DVANCE IN ADIOLOGY AND COLOGY Editors Tomaž Benulic Gregor Serša Viljem Kovac ADVANCES IN RADIOLOGY AND ONCOLOGY Editors Tomaž Benulic, M. D. Department of Radiotherapy Institute of Oncology Ljubljana, Slovenia Gregor Serša,. Ph. D. Department of Tumor Biology and Biotherapy Institute of Oncology Ljubljana, Slovenia Viljem Kovac, M. D. Department of Nuclear Medicine and Department of Radiotherapy Institue of Oncology Ljubljana, Slovenia Research and review papers th contributed on the 25. anniversary of publishing Radiologia lugoslavica. Alf rights reserved Radiologia Iugoslavica, Zaloška 2, 61000 Ljubljana, Slovenia Reader of English language Olga Shrestha Design Monika Fink-Serša dipl. ing. arch. UDC and Key words dr. Eva Klemencic Secretary Milica Harisch, Betka Savski Printed by Tipograf, Rijeka, Croatia CIP -katalogizacija v knjigi Narodna in univerzitetna knjižnica. Ljubljana 615.849(082) 616-006-073. 75(082) ADV ANCES in radiology and oncology / (uredniki. editors Tomaž Benulic. Gregor Scrša. Viljem Kovac). -Ljubljana : Radiologia Iugoslavica. 1992 l. Benulic. Tomaž 2. Serša. Gregor 28392960 ADV ANCES IN RADIOLOGY AND ONCOLOGY SCIENTIFIC BOARD Sonja Bebar, Ljubljana, Slovenia Tomaž Benulic, Ljubljana, Slovenia Matija Bistrovic, Zagreb, Croatia Erika Brencic, Ljubljana, Slovenia Ivo Drinkovic, Zagreb, Croatia Jure Fettich, Ljubljana, Slovenia Durila Horvat, Zagreb, Croatia Berta Jereb, Ljubljana, Slovenia Vladimir Jevtic, Ljubljana, Slovenia Viljem Kovac, Ljubljana, Slovenia Ivan Lovasic, Rijeka, Croatia Miljenko Marotti, Zagreb, Croatia Dušan Pavcnik, Ljubljana, Slovenia Stojan Plesnicar, Ljubljana, Slovenia Miran Porellta, Ljubljana, Slovenia Gregor Serša, Ljubljana, Slovenia Josip Stojanovic, Zagreb, Croatia Slavko Šimunic, Zagreb, Croatia Janez Škrk, Ljubljana, Slovenia FOREWORD The book »Advances in Radiology and Oncology« has been published by the journal »Radiologia Iugoslavica« to celebrate the appearance of the 25th vol ume. Since its founding in 1964, the journal has featured scientijic papers dealing with X-ray diagnosis, nuclear medicine, racliotlzerapy, oncology, racliobiology, racliophysics, racliation pro/ection as well as with recently clevelopecl .fielcls i11c/ucli11g i11terve11tio11a/ radio!ogy, computed tomography, magnetic resonance inwging, u!trasou11d ancl experimenw/ 011co!ogy. With continuing publication of the resu!ts of increasing!y comprehensive scope of research conducted at home and ahroad, the journa/ succeeded in kecping ahreast 11•ith tlze !atest achievements in these branc/zes of medicine. The fact that the papers published were not restricted to just a few topics or even a sing!e subject, inevitably led to fragmentation of particular topics which were dealt with in severa! different issues or even entire volumes. Far the sake of consistency, and to give a more comprehensive overview of formerly published materials, we decided on the occasion of the 25th anniversary of the journal to issue a comprehensive book which would combine both topic-related papers ancl specific thematic fields. Many authors from Slovenia and ahroad responded to our invitation by contributing their papers for this issue. The book has been created over a longer period of time, cluring the years when the links between the former Yugoslav republics were beginning to break up. Nevertheless, thanks to the joint efforts of the authors, institutions ancl sponsoring producers of medica! equipment we finally overcame all obstacles to the publication of this boo k. We trust that it will be greeted with interest by a large reading public. Despite the rapid progress made in medica! technology, it must be aclmitted that human distress, anxiety and pain related to disease have unfortunately remainecl unchanged. Nevertheless, a number of modern diagnostic and treatment devices and modalities enable us to help the patient in a faster, much more effective and less painful way that was possible in the past. If in these times of highly developed technology involving complex graphs and figures, we have also succeeded in directing due attention to the patient, the purpose of this book will have been fully justified. T. Benulic, MD T ABJLE OF CONTENTS Chapter I. INTERVENTIONAL AND DIAGNOSTIC RADIOLOGY Percutaneous transcatheter placement of the aortic val ve Pavcnik D, Wright K, Wallace S 15 An injection technique for repositioning subcvlavian catheters Mafzon T, Lawrence D, Sr. 22 Properties of biodegradable polymers for 5-fluorouracil and tamoxifen microcapsules Yang Dl, Byun HS, Kuang LR, Yamashita Y, Mou!t RG, Tansey W, Wright KC, Wallace S 25 Intravenous trombolysis of acute coronary occlusion after percutaneous transluminal angioplasty Kranjec/, Cijan A, Pavc'nik D 30 Current diagnostic of pituitary disorders by CT Lovrencic M 35 Chest CT in sarcoidosis Dalagija F, Dizclarevic Z, Be.lilic S 41 Computed tomography during arterial portography of the !iver without interslice gap Yasumori K, Hasuo K, Baba H, Yoshida K, Mizushima A, Hirakaw R, Kuroiwa T, Onitsuka H, Masuda K 47 CT -guided percutaneous drainages of abdominal abscesses Lincender L SI Computerized tomography of the spine Papa l 56 Magnetic resonance imaging of kidney diseases Marotti M, Hricak H, Kovacevic D 60 Magnetic resonance imaging of hand joints in patients with rheumatoid arthritis, psoriatic arthritis and nondifferentiated seronegative polyarthritis Jevtic V, Rozman B, Kos-Golja M, Jarh O, Dem.for F 66 Preoperative evaluation of long bone osteosarcomas with magnetic resonance imaging Poleksic Lj, Zdravkovic D, Atanackovic M, Mitrovic M, Bacic G 72 Chapter II. ULTRASONOGRAPHY Ultrasound diagnostic of the neck: A ROC study Pichler E, Breyer B, Mihajlovic L, Boka H, Kralj Z 81 Position of nodules in echographically multinodular thyroid in cQmparison with patohistologic nature of nodules Brkljacic B, Drinkovic I, Delic-Br.kljacic D, Boka H, Vidjak V 86 Parameters for echographic evaluation of gallbladder contractions Miric S, Sabljak I 90 The treatment of appendiceal abscess by ultrasonically guided drainage Drinkovic I, Brklja.ic B, Boka H, Odak D, Vidjak V, Anic P 96 Ultrasound in diagnostics of muscular injuries Vidjak V, Drinkovic I, Brkljacic B, Krmpotic T, Boka H, Odak D 99 Ultrasound as a diagnostic tool in malignant bone tumours Babic M, Matovinovic D 102 Chapter III. NUCLEAR MEDICINE Labelling leucocytes with Tc-99m HMP AO for imaging inflammation 111 PetersAM COST B2 -Cooperation in science and technology: A european programme on the quality assurance of nuclear medicine software Britton KE 116 New aspects in nuclear cardiology -PET, NMR and SPECT Henze E, Milcinski M, Lietzenmayer R, Clausen M 123 Reflections on immunoscintigraphy and radioimmunotherapy Roos JC, van Lingen A, Teule GJJ 135 lnterlaboratory comparison study on quality performance of nuciear medicine imaging devices Karanfilski B 142 Chapter IV. CLINICAL ONCOLOGY Radiosurgery: A review of clinical aspects · Souhami L, Podgoršak EB Medulloblastoma, results of treatment at the Institute of Oncology, Ljubljana Petric-Grabnar G, Župancic N, Klun B, Umek B, Stare 1, Kopac š, Kragelj B, Cindro L, Škrbec M, Jereb B lntrapleural application of human leukocyte interferon (IFN-CI') in breast cancer patients with pleural carcinosis Jereb B, Štabuc B, Us-Krašovec M, Cerar O, Stare J 175 Soft tissue sarcomas in childhood. Experience of the Austrian cooperative rhabdomyo­sarcoma study (A-RMS 82). A final report Ladenstein R, Grumayer ER, Hawliczek R, Krepler R, Fink FM, Haas H, Mutz /, Ploier R, Stolinger O, Thun-Hohenstein L, Tulzer W, Urban C, Gadner H 181 Our experience in treating children with osteogenic sarcoma Benedik-Dolnicar M 196 Recent strategies in the treatment of malignant germ celi tumour Clemm Ch 202 Thyroid status following irradiation for laryngeal cancer Dimitrovska A, Karanftlski B, Georgieva B, Velkov K, Jovanovski D, Nikolova L 208 Tumour markers in clinical oncology Novakovic S, Serša C 214 The predictive significance of CA 15-3 for the first relapse in early breast cancer patien(s Neškovic-Konstantinovic Z, Vuletic L, Tasic S, Ogrizovic N, Šušnjar S 221 Chapter V. EXPERIMENTAL ONCOLOGY Prostaglandins in malignant tumours: Role in tumour growth and therapy Milas L 229 Problems in tumour celi repopulation during irradiation treatment of squamous celi carcinoma of head and neck Budihna M, Škrk J 236 Dipyridamole enhances the cytotoxicity of drugs and radiation in HeLa and V-79 cells in vitro Ban J, Soric J, Bistrovic M, Dujmovic /, Miletic Z 241 Stress, tumour metastasis and antitumour chemotherapy in mice Giraldi T, Perissin L, Rapozzi V, Zorzet S 249 Combining photodynamic therapy with immunotherapy: Will it work? Krošl C, Korbelik M 261 Role of DNA flow cytometry and cytomorphology in the search of effective chemothe­rapy for chemoresistant tumours (Renat celi carcinoma) Auersperg M, Us-Krašovec M, Stanovnik M, Cufer T, Bešic N, Goehde W 268 Flow-cytometric DNA ploidy and clinicopathologic variables in primary breast carcinoma Us-Krašovec M, Brac ko M, Cu/er T, Goe/ule W, Ko.{melj K, L{{n/01•·ec J, Pogacnik A 275 Correlation of DNA-ploidy and estrogene and progesterone receptor content in primary breast cancer Cufer T, Bracko M, Goehde W, L{{movec J, Ko.1'1nelj K, Pog{{(11ik A, Us-Krašovec M 280 Potentials of hyperthermia in clinical use Lešnicar H, Budihna M 284 Chapter VI. RADIOPHYSICS Radiosurgery: A review of physical aspects Podgon.'ak EB, Souhami L 301 A survey of radiation biophysics for the radiotherapist Bistrovic M 311 A practical method of TBI in vivo dosimetry based on real scatter contributions Vrtar M 323 Subtotal-skin electron irradiation of the torso Umek B, Habic M, Jereb B, Štabuc B 330 Scanning slit radiography apparatus with a radiographic grid Miklavcic L Chapter VII. RADIA TION PROTECTION Incidence of structural chromosome aberrations in medical staff occupationally exposed to ionizing radiation Horvat D 343 The effect of ionizing radiation on the peripheral blood flow of occupationally exposed medical personnel Brumen V, Bonic I 348 Biological effects of microwave radiation Garaj-Vrhovac V 356 Chapter l. ,· INTERVENTIONAL AND DIAGNOSTIC RADIOLOGY Adv Radio/ Oncol 1992; 15-21. Percutaneous transcatheter placement of the aortic valve Pavcnik D, Wright K and Wallace S From the John S. Dunn Research Foundation Center for Radiological Sciences, University of Texas, M. D. Anderson Cancer Center and from the Institute Diagnostic and lnterventional Radiology, University Medica! Center, the University of Ljubljana, Slovenia. Twelve adult mongrel dogs have received prosthetic aortic valves consisting of a ring, cage and bali. Ali valves were percutaneously placed in the ascending aorta as the first step in the investigation of transcatheter delivered prosthetic aortic valves for the treatment of aortic insufficiency. Results of these studies have led to severa! design modifications. At present, prosthetic valve is a ball-in-cage design which functions as a simple one-way bali valve. Competency of the val ves was tested by injecting contrast media. The ultimate goal of this project is to construct and experimentally evaluate an alternative device which can be placed percutaneously, obviating the need for general anesthesia and open heart surgery. Additional studies are now in progress to evaluate the current design wiht regard to thrombogenicity, hemodinamic function, durability and biocompatibility. Key words: aortic valve-surgery, heart valve prosthesis, catheterization. percutaneous Introduction As a result of trauma, disease, or congenital abnormalities, the valves of the heart may be­come incompetent and blood can either regurgi­tate or leak back against the normal direction of the circulation. Prosthetic valves are now manu­factured which can be used to replace the natura! valves of the heart. Heart valve replacement began thirty-seven years ago when Hufnagel surgically implanted the first valve prosthesis in the descending aor- Correspondence to: Dr. Dušan Pavcnik, M.O. Asso­ciate Professor of Radiology Institute for Diagnostic and Interventional of Radiology, University Medica) Center, Zaloška 7, 61000 Ljubljana, Slovenia. Supported in part by a grant from the John S. Dunn Research Foundation and the George A. Cook Memo­rial Fund. UDC: 616.126.52-089.28 ta. 1 Since then, over twenty different mechanical and biological valves have been developed. 2-4 However, ali of the prosthetic cardiac valves used clinically have to be implanted surgically. Our objective in this project is to construct and experimentally ( canine model) evaluate an alter­native val ve which can be placed percutaneously, obviating the need for general anesthesia and open heart surgery. Materials and methods Device description The prosthetic aortic valve for percutaneous insertion includes a cage, a ring and a bali (Figure 1). The cage consists of a Gianturco self-expanding stainless steli stent5. with barbs and four to six lenghts of fiat, flexible stainless steel wire attached at one end to form the top of the cage. Pav(nik D cl a/. Cage Ball Ring Figure 1. Prosthetic aortic valves -cross section The ring is constructed of two stainless steel wires coiled together in a spring like configura­tion and covered with nylon mesh. Barbs are located at various points around the ring for stabilization following placement. The ring is attached to the cage assembly with a length of stainless steel tubing (Figure 2). The bali is a detechable latex balloon made out of the front part of 6 or 8 Foley catheters with a 3 cc balloon placed at the tip of a 5-French high-flow catheter. It is filled with liquid silicone rubber6 after placement in the cage assembly (Figure 2). For placement in dogs, the valve was made in different sizes. The cage was either 2.8 or 3.0 cm. in diameter, the outside diameter of the ring varied in 2 mm incriments from 2.4 cm. to 3.4 cm. For each valve the size of the bali was predetermined as well as the required volume of silicone (Table 1). The valve system can be collapsed and pushed through an 11-12 French catheter system (Figure 4). Table l. Predeterrnined volurne of silicone. CM Air Silicone Balloon ml ml l.O 3.0 1.8 1 + 0.8 1.2 3.2 2.1 1.3 + 0.8 1.3 3.4 2.6 1.8+0.8 1.4 3.8 2.8 2.0+0.8 1.5 4.0 3.0 2.2+0.8 1.6 4.2 3.2 2.4+0.8 1.7 4.5 3.3 2.5 +0.8 1.8 5.0 3.6 2.8+0.8 l.9 5.5 3.8 3.0+0.8 2.0 6.0 4.1 3.3+0.8 pushed frorn a 12-F sheat. Figure 3. lnflated detachable latey balloon placed at the tip of a 5 French catheter. Animal studies Initial in vivo testing of the valve has been carried out in twelve dogs. After induction of general anesthesia, the right common carotid artery was surgically isolated and an 11-12 French Teflon sheath was introduced. This sheath was advanced into the ascending aorta under fluoroscopic monitoring. / Percwaneous 1ransca1he1er p/acemenl o{ 1he aor1ic vafi,e Figure -t. C"ll.tp,cd prosthetic aortic valve within a dist;il L'rHI "r" 1 .-r-,hcath, ' Figure 5. Norm.ti aor1ogra111 = ldt latcr.tl po,ition, With the animal in the left lateral position, an aortogram was performed to determine the dia­meter of the sinus Valsalva and a_scending aorta (Figure 5), This is necessary in order to select the proper size of the prosthetic valve, Following the aortogram, the tip of the deli­very sheath was positioned in the posteJior non cornonary cusp. A small amount of contrast materal was hand injected to check the position of the sheath, and this was marked by placement of a needle in the overlying skin, The prosthetic valve was then delivered through the sheath using a pusher catheter. As the ring exited the sheath, it opened and was 'anchored against th_e annulus by the barbs. Once certain that the ring was positioned correctly, the en tire device was extruded into the aorta. The ring was placed below the ostia of the coronary arteries in such Pad11ik D e1 a/. Figure 7. a) Movernent of ti,..: bali -diastole b) Movernent of the bali -sistole a way that the cups of the aortic valve were compressed. This created a huge regurgitation and an appropriate animal model for evaluating the new device (Figure 6). The detachable bal­loon was then placed into the cage. After infla­ting a predetermined volume of silicone, it was detached from its delivery system. The move­ment of the bali was checked by fluoroscopy in an. anterior and lateral position (Figure 7). The position of the ring and the competency of the valve were tested by injecting contrast media directly above the cage (Figure 8). Results To date, we have placed prosthetic aortic valves in 12 dogs. Results of these studies have led to The first four aortic valves were placed in dogs with the ring constructed out of stainless steel coil wire and latex tubing covered with nylon mesh. Latex tubing was connected through a one-way valve with a 2-F catheter to fill the ring with liquid silicone. Ali attempts to inflate the ring with silicone failed. The ring made out of latex tubing required a 14-F delivery system. The ring was simplified. A new nylon mesh design was developed and the size of the delivery system was decreased from 14 to an 11-F cathe­ter. In ten animals, the ring was successfully placed below the ostia of the coronary arteries. Post­morten examination showed that the ring was anchored against the annuls of the heart by the barbs in such a way that cusps of the aortic valve were compressed. This created a huge regurgita­ severa! design modifications. tion and an appropriate model for evaluating the Percutaneous transcatheter placement of the aortic val ve Figure 8. Cumpctcncc of the prosthetic valve. Ring is placed below the ostia of the coronary arteries (filling defect). new device. In ali 12 cases, a huge insufficiency was created. In the first two experiments the cage was constructed out of a Gianturco Z stent, 2.5 cm in lenght x 2.5 cm in diameter, from stainless steel wire (0.018 inch) bent in a zig zag pattern and two lengths of stainless steel guide wire soldered at one end to form the top of the cage. Animal experiments showed that the cage was too long and that the guide wire was not appro­priate for the top of the cage. At necropsy, thrombus was found. It was attached to both lengths of the guiding wire. , To decrease t.e length of the cage, stents were constructed out of 0.014 stainless steel wire with 8-9 bends and l. 7 cm in length. Instead of guide wire, four lengths of fiat, flexible stainless steel wire were attached to the bends. Once the ring and cage assembly were in place, the balloon was inserted within the valve between the ring and cage. In the first nine experiments, the balloon was filled with air or contrast medium. Severa! in vitro experiments were performed with a DOW CORNING 3110 R. T. V. silicone rubber to which barium sulfate (250% WN, E-Z-Hd, E-2-EM Inc., Westbury, NY) Ethidiol (Savage Laboratories, New York), DOW COR­NING 4 catalyst and DOW CORNING 200 silicone fluid were added. These experiments led to preparation of silicone formulation consisting of 5 ml of 3110 silicone, 1 ML of 200 silicone, 0.3 ml of barium sulfate and 0.20 ml of DOW CORNING 4 catalyst. This silicone formulation had low enough initial viscosity to be injected through a 60 cm long 5 French high flow P. E. catheter with a 1 ml syringe. Polymerisation of the mixture occurred within 25 minutes after mixing and, when fully cured, had the consi­stency of soft rubber. The silicone mixture was used in three dogs for balloon inflation. In ali three cases, a small amount of air stayed trapped in the balloon. The three most recently placed valves showed excellent results for a short period of tirne. The ring and surrounding structures were sufficiently close together to prevent leakage of blood bet­ween them. In ali cases, the coronary arteries were patent. In the last dog, the bali escaped out of the cage after three hours. Migration of valves was noted in one animal. Discussion Mechanical heart valves are generally of two designs, either a ball-in-cage valve or a slim valve. At present, the ideal valve does not exist. 1 With regard to cu'rrently aviable prosthetic val­ves, the doctor and patient must · weight the advantages and disadvantages of each type of prosthetic valve. Currently, placement df prosthetic valves re­quires general anesthesia and open heart surgery before, and the recipient of the prosthetic valve :rn Pavcnik D ct al. is subjected to the inherent risk of mortality associated with surgery. A need therefore exists for a prosthetic heart valve which'does not require surgery for installa­tion and placement and, therefore, eliminates the risk associated with surgery. When we started with construction of the prosthetic valve for the percutaneous transcathe­ter technique, we thought that the valve with the ball-in--cage design would be the best to reach te goal. A factor which limits the coristruction of the prosthetic valve for the transcatheter technique the most is the size of the delivery system. Ali three parts of the valve, ring, cage and bali should be small enough to be collapsed and pushed through the catheter sheath of reasonable s1ze. At present, our prosthetic valve is a ball-in­cage design which functions as a simple one­way bali valve. The valve can be collapsed and pushed through an 11-12 French Teflon sheath. Since the assembly is self-expanding, it opens as it exits the introducer. The fina! step is deployment of the bali. The essential feature of val ve stability is mechanical fixation provided by the ring with the barbs and cage with the barbs. The ring and cage are attached by stainless steel tubings. Also assisting fixation of the valve is the stent. When fully e)(tended, a combination presure/friction effect which tends to help the stent wires in contract with surrounding tissue. The tissue in­growth may also occur to prevent migration of the valve. For good hemodynamic function of the prost­hetic valve, the position of the ring without regurgitation and periprosthetic leaks is impor­ _ tant. The use of 4-5 barbs holds the ring in place below the ostia of the coronary arteries. The ring is covered with a fine mesh of a woven. nylon material. Yoshioba et -al.7 and Uirich et al.x proved that nylon mesh can provide a framework for neointimal encasement. Our experiments de­monstrated that nylon mesh with film deposits, which occur immediately after valve placement into the animal, provides effective blood flow blockage so that the blood flow in the sistoli passes through the central opening of the ring when the bali is unseated and prevents leakage back during diastoli. Woven nylon material is thin enough so that the ring can be easily passed through an 11-F sheath. We have modified the front part of the Foley catheter for a bali. The balloon includes a simple one-way valve which allows after-inflation for detachment. The balloon was placed at the top of a 5-F high flow catheter. The catheter con­tains mandril for better pushability and insertion. After placement, the mandril is withdrawn and the air within the catheter and balloon was removed by a 20 cc syr_inge. Then the balloon was filled with a silicone mixture and detached from the catheter. Once the silicone has been injected and the balloon detached, a one way valve sealed the silicone within the latex balloon to maintain the ballon's spherical shape. In ali cases, we had a problem. A small balloon of air always stayed trapped in the bali. Conclusion The limited experience reported prevents conclu­sive opinions. However, encouraging initial re­sults from the animal trials have been one more step toward transcatheter valve replacement in humans. Additional studies are now· in progress to evaluate the current design with regard to throm­bogenicity, haemodinamic function, durability, and biocompatibility. Acknowledgments The authors wish to thank Raquel Collins, B.S. and Irene Szwarc, R. T., for their invaluable technical assistance and Peggy Bergmanson for her secretarial help in the preparation of this manuscript. Perc111aneo11s 1ra11sca1herer p/ace111e111 of rhe aorric ,·ali·e References 1. Hufnagel CA, Harvey WP, Rabil PJ, McDermott TF, Washington D. C. Surgical correction of aortic insufficiency. Surgery 1954 ;35 :673-83. 2. Rabago G. A worldwide overview of valve usage. In: Rabago G .Cooley DA. eds. Hearr volve repla­cemenl and fu1ure frends in cardiac surgery. New York, Futura Publishing Co., !ne. 1987;3-9. 3. Morse D. Steiner RM. Fernandez J. Cuide 10 Prosrheric Carcliac Valves. New York. Springer­Yerlag, 1985;24-69. 4. Cooley DA. Technique in Carcliac Surgery. Phila­delphia, Saunders, 1986: 11. 5. Wright K, Wallace S, Charnsangavej C. Carrasco CH, Gianturco C. Percutaneous endovascular stents: An experimental evaluation: Racliology 1985 ;156:68-72. 6. Mahon TG, Wright KC, Armeniades CD. lnjection of a silicone prepolymer system into the gallbladder of rabbits. lnves1iga1ive Raclio/ogy 1991; (in press). 7. Yoshioka T, Wright K, Wallace S. Lawrence Jr. D. Gianturco C. Self expanding endovascular graft: An experimental study in dogs. AJR 1988;151:673­6. 8. Mirich D, Wright K, Wallace S, Yoshioka T. Lawrence Jr. D. Charnsangavej C, Gianturco C. Percutaneously placed endovascular grafts for aortic aneurisms: Feasibility study. Radiology 1989 ;170: 1033-7. Ad,· Radio/ 011col 1992: 22---4. An injection technique for repositioning subclavian catheters Mahon T and Lawrence D, Sr. Department of Diagnostic Radiology The University of Texas M.D. Anderson Cancer Center 1515 Holcombe Blvd. Houston, TX 77030 Malposilioned central venous catheters need to be repositioned so as to avoid local toxicity from chemotherapeutic and other agents and to prevent venous thrombosis. We describe a simp/e saje and ejfective technique for repositioning silicone central venous catheters, by using a hand injection of sterile saline. It was successful in alf 9 patients in whom it was attempted, with no complications. Five catheters were single lumen and 4 were double lumen. We jee/ tirni rhis method should be attemp!ed prior to lhe use of more invasive techniques. Key words: catheterization central venous, catheters, indwelling, injections Introduction The insertion of central venous catheters is a common procedure, particularly in patients re­quiring chemotherapy, total parenteral nutrition or long term intravenous antibiotics. Occasio­nally the catheter may be malpositioned, mo.t often into the ipsilateral interna! jugular vein. The catheter can be repositioned using a variety of invasive techniques. We assessed a new mini­mally invasive method to reposition silicone catheters. Methods Ten patients presented between April 9, 1990 and June 30th, 1990 for repositioning of central Correspondence to: Dr. Thomas Mahon, New En­gland Deaconess Hospital, Harvard Medica! School Department Diagnostic Radiology, 185 Pilgrim Rd. Boston, MA 02215. L!DC: 616.141-072.2 venous catheters. A limited venogram was per­formed by injecting 10 to 20 mls of Angiovist-292 (diatrizoate meglumine and diatrizoate sodium, Berlex Laboratories) through the catheter. In one patient the catheter tip was in a tributary of the interna! jugular vein and the patient was excluded from the study. Nine patients were studied, in whom venography demonstrated in­traluminal placement of the catheter tip and a patent superior vena cava. Two catheters were inserted via the left subcla­vian vein. The tip was in the left interna! jugular vein in 1 patient and in the right interna! jugular vein in the other Ratient. The former was a single lumen catheter and the latter a double lumen catheter. Seven central venous catheters were inserted into the right subclavian vein. The tip was in the right interna! jugular vein in 3 patients, the left subclavian vein in 2, the left interna! jugular vein in 1, and the azygos vein in 1 patient. The two catheters in the left subclavian vein and one in the right interna! jugular vein were 7 French An injcction tcchnique far repositioning subclavian catheters Figure l(a). Double lumen right subclavian catheter (arrowheads) with tip in left subclavian vein. double lumen silicone catheters (Cook Inc.) the others were 5 French single lumen silicone cathe­ters (Da vol Inc.) Following the venogram, the catheter was flushed with a brisk hand injection of 5 mls of normal saline in a 5 ml syringe. Moderate effort was used over about 1 second. This was done in the erect position in 6 patients, and in the supine or semi-erect position in 3 patients. A repeat venogram was then performed to confirm sati­sfactory position of the catheter. Figure l(b). After a single injection of saline the tip (arrowhead) is in the right atrium. Results In ali 9 cases the catheter was repositioned, with its tip in the superior vena cava or right atrium. In 2 patients a second injection of saline was necessary. One was a single lumen right subcla­vian catheter with the tip in the left interna! jugular vein, the other was a double lumen left subclavian catheter with the tip in the right interna! jugular vein. There -w:as no evidence of any complications, patient discomfort or trauma to the catheters. Mahon T and Lawrence D Discussion It is important that central venous catheters are properly sited with their tips in a major central vein. The optimal position we feel is in the superior vena cava below the azygos vein. This is so as to provide maximum concentration on first pass of chemotherapy and to have a high flow rate so as to avoid local toxicity from chemotherapeutic and other agents. The malpo­sitioned catheter may also initiate venous throm­bosis if it is in a minor vein or is in a position to traumatize the vessel wall. Traditional methods for repositioning central venous catheters are invasive and may entail the use of multiple guide wires, tip-deflector wires and balloon catheters (Lois et al, 1987). In our department, catheters were often repositioned by using a deflector guidewire which was introdu­ced into the catheter. By using a hand injection of 5 mls of normal saline in a 5 ml syringe, we have successfully repositioned the catheter in ali 9 patients in whom it was attempted. The technique is quick and easy to perform, avoids patient discomfort and is minimally invasive. Olcott, et al (1989) describes central venous catheter repositioning using an injection techni­que in 3 patients. However, ali their patients had single lumen right subclavian catheters only. Furthermore, we placed the patient in the erect position, when possible, so as to use gravity to aid in repositioning. We also believe that perfor­ming an initial venogram is essential. This will confirm satisfactory intraluminal position of the catheter, the absence of thrombus and normal venous anatomy. Due to the possible risk of intravascular trau­ma, only floppy silicone catheters were injected and patients with triple lumen catheters were excluded from the study. Similarly no attempt was made to reposition long venous lines, as injection of these may traumatize the catheter. We feel that this method is a safe and useful technique for the treatment of malpositioned single or double lumen silicone subclavian cathe­ters. While it was successful in ali our patients, if it fails, other methods may then be used to reposition the catheter. Acknowledgement The authors wish to thank Patricia Eugene for manuscript preparation. References 1. Olcott E. Gordon R, and Ring E. The 1n1cct1on techniquc for repositioning central venous cathe­ters: Technical note. Cardiovascu/ar and lnterven­tiona/ Radiology 1989 ;12:292-93. 2. Lois J. Gomes A. Pusey E. Nonsurgical Repositio­ning of Central Yenous Catheters.Radiology 1987;165:329-33. Adv Radio/ On col 1992; 25-9. Properties of biodegradable polymers for 5-fluorouracil and tamoxifen microcapsules Yang DJ, Byun HS, Kuang LR, Yamashita Y, Moult RG, Tansey W, Wright KC and W allace S Division of Diagnostic lmaging, Box 57, 1515 Holcombe Boulevard, University of Texas M. D. Anderson Cancer Center Houston, Texas 77030 U.S.A. Selective intraarterial infusion of microcapsules exerls therapeutic effects through both infarction and sustained drug activity; such use of microcapsules has also been applied to chemoembolization use. This study is aimed. at developing biodegradable microcapsules which contain the widely used tumor chemotherapeutic agents such as 5-fluorouracil (5-FU) and tamoxifen (TX) for drug delivery. Poly-d, 1-lactic acid (PLA), polycaprolactone (PCL), and polycaprolactone dio/ (PCLD) were the polymers chosen for preliminary evaluation. Drug to polymer ratios of 1 :1 and 1 :3 were prepared with mean capsule size of 100 µm. We then compared the ejfects of the drug: polymer ratios and incubation tirne on in vitro dissolution rale. Our study demonstrated that the type of polymer used had a direct relationship to the amount of drug released. The following polymers are listed in descending orcler according to the effectiveness of each in facilitating the release of contenls at 48 h incubation from TX-loaded microcapsu/es: PLA > PCL > PCLD, with 5-FU /oaded microcapsules, efficacy of polymers was also rated: PCL > PCLD > PLA. The percent drug yield compared to drug: polymer ratios were, on average (W/W): from TX microcapsu/es, 14% at 1:3 and 30% at 1:1; from 5-FU microcapsules, 8% at 1 :3 and 10% al 1: 1. The resu/ts indicate that 5-FU and TX microcapsules produce sustained release propenies and are useful in tumor chemotherapy. Key words: fluorouracil; tamoxifen; capsules; infusions, intra-arterial; biodegradation Introduction The development of microencapsulation as a technique for administering sustained release dosages has been successfully applied to tumor chemotherapy. 1-4 Selective intraarterial infusion of ethylcellulose microcapsules loaded with mito­mycin C has been used in the treatment of primary or secondary carcinoma of the kidney, !iver, bone, intrapelvic and retroperitoneal or­gans. These capsules exert therapeutic effects Correspondence to: Yang OJ. Ph. D. UDC: 615.277 3.014.6.032. 13 through both infarction and sustained local drug activity during the chemoembolization process. Furthermore, microcapsules also alter the syste­mic toxicity that is produced by the parent drugs_'i-7 In this study, we will provide informa­tion on sustained release properties as they apply to different biodegradable polymers. Such information which will facilitate tumor chemo­therapy evaluation (e. g. reduce toxic effects on non-target tissues). 5-fluorouracil (5-FU), a fluorinated primidine analog, shows a close correlation with metabo­lism of nucleic acid and has been used in primary breast and metastatic !iver chemotherapy. 8 Ta­ Yang DJ et a/. moxifen (TX), a potent nonsteroidal antiestro­gen, which is more lipophilic than 5-FU has also been used in treatment of breast tumor. 9 Both drugs were chosen as model drugs in this evalua­tion. The use of biodegradable polymers for a drug delivery system has been reported. 111-1.1 Because they are non-toxic, biodegradable poly­mers provide the ideal coating for the prepara­tion of microcapsules. Anticancer drugs can then be sent to the target site in sustained dosages released by microcapsules. The release patterns for these loaded microcapsules vary according to the different polymers used. However, little is known about the release rates of various poly­mers. For that reason, three commonly used biodegradable polymers, each with different che­mical structures and properties, were chosen for preliminary evaluation: poly-d, 1-lactic acid (PLA), polycaprolactone (PCL), and polycapro­lactone-diol (PCLD). PLA is a weakly ionic polymer. whereas PCL and PCLD are more non-ionic in character. In order to examine the release properties of these polymers, we used drug: polymer ratios of 1: 1 and 1 :3 (w/w) and a mean capsule size of 100 µm. Materials Poly-d. 1-lactic acid (M. W. 2000-50000), polyca­prolactone (M. W. 35000-45000), polyvinyl alco­hol and polycaprolactone diol (M. W. 3000) were purchased from Polysciences Inc. (Warrin­gton. PA). Tamoxifen and 5-FU were purchased from Sigma Chemical Company (St. Louis, MO). Preparation of microcapsules The microcapsules were prepared using the sol­vent evaporation method, with drug: polymer ratios of 1: 1 and 1 :3 (W/W) and polyvinyl alcohol as an emulsifier. Drugs were dissolved in methylene chloride. then added with the emulsi­fier to a stirred at emulsifier 400 rpm water solution. After 6 h, the capsules were washed with water and dried in air. The capsules ranging in size from 106 to 212 µm in diameter were collected from mesh screens. Microscopic examination The microcapsules were observed with a binocu­lar research microscope (Zeiss, Germany). The microcapsule's size was measured by an eyepiece micrometer. Following the observation by the light microscope, microcapsules (100 µm) were scanned with a scanning electron microscope to eval uate the surface characters of microcapsules. Assay of the drug content Microcapsules (5 mg) were weighed and dissol­ved in 5 ml of methanol. The solution was centrifuged and the supernatant (100 µ.!) was diluted with methanol (3 ml) and analyzed spec­trophotometrically at 238 nm by a single beam spectrometer. A standard curve was produced using the same procedure by adding known amounts (2 mg) of both TX and 5-FU. The drug content was calculated as a percent of the total capsule weight. The experiment was performed in triplicate. Dissolution studies A set of capped test tubes was filled with 5 ml phosphate buffered saline solution of pH 7.4 (O.OS M) and placed in a water bath shaker sel at 100 rpm with a constant temperature of 37°C. Microcapsules (5 mg) were added to each test tube, and sample solutions of 3 ml were collected at different tirne intervals during centrifugation. After each determination, the sample solutions were returned to each test tube. The concentra­tions of the drug released from microcapsules were determined by comparison with the stan­dard drug (2 mg) in the same dissolution solution for the controls and measured spectrophotome­trically at 238 nm. The experiment was perfor­med in triplicate. Statistical analysis A student's t-test 1 -1 was used to compare the significant difference (p < O.OS leve!) between the sample at 1 h and the corresponding sample at different incubation tirne interval. Properlies of biodegrac/a/,/e polymers for 5-.fl11oro11racil a11d 1,11110.rif'<:11 111icroacaps11/es Results and discussion In general, the encapsulated yield from the TX microcapsules was 30 to 36% far the 1: 1 group and 13 to 23% far the 1 :3 group; yield far S-FU microcapsules was 8 to 10% far the l: l group and 6 to 9% far the 1:3 group (Table 1). Since S-fluorouracil is hygroscopic and hydrophilic, some drugs may diffuse into water from the capsules during the encapsulation process. The. refore, the yield of S-FU loaded microcapsules is lower than that of TX microcapsules. Scanning electron microscopy showed that ali the micro­capsules prepared were spherical in shape with smooth outer surfaces (Figure 1). Although the release rate of TX from the PCLD capsules at 48 h the different drug: polymer, there is a similar pattern of release. The release rate of TX at 48h incubation tirne was faund to decrease in the order: PLA > PCL > PCLD (1:1) (Figure 3). The release rate far PCL capsules was slightly faster than PLA in 1 :3 group at 24 and 48 h. The release rate of TX from the PCLD capsules at 48h incubation time was 10% which remained significantly (p < O.OS) lower than far PLA and PCL microcapsules. In general, TX was released from PLA capsules at a rate of 3S% at 48 h incubation tirne far both 1: 1 and 1:3 ratios. The drugs in the standard had already dissolved by that time. The release rate of S-FU microcapsu­les at 48h incubation time was faund to decrease in the order: PCL > PCLD > PLA (Figure 4). 5-FU was released from PCL capsules by 4S% far the 1 :1 and 60% far the 1 :3, a yield that was significantly higher (p < O.OS) than the yield far PLA and PCLD capsules. The differen­ces among the release rates of the capsules could be attributed to the lipophilic character of the drugs; TX is more lipophilic than is S-FU. The properties of various polymers also my alter the release rates. Figure l. Scanning clcctron microscope PCL micro­capsules loaded with 5-FU at the rations of 1 :1. Our preliminary studies indicate that biodegra­dable polymers can be used to prepare sustained release microcapsules of hydrophobic -and hy­drophilic-type drugs. A lipophilic drug coated with PLA showed a faster release rate than did the same drug coated with other polymers; a hydrophilic drug coated with PCL similarly sho­wed a faster release rate pattern than when other polymers were used. This faster release rate would be appropriate when administering the initial dose of the sustained release farmulation. Table l. % of drug contents in microcapsules. DRUG POLYMER DRUG: POLYMER 1:1 1 :3 Tamoxifen Polylactic acid Polycaprolactone Polycaprolactone diol 30.0 30.7 36.4 22.5 13.0 14.9 5-Fluorouracil Polylactic acid Polycaprolactone Polycaprolactone diol 8.8 9.9 7.6 8.5 6.6 7.6 Yang DJ et a/. S•FU Microcapsules(l :1) Tamoxifen Microcapsules(1 :1) so,---------------­ JO ¦ PU. 20 Raleaud ra Pa. ¦ FCU) 10 ,. .. 20 Tamoxilen Microcapsulcs(l :3) so ,o .. JO ¦ PU. n,1,u,d r.i Pa. E!I FCU) 20 10 ' ,. .. J 20 El,pnd llme(hourt) Figure 2. Release rate of microcapsulcs loaded with tamoxifen. We believe that the sustained release proper­ties of both 5-FU and TX microcapsules demons­trate potential for use in tumor chemotherapy. The information in this study would be useful in predicting the sustained release properties of drugs and reducing the toxic effects on non-tar­get tissues. Acknowledgements This work is supported in part by John S. Dunn Chair in Diagnostic Radiology, John S. Dunn Foundation and Cancer Fighters of Houston. We thank Dianne Perez Onuogu for preparing the manuscript. References l. Bechtel W, Wright KC. Wallace S, Mosier B, Mosier D, Mir S, Kudo S. An experimcntal evaluation of microcapsules for arterial chemoem­bolization. Radiology 1986; 161 :601-4. .. JO l!I FCU) R•leaud 13 Pa. ¦ PU. 20 ' . ,o 2• .. 5-FU Microcapsulcs(1 :3) 80 GO ¦ PLA Released 13 PO. S] PCLD .. i 1 1 1 1 1 20 .l . lJIJlalJ J ' ,o ,. " Elapsod tlmo\hour,) Figure 3. Release rate of microcapsules loaded with 5-FU. 2. Brunning JL. Kintz, BL. In: Computational Han­dbook of Statistics. Scott, Foresman and Compa­ny, Glenview, Illi .. 1977; 13-5. 3. Jalil R. Nixon. JR. Microencapsulation using poly ( 1-lactic acid) I: Microcapsule properties affected by the preparative technique. 1 Microencapsula­tion 1989; 6(4):473-84. 4. Kato T. Nemoto R, Mori H. Kumagai I. Sustai­ned-release properties of microencapsulated mito­mycin C with ethylcellulose infused into the rcnal artery of the dog. Cancer 1980; 46: 14-21 . 5. Kato T, Nemoto R, Mori H. Takahashi M, Ha­rada M. Arterial chemoembolization with mitonw­cin C microcapsules in the treatment of primary iir secondary carcinoma of the kidney. liver, bone and intrapelvic organs. Cancer 1981 ; 48 :674-80. 6. Kato T. Nemoto R. Preparation and properties of microencapsulated mitomycin C. IRCS Medica/ Science 1978; 6:31 l. 7. Leelarsamee N, Howard SA. Malanga CJ. Ma JKH. A method for the preparation of polylactic acicl microcapsules of controlled particle sizc and drug loading. 1 Microencapsulation 1988; 5(2): 147-57. 8. Leclarsamcc N, Howard SA, Ma JKH. Effcct of surface active agents on drug releasc from polylac­tic acid hyclrocortisone microcapsules. J Microen­capsulation 1988; 5(1):37-46. Properties of hiodegradahle polv111ersjrH 5-j/11oro11racil a11d 111111oxi(e11 111icroarnps11/e.1· 9. Loscr R. Seibel K. Roos W. Eppnbcrgcr U. In vivo and in vitro antiestrogenic action of 3-hydro­xytamoxifen. tamoxifen and 4-hydroxytamoxifcn. Eur J Cancer Ciin Oncol 1988; 21(8}:988-90. 10. Miyazaki S. Hashiguchi N. Sugiyama M. Takada M. Mori moto Y. Fibrinogen microsphercs as novel drug delivery systems for anticancer drugs. Che111 Pharm 811/11986; 34:1370-75. 11. Spcnlehauer G. Yeilland M. Bcnoit J-P. Forma­tion and characterization of cisplatin loadcd poly­ d. 1-lactidc microspheres for chcmoembolization. J Pharrn Sci 1986; 75:750-5. 12. Tsurumi Y. Kamcyama M. lshiwata K. Katakura R. Monma M. Ido T. Suzuki J. F-18-Fluoro-2-dco­xyuridinc as a tracer of nucleic acid metabolism in brain tumors. J Ne11ros11rg 1990; 72: 110-3. 13. Wallace S. Chung YP. Carrasco CH. Charnsanga­vej C. Bechtcl W. Wright KC. Gianturco C. Physio-anatomic conccpts and radiologic tcchni­ques for intraartcrial delivery of therapcutic agents. Cancer B11// 1984; 36:6-14. 14. Wright KC. Wallacc S. Mosicr B. Mosicr D. Microcapsules for arterial chemoembolization: ap­pearancc and in vitro drug rclcase charactcristics. J Microencapsulation 1988; S( 1): 13-20. Adv Radio/ Oncol 1992; 30---4. Intravenous thrombolysis of acute coronary occlusiou-after percutaneous transluminal agnioplasty Kranjec I, Cijan A, Pavcnik D Department of Cardiology, University Medica[ Centre, Ljubljana Institute for Diagnostic and lnterventional Radiology, Ljubljana Thrombolytic therapy can restore vessel patency in some patients with acute coronary occlusion after percutaneous transluminal coronary angioplasty. A case is presented in which prolonged intravenous application of thrombolytic agent appeared as effective and safe as intracoronary treatment. The potential use of fibrin-specific enzymes in simi/ar circumstances is discussed. Key words: coronary thrombosis, angioplasty, transluminal, percutaneous coronary thrombolytic therapy lntroduction Acute coronary occlusion (ACO) of the dilated vessel is the most apprehended complication after percutaneous transluminal coronary angio­plasty (PTCA) as it may lead to myocardial infarction or even death. ACO develops mostly within the first 24 hours after the procedure. In a recent series at the University Hospital of 1 Geneva it occured in 4% of PTCA procedures, compared to a rate of 5% seen in our last 100 patients. A variable combination of coronary wall dissection, thrombosis and vasoconstriction is involved in the pathogenesis of ACO. Therefo­re, mechanical recanalization, thrombolytic the­rapy and nitrate administration have been propo­sed as measures to improve the jeopardized coronary perfusion. Repeated PTCA is conside­red the treatment of choice, especially if the guide wire is stili in position. Intracoronary Correspondence to: Igor Kranjec M.D. Kotnikova 19, 61000 Ljubljana, Slovenia. UDC: 616.132.2-005.6-089 application of thrombolytic agents may prave effective if PTCA or other mechanical means fail to achieve durable patency and when rapid transfer for bypass surgery is not necessary. However, this type of therapy is tirne consu­ming, 1 the success may be difficult to assess, 2 and a significant haemostatic defect with a blee­ding potential is often induced.' Despite the aforementioned disadvantages severa! published articles have confirmed the efficacy of the intra­coronary streptokinase4 or urokinase5 in resto­ring patency of the occluded coronary artery. In this report we describe a case with ACO after PTCA in which the thrombolytic agent was applied first into the coronary artery and then also intravenously. Case report , A 58--year-old man was referred to our hospital for coronary angiography. Five months before the admission he began to experience a chest discomfort occuring initially with physical acti­vity and in the last two weeks also at rest. His Inrravenou.1· thrombolysis of acute coronary occlusion after percwaneous translwninal angioplasty ,,-,:";''.._llf-[IJI• ,., -l' -t-•-+:' rt -t-• 1 Ai· ta- I+ mi 1 1 I ;.:::J:T".:llL Figure l. ECG tracing_ at the hospital admission showing Q-waves and inversion of T-waves in inferior leads: Figure 2. Left anterior oblique angiogram of the right coronary artery before the PTCA. There is 83% stenosis in the midportion (arrow) and 76% in the 120 sec (normal values 7-11 sec) and fibrino­gen was unmeasurable toward the end of throm­bolysis (normal values 1.6-4.5 g/1). The chest discomfort soon disappeared and the ECG tra­cing reverted to the previous pattern again (Fi­gure 8). However, the total plasma CK activity rose to 7 .61 µkat/1 (normal values 0.17-2.08 µkat/1) with 8% of the MB-CK fraction. These findings indicated a small inferior wall reinfarc­tion. The fina! coronary angiography performed next day revealed a patent RCA with narrowings of 57% and 47% in its medial and _,.,, ... 4.: ,. <#i.-."; 7,1 ·:.--.-... .:!') ..'{/'. .i .. ., .) i,:· ....... :. '. . 'll.: . ' ­ ,. .. .·.ii:'f ;;.·:.:. ,. l»fi . ľ'.(·-< f, .• ·1;. .. 4;(..'.; ,w •.• +t;,,.;>-.... . ;_ . • .t':'<;,.,;-. •· • ... } ... : '-'· Figure 2. TI W (a) anJ T2W (bJ axial i111agcs or hc111orrhagic cystic formations in sol\ tissuc parl of thc <:".._ '--•• // '-.. .// ·, • ./ -----./ .• / o + + o ++ Contraction degree Graph l. The curves of gallbladder contractions at 30,45 and 60 min after stimulation by two dried eggs. Number 40 \ \ \ \ \ \ 30 \ \ \ \ \ \ o--·-· -.-l_o_ 20 \ .. ._ . \ .. --,--.l.---60 C \ ;· . ' ....... ................. ....... '<>._ -..... 10 ..... --..... \45 30 + ++ o Contraction degree Contraction degree Graph 2. The curves of gallbladder contractions at 30,45 and 60 min after stimulation by two fresh egg yolks. :-:umber 40 . \ \ \ \ 30 \ \ 0 45 # \ :.