ADIOLOGY 1,.11 NCOLOGY December 2001 Vol. 35 No. 4 Ljubljana ISSN 1318-2099 Editorial office Radiologij and Oncologtj Institute oj Oncology Zaloška 2 SI-1000 Ljublja11a Slovenia Plzone: +386 1. 4320 068 Phone/Fax: +386 1 4337 410 E-mail: gsersa@o11ko-i.si Aims and scope cillTI. December 2001 Vol. 35 No. 4 Pages 23 7-334 ISSN 1.318-2099 UDC 616-006 CODEN: RONCEM Radio/ogy a11d Oncology is a joumal devoted to p11blicatio11 oj original contri/Jlltions in diagnostic and i11terve11tional radiology, comp11terized tomograpliy, ultrasound, magnetic resona11ce, nuclem· medicine, radiot/1erapy, cli11ical mzd experimental oncology, rndiobiology, rndioplzysics and rndiation protection. Editor-in-Chief Gregor Serša Ljubljana, Slove11ia Executive Editor Viljem Kovac Ljubljana, Slove11ia Editorial board Marija Auersperg Lj11bljana, Slovenia Nada Bešenski Zagreb, Croatia Karl H. Bolmslavizki Ha111b11rg, Germom; Haris Boka Zagreb, Croatia Nataša V. Budi/zna Lj11blja11a, Slovenia Maijan Budilma Lj11bljana, Slovenia Malte Clausen Hamb11rg, Germany Christoph Clemm Miinclze11, Germm11; Mario Corsi Udine, Italy Ljubomir Diankov Sojin, Bulgaria Christian Dittrich Vie1111a, A11stria Ivan Drinkovic Zagreb, Croatia Gillian Duchesne Melbourne, Australia Editor-in-Chief Emeritus Tomaž Benulic Liubljana, S/ove11ia Editor Uroš Smrdel Ljubljana, Slovenia Bela Fornet Budapest, Hzmgan; Tullio Giraldi Trieste, Italy Andrija Hebrang Zagreb, Croatia Laszl6 Horvath Pecs, Hungary Berta Jereb Ljubljana, S/ovenia Vladimir Jevtic Ljubljana, Slovenia H. Dieter Kogelnik Salzburg, A11stria Jurij Lindtner Ljubljana, Slovenia Ivan Lovasic Rijeka, Croatia Marijan Lovrencic Zagreb, Croatia Luka Milas Houston, USA Metka Milcinski Ljubljana, Slovenia Maja Osmak Zagreb, Croatia Branko Palcic Vancouver, Canada ]urica Papa Zagreb, Croatia Dušan Pavcnik Portland, LISA Stojan Plesnicar Lj11blja11a, Slovenia Ervin B. Podgoršak Montreal, Canada Jan C. 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Reader for English Mojca Cakš Key words Eva Klemencic Secretaries Milica Harisclz Mira Klemencic Design Monika Fink-Serša Printed by Imprint d.o.o., Ljubljana, Slovenia Publislzed quarterly in 700 copies Bank account nwnber 02010-0090006751 Foreign currency accozmt number 010-7160-900067 /4 NLB d.d., Podružnica Ljubljana Center, Ljubljana SWIFT: LJBASI2X Szzbscription fee far i11stitutio11s $ 100 (16000 Sf/), individuals $ 50 (5000 SIT) The publication of this jozmzal is subsidized by the Ministry of Scirnce and Teclwology of t!ze Republic of Slovrnia. lndexed and abstracted by: BIOMEDICINA SLOVENICA CHEMICAL ABSTRACTS EMBASE / Excerpta Medica Sci Base Tlzis journal is printed on acid- free paper Radiology mzd Oncology is available 011 the intemet at: http:j /www.011ko-i.sijradiolog/mo.lzt11z/ Ljubljana, Slovenia December 2001 Vol. 35 No. 4 CONTENTS DIAGNOSTIC RADIOLOGY .TI. ISSN 1318-2099 UDC 616-006 CODEN: RONCEM Diagnostic value of pneumoperitoneum on plain abdominal film Frkovic M, Klapan T, Moscatello I, Frkovic M 237 ONCOLOGY Can we rely on cancer mortality data? Checking the validity of cervical cancer mortality data for Slovenia Primic Žakelj M, Pompe Kirn V, Škrlec F, Šelb J 243 Communication after laryngectomy Hocevar- Boltežar I, Žargi M 249 Combined therapy for oral cavity and oropharyngeal squamous cell carcinoma: Depth of invasion as prognostic factor Cizmarevic B, Lanišnik B, Didanovic V, Kramberger K 255 Evolving strategies in the treatment of childhood rhabdomyosarcoma: Slovenian experience Pohar-Marinšek ž, Anžic], Jereb B 259 The technique of craniospinal irradiation of paediatric patients in supine position Šlanzpa P, Seneklova Z, Simicek ], Soumarova R, Bur/con P, Burianova L 267 Anal ultrasound in the diagnosis of anal carcinoma Sudol-Szophiska I, Szczepkowski M, Jakubowski W First experience with a novel luminescence-based optical sensor for measurement of oxygenation in tumors Jarm T, Lešnicar H, Serša G, Miklavcic D 277 Persistent chromosomal aberrations in somatic cells in testicular cancer patients after different therapies Bi/ban-Jakopin C, Bi/ban M 293 PAPERS FROM THE SEMINAR "LABORATORY ANIMALS IN BIOMEDICAL RESEARCH" Legislation on the protection of experimental animals Ornik D, Pogacnik M 303 Standardisation of laboratory animals for biomedical research in Poland Szymmiska H, Krysiak E, Piskorowska ], Woszczyriski M, Czarnomska A 309 SLOVENIAN ABSTRACTS 317 NOTICES 325 REVIEWERS IN 2001 330 AUTHOR INDEX 2001 330 SUBJECT INDEX 2001 Diagnostic value of pneumoperitoneum on plain abdominal film Marija Frkovic, Tajana Klapan, Ines Moscatello, Marijan Frkovic Clinical Institute of Diagnostic and Interventional Radiology Rebro, Clinical Hospital Center Zagreb, University of Zagreb, Zagreb, Croatia Background. Pneumoperitoneum is the presence of air outside the gut lumen as the hallmark of alimenta­ry tract perforation. It can be spontaneous or traumatic in origin. The most frequent cause of spontaneous pneumoperitoneum is the perforation of gastric or duodenal ulcer and the aim of the study was to assess the diagnostic value of pneumoperitoneum on plain abdominal film. Patients and methods. This is a retrospective study based on the diagnostic value of pneumoperitoneum on plain abdominal film, with the patient in upright, supine and sometimes left lateral decubitus position. The study included 79 patients who were admitted to our hospital during a 2-year period of time (1998— 1999) and operated on for perforated gastroduodenal ulcer. Results. Ten (12.66 %) of 79 patients underwent operation without radiological procedure. Sixty-nine (87.34 %) patients were examined radiographically and 53 (76.81 %) of them had signs of pneumoperi­toneum initially on the plain film. Conclusions. The most common cause of pneumoperitoneum was perforated duodenal ulcer in elderly male patients. The most frequent sign of pneumoperitoneum was the crescent shaped free air beneath the di­aphragm. Key words: peptic ulcer perforation; pneumoperitoneum – radiography Introduction Pneumoperitoneum is the presence of air out­side the gut lumen as the hallmark of alimen­tary tract perforation on plain film.1 It can be spontaneous or traumatic in origin. The most Received 25 September 2001 Accepted: 6 October 2001 Correspondence to: Marija Frkovic, M.D., Ph.D., Maksimirska str. No 12, 10000 Zagreb, Croatia; Phone: +385 1 238 8454; Fax: +385 1 233 3725; E-mail: marijan.frkovic@zg.tel.hr frequent cause of spontaneous pneumoperi­toneum is the perforation of gastric or duode­nal ulcer2 and that is the reason why we in­cluded these patients in our study. The aim of the study was to assess the diagnostic value of pneumoperitoneum on plain abdominal film. The traditional sign of pneumoperitoneum is the crescent shaped free air beneath the di­aphragm on erect chest seen on abdominal plain film. In this position, it is possible to de­tect as little as 1 to 2 ml of free air.1,3 The signs of pneumoperitoneum on supine abdominal film are: • “the dog’s cap sign” – air in Morison’s pouch,4 • free air occurring subhepatically inferior and anterior to the liver as a linear collection of air parallel to the lower edge of liver,5 • the falciform ligament sign as a vertically oriented soft-tissue band parallel to the right border of the spine in the region of the thoracolumbar juncture,6 • “Wind’s sign” or “lucent liver sign”,1 • “the dome sign” – free air trapped under the middle part of the diaphragm,7 • “the double wall sign” or Riegler sign – when there is air in the peritoneal cavity and in the bowel lumen, the mucosal and seros­al surfaces are simultaneously outlined,1 • “the sign of triangle” – the air between the loops of bowel,7 • “the football sign” – the air-distended peri­toneum beneath the anterior abdominal wall,1 • and finally, rare signs of pneumoperitone-um, like visible lateral umbilical ligaments or urachus, free air in an inguinal or femo­ral hernia sac and pneumoscrotum.8-10 Some of the described signs are visible on Figures 1 and 2. Pneumoperitoneum may be a solitary plain film finding or it may coexist with pneu­momediastinum or pneumoretroperitoneum, or both.1,11,12 It should be emphasized that there is a condition known as “benign” or “internisti-cal” pneumoperitoneum – spontaneous pne­umoperitoneum without peritonitis which usually has no clinical signs13 and can be di­agnosed only by plain abdominal film.14,15 “Benign pneumoperitoneum” was described with gastric distension,14.15 jejunal diverticulo-sis,16 pneumatosis intestinalis17 and sclero­derm,18,19 and immunosuppressive thera­py.11,15 Such patients are treated by conserva­tive therapy and very rare by surgical ope­ration.20,21 The other conditions which can mimic the signs of pneumoperitoneum are: interposi­tion of the colon or Chilaiditi syndrome, fat depositions, artifacts, intraabdominal ab­scess, intraperitoneal or internal hernia and volvulus, especially of the mobile caecum. Pneumoperitoneum is usually diagnosed on plain abdominal film with the patient in upright, supine or left lateral position. Also, it can be diagnosed on erect chest film or using ultrasound (US),22 or computed tomography (CT).23 Patients and methods During a 2-year period of time (1998—1999), 79 patients were admitted to our hospital and operated on for perforated gastroduodenal ulcers. The abdominal plain films were taken in 69 (87.34 %) patients preoperatively, with the patients in upright (62 cases), supine (3 cas­es) and left lateral decubitus position (4 cases)). In 5 (7.25 %) of 16 patients with suspected ulcer perforation but with normal findings on plain film, the additional contrast-study un­der diascopic control was performed. We divided our patients in several groups, according to their age, sex, type of ulcers and signs of pneumoperitoneum. Results We reviewed retrospectively the hospital data of 79 patients who were admitted to our hos­pital in the 2-year period of time from 1998 to 1999 and operated on for perforated gastro-duodenal ulcer. The data are given in Table 1. Ten (12.66 %) of them underwent an oper­ation without radiological procedure. We noticed that men had higher incidence of both ulcers than women, especially duode­nal (M:F = 55:24). Also, it was interesting to note that women had equal incidence of both ulcers (Figure 3). The distribution of pathologic findings, ac­cording to the age of patients is shown by lin­ear chart (Figure 4). Apparently, most of the patients were 40 to 60 years old. The most frequent sign of pneumoperi­toneum was the crescent shaped free air be­neath the diaphragm (49 cases or 92 %), whe­reas other known signs of pneumoperi­toneum were mentioned and identified very rarely (Figure 5). Table 1. Operated patients because of perforated gastroduodental ulcers and radiologic examins performed in emergency department Figure 4. The age incidence of patients with perforat­ed ulcers shown by chart. Figure 5. Identified signs of pneumoperitoneum on plain abdominal films graphically. The other signs were: subhepatic collec­tion (2 cases or 4 %), “doge’s cap sign” or free air in Morison’s pouch (1 case or 2 %) and atypical collection of intraperitoneal air be­tween the bowel loops (1 case or 2 %). Discussion Most of radiologists describe the findings of pneumoperitoneum as positive in 60-85 % of cases.13,24-26 So, our results are in corellation with their findings. Also, we have to emphasize that abdominal plain films were analyzed by different radiolo­gists in Emergency Department. Maybe, these results would be better if the plain films were analyzed by radiologists who are subspecialists in gastroenterology. The most frequent sign which we found was crescent shaped free air beneath the diaphragm. We think that the rea­son why it is so, is probably the fact that most of radiologists (ours and others) prefer that, whenever is possible, the abdominal plain film are made with the patients in upright position. It is the least dependent part of peritoneal cav­ity and free air can be easily detected. We be­lieve that, if radiologists were able to recognize the signs of pneumoperitoneum, the position of patient would not matter at all. In that case, other signs of pneumoperitoneum, not only the crescent shaped free air beneath the di­aphragm (Figure 5), would be recognized and identified on the plain abdominal films. Since the policy of most surgeons is to rec­ommend surgery in any patient with abdomi­nal symptoms and suspected pneumoperi­toneum, it is evident that the plain abdominal films can give valuable information.24 Should we perform the plain abdominal film in all patients? The “board – like” rigidity which generally indicates an abdominal catastrophe needing laparotomy can be found in 83-93 % of these patients.10 It means that a substantial number of patients with perforated ulcer are referred to roentgen examination even though laparo­tomy is indicated whatever the radiologic findings. Some proportion of patients with normal plain films operated on directly and without further investigations may support the impression that the plain films were tak­en in many patients “just to be sure”.24 Very often, less experienced surgeons work in emergency departments and they need a plain abdominal films as a diagnostic support. One argument that may support the prac­tice of obtaining X-ray films in nearly all pa­tients is related to the fact that the perfora­tion occurs in elderly patients with often atypical clinical findings.25,27,28 Our investigation also confirms this (Figure 4). The influence of age on a low proportion of pneumoperitoneum in young patients is diffi­cult to explain. Seely29 and Taylor30 have sug­gested that the acute ulcers, which may be more common in young patients, can be ex­pected to heal spontaneously. Even in centers with unlimited resources, plain films supplemented by gastrointestinal contrast studies as needed, remain the mod­ern standard for evaluation of patients who have suspected gastrointestinal perforation. These widely available, easy to perform, and relatively inexpensive procedures are rel­atively sensitive and specific for evaluation of this problem. It is, therefore, crucial for radi­ologists to be familiar with the often subtle signs of gastrointestinal perforation on plain abdominal films. Skills of plain film interpretation should not be permitted to erode in the environment of newer technologies.31 Although newer technologies like US22 and CT23 give possibillity for detection of pneumoperitoneum, they are not routinely used in emergency departments so that the signs of pneumoperitoneum detected by them are significant, but usually an incidental finding. References 1. Shaffer HAJr. Perforation and obstruction of the gastrointestinal tract. Assessment by conventional radiology. Radiol Clin North Am 1992; 30: 405-11. 2. Sibbald WJ, Swenny JP, Inwood MJ. Portal venous gas (PVG) as an indication for heparinization. Am J Surg 1972; 124: 690-3. 3. Miller RE, Nelson SW. The roentgenological demonstration of tiny amounts of free intraperi­toneal gas: Experimental and clinical studies. Am J Roentgenol 1971; 112: 574-85. 4. Hajdu N, De Lacey G. The Rutherford Morison pouch: a characteristic appearance on abdominal radiographs. Br J Radiol 1970; 43: 706-9. 5. Menuck L, Siemers PT. Pneumoperitoneum: Importance of right upper quadrant features. Am J Roentgenol 1976; 127: 753-6. 6. Millerr E. Perforated viscus in infants: a new roentgen sign. Radiology 1960; 74: 65-7. 7. Frkovic M. Pathological conditions of gastroin­testinal tract in children. In: Radiological atlas of children’s gastrointestinal tract. Chapter 3. Zagreb: Informator; 1998. 8. Bray JF. Pneumoscrotum with testicular delin­eation – a new sign of pneumoperitoneum. Br J Radiol 1982; 55: 867-8. 9. Jelaso DV, Schultz EH Jr. The urachus – an aid to the diagnosis of pneumoperitoneum. Radiology 1969; 92: 295-8. 10. Weiner CI, Diaconis JN, Dennis JM. The “inverted V”: a new sign of pneumoperitoneum. Radiology 1973; 107: 47-8. 11. Pear BL. Pneumatosis intestinalis: a review. Radiology 1998; 207: 13-9. 12. Stahl JD, Goldman SL, Minkin SD, Diaconis JN. Perforated duodenal ulcer and pneumomedi­astinum. Radiology 1977; 124: 23-5. 13. Wellwood JM, Willson AN, Hopkinson BR. Gastrografin as an aid to the diagnosis of perfo­rated peptic ulcer. Br J Surg 1971; 58: 245-9. 14. Felson B, Wiot JF. Another look at pneumoperi­toneum. Semin Roentgenol 1973; 8: 437-43. 15. Frkovic M, Mandic A, Jonic N, Labar B, Mrsic M. Pneumatosis intestini in acute lymphoblastic laeukaemia. Radiol Oncol 1996; 30: 41-5. 16. Dunn V, Nelson JA. Jejunal diverticulosis and chronic pneumoperitoneum. Gastrointest Radiol 1979; 4: 165-8. 17. Miller RE, Becker GJ, Slabaugh RD. Nonsurgical pneumoperitoneum. Gastrintest Radiol 1981; 6: 73­4. 18. Clavadetscher P, Binkert D., Wellauer J. Compli­cations of cutaneous scleroderma. JAMA 1975; 232: 390-1. 19. Yerella JT, McCullough JY. Pneumoperitoneum in infants without gastrointestinal perforation. Surgery 1981; 89: 163-7. 20. Baker SR. Plain film radiography of the peritoneal and retroperitoneal Spaces. In: The abdominal plain film. Norwalk, Connecticut: Appleton & Lange; 1990. p. 71-125. 21. Knechtle SM, Davidoff AM, Rice RP. Pneumatosis intestinalis: surgical management and clinical out­come. Ann Surg 1990; 212: 160-5. 22. Kuang-Chau Tsai, Hsiu-Po Wang, Guang-Tang Huang, Shih-Ming Wang. Preoperative Sonogra­phic Diagnosis of Sealed-Off Perforated Gastric Ulcer. J Clin Ultrasound 1998; 26: 269-71. 23. Ranschaert E, Rigatus H. Confined gastric perfo­ration: ultrasound and computed tomographic di­agnosis. Abdom Imaging 1993; 18:318-9. 24. Svanes C, Salvesen H, Bjerke Larsen T, Svanes K. Trends in value and consequences of radiologic imaging of perforated gastroduodenal ulcer. A 50­year experience. Scand J Gastroenterol 1990; 25: 257-62. 25. Winek TG, Mosely HS, Grout G, Luallin D. Pneumoperitoneum and its association with rup­tured abdominal viscus. Arch Surg 1988; 123: 709­12. 26. De Bakey M. Acute perforated gastroduodenal ul­ceration. A statistical analysis and review of the literature. Surgery 1940; 8: 852-84. 27. MacKay C. Perforated peptic ulcer in the west of Scotland: a survey of 5343 cases during 1954-63. Br Med J 1966; 240: 701-5. 28. Svanes C, Salvesen H, Espehaug B, Soreide O, Svanes K. A multifactorial analysis of factors re­lated to lethality treatment of perforated gastro-duodenal ulcer 1935-1985. Ann Surg 1989; 209: 418-23. 29. Selly SF, Campbell D. Non-operative treatment of perforated peptic ulcer: further report. Int Abstr Surg 1956; 102: 435-46. 30. Taylor H. Non-surgical treatment of perforated peptic ulcer. Gastroenterology 1957; 33: 353-68. 31. Young WS, Englebrecht HF, Stoker A. Plain film analysis in sigmoid volvulus. Clin Radiol 1978; 29: 553-60. Can we rely on cancer mortality data? Checking the validity of cervical cancer mortality data for Slovenia Maja Primic Žakelj1, Vera Pompe Kirn2, Fani Škrlec2, Jožica Šelb3 1Epidemiology Unit, 2Cancer Registry of Slovenia, Institute of Oncology, Ljubljana, Slovenia, 3Institute of Public Health. Ljubljana, Slovenia Background. Valid inference on cervical cancer mortality is very difficult since – on the basis of death cer­tificates – it is not always possible to distinguish between cervix, corpus and unspecified uterine cancer deaths. Our aim was to estimate the extent to which cervical cancer as the official cause of death reflects the true mortality from cervical cancer in Slovenia. Material and methods. The data on 2245 deaths from cervix, corpus uteri, and uterus-unspecified cancers for the period 1985-1999 were linked to the Cancer Registry of Slovenia database from the mortality data­base of Slovenia. Results. Officially, in the period 1985-1999, there were 878 cervical cancer deaths. The comparison of these causes of death with the cancer sites registered in the Cancer Registry revealed that they include only 87.7 % patients with a previous diagnosis of cervical cancer. Of 650 corpus uteri cancer deaths, 17. 