch Surg 2003; 138:26-33. 50. Scuderi S, Costamagna D, Vaira M et al. Treatment of pseudomyxoma peritonei using cytoreduction and intraperitoneal hyperthermic chemotherapy. Tumori 2003; 89(Suppl 4):43-45. 51. Rossi CR, Pilati P, Mocellin S et al. Hyperthermic intraperitoneal intraoperative chemotherapy for peritoneal carcinomatosis arising from gastric adenocarcinoma. Tumori 2003; 2(5):S54-S57. 52. Pilati P, Mocellin S, Rossi RC et al. Cytoreducitve Surgery combined with hyperthermic intraperitoneal intrapoperative Chemotherapy for peritoneal carcinomatosis arising from colon adenocarcinoma. Ann Surg Oncol 2003; 10(5):508-513. 53. Elias D, Pocard M. Treatment and prevention of peritoneal carcinomatosis from colorectal cancer. Surg Oncol N Am 2003; 12:543-559. 54. Cavaliere F, Peri P, Rossi CR et al. Indications for integrated surgical treatment of peritoneal carcinomatosis of colorectal origin: Experience of the Italian Society of Locoregional Integrated Therapy in Oncology, Tumori 2003; 89:21-23. 55. Rossi RC, Deraco M, de Simone M et al. Hyperthermic intraperitoneal intraoperative chemotherapy ager cytoreducitve surgery for the treatment of abdominal sarcomatosis. Cancer 2004; 100(9):1943-1950. 56. Glehen O, Kwiatkowski PH, Sugarbaker D et al. Cytoreducitve surgery combined with perioperative intraperitoneal chemotherapy for the management of peritoneal carcinomatosis from colorectal cancer: A multi-institutional study. J CHn Oncol 2004; 22(16):3284-3292.
CHAPTER 18
Whole Body Hyperthemiia at 43.5-44''C: Dream or Reality? Alexey V. Suvernev, Georgy V. Ivanov, Anatoly V. E£reinov and Roman Tchervov* Abstract
A
high level of body temperature (43°C) is needed for effective use of whole body hyperthermia. Such a high level hyperthermia can only be safely used taking into account a theory of developing post-aggressive hyperproteolysis.^'^ Besides the control of proteolysis, it is also necessary to apply total phentanyl anesthesia, high-frequency lung ventilation and a high rate of heating.^ Clinical application of this method allows inducing the apoptosis of malignant cells, decreasing the viral load in HIV and HCV-infected patients and also causing a general sanitary effect. Use of water immersion makes the technology noninvasive and "physiological**. Application of this whole body hyperthermia technology reduces ventilation time and complications.
Introduction It is known, that therapeutic opportunities of the thermal factor are used since ancient times. However, in the 20th century scientists and physicians have reached essential results in application of hyperthermia in treatment of oncological, immunological, viral and other diseases. Necessity of development and improvement of hyperthermic technologies was predetermined because of insufficient efficacy of commonly used surgical, pharmacological, immunobiological and other methods of treatment of dangerous (oncological, viral, immunological) diseases and detection of unknown clinical effects of a hyperthermia. Those clinical significant effects are: 1. Destruction of malignant cells due to induction of necrobiosis and apoptosis by the thermal factor. 2. Elimination of tolerance of malignant cells to chemotherapy 3. Potentiation of medical effects of chemotherapy in combination with hyperthermia. It allows to reduce dosages of chemotherapy, decreasing damaging effect to the healthy tissues and keeping antineoplastic activity. 4. Activation of immunomodulating effects of hyperthermia, increase of protective forces of the patient, which important and never occurs after chemo- and radiotherapy.
Necessity of High-Level Whole Body Hyperthemiia It is known that there are no universal solutions for technical realization of artificial hyperthermia and for choosing its rational temperature range. Different variants of local and whole •Corresponding Author: Roman Tchervov—Siberian Institute of Hyperthermia, Nagornaya 14a, lskitim-5, 633205, Russia. Email:
[email protected]. Hyperthermia in Cancer Treatment: A Primer^ edited by Gian Franco Baronzio and E. Dieter Hager. © 2 0 0 6 Landes Bioscience and Springer Science+Business Media.
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body hyperthermia (wave, immersion and perfusional) should not be opposed to each other. It is necessary to take into account the concrete cHnical situations to achieve the maximal medical effect. Below we summarize arguments which were taken into account at studying and applying a high level (extreme) hyperthermia (up to 44.0°C). First of all, it is important to reply: why is it necessary to achieve WBH above 43 "C? The answer to this question is obvious not only for oncology, but also for those areas of medical practice where a selective cell damaging effect of heat is required. In particular, it is actual for oncological, virological and allergological practice, when it is necessary to initiate a necrobiosis and apoptosis of malignant cells, to suppress the HlV-infection or to destroy para-proteins and pathological antibodies. Some inspiring results in this field have been already received. So E. Kano (1987) has established, that "... energy of activation of heat cell killing at temperatures from above 43.0''C and below 43.0 "C is equal to 150 kcal l[i and560 kcal l\i accordingly..." Thus, on the one hand it is possible "to examine" an organism of the cancer patient protractedly on its nonspecific resistance to temperature up to 4 r C during 1-3 hours per hope that cancer cells in his organism appear less steady against the increased temperature. On the other hand, in conditions of adequate anesthetic protection, rapidly provide the 43.0-43.5-44.0°C level of hyperthermia and to start the biological mechanism of apoptosis in cancer cells. We shall note, that doctor Kano with colleagues within the next 10 years repeatedly confirmed reliability of the phenomenon registered by them certainly accepted by us in attention. Also it is necessary to note, that Mathe G. on XXIVth Congress in Rome (September 2001) also has confirmed, that"... apoptosis ofcancer cells is started only at achievement oftemperature in 43.0''C..." The authority of these and other known scientists allows to consider that high level hyperthermia (above 43 °C) is basic for oncological practice. Moreover, application of whole body hyperthermia in an interval of 40-42"C is fraught with a potential danger of dissemination of malignant cells and stimulation of their growth.
Risk Factors of Whole Body Hyperthermia Over 43**C and Pathogenetic Substantiation of Their Overcoming It is known, that homoiothermic organisms ".. .are sheltered right at the threshold of thermal death..." Artificial realization of whole body hyperthermia even in an interval of 41.8-42.0°C is bound to the risk of development of dangerous complications. Those are: • Thermal shock • Brain edema • Acute circulatory insufficiency • Hepato-renal syndrome • Acute respiratory distress syndrome (ARDS) • Disseminated intravascular coagulation The probability of occurrence of the specified complications is especially great in patients with oncological pathology; in elderly and senile age, when hyperthermia application is compelled on a background of multiple organ failure and the general bad state of health. In this connection at the 32nd Congress in Okayama (1994)^^ it was noticed, that whole body hyperthermia up to 43"C is desired for clinical practice, but mortality reaches a level of 17%. However our experience in whole body hyperthermia over 43°C with more than 500 patients, who successftdly and repeatedly undergone this procedure with no complications and multiple organ failure, is a basis to assert about a basic opportunity of safe extreme hyperthermia. Below there are some pathogenetic positions by which we were guided during development and perfection of high level whole body hyperthermia (43.5-44.0°C).
Whole Body Hyperthermia at 43.5-44 "C: Dream or Reality?
37°C
Normal protein
229
43°C
Partial denaturation
37°C
Normal protein
Figure 1. The process of reversible temperature disintegration of protein structure.
It is known, that".. .during cell reactions to damaging agents, plasmic proteins undergo the reversible structural changes keeping nuclear composition invariable..." ^ Such changes are happen by turns of nuclear groups around the single bonds. The essence of conformational changes of proteins is in redistribution of binding energy which can result in breaks of existing and establishment of weak bonds supporting secondary, tertiary and quaternary structure of a protein molecule. ^"^ It is known, that the spatial structure of a protein molecule is supported by various forces. Hydrogen bridges are established between oxygen of carbonyl groups of one amino acid with the imine nitrogen of another (N-H 0=C). They basically support the secondary structure of protein molecules. Covalent disulfide bonds (-S-S-) can participate in architectonics of tertiary structure. Influence of hydrophilic and hydrophobic sites of a protein molecule on an arrangement of water in their nearest environment creates quaternary structure determining stability of a protein macromolecule. It is also known, that the temperature maximum of stability of the most of proteins is much lower than a temperature of their vital optimum. Hence, the rise of temperature over an optimum should reduce stability of the protein molecules. And, the rise of temperature is more intensively destabilizing the periphery, the most reactive parts of macromolecules which are remote from an internal hydrophobic nucleus globule. Process of denaturation is more or less a complete destruction of quaternary, tertiary and secondary structure without hydrolytic splitting of peptide bonds. Increased temperature, in comparison with others well-known denaturation agents (urea, alcohol, acetamide) is the most universal "destructor" of macromolecular structures. Certainly, increasing a temperature, depending on its level and time of exposure, macromolecule changes occur step-by-step: from light disturbances of a stereochemical configuration to the formation of more or less chaotic clews of polypeptide bonds with a complete loss of function (Fig. 1). If the disturbances in macromolecular structure are incomplete, the termination of heating and decrease of temperature to an initial level enables renaturation. Restoration of initial, native structure and renewal of function is considerably longer than damage and depends on both a depth of reversible denaturation and the type of protein macromolecule. In our experience, after heating the patient up to 44°C, a process of functional restoration of various protein structures lasts from 2 until 8-16 hours. In this period, meeting the certain conditions, the structure and function of protein macromolecule are gradually restored. However, the given pattern has an exception relevant to the unique enzyme trypsin. It is known, that this enzyme with rather simple structure and small molecular weight, can completely
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37°C
43X
37X
Products of proteolytjc destruction of proteins causing severe intoxication
Trypsin
Figure 2. Participation of trypsin in hyperthermic proteolysis. restore its structure and ftinction within 10-15 min. after heating up to 43-44°C! Thus, a dangerous pathogenetic situation appears. A huge amount of partially denaturated proteins (substratum) and an increase in active, nonspecific exo-endoproteinase (trypsin). Not without reason, Lehninger^^ and Szent-Gyorgyi compared it to a "spring" or a "zipper". Proteinases also are denaturising agents. They destruct not only complicated structures but also even peptide bonds, breaking a primary structure of protein. The high activity of proteolysis in an overheated organism causes accumulation of a plenty of oligopeptides inducing endotoxemia. In other words, for many protein structures: the high temperature causes partial denaturation, and proteolysis destroys macromolecules (Fig. 2). In our opinion, a pathogenetic pattern mentioned above, is the one of key positions that should be taken into account for safe performance of extreme whole body hyperthermia. We established, that the phenomenon of hypertrypsinemia is a typical, nonspecific reaction of an organism to any stress and shock (Fig. 3). The data given in Figure 3, shows that high trypsin activity in blood of experimental animals was registered within 1 hour after any stress. We revealed, that the source of trypsinemia are the zymogenic granules of the pancreas, from which the shock enzyme "evades" into the bloodstream and causes a hyperproteolysis and endotoxemia. Hence, at anaesthetic management of high-level artificial hyperthermia is necessary to prevent hypertrypsinemia and to control proteolysis.