-----\------·,, //60 20 / \ '-/ . / ', ·, 0 ,..;L.. __ •30 10 ·­ o + ++ Contraction degree Graph 3. The curves of gallbladder contractions at Graph 4. The curves of gallbladder contractions at 30,45 and 60 min after stimulation by 25 gr »Eurocre­30,45 and 60 min after stimulation by 10 gr chocolate. ma«. Miric Sand Sabljak I fn the first group of 78 patients, two dried eggs were ingested as astimulatory meal. The degree , of gallbladder contraction and tirne of the evalua­tion of contractility is presented in Table 1 and Graph l. The greatest number of the most intensive contractions 44/78, was obtained 1lfter 60 min,. but the curve of the contractions intensity with the least oscillations was marked after 45 min. In the second group of 78 patients, two fresh egg yolks were ingested as a stimulatory meal. The degree of gallbladder contraction relating to the tirne of 30,45 and 60 min. is presented in Table 2 and Graph 2. The greatest number of the most intensive contractions 38/78 was obtai­ned after 60 min. The most optiinal arrangement of the degree of contractions is evidented from the curve after 45 min. In the third group of 75 patients ingesting 25 gr of »Eurocreme« for stimulation, the low degree of gallbladder contractions was eviden­ted. The greatest number of cases 40/75 without contractions and the least number 7/75 with the highest degree of contractions were evidented after 30 min (Table 3, Graph 3). In the fourth group of 75 patients 10 gr of chocolate was ingested as a stimulatory meal. The obtained degrees of contractions are presen­ted in Table 4 and Graph 4. Gallbladder contrac­tions were higher than by »Eurocreme« stimula­tion, but lower than by eggs stimulation. Discussion Gallbladder contractions and emptying in pero­ral cholecystography improve the presentation of gallbladder and visualization of small stones, polyps in hyperplastic cholecystosis, gallbladder deformities, acalculous inflammations, as well as functional and organic disorders of the Oddi's sphincter. 14-16 Echography has enabled a rapid and simple imaging of the structural pathologic changes in dependently from the possibility of contrast pre­sentation of the gallbladder, which is limited by many other factors. In normal structural findings of the gallbladder a change of contractions and emptying may be caused by the acalculous and unmanifest inflammation or functional changes 7 911 17 18 of the gallbladder and biliary ducts. 5•••-• According to Hopmann (1.85), the effects of gallbladder contractions after a fatty stimulatory meal were not significantly different relating to the intravenous use of cholecystokynin, whi°ch was used by many authors. 5•12 •19•20 Ultrasonographically, for the evaluation of changes in size, some authors measure the vo­lume by method of the sum of cylinders using computer data processing. 10•12 ·19 The other aut­hors report that the measuring of volume is as successful as by the method of calculating the volume of elypsoide. 11 •21 Besides, there are aut­hors who evaluate the gallbladder size, based on the length and width of gallbladder shadow20 or its cross section. 5 In our out-patient investigation, there was no possibility of the computerized calculation of volume. Being identical with the roentgenologic image after opacification by contrast material, visual evaluation of the size at echography can be used as a substitute for roentgenologic evalua­tion of g_allbladder contraction. Figure 1 shows a medium gallbladder contraction (»+«) after sti­mulation. Figure 2 shows a mild (»-«) contraction, Figure 3 very marked (»++«) contraction. Gall­bladder in Figure 4 failed to show the signs of contraction (»O«). Measuring the length and width of the greatest section in millimeters, the evaluation of contrac­tion degree, agreed with the visual evaluation of gallbladder size changes. Measurement can re­place imaging in the cases of the Jack of films. The stimulatory meals used: dried eggs, fresh egg yolks, »Eurocreme« and chocolate, were selected because of the availability and possibi­ lity to fi_nd them in the town and its surroundings. Conclusion The changes in gallbladder emptying and con­traction intensity are indicative for the diagnosis of the structural and functional pathologic condi­tions of the gallbladder. Echography enables, a Parametcrs for echographic e\'C1/uation of gallh/adda contractions rapid and simple evaluation of the size changes after stimulatory meal, or measuring the width and length in the image of the cross section. The parameters for evaluation of the contraction intensity are the kind of stimulatory meal and tirne of evaluation of the degree of contractions after stimulation. On the basis of performed investigation, the best contractions were obtai­ned after the ingestion of two fresh egg yolks, and the most optimal tirne for evaluation of the degree of contractions is 45 min. after stimula­tion. References 1. Boyden EA. Study of behavior of human gallbla­dcr in rcsponsc to ingcstion of food. Anat Rec 1926; 33:201-39. 2. Sussman ML. Emptying of normal gallblader. AJR 1937; 38:867-71. 3. Ivy AC. Motor dysfunction of biliary tract; analy­tical and critical consideration. AJ R 1947; 47: 1-11. 4. Everson GT, Braverman DZ, Johnson ML, Kern F Jr. Acritical evaluation of real-tirne ultrasono­graphy for the study of gallblader volume and contraction. Gastroenterology 1980; 79:40-6. 5. Davis GB, Berk RN, Scheiblc FW, Witztum KF, Gilmore IT, Strong RM, Hofmann AF. Cholecy­stokinin Cholecystography, Sonography, and Scin­tigraphy: Detection of Chronis Acalculous Chole­cystitis. AJR 1982; 139: 1117-21. 6. Marzio L, Di Giammarco AM, Schiavone C. Curccurullo F. Realtime ultrasonography of gall­blader emptying in normal subjects. U/trasound in med and bio/ogy 1982; 8:123. 7. Kinugasa T. Fukano S, Sekimoto K. An operative indication of asymptomatic gallstone using real­timc ultrasonography. Ultrasound in Med wul Bio/ogy 1982; 8:%. 8. Ono V, Tomiyama H, Ochiai Y. Ito T, Shinagawa A, Kawashima Y, Honda T. Observation on gallblader contraction by means of an ultrasound electron scanncr. Ultrasound in Med wul Biolog_,. 1982; 8:145. 9. Bobba VR, Krishnamurthy GT, Kingston E, Tur­ner FE, Brown PH, Langrell K. Gallbladaer Dynamics Induced by a Fatty Mcal in Normal Subjects and Patients With Gallstones. J Nuc/ Med 1984; 25:21-4. 10. Hopman WPM, Kerstens PJSM. Jansen JBMJ. Rosenbusch. Lamers CBHW. Effect of Graded Physiologic Doses of Cholecystokinin on Gallblad­der Contraction Measured by Ultrasonography. Gastroenterology 1985; 89: 1242-7. II. Kishk SMA, Darwesh RMA, Dodds WJ, Lawson TL, Stewart ET. Kern KM. Hassanein EH. Sono­graphic Evaluation of Resting Gallbladder Volume and Postprandial Emptying in with Gallstones. AJR 1987; 148:875-9. 12. Masclee AM, Hopman WPM. Corstens FHM. Rosenbusch G, Jansen JBMJ, Lamcrs CBHW, Simultaneous Mcasurement of Gallbladder Emp­tying with Cholescintigraphy and US during Infu­sion of Physiologic Doses of Cholecystokinin: A Comparison. Radiology 1989; 173:407-10. 13. Miric S. Cengic F, Linccnder L. Pamucina P, Bajgoric M, Klancevic M. et al. Ehografska proc­ jcna kontraktilnosti holccistc In: Glas CCCLXIII. Srpska akademija nauka i umjetnosti, Odjcljenjc medicinskih nauka. Beograd 1991; 40:133-9. 14. Berk RN. Oral cholecystography. l. Berk Rn, Fcrrucci .JT Jr. Leopold GR cd. Radiology of the gallbladder wul hile duct.1·. Philadelphia. Saunders. 1983; 83-163, 15. Laufer L Gledhill L. The value of the fatty meal in oral cholccystography. Radiology 1975; 114:525­7. 16. Harvey IC, Myo T. Low-Becr TS. The value of the fatty meal in oral cholccystography. Ciin Ra­dio/ 1976; 27:117-21. 17. Weinberger H. Blumhagen JD, Odeli JM. Gall­bladder Contraction in Biliary Atresia. AJR 1987; 149-402. 18. Goldstein F. Grunt R, Margulies M. Cholecystoki­nin Cholecystography in the Differential Diagnosis of Acalculosis Gallbladder Disease. Am J Dig Dis 1974; 19:835-49. 19. Hopman WPM, Rosenbusch G, Jansen JBM. de Jong AJL. Lamers CBHW. Gallbladder Contrac­tion: Effects of Fatty Meals and Cholecystokinin. Radiology 1985; 157:37-9. 20. Nathan MH. Newman A. Murray DJ. Normal Findings in Oral and Cholecystokinin Cholccysto­graphy. JAMA 1978; 21:2271-2. 21. Dods WJ, Gron WJ. Darweesh RMA. Lawson TL. Kishk SMA. Kern KM. Sonographic Measurc­ment of Gallbladdcr Vol ume. AJR 1985; 145: 1009­1. Adv Radio! Oncol 1992; 96-8. The treatment of appendiceal abscess by ultrasonically guided drainage Drinkovic I, Brkljacic B, Boko H, Odak D, Vidjak V, Anic P University Hospital »Dr O. Novosel« Zagreb Center for Ultrasound Appendiceal abscess is one of the possible complications of surgically non-treated acute appendicitis. It is presented by palpable, tumourous mass, usually in right lower quadrant, local tenderness, fever and leukocytosis. Therapy of the disease consists in abscess drainage, antibiotics therapy and postponed surgical operation. In the treutment of appendiceal abscess we use ultrasonically guided percutaneous thin catheter drainage which has considerable advantages in comparison with surgical drainage, and its proper performance enhances success of the procedure. Key words: appendicitis; abscess-therapy; drainage; ultrasonography Introduction Acute appendicitis is the most common abdomi­nal surgical emergency. It results from bacterial infection of the vermiform appendix. Contribu­tory factors include intraluminal obstruction by fecaliths, lymphoid hyperplasia, rarely by parasi­tes, or even a carcinoid tumour, or a tumour in the coecum. O,bstruction is followed by inflam­mation which causes edema and ischemia in ali layers otappendix and can progress to gangrene and perforation as a result of increased intralumi­nal pressure. Perforation may occur early in the course of the disease (within 24 to 48 h) and may produce either a localized or general peritonitis which is the result of non-treated peritonitis. 1 The treatment of appendiceal abscess can be conservative by administering antibiotics, or Correspondence to: Doc. dr. sci. Ivan Drinkovic, KB »Dr O. Novosel«, Zajceva 19, 41000 Zagreb, Croatia. UDC: 616.346.2-002.3-089.48:534-8 agressive -surgical or ultrasonically guided drai­ 23 45 nage. •••In our department, ultrasonically gui­ded percutaneous thin catether drainage is being performed for the last four years in the treatment of apendiceal abscesses. 6 Material and methods Ultr'asonically guided percutaneous appendiceal abscess drainage, using thin catether was perfor­med in 21 patients, in the period of 4 years. Ali patients were hospitalized on the Department of Surgery, University Hospital »Dr O. Novosel«, Zagreb. Their age was from 29 to 62 years. Ali abscesses were localized in the region of right lower abdominal quadrant. The smallest abscess was measuring 3 cm in the longest diameter, and the biggest was measuring 15 cm. Ultrasonic images of abscess contents were sonolucent, representing liquid, as seen in Figure l. Drainage was performed by using 5 F pigtail catheter. After local anesthesia and skin incision The treatment of appendiceal abscess by ultrasonically guided drainage cathcter was positioned by trocar technique and tip of the cateteher was constantly monitored by ultrasound, as scen in Figure 2. Duration of drainage, employing continuous negative pressure, was up to seven days. Material obtained from abscess is routinely being subjec­ted to culture (antibiogram) analysis. Ultrasonic equipment we used had linear, convex and sector probes of 5 MHz, 6 MHz and 7.5 MHz, respecti­velly. Results Of 21 patients in whom appendiceal abscess drainage was performed, pus was obtained and successfully evacuated in 16 patients. In 5 pa­tients it was necessary to perform additional isotonic solution lavage through the catheter. Serious complications were not observed. One catheter displacement occured a day after proce­dure, but reintervention was not necessary. Ali patients were under antibiotic therapy. Sanation of abscess was accomplished in ali patients, and deffinitive surgery was performed in the period of up to 3 months. Discussion Acute appendicitis in its natura! course has three phases. In up to first 8 hrs after luminal obstruc­tion integrity of appendiceal wall is being gra­dually !ost. Between 2-24 hours of the disease course occurs inflammation, and after that com­plications in the forms of diffuse peritonitis or appendiceal abscess can take place. Appendiceal abscess presents itself. following symptoms of acute appenciditis, by palpable tumourous mass, usually in right illeal fosa, local tenderness, fever and leukocytosis. The patients are usually in bad general condition. While acute appendicitis requires operative treatment, occurence of appendiceal absccss re­quires drainagc, since antibiotics therapy alone is not always sufficient. When appendectomy is deffered under thcse circumstances, it may bc performed a number of weeks after recovery from the acute process. 1 Treatment of appendical abscess by surgical drainage can be accompanied with some compli­cations2 · 3, whereas the ultrasonically guided per­cutaneous thin catheter drainage has the advan­tage of only local anesthesia and thin catheter causes less traumatization. Also trocar technique of catheter implacement reduces duration of the whole procedure to the period of only few minutes. 4• 5 Ultrasound enables visualisation of the exact Iocalization of abscess and provides information on abscess contents on the basis of echographic st1ycture of abscess interna! echos. Figure l. Ultrasonic imagc of inflammcd appcndix with thick appendiceal wall and large sonolucent adja­cent abscess formation with some interna! echoes. Drinkovic I et al. Once placernent of the catheter is perforrned succesfully, drainage of pus and gass pressure dirninishrnent cause evident irnprovernent of pa­tients general condition, as well as reduction of fever and pain. 2•3 . 4 This rnethod is quick, cheap and without cornplications that rnight follow surgical draina­ge. Longer illness duration occuring with only conservative treatrnent is reduced as well. It is neccessary to ernphasize that cornplete sanation of process cannot be achieved by this rnethod, and appendectorny in the period of up to three rnonths is rnandatory, because of the possibility of recurrence and_ its cornplications. 1 We conclude that ultrasonically guided percu­taneous thin catether drainage of appendiceal abscess is relatively new and successful rnethod in the treatrnent of this disease, which enables us to observe progression of abscess and to deter­rnine tirne to perforrn drainage. Uncertain result of antibiotic therapy and cornplications that rnay accornpany surgical drainage are therefore being avoided, and patients condition is being irnpro­ved. The rnethod can be applied in ali hospitals that are equipped with adequate ultrasonic equiprnent. References l. Berkow R, ed. The Merek manual of diagnosis and therapy. 15th ed. Rahway NJ Merk & Co .. 1987, 757-9. 2. Glass C et al. Drainage of intraabdominal abscesses. Am J Surg 1984; 147:315-7. 3. Gary R et al. Percutaneous abscess Drainage. Ga­strointest Radio! 1985; 10 :79-84. 4. Froelich E et al. Ultrasound guided percutaneous drainage of abdominal abscesses. Radio! lugosl 1990; 24:393-5. 5. Puylaert J. The appendiceal mas: new perspectives by ultrasound. Proceedings of the 17th international congress of radiology, Paris, 1989. 6. Drinkovic I et al. New catheter set for US-guided procedures. Radio! Iugosl 1990; 24:409-11. Adv Radio/ On col 1992: 99-10 l. Ultrasound in diagnostics of muscular injuries Vidjak V, Drinkovic I, Brkljacic B, Krmpotic T, Boko H, Odak D University Hospital »Dr. O. Novosel« Zagreb Center for Ultrasound Ultrasonic investigation of 38 patients with muscular injury of thigh, referred to our department was performed. Ali patients were sportsmen, aged from 21-43 years. Ultrasonic findings were arranged in five categories according to the extent and echostructure of visualized alterations. The most often encountered pattern of echogenicity, in changes observed within 4 days after injury, showed anechoic, inhomogenous intramuscular areas with muscular contours irregularity. The authors conclude that ultrasound can, in wide extent, provide information about type and extent of trauma and about s11cce.1jii/ness of treatment of musc11/ar injuries of thigh. Key words: muscles-inj uries; ultrasonography Introduction Ruptures and minor muscle injuries have be­come more common with expansion of sports, and are of special interest for both sportsmen and phyisicians. Muscular injuries are the conse­quence of direct hit, muscular hyperextension or puncture. Clinical recognition of ruptures is ba­sed on following symptoms: pain in the site of injury, pain on palpation, painful active and. passive movements, swelling that may evolve into tumorous formation, as well as functional impairment. 1.2 Ultrasound provides insight into normal ana­tomy of extremities, as well as into pathological alterations. It is possible to delineate muscular contours, by visualizing fascia. Longitudinal and Correspondence to: Dr. Vinko Vidjak, KB »Dr O. Novosel«, Zavod za radiologiju, Zajceva 19, Zagreb, Croalia. transverse sections enable visualization of con­tours and interna! muscular echostructure. 3A Ultrasonic images of traumatical muscular in­juries are cathegorized in three levels (according to Burgeois)3 · 5 : I: no changes visible by ultrasound; II: small rupture areas, measuring less than 2 cm in diameter, with inhomogenous interna! echostructure; III: rupture visible as irregular anechoic zone, measuring more than 2 cm in diameter, with concomitant major muscular injury. Soft tissues of thigh are very often exposed to traumatic injuries. We present cases of examined patients, referred to our ultrasonic department in the period of three years, on clinical suspicion of thigh muscles injuries. Material and methods In the period of 3 years, 38 patients with muscu­ UDC: 616. 74-00 l.4-073 :534-8 lar trauma of thigh were examined in our depart­ Vidjak Vet al. ment, ali male, aged from 21 to 43 years, referred to us by the sport-medicine specialist. after clinical examination. Patients were ali acti­ve or former 7 sportsmen. Ultrasonic examina­tion was performed by General Electris -RT 4000 machine, having linear -7.5 MHz probe. The examinations were performed by longitudi­nal and transverse scanning of injured muscles and of the contralateral thigh. Results Ultrasonic findings were cathegorized into five types: 1) Seven patients had anamnestic data of in­jury within 4 days previous to examination, and some clinical indicators (pain on palpation) as well, but it was not possible to visualise ultrasoni­cally any pathological signs in muscular echo­structure. 2) Nineteen patients with anamnestic data of injury within 4 days previous to examination had ultrasonic findings indicating certain muscular irregularity with focal anechogenic areas contai­ning no interna! echoes (Figure 1). These patients had haematomas, measuring up to 2 cm. In 16 patients, injury of m.vastus lateralis was found, and in 3 patients m.rectus femoris injury was found. 3) In two patients large muscular .separa,tion was found, represented ultrasonically by the _ wider hypoechoic zone with almost no inter_nal echoes, and clear muscular contou"rs delineation (Figure 2); the finding was a result of fresch haematoma caused by muscular rupture and ultrasonically measured dimensions were bigger than 2 cm in diameter (46 mm and 39 mm, respecitvely). In both cases m.rectus femoris was traumatized. 4) In eight patients, who were examined. 3-5 weeks following trauma, inhomogenous, predo­minantly hyperechoic area inside the muscle was found. Delineation was. vague (Figure 3). The fin­ ding represented organized haematomas. Muscu­lar contours delineation was unclear, and dimen­sions of haematomas were smaller than 3 cm in Figure l. Ultrasonic irnagc showing arca of rnuscular irrcgularity with srnall focal ancchogcnic arcas. Figure 2. Ultrasonic irnag_c of widc hypocchoic zone with no interna! echoes, and clear muscular contours dclincation. Ultraso1111d in diagnostics ofmuscular injuries diameter. Four patients had m.rectus femoris and four m.vastus lat. injury. 5) In two patients, no ultrasonic findings were visualized which could correlate with site of injury occuring 9-12 months prior to examina­tion. Discussion Ultrasonic examination proved to be uscful met­hod in confirmation of existence or nonexistence of posttraumatic haematomas, as well as for differentiation with other processes that may mimic haematoma (abscess) in some degree. Using ultrasound it is possible to categorize muscular injuries in three forms, according to Burgeois. We formed simillar elassification ad­ding cathegories of injuries examined few weeks Figure 3. Ultrasonic imagc of inhomogcnous, prcdo­ minantly hypercchoic arca insidc thc musclc with after trauma, as well as old trauma with no vaguc dclincation 'of muscular contours. visible ultrasonic changes, stili existing. Standard radiological methods offer little data References about traumatic muscular alterations. while CT provides valuable data but is eostly and often l. Siwck CW. Rao JP. Rupturcs of thc cxtcnsor inaccessible, not to mention harmfulness of ioni­ mcchanism of thc kncc joint. J Rone Joint Surg 1981; 63:932-7. zing radiation. 2. Robbins SL. l'awlogijske os1101·e bolesti. Zagreb: Školska knjiga, 1985-; 15..\1-99. Ultrasound provides possiblity of insight into 3. Bourgcois JM, Wagner P, Harris A. Ultrasonograp­ state and type of muscular injury, thc degree of hic evaluation of musclc injurics in sportsmen. J Ultrasound Med 1983; 2:32. injury and successfulness of treatment. Apart of 4. Fornagc BO. Touchc DH. Segal P, Rifkin MD. that, ultrasound is easily accessiblc, cheap and Ultrasonography in thc cvaluation of musculoscclc­tal traurna. J Ultrasound Med 1983; 2:5..\9-54. simple to perform, presenting no harm to the 5. Matasovic T. ct. al. Ultra:\'1/l'na dijag11os1ika s11­patient. s1m·a za krewnje. Školska knjiga 1988. Adv Radio/ Onco/ 1992; 102-8. Ultrasound as a diagnostic tool in malignant bone tumours Babic M and Matovinovic D Special Clinical Orthopaedic Hospital -Lovran Morbidity of malignant osseous tumours has been present in orthopaedic casuistics with 0,001%. in the available literature we could not find information about the application of this diagnostic method in detecting the intraosseous tumours. The work connects JO years of experience that has been made while working with this casuistics. Twenty-five persons were examined with intraosseous malignant formations. Forty healthy persons were »controling group«. Standard linear probes of 3,5 MHz and 5 MHz were used. The examinations were performed in the transversal and longitudinal axis of the bone. The echophenomenal degree was observed, and according to the results obtained, it was possible to speak about the intraosseous tumour existence. Tumour smaller than 1 cm are not diagnosable by this method. According to characteristic echophenomenal findings it was possible to differentiate the findings whether they are benign or malignant, solid or cystic. It is possible under the ultrasound guidance to perform the puncture of the intraosseous tumour in some cases. Key words: bone neoplasms; ultrasonography Introduction Bone neoplasms account only for 1 % of the whole orthopaedic casuistics. Fortunately less than 0,001 % 7 are malignant bone tumours (pri­mary and metastatic bone tumours). Today certain problems stili exist in the identi­fication and detection of osseous malignant neo­plasms, especially in the early stages. It is of great importance to detect and treat the mali­gnant bony tumours in the stage when the malignant tissue growth has not yet invaded the surrounding soft tissue. Taking ali diagnostic methods at disposal to­day, with the exception of magnetic resonance Correspondence to: dr. Babic M. UDC: 616.71-006.6-073:534-8 imaging, this aim is very difficult to achieve with high probability and accuracy. Therefore we came to the idea that some diagnostic problems could be solved by ultrasound. Ultrasound as a diagnostic tool becomes inevi­table in many fields of orthopeadics. It is preffe­red in the diagnostics of the congenital disloca­tion of the hip in children and of certain soft tissue pathological abnormalities of the musculo­skeletal system. 1• 2• 4• 5 The aim of the study Having in mind the characterictics of the ultra­sound we tried to use this method in detecting malignant (expansive) processes inside the bone. According to our knowledge there are no recent reports dealing with the diagnostic possibilities of the ultrasound in revealing the pathological Ultrasound as a diagnostic tool in malignant bone tumours growth inside the bone. Therefore it seemed necessary to apply this diagnostic technology developing at the same tirne our own approach and method. Patients and methods In our ten-year experience we had 16 patients (22 -60 years old) with malignant bone tumours. In 14, detailed orthopaedic clinical investigation (history, physical examination, imaging studies: plain film roentgenography and tomography, skeleta! scintigraphy, angiography and recently CT scan) was made. In two patients the diagnosis of neoplastic growth was confirmed by ultra­sound after history and physical examination. These patients were subjected to the echosono­graphic examination because abnormalities in other organic systems were suspected. With this method we succeded in detecting a metastatic spread in lumbar spine region in 6 patients, as well as 3 metastatic spreads in the pelvic bone area. In the beginning of our work we examined 40 healthy persons (20 men and 20 women) in order to acquire experience and skills in using this new diagnostic possibility. In this study we shall not report our experience in applying this diagnostic method in detecting benign tumours of the musculoskeletal system. According to literature it was not possible to perform an echosonographic examination of the musculoskeletal system. Therefore, after first positive experiences, we had to develop our own technique and methodology of the study. 3• 11 In this study we used standard linear probes of 3,5 and 5 MHz. It seems that a linear probe of 3,5 MHz, 10 cm in length was preferred in the examination of the musculoskeletal system espe­cially in the process of detection. With this probe it is possible to examine greater areas, and the distinction between normal and abnormal finding becomes more evident. After this first step we proceeded examining using a smaller 3,5 MHz linear probe. In a few patients we used a 5 MHz linear probe with an amplification of the exami­ned structures, which enabled us to make a more distinct differentiation of the examined structu­res. It is mandatory to examine the normal and abnormal extremity as well as the normal and abnormal spina! area at the same tirne. The investigation always began in the longitudinal axis in relation to the extremity or spine, in the anteroposterior direction while the patient was lying. The investigation of the extremity was performed with a gentle slipping of the probe along the whole circumference. While examining the spine we gradually moved away to the medial line, to the right and to the left (so called dorsal aproach). After the initial examination was completed the probe was positioned in the sagital axis and afterwards gently moved proxi­mally and distally along the extremity, cranially and caudally along the spine respectively (the so called ventral and dorsal approach). Results In the preliminary part of this study we tried to acquire experience in the investigation of the musculoskeletal system among healthy persons as well as among persons with neoplastic growths inside the bone. After these first experiences a differentiation of skin, subcutis, muscles and periost among healthy persons ( controls) as made possible du­ring investigation. The depth of the soft layer depends on the adipous tissue and the muscular mass with the echogenic characteristics of the soft tissue. When the probe is positioned in the longitudi­nal axis a line with a distinct hyperechogenic zone comprising 1.3 mm can be seen underneath the soft layer which corresponds to the periost. Underneath this hyperechogenic zone another hyperechogenic homogenous zone can be found corresponding to the cortical and medullary part of the bone (Figure 1, 6, 7). It is seldom possible to find the hyperechogenic zone of the periost on the opposite side again. Among youngsters, especially children, we almost always succeded in showing the other Babic M and Matovinovic D Figure 1. Echosonographic image of a normal bone (5MHz probc longitudinally positioned along the axis of the bone). A -skin and subcutis, B muscular la\'er. C periost and bon\' structun:. ,il ff'\ . i:', . ;t. ' 1 1 ­ •it f,i Figure 6. Thc same paticnt shmrn o! ligurcs ., .. .J .. :i .. !,ix months aftcr the opcration. Homotransplant. Figure 7. Echosonographic imagc of thc patinet shown of Fig. 6. A -skin and subcutis, B -muscle, C -pcriost and the cortical part of the bone, D -the pace of junction of homotransplant and the remaining part of the bone, E -growth of a new bone into the homotransplant, F alenthesis, E -phenomenon of reveberation side. Great caution in the evaluation of a picture issued in this way is needed, because of the reveberation phenomenon and the possibility that an artefact issued in this way might be taken as the periost from the opposite side. To avoid any danger of interpreting and evaluating the picture in this way, we must measure the distance from the severa! analyzed structures in relation to the sufrace, periost-periost to the other side, espectively. Any doubt of the exsistence of the reveberation phenomenon can be releaved or confirmed in this way. It is necessary to perform the evaluation and analysis of the b0ny structures while the muscles are maximally relaxed or contracted, ignoring in this way the possibility that fascia is taken for periost, which is sometimes possible. In the cases when the probe is positioned transversally to the bony axis, a crescent-like Ultrasound as a diagnostic too/ in malignant bone tumours slightly hyperechogenic zone corresponding to the periost can be seen under the soft structures ( normoechogenic zone). The ringlike structure of the whole bone can be shown only among children. Under the crescent-Iike slightly hypere­chogenic zone of periost there is a hypoechogenic zone corresponding to the cortical and medullary part of the bone. 3 Following the ventral approach to the spine it is possible to show a vertebral body projecting itself as a horseshoe.s haped hypoechogenic zone at the bottom of which a triangle-like hyperechogenic zone with an anechogenic center corresponding to the spina! canal can be found. Amplifying the strength of the ultrasound beam a certain degree of echogenicity is reached inside the vertebral body, but in this case it is impossi­ble to analyze structures which could possibly be found inside the body. Spinous and transverse processes and the vertabral arci are examined with more precision and ease following the dorsal approach (with the patients prone), and appear 2• 3 with Iess echogenicity.1• After the necessary experience was acquired with ultrasound examinations of bony structures and the spines of tealthy persons, an investiga­tion of the abnormalities inside the bony system could be performed. In the beginning of our study, before ultra­sound as a diagnostic tool was applied, we applied ali other available diagnostic methods (radiography-tomography, biopsy, CT scan) to get a better insight in the possibilities of ultra­sound in this area. During the study we reached the conclusion that a neoplastic mass can be seen more easly when the probe is positioned longitudinally, along the axis of the bone examined. Intraosseal­ly, in the site of the tumours mass, the degree of echogenicity changes, depending on the mass itself. Using ultrasound as a diagnostic tool we primarily tried to locate the tumour, and to find out whether this mass was localized inside the bone or was also invading surrounding soft tissue (Figure 2). Figure 2. Malignant tumour of the upper and the middle third of the humerus A -skin and subcutis, B -tumour invading soft tissucs, C -thc pace of the tumour invasion into the soft tissue, D -tumourous mass inside the bone. Tumour 14 cm. Cleary discerni­ble hyper and hypoechogenic zone inside changed structure of the bone, corresponding the tumourous mass. The character of the finding corresponds to the character of the intraosseal tumour, i.e. whether it is malignant or benign. In the case of a malignant process the echogenity inside the tumours mass has different degrees from anecho­genic to hyperechogenic, the borders are not defined and saw-like in respect to healthy bone. A clear border between the bone invaded with the tumour and the healthy bone, can not be found in spite of the fact that this border can be guessed. Inside the tumour itself the hyperecho­genic zones correspond to the zones of necrosis and to the zones of calcifications inside the tumour itself. A clear distinction between the tumour itself and the healthy tissue can be made according to the different degrees in echogenicity (Figure 2, 3). A break in the continuity of the periostal hyperechogenic zone points to the inva­ Babic M and Matovinovic D sion of the tumours mass into the surrounding soft tissue (Figure 3). The invasion of the mali­gnant mass into the healthy tissue changes in the Figure 3. Echosonographic image of femur condyles of a grown-up person. A -skin and subcutis, B ­invasion into the soft tissues, C -the pace of invasion of the tumour into the soft tissues, D -tumorous mass, E -periost and the cortical part -healthy tissue. same the degree of echogenicity and points to a clear distiction between the healthy and diseased tissue (Figure 3, 4, 5). In the cases of metastatic spread in the region of the vertebral body, zones of increased echoge­nicity appear, not clearly limited from their surroundings, while the remaining healthy tissue has an anechogenic projection (Figure 8, 9) In the past ten years in a total of 16 patients malignant intraosseal neoplasms were revealed using ultrasound as a diagnostic tool. Among 6 patients metastatic spread Iocalized in the lumbar spine and among 3 inside the pelvic bone was found. Discussion The theoretical background for the first diagno­stic possibilities for revealing tumours inside the bony system was presented by J M. Heveri 1983. His theoretical work could not find a practical application due to the Jack of a suitable ultra­sound apparatus.8 Figure 4. A -pathoanatomical prcparation of a tumour gaincd during the opcrativc trcatment. Thc ,arnc tumour is shown hy thc same tcchniqt1c on Fig. 3 Figure 5. Thc same tumour Shown by a CT scan Ultrasound as a diagnostic too/ in malignanl bone tw11011rs Taking the standpoint that ali tissues could be examined by ultrasound under the conditions that the ultrasound wave lenght during penetra- Figure 8. Tile vcrtcbral hacmangioma. A -body of the vertcbra changed by thc haemangioma B spina! channel Figure 9. CT scan of thc same paticnt. tion through the tissues, acoustic impedence, reflexion of the ultrasound on the borders of certain tissues, the velocity of the ultrasound during the spread through the tissues and specific gravity as well as density of the tissues must be known, we tried to create such relations under which the study of the structures inside the bone with ultrasound an a diagnostic tool would be enabled.9 To enable the interpretation of achieved re­sults, it was necessary to study the specific physical characteristics of the ultrasound beam when in contact, or during the spread through the bony tisue. While passing through the bony tissue, the ultrasound behaves quite differently than when spreading through soft tissues, homo­genous tissues or liquid. On the leve! of periost, because it is so smooth, there is reflexion of a part of the ultrasound waves, for instance when the ultrasound hits a mirror or any other smooth surface. The second important factor which cau­ses reflexion of a part of the ultrasound waves is the actual difference in acoustic impedence bet­ween the muscular mass 1,70 C 10 kg/cm2) and the bony structures 7 ,80 C 10 kg/cm2). Inside the bony system itself the ultrasound beam behaves in a different way than when passing through the muscular or homogenous tissues. This is the result of the special hive-Iike structure of the bone (bone trabeculae). A part of the ultrasound beam is !ost because it is prone to uncontrolled movements through the bone, as well as its reflexions, that is its absorption which is very important. When passing through the bone the ultrasound beam increases, which is a result of the difference in the tissue quality. When compa­ red to the movement of the ultrasound beam through the soft tissue this acceleration is three 1.io times greater. This must be kept in mind when measuring severa! distances inside the bone, since the obtained results are not identical to its actual dimension. It can be seen that during the study of the bone by ultrasound severa! problems emerged that could virtually not be solved. This is why only a small amount of information can be obtained from the study of healthy bone. Babic M and Matovinovic D 108 In the healthy bone periost and structures directly underneath can be analyzed (Figure 1). Other structures of the healthy bone can usually not be presented with an ultrasound, except on very rare occasions, depending on structure and the mineral composition of the bone itself. Ac­cording to actual degree of the ultrasound tech­nology pathological mass smaller than 1 cm can not be presented. Only a smaller part of the ultrasound beam is reflected from the healthy bone and this part is accesible for analysis. Due to the fact that tumorous tissue changes the quality, composition and structure of the bone, the bone becomes more accesible to ultra­sound beam. New conditions emerge that allow us to study the primarily diseased bone. Equally, the difference in the echogenicity of the tissues enables us to find the limit between healthy and diseased bone. The early detection of tumours masses which expaned out of the bony system into the soft tissues is based on the same princi­ples. This is of significant help for future treat­ment and for decisions about the plan of the treatment itself. Due to interventional ultrasound as a diagno­stic tool, it is possible to perform a biopsy and obtain material for pathohistological analysis. Conclusion According to known research, diagnostic proce­dures and follow-up of tumours inside the bony system, some important conclusions have to be made. In view of obtained experience and the evaluation of results, it is obvious that the analysis of tumours greater than 1 cm located intraosseally was possible. It is primarily possible to prove the existence of a mass with osteolytic characteristics. Depending on degree and charac­ter of echogenicity a differentiation between tumours (solid -cystic) was made possible. Based on these results it is possible to raise a doubt whether a malignant intraosseal mass is in question. By using this method we can easily confirm the invasion of the tumour into soft tissues which has a specific impact in planning further treat­ment. It is possible to perform a biopsy during the investigation if an interventional probe is availa­ble. Material for pathohistological diagnosis is obtained in this way. The method is quite simple if certain guidlines are accepted. It is generally recommended as a screening method. References l. Babic M. Mogucnost dijagnostike hernije inter­vertebralnog diska metodom ultrazvuka. Magi­starski rad. Medicinski fakultet Rijeka, 1980;18­28. 2. Babic M. Ultrazvuk u evaluaciji kompresije u lumbosakralnom segmentu. Lijec Vjesn 1982; 104:354-7. 3. Babic M. Matovinovic D. Mogucnost primjene ultrazvuka u dijagnostici intraosalnih tumora. Me­dicina 1990;26:141-7. 4. Matanovic T, Vrdoljak J. Ultrazvucna dijagno­stika kuka i natkoljcnice. In: Kurjak A. ed Ultra­zvuk u klinickoj medicini, Zagreb, Medicinska biblioteka, 1989 ;779-89. 5. Matanovic T. Dijagnosticki ultrazvuk u ortopedi­ji. In: Matanovic T. ed U/trazvu(na dijagnostika .1·ustava za kretanje, Zagreb. Školska knjiga 1989: 23-47. 6. Andric J, Babic M. Pokušaj dijagnostike migracije totalne endoprotczc pomocu ultrazvoka. In: Mikic z. ur. IX. Kongres ortopeda i traumatologa Jugo­slavije -JUOT 1986. Novi Sad 1987;459-63. 7. Richend S, Marcus R.M, Spaiser S.S, Enaeking E.W Surgical treatment for osteosarcoma. J Bone Joint Surg, 1988;70:1119-24. 8. Hevezi J .M. Physical principlcs of diagnostic ultra­sound. In: Anderson E. ed. Radiologic mul other biophysics methods in twnour diagnosis. Chicha­go. 1975: 99-107. 9. Brcyer B. Andrcic ž. Fizika ultrazvoka. In: Kurjak A. ed. Ultrazvuk u klinil'koj medicini. Zagreb. Mcdilcinska biblioteka. 1989: 1-3. 10. Breyer 13. Fizikalne osebine ultrazvucne dijagno­stike. In: M,1tasovic T. ed. Ultrazvul'na dijagno­stika sustava za kretanje, Zagreb, Školska knjiga 1989;9-21. 11. Babic M. Pokušaj dijagnostike intraosalnih tumora ultrazvukom, In: Kurjak A., Sretenovic Z. cd III. jugoslavenski kongres o primjeni ultrazvuka u medicini i veterini. Beograd:, Sekcija za ultra­zvucnu dijagnostiku Jugoslavije i Srbije, 1989; 193-4. Chapter III. NUCLEAR MEDICINE Adi· Radio/ Onco/ 1992: 111-15. Labelling leucocytes with Tc-99m HMP AO for imaging inflammation Peters AM Royal Postgraduate Medica! School, London, United Kingdom Tc-99111 HM PA O is w1 important new development in the imaging of inflammatory diseases and now lzas an establic!zed role in Nuclear Medicine. We are only just beginning to appreciate the appro­priate indications of Tc-99m HMPAO and when to use In-! 11. There is no reason why both agents should not be offered at a diagnostic leve!. Thus, at least at present, In-111 chloride and HM PA O are about the same price. HMPAO can be kept on the shelf and used at short notice which is the usual requirement for acute sepsis. In-111, on the other hand, will usually be indicated for more chronic processes, which can be imaged more electively, thereby allowing sujficient time for the isotope to be ordered. Key words: scpticcrnia-diagnosis: lcucocytes: tcchnetiurn Introduction In-111 labelled leucocytes have now been in routine use for irnaging inflammatory disease for many years. However, since McAfee and Thakur first described the technique of celi labelling with In-111 in 1976, there have been many attempts to label leucocytes wfth Tc-99m. 1 In­deed, Me Afee and Thakur (1976) investigated Tc-99m oxine for its celi labelling efficiency but found it too unstable. 1 Shortly afterwards, Schroth et al. described a technique for labelling phagocytic cells with Tc-99m colloids but this never gained a wide clinical acceptance except in 3 Australia. 2· Other efforts, based on Tc-99m Correspondence to: A. M. Peters, Reader in Nuclear Medicine, Royal Postgraduate Medica! School Ham­mersmith Hospital, Du Cane Road, London, 12 ONN, Unitcd Kingdom. UDC: 616.94:616.155.3:539.163 pyrophosphate and Tc-99m labelled porphyrins 5 have also failed to gain acceptance. 4· The advantages offered by a Tc-99m agent over In-111 include better image resolution, greater convenience, lower radiation dose, and it should be cheaper. In 1986, in collaboration with Amersham Interantional pic and RJ Haw­ker of the Queen Elizabeth Hospital Birmin­gham, we described the first clinical results with a new Tc-99m agent, HMPAO, which was ini­tially developed for imaging cerebral perfusion but, because of its high lipophilicity, also pene­trates blood cells. The labelling of cells is relati­ vely stable because the agent is converted intra­cellulary from lipophilic to hydrophilic secondary species, which become effectively trapped within the celi. Labelling Since Tc-99m HMPAO is, like In-111 oxine and In-111 tropolonate, a lipophilic complex, the 112 PetersAM technique of labelling cells with it is essentially the same as that of labelling with In-111. Thus, the red cells first have to be sedimented leaving a supernatant from which the leucocytes and platelets are then concentrated. The labelling efficiency of Tc-99m HMPAO is satisfactory in a medium containing 20% plasma and this is an advantage from the point of view of ultimate cellular integrity. 6 The agent labels neutrophils with some degree of selectivity, similarly to In-111 oxine and In-111 tropolonate. Although Tc-99m is not as firmly held by the celi as In-111, the stability is adequate for clinical imaging. We initially thought that neutrophils became labelled with Tc-99m with more stability than other celi types but there is some question about this and, indeed, Tc-99m labelled platelets are now being used on a wider scale and are replacing In-111 for imaging thrombus. 7 Biodistribution The biokinetics of Tc-99m labelled leucocytes are broadly similar to those of In-111 labelled neutrophils. Thus there is minimal first pass hold-up in the lungs, and although this may appear quite striking on imaging, the cells are rapidly cleared from the Jung. The labelled cells enter the splenic pool and display a transit tirne through it similar to In-111 granulocytes. The !iver remains cool while the bone marrow beco­mes very prominent, more prominent indeed than it does with In-111 granulocytes. The inten­sity of bone uptake led us to suspect that free Tc-99m HMP AO may be taken up by bone as opposed to bone marrow. However, when we injected supernatant from a medium in which Tc-99m HMPAO labelled cells had been suspen­ded for 30 minutes, we saw urinary activity (and some splenic activity thought to be due to conta­minating labelled platelets) but no skeleta! activi­ty. Furthermore, comparison of the Tc-99m HMP AO white celi scan with the bone scan in patients with metastatic disease showed that the hot spots on the bone scan were matched by cold areas on the white celi scan, indicating destruc­ tion of marrow and abolition of uptake. Figure l. Inflammatory bowel discase with involvc­ment of small bowel. Anterior gamma camcra imagc taken 3 hours following injection of Tc-99111 HMP AO labelled leucocytes. Abnormal migration to loops of small bowel can be scen in the right iliac fossa (reprinted with permission from Radiology). The stability of Tc-99m in neutrophils is not high enough for quantitative studies of neutrop­hil kinetics. In vitro studies indicate an elution rate from neutrophils of about 5% per hour. The half tirne of disappearance from blood of native neutrophils is thought to be about 7 hours. 8 In-111 labelled neutrophils have, in our hands, a half tirne approaching 7 hours.9 Whilst our Tc­99m HMPAO labelled neutrophils ha'.c a half tirne of only 4.5 hours it increases to 6.5 hours when a correction is made for isotope elution. 