1 % of patients were registered to have cervical cancer, and of 717 unspecified uterine cancer deaths, 31.4 % were registered. Taking into account the correctly identified cervical cancer cases among cervical cancer deaths and misclas­sified cervical cancer deaths as corpus uteri and unspecified uterine, the corrected number of deaths would be 1106. Conclusions. When evaluating the impact of cervical cancer mortality from national mortality rates, the stated underestimation should be taken into account. However, this does not hold for some other cancers. Key words: cervix neoplasms – mortality; death certificates; registries; Slovenia Received 5 November 2001 Accepted 25 November 2001 Correspondence to: Maja Primic Žakelj, M.D. Ph.D., Epidemiology Unit, Institute of Oncology, Zaloška 2, SI-1000 Ljubljana, Slovenia. Phone: +386 1 43 22 316; Fax: +386 1 43 10 271; E-mail: mzakelj@onko-i.si Introduction In our everyday practice, we often meet clini­cians seeking the information on the official cause of death of their patients believing that this source is the most reliable. To prove that this is not always true we present our analy­sis of the data on cervical cancer mortality in Slovenia. Their validity is of special impor­tance because mortality is the final measure of the effectiveness of cervical cancer screen­ing programs in a country.1 It has already been pointed out that a valid inference on cervical cancer mortality is very difficult since – on the basis of death certifi­cates – it is not always possible to distinguish between cervix, corpus and unspecified uter­ine cancer deaths.2 To estimate the extent to which cervical cancer as the official cause of death reflects the true mortality from cervical cancer in Slovenia, all causes of death from uterine cancers (ICD 8 codes 180 and 182) were matched with the diagnosis registered in the Cancer Registry of Slovenia (CRS). The CRS has been operating since 1950 and is a unique basis for such epidemiological analyses.3 The official crude mortality rate from cer­vical cancer increased from 12.5/100,000 in 1953 to 16.3/100,000 in 1962. Since then, it was slowly decreasing (Figure 1). In 1968, it was the first time below 10/100,000. In 1997, it was 6.2/100,000 and in 1999, 5.1/100,000.4,5 Material and methods Data on 2245 deaths from cervix, corpus uteri, and uterus-unspecified cancers in the period 1985-1999 were linked to the Registry database from the mortality database of Slovenia.5 During the calendar period under study, the eighth revision of the International Classification of Diseases (ICD) was used in the CRS, while the ninth and tenth in coding cancer deaths.6,7,8 The classification of cancer deaths was thus re-coded according to the eighth revision. The linkage was based on PIN. The cause of death was compared to the diagnosis registered in the Registry. In case more than one cancer had been registered, the gynaecological cancer (ICD 8 codes 180­ 184) was considered as the cancer of interest irrespective of the status of cervical cancer at death of the patient. Results Officially, in the period 1985-1999, there were 878 cervical cancer deaths. The comparison of these causes of death with the cancer sites registered in the Cancer Registry revealed that they include only 770 (87.7 %) patients with a previous diagnosis of cervical cancer. But of 650 corpus uteri cancer deaths, there were 111 (17.1 %) patients registered to have cervical cancer and of 717 in whom unspeci­fied uterine cancer was recorded as the cause of death, 225 (31.4 %) were registered to have cervical cancer. Taking into account the cor­rectly identified cervical cancer cases among cervical cancer deaths and misclassified cer­vical cancer deaths as corpus uteri and un- Figure 2. Uterine cancer as official cause of death by subsite, by cancer site registered in CRS, and by age; 1985-99. specified uterine, the corrected number of deaths would be 1106. Hence, the official cervical cancer mortali­ty would be underestimated by 26 %. This un­derestimation differs by age groups. In women, aged 20-44 years, nearly all deaths from cervical cancer (158 of 162) were con­firmed at the Registry. However, the death of 33 young patients who had been registered as cervical cancer cases was attributed to the corpus uteri (8) and uterus unspecified cancer (25). The official data on cervical cancer mor­tality would thus be underestimated by 17 % in this age group, while in the age group 45­64 years and in the oldest one for 23 and 33 %, respectively (Figure 2). The trend of official and corrected cervical cancer mortality in the period 1985-1999 is presented in Figure 3. The shape of the time trend was not appreciably affected by the un­derestimation of mortality. Discussion Though mortality data are available for more countries than incidence data, they have sev­eral disadvantages. Death certification is less precise in terms of cause of death than the in­cidence data recorded by cancer registries.9,10 In cervical cancer we face also the problem of erroneous cause of death statements and of a varying proportion (20-65 %) of cervix cancer deaths that are coded to “uterus not other­wise specified”.2,11 Our study revealed that among “uterus not otherwise specified” cancers about a third are attributable to cervical cancer. Also, among cervical cancer deaths not all cancers have been diagnosed as this cancer. After combin­ing the erroneous cause of death statements and inexact coding, the official mortality sta­tistics underestimates cervical cancer deaths for about a quarter. In mortality statistics the underlying cause of death is coded according to the rules of the International Classification of Diseases cur­rently in use. In most patients who have ever been diagnosed with cancer, this disease is recorded on death certificate as the underly­ing cause of death. But it depends on the doc­tor, completing the death certificate, whether cancer site is determined properly and, con­sequently, properly coded in mortality statis­tics. It has been shown that the degree of mis­classification varies with cancer site, being greater for those that are more difficult to di­agnose.10 It is well known that the reliability of diagnoses recorded on death certificates depends on the place of death, being more ac­curate for those who died in hospitals and where the autopsy had been performed. It was assumed that, in women below the age of 45 years, most deaths from uterine can­cers are due to cervical neoplasm; so, many international comparisons take into account all uterine cancer deaths.2,12,13 Our study pro­vides a numerical estimate for this hypothe­sis: officially, there were 210 uterine cancer deaths in this age group in the period under study, of which only 192 were identified as cervical cancer cases in the CRS. Older women less often dye in hospitals than younger ones. This could also explain a greater proportion of misclassified cervical cancer deaths in older age group. Namely, in case of the patients who did not dye in the hospital, the physician, certifying death, was not always the family doctor or the doctor who treated the patient, but the one who was on duty and did not know the medical histo­ry of the deceased in details; so, the cause of death may be less precise. Our study suggests that the extent of mis-classifications and improper or less precise coding of death causes have not changed ap­preciably in the time period observed, and the curve shape of the time trend in cervical cancer mortality was not considerably affect­ed. But it may happen that by improving death certification reliability, changes in mor­tality from cervical cancer may lead to an ap­parent increase, as already experienced in some countries.11 Conclusion When evaluating the impact of cervical can­cer mortality drawn from national mortality rates, the underestimation as stated above should be taken into account. However, this is not true for some other cancers. In lung cancer, in the CRS during the data cleaning proces, we observe, each year, an overestima­tion of death officially attributed to lung can­cer. Due to misclassifications described in can­cer mortality data and similar deficiencies in the data on suicide and other injuries and poisonings, infant mortality data and mater­nal mortality, the Institute of Public Health plans to delay the publishing of the official edition on mortality for at least one year. It is hoped that this time will be enough to link mortality data with other databases to render mortality data more reliable. But in monitor­ing long term trends in mortality this change will have to be taken into account. Acknowledgement We appreciate the financial support from the European Commission, contract grant num­ber SI2.305757 (2000 CVG2-029). References 1. Prorok PC, Kramer BS, Gohagan JK. Screening theory and study design: the basics. In: Kramer BS, Gohagan JK, Prorok PC, editors. Cancer scree­ning. Theory and practice. New York: Marcel Dekker Inc., 1999. p. 29-53. 2. Levi F, Lucchini F, Negri E, Franceschi S, la Vec­chia C. Cervical cancer mortality in young women in Europe: patterns and trends. Eur J Cancer 2000; 36: 2266-71. 3. Pompe-Kirn V et al. Cancer incidence in Slovenia 1998. Ljubljana: Institute of Oncology, 2001. p. 5­6. 4. Pompe-Kirn et al. Cancer incidence in Slovenia 1993. Ljubljana: Institute of Oncology, 1996. p. 14. 5. Mortality data-base. Ljubljana: Institute of Public Health of the Republic of Slovenia, 2001. 6. World Health Organisation. International classifica­tion of diseases: 8th revision. Geneva: World Health Organisation, 1967. 7. World Health Organisation. International classifica­tion of diseases: 9th revision. Geneva: World Health Organisation, 1977. 8. World Health Organisation. International classifica­tion of diseases: 10th revision. Geneva: World Health Organisation, 1992. 9. Parkin DM, Whelan SL, Ferlay J, Raymond L, Yo­ung J. Cancer incidence in Five Continents. IARC Sci Publ 1997; 143: 45-53. 10. Dos Santos Silva I. Cancer epidemiology: principles and methods. Lyon: IARC, 1999. p. 31-2. 11. Smith J, Parkin DM. Evaluation and monitoring of screening for cervix cancer: time trends. In: Sanki-la R, Demaret E, Hakama M, Lynge E, Schouten LJ, Parkin DM, editors. Evaluation and monitoring of screening programmes. European Commission, Europe against cancer programme. Luxembourg: Office for Official Publications of the European Communiti­es, 2001. p. 59-76. 12. Cuzick J, Boyle P. Trends in cervix cancer morta­lity. Cancer Surv 1988; 7: 417-39. 13. Levi F, Lucchini F, Negri E, Boyle P, La Vecchia C. Cancer mortality in Europe, 1990-1994, and an overview of trends from 1955 to 1994. Eur J Cancer 1999; 35: 1477-516. Communication after laryngectomy Irena Hocevar-Boltežar and Miha Žargi Department of Otorhinolaryngology and Cervicofacial Surgery, University Medical Center, Ljubljana, Slovenia Background. Laryngectomy is the mode of treatment of the patients with advanced laryngeal and hy­popharyngeal cancer. It affects many important functions, including speech. Patients and methods. Various alaryngeal speech modes are available so that no laryngectomee should be left without a means of communication. Results. There is a variety of artificial devices, including electronic ones that produce their own battery driv­en sound. Alternatively, the patient can learn a new form of voicing using a muscular segment of the upper esophagus as a source of sound (esophageal speech). A puncture can be created surgically through the esophageal wall and a prosthesis placed in it to divert pulmonary air into the esophagus and through the same muscular segment to produce sound. Conclusions. Many factors influence the choice of an alternative to be used with a particular patient. In Slovenia, esophageal speech is the most frequently used alaryngeal speech mode. Key words: laryngeal neoplasms; laryngectomy; speech, alaryngeal; speech, esophageal Epidemiology and etiology of laryngeal and hypopharyngeal cancer Laryngeal and hypopharyngeal cancers are quite common in Slovenia. In 1995 they rep­resented 1.9 % of all new malignant diseases in Slovenia. The incidence of laryngeal cancer was 9.1/100.000 inhabitants in men, and 0.5/100.000 inhabitants in women. The inci­dence of hypopharyngeal cancer was 4.5/100.000 inhabitants in men, and 0.3/100.000 inhabitants in women. In 55 % of patients with laryngeal cancer and only 12 % of patients with hypopharyngeal cancer, the Received 15 October 2001 Accepted 10 November 2001 disease was discovered in a localized stage. In all other patients, the malignant disease was in an advanced stage and required more ag­gressive treatment.1 Laryngeal and hypopharyngeal cancers usually occur in men aged 50-65 years with a long history of tobacco consumption fre­quently associated with alcohol abuse. The al-cohol-related nutritional deficiencies could be involved in the etiology of these cancers.1,2 As a result, the patients often present with no­table co-morbidities. In addition, the socio- Correspondence to: Irena Hocevar-Boltežar, MD, PhD, Department of Otorhinolaryngology and Cervicofacial Surgery, Zaloška 2, SI-1000 Ljubljana, Slovenia. Phone: +386 1 522 24 65; Fax: +386 1 52 24 815; E-mail: irena.hocevar-boltezar@guest.arnes.si cultural level is rather poor in the majority of cases. This particular characteristic of laryn­geal and hypopharyngeal cancer patients ex­plains the delay in diagnosis and the prob­lems linked to treatment compliance. Laryngectomy and its consequences Laryngectomy is a surgical procedure usually reserved for patients with advanced laryngeal or hypopharyngeal carcinoma or patients who fail radiation treatment.3 Loosing the lar­ynx means adapting to a living without some basics that characterize us as human. Respiration and speech are altered for ever; swallowing needs to be re-learned; smell and taste are attenuated; lifting, straining and coughing (all of which are dependent on a closed glottis) are compromised. Although there are numerous potential problems (emo­tional, psychological, physical, economic, so­cial, surgical, and communicative), the inabil­ity to speak is considered the greatest of the difficulties the patient is faced by.4 Voice restoration after laryngectomy After the removal of the larynx, the patient no longer has a source of sound for speaking. Currently, there are two categories of sound restoration: alternative “natural” sound sour­ces and mechanical speech aids. The former category utilizes esophageal and tracheoeso­phageal speech, whereas the latter an elec­tronic artificial larynx.5 Esophageal speech (ES) ES traditionally has been the dominant ap­proach to laryngeal speech rehabilitation. Some retrospective studies demonstrated a range of success from 12 % to 97 %.6-11 In 1982, Gates et al. published the results from the first prospective investigation of ES ac- Radiol Oncol 2001; 35(4): 249-54. quisition, which showed that 26 % of their la-ryngectomy study group were able to acquire ES.6 In a more recent prospective study, Hillman et al. found that only 6 % of their pa­tients developed usable ES.12 ES is produced by compressing the air into the esophagus; the released air vibrates the pharyngeal-esophageal segment and pro­duces the esophageal tone used for speech. The sound produced enters the oral cavity where it is articulated and shaped into words. Generally, there are three primary meth­ods used to teach esophageal speech: conso­nant injection, glossopharyngeal press, and inhalation. Regardless the method used, the goals are for the patient to be able to im­pound rapidly the air into the esophagus, ex­pel it from there in a controlled manner, and produce fluent ES. Esophageal speakers have a much lower air reservoir (less than 100 cm3) than is available to laryngeal speakers from the lungs (even > 5 litres). The small air sup­ply will limit the esophageal speaker’s ability to produce long utterances on a single charge of air. The advantages of the ES are: • The sound of ES is more natural and closer to the laryngeal voice. • ES requires no dependence on mechanical instrument. • The patient is able to achieve some meas­ure of pitch and loudness control, and good esophageal speakers are able to vary these dynamically during speech. • Both hands are free during speech. ES has also some disadvantages: • ES must be learnt and may take a long time to master it. Some patients may nev­er learn to produce functional ES even af­ter much effort. • A person’s ability to articulate clearly must be good, otherwise the intelligibility of ES may be poor. • The patient may have difficulty being heard above back-ground noise.5 Tracheoesophageal speech (TES) The tracheoesophageal puncture method, cou­pled with the use of the voice prosthesis, was introduced by Singer and Bloom in 1980.13 The surgery may be performed at the time of the la-ryngectomy (primary procedure), or it may be performed at a later date (secondary proce­dure). Early studies, focused on carefully se­lected groups of patients who underwent the insertion of a prosthesis as a secondary proce­dure, reported success rates ranging from 56% to 93 %.14-16 More recent studies, which have focused on the insertion of a prosthesis as a primary procedure, have reported acquisition rates ranging from 30 % to 93 %.17-19 In this approach, a small, silicone, valved prosthesis is inserted into a surgically created midline tracheoesophageal fistula. The uni-di­rectional valved prosthesis is designed to maintain tract patency and protect against as­piration. The patient can divert pulmonary air from the trachea (by occluding the tra­cheostoma with a finger) through the prosthe­sis, thereby creating a sound in the pharyngo-esophageal segment. The air pressures required to force open the slit of the valve range between 2 and 25 cm H2O and depend on the rate of airflow from the lungs and the type of the device used.20 Some patients may have considerable difficulty producing the pressures. In these cases, a lower resistance prosthesis is suitable. Special valves are avail­able to avoid manual occlusion of the stoma. These valves close automatically when greater than normal thoracic pressures are present as when the patient wishes to produce speech. There are still contraindications in the se­lection of patients for the prosthesis inser­tion: inability to care for the stoma, poor man­ual dexterity, a stenotic stoma, poor eyesight, esophageal stenosis, and poor patient’s moti­vation. The advantages of TES are: • This technique can provide the most rapid restoration of nearly normal speech in most of the laryngectomized patients. • TES is smooth and fluent because of the availability of pulmonary air. • Loudness and pitch variation is possible. • The approach is feasible in most of the la-ryngectomized patients and is also re­versible if so desired. The disadvantages of TES are: • The insertion of the voice prosthesis re­quires another surgical procedure if not done together with the laryngectomy. • Occasional aspiration due to poorly seated prosthesis, or poorly functioning prosthe­sis is possible. • A buildup of candida deposits requires fre­quent cleaning. • The functioning period of the prosthesis is limited.5,21 Artificial larynx (AL) Previous reports of AL use among laryngecto-my patients vary in many aspects. The esti­mates of AL use range from 5 % to 66 %.6,22,23 This device uses electric power to drive a vi­brator that provides the sound source. It gen­erates a sound with approximately the same frequency as is the fundamental laryngeal fre­quency. One type of the device consists of a tube that delivers sound from the vibrator to the mouth. the sound is then articulated in the normal way. Another version consists of a hand held vibrator that is designed to deliver the sound through the skin when placed on the neck. Until recent years, the AL was con­sidered to be the method of choice only for those patients who were unable to learn ES. Clinical experience has demonstrated that AL actually may be helpful in the acquisition of ES. AL may serve as a communication bridge until ES or TES training is initiated. Recent studies from the USA report that a majority of laryngectomees use AL – 55 %.12 The advantages of AL: • It is easy to learn how to use it. • AL provides adequate volume to be heard in noisy places. • Volume and pitch control is possible. • The speech with AL is intelligible when properly used. Speech with AL has some disadvantages: • AL has a noisy electronic sound that at­tracts attention. • The neck type device cannot be used in the patients with heavily scarred neck. • Moderate initial purchase cost and occa­sional additional cost for repair. • AL requires very clear articulation to as­sure intelligibility.5,21 Characteristics of alaryngeal speech Fundamental frequency (F0) Most electronic speech aids have a manually adjustable F0. These are typically set to a low pitch for a male voice (about 100 Hz) and to a higher one for a female voice (about 200 Hz). Some have a variable frequency adjustment. The F0 of the ES is usually lower than the average laryngeal F0. The results of different studies range from 57 Hz to 136 Hz.24,25 The reasons for such a variety of the results are probably different measuring instruments used in different speech samples. The F0 in TES is reported to be closer to normal laryngeal speakers, at least for male speakers, and ranges from 72 Hz to 134 Hz.24,26 Some authors measured higher F0 in TES than in ES.27 Vocal intensity(VI) The level of VI in users of AL is typical of nor­mal laryngeal speakers during normal con­versation or reading, ranging between 75 and 85 dB. The intensity range is somewhat re­duced.28 The intensity of the electronic vibra­tor is largely determinated by the design of the instrument. The intensity of ES is usually lower in over­all loudness than normal. The range of VI that esophageal speakers are able to produce is much lower than the intensity range of nor­mal speakers (about 10 dB vs. 30 dB).29 The intensity of TES appears to be only slightly lower than the levels produced by la­ryngeal speakers. In some speakers, variation of intensity may be greater than normal.30 Temporal characteristics There are little data on the temporal charac­teristics of speech with an AL. The few stud­ies available indicate that reading rates are slower when using an AL compared to nor­mal phonation or TES.28 We might expect longer reading times because of the need for precise articulation to maintain an acceptable level of intelligibility. In general, the patients using ES read slow­er than normal laryngeal speakers. Their reading rates are about 60-70 % of the rate of a normal speaker. They spend 30-45 % of their reading time in silence because they need to recharge air supply more frequently. They have a much shorter phonation time, typical­ly less than 6 s, which is no doubt due to a small volume of air in the esophagus.24,29,30 Patients using TES also read at a slower rate than normal speakers but faster than esophageal speakers. Their slower rate may be due to difficulties in controlling the pharyngo-esophageal segment and the need to articulate precisely. These speakers spend about 10-30 % of their reading time in silence. Their phona­tion time is longer than in esophageal speak­ers – about 12 s, but shorter than in normal speakers (in average 15-20 s).24,29,30 Intelligibility Few studies have compared all three forms of alaryngeal speech. The intelligibility of speak­ers using AL ranges between 30 and 90 %. The average intelligibility is reported to be about 60 %. The major cause of relatively low intelligibility is the failure to maintain the voicing distinction. Voiceless consonants tend to sound like voiced consonants. The intelligibility of ES varies consider­ably, but in general, it is somewhat higher than in AL users. The average is about 70 %. Most of the deficiencies committed by esophageal speakers were voicing errors. Like in users of an AL, voiceless consonants were perceived as voiced. TES also show considerable variation in in­telligibility, but in general, they produce the most intelligible speech of the three forms of alaryngeal speech. The patients using TES do not have to use any structures in the vocal tract to insufflate the pharyngo-esophageal segment; therefore, they can maintain their normal (or develop near normal) patterns of articulation and more normal flow of speech.31-33 Speech rehabilitation after laryngectomy in Slovenia According to a study performed in a group of members of an Association of laryngec­tomized subjects in Slovenia, 62 % of the sub­jects use ES, 8 % use TES, and 9 % of subjects use AL in every-day communication. About 18 % of 113 subjects who answered the ques­tionnaire communicate using pseudo-whis­per or writing. Only 2 % of subjects use AL and ES or TES. The laryngectomees assessed their satisfaction with their mode of commu­nication using longitudinal analogue scale – 85 %.34 The results differ from the studies per­formed in the USA or Australia. Hillman et al. report that only relatively small percentages of laryngectomees developed usable ES (6 %) or remained nonvocal (8 %), and that a major­ity of the patients ended up as users of AL (55 %) or TES (31 %).12 In Australia, the mode of communication after laryngectomy is about the same: 5.2 % patients use ES, 50 % AL, and 31.6 % TES.35 The reason for the difference in communi­cation mode is a good access to speech thera­py in Slovenia. Patients start learning ES two or three weeks after the surgery. The Association of laryngectomized subjects or­ganizes free courses for better communica­tion skills twice a year. Perry and Shaw report on very low referral rate to speech pathology in Australia.35 In Slovenia, the voice prosthe­sis is inserted only in the patients who cannot learn ES. Many factors influence the choice of the mode of speech rehabilitation in a particular patient. For a successful rehabilitation, a team approach is necessary. A team of pro­fessionals (ENT surgeon, phoniatrician, speech therapist, psychologist) meets the pa­tient before laryngectomy to explain him/her the possibilities of voice restoration after the surgery. The rehabilitation is started as soon as the wounds are healed and continues in the following years. The whole team and the patient are aware that only a successful speech rehabilitation enables a good quality of life after laryngectomy. References 1. Cancer incidence in Slovenia 1995. Ljubljana: Institute of Oncology, Cancer Registry of Slovenia; 1997. 