What Are Possible Ways to Suppress the Activity of Trypsin during Hyperthermia? It appears to be a problem because polyvalent proteinase inhibitors, commonly used in clinical practice, have a protein nature and denaturise at high temperature. In other words, the target was an alternative way of proteinase inhibition. Searching for a pharmacological preparation, capable to inhibit the activity of trypsin, the attention was drawn to clinical application of formaldehyde, described in V.V. Kovanov's works (1980-1982).^"^ It was established later, that formaldehyde blocks the action of proteolytic enzymes of trypsin type and can essentially reduce a proteolytic activity of the blood. As intravenous introduction of formaldehyde is not permitted by the pharmacopoeia, an alternative variant of its application was found. It lies in the application of hexamethylenetetramine which is commonly used in medical practice. This preparation introduced to the blood can split into formaldehyde and anmionia, on condition that liver and kidneys are ftmctioning normally.
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Blood level of trypsine in rats (MED) 0.0
2.0
4.0
6.0
8.0
10.0
12.0
Physical training Anaphylaxia Imnrtobilisation Hyperthermia Ishaemlareperfusion stress 30* occlusion of V, Portae
Haemorrage Burn Pyrogenal Trauma Adrenaline
Figure 3. Activity of trypsin in blood of rats at 1 hour after aggression.
Considering the above arguments, hexamethylenetetramine can be validly used in patients with a hyperactivity of trypsin. Using this preparation a relevant decrease of trypsin activity from levels of 2.34-9.72 MED to 0.2-0.3 MED was noted. The optimum doze of hexamethylenetetramine (80 mg/kg) and its half-time excretion (4-6 hours) were found empirically using recommendations of the pharmacopoeia. It is a very important point of a problem, taking into account an essential circumstance: most frequently antiproteinase preparations used in medical practice (Trasilol, Contrical, Gordox etc.) are ineffective in trypsinemia and absolutely not effective at high temperature.^^ It happens because the specified polyvalent trypsin inhibitors have protein structure and are subjected to disintegration by temperature. On the contrary, the formaldehyde formed in blood at hydrolysis of hexamethylenetetramine, keeps the antiproteinase properties even at 44°C. Thus, one of the basic conditions of a safe whole body extreme hyperthermia (> 43 °C) was met by a maximal suppression of proteolytic activity in blood. This became possible in view of
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Figure 4. A general view of aaive immersion-cx)nveaional heating of the patient. the concept about a negative role of postaggressive trypsinemia and the finding of an alternative way to inhibit the trypsin activity.
Technological and Anaesthetic Features of Whole Body Severe Hyperthermia To achieve a condition of controlled whole body extreme hyperthermia, we use a simple variant of immersion-convectional for physical heating of a patient s body. Patient is under general anaesthesia, heating with a water, warmed up to 44.0-47.0°C. The process of active physical heating is carried out in a special bath, for example "CHIRANA'' (Fig. 4). The patient's body and extremities immersed in warmed water except the head. No cranio-cerebral hypothermia is performed during the process of heating. Process of active heating should be fast enough (0.2-0.5''C/min.). Registration of the body temperature of the patient is made with thermal sensors, installed in a gullet. Process of heat transfer is reached by the relevant peripheral vasodilatation and hyperkinetic reaction of circulatory system. That provides equalization of a temperatm-e gradient between peripheral and the central organs. After achievement of a necessary level of hyperthermia, the process of heating stops and the patient is takenfiroma bath. The further process of normothermia restoration occurs passively and usually lasts 35-45 minutes. The specified method of whole body hyperthermia is achieved under conditions of special anaesthetic protection, whose goal is prevention of probable complications. To avoid such complications it is necessary to meet a number of indispensable conditions: 1. Special preprocedural preparation 2. Total intravenous anaesthesia
Whole Body Hyperthermia at 45.5-44"C: Dream or Reality^
235
Figure 5. Disappearance of multiple melanoma metastases in liver. 3. Control of proteolysis 4. Application of high-frequency artificial lung ventilation 5. Maintenance of high rate of active heating 6. Special monitoring of homeostasis. Clinically relevant effects of whole body hyperthermia technology (43-44 "C), can be illustrated by the following facts: 1. Disappearance of multiple melanoma metastases in liver (Fig. 5). 2. Fast decrease of HIV-1 RNA plasma concentration in HIV-infected patient (Fig. 6). 3. Removal of abstinence syndrome and total elimination of physical addiction in patients with drug abuse (Heroin, Methadone). 4. Elimination of an allergen (chloropicrin) and circulating immune complexes from the plasma of a patient with atopic bronchial asthma (Fig. 7). 5. Normalization of lung function parameters in a patient with atopic bronchial asthma (Fig. 8).
500 000,
Figure 6. Fast decrease of HIV-1 RNA plasma concentration in HIV-infected patient.
Hyperthermia in Cancer Treatment: A Primer
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19,0
I before WBH I after WBH
15,0
10,0
5,0
A
0,0
300.0 before WBH I after WBH
E
200,0
•5
100,0
i
Figure 7. Elimination of an allergen (chloropicrin) and circulating immune complexesfromthe plasma of a patient with atopic bronchial asthma. Fast decrease of HIV-1 RNA in FIFV-infected patient after WBH (Fig. 6) gave us a good reason forftirtherinvestigations in thisfield.We conducted a small pilot study to reveal HIV-1 RNA dynamics after hyperthermia and it brought interesting resiJts.
Method The group of investigation included 10 patients with progressive viral load. Whole body hyperthermia with general anesthesia used to achieve patients core temperature of 43.0-43.7'*C. Each patient had four procedures of hyperthermia within 12 months. All patients were
Whole Body Hyperthermia at 45.5-44"C: Dream or Reality^
255
W before WBH 11 after WBH
Figure 8. Normalization of lung function parameters in a patient with atopic bronchial asthma. observed not less than 12 months after the first procedure. Three of those patients observed over 15 months. One patient observed 33 months.
Results Within 12 months viral load in all patients decreased to less than 10% of initial level. Four patients with longer follow-up had viral load less than 7% of initial level at the moment of last observation. One patient with 33 month observation had viral load 0.9% of initial level (Fig. 9). None of the patients used known antiretroviral chemotherapy.
Discussion Such stable long-term depression of viral load in investigation group brought up a number of questions which have no answers yet. It s still unclear why viral load is not rising when absolute C D 4 count increases to normal values. At the same time stable persistence of minimal viral load is also not understood. There is an impression that those patients are quite similar to true long-term "nonprogressors*'. If it will be proven, there will be a reason to conclude that hyperthermic treatment with high level whole body hyperthermia (over 43'C) converts HIV-progressors to nonprogressors.
Conclusion 1. Until now the relevant negative activity of proteolysis wasn't taken into account when dealing with the problem of endotoxication caused by extreme thermal exposure. Developing a trypsin inhibition method during thermal exposure can prevent endotoxication and significantly decrease hyperthermic complications. 2. Premature conclusions about inefficiency of hyperthermia in HIV-infection stated in 90s formally "closed" research work in that direction. Development of intensive and safe hyperthermia can possibly resurrect interest in thermomedicine for HIV/AIDS.
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Figure 9. Long-term dynamics ofHIV-1 RNA after hyperthermia. Decrease ofviral load after extreme whole body hypenJiermia.
References 1. Suvernev AV, Meshalkin EN, Sergievskii VS et al. Trypsinemia in stress reactions of an organism. Novosibirsk, Nauka: Siberian branch 1982. 2. Suvernev AV. Primary prophylaxis allows the portabiUty of general hyperthermia up to 44°C in dogs. Human adaptation and primary prophylaxis. Novosibirsk, 1986:1:117-118. 3. Suvernev AV, Gleim GK. Pancreatogeneous intravascular hyperproteolysis. New methods of diagnostics, treatment and prevention of diseases. Novosibirsk, 1987:200-201. 4. Suvernev AV, Rodionov SU, Plyaskin KP et al. Application of whole body hyperthermia in treatment of oncological pathology Organization of palliative care and treatment of severe forms of malignancies. Moscow, 1995:113-114. 5. Suvernev AV. An experience of whole body hyperthermia in treatment of oncological pathology. Visceral Tumors. Tomsk, 1995:97-99. 6. Suvernev AV, Gleim GK, Takach GL et al. Problems of whole body hyperthermia over 42°C. Hyperthermia in Oncology. Minsk, 1990:115. 7. Suvernev AV, Patoka AV, Gleim GK. Experience of whole-body hyperthermia for treatment of oncological patients. Materials of the 2nd Far-eastern International Congress of Multimodal Cancer Treatment. Vladivistok, 1994:45. 8. Kano E. Fundamentals of thermochemotherapy of cancer. Can No Rinsho 1987; 33(13):l657-63. 9. Mathe G. From mechanisms of action to relation indications of hyperthermia at 40°C of 43°C in cancer treatment. XXIV International Congress on CHnical Hyperthermia. Rome, 2001. 10. Proc. 32nd Annual Congress of Japan Society of Cancer Therapy Meeting, Japan: Okayama, 1994. 11. Nasonov DN et al. Biological reaction on external influence. Moscow-Leningrad, 1940. 12. Koshland DE. Federat. Proc 1964; 23(3 pt l):719-726. 13. Lehninger L. Biochemistry Moscow, 1974. 14. Kovanov W et al. Proc. of the 1st Moscow Medical Institute Moscow, 1982. 15. Litvinov IV. Choosing the method of ventilation at whole body hyperthermia in cancer patients. Novosibirsk, 1998:19, (Ref Type: Dissertation). 16. Stebbing J, Gazzard B, Kim L et al. The heat-shock protein receptor CD91 is up-regulated in monocytes of HIV-1-infected "true" long-term nonprogressors. Blood 2003; 101(10):4000-4.
CHAPTER 19
Extreme Whole-Body Hyperthemiia with Water-Filtered Infrared-A Radiation Alexander von Ardenne* and Holger Wehner Abstract
T
he testing of various methods to realise extreme whole-body hyperthermia (eWBH) finally led to the utilisation of radiative systems. Among these the application of water-filtered infrared-A radiation (wIRA) distinguished itself by its high penetration, all the way into the capillary bed of the skin. With wIRA the interfering infrared-B and the infrared-C is eliminated from the heat radiation. Thus a clearly higher radiation power can be applied at a tolerable level than by applying unfiltered heat radiation. In two independent phase I clinical studies the high tolerance of eWBH (approx. 42°C/60 min) was proven in the scope of the so-called systemic cancer multistep therapy (sCMT) while applying wIRA. First proof of the retardation of tumour progression could be carried out by a retrospective observation study of over 490 sCMT treatment courses of cancer patients with various different tumour entities at an advanced stage. A phase I/II clinical study on the treatment of 19 patients with metastatic colorectal cancer with sCMT and wIRA in combination with chemotherapy suggests that sCMT may enhance the effect of chemotherapy. In a prior study on the treatment of patients with metastasised adenocarcinomas only 3/19 patients remained in progression.