111 Stability of Tc-99m at the normal sites of labelled neutrophil uptake, i. e. !iver, spleen, and bone marrow, is also reduced compared with In-111. Thus, bone marrow activity, which Labelling leucocytes with Tc-99m HMPAO far imaging inflammation Figure 2. Intlammatory bowcl discasc with pancolitis. Makcd upt<)ke ofTc-99m HMPAO labelled white cells can bc seen throughout the colon on this anterior gamma camera image taken 3 hours following injection of labelled cells. Normal uptake is seen in the !iver and splcen. Note the urinary activity in thc bladdcr. rises steeply up to 4 hours after injection of In-111 granulocytes, continues to rise until 24 hours when it reaches a plateau. Tc-99m HMPAO, on the other hand, although showing the same steep rise up to 4 hours, does not show any further increase beyond this tirne, even though labelled granulocytes continue to circula­te. This suggests elution of label from bone marrow reticulo-endothelial cells. More obvious evidence of elution (though giving no clues to its origin) is seen in the form of urinary activity. The lipophilic nature of Tc­99m HMP AO allows its entry into cells. Once inside the celi, however, the agent undergoes biocheinical alteration to secondary species which are hydrophilic and cannot therefore escape from the cells. Although this forms the basis of the relative stability of Tc-99m HMPAO, it also accounts for the appearance of activity in the urine. There is, in addition, significant reten­tion of activity in the renal parenchyma. There is also evidence of biliary excretion of complex and, indeed, the gall bladder is visualised occa­sionally. Biliary excretion, which may be of a lipohilic complex, is almost certainly the basis for the non specific gut activity that is seen from about 3-4 hours onward. This gut activity is a serious disadvantage of Tc-99m labelled leucocy- Figure 3. Prc-sacral ahsccss in a paticnt with an ilcosto111y imagcd wilh Tc-l/ated between the committees of the American Colle­ges of Radiologists (ACR/NEMA) which had not previously considered Nuclear Medicine but had made proposals suitable for image transfer in general radiology, computer tomography and other aspects of radiological quality control. To improve liaison, Dr. Briandet, representing ACR/NEMA, has attended the working Group 1 workshop and Andrew Todd-Pokropek has attended the Working Group 1 workshop and Andrew Todd-Pokropek has participated in the ACR/NEMA meetings in the USA. The aims are to finalise version 4.0 of the INTERFILE System and propose validation and conformance testing procedures for the translation program­mes, probably at three levels of conformance. Finally, pilot conformance testing centres will be set up to complete this work. Working Groups 2, 3 and 4 are concerned with thc 4uality assurance of organ software. Working Group 2 is on the quality assurance of cardiac software, Chairman Ellinor Busemann-Sokole 120 Britton KE (NL), Secretary Michel Bourguignon (F). This Working Group aims to define standard criteria for the selection of software phantoms for the main cardiac studies of interest. Clinical valida­tion criteria have been defined for a selection of normal patients, patients with coronary artery disease including transmural myocardial infarc­tion pattern, myocardial ischaemia pattern, pre and post angioplasty patients and idiopathic cardiomyopathy. Data acquisition and analysis criteria are defined for Thallium 201 SPECT studies and Technetium 99m gated equilibrium blood pool studies. The group is drawing on national work from Finland on SPECT myocar­dial software phantoms and from the Nether­lands, the IAEA gated cardiac blood pool phan­toms and from Canada, mathematical software phantoms, from France, the first pass shunt software phantoms, from Germany gated cardiac blood pool software phantoms and from Spain, SPECT myocardial exercise and rest, Thallium and Technetium-99m MIBI studies. Very impor­tantly, this group has coordinated with the Euro­pean Association of Nuclear Medicine Cardiac Task Group, the European Society of Nuclear Cardiology Task Group and the International Atomic Energy Agency, software cardiac phan­ toms for developing countries and WHO. Working Group 3 is on the quality assurance and standardisation of kidney software. The Chairman is Helmar Bergmann (OS) and the Secretary, Phil Cosgriff (UK). This Working Group has defined and published criteria for normal studies, for unilateral renal artery steno­sis, and for obstructive nephropathy. They are advanced in the collection of software phantoms for these three studies. These are to be vetted by an international panel of experts before their distribution to selected test centres to test them out in practice. Working Group 4 is on the quality assurance of brain software. Chairman Dr. Pedro Domin­guez-Montero (E), Co-ordinator Dr. Per Asard (SW), Secretary Dr. K. E. Britton (UK). This Working Group has defined the criteria for acceptance of normal brain SPECT studies and patients with cerebral infarct in the right or left middle cerebral artery territory. The collection of SPECT data with Technetium HMP AO is awaiting the results of Working Group 7 on SPECT to finally confirm the methods of data collection, then software phantoms will be collec­ted. Already Sweden has produced a magnetic resonance based normal brain map, future areas include typical findings of temporal lobe epilepsy and Alzheimer's-type dementia. Working Group 5 is on knowledge based sy­stems, Chairman Professor D.P. Pretschner (D), Secretary Dr. J.H.C. Reiber (NL). The goals of this working group are the application, develop­ment and evaluation of knowledge-based system methodologies such as human experts, tools, shells, models and programming languages fo_r solutions of clinical problems in Nuclear Medici­ne. They have identified areas in Nuclear Medi­cine suitable for knowledge based systems, in particular bone, kidney, heart and thyroid. Their aim is the assessment and transfer of artificial intelligence methodology and concepts for Nu­clear Medicine, in particular to specify data structures and procedures used in KBS ( object orientated model) to specify the human compu­ter interface to specify the terms used in KBS and to specify data and case exchanges. A large number of publications and workshops have been produced by this group. Working Group 6 is on the bone phantom data base, Chairman Professor D.P. Pretschner (D), Secretary Dr. P. Vassilikos (Gr). The goal of this working group is to evaluate the data struc­ture needed to develop an integrated electronic archive of scintigraphic studies combined with the relevant clinical data in imaging the skeleta! system. In this group, the work of Dr. J. Mark­wardt on quantitative bone imaging and the transmission phantom developed by Dr. A. Skretting (Norway) are import.nt features. COST B2 121 Working Group 7 is on the quality assurance of SPECT. The Chairrnan is Dr. P.H. Jarritt (UK), Secretary Andrew Todd-Pokropek (UK). The goal of this working group is the provision of expert inforrnation regarding the specification of various nuclear medicine syste·ms with respect to single photon emission computed tomography acquisition and analysis. It is involved in the generation and testing of data transfer program­mes from INTERFILE to specify systems by members of the working group in relation to cardiac and brain SPECT data and it is concer­ned with the evaluation of SPECT system recon­struction software through the use of software phantoms. A particular problem concerns the centre of rotation and uniformity and other corrections required for software phantoms ob­tained frorn different manufacturers'data. A pro­posed solution is to define two alternative met­hods of transfer of such studies via INTERFILE. In the first group, data are supposed to be fully corrected with a flag to indicate this and with some pararneters passed on for information. In the second group, key words will be defined to cover basic reconstruction correction met­ho\:ls such as the centre of rotation offset in milllimetres as a function of angle, pointers to the uniformity correction matrix and such-like. In addition, the SPECT working group is evalua­ting Jaszczak software phantoms, the non-atte­nuated Jaszczak phantom as an appropriate test object for indicating errors in straight forward reconstruction software and as a test bed for evaluating the INTERFILE format for SPECT data and the attenuated Jaszczak software phan­tom. This revised phantom using Monte Carlo photon transport methods is in the process of being evaluated. Working Group 8 is on minimum user require­ments for data systems for nuclear medicine. The Chairman is Andrew Todd-Pokropek (UK), Secretary K.E. Britton (UK). This working group has completed a document on user requi­rements for data systems for nuclear medicine and Andrew Todd-Pokropek is developing a software design ideal computer. This design is thought about in terms of its architecture, its platform and its implementation. The architec­ture rnust be as far as possible machine indepen­dent so that it can migrate on to different hardware configurations. The software should conform as far as possible to existing and future standards and make use of existing tools to minimise work. The platform for ali but real tirne applications, the UNIX system is proposed and for real tirne applications the system running under MS-DOS is proposed. The implementa­tion on appropriate hardware requires that it should be capable of at least 10 MIPS in terms of processing. Three hardware platforms are reasonable, INTEL, such as 1860, Motorola such as 86030 and RISC as in SP ARC, MIPS and RS 6000. A gamma camera interface is nuclear medicine specific and should therefore be isola­ted. Finally, the hardware platform including processing display and acquisition modules should not cost more than $US40,000. Working Group 9 is a computer conference coordinating centre. This coordinating centre is set up to accept the software phantoms from Wor­king Groups 2,3 and 4 once they have been valida­ted and tested, to store them and to then send out appropriate compilations of software phantorns to nuclear medicine departments and to industry so that their software systems can be tested. These quality control objects will be licensed to users, the whole set of data phantoms will be stored on CD ROM and subsets distributed as required. The intention is to update the software phantom package each year and always to hold back a reserve set of software phantoms so that programmes are not designed just for the soft­ware phantom. The coordinating centre will collate the information returned and develop statistical methods for evaluating the distribution of results for each organ and each disease system tested by the software phantoms. In conclusion, it can be seen that the COST B2 Project is ambitious and wide-ranging, that it addresses the problem of quality assurance in 122 Britton KE nuclear medicine software not only in a theoreti­cal way but by offering practical solutions. The work of COST B2 is mainly voluntary and the efforts of COST B2 in this field need the support of nuclear medicine societies in every country so that we can continue to improve, monitor and audit the performance of our techniques. References l. Bernauer JA. Controlled Vocabulary Framework for Report Generation in Bone-Scintigraphy. In: Millar RA ed. Proc. 14th Symposium on Compu­ter Applications Medica! Care 90. Washington: IEEE Computer Society Press, 1990. 2. Bernauer J. Designing a terminology controlled interface for report generation. In: van Bemmel JH et al eds. Proceedings of the software enginee­ring in medica! informatics (SEMI-IMIA) Work­shop. Amsterdam: Elsevier Scientific Publishing. 1990. 3. Bernauer J, Pretschner DP. Knowledge based report generation for quality assurance in bone scintigraphy. In: Knowledge-based systems to aid medica! image analysis. Vol l. Pretschner DP, Urrutia B, Eds Luxembourg: European Commu­nities -Commission: EUR 12797:164-86. 1990; 4. Busemann-Sokole E. Quality assurance in nuclear medicine imaging: hardware and software aspects. PhD Thesis, University of Amsteraam (Decem­ber) ISBN 90-9003872-8. 1990. 5. Busemann-Sokole E, Cradduck TD, Erickson JJ. Dynamic cardiac phantoms for use in computer software quality control. In: Dvnamic functional studies in nuclear medicine in dn,·loping countries. Vienna: International Atomic Energy Agency, 1989; 493-8. 6. Busemann-Sokole E., Cradduck TD, Erickson JJ. Experience with gated cardiac software phantoms for quality control of applicatione programmes. Eur J Nucl Med 1990; 17:106-10. 7. Cradduck TD, Bailey DL, Hutton BE, Deconinck F, Busemann-Sokole E, Bergmann H, Noelpp U. A standard protocol for the exchange of nuclear medicine image files. Nucl Med Commun 1989; 10:703-13. 8. Cradduck TD, Busemann Sokole E, Erickson JJ Development of software phantoms for software validation. In: Dynamic functional studies in nu­clear medicine in developing countries. Vienna: 1989; 483-92. International Atomic Energy Agen­cy. 9. Kotzke· K, Pretschner DP. Quality assurance of knowledge-based systems -experience and results from evaluation of the automatic diagnosis of cardiac motility. In: Pretschner DP, Urrutia B Eds. Knowledge-based systems to aid medica{ image analysis. Vol l. Luxembourg: European Communities Comission, 1990; EUR 12797:96­112. 10. Pretschner DP, Vauramo E. Editorial. New COST project in the field of nuclear medicine software. Eur J Nucl Med 1986; 12:1. 11. Pretschner DP, The current situation, aspects and projects conceming nuclear medicine software in Europe. Eur J Nucl Med 1986; 12:2-4. Adv Radio/ Onco/ 1992; 123-34. New aspects in nuclear cardiology -PET, NMR and SPECT Henze E, Milcinski M, Lietzenmayer R an4 Clausen M Divisions of Nuclear Medicine, Universities of Kiel and Ulm (Germany) and Ljubljana (Slovenia) Single photon emission computed tomography (SPECT) represents advanced technical possibilities in myocardial pe,fusion assessment. Thallium 201 and new technetium labeled radiopharmaceuticals enable the detection of resting or stress ischemia, and quantify the extent of perfusion abnormalities. SPECT in radionuclide ventriculography helps to define abnormal contraction pattern in preexcitation syndromes or other severe arrhythmias and accurately evaluates regional wall motion abnormalities. Nuclear magnetic resonance (NMR) imaging provides a noninvasive assessment of cardiac anatomy and ventricular function, the information simi/ar to that obtained by echocardiography and radionuclide ventriculography. lts unique role, however, can be in the study of biochemistry and blood flow. The real tirne three-dimensional display of the heart is a realistic prospect. Magnetic resonance spectroscopy (MRS) has its well established role in cardiac research. Possible aid in the diagnostic proces.1· and treatment of patients with cardiac disease remains an exciting challenge for future research of this technique. Positron emission tomography (PET) is a new tomographic technique in nuclear medicine, allowing quantitative in vivo studies of metabolism and blood flow after labeling any organic substrate with short living isotopes e. g. C-11, %-15, N-13 or F-18. lts cost is very high and this limits the use of PET at the moment almost exclusively to cardiologic research. The clinical aspect of vitality of akinetic myocardium can be explained by combining the blood flow tracer N-13 ammonia with the metabolic indicator F-18 deoxyglucose. Myocardial metabolism of isolated substrates -glucose or fatty acids can be analysed as well. Thus, with these three new imaging modalities PET, NMR and SPECT-the noninvasive diagnostic work-up of patient with heart disease will be definitely improved, in particular with a new insight into the metabolism and quantification of blood flow and function. Key words: heart diseases-radionuclide imaging; tomography, emission computed; NMR Introduction Positron em1ss1on tomography (PET), nuclear magnetic resonance imaging (MRI), nuclear magnetic resonance spectroscopy (MRS), and single-photon emission computed tomography Correspondencc to: Prof. dr. Ebcrhard Hcnzc. Dircc, tor, Abteilung Nuklearmedizin, Arnold-Hcllcr Stral3e 9, D-2300 Kicl, Germany UDC: 616.12-073:539.163 (SPECT) are today the most exciting techniques in nuclear cardiology with most promising pro­perties for future development. These techniques and their impact on cardiological research and clinical practice are described and discussed in this review. PET is a tomographic scanning technique in nuclear medicine, which enables noninvasive investigation of myocardial blood flow, its meta­bolism and function. Up to now, there are only few PET centers available worldwide. PET is Henze E e/ al. based on both the chemistry and physics inherent in positron emitting nuclides which allow ideal labeling of nearly ali important biosubstrates with short half-life. Disadvantages of the method are considerably high costs and the necessity of a cyclotron for isotope production and a radio­pharmaceutical laboratory for fast synthesis of radiotracers nearby to the PET scanner. Magnetic resonance imaging (MRI) has two important qualities. in addition to its high spatial resolution. that are likely to dominate future developments in cardiological applications. First. the nuclear magnetic resonance (NMR) pheno­menon is extremely sensitive to motion and flow, which makes it potentially an ideal tool for producing images of cardiovascular function in addition to depicting cardiovascular morphology. Second, MRI is considerably more sensitive to paramagnetic substances introduced into the body than e.g. computed tomography (CT) to radiologic contrast agents. This, combined with the fact that MRI is harmless and thus makes it possible to obtain multiple seans at the same leve! of the body, permits the acquisition of information on the temporal evolution of the distribution of injected contrast agents. For example, high-resolution dynamic scanning of the heart after bolus injection of a contrast agent is possible, providing a combination of morpho­logical and functional images of the cardiovascu­lar system. Magnetic resonance spectroscopy (MRS) of­fers a unique possibility to detect important metabolites noninvasively in living tissue. In recent years, an intense research effort has been made into the use of this technique for studying the metabolism of a variety of organs. Most of the cardiac spectroscopy that has been perfor­med so far has involved analysis of high-energy phosphate metabolism in vitro and in vivo. Technical advances such as SPECT in radio­nuclide imaging have important implications for the management of patients with acute myocar­dial infarction. SPECT with thallium-201 offers greater accuracy than planar imaging in detec­ting, localizing and sizing myocardial perfusion defects. The use of SPECT with Tl-201 should allow for a more accurate assessment of progno­sis after myocardial infarction. A new radiophar­maceutical, Tc-99m methoxyisobutyl isonitrile, provides a numbcr of advantages over Tl-20 l. including higher quality images. lack of redistri­bution, and the ability to assess first-pass ventri­cular function. Thc use of SPECT with radionuc­lide ventriculography provides improvements such as reconstruction of planar views oriented according to the long axis of thc heart for easier interpretation and more accurate localization of regional wall motion abnormalities. PET Positron emission tomography is now for about 15 years a technically realizable nuclear medicine technique providing slice images, which opens due to the physical properties of positrons and the possibility of labeling any substrate a new dimension in tcrms of an in vivo autoradiogra­phic method for noninvasive cardiological diag­nostic studies of cardiac blood flow. metabolism 1 ·2"1 and function. One of the main advantages of PET lies in the physical propcrties of positrons. which -emmit­ted from the nuclei of appropriate atoms ­bounce against electrons of any matter. destroy each other according to the laws of atomic physics and convcrt into electromagnetic radia­tion. This radiation consists characteristically of two 511 keV photons moving on the same axis in the opposite way and thercfore allow ideal axial tomographic detection for image recons­truction, comparable to an x-ray computcd to­mography device. Accordingly, in a PET camera there are circular arrangcd radiation detcctors switched in axial coincidence connection, and therefore the external appearance of a PET scanner closely rcsembles an x-ray scanner. The second important advantage of PET is determined by thc available positron emitting isotopes, such as N-13, 0-15, C-11, F-18 or Rb-82. The first three mentioned are isotopes of elements that occur naturally in organic molecu­les. Thus, radiopharmaceutical synthethis by a PET, N1\!IR and SPECT New aspi'Cts in nuclear cardiology specialized laboratory is possible. and the tracer principle (»make as small a change in the mole­cule to be traced as possible«) is ideally satisfied. Each interesting organic substrate, e.g. natura! and synthctic metabolites, pharmaceuticals etc. can be labeled optimally without changing their biological and chemical properties. In addition, the short half-lives of the radionuclides C-11: 20 min; N-13: 10 min; 0-15: 2 min; F-18: 110 min -perm it the acquisition of severa! studics on the same day with minimum radiation burden to the patient and without background activity from prior injections interfacing with the measure­ments. allowing repeated studics, e.g. before and after pharmaccutical or invasive interven­tion, which is of big importance in diagnostic and therapcutic cardiology. The advantage of short half-lifc neverthelcss implies thc main disadvantage, i.e. the necessity of having a cyclotron for continuous isotope production and a po\\ierful radiochemical labora­tory for immediate production of radiotracers ncarby. Thercfore, PET is very cxpcnsivc with primary costs of about 10 million US$ for a complctc PET center and costs of abotlt 1000 US$ per invcstigation. Up to now. thc worldwide only fcw functio­ning PET centers havc focussed in cardiologic rcsearch on thc potential applications of this new techniquc in animal cxpcrimcnts rcgarding the noninvasivc investigation of problems conncctcd with myocardial ischemia and cardiomyopa­thics. -1 •• uJ,.7 Conclusivc results of clinical cardio­logical PET studics performcd mainly in USA are availablc now for only fcw ycars and a rclativcly limited numbcr of about 30 original papers conccrning 200-300 paticnts. Table 1 gives a summary of the most important tracers which have been used for monitoring myocardial blood flow, metabolism anci adreno­neuronal reccptors. These tracers have been used in clinical pilot studies. Because of its possible clinical importance, at present only specific PET techniques have to be discussed, which allow imaging and quantification of mar­ked known changes in heart energy metabolism during hypoxia (stop of fatty acid metabolism Table l. Positron emitting tracers for cardiological PET Studies Blood flow N-13NH3 Rb-82 0-15 H20 Metabolism C-11 palmi tate mctabolism of fatty acids F-18 deoxyglucose glucosc metabolism C-11 acetate 02 consumption in Krebs cyclc C-11 and N-13 amino acids protein synthesis Neuronal receptors C-11 quinucyclidine S-receptor ligand F-18 mctaraminol adrenergic innervation Miscellaneous 0-15 or C-11 C0 blood pool labelling F-18 misonidazole demonstration of hypoxia Rh-81 monitoring the potassium pool and switching over to aerobic and anaerobic glycolysis) simultaneously with blood flow meta­bolism studies. Ions of Rb-82 and N-13 ammonia proved to be good blood flow tracers analoguou­sly to Tl-201 scintigraphy, whereas F-18 labellcd deoxyglucose, F-18 DG, as a tracer of regionally increased glucose utilization, can be regarded as an established positive indicator of regional myo­cardial ischemia. With the use of this double tracer technique it seems possible to differentiate unequivocally normal myocardium, characteri­zed by normal blood flow and low glucose utilization under fasting conditions, from ische­mic but still vita! myocardium with decreased blood flow and increased glucose utilization, by this so-called »mismatch«. Additionally, infarc­ted myocardium (scar tissue) can be identified by F-18 DG and C-11 palmitate as a region of decreased flow and metabolism and quantifica­tion of infarct size is possible. 1.2 A typical exam­ple of a combined normal, ischemic, and infarc­ted myocardium is shown in Figure 1. Nearly all clinically relevant studies deal with the use of PET for specific and at least semiquan­titative evaluation and differentiation of normal, ischemic and infarcted myocardial tissue, e.g. after myocardial infarction or for follow-up be­fore and after coronary artery bypass grafting, angioplasty or pharmaceutical interven­ · 10•11 tion. 7•8•9 Unexpected and partially spectacu­ 126 Henze Eetal. Figure 1. PET tomograms of mi c: 2:! 50 ·: B .. . 40 . . 26 ,o 26,0 "' 20 1 10 4,3 '"L 17 ,3 r. : · n Target location A-3 B-2 0-3 0-2 D-4 B-4 Figure 4. Summary of target detectability in the thyroid phantom. 8/11 Location 62 D4 €3 C2A . 01 Al C1 E2 C:3 Size(mm} 20 16 M 11 10 9 9 7 7 Figure 5. Summary of target detectability in the brain phantom. routinely performed and observations of the phantom images. Fifteen laboratories carried out 23 studies with thyroid phantom, where 13 were performed with cameras and 10 with scanners. The scanners were generally older than cameras mostly which were manifactured in the 1970s. Five-inch crystal instruments represented the majority of scanners employed. Figure 4 depicts the rate of target detection according to target location, size and intrinsic contrast. This shows that the relationship bet­ween the three parameters identified with these techniques as determining detectability of targets are such that if two drop below a certain critical leve!, the target become undetectable regardless of the value of the third parameter. Eleven laboratories performed 11 studies wit.h brain phantom, by mearrs of gamma cameras. Figure 5 summarizes target detectability results in terms of target diameter for brain phantom. From the histogram in Figure 5 it is clearly demonstrated that detectability, as exp.cted in­creases with target diameter. In eight of 11 studies targets were correctly identified with diameter between 10 and 20 mm, but detectabi­lity significantly decreased for a target diameter 7 mm or smaller. A total of 15 laboratories participating in the !iver study provided results from 18 evaluations, 146 Karanfilski B 10 17 17 16 16 16 15 14 12 to 10 ) 1 2 A2@A4flS @A7 01@03 0<1@)06 n1@c2@ct1 cs C6 01@03@)05 D6 07 El E2 (3 [,1fS r1@FJ@r 5 G1 G2 r,. r.4 H1 K -. 2 . i . 4 3 :e 6 8.8 8 .10 Figure 6. Summary of target detectability in the !iver phantom. Tota! number of true pos1t1ve and false Figure 7. The heart phantom. Results obtained for ejection fraction. Tota! number of studies 18. ali performed with gamma cameras. Figure 6 depicts the rate of target detection according to target location and intrinsic contrast, presented as true and false positive reports. A decrease in detectability occurred for target to background ratios greater than 0.20:1. Ten participating laboratories submitted 18 studies performed on cardiac phantom. The mean ejection fraction for the entire group was 45.4 with standard deviation 6.3, coefficient of variation of 13.9% and a range of values from 33 to 55% (Figure 7). The corresponding ejection fraction for our heart phantom according to producer (Medica! Designs Inc., Danbury, USA) was 51.2%. Discussion The purpose of the intercomparison study on quality performance of nuclear medicine imaging devices is to allow as many as possible interested laboratories to participate within a resonable tirne period. Participation was voluntary. Ali laboratories interested were included in the stu­dy, without any selection. The study was found to be of great interest to ali participants. Effec­tive feed-back between the country coordinator and individual participating laboratories, and presentations of the results received from natio­nal nuclear medicine meetings are ali an integral part of the organizational mechanism of the Interlaboratory compar.ison study on quality performance of nuc/ear medicine imaging devices inter-comparison, and play an important role in the improvement of diagnostic possibilities. This inter-laboratory comparison study has provided an opportunity to study the leve! of total perfor­mance in thyroid, brain and !iver imaging in most of Yugoslav nuclear medicine institutions. Results of this study do not differ substantially from those obtained with thyroid, brain and !iver phantoms un Europe, 1• 2 but there has been relatively low degree of accuracy in target detec­tability in our studies. Herrera3 demonstrated that three of the tar­gets' physical characteristics appeared' to play a role in the ability of the participating laboratories to detect the constructed targets. These three parameters were the target diameter, the target­to-background ratio or intrinsic contrast, and the depth of the target within the phantom »body«. Based on these observations, it was concluded that the limit of detectability of low contrast targets using the existing instruments was as follows: minimum size of approximately 10 mm; depth within phantoms less than 7 cm; target to background ratio within 20% of the background radioactivity. Statistical analysis of the results, including ali targets and phantoms used up to 1978, confirmed these findings. Futhermore, the analysis demon­strated that relationship of these factors was such that if two of these parameters were below the critical leve!, it led to non-visualization of the target regardless of the value of the third one. 4 When a study of resolution is to be carried out, then the targets must vary in size (brain phantom). If a study of contrast is to be carried out then the lesions must vary in their relative absorbance of the targets to the activity over the background area(liver phantom). In studies of both contrast and resolution, the number of lesions must be included, some varying in ab­sorpance and others in size (thyroid phantom). Whatever parameter is chosen, it is important that some of the targets can be detected by ali laboratories and some of the targets by none, so that a complete range of abilities is tested. Since almost every camera or scanner can detect a 2 cm lesion and few can detect a 0.2 cm lession at a distance from the surface of the collimator, the range is fairly obvious. If using the survey to assess tqe ability to detect contrast changes; the size must be kept constant, as contrast and resolution are not independent variables. In this case the target size must be large enough to be detected by poorly resolving instruments. Then the change in the count density caused by the targets must be varied: Almost no camera system can detect a change of 5 percent but ali ,should certainly detect a change of 50%, or even 25%. The brain phantom was designed to investigate the relation betweerf 'target detectability aij,kl target size. Figµre 5 clearly d.emonstrates that detectability, as expected, increases with target diameter. With the !iver phaniom, the variable is target to background ratio and fiugre 6 shows that target detectability is inversely related to target to background ratio. The number of studies with rectilinear scanner was small with only 10 thyroid studies performed using this type of instrument. Thus, although the number of studies is too small to permit a detailed comparison between camera and scan­ner, it is of interest to record that for both types of instruments the leve! at which about 50% of the studies detected a target was similar. Quality control of nuclear medicine techniques to evaluate left ventricular ejection fraction is hampered by the lack of inexpensive, conve­nient, commercially avilable phantoms, and the absence of a generally accepted gold standard for the procedures. This reflects the fact that participants of our study were not performing quality control of this procedure in their labora­tories. In many instances this technique is imple­mented on the basis of comparison of results in patients having cardiac cateterization procedures of patients expected to have normal ejection fraction. The data illustrated in the figure 7 clear reveal considerable variation in the results obtained by participants in this study. This outcome is more striking considering that they were obtained using a simple mechanical device with invariant physical characteristics, uniform background and unhampered by cardiorespiratory movements 148 Karanfilski B and other, ,patient; characteristics. In view of this, the .data suggest at leas't equally serious problems in the clinicaL application of this technique. The causes, of the variation ,demonstrates that, alt­hough obviously related to kchnical procedures, equipment and software employed cannot rea­dily be established by the interlaboratory compa­rison method. This,is a well known limitation of thisqechnique. Also, artificial characteristics of phantom cannot replicate the clinical situation. References l. Volodin V, Souchkevitch G, Racoveanu H, Buse­mann-Sokole E. Deleloye B, Dermentzoglou F, :Georgescv G. Hcrrera N, Jasinski W, Kasatkin Y, Paras P, Mould R, World health organisation inter­laboratory comparison study in 12 countries on quality performance of nuclear medicine imaging devices Eur J Nucl Med 1985; 10:193-7. r• ) ·. ; ' 'il ;r; , 1 . ) ') :n ·-':11, ,-}',l 1• 1 1; ,. , . 2. Souchkevitch G, Asikainen M, Bauml A, Berg­mann H, Busemann-Sokolc E, Carlsson S, Delaloye B, Dermentzoglou F, Herrera N, Jasinski W, Ka­ranfilski B, Mester J, Oppelt A, Perry J, Sketting A, van Herk G, Volodin. V, Wegst A, Mould R. The World Health Organization and International Atomic Energy Agency second interlaboratory comparison study in 16 countries on quality perfor-. mance of nuclear medicine imaging devices. Eur J ,'Nucl Med 1988; 3:495-501. 3. Herrera N. History of the Interlaboratory Compari­son Programme. IAEA/WHO Newsletter on Qua­ . lity Assuarance Programmes in Nudear Medicine, IAEA, Vienna 1987; 2:4-6 . 4. Herrera N, Hermann GA, Hauser W, Paras P. College of American Pathologists Program Series X Survey Program. In: Medica/ Radionuclide lma­ging, 1980, IAEA, Vienna 1981; 2:177-87. .. ;r .r .p, t",L 1' Chapter IV. CLINICAL ONCOLOGY Adv Radio/ Oncol 1992; 151-66. Radiosurgery: A review of clinical aspects Souhami L1 and Podgoršak EB2 1 Departments of Radiation Oncology and 2 Medica! Physics Montreal General Hospital, McGill University Montreal, Quebec, Canada Radiosurgery is defined as the delivery, usually in a single session, of a high dose of radiation to a stereotactically localized small intracerebral lesion, while only minimally irradiating adjarent normal brain tissue. The aim of radiosurgery is to destroy or else to affect the function of the targeted volume. Although it was initially developed for the treatment of functional brain disorders, radiosurgery became a major treatment modality for a wide variety of selected malignant or nonmalignant intracranial lesions, including arteriovenous malformations, acoustic neurinomas, meningiomas, and small, solitary metasta­tic brain disease. Severa! thousand patients have been treated successfully with radiosurgery to date and the clinical interest in this therapeutic approach is growing rapidly. In this paper an overview of the clinical treatment results on the use of radiosurgery in severa! intracranial lesions is presented. Proton, charged particles, gamma unit, and linear accelerator-based techniques appear to be equivalent in terms of clinical results. Radiosurgery is an effective and well tolerated procedure for selected patients, it is, however, a complex procedure and should only be perf ormed in institutions with an expert inter-discipli­nary team of professionals. Key words: brain neoplasms-radiotherapy; radiosurgery, stereotactic radiotherapy Introduction Stereotactic radiosurgery was developed in Swe­den by Leksell more than 30 years ago1 with the primary intent of treating functional disorders of the brain. Over the years, the technique evolved to become an attractive therapeutic modality for a number of small, selected malignant or non­malignant intracranial lesions. The well proven efficacy of the technique has led to the develop­ment of a large number of radiosurgical facilities Corespondence to: Dr. L. Souhami, Department of Radiation Oncology, Montreal General Hospital, 1650 evenue des Cedres, Montreal, Quebec, CANADA H3GIA4. The growing interest and enthusiasm for radio­surgery were certainly stimulated by the excellent treatment results reported so far with the various radiosurgical approaches. These results have established radiosurgery as a major treatment modality for selected arterio-venous malforma­tions (AVMs), acoustic neurinomas, meningio­mas, pituitary adenomas, and small, solitary metastatic brain tumors. 2-27 To date more than 10 000 patients have been treated by radiosur­gery for a wide variety of intracranial lesions. However, in many lesions the number of patients treated is very small and foJlow-up is inadequate, UDC: 616.831-006.6:615.849.5 Souhami L and Podgoršak EB so that definitive conclusions regarding treat­ment efficacy cannot be made yet. In this paper, we review the clinical aspects and treatment results on the use of stereotactic radiosurgery in benign and malignant intracranial tumors. The physical aspects of radiosurgery and the various clinically used radiosurgical techniques are· dis­ cussed in the Chapter VI. Radiophysics. Clinical considerations in radiosurgery Radiosurgery is usually performed on an outpa­tient basis, although some institutions observe patients overnight following the treatment. In our departm > "' => = LEGEN OS 25 --= < 2 yrs ( n , 4 ) -·-·-·= . 10 yrs (n, 6) 25 50 75 100 125 150 175 200 225 TIME -M0HTHS Figure 3. SURVIVAL VERSUS GROUP - ALL PATIENTS ( n=37) GROUP I ( n = 15 ) 6ROUP ll ( n = 17 ) 6ROUP III ( n = S ) 125 150 Figure 4. Petric-Grabnar G et al. SURVIVAL 1H GROUP I PATIEHTS 1i'o1 .-----7 l______________________________ -­ 751 10 LEGEN OS 15 --no pos\op. CT, no SF cy\ology (n:15) ----pos\op. Cl= neg , S F, neg ( n: 9 ) ll so 71 100 115 110 TIME· M0KTHS Figure 5. Table 3. Results in relation to group and chemotherapy of the patients properly classified as Group I, protocol one died in tumour (Figure 5). Group SIOP HD Cytoxan No Cht TOT AL Of the 17 patients classified as Group II (8 of them without CSF cytology), 10 are alive. Of the 15 patients who had preradiation ChT, 9 are ChT I -II 7;5+ 4/2 513+ 16/10 alive and well. It could not be concluded that III 1/1 3/1 1/0 5/2 ChT had a favourable effect on the survival in TOT AL 8/6 7/3 22/14 37/23 this small series, it is, however, interesting to note that the 2 survivors with Group III disease both had preradiation ChT (Table 3). - 16/11 16/11 + -one pt had adjuvant ChT Table 4. The pattern of recurrenccs according to prognostic factors Ali failures LOW RISK FAILURES 16 pts -supratentorial & CSF-posterior fossa only 2 1 HIGH RISK FAILURES In sixteen patients the tumour recurred, 2 of these are alive, one stili being treated, and one without sign of tumour more than one year afte; treatment for recurrence. The pattern of recurrence is shown in Table 4. Most common was supratentorial recurrence, we could not find, any correlation with a lower dose in the anterior part of the brain. -supratentorial & CSF-CSFonly 3 30 -supratentorial onlyTen of the_ 16 ( 60%) recurrencies appeared -posterior fossa only 2 during the first 2 years, 13 (80%) during the first -posterior fossa & CSF 1 3 years, another during the 4th year and one recurrence in the posterior fossa more than 7 +2 -recurrences at the junction o2 -suprasellar recurrences years after treatment. Medulloblastoma, results. of treatment at the Institute of Onco/ogy, Ljubljana Discussion and conclusion The reports on significant improvement clue to treatment could not be confirmed in our study The survival rate in the group treated during the recent period was not better from the survival earlier, despite the improved technique and addi­tion of chemotherapy. One explanation, of course, is that the 2 groups are not comparable. Ali small children up to 2 years of age known to have a poor prognosis5 •17 are from the recent group. The most important factor influencing the outcome is the extent and resectability of the tumour. _Patients with completely resected tu­mours without spread do very well, an observa­tion reported from experience on larger series and confirmed in ours. 4•5 •6 In the more advanced -non resectable (Group II) tumours and in those with malignant cells in the CSF our results are poor and chemotherapy did not improve the survival rate, possibly the survival tirne. It cer­tainly did not contribute to a better survival in those small children treated with a smaller radia­tion dose and preradiation ChT (HD Endoxan). Both boys less than 2 years at diagnosis had, however, a normal development during treat­ment and remissions. They were repeatedly in complete clinical remissions after HD Cytoxan and lived more than 3 respectively 4 years from diagnosis. While lower doses of radiation seem to be suficient for cure in some low risk group patients8 , they are insufficient in the high risk group patients comprising children up to 2 years of age who are most in need of less treat­ment. 9, 15 , 19,22 The fact that in our series the most common site of recurrence was supratentorial, is unusual, especially the 3 suprasellar recurrences. We have not found any correlation with a possible lower dose in the supratentorial region with the recur­rences (less than 3600). As neither daily checking of setups nor consistent checkings with portfilms were possible this finding could allow for suspi­cion of occasional too generous blocking. Although the series is small, the observations allow us to conclude the following. As in other series: children of 2 years or less have poor prognosis, as have the patients with incomplete tumour excision and/or malignant cells in the CSF. 5 • 17 Lower doses of radiation to the CNA in combination with HD Cytoxan do not re.uli in cures in young children. Chemotherapy has not significantly impr-6ved the cure rate, but seems to prolong the survival, and possibly postopone recurrence. At present the prognosis in patients with advanced medullo­blastoma (more than Group I) is stili poor. More effective ChT regimens than those used in our series are needed. References 1. Schulte FJ. Intracranial tumours in childhood ­concepts of treatment and prognosis. Neuropedia­trics 1984; 15 :3-12. 2. Incidenca raka v Sloveniji 1985. Ljubljana: Onko­loški inštitut, Register raka za SR Slovenijo 1989. 3. Dietz R, Graf N. Zur Therapie des Medullobla­ stoms -Ergebnisse aus den Jahren 1968 bis 1985. Strahlenther Oncol 1990; 166:683-7. 4. Bloom HJG, Glees J, Bell J. The treatment of longterm prognosis of children with intracranial tumours, a study of 610 cases at the Royal Mar­sden Hospital, 1950-1981. Int J Radiat Onco/ Biol Phys 1990; 18:723-45. 5. Hughes EN, Shillito J, Sallen SE, Loeffler JS, Cassady JR, Tarbell NJ. Medulloblastoma at the joint center for radiation therapy between 1968 and 1984. Cancer 1988; 61:1992-8. 6. Jereb B, Reid A, Ahuja RK. Patterns of failure in patients with medulloblastoma. Cancer 1982; 50:2941-7. 7. Jereb B, Krishna.wami S, Reid A, Allen JC. Radiation for medulloblastoma adjusted to pre­vent recurrence to the cribriform plate region. Cancer 1984; 54:602-4. 8. Halberg FE, Wara WM, Fippin LF et al. Low­dose craniospinal radiation therapy for medullo­blastoma. Int J Radia/ Oncol Biol Phys 1991; 20:651-4. 9. Kun LE, Constine LS. Medulloblastoma -caution regarding new treatment approaches. Int J Radiat Onco/ Biol Phys 1991; 20:897-9. 10. Tomita T, McLone DG. Medulloblastoma in child­hood: results of radical resection and low-dose neuraxis radiation therapy. J Neurosurg 1986; 64:238-42. 11. Levin V A, Rodriguez LA, Edwards MSB et al. Treatment of medulloblastoma with procarbazine, hydroxyurea, and reduced radiation doses to whole brain and spine. J Neurosurg 1988; 68:383-7. Petric-Grabnar G et al. 12. Allen JC, Helson L, Jereb B. Preradiation chemot­herapy for newly diagnosed childhood brain tu­mours: a modified phase II tria!. Cancer 1983; 52:21-6. 13. Kovnar EH, Kellie AJ, Horowitz ME and al. Preirradiation ciscplatin and etoposide in the treat­ment of high-risk medulloblastoma and other mali­gnant embryonal tumours of the central nervous system: a phase II study. J Ciin Oncol 1990; 8:330-6. 14. Lefkowitz IB, Packer RJ, Siegel KR, Sutton LN, Schut L, Evans AE. Results of treatment of children with recurrent medulloblastoma / primi­ tive neuroectodermal tumours with lomustine, ci­splatin, and vincristine. Cancer 1990; 65:412-7. 15. Loeffler JS, Kretschmar CS, Sallan SE et al. Pre-radiation chemotherapy for infants and poor prognosis children with medulloblastoma. lnt J Radiat Oncol Biol Phys 1988; 15:177-81. 16. Tait DM, Thornton-Jones H, Bloom HJG, Le­merle J, Morris-Jones P. Adjuvant chemotherapy for medulloblastoma: the first multicentre control tria! of the International society of Paediatric oncology (SIOPI). Eur J Cancer 1990; 26:464-9. 17. Deutsch M. Mudulloblastoma: staging and treat­ment outcome. lnt J Radiat Oncol Biol Phys 1988; 14:1103-7. 18. Harisiadis L, Chang CH. Medulloblastoma in chil­dren: a correlation of staging and results of treat­ment. lnt J Radiat Oncol Biol Phys 1977; 2:833-41. 19. Neidhardt MK, Bamberg M, Riehm H. Medullo­blastoma: strategies for therapy. Monogr Paediat 1986; 18:296-315. 20. Kaplan EL, Meier P. Non-Parametric estimation for incomplete observation. J Am Statis Assoc 1958; 53:457-81. 21. Mrilhern RK, Horowitz ME, Kovnar EH et al. Neurodevelopmental status of infants and young children treated for brain tumours with preirradia­tion chemotherapy. J Ciin Oncol 1989; 7:1660-6. 22. Shalet SM, Beardwell CG, Morris-Jones PH et al. Pituitary function after treatment of intracranial tumours in children. Lancet 1975; 2:104-7. Adv Radio! Oncol 1992; 175-80. Intrapleural application of human leukocyte interferon (IFN-a) in breast cancer patients with pleural carcinosis Jereb B1 , Štabuc B 1 , Us-Krašovec M1 , Cerar 01 , Stare J2 1 The Institute of Oncology, Ljubljana, 2The Institute for Biomedical Informatics Medica! Faculty, Ljubljana Ten patients with breast cancer and pleural carcinosis, receiving IFN-a intrapleurally in addition to conventional treatment, were compared with 20 control patients who had no IFN-a. The effect of IFN-a. was evaluated with cellular morphology of the pleural fluid and with the patients'survival. After application of IFN-a intrapleurally reactive cells appeared and malignant cells disappeared eventually. The mean survival time after onset of pleural carcinosis was 15 months in the experimental group and 9 months in the control group. Three patients out of the JO in the experimental group are alive, 2 of them (both had inflammatory cancer) are NED off treatment more than I respectively 2 years. One patient of the 20 in the control group is alive with disease. There were no complications. IFN-a looks promising as supplementary treatment in these patients. Key words: breast cancer, pleural carcinosis, IFN-a Introduction In a previously reported series of 7 patients with. breast cancer and ipsilateral pleural carcinosis it was observed that a crude product of IFN-a was locally highly effective. 1 Malignant cells disap­peared from th. pleural fluid, but aplications of IFN-a were painful and also produced pleural adbesions. The effect on survival could not be evaluated. We therefore continued the investiga­tion with a more purified product, and tried also to find out whether local applications of IFN-a do affect the survival of patients with breast cancer. Correspondencc to: Berta Jereb, M.D., The Institute of Oncology, Zaloška 2, 61000 Ljubljana, Slovenia. UDC: 618.19-006.6-085:616.25-006.6-085 Material and methods Ten women with breast cancer of both the experimental (10 pts) and the control group had pleural effusion diagnosed during 1985 through 1989. Ali had had the diagnosis of pleural carci­nosis proven by cytology. In both groups the treatment was decided upon by the caring physician, regardless of whether the patient was to receive IFN-a or not. The patients of the experimental group were chosen at random. The systemic treatment for breast carcinoma either primary or for recurren­ces was rather uniform through the years unper investigation. The age of the patients was between 37-70, and the mean for the experimental group was 49, and 57 for the control group. The control group consisted of 20 patients, .with ipsilateral effusion in 18 and bilateral in 2. Jereb Bet al. Eleven patients had locally advanced (T 3 and T 4) and 9 had operable (T 1 T 2) bre.st cancer at diagnosis. The primary treatment was radical mastectomy in 15 (with adjuvant chemotherapy in 7 and hormona! treatment in 2,6 had radiation therapy, 5 of which also had chemotherapy). Pleural carcinosis was found at diagnosis in 5 patients, 16 had bone and soft tissue metastases, 10 had Jung and 4 had !iver metastases at onset of pleural carcinosis. Ali patients had systemic treatment and 9 had also 1 or 2 instillation of Bleomycin (30-60 mg) into the pleural cavity. The clinical = = . . 0.80 . > . ::,: = 0.60 0.10 0.20 1 ', 1 L7 1 ', 1 L---7 1 1 L--, --­EXP!:RIMEUAL GRDUP ----­tllKIRDL GRDUP 1 1 1 _ll_____________ -­ O.O 10 10 JO MOKTHS • ··• • (1 • .... Discussion 1 Figure 3. Same patient. Pleural effusion after 3"How much IFN-a given intrapleurallyl, is re­application of IFN-a Lymphocytes and few degene­ sponsible for the changes in cytology of the rated (malignant ?) cells. Giemsa. original magnifiction 25 X 4. pleural fluid is difficult to establish. There might 179 /1111.111!,·uml II/JJJ/ic11tio11 of lu111w11 leukocytl' i111cr/1·ro11 /Fi\'-a in /m•1111 n111cer 1111tie111s Table 2. Clinical data at onset of pleural carcinosis for experimental (10 patients) and control group (20 pa­tients) CLINICALDATA GROUP expcrimental control nr. of pts nr. of pts lnflammatory cancer at diagnosis 4 5 pleural carcinosis at I st treatment 2 2 plcural carcinosis at recurrence 2 3 Plcural carcinosis at rccurrence 8 15 1" 4 6 2nd 4 9 Ipsilateral pleural carcinosis and involvement of chest wall 10 Bilateral pleural carcinosis and lung carcinosis 5 10 Involvcment of othcr organs 4 13 !iver 2 4 bone 4 10 lymph nodes 2 7 Table 3. Systcmic trcatmcnt for plcural carcinosis for cxpcrimcntal (10 paticnts) and control group (20 paticnts) CHEMOTHERAPY GROUP experimcntal control nr. of pts nr. of pts Mitomycin & Adriablastin 3 8 (5) Methotrexate & 5-Fluorouracil 2 5 (1) Mitomycin & Vinblastinc 1 Epidoxarubicin & Cytoxan & 5-Fluorouracil Epidoxarubicin & Vincristine THIO-TEPA & Vinblastine CCNU-Nitrosurea & Dacarbazin & 5-Fluorouracil 2 Hormoncs 2 (2) None 3 (3) ( ) also Blcomycin locally be other reasons for changes in the appearance of celi, f.i. concomitant chemotherapy. On the whole, however, it can bc said that during trcatment with IFN-a malignant cells degenera­ted and finally disappeared, while at the same tirne reactive cells were observed. Allthough this effect might vary in degree, it is rather unifor­mely observed in a previous and in this group of patients. In this series, however, the impression was, that the appearance of reactive cells was less expressed in the more recently treated group of patients. This could be a consequence of more purified interferon as well as of concomitant chemotherapy. It is generally agreed upon that pleural carci­nosis is associated with poor prognosis and has to be treated with aggressive chemotherapy even in the abscnce of othcr evidence of metastasis is. 2-5 The kind of additional local treatmcnt might, however. stili be a matter of different choices. There are few randomized studies, and a clear superiority of one modality or agent has not been established. Bleomycin seems to be one of the most effective agents but its severe toxicity was reported. 