2. Michaels L. Larynx. In: Michaels L. Ear, nose and throat histopathology. London: Springer-Verlag; 1987. p. 371-84. 3. Fried MP (ed). The larynx: a multi-disciplinary ap­proach. Boston: Little, Brown; 1988. 4. Greene MCL, Mathieson L. Voice rehabilitation af­ter laryngectomy. In Greene MCL, Mathieson L. The voice and its disorders. London and New Jersey: Whurr Publishers; 1989. p. 326-32. 5. Casper JK, Colton RH. Rehabilitation after laryn­gectomy. In: Casper JK, Colton RH. Clinical manu­al for laryngectomy and head and neck cancer rehabili­tation. San Diego, California: Singular Publishing Group, INC.; 1993. p. 55-78. 6. Gates G, Ryan W, Cooper J, Jinks P. Current sta­tus of laryngectomee rehabilitation: I. Results of therapy. Am J Otolaryngol 1982; 3: 1-7. 7. Johnson C. A survey of laryngectomee patients in Veterans Administration Hospitals. Arch Otolaryn­gol 1960; 72: 768-73. 8. King P, Fowlks E, Pierson G. Rehabilitation and adaptation of laryngectomy patients. Am J Phys Med 1968; 47: 192-203. 9. Volin R. Predicting failure to speak after laryngec­tomy. Laryngoscope 1980; 90: 1727-36. 10. Schaefer S, Johns D. Attaining esophageal speech. Arch Otolaryngol 1982; 108: 647-9. 11. Hunt R. Rehabilitation of the laryngectomee. Laryngoscope 1964; 74: 382-95. 12. Hillman RE, Walsh MJ, Wolf GT, Fisher SG, Hong WK. Functional outcomes following treatment for advanced laryngeal cancer. Ann Otol Rhinol Laryngol 1998; 107: 2-27. 13. Singer MI, Bloom ED. An endoscopic technique for restoration of voice after laryngectomy. Ann Otol Rhinol Laryngol 1980; 89: 529-33. 14. Donegan JO, Gluckman JL, Singh J. Limitations of the Blom-Singer technique for voice restoration. Ann Otol Rhinol Laryngol 1981; 90: 495-7. 15. Singer MI, Blom ED, Hamaker RC. Further experi­ence with voice restoration after laryngectomy. Ann Otol Rhinol Laryngol 1981; 90: 498-502. 16. Wood BG, Rusnov MG, Tucker HM, Levine HL. Tracheoesophageal puncture for alaryngeal voice restoration. Ann Otol Rhinol Laryngol 1981; 90: 492­4. 17. Kao W, Mohr R, Kimmel C,Getch C, Silverman C. The outcome and technique of primary and sec­ondary tracheoesophageal puncture. Arch Otolaryngol Head Neck Surg 1994; 120: 301-7. 18. Lau W, Wei W, Ho C, Lam K. Immediate tra­cheoesophageal puncture for voice restoration on laryngopharyngeal resection. Am J Surg 1988; 156: 269-72. 19. Quer M, Burgues-Vila J, Garcia-Crespillo P. Primary tracheoesophageal puncture vs esophageal speech. Arch Otolaryngol Head Neck Surg 1992; 118: 188-90. 20. Weinberg B, Moon J. Aerodynamic properties of four tracheoesophageal puncture prosthesis. Arch Otolaryngol Head Neck Surg 1984; 110: 673-5. 21. Prater RJ, Swift RW. Manual of voice therapy. Boston: Little Brown; 1983. 22. Lopez M, Kraybill W, McElroy TH, Guerra O. Voice rehabilitation practices among head and neck surgeons. Ann Otol Rhinol Laryngol 1987; 96: 261-3. 23. Gray S, Conrad H. Laryngectomy: postsurgical re­habilitation of communication. Arch Phys Med Rehab 1976; 57: 140-2. 24. Weinberg B, Bennet S. Selected acoustic charac­teristics of esophageal speech produced by female laryngectomees. J Speech Hear Res 1972; 15: 211-6. 25. Singh W, Ainsworth WA. Computerized measure­ments of fundamental frequency in Scottish neoglottal patients. Folia Phoniatr 1992; 44: 231-7. 26. Debruyne F, Delaere P, Wouters J, Uwents P. Aco­ustic analysis of tracheo-esophageal versus eso­phageal speech. J Laryngol Otol 1994; 108: 325-8. 27. Moon JB, Weinberg B. Aerodynamic and myoelas-tic contribution to tracheoesophageal voice pro­duction. J Speech Hear Res 1987; 30: 387-95. 28. Weiss MS, Yeni-Komshian GH. Acoustical and perceptual characteristics of speech produced with electronic larynx. J Acoust Soc Am 1979; 65: 1298-308. 29. Baggs T, Pine S. Acoustic characteristics: tracheo-esophageal speech. J Commun Dis 1983; 16: 299­307. 30. Trudeau MD, Qi YY. Acoustic characteristics of fe­male tracheoesophageal speech. J Speech Hear Dis 1990; 55: 244-50. 31. Mervin G, Goldstein L, Rothman H. A comparison of speech using artificial larynx and tracheoe­sophageal puncture with valve in the same speak­er. Laryngoscope 1985; 95: 730-4. 32. Blom ED, Singer MI, Hamaker RC. A prospective study of tracheoesophageal speech. Arch Otolaryngol Head Neck Surg 1986; 112: 440-7. 33. Sanderson RJ, Anderson SJ, Denholm S, Kerr AIG. The assessment of alaryngeal speech. Clin Otolaryngol 1993; 18: 181-3. 34. Jelicic M, Hocevar-Boltežar I, Novak C. Uporaba nadomestnih govornih metod pri laringektomi­ranih osebah v Sloveniji. Ustvarjalnost v logopedi­ji. Zbornik prispevkov. Nova Gorica: Aktiv lo-gopedov Severnoprimorske regije, 1999: 186-9. 35. Perry AR, Shaw MA. Evaluation of functional out­comes (speech, swallowing and voice) in patients attending speech pathology after head and neck cancer treatment(s): development of a multi-cen­tre database. J Laryngol Otol 2000; 114: 605-15. Combined therapy for oral cavity and oropharyngeal squamous cell carcinoma: Depth of invasion as prognostic factor Bogdan Cizmarevic1, Boštjan Lanišnik1, Vojko Didanovic1, Kristina Kramberger2 1Department of Otorhinolaryngology and Cervicofacial Surgery, 2Department of Pathology, Teaching Hospital Maribor, Maribor, Slovenia Background. The aim of the study was to emphasize the importance of surgical management of squamous cell carcinoma (SCC) in the head and neck and to find the most important predictive factor for cervical lymph node metastasis and prognostic factor for survival. The use of multimodality therapy is being dis­cussed as well. Patients and methods. From June 1st, 1992 to May 31st, 1998, 154 patients with oral cavity and oropha­ryngeal SCC were admitted to the Department of Otorhinolaryngology and Cervicofacial Surgery in the Teaching Hospital of Maribor. The criteria for inclusion into the study were met by 142 patients, but only 62/142 patients entered the multimodality protocol (surgery and postoperative radiotherapy). These 62/142 patients were treated surgically and 49 of them were postoperatively irradiated, while 13/62 de­clined postoperative radiotherapy. Surgical specimen was evaluated for positive or negative lymph nodes, tumor margins and the depth of invasion. Tumor cells were stained for Ki67 proliferative factor. Results. The depth of invasion was the most important predictive factor for the neck metastases in multi­variate model including also the grade, pT and T. pN was found to be important in determining the overall survival using Cox regression model (p < 0,05). A statistically important discrepancy between N and pN classification was found. In 23 cases N was overrated and in 3 cases underrated. The overall 5-year disease specific survival was 55 %. Ki67 correlated with the grade of tumor differentiation. No statistically signifi­cant correlation was found with lymph node metastases. Conclusions. The depth of invasion is the most important factor determining the occurrence of the neck metastases whereas the N status determines the survival. Key words: mouth neoplasms – therapy; oropharyngeal neoplasms – therapy; combined modality thera­py; carcinoma, squamous cell; neoplasm invasiveness; survival analysis Received 7 July 2001 Accepted 10 August 2001 Correspondence to: Bogdan Cizmarevic, M.Sc., M.D., Otorhinolaryngologist, Maxillofacial Surgeon, Department of Otorhinolaryngology and Cervicofacial Surgery, Teaching Hospital Maribor, Ljubljanska 5, 2000 Maribor, Slovenia. Phone: +386 (0)2 321 1592; E­mail: bogdan.cizmarevic@sb-mb.si Introduction The modality of treatment of oropharyngeal and oral cavity squamous cell carcinoma (SCC) is still a matter of debate. Almost all authors use the multimodality therapy ap­proach, and only a few are willing to take a chance with the single modality therapy. Chemotherapy is becoming more and more used in the therapy of head and neck SCC, al­though its results are inconsistent and bene­fits not proven. Regardless of the modality used, the sur­vival of patients with head and neck SCC in the last 50 years has not improved as much as we would like, but the quality of life has, es­pecially with the use of reconstructive proce­dures. In the management of the head and neck SCC, two questions emerge: 1. How to treat? 2. How much is enough (ultraradical multimodality therapy, close margin sur­gery)? The answers to these two questions should bring us closer to the final goal: the cure of the patient with less therapy and bet­ter quality of life. We wish to present the results of multi-modality approach to the treatment of pa­tients with oral cavity and oropharyngeal SCC at the Department of Otorhinolaryngo-logy and Cervicofacial Surgery in Maribor Teaching Hospital. We tried to identify the most important predictive factors determin­ing regional metastasizing and locoregional prognosis as well as prognostic factor for sur­vival. Patients and methods From June 1st, 1992 to May 31st, 1998, 154 pa­tients with oral cavity or oropharyngeal SCC were admitted to our department. The criteria for the inclusion into the analysis were: 1. histologically proven SCC in the oropharynx or oral cavity, 2. no meta- or synchronous tumors found at panendoscopy, 3. no prior oncological treatment, 4. re-sectable tumor. The inclusion criteria for entering the mul­timodality protocol were satisfied by 142 pa­tients. In 23 of 142 patients, the carcinoma was medically inoperable because of a signif­icant co-morbidity or extent of the disease, whereas 57 patients declined primary radical surgical therapy. Sixty-two (44 %) of 142 patients were treat­ed surgically and 49/62 (80 %) under multi-modality protocol: surgery and postoperative teleradiotherapy with doses ranging from 52 to 56 Gy, while 13/62 declined postoperative radiotherapy. There were 58 (93 %) males and 4 (7 %) fe­males. The average age of patients was 55.6 years. The youngest one was 31 and the old­est 78 years old. All patients were preoperatively submitted to screening tests, panendoscopy and neck ultrasound, and had the tumor (re)classified according to cTNM.1 The radicality of tumor excision was proven with intraoperative frozen section. Tumor and neck specimens were sent to the Pathohistology Department for the final macro- and microscopical analysis. The fol­lowing parameters were determined: 1. Depth of invasion measurable from virtual line connecting uninvolved tissue margins to the deepest tumor line. 2. Immunohistochemical detection of Ki-67 proliferating factor. 3. Thickness of tumor measured from the most superficial tumor line to the deepest line of invading tumor. 4. Lymph nodes identified in fibrofatty tissue obtained during neck dissection; the nodes were searched for metastatic cells and ex-tracapsular spread by conventional histo­logical technique and serial sections through lymph node. 5. Tumor classified according to pTNM. Ki-67 was detected by Immunotech mono­clonal antibody Ki-67 antigen (MIB-1) using standard immunochemistry methods.2 The average proportion of Ki-67 positive nuclei was calculated for each sample. The data were analyzed using Statistica for Windows with Student t test, multivariate lin­ear regression models, Kaplan Meier survival analysis and Cox regression analysis. Results Intra/transoral excision and comprehensive neck dissection were performed in 41/62 (66 %) patients, and commando operation in 21/62 (34 %) patients. All resections were R0. The defect was reconstructed with myocu­taneous flaps (platysma, PMMF, infrahyoid) in 10/62 cases (16 %), with forearm free fas-ciocutaneous flaps in 8 cases, local flaps in 13 cases, and primary suture or split thickness skin graft in 31 cases. Of 62 patients, 51 (83 %) were with tumors classified as stage III or IV. Bilateral and multilateral neck dissections were performed in 55 and 7 cases, respective­ly. In each neck specimen, more than 30 lymph nodes were isolated (average 39). N+/pN0 conversion was observed in 15/62 (24 %) cases and overall 23/62 (37 %) necks were overrated (cN > pN). Occult metastases were found in only 3/62 (5 %) cases. Using Student t test, we found statistical difference between N and pN for the same cohort of patients (p = 0.019). Kaplan Meier survival curves for the pa­tients treated with multimodality approach (n = 49) according to N stage are presented in Figure 1. In order to identify factors influencing the rate of metastases in regional lymph nodes, we analyzed clinical and pathognomonical factors using multivariate linear regression. The depth of invasion, T stage, thickness and differentiation grade were analyzed. The only statistically important factor was the depth of invasion > 4 mm (p = 0.0237). Figure 1. Kaplan Meier survival analysis according to pN stage. The patients’ survival was dependent on pN status (p = 0.056) in the model that includ­ed pN, pT, G and depth of invasion, using Cox regression for analysis. A 5-year disease specific survival of the group of patients with oropharyngeal cancer was 74% and those with oral cavity 47 %. The overall disease specific survival was 55 %. Of 62 patients, 16 died of disease, 3 were lost to follow up, 5 died from locoregional re­currence, 7 from distant metastases and 4 had regional recurrence without any evidence of primary tumor. All patients with regional recurrence (9/16) had positive neck status at the first treatment. The incidence of locoregional recurrence in combined modality group and in single modality (surgery only) group was 16.3 % and 7.5 %, respectively. The distribution of ad­vanced stage in both groups was statistically similar. Ki-67 expression correlated to the grade of differentiation and had no statistically signif­icant influence on node metastases. The re­sults are preliminary and the independent re­search is being performed. Discussion In our analysis, we presented a “classical” treatment concept of SCC of the oral cavity or oropharynx. The most surprising result is low incidence of occult metastases and a high rate of overrated necks. In 24 % of all cases, the clinical suspicion for metastases didn’t prove correct. Regional disease is an important sur­vival prognostic factor and Kaplan Meier sur­vival curves are significantly different for pN0 and pN+ status. The depth of invasion was found to be one of the most important factors determining the occurrence of regional metastases. The depth of invasion > 4mm means a 3 times higher probability of positive neck.3,4 The depth of invasion is not included in TNM system. The invasion of tumor cells in the surrounding tis­sue is followed by angiogenesis and matrix ly­sine. Invasive and antigenic tumor potentials are connected and only together they have prognostic value. The depth of invasion is simple and evident in any gross evaluation of a tumor specimen. The grade of differentiation does not prove to be the decisive factor in metastasis prog­nosis. As indicated by our preliminary re­sults, the same holds true for Ki-67. In our series, a large proportion of admit­ted patients with resectable and medically op­erable tumors (57/119) declined further surgi­cal therapy. The reason for their decision is not clear from retrospective analysis. According to the results of some other au­thors, surgery as single modality treatment for advanced oral cavity or oropharyngeal cancer is definitely more successful than pri­mary radiotherapy.5,6 Most authors advocate postoperative irradiation in the cases of posi­tive margins, perineural and perivascular spread in the case of extracapsular spread or in the case of multiple positive nodes.7 Conclusions We believe that the depth of invasion should be included in every pathologist report of oropharyngeal and oral cavity SCC because it may influence the decision for further treat­ment and postoperative irradiation. The depth of invasion is as important for the prognosis as TNM classification. In our study, Ki-67 proliferative factor didn’t correlate with metastatic potential of the SCC of the oral cavity and oropharynx. References 1. UICC International Union Against Cancer. TNM classification of Malignant Tumours. Fifth edition. New York: A John Wiley & Sons, Inc., Publication; 1997. 2. Zidar N, Gale N, Cor A, Kambic V. Expression of Ki-67 antigen and proliferative cell nuclear antigen in benign and malignant epithelial lesions of the larynx. J Laryngol Otol 1996; 110: 440-5. 3. Fukano H, Matsuura H, Hasegawa Y, Nakamura S. Depth of invasion as a predictive factor for cer­vical lymph node metastasis in tongue carcinoma. Head Neck 1997; 19: 205-10. 4. Ambrosch P, Kron M, Fischer G, Brinck U. Micrometastases in carcinoma of the upper aerodigestive tract: detection, risk of metastasiz­ing and prognostic value of depth of invasion. Head Neck 1995; 17: 473-9. 5. Hicks WL Jr, Kuriakose MA, Loree TR, Orner JB, Schwartz G, Mullins A, et al. Surgery versus radi­ation therapy as single modality treatment of ton-sillar fossa carcinoma: the Rosewell Park Cancer Institute experience (1971-1991). Laryngoscope 1998; 108: 1014-9. 6. Suarez C, Rodrigo JP, Herranz J, Rosal C, Alvarez JC. Extended supraglottic laryngectomy for pri­mary base of tongue carcinomas. Clin Otolaryngol 1996; 21: 37-41. 7. Houck JR, Medina JE. Management of cervical lymph node in squamous cell carcinomas of the head and neck. Semin Surg Oncol 1995; 11: 228-39. Evolving strategies in the treatment of childhood rhab­domyosarcoma: Slovenian experience Živa Pohar-Marinšek1, Jožica Anžic2, Breta Jereb3 1Department of Cytopathology, Institute of Oncology, 2Hemato-oncological unit, University Children’s Hospital, University Medical Center, 3Institute of Oncology, Ljubljana, Slovenia Background. Neoadjuvant chemotherapy (Cht) has changed the treatment of rhabdomyosarcoma (RMS) in children. The purpose of our study was to review the children treated for RMS between 1974 and 1996. Patients and methods. Fifty-one children, 1-15 years old, were included. Primary sites of tumour were: head and neck 15, orbit 6, genitourinary 12, extremity 9, torso 5 and paratesticular 4. Twelve patients were in stage I, 10 in stage II, 26 in stage III and 3 in stage IV. Of 43 histologically confirmed RMS 25 were em-bryonal, 13 alveolar, 1 botryoid, 1 spindle cell and 3 sarcoma NOS. In 8 patients, only fine needle aspira­tion biopsy (FNAB) was available. All patients had Cht, 29 neoadjuvant, 20 had surgery first, 40 had irra­diation (RT), 2 stage IV patients had bone marrow transplant (ABMT). Multidrug Cht varied: VCR, AMD, and cyclophosphamide (VAC) were used in the 1970s, with Adriablastine (T2), methotrexat (MTX) and/or other drugs (T6, T11) in the 1980s, and in the 1990s, cyclophosphamide was replaced by ifosfamide (VA­IA). The treatment was started with Cht in orbital and head and neck tumours and in the majority of geni­tourinary tumours, but surgery was first in paratesticular and in the majority of extremity tumours. Results. The 3 patients with stage IV disease died. Of those with localised tumour, 34 (70%) were alive and well 5 years after treatment, 80% stage I, 75% stage II and 61% stage III. One patient died of heart failure, 3 of Cht toxicity and 1 of intercurrent disease. Conclusions. The survival of our patients has improved during the last 2 decades and increased from 57 % to 70 % for patients treated after l985. It is now comparable to that in other centres. With the introduction of neoadjuvant Cht, surgery and RT have become more conservative and could sometimes even be aban­doned, thereby reducing considerably the risk of late sequels. Orbital, genitourinary and paratesticular em-bryonal RMS of low stages have very good prognosis. Primary tumours of the extremities and head & neck, mainly of alveolar type, have poor prognosis. For alveolar type of RMS and stage IV tumours, the present treatment modalities, including ABMT, are not effective. Key words: rhabdomyosarcoma – therapy, child, survival analysis Correspondence to: Prof. Berta Jereb, MD, PhD, Institute of Oncology, 1000 Ljubljana, Zaloška 2, Received 3 October 2001 Slovenia; Phone +386 1 522 42 49; Fax +386 1 431 41 Accepted 20 October 2001 80, E-mail: zpohar@onko-i.si Introduction When treating a child with a malignant tu­mour we try to achieve a cure with minimal risk for early, as well as late toxic effects of treatment.1 This aim is easier to reach in ear­ly stages of the disease. The cure rate has sig­nificantly improved in the last decades with the introduction of multidrug chemotherapy (Cht). This is now used practically in all solid malignant tumours in children.2,3 With the introduction of neoadjuvant Cht, surgery and radiation therapy have acquired more the role of adjuvant treatment may be more conserva­tive and even be omitted in some cases after a complete response to Cht. The advantages are twofold: the response of the tumour can be evaluated in each particular patient, but not only statistically. Therefore, it can contin­ue and be successful also after surgery. Moreover, Cht can be sufficient as the only treatment.4,5 A morphological diagnosis to confirm malignancy is required before treat­ment as well as for the choice of proper Cht.6 The aim of this study was to review the chil­dren with rhabdomyosarcoma (RMS), treated at the Hemato-oncological unit of the University Children’s Hospital and at the Institute of Oncology in Ljubljana during a 23-year period. Patients and method Between l974 and l996, 5l children aged from l to l5 years (median 6) and registered at the Cancer Registry of Slovenia with the diagno­sis of RMS, were managed by a team of physi­cians at the Hemato-oncological unit of the University Children’s Hospital and the Institute of Oncology in Ljubljana. Five chil­dren with RMS, who were first treated and /or followed outside Slovenia, are not includ­ed. Evaluation of the tumour extent included clinical examination, plain radiography com­puted tomography (CT), magnetic resonance imaging (MRI), ultrasound, technetium bone scan, bone marrow biopsy, spinal tap in pa­tients with parameningeal disease and those with stage IV tumours. The tumours were staged according to the SIOP-UICC7 as fol­ lows: Stage I: Tumour restricted to the organ of origin 12 Stage II: Tumour invasion beyond the organ of origin 10 Stage III: Tumour with regional metastases 26 Stage IV: Tumour with distant metastases. 