Introduction Lamps with water-filtered infrared radiation for therapeutic applications had already been produced at the beginning of the 20th century.^ In 1931 A. Bachem^ could already prove that skin only featured a high transmission depth or penetration for heat radiation from the spectral region of infrared-A (760 nm ... 1,400 nm). In the spectral regions of infrared-B (1,400 nm ... 3,000 nm) and the close infrared-C (3,000 nm. 1 mm) on the other hand, the transmission is very slight which leads, at an application of a high radiation power, to an overburdening of the skin (Fig. 1). As standard electric light bulbs and halogen lamps radiate approx. half of their energy in the spectral region of infrared-B and -C, this share of radiation has to be eliminated. This is possible with a water layer, which placed in the ray path, acts like an edge filter and eliminates the infrared-B and -C share from the heat radiation (Fig. 2). If a water layer of appropriate thickness is placed, for example, in front of a halogen lamp with approx. 2600°K filament temperature, this results in the spectral radiation distribution displayed in Figure 3. In this way heat radiation is generated with a spectral distribution, the focus of which is in the area of highest transmission of the skin. This is defined as water-filtered infrared-A radiation (wIRA). *Corresponding Author: Alexander von Ardenne—Von Ardenne Institute, Zeppelinstr. 7, D-01324 Dresden, Germany. Email:
[email protected] Hyperthermia in Cancer Treatment: A Primer^ edited by Gian Franco Baronzio and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.
Hyperthermia in Cancer Treatment: A Primer
238
.9 1.0-
'liilWilifiltft' ^ * "
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f
to;
1 « | ; 1500 1800 2100 2400 2700 3000nm wavelength
Figure 1. Transmission of electromagnetic waves in the spectral region of infrared-A, -B and -C through a human skin layer of a thickness of 1.4 mm. U. Henschke^ provided an early quantitative substantiation for the tolerance of water-fdtered infrared-A radiation. He publicised that for long-term application the maximum toleration of radiation intensity for water-filtered heat radiation was double as high as for standard bulb radiation. Unfortunately this knowledge did not lead to a wider range of application in the following decades in clinics. For patients with advanced and metastasised malignant tumours, only a systemic therapeutic approach is adequate. Such a prerequisite is, among others, offered by the extreme whole-body hyperthermia (eWBH) with target temperatures around 42°C, which is usually combined with adapted chemotherapy today. After ten years of research and development in the field of radio frequency hyperthermia at 27 MHz to bring about a noncontact whole-body hyperthermia in combination with an increase of temperature locally^ (Fig. 4), M. von Ardenne 1985 recognised the limits of the application at that time: The development of hot spots, impossibility of temperature mapping, high demand made on technology and shielding. A way out of this dilemma was offered by the idea of thermal treatment of the outer body shell. As even according to the law of thermo-dynamics, it is only a question of time until the innermost point of an entity that is surrounded by an isothermal outer body shell is at the same temperature level as the outside body shell itself. To achieve this, a thermal source is required, the radiation of which only penetrates the millimetre area of the skin and is then almost completely absorbed. If this is successful, simple two-step temperature monitoring is possible by which the temperature of the skin (outside body shell) and, on the other hand, the body core
Water \
!i500
I
\
I
I
I
1800 2100 2400 2700 3000 nm wavelength
Figure 2. Transmission of electromagnetic waves in the spectral region of infrared-A, -B and -C through a water layer of defined thickness.
Extreme Whole-Body Hyperthermia with Water-Filtered Infrared-A Radiation
^ a> 1.0
259
Visual
Halogen Lamp + Water Filter T 1 1 r 1500 1800 2100 2400 2700 3000 nm wavelength good penetrallon of sidn + absorption of H20«specHic frequencies
Figure 3. Radiation spectrum of a halogen lamp after passing a water filter (water-filtered infrared-A radiation). temperature, is observed during hyperthermia treatment by inserting a temperature sensor into a body opening, e.g., the rectum.
Technical Realisation of Water-Filtered Infrared-A Radiation Inspired by E. Braun, in 1985 M. von Ardenne once again took up the principle of water-filtered infrared-A radiation whereby he was able to implement the idea of heating up the body shell. The water-filtered infrared-A radiation penetrates into the capillary bed of the corium, where the direct transmission of heat into the blood circulation system takes place (Fig. 5) and thus an equalisation of the temperature takes place in the whole organism.
Figure 4. Radio frequency hyperthermia with a 27 MHz-SELECTOTHERM system for the noncontact combined whole-body and local hyperthermia with an applicator that can be focused on the tumour (1987).
Hyperthermia in Cancer Treatment: A Primer
240
1.0 (0
60H
o
40H
Log-Rank: p=0.44 Wilcoxon: p=0.87 chemotherapy + sCMT
chemotherapy alone
2
20i
•~T-
10
20
30
40
50 weeks
60
—r~ 70
80
90
Figure 9. Progression-firee survival in test and control group. Note: The graph does not represent a comparison of similar patient groups: all patients treated with sCMT plus ChT previously had not responded to three courses of conventional ChT. All patients treated with ChT alone had a PR after three courses. (From Hildebrandtetal.^5)
Extreme Whole-Body Hyperthermia
with Water-Filtered Infrared-A
Radiation
245
5- I
8 &S
8^i
Figure 10. Temperature/pulse - time chart of a therapy with water-filtered infrared-A radiation between fever range hyperthermia and extreme whole-body hyperthermia. Hyperthermia equipment I R A T H E R M 1000 (Therapy No. 2, Clinic Eubios Griinhain, Germany).
Even though this contribution primarily deals with whole-body hyperthermia, it should not remain unmentioned that the water-filtered infrared-A radiation is also suitable for the implementation of moderate hyperthermia or fever range hyperthermia. In this case the patients are treated with a "temperature dose" of 39°C/3 h up to 40°C/6 h. Currently research groups around J. Bull, and W. Kraybill are working in this field with unfiltered heat radiation, whereby the patients are heated in a chamber. The water-filtered infrared-A radiation in an open equipment design, on the other hand, allows for the one-sided heating of the patient - fever range hyperthermia. Nevertheless, a quick increase in temperature at good tolerance and controllability is given of the temperature level aimed at. The temperature/pulse time chart with a fever range hyperthermia above the aimed at temperature displayed in Figure 10, shows the good controllability of the hyperthermia with water-filtered infrared-A radiation. Furthermore, the heat radiation concentrated on the patient can be individually adjusted to spot and intensity.
Conclusions The tolerance of the water-filtered infrared-A radiation to realise a whole-body hyperthermia in the form of sCMT in clinical routine could be proven by phase I studies. Initial clinical results allow for the assumption that this method could intensify the effect of ChT, as could be displayed in the example of patients with metastatic colorectal cancer and adenocarcinomas refractory germ cell tumours with small case numbers. The still unsatisfactory situation of available data for the proof of effectiveness and first positive results are an encouragement to carry out higher-grade studies. It is still absolutely unanswered as to which extent this method could decrease the chance of formation of metastases if, for instance, implemented post-operatively.
References 1. Malten H . Die Lichttherapie. Munchen: Bergmann, 1926:40-60. 2. Bachem A, Reed CI. The penetration of light through human skin. Amer J Physiol 1931; 97:86-91. 3. Henschke U. Biologische und physikalische Grundlagen der Rot- und Ultrarotstrahlentherapie. Strahlentherapiel939; 66:646-662. 4 Wust P, Hildebrandt B, Sreenivasa G et al. Hyperthermia in combined treatment of cancer. Lancet Oncol 2002; 3:494. 5. Ardenne Mvon, Kriiger W. Combined whole-body and local hyperthermia for cancer treatment: C M T selectotherm technique. In: Gautheria M, Ernest Albert U, eds. Proc Sympos Biomed Thermology, Strasbourg 1981. New York: Allan Liss, 1982:705-713.
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6. Borchert R, Jubitz W. Infrarottechnik. Berlin: Verl Technik,1958. 7. MefFert H, Hecht HC, Gunther H et al. Biophysikalische Ergebnisse des Klinischen Tests der IRA-Therm-Hyperthermietechnik der 2. Generation ThermoMed 1990; 6:71-78. 8. Wust P, Riess H, Hildebrandt B et al. Feasibility and analysis of thermal parameters for the whole-body hyperthermia system IRATHERM 2000. Intl J Hyperthermia 2000; 4:325-339. 9. Ardenne Mvon. Principles and Concept 1993 of the systemic Cancer Multistep Therapy (sCMT). Strahlenther. Onkol 1994; 170:581-589. 10. Ardenne Mvon. Systemische Krebs-Mehrschritt-Therapie. Stuttgart: Hippokrates Verls^, 1997. 11. Steinhausen D, Mayer WK, Ardenne Mvon. Evaluation of systemic tolerance of 42.0''C infrared-A whole-body hyperthermia in combination with hyperglycemia and hyperoxemia: A phase-I study. Strahlenther Onkol 1994; 170:322-334. 12. Kerner T, Deja M, Ahlers O et al. Whole-body hyperthermia: A secure procedure for patients with various malignancies? Intensive Care Med 1999; 25:959-965. 13. loccit. 10: 219. 14. loccit. 10: 195 ff. 15. Hildebrandt B, Drager J, Kerner T et al. Whole-body hyperthermia in the scope of von Ardenne's systemic cancer multistep therapy (sCMT) combined with chemotherapy in patients with metastatic colorectal cancer: A phase I/II study. Intl J Hyperthermia 2004; 3:317-333. 16. Bremer K, Meyer A, Lohmann R. Pilot study of whole-body hyperthermia combined with chemotherapy in patients with metastasised pretreated progressive breast, ovarian and colorectal carcinomas. Tumordiagnostik and Therapie 2001; 22:115-120. 17. Hildebrandt B, Wust P, Loffel J et al. Treatment of patients with refractory germ cell tumors with whole-body hyperthermia and chemotherapy. In: ESHO 1999 September 1-4. Rotterdam, 1999:66. 18. web site of "Interdisziplinare Arbeitsgruppe Hyperthermie / lAH" (sub organisation of German Cancer Society), 2004. www.hyperthermie.org. Zentren / Suche nach Karzinomen. 19. Bull JMC, Nagle VL, Scott G et al. A phase I study of optimally-timed Gemcitabine + Cisplatin/ Interferon-a combined with long-duration, low-temperature whole-body hyperthermia. In: ESHO 2001 May 30 - June 2. Session V Verona, 2001:67. 20. Bull JMC, Glenna LS, Strebel FR et al. Update of a phase I clinical trial using fever-range whole-body hyperthermia + Cisplatin + Gemcitabine + Metronomic, low-dose Interferon-a. In: 9th ICHO April 20-24. St Louis Missouri 2004:68. 21. Kraybill WG, Olenki T, Evans SS et al. A phase I study of fever-range whole body hyperthermia in patients with advanced solid tumours: Correlation with mouse models. Intl J Hyperthermia 2002; 3:253-266.
CHAPTER 20
Meets of Local and Whole Body Hyperthermia on Immunity Gian Franco Baronzio,* Roberta Delia Seta, Mario D'Amico, Attilio Baronzio, Isabel Freitas, Giorgio Forzenigo, Alberto Gramaglia and E. Dieter Hager Around every tumor there is a patient—Blacky Natl Cancer Inst Mono^ 1972; 35:276.
Abstract
I
n this review, we summarized the historical and experimental basis of cancer immunity and the role of fever and of artificial elevation of temperature on immunity. The interactions of heat in vitro and in vivo on cytotoxicity of immune competent cells are discussed as their positive contribution on the various cancer immunotherapeutic strategies. Furthermore we have described the link existing among heat shock proteins, Toll like receptors and innate immunity justifying the use of temperature elevation for treating cancer. The disputed and life-threatening effect of local and whole body hyperthermia on metastasis is also reviewed.