6· 7 As addition to systemic therapy in patients who already have been, in the majority of cases, treated previously with chemotherapy and there­fore at risk for toxicity, an approach with a less toxic agent would be more advisable. IFN-a as it was used in this series is a harmless and effective tool for palliative treatment of pleural carcinosis in breast cancer. The series is too small to allow conclusions as to its effect on the survival; encouraging are the 2 patients without evidence of active disease. In women with inflammatory cancer at diagnosis and pleural effusion IFN-a should be tried. References l. Jereb B, Us-Krašovec M, Cervek J, Šooš E. Intra­pleural application of human leukocyte interferon (HLI) in breast cancer patients with ipsilateral pleural carcinomatosis. I Interferon Res 1987; 7:357­63. 2. Raju RN, Kardinal CG. Plcural effusion in breast carcinoma: analysis of 122 cases. Cancer 1981; 48:2524-7. 3. Izbicki R, Weyhing BT III, Baker I, Caoili EM, Vaitkevicius VK. Pleural effusion in canccr pa­tients: a prospcctive randomizcd study of drainage with the addition of radioactivc phosphorous to the pleural spacc vs. pleural drainage alonc. Cancer 1975; 36:1511-8. 4. Fentiman IS, Rubens RD, Hayward JL. Control of plcural cffusions in patients with brcast canccr. Cancer 1983; 52:737-9. 180 Jereb Beta/. 5. Rosso R, Rimoldi R, Salvati F et al. Intrapleural 7. Ostrowski MJ, Priestman TJ, Houston RF, Martin natura! beta interferon in the treatment of mali­WMC. A randomized tria! of intracavitary bleomy­gnant pleural effusions. Oncology 1988; 45:253-6. cin and corynebacterium parvum in the control of 6. Ostrowski MJ, Halsall GM. Intracavitary bleomycin malignant pleural effusions. Radiother Oncol 1989; in the management of malilgnant effusions: a multi­14:19-26. center study. Cancer Treat Rep 1982; 66:1903-7. Adv Radio! Oncol 1992; 181-95. Soft tissue sarcomas in childhood Experience of the Austrian cooperative rhabdomyosarcoma study (A-RMS 82) A finai report Ladenstein R, Griimayer ER, Hawliczek R, Krepler R, Fink FM, Haas H, Mutz I, Ploier R, Stollinger O, Thun-Hohenstein L, Tulzer W, Urban C and Gadner H Fram the Austrian Rhabdamyasarcama Study Graup. Caaperating centers: St. Anna Children's Haspital, Vienna (R.L., E.R.G., H.G.); Clinic af Radiatian Therapy and Radiabialagy, University Vienna (R.H.), Institut far Pathalagy, University Vienna (R. K.), Departments af Pediatrics: University af Innsbruck (F.M.F.), Kardinal Schwarzenberg'sches Krankenhaus, Schwarzach im Pangau (H.H.), Landeskrankehaus Leaben (I.M.), Landes­krankenhaus Steyr (R.P.), Krankenhaus der Barmherzigen Schwestern Linz (O.S.), Univer­sity af Vienna (L. TH.), Landeskrankenhaus Linz (W. T.), University af Graz (C. U.); Fifty-two children with soft tissue sarcomas were treated according to the Austrian Study A-RMS 82. First surgery included non-mutilating primary turno,-removal or biopsy only. Stratification to treatment arms was based on site of primary tumor, stage of disease and response to initial multi-agent chemotherapy. Treatment duration was 36 weeks for clinical groups I to IIA and 56 weeks for clinical groups IIB, III and IV, respectively. Median age at diagnosis was 5.5 years, the median observation tirne 65 months. The overall complete response rate was 82.5%. The probability far overall survival was 71%, for event free survival (pEFS) 63% at 65 months. Protocol patients had a pEFS of 86% in clinical groups I to IIA and of 64% in clinical group III. Rapid tumor response to initial chemotherapy was a positive prognostic indicator. Extremity sites had an excellent 74% pEFS rale. Preservation of involved urogenital tract organs was achieved in all patients with this primary site. Early shifting of nonresponclers to enhancecl, six drug chemotherapy resultecl in aclclitional complete remissions. However, prognosis of patients with retroperitoneal sites ancl metastatic clisease was poor. Key words: rhabdomyosarcoma; child Introduction Soft tissue sarcomas (STS) represent about 10% '6f ali malignant solid tumors in children 1 with rhabdomyosarcoma (RMS) as the most frequent. Treatment of RMS with surgery of radiotherapy Correspondence to: Helmut Gadner, MD, (senior invcstigator) St. Anna Children's Hospital, Kinderspitalgasse 6, A-1090 Vienna, Austria. Phone: 0043/222/40170/250 UDC: 616.74-006.364.04-053.2 alone or even a combination of both had been disappointing illustrated by an overall survival (SU) of 10%. 2 The addition of single3A and later of multidrug chemotherapy to local treatment improved overall SU in a range from 28% to 37%. 5· 6 Consequently, multimodality aproach­es to treatment developed. They included surgi­cal resection of primary tumors, radiotherapy to the tumor bed if microscopic disease remained and chemotherapy, reflecting the advancement through a variety of drug schedules. In this way considerable further improvement of survival Ladenstein R et al. rates within the range of _45% to· 54% was 11 - achieved. 7Various prognostic factors like pri­ mary site, stage of disease, histology and tumor 16 - response to chemotherapy were identified. 12 Compared to RMS less data are available about chemosensitivity and optimal treatment approac­hes for other STS in childhood. 17-21 We report on 52 patients treated according to the cooperative Austrian Rhabdomyosarcoma Study A-RMS 82, which was designed in close collaboration with the German Soft Tissue Sar­coma Study Group. The objectives of the study were: l. Enrolling of ali children with soft tissue sarcomas to a common treatment concept. 2.. Avoidance of pri­mary mutilating surgery, in particular in children with involvement of the urogenital tract. 3. Treat­ment intensification for extremity tPmors. 4. Intro­duction of a six-drug salvage therapy in the early treatment phase in case of non-response to initial chemotherapy and for clinical group IV. Patient and methods A total of 52 patients were registered from January 1982 til! September 1987 and received treatment in 9 pediatric centers in Austria. There were 29 male and 23 female patients with an average age of 5.5 years at diagnosis (range: 2 weeks to 18 years). Full evaluability required review of histopathology, chemotherapy, surgery and radiotherapy data. Eligibility requirements have been fullfilled by 39 protocol patients (P pts). Thirteen patients were excluded from detailed survival analysis because of different pretreatment or violations of guiding principles, but have been followed as study patients (S pts). Histology Fourty patients (77%) presented with RMS. The distribution of histologies in the other 12 patients (23 % ) was as given: 2 primi ti ve neuroectodermal tumors (PNET), 2 synovial sarcomas (SyS), 2 angiosarcomas, 2 spindle-cell sarcomas, 1 extra­sceletal Ewing's sarcoma (EES), 1 hemangiope­ricytoma, 1 fibrosarcoma and 1 rhabdoid tumor. Within the RMS group 24 patients displayed the embryonal, 10 patients the alveolar and 2 pa­tients the pleomorphic subtype. Four patients presented with undifferentiated RMS. Seven out of 15 patients with extremity sites exhibited the alveolar subtype. Primary tumor sites and clinical groups Details on the primary tumor sites and clinical groups of the 52 patients are shown in Table l. treatment and course of disease are given in Table 2. The percentage of patients within clini­cal groups I to IV was 17%, 15%, 56%, and 12%, respectively. The most common site was the extremity (29%), followed by UG (17%). An equal number of patients was found for tumors of the orbit, other head and neck loca­tions ( orbit excluded) and retroperitoneal sites (UG and paratesticular sites excluded), each accounting for 11 % . The remaining patients had tumors arising from paratesticular sites (8 % ) and paraspinal sites ( 4 % ) . Diagnosis Clinical evaluation. Initial stage of disease was assessed by a complete physical examination, x-ray, ultrasound and CT of primary tumor site to evaluate initial extent and size of tumor; complete blood count, complete serochemical Table l. Primary sites and clinical groups Number Clinical Groups Primary Sites of after First Surgery Patients I II(A/B) III IV Head& Neck 6 1 4 1 Orbit 6 1(1/0) 4 1 Thorax 4 1 2(2/0) 1 Paraspinal Sites 2 1(1/0) 1 Urogenital Tract 9 1 1(1/0) 7 Paratesticular 4 2 2 Retroperitoneal 6 4 2 Extremity 15 4 3(3/0) 7 1 Tota! 52 9 8 29 6 Soft tissue sarcomas in childhood, austrian multicenter study A-RMS 82 screening, bone marrow aspirate from the iliac A-RMS 82 crest, CT of the chest, abdominal ultrasono­graphy and 99 Tc-total body scintigraphy, i.v. urography and lumbar puncture when indicated. Ali patients were grouped according to the modi­fied Intergroup Rhabdomyosarcoma Study cla­sification 10 based on the extent of disease and type of primary surgery performed (Table 3). Table 3.,Clinical grouping. GROUPS CLASSIFICATION I: Localised disease, completely resected, regional lymphnodes negative. IIA: Grossly resected tumor with microscopic residual disease, regional lymph nodes negative. IIB: Grossly resected tumor ( completely resected or microscopic residual disease), regional lymph nodes involved but resected. III: Incomplete resection or biopsy with gross residual disease with or without lymph node involvement. IV: Metastatic desease present at onset. Definition of response Response evaluation was based on local CT and/or ultrasonography. Second look surgery provided analysis of histological response. Com­plete response (CR) was defined as non evidence of disease. Partial response (PR) criteria were reduction to one third of the maximum tumor diameter after the first chemotherapy cycle and at least 50% reduction after the second course including dissappearance of ali metastatic le­sions, eventually. Non-response (NR) included persistence as well as progression of disease. Study design (Figure 1) Assignment to therapy arms. Patients were classified first to clinical groups I-IV according to the result of first surgery (biopsy only or resection) or due to metastatic disease. Second, ali patients with localised extremity or urogenital (UG) tract tumors were assigned to the more intense treatment of clinical group III irrespec­tive of initial post surgical grouping to reduce the [ rc\cction or hiopsy 1„ .; ' ' 1, lin llh.111 IV . . . 10 " . ' . ' [X} .; l rcscction or biupsy 1 ' ' ,: im1dirnion primnry site : 40 • 50Gy J lymphnode!> : :w.<;y f lO 'III "II . [I]] 'III 26 "II 30 'III JO "II -)(, . . 36 «I v v [z;] 4(, _, v v l-0 ,. v v OJ] wwb ".':l, OvACA 0vAPAC [ID VECA "II VA V VAC Figure 1 Schematic presentation of the Austrian Rhab­domyosarcoma Study 82. Treatment plan. V ACA: vincristine (VCR), doxorubicin (ADR), cyclophospha­mide (CYC), dactinomycin (AMD); VAPAC: VCR, ADR, cisplatin (CDDP), AMD, CYC; VECA: VCR, etoposid (VP16), CYC, AMD; VA: VCR, AMD; VAC: VCR, AMD, CYC; known high risk of relapse in the former and to increase the chance of preserving UG organs in the latter. Patients with parameningeal tumor sites received intrathecal therapy in addition22 . In case of tumor non-response to initial chemoth­erapy a third classification step was employed. These patients were shifted to the enhanced treatment arm as used for clinical group IV. Systemic therapy. The initial chemotherapy for ali patients was one V ACA cycle (Figure 2) inclu­ding vincristine (VCR), doxorubicin (ADR), cyclophosphamide (CYC) and dactinomycin (AMD). In clinical groups I-III a second VACA Table 2. Paticnts charactcristics at diagnosis. trcatmcnt stratcgy. rcsponsc and outcomc Clinical B/R Ciin. Status at CT end T Pts Follow/ Age Primary Group / Re.onse Stage Irradiation Gy J/Jf, No. at Primary Tumor Treatment at eeks after (local + Iy.no. Site of L or OCD P/S Sex Dx Site Histology Size Arm 9 and 16 S.L.S. /+ met. /+ Iungs) (mo after Dx) 50 + 30 CR / DOD / L.F.(23) CR / DOD / met.(29) 14P 17S 14.llyr m f m H&N H&N alveolar > 10 cm III / III PR/CR n.d pleomorph > 10 cm III/IVNR NR/PR H&N undiff. 3 -5 cm III/ III CR/CR 3 -5 cm III / III PR/NR 24P 37P m 0.Syr llyr embryonal em.ryonal B/IIA B/I 70 (earlier) n.d. CR/NED PD/DOD H&N H&N 5 -10 cm IV /IV < 3 cm I /I PR/CR R/I 40 /+25 /+15 CR/NED 47P f 13.llyr 48P m 2.llyr H&N ang10 8P m 20S m 2weeks scar CR/NED CR/NED orbit orbit 8.lyr 12.9yr 5yr orbit embonal 3 -5 cm III / III PR/CR n.d. 40 rhab oid II /IVNR orbit 4< 3 cm NR/PR 50 CR/CR NR/PR III /i III III IVNR < 3 cm 50 CR/NED 50 29P m 32S 40P orbit orbit embryonal hemangio embryonal embryonal B/IIA R/1 n.d. m PR/PR NR/NR < 3 cm < 3 cm IV/IV < 3 cm III/IVNR f CR/NED 50 /+40 CR / OCD (MCCL(8)) 50 PD/DOD R/IIA 52S f 2P f 18thorax PNET 5 -10 cm I/1 CR/CR n.d. n.d. CR/NED 25S f l. ?;r thorax alveolar 5 -10 cm IVlV PR/CR n.d. 50 + 30 /+50 CR/NED 0.4yr thorax undiff. > 10 cm II III CR/CR n.d. n.d. CR/NED r, s:, ;, 30P f n.d. CR/NED 39S m 0.8yr thorax embryonal 3 -5 cm II/II CR/CR n.d. 21P f 11.lOyr paraspinal undiff. < 3 cm II/II CR/CR n.d. 40 CR / DOD / L.F.(10) . f 1.4yr paraspinal EES 5 -10 cm III/III CR/CR n.d. 40 CR/NED 23P s:, 5S m 17yr retroperit angio > iD cm III/III PR/PR n.d. experimental CR/LTFU 7P f 17.4yr retroperit embryonal > 10 cm IV /IV NR PD/DOD 15P m 12.?yr retroperit alveolar > 10 cm III/III CR/CR B/1 40 + 30 CR/NED 33P m 2yr retroperit embryonal 5 -10 cm III/III PR/CR B/I 40 + 30 OCD (ARF(12)) 44P f 11.lyr retroperit undiff. > 10 cm HI/III PR/NR PD/DOD 50S m 4.7yr retroperit embryonal > 10 cm IV /IV PR/PR R/IIA 40 PD/DOD Table 2. Patients characteristics at diagnosis, treatmcnt stratcgy. rcsponsc and outcomc Clinical B/R Ciin. Status at CT end /Pts Age Primary Group / Response Stage Irradiation GyFollowUŠ / No. at Primary Tumor Treatment at Weeks after (local + ly.no. Site of R L or OCD P/S Sex Dx Site Histology Size Arm 9 and 16 S.L.S. / + met./+ lungs) ( mo after Dx) m urogenital CR/CR B/1 B/1 6P m 5.6yr urogenital embryona 15-lOcm ITI/III PR/PR B/I 50 + 30 CR/NED 9P m 0.9yr urogenital embryonal > 10 cm III/IVNR NR /PR R/IIA 50 + 30 CR/NED11P 2.3yr < 3 cm 50 + 30 embryonal embryonal III/III II˝IVNR 13P 27P 5 -10 cm > 10 cm 50 + 3O(earlier) n.d. s CR/NED CR/NED f f 36P 45P II III NR /CR CR/CR B/I CR/NED PD /DOD 15.lyr urogenital urogenital embryonal embryonal > 10 cm 5 -10 cm :;.· m PR/NR NR/PRPD /DOD PR/PR 50 + 30CR/LTFU n.d. 46P 34P m l.2yr l.9yr urogenital urogenital uro.enital embryonal embryonal "' III/III III/IVNR '< m f R/IIA n.d. > 10 cm < 3 cm r, o CR/CR penanal embryonal nyrn I/ CR/NED s "'"' lP 19P m paratest. embryonal 3 -5 cm I/I paratest. embryonal 3 -5 cm CR/CR n.d. n.d.CR/NED CR/CR n.d. n.d. CR/NED40 + 30 s· m 35S 43S I/I 3 -5 cm III/III PR/PR < 3 cm III/III CR/CR :::.: ­ ­ o o CR/NEDCR/NED paratest. paratest. embryonal spindle-c. m 16.4yr 6.6yr m R/I B/I n.d. n.d. 40 + 30CR/NEDR/I 40 + 30CR/NED 40 + 30 3P f f 4P 10P m 15 2. rv; yr extremity PNET 3 -5 cm II{III CR/CR extremity alveolar 5 -10 cm II /III PR/PR > 10 cm .­ "' f 5.2yr extremity NR/PR PR/CR R/I n.d. embryonal alveolar alveolar III/IVNR 5 -10 cm III / III 3 -5 cm CR/NED 50 + 30 12P 16P m extremity extremity extremity extremity extremity CR/CR PR n.d. CR / DOD / met.(42)CR/NED 40 + 30 f spindle-c fibro I / III 5 -10 cm III/ III 3 -5 cm III/ III 7.6yr 0 3yr ::, 18S 22P :;:: /DOD s PR/CR R/I 40 + 30CR/NEDPR/CR B/1 50 +30CR/NED40 + 30 m alveolar 5 -10 cm < 3 cm III / III I / III ­ 26P 28P f B/I n.d. ::s· m f 7.2yr 9.llyr 16 extremity extremity embryonal SyS .s < 3 cm III /IVNR I / III CR/CR NR/PR 50 + 3050 + 30 CR/NED 31P 38P m CR/CR n.d. PR/NR '< =:?. f 8. tv; yr extremity veolar 3 -5 cm 5 -10 cm 5 -10 cm IV /IV NR II IVNR CR / NED / met.(1O)CR / DOD / met.(28) l 41S 42P m PD /D0D NR /CR earlier 50 + 30CR/NED CR/CR n.d. 40 + 30 L.F-?U: NED B/I 40 + 30 extremity extremity extremity alveolar alveolar pleomorph > 10 cm 3 -5 cm 2yr 13.llyr lyr 49S 51P f f extremity II / III I / III CR/CR :i. CR D . 1 2:: vi Pts No: patients number, P/S: prot9col or study patient, DX: diagnosis, S.L.S.: second look surgery, B/R: biopsy or resection, Iy. no.: Iymph nodes, met: metastases, mo: months, m: C:; male. f: fcmale, yr: ycars, H&N: hcad and nc.k • undiff.: undiffcrcntiated RMS. angio: angiosarcoma. rhabdoid: rhabdoid tumor, hemangio: hcmangiopcricytoma, Pnct: pcriphcral N . _ neurocctodcrmal tumor, EES: cxtra-osseous Ewmg s sarcoma, spmdle-c: spmdle-cell sarcoma. f1bro: f1brosarcoma. SYS: synov,al sarcoma, CR: complctc responsc. PR: partial response. NR: non-responsc, NED: no evidence of disease, L.F.: local failure, PD: progrcssing discase, DOD: dead of disease, OCD: other cause of death, LTFU: !ost to follow up, ARF: acute renal failurc, MCCL: megacaryocytic Ieukemia u, Ladenstein Reta/. VACA CYC o 1201> m,:/m1 ... CYC o 400 m,:/m! fa . ne " J.!00 m,:/m1 ADR CI JO mltfm1 -\\tl) • U.5 m,:/m ! .\f>R O .lO rn,:/m1 1 1 1 1 \('R 1.5 m,e./m. :,C „n-J..\ Figure 2. V ACA Trcatmcnt schcdulc. V ACA: vincri­stine (VCR), doxorubicin (ADR), dyclophosphamide (CYC). dactinomycin (AMD). cycle was applied. Clinical group IV and patients with non-response to the first V ACA cycle received as second cycle V APAC, a modification of VACA, in which CYC was replaced by cisplatin (CDDP, 120mg/sqm, day 3) at weeks 1 and 7. Further chemotherapy for clinical groups I and IIA consisted of six pulses of V A (VCR-1.5mg/sqm and AMD: 1.5mg/sqm) in a three week interval. Clinical groups IIB and III recei­ved two more V ACA cycles and 6 further V AC pulses (VCR: 1.5mg/sqm, AMD: l.5mg/sqm and CYC: 20mg/kilogramm). In clinical group IV ADR was replaced by etoposide (VP16: 150mg/sqm, days 1-3) at weeks 1 and 7 in the third cycle called VECA. The first three cycles were then repeated in this group once more. Thus treatment duration was 36 weeks for clini­cal groups I and IIA and 56 weeks for clinical groups IIB, III and IV, respectively. Patients with parameningeal tumors received additional intrathecal chemotherapy eight times during the first 16 weeks. The age adapted dosage was for infants, children of 1-2 years, 2-3 years and for those older than 3 years for methotrexate: 6mg/ Smg/ lOmg/ 12mg, for cyto­sin-arabinosid: 20mg/ 26mg/ 34mg/ 40mg and for hydrocortisone: 4mg/ 6mg/ Smg/ 10mg, respecti­vely. Local Treatment. The issue of initial surgery was non-mutilating tumor removal or assessment of histology by biopsy only. Second lo?k surgery was demanded for patients with residual disease at week 16 and had the purpose of radical resection. Patients in clinically complete remis­sion at week 16 were desired to have biopsies of the primary tumor region to assess their histolo­gical remission. Radiotherapy was timed after second look surgery for local tumor control as well as distant tumor sites, using super-voltage equipment with wide margins. Patients in clinical groups I and IIA received non radiation therapy except for tumors of extremity and urogenital sites or in case of proven microscopic residual disease for clinical group IIA at second look surgery. In ali other patients local irradiation with 40 Gy (frac­tion size 1.8 Gy, 5-6 times per week) was applied within 5 to 6 weeks. If residual disease remained at second look surgery, a boost to 50 Gy was given. The first, non-involved lymph node leve! was irradiated with a dose of 30 Gy in ali patients. In cases of primary lymph node invol­vement irradiation with 40 to 50 Gy was given, depending on histological response. The next negative lymph node leve! was irradiated to 30 Gy in these cases. Metastates were irradiation up to 40 to 50 Gy, depending on local organ tolerance as dose limiting factor. In paramenin­geal sites the base of the scull was included into the radiation field for the high risk of tumor extension to the meninges and brain. In patients with documented involvment of the base of the skull the neurocranium was irradiated to 30 Gy in addition. In case positive liquor cytology the neuroaxis was also irradiated to 30 Gy. Statistica! methods Probability estimates of overall survival (SU) and event-free survival (EFS) were obtained by the Kaplan-Meier method. 23 For the latter, the events of interest were failure to achieve com­plete reponse, relapse, or death from any cause. The starting point for survival tirne was the date of primary treatment; statistical analysis was performed on February 15, 1990 at a median Soft tissue sarcomas in childhood, austrian multicenter stucly A-RMS 82 Table 4. Rcsponse analysis in clinical group II & IV paticnts Respunse Respunse NoofPtsl Noof Pts/ Ciin. Week 7 Treatm. Weekl6 S.L.S.(BIR) Follow Up Group Noof Pts Arm Noof Pts (Histol. Noofpts Res ult) III CR/6 lII CR/6 4B (4 l)/1 n.d. 6NED 1B(IIA) PR/15 III CR/6 2 B(I) 1 NED/1 OCD R(I) INED 3n.d. 1 NED/2D0D PR/5 1 B (1)/1 n.d. 1 NED/1 LTFU 2R( lLIIIA) 2NED/l LTFU NR/4 400D NR/8 IVNR CR/1 IB(I) INED PR/6 3R( 21,l IIA) 3NED IR(IIA) IDOD 2n.d. INED/1D0D NR/1 IR(IIA) IDOD IV PR/5 IV CR/2 PR/2 NR/1 1 R (1)/1 n.d. 1 R (IIA)/1 n.d. 1 n.d. 2 NED 1 DOD. IOCD 1 DOD NR/1 IV DOD No of Pts: number of paticnts, S.L.S.: second look surgery, DOD: dead of disease, OCD: other cause of death, LTFU: 1 B/R: biopsy or resection, CR: complete response, PR: partial ost to follow up response, NR: non-response, NED: no evidence of discase, follow up of 65 months, range 32 to 97 months. Differences between survival patterns were eva­luated by the log-rank test. Results Primary tumor sites Orbit Six patients had tumors of the orbit. Embryonal RMS was found in 4 patients, a hemangiopericy­toma and a rhabdoid tumor in two further ones. One girl (no. 52) with embryonal RMS neither responded to the intensified treatment arm and irradiation nor surgery was effective in terms of tumor control. She died with progressing disease and brain involvement. One girl (no. 40) develo­ped megakaryocytic leukemia 1 month after termination of treatment. Four patients are in remission with preserved eyes and were ali trea­ted according to protocol criteria. Head and neck Six patients presented with the head and neck as primary site, two of them with parameningeal locations (patients no.37 and 17). Three patients with undifferentiated sarcoma, angiosarcoma and embryonal RMS, respectively, fared well. The boy (no.37) with embryonal RMS of the epipharynx developed Jung metastases before local treatment was iniated after a very good local response to the first two V ACA cycles. The girl (no.17) with pleomorphic RMS of the nasal cavity responded to the NR treatment arm, but suffered a distant relapse 29 months after diagno­sis. A boy (no.14) with alveolar RMS of the right cervical side sustained a local relapse 23 months after diagnosis. Urogenital tract Embryonal RMS was found in ali 8 patients with the urogenital tract as primary site. An adoles­cent (no.36) with embryonal RMS of the blad­der/prostate achieved PR with initial chemothe­rapy, but died with cerebral metastases, which were not observed on the initial cranial cat scan. Another patient with involvement of the bladder (no.45) developed again progressing disease af­ter a PR to the enhanced treatment arm. Five patients, 3 with the bladder and 2 with the prostate as primary tumor site, are long term survivors. One patient with the bladder as pri­mary site was !ost to follow up. None of them required radical surgery at week 16. Urinary bladder dysfunction was not observed in these patients. One girl (no.34) had a primary perianal tumor resembling embryonal RMS. Paratesticular sites Four patients were found with this primary site, 3 tumors were classified as embryonal RMS and 1 as spindle-cell sarcoma. All of them are in continuous complete remission. Ladenstein Ret al. Thorax Three patients presented with tumors of the chest wall classified as PNET, alveolar RMS and undifferentiated sarcoma, respectively. An in­fant (no.30) was the only patient with an intra­thoracal tumor involving the mediastinum. In this case radiation therapy was omitted due to the risk of heart disease. Ali these patients are alive with no evidence of disease. Retroperitoneal sites Patients with retroperitoneal tumors faired poor­ly. Five children presented with hughe tumors of more than 10 cm in diameter, one with a tumor of 5-10 cm. The histologies in this group were 3 embryonal RMS, 1 alveolar RMS, 1 undifferen­tiated sarcoma and 1 angiosarcoma. The boy (no. 5) with angiosarcoma had involvement of muscles of the gluteal and iliac region. He suffered of associated symptoms like loss of stool, dysuria and palsy of one leg. He achieved PR after initial chemotherapy. Tumor related symptoms had improved. He was selected for experimental radiation with heavy particles (ne­gative pions) in Villingen, Switzerland, because surgery for residual disease would have caused paraplegia. At the end of treatment he had a normal walking function, but his bladder func­tion remained impaired. There was no evidence of disease at treatment termination, but he was then !ost to follow up. Three patients died with progressing disease, another one (no.33) in CR with treatment related acute renal failure at the end of therapy. Extremities Extremities were the most common primary site and were observed in 15 children. Pathohistology revealed 10 RMS (7 alveolar, 2 embryonal, 1 pleomorph), 2 SyS, 1 PNET, 1 fibrosarcoma and 1 spindle-cell sarcoma. Eleven patients are alive with no evidence of disease. A girl (no.49) with trisomy 21 experienced the only early local re­lapse 7 months after diagnosis while stili on therapy. This relapse appeared at the margin of the radiation field and higlights the importance of safety clearance of tumor margins. Patient no.31 had synovial sarcoma of the right hand. She suffered distant relapses. The latter two patients responded to relapse chemotherapy and are alive in complete remissions. A girl (no.18) with spindle-cell sarcoma of the right forearm achieved PR after 16 weeks. Further treatment was omitted by her parents and she died with lung metastases after palliative amputation. A girl with alveolar RMS of the forearm (no.12) suffered a late distant relapse 42 months after diagnosis. Another girl (no.41) with alveolar RMS of the thigh developed progressing disease after intermediate PR to enhanced chemothera­py. Others Two patients had tumors originating from para­spinal sites. One of them (no.21) experienced a local relapse 10 months after diagnosis and died. Response to treatment Forty-three patients (pts) achieved complete remission at various points of tirne during the treatment course. Thus the overall complete response rate was 82.5%. The response beha­viour of clinical grm,ips during the first 16 weeks is demonstrated in detail on Tables 2 & 4. A total of 35 children (67.3%) responded to treat­ment well. After initial surgery and the first V ACA cycle 15 children in clinical groups I and II were in CR and 6 children in clinical group III. Eight further patients achieved CR ( clinical groups III: 6 pts and IV: 2 pts) after the second V ACA cycle. Second look surgery obtained CR in 3 patients in clinical group III. Finally, radia­tion therapy attributed to CR in 3 children in clinical groups III (2 pts) and IV (1 pt). Eight patients (15.4%) with initial poor or no-response achieved CR after shifting to enhan­ced chemotherapy. Three patients received addi­tional local treatment before week 16. Two of them (no.31,42) had early second look surgeries for tumor progression, the other one (no.13) Soft tis.me sarcomas in childhood, austrian multicenter study A-RMS 82 p 60 72 84 96 Figure 3. Probability (p) of ovcrall and cvcnt-frec survival for ali 52 childrcn with soft tissue sarcomas in thc A-RMS 82 Study. IIA comparcd to 22 protocol paticnts in clinical group III, ali trcatmcnts combincd. p Figure 4. early irradiation. Out of the latter patient group, 2 children of clinical groups II an III (no.13, no.43) were in CR by week 16. Second look surgery at week 16 assisted to obtain CR in 2 patients belonging to clinical group III. Radia­tion therapy resulted in CR in further 3 patients of clinical groups II (pt no.20) and III (pts no.9, no.31). One patient (no.17) was in CR at termi­nation of chemotherapy. Seven patients (13.5%) of clinical groups III (5 pts) and IV (2 pts) achieved only partial remissions. Four of them died during initial treatment, the other three after week 16. Two Ladenstein R et al. Figure 5. Probability (p) of evcnt-free survival for the J90% TI 0B,(HDM, 9 5imoNED open biopsy Chondroblastic BCD,ADR) component 6 14.5 F NOV., 1987 distal Femur OS/-4xHDM Amputation Osteoblastic 70% TIOA(ADR, 10 44moNED ;," FNAB CDDP, BCD) 2. 7 15.5 M AUG., 1987 pr. Femur EWING/OS 4xHDM Hemipel-Osteoblastic 40% TIOA(ADR, 10 26moRPM open biopsy vectomy high fibroblast-CDDP,BCD) 36 mo OP a:: ic component now !Orno NED 8 12 M AUG., 1987 pr. Tibia OS/-4xHDM Amputation Osteoblastic 10 0% 2xHDM, 8 19mo DOD open biopsy BCD,ADR 9 10 M MARCH, 1988 distal Femur OS/-3xHDM None * Telangiect-0% NONE 2 2mo died FNAB 3x IA CDDP tatic, FNAB xx Rea chemo. 10 12 F JAN., 1989 pr. Humerus OS/-4xHDM Amputation Osteoblastic 10% TIOA(ADR, 9 9mo DOD FNAB CDDP,BCD) pr: proximal; FNAB: fine nedle aspiration biopsy; OS: osteogenic sarcoma; ALL: acute lymphatic leukemia; HMD: high-dose methotrexate with leucovorin rescue; BCD: Bleomycin, Cyclophosphamide, Dactinomycin; ADR: Adriamycin; CDDP: cisplatin; IA: intraarterial; NED: no evidence of disease; DOD: died of disease; RPM: right pulmo metastasis; OP: operation-right pneumoectomy; Rea: reaction Our experience in treating chi/dren with osteogenic sarcoma shortage. Patient No.S, who had 90% tumour necrosis, received chemotherapy according to protocol T 10 B with HDM and BCD. Patient No.4 who had 50 -90% tumour necrosis received also CDDP and radiotherapy for metastases discovered at diagnosis (Table 1). Patient No.3, who also had 50 -90% tumour necrosis, received Ifosfamide, CDDP and only 2 treatments of HDM due to a low tumour necrosis (Table 1). In 8 patients the whole course of treatment lasted 8 -11 months. One patient with metasta­ses at diagnosis was treated for 16 months and one patient who had lethal toxic reaction to chemotherapy expired two months after diagno­sis during of preoperative treatment. Results The survival rate has been computed from the date of the diagnosis to the date of the last follow up or patient death. The follow-up period was concluded June 1991. Six patients are alive 44 to 66 months after diagnosis; 4 had primary tu­mours and 2 secondary OS. One of the latter two had multiple right pulmonary metastases 3 years after diagnosis of secondary osteosarcoma and is now disease free 10 months after pneumoecto­my. The tumour necrosis was 10% in one patient, 40% in one, between 50% and 90% in 6, 90% in one and 100% in one (Table 1). Three patients have died of metastatic OS, two of them with pulmonary metastasis 21 and 19 months respectively after diagnosis. The third had pulmonary and bone metastases 11 months after diagnosis. The fourth patient died preope­ratively because of toxic effects of chemothera­py. Discussion Long-term survival is a realistic prognosis for about 60-80% of patients with OS due to effec­ 7912 18 tive adjuvant chemotherapy. 3-• · , The qua­ lity of life is also improved in these patients since a limb-salvage operation with adequate margins carries the same risk for local recurrence (less than 5%) as the transmedulla{ amputation. 6 Limb salvage surgery is ussually contraindica­ted in cases of neurovascular structure in vasi on, pathological fracture, infected biopsy site, signi­ficant tumour invasion of soft tissues, afld skele­ta! immaturity. 6 Arteriography, which correlates remarkably well with the degree of tumour necrosis, is essential in choosing the type of surgery. 6• 20 It is not yet clear whether intra-arte­rial or intra-vencius CDDP is more efficacious. Recent studies however, have demonstrated that CDDP is highly effective when administered by the IA ruote, rendering many patients suitable candidates for safe surgical limb-salvage proce­dure. 22 IA CDDP does not prolong disease free survival and does not reduce the occurrence of metastasis. 6 Good team-work in a center with accumulated experiences is essential for effective treatment. It is also important for the patient to have the biopsy properly done in this center, since a poorly chosen biopsy site can preclude later limb-salvage surgery. 6 FNAB has shown to be adequate for diagnosis in our institution. Open surgical biopsy is only necessary when FNAB has not yielded sufficient material. Both have to be performed at the site which will later be removed. Three of our patients have died of metastatic disease 11-21 months from diagnosis. This corre­ sponds to other studies showing that about 90% of relapses occur within two years of diagnosis ;4• 10 first relapses after five years are unusual. 4 Patient No.4 had at diagnosis bilateral pulmo­nary metastases, metastasis in thorac1c vertebral body 11 and an unusually large primary tumour in the distal femur. Furthermore, her surgery was delayed for 5 months due to parental indecision. Other centers also have poor survival rates of patients with metastasis at diagnosis. 4 For patient No.S one would expect a favorable outcome, based on tumour necrosis of 100%, which usually correlates very well with survival, 6• 8• 21 We can only surmise that he died because he only reccl­ Benedik-Dolnicar M ved HDM twice, owing to a shortage of metho­trexate. Patients No.10 was resistant to HDM; her disease progressed during pre-operative che­motherapy and the tumour necrosis at surgery was only 10%. Poor response to preoperative chemotherapy identifies patients at a high risk of early relapse. 7 Good responders to pre-opera­tive chemotherapy have a much better prognosis (81 % at 5 years) than poor responders ( 45% ). 18 The patient No.7 had right pulmonary metastasis 3 years after diagnosis of secondary OS and is now disease free 10 months after right pneumo­nectomy. About one third of the patients with pulmonary metastasis are curable with metasec­tomy but there is no evidence of a ·beneficial effect of postoperative chemotherapy. 6 A combination of complete (wide) surgical resection or amputation and aggressive chemot­herapy offers the best chance of long term survival. 23 From our study it seems that secon­dary osteogenic sarcoma does not have a poorer prognosis that primary osteogenic sarcoma. Acknowledgements The author wishes to thank the orthopedic sur­geon Dr Boštjan Baebler, the oncologic surgeon Dr Janez Novak, the pediataricians Dr Nasta Mihevc, Dr Jožica Anžic and many other doctors of severa! specialities who collaborated in the diagnosis or treatment of these patients. The author also wishes to thank Dr Berta Jereb for her advice and support and Nada Pušnik for preparing the manuscript. References l. Altman AJ, Schwartz AD. The Cancer Problem in Pediatrics: Epidemiologic Aspects. Major Probl Ciin Pediatr 1983;18:1-21. 2. Barjaktarevic ž, Kezic J, Mitrovic M, Višacki R, Aleksandrovic S, Subotic Z, Atanackovic M, Olu­jic D, Brzakovic P, Opric M. Treatment of Chil­dren 'with Osteosarcoma. Acta Orthop Jugosl 1988,19:52-8. 3. Gillespy T, Mnfrini M, Ruggicri P, Spanier S, Petterson H, Springfield D. Staging of lntraos­seous Extent of Osteosarcoma: Corrclation of Preoperative CT and MR, lmaging with Patholo­gic microslides. Radiology !988;167:765-67. 4. Goorin AM, Abelson HT, Frei E. Osterosarcoma: Fifteen Years Later. N Engl J Med 1985;313:1637­43. 5. Jiirgens H. Bone Tumours, Diagnostic Mcthods and Treatment Possibilities. Europcan School of Oncology, Postgraduatc Course on Pediatric On­cology, June 1990, Višegrad, Hungary. 6. Meyer WH, Malawer MM. Osteosarcoma. Pediatr Ciin Norih AM 1991 ;38:317-48. 7. Winkler K, Beron G, Kotz R, Salzer-Kuntschik M, Besk J, Beck W, Brandeis W, Ebell W, Erttmann R, Gobe! U, Havers W, Henze G, Hinderfeld L, Hocker P, Jobke A, Jiirgens H, Kapisch H, Preusser P, Prindull G, Ramach W, Ritter J, Sekera J, Treuner J, Wiist G, Landbeck G. Neoadjuvant Chemotherapy for Osteogenic Sarcoma: Results of a Cooperative German/Au­stria Study. J Ciin Onco/ 1984;2:617-24. 8. Bacci G, Picci P, Ruggieri P, Mercuri M, Avella M, Capanna R, Brach del Prever A, Mancini A, Gherlinzoni F, Padovani G, Leonessa C, Biagini R, Ferraro A, Ferruzzi A, Cazzola A, Mnfrini M, Campanacci M. Primary Chemotherapy and De­layed Surgery Neoadjuvant Chemotherapy for Osteosarcoma of the Extremities. Cancer 1990 ;65:2539-53. 9. Elomaa J, Siimes MA, Blomqvist C, Karaharju E, Ryoppy S, Laasonen E, Halmstrom T. Ten Years' Experience in Patients with Osteogenic Sarcoma in Finland. Eur J Surg Oncol 1990;16:147-52. 10. Hudson M, Jaffe MR, Ayala A, Raymond AK, Carrasco H, Wallace S, Murray J, Robertson R. Pediatric Osteosarcoma: Therapeutic Strategies, Results, and Prognostic Factors Derived From a 10-year Experience. J Ciin Oncol l990;8:l988-97. 11. Bacci G. Picci P, Avella M, Dallari D, Ferrari S, Prasad R, Di Sciascio M, Malaguti C, Caldora P. The Importance of Dose-intensity in Neoadjuvant Chemotherapy of Osteosarcoma: Retrospective Analysis of High-dose Methotrexate, Cisplatinum and Adriamycin used Preoperatively. J Chemother 1990;2:127-35. 12. Newton WA, Meadows AT, Shimada H, Bunin GR, Vauter GF. Bone Sarcomas as Second Mali­gnant Neoplasms Following Childhood Cancer. Cancer 1991 ;67:193-201. 13. Tucker MA D'Angio GJ, Boice JD, Strong LC, Li FP, Stovall M, Stone BJ, Green DM, Lambardi F, Newton W, Hoower RN, Fraumeni JF. Bone Sarcomas Linked to Radiotherapy and Chemothe­rapy in Children. N Engl J Med 1987;317:588-93. 14. Beattie EJ, Harvey JC, Marcove R, Martini N. Results of Multiple Pulmonary Resections for Metastatic Osteogenic Sarcoma After Two Deca­dcs. J Surg Oncol 1991; 46:154-55. 15. Rosen G, Caparros B, Huvos AG, Kosloff C, Nirenberg A, Cacavio A, Marcove RC, Lane JM, Mehta B, Urban C. Preoperative Chemotherapy for Osteogenic Sarcoma: Selection of Postopera­tive Chemotherapy Based on the Response of the Primary Tumour to Preoperative Chemotherapy. Cancer 1982;49:1221-30. 16. Rosen 6, Huvos AG, Marcove R, Nirenberg A. Telangiectatic Osteogenic Sarcoma. Ciin Orthop 1986;207:164-73. Our experience in treating children with osteogenic sarcoma 17. Brunat-Mntigni M, Biron P, Kohler R, Blondet R, Berard J, Chauvod P, Bouffet E, Carret JP, Jonas P, Patricot LM. Developments in the Treat­ment of Osteosarcoma since 1979. Report of the Statistics at the Centre Leon-Berard. Buli Cancer 1990;77:933-40. 18. Kashdan BJ, Sullivan KL, Lackman RD, Shapiro MJ, Bonn J, Weiss AJ, Gardiner GA. Extremity Osteosarcoma: Intraarterial Chemotherapy and Limb-sparing Resection with 2-year Follow-up. Radiology 1990;177:95-99. 19. Bilbao JI, Martin Algarra S, Martinez de Negri J, Lecumberri F, Longo J, Sierrasesumaga L, Cana­dell J. Osteosarcoma: Correlation between Radio­ logical and Histological Changes after Intra-arte­rial Chemotherapy EurJ Radiol 1990;1:98-103. 20. Delepie N, Delepine G, Trifaud A, Voisin MC, Mazabraud A, Jasmin C. Evaluation of the optimal length of neo-adjuvant chemotherapy in osteoge­nic sarcoma. Neoadjuvant Chemother 1986;137:581-86. 21. Epelman S, Estrada J, Jaffe N, Bianci A. Pediatric Osteosarcoma. Successful Retreatment of Relap­sed Primary Tumour and Soft Tissue Recurrence With Intraarterial Cis-Diamni nedichloroplatin-II. Cancer 1990;66:801-5. 22. Kozakewich, Perez Atayde AR, Goorin AM, Wil­kinson RH, Gebhardt MC, Vawter FG. Osteosar­coma in Young Children. Cancer 1991 ;67:638-42. Adv Radio! Oncol 1992; 202-7. Recent strategies in the treatment of malignant germ celi tumour Clemm Ch Dept. of Interna/ Medicine III, Klinikum Gro.hadern, University of Munich, FRG Since cisplatin was introduced into chemotherapy in 1979, there has been a radical improvement in the treatment of malignant testicular tumour. Cure rates of over 80% are now possible. Far seminoma, the treatment strategy has changed from radiotherapy to chemotherapy in advanced stages of disease with long term remission of 90%. The introduction of carboplatin more recently will possibly bring about further good results. HCG-positive seminoma has no worse prognosis than others. In the case of non-seminoma, tumour markers play an important role in prognosis. Early detection as well as the earliest possible beginning of consequent therapy that is stage conform is of primary importance in achieving long term remission far our patients. Beware of «wait-and-see» strategies. In stage 1 we carry out RLA, in stage IIA and IIB good results have been achieved with adjuvant chemotherapy, in future possible neoadjuvant. Stages IIC-IV require intensive therapy strategies using standard therapies but with questionable bleomycin reduction. Very advanced stage of disease demands therapy intensification, treatment with high dose protocols plus cytokines and in some cases autologous bone marrow transplantation, which can only be carried out in a few specialized centers. Key words: testicular neoplasms-therapy; dysgerminoma lntroduction The therapy of malignant testicular tumours has changed radically during the past few years. While in 1960 only 5% of ali patients reached long term remission, more than 60% of patients even in metastasized stages of disease have been curable since 1980. The incidence is slightly rising, at the moment 7 of 100.000 male inhabi­tants are afflicted per year. In addition to impro­vements in therapy, diagnosis has also been highly improved during the last years. Histologi­cal examination has been made more accurate Correspondence to: Doc. dr. Christoph Clemm, Lud­wig-Maximilians-Universitiit Miinchen Klinikum Grof3hadern, Postfach 701260, 8000 Miinchen, Deuts­chland. UDC: 616.681-006.884-08 by using immune peroxidase technique. The analysis of the tumour markers AFP (alphafoeto­protein) and hCG (human chorionic gonadotro­pine) in the serum has led to a more differentia­ted diagnosis as has the use of radiological techniques such as cat-scans and more recently nuclear magnetic resonance. In spite of ali efforts, and even with improved polychemotherapy as a result of the introduction of cisplatin in 1979, more than 200 patients die yearly in the FRG.1 This shows quite clearly that theoretical improvement of therapy as described in studies has not yet been fully put into practice. It is thus highly desirable that patients with this curable tumour are diagnosed as early as possible and then treated consistently. In order to do so, the most complete possible survey of these pa­tients as well as consistent treatment within a Recent stratei;ies in the treatment of malignalll germ celi twnow 203 network of controlled studies is necessary. With this as a prerequisite, the complete remission rates are at about 80% at the moment and stili tend increase. 2 It has also proved favorable if these patients are treated at a tumour center, and therapy is coordinated or monitored from there. This goes as well for the follow-up treat­ment. While previously the treatment of semi­noma consisted of radiotherapy, teratoma and combination tumours had always been treated surgically and chemotherapeutically. In recent years, the treatment strategy of seminoma has changed, as has the use of chemotherapy in the case of non-seminoma patients. Both tumour entities shall now be discussed. Seminoma In earlier years, seminoma patients received radiation even in advanced stages of disease. Nowadays, due in part to the success of chemot­herapy in the treatment of non-seminomas, che­motherapy has been implemented for the treat­ment of seminomas. Since the relapse rates for advanced stage of disease with lymph node metastases larger than 5cm as well as organ metastases increase to approximately 30%, a primary radiation therapy should not be indica­ ted here. 3 The results achieved here with poly­chemotherapy reach 90% in these _stages. Small lymph nodes (less than 5 cm) can be treated successfully with radiotherapy alone, here long term results also range at about 95%. In addition to the combination therapy with cisplatin, etopo­side and bleomycin, ifosfamide is eff.ctively used as a bleomycin substitute. Our results here with 45 patients are very good, showing a com­plete remission rate of 91 % (Table 1). Stable remission rates of 68%-100% can be reached with other combination therapies as well. One must pay attention to the toxicity of the therapy, since patients with seminomatous tumour are as a rule 10 years older than patients with non-se­minoma. Combination therapies show increased bone marrow toxicity and require strict surveil­lance and possibly dose reduction. Therapy must be carried out with continous hyperhydration to prevent a cisplatin-induced nephrotoxicity. In recent times we have seen the results of monotherapy treatment, at first with cisplatin and more recently with carboplatin. Here long term NED was observed in almost 90% of the patients. But the rate of complete remissions in a carboplatin monotherapy seems to be somew­hat lower (Table 2). In Great Britain and Ger- Table l. Chemotherapy results in literature (Polychemotherapy) seminoma. Author Year Patients Therapy CR Remission duration Einhorn 1980 19 PVB+A 63% 17+months Vugrin 1981 9 PC­89% 19 ,5 + months Samuels 1983 32 P+C 94% 24 + months Peekham 1983 7 BEP l00% no data Schmoll 1984 15 PVB + I 80% no data Wettlaufer 1984 12 VinPC 92% 24+months Stanton 1985 28 PVBCD 85% 32+months Schuette 1985 28 PVB!Tu]A/I 89% 28+months Friedman 1985 20 PVB or PE 90% 24+months Pizzocaro 1986 31 PVBor PEB + A 84% 34+months Fossa 1987 54 PVB/PEB 91% 36+months Loehrer 1987 60 PVB + A/PEB 68% no data Einhorn 1989 111 multiple 84% no data Clemm 1991 45 VIP/EIP 91% 45+months P = Cisplatin, V= Vinblastine, B = Bleomycin, A = Adriamycin, C= Cyclophospahmide, E = Etoposide, I = Ifosfamide, V= Vincristine, D = Dactinomycine, Carboplatin, CR = complete remission, PR = partial remission 204 ClemmCh Table 2. Monotherapy results. Author Year Patients Therapy CR Remission duration Oliver 1984 14 p 71% 15+months Horwich 1989 34 Carboplatin 42% 2.6+months 55% PR Harstrick 1990 41 Carboplatin 71% 22+months 17% PR many more than 40 patients have been studied and showed a good response to this therapy regimen, whereby it is not necessary to hospita­lize the patients. or carry out the therapy with continuous hyperhydration, but patients are trea­ted ambulatory. Our present experiences are not yet long term and have not been verified for a longer period of tirne, thus there might be a higher rate of relapse. 4 In order to compare this very well tolerated and potentially very effective therapy with better known combination thera­pies as far as effectiveness and tolerability are concerned, a study is being carried out presently in the FRG. Here a monotherapy using carbopla­tin is being compared with a combination therapy consisting of etoposide, ifosfamide and cisplatin. This study is meant to disclose information about primary risk factors, the tolerability and effecti­veness of therapy as well as the relapse rate and information about possible indications for secon­dary surgery in the case of residual tumour. 5 In patients with pure seminoma one finds vita! tumour remains in much fewer cases which is one of the reasons why a number of authors recommend; omission of surgical intervention, or operating only if lymph nodes are larger than 3 cm. In addition to organ metastases e.g. in the bone, as well as visceral metastases in the lung or !iver, possible risk factors can also be tumour entities that show hCG producing seminoma cells or synciotrophoblastic cells. These are also seen in pure seminoma, but an hCG leve! higher than 1.000 U/1 should give rise to the suspicion that one might be dealing with a chorion carci­noma and thus these patients should not be treated as pure seminoma patients. 6 Non-seminoma An important difference between non-seminoma and pure seminoma can be established by histolo­gical analysis of the tumour markers hCG and AFP as seen in the histological section. Determi­ning these two markers as well as lactate dehy­drogenase is a must in serum. While hCG and LDH increase can be seen in pure seminoma as well, AFP is always consistent with the diagnosis is of non-seminomatous tumour. These patients may not be treated as pure seminoma patients but must be consistently and intensively treated in the same way as teratoma or combination tumour patients (Table 3). In Munich we have achieved good results with a stage conform therapy. In stage I the turno ur is only in the testis, stage II shows lymph nodes below the diaphragm, IIA with lymph nodes up to 2 cm, IIB lymph nodes 2-5 cm, IIC lymph nodes 5-10 cm, and IID lymph nodes exceeding 10 cm in diameter. Stage III implies lymph nodes above the diaphragm and st,age IV organ metastases. Depending on the tumour stage we consistenly operate in the case of non-seminoma, if the histology shows no evidence of ch'orion carci­noma and hCG levels are not extremely elevated _ (over 1.000 U/1) and indicate a rapidly prolifera­ting trophoblastic tumour. Following an inguinal semicastration, these patients are treated prima­rily with chemotherapy, which is also documen­ted in other studies. 