3 The following primary sites were defined: Head & Neck 15 Parameningeal 8 pterigopalatinal fossa 1 nasopharynx 3 maxilla 4 face 5 ear lobe 1 oral cavity 1 Orbit 6 Genitourinary 12 vagina 4 prostate 1 (para)vesicular 7 Paratesticular 4 Extremity 9 lower 5 upper 4 Torso 5 There were 27 boys and 24 girls less than l6 years old at diagnosis (mean 6.9, SD 4,2). The diagnosis was provided by fine needle as­piration biopsy (FNAB) in 20 and by surgical biopsy in 31 patients prior to treatment. Of 43 histologically confirmed RMS, 25 were em-bryonal, 13 alveolar, 1 botryoid, 1 spindle cell and 3 sarcoma NOS. Multidrug chemothera­py varied. Vincristine, actinomycin D and cy­clophosphamide (VAC) were used in the 1970s, VAC in combination with adriablas- Table 1. Chemotherapy according to stage (51 pa­tients) CHT/ Stage VAC T2, VACA & VAIA T6, T9 & T11 NP* I II III IV Total 2 3 2 / 7 6 5 10 / 21 3 1 14 / 18 2 / / 3(ABMT) 5 *CIVADIC tine (T2 -protocol, VACA) and methotrexate (T6 or T11-protocols) in the 1980s. VACA or VAIA (cyclophosphamide replaced by ifos­famide) were used in the 1990s (Table 1). All 5l patients received Cht; 29 neoadju­vant, 20 had surgery first, in 40, Cht was combined with irradiation (RT). Of the 3 stage IV patients, 2 had bone marrow trans­plant (ABMT) at first treatment and one had half body RT in combination with Cht (VAIA alternating with VP 16 and cisplatinum) and RT to all involved areas. The treatment was started with Cht in or­bital and head and neck and in the majority of genitourinary tumours, whereas surgery was the first treatment in paratesticular and in the majority of extremity tumours. Twenty-one patients had no surgery (2 stage I, 1 stage II, 15 stage III and the 3 stage IV patients). Surgery was delayed in 10 patients (5 geni­tourinary, 1 orbital recurrence, one extremity and one torso) (Table 2). Forty patients received RT to primary or metastatic site (including the 2 who had ABMT). In 2 patients with maxillary primary tumours, RT was given at first treatment, while in 24 patients, after Cht and in l6 also after surgery. The doses of RT varied between 30 Gy and 50 Gy, depending on tumour stage and response to Cht as well as on the experi­ence of an earlier study.8 Two patients re­ceived 60 Gy. The patients were followed between 5 and 26 years (median 12) from the diagnosis. None were lost from the follow-up. The sur­vival was calculated from the date of the di­agnosis until December 3lst 2000, when the study was concluded, or until the date of death. It was presented in survival curves ac­cording to the Kaplan-Meier method.9 The statistical significance was calculated using the log rank test. Results Sixty-seven % of all patients included in the study and 70 % of those with locoregional dis­ease were alive and well more than 5 years from diagnosis. The survival of patients treat­ed before l985 was 57 % (12/21) and 70 % (21/ 30) of those treated after 1985. Of the l7 pa­tients who died, l3 died of tumour, 4 of com­plications and one, l8 years after diagnosis, of an intercurrent disease without tumour. Toxicity due to Cht was the cause of death in Table 2. Treatment according to primary site (48 patients, stage IV excluded) Site Ctotal hemotherapy 1th 2nd total Surgery 1th later RT* total Orbit 6 5 1 2 1 1 6 Head& Neck 13 5 8 6 6 / 13 Genitourinary 12 7 5 10 5 5 8 Extremity 7 4 3 6 3 3 6 Torso 6 5 1 2 1 1 4 Paratestis 4 / 4 4 4 / / Total 48 26 22 30 20 10 37 *radiotherapy 2 patients treated for recurrence and in one stage IV patient. Cardiotoxicity was the cause of death in one patient with stage III alveolar RMS of the anterior thoracic wall 8 years af­ter completed treatment. At the time of death, a recurrence was also present. She was 3 years old at diagnosis. She was treated by Cht according to T2 protocol, including adri­ablastine and concomitant RT to the thoracic wall (4500 Gy given in split courses during 3 months). The survival of the patients with stage I and stage II was very good, while it was sig­nificantly worse for the patients with stage III and stage IV (Figure 1). None of the stage IV patients survived, all had multiple bone metastases. All patients with orbital tumours and those with paratesticular tumours sur­vived. Of the l2 patients with genitourinary tumours, one with primary tumour of the prostate died. Patients with primary tumours of the head and neck, torso and extremities did poorly. The patients with paratesticular, orbital and genitourinary primary tumours did significantly better than those with tu­mours of other primary sites (Figure 2). The survival was not different in those patients in whom treatment was started with Cht com- PA = paratesticular, OR = orbit, GU = genitourinary, TO = torso, EX = extremity Figure 2. Disease free survival by tumor site in 48 pa­tients Figure 3. Difference in disease free survival between patients with adjuvant and neoadjuvant chemothera­py (48 patients) pared to those who received Cht postopera­tively (Figure 3). Furthermore, there was no difference in survival between those who had no surgery, delayed surgery or surgery as first treatment (Figure 4). The outcome of the dis­ease in patients who had more intensive Cht Figure 1. Disease free survival by stage in 51 patients. Figure 4. Disease free survival by surgery in 48 pa­tients Figure 5. Disease free survival by type of chemothera­py in 48 patients. was not better compared to those who had VAC (Figure 5). The morphological type of RMS did not significantly influence survival (Figure 6). There were two late deaths after re­currence, both in patients with alveolar type of RMS. Discussion In our series of patients with RMS the distri­bution of sex, age and primary sites is similar to that of other reported studies. The same is true for the overall 5-year survival, which has improved in Slovenia during the last two decades from 57 % to 70 %.10,11 The stage of the disease and the site of the primary tu­mour significantly influence the outcome, as known. Recently, a staging classification, based on the experience of the IRS group, was proposed, with some low stage tumours classified as unfavourable and some tumours of higher stages with expected favourable outcome.12 In our experience, this has not been entirely confirmed. Patients with pri­mary parameningeal tumours, classified as unfavourable, regardless of the stage, do ex­tremely poorly (only one of the 8 survived) as also reported by others. All 7 patients with vesicular and paravesicular primary tumours, however, are among survivors (Table 3). Only 2 of them had tumours with less than 5 cm in diameter, all embryonal RMS. All patients with genitourinary primary tumours, except one with a primary tumour of prostate, sur­vived. The influence of tumour morphology on survival could not be statistically confirmed in our small series; however, it was noted that the patients with the alveolar type of tumour did poorly (Figure 6). This finding is consis­tent with other reports.13,14 Fourteen patients had alveolar RMS, including 3 stage IV pa­tients and only 5 out of the remaining 11 with locoregional disease are among survivors. Of the 6 who died, 5 had local recurrence (one died of cardiotoxicity with the tumour still present). They died 3, 4, 6 and 8 years after diagnosis. The results of the IRS studies II and III differ regarding the influence of tu­mour morphology on survival. It has there­fore been suggested that the primary site, cor­related to the histological type, is the prevailing and independent prognostic fac- Table 3. Patients with primary tumours of the bladder or tumours invading the bladder Pt I Sex Age in Year of Prim. site Stage Type Surgery RT Cht Late effects years diagnosis I.P. M 5 1976 paravesicular T2 N1 M0 S TR 48Gy VAC elevated FSH, soft tissue atrophy, recurrent cystitis M.P. M 2 1978 bladder TlN0M0 E Cystectomy none VAC stoma, recurrent uroinfections K.Ž. F 5 1984 paravesicular T2N0M0 E STR 46 T11 soft tissue atrophy, amenorrhoe M.J. F 1.5 1990 paravesicular T2N0M0 E STR none T2 none R.U M 7 1993 paravesicular T2N0M0 E STR 30* VACA none B.K. M 2 1994 bladder T2N0M0 E none 37.5 VACA none A.M. F 2 1995 bladder TlN0M0 E TUR 39** VACA none *Hyperfractionation, **Transperineal implant, Ir 192, TR = Total resection with partial cystectomy, TUR Transuretral resection, STR Subtotal resection, S = spindle cell RMS, E = embrional RMS, FSH = follicle simulat­ ing hormone Figure 6. Disease free survival by morphological type in 51 patients. (A = Alveolar, E = Embryonal, B = Botroyd, S = spindle cell, NOS= RMS not otherwise specified) tor.15 It might also be possible that the mor­phology of the tumour did not influence the outcome significantly because the patients with alveolar tumours received more inten­sive Cht. In our series, the primary site and the type of the tumour were correlated (Table 4). All tumours of the extremities were of the Table 4. Primary site versus morphological type Primary site Alveolar Embryonal and other Orbit 1 5 Head & neck 6 9 Genitourinary 0 16 Paratestis 0 4 Extremity 5 0 Torso 2 3 Total 14 37 alveolar type and all genitourinary and parat­esticular tumours were of nonalveolar type. The survival of patients in our study was not influenced by different schedules of Cht (Figure 5), which was probably too aggressive for some groups of patients. At least 3-drug combinations were used, often 4 or even more. ABMT has not been reported as suc­cessful in patients with RMS,16 which was al­so true in our 3 stage IV patients who all had bone metastases. The group of patients with bladder and paravesicular tumours, presented in Table 3, reflects the development of treatment ap­proaches during the two decades, for the en­tire series. Radical surgery and high dose RT were applied in the 1970s and very aggressive and Cht with more conservative surgery and lower doses of RT in the 1980s. Later, surgery and RT were more conservative, while 4-drug Cht was still used. With equal results in terms of survival, the consequences of treat­ment are considerably reduced. The survival curves for those patients who were first treated with surgery and those who had neoadjuvant Cht and delayed, more con­servative surgery are identical (Figure 6). The patients who had no surgery at all did not do significantly worse, even with the inclusion of the patients with high-risk parameningeal tumours. Conclusions During the period under investigation, the methods of treatment of children with RMS have evolved. With the introduction of neoadjuvant Cht, surgery and RT have be­come more conservative or were even aban­doned, thereby reducing the risk of late se­quelae. The survival of our children with RMS has improved during the period under investigation and is now comparable to that in other centers. Orbital, genitourinary and paratesticular embryonal RMS of low stages have very good prognosis and may be cured with conservative treatment and less toxic chemotherapy. Primary tumours of the ex­tremities and head and neck, mainly of alveo­lar type, have poor prognosis with present treatment modalities. Hopefully, new Cht schedules, will prove more effective. For alve­olar type of RMS and stage IV tumours the present treatment modalities, including ABMT, are not effective; therefore, new ap­proaches should be considered.17 References 1. Tefft M, Lattin PB, Jereb B, Cham W, Ghavimi G, Rosen G, et al. Acute and late effects on normal tissues following combined chemo- and radiother­apy for childhood rhabdomyosarcoma and Ewing’s sarcoma. Cancer 1976; 37: 1201-13. 2. Taub JW. Factors in improved survival from pae­diatric cancer. Drugs 1998; 56: 757-65. 3. Donaldson SS, Anderson J. Factors that influence treatment decisions in childhood rhabdomyosar-coma. Intergroup Rhabdomyosarcoma Study Group of the Children’s Cancer Group, the Pediatric Oncology Group, and the Intergroup Rhabdomyosarcoma Study Group Statistical Center. Radiology 1997; 203: 17-22. 4. Oberlin O, Rey A, Anderson J, Carli M, Raney RB, Treuner J, et al. Treatment of orbital rhab­domyosarcoma: Survival and effects of treatment – Results of an international workshop. J Clin Oncol 2001; 19: 197-204. 5. Hays DM. Bladder/Prostate Rhabdomyosarcoma: Results of the multi-institutional trials of the Intergroup Rhabdomyosarcoma Study. Sem Surg Oncol 1993; 9: 520-3. 6. Sommelet D, Pinkerton R, Brunat-Mentigny M, Farsi F, Martel I, Philip T, et al. Standards, options and recommendations (SOR) for clinical care of rhabdomyosarcoma (RMS) and other soft tissue sarcoma in children [Article in French]. Bull Cancer 1998; 85: 1015-42. 7. Rodary C, Flamant F, Donaldson SS. An attempt to use a common staging system in rhab­domyosarcoma: A report of an international work­shop initiated by the International Society of Pediatric Oncology (SIOP). Med Pediatr Oncol 1989; 17: 210-5. 8. Jereb B, Ghavimi F, Exelby P, Zang E. Local con­trol of embryonal rhabdomyosarcoma in children by radiation therapy when combined with chemotherapy. Int J Rad Oncol Biol Phys 1980; 6: 827-33. 9. Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. J Am Stat Assoc 1958; 53: 457-81. 10. Dagher R, Helman L. Rhabdomyosarcoma: an Overview. Oncologist 1999; 4: 34-44. 11. Crist W, Gehan EA, Ragab AH, Dickman PS, Donaldson SS, Fryer C, et al. The Third Intergroup Rhabdomyosarcoma Study. J Clin Oncol 1995; 13: 610-30. 12. Baker KS, Anderson JR, Link MP, Grier HE, Qualman SJ, Maurer HM, et al. Benefit of intensi­fied therapy for patients with local or regional em-bryonal rhabdomyosarcoma: Results from the Intergroup Rhabdomyosarcoma Study IV. J Clin Oncol 2000; 18: 2427-34. 13. Pappo AS, Anderson JR, Crist WM, Wharam MD, Breitfeld PP, Hawkins D, et al. Survival after re­lapse in children and adolescents with rhab­domyosarcoma: A report from the Intergroup Rhabdomyosarcoma Study Group. J Clin Oncol 1999; 17: 3487-93. 14. Wolden SL, Anderson JR, Crist WM, Breneman JC, Wharam MD Jr, Wiener ES, et al. Indications for radiotherapy and chemotherapy after complete resection in rhabdomyosarcoma: A report from the Intergroup Rhabdomyosarcoma Studies I to III. J Clin Oncol 1999; 17: 3468-75. 15. Kodet R, Newton WA Jr, Hamoudi AB, Asmar L, Wharam MD, Maurer HM. Orbital rhabdomyosar-comas and related tumors in childhood: relation­ship of morphology to prognosis—an Intergroup Rhabdomyosarcoma Study. Med Pediatr Oncol 1997; 29: 51-60. 16. Boulad F, Kernan NA, LaQuaglia MP, Heller G, Lindsley KL, Rosenfield NS, et al. High dose in­duction chemotherapy followed by Co autologous bone marrow transplantation as consolidation therapy in rhabdomyosarcoma extra-osseous Ewing’s sarcoma, and undifferentiated sarcoma. J Clin Oncol l998; l6: l697-706. 17. Pappo AS, Lyden E, Breneman J, Wiener E, Teot L, Meza J, et al. Up-front window trial of topotecan in previously untreated children and adolescents with metastatic rhabdomyosarcoma: An Inter­group Rhabdomyosarcoma Study. J Clin Oncol 2001; 19: 213-9. The technique of craniospinal irradiation of paediatric patients in supine position Pavel Šlampa1, Zuzana Seneklova1, Jiri Simicek1, Renata Soumarova1, Petr Burkon2, Ludmila Burianova2 1Masaryk Memorial Cancer Institute, 2St. Anneis University Hospital, Brno, Czech Republic Background. Postoperative radiation therapy has significant impact on local control and overall survival of paediatric patients with brain tumours but an irradiated volume is often a controversial issue. Our aim was to describe a new technique of craniospinal irradiation as a postoperative treatment in patients with the risk of relapse of brain tumours as well as to estimate the side effects of such craniospinal irradiation. Patients and methods. In the last 4 years, 17 paediatric patients under 15 years of age with medulloblas­toma (8) ependymoma (6) and glioblastoma (3) received postoperative craniospinal axis radiotherapy by a new technique developed in our departments. This technique is based on irradiation in supine position with the use of asymmetric jaws of the linear accelerator. Results. Radiotherapy was well tolerated and dose-reduction was not needed in any case. Skin reactions were mild in all patients. The gastrointestinal and haematological toxicity was mild to moderate (WHO grade I-II). Conclusion. The proposed new technique of craniospinal irradiation is advantageous in terms of side ef­fects and could be recommended to be widely used. Craniospinal irradiation in supine position is an alter­native method to the treatment in prone position. The evaluation of the effectiveness was limited by a short follow-up interval. Key words: brain neoplasms – radiotherapy; radiotherapy – methods; supine position; child Received 26 October 2001 Accepted 3 November 2001 Correspondence to: Pavel Šlampa, M D, PhD, Head, Department of Radiation Oncology, Masaryk Memorial Cancer Institute, Zl. kopec 7, 656 53 Brno, Czech Republic; Fax: +420 5 4313 1117; E mail: slampa@mou.cz Introduction The primary treatment of choice of the brain tumours is surgery. Combination of treat­ment modalities is preferred in the treatment of medulloblastomas, ependymomas and gliomas. Postoperative radiation therapy has significant impact on local control and overall survival. In general, postoperative radiothera­py has been reported to improve the out­come. Meta-analyses of the patients irradiat­ed postoperatively have revealed a prolonga­tion of the 5-year survival rate by 20-30 % (ependymomas) and 60 % (medulloblas­tomas). An irradiated volume is often a con­troversial issue, especially regarding irradia­tion of the spinal axis after resected intracra­nial ependymomas and gliomas. In this study, we report a new technique for cran­iospinal irradiation developed in our depart­ments.1-6 Patients and methods Between January 1997 and March 2001 a total number of 17 (medulloblastoma 8, ependy­moma 6, glioblastoma 3) paediatric patients aged under 15 years (median age 6.6 years) were treated in the Departments of Radiation Oncology, St. Anne’s Hospital and Masaryk Memorial Cancer Institute in Brno. Surgical resection was performed in all patients. All tumours were histologically proved and were localized infratentorially in the posterior fos­sa. In the indicated cases, chemotherapy was administered after radiotherapy. All of the patients were irradiated with a dose of 24-36 Gy to the whole craniospinal axis and with a dose of 50-54 Gy to the tumour bed (30-36 Gy “high risk”, 24-30 Gy “standard risk” group). In cases with residual tumour a total irradia­tion dose up to 58-60 Gy was delivered. A new radiotherapy technique using asymmet­ric jaws of the linear accelerator was em­ployed in all patients. The development of modern accelerators enables the use of asymmetric jaws in whole brain irradiation in order to minimise the risk of damage of critical areas in close proximity to the target volume, and to diminish the risk of overdosage at the border of the adjacent fields in the area of the irradiated spinal canal (Figure 1).7-10 The definition of the planning the target volume: 1) Whole brain: this volume encompasses the whole brain with 1 cm safety margin. The lower limit of the frontal area must be 5 mm below the frontal sinus and 1 cm be­low the temporal lobes. In front of vertebra C2, 5 mm are required. 2) Spinal axis: the inferior limit must be ver­tebra S2-4. The lateral safety margin of 5 mm is required regarding the lateral process. ICRU 50 point of the whole brain is on the mid axis of the target volume and that of the spinal axis is on the axis of medullar cord (Figure 2). Treatment plan­ning was based on a series of about 20-25 consecutive CT slices. The use of three-di­mensional treatment planning is a stan­dard method. In the whole brain and cervical spine irradi­ ation (with the caudal border C3-C4 ), two op­posite lateral fields were chosen with shield­ing blocks of the eye bulbs; the spinal cord was irradiated with two direct posterior fields. After reaching 33 % and 66 % of the planned dose, the size and the borders of the adjacent fields in the area of the spine were modified (Figure 3). It was necessary to include the whole vertebral volumes in the irradiated vol­ume in order to diminish the risk of postirra­diation scoliosis of the spine. Patients were ly­ing in supine position and were fixed by a vac­uum body immobiliser and ORFIT head mask. With the use of the vacuum body immobiliser, the applied depth dose decreased by 1-2 %. This irradiation technique caused a tolerable increase of the superficial skin dose with re­gard to the total dose applied to the planning Figure 1. Modified technique of craniospinal irradiation with the use of two opposite lateral fields (individual shielding blocks of the eye bulbs and the face part of the skull) (a), and two direct fields (b), with the use of the asymmetric jaws (c). (Linear accelerator, X-ray, 6 MV). target volume. Irradiation was performed us­ing standard fractionation (5 fractions per week) with a single dose of 1.5-1.8 Gy for cran­iospinal axis by photon beam (6MV) of the lin­ear accelerator. It was necessary to determine exactly the position of the child on the treat­ment table by laser beams and the optical pointer of the irradiation device. Results The prescribed dose of irradiation was deliv­ered to all patients; dose reduction was not necessary in any of them. In October 2001 (date of evaluation), 8 out of 17 patients (47 %) were without any sign of disease and had no serious problems. Two patients pre­sented significant neurological symptoms. Local relapse occurred in one patient, but was successfully treated by surgery. Six patients (35 %) died of local recurrence (5 of these 6 patients had partial resection). Radiotherapy was well tolerated. Skin reac­tions were mild and were of grade 1 (WHO) in 15 (88 %) patients and of grade 2 in two (12 %) patients. Within three months after the com­pletion of radiotherapy, these reactions disap- Figure 2. Isodose plan of irradiation spinal axis with two changes of the borders of the adjacent fields. peared. The gastrointestinal and haematologi-de I in 47 % and grade II 23 %, nausea in 47 %. cal toxicities were mild to moderate in all pa-Further evaluation of the effectiveness of tients (WHO 1-2 gr.). Leucopenia grade I oc-our therapy is not feasible due to the small curred in 70 % of patients, grade II in 18 %, number of patients and short follow-up inter-trombocytopenia grade I in 35 %, diarrhoea gra-val. Discussion Irradiation of the craniospinal axis is a part of the treatment modality of a number of malig­nant diseases. Patients are usually treated in prone position which is not as comfortable, reproductable, and as easily maintained as supine position.5-7 Acute skin, haematological and gastroin­testinal reactions were comparable with those in patients irradiated in prone position.11 To minimise anaesthesia related risks, the irradiation in supine position would be preferable to standard prone position. The treatment in supine position would be more comfortable for adult patients as well.12 In this study, we presented a technique of craniospinal axis radiotherapy with the use of asymmetric jaws of the linear accelerator in order to minimise the risk of irradiation in­duced toxicity to healthy tissue and to over­come the risk of overdosage at the adjacent fields. It is necessary to use a three-dimen­sional treatment planning. The assessment of the effectiveness is limited by the short fol­low-up interval.13,14 In conclusion, our technique of cran­iospinal axis irradiation is advantageous in terms of tolerability and side effects. We be­lieve that it deserves to be widely used. References 1. Bloom HJ, Glees J, Bell J. The treatment and long­term prognosis of children with intracranial tu­mors. A study of 610 cases, 1950-1981. Int J Radiat Oncol Biol Phys 1990; 18: 723-45. 