Introduction The role of the immune system in eradicating malignant cells is not yet clarified; however spontaneous regression of some cancers has been demonstrated to be associated to the induction of fever and activation of immunity. ^'^ The crucial importance of fever in these regressions justifies the attempt to induce artificial thermal elevation of body temperature (hyperthermia) for mimicking natural fever effects on cancer.^'^
Tumor Immunity Background Tumor regression in vivo is mediated by a complex interplay between two main mechanisms: innate and adaptive immune response (Tables 1,2), that is involved with the immune recognition of cancer cells. Innate mechanism [involving soluble and cellular components, (Table 1)^'^^ may trigger inflammatory events in the tumor microenvironment and in presence of a local adequate cytokine combination (IL-2, IL-12, IL-18, IL-23), stimulate dendritic cells (DCs),^ the most specialized antigen presenting cells (APCs), to react against tumor specific surface antigens (TAAs).^"^' After engulfment by DCs, TAAs are presented to naive T cells associated to Major Histocompatibility complex (MHC).^^ Naive T cells activation occurs when the antigenic peptide-MHC complex interacts with the T Cell Receptor (TCR). However TCR receptor binding is not sufficient for a full activation ofT cells unless *Corresponding Author: Gian Franco Baronzio—Family Medicine Area, ASL-01 Legnano; Radiotherapy Unit, Policlinico di Monza, Via Amati 11, 20052 Monza (Mi), Italy. Office address: P.O.B. 5, 20029 Turbigo (Mi), Italy. Email:
[email protected] Hyperthermia in Cancer Treatment: A Primer, edited by Gian Franco Baronzio
and E. Dieter Hager. ©2006 Landes Bioscience and Springer Science+Business Media.
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Hyperthermia in Cancer Treatment: A Primer
Table /. Principal cellular and soluble antitumoral components of innate immune system • • • • • •
Dendritic cells Neutrophils Macrophages NK cells Cytokines Chemokines
• Complement • Fever • Defensins • Interferon producing cells • B cells • 7 6 ! cells
costimulatory molecules interact with the specific ligands on the surface of APC. The presence or absence of costimulatory signals like B7-1(CD80), B7-2(CD86) and CD40/CD40L), determines whether immune response becomes anergic or tolerant. CD40/CD40L is expressed transiently following TCR activation on the surface of CD4^ cells and it is a key molecule in mediating the activation of B cells and in controlling CDS T cells. Antigens can be associated to M H C I or MHCII class complexes and are presented by DCs to TCR of CD8^ and CD4^ T cells, respectively associated to the proper costimulatory molecules (Fig. 1). Both CD4^ and CDS"^ cells after activation and costimulation produce a series of cytokines that differentiate T- Helper (CD4+) lymphocytes in two subpopulations (TH 1, T H 2 cells). T H 1 cells produce IL-2, IFN-y, TNF-a and granulocyte macrophage colony stimulating factor (GM-CSF) that increase the activity of macrophages, and the expression of MHC class 1 molecules on the surfaces of CD8+ cells. T H 2 secretes another group of cytokines IL-4, IL-5 and IL-10, that induces naive B cells to produce antibodies. The shifting towards T H 2 pattern has recently been associated with an increased tumor metastasisation and a decreased survival in many human and animal neoplasia (Fig. 4). 12,14,17.18 CD8+, cytotoxic T cells (CTLs) are the major effectors of tumor regression; ^^ however CD4+T cells collaborate to their activation. Once activated CTLs do not need costimulation, since MHC-1-bound antigen is sufficient. For eliminating target cells (neoplastic cells) CTLs use three effector molecules: Perforins, Granzyme and Fas ligand. Associated to these killing mechanisms, CTLs secrete specific cytokines, such as: IFN-y, TNF-a andTNF-p. This pattern of cytokines plays an important role in the activation of macrophages which can exert a direct tumor cytotoxic or, conversely, stimulate tumor progression, depending on the tumor microenvironment.^^ Other cells morphologically and fiinctionally distinct, such as natural killer cells (NKs), macrophages and neutrophils, use pattern-recognition receptors and other cell-surface molecules to detect tumor cells directly. '^° Differently from T cells, NKs inhibit tumor growth in a MHC-non restricted manner. Frequently tumor cells (like stressed cells) express on their surfaces different glycoproteins (MICA and MICB) which ftmction as ligands for NKG2D receptors on NK cells. Once activated, these receptors stimulate NK cell activity.^^ By contrast, DCs use CD36 and (XvP5 int^rin to recognize and phagocytize apoptotic tumor cells. Apoptotic tumor cells in turn release heat shock proteins that, after specific interaction with CD91 receptors on DCs, induce their maturation: thus providing a tailored inunune response (see Figs. 1,4).^^
Table 2. Principal antitumoral components of adaptive immune system • • •
Dendritic cells Neutrophils Macrophages
• B Cells • CD4^ • CDB^
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249
INNATE ADAPTIVE IMMUNITY INTERACTIONS AND T-CELLS MEDIATED IMMUNE RESPONSE
iNNATE IMMUNITV'
ADAPTIVE IMMUNITY
» Inhibitory effect > Stimulatory effect
Figure 1. In this diagram the various mechanisms eUcited by stress for stimulating innate and adaptive immunity against cancer are illustrated. DCs: dendritic cells; CTL: cytotoxic T lymphocytes; TCR: T-cell receptor; MHC: major histocompatibility complex; Abs: antibodies; FRs: free radicals; TGFB: transforming growth factor-P; PGE2: prostaglandins of E2 type.
Tumor Microenvirontnent as Regulator of Cancer Immunity and Immune Evasion The switch to an angiogenic phenotype is a fundamental determinant of neoplastic growth and tumor progression. This occurs following the local secretion of specific angiogenic cytokines, especially vascular endothelial growth factor (VEGF).^^'^^ Recently VEGF has been demonstrated not only to promote angiogenesis but to suppress anti-tumor immune response principally by hampering leukocyte recruitment^^ and by inhibiting CD34'^ cell differentiation into dendritic cells."^^' Tumor immunotherapy success or failure is strongly dependent from leukocyte migration into tumor area.^'^^'^^ In fact, leukocytes and macrophages, before reaching the target tissue (tumor site), undergo a series of sequential steps during extravasation from blood into tissues: tethering, rolling, adhesion and diapedesis. Among these steps leukocytes adhesion to tumor endothelium is critical and it occurs through the expression of specific adhesion molecules, such as: L-selectin ligands, alpha-4beta-7 integrin adhesion receptors (a4b7) and mucosal addressin cell adhesion molecule-1 (CAM-1). Several animal experiments have shown that in presence of VEGF a significant decreased expression of these adhesion molecules occurs, determining a decline in leukocyte infiltration into the tumors mass.'^^''^^'^ Besides, this impaired tumor infiltration by immune competent cells, tumor environment (hypoxia, acidic pH) itself unfavourably modifies T lymphocytes, NK cells and macrophages activity.^^ In fact, this kind of tumor environment alters the pattern of secreted cytokines towards an immunosuppressive T H 2 pattern, permitting tumor escape from immune surveillance.'^^'^'^
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Heat Shock Proteins (HSPs), Their Role in Antigen Presentation and in Cancer Immunity When cells are submitted to a variety of stressful events (e.g., heat, hypoxia, glucose deprivation), there is a rapid and coordinated increase in the expression of a group of proteins, the so-called heat shock proteins (HSPs).^ HSPs are one of the most conserved groups of proteins throughout evolution and are classified into several families according to their molecular weight in kilodaltons (e.g., HSPs 100, 90, 70, s60, s40) and their compartmentalization inside the cell (cytosol or endoplasmatic reticulum, mitochondria).^^'^^^® HSPs fulfil different important intracellular processes, such as protein synthesis, folding and they are activated by a specific set of genes induced by different physical stress such as elevated temperature, hypoxia, glucose deprivation and oxidative reagents.^^ Linearly at molecular level, heat stress increases the synthesis of HSP 70 until a certain threshold temperature that varies according to cell type. Beyond this threshold temperature their synthesis is inhibited and an exponential cell death follows. ^' Initially, the role of HSPs, peculiarly of HSP70, appeared to be implicated in the thermotolerance."^ ^ Recendy, they have been recognized to activate the immune system becoming a specialized carriers of antigenic peptides in vivo as well."^^' In fact, Srivastava et al have established that HSPs are not immunogenic per se, but when they are complexed with antigenic peptides, become powerful immunogens. In fact it has been found that cancer derived HSPs are highly specific and this specificity is associated with the agglomerate HSP/peptide. Once HSP-complexes (MCHl/MCH2+HSPs) are exposed at the outer surface of cancer cells, they interaa with macrophages and dendritic cells through specific surface receptors.^' ^ HSP70 binds to the surface of monocytes through the Cluster of Differentiation (CD) 14 (CD 14) receptor, whereas gp96 binds with the a-2 macroglobulin/LDL receptor related protein or CD91. Furthermore HSP60 has been demonstrated to be a ligand for the Toll-like receptors 4 (TLR4 and TLR2) complex on macrophages."^'"^ These data support the data that APCs (macrophages, dendritic cells) have evolved receptors for detecting danger signals (HSPs complexes) released during neoplasia. The exposure of the HSP chaperoned peptides by the MCHl and MCH2 molecules to macrophages or dendritic cells triggers a secretion of inflanunatory cytokines and costimulatory molecules, such as: IL-6, IL-12; TNF-a, B7 that induce the maturation of DCs towards theTn 1 phenotype. '^' ^ Their association, with a broad array of peptides generated within cells, make HSPs a good candidate for cancer vaccines."^^ In fact HSP-peptide complexes, isolated from a patients tumor, can be utilized as tailored patient specific antigens, which would avoid the search for specific epitopes. HSPs, isolated from cancer cells but not those derived from normal cells, can generate an immune response, as observed by Tamura and Srivastava. The potentiality of HSPs in tumor eradication has been validated in more than ten types of tumor models of different histologies and in different animal species, demonstrating that: (a) microgram quantities of HSPs are sufficient to generate substantial inunune response; ' (b) the immunogenicity of antigens expressed by dying cells occurs via necrosis or apoptosis. Among the two dying mechanisms only necrotic cells or heat stressed cells have been demonstrated to be able to elicit a tumor-specific inmiunity.^^'^^ Several non randomised clinical trials with heat shock protein-peptide complexes on human cancers are actually in progress.
Therapeutic Modalities Immunotherapy may be either active or passive, specific or nonspecific depending on the process of host immune system stimulation (see Table 3).