7 Yet other authors observe good results in stage I with «wait and see» strategy, i.e. forfeiting additional surgical or chemotherapeutic measures after inguinal semi­castration. Peckham et al. have reached very good long -term results with 137 patients, but a Recent strategies in the treatme111 of malignant germ celi 111mo11r Table 3. Trcatmcnt stratcgy for malignant gcrm celi tumour High inguinal semicastration. pure seminoma malignant teratoma TD, MTI, MTU MTT with high hCG or MTT with low hCG pure chorion carcinoma lr stage stage stage stage stage stage stage IIC III, IV I, IIA IIB IIC, I-IV III, IV 1 1 Radiation Rroperitoneal lymphadenektomb y Control Control l_ AdjuvantChemotherapy Combination Chemotherapy Table 4. Definition for »Bulky« tumour. 1.Metastases size: Lymph nodes?: 10 cm diameter ( abdominal/mediastinal) Lungfiliae ?: 5 cm diameter 2. Metastases number: more than 10 lungfiliae 3. Metastases location: CNS/Liver/Bone severa! organs simultaneously 4.Tumormarker: hCG higher than 10.000 U/1 AFP higher than 1. 000 ng/ml LDH higher than 500 U/1 5.Primary extragonadal germ celi tumours relapse rate of 30% must be taken into account. If the relapse is discovered early, long -term results and chances of survival are good with 96%. These results have been confirmed in severa! studies. 8 We do not treat in accordance with these studies because we prefer primary surgery due to possible lack of compliance on the part of the patients as well as the difficulties of discovering a relapse in tirne. The possible advantage of maintaining ejaculation by doing without surgery is weakend by the relapse rate of 30%. We have thus decided to carry out a modified retroperito­neal lymphadenectomy even if there is a negative cat-scan of the abdomen in stage l. This modi­fied surgery has the advantage that in contrast to earlier operating techniques where there was a loss of ejaculation in over 80% of the patients (or retrograde ejaculation), we now have only 15 to 40% patients suffering from loss of ejacula­tion. The relapse rate has not risen decisively because of the modified surgery and dispensing with contralateral resection. 9 If lymph node me­tastases are found during the operation, a radical resection is carried out depending on the size of the nodes. If histologically the lymph nodes are smaller than 2 cm, the patient will only be controlled after RLA. If, on the other hand, the lymph nodes exceed 2 cm in diameter, we admi­nister adjuvant chemotherapy. This is also done when there are severa! small lymph nodes or negative tumour markers indicating a possible relapse that might be discovered too late. The indication for adjuvant chemotherapy is not considered as strictly in other tumour cen­ters. Thus Williams et al. have published data where in relinquishing adjuvant therapy, patients who relapsed (up to 50% of the patients) they stili achieved good cure rates by administering chemotherapy to treat the relapse. 10 We consider this mode of treatment as too risky to be carried out outside a tumour center, even though it has been tried out in studies. Patients with !iver metastases of extended lymph node metastases are often discovered late, leading to poor pro­gnosis. Many studies propagate a reduction of adjuvant chemotherapy cycles from 4 to 2 cycles. We have not carried out these studies, but advocate a median adjuvant therapy of 3 cycles. 206 C/emm Ch The experience and results with our patients with this therapy are very good. In 65 patients only one showed new tumour and the relapse was successfully treated with renewed intensive chemotherapy. This patients is now tumour ­free since more than 4 years. On the other hand, patients who had only been operated on primarily and did not receive chemotherapy until they relapsed, had a clearly worse prognosis and in many case also CNS metastases. This important restriction when tran­sposing study results and bringing them into general practice must be mentioned, since the results could be influenced negatively hereby. A new method of reducing the side effects of surgery consists in the so-called neoadjuvant chemotherapy proceedings; this is implemented if lymph node metastases of 2-5 cm are already evident in the cat-scan. If they exceed 5 cm, chemotherapy should be carried out by ali means (Table 4) prior to surgery. Some authors recommend prior chemotherapy even if lymph nodes are smaller than 5 cm but exceeding 2 cm, because adjuvant chemotherapy would have been carried out after surgery anyhow. This neoadjuvant procedure is also carried out outside the studies and has the advantage that if the lymph nodes disappear completely, in 80% of the cases it is possible to do without lymphade­nectomy. We do not share this view because of the unreliability of cat-scan checks , and are awaiting the results of a study that is presently being carried out in Germany. Further attempts at minimalizing therapy in low tumour stages consist in doing without the fourth cycle of chemotherapy, which Einhorn has shown as being feasible in a randomized study. Other studies that leave out bleomycin show a clear risk, which is the reason why we could not decide to leave out bleomycin per se. New data about a combination of cisplatin /eto­poside as opposed to carboplatin/etoposide show equal effects and results for both regimens, but a higher relapse rate for carboplatin, which is why we do not recommend this treatment stra­tegy outside of studies. Highly advanced tumour stages While in Iow tumour stages a minimization of therapy is documented, whereby therapy success must be maintained, ali efforts concentrate on a maximization of therapy and improvement of results in the case of advanced tumour stages. Some studies with double dose cisplatin showed very little improvement in results, combination therapy with cyclophosphamide and ifosfamide brought some improvement, but this was not reliably proven in statistics. Patients with very advanced stage of disease should not be treated with the conventional regimen (PEB) in individual cases, but with more intensive therapy. A German study is presently testing the dosis escalation of cisplatin, etoposide and ifosfamide with the addition of a granulocyte-macrophage-stimulating factor. Re­sults also seem very positive with autologous bone marrow transplantation, but toxicity is strong and the Iong -term improvement contro­versial.11 Efforts must be concentrated on early diagnosis and consistent therapy in Iow tumour stages, in order not to risk any chances for a complete remission. It is important to note that the primary extragonadal tumour, which makes up 5% of ali advanced germ celi tumours, is connected to the absence of a palpable testis tumour. This accounts for the fact that these patients are often diagnosed in a Iater stage and the disease is already well advanced. It is useful in this context to remember that as far as young men between 20 and 30 years of age are concer­ned, germ celi tumours are (besides lymphoma) the most frequent tumours. In 2/3 of these patients the AFP and hCG Ievels can already be elevated. A major surgical diagnosis should give way to a small mediastinoscopy and quick and consistent treatment, in order to give these patients a good chance of reaching complete remission and long term survival. 12 References l. Clemm Ch, Sauer H, Hartenstein R. Behandlung nicht-seminomatoser Hodentumoren im Stadium I-IIB. Dtsch Med Wchschr 1989; 114:1276-82. Recent strategies in the treatment of malignant germ celi tumour 2. Einhorn LH. Treatment of advanced disseminated germ cell tumours. Annals of Oncology 1991; 2:167f. 3. Clemm Ch. Hartenstein, R. Willich et al. Vinbla­stine-ifosfamidecisplatin treatment of bulky semino­ma. Cancer 1986; 58:2203-7. 4. Horwich A, Dearnaley D P, Duchesne G M. et al. Simple Nontoxic Treatment of Advanced Me­tastatic Seminoma with Carboplatin. J Ciin Oncol 1989; 7:1150-6. 5. Clemm Ch. Phase III-Studie: Cisplatinkombina­tionstherapie vs. Carboplatinmonotherapie bei metastasierten Seminom. Urologe (A) 1991; 30:75f. 6. Mann K. Tumormarker beim Hodenkarzinom. In: Urogenitaltumoren, Hrsg. Ch. Clemm, W. Zuckschwerdt Verlag, Mi.inchen (1991) 3-18. 7. Pont J, H6ltl W, Kosak D et al.: Risk-adapted Treatment Choice in Stage I Nonseminomatous Germ Cell Cancer by Regarding Vascular Invasion in the Primary Tumour: A Prospective Trial. J Ciin Oncol 1991: 8:16-20. 8. Stephenson R A. Surveillance for Clinical Stagc I Nonseminomatous Testis Carcinoma: Rationale and Results. Uro! Int 1991; 46:290-3. 9. Wei.bach, Bussar-Maatz. Operative Ma.nahmen beim Hodentumour. Onkologisches Forum 3 (1988) 19-28. 10. Williams, S D, Stablein D M, Einhorn L H et al.: Immediate Adjuvant Chemotherapy Versus Ob­servation with Treatment at Relapse in Pathologi­cal Stage II Testicular Cancer. N Engl J Med 1987; 317:1433-8. 11. Nichols C R, Tricot G, Williams S D et al.: Dose-Intensive Chemotherapy in Refractory Germ Celi Cancer -A Phase I/II Trial of High-Dose Carboplatin and Etoposide with Autologous Bone Marrow Transplantation. J Ciin Oncol 1989; 7:932-9. 12. Clemm Ch, Mair W, Voigt G et al.: Chemothe­rapy in extragonadal germ celi tumours. J Cancer Res and Ciin Oncol 1990; 116: suppl 589. Adv Radio! Oncol 1992; 208-13. Thyroid status f ollowing irradiation f or laryngeal cancer Dimitrovska A 1 , Karanfilski B2 , Georgieva B2 , Velkov K1 , Jovanovski D1 , Nikolova L1 1 Institute of Radiotherapy and Oncology and 2 Institute of Pathophysiology and Nuclear Medicine, Skopje The effect of Co-60 irradiation on the thyroid gland was studied in 174 patients, 75 previously operated. While clinically manifest hypothyroidism showed 5 (3%) cases, a significant proportion of patients were noted to have vague, nonspecific symptoms (7%) or latent hypothyroidism (25%). Low radioiodine 24 hour uptake was measured in 16%, low thyroxine and triiodthyronine in 7% and 15% respectively, elevated serum thyroid-stimulating hormone (TSH) in 31%. The thyrothropin releasing hormone test revealed augmented TSH response in a further 14 patients. 33% of patients had elevated titers of thyroid antibodies. 42% showed J-131 scan abnormalities. The data suggest that standard doses of external irradiation induce thyroid functional and morphological changes which signals the need for further evaluation of the gland in patients successfully treated f or head and neck cancer. Key ,words: laryngeal neoplasms -radiotherapy; thyroid function tests Introduction Thyroid dysfunction following irradiation of the neck region has been found in a rather high percentage of Iymphoma patients. 1•2•3•4•5 •6 In pa­tients with head and neck tumors who receive higher doses of irradiation the incidence of the thyroid damage varies from 0-100% which de­pends on the duration of the follow-up and the 578,910 11 1213 sensitivity of the tests employect. 1 ,,, ,,,, In 1961, Felix8 first reported a case with myxe­dema appearing 6 years after radiotherapy (RT) for laryngeal cancer. In 1965, Markson and Flatman 12 described 5 cases with hypothyroidism 4-36 months after RT for different neoplasms in Corespondence to: Doc. dr Aneta Dimitrovska, Insti­tute of Radiotherapy and Oncology, Vodnjanska 17, 91000 Skopje, Macedonia. UDC: 616.22-006.6:615.849.06:616.441 the neck region. In one of the first systematic examination of the thyroid, Cocina and associa­tes 7 found normal J-131 uptake in 50 patients soon following doses of 60 Gy to the neck. Koulumies and associates 11 also did not find thy­roid dysfunction in 118 patients with laryngeal cancer in tile_ first year after RT judged by normal RAi uptake, but 17 of 34 patients had displayed low PBI after more measurements. Greig10 determined normal RAi uptake and PBI in 20 patients following doses of 37-65 Gy. Latter studies, however, using more sophistica­ted tests currently available refer considerably higher percentage of thyroid dysfunction due to 9 13,14,15 16 irradiation. 1 ,, , This paper is a continuation of the investiga­tion of the thyroid gland after radical RT for Iaryngeal cancer started 1980. The findings obtai­ned for 146 patients tested once have previously been described. 17 Since 1982 a half of the obser­ Thyroid status following irradiation far laryngeal cancer ved patients have been followed for thyroid abnormalities including 30 new cases. The results after more than one examination of the pati.nts will be presented. Material and methods 174 patients followed for laryngeal cancer were evaluated for thyroid abnormalities. They had received 60-70 Gy in 6-7 weeks Co-60 irradiation 2 months to 18 years prior to the examination of the gland. The patients were grouped and analy­sed according to the tirne interval between irra­diation and the tests performed. The first group consisted of patients examined 2 and 6 months after completion of treatment, seen on the first and the third follow-up, the second after 1 year, the next after 2,3,4,5 and more than 5 years after RT. None of the patients had any known disor­ ders of the thyroid before treatment. In 99 99 (57%) received RT alone. To assess the thyroid status after clinical exa­mination the patients were subjected to the following tests: 24-hour radioactive iodine (RAi) uptake, serum total thyroxine (T 4) using a modi­fication of the competitive protein-binding met­hod of Murphy, 18 triiodthyronine (T 3) and thy­roid stimulating hormone (TSH) measured by radioimmunoassay, 19•20 serum TSH response to the thyrotropin-releasing hormone (TRH test,2 1.22 the determination of thyroid antibodies by the modified method of Boyden23 and J-131 seans obtained 24 hours after oral administration of 50 mCi radioiodine by means of a commercial scanner Nuclear Chicago. Results Clinically manifested hypothyroidism was recog­nized and later proven by laboratory tests in 5 (3%) of 174 patients. Three of them have had surgery prior RT and 2 have received irradiation as the sole therapy. The following symptoms were recorded: fatigue, sensitivity to cold, con­stipation, muscle aches, weakness. Despite the poor appetite 3 of the patients gained in weight and complained of requiring more sleep. The puls was slow and the skin dry and ro_ugh. One patient (woman) had a swelling of the eye-lids and hands and the other only of the eye-lids. Electrocardiogram showed bradicardia in 3 of 5 examined. In ali of them replacement hormone therapy with T 4 was prescribed. Substitution (tab. Novothyral and later Vobenol) was succes­sful in 4 cases. Besides clinical improvment the serum TSH and T 4 normalized their values. The Table l. Subnormal T4 in different intervals after radiotherapy Operated Non-operated Both < 1 year 0/12 0/25 1/25 0/37 5% 1 year 1/12 2 year 0/14 0/12 0/10 2/10 (57%) patients some of the thyroid tests were 0/26 2/20 10% repeated 2-5 times with the interval from the 3 year 4year 0/5 0/7 2/8 0/12 3/10 30% 5/32 first to the next examination of minimum 1 year. 75 (43%) were treated with surgery and RT and 5 year 1/2 > 5 year 4/20 1/12 16% Tota! 6/75 (8%) 6/99 (6%) 12/174 (7%) fifth patient developed lung metastases 6 month after commencement the substitution, the the­rapy was discontinued and he died a year later. Others are under the control of an endocri­nologist and are doing well. However, laboratory tests revealed pathologi­cal findings in a great proportion of asymptoma­tic patients. Reduced RAi uptake was recorded in 16% (18% operated and 14% non-operated) of the patients. 12 (7%) patients has subnormal T4 (Table 1). The mean value was 42 ± 11,96 nmol/1, 40 ± 13,53 nmol/1 in the group with surgery and 44 ± 12,05 nmol/1 in the group without surgery. There was no difference bet­ween the two groups in regard to the incidence of the subnormal T4 (8% vs, 6%). First cases with low T4 appeared one year after RT. The longer the follow-up the greater was the number of the hypothyroid patients. Table 2 shows the Dimitrovska A et al other laboratory findings in patients with subnor­mal T 4• The first 5 are clinieally and bioehemi­eally hypothyroid. Nine (75%) of 12 patients had a raised TSH leve!, 5 had low T 3, 9 showed pathologieal sean. The serum T3 leve! varied in a great number of patients from one to another measurement. After 1-5 tests 15% showed inereased and 25% redueed leve! of T 3• The TSH was determined in 158 patients and 49 (31 % ) had elevated leve! (Table 3). It was noted the tendeney of the TSH to raise with the tirne of follow-up. As seen from the table 16% and 18% of the examined patients displayed elevated TSH eoneentration within the first year after RT. After the seeond year of irradiation elevated TSH ranges from 32%-55%. Some of the patients tested in the first year after RT showed transitory elevation of TSH. Of 49 pa­tients having elevated TSH leve! 9 and 22 exhibi­ted subnormal T 4 and T 3 respeetively. Five of these had both redueed leve! of T 4 and T 3. Forty (25%) eases were reeorded with elevated TSH and normal T 4• The findings of thyroid seans are seen in Table 4. In 73 ( 42%) of 174 patients one or more abnormalities were noted, most eommonly a redueed size of the gland. The number of the pathologieal seans was higher in the group with surgery ( 52 % ) . Thyroid antibodies were determined in 83 patients who had displayed subnormal T 4 and T 3 and/or elevated TSH. Positive antibody titers were found in 27 (32,5%) eases, more often antimierosomal (in 25 or 30% of the patients) than antithyroglobulin (in 6 or 7% of the pa­tients) antibodies. Ali, exept one showed low titers. The TRH test eonfirmed hypothyroidism in 14 of 17 eases suspeeted as hypothyroid on the basis of the previously doeumented borderline values of T4 and TSH. The results of ali tests earried out in 174 patients are summarized on the Table 5. Discussion Early and late histopathologieal ehanges in the irradiated thyroid had been widely stu­died. 10•24•25•26•27•28 While follieular epithelial eells and follicles tolerate high doses of irradiation, small blood vessels ean be damaged with mode­rate doses which results with their degeneration, neerosis, thrombosis and obliteration as well as an early interstitial fibrosis. 26•27 At doses of 60-70 Gy fine vaseular damage and impairment of cireulation eause seeondary parenehymal atrophy and a deerease of the funetional aetivity of the gland. Minimum tirne required for late Table 2. Laboratory findings in paticnts with low T-1 Patient/ T4 T3 TSH RAI Thyr. Antib. SCAN Interval Treatm. uptake MG TG from irrad. 1/oper. 25 0,98 70 12% 1:80 0 small size 1 year 2/nonop. 48 0,9 18 31% 0 0 cold nod. 1 year 3/nonop. 20 1,4 13,5 13% 0 0 small size 5 year 4/oper. 20 2,4 70 22% 0 0 small size 9 year 5/oper. 39 1,3 40 23% 0 1:40 normal 11 year 6/nonop. 44 1,6 8,4 15% - small size 3 year 7/nonop. 46 1,8 5 34% 0 0 small size 5 year S/oper. 49 1,4 40 25% 1 :20 0 one lobe 10 year 9/ oper. 52 2,2 0,5 23% - - normal 5 year 10/nonop. 51 2,3 35 18% 0 0 one lobe 10 year 11/oper. 46 2,1 60 23% 0 0 small size 7 year 12/nonop. 53 1,3 3,7 33% 0 0 normal 3 year Normal values: T4-54--160 nmol/1, TTl,5-3,4 nmol/1, TSH-0-5 nU/ml, RAI uptake 20% -50% Thyroid status following irradiation for laryngeal cancer Table 3. Elevated TSH in different inteervals after Table 4. Scan abnormalities in different periods after radiotherapy radiotherapy Operated Non-operated Both Operated Non-operated Both < 1 year 1/10 4/20 5/30 16% < 1 year 2/12 3/25 5/37 13% 1 year 2/9 3/19 5/28 18% 1 year 8/12 13/25 21/37 57% 2 year 5/13 3/12 8/25 32% 2 year 6/14 3/12 9/26 35% 3year 3/10 6/10 9/20 45% 3year 8/10 7/10 15/20 75% 4year 2/6 3/8 5/14 36% 4year 0/5 2/12 17°/4, 5 year 1/1 55% 5 year 1/2 2/8 3/10 30% > 5 year 8/20 4/12 12/32 37% > 5 year 14/20 4/12 18/32 Tota! 22/69 (32%) 27/89 (30%) 49/158 (31 % ) Table 5. End results after 1-5 thyroid tests following irradiation Test Patients Normal Pathological Findings Findings Clinical exarn. 174 169 (97%) 5 (3%) Hypothyroid RAJ uptake 168 136 (81%) 27 (16%) Low 5 (3%) high T4 174 159 (91,3%0) 12 (7%) Subnormal 3 (1,7%) high T3 161 96(60%) 41 (25%) Subnormal 24 (15%) high TSH 158 109 (69%) 49(31%) Elevated Thyr. antibodies 83 56(67,5%) 27 (32,5%) Scan 174 101 (58%) 73(42%) effects of irradiation to be manifested is 6 months or more. In our material first hypothyroid pa­tients and most of scan abnormalities appeared after the first year of RT. Some of the pathologi­cal tests done 2 or 6 months following irradiation have tendency to be transitory, particularly TSH and T3 . Obtained results in this study confirm reports which indicate that a high percentage of patients who had received RT in the neck region have abnormal thyroid tests. 1 •2 •3•5 •9 •13• 16 Clinically evi­dent hypothyroidism showed 5 (3%) patients, slightly less than in the series of Einhorn and Wikholm. 9 They detected 3 (7 ,3%) hypothyroid patients and in the rest 38 out of 41 patients reduced thyroid response 24 hour after TSH administration. The mean follow-up had been 18 years. They have pointed out the state of the reduced thyroid reserve after high doses of irra­diation if the period of follow-up is sufficiently Ion g. 7% of the examined patients had low T 4 and 25% had low T 3, most of them having low RAi uptake and elevated TSH. The data are similar with those reported by Fuks1 , Murken15 and Brase.29 Elevated TSH had 49 (31%) cases. 40 (25%) of the patients having raised TSH but normal T 4 were defined as being in latent hypot­hyroidism. In these cases a reduction in thyroid activity has been compensated for by an increa­sing output of TSH to maintain an euthyroid state. 30• 31 Our findings are identical with those of Fuks1 who also found 25% (13 of 52) patients with elevated TSH and normal T 4• There was no correlation between the pre­sence of antibodies and the onset of manifest of latent hypothyroidism. Elevated TSH was obser­ved in 11 (41%) of 27 patients with antibodies and in 49 (31 % ) of the whole group, while reduced leve! of T 4 was found in 3 (11 % ) in the group with antibodies and in 12 (7%) in the whole group of 174 patients. The difference is not statistically significant. An autoimmune pro­ Dimitrovska A et a/ cess does not appear to be a major factor in the development of hypothyroidism. Scan abnormalities appeared in 42% of 174 patients. In the literature available only Nelson4 reports the thyroid seans findings in lymphoma patients using Tc-99m. She detected pathological scan in 17 of 45 (38%) cases, the data that are similar with ours. There was no difference in obtained results between patients treated with or without surgery exept for 2 cases having hemythyroidectomy with total Iaryngectomy and one patient with hemityroid who were hypothyroid. In this re­spect our findings correspond .with ot­hers. 14, 15, 16,32 The presence of some abnormality in more than one half of the examined group signals the need for close evaluation of the thyroid in patients successfully treated for head and neck tumors. Periodically measurement of TSH as a sensitive index of subtle impairment of the gland seems to be the most appropriate test for evalua­tion of the thyroid damage due to external irradiation. References l. Fuk. T, Glatstein E, Marsa GW, Bagshaw MA,Kaplan HS. Long term effects of external radia­tion on the pituitary and thyroid glands. Cancer 1976; 37;1152-61. 2. Glatstein E, McHardy-Young S, Brast, Eltrin­ghalm JR, Kriss JP. Alterations in serum thyrotro­pin (TSH) and thyroid function following radiot­herapy in ,patients with malignant lymphoma. J Ciin Endocrinol Metah 19071; 32:833-41. 3. Kini YH, Fayos JV, Sisson JG. Thyroid functionfollowing neck irradiation for malignant lympho­ma. Radiology 1980; 134:205-8. 4 .. Nelson DF, Reddy KV, O'Mara R.E., Rubin P. Thyroid abnormalities following neck irradiation for Hodgkin's disease·. Cancer 1978; 42:2553-62. 5. Schimpff SC, Diggs CH, Wiswell JG, Salvatore,PC, Wiernik PH. Radiation related thyroid dy­sfunction: implications for the treatment of Hodg­kin 's disease. Ann Intern Med 1980; 92:91-8. 6. Tamura K, Shimaoka K, Friedman M. Thyroidabnormalities associated with treatment of mali­gnant lymphoma. Cancer 1981; 47:2704. 7. Cocina KN, švarcberg EM. Issledovanie funkciištitovidnoi železi pri pomošti radioaktivnogo joda pri lucevoi lecenii raka gortani. Med Radiol 1960; 8:14-8. 8. Felix H, Durpe N, Drape M, Court L. Incidence a long terme d'une radiotherapie pour cancer dularynx sur l'apparition d'un myxoedeme. Lyon Med 1961; 93:1043-50. 9. Einhorn J, Wikholm G. Hypothyroidism afterexternal irradiation to the thyroid region. Radio­logy 1967; 88:326-8. 10. Greig WR, Boyle JA, Buchanam WW, Fulton S.Clinical and radiobiological observations on lattenteffects of X irradiation on the thyroid gland. J Ciin Endocr 1965; 25:1009-14. 11. Koulumies M, Voutilainen A, Koulumies R, Ef­fect of X ray irradiation of laryngeal cancer on thefunction of the thyroid gland. Ann Int Med Fenn 1964; 53:89-96. 12. Markson JL, Flatman GE. Myxoedema after deep X ray therapy to the neck. Br Med J 1965; 1:1228-30. 13. Samaan NA, Vieto R, Schultz PN, Maor M, MeozRT, Sampiere VA, Cangir A, Ried HL, Jesse RH.Hypothalamic, pituitary and thyroid dysfunctionafter radiotherapy to the head and neck. Int J Radiat Oncol Biol Phys. 1982; 8:1857-67. 14. Lavelle RJ. Thyroid function after radiotherapyand total laryngectomy in the treatment of carci­noma of the larynx. Ann Oto/ Rhinol Laryngol 1971; 80:593-8. 15. Murken RE, Duvall AJ. Hypothyroidism follo­wing combined therapy in carcinoma of the laryn­gopharynx. Laryngoscope 1972; 82:1306-14. 16. Shafer RB, Nuttal FQ, Pollak K, Kuisk H. Thyroidfunction after radiation and surgery for head andneck cancer. Arch Intern Med 1975; 135:843-64. 17. Dimitrovska A, Georgievska B, Jovanovski D, Velkov K. Thyroid function after irradiation and surgery for laryngeal cancer. Radio/ Iugosl 1982; 16:331-5. 18. Aleksic ž. Šestakov D. Comparative measuring ofthyroxine by.radioimmunologic method (RIA) andcompetitive method. Radio/ Iugosl 1978;2:450-1. 19. Šestakov D, Ivanovski R. Radioimmunoassay oftriiodthyronin using thermo-denaturated serum and scparation on charcoal. Radio/ lugos/. 1978; 2:451-2. 20. Šestakov D, Ivanovski R. Radioimmunoassy ofTSH -experience with labeling, storage and deter­mination with calbiochem kit. Radio/ Iugosl 1978 ;2 :453-4. 21. Oliver C, Chartet JL, Codaccioni J, Vague J.Radioimmunoassay of thyrotropin-releasing hor­mone (TRH) in human plasma. J Ciin Endocrinol Metah 1974; 39:406-9. 22. Ormstron BJ, Garry R, Cryer RJ, Besser GM,Hall R, Thyrotropin-releasing hormone as a thy­roid function test. Lancet 1971 ;2:10-4. 23. Boyden SV. The absorption of proteins on erytro­cytes treated with tannic acid subsequent haemag­glutination by antiprotein sera J Exp Med 1951 ;93:107. 24. Aubin PM, Kinsley RM, Andrews GA. Externalirradiation of the thyroid gland in dogs. Effects oflarge doses of roentgen rays upon histologic strne­ Thyroid status following irradiation for laryngeal cancer ture and J-131 metabolism. Am J Roengenol Ra­dium Ther Nucl Med 1957; 78:864-75. 25. Lindsay S, Dailey ME, Jones MD. Histologic effects of various types of ionizing radiation on normal and hyperplastic human thyroid glands. J Ciin Endocrinol 1954; 14:1179-218. 26. Michaelson SM, Quinlan W, Casarett GW, Mason WB. Radiation induced thyroid dysfunction in the dog. Rad Res 1967; 30:38-47. 27. Rubin, Casarett GW. The endocrine glands. In: Clinical radiation pathology, vol 2, Philadelphia, W.B. Saunders 1968; 721-67. 28. Shively JN, Epling GP, Early changes in the fine structure of the midlethally irradiated thyroid dogs. Rad Res 1969; 37:71-82. 29. Brase AR, Sippel R. Zur hypothyreosehiiufigkeit nach perkutaner telekobaltbestrahlung 7o 1 lil r-i cx: So u 33 2o lo adj. Th Figure 2. CA 15-3 in patients stili in remission: A -Patients with occasional moderately increased CA 15-3. B -Patients with continuously increased CA 15-3. C -One patient who developed ovarian carcinoma without a relapse of BC 225 The predictive significance of CA 15-3 for the first relapse in early breast cancer patients Table l. CA 15-3 at the tirne of relapse and prior to Therefore, we analysed the quantitative values the first relapse. of CA 15-3 in disease-free patients and in relap­sed patients at the tirne of relapse and before it, CA 15-3 using in the first and latter case the maximal Normal Tota! registered value (Figure 3). There was no statisti­cally significant difference between the quantita­ 18 43 No of relapsed pts. 25 tive marker levels at the tirne of relapse, _and the No of pts. in remission 13 35 maximal CA 15-3 levels preceding the relapse Tota! 38 53 91 No of pts. with later remission 13 35 48 Sensitivity for the presence of metastatic disease =0.58 Specificity for the present metastatic disease =0.73 PPV for metastatic disease =0.65 NPV for metastatic disease =0.66 Sensitivity for the prediction of metastatic disease =0.53 sensitivity and prognostic value of this tumour marker. Sensitivity, defined as the proportion of patients with increased marker value in relapse (trne increase) was 0.58. Specificity, the propor­tion of patients without relapse who had trne negative marker values, was 0.73. The positive predictive value (PPV), representing the proba­bility that a pt. had the relapse if marker was increased, was 0.65, and the negative predictive value (NPV), representing the probability that a patient did not have the disease if marker value was normal, showed the value of 0.66. Clinical relevance of CA 15-3 in the prediction of the first relapse is possibly greater than the importance of this tumour marker in diagnosis of already manifested metastatic disease. There is no doubt about the usefulness of this tumour marker in screening for subclinical metastatic disease. Nevertheless, the possible use of systemic treat­ment in disease-free patients with increased CA 15-3 is stili questionable. The reason is not only the chance of a false increase in tumour mark er, which actually exists, but also the impossibility of predicting the localization of the first relapse, which could influence the choice of systematic or local treatment. On the other hand, the arbitrary choice of cut-off values is influenced by the tumour marker concentration in normal healthy persons. The crncial question here is the diffe­rence between trnly disease-free breast cancer patients and subclinically metastatic patients. occurrence. However, both of these two marker values were significantly higher than the maximal tumour marker values in non-relapsed patients. The same result was obtained when only the marker levels were tested, representing patients whose tumours seemed to produce this tumour marker at any tirne. We also noted that in 95% of disease-free patients the maximaf values never .--. o-i :::, -....., C') 1 U1 o-i C,: o • " • • looo ! • .. • " ' • .. o ... " • • .. . o II loo \C 1 • • 57 ----.. -------o---­ ----. ·-:· ­ 33 !: -i -i--r i' • • " lo • "" • " a b C Figure 3. Quantitative CA 15-3 values in paticnts with relapse (a -prior to relapse maximal values, b -at the tirne of relapse) and c -in diseasc-free patients ­maximal values. Logarithmic scale is used. Neškovic-Konstantinovi{; Zet al. exceeded 57 U/ml. Using this as a cut-off value, the obtained sensitivity was 0.44, specificity 0.93, positive predictive value 0.86, and negative pre­dictive value 0.65. The obtained sensitivity in relapse prediction was even lower (0.39), but predictive values were nearly the same (0.85 and 0.63, respectively). Further follow-up of this particular group of patients is necessary to con­firm these results, and to propose the rise of the upper cut-off limits to 57 U/ml. In any case, quantitative marker values in addition to qualita­tive data, seem to be significant for relapse prediction. Concerning the pattern of the first relapse, it seems that an early increase of CA 15-3 is more frequent in BC patients who would develop bone metastases: in 16/24 patients the CA 15-3 preceded the bone relapse. The increased marker values preceded the soft tissue relapse in 1/10 pts., pleuro-pulmonal in 1/4 pts., !iver meta­stases in 2/3 pts., and the multiple site of relapse in 2/2 pts. So, the increased CA 15-3 values in disease-free patients suggested the need for a more careful search for metastatic disease, parti­cularly i. the bones. In conclusion, CA 15-3 has been confirmed as a sensitive tumour marker both for diagnosis of metastatic disease and for predicting the first relapse. References l. Hayes DF, Sekine H, Ohno T, et al. Use of murine monoclonal antibody for detection of circulating plasma DF3 antigen levels in breast cancer. J Ciin Invest 1985; 75:1671-8. 2. Martoni A, Ercolini L, Bellanova B, et al. CA 15-3 and CEA plasma leve! monitoring in patients with breast cancer. Int J Biol Markers 1988; 3:154-8. 3. Tondini C, Hayes DF, Kufe DW. Circulating tu­mour markers in breast cancer. Hematology!Onko­logy Clinics of Norih America 1989;3:653-74. 4. Kallioniemi OP, Oksa H, Aaran RK, at al. Serum CA 15-3 assay in the diagnosis and follow-up of breast cancer. Br J Cancer 1988; 58:213-5. 5. Malkin A. Tumour Markers. In: Tannock JF & Hill· RP ed. The Basic Science of Oncology, Pergamon Press 1987; 192-204. 6. Safi F, Kohler I, Rottinger E et al. Comparison of CA 15-3 and CEA in diagnosis and monitoring of breast cancer. Int J Biol Markers 1989; 4:207-14. 7. van Dalen A. Pre-operative tumour marker levels in patients with breast cancer and their prognosis. Tumor Biol 1990; 11:189-95. 8. Kiang DT, Greenberg LJ, Kennedy BJ. Tumor marker kinetics in the monitoring of breast cancer. Cancer 1990; 65:193-9. 9. McGuire WL, and Clark GM. Prognosis in Breast Cancer. In: Senn HJ, Goldhirsch A, Gelber RD and Osterwalder B, eds. Adjuvant therapy of pri­mary breast cancer. Springer-Verlag Berlin Heidel­berg 1989; 170-4. Chapter V. EXPERIMENTAL ONCOLOGY Adv Radio! Onco/ 1992; 229-35. Prostaglandins in malignant tumors: Role in tumor growth and therapy Milas L University of Texas M.D. Anderson Cancer Center, Department of Experimental Radiotherapy, 15 I 5 Holcombe Boulevarcl, Houston, Using a number of murine tumors we have demonstrated that prostaglandins (PGs) are important in regulating tumor growth and response to radiotherapy. Tumors may or may not produce PGs; those that produce them will respond to the inhibiton of PGs by slowing their growth rate. Inhibition of PGs was achieved by treatment of tumor hosts with indomethacin, an inhibitor of PG-synthase. The antitumor activity of indomethacin was found to be mediated mainly through inhibition of tumor neoangiogenesis. Indomethacin also potentiates tumor response to ionizing radiation, but by eliciting or augmenting the antitumor immune reactions. In contrast, indomethacin either does not affect normal tissue radioresponse or it protects some normal tissues against radiation injury. Thus, indomethacin can greatly improve the therapeutic ratio of radiotherapy, which can be further improved by combining indomethacin with other radiosensitizers or radioprotectors that have different mechanisms of action from those of indomethacin. These studies are important for understanding the role of PGs in the growth regulation of malignant tumors and their response to therapy, and may have potential implications to clinical radiotherapy. Key words: neoplasms-therapy; prostaglandins Introduction Eicosanoids are metabolites of unsaturated fatty acids, principally of arachidonic acid, that play a regulatory role in many biological processes. Virtually ali mammalian cells produce eicosa­noids, which are further classified into two major groups: prostaglandins (PGs) and leucotrienes. These are produced via the cyclooxygenase and lypooxygenase pathways, respectively. Howe­ver, advances in eicosanoid research have come mostly from studies on PGs. The biological Corespondence to: Luka Milas, M.D., Ph.D., Profes­sor and Chairman, Department of Experimental Ra­diothcrapy 66, M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030 USA. UDC: 616-006.6-08:615.277.3.015.43 activities of PGs are many and include immuno­modulation, vasoregulation (constriction or dila­tation), platelet aggregation, celi growth and differentiation, and tumor development and growth. 1 With regard to the latter, PGs regulate both the initiation and promotion steps of carci­nogenesis, rate of tumor growth and tumor dissemination. 1 The discovery that PGs play a role in tissue response to ionizing radiation represents a recent development in research on eicosanoids. 2•The reported findings have, however, been conflic­ting because both the addition of PGs, 2•3 as well as their inhibition, 45 have been found to protect a number of normal tissues from radiation dama­ge. Our own research has been aimed at increa­sing the therapeutic efficacy of radiotherapy through modulation of PGs production, prima­ Milas L rily of that in tumors. Our initial hypothesis was that inhibition of PGs in tumors would increase tumor radioresponse presuming that PGs act radioprotectively. We have already reported a number of observations made in rodent tumors and normal tissues on the role of PGs inhibition in tumor radiotherapy. 6-11 This repo rt is a brief account of our major findings. PGS synthesis by tumors and their inhibition It has been well documented that malignant 13 tumors in manl . 12 and in experimental animals1 . produce PGs. The production varies both quanti­tatively and qualitatively among tumors of diffe­rent histological types as well as among indivi­dual tumors with the same histology. An illustra­tion of this wide variability in PG and other eicosanoid production is shown in Table 1, which contains our own analysis of eicosanoids produ­ced by five different murine tumors. 6 Of five tumors analyzed, only three produced PGs. Ho­wever, even among the three PG-producing tumors there was a marked difference in the type and amount of individual PGs produced. Similar­ly, there were significant differences in produc­tion of leukotrienes. Most PG production was by tumor cells. However, it is likely that host cells that infiltrate these tumors, such as macrophages and lymphocytes, also participated in PG pro­duction. Macrophages are known to be good producers of the PG classes PGE2 and TXB2. 14 Also, endothelial cells of tumor blood vessels could participate in PG production, and espe­cially in the production of PGI2. 15 Because different types of PGs exhibit different biological actions, the action of PGs in regulation of tumor behaviors will depend on the type and quantity of a given PG produced by the tumor. For example, while tumors that produce high quanti­ties of TXB2 are expected to be prone to the clogging of their blood vessels, tumors that are high producers of PGI2 should have good blood flow. This is because TXB2 stimulates and PGI2 inhibits platelet aggregation, and through this process regulate blood coagulation. That PGs participate significantly in the ove­rall regulation of tumor growth and its spread has been shown in many ways. Inhibition of PGs is a prominent method that has frequently been used. 11 Inhibition of PGs is readily achieved using indomethacin or other nonsteroidal anti­inflammatory agents that block the cyclooxyge­nase pathway in arachidonic acid metabolism. This inhibition is frequently associated with a retardation in tumor growth, which indirectly implies that PGs are stimulators of tumor growth. The antitumor action of indomethacin is Table 1. Eicosanoid mctabolites of mouse tumors". Mouse tumors Eicosanoid FSA NFSA SA-NH MCA-K HCA-I 6KPGF1" 102.8b 135.1 NDC 10.3 TXB2 46.2 31.9 ND ND 28.6 PGF2a ND 11.3 ND ND 24.3 P.GE2 336.1 36.6 ND-ND 14.5 PGD2 ND ND ND ND 6.2 LTB4 ND 28.9 10.7 64.0 ND HHT 83.5 2.8 14.7 di-HETEs ND ND 12.1 65.6 ND 15-HETE ND 19.3 ND ND ND 5-or 12-HETE 146.7 30.5 26.6 86.9 115.7 Tota! metabolites 715.3 329.0 55.9 231.2 245.0 "Tumors were generated by 5 x 105 tumor cells injected into the muscles of the right thighs. Tumors were assayed for eicosanoid metabolites when approximately 9 mm in diameter. hValues in ng/gm are given as the mean of two samples. Each sample was obtained from a pool of 4 to 6 tumors. 'ND, not detectable ! Reprinted from Furuta et al.,6 with permission. Prostaglandins in malignant tumors: Role in tumor growth and therapy variable, with · only a fraction of the tumors tested showing a good response. Most frequen­tly, only a retardation in tumor grnwth rate is seen after indomethacin treatment. Complete and long lasting tumor regressions have been extremely rare. The mechanisms which mediate the antitumor effects of indomethacin are stili a matter of consi'derable contrnversy. The effect has most commbnly been attributed to the ability of indo­methacin to elicit or potentiate antitumor im­ mune mechanisms thrnugh the inhibition of prn­duction of immunosuppressive PGs, notably PGE2. 1 •16•17 Our own studies8 question the invol­vement of the immunological reactions and point to other, probably more important, mechanisms. We have observed that the antitumor activity of indomethacin is independent of both tumor im­munogenicity and host immunocompetence.6•8 Tumors in mice that are generally immunocom­petent or lacking T-lymphocytes respond to indo­methacin as well as tumors in normal mice. 8 Importantly, however, the effect of indometha­ cin depended entirely on PGs production. Only tumors that produce PGs respond to indometha­cin. 6 This discovery has a valuable clinical impli­cation in that it may be possible to predict which tumors will respond to this therapy by assaying the PG profile of individual patients prior to therapy. Indomethacin has no direct cytostatic or cyto­toxic effect for tumor ce11s6, but it slows tumor cells proliferation6 by indirect means. A possibi­lity that we recently investigated was that indo­methacin inhibits tumor vascularization, and thus reduces the blood supply to tumor cells. Tumor cells would then become deficient in oxygen and other nutrients, which would slow their prnlifera­tion. This hypothesis is based on the evidence that PGs stimulate tumor angiogenesis. 18 Using an intradermal assay that we recently developed for studying tumor angiogenesis in mice 19 , we demonstrated that indomethacin reduced the formation of new vessels at the site of tumor celi injection, which preceded the slowing of tumor growth8 (Figure 1). Based on this evidence, we conclude that inhibition of tumor neovasculariza­ tion is a major mechanism by which indometha­cin exerts its antitumor action. 100 .Q T / I / 1 (f) .,,..-/ w ­ lfl n E (f) /5 •J. --/·-· . s _ /Q • /!f o ..,.-,,,, ,,/1 ,l ow 1 O o2 3 ::::, (D --' ./ i/ / !J.. §; o o:: / o:: o ;· o/d -/ ;-/' w :::;; (D ::::, :::;; ­ T o 2 4 6 8 10 DAYS AFTER TUMOR CELL INOCULATION Figure l. Effect of indomethacin on tumor angiogene­sis. The mice were given intradermal inoculation of 106 NFSA cells, and the number of vessels at the injection site was determined on the indicated days thereafter in indomethacin-treated ( closed circle) and control ( open circle) mice. Tumor volume in indomethacin-treated ( closed square) and indomethacin-untreated ( open square) mice was also plotted. Vertical bars are S.E. of the mean values. Treatment with indomethacin, 35µ,g/ml in drinking water, was started 1 day after tumor celi inoculation and was continued daily for 9 days. Reprinted from Milas et al., 8 with permission. Indomethacin-induced potentiation of tumor radioresponse The hypothesis was that if PGs act as radiopro­tectors, a reduced leve! of PGs in tumors should make the tumors more responsive to radiation. Indeed, we observed that inhibition of PGs in tumors by using indomethacin was accompanied by an augmentation of the radioresponse of PG-producing tumors7•8•10 as assayed either by the tumor growth delay or the TCD50 assay (radiation dose yielding 50% local tumor con­trol). The degree of indomethacin-induced aug­mentation of tumor radioresponse ranged from a factor of 1.3 to > 2, and depended on many factors including tumor type, choice of fractiona­tion in radiation treatment schedule, timing of indomethacin administration in relation to tumor irradiation, and the endpoint of tumor response. Milas L The augmentation was greater with fr.actionated than with single dose irradiation. 8 Figure 2 illu­strates the indomethacin-induced potentiation of radioresponse of a murine sarcoma. Figure 2. Radiation dose response curves for Iocal tumor control of 8-mm FSA tumors growing in the Iegs of mice treated with radiation alone (7) or with radiation plus INDO (...,..), MISO (.), or both INDO and MISO ( .). Error bars, TCD50, 95% confidence limits. Treatment with INDO, 35 JLg/ml in drinking water, was started when FSA tumors were 6 mm and continued for 10 days. MISO (1 mg/g) was given i.p. 30 min before local tumor irradiation. Points, data presented here are from 2-6 separate experiments. Reprinted from Milas et al., 10 with permission. Our studies on the mechanisms by which indomethacin potentiated tumor radioresponse failed to support the initial hypothesis that the potentiation was the result of lowering the leve! of PGs. 8 This was established by showing that ind6methacin augmented tumor radioresponse when given after irradiation was completed, indicating that in order to potentiate tumor radioresponse, the hypothetical radioprotective PGs need not be removed or reduced at the tirne of radiation exposure. This finding is also incon­sistent with a second mechanism which we advanced earJier7 , nameJy that indomethacin potentiates tumor radioresponse by arresting tumor cells in the G2/M and Gl celi cycle phases which are more sensitive to radiation than cells in the S-phase. However, the radiopotentiating effect of indo­methacin was greatJy reduced or abolished in mice immunosuppressed by whoJe body irradia­tion or in nude mice that seJectively Jack T-Jym­phocytes. 8 This implies that the potentiation of tumor radioresponse was mainly, if not exclusi­veJy, mediated through the antitumor immune mechanisms eJicited or augmented by indomet­hacin. As discusscd above, this is in contrast to the nonimmunoJogicaJ mechanism of antitumor activity found to be operative when indometha­cin is appJied aJone. The existence of two diffe­ mechanisms of the indomethacin action which appear to operate under different experi­mentaJ conditions is difficult to expJain on the of currentJy avaiJable data. A plausible expJanation may be that, in combination with radiotherapy, the antitumor immune reaction induced by indomethacin, even if it is weak, may be effective in eliminating those few viable tumor cells that survived radiation. Such a process is, however, unlikely to be effective in slowing the growth of established tumors that contain large numbers of tumor cells, as exemplified by tumors treated with indomethacin alone. It is logical then that in this case the antiangiogenic mecha­nism of indomethacin would predominate. Modification of normal tissue radioresponse by indomethacin To render a therapeutic advantage, indometha­cin must be more effective in augmenting tumor radioresponse than that of normal tissues within the radiation fieJd. For this purpose we have investigated the effect of indomethacin on the radioresponse of the following tissues and or­gans: hematopoietic tissue, Jung, esophagus, je­junum, coJon, hair follicles and tissues responsi­bJe for radiation-induced leg contractures. 7,9-11 In contrast to its potentiating effect on tumor radioresponse, indomethacin did not aJter radio­response of most of the listed tissues and actually protected two of them: hematopoietic tissue and the Jung. Protection of hematopoietic tissue was Prostaglandins in malignant tumors: Role in tumor growth and therapy 233 demonstrated by the increase of hematopoietic spleen colonies (Figure 3) and by the reduction of mouse Iethality.9 Protection, which in the spleen colony assay was by a factor of 1.3 (Figure 3), was achieved only when indometha­cin was given before irradiation. Indomethacin did not alter the radiosensitivity of hematopoietic stem cells; rather, it exerted its radioprotective action indirectly by stimulating the proliferation of hematopoietic cells. The mechanism(s) of indomethacin induced lung radioprotection re­mains unknown at present. This information, together with that on tumor radioresponse, 100 rn Q) • C 1 o o .-o o • : • u 10 o • • • C • Q) o • Q) o.. o (/) o "'-cs:>o o o • ..o Q) o . o 1 r o E :s z o 0.1 6 7 8 9 o;•1 o­ ' f'{'. Radiation Dose (Gy) Figure 3. Effect of indomethacin on endogenous ipleen colony survival after total body irradiation (TBI): Vehicle or indomethacin was given for 6 days before · TBI. Mice were sacrificed S or 9 days after TBI, spleens were removed and fixed in Bouin's solution, and the number of colonies were counted. A higher number of spleen colonies was observed in the indo­methacin-treated mice ( closed circle) than in the con­trol mice (open circle). The protection factor at leve! of 10 spleen colonies was 1.3. The data are from six separate experiments; each symbol is a mean value of number of spleen colonies obtained within individual experiments. The lines were fitted by least-squares regression analysis. Reprinted from Nishiguchi et al., 9 with permission. clearly shows that indomethacin can significantly increase the therapeutic ratio of tumor radiothe­rapy. Combination of indomethacin with true radiosensitizers and radioprotectors Our observations that indomethacin augments tumor radioresponse through immunological reactions and protects some normal tissues by stimulating celi proliferation suggested the possi­bility of combining indomethacin with agents that affect tumor or normal tissue radioresponse through mechanisms different from those of indomethacin. Hypothetically, this combination should further increase the therapeutic ratio of radiotherapy. Figure 2 shows that combining indomethacin with misonidazole, a prototype of those agents that specifically sensitize hypoxic cells to radiation, increased tumor radioresponse significantly more than indomethacin or misoni­dazole alone. Likewise, combining indomethacin with WR-2721, a trne radioprotector, was signifi­cantly more radioprotective for hematopoietic tissue than were the individual drugs alone. 