2. Carrie C, Mottolese C, Bouffet E. Non-metastatic childhood ependymomas. Radiother Oncol 1995; 36: 100-6. 3. Sterba J, Pavelka Z, Skotakova J, Kepak T. Current chemotherapy options in the treatment of malig­nant gliomas of the CNS in children. Czech-Slov Pediatrie 2001; 56: 537-41. 4. Perez CA, Brady LW, editors. Principles and practice of radiation oncology. Third edition. Philadelphia: Lippincott-Raven Publ; 1998. 5. Miralbell R, Bleher A, Huquenin P, Ries G, Kann R, Mirimanoff RO, et al. Pediatric medulloblas­toma: Radiation treatment technique and patterns of failure. Int J Rad Oncol Biol Phys 1997; 37: 523-9. 6. Slampa P, Spurny V, Cervena R. Radiotherapy and prognosis of ependymomas [Czech]. Clin Oncol 1997; 10: 139-42. 7. Dobbs J, Barret A, Ash D. Practical radiotherapy planning. Third edition. London: Arnold; 1999. 8. Griffiths, S.E., Short, Ch.A, et al. Radiotherapy: principles to practice. First edition. Edinburgh: Churchill Livingstone; 1994. 9. Vôute PA, Barrett A, Lemerle J. Cancer in children. Clinical management. Third edition. Berlin: Springer-Verlag; 1992. 10. Gunderson LL, Tepper JE. Clinical radiation oncolo­gy. First edition. Philadelphia: Churchill Livingstone; 2000. 11. Hawkins RB. A simple method of radiation treat­ment of craniospinal fields with patient supine. Int J Radiat Oncol Biol Phys 2001; 49: 261-4. 12. Rades D, Baumann R, Bremer M, Leuwer M, Karstens JH. Application of a new verification technique allowing craniospinal irradiation in supine position. Radiother Oncol 2001; 58: 215-7. 13. Slampa P, Seneklova Z, Sterba J, Dembicka D, Burianova L. The new technique of craniospinal irradiation. Med Pediatr Oncol 2000; 35: 318. 14. Taylor RE. The SIOP/UKCCSG PNET strategy and future SIOP medulloblastoma studies. Med Pediatr Oncol 2000; 35: 220. Case report Anal ultrasound in the diagnosis of anal carcinoma Iwona Sudol-Szopinska1, Marek Szczepkowski2, Wieslaw Jakubowski1 1Department of Diagnostic Imaging, Second Faculty of Medicine, 2Second Surgical Department, Bielany Hospital, Warsaw, Poland Background. We present a case of adenocarcinoma of the anal canal to demonstrate the usefulness of the anal ultrasound (AUS) in the diagnostics. Case report. AUS was performed with the use of Bruel&Kjaer scanner type 3535 with an axial 7.0 MHz endoprobe. Examination was performed in left lateral position. AUS allowed for exact assessment of the depth of infiltration of the anal wall by the tumor, and precisely visualized its location and echotexture. Assessment of the perianal lymph nodes and tumor spread into ad­jacent tissues was also possible. Conclusions. AUS is a valuable imaging method in assessing the depth of invasion of anal carcinoma and gives valuable information before deciding on the choice of treatment. Key words: anus neoplasms – ultrasonography Introduction Case report Anal tumors account for about 2.5-5 % of all malignant tumors of the colon. Their precise staging is important with regard to different methods of treatment. The depth of penetra­tion within the wall of the anal canal is poor­ly recognized by digital examination. We present the ultrasound images and useful­ness of anal ultrasound (AUS) in staging of anal canal carcinoma. Received 13 September 2001 Accepted 30 September 2001 Correspondence to: Iwona Sudol-Szopinska, MD, PhD, Zaklad Diagnostyki Ultrasonograficznej, Wojewódzki Szpital Bródnowski, 03-285 Warszawa, ul. Kondratowicza 8; Phone/Fax +48 22 811 95 91; mo­bile 0 501 716 407; e-mail: mdyvonne@poczta.wp.pl An 83 year-old man complaining of anal bleeding and constipation observed for one year was admitted to proctologic outpatient clinic. Per rectum examination revealed an abnormal, mobile mass on the anterior wall of the anal canal, with the diameters of 2.5×2 cm. During anoscopy, a specimen was taken and the histopathologic diagnosis revealed adenocarcinoma. Prior to AUS, an enema was done. AUS was performed to assess the stage of the tumor that was essential to choose a treatment method. For anal ultrasonography, Bruel&Kjaer scanner, type 3535 with axial endoprobe of a frequency of 7.0 MHz and covered by a plas­tic cone with the external diameter of 17 mm was used. The cone was filled with a few mil­liliters of degassed water, covered with a con­dom, and then introduced into the anal canal up to the depth of 5 cm. At the mid anal lev­el, at a depth of 2 cm, a tumor was visualized originating from the mucous of the anterior and right wall, infiltrating a distal end of the internal anal sphincter (Figure 1). The tumor mass was getting larger towards the anal ori­fice and was infiltrating the subcutaneous part of the external anal sphincter at its ante­rior wall. At the low anal level, its diameter on axial image reached 15×13mm (Figure 2). The tumour’s echotexture was not homogenous with anechoic areas representing most likely degeneration. No enlarged lymph nodes were visualized in the perianal tissues and the sur­rounding structures were not invaded. According to the sonographic classifica­tion (uTN), the stage of the disease was de­fined as uT2N0. The tumor’s size less then 2.5 cm, its mobility on rectal examination and lack of enlarged lymph nodes in perianal tis­sues were all the signs that spoke in favor of the local excision of the tumor. A polypoid le­sion on the anterior wall at the low anal level, 1-2 cm from the anal orifice, of the size of 2.5×1cm and degenerating in the center was visible during operation. The result of the histopathologic examination of the interoper­ative specimen was adenocarcinoma muci­nosum partim gelatinosum in adenoma villo­tubulare ani. A malignant infiltration was found in the superficial muscular layer with­in a transition zone of the anal canal. Discussion The presented case of adenocarcinoma of the anal canal is rare, not only because of its prevalence but also its histologic type. Among the most frequent epithelial neo­plasms, such as carcinoma planoepitheliale, basocellulare and mucoepidermale, adeno-carcinoma is the most rarely diagnosed.1 Additionally, anal canal carcinomas most fre­quently occur in patients between 55-60 years of age, and mostly affect women (ratio 2:3). The staging of the anal canal cancers is im­portant in planning treatment strategies (which include local excision, abdomino-per­ineal resection of the rectum, radiotherapy, chemotherapy).1-3 The TNM classification system currently used is based on the result of rectal examination, where only the mar­gins of the tumor around the anal circumfer­ence and its proximal and distal ends, are as­sessed without assessing its mobility and evaluating the depth of penetration of the tu­mor into the canal wall. There is a small num­ber of reports presenting the results of imag­ing methods for anal tumors and they are mostly addressed to low rectal cancers invad­ing the anal canal. AUS in anal tumors diag­nostics enables to define precisely the loca­tion of the tumor and its relation to the anal levels and walls. It is possible to assess local advancement with AUS because the layered structure of the anal canal is visible on AUS image.4-8 Similarly as rectal tumors, anal car­cinomas are also staged according to uTN classification, where Au” means that ultra-sonography was used to determine the stag­ing. In: 1) uT1 tumor is limited to submucosa and mucosa 2) uT2 is limited to sphincters 3) u T3 infiltrates perirectal tissues, 4) u T4 invades surrounding structures. N0 and N1 mean lack or metastatic re­gional lymph nodes, respectively. An anal carcinoma in AUS image appears as hypoechoic mass, most commonly not ho-mogenous with areas of degeneration and with irregular outlines. Biopsy is necessary to confirm final diagnosis. In literature, single publications can be found on the accuracy of AUS in anal tumors staging, which is said to be almost 86 %.2,3,9 In a study by Novell F. et al.3 accuracy of AUS in local staging of anal canal cancers was 85.7 %: in a group of seven patients with anal carcinomas, sonographical assessment caused downstaging in only one case. In another study9, AUS was performed on a group of 30 patients with anal cancers. AUS accurately assessed the depth of inva­sion by tumors and their relation to the sur­rounding structures. The sonographic diagnosis influenced the choice of treatment methods. In the present­ed case, the staging of the anal cancer and its relation to the anal sphincters was not prob­lematic at all. Lymphadenopathy is found in 25 % of cases with anal tumors. It is said that sensitivity of endosonography in visualizing enlarged lymph nodes is 83 %. Lymph nodes larger than 3 mm are already visible on AUS.5,8 The accuracy of endosonography and endoluminal magnetic resonance imaging (MRI) for lymph node staging is 62-83% and 39-95 %, respectively.10 The accuracy of com­puted tomography (CT) is between 22-73 %.10 The studies comparing AUS and MRI show that MRI is inferior to AUS in N staging, al­though the specificity of both is low.10-12 The size appeared to be unreliable criterion10,12 whereas their echogenicity appeared to be more reliable. Hypoechoic lymph nodes rep­resenting metastases were predicted with a sensitivity of 72 %.6 When the diagnostics of malignant dis­eases is concerned, AUS is currently used be­fore surgery of rectal tumors. Its ability to as­sess the contraction activity of striated muscle in the so-called “dynamic examina­tion” is important.13 For the anal canal tu­mors diagnostics, it is of a great value and, comparing to the endosonographic diagnos­tics of rectal tumors, AUS will probably have greater value due to less problematic and more equivocal criteria of diagnosis for each stage of the anal carcinomas. In postoperative follow-up, AUS may allow an early diagnosis of local recurrence in perianal tissues before they are evident on clinical examination.14 In case of primary radiation or chemotherapy, AUS may be used to assess the tumor re­sponse to treatment from the changes of its size and echotexture.2,3 A fine needle aspira­tion biopsy under sonographic control of an abnormal lesion is also possible to perform.3 Simplicity, availability, non-invasiveness of AUS together with excellent images of anal cancers and a precise assessment of their in­vasiveness are the advantages of this method, which should be carried out in addition to digital and anoscopy examinations and should have its place in the diagnostic algo­rithms of the malignant diseases of the anal canal, as it already has for rectal cancers. References 1. Góral R. Chirurgia odbytnicy i okreznicy. Warszawa: PZWL;1993. 2. Herzog U, Boss M, Spichtin HP. Endoanal ultra-sonography in the follow-up of anal carcinoma. Surg Endosc 1994; 8: 1186-9. 3. Novell F, Trias M. Intraluminal anorectal ultra-sonography in the staging of anal canal cancer. Rev Esp Enferm Dig 1993; 84: 153-5. 4. Beynon J. An evaluation of the role of rectal en-dosonography in rectal cancer. Ann Royal Coll Surg Engl 1989; 71: 131-8. 5. Beynon J, Mortensen NJ, Foy DMA, Channer JL, Rigby H, Virjee J. Preoperative assessment of mesorectal lymph node involvement in rectal can­cer. Br J Surg 1989; 76: 276-9. 6. Hildebrandt U, Feifel G, Ecker K-W. Rectal en-dosonography. Bailliere’s Clin Gastroenterol 1989; 3: 531-42. 7. Mortensen N. Rectal and anal endosonography. Gut 1992; 33: 148-9. 8. Zainea GG, Lee F, McLeary RD, Siders DB, Thieme ET. Transrectal ultrasonography in the evaluation of rectal and extrarectal disease. Surg Gynecol Obstet 1989; 169: 153-6. 9. Magdeburg B, Fried M, Mayenberger C. Endo­scopic ultrasonography in the diagnosis, staging, and follow-up of anal carcinomas. Endoscopy 1999; 31: 359-64. 10. Heriot AG, Grundy A, Kumar D. Preoperative stag­ing of rectal carcinoma. Br J Surg 1999; 86: 17-28. 11. Kim NK, Kim MJ, Yun SH, Sohn SK, Min JS. Comparative study of transrectal ultrasonography, pelvic computerized tomography, and magnetic resonance imaging in preoperative staging of rec­tal cancer. Dis Colon Rectum 1999; 42: 770-5. 12. Gualdi G, Casciani E, Guadalaxara A, d’Orta C, Polettini E, Pappalardo G. Local staging of rectal cancer with transrectal ultrasound and endorectal magnetic resonance imaging. Comparison with histologic findings. Dis Colon Rectum 2000; 43: 338-45. 13. Rieger NA, Downey PR, Wattchow DA. Short communication: endoanal ultrasound during con­traction of the anal sphincter-improved definition and diagnostic accuracy. Br J Radiol 1996; 69: 665­7. 14. Beynon J, Mortensen NJ, Foy DMA, Channer JL, Rigby H, Virjee J. The detection and evaluation of locally recurrent rectal cancer with rectal en-dosonography. Dis colon Rectum 1989, 32: 509-17. First experience with a novel luminescence-based optical sensor for measurement of oxygenation in tumors Tomaž Jarm1, Hotimir Lešnicar2, Gregor Serša2 and Damijan Miklavcic1 1University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia, 2Institute of Oncology, Ljubljana, Slovenia Background. The purpose of this preliminary study was to evaluate a novel luminescence-based fiber-op­tic sensor (OxyLite system) for the measurement of partial pressure of oxygen (pO2) in tumors and for the detection of changes in pO2 as a function of time. The new method was used simultaneously with the laser Doppler flowmetry method for the measurement of relative tissue perfusion. Materials and methods. Blood perfusion and pO2 were measured continuously via fiber-optic sensors in­serted into SA-1 tumors in anesthetized A/J mice. The changes in blood flow and oxygenation of tumors were induced by transient changes of the parameters of anesthesia and by injection of a vasoactive drug hy­dralazine. Results. Both optical methods used in the study successfully detected the induced changes in blood flow and pO2. The measurements of pO2 were well correlated with measurements of microcirculatory blood perfu­sion. In the majority of pO2 measurements, we observed an unexpected behavior of the signal during the stabilization process immediately after the insertion of the probe into tumor. This behaviour of the pO2 sig­nal was most probably caused by local tissue damage induced by the insertion of the probe. Conclusion. The novel luminescence-based optical oximetry can reliably detect local pO2 changes in tumors as a function of time but some aspects of prolonged pO2 measurement by this method require further in­vestigation. Key words: sarcoma experimental-blood supply; laser-doppler flowmetry; oxygen; luminiscence Received 3 August 2001 Accepted 28 August 2001 Correspondence to: Prof.dr. Damijan Miklavcic, Faculty of Electrical Engineering, University of Ljubljana, Tržaška 25, SI-1000 Ljubljana, Slovenia. Tel.: +386 1 4768-456; Fax: +386 1 4264-658; E-mail: damijan@svarun.fe.uni-lj.si Introduction Over many years, abundant evidence has been accumulated that the oxygenation status in ex­perimental and clinical tumors can influence the response of these tumors to various thera­pies, e.g. radiotherapy, hyperthermia, oxygen-dependent chemotherapy, photodynamic therapy and cell-mediated immunotherapy.1-3 The oxygenation status and hypoxia in partic­ular are also important for the development of malignant growth and for progression and outcome of the disease.3 The ability to meas­ure oxygenation status in individual tumors would be very valuable for the selection of ap­propriate therapy, treatment planning, and for prediction of the treatment outcome. Many different methods have been devel­oped to measure different physiological pa­rameters related to tumor oxygenation or to tumor blood flow.4,5 In general, tumors are poorly oxygenated in comparison to normal tissues.1,3,5 The measurement methods should therefore enable an accurate assess­ment of very low oxygen levels. Extreme in-tratumoral heterogeneity in oxygenation and blood flow found in many experimental and clinical tumors require an evaluation of oxy­genation and blood flow in different regions within the same tumor. Methods suitable for continuous measurement are particularly useful. They enable monitoring of changes in oxygenation at one location. This could be useful for at least two reasons. First, the ef­fectiveness of treatment procedures targeting tumor blood flow and oxygenation in individ­ual tumors could be evaluated. Second, the susceptibility of individual tumors to oxy­genation- and blood flow-dependent therapy could be evaluated before the therapy is ap­plied. It is also desirable that the measure­ment method of choice is noninvasive. In the present preliminary study, we used two optical measurement methods, which fulfill some of the requirements mentioned earlier in this text. One of them, the time-re­solved luminescence-based optical oximetry, presents a new approach for the measure­ment of oxygen partial pressure in a tissue and is an alternative to the well-established polarographic needle oximetry technique.6,7 This method has only recently become com­mercially available (OxyLite instrument, Oxford Optronix, U.K.). There are two major advantages of this new method over standard polarographic method. First, the lumines-cence-based sensor does not consume oxy­gen; therefore, to monitor the changes in oxy­genation as a function of time, it could be kept in one place in a tissue. Second, its ac­curacy is inversely proportional to oxygen content in the tissue, which makes this method of particular interest for the measure­ment of oxygenation in tumors where low oxygen content is typically encountered. The other optical method used in our study, the laser Doppler flowmetry, is not a new tech­nique, but it has not been used extensively for measurements in tumors. Laser Doppler flowmetry enables the monitoring of micro-circulatory blood perfusion changes. Although microcirculation and oxygenation in a tissue are related, simultaneous use of both methods can give more information about oxygen supply to a tissue than separate use of a single method. Both methods are minimally invasive and require only thin op­tical fibers to be inserted into tissue. Before a new method can be used with confidence in experimental or clinical stud­ies, its characteristics and limitations need to be known and understood. The use of lumi­nescence-based oximetry has been so far doc­umented in very few reports. The main goal of the present study was to get the first expe­rience and to evaluate the usefulness of this new optical method for a continuous meas­urement of tumor oxygenation and for the de­tection of short-term oxygenation changes. The measurements were performed on SA-1 tumors in A/J mice under different conditions modifying blood flow- and oxygenation. Materials and methods Animals and tumors The female A/J mice purchased from Rudjer Boškovic Institute, Zagreb, Croatia, were used in our study. The mice, 10 to 12 weeks old, were kept in standard animal colony at 22°C and were fed and watered ad libitum. The experimental tumor line that we used was SA-1 fibrosarcoma (The Jackson Labo­ratory, Bar Harbor, USA). Tumor cells for the inoculation of solid tumors were obtained from the ascitic form of SA-1 tumor in A/J mice. Approximately 5×105 of viable cells were re-suspended in 0.1 ml NaCl solution (0.9%) and transplanted under the skin. Solid subcutaneous tumors were grown dorsolater-ally on the right flank of mice. Experiments were performed 8 to 10 days after the trans­plantation when the tumors reached the size of approximately 100 mm3. The size of the tu­mors was calculated using the ellipsoid for­mula as V = pabc/6 where a, b, and c are three mutually perpendicular tumor diameters measured by a vernier caliper. At the end of the experiments, the mice were euthanized under anesthesia by cervical dislocation. The experimentation on mice was conducted in accordance with the pertaining legislation and was approved by the Veterinary Administration of Ministry of Agriculture, Forestry and Food of Slovenia (permit num­ber 323-02-156/99). Anesthesia All experimental procedures and measure­ments were conducted on anesthetized mice in order to eliminate pain and discomfort in mice and to minimize movements of non-re­strained mice during long-lasting measure­ments. Anesthesia was induced and main­tained by inhalant anesthetic isoflurane (Flurane-Isoflurane, Abbot Labs, U.K.). The gas mixture of oxygen O2 and nitrous oxide N2O (flow of each 0.6 l/min) containing isoflurane at 1.7 % concentration was deliv­ered to the mouse via a miniature facemask. While anesthetized, the animals were kept on an automatically regulated heating pad to prevent hypothermia. Rectal temperature was kept as close as possible to 37°C with variations of up to 0.5°C during single meas­urements and with the contact surface tem­perature of the heating pad below 