Passive Nonspecific Immunotherapy (LAK Cells, TIL) Passive adoptive immunotherapy involves the transfer of immune cells into patients from an external source. This cellular adoptive transfer consists in the generation of cells with antitumor activity obtained in vitro in presence of IL-2. According to their derivation they
Effects ofLocal and Whole Body Hyperthermia on Immunity
Table 3. Classification
of cancer
Specific
251
immunotherapy Nonspecific
Active Tumor cells based vaccines Tumor antigen vaccines DCs vaccine
Bacille Calmette-Guerin (BCG) Corynebacterium parvum Detox Viral vectors: Levamisole Retroviruses Coley's Toxins [CTs] Adenoassociated Cytokines: Viruses (AAV) Interferon-a (I FN-a) Gene therapy Non viral vectors lnterleukin-2 (iL-2) Naked DNA Tumor necrosis factor-a (TNF-a) Liposomes GM-CSF (Cationic lipids [lipoplexxes] and Pegylated Liposomes) HSPs Passive Monoclonal antibodies LAK cells (Lymphokine-activated natural killers) Tumor-infiltrating lymphocytes TIL (tumor infiltrating lymphocytes) Activated T cells Allogenic stem cell trabsplantation cytokines : Interferon-a (I FN-a) lnterleukin-2 (IL-2) GM-CSF Tumor necrosis factor-a (TNF-a) Flt3-L
can generate Lymphokine-Activated Killer Cells (LAK cells) or Tumor Infiltrating Lymphocytes (TIL). LAK cells are generated by culturing in vitro patient peripheral blood leukocytes with IL-2. These LAK cells are injected back into the patient with IL-2. Adoptive LAK therapy has been applied to advanced cases of renal carcinoma and melanomas with variable efficacy. TIL cells are generated fi-om mononuclear cells obtained firom infiltrates around the tumor after surgical resection. This approach, even if more specific, has yielded only limited success. ^^
Active and Passive Nonspecific Immunotherapy (Cytokines, IL-ly INF-CXy GM-CSF) IL-2 has been the first cytokine used alone or in combination with LAK cells for the treatment of different types of metastatic cancers. Patients with metastatic renal cell carcinoma and melanoma receiving IL-2+LAK had a higher rate of complete response. IL-2 has been administered with different regimes and doses demonstrating an elevated toxicity. ^"^'^"^ Numerous other cytokines have been identified and tried clinically, however only Interferon-a (INF-a) and Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) are currently used given the high toxicity of Interferon-y and IL-12.^'^'^'
Active Specific Immunotherapy (Vaccine, Gene Therapy, Heat Shock Proteins) Serological identification of antigens by recombinant expression cloning technique (SEREX) has recently permitted to detect more than 1500 TAAs holding a specific antitumor activity. ^^'^
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Hyperthermia in Cancer Treatment: A Primer
Tumor vaccination (TV) is a therapeutic form of therapy involving patients with detectable disease and it is used for triggering a robust, appropriate and specific immune response towards a well characterized TAAs, avoiding immune-tolerance and providing a long lasting immune response. ^'^^ The first tumor vaccines were obtained by irradiating tumor cells. The obtained results were not enthusiastic, inducing several authors to combine tumor vaccines with nonspecific immune modulators, such as BCG, New Casde Disease virus (NDV) and Detox; however the survival rate was comparable to the group treated with chemotherapy. To ameliorate the results, newer vaccines, including allogenic or autologous tumor cells, were genetically manipulated in order to produce a stronger inmiune response. Tumor cells, were encoded with different cytokines, however only those engineered to produce GM-CSF proved to induce tumor rejection.^^ A large number of clinical trials, involving different strategies, are actually being conducted and have been reviewed elsewere. '^^ Some of them will however be discussed for a positive interaction with hyperthermia treatment (see Table 7). These are: dendritic cells, gene therapy and heat shock proteins. Dendritic Cells Dendritic cells (DCs) are a distinct population of leukocytes that play a crucial role as antigen presenting cells and as initiator of antitumor immunity.^^ DCs reside mainly in peripheral nonlymphoid tissue, after antigen uptake; however they migrate to the draining lymph node to present antigens to T cells. Preclinical studies have demonstrated that antigen pulsed DCs can generate a potent antitumor activity. DCs are artificially loaded with antigens by different techniques and can stimulate both innate and adaptive immune system.^'^ Many clinical trials, testing dendritic cell-based vaccines are in progress and have proved a partial clinical efficacy. ' In every case, preclinical studies have also demonstrated that the direct intratumoral injection of ex vivo-generated DCs can avoid the need for a tumor antigen loading ex vivo by improving antigen presentation or migration to lymph nodes. A required condition is that injected DCs must be genetically modified in vitro to produce cytokines and chemokines, such as: IL-2, IL-7, IL-12, CD40L, GM-CSF, lymphotactin or secondary lymphoid tissue chemokine (SLC).^^'^^ The presence of these cytokines may render DCs more able to capture and process TAAs. This overcomes the increased immune suppressive factors (IL-10, VEGF andTCFp PGE2) present in the tumor medium.69-^2 Gene Therapy Gene therapy is defined as the method of delivering genetic material into a cell, by altering the cellular phenotype permanendy or transiendy.^^ The efficient and precise targeting of a gene to specific cells or tissues remains the technical hurdle of gene therapy. The delivering methods consist in use of viral or nonviral vectors and in vivo or ex vivo gene therapy approach.^"^ The ex vivo approach involves the removal of patient's own cells and the readministration after genetic modification. By contrary, in vivo, therapy involves the direct injection of genetic material into patients. The most promising designed vectors for in vivo therapy are viral vectors. The viral vectors enter into cells by receptor mediated endocytosis and escape from endosomes after DNA delivery to nuclei. They have some disadvantages such as: (a) patient inmiune recognition of viral proteins with a consequent immune attack and destruction of infected cells, (b) restriction in size and quantity of transgene, (c) inability of repeated viral administration.^^'^ Most clinical studies have been carried out using retroviruses or adenoviruses as transfer vector with various advantages and disadvantages.^'^^ The targets of viral therapy can be: inactivation of oncogenes (i.e., ras, c-myc, c-erB-2, abl, bcl-2);'^'^ genes involved in tumor progression (i.e.. Triple-helix formation); immunomodulatory genes encoding cytokines (TNF-a, IL-2; GM-CSF, INF-y) costimulatory molecules (i.e., MCH molecules, CD40ligand, B7, Cd28); angiogenic factors (i.e., VEGF)^°'^5 ^ j tumor associated antigen genes.
Effects ofLocal and Whole Body Hyperthermia on Immunity
253
Non viral vectors (naked DNA, lipids, liposome) make use of physical mediods of gene transfer. They are free of the side effects of viral vectors, but they suffer of specificity/^ Ex vivo gene delivery is the best system for such vectors. Naked DNA in the form of plasmid can be injected into muscle tissues or conjugated with gold particles and bombarded into the tissues. Muscle tissue injection is the most effective delivery method of administration. Repeated injections determine an improvement in therapeutic gene expression but the effect does not last for long.'^^'^'^ Another route of DNA administration is the use of lipid vehicles. Two classes of lipid aggregates have been used: (1) cationic lipids [lipoplexes]; (2) stabilized liposomes. Lipoplexes are positively charged and interact with negatively charged DNA forming a stable complex. As outlined by Clark,^^ cationic lipids have many benefits, such as: easy and inexpensive way of production, non toxicity and potential of delivering large quantity of polynucleotides into cells. However, in order to develop commercially these vector systems, several barriers must be overcome, especially formulation and stability of manufacture.^^ The presence of an excessive number of particles positively charged favour precipitation and aggregation. To circumvent this problem of flocculation, hydrophilic polymers like polyethylene glycol polymers (PEG) have been created. ^^ These sterically stabilized liposomes (PEG liposomes) allow efficient encapsulation of DNA and oligonucletides. They also permit to prepare well-defined and uniform particles suitable for drug carrier.^^'^^ Furthermore PEG liposomes show an improvement in half life time due to a reduced renal and cellular cleareance. Also they show an enhanced protection from proteolysis with reduction of toxicity.^ ^'^'^ Other types of liposomes based on polyethyieneimine (PEI) have proved to be stable to nebulization and to be efficiently delivered as aerosol DNA plasmids to lung parenchyma,^^'^ exhibiting a greater concentration into lungs when compared to other route of administration such as intravenous route.^'^^ Concluding, vaccine therapy is a tailored specific therapy. However, critical issues to overcome are the choice of the delivery system and the identification of singular epitopes. Heat Shock Proteins Heat shock proteins meet the request for searching specific epitopes. In fact, they have been implicated at multiple points in the immune response, including initiation of proinflammatory cytokine production, antigen recognition and processing, and phenotypic maturation of DCs. ^ A preliminary clinical study on colorectal tumor metastasised to liver treated with autologous HSP-96, has clearly demonstrated important points.^^ Namely, HSP-complex induced a significant increase in committed lymphocytes, as response patients have a better clinical outcome as overall survival and as disease free survival and no toxicity was observed. '^^ The importance of hyperthermia in generating HSPs with the aim of immunization will be described later.
Active Nonspecific Immune Therapies: Coley Toxins (CTs) In 1868 W. Busch in Germany concluded that fever induced by certain bacteria from erysipelas can cause tumor regression or cure cancer. This was suggested after the observations on a patient with a soft tissue sarcoma of the neck infected by erysipelas. The causative agent (streptococcus) at that time was not identified. Subsequendy, in 1892, a young American surgeon W B. Coley, unaware of Busch's findings, observed a regression of soft tissue sarcomas in a patient infected by erysipelas. Stunned by that finding, he searched the medical literature and found many publications confirming his observation. '^ Coley prepared initially a culture of streptococci that he injected at the tumor site with encouraging results. He even noted that the presence oiSerratia marcenscens could enhance the virulence of streptococci and an injection remote from the tumor site coidd equally result in tumor regression. After these observations Coley incorporated Serratia marcenscens into the streptococcal vaccine just obtaining the so called "Coley s toxin" or "Mixed Bacterial Vaccine" (MBV). The intravenous route was the most effective; the toxin dose was considered sufficient only if accompanied by fever (39-40°C). Fever and the sustained pyrexia, now recognized to be elicited by tumor necrosis alpha (TNF-a) and by other cytokines, ^'^'^ were considered the critical points in the tumor regression. ' It was also observed that those who developed the highest fever were most often the ones with the longest survival.
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Hyperthermia in Cancer Treatment: A Primer
Table 4, Effects of thermal component of fever on innate and adaptive immunity Immune Response
40°C
Animal Studies
T-cells CD4 CD4/CD8
t T
X
X
t(t++)
X
X X X X X X X X X X X
t
B cells DCs HSPs
U U CD3«i
»
r
» =4. » T t
Ref. 97,129
(T++) NK cells
Human Studies
tt
X X
X
97 133 97129 132 112 116 107 107 121 107 121 107 186,194 223
t : increased response; i: decreased response; PMN: polymorphonuclear leucocytes; **Nottrue for all bacterial species; and antibodies secretion is increased for a beneficial effect on T helper cells; t + : means increased response in presence of a biological response modifier; «: no variations.
Coley should be considered the first scientist to apply induced hyperthermia (fever) as immune treatment. ^'^ He noted that tumor regression was obtained only in presence of fever. Since then the interaction between hyperthermia (in whatever way obtained) and inmiunity has not been completely elucidated.^ To our opinion, a better comprehension of the effects of heat on inmiunity is obtained by understanding the effects of thermal component of fever on immunity (see Table 4).