9 More detailed information about these combina­tions is given in our recent reports. 9-11 This is a new line of our research that is currently under extensive investigation. Conclusions Malignant tumors may or may not be good producers of PGs. PGs regulate the growth of malignant tumors mainly through stimulation of tumor neovascularization. Inhibition of neovas­ cularization by indomethacin restrains tumor celi proliferation and consequently retards tumor growth. The antitumor activity of indomethacin is independent of tumor immunogenicity and of the ability of the tumor host to mount antitumor immunological response. Indomethacin treatment results in inhibition of PG production by tumors, and in a significant potentiation of tumor radioresponse. Only PG­ producing tumors exhibit this potentiation. The 234 Milas L extent of potentiation varies between 1.3 and > 2.0 and depends on a number of factors and experi­mental conditions. The radiopotentiating effect of indomethacin is greatly dependent on tumor host immunocompetence, and was reduced or abolished by preventing the host to mount immu­nological reaction to the tumor. Therefore, whe­reas the antitumor activity of indomethacin alone is mediated primarily through inhibition of tumor angiogenesis, the radiopotentiating effect of in­domethacin is mainly an immunological pheno­menon. In contrast to its effect on tumor radiorespon­se, indomethacin does not influence the radiore­sponse of some normal tissues and it protects some, notably hematopoietic tissue and lung. The protection of hematopoietic tissue was me­diated indirectly via stimulation of stem-cell proliferation. Therefore, while indomethacin renders a signi­ficant increase in tumor radioresponse, it either does not affect or reduces the response of normal tissues, thus providing a significant increase in the therapeutic gain of radiotherapy. This gain can be improved even further by combining indomethacin with either hypoxic cel radiosensi­tizers, such as misonidazole, or trne radioprotec­tive agents, such as WR-2721, which modulate tumor or normal tissue radioresponse by mecha­nisms different from that of indomethacin. It should be noted here that there already exists some evidence that the inhibition of PGs by nonsteroidal anti-inflammatory agents can im­prove the therapeutic efficacy of clinical radio­therapy. 20.21 References l. Honn VK, Bockman RS, and Marnett LJ. Prosta­glandins and cancer: A review of tumor initiation through tumor metastasis. Prostaglandins 1981; 21 :833-64. 2. Hanson WR, Ainsworth EJ. 16-16 Dimenthyl prostaglandin E2 induces radioprotection in mu­rine intestinal and hematopoietic stem cells. Ra­diat Res 1985; 103:196-203. 3. Walden TL Jr, Patchen M, and Snyder SL. 16-16 Dimethyl prostaglandin E2 increases survival in mice following irradiation. Radiat Res 1987; 109:440-9. 4. Northway MG, Libshitz HI, Osborne BM, Feld­man MS, Mame! JJ, West HH, and Szwarc IA. A radiation esophagitis in the opossum: radioprotec­tion with indomethacin. Gastroenterology 1980; 78:833-7. 5. Suzuki JB, Emmering TE, Kishiby JS, Schonfeld S, Sowle J, Tatum RC, and Van Dyke T. Protec­tion of irradiated parotid by prostaglandin synte­sis inhibitors. J Am Dental Assoc 1986; 112:179­81. 6. Furuta Y, Hall ER, Sanduja S, Barkley HT Jr, and Milas L. Prostaglandin production by murine tumors as a predictor for therapeutic response to indomethacin. Cancer Res 1988; 48:3002-7. 7. Furuta Y, Hunter N, Barkley HT Jr, Hall E, and Milas L. Increase in radioresponse of murine tumors by treatment with indomethacin. Cancer Res 1988; 48:3008-13. 8. Milas L, Furuta Y, Hunter N, Nishiguchi I, and Runkel S. Dependence of indomethacin-induced potentiation of murine tumor radioresponse on tumor host immunocompetence. Cancer Res 1990; 50:4473-7. 9. Nishiguchi 1, Furuta Y, Hunter N, Murray D, and Milas L. Radioprotection of hematopoietic tissues in mice by indomethacin. Radiat Res 1990; 122:18­92. 10. Milas L, Ito H, Nakayama T, and Hunter N. Improvement in therapeutic ratio of radiotherapy for a murine sarcoma by indomethacin plus misoni­dazole. Cancer Res 1991; 51:3639-42. 11. Milas, Nishiguchi 1, Hunter -N, Murray D, Fleck R, Ito H, and Travis E. Radiation protection against early and late effects of ionizing irradiation by the prostaglandin inhibitor indomethacin. In: Advances in Space Research 1992; 12:265-71. 12. Rolland PH, Martin PM, Jacquemir J, Rolland AM, and Toga M. Prostaglandin in human breast cancer: Evidence suggesting that an elevated pro­staglandin production is a marker of high metasta­tic potential for neoplastic cells. J Nat/ Cancer lnst 1980; 64:1061-70. 13. Fitzpatrik FA, Stringfellow DA. Prostaglandin D2 formation by malignant melonoma cells correlates inversely with cellular metastatic potential. Proc Natl Acad Sci USA 1979; 76:1765-9. 14. Goldyne ME, Stobo JO. Immunoregulatory role of prostaglandins and related lipids. CRC Critica/ Reviews in /mmunology 1981; 2:189-223. 15. Vermylen J, Chamone DAF, and Verstraete M. Stimulation of prostacycline release from vessel wall by Bay g 6575, an antithrombotic compound. Lancet 1979; 1 :518-20. 16. Plescia OJ, Smith AH, and Grinwich K. Subver­sion of immune system gy tumor cells and role of prostaglandins. Proc Natl Acad Sci USA 1975; 72:1848-51. 17. Fulton AM. Interactions of natura! effector cells and prostaglandins in the control of metastasis. J Natl Cancer lnst 1987 ;78:735-41. 18. Ziche M, Jones S, and Gullino PM. Role of prostaglandin E1 and copper in angiogenesis. J Natl Cancer /nst 1982; 69:475-81. 19. Runkel S, Hunter N, and Milas L. An intradermal assay for quantitation and kinetics studies of tumor angiogenesis in mice. Radiat Res 1991 ;126:237-43. Prostaglandins in malignant tumors: Role in tumor growth and therapy 235 20. Weppelmann B Monkemeier D. The influence of 21. Pillsbury HC, Webster WP, and Rosenman J. prostaglandin antagonists on radiation therapy of Prostaglandin inhibitor and radiotherapy in advan­carcinoma of the cervix. Gynecol Oncol 1984; ced head and neck cancers. Arch Otolaryngol 17:196-9. Head Neck Surg 1986; 112:552-3. Adv Radio/ Oncol 1992; 236-40. Problems in tumour celi repopulation during irradiation treatment of squamous celi cardnoma of head and neck Budihna M and Škrk J The Institute of Oncology, Ljubljana The repopultion rate of clonogenic cells is discussed in the light of our patients with head and neck squamous cel! carcinoma and reports in the literature. Saunders' data of survival of patients with head and neck carcinoma treated by continuous hyperfractionated accelerated radiotherapy (CHART) and conventional irradiation treatment were used to estimate when in the course of the treatment the clonogenic cells accelerate their division rate. By means of Overgaard's dose response curves far laryngeal carcinoma the TCD50 far CHART and conventional treatment was estimated. The difference of TCD50 and overall treatment tirne between CHART and conventional treatment was used far calculation when the repopulation begins. Assuming that the maximum repopulation rate expressed in Gy is 0.6 Gy per day as obtained by Withers it was calculated that the repopulation begins toward the end of the second week of irradiation treatment in advanced tumours and probably sooner in early tumours. Key words: head and neck carcinoma; head and neck neoplasms-radiotherapy; clonogenic cells, repopulation; Introduction The cure of carcinoma is achieved when ali clonogenic tumour cells are killed in the course of the irradiation treatment. 1 Usually clonogenic cells represent a small proportion of tumour cells, i.e. aproximately 1 % of the tumour popula­tion. 2 Their properties cannot be determined by microscope or flow cytometry. According to the sim plest radiobiological theory, the curative ra­diation dose depends only on the radiosensitivity of the clonogenic tumour cells and on their number. If the number of tumour clonogens Correspondence to: Budihna Marijan M. D., Ph. D; Institute of Oncology, Zaloška 2, 61000 Ljubljana,. Slovenia. UDC: 616-006.6-08:615.849 :611.92/.93 which are not killed by irradiation increases in the course of treatment a higher radiation dose is needed for cure. Thus, the speed of repopula­tion of clonogenic cells is extremely important. The only available method to measure it during a certain period of treatment is based on the determination of dose difference in two radiation treatment schedules of different duration, nor­malized to the same tumour dose per fraction, both giving the same tumour response. The aim of this paper is to discuss the repopu­lation of clonogenic cells in the tumour. There are three questions to be answered in this re­spect: how fast is the repopulation of the clono­genic cells? Does the speed of the repopulation change during the irradiation treatment and when does the change occur? Problems in tumour celi repopulation during irradiation treatment of squamous celi carcinoma... Material, methods and results How fast is the repopulation of the clonogenic cel/s? The division rate of clonogenic cells in favora­ble living conditions is best correlated with the potential doubling tirne (T pot). T pot can be measured quickly by monoclonal antibodies to BUdR or IUdR on denaturated DNA in which the thymidine analog Iodo-or Brorno-deo­xyuridine is incorporated into DNA3. The length of T pot was less than 6 days in half of the head and neck carcinornas as rneasured by McNally4. As defined by Stee!2 T pot is a cornbined inforrna­tion resulting frorn celi cycle tirne and · growth fraction (the latter being very rnuch in correla­tion with the clonogenic fraction), and it repre­sents the doubling of the celi population if there was no celi loss. A celi loss factor of 90 or 95% would rnean that the turnour volume doubling tirne is 10 or 20 tirnes greater than T po/. This, in fact, can be seen in untreated squarnous celi carcinorna of the head and neck which doubles its volurne in aproxirnately 2 to 3 rnonths. Assu­ming that the growth fraction and cell-loss factor change little over a srnall nurnber of volurne doubling tirnes, the preirradiation volurne dou­bling tirne is also approximately the doubling tirne of turnour clonogens. 7 Does the speed of the repopulation rale change during the irradiation treatment? In 1980 Budihna et al. 8 calculated, on the basis of the difference between the total tumour dose in the split-course and conventional irradia­tion treatment of the laryngeal carcinoma and the duration of the split-course interval, that the doubling tirne of clonogenic cells in the rest interval is 3.5 ± 0.5 days; 0.4 Gy were needed to kili the daily regrowth of the clonogenic cells. It was assumed that cells grow exponentially. This was regarded as an acceleration of the pretreatment tumour growth and accounted for by better living conditions of the clonogenic tumour cells surviving the first part of the split­course irradiation treatment. This, in fact, was also the first observation of the kind on human data reported in the literature. Frorn the data of Galante9 published in 1982 it can be seen that the median value for the doubling tirne of the postoperative recurrences of head and neck carcinornas was 7.8 days, assurning the exponential growth of turnour. Maciejewski in 198310 noted that the total turnour dose needed for the 50% control of laryngeal carcinorna (TCD50) had to be increased for 8 Gy if the overall tirne of irradiation treat­rnent was prolonged for 16 days. He assurned that 2 Gy are necessary to cornpensate for one doubling of clonogenic cells. Based on that, he calculated that the doubling tirne of clonogenic cells during irradiation treatrnent did not exceed 4 days. Budihna et al. in 198611 found on the basis of the difference between the total turnour doses of conventional and split-course irradiation treat­ rnent and the duration of split-course rest inter­val that the doubling tirne of the clonogenic cells of nasopharyngeal carcinoma in the rest-interval of split-course irradiation treatment is 3.5 -4.5 days. Here again the exponential tumour growth was assurned; 0.4 Gy were needed to kili the daily regrowth of clonogenic cells. Overgaard in 198812 dernonstrated by means of dose-response curves for conventional and split-course irradiation the need for an increase in the total dose of approximately 12 Gy in the split-course irradiation regirnen to obtain an equal effect in both treatrnent regirnens. He hypothesized that if 6 Gy were necessary to reduce the clonogenic celi population with a factor 10, 12 Gy meant a hundredfold increase of clonogenic cells in three weeks of the rest interval. Frorn the above data he calculated that the clonogens of laryngeal carcinorna, in the rest interval of the split-course treatment doubled their nurnber in 3-4 days. Withers in 19886 noted that TCD50 normalized to 2 Gy, had to be increased for 0.6 Gy per each day of treatment prolongation. Assurning that 2.4 Gy in a 2 Gy fraction regirnen were required to cornpensate for each doubling in clonogen nurnber, he calculated that the doubling tirne of Budihna M and Škrk J the clonogenic cells during the irradiation was 2.4 Gy : 0.6 Gy/day = 4 days. Thus, from the presented studies, it can be seen that the repopulation rate of the clonogenic cells of squamous celi carcinoma changes very much in the course of irradiation: it accelerates up to 10-20 times of its pretreatment value. The doubling tirne of clonogenic cells during the course of irradiation and in the split course rest-interval is, therefore, close to the T pot· This means that the celi loss factor of the tumour falls during the irradiation treatment to zero. 5 When does the acceleration of the repopulation begin? Withers6 suggested that there is a lag period of about 4± 1 week after the beginning of treatment during which there is on average little change in TCD50 (i.e.: before a burst of rapid repopulation of head and neck squamous celi carcinoma be­gins). Saunders et al. 13, analysed the survival of 2 groups of patients with head and neck carcinoma irradiated conventionally in the duration of 6-7 weeks or with continuous hyperfractionated ac­celerated radiotherapy (CHART) in the duration of 12 days. Two-year survival of patients treated by CHART was almost 2 times better than that of those treated conventionally. Because of that he suggested that the accelerated repopulation of clonogenic cells might occur within a short while of the initiation of irradiation therapy. However, she did not give an explanation why it is so. Yet, Trott14 in the same Saunders' data could only see that the tumour control rates as achieved by CHART were at least as good as those achieved elsewhere with conventional treatment by giving 70 Gy in 7 weeks in head and neck cancer. Since it was not quite apparent from Sounders' data when the rapid repopulation began we tried to estimate what could be the TCD50 from his data and present it in relation to the overall treatment tirne as Withers did6 in order to see when the burst of repoulation started. Our approach using Saunders' 13 data. In conventional treatment of Saunders' stu­dy 13, 60 to 70 Gy, mostly 2 Gy per fraction, were applied 5 times weekly. In CHART 3 x 1.4 to 1.5 Gy per day in 12 consecutive days were given, the total tumour dose being 50.4-54 Gy. But in fact almost always the tumour dose was 1.5 Gy per fraction. 15 Patients in both groups were matched by tumour stage. The two year survival of patients, obtained by CHART scheme was 63% for ali patients, 63% for T-3 and T-4 tumours taken together and 57% for T-4 tumours alone as estimated from the graphs. The survival of patients treated by con­ventional irradiation was as follows: ali patients survived 45%, whereas those with T-3 and T-4 tumours in 33%, and the patients with T-4 tumours alone survived in 23%. Our working hypothesis was that the tumor dose per fraction in CHART was 1.5 Gy, and to tal tumor dose 54 Gy. In the conventional treatment the tumor dose was 2 Gy per fraction, and the total tumor dose 65 Gy. The equivalent total tumor dose in CHART scheme (D2 Gy) if the tumor dose per fraction were 2 Gy would be 53 Gy as calculated by formula: D2 Gy = D1.5 Gy x (a/{3 + 1.5) : (a/{3 + 2) where D1.5 Gy is the total tumour dose in CHART, and a/{3 was taken as 25 Gy (as Withers et al. did in their paper 1988). For further analysis it is necessary to deter­mine TCD 50 for both irradiation schemes under discussion. But it is impossible to derive TCD50 from Saunders' data 13 because we cannot cons­truct a dose response curve. To get an approxi­mate estimation of what could be TCD50 we borrowed the dose response curve for laryngeal carcinoma from Overgaard et al. 12 Drawing this curves (which are taken parallel because the groups of patients are matched) through survival values of patients as obtained by Saunders we estimated TCD50 for each treatment group of patients whose survival is being compared (Fi­gure 1). The TCD50 values obtained in this way are shown in the Table l. Problems in tumour celi repopulation during irradiation treatment ofsquamous celi carcinoma . . . Figure l. Thc cstimation of TCD50 for Saunders, 13 survival data (oper circles) for T-3 & T-4 and T-4 stages treated by CHART and conventional irradiation by means of Overgaard's dose response curves. 12 Table l. TCD50 for patients of different stages (T) treated by CHART normalized to 2 Gy daily dose, and convcntional trcatmcnl. Irradiation scheme T-3 & T-4 T-4 CHART2c;y 50.0 Gy 51.5Gy Conventional2 Gy 70.0 Gy 73.0 Gy Assuming that 50 Gy the CHART2 oy were applied in about 11 days and 70 Gy of conventio­nal treatment in 47 days we would get a dose increase 0.56 Gy/day due to repopulation effect (20.0 Gy divided by 36 days) in the group ofT-3 & T-4 tumours. Similarly, for T-4 tumours we calculate that the daily increase of dose is 21.5 Gy divided by 37.5 (assuming that 73.0 Gy would be delivered in 49 days and 51.5 Gy of CHART2 Gin 11.5 days) which amounts to 0.57 y Gy/day. Dividing the difference between the total tu­mour dose applied by CHART2 Gand the total y tumour dose applied by conventional irradiation treatment by 0.6 Gy/day (as obtained by Wit­hers) the duration of accelerated repopulation in conventional treatment is obtained. Subtracting this value from the overall treatment tirne of the Saunders' conventional irradiation it can be esti­mated when the accelerated repopulation begins: it appears, that the tumour repopulates with full speed already on the 13-th or 14-th day after the beginning of irradiation treatment. Discussion These figures for daily repopulation dose are obtained assuming that the slope of dose re­sponse curve is such as reported by Overgaard for laryngeal carcinoma 12 and that the total tumour dose of CHART was 54.0 Gy, whereas that of the conventional treatment was 65.0 Gy. If the slope of the curve was less steep, the difference between TCD50 for CHART and con­ventional irradiation treatment would be more pronounced which would mean a full speed repopulation, already on about the 14-th day of treatment. If, on the other hand, the slope of the dose response curves was steeper, the differences between TCD50 for CHART and conventional treatment would be smaller. Assuming that the maximum speed of repopulation in tumor is 0.6 Gy/day as calculated by Whithers6 , the start of the repopulation would begin to accelerate somewhere in the middle of the third week. But steeper dose response curves are probably less likely to occur because a considerable heteroge­neity in the human tumours exists already within one stage. If the average total tumour dose in conventio­nal irradiation approached 70 Gy, the values of repopulation of clonogens in the tumour would stili be about 0.6 Gy/day; again, this would indicate that the repopulation begins toward the end of the second week of irradiation treatment. The quick repopulation of clonogenic cells is probably caused by better living conditions of cells surviving the irradiation and a consequent drop in cell loss. In clinical practice we often see smaller tumours in the head and neck area shrinking and disappearing earlier in the course of irradiation than the bulkier ones. It would be logically to expect that in small tumours the events which lead to the burst of repopulation take place sooner than in bulky tumours. This would in turn mean that short-course irradiation treatment would be relatively less advantageous in early than in advanced tumours. This, in fact Budihna M and Škrk J becomes apparent in the Saunders' study: 13 the statistical significance in survival difference in favour of CHART was the largest in T--4 tu­mours (p = 0.007), less pronounced if T-3 and T--4 tumours were taken together (p = 0.018) and was nonsignificant when T-2, T-3 and T--4 tumours were compared together (p = 0.097). This supports the belief that in early tumours the repopulation is accelerated well before the end of the CHART scheme. Conclusions The repopulation rate of clonogenic cells increa­ses during the course of radiotherapy. Using Saunders' data we calculated that the clonogenic cells begin to accelerate their division rate at the end of the second week of the treatment in T-3 and T--4 tumours and probably earlier in T-1 and T-2 tumours. References 1. Trott K-R, Kummermehr I. What is known about tumour proliferation rates to choose between acce­lerated fractionation or hyperfractionation? Ra­diother Oncol 1985; 3:1-9. 2. Steel GG. Celi proliferation kinetics in tumours. In: Steel GG, Adams GE, Horwich A eds. The biological basis of radiotherapy. e. ed. Amster­dam-New York-Oxford: Elsevier, 1989; 77-88. 3. Nias AHW. An introduction to radiobiology. Chi­chester: John Wiley & Sons, 1990; 29-45. 4. McNally NJ. Can celi kinetic parameters predict the response of tumours to radiotherapy? lnt J Radiat Biol 1989; 56:777-86. 5. Fowler JF. Fractionation and therapeutic gain. In: Steel GG, Adams GE, Horwich A. The biological basis of radiotherapy. 2. ed. Amster­dam-New York-Oxford: Elsevier, 1989; 181-207. 6. Withers HR, Taylor JMG, Maciejewski B. The hazard of accelerated tumour clonogen repopula­tion during radiotherapy. Acta Oncol 1989; 27:131-46. 7. Withers HR, Thames HD. Dose fractionation and volume effects in normal tissues and tumours. Am J Ciin Onco/ 1989; 11:313-29. 8. Budihna M, Škrk J, Šmid L, Furlan L: Tumor celi repopulation in the rest interval of split-course radiation treatment. Strahlentherapie Onkol 1980; 156:402-8. 9. Galante E, Gallus G, Chiesa F, Bono A, Bettoni I, Molinari R. Growth rate of head and neck tumours. Eur J Cancer Ciin Oncol 1982; 18:707­12. 10. Maciejewski B, Preuss-Bayer G, Trott K-R. The influence of the numer of fractions and of overal treatment tirne on local control and late complica­tion rate in squamous celi carcinoma of the larynx. lnt J Radiat Oncol Biol Phys 1983; 9:321-8. 11. Budihna M, Šmid L. Deljeno (split-course) obse­vanje karcinoma epifarinksa. Radio/ lugosl 20:4, 391-398, 1986. 12. Overgaard J, Hjelm-Hansen M, Johansen LV, Andersen AP. Comparison of conventional and split-course radiotherapy as primary treatment in carcinoma of the larynx. Acta Oncol 1988; 27:147­52. 13. Saunders MI, Dische S, Hong A et al. Continuous hyperfractionated accelerated radiotherapy in lo­cally advanced carcinoma of the head and neck region. lnt J Radiat Oncol Biol Phys 1989; 17:1287­93. 14. Trott K-R. Celi repopulation and overall treatment tirne. Jnt J Radiat Oncol Biol Rhys 1990; 19:1071­75. 15. Saunders MI. (personal communications) 1991. Adv Radio! Oncol 1992; 241-8. Dipyridamole enhances the cytotoxicity of drugs and radiation in HeLa and V79 ceHs in vitro Ban J, Soric J, Bistrovic M, Dujmovic I, Miletic Z Central Institute for Tumors and Allied Diseases, Zagreb Laboratory for Experimental Cancerology and Department of Radiotherapy Dipyridamole (DP) was utilized as the nucleoside transport inhibitor in combination with anti-cancer drugs which directly damaged DNA. It was also used in combination with radiation. Cytotoxic action of DP alone on HeLa cells was concentration and tirne dependent. DP, on incubation far 10 days, killed human tumor HeLa cells with an IC5rF 15 µM measured by clonogenic assay. In combination with doxorubicin or epirubicin, DP significantly enhanced their killing action on HeLa cells. Synergistic cel! kil! was also obtained with bleomycin or BCNU, producing a 2.2 ar 2.6-fold reduction in 10% inhibitory concentration at a DP concentration of 15 µM, respectively. Combination of mustargen or dacarbazine plus DP gave only an additive cytotoxic effect. DP intensively influenced the killing action of radiation. Using isobologram method, it was demostrated that the interaction was truly synergistic. This combination was also slightly synergistic in normal V79 cells. These cells are less sensitive to both DP and irradiation than tumor cells. DP killed V79 cells with IC5rF 23 µM after 5 days of incubation. DP together with particular anticancer drugs on V79 cells was tested far comparison. The magnitude of cytotoxic effects on normal cells was examined. Key words: HeLa cells-drug effects; V79 cells-drug effects; dipyridamole, radiation Introduction Dypyridamole (DP) is used in this study on the basis of in vitro and in vivo studies which Abbreviations: DP, dipyridamole; DX, doxorubicin; EPI, epidoxorubicin; BCNU, carmustine; DTIC, Da­carbazine; MS, Mustargen; BLEO, Bleomycin; PBS, phosphate buffered saline; FCS, fetal bovine serum; MEM, minimal essential medium; cAMP, cyclic ade­nosine monophosphate; Do, mean lethal ----'X u ' 10 O 8 16 24 48 if-jCUBATION (hr) Figure 2. The test for reversibility of DP effect on celi growth of chinese hamster V79 cells. 100 JLM DP was added to celi cultures (lxl05 cells/Petri dish) for 2 (O); 4 (o); 6 (L) and 12 (X) hr, followed by washing of cells and incubation with drug-free medium. At indicated intervals the celi number was determined. Untreated control ( e ). Points are mean values for triplicate assays. 244 Bani et al. or 72 hr, respectively. The degree of •the celi growth inhibition was concentration and time-de­ pendent. We also examined the reversibility of effects caused by DP. Por this purpose V79 cells were incubated with 100 µM of DP for 2, 4, 6 and 12 hr as indicated in Figure 2. After treatment DP was washed out and cells were grown in medium up to 48 hr. Cells were counted after varying tirne intervals. Proliferatiog V79 cells grew in culture with a doubling tirne of about 15 hr. Exposure of cells to DP resulted in a dose dependent decline of celi proliferation. Cells treated with DP for 2 and 4 hr were not dividing for the first 24 hr but after that the celi division became of the same rate as in untreated control. The presence of DP for 6 or 12 hr inhibited the celi growth for the next 12 hr but further incubation without the drug increased the doubling tirne to approxima­ tely 18 or 24 hr, respectively. We concluded that the action of DP on celi growth was reversible. Action of DP on cel! survival In order to examine the killing action of DP on normal and tumor cell lines we tested the cytoto­xicity of the drug on celi survival. Thus, the proliferating V79 cells were exposed to increa­sing concentrations of DP for 5 days to form colonies. The survival of treated cells was evalua­ted by colony counts. The survival curve was of the threshold-exponential type with IC5o= 23 µM (Figure 3). Minimum effective dose was lµM, D0 =8 and Dq20 µM. Por comparison human = tumor HeLa cells were also treated with different concentrations of DP for 10 days. The threshold­exponential type of dose-response survival curve with IC5o=15 µM, D03 µM and Dq=15 µM is = shown in Figure 3. The .curves show that HeLa cells were 1.5-fold more sensitive as compared to the killing action of DP on normal V79 cells. Stability of DP in different solutions It was relevant to examine the stability of DP under experimental conditions because of the duration of some experiments. Por this rea­son the drug was tested for the stability of its activity in different solutions. The drug (20 and o DP (µM) Figure 3. Survival curves of proliferating chinese hamster V79 (o) and human cervix carcinoma HeLa cells (O) exposed to increasing concentrations of DP. Cells were seeded (500/Petri dish) and supplemented with the drug for 5 (V79) or 10 (HeLa) days. Colonies were counted and the counts expressed as % of colonies of untreated cells. Each point is the average of triplicate samples of 3 experiments. 50 µM) was dissolved and preincubated at 37° C in complete medium containing FCS and in PBS (in the absence of FCS) for various periods of tirne; it was then added to proliferating V79 cells Dipyridamole enhances the cytotoxicity of drugs and radiation in HeLa and V79 cel/s and after 5 days of incubation the percent survi­val was measured by colony counts. Surviving fractions of cells treated more than 4 hr with preincubated DP in both solutions were about 60% higher than those of cells treated immedia­tely (Table 1). There were no differences bet­ween the survivals of cells treated with DP preincubated in complete medium or in buffer solution. DP !ost some of its killing activity with the duration preincubation tirne. The above results do not allow to conclude that drug has been bound to serum proteins in medium. Our in vitro data do not contradict the data obtained in vivo. D binds to the specific human serum protein, a1-acid glycoprotein23, which can signifi­cantly influence the inhibition of adenosine up­ . I0,23 take in vivo. s.9 Influence of DP on the cytotoxicity of various anticancer drugs In order to test the hypothesis that DP enhan­ces the cytotoxicity of drugs which directly da­mage DNA, proliferating HeLa cells were expo­sed to specific drug combination. Cells were treated with incresing concentrations of tested antitumor agents alone or in combination with DP added in its IC50 value (15µM). Cells were then incubated for 10 days to form colonies. To test the influence of intercalating agents, cells were exposed to various concentrations of DX or EPI. Results are shown in Figure 4. Threshold exponential type of dose-response survival cur­ves were obtained with DX or EPI, with D0 = 0.014 or 0.009 µM and D9=0.0l or 0.022 µM, respectively. DP significantly enhanced the kil­ling ability of both anthracyclines. Observed dose response curves also are of a threshold exponential type with D0=0.0l8 or 0.007 µM and D=0.004 or 0.006 µM for DX or EPI plus DP, respectively. The predicted dose-response curves are identically shaped as those of DX or EPI but shifted downwards for the amount corresponding to the DP toxicity. This shift indicates the multi­plication only. The translated dose-response curve represents the leve! of expected survival if no mutual potentiation of drugs occurred. The predicted survivals and possible synergism were 100. _ O DP(15JJM) v. DP(15JJM) -o . o-o-o ...-o '"){, \'.\EPI \\\ ° ,o 10-j \ '\\ ''° ' \ Pcedkted • EPl+DP j'-t- Predicted DX+DP • ,_J > O:: ;,e /\ EPI+ DP • 0.1 , , , 0.01 002 0.03 00. O 0.01 0.02 0.03 . DX(µM) EPl{µM) Figure 4. Survival curves of proliferating human tumor HeLa cells exposed to increasing concentrations of DXor EPI and a fixed dose of DP (15 µ,M), usedseparately or in combination. Cells were treated for 10days, colonies were counted and results calculated as inFigure 3. Each point is the average of triplicate samples. The result is from a representative experi­ment. Predicted survivals = (mean % of drug survival)x (mean % of DP survival)/100. Observed survivals areall significantly lower than predicted (p < 0.001). calculated according to Webb. 24 Mean survival values by corresponding doses of the single agents were used for calculating predicted survi­val. Ali experimentally obtained survivals were significantly lower. Similar dose-response survival curves of nor­mal V79 cells exposed to various concentrations of DP alone or with added DX or EPI were obtained. The isoboles were constructed22 • , and those for 10% survivals showed a concave up shape i.e. DP enhanced the sensitivity of 246 Ban] etal. tumor and normal cells toward DX or EPI (Figure 5). DP also enhanced the sensitivity of tumor or normal cells to BLEO producing a 2.2 or 2.5-fold reduction in 10% inhibitory concentrations, re­spectively. This combination could be of interest for in vivo experiments because HeLa cells were more sensitive to DP and nearly 250 times more sensitive to BLEO than normal cells (Table 2). HeLa cells were also treated with some other antitumor drugs as indicated in Table 2. DP enhanced the sensitivity of HeLa cells to BCNU producing a 2.6-fold reduction in 10% inhibitory concentration. Synergistic cell kili was observed only when higher concentrations of BCNU were combined with DP, indicating that this combina­tion was concentration dependent. This interaction could be of interest for in vivo treatment. Namely, highly synergistic inte­raction could be expected in the peritoneal cavi­ty, but much lesser interaction in the plasma where drug concentrations are much lower. 23 V79 cells were more sensitive to BCNU and 3.0-fold reduction in IC 10 value was obtained (Table 2). This fact has to be taken into account for in vivo experiments. Mustargen or DTIC applied in combination with DP gave only summation in HeLa cells but synergistic interaction was observed in normal cell population (Table 2). These data suggest that above combinations could not be recom­mended for ,clinical use since normal cells were killed at a relatively higher rate. The effect of DP on radiation damage DP affects the killing ability of radiation. HeLa cells were first irradiated with increasing doses of radiation and immediately after-wards treated with various concentrations of DP for 10 days. The results of interaction (10% survivals) are shown in Figure 6. A concave-up isobole was obtained indicating a slight synergism. For the comparison, V79 were also treated with the combination of DP and radiation. The result was similar: a slight synergism. Data not presented here also showed that DP combined with higher 0.2-11 1 1 1 1 1 1 1 -1 2 =i.. f,' 1 C) => 0.1 • EPI ,,­ 0::: \1 o 1 ., ,o •----· o--=====• o.o 1 i i 1 -, O 10 20 30 -40 DP(JJM) Figure S. Combined cytotoxic effect of EPI or DX plus DP on V79 cells. Isoboles are based on the mor­phological studies for planning of treatment in a variety of human solid tumours in vivo. 8•9• 10 In the present on-going study on the use of FC DNA measurements for individualisation of chemothe­rapy we have enrolled 63 patients with chemore­sistant tumours who failed standard treatment. 15 The detailed results will be reported elsewhere. The results of these methods in a case of chemo­resistant metastatic renal celi carcinoma will be presented to illustrate the clinical application of FC DNA measurements and cytomorphological studies for planning chemotherapy. Rena! celi carcinoma responds poorly to CHT. The response rate to single agents or combined CHT are mostly under 20%. 17· 18· 19 A combina­tion of interferon (IFN) and vinblastine (VLB) improved the response rate according to some reports20 but this issue is stili controversial. 17 Material and methods A 68-year old male patient with metastatic renal celi carcinoma entered the study on individuali­sed CHT in September 1989. The patient under­went three surgical interventions for liposarcoma in the left gluteal region from 1985-87. In De­cember 1988 a tumour was discovered in his left kidney by ultrasound (US). Fine needle aspira­tion biopsy (FNAB) of the tumour showed a renal celi carcinoma. A left nephrectomy and removal of regional lymph nodes was performed. On histology there was moderately differentiated renal celi carcinoma with metastatic involvement of the lymph nodes in renal hilus and of the paraaortic lymph nodes. Because of lymph node involvement postoperative irradiation was deli­vered to the region of the left kidney and involved lymph nodes (180 cGy per day, TD 4500 cGy, linear accelerator) concomitantly, im­munotherapy with IFN 6 x 106/day x 8 in 3-week intervals was applied after surgery. Five months after nephrectomy during immunotherapy the tumour relapsed in the supraclavicular lymph nodes and was treated with concomitant chemo­immunotherapy (VLB 7 mg in bolus and IFN 6 x 66/day x 8 in 3-week intervals) and irradiation (200 cGy/day; TD 3000 cGy). This treatment resulted in stabilisation of the disease for 3 months. In August 1989 the tumour escaped: progression of supraclavicular metastasis, appea­rance of new metastases in the mediastinal lymph nodes and in a paraaortic lymph node were diagnosed. As the mediastinal tumour caused respiratory distress this tumour site was treated by irradiation in combination with low doses of adriamycin (Adria 40 mg and 20 mg) whereas the supraclavicular metastasis was used for te­sting the efficacy of adriamycin. For evaluating the effect ot Adria, FNAB of supraclavicular metastasis were performed before and at uneven intervals after Adria 40 mg and Adria 20 mg. The FNAB specimens were Giemsa stained for morphological studies. FNAB samples were ta­ken for FC DNA and protein measurements as well. Specimens were fixed in 75% ethanol and 'stored at -20°C until measuring. Fixed specimens were centrifuged at 1500 rpm for 5 min. One ml of 0.5% Pepsin (Serva 31855, Heidelberg) in HCI (1 mol/L) were added to one drop of_pellet and . shaken gently for 5 minutes. Eight ml of a mixture of fluorochromes DAPI (Serva No. 18860) and SR 101 (Serva No. 3598-Heidelberg) were added. After 2 hrs of staining the specimen was filtered through a nylon mesh (50 microns). The measurements of DNA and proteins were carried out on a PAS II (Partec Muenster) cytophotometer. For the excitation of both fluo­rochromes a mercury high pressure lamp HBO­100 (Osram, Muenchen) and UGl excitation filter (Schott, Meinz) were used. For the fluore­scence of DAPI a GG 455 barrier filter and for SR-100 fluorescence a RG 590 barrier filter were Auersperg Metal. used. Up to 50.000 cells were measured in Si,imples. The CV of samples before chemothe­rapy were below 5%. Results Clinical results Treatment with the combination of irradiation (3000 cGy) and Adria ( 40 mg and 20 mg in 14-day interval) resulted in a stabilisation of mediastinal tumour and a minor response in supraclavicular tumour with the relief of clinical symptoms. Because of poor condition of the patient who had repeated urinary and respiratory infections, poor nutritional status, elevated crea­tinine and urea, CHT could be repeated only 5 weeks later. At this tirne the patient had perito­neal carcinosis with ascites, minor progression of supraclavicular metastasis and new metastases in the axillary lymph nodes measuring 5 and 3 cm respectively. FNAB of supraclavicular and axil­lary lymph nodes were performed and the effi­cacy of the infusion of 2 mg of VLB over 12 hrs was tested by FNAB 12 and 36 hrs after the termination of infusion. On the basis of FC dual parameter analysis of DNA and proteins and cytomorphological studies of specimens after a previous Adria application, and testing the effi­cacy of VLB by the same methods, a combina­tion of infusions of VLB and Adria was planned with individual intervals according to FCM mea­surements (Figure 1). Eight days after this CHT a painful enlargement of supraclavicular metasta­sis occurred. FNAB demonstrated a complete necrosis of tumour cells in the supraclavicular metastasis and more than 50% of damaged cells in the axillary lymph node. Clinically, supraclavi­cular and one of the axillary metastases regressed completely, whereas the other axillary metasta­ses showed a partial response. The disease in the abdomen was stabilised. After 5 weeks the di­sease progressed in the lungs and abdomen whereas the response in the axilla and supraclavi­cular region stili persisted. A second cycle of individualised CHT was started with VLB in December 1989, but the patient refused further VlB 2mg in!erval 1.ADRIA 20mg inter1al .?.AORiA 40m, r 12b 36h 2h 70h l 4b l Figure l. Chemotherapeutic schedule for a metastatic renal celi carcinoma planned individually on the basis of FC dual parameter analysis of DNA and protein and cytomorphological studies. treatment, was discharged from the hospital and died in February 1990 with progressive disease in the abdomen and lungs. An autopsy was not performed. Cytomorphological and flow-cytophotometrical results Cytomorphological examination of FNAB smears after Adria 40 mg showed no major changes up to 90 hrs after Adria application. Only at 90 hrs after Adria enlargement of tumour celi nuclei and degenerative changes in tumour cells became apparent. In the specimen taken 12 days after Adria 40 mg the changes of tumour cells were more expressed than in samples 90 hrs after Adria. A second dose of Adria 20 mg was applied 14 days after the 1st dose of Adria 40 mg. Cytomorphology showed enlargement of cells and degenerative changes on a part of tumour celi population. These changes appeared as early as 24 hrs after Adria 20 mg. Morphologi­cal changes appeared earlier and were more expressed than after the first application of Adria, despite the lower 2nd dose of Adria. Cytomorphological examination of FNAB sam­ples showed that Adria was effective in this particular tumour. After VLB 2 mg in a 12-hour infusion morphological changes in tumour cells were detected already 12 hrs after the termina­tion of infusion and were more expressed in specimen taken 36 hrs after VLB. Dissociation of tumour cells, nuclear enlargement, multinu­cleated cells, micronuclei and mitoses were de­monstrated (Figure 3). Samples taken 72 hrs after CHT with VLB and Adria in combination showed degenerative changes in nuclei, enlarge­ Role of DNA flow cytometry and cytomorpho/ogy in the search of effective chemotherapy * Figure 2. FNAB celi sample of a metastatic renal celi carcinoma -supraclavicular lymph node metastasis bcfore CHT. Gicmsa, orig. magn. 200x. Figure 3. FNAB of the same tumour site 12 hrs after the termination of VLB infusion (2 mg over 12 hrs). Giemsa, orig. magn. 200x. Enlarged nuclei can bc scen. Figure 5. FNAB of the same tumour 8 days after CHT. Giemsa, orig. magn 200x. Complete disintcgration of tumour cclls; macrophagcs are presen t. ment or pyknosis, many cells were in mitoses. Eight days after chemotherapy a complete necro­sis of the tumour was demonstrated by cytology (Figure 5). The sequence of morphological chan­ges in FNAB samples before and after CHT are demonstrated in Figure 2-5. Flow-cytophotometric measurement of DNA and protein FC measurement before CHT showed a hypodi­ploid tumour (Figure 6A); 12 hrs after VLB a slight increase in protein content reflecting in­creasing celi volume was seen. As a result of celi disintegration debris appears in the histograms. A slight increase in the S celi compartment is also demonstrated (Figure 6B). At 36 hrs after VLB the amount of debris and S cells increased (Figure 6C). The DNA and protein distribution pattern remain unchanged 69 hrs after the 1 st Adria in the CHT schedule (Figure 6D) whereas 72 hrs after the completion of CHT with VLB and Adria 1 + Adria 2 the amount of protein was increased as well as the S phase compartment and debris (Figure 6E). Eight days after CHT only debris was demonstrated in the histogram (Figure 6F). Auersperg Metal. DNA content B Go/G, TU }Hl:' .!!! ,; " o o z p. FlM 1:.. ""' ,,,. JJ2'8 JU ,; .. IJ . .,, 1 ... 1;,,. h, 1, .. };. m !Ji' LM ,Jl1i{i.:, ::<. !._.,..;;;,':: ii,tli i:\f: " Jff. --·:1:1i E o ·====:.::=:-_--.:====::=:­ D E F ""' -•'·") J'l. .•·" Go/G, TU 111t:1 )IM .!!! ,; _ ;l ':'(-ti ]'.-f.l ,... _f'X' ,,_IJ 0 lf.I) o 1 -t<>f\ _17n .•t ... 11,., 1i>1 f;:.i­ 11-n <...!'"' 2.. 1_?2' 1_?7 c c " .·;·: \//;J:!;2.·:: 10:' . !]'i' ·;;; '2 o .'.' ' \j,:i;;·\ 1,·.< .. a: C ,,.--.1....;.1-· ... -.:: . . . .\:.•:·.. -i ; :, . i.; -· ._ ._ ,f:"..11',/i:: ­ J_? " [: .. ----------­ DNA conlcnl Figure 6A-F. DNA and protein dual parameter FC measurement of renal celi carcinoma metastas1s m the supraclavicular lymph node. DNA measurements are shown in the upper part of Figs. A-F. Underneath a scattergram representation of DNA vs. protein analysis is illustrated. DNA is displayed on the horizontal and protein on the vertical axis. GJG 1 peak of the tumour cells is marked with Tu, the corresponding peak of normal cells with N (Fig. 6A), the S phase compartment is marked with arrows (Fig. 6E): A) DNA and protein distribution before CHT showing a hypodiploid tumour with a rather high protein content. The 1st peak represents GJG1 of the tumour, the 2nd peak GJG1 of normal cells, the 3 rd peak G2 + M of the tumour cells. B) The same tumour site 12 hrs after VLB infusion 2 mg/12 hrs -a slight increase in protein content, S phase compartment and debris. C) 36 hrs after VLB -a large amount of debris (left to the 1st peak -increase in S phase compartment. D) 69 hrs after VLB and l. Adria. The DNA and protein distribution unchanged as compared to C; E) 72 hrs after completed CHT -increase in S phase compartment and protein content. F) 8 days after CHT only debris is demonstrated. Role of DNA flow cytometry and cytomorphology in the search of effective chemotherapy Discussion For planning effective CHT the knowledge· of celi kinetic changes of tumours evoked by single chemotherapeutic drugs or their combinations would be very important. There have been ex­tensive studies on the effect of CHT drugs performed on experimental tumours as well as in human tumour celi lines in vitro and in ani­ 22• 23, 24• 25• 26 mals. 2 1, In contrast, there is little knowledge on celi cycle perturbation as a result of treatment in human solid tumours in vivo. Dyson11 used FC DNA measurements for stu­dying the radioresponsiveness of cervical carci­noma and found that aneuploid tumours were more radiosensitive than diploid tumours. Fiet­kau studied celi kinetic changes in head and neck tumours during irradiation and found an increase in S and G2 + M compartment after 10 and 20 Gy. 12 Recently, Hemmer used in vitro Bromo­deoxyuridine (BUdR) labelling method and FC DNA measurements in surgical biopsies of 6 patients treated with intraarterial cisplatinum and epirubicin for oral carcinomas for the evalua­ 14 tion of the response to CHT. 13• In effective CHT elimination of aneuploid tumour clones was demonstrated by DNA measurements and an interruption of celi cycle progression by a lack of BUdR incorporation into cellular DNA. 13• 14 Lack of reports on monitoring treatment could be attributed to the ethical problems in taking repeated surgical biopsies of tumours for FC DNA measurements. Modem development of techniques in FC enabled reliable and reproduci­ble measurements of DNA on FNAB obtained samples. FNAB is much less traumatic than surgical biopsy and causes minimal discomfort to the patient. The danger of sampling different tumour populations by FNAB can be minimised by regular comparison of DNA measurements and cytomorphological pictures of the sameles. In our previous studies we successfully used single-cell DNA measurements of FNAB sam­ples for monitoring and planning CHT in various solid tumours. 8• 9• 10 The gain of such a study for the benefit of a particular patient to our opinion greatly outweighs the minimal discomfort of FNAB. According to our experience DNA mea­surements combined with cytomorphological stu­dies of FNAB samples have great individual predictability for the outcome of treatment in a particular patient. As FC DNA measurements are much faster than single-cell measurements the monitoring of CHT will be applicable to a greater number of patients. Since 1988 we stu­died the effect od CHT in 63 patients with various solid tumours. This study suggests that a block of cells in S phase or elimination of aneuploid cells predicts a good effect of CHT. 15 More data have to be collected before any fina! conclusions can be drawn. Renal cell carcinomas are particularly CHT 18• 19 resistant. This was proved in patients17• as well as by in vitro experiments. 