39°C. Oxygenation and blood flow measurement Partial pressure of oxygen, pO2, was meas­ured by the OxyLite 2000 instrument (Oxford Optronix Ltd., Oxford, U.K.), a commercially available implementation of a novel time-re­solved luminescence-based optical oximetry. The instrument has two independent chan­nels for measuring pO2 and temperature. The diameter of precalibrated optical probes is 230 µm. A thin wire thermocouple tempera­ture sensor (diameter less than 100 µm) is at­tached to each pO2 probe, which allows on-line temperature correction of pO2 measu­rements. The principles of this luminescence-based optical method are described in more detail elsewhere.6,8 Briefly, pulses of blue light emitted by a LED diode are carried via an optical fiber to ruthenium chloride lu­minophore, which is incorporated in a sili­cone rubber that is used to immobilize the tip of the probe. The tip of the probe is placed in­side the tissue where oxygenation is to be measured. The incident light pulses induce pulsatile fluorescence of ruthenium mole­cules. The fluorescence decays in time be­cause of collisions between the oxygen and the ruthenium molecules. The life-time of the excited fluorescence is inversely proportional to pO2 in the part of the tissue that is in con­tact with the tip of the probe. The pO2 can therefore be calculated from the measured life-time of the fluorescence using the so-called Stern-Volmer relation.8 Relative blood perfusion was monitored using a two-channel OxyFlo 2000 laser Doppler instrument (Oxford Optronix Ltd., Oxford, U.K.). Even though laser Doppler flowmetry (LDF) can be applied entirely non-invasively, we used thin invasive probes (di­ameter 200 µm) in order to assess the perfu­sion inside the tumor. LDF is an optical method used to monitor local microvascular blood flow in a tissue. Extensive literature ex­ists on its theory and application.9,10 Briefly, when a tissue is illuminated by a coherent laser light, the light scatters on different tis­sue structures. When photons are scattered on moving structures, their wavelength is slightly changed. This is the so-called Doppler shift effect, which can be measured. The predominant moving structures in a tis­sue at rest are red blood cells. Their move­ment, which results from blood flow, can be detected by means of the Doppler shift effect. The output signal of LDF is proportional to the red blood cell perfusion which is defined as the number of the red blood cells multi­plied by the mean velocity of these cells that move in the tissue sampling volume. The con­stant of proportionality between the perfu­sion and the detected LDF signal is unfortu­nately different for each location even within the same tissue. This means that all LDF measurements are intrinsically of relative na­ture and are quantified in arbitrary blood per­fusion units (BPU). Both instruments OxyLite and OxyFlo were connected to OxyData data acquisition unit (Oxford Optronix Ltd., Oxford, U.K.), which enabled data storage to a PC via a SC­SI connector. All signals were sampled and stored at the frequency of 20 Hz. Measurement protocol Anesthesia in the mouse was started in the induction chamber at a concentration of isoflurane of 3 %. The mouse was then placed on the heating pad in prone position. No physical restriction was used. The anesthetic gas was delivered via a miniature facemask. The concentration of isoflurane was reduced to 1.7 %, which provided stable anesthesia. Rectal and surface temperature probes were attached for control of the core temperature of the mouse and of the surface temperature of heating pad. Approximately four minutes after the induction of anesthesia, the pO2 and the LDF probes were inserted into the tumor through small superficial incisions in the skin. The probes were inserted through the incisions, pushed a few millimeters fur­ther into the tumor and then slightly with­drawn in order to minimize the pressure of the tip of the probe on the surrounding tis­sue. Although an exact positioning of the probes was not possible, one pair of pO2 and LDF probes was inserted in a peripheral re­gion of the tumor and the other in a central region of the tumor. Data recording was started normally about five minutes after the beginning of anesthesia. Special care was taken throughout the measurement not to move the probes or the mouse in order to minimize the movement artifacts in recorded signals. A typical measurement lasted between one and two hours. In order to evaluate the appli­cability of both measurement methods for the detection of the changes in the blood perfu­sion and oxygenation in tumors, different procedures were applied. These procedures to induce changes in blood flow and oxy­genation were performed only after the recorded signals had been stable for at least ten minutes. Normally, the stability of all sig­nals (two pO2 and two LDF) was reached be­tween 20 and 40 minutes after the start of anesthesia. The effect of hydralazine An arteriolar vasodilator hydralazine (HYZ) was injected i.v. at a dose of 2.5 mg/kg of mouse weight. The solution for injection was prepared from powdered HYZ (Hydrazino­phthalazine, Sigma Chemical Co., U.S.A.) by dissolving it in sterile physiological saline (0.9 % NaCl). The mice in the control group were injected with sterile physiological saline only. The effect of anesthetic concentration Normal anesthesia was maintained by deliv­ering isoflurane at a concentration of 1.7 % in a steady flow of O2 (0.6 l/min) and N2O (0.6 l/min). For the purpose of evaluation of the effect of anesthetic concentration on the blood flow and oxygenation in tumors, the concentration of isoflurane was increased to 3% for three minutes and then returned to the normal level of 1.7 %. The effect of euthanasic procedure In order to evaluate the validity of measure­ments, pO2 and blood perfusion were also monitored during euthanasia of mice at the end of experiment. First the delivery of O2 was eliminated from anesthetic gas mixture while maintaining the flow of nitrous oxide and isoflurane. Within two minutes after this procedure, the mice stopped breathing. Results A typical measurement Both pO2 and blood perfusion were measured at two locations in each tumor. In most cases the data recording was started immediately after all four probes were inserted into the tis­sue and, in few cases, the data recording was started just before the probes were inserted. All this occurred within five minutes after the start of anesthesia. The measurement lasted between one and two hours. Figure 1 shows an example of typical signals recorded simul­taneously from one tumor during the first 40 minutes of measurement. Several important features characteristic of all pO2 and LDF measurements can be observed. After the insertion of the probes, the value of pO2 in all measurements varied rapidly (within one minute). It decreased from initial­ly high level to zero or close to zero value. We call this a decrease phase which cannot be ob­served in Figure 1 because, in this case, the da­ta recording was started after the insertion of the probes. Following the decrease phase, two different types of pO2 recordings were ob­served. An example of the first type, we shall call it a type I pO2 signal, is shown in Figure 1a. In the type I measurements, the pO2 value re­mained at zero or close to zero for a period of time which varied between different measure­ment locations, but in the majority of meas­urements, it lasted about five minutes. We call this a zero pO2 phase. Following the zero pO2 phase, the value of pO2 in the type I measure­ments entered the increase phase during which the value of pO2 was slowly increasing. The in­crease phase lasted on average between 15 and 20 minutes. After the increase phase, pO2 val­ue stabilized and remained mostly unchanged thereafter unless some oxygenation-modifying procedure was applied. We shall refer to this final stage in type I pO2 measurements as to a plateau phase. In most type I measurements, pO2 continued to increase very slowly even during the plateau phase but this increase was much slower than the increase during the in­crease phase. The type I pO2 measurement was found in approximately 70 % of all measure­ment locations in tumors. In about 30 % of all pO2 measurements performed, pO2 in tumors remained at zero or close to zero level for the entire period of observation after the initial decrease phase. An example of this second type of pO2 recording, we shall call it a type II pO2 signal, is shown in Figure 1b. In comparison to the type I measurements, the type II measurements are characterized by a complete absence of the in­crease and the plateau phases (compare the top two graphs in Figure 1). It is very important to note that, in some cases, both the type I and the type II pO2 Figure 1. A typical measurement consisting of two pO2 (a, b) and two LDF measurements (c, d) recorded simulta­neously in the same tumor. The two distinct types of pO2 measurements can be identified (a, b). The first 40 min­utes of recording from a single tumor are shown. measurements were encountered within the riod of five minutes. It is again important to same tumor, as in the case presented in note that these averaged pO2 values in the Figure 1. In other cases, both pO2 measure-type I measurements varied extremely be-ments in tumor resulted in either of the two tween tumors and also from one location to types, the type I or the type II measurement. another within the same tumor, from as little In order to obtain a representative pO2 value as 1 mmHg to more than 40 mmHg in a few for each type I measurement, we averaged the cases. All tumor pO2 values at rest including raw pO2 signal in the plateau phase over a pe-the averaged type II values were pooled to-gether and are presented in a histogram in Figure 2. The median pO2 value of 40 meas­urements from 28 tumors was 10.3 mmHg. A fraction of pO2 values below 2.5 mmHg was 40 %. Contrary to the pO2 signals, only one type of LDF measurements was identified in tu­mors (Figure 1c and 1d). All recorded LDF signals became stable soon after the insertion of probes. A zero or close to zero blood per­fusion was never encountered, which again is contrary to the pO2 measurements. Due to the nature of LDF technique, the blood perfu­sion measurements are extremely sensitive to any kind of movement of probes relative to the surrounding tissue. The “smeared” blood perfusion signals in Figure 1 is a movement artifact caused by a quasi-periodic breathing of the mouse. This is shown in more detail in Figure 3. The spikes in Figure 3 correspond to inhalation and the “valleys” in between the spikes correspond to exhalation. The true blood perfusion is at the lower edge of the “smeared” blood perfusion signals in Figure 1. The amplitude of the quasi-periodic com­ponent in LDF signal, which could be even bigger than the true perfusion component of the signal, depended on the position of indi­vidual LDF probes relative to the direction of tumor movement caused by respiration. Because of this, substantial differences in the relative amplitude of the quasi-periodic com­ponent in comparison to the true perfusion level in different locations within the same tumor were common (compare Figures 1c and 1d). Figure 3. A close-up of a typical blood perfusion signal measured by LDF. The movement artifact caused by qua-si-periodic respiration of the mouse is shown. The effect of hydralazine Within one minute after the injection of HYZ, all type I pO2 and all blood perfusion signals in tumor started to decrease (example in Figure 4b, c, d). In five to ten minutes after the injection, these signals reached the lowest level. On average, pO2 decreased by 80 % (n = 13) and blood perfusion decreased by 50 % (n = 17). HYZ also induced a decrease in respiration rate and an increase in depth of breathing in mice, thus resulting in increased amplitude of the movement artifact in LDF signals. In most tumors treated with HYZ, type I pO2 signals and blood perfusion sig­nals started to recover very slowly approxi­mately half an hour after the injection of HYZ. In the type II pO2 signals (Figure 4a) no change was observed after the injection of HYZ. No significant changes were observed either in the type I pO2 signals or in the blood Figure 4. The effect of hydralazine on tumor pO2 (a, b) and blood perfusion (c, d). The vertical line shows when hydralazine (dose 2.5 mg/kg) was injected. All recordings are from the same tumor. perfusion signals in control animals which were injected with physiological saline (ex­ample not shown). The difference in the de­crease of type I pO2 values in tumors between HYZ-treated and control mice was highly sig­nificant (p < 0.001; Mann-Whitney rank sum test) as was also the difference in the de­crease of blood perfusion between HYZ-treat­ed and control mice (p < 0.001; Mann-Whitney rank sum test). The effect of anesthetic concentration An attempt was made to modify the blood per­fusion and oxygenation in tumors by a tran­sient increase and subsequent decrease of isoflurane concentration in the inhalation mix­ture. An example of the results in one tumor is shown in Figure 5. About one minute and a half after the increase of isoflurane concentra­tion from 1.7 % to 3 %, the blood perfusion started to decrease, as can be seen in both LDF signals in Figure 5c and d. With the increase of isoflurane concentration the respiration in mice to become slower and jerkier, which re­sulted in an increased amplitude of the move­ment artifact in LDF signals. The decrease of blood flow was closely followed by a decrease in the type I pO2 signals (Figure 5a). When isoflurane concentration was returned to nor­mal level of 1.7 %, the blood perfusion started to increase within a few seconds. After a delay of about 1.5 minutes, the type I pO2 signal al­so started to increase. While the LDF signals asymptotically approached the pre-treatment value, the type I pO2 signals usually ap­proached the pre-treatment level after an over­shot as can be seen in Figure 5a. No changes at all were seen during the described proce­dure in the type II pO2 signals (Figure 5b). The effect of euthanasic procedure For the purpose of validation of the measure­ments, pO2 and blood perfusion were also monitored during euthanasic procedure. At the end of each measurement, the flow of oxygen to the inhalation mixture was termi­nated while maintaining the flow of nitrous oxide and isoflurane. A typical example of the effect of this procedure on pO2 and blood perfusion can be observed in Figure 6 which shows the last four minutes of recorded sig­nals in one tumor. The type I pO2 signals and blood perfusion signals rapidly decreased. This occurred within one minute after the shutdown of oxygen flow to the vaporizer. The decrease of pO2 always occurred approx­imately ten seconds before the decrease of blood perfusion. The bottom level of pO2 reached after the death was always close to zero in the range of -0.6 to +0.3 mmHg. The type II pO2 signals (Figure 6b), which were close to zero value during the whole period of observation, remained unchanged during this procedure. In the majority of cases, the bot­tom level of blood perfusion reached after death was slightly above zero value. Discussion The time-resolved luminescence-based optical method used in our study is a new method that has only recently become available on the mar­ket. Its use has been reported by few authors so far.6,7,11-13 Most authors report on evalua­tion of the new method by comparing this method to other techniques for measuring tis­sue oxygenation, in particular to the well-es­tablished polarographic oximetry. By many re­searchers, the polarographic oximetry and its implementation in Eppendorf Histograph in­strument is considered to be the “golden stan­dard” for pO2 measurements in experimental and clinical tumors. A generally good correla­tion between the results of polarographic method and the new optical method was found in tumors,6,13 but there were also dis­crepancies between the two methods in cer­tain conditions and in different tissues.11,13 These discrepancies arose from differences be- Figure 5. The effect of transient change in anesthetic concentration on tumor pO2 (a, b) and blood perfusion (c, d). The two vertical lines show when concentration of isoflurane was changed. All recordings are from the same tumor. tween the two methods such as the underlying physical principle of measurement, dimension of the probe, tissue sampling volume, and con­sumption of oxygen by the sensor. A typical measurement In our study, individual values of pO2 meas­ured with the novel luminescence-based method at rest prior to any blood perfusion-and oxygenation-modifying procedure were scattered in the range of 0 to above 40 mmHg. The great inter- and intratumoral variability in oxygenation observed in our study is in agreement with the well-docu­mented inter- and intratumoral variability of many experimental and clinical tumors.14 The histogram of all measured pO2 values at Figure 6. The effect of euthanasic procedure on tumor pO2 (a, b) and blood perfusion (c, d). The vertical line shows when oxygen supply to inhalation mixture was terminated. All recordings are from the same tumor. rest (Figure 2) shows a commonly encoun­tered distribution of pO2 values in tumors with the majority of pO2 values close to zero. However, we did not expect the multiphase behavior found in the type I pO2 measure­ments (Figure 1a). The most probable reason for the observed phenomenon is the direct effect of the inser­tion of the probe on the tissue oxygenation. Steinberg et al. evaluated the injury caused by the insertion of polarographic pO2 probe into tumor tissue in different tumor models.15 They found clear histological evidence of tis­sue destruction and extravasation of erythro­cytes around the insertion channel in tumor tissue caused by the insertion of probe. On the other hand, Schramm et al. provided his­tological evidence for compressed microves-sels in the vicinity of the tip of polarographic sensor inserted into the rat muscle tissue.16 The capillary compression increases the per­fusion resistance of the passing erythrocytes, decreases the oxygen-carrying capacity of capillaries and, thereby also the oxygenation of the tissue in direct contact with the probe. It is also possible that the insertion of the probe into the tissue causes the vasoconstric­tive reaction and subsequent deoxygenation of the tissue.13 The following hypothesis may explain the type I pO2 signals. The insertion of the probe undisputedly injures the tissue in the imme­diate vicinity of the insertion channel. Some capillaries are ruptured and others can be oc­cluded; a vasoconstrictive reaction in the tis­sue can occur. All these physiological chan­ges can decrease the oxygen delivery to the tissue in the immediate vicinity of the tip of the probe. This tissue might therefore be se­verely deoxygenated immediately after the in­sertion of the probe. This deoxygenated state probably corresponds to the zero pO2 phase in the type I pO2 measurements (Figure 1a). In the period that follows, this tissue could be re-oxygenated due to gradual restoration of microcirculation. Gradual reoxygenation is probably reflected in the increase phase in the type I pO2 measurements. In case of the type II pO2 measurements, pO2 of the tissue is most probably so low that no further deoxy­genation and subsequent reoxygenation could be observed after the insertion of the probe. The multiphase behavior of the type I pO2 signals was never experienced in our blood perfusion measurements. This difference be­tween generally well correlated LDF signals and type I pO2 signals can be explained by a much larger tissue sampling volume in case of LDF measurement than in pO2 measure­ment. LDF samples a tissue volume of the or­der of a cubic millimeter. The actual tissue sampling volume for the new time-resolved luminescence-based method is unknown, but is much smaller than the tissue sampling vol­ume of LDF. The measured pO2 values reflect local oxygenation in a very small part of tis­sue surrounding the tip of the probe. It is be­lieved that this method samples pO2 in the cells and intercellular space in direct contact with the tip of the probe, which means a vol­ume of about 400 cells only.7 The tissue far­ther away from the insertion channel, which is not affected by the insertion itself, should therefore contribute significantly to the LDF signal but not to the pO2 signal. To our knowledge, the unexpected multi­phase behavior of pO2 has been so far report­ed only in a recent paper by Seddon et al..13 Our results are in excellent agreement with their results even though Seddon et al. per­formed their measurements on non-anes­thetized and physically restrained mice. Other authors using OxyLite system have not reported this phenomenon.6,7,11,12 The effect of hydralazine The results of our study showed that hy­dralazine at a dose of 2.5 mg/kg significantly reduced blood flow and oxygenation of SA-1 fibrosarcoma tumors in A/J mice. The effect was seen in all tumors in case of LDF meas­urements and in case of all type I pO2 meas­urements (Figure 4). The amplitude and dy­namics of the decrease in blood perfusion after the injection of HYZ obtained in our study are in direct agreement with the results obtained with LDF by other authors in vari­ous mouse tumor models after the injection of HYZ.17-20 Our measurements of pO2 by means of the novel time-resolved lumines-cence-based optical oximetry showed a pro­nounced decrease of pO2 after the injection of HYZ. On average, pO2 decreased by 80 % from the pretreatment level. This decrease was well correlated to the decrease in blood flow measured by LDF. Our results are also in agreement with the results of Okunieff et al.21 who showed that metabolic rate in experi-mental mouse tumors as measured by 31P­NMR spectroscopy was significantly de­creased by HYZ at a dose similar to ours. In their study the decreased metabolism caused by the lack of oxygen was demonstrated by the decrease of organic phosphates and in­crease of inorganic phosphates. Hydralazine is an effective peripheral vasodilator that has been used in the treatment of hypertension in humans.22 It relaxes arteriolar smooth mus­cle, thereby effectively reducing the peripher­al vascular resistance and decreasing blood pressure. In these conditions, the organism is trying to maintain normal blood flow in vital organs and tissues by “stealing” the blood flow in less vital tissues.21 This “steal phe­nomenon” is responsible for the demonstrat­ed decrease in blood