EfFects of Thennal Component of Fever on Immune System Often in the presence of tumor and microrganisms, the host responds by increasing body temperature.^'^ Fever is a complex neuroendocrine adaptive response due to an increase in the set-point temperature regulator found in hypothalamic area. After their entry into organism, bacteria or viruses induce macrophages to produce a series of proinflammatory cytokines such as: interleukins (ILs) -1,2,6, tumor necrosis factor alpha (TNF-a), Interferon-a (IFN-a), IFN-y. These cytokines, with an additional mediator [prostaglandin E2 (PG E2) cyclooxygenase 2 prostaglandins derivate (COX-2)], act on the thermoregulatory area and reset it to a higher level of temperature, producing the febrile response.^^'^^ Temperature elevation has been suggested to be beneficial for the host.^^'^^ It is unlikely that evolution has maintained such an expensive defensive metabolic mechanism, without a role.^'^ However, the role of temperature on immune defence is not completely understood. Different studies support the idea that temperature rise has a beneficial role, such as an improved efficiency of macrophage killing activity and an increase on survival in mice infected by herpes virus or with rabies.^ ^ Leukocyte adhesion to endothelium and emigration to the site of inflammation are positively affected by heat as the antigen-non specific defence systems (chemotaxis, phagocytosis.
Ejfects ofLocal and Whole Body Hyperthermia on Immunity
255
120i
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'
0
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Figure 2. Time-dependent exemplary induction of IL-1 in a patient after induction of fever up to 39.8°C with a biological pyrogen (Vaccineurin®). complement haemolysis) (see Figs. 2,3). Even antigen-specific activation, proliferation, cytokine expression diflFerentiation and antibody secretion by lymphocytes are affected by temperature. T cell responsiveness to mitogens, IL-1, IL-2 and antigens increase linearly until a temperature of 39°C; beyond this limit a decline is observed.^ Furthermore, it has been shown that antibody secretion in vitro is temperature-dependent and this effect declines in the absence of T-helper
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256
Hyperthermia in Cancer Treatment: A Primer
Table 5. Effects of WBHT, and LHT on immune response Immune Response
WBHT
LHT
Endothelial T[L-Selectin,a ^P7] t(±IFN-^ adhesion molecules [ICAM-1]
t T T
Lymphocyte infiltration into tumor
t i(RT)«
t T T t TGCSF
t(IL-2, PHA)
t leukocytes
(40T-12h) (4rC-12h) (43X • 2 h) (42.5-44°-1/2 h) (45°C • 1/2 h)
THS I I I
Human Studies
Animal Studies X X
X X X X
t(PMCT)
Antigen presentation Neutrophils increase
TH
(39.8°C • 6-8 h) (42.5°C • 6 h) (42.5°C-3h) (43°C • 3 h) (39.5°C • 2 h) (38-39.5T • 2 h) (43°C • 3/4 h) (39.5°C • 2 h) (38-39.5°C • 2 h (41.8° • 2 h) (39.5-40°C • 6 h)
X X X X X X X X X X
Ref. 117 131 136 129 130 135 173 137 138 140 107 132 157 107 132 134 134
TH: time of exposure to hyperthermia and heating degree in "C; PHA: phytohemmagglutinin; GCSF: granulocyte colony stimulating factor, PMCT: percutaneous microwave coagula tion therapy; RT: radiotherapy; LL: lowtemperature long duration WBH; HS: High temperature short duration WBH;0: macrophages.
A summary of temperature effect on specific and non specific inmiunity is presented in Table 4. From it you can conclude, that temperature on the order of fever range (39-40°C) is the most favourable ones for the immune response.^"^'^^'^^ The effects of hyperthermia on the immune system are complex and pleiotropic and are dependent on temperature, time and microenvironment.
Effects of Induced Thermal Elevation (Hyperthermia) on Immunity Temperature application above the physiologic range (> 42.5°C) has been demonstrated to induce various effects on immune system (see Tables 5, 6).^'^'^^'^^^
In Vitro Studies NK and LAK cell cytotoxicity was determined in a standard 4 h chromium releasing assay using K-562 human erythroleukemic cells as target. Cell viability was measured by esclusion of Trypan Blue dye.^^ Some studies have analysed the leukocyte function in presence of temperature associated with IL-2, TNF-cx or Interferon.^®^'^^^ The purpose was to verify the possible abrogation of the immunosuppressive effects of temperature and the rescue on their activity by these cytokines.^^^'^^^ Fuggetta et al^^^ have evaluated in vitro the influence of hyperthermia (HT) (1 h, 42°C) on the cytotoxicity of IL-2 activated NK cells. Hyperthermia reduced the lytic activity of NK cells profoundly. The inhibition of this lytic activity has been demonstrated to be transient and not due to an aptosis but rather to an induced reduction of the effector cells. ^^^ Furthermore these authors, in agreement with others have observed that the heat treatment of target cells alone (K 562 and Daudi cells) did not alter their sensitivity to lysis. ^ ^"^
Ejfects ofLocal and Whole Body Hyperthermia on Immunity
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Hyperthermia in Cancer Treatment: A Primer
LAK cell cytotoxicity has been studied by Shen et al.^ ^^ They have demonstrated that LAK cell induced cytotoxicity was temperature-dependent. It enhanced in presence of febrile temperature (< 40°C) but decreased by exposure to 1 h at 42°C.^^^ In accordance, investigations by Singh et al have shown that LAK cell cytotoxicity, as with NK cell induced cytotoxicity, decreased at temperature above 38.5°C. TNF mediated cytotoxicity however was significantly enhanced at 40°C.^^^ By contrast, Fritz et al have obtained a certain variability on LAK cell mediated lysis in presence of heat and INF-y.^^^ From the various studies analysed it appears that the immune suppressive effects of heat on NK and LAK cells become evident at temperatures above 39°C. The response of NK and LAK cells to hyperthermia is temperature and dose-dependent and not related to cell viability or to the absolute number of cells. ^^^'^^^'^^^ In fact, several authors have noted litde loss of cell viability at 39°C,^^'^^'^^^ whereas a decrease was demonstrated at temperature above 42°C.^^ The lytic function is more heat sensitive than the recognition and binding functions. The extent of recovery of activity after exposure to heat is inversely correlated with the temperature and the recovery time can be complete.^ ^^'^^^ Human and animal cells show a different behaviour during heat exposure. Human and animal NK cells show similar inhibitory behaviour after heat and seem the most sensitive among the immune competent cells. On the contrary, B cells are less affected by heat while murine lymphocytes, compared to the human counterpart, appear to be more heat sensitive.
In Vivo Studies Conditions in vitro cannot simulate those in vivo; therefore Kappel et al^^ investigated the behaviour of NKs and other immune competent cells of healthy volunteers during whole body hyperthermia (WBHT) at 39.5°C for 2 h in vivo conditions. NK cells cytotoxicity increased in temperature-dependent way. Cells incubated with IL-2 or INF-a had an enhancement in cytotoxicity and nmnber compared with control values. At temperature above 39.5°C a slow decrease in number of CD3^ was evident with no variations regarding CD 19"^ cells (B cells).^° More recendy, Atanackovic et al^'^^ have examined the following inmiunological parameters during WBH treatment: (A) behavior of CTLs with a panel of Cluster of Differentiation (CD) activation markers; (B) serum cytokines; (C) intracellular cytokine levels (D) capacity of these cells to proliferate.^^ They have classified the effects of WBH on patients immunity in different arbitrary phase defined by post treatment time. Immediately after WBH, a drastic increase of NK cells and CD56 CTLs was noted in peripheral blood. This phenomenon was transient and followed, after 3 h post WBH, by a short period of reduced T cell activity, indicated by diminished serum levels of soluble interleukins 2 receptors (sIL-2R). In this first phase, a short-lived increase (for the first 5 hs following treatment) in the serum concentration levels of IL-6 was found with a recovery to normal values after 24h. TNF-a increased significandy during the first 24 h, associated to a marked increase in the peripheral percentage of CTLs and CD56. CTLs, CD56, sIL-2R and lymphocytes expressing CD 69 markers reached their maximal concentration 48 h post WBH. CD69 is an antigen expressed early in the activation of lymphoid cells, and it is considered restricted to activated lymphocytes and undetectable on resting lymphocytes. CD 69 is normally induced, after stimulation, with mitogens or cytokines like INF-a, INF-y or TNF-a. In agreement, the intracellular concentrations in CDS cells and in serum of INF-y and TNF-a were found elevated 24 h post WBH in the 80% of the patients. Other authors have studied the production of other cytokines in the serum of patients treated with local hyperthermia (LHT) and WBHT or perfiisional hyperthermia (PHT). The studies are summarized in Table 6. During LHT no detectable increase in IL-1, IL-6 or TNF-a was observed^^^ whereas after WBHT serum levels of IL-1 and IL-6 were increased. ^^^ Robins et al^^"^'^^^ investigated why, during WBHT at 4l.8°C, an enhancement of radiation or chemotherapy response was found without a concomitant increase in myelosuppression. For this reason, they studied an expanded panel of serum cytokines in different patients and found an increase of IL-lp,IL-6, IL-8, IL-10, G-CSF and TNF-a within hours after WBHT.^^'^^^'^^^'^^^ Moreover, they reported that bone marrow cells stimulated the production of IL-1 p^ IL-6 and
Effects ofLocal and Whole Body Hyperthermia on Immunity
259
that TNF-a increased further their plasma levels. This interaction between tumor cells and cytokines, such as interleukin IL-6 resulted in a secondary induction in the bone marrow (BM) of IL-3 and GM-CSF. These factors are produced by BM stromal cells and byT lymphocytes. The plasma levels of the cytokine panel increased 1 h following WBH and diminished after every WBH application reaching the minimun concentration after 4 cycles. ^^^ Alonso et al reported a similar cytokine increase with the addiction of IL-2, TGF-p, INF-y and INF-a in patients undergoing extracorporal perfusion. ^^® A disagreement between the two groups about the TGF-p production has not been completely clarified. Some other aspects of immune response are affected by WBHT and LHT and are illustrated in Table 5. The concentration of some adhesion molecules such as ICAM-1, L-selectin and a P? have been demonstrated to be induced and increased both by LHT and WBHT, augmenting the homing effect into the tumor area of lymphocytes, leukocytes, neutrophils and macSeveral authors have tried to verify the possible modification of tumor cell immunogenicity after exposure in vitro to hyperthermia treatment. ^^'^'^^^ Dickson concluded that immunization and survival of mice with Ehrlich ascites did not increase after HT.^^'^ By contrast Mondovl obtained an increase in the immunogenicity and found that heated cells had an higher immunogenicity compared to cells treated with irradiation. The phenomenon was dependent on the length of treatment and the choice of temperature was extremely important. ^^'^^^ Recent studies on ratT-9 glioma cells by Ito et al^"^ seem to confirm Mondovis experiments. In fact, these authors have shown a significant increase in MHC class 1 antigen on the surface of heated cells 24 h after heating associated to an increased expression of HSP70. No modifications of other surface immunologic mediators, such as ICAM-1 or MHC class II antigen were noted. The in vivo growth of T-9 glioma cells, using immtmocompetent syngeneic rats (F344) was significandy inhibited and associate to a an increased cytotoxic specificity.
Specific Effects of Hyperthermia on Immune Therapeutic Modalities Several recent studies, suggest that hyperthermia is suitable as a complementary therapeutic modality for immunotherapy (see Tables 6, 7).