28 The patient presented in this report had an aggressive tumour which progressed during immunotherapy with IFN as well as during chemo-immunotherapy with VLB and IFN. Nevertheless, a complete regression of the supraclavicular metastasis and a partial regression of axillary lymph node meta­stasis was achieved with a combination of infu­sions of VLB and Adria which were planned according to DNA measurements and cytomorp­hological findings. Approximately three times lower doses of VLB and two times lower dosis of Adria than applied in standard schedules were used in this patient in individually planned CHT (according to Figure 1). We assume that the dramatic response in the supraclavicular metasta­sis was obtained because of the appropriate timing of VLB and Adria infusions. Adria is particularly effective for cells in S, G2 + M phases. Therefore, two doses of Adria during the tirne of increased number of cells in S phase compartment (detected by FC DNA measure­ment) resulted in a complete necrosis of the tumour. Despite very efficient chemotherapeutic schedule, this particular patient did not gain much of CHT. Because of his poor general condition, nutritional status, repeated urinary and pulmonary infections the interval between individually planned CHT was too long (7 in­stead of 2-3 weeks). During that interval a repopulation in the tumour must have occurred which resulted in the ultimate treatment failure. Auersperg Met a/. More experience is needed in the interpreta­tion of FC DNA and protein measurements during CHT. Our preliminary experience indica­tes that a combination of DNA measurements and cytomorphological studies can represent an important tool in individually tailored treatment strategies adapted to the individual cytokinetic characteristics of a particular tumour. References l. Gray JW, Dolbeare F, Pallavicini MG, Beisker W, Waldman F. Celi cyc!e analysis using flow cytometry. Int J Radiat Bio/ 1986;49:237-55. 2. Williams NN, Daly JM. Flow cytometry and prognostic implications in patients with solid tu­mours. Surg Gynec Obstet 1990;171:257-66. 3. Beerman H, Kluin PM, Hermans J, van de Velde VCJ, Cornelisse CJ. Prognostic significance of DNA ploidy in a series of 690 primary breast cancer patients. Int J Cancer 1985;45:34-9. 4. Ferno M, Baldetorp B, Akerman M. Flow cyto­metric DNA ploidy analysis of soft tissue sarco­mas. A compatative study of preoperative fine needle aspirates and postoperative fresh tissue and archival material. Ana/ Quant Cytol Histol 1990;12:251-8. 5. Alvegard TA, Berg NO, Baldetorp Bet al. Cellu­lar DNA content and prognosis •Of high-grade soft-tissue sarcoma. The Scandinavian sarcoma group experience. J Ciin Onco/ 1990;8:538-47; 6. Grignon DJ, Ayala AG, El-Naggar A et al. Rena! c::ell carcinoma: a clinicopathologic and DNA flow cytometric analysis of 103 cases. Cancer 1989;.:2133-40. 7. Ljungberg B, Stenling R, Ross G. DNA content and prognosis in renal celi carcinoma: a compari­son between primary tumours and metastases. Cancer 19,86;57:2346-50. 8. Auersperg M, Porenta O, Us-Krašovec M,. Oblak M, Furlan L. Cytophotometric DNA studies in human head and neck tumours after cis-platinum infusion. 13th International congress of chemothe­rapy, Vienna 1983. Proceedings:280/10. 9. Auersperg M, Zorc R, Us-Krašovec M et al. DNA measurements used for planning of multi­modal · treatment in sarcomas. 14th international cancer congress, Budapest 1989. Abstracts 2:637. 10. Auersperg M, Zorc R, Us-Krašovec M, Pogacnik A, Petric G. Porenta-Vraspir O. Chemotherapy · 'for Huerthle celi' carcinoma based on sequential DNA measurements. Radio/ !ugasi 1988;22:269­75. 11. Dyson JED, Joslin CAF, Rothwell RI, Quirke P, Khoury GG, Bird CC. Flow cytofluorometric evi­dence for the differential radioresponsiveness of aneuploid and diploid cervix tumours. Radiother Onco/ 1987;8:263-72. 12. Fietkau R, Langer E, Iro H et al. Impulszytopho­tometrische Messungen vor und wahrend der Strahlentherapie bei Kopf-Hals-Tumoren. Strah­lenther Oncol 1989;165:34-42. 13. Hemmer J. Rapid in vitro bromodeoxyuridine labeling method for monitoring of therapy· re­sponse in solid human tumours. Cytometry 1990;11:603-9. 14. Hemmer J. Cellular proliferation of oral carcino­mas during chemotherapy assessed by DNA flow cytometry and bro)nodeoxyuridine labeling. Tu­mourdiagn Ther 1991 ;12:16-20. 15. Auersperg M, Us-Krašovec M. Goehde W et al. DNA and protein flow cytometry in human solid tumours used for planning chemotherapy. 13th European congress of pathology, Ljubljana 1991. Abstracts. Pathol Res Pract 1991 ;187:651. 16. Auersperg M, Us-Krašovec M, Pogacnik A, Pohar Ž, Goehde W, Jaffe N. DNA flow cytometry and cytomorphology for evaluation of chemotherapy. 15th International cancer congress Hamburg 1990. Poster Abstracts. J Cancer Res Ciin Oncol 1990;116.478: Supl. 17. Figlin RA, deKernion JB, Maldazys J, Sama G. Treatment of renal celi carcinoma with alpha (human leucocyte) interferon and vinblastine in combination: a phase I-II tria!. Cancer Treat Rep 1985;69:263-7. 18. Dexeus FH, Logothetis CJ, Sella A, Finn L. Interferon alternating with chemotherapy for pa­tients with metastatic renal celi carcinoma. Am J Ciin Oncol 1989;12:350-4. 19. Kish JA, Ensley JF, Al-Sarraf M. Phase II evalua­tion of 4'deoxydoxorubicin in advanced renal celi carcinoma. Am J Ciin Onco/ 1990;13:17-8. 20. Bergerat JP, Ford J, Herbrecht Ret al. Recombi­nant alphainterferon plus vinblastine in metastatic renal-cell cancer: analysis of response and survival in 58 evaluable patients. Prog Ciin Biol Res 1988;348:137-50. 21. Lanks KW, Lehman JM. DNA synthesis by L929 cells following doxorubicin exposure. Cancer Res 1990;50:4776-8. 22. Frankfurt OS, Seckinger D, Sugarbaker EV. Flow cytometric analysis of DNA damage and repair in the cells resistant to alkylating agents. Cancer Res 1990;50:4453-7. 23. Severin E, Hagenhoff B. Die Synchronisation von Tumorzellen mit 5-Fluorouracil plus Uradi und mit vinblastin und die Bestrahlung synchronisierter Kulturen. Strahlenther Onkol 1988 ;164:165-72. 24. Bara! E, Auer G. In vitro effect of doxorubicin on nonproliferating and proliferating epihtelial cells. Int J Radia/ Onco/ Biol Phys 1990;19:963-5. 25. Wagner W. DNA index -parameter for the prediction of prognosis and primary radioresistan­ce. J Cancer Res Ciin Onco/ 1990;116:315-7. 26. Jacke! M, Kopf-Maier P. Influence of cisplatin on cell-{;ycle progression in xenografted human head and neck carcinomas. Cancer Chemother Pharma­col 1991 ;27 :464-71. 27. Gohji K, Maeda S, Sugiyama T, Tshigami J, Kamidono S. Enhanced inhibition of anticancer drugs by recomibant gammainterferon for human renal celi carcinoma in vitro. J Uro/ 1987;137:539­43. 28. Bazeed MA, Scharfe T, Recht E, Schmidt J, Jacobi GH, Thuroff JW. In vitro chemosensitivity testing of renal celi cancer. Short-term culture technique. Urology 1988;31:240-4. Adv Radio/ Oncol 1992; 275-9. Flow-cytometric DNA ploidy and clinicopathologic variables in primary breast carcinoma Us-Krašovec M1 , Bracko M1 , Cufer T1 , Goehde w2 , Košmelj K3 , Lamovec J1 , Po2acnik A1 1 The Institute of Oncology, Ljubljana, 2/nstitute of Radiobiology, University Muenster, Germany, 3 Biotechnical Faculty, University Ljubljana DNA ploidy was investigated by flow cytometry in 108 primary invasive breast carcinomas. Aneuploidy was found in 74% of the tumours. Whereas DNA ploidy was not associated with age, menopausal status, clinical T stage, lymph-node involvement or ER status, significant correlation was found between DNA ploidy and PR status (P=0.03), tumour size (P=0.03), histologic type (P=0.003) and histologic grade (P=0.000001). Key words: breast neoplasm-pathology; flow cytometry; ploidy Introduction The prognosis of breast cancer patients is deter­mined by clinical TNM staging, histologic subty­ping and histologic malignancy grading. The axillary lymph-node status is considered the single most important prognostic factor. In spite of these prognostic indicators, the clinical course of breast cancer remains variable and unpredicta­ble. Approximately 30% of patients having nega­tive axillary lymph nodes, i.e. good prognosis, will develop metastases and will not survive 10 1 years. Current knowledge regarding the biology and epidemiology of breast cancer indicates that the disease is not a homogeneous entity. Besides, both clinical staging and histologic grading sy- This work was supported by the Slovenian Research Council under contract URP No. 16/B Correspondence to: prof. dr. Marija Us-Krašovec. The Institute of Oncology, Zaloška 2, Ljubljana, Slovenia. UDC: 616.19-006.6-091.83 stems are subjective and have low inter-and intra-observer reproducibility. 2• 3 Therefore, ad­ditional and more objective prognostic criteria are required for identification of patients who are at higher risk for tumour recurrence, espe­cially among lymph-node negative patients. Flow cytometry (FCM) and celi image analysis (IA), two quantitative analytical methods· deve­loped in the past decades, enable a rapid and objective measurement and quantification of 5 cells and cellular constituents. 4• Investigators, having two new instruments and following the conceptual idea that the main characteristic of malignant tissue is abnormal genome, have focu­sed their interest on deoxyribonucleic acid (DNA) content of malignant tumours. DNA is one of the celi constituents which nas been currently most extensively studied by FCM and IA as a potential prognostic parameter. During recent years a substantial number of papers reporting results of DNA analysis of breast cancer have been published. Both retro­spective and prospective studies have provided rather conflicting data regarding the value of Us-Krašovec Metal. DNA analysis for determining the prognosis of women with invasive breast cancer. 6 Material and methods The present study was based on 108 cases of breast cancer selected at random from patients who underwent mastectomy or lumpectomy with axillary node dissection at the Institute of Onco­logy, Ljubljana, in 1989 or 1990. Cases of nonin­vasive or minimally invasive carcinoma were excluded from the study, as were patients with recurrent tumors and those who were given preoperative radiotherapy or chemotherapy. The information on age, menopausal status, clinical TNM stage, tumour size, nodal status, estrogen receptor (ER) and progesteron receptor (PR) status were retrieved from the patients' records. The original histologic tumor material was revie­wed independently by two of the authors. Classi­fication and grading was performed according to a modification of the Bloom and Richardson grading system. 7 For FCM analysis, tissue slices of approxima­tely 50 .m 3 were taken from fresh unfixed surgical specimens and stored at -20° C. Before the measurement, the tissue was thawed, minced with scalpel and treated in 2 ml of 0.5% pepsin solution (Serva, Heidelberg) at pH 1.8 with light agitation at room temperature for 5 to 10 minu­tes. The celi suspension was filtered through a 50 nm nylon mesh and stained with a combination of 4,6-diamidino-2-phenylindole (DAPI, Serva, Heidelberg) and sulforhodamine (SR 101, Serva, Heidelberg). DNA content was measured on a PAS·II flow cytometer (Partec, Muenster) equip­ped with a 100. W high pressure mercury lamp, and analysed using a Partec software package. On average more than 10 000 cells were measu­red from each tumor celi suspension. The mean coefficient of variation was 4.37 ± 1.66 (range, 2.28-9.53). Tumours showing only one Goi peak were classified as diploid and those with one or more additional Goi peaks as aneuploid (Figure 1). In the · Jatter, the leftmost peak was considered. to represent the nonneoplastic diploid population. Tumour ploidy was expressed by DNA index (DI) representing the ratio between the moda! chan­nel number of the tumour .Goi peak and the diploid G01 peak. A diploid tumor has thus, by definition, a DI of . .O. Statistical evaluation was based on the analysis of contingency tables using Chi-square test. Go1IO) Go1(D) rj 1440 „tZCI 1<00 _!101.'l J280 §.O 112e ,60 .JU' _ _:(80 _!OO .l)J) . .S·IO ]20 G01{A) ..AO }.O .JE-O ·' II.S -lllt!:i.z -11.1lUI' ONA contont Figure l. DNA histograms of (A) a diploid tumour (DI= 1) and (B) an aneuploid tumour (DI= 1.44). Results Clinico-pathologic variables The mean age of patients included in this study was 54.4 years, 62 (57%) of them were older than 50 years. Sixty (56%) patients were postme­nopausal. CJinical estimation of tumour size was as fol­lows: Ti in 10 patients, T2 in 65 patients, T3 in 23 and T 4 in 10 patients. Steroid receptor analysis revealed positive ER in 54 (50%) patients and positive PR in 47 (44%) patients. On gross pathologic examination, only one tumour was less than 10 mm in size, 22 measured 10-19 mm, 77 measured 20-49 mm and 8 measu­red more than 50 mm. Eighty-five (79%) tumours were ordinary in­vasive dueta! carcinomas, 12 (11 % ) lobular, 5 papillary, one mucinous, one medullary and 4 unclassified carcinomas. Dueta! carcinomas were graded on a three-grade scale. There were 10 grade I (well differentiated) tumours, 34 grade II (moderately differentiated) tumours and 41 grade III (poorly differentiated) tumours. Flow-cytometric DNA ploidy and clinicopathologic variables in primary breast carcinoma On average, 18 axillary lymph nodes (range, 5-39) were examined in eaeh ease. Mieroseopie examination revealed metastases in 59 pa.tients (55%); 30 of them had 1-3 metastatic nodes and 29 more than three. DNA values Eighty (74%) of 108 examined breast eareinomas were aneuploid. DI of aneuploid tumours ranged from 1.09 to 3.72 (Figure 2). No signifieant differenees in pereentage of aneuploid tumours were found between patients younger or older Figure 2. Frequency distribution of DI values in 108 breast cancer samples. than 50 years nor between premenopausal and postmenopausal patients. Aneuploid DNA values were observed in 60% of stage T1 tumours, 74% of stage T2, 83% of stage T3, and 70% of stage T4 tumours. Seventy-eight pereent of ER negative and 70% of ER positive tumours were aneuploid. Aneuploidy was found in 82% of PR negative and 65% of PR positive tumours. The proportion of aneuploid tumours was higher in larger tumours: 55% in tumours 10-19 mm, 79% tumours 20-49 mm and 88% tumours more than 50 mm of size. Eighty pereent of dueta! eareinomas were aneuploid. DNA aneuploidy was found in ali 5 papillary eareinomas, in single medullary and in 2 out of 4 unclassified eareinomas. Of 12 lobular eareinomas only 4 (33%) were.aneuploid. In the group of dueta! eareinomas, 20% of grade I tumors, 79% of grade II and 95% of grade III tumours were aneuploid. Sixty-nine pereent of lymph-node negative pa­tients had aneuploid tumours, eompared to 73% of patients with 1-3 positive lymph nodes· and 83% of patients with more than 3 positive lymph nodes. Discussion In the present series 74%. of the tumours were aneuploid. This finding is in agreement with the results obtained in some other studies. In most of them a minor fraetion of malignant tumours had diplod DNA eontent. The lowest proportion of aneuploid tumours ( 41 % ) has been reported in a series of 155 lymph-node negative patients. 8 In the majority of studies the pereentage of aneuploid tumours was higher, At present, the question why ali malignant tumours are not aneuploid and why there are sueh differenees in DNA prifiles remains i:man­swered. The differeriees in FCM methodology and examined specimens·, different eriteria for identifieation of diploid peak and seleetion of patients for the study are some of the reasons whieh may aeeount for differ.nt DNA distribu­tion pattern in breast eaneer obtained so far. Aneuploidy has been reported initially as highly speeifie for malignant tissue. However, aneuploid pattern has also been demonstrated in non-invasive breast eaneer as well as in mild and atypieal dueta! hyperplasia. There were no signi­fieant differenees between DI values of benign lesions and well differentiated non invasive ade­ 20• 21 noeareinoma. 19• Thus, it seems that aneu­ploidy, espeeially of minor degree, eannot be used alone as an independent eriterion of mali­gnaney in breast tumors. In aeeordanee with most previous studies we have also not found any signifieant eorrelation between DNA ploidy and age, menopausal sta­tus and clinieal tumour stage (Table 1). 8• 12, 13 -15 16, 17,22 Us-Krašovec Met al. Table l. DNA ploidy, clinical variables and steroid Table 2. DNA ploidy and pathologic variables. receptors. Diploid Aneuploid Diploid Aneuploid No. No. (%) No. (%) No. No. (%) No. (%) Tumoursize Age < 50 46 12 (26) O-9mm 1 1 o >50 62 16 (26) 10-19 mm 22 10 (45) 12 ( 55) 20 -49mm Premeno -> 50mm P=NS 16 (21) 61 ( 79) 8 1 (12) 7 ( 88) pausal 12 (26) P= 0.03 Tumourtype Postmeno-Dueta! Ca. 85 17 (20) 68 ( 80) pausal 60 16 (27) 44 (73) Lobular ca. 12 8 (67) 4 ( 33) Papillary ca. 5 o 5 (100) P=NS Clinical tumour Mucinous ca. 1 1 o stag e Medullary ca. 1 o 1 T1 10 4 (40) 6 (60) Unclassified ca. 4 2 2 Tz 65 17 (26) 48 (74) P = 0.003 T3 23 4 (17) 19 (83) Tumour grade (dueta! ca.) T4 10 3 (30) 7 (70) I 10 8 (80) 2 ( 20) 7 (21) 27 ( 79) P=NS II ER III 41 2 ( 5) 39 ( 95) negative (-) 12 (22) 42 (78) P=0,000001 positive ( +) 16 (30) 38 (70) P=NS Lymph node status PR Negative negative(-) 61 11 (18) 50 (82) 15 (31) 34 ( 69) 1-3 positive 30 8 (27) 22 ( 73) positive (+) 17 (36) 30 (64) 5 (17) 24 ( 83) 3· positive P = 0.03 There are controversial data concerning ste­roid receptor status correlated to the ploidy. 10, 15 • 23• 24 In our study group we have found a significant association between ploidy and proge­sterone receptors (P = 0.03) but not estrogen receptors. Correlation between DNA ploidy and patholo­gic variables is' presented in Table 2. Axillary lymph-node status, which is conside­red the most important prognostic factor in breast cancer, did not correlate significantly with ploidy: 69% of lymph node negative tumours had aneuploid values as compared to 73% of tumours with 1-3 and 83% of tumours with more than three positive lymph nodes. In the majority of FCM studies presence or absence of lymph­node metastases was also not found to be related to ploidy. 13,22 ,23,25 Three pathologic parameters, i. e. tumour size, tumour type and tumour grade were found to correlate with tumour DNA content. In severa! studies which have analysed the prognostic relevance of established pathologic P=NS parameters, tumour size has been found to con­tribute most important information about early recurrence. 26 In our study group a significant increase of aneuploidy incidence was found in Iarger tumours (P = 0.03): 79% of tumours measuring 20 to 49 mm and 88% of tumours measuring more than 50 mm were aneuploid, as opposed to 52% of tumours measuring Iess than 20mm. In a majority of FCM studies, however, this association has --;;-ot be. observed. 12·13-17-27 Statistically important differences in DNA va­lues were found among different tumour types (P = 0.003): 80% of dueta! carcinomas were aneuploid, whereas only 4 (33%) out of 12 Iobular carcinomas had abnormal DNA values. A Iow proportion of aneuploid lobular carcinoma has also been reported in other studies (14,28). In contrast to other series where only about 30% of papillary carcinoma had aneuploid values6 , in our group ali five tumours of this type were aneuploid. Tumour grade, one of the important progno­stic parameters, and DNA ploidy correlated Flow-cytometric DNA ploidy and clinicopathologic variables in primary breast carcinoma significantly. An aneuploid pattern was found in 79% of grade II tumours and in 95% of grade III tumours, whereas only 20% of grade I tumours were aneuploid (P = 0.000001). Good correla­tion between ploidy and histologic grading accor­ding to Bloom and Richardson method has been observed by the majority of investigators. 6 There is stili a Jack of agreement about corre­lation between ploidy leve! and established pro­gnostic variables. Some data indicate that DNA profile alone or in combination with proliferative activity may have independent prognostic signifi­cance. Further prospective and cooperative stu­dies are needed to evaluate the prognostic value of DNA ploidy and to confirm whether DNA ploidy and celi cycle analysis can provide additio­nal prognostic information to the established prognostic parameters in invasive breast cancer. References l. deVita VT. Breast cancer therapy: experiencing ali our options. New Engl J Med 1989; 320:527-9­ 2. Stenkvist B, Westtnan-Naeser Set al. Analysis of reproducibility of •subjective grading systems for breast carcinoma. J Ciin Pathol 1979; 32:979-85. 3. Delides GS, Garas G, Georgouli G et al. Interla­ boratory variations. in the grading of breast cancer. Arch Pathol Lab Med 1982; 106:126-8. 4. Shapiro HM. Multistation multiparameter flow cytometry: a critical review and rationale. Cytome­try 1983; 3:227-43. 5. Wied GL, Bartels PH, Bibbo M, Dytch HE. Image analysis in quantitative cytopathology and histopathology. Hum Pathol 1989; 20:549-71. 6. Frierson HF Jr. Ploidy analysis and S-phase frac­tion determination by flow cytometry of invasive adenocarcinomas of the breast. Am J Surg Pathol 1991; 15:358-67. 7. Elston CW. Grading of invasive carcinoma of the breast. In: Page DL, Anderson TJ eds. Diagnostic histopathology of the breast. Edinburgh: Churchill Livingstone, 1987: 300-311. 8. Lewis WE. Prognostic significance of flow cytome­ tric DNA analysis of node-negative breast cancer patients. Cancer 1990; 65:2315-20. 9. McDivit RW, Stone KR, Craig RB, Meyer JS. A comparison of human breast cancer celi kinetics measured by flow cytometry and thymidine labe­ling. Lab lnvest 1985; 52:287-91. 10. Bichel P, Poulsen S, Andersen J. Estrogen recep­tor content and ploidy of human mammary carci­noma. Cancer 1982; 50:1771-4. 11. Kute TE, Muss HB, Hopkins M, Marshall R, Case D, Kammire L. Relationship of flow cytometry results to clinical and steroid receptor status in human breast cancer. Breast Cancer Res Treat 1985; 6:113-21. 12. Dressler LG, Seamer LC, Owens MA, Clark GM, McGuire WL. DNA flow cytometry and progno­stic factors in 1331 frozen breast cancer specimens. Cancer 1988; 61:420-7. 13. Dowle CS, Owainati A, Robins A et al. Prognostic significance of the DNA content of human breast cancer. Br J Surg 1987; 74:133-6. 14. Feichter GE, Mueller A, Kaufmann M et al. Correlation of DNA flow cytometric results and other prognostic factors in primary breast cancer. Int J Cancer 1988; 41:823-8. 15. Hedley DW, Rugg CA, Ng ABP, Taylor IW. Influence of cellular DNA content on disease-free survival of stage II breast cancer patients. Cancer Res 1984; 44:5398-8. 16. Beerman H, Kluin PM, Hermans J, van de Velde CJH, Cornelisse CJ. Prognostic significance of DNA-ploidy in a series of 690 primary breast cancer patients. Int J Cancer 1990; 45:34-9. 17. Taylor IW, Musgrove EA, Friedlander ML, Foo MS, Hedley DW. The influence of age on the DNA ploidy levels of breast tumours. Eur J Cancer Ciin Oncol 1983; 19:632-8. 18. Olszewski W, Darzynkiewicz Z, Rosen PP, Schwartz MK, Melamed MR. Flow cytometry of breast carcfooma: I. relation of DNA ploidy level to histology and estrogen receptor. Cancer 1981; 48:980-4. 19. Norris HJ, Bahr GF, Mike! VU. A comparative morphometric and cytomorphometric study of in­traductal hyperplasia and intraductal carcinoma of the breast. Analyt Quant Cytol Histol 1988; 10:1-9. 20. Teplitz RL, Butler BB, Tesluk H et al. Quantita­tive DNA patterns in human preneoplastic breast lesions. Analyt Quant Cytol Histol 1990; 12:98-102. 21. Erhardt K, Auer GU. Mammary carcinoma com­parison of nuclear DNA content from in situ and infiltrative component. Analyt Quant Cystol Histol 1987; 9:263-7­ 22. Ewers S-B, Langstrom E, Baldetorp B, Killander D. Flowcytometric DNA analysis in primary breast carcinomas and clinicopathological correlations. Cytometry 1984; 5:408-19. 23. Muss HB, Kute TE, Case LD et al. The relation of flow cytometry to clinical and biologic characte­ristics in women with node negative primary breast cancer. Cancer 1989; 64:1894-900. 24. Meckenstock G, Bojar H, Hort W. Differentiated DNA analysis in relation to steroid receptor status, grading, and staging in human breast cancer. Anticancer Res 1987; 7:749-54. 25. O'Reilly SM, Camplejohn RS, Barnes DM et al. DNA index, S-phase fraction, histological grade and prognosis in breast cancer. Br J Cancer 1990; 61:671-4. 26. Wallgren A. Prognostic factors in operable mam­mary carcinoma. Doctor thesis. Stockholm 1976. 27. Baildam AD, Zaloudik J, Howell A et al. DNA analysis by flow cytometry, response to endocrine treatment and prognosis in advanced carcinoma of the breast. Br J Cancer 1987; 55:553-9. 28. Horsfall DJ, Tilley WD, Orell SR, Marshall VR, Cant ELK. Relationship between ploidy and ste­roid hormone receptors in primary invasive breast cancer. Br J Cancer 1986; 53:23-8. Adv Radio! Oncol 1992; 280-3. Correlation of DNA-ploidy and estrogene and progesterone receptor content in primary breast cancer Cufer T1 , Bracko M1 , Goehde W2, Lamovec J1 , Košmelj K3 , Pogacnik A 1 , Us-Krašovec M1 1 -The Institute of Oncology, Zaloška 2, Ljubljana, Slovenia 2 -Institut fur Radiobiologie, Universitiit Muenster, Germany 3 -Biotechnical Faculty, University of Ljubljana, Slovenia Our study was aimed to establish possible correlation between hormone receptor status and DNA-ploidy in 108 patients with invasive breast cancer. Approximately 74% of tumours were found to be aneuploid. A statistically significant correlation was found between DNA ploidy and PR-status (p = 0.03). There were 82% of aneuploid tumours found among PR-negative and 64% among PR-positive tumours. A higher percentage of aneuploidy was established in ER-negative group in comparison with ER-positive group (78% vs. 70%), though the difference was not statistically significant. After the stratification of patients into node-negative and node-positive groups, no statistically significant correlation could be established between DNA-ploidy and ER or PR status respectively. Key words: breast neoplasms; flow cytometry; receptors, estrogen; receptors, progesterone; ploidy Introduction The course of disease in patients with primary breast carcinoma depends on severa! clinicopat­hological factors known to be of prognostic significance, .such as tumor size, axillary lymph node involvement, histological type and patholo­gical grade of tumour. 1 These are known as classical prognostic indicators. Recently, it has been attempted to find possible new prognostic factors which would help to identify more aggres­sive breast cancer tumour types, i. e. DNA-ploi­dy, S-phase fraction, oncogene amplification, proliferation antigen (e. g. Ki67) and others. 1 The latter proved of decisive importance particu­larly in patients lacking the classical prognostic factors, for example in patients with negative axillary Iymph nodes, and those in whom neoad­juvant CHT represents a primary treatment. In order to determine the value of these new prognostic factors it is essential to establish whether, and to what extent, they correlate with the classical ones. Thus, the prognostic value of hormona! recep­tors has been based on the fact that the patients with positive estrogen (ER) and progesterone (PR) receptors had a better disease-free survival as well as overall survival in comparison with hormonal-receptor-negative patients. There have been many studies on the correlation bet­ween hormona! receptor status and DNA-ploidy reported in the existing literature. The informa­tion, however, is controversial, since in contrast to the authors who have not found any such Correspondence to: dr. Tanja Cufer, The Institute of 3• 4 5 7• 89• Oncology, Zaloška 2, 61000 Ljubljana, Slovenia. correlation, 2• • • there are many others6 • • 10•11 •12•13•14 who claim that such a relation exists: UDC: 616.19-006.6-074 Correlation of DNA-ploidy and estrogene and progesterone receptor content in primary breast cancer 281 this has been confirmed particularly in the studies done on large series of patients. 6·7 ·8 Therefore, it was our intention to study the correlation between hormona! receptor status and DNA-ploidy on a group of 108 primary breast cancer patients treated at the _Institute of Oncology in Ljubljana. Material and methods So far, 108 patients with primary breast cancer have been included in our study. All tumours were histologically confirmed as invasive carcino­mas. For flow-cytometric analysis and hormona! receptor assay, tissue segments were taken from fresh unfixed surgical specimens and stored at -20°C. DNA values of tumour cells were measu­red on a PAS II (Partec, Muenster), a high-pres­sure-mercury-lamp based flow cytometer. DNA values were expressed by DNA index which represents the ratio between moda! channel number of aneuploid G0 peak and diploid G0 peak. Tumours with DNA index 1.00 were clas­sified as diploid. For ER and PR assay a Dextran-coated char­ coal technique was used. ER values > 10 fm/mg protein and PR values > 20 fm/mg protein were considered positive. Statistical analysis has been based on contin­ gency tables. Pearson's chi-square statistics has been calculated to determine the correlation between DNA-ploidy and other variables of interest. Results significant correlation (p = 0,03) was found between DNA-ploidy and the absence of proge­sterone receptors i. e. aneuploidy was found in 82% of PR-negative and in 64% of PR-positive tumours (Table 4). For the combined receptor status the absence of one or both receptors was found to be significantly associated with the presence of DNA-aneuploidy (Table 5) After the stratification of patients according to axillary lymph-node involvement, no significant correla­tion between DNA-ploidy and estrogene and progesterone receptor content respectively was found either for negative or positive patients (Table 6), despite the fact that especially in node positive groups the percentage of DNA-aneu­ploidy is much higher in PR negative than in PR positive group of patients. Table l. Ploidy analysis in primary breast cancer. No.of DNA-diploid DNA-aneuploid cases No. (%) No. (%) 108 28 (26) 80 (74) Table 2. Frequency of ER and PR combinations in primary breast cancer. Receptor Status No. of cases (%) ER+PR+ 31 29 ER+PR­ 23 21 ER-PR+ 16 15 ER-PR- 38 35 Table 3. DNA-ploidy in relation to the content of estrogen receptors. Estrogen No. of DNA-aneuploid receptor cases No. (%) Our analysis for DNA ploidy and hormone receptor status included 108 invasive breast can­ ER negative ER positive 54 54 42 78 38 70 p=0,37 ,cers. Among these, 80 (74%) showed measura­ble aneuploidy (Table 1). The same samples were analysed for hormona! receptor status which is presented in Table 2. The rate of DNA-aneuploid tumours was slightly higher in ER-negative than in ER-positive tumours (78 vs 70% ), but the difference was not statistically significant (Table 3). In contrast, a statistically Table 4. DNA-ploidy in relation to the content of progesterone receptors. Progesterone No. of DNA-aneuploid receptor cases No. (%) PRnegative 61 50 (82) PRpositive 47 . 30 (64) p=0,03 282 cufer Teta!. Table 5. DNA-ploidy in relation to the com6ined patients. 6• 7• 8 A review of so-far published re­receptor status. ports also indicates that the correlation between ER and DNA-ploidy was invariably associated Hormone No. of DNA-aneuploid with PR and DNA-ploidy correlation. 6• 7• 8• 9• 10• Receptor Status cases No. (%) 12• 13• 14 In this respect our results differ from ER+PR+ 31 18 (58) those reported by other authors. Namely, in our ER+PR­ ER-PR+ 32 (82) study, a significant correlation was found only ER-PR-38 30 (78) p=0,052 Table 6. DNA-ploidy in relation to the hormone receptors by lymph node status. Node negative pts Hormone No. of D N A-aneuploidy Receptor Status cases No. (%) ER negative 21 15 (71) ER positive 28 19 (67) p=0,78 PR negative 23 18 (78) PR positive 26 16 (61) p=0,20 Node positive pts Hormone No.of DNA-aneuploid Receptor Status cases No. (%) ER negative 33 27 (81) ER positive 26 19 (73) p=0,42 PR negative 38 32 (84) PR positive 21 14 (66) p=0,11 for PR and DNA-ploidy, though the percentage of aneuploid tumours found among ER-negative tumors was somewhat higher. The only author who established a statistically more significant correlation between PR and DNA-ploidy (p = 0.0006) than between ER and DNA-ploidy (p = 0.04) was Kallionemi. 13It is possible that the difference between our and his results could be attributed to the fact that his series of patients was approximately three times larger than ours. Comparing the DNA-ploidy and combined receptor status, we found a statistically signifi­cant correlation between the presence of both receptors and DNA-ploidy. Nearly equal values of aneuploidy were found in patients with both receptors negative as well as in those with only one negative receptor. Discussion In our group of 108 breast cancer patients, DNA aneuploidy was found in 74% of tumours. These results are in agreement with those reported by other authors according to which DNA aneu­ploidy has been established in 50-92% of tu­mours (10). As to the correlation between DNA­ploidy and hormone receptor status, the results of different authors are controversial. In a majo­rity of studies performed so far, possible correla­tion between DNA-ploidy and hormona! recep­tors could not be confirmed, whereas in one third of these reports a statistically significant correlation was found between DNA-ploidy and ER, and in one fourth between DNA-ploidy and PR.15 It should be pointed out, however, that positive correlation results were established in studies with the highest number of included Considering the known fact that ER status is prognostically more relevant in node-negative patients and PR status in node-positive ones 1, our patients were distributed into node-positive and node-negative groups. After this stratifica­tion, no statistically significant correlation could be established between DNA-ploidy and ER or PR status respectively. It should be pointed out, however, that the rate of aneuploidy was gene­rally higher in the receptor-negative groups. The difference in the percentage of aneuploid tu­mours between PR negative and PR positive patients was particularly great (18%) in the node-positive group, i. e. in the group where PR status has proved to be prognostically most relevant. We believe that the reliability of our results could be significantly improved by increa­sing the number of patients included in the subgroups. The suggested inclusion of a larger number of patients would also help to solve the problem whether a statistically significant correlation exists only between PR status and DNA-ploidy or also between ER status and DNA-ploidy. Correlation of DNA-ploidy and estrogene and progesterone receptor content in primary breast cancer 283 References 1. McGuire WL. Prognostic factors for recurrence and survival in human breast cancer. Breast Can­cer Res Treat 1987; 10:5-9. 2. Abondowitz HM, Ow KT, Hardy D, Keightley DD, Sarfaty GA, Nash AR, Relationship between flow cytometric parameters, steroid receptors, and menopausal status in breast cancers. Oncology 1987; 44 :24-9. 3. Dowle CS, Owainati A, Robins A, Burns K, Ellis 10, Elston CW, Blamey RW. Prognostic signifi­cance of the DNA content of human breast cancer. Br J Surg 1987; 74:133-6. 4. McDivitt RW, Stone KR, Craig RB, Palmer JO, Mayer JS, Bauer WC. A proposed classification of breast cancer based on kinetic information. Derived from a comparision of risk factors in 168 primary operable breast cancers. Cancer 1986; 57:269-76. 5. Muss HB, Kute TE, Case LD, et al. The relation of flow cytometry to clinical and biologic characte­ristics in women with node negative primary breast cancer. Cancer 1989; 64:1894-900. 6. Cornelisse CJ, van de Velde CJH, Caspers RJC, Moolenaar AJ, Hermans J. DNA ploidy and survival in breast cancer patients. Cytometry 1987; 8:225-34. 7. Hedley DW, Rugg CA, Gelber RD. Association of DNA index and S-phase phraction with progno­sis of nodes positive early breast cancer. Cancer Res 1987; 47:4729-35. 8. Dressler LG, Seamer LC, Owens MA, Clark GM, McGuire WL. DNA flow cytometry and prognostic factors in 1331 frozen breast cancer specimens. Cancer 1988;61:420-7. · 9. Moran RE, Black MM, Alpert L, Straus MJ. Correlation of cell -cycle kinetics, hormone re­ceptors, histopathology, and nodal status in hu­man breast cancer. Cancer 1984; 54:1586-90. 10. Feichter GE, Mueller A, Kaufmanu M, et al. Correlation of DNA flow cytometric results and other prognostic factors in primary breast cancer. lnt J Cancer 1988; 41:823-8. 11. Stal O, Wingren S, Carstensen J et al. Prognostic value of DNA ploidy and S-phase fraction in relation estrogen receptor content and clinicopat­hological variables in primary breast cancer. Eur J Cancer Ciin Oncol 1989; 25:301-9. 12. Horsfall DJ, Tilley WD, Orel! SR, Marshall VR, McCant EL. Relationship between ploidy and steroid hormone receptors in primary invasive breast cancer. Br J Cancer 1986; 53:23-8. 13. Kallionemi O, Blanco G, Alavaikko M et al. Improving the prognostic value of DNA flow cytometry in breast cancer by combining DNA index and S-phase fraction. Cancer 1988; 62:2183­90. 14. Kute TE, Muss HB, Hopkins M, Marshall R, Case D, Kammire L. Relationship of flow cytometry results to clinical and steroid receptor status in human breast cancer. Breast Cancer Res Treat 1985; 6:113-21. 15. Frierson HF. Ploidy analysis and S-phase fraction determination by flow cytometry of invasive ade­nocarcinomas of the breast. Am J Surg Pathol 1991; 15:358-67. Adv Radio! Oncol 1992; 284-98. Potentials of hyperthermia in clinical use Lešnicar H and Budihna M The Institute of Oncology, Ljubljana More than 10.000 articles have been published around the world in recent 10 years constantly showing the possible benefit of hyperthermia in the treatment of cancer. Despite of ali this accumulated knowledge on this subj<:ct there is stili a lot of problems in introducing hyperthermia as an conventional treatment modality. The biological rationales that strongly support the clinical use of hyperthermia are: direct cytotoxicity of heat and its radiosensitizing effect. However, difficulties with determination of thermal dose and construction of really safe and low cost equipment currently inhibit the definite promotion of thermotherapy. Nevertheless, a number of encouraging clinical results suggest that hyperthermia in the future has to be considered as a topic of general interest. Key words: neoplasms-therapy; hyperthermia, induced Introduction Hyperthermia can be defined as an increase in temperature beyond that normally found in the body. For the therapeutic intent elevated tempe­rature may be limited to local or re.ional area, or it may involve the entire body. Therefore, the terms Iocal, regional and systemic hyperthermia treatment are used in clinical practice. With regard to the way of access to the body area of interest we distinguish between percutaneous or external, intraluminal, intracavitary, perfusional and interstitial type of thermal therapy. 1 Althoug cauterization for local tumor destruc­tion in old Egiption medicine, 2 as well as syste­mic hyperthermia in ancient lndia3 has been widely used even millenniums B.C., the first documented reports on the therapeutic effect of Correspondence to: Hotimir Lešnicar M.D., The Insti­tute of Oncology, Zaloška 2, 61105 Ljubljana, Slovenia. UDC: 616-006.6-073:615.832 elevated body temperature dated from very end of the 19-th century. 4 Already at that tirne the importance of distinction between the two ap­proaches (local and systemic) to the cancer therapy using hyperthermia was recognized. For whole body treatment the proposed temperature rarely exceeded 40 ° C, in order to be well tolera­ted by the organism. On the other hand, Iocal hyperthermia required considerably higher tem­peratures, usually beginning with 42 ° C. 1 With later investigations it was presumed that two different mechanisms play role in the tumoricidal effect of heat. Whereas the host's defence me­chanism, most likely through the immune sy­stem, should be responsible for the effect of whole body hyperthermia on cancer cells,5 in local application the heat itself, with a number of various damaging effects on cell constituents directly induces lethal celi injury. 2 Despite of this early reports on the usefulness of hyperthermia in clinical practice, the Jack of reports with similar results confirmed by other Potentials of hyperthermia in clinical use therapists has driven the whole hyperthermia business into the past. 6 Many decades had to pass before the renewed interest for thermothe­rapy was born. In early 7O's much of the pioneer work on studying the effects of ionizing radiation on cell reproductive capacity was done. In some of these studies it was found that heating combi­ned with radiation increased the cell-killing effect of radiation applied alone. 7-10 Based on these observations numerous clinical trials were car­ried out mostly on comparable superficial mali­gnant lesions treated by either radiotherapy alone or in combination with hyperthermia. There is a strong evidence that adjuvant thermot­herapy considerably increases the probability of a good tumor control. 11-23 The rationales for clinical use of hyperthermia In last decade, the greatest effort of radiothera­pists bas been focused on the problem how to augment the limited effect of radiotherapy on hypoxic fraction of tumor cells. The marked radioresistancy of this specific segment of tumor burden, with no doubt, is highly responsable for the local treatment failure. Hyperoxygenation, accelerated charged particles with high LET, electron affinic drugs and cytotoxic drugs, as well as many unorthodox fractionation regims have been tested to overcome the problem of hipoxia. 24 Many of them have failed to prove its suitability for daily exploitation either on basis of high cost or major toxicity to normal tissue. Although the first documented clinical results on hyperthermia used as an adjuvant to radiothera­PY, appearing in the initial stage of clinical usage of X-rays,25 indicated a significant improvement of the irradiation effect, many decades had to pass for hyperthermia to be recognized as one of the modern experimental clinical techniques. Soon after the first few experimental 3 cm) 2 63 33 42 80 65 * Percent of tumors with complete response after therapy technology, unable of heating at greater depths, Table 2. Aproximate values of treatment tirne for various clinically relevant treatment temperatures. omit this advantage. This problem is much more noticeable in deep-seated tumors, 33-35 although it Temperature 43* 44 in"C 41 may play an important role also in the treatment Treatment tirne in minutes 2160 360 60 30 15 of the superficial lesions. 86 Different histologic subtypes, on the other hand, do not greately affect the response rates. 87-88 The administered total radiation dose, howe­ver, is remarkably important. In brief, higher the total dose, better the outcome of the combi­ 90 ned treatment. 39 -Dose per fraction and num­ber of fractions do not seem to have such an important impact on the therapy outcome, ex­cept for malignant melanoma. 78•91 Some experi­mental data suggest that only very high dose-ra­tes (> 300 cGy/min) as well as extremely low dose­rates ( < 0.5 cGy/min) potentiate heat radiosen­sitization, and practically no benefit is to be expected from using conventional radiotherapeu­tic dose-rates. 92 At this place it is important to stress that practically ali studies with hyperther­mia mainly included reccurences of previously irradiated tumor lesions. Thereafter reirradiation mostly consisted of lower total tumor doses. Yet, the radiotherapy variables in the combi­ned treatment are stili much easier to determine in comparison to thermal variables. It is so because in hyperthermia currently no functional equivalent to the absorbed energy per unit mass * Transition point ( as Gray in radiotherapy) existed. 23 Many at­tempts had been made to quantitate the thermal dose. Mainly the concept of thermal dose bases on biological isoeffect relationship between treatment tirne an temperature, frequently ex­pressed as »equivalent heating tirne at 43 °C«. 93 From numerous experimental studies done on various celi cultures and different types of tissue it was found that this relationship is not a simple linear product of the two observed parameters. There is a transition point which normaly occurs between 42°C and 43°C. For the production of the same degree of thermal injury each step of 1 °C above this point results in reduction of the heating tirne by factor 2. In other words if 1 hour of heating at 43°C is needed for acquireing a certain thermal damage, only 30 min at 44°C enables the development of the same effect. On the other hand, if temperature below this point is used 6 x longer heating tirne is required for each decrease of 1 °C. In Table 2 approximate Potentials of hyperthermia in clinica/ use values of treatment tirne for various treatment temperatures are listed according to the data published by Field and Morris. 94 Since the biological rationals for thermal events occurring at the breaking point (between 42°C and 43°C) are not fully understood, it seems that the development of thermotolerance during heat treatment with lower temperatures is somehow responsible for the big difference in treatment tirne needed for same treatment re­sults as obtained with temperatures above transi­tion point. 95 Further research is needed for real safe application of thermal isoeffect dose concept in clinical practice, 96· 97 although for the moment tirne/temperature formula provides a resonable method for comparing clinical hyperthermia treatments. 98 From severa! clinical trials it can be concluded that the possibility of obtaining a high local tumor control rate especially corelates with mini­mum tumor temperatures. If these temperatures were stili in the cytotoxic range much better 99 treatment results were acquired. 23· That is the reason why good multiple point intratumoral thermometry is an essential demand in clinical hyperthermia. The fractionation in hyperthermia is a constant subject of discussion. Slow rate of decaying of thermotolerance dictates at least 72 hours of interval between two hyperthermia sessions, thus permitting one or at most two treatments weekly. Greater problem is a total number of fractions. Retrospective analyses failed to show an impro­vement of local control when multifraction cour­ses of hyperthermia were used in clinical trials. 45•91 Moreover, in recently published ran­domized prospective study, including matched paired lesions in the same patient, there were no demonstrable difference found in two arms com­paring six vs. two hyperthermia treatments. 100 The other problem which needed to be solved is sequencing of hyperthermia and radiotherapy. From the results of thermobiological investiga­tions (see above) one can conclude that simulta­neous treatment with both modalities guarantees the best treatment results. However, presently available technique does not allow a real simulta­neous therapy. We must not overlook the fact that hyperthermia should have an opposite effect regarding radiosensitivity of normal and tumor tissue. Mild hyperthermia may cause an increa­sed blood flow through the normal tissue resul­ting in improved oxigenation and therefore an enhanced sensitivity to the subsiquent radiothe­rapy. On the other hand higher temperatures in tumour tissue may result in vascular stasis with subsequent tumor hypoxia and decreased radia­tion sensitivity. 