perfusion in tumors. In our preliminary study we provide direct evi­dence of markedly decreased tumor oxygena­tion caused by the decrease in blood perfu­sion after the injection of hydralazine. The effect of anesthetic concentration Anesthetics undoubtedly affect a number of parameters of physiological conditions in mice. Isoflurane used in our study is a recom­mended anesthetic for small animals due to its minimum side effects, stable anesthesia, and wide safety margin. Isoflurane produces little or no depressant effect on cardiovascu­lar system but it causes some respiratory de­pression.23 Nitrous oxide, which was used to­gether with oxygen to deliver isoflurane to anesthetized mice, has no significant effects neither on cardiovascular nor respiratory sys­tem.23 Despite these facts, it was shown that a transient increase in concentration of isoflu­rane from 1.7 % (concentration used for main­tenance of long-term stable anesthesia) to 3 % (concentration used for induction of anesthe­sia) produced a significant decrease both in the blood perfusion and in oxygenation of tu­mors (Figure 5). Both variables decreased with similar dynamics. When isoflurane con­centration was returned to normal level, there was a delay between the increase of blood perfusion increase and that of pO2. This can be explained by the delivery-limited oxygen consumption in low pO2 conditions. All addi­tional oxygen delivered by the increasing blood flow was readily consumed until oxy­gen delivery became abundant. When this happened, pO2 also started to increase. The demonstrated effect of the change of anes­thetic concentration indicates that: i) anes­thetic conditions should be kept as constant as possible during the prolonged measure­ments of tumor blood perfusion and oxy­genation; and ii) the values of tumor pO2 measured under anesthesia are probably not entirely representative of the pO2 in non-anesthetized conditions. The effect of euthanasic procedure Tumor pO2 and blood perfusion were moni­tored during euthanasic procedure in order to verify if the measurements were valid. The re­sults were as expected: the blood perfusion and type I pO2 signals both decreased when oxygen supply to inhalation mixture was ter­minated (Figure 6). It is noteworthy to men­tion that the decrease in pO2 preceded the decrease in blood perfusion by several sec­onds. This is a consequence of the decreased oxygen delivery in the presence of the still functioning blood flow. The value of type I pO2 signals in all measurements dropped to zero level (as it should at death). However, in the majority of LDF measurements, there was still some residual blood perfusion signal present after death. This fake “blood perfu­sion” signal experienced in the absence of true blood flow is the so-called biological zero signal which is usually observed in laser Doppler measurements. The principle of laser Doppler flowmetry is based on the measurement of movement of red blood cells. In case of biological zero, the movement de­tected is predominantly Brownian motion (thermally induced random motion) of vari­ous structures in the tissue.9 In the laser Doppler signal picked up from the living tis­sue, the ever-present biological zero compo­nent is outweighed by a much stronger com­ponent originating from the true blood flow. In our measurements in tumors, the biologi­cal zero level detected after the death of the mice was normally less than 5 % of the total signal level detected in tumor at rest. Conclusions The first very important finding of the pres­ent study is that two distinct types of pO2 sig­nals were encountered in tumors. Only the type I measurements resulted in pO2 values different from zero and, only in the type I measurements, the effect of oxygenation-modifying procedure could be seen. It is how­ever possible that a procedure, which in­creases oxygenation, might convert some of the type II pO2 measurements to the type I measurements. The second very important finding is that it takes a considerable amount of time before the type I pO2 signals stabilize in the plateau phase. Reliable measurements of pO2 changes can only be performed after the signal has entered the plateau phase. But it remains to be seen whether and to what ex­tent the pO2 value measured in the plateau phase represents the true pO2 as it was before insertion of the sensor. In our preliminary study using a novel time-resolved luminescence-based method for measuring the tissue oxygenation in com­bination with a well-established laser Dop­pler flowmetry, we have shown that both methods can be effective in the detection of local oxygenation and blood perfusion changes in tumors. Good correlation between the signals of both methods was found as it should be found since oxygenation in tissue depends on tissue microcirculation. It is im­portant to note that these two methods are es­sentially showing different things and that their respective tissue sampling volumes are very different. Therefore, the results of one method can only supplement, but not replace the results of the other. Based on our results, we conclude that the interpretation of some aspects of pO2 measurements with the novel luminescence-based method requires further investigation. Acknowledgements This study was supported partly by the Ministry of Education, Science and Sport of the Republic of Slovenia (grants J2-2222­1538, J3-3489-0302-01) and partly by the European Commission and the 5th Frame­work Programme (grant QLK 3-99-00484, CLINIPORATOR project). The authors wish to thank Simona Kranjc, M.Sc., of the Institute of Oncology, and Blaž Podobnik, B.Sc., of the Faculty of Electrical Engineering for their technical support in the course of the study. References 1. Horsman MR. Measurement of tumor oxygena­tion. Int J Radiat Oncol Biol Phys 1998; 42: 701-4. 2. Höckel M, Vorndran B, Schlenger K, BauRmann E, Knapstein PG. Tumor oxygenation: a new predic­tive parameter in locally advanced cancer of the uterine cervix. Gynecol Oncol 1993; 51: 141-9. 3. Vaupel P, Kelleher DK, Thews O. Modulation of tumor oxygenation. Int J Radiat Oncol Biol Phys 1998; 42: 843-8. 4. Horsman MR, Nordsmark M, Overgaard J. Techniques to assess the oxygenation of human tumors. Strahlenther Onkol 1998; 174(Suppl.IV): 2­5. 5. Raleigh JA, Dewhirst MW, Thrall DE. Measuring tumor hypoxia. Semin Radiat Oncol 1996; 6: 37-45. 6. Collingridge DR, Young WK, Vojnovic B, Ward-man P, Lynch EM, Hill SA, Chaplin DJ. Measu­rement of tumor oxygenation: a comparison be- tween polarographic needle electrodes and a time-resolved luminescence-based optical sensor. Radiat Res 1997; 147: 329-34. 7. Griffiths JR, Robinson SP. The OxyLite: a fibre-op­tic oxygen sensor. Br J Radiol 1999; 72: 627-30. 8. Oxford Optronix. OxyLite user manual ver. 1.12. Oxford: Oxford Optronix Ltd.; 1998. 9. Oxford Optronix. OxyFlo user manual ver. 1.03. Oxford: Oxford Optronix Ltd.; 2000. 10. Shepherd AP, Oberg PA, editors. Laser-Doppler blood flowmetry. Boston: Kluwer Academic Publishers; 1989. 11. Braun RD, Lanzen JL, Snyder SA, Dewhirst MW. Comparison of tumor and normal tissue oxygen tension measurements using OxyLite or micro-electrodes in rodents. Am J Physiol, Heart Circ Physiol 2001; 280: H2533-44. 12. Bussink J, Kaanders JHAM, Strik AM, van der Kogel AJ. Effects of nicotinamide and carbogen on oxygenation in human tumor xenografts meas­ured with luminescence based fiber-optic probes. Radiother Oncol 2000; 57: 21-30. 13. Seddon BM, Honess DJ, Vojnovic B, Tozer GM, Workman P. Measurement of tumor oxygenation: in vivo comparison of a luminescence fiber-optic sensor and a polarographic sensor and a polaro-graphic electrode in the P22 tumor. Radiat Res 2001; 155: 837-46. 14. Thews O, Kelleher DK, Vaupel PW. Intra- and in-ter-tumor variability of oxygenation: quantifica­tion and evaluation of minimum number of elec­trode tracks for representative measurements. In: Thews O, Kelleher DK, Vaupel PW, editors. Tumor oxygenation. Stuttgart: Gustav Fischer Verlag; 1995. p. 39-47. 15. Steinberg F, Hildenhagen-BrLggemann E, Konerding MA. Oxygen electrode injury in tu­mour tissue. In: Thews O, Kelleher DK, Vaupel PW, editors. Tumor oxygenation. Stuttgart: Gustav Fischer Verlag; 1995. p. 186-93. 16. Schramm U, Fleckenstein W, Weber C. Morpho­logical assessment of skeletal muscular injury caused by pO2 measurements with hypodermic needle probes. In: Ehrly AM, Fleckenstein W, Hauss J, Huch R, editors. Clinical oxygen measure­ment II. Berlin: Blackwell Ueberreuter Wissenschaft; 1990. p. 38-50. 17. Trotter MJ, Acker BD, Chaplin DJ. Histological ev­idence for nonperfused vasculature in a murine tu­mor following hydralazine administration. Int J Radiat Oncol Biol Phys 1989; 17: 785-9. 18. Zlotecki RA, Baxter LT, Boucher Y, Jain RK. Pharmacologic modification of tumor blood flow and interstitial fluid pressure in a human tumor xenograft: network analysis and mechanistic in­terpretation. Microvasc Res 1995; 50: 429-43. 19. Kalmus J, Okunieff P, Vaupel P. Dose-dependent effects of hydralazine on microcirculatory func­tion and hyperthermic response of murine FSaII tumors. Cancer Res 1990; 50: 15-9. 20. Horsman MR, Christensen KL, Overgaard J. Relationship between the hydralazine-induced changes in murine tumor blood supply and mouse blood pressure. Int J Radiat Oncol Biol Phys 1992; 22: 455-8. 21. Okunieff P, Walsh CS, Vaupel P, Kallinowski F, Hitzig BM, Neuringer LJ et al. Effects of hy­dralazine on in vivo tumor energy metabolism, hematopoietic radiation sensitivity, and cardiovas­cular parameters. Int J Radiat Oncol Biol Phys 1989; 16: 1145-8. 22. Albrecht RF, Toyooka ET, Polk SLH, Zahed B. Hydralazine therapy for hypertension during the anesthetic and postanesthetic periods. Int Anesthesiol Clin 1978; 16: 299-312. 23. Short CE, Paddleford RR, Harvey RC. Induction and maintenance of general anaesthesia in small animal practice. Wien Tierärztl Mon Schr 1993; 80: 177-87. Persistent chromosomal aberrations in somatic cells in testicular cancer patients after different therapies Cvetka Bilban-Jakopin1 and Marjan Bilban2 1Department of Radiation Oncology, Institute of Oncology Ljubljana, 2Institute of Occupational Safety, Ljubljana, Slovenia Background. The damage due to radiation or chemotherapeutic agents has been estimated successfully for the last 35 years from the numbers of the chromosome changes. This finding may serve as biological dosime­ter. The aim of the study was to find persistent chromosomal aberrations in somatic cells in testicular can­cer patients after different therapies. Patients and methods. This prospective study includes 60 patients with testicular tumours. With respect to the histological results and various therapies that they were given they were divided into four groups. Prior to treatment, we did not detect any deviations either in the genome picture of our patients or in that of the subjects of the control group without malignant disease. The changes in the genome of individual cells after therapy were detected by the following tests: structural chromosomal aberrations (SCA) test, sister chromatid exchange (SCE) test and micronucleus (MN) test performed on binuclear lymphocytes. Results. Immediately after the completion of treatment, chromosomal aberrations were inhibited, with the exception of dicentrics which persisted. Chemotherapy is less detrimental to the genome picture than radio­therapy and causes different types of chromosome changes. From the cytologic and mutagenetic points of view, irradiation proved to be more aggressive to patients than chemotherapy. Six months after the com­pleted treatment, the mitotic activity was found to be nearly normal; but the chromosome damage persisted and was higher than before therapy and in the fourth group of patients who had been only operated on. Conclusions. After irradiation as well as after chemotherapy the genome was repaired, because the dam­aged cells had died away. Considering that in the observed patients, only small tissue-cellular complex re­sponded to radiation and that the number structural chromosome changes, predominantly unstable aberra­tions, such as dicentrics, was rather high, we assume that the repair of the genome will be faster in these patients. Key words: testicular neoplasms – therapy; chromosomal aberations; micronuclei Correspondence to: Cvetka Bilban-Jakopin, M.D., Ph.D., Department of Radiation Oncology, Institute of Received 19 September 2001 Oncology, SI 1000 Ljubljana, Zaloška 2, Slovenia; Fax: Accepted 27 September 2001 +386 1 43 14 180; E-mail: tina.bilban@siol.net Introduction Patients with seminoma tumours received postoperative irradiation1,2 or postoperative adjuvant chemotherapy with paraplatinum.3,4 Patients with nonseminoma tumours may be treated by postoperative chemotherapy with BEPV5 or by surgery alone. The choice of treatment modality depends on the stage of disease, and toxic effects of treatment.1,6-9 The damage due to radiation or chemo­therapeutic agents has been estimated suc­cessfully for the last 35 years from the num­bers of the chromosome changes in the culture of lymphocytes in the peripheral blood.10 The exposure of cells to ionising ra­diation can be recognised from a higher num­ber of structural chromosome changes. This finding may serve as biological dosimeter. The technique most often used for the identi­fication of these changes uses the following steps: preparing the cells exposed to radia­tion under in vitro circumstances and count­ing the chromosomal changes.11-14 Patients and methods Subjects This prospective study included 60 patients with testicular tumours, aged between 15 and 35 years, who had not been previously treat­ed for any malignant disease. With respect to the histological test results and various thera­pies applied (surgery, radiotherapy, chemo­therapy) we divided them into four groups. Mutagenetic tests were performed at diagno­sis, immediately after the completion of treat­ment and six months after it. The stage of the disease in patients with testicular tumour was defined according to TNM classification from the year 1992 (29). Treatment approaches Group I All patients in the first group received post­operative irradiation. Radiotherapy was per­formed by an 8 MeV linear accelerator, using X rays. The irradiated area involved the iliac and paraaortal lymph nodes on the same side as the malignant process. The radiation field comprised an area from Th 10 to L5 and had a width of 10-11cm, AP and PA, and the in­guinal lymph nodes on the same side, from the upper edge of L5, 1 cm medially, involv­ing also the entire postoperative scar only up to a depth of 3 cm and only AP. On the back, the radiation field was the same, extending up to L5 and from there to the sacrococcygeal bone. The total radiation dose for Stage I was 30 Gy and for Stage II 36 Gy; the daily dose was 1.5 Gy. Thirteen patients were irradiated with 30 Gy and only two with 36 Gy. Group II The patients of the second group had a post­operative adjuvant chemotherapy with para-platinum. A single dose (paraplatinum 300­400 mg/m2) was administered in short infusion. The patients in the second group re­ceived one, two or three cycles of paraplat­inum in doses from 450 mg to 750mg, de­pending on the body surface area. The total dose of paraplatinum thus ranged between 450 mg and 2250 mg per individuum. Group III Patients with nonseminoma tumours under­went surgery, orchidectomy of the affected tes­tis followed by the adjuvant chemotherapy ac­cording to BEPV schedule: cisplatin 20 mg/m2 i.v., days 1-5; bleomycin 15 mg/m2 i.v., 1st and 2nd day; etoposide 100 mg/m2 i.v., days 1-3; -vinblastine 3 mg/m2 i.v,. 1st and 2nd day. Patients had two, three or four cycles of chemotherapy according to BEPV schedule. Seven patients had four cycles, four patients had three cycles and four patients had two cy­cles of chemotherapy. Group IV All patients in this group underwent only sur­gery, i.e. orchidectomy of the affected testis, followed by lymphadenectomy or observa­tion only. Cytogenetic studies We used peripheral blood lymphocytes as tar­get material. Blood samples were taken si­multaneously for the following three tests. Structural chromosomal aberrations (SCA) We used standard in vitro lymphocyte cul­tures for structural chromosomal aberration analysis. We added 0.3 ml of heparinised whole blood to 5 ml of Chromosome med 1A­GIBCO culture medium. The first in vitro cell division cycle was established with the addi­tion of 5 µg/ml of BrdU-Sigma. The hypoton­ic procedure was performed with 0.075 mol/l potassium chloride, whereas fixing was car­ried out in a mixture of glacial acetic acid and methyl alcohol at a ratio of 1:3. The cell sus­pension was pipetted onto cold glass slides. The specimens were air-dried and stained with Giemsa-Sigma. The maximum analysed for every test subject were the very first 200 in vitro metaphases. The chromosome analy­sis was carried out exclusively in the metaphases of the first division cycle, identi­fied by homogeneously stained chromo­somes. Structural damages to chromosomes were categorised as chromosomal breaks, acentric fragments, dicentrics and ring chro­mosomes. Gaps were not included in the to­tal number of chromosomal aberrations.16 Sister chromatid exchange (SCE) The same culture medium was used as in the first test. We prepared in dark 72-hour lym­phocyte cultures with the addition of 10 µg/ml BrdU and carried out the procedure ac­cording to Kato.17 We analysed 50 cells per subject, counted SCE and presented them as average numbers per cell. The range of SCE frequencies was also recorded for every sub­ject. Micronucleus test (MN) For this test, 3 µg/ml of cytochalasin B-Sigma was added to each in vitro lymphocyte cul­ture in the 43rd hour of cultivation. We used the Fenech-Morley method. Hypotonic proce­dure was omitted. The specimens were stained with May-Grunwald and Giemsa. We analysed the cells with clearly blocked cy­tokineses (CB cells), i.e. binuclear cells. We examined 500 cells per person and presented the results as the number of micronuclei per 500 CB cells. For technical reasons, the MN test was not performed on the control group – environmental exposure.18 Statistical data processing The median value of all parameters in the four groups of patients and for all three measurements was presented graphically (Windows Microsoft Excel). The changes in the genome picture were analysed for each treatment modality using the overall analysis of all three measurements (Friedman’s test)19 and the analysis of two measurements (Wil­coxon’s test).19 The Kruskal-Wallis (KW) test and Mann-Whitney (MW) test were used for the comparison of the median values of the areas between the two groups; the difference between groups is statistically significant if p < 0.025, since Bonferroni’s correction must be taken into account.20 A personal comput­er and the SPSS programme package (SPSS for Windows, Version 8.0.1) was used for sta­tistical data processing that was carried out at the Institute for Biomedical Informatics of the Faculty of Medicine, University of Ljubljana. Results Prior to treatment, we did not detect any de­viations neither in the genome picture of our patients nor of the subjects of the control group without malignant disease. (Table 1, Fig. 1-6). Immediately after the completion of treat­ment, the mitotic activity was observed as well as a significant increase in the frequency of chromosomal aberrations. In the patients treated with radiotherapy, this was mainly due to an increased number of dicentrics (Fig. 2). Chemotherapy affects the genome to a lesser degree than radiotherapy. Usually, the percentage of structural chromosome chan­ges is higher after chemotherapy (p = 0.005) (Fig. 1A), but still lower than after irradiation p = 0.005 (Fig. 1). The types of chromosomal changes in the observed patients were differ­ent, with the unstable chromosome changes predominating (p = 0.005) (Fig. 2). After che­motherapy, the number of dicentrics is not significantly different (p = 0.08) (Figure 2). The number of MN is higher after irradiation than chemotherapy (p = 0.005) (Fig. 5), al­though after chemotherapy according to BEPV schedule and chemotherapy with para-platinum, the numbers of micronuclei differ significantly (p = 0.005) (Fig. 5). A significant­ly higher number of SCE was noted after chemotherapy than after radiotherapy (p = 0.001) (Fig. 6). Six months after the completion of treat­ment, the mitotic activity was found to be mainly normal, but in comparison to the fourth group that was treated only by sur­gery, a large percentage of chromosomal aberrations persisted (Fig.1). The analysis of genome changes immedi­ately after the completion of therapy and six months later showed statistically significant difference in chromosome changes in the first group (p = 0.01) and in the third group Table 1. The median value of all parameters in four groups of patients and for all three measurements measurement group I group II group III group IV Me of % SCA/200 cells 1 2 1,5 1,5 1,5 2 50 4,5 6 1,5 3 9 3 3,5 1,5 Me of dicentrics/200 cells 1 0 0 0 0 2 17 0 0 0 3 6 0 0 0 Me of acentric fragments/ 1 1 0 0 0 200 cells 2 21 2,5 2 0 3 8 2 2,5 0 Me of chromosomal breaks/ 1 3 3,1 2 2,9 200 cells 2 5 5,1 4,9 3 3 5 4,1 4 3 Me ofMN/500CB cells 1 5 6 6 4 2 19 8 12 4 3 12 7 10 4 SCE/50 cells 1 6,2 6,4 6,4 6,2 2 6,4 7,9 8,3 6 3 6,5 7,4 7,2 6 Me = median value, No = number, SCA = structural chromosomal aberrations, MN = Micronucleus test, SCE = sis­ter chromatid exchange Figure 1. Median value of the chromosomal aberrations of patients with testicular tumour (p = 0.005). Figure 1a. Median value of the chromosomal aberrations of patients with testicular tumour (p = 0.005). Figure 2. Median value of the dicentrics of patients with testicular tumour (p = 0.005). Figure 3. Median value of the acentric fragments of patients with testicular tumour (p = 0.005). Figure 4. Median value of the chromosomal breaks of patients with testicular tumour. Figure 5. Median value of the MN of patients with testicular tumour (p = 0.005). Figure 6. Median value of the SCE per cell of patients with testicular tumour (p = 0.001). (p = 0.004). The comparison of numbers of chromosomal changes prior to the therapy and six months later was statistically signifi­cant in groups I to III (p = 0.001). The number of micronuclei was also sig­nificantly higher in all the first three groups. It was the highest in the first group of pa­tients treated by radiotherapy. Only in this group, the number of micronuclei was found to be significantly decreasing six month after the therapy (p = 0.001). Six months after ther­apy, MN values in