Radioimmunotherapy and Monoclonal Antibody Therapy An enhancement of therapeutic outcome of radio-immunotherapy and monoclonal antibody (MAbs) activity in combination with WBH and LHT respectively has been demonstrated. The tumor uptake of MAbs against different experimental tumors (colon cancer, ^ glioma, breast and prostate carcinomas^ ) increased from 12% in non heated tumors to 42% in tumors heated with local hyperthermia (41.8°C x 4 h) and persisted in the heated tumors over 48 and 96 h. The increased uptake of MAbs obtained using local hyperthermia, seems not so be induced by a decrease in tumor interstitial fluid or modification of kinetic parameters, but rather to an increased extravasation.^
Gene Therapy The use of gene therapy as potential therapeutic method is increasing. Different authors have demonstrated that HT enhances the effect of viral gene vectors encoding IL-12 and the treatment was effective, devoid of systemic side effects and associated to a substantial tumor growth delay, as compared to animals treated without HT.^"^^'^^^ Mutated p53 genes are found in over 50% of all cancers and they are responsible for the decreased HT induced apoptosis. Adenovirus transfer of p53 associated with HT seems attractive for overcoming this resistance. In fact glioma and human salivary gland adenocarcinoma treated with this combination demonstrated a higher sensitivity to hyperthermia and radiotherapy. Other useful methods to obtain an efficient gene transduction is the use of liposomes. Several authors have demonstrated that the transduction of DNA plasmid complex (lipoplex) was more efficient under conditions of hyperthermia than at 37°C. An amelioration of this
Hyperthermia in Cancer Treatment: A Primer
260
Table 7. Effects of WBHT and LHT on different immune treatment modalities Therapeutic Modalities
Human Studies
WBHT
LHT
TH
MAbs Activity Vaccine Therapy
TRI
t
T
T T
HSPs
THSP110 TtHSP70, 90
T
(40°C X 3-6 h) (41.8°Cx4h) (39.5°C X 2 h) (42.5°Cx3 h) (41-45° X 1/2-1 h) (39.5°C X 1 h) X (41.8°C X 1 h)
DCs
T
T(HSP 70)
X X X X X X X
t(HSP 70) Gene Therapy
Animal Studies
X
T T
X X
(42°C X 3/4 h) (42°Cx1/2h) (43°C X 3/4 h; THSG 44°Cx1/3 h) T U251 glioma (43-44°C x 1 h) T N I H 3T3
(4rCx1 h)
tA549
(41°Cx1 h)
X X X
Ref. 142 143 120 138 151 120 197 47 194 195 148 149 150 151 153 153
TH: time of exposure to hyperthermia and heating degree in "C; HSG: human salivary gland adenocarcinoma cell line; DCs: dendritic cells; MAbs: monoclonal antibodies; NIH3T3: murine fibroblast; A549: human lung cell line; Rl: radioimmunotherapy.
effect has been demonstrated using thermosensitive liposomes or magnetic cationic liposomes, recently developed. ^^^'^^^'^^^
Cytokine Therapy The treatment of tumors with cytokines such as IL-2, TNF-a, INF-a and GM-CSF, has been disappointing. This has induced many researchers to use IL-2, GM-CSF, Interferons and TNF-a with WBHT and LHT^^"^^^^ Generally, an additive effect has been demonstrated without an increase in toxicity. The majority of studies have been conducted on animals using IL-2 and TNF-a. ^^^'^^"^ Human studies have been conducted, primarily with WBH and perfusional hyperthermia (PH) associated to TNF-a. ^^^'^^'^ IL-2 administered before local hyperthermia treatment has demonstrated to be additive and useful to treat mice with lung metastases.^ ' The same additive effect has been demonstrated by Geehan regarding melanoma and sarcoma. The response was obtained using IL-2 simultaneously to WBHT application. The doses and toxicity were lower than those usually reported. ^^^'^^ Fritz et al have demonstrated that the potentiation of IL-2 combined wdth HT is mediated by TNF-a induction. In fact, the effect of IL-2 was abrogated by anti-TNF-a antibodies. ^^® Whole body hyperthermia or local hyperthermia combined with low-dose IL-2 was more effective on reducing tumor growth than each modality alone, and the response was more pronounced for macroscopic tumor than for microscopic one. Geehan et al surest that this phenomena is to be ascribed by two factors: (A) selective increase in permeability in tumor vessels, as compared to normal tissues with a consequent accumidation of drug, (B) an augmented expression by HT of intercellidar adhesion molecide-1 (ICAM-1) followed by an increased homing into tumor tissue of LAK cells.^^^'^^°'^^^
Ejfects ofLocal and Whole Body Hyperthermia on Immunity We refer to the review of Klostergaard et al^^^ for studies on combination of hyperthermia with TNF-a and INF-a. In brief in vitro and in vivo studies on human and animal tumors indicate a sensitization of TNF-a effect combined with heat. Sensitization was greater when tumor targets were treated with TNF-a prior heating treatment and the effect in vivo can be reached with lower dosage and with less toxicity. It appears that the effect of TNF-a in vivo is partially due to an increase on plasma membrane receptor expression or affinity and on tumor vasculature. Klostergaard et al^"^^ reported similar effect using INF-a and INF-P both in vitro and in vivo. The maximum effect was observed with intratumoral administration and the time of administration with HT was less important. A combination of TNF and INF is possible with additive effect and decreased dosage. As for TNF-a the antiproliferative effect seems to be ascribed to a direct effect on plasma membrane receptor expression or affinity. Recent studies are more oriented to use TNF-a combined with chemotherapy and WBHT or with limb or organ isolated perfusional HT. As reported by many authors a synergism among hyperthermia, melphalan (L-PAM) and TNF-a in the clinical setting of limb perfusion for malignant melanoma and sarcoma has been demonstrated. ^^^ A TNF-a concentration superior to that achieved by bolus administration (10-20 )lg/ml) can be given locallv (1-2 ^ig/ml) associated only to mild toxicity (grade lor 2) in 25% of patients treated.^^^''^^^ A similar combination of therapeutic regimen for treatment of unresectable liver malignancies (confined to liver) by using isolated hepatic perfusion (IHP) has been studied by Alexander et al. According to a critical evaluation by these authors IHP with L-PAM and TNF-a is the best combination regimen for obtaining a good response rate as compared to other chemotherapeutic regimens using drugs such as FUDR.
Effects of Hyperthermia on Lymphocyte Homing As previous mentioned, leukocyte infdtration into tumor mass is mediated by the expression of various adhesion molecules and cytokines. ^'^^'^'^ Among these adhesion molecules, ICAM-1 and a4p7 integrin are pivotal in r^ulation of the migration of leukocytes from the blood vessels.^^ Their expression has been demonstrated to decrease in presence of VEGF, ' to increase in presence of febrile range temperature (38-40°C) and INF-a. ^^^'^^"^ In fact, there is an increasing evidence that local and whole body hyperthermia can enhance both L-selectin lymphocyte-endothelial cell adhesion. ^^^'^^^'^'^^ Regarding increase of adhesion molecules it appears that hyperthermia differs compared to other therapies. In fact the increase has been detected only on tumors microvasculature and on peritumor lymphatic not on surrounding normal vasculature. ^^^ The mechanism underlying HT control of L-selectin have revealed that febrile temperature do not increase lymphocyte L-selectin surface density or L-selectin dependent recognition of soluble carbohydrates but the avidity of preexisting adhesion molecules for physiologic ligands.^ This up regulation of adhesion process su^ests the use of LHT and WBHT in clinical settings for delivering selectively cytotoxic T-cells or gene armed lymphocytes only into the tumor area.
The Danger Model and Hyperthermia-Effects on Dendritic Cell Maturation and Stimulation of Innate-Adaptive Immunity During the last decade, we have learned a great deal about the molecular mechanisms responsible for the modulation of tumor immunity; however, litde advances have been made clinically so far. The reasons for this failure may be the result of several mechanisms: L not complete control of tumor microenvironment^^^ 2. non appropriate presentation of antigens^^^ 3. innate immunity not adequately stimulated or suppressed^^^ 4. tumor immune evasion. Hyperthermia has a role in controlling the first three mechanisms. Inside a tumor mass, it exists at least partially a stressful hypoxic and acidic micro milieu which hampers antigen presentation or immune effectorsftinction."^^'^^^On one hand, this kind of stressful envi-
261
Hyperthermia in Cancer Treatment: A Primer
262
CELL DEATH IMMUNITY
ANTITUMOR IMMUNITY
MACROPHAGE PHAGOCYTOSIS
/•-\'
A.
cytokines
DANGER SIGNAL
i"
MACROrMAOeS LYMPNOCTTES RBCRUiTMeNT
INNATE IMMUNITY
ADAPTIVE IMMUNITY
Figure 4. After heat stress the HSPs can induce tumor immunity by two mechanisms: Increasing Antigen presentation through CD91 receptors, or by TLRs receptors. ronment can modify the response of lymphocytes [in vitro] to synthesis and release of mitogens and cytokines during HT treatment,^^^ on the other part it is the more suitable for killing tumor cells by heat, and for the generation of the "Danger Signal". ^^^
Danger Model According to Matzingers danger model,^^^'^^^ tissue damage in general provides a stimulus for initiating protective inmiune response. Data by Feng et al^^ indicate that heat stressed tumor cells are capable of providing the necessary danger signals, likely through increasing surface expression of heat shock proteins (HSPs), '^^^ resulting in activation and for maturation of dendritic and natural killer cells. ^^^'^^"^ Heat stressed apoptotic 12B1-D1 cells, compared to not heat stressed cells, were more effective in stimulating dendritic cells to secrete interleukin-12 (IL-12) and in enhancing their immunostimulatory functions in mixed leukocyte reaaions. Thus, DCs are able to distinguish between stressed and non stressed cells undergoing programmed cell death. In conclusion, a tumor tissue that undergoes a stress response (i.e., heat) and goes in apoptosis increases the synthesis of stress proteins on their surfaces or releases products during tissue damage that are recognized by tumor-infiltrating lymphocytes as not-self This may generate potent antitumor T-cell responses. ^^^ In contrast, non stressed apoptotic tumor cells are recognized by the immune system as a physiologic process, critical to normal development and able to elicit only a non inflanmiatory/ or even tolerigenic bland danger signal (Fig. 4). Furthermore, HT stimulates DCs migration,^^^'^^^ lymphocyte homing^^^ and HSPs synthesis. ^^^'^^^ DCs emigration from peripheral organs to lymph organs, is crucial for their maturation and their shifting from an antigen-capturing mode to a T cell-sensitising mode. In this sense, Ostberg et aT^^'^^^ have observed an enhancement of antigen-dependent immune responses at skin level and the stimulation and emigration of epidermal DCs to draining
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lymph nodes following WBHT. Concluding, cell deadi occurring with a concomitant production of HSPs, such as during HT treatment, is highly immunogenic. Tumor immunogenicity is also enhanced in cancer cells over-expressing HSP70 and these cells induce a marked T H l type immune response compared to cells dying via apoptotic mechanism. ^^^'^^^ In Figure 4, the various mechanisms of cell death and the consequences on tumor immunity are illustrated. It is important to note that apoptosis after macrophage phagocytosis induce the production of the immunosuppressive cytokine IL-10.^^^ This can permit tumor tolerance.