45 That is why severa! studies were done to confirm which of two treatment strategies (radiation before or radiation after hyperthermia) gives better therapeutic •45 •91 gain. 17From these studies a treatment plan can be derived prefering radiotherapy folowed by hyperthermia with a possibility of normal tissue (skin) cooling. Although there are some reports in literature which proved a role of hyperthermia as a pallia­tive method in combination with moderate ra­diotherapy 101 ·1 03, or even used alone104 in treat­ment of previously irradiated tumor lesions, the results of recent RTOG study suggest a real benefit from combination of hyperthermia with definitive radiation as a primary therapy21 . Finaly if we want to summerize an answer to few questions presented previously in our text, we have to determine that at the present tirne we have no general propositions regarding the com­bination of hyperthermia and radiation treat­ment. In the majority of lately designed clinical trials 'the most often proposed protocol consists of few large heat treatments (minimum tumor temperature > 42.5°C, treatment duration tirne approximately 45 min.) given once or twice weekly after radiotherapy. Expected results should be better if normal full dose radiotherapy is used46. It is important to accent that previous data dealing the combination of hyperthermia and irradiation reffered only to the local appliance of both treatment. The problems regarding whole body hyperthermia are far more complex. Alt­hough tumour regression after deliberate induc­tion of erisipelas causing elevated body tempera­ture, reported by Coley in 1893, was recognized Lešnicar H and Budihna M 290 as one of the first published articles of hyperther­mia effect on cancer patient, the problem of severe side effect gave the priority to the deve­lopment of the !ocal type of treatment105 . Lately a variety of methodologies has been innovated which permit somehow safer delivering of eleva­ted temperature to the entire body. Whole body hyperthermia can be induced by energy input via the surface of the patient, such as by using 106107 108 paraffin wax•, hot air and radiofrequency 109•110 or water perfused blankets and suits. Anot­her method is warming the patient by heating the blood circulating in an extracorporeal cir­ 111 cuit . The greatest problem in systemic use is maintaining constant body temperature which should not exceed 41.8-42 ° C, otherwise causing deleterious and even fatal side effects. With that purpose numerous rectal, oesophageal, nasopha­ryngeal, intramuscular, temperature probes has to be placed for temperature monitoring112• Alt­hough not all 113, most of currently used whole body hyperthermia systems has to be given under general anaesthesia, that is why also an introduction of the bladder catheter is needed. Inspite of all precautions the probability of get­ting various cardio-pulmonary, !iver and neuro­Iogic complications, whether they are transient or not, is rather high114•115• Because temperatu­res used in whole body hyperthermia have to be tolerated by some critical tissues, they are rather low. Therefore considerable prolongation of the treatment tirne (approximately 2 hours) is neces­ 112 sary to obtain some cytotoxic effect. If even lower temperatures were used (39.5 -40 ° C), at least in animal experiments, increased natura! killer (NK) cells activity was observed, sugge­sting that antitumoral effect of whole body hyperthermia with mild temperatures is media­ted through the immune system116• An imme­diate pain relief after the whole body hyperther­mia, which was most oftenly one of the leading treatment issues, was associated with marked Table 3. Types of non-invasive (percutaneous) hyperthermia techniques Radiofrequency Microwave Ultrasound inductive coupling capacitive coupling (electric) (magnetic) References 122-124 125,126 127,128 129 ,130 131,132 Frequency range 8-!00 MHz 433,915 and 0,3-3 MHz and2450 MHz Applicator l. Two electrodes l. Pancake coil 1. Large electrodes l. Direct contact l. Direct contact positioning positioned oppositely positioned ca 3cm (diameter 20 -25 cm) applicators with or use of water 2. Bolus is required above the skin Concentric, coaxial or without bolus immersion 2. Bolus not requi -or helical coils red and annular phased array Advantages 1. Ability to heat large l. Penetration of l. Treatment of l. Pennetration l.Better pe- volumes at depth 3-4 cm in tumour larger volumes at better for frequencies netration or muscle tissue greater depth Iower than l 000MHz than micro­ 2. Size and shape (ca 6cm) 2. Possible treatment waves of applicators may of larger areas by 2. With multi be changed using more than one transducers applicator system tissue depth of7-10 cm could be obtained Disadvantages 1. Production of cold l. Excessive heating 1. Poor penetration 1. Large reflec- and hot spots due to of superficial for small applicators tions between tissue inhomogeneitis tissue soft tissue 2. Excessive heating of 2. Poor penetration and gas or bone superficial and fatty in tissue at high tissue frequencies 3. Stray microwave radiation Potentials of hyperthermia in clinical use rise in plasma endorphin levels117 • As hyperther­mia is nonmyelosuppressive, its use in multimo­dality treatment of systemic cancer, potentiating the effect of radiotherapy, chemotherapy or immunotherapy is attractive. However, clinical trials using whole body hyperthermia are to be planned only in selected institutions with appro­priate technical equipment and well trained per­sonne! I 18. I 19 _ Heating techniques and thermometry Heating technique system capable of delivering reproducible treatment presents a major chal­lenge for physicists and engineers dealing with clinical hyperthermia. The problem is far from being solved. A detailed information on this subject is beyond the scope of present paper and one can get it elsewhere in the literature120• 121 . Regardless to the problem of whole body hypert­hermia, in general, clinical hyperthermia techni­ques can be classified into two groups: non-inva­sive or percutaneous and invasive or interstitial. In both of them radiofrequency and microwave applicators are mostly used. In some centers ultrasound applicators has been constructed for percutaneous deep tissue heating. In Table 3 characteristics of non-invasive heating techni­ques are listed according to some published datas. The currently used methods for interstitial hyperthermia can be devided on those using electromagnetic waves (implantable microwave antennas and applicators using localised current fields) and so called »hot sources« (ferromagne­tic seed technique and circulating hot water technique). In Table 4 some of the characteristics of invasive techniques are listed together with available literature. Table 4. Typcs of invasive (interstitial) hyperthermia techniqucs. Elcctromagnetic Hot sources radiofrequency (localised current ficlds) microwave ( coaxial cables) ferromagnetic seeds circulating hot water Referenees 133 -137 133.134,138-141 l33.134,142-144 145-148 Opcrating frequencics 0.2 -l MHz 0-5-lGHz / / Optimum spaeing of applieators/ncedlcs Jem 1.5 cm Jem Jem Advantages 1. Less prominent thermo­metry artefaets when ! . Anten nas eould be flexible l. Together with I-125 seeds ferromagnetie seeds 1. Hot water should be eonsidered as a using low frequeney are usable for permanent eonstant temperature eurrents implants 2. Heating of deep-seated tumors is possible 3. Reapeted heatings are possible souree 2. The fall-off the temperature along the tube is.minumum 3. Maximum temperature 4. Seeds can be placed within movable catheters in tissue eannot exeeed water temperature 4. Thermometry is easy to perform Disadvantages 1. Elcctrodes are rigid 2. Spacing of elcetrodes needs to be more dense Ihan in thc case of mierowaves 3. There is a need for 1. Temperature distri­bution on thc longitu­dinal axis is non-uni­form individual designed antennas l. The real eonstant tem­perature seeds are stili under investigation 2. Adjustment of seeds impossible (for pcrma­nent implants) 5. Cooling of normal tissue is possible 1. Good geometry and parallelism are of major importance 2. Rigi) and couch or chair (angle 8) of linacs is shown in Figure 5. Developed by Houdek and co-workers at the University of Miami 12, the single plane rotation is the simplest radiosurgical technique. It is similar to rotational techniques used in standard radiotherapy, except that in radiosurgery the radiation field is very small, the dose is given in a single session, and a stereotactic frame is used for treatment setup and patient immobilization during the treatment. The patient is placed supine on a stationary couch ( = 0°), and the gantry rotates from 0 = 0° to 0 = 360° during the treatment. In the direction perpendicular to the plane of rotation, the dose falloff outside the targeted volume is very steep, but in the plane of rotation (transverse plane), it is very shallow as a result of the dose superposition outside the target of an infinite number of parallel-opposed beams. The shallow dose falloff in the transverse plane produces dose distributions which compare unfavorably with those obtained for the gamma 307 Radiosurgery: A review ofphysical aspects 1ao· Plane of gantry rotation -oo· go• Plane of couch rotation o Figure 5. Schematic representation of the gantry ( angle 0) and couch ( angle ) rotational motions for linac­-based radiosurgical procedures. unit. This then precludes the use of single plane rotation in eontemporary radiosurgery. The beam entry traee for a full single plane rotation is in a transverse plane and eoineides with the beam .exit traee, as shown in Figure 3b. To improve the dose falloff outside the target­ed vol ume, Betti and Dereehinsky3 , Colombo and eolleagues4, and Hartmann and eolleagues5 , devised the multiple noncoplanar converging arcs teehnique on isoeentrie linaes. A series of ares, eaeh with a different stationary position of the 5 treatment ehair3 or eoueh 4•, is used to spread the dose outside the targeted volume over as large a volume as possible. The angles of ares are usually smaller than 180 ° to avoid parallel-op­posed beams in the plane of the are. Hartmann's group in Heidelberg has used up to 11 ares, eaeh either from 20 ° to 160 ° or from 200 ° to 340 ° , depending on the treatment eoueh position. The beam entry traces on the patient's head for the Heidelberg teehnique are shown in Figure 3c. Lutz and eoworkers at the Joint Center for Radiotherapy in Boston have shown that reason­able dose fall'offs ean be obtained with only 4 ares13 . The stereotaetie frame is attaehed to a floor stand, as shown in Figure 4a, and one 260 ° are is given in the transverse plane ( eoueh angle ep = 0 ° ) from 0 = 50 ° to 0 = 310 ° , and 100 ° ares are given with eoueh angles ep of 90 ° , 45 ° , and -45 ° . The beam entry traees on the patient's head for this teehnique are shown in Figure 3d. Currently, in Boston four to nine noneoplanar ares are used in clinieal radiosurgery, depending on the size, type, and loeation of the target treated. The dynamic radiosurgery method was develo­ped by Podgorsak and eolleagues at MeGill University in Montreal. 14•15 A 10 MV isoeentrie linae is used, with the patient in the supine position on the treatment eoueh. The stereotaetie frame is attaehed direetly to the eoueh, as shown in Figure 4b. The main feature of the dynamie rotation is the eontinuous and simultaneous rota­tion of the gantry and eoueh during the treat­ment. The gantry rotates 300 ° from 0=30 ° to ep= 330 ° and the eoueh 150 ° from ep= 75 ° to our error ep = -75° . Thus, eaeh degree of eoueh rotation eorresponds to two degrees of gantry rotation. The beam entry traee on the patient's skull has a peeuliar pattern (see Figure 3e) but always lies in the upper hemisphere, eausing all beam exit points to lie in the lower hemisphere. Thus, even though all beams interseet in the targeted volume and the gantry travels almost a full eircle (300 ° ), there never is a parallel-op­posed beam situation whieh would degrade the steepness of the dose falloff outside the target. Reeently, MeGinley and assoeiates from Emory University in Atlanta16 originated a linae­based radiosurgieal teehnique in whieh the pa­tient rotates on a speeial treatment ehair from cp=0 ° to ep=360 ° , while the gantry is stationary at a given angle e between 90 ° and 180 ° . The stereotaetie frame is attaehed to a pedestal, whieh in turn is mounted to the base plate of the patient support assembly. Up to three gantry positions (0=100 ° , 120 ° , and 145 ° ) are used for a typieal treatment, resulting in eoaxial eircles for beam entry traees in the upper hemisphere and a eonieal irradiation pattern, as illustrated for two gantry positions in Figure 3f. Computerized treatment planning for radiosurgery In radiosurgery, as diseussed above, the goal of minimizing the dose to tissues surrounding ihe Podgoršak EB and Souhami L targeted volume for a given target dose is achie­ved by aiming the radiation beam toward the i,u-g.tJmm . 4u:ge_n.mher of UQP.CQRL111• Figure 6. lsodosc distributions cakulatcd with thc McGill treatment planning system 17 for various linac­based radiosurgical techniques. The calculated dose distributions are normalized to 100% at the isocenter and directly superimposed onto the transverse, coro­nal, and sagittal MRI slices of a typical patient. The radiation beam energy and diameter were 10 MV and 1.5 cm, respectively. The 90% isodose surface covers the spherical target volume with a diameter of 1.5 cm, the other isodose surfaces displayed are 50% and 10%. techniques in clinical use: Boston 4 noncoplanar arcs, Heidelberg 11 noncoplanar arcs, McGill dynamic rotation, and Emory conical rotation. The beam energy and diameter were 10 MV and 1.5 cm, respectively, the lesion, located in the left deep parietal region, was spherical with a diameter of 1.5 cm. The dose distribution is normalized to 100% at the isocenter, the isodose surfaces dispayed are 90%, 50% and 10%, direct­ly super-imposed on the patient's transverse, coronal and sagittal MR slices through the target center. Radiosurgery: A review of physical aspects Several features of the dose distributions of Figure 6 are immediately apparent: (i) the dose falloff estimated from the distance between the 90% and 10% isodose surfaces is reasonably sharp for all techniques, however, the falloff depends on the direction of measurement, (ii) the 90% isodose surface is spherical and covers the targeted volume, (iii) the 50% isodose surfa­ce, similarly to the 90% isodose surface, is essentially isotropic, irrespective of the treat­ment technique, (iv) for all techniques the dose falloff from the 90% to the 50% isodose surface is very sharp, on the order of a few mm, (v) from the 90% at the edge of the targeted volume down to the 50% isodose surface, the four radiosurgical techniques give essentially the same isodose distributions in spite of the considerable differences in methods they employ for dose delivery, and (vi) the 10% isodose surfaces are anisotropic and their shapes reflect the particular dose delivery technique used. Each radiosurgical technique may be character­ized by the sharpest and shallowest dose falloffs outside the targeted volume, with ali other fall­offs situated between the two extremes. The closer the two are to each other, the more isotropic is the dose distribution and the better satisfied is the radiosurgical requirement for a sharp dose falloff outside the target. From Figure 6 one may conclude that the Heidelberg techni­que gives the most isotropic dose distribution, i.e., the sharpest dose falloff outside the target. It turns out, however, that from 90% down to 20%, ali clinically used linac-based techniques exhibit very similar dose falloff characteristics and that only in the dose region below the 20% surface the dose falloffs for techniques with a larger number of arcs are sharper. However, the 20% isodose surface is usually in the range of normal brain tissue tolerance dose and a sharp dose falloff in this region is no longer of vita! importance. The dose falloff characteristics are therefore not a determining factor in the choice of a particular linac-based technique for clinical use. Other factors, such as simplicity of opera­tion, availability of technical help, availability of an appropriate target localization and treatment planning system, and the ingenuity of staff, decide on which technique to introduce clinical­ A comparison of dose falloffs for linac-based techniques to those obtained recently for the gamma unit18 , shows that from the dose falloff point-of-view, radiosurgery with noncoplanar arcs, dynamic rotation, or conical rotation is comparable to radiosurgery with the gamma unit. A detailed comparison among various pho­ton beam radiosurgical techniques and a study of the adequacy of linacs for radiosurgery have been published recently.19• 20 Conclusions During the past five years, radiosurgery has developed from a relatively obscure neurosurgi­cal technique, for three decades practiced in only a few specialized medica! centers around the world, into a sophisticated radiotherapeutic technique that most major medica! centers use or plan to use in the near future. The recent rapid proliferation of radiosurgery was timulat­ed by the following: (i) excellent treatment results, both short term and long term, were obtained during the past three decades by the pioneers in the field, (ii) new imaging modalities that greatly facilitated the target localization were introduced in the 1970s ( computerized tomography) and 1980s (magnetic resonance imaging and digital subtraction angiography), (iii) radiosurgical techniques were developed on linear accelerators making radiosurgery much iess expensive and therefore more widely avail­able in comparison with the traditional techniques based on gamma units or protons from cyclo­trons, and (iv) radiosurgical target localization programs based on stereotactic fram_es and 3-di­mensional treatment planning systems became commercially available. The basic requirement for radiosurgery, which in essence is the delivery of prescribed dose to the targeted volume with a high degree of spatial and numerical accuracy combined with a sharp dose falloff outside the targeted volume, can be adequately met by isocentric linear accelerators. Podgoršak EB and Souhami L It should be remembered, however, that techni­cal and clinical requirements for radiosurgery are far more stringent than those applied to standard radiotherapy. No matter what equip­ment it employs, radiosurgery is a complex treatment modality for which a successful clinical outcome requires a collaborative team effort by severa! hospital-based professionals, including neurosurgeons, radiation oncologists, neurora­ diologists and medica! physicists. The clinical aspects of radiosurgery are discus­sed in the Chapter IV Clinical oncology. References 1. Leksell, L. The stereotaxis method and radiosur­gery of the brain. Acta Ciin Scan 1951; 102:316­9. 2. Larsson B, Liden K, Sarby B. Irradiation of small structures through intact skull. Acta Radio! TPB 1974; 13:513-34. 3. Betti 00, Derechinsky VE. Hyperselective en­cephalic irradiation with linear accelerator. Acta Neurochirurgica 1984; Supl. 33:385-90. 4. Colombo F, Benedetti A, Pozzo F, Avanzo RC, Marchetti C, Chierego G, Zanardo A. External stereotactic irradiation by linear accelerator. Neu­rosurg'ery 1985; 16:154-60. 5. Hartmann GH, Schlegel W, Sturm V, Kober B, Pastyr O, Lorenz WJ. Cerebral radiation surgery using moving field irradiation at a linear accelera­tor facility. Int J Radiat Oncol Biol Phys 1985; 11: 1185-92. 6. Lyman JT, Kanstein L, Yeater F, Fabricant JI, Franke! KA. A helium-ion beam for stereotactic radiosurgery of CNS disorders. Med Phys 1986; 13:695-99. 7. Griffin BR, Warcola SH, Mayberg MR. Stereo­tactic neutron radiosurgery for arteriovenous mal­formations of the brain. Medica! Dosimetry 1988; 13:179-82. 8. Larsson B, Leksell L, Rexed B, Sourander P, Mair W, Anderson B. The high energy proton beam as a neurosurgical tool. Nature 1958; 182:1222-3. 9. Lawrence JH, Tobias CA, Bom JL, Wang C, Linfoot JA. Heavy particle irradiation in neopla­stic and neurologic disease. J Neurosurg 1962; 19:717-22. 10. Kjellberg RN, Shintani A, Frantz AG, Kliman B. Proton beam therapy in acromegaly. N Eng J Med 1968; 278:689-95. 11. Leksell L. Cerebral radiosurgery I. Gamma thala­motomy _in two cases of intractable pain. Acta Ciin Scand 1968; 134:585-95. 12. Houdel< PV, Fayos JV, Van Buren JM, Ginsberg MS. Stereotaxis radiotherapy technique for small intracranial lesions. Med Phys 1985; 12:469-72. 13. Lutz W, Winston KR, Maleki N. A system for stereotactic radiosurgery. Appl Neurophysiol Int J Radiat Oncol Biol Phys 1988; 14:373-81. 14. Podgorsak EB, Olivier A;:Pla M, Hazel J, deLot­biniere A, Pike BG. Phystcal aspects of dynamic stereotactic radiosurgery. Appl Neurophysiol 1987; 50:263-8. 15. Podgorsak EB, Olivier A, Pia M, Lefebvre PY, Hazel J. Dynamic stereotactic radiosurgery. Int J Radiat Oncol Biol Phys 1988; 14:115-25. 16. McGinley PH, Butker EK, Crocker IR. A patient rotator for stereotactic radiosurgery. Phys Med Biol 1990; 35:649-57. 17. Pike BG, Podgorsak EB, Peters TM, Pia C. Dose distributions in dynamic stereotactic radiosurgery. Med Phys 1987; 14:780-9. 18. Walton L, Bomford CK, Ramsden D. The Shef­field sterotactic radiosurgery unit: physical charac­teristics and principles of operation. Br J Radio! 1987; 60:897-906. 19. Podgorsak EB, Pike GB, Olivier A, Pia M, Sou­hami L. Radiosurgery with high energy photon beams: a comparison among techniques. Int J Radiat Oncol Biol Phys 1989; 16:857-65. 20. Podgorsak EB, Pike GB, Pia M, Olivier A, Sou­hami L. Radiosurgery with photon beams: physical aspects and adequacy of line ar accelerators. Ra­diother Oncol 1990; 17:349-58. Adv Radio/ Oncol 1992; 311-22. A survey of radiation biophysics for the radiotherapist Bistrovic M Central Institute for Tumours, Zagreb The present paper is a short introduction into the contemporary radiobiology from the point of interest of a radiotherapist. The basic concepts of cellular radiobiology were represented on the experimental mode'! of cells cultivated in dishes under specific conditions. The linearquadratic model of Geli survival 'and the concept of the repair of sublethal damage ( Elkind repair) were explained by the theory of dual radiation action. Assuming that there is no essential difference between cells in a tissue and those in a dish, the difference between early and late responding tissues was deri ved from the shape of the survival curve. Since the total dose in a radiotherapy treatment is delivered either in fractions or continuously in the due course of tirne, particular attention was paid to the repair and proliferation phenomena playing an important role regarding the fina! effect on cells. Key words: radiotherapy; biophysics Introduction The LQ-model of celi survival, although not revolutionary from the point of view of standard fractionation schedules, allows the the rapist to understand better which alterations can be expected in tumour and normal tissue reac­tions after the schedule has been modified. Namely, the new model compares the effects of radiation on various types of tissue characterized by specific parameters. Therefore the new con­cept requires a better knowledge of radiobiology. This paper aims to enlighten the approach to the radiobiological background involved in various types of fractionation schedules. Correspondence to: Prof. dr. Matija Bistrovic, Central Institute for Tumours, !lica 197, 41000 Zagreb, Croatia. UDC: 615.849:539.1 Normal tissue, tumours and the response to radiation The radiatherapist must distinguish two types of normal tissue. One, H-type10, consists of hierar­chically organized cells growing from stem cells. Stem cells divide and after a few generations of differentiation they mature for the role they will play within the organism. The original stem cells have a long doubling tirne because their role is to save the genetic material for any later need for renewal of the functional tissue. However, the transit cells which participate in the matura­tion sequence have a short doubling tirne. So they will rapidly renew the functional strain of cells which are usually short living. The entire celi system is balanced by a feed-back mechanism causing the enhancement of stem cells' division . ;f the rate of mature celi production is too low. H-type tissues are bone marrow (haematopoietic system), epidermis, gastrointestinal mucosa and other mucosae. 312 Bistrovic M The other type of tissue, flexible or F-type, consists of highly differentiated functional cells with very long doubling tirne or not dividing at ali i.e. resting in G0 piIBse. The damaged tissue will substitute the !ost number of cells very slowly and with diffitulty. If there is any celi division, it will preserve the degree of differentia­tion of the mother cells. F-type cells are those of the Jung, brain, !iver, kidney, urether, spina! cord etc. The irradiated cells do not necessarily die and can repair radiation damage i.e. save their repro­ductive ability. The severely damaged cells will die after the first division or will lose an entire posterity after a few divisions. Therefore, every tissue has a characteristic latency period after which the celi damage will become visible on tissue. In H-type tissue the transit cells rapidly divide and the degradation of functionality will take place after the entire maturation sequence is finished. After the latency period repopulation, an accelerated division in stem cells will start, in order to compensate the Ioss of functional cells. In the bone marrow and in the gastrointestinal mucosa the latency takes 1-2 days, and in the epidermis and other mucosae 14-21 days. Since these latencies are relatively short, H-type tissues are also known as early responding tissue. On the other hand, in F-type tissues the latency period is very long because of a large number of cells resting in G0 phase. In this way functional damage can appear after many months and even years. These are late responding tissues. Tumour cells have a relatively short dividing cycle and die soon after irradiation. Therefore, they may be considered to be early responding tissue. In so far as tumour tissue is not functional at ali, the question of differentiation is pointless. Radiotherapists who irradiated head and neck tumours using the split course techniques3 obser­ved that an additional dose was required to obtain a cure rate equivalent to that achieved with conventional fractionation. This showed that tumour cells divided during the therapy. Thus, if we whish to kili the tumour we have to kili the most rapidly growing cells and our fractionation schedule must overtake the growth rate. The effective doubling tirne of that celi line is usually called potential doubling tirne and denoted by Tpot or Tp; it can be determined either by a careful analysis of results of radiothe­rapy or by specific measurements on tumour samples in laboratory12• Recently, efforts have been made to introduce the routine measure­ment of T P into the clinical practice. The linear-quadratic model of survival curve Most of our knowledge about the irradiation of human cells originates from the experiments with mammalian cells in culture. Various celi lines of animal and human origin are cultivated for years in an adequate liquid medium in Petri dishes. They can be separated in single cells, implanted in new dishes where they will attach to the bottom. In convenient circumstances (37 ° C and humidified atmosphere with 5% C02 in a thermostat and adequate growth medium) they will grow exponentially. This means that the number of cells will be reduplicated after the doubling tirne Tr. The factor of celi multiplica­tion is given by o.693'Tffr) F = e [1] = where e 2.713. 10. 5 HeLa 4 lj-= 24 h 3 2 T[h} 10 20 30 40 Figure l. Celi number growth. After a short delay the number of cells increases exponentially. A survey of radiation biophysics far the radiotherapist In a semilog coordinate system (T, F) the exponential growth is represented as a climbing straight line (Fig. 1). In this representation equal shifts along the ordinate mean multiplication by a factor > 1. Thus, if T increases by T„ the ordinate F will always increase by an equal shift meaning multi­plication by two. Typical mammalian cells are HeLa cells and V79 cells with respective dou­bling times of 24h and lOh. Experiments are usually carried through by irradiating the cells immediately after they have been attached to the dish bottom. After the experiment, samples are incubated in a thermo­stat for 5-10 days. During that period of tirne the surviving cells will form visible colonies which will be counted in irradiated samples as well as in untreated ones. The surviving fraction of cells S < = 1 will be represented by the ratio of celi count in the treated and those in the untreated dishes. If we represent the values of S as they depend on the =t112. In the latter case the effect of radiation will be somewhat weakened due to partial SLDR. As said, ali sparsely ionizing radiations exhibit a shouldered survival curve, but its shape and values of ct and /3 change. For instance, 20 kV X-rays exhibit a steeper survival curve than Co -y-rays do. This is explained by an increase in the efficiency of production of both types of damage. However experiments show2 that at equal survi­val S the repair ratios F (formula [3]) do not differ. This means that the amount of one event­damage versus two-event damage is the same at the same S. Thus, X-rays, -y-rays and electrons would cause an effect fully equivalent to that of a certain referent quality, provided the doses are corrected by a certain correction factor for obtai­ning the same survival. The correction factor is the well known radiobiological efficiency, RBE. If Co -y-rays are chosen for the referent quality with RBE=l, then D1 * RBE1 = D2 * RBE2 = D (Co -y-rays), [4] where subscripts indicate different radiation qua­lities (Figure 8). Aproximate values of RBE's are given in table 1. In the further text we shall consider various modes of radiation always refer­ring to the reference quality i.e. to Co -y-rays. Table l. RBE for various qualities of radiation. Radiation quality RBE Co-60 -y-rays 1 5-40 Me V electrons 1 3-42 MV X-rays 1 200 kV X-rays 1.10 70 kV X-rays 1.20 20 kV X-rays 1.40 A survey of radiation biophysics for the radiotherapist Due to the equality of the damage, results of our considerations will be always translatable to any other quality by dividing ali doses by the appro­priate RBE. Extrapolted response dose with complete SLDR The most important radiobiological task of ra­diotherapy is to divide radiation into small acu­tely given fractions in order to kili the tumour at a relatively low cost to the surrounding functio­nal tissue. In other words, the question is how to compare various regimes of fractionation regar­ding both the therapeutical effect and the tole­rance of normal tissue of both types. Earlier approaches to this problem were based more on clinical experience than on assumed in vitro systems as confident models for the actual in vivo situation. However, in the early eighties the LQ-model was recognized 1• 7 as essential for explaining the early and late effects in radiothe­rapy. A basically new assumption is that the true measure of radiation damage is the surviving fraction of stem cells. Thus the logic of the cells within a tissue is analogous to the logic of the cells cultivated within the dish. This means that the same LQ-type of survival curve will remain valid and the same effect will mean the same leve! of S. Thus, the difference between the tissues (early-, late-responding and tumour) must be reflected in tissue parameters a and /3. Por the sake of simplicity, however, the effect will be represented by the nagative natura! logarithm instead by S. This step is necessary in order to obtain an additive quantity increasing with the actual effect. Thus, after a single fraction d, the efect is represented by ef = -In S= ad+/3d2 = d*(a+/3d). [5] If there is a sufficiently long tirne interval between fractions, and 24 h is more than suffi­cient, cells will completely repair SLD. If we neglect the proliferation of cells, ef will be a purely additive quantity. Namely, if N equal fractions are given (Figure 9), the total effect EF will be EF = N*d*(a+/3d). [6] Now the extrapolated response dose, ERD, the quantity which is the measure of the overall radiation effect, is defined as d ERD=EF/a=D*(l + _ _)=D*RE, [7] where D=N d is the total dose. It is useful to note that ERD differs only by the factor a from the logarithm of the total survival S i.e. ERD directly reflects the leve! of the fina! celi survival. The factor d RE =l+--> 1 al/3 is called relative efficiency, RE, and is similar to the RBE concept. It expresses the efficiency of the to tal dose D: D must be multilplied by RE in order to obtain the total effect ERD. RE is essentially dependent on the size of the single fraction d and on the size of parameter a/ f3 which has various values for various types of tissue: its value is 8 -20 Gy for tumours and early responding tissues, and 2-4 Gy for late respon­ding tissues (see e.g. 11). ,,(/(3=2 Gy \ \ 'i LATE ffFECTS 318 BistrovicM •d 1 fu .o(/ ;3 = 2 Gy , LATE EFFECTS \ \ \ 2 16 . \ \ \ \ . rl/fd = 10 Gy \ EARLV EfFECTS \ \ 1 \ s \NEUTRDNS Figure 10. Difference between the survival curves of the late responding tissue ( o./ f3 = 2Gy) and early re­sponding tissue (o.//3 = lOGy) cells. The dotted straight line represents the survival curve for neutrons. =-------------,d D,RE= D2 ·RE = ERD 16 1 and a/. = 10 Gy as representative of the early responding tissue are compared in Figure 10. Grap­hically, a smaller a of the late responding tissue means a smaller initial slope of the survival curvy and expresses the fact that a small single a/{3 the selfenhancing two-event damage becomes dominant. Thus if we change the size of a single 100h: d 2R 1 RE=l +-­ ..!.. Example 3: Determine the RD for D = 60 Gy, given by protracted mode during the tirne intereval T =168h i.e. with dose rate R = 60/168 = 0.36 Gy h-1. Find the formula for the exposure expressedas the function of dose rate, requiring a constantlate effect ERD (a/. = 4Gy, µ = 0.46 h-1). Solution: From formula [13] one obtains RE = 1.391 and ERD = 60*1.391 = 83.5Gy. Using the same for­ .L1D­ D 1 Dpl mula and equation ERD = RE*D = RE*R*T, one obtains T = ERD/(RE*R), namely T = 83.5/(R*(l+l.08696)). Using this formula for the Figure 14. a correctionThe proliferation of tumor cells requires. A ·supplementary dose D = Dp-D is dose rates 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 Gy h-1, necessary to compensate it. 322 Bistrovic M knowledge of T P allows to modify the concept of ERD in the following way: since the number of cells multiplies by the factor F given in equation [1], the corresponding proliferative effect will be given by ef p = -In F = -(ln2)*T(fp · Using [6] and taking into account that efp is a negative quantity, the entire corrected effect will be: EF p = N*ef -I ef P I, where II denotes the absolute value. Dividing by a one obtains the corrected ERDP : ERD p = EFJa = ERD -(0.693/a) * (T/Tp)-(14] Thus the expression for ERD can now be correc­ ted for the proliferation of tumour cells, provi­ded parameters a and T P are known. The above equation shows that the total ERD is diminished by a term proportional to the total duration of therapy (Figure 14). Therefore, the proliferation has for the consequence the necessity for additio­nal dose in order to compensate the slow repair effect. Example 4: According to Budihna et al. (1985) the 'split course' radiotherapy of glottis and supraglottis ­total dose D1 = 60y during T1 = 40 days was supplemented up to D2 = 75Gy due to the pro­longation up to T2 = 70 days together with a 3.5 week pause and unchanged daily fractionation d = 2Gy. Determine T P of the clonogenic celi fraction ( a = 0.3 Gy-1, a/. = lOGy). Solution Using [14] one obtains for the conventional regime: Erd p1 =D 1 * RE -(0.693/a) * (T 1/T); p and for the 'split course' regime one obtains: ERD p2 = D2 * RE -(0.693/a) * (T2/Tp): since ERDP 1 = ERD p2 , after subtraction of equations and using notation AD = D2 -D 1 and AT = T2 -T1 one obtains: T p = 0.693/ (a * RE * (AD/AT)); since AD/AT=(75-60)/70-40) = 0.5 Gy/day and RE = 1.2, T P is T P = 0.693/(0.3 * 1.2 * 0.5) = 3.85 days. References l. Barendsen GW. Dose fractionation, dose rate and isoeffect relationships for normal tissue re­sponse. lnt J Radiat Oncol Biol Phys 1982;8:1981­97. 2. Bistrovic M, Bišcan M, Viculin T. RBE of 20 kV and 70 kV X-rays determined for survival of V79 cells. Radiother Oncol 1988; 7:175-80. 3. Budihna M, Škrk J, Šmid L, Furlan L. Tumour celi repopulation in the rest interval of split-course radiation treatment. Strahlentherapie 1980; 136:402-8. 4. Chadwick KH, Leenhouts HP. A molecular theory of celi survival. Phis Med Biol 1973; 18:78­87. 5. Dale RG. The application of the linear-quadratic dose-effect equation to fractionated and protrac­ted radiotherapy. Brit J Radiol 1985; 58:515-28. 6. Dale RG. The application of the linear-quadratic model to fractionated radiotherapy when there is incomplete normal tissue recovery between frac­tions and possible implications for treatments involving multiple fractions per day. Brit J Radio! 1986; 7:175-80. 7. Fowler JF. Tota! doses in fractionated radiothe­rapy -implications of new radiobiological data. lnt J Radiat Bio/ 1984;46:103-20. 8. Habic M. Personal communication, 1990. 9. Kellerer AM, HH Rossi. The theory of dual radiation action. Current topics in radiation re­search quarterly 1972; 8:85-158. 10. Nias AHW. Clinical radiobiology. Churchill Li­vingstone, Edinburgh 1988. 11. Thames HD, Bentzen SM, Turesson I, Overgaard M, Van den Bogaert W. Time-dose factors in radiotherapy: a review of human data. Radiother Oncol 1990; 19:219-35. 12. Trott KR, Kummermehr J. What is known about tumour proliferation rates to chose between acce­lerated fractionation or hyperfractionation? Ra­diother Oncol 1985; 3:1-9. Adv Radio/ Onco! 1992; 323-9. A practical method of TBI in vivo dosimetry based on real scatter contributions Vrtar M Institute of Oncology and Radiotherapy, KBC Rebro, Zagreb The use of in-vivo dosimetry in anterior-posterior (AP) 6°Co total body irradiation (TBI) makes possible to determine the absorbed dose in the patient's mid plane point by means of entrance and exit dose readings for the direction of certain beam ray. The arithmetic mean of this two readings far each location must be corrected by a factor which depends on geometrical and physical conditions of the applied TBI. Here, the local tissue air ratio method (local TAR) was used. The theoretical method, which included different sizes of TBI phantoms in length, widths and lung inhomogeneity dimensions, was established. The practical tables of correcting factors for a number of important location§ in TBI in-vivo dosimetry, based on real scatter contributions (i. e. including the influence of the local field for dose determination points), were presented. The correlation to the literature data was also discussed. Key words: whole body irradiation; radiotherapy dosage Introduction The continuation and enlargement of the method of local dosimetric functions, as earlier introdu­ced in TBI in-vivo dosimetry, 1 must lead to such possibilities of application which would cover the whole practical range of real irradiation procedures. Experimental and theoretical ap­proach were performed on the assumption that an effective field in TBI phantom in the surroun­dings of location, where the absorbed dose has to be determined, can be defined by the range of scattering contributions. Aside from the primary dose, scattering reflects the shape, characteristic dimensions and mass arrangement in the nearest Correspondence to: Dr. Mladen Vrtar, Institute of Oncology and Radiotherapy, KBC Rebro, Kišpaticeva 12, 41000 Zagreb, Croatia. neighborhood of single location. The importance and validity of these assumptions was confirmed in the investigation on an anatomic phantom,2 so that the use of standard central axis data, 3 even taking into account the finite phantom dimen­ 5 sions, 4• can not be recommended in TBI. It can be best seen from the behavior of local TAR. Further, during in-vivo TBI, as in 6°Co gamma AP -PA irradiation at extended distance of 4 m, the absorbed dose in the mid-plane point of some location is not a simple arithmetic mean of entrance and exit readings (measured with semi­conductor or thermoluminescent dosimeters) for places in certain beam ray direction. It must be corrected with a factor which depends on geome­trical and physical conditions of the considered TBI method. Therefore, each location at AP/2 depth has its own factor as a consequence of ali the irradiation conditions. Also, the effects of UDC: 615.849.5 the »finite« local phantom AP widths would be 324 Vrtar M included in this model as well as contours and dimensions of the whole phantom which is situa­ted in a large field. Material and methods The results of measurements of local TAR in an anatomic-cubical phantom, which was construc­ted to represent the patient lying on his side (as in parallel opposite AP-P A irradiation techni­que), indicated a close agreement with the theo­retical model based on TBI adapted sector inte­gration method, i. e. better than 2% for ali depths of AP/z on average. But, the model was restricted to a 70 kg-weight water phantom, 150 cm in length and with carefully selected dimen­sions of local AP widths, which was further used as a reference. In order to cover the applicability of the presented method to various real situa­tions, a generalization to variable phantom di­mensions has been imposed to it. Therefore, the characteristics of 11 proportional phantom di­mensions, including the referential one, were theoretically investigated. The lengths of phan­toms were (in cm) from 100 to 200 with step 10. The associated factors of proportionality (re­ferred to 150) were applied to the height too, so that from the source eye view, the phantoms were similar in a geometrical sense. The appro­priate widths corresponded to the considered locations, so that the factors of proportionality in this dimension were defined from the follo­wing AP widths (cm): head (H), 11, 12, ... 21; Neck (N), 7, 8, ... 17; Shoulder (S), 5, 6 ... 15; Lung (Lg), 12, 14, ... 32; Mediastinum (M); 12, 14, ... 32; Abdomen (A), 10,. 12, ... 30; Thigh (T), 10, 12, ... 30; Leg (L), 7, 8, 9, 11, ... 21, 22, 23; Knee (K), 6, 8, ... 14, 15, 16,18, ... 24; Foot, 5, 6, ... 15. The reference AP values of the respective locations were H(16), N(12), S(lO), Lg(22), M(22), A(20), T(20), L(15), K(15), F(lO), and their corresponding coordinate sy­stem), H(65,4), N(50,2), S(40,21), Lg(30,8), M(30,1), A(0,0), T(-10,10), L(-30,3), K(-42,­2,5), S(-70,-7.5). So for example, the phantom No. 4 in the case of Jung had the following factors of proportionality 0.867, 0.867, 0.818. In order to find the reduction factor for 0.05)for the field Subjects Distribution o 12 3 10 13 21 25 33 39 42 47 48 188 8 191 9 193 5 2 191 4 2 3 194 2 4 192 8 188 11 1 179 2 6 3 10 Table 3. Distribution of acentrics. Subjects Distribution o 12 3 10 13 193 4 3 21 197 3 197 1 2 of chromatid breaks and chromosome breaks between the exposed group and the control one. On the contrary, Fischer's test showed signifi­cant difference (p < 0.02) for the yield of acentrics and dicentrics between the exposed and control group, while chi-square value for the same case was on the limit (p = 0.05). Discussion and conclusion Problem of exposure to non-iomzmg radiation 42 195 5 47 194 5 1 48 189 3 1 Table 4. Distribution of chromosome breaks. Subjects Distribution o 1 2 3 10 13 197 3 21 197 3 25 199 1 33 198 2 39 199 1 42 199 1 47 197 3 48 198 2 sources is getting more important in the modem world. Setting standards for limitation of the field strenght during microwave radiation has become a priority task due to an increased application of electronic devices and techniques in various fields of industry, medicine and for military purposes. Ali of these increase the rate of exposure of general population, and especially of the exposed individuals, to the »pollution« Table 5. Distribution of dicentrics. caused by the n onionizing radiation. Subjects Distribution _ o 1 2 3 Biologically detrimental effect of the foremen­ 10 tioned radiation in occupationally exposed per­ sons has not yet been sufficiently explored, and 13 21 199 199 1 the existing data are often contradictory. Accor­ 25 199 1 ding to the existing norms of protection against 33 39 199 198 2 1 microwave radiation, which are nowadays being 42 199 1 used in certain countires, it can be said that ali 47 199 1 the values are based on the results of thermal 48 193 3 1 Garaj-Vrhovac V effects. Radiation duration has a considerable effect on human health. In this connection, the permissible human exposure tirne has been suge­sted and determined. The American National Standards Institute (ANSI) stipulates conditions of the occupational exposure to frequencies from 300 MHz to 300 GHz, as well as the upper microwave radiation intensity limit to 10mW/cm2. The total daily duration of exposure has also been limited to 8 hours. 47 These conditions are considered a harm­less leve! of exposure to microwave radiation. It is considered that power density of 100 mW/cm2 has detrimental effects on human health, while at the power density surpassing 25 mW/cm2 it is forbidden to stay in the radiation zone. Russian and East European standards for the occupational exposure in the microwave radia­tion zone of 300 mMHz to 300 Ghz should not exceed 10 µ, W!cm2 in an 8-hours working day, 100 µ,W/cm2for 2 hours a day and 1 mW/cm2 for not more than 20 minutes a day. 48 For the general population Russian standards set values 49 of 5 µ,W/cm2 • According to our regulation, a standard of 0.1 to 1 mW/cm2 has been set, but without any real experimental values support. This Yugoslav stan­dard is considered safe enough for all the fre­quencies, throughout a working day. Though both Russian and American standards stipulate the permissible exposure tirne, our examinees work in 12-hour shifts. There are also temporary situations during which radar radiation exceeds even the very liberal American standards, not to mention the Russian ones. As a result of those, there is evidence of structural chromosome aberrations found in our examinees. Severa! recent research projects carried out in the last few years on radar centres personnel show their protection as being often inadequate. Protection of the occupationally exposed per­sonnel is made more difficult due to the fact that there are no personal dosimetres similar to those carried by those working in the zone of ionizing radiation. Prevention of occupational impair­ments caused by microwave radiation should be based primarily on individual protection measu­res (protective clothes, protective eyefold etc.) during the direct work with the equipment, -limited duration of stay in the zone of micro­ wave radiation, and -periodical medica! check-up for all the persons working with microwave radiation sources. Until recently it has heen argued that th.ermal effect is the primary effect of microwave elctro­magnetic field. But nowadays non-thermal ef­fects are being paid more attention to, since biological consequences (headache, fatigue, me­mory loss, cardiovascular and various other changes) occur even in low power densities. Our examinees also complained of a series of subjec­tive symptoms like fatigue, neurastenic condi­tions, headache and others, which can be charac­terised as »neurostenic syndrome« or »micro­wave disease« as it is sometimes referred to. Subjective difficulties, headache and exhaustion, as well as the appetite loss, dizziness, emotional instability, have been described by other aut­hors. 20-30•31 Our examinees underwent a series of other analyses which disclosed hematologic chan­ges as a most common findings. From the perso­nal history it could be concluded that some of the examinees had fertility problems, accompa­nied by the spermiogram changes, which indica­ted reproductive cells impairments. Similar data were presented by Lancranjan et al, Manikowska et al, Rosenthal and Beering. 36•50•51 However, there are also some contradictory results. 52 Savitz et al draws attention to an increased risk of leukemia in persons occupationally expo­sed to the electromagnetic field. 53 Our study also shows that microwave radiation has detri­mental effects on cel! structures like chromoso­mes. Chromosome aberrations in occupationally exposed persons range from 1 to 10.5%, and besides structural changes in the number of chromosomes, some of the examinees also show typical structural aberrations. Some of the obtai­ned results show borderline (p = 0.05) or increa­sed values (p > 0.02) for acentric fragments and dicentric chromosomes. However, Bauchinger et al, surveying the incidence of chromosome aberrations in persons Biological effect of microwave radiation occupationally exposed to 50 Hz electromagnetic field, did not find a significant increase in the structural changes in comparison with the control group and a parallel group of subjects exposed to ionizing radiation. 54 In our examinees, results of structural chromo­some aberrations analyses are in correlation with the results of micronucleus test for the same examinees, which confirms, without any doubt, the presence of changes in the celi genome of persons exposed to microwave pulse radiation. 55 Number and structure of chromosome aberra­tions can be regarded in the light of the effect of microwave radiation on the DNA molecule structure. Recent studies suggest that micorwave radiation could result in one-strand and double­strand breaks of the DNA molecule,56 as well as in changes of the deoxiribonucleine acid synthe­sis and structure. 44 The analysed mutagenic changes of somatic cells in our examinees can be attached to their occupational exposure to microwaves. According to other authors, consequences of these muta­tions are related to neoplasms. Out of 29 epidemiological studies published in the last decade, 22 suggest a relation between various neoplasms and exposure to electroma­gnetic field. 57 Therefore, this study, followed by further research of biological effect of microwaves, should shed more light on numerous problems caused by the occupational exposure. Since neit­her any real national standards nor safety regula­tions concerning non-ionising radiation have been set in our country, the presented results are our contribution to the notion that protection is a most important in this field, and that our national standards should be set as soon as possible. Acknowledgment The author would like to thank Mrs Jadranka Racic for technical assistance. References l. Meisel N. Applications industrielles: Les microon­des dans les industries alimentaires, Symposium International: Ondes electromagnetiques et Biolo­gie. Jouy-en-Josas France 1980: 93-101. 2. WHO: Nonionizing radiation protection, Copen­hagen, World Health Organization Regional, Of­fice for Europe, WHO Regional Publication Euro­pean Series No 10: 1982; 97-179. 3. Villforth J C. 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