Innate Immunity Heat Shock Proteins Link Hyperthermic treatment can induce and elicit a variety of innate inmiune responses. Innate immune responses have been shown to contribute to the control of tumor in mice and there is indirect evidence that it contributes to the control of cancer in humans too. This response is dependent on the stimulation of TLRs expressed on DCs surface as confirmed by recent studies by Hilf^° and Valubas.^^ In fact they have demonstrated that the *ER-resident chaperone Gp96 (*ER: endoplasmaticum retictdum) is a potent tumor vaccine in animal models, and it induces both innate and specific immunity in a high efficient way interacting with TLRs pathway. ^^ Intriguingly, many other HSPs and substances from nonmicrobial sources have been recendy shown to signal through TLRs. There are ten members of the TLR family identified to date,^'^^^ in particular TLR4 is involved in activation by HSP 60, TLR2/4 by HSP 70 and GP96, and TLR2 by necrotic cells.^^^ Furthermore, recent findings show that TLRs are expressed in a multitude of different cell types, such as APCs, macrophages and DCs. The best characterized regulator of TLR signalling is the NF-KB transcription factor, which controls the expression of many genes involved in the inflammatory response. ^^^'^ This engagement triggers the induction of proinflammatory cytokines and chemokines which contribute to the maturation of DCs and the activation of naive T cells (see Fig. 4). Between the cytokines and chemokines released IL-12, IL-18 and TNF-a have a crucial role in directing THl response.^^ IL12 stimulates, also, NK cells that have been demonstrated to be required for obtaining an adequate antitumor activity by HSPs. Recent studies by Tournier et al have confirmed that fever plays a role in activation of DCs through the release of different temperature sensitive cytokines. Another confirmation comes from a recent work by Basu et al^^ that have demonstrated that elevated temperature in the range of 39.5°C-4l°C causes immature DCs to mature, specifically through elevation of intracellular levels of HSP 90. The HSPs released from cells imdergoing necrotic death cause translocation of NFkB into the nucleus and maturation of DCs. Concomitandy, the elevations of the aforementioned cytokines (IL12 and TNFa) contributes further to DC-maturation. Additional study has outlined the importance of DCs maturation as an essential prerequisite for a successful vaccination. ^^^ Studies by Wang et al^"^^ have shown that fever, as wells as, HT combined with tumor derived HSP (HSPs 110 and 70) significandy enhanced the vaccine efficiency on mole-to-mole basis and reduced tumor volume. Okamoto^^^ and Schuller^^'^ reported that LHT induced tumor-specific CTL response on colon carcinoma and necrosis of hepatocellular carcinoma cells. Heat shocked activated DCs are more able to stimulate T cells than non heat shocked DCs, indicating that temperature elevation can be exploited to generate a powerful immune-activity. ' Hyperthermia stimulates also neutrophils and macrophages recruitment. '^^^ Two cells subset of innate immunity that have demonstrated to play an important role in host defence against tumors. ^^^ As demonstrated by Gough et al,^^ macrophages can distinguish between tumor cells dying through classical apoptosis or cells engineered to die through non apoptotic mechanisms. In a certain sense, the immune system is able to read in which way tumor cells die and to react consequently. Additionally the presence of HSP 70 acts as one component of a bimodal alarm signal that activates macrophages, in the presence of stressful-immunogenic tumor cell killing to become activated in an antitumoral way. Different studies have demonstrated that tumor immunogenicity is enhanced in cancer cells over-expressing HSP70 and these cells induce a marked T H l type immune response compared to cells dying via apoptotic mechanism (Fig. 4,)}^'^'^^^ The danger signal and theTRL activation can work in concert and
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Hyperthermia in Cancer Treatment: A Primer
can augment the tumor immune response. In every case, this mechanisms are to be completely elucidated and clinical demonstrated.
Streptococcal Preparation OK'432 (Picibani^)-Corynebacterium Parvum Following the abandonment of Coleys Toxin, different authors, unaware of the importance of innate immunity in tumor rejection, demonstrated that cell wall bacteria products such as streptococcal preparation OK432 (Picibanil®) and Corynebacterium Parvum, can strengthen tumor lysis cells by lymphocytes when used with hyperthermia."^^"^^^ OK-432 is a lyophilized streptococcal preparation made by penicillin treatment of the Su-strain of A-group streptococcus and it has been used in Japan since 1975 as cancer agent. Picibanil® associated with hyperthermia has demonstrated in different animal strain to induce NK cell activity and to enhance tumor tissue response to heat.^^^'^^^ Corynebacterium Parvum is an aspecific immimotherapeutic agent constituted by bacterial cell wall glycoproteic and lipid fractions. In presence of hyperthermia it has demonstrated similar immune and thermal effects than OK-432.^^^'^ For Picibanil an involvement of TLR receptor 4 in its antitumor effect has been recendy found demonstrating a link between innate immunity and anticancer activity.^ At the light of these studies, heat can be considered as an aspecific innate immunity booster. The use of bacterial product extracts increase further this stimidation.
££Fects of Hyperthermia on Metastatic Process Metastatic process is the most fearsome aspect of cancer. The formation of metastases is a complex multistep process influenced by the host and by selection and resistance induced by the different therapeutic approaches used.^°^'^°^ Shah oudined in animal experiments that a sublethal heating (< 42.5°C) of tumors with a non-complete destruction of all cancer cells, may enhance metastases formation, in a way not dissimilar to chemo- and radiotherapy. In 1974, studies by Dickson and Ellis^^° opened a great controversy on the efficacy of hyperthermia treatment, in fact they reported an increased metastasization to the liver following heating of a Yoshida sarcoma in rats. Further studies reported by Shah^^^ showed an increased metastasization preferentially to lymph nodes and lungs following a non appropriate heating. A curative treatment with appropriate temperature (> 42.5°C) was associated to regression of primary tumor and was accompanied by increased host immunocompetence. An increase on metastization has been reported by some authors and attributed to a decreased activity of NK cells.6.7ao6 Recent studies using WBHT in humans and animals"^^ ^ did not found a decreased activity of NKs and CD4 cells in vivo, contradicting old studies in vitro. ^^^ Urano,^^^ Dickson and Shah^ observed that WBHT may induce metastases at a frequency higher than LHT. Urano et al revealed that the size of the tumor and not its immunogenicity was critical for metastasization during WBHT.^^^ To reduce this incidence Oda et al^^^ suggest the use of anticancer drugs associated to WBHT. Dickson and Shah^ reported the abrogation of the effects on immune response generated by curative local heating providing that WBHT was followed or was associated with local hyperthermia. More recent clinical studies on animals with melanoma, treated with Radiation alone or combined with LHT, have demonstrated no significant difference in metastasization between the two groups, though local recurrence was a common event and was associated with metastases appearance.^^ '^^^ Similar phase III trial on human melanomas treated using similar design has shown an improved local control and a reduced metastasization with combined RT+LHT.^^^ A recent randomised study on soft tissue osteosarcoma has demonstrated that the rate of metastasization with thermoradiotherapy is similar to that seen with preoperative radiation therapy alone.^ Similar conclusions about dissemination of malignant cells during WBHT in combination with chemotherapy for epithelial malignancies have been reached by Hegewisch-Becker and collaborators. In order to obtain a maximum benefit from tumor heating a functioning host immune response seems necessary. In fact, studies conducted in the late 1990 by Ponti^ia et al"^^^ have clearly demonstrated that patients with higher values of inflanmiatory tests {al glycoprotein > 1.0 g/1; high sedimentation rate > 50/Ih, and with a lymphocytes count < 500/mc, and a ratio
Effects ofLocal and Whole Body Hyperthermia on Immunity CD4/CD8 < 1.2) failed to obtain remission. The same authors outlined that patients responsive to BRM (i.e., INF-a) show an increase in NK cell number and responders have a better prognosis as compared to non responders.^^^ Another important phenomena to take into consideration is the abscopal response. It consists in a regression of tumor at other anatomic sites following curative heating of primary tumor. This phenomena has been described in animals and humans.^^'^^^ Disappearance of primary tumor and regression of distant metastases after hyperthermic limb perfiision in both sarcoma and melanoma has been ascribed to a non specific immune reaction, such as inflammatory reaction with an increased macrophage infiltrate."^^'^^'^^'^^^'^^ Clinical studies by Shah^^^ and Dickson^ ^® have not demonstrated distant metastases regression after primary tumor treatment with LHT. Abscopal phenomena seems not pertinent to WBH treatment.
Conclusions and Comments Tumor regression of some type of human tumors following HT has been reported. The evidence of an involvement of tumor inmiune response in this regression due to temperature elevation is increasing step by step. The effects of hyperthermia on the inmiune system are pleiotropic.^ First of all, the compartment shifting and homing of immune competent cells (T-lymphocytes) and neutrophils to tumor area is increased whereas cytotoxicity is only transitorily affected. ^^^ The increase of temperature correlates positively with an increased phagocytosis of leukocytes and macrophages within fever range temperatures. ^^^'^^^ B-cells are activated by heat which brings about an increase of the production of immunoglobulins. On the contrary NK cells cytotoxicity is suppressed and their activity more affected in yitro.^ '^^^'^^^'^'^^ This suppression is however temporary. In the range of temperature beyond 39° after a period of stunning a complete recovery follows. Some authors have also demonstrated that recovery can be augmented or cytotoxicity partially spared by using interferons^"^^'^^^ or antioxidants^ ^^ such as superoxide dismutase. A summary of the effects of LHT and WBH on LAK cell and NK cells cytotoxicity can be found in Table 8. From these studies some aspects become evident: 1. temperature is crucial 2. WBH affects less the cytoxicity activity of both immunocompetent cells than LHT 3. times of exposure to heat in vitro studies are not clinical comparable. In fact different parameters can influence lymphocyte reaction in vitro compared to in vivo situation. They are: (a) treatment time that is too long (3-4 h; 18 h) or too short (1/2 h), (b) the different lytic E:T ratio, (c) the nonuniform distribution of heat easily obtainable in vitro but not in vivo and (d) the pH of the medium. Studies by Skeen et al have tried to reproduce tumor pH microenvironment and its effect on lymphocytes. They have demonstrated that the pH of the medium (obtained adding lactic acid) had no effect at 37°C but showed a synergistic impairment with heat at 41, 42 and 43°C.^^^ These examples indicate experiments are to be conducted with appropriate methods for assessing in vitro and in vivo cellular immune response. We also suggest to employ experimental conditions well defined, standardized and nearer the clinical simation for better understanding HT effect on immunity. In particular: pH of medium, cytokine assay in situ, activation and impairment of cellular response by DCs use. Comprehensive clinical studies on W ^ H T are to our opinion those of Robins and Atanactovic.^^^'^^ ,123,125,127 CD4+ / CD8+ lymphocytes ratio does not change in vivo, whereas activation and maturation of dendritic cells is positively affected.^^ In fact, in animals experiments, febrile range temperature elevation (39.5-4l°C) have demonstrated to elicit DCs maturation through HSPs induction. Furthermore, for this order of heating WBHT induces a panel of cytokines similar to that induced during fever;"^^^'^^^ on the contrary LHT is not able to elicit any cytokines production (see Table 6).^^^ The induction of HSPs is rapid and occurs immediately following exposure to only a few degrees above normal physiological temperature stimulation.'^'^^ HSPs induction occurs during
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Hyperthermia in Cancer Treatment: A Primer
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