the patients treated by ra­diotherapy and in those treated by chemo­therapy according to BEPV schedule were significantly higher compared to pre-expo­sure levels. Six months after chemotherapy, the num­ber of SCE was still higher than the starting values (p = 0.001). Discussion Prior to treatment, we did not detect any de­viations neither in the genome picture of our patients nor of the subjects of the control group without malignant disease.21 The changes inside the genome were observed af­ter chemotherapy as well as after radiothera­ py.22,23 From cytologic and mutagenetic points of view radiotherapy is more aggres­sive than chemotherapy, mostly because of a much higher level of unstable chromosomal changes, such as dicentrics. In irradiation, the mitotic activity in lymphocytes is significant­ly, yet only temporarily inhibited, whereas in chemotherapy, the inhibition of mitotic activ­ity in vitro depends on the chemotherapeutic treatment scheme. In a certain lapse of time after treatment, the genome picture is repaired. The time of repair depends on the length of exposure of the tissue-cellular complex and the type of damage. The genome is repaired because the damaged cells have died away. A great num­ber of unstable aberrations, such as di­centrics, predominate in the patients treated by radiotherapy. These unstable cells are so severely damaged after radiotherapy that they are unable to survive and die shortly. In irradiated patients, a fast repair of the genome is mainly due to the following two reasons: the first is that a small tissue-cellu­lar complex responds to radiation, and the second is that the structural chromosome damages predominate, especially unstable aberrations, such as dicentrics. The differ­ences in the genome of individual cells are still possible. They may induce the develop­ment of oncogene and, consequently, of sec-ondary tumour due to primary tumour treat­ment.24 The analysis of the treatment results in pa­tients treated by chemotherapy showed that the difference between the chemotherapy ac­cording to BEPV schedule and the chemother­apy with paraplatinum was very small. In our patients, the only difference between both chemotherapy schedules was the number of micronuclei. This difference was statistically significant at all parameters in comparison to the first group been treated only by radio­therapy or the fourth group treated only by surgery. Similar results were published also in oth­er studies reporting of significantly higher ef­fect of irradiation on the genome than that of chemotherapy 25,26 and of significantly higher effect of chemotherapy on the genome in comparison to the genome of the patients treated by surgery alone.27 Six months after the completed therapy, the number of cytogenetic changes was lower and not statistically significant any more. Monochemotherapy with paraplatinum is a new method of adjunct treatment of patients with an early stage of seminoma and is just being introduced at our Institute. The pa­tients included in this study are the first to be treated with this method in Slovenia.28,29 The advantages of paraplatinum monotherapy, lie primarily in short hospitalisation time, there­by also in shorter overall treatment time, as well as short-lived and mild side effects of chemotherapy,3,4 as was also confirmed in our study. In conclusion, the following question re­mains: Can the chromosome changes after chemotherapy, such as chromosome and chromatide breaks that take a longer time for the genome repair, also induce the develop­ment of oncogene and, consequently, of sec­ondary tumours, considering that the cells with that kind of damage have more possibil­ities for surviving? References 1. Horwich A, Bell J. Mortality and cancer incidence following radiotherapy for seminoma of the testis. Radiother Oncol 1994; 30: 193-1. 2. Niewald M, Waziri A, Walter K, Nestle U, Schnabel K, Humke U. Low-dose radiotherapy for stage I seminoma: early results. Radiother Oncol 1995; 37: 164-6. 3. Oliver RT. Testicular cancer. Curr Opini Oncol 1991; 3: 559-64. 4. Oliver RT. Germ cell cancer of the testes. Curr Opin Oncol 1995; 7: 292-6. 5. Gutierez-Delgado F, Titov DA, Tjulandin SA, Garin AM. Drug dose delivery and treatment out­come relationship in standard bleomycin, etopo-side and cisplatin combination chemotherapy in nonseminomatous germ cell tumor patients. Neoplasma 1999; 46: 190-5. 6. Babosa M, Baki M, Gundy S, Bodrogi I. Children fathered by men treated for testicular cancer con­ceived before, during and after chemotherapy – examination for evidence of congenital malforma­tions, malignancies and immunological defects. Acta Paediatr Hung 1992; 32: 11-30. 7. Fossa SD, Langmark F, Aass N, Andersen A, Lothe R, Borresen AL. Second non-germ cell ma­lignancies after radiotherapy of testicular cancer without chemotherapy. Br J Cancer 1990; 61: 639­43. 8. Martin RH, Ernst S, Rademaker A, Barclay L, Ko E, Summers N. Analysis of human sperm kary­otypes in testicular cancer patients before and af­ter chemotherapy. Cytogenet Cell Gen 1997; 78: 120-3. 9. Martin RH, Ernst S, Rademaker A, Barclay L, Ko E, Summers N. Chromosomal abnormalities in sperm from testicular cancer patients before and after chemotherapy. Human Gen 1997; 99: 214-8. 10. Bender MA, Awa AA, Brooks AL, Evans HJ, Groer PG, Littlefield LG, et al. Current status of cytoge­netic procedures to detect and quantify previous ex­posures to radiation. Mutat Res 1988; 196: 103-59. 11. Garaj-Vrhovac V, Kopjar N. Micronuclei in cytoki­nesis-blocked lymphocytes as an index of occupa­tional exposure to antineoplastic drugs. Radiol Oncol 1998; 32: 385-92. 12. Johansen J, Streffer C, Fuhrmann C, Bentzen SM, Stausbol-Gron B, Overgaard M, et al. Radiosensiti­vity of normal fibroblasts from breast cancer pa­tients assessed by the micronucleus and colony as­says. Int J Radiat Biol 1998; 73: 671-8. 13. Muller WU, Nusse M, Miller BM, Slavotinek A, Viaggi S, Streffer C. Micronuclei: a biological indi­cator of radiation damage. Mutat Res 1996; 366: 163-9. 14. Shibamoto Y, Shibata T, Miyatake S, Oda Y, Manabe T, Ohshio G, et al. Assessment of the pro­liferative activity and radiosensitivity of human tu­mours using the cytokinesis-block micronucleus-assay. Br J Cancer 1994; 70: 67-71. 15. UICC International Union Against Cancer. TNM Classification of malignant tumours. 4th ed. 2nd rev. Berlin: Springer-Verlag; 1992. p. 145-7. 16. International Atomic Energy Agency. Biological dosimetry. Chromosomal aberration analyses for dose assessment. Technical Report Series No.260. Vienna; 1986. p.59-65. 17. Kato H. Spontaneous sister chromatid exchanges detected by a BrdU-labeling method. Nature 1974; 251: 70-2. 18. Fenech M, Morley AA. Measurement of micronu­clei in lymphocytes. Mutat Res 1985; 147: 29-36. 19. Siegel S, Castellan NJ. Nonparametric statistics for the behavioral sciences. International edition. 1988. p. 128-16. 20. Altman DG. Practical statistics for medical research. London: Chapman & Hall; 1991. p. 218-20. 21. Bilban M, Vrhovec S. Incidence of spontaneous cy­togenetic changes in peripheral blood lympho­cytes of a human population sample. Radiol Oncol 1996; 30: 297-304. 22. Gundy S, Baki M, Bodrogi I, Czeizel A. Persistence of chromosomal aberrations in blood lymphocytes of testicular cancer patients. I. The effect of vin­blastine, cisplatin and bleomycin adjuvant thera­py. Oncology 1990; 47: 410-4. 23. Gundy S, Baki M, Bodrogi I, Czeizel A. Persistence of chromosomal aberrations in blood lymphocytes of testicular cancer patients. II. The effect of chemotherapy and/or radiotherapy. Oncology 1992; 49: 376-80. 24. Perera FP, Motzer RJ, Tang D, Reed E, Parker R, Warburton D, et all. Multiple biologic markers in germ cell tumor patients treated with platinum-based chemotherapy. Cancer Res 1992; 52: 3558­65. 25. Rigaud O, Guedeney G, Duranton I, Leroy A, Doloy MT, Magdelenat H. Genotoxic effects of ra­diotherapy and chemotherapy on the circulating lymphocytes of breast cancer patients. II. Alternation of DNA repair and chromosome ra­diosensitivity. Mutat Res 1990; 242: 25-35. 26. Sala-Trepat M, Cole J, Green MH, Rigaud O, Vilcoq JR, Moustacchi E. Genotoxic effects of ra­diotherapy and chemotherapy on the circulating lymphocytes of breast cancer patients. III: Measurement of mutant frequency to 6-thiogua-nine resistance. Mutagenesis 1990; 5: 593-8. 27. Smolarek TA, Blough RI, Foster RS, Ulbright TM, Palmer CG, Heerema NA. Cytogenetic analyses of 85 testicular germ cell tumours: comparison of postchemotherapy and untreated tumors. Cancer Genet Cytogenet 1999; 108: 57-69. 28. Bilban-Jakopin C: Chromosomal aberrations in so­matic cells after administration of fractionated doses of irradiation or antineoplastic drugs in treating patients with seminoma. European Radiation Research 98, Capri; 1998. p. 42. 29. Bilban-Jakopin C. Chromosomal changes in so­matic cells in seminoma patients after treatment with ionising radiation or cytostatics. Neoplasma 2000; 47: 48-55. Legislation on the protection of experimental animals Dragica Ornik and Milan Pogacnik 1Ministry of Agriculture, Forestry and Food, Veterinary Administration of the Republic of Slovenia, Maribor, 2Veterinary Faculty of the University of Ljubljana, Institute of Pathological Morphology, Forensic and Administrative Veterinary Medicine, Ljubljana, Slovenia The aim of this paper is to establish the current situation in the field of legislation on the protection of ex­perimental animals in Slovenia. The protection of experimental animals has been regulated by the provisions of the Protection of Animals Act.1 On the basis of this act, the Instructions on Conditions for the Issuing of Authorisations for Experiments on Animals for Scientific and Research Purposes2 and the Rules on the Ethics Commission for Experiments on Animals 3 have been used. The basic protection of experimental animals is provided for by a system of permits for experiments on an­imals. Permits for experiments on animals are granted by the administrative authorities responsible for vet­erinary medicine in cases where experiments are urgently required for medical, veterinary medical, or sci­entific and research purposes and the results are expected to produce important new knowledge, or when the suffering of animals is ethically acceptable in comparison with what the experiment is expected to achieve; where, in cases of basic research, experimental aims cannot be achieved by any other method or procedure, the experiment is performed on the minimum possible number of animals of the lowest neuro-physiological sensitivity and a method is used that causes the minimum level of suffering, pain or lasting harm. Staff involved in the execution of experiments or in the care and nursing of animals, the premises for the accommodation or rearing and provision of animals, and the installations and devices used must all com­ply with the set conditions. With the adoption of the Act, which has been harmonised with EU regulations, legislation on the protection of experimental animals has been put into effect. As laid down by the act1, the implementing regulations will lay down the conditions for the issuing of permits for experiments on animals, the procedure, documenta­tion, records, reports and responsibilities of experts on the protection of animals, and staff and other condi­tions relating to the execution of experiments and procedures involving animals. Key words: animals, laboratory; animal husbandry; animal rights; legislation Received 19 October 2001 Accepted 25 October 2001 Correspondence to: Dr Dragica Ornik, Dr vet. med., Ministry of Agriculture, Forestry and Food, Veterinary Administration of the Republic of Slovenia, Maribor Unit, Ulica heroja Tomšica 2, 2000 Maribor, Tel: + 386 2 22 01 673, Fax: + 386 2 25 26 758, E-mail: Dragica.Ornik@gov.si This paper was presented at the “Laboratory Animals in Biomedical Research” seminar, Ljubljana, 27 October 2001. Introduction The above cited parts of the legislation on the protection of experimental animals in Slovenia have been reviewed. The protection of animals used for experimental purposes is an increasingly important area. In most de­veloped European countries, extensive leg­islative activities in this area began back in 1986. With the adoption of the Protection of Animals Act1 (which came into effect on 18 December 1999), legislation regulating the protection of experimental animals finally en­tered into force in Slovenia. The act not only provides protection for animals against cruelty but also lays down the principles of animal welfare. In the gener­al provisions section, the act defines public responsibility for the protection of animals and their life, health and welfare by stressing the following basic principle: “No man shall be allowed to cause suffering, illness or death to an animal without a well-substantiated reason.” Data from the legislation in force in Slovenia The field of protection of experimental ani­mals in Slovenia is regulated by the Protection of Animals Act1, the Instructions on Conditions for the Issuing of Authorisa­tions for Experiments on Animals for Scientific and Research Purposes2, and the Rules on the Ethics Commission for Experiments on Animals.3 All animals with a developed nerve system that enables them to feel painful external influences must be dealt with under the terms of this act. The provi­sions of the act must always be taken into consideration when working with vertebrate animals and, depending on their level of sen­sitivity, with all other animals. There is a spe­cial chapter in the act: Experiments on Animals (Articles 21-24). Basic protection for experimental animals is provided for by a sys­tem of permits for experiments on animals which are issued by the administrative au­thorities, competent for veterinary medicine. Experiments on animals may only be car­ried out by organisations registered to per­form experiments on animals and granted permission by the Veterinary Administration of the Republic of Slovenia (VARS). Any or-ganisation performing experiments on verte­brate animals must appoint an animal protec­tion expert with the appropriate quali­fications. The expert provides the explana­tions needed in order to obtain approval for the experiment and is responsible for ensur­ing that experiments are carried out in com­pliance with the law. Permission for an experiment is granted on­ly if the experiment is truly necessary, and if: • the experiment is required for medical, veterinary or general, scientific and re­search purposes, it is expected that the re­sults will yield new and important find­ings, and it is expected that animal suffering will be ethically acceptable in comparison with what the experiment is expected to achieve; • it is basic research; • the goal of the experiment cannot be achieved by any other methods and proce­dures; • the lowest possible number of animals with minimal neuro-physiological sensitiv­ity and methods that cause the lowest amount of suffering, pain or permanent in­jury are used in the experiment; • the animal is anaesthetised before the start of the experiment, unless the pain caused by the experiment is lower than the pain caused by anaesthesia or the anaesthetisa­tion of the animal is against the purposes of the experiment; • the animal will be properly treated or killed after the experiment is completed, if suffering cannot be prevented; • the persons who carry out experiments or take part in them and the persons who take care of the animals, including duties of a supervisory nature, have attained the appropriate level of education; • the animals used in the experiment come from well-organised and registered breed­ing establishments; in exceptional cases, when the experiment is necessary for the preservation of an animal species and the animal is the only one suitable for the ex­periment or when it cannot be bred, the competent authority may issue permission for the experiment in compliance with the Regulation on the Protection of Endange­red Animal Species.4 Experiments on equine animals, bi-ungulates, dogs or cats may only be performed when it is not pos­sible to achieve the envisaged aim by ex­perimenting on other animals.2 A special permit from VARS is required for each individual experiment. The person who will conduct the experiment must have the proper qualifications.1 Only persons with adequate qualifications in veterinary and hu­man medicine, biology and animal husbandry may perform experiments on animals.2 An authorisation is not necessary for experi­ments that have been prescribed by a legal act or ordered by a court of law or a competent inspector on the basis of a legal act, or in the execution of vaccinations or diagnostic inves­tigations, the collection of blood or other ma­terial, or the detection of injuries and dis­eases.1 The performance of experiments on animals in order to test chemical agents used in war, cosmetic preparations, alcohol or to­bacco products, or of experiments in which muscle-paralysing agents are used and per­formed without the use of anaesthesia are prohibited.1 Surgical operations for educa­tional purposes which cause suffering and harm to or even the death of the animal are al­so prohibited. These can be performed only exceptionally by university or scientific and research institutions in cases where they are necessary in the course of the regular training of doctors of human or veterinary medicine, biologists or pharmacists, and where their ob­jective cannot be achieved by any other means of training (films, photographs, mo­dels, etc).1,2 The Rules on the Ethics Commission for Experiments on Animals lay down the proce­dure of examining and processing opinions on the necessity of a certain experiment in or­der to grant permission to carry it out on an animal.3 The rules lay down in detail the composition, tasks, competence and method of work of the ethics commission for experi­ments on animals. Immediate inspection and control of the implementation of the law and other regula­tions and international agreements on the protection of animals against suffering is car­ried out by veterinary inspection and control services. The Veterinary Practice Act5 and the Protection of Animals Act1 define the competence of the veterinary inspection and control service to order measures to protect animals against suffering, and the penalties for violations of the legislation. A veterinary inspector has the right and duty to prohibit the performance of any procedures on ani­mals which are not permitted or which vio­late the provisions of the Protection of Animals Act, order such experiments to be discontinued, or prohibit the carrying-out of experiments on animals which are carried out in violation of the provisions of the act, or when any existing deficiencies have not been eliminated by the prescribed deadline. The performance of experiments on animals in violation of the regulations is punishable by a fine under the Protection of Animals Act1 and, when premeditated, is considered as having caused suffering to the animal in question, which as such is punishable under the Penal Code.6 The penalty envisaged for such an offence is a prison sentence of up to three months. A fine of between SIT 100,000 and 150,000 under the act1 is imposed on an individual carrying out an experiment on animals in vio­lation of Article 21 of the Act (without per­mission and in an organisation that has not been registered for such an activity) or when experimenting on animals in violation of Article 22 of the act (procedures for educa­tional purposes without an approval). A fine of between SIT 150,000 and 10 million is im­posed on a legal entity for the same violation. A fine of between SIT 150,000 and 10 mil­lion is imposed on an individual who con­ducts painful procedures on vertebrate ani­mals without the use of appropriate anaesthesia or when conducting experiments on vertebrate animals in violation of Article 23 of the Act (when the organisation in ques­tion fails to appoint an expert on the protec­tion of animals). A fine of between SIT 25,000 and 500,000 is imposed on the responsible person of the legal entity which committed such a violation. Discussion There is direct and indirect links between an­imals and human beings – they are of vital importance to us. We should never forget that animals are living creatures with senses and are capable of expressing their feelings. We should handle them in a humane manner and with a due level of responsibility. Well-regulated legislation is of great importance for animal protection. Major progress at the international level in the legal regulation of the protection of experimental animals was made in the 1980s. An important turning point was the 1986 Convention of the Council of Europe on the Protection of Animals Used for Experimental and Other Scientific Purposes.7 A contribution towards increased protection for experimental animals was also made by the European Union (then the European Economic Community) with the adoption of Directive 86/609/EEC8, which is essence does not differ from the European Convention. The objective of the Directive and of the European Convention is to ensure that the provisions on the protection of ex­perimental animals are harmonised in the na­tional legislation of the member states. The European Convention7 and the European Union Directive8 define in different chapters the essential principles of use of animals in experiments and the conditions for rearing and accommodating the animals, with the minimum recommended housing areas for individual animal species and the micro-cli­matic conditions. By incorporating the pro­posed standards, any departures from the norm will be eliminated; by prescribing con­ditions for the rearing, accommodation and use of animals in experiments, measures for the protection of experimental animals will be made uniform. Most importantly, by granting mutual recognition to the test results of experiments performed on animals, the unnecessary duplication of experiments will be prevented. Slovenia is undergoing accelerated legisla­tive activities to protect experimental ani­mals. Experiments on animals carried out for scientific and research purposes have been regulated and subject to restrictions in Slovenia since 1985, when the Instructions on Conditions for Granting Authorisations for Experiments on Animals for Scientific and Research Purposes were issued.2 These in­structions include certain provisions that were laid down later on by the European Convention 7 and EU Directive.8 According to the instructions, any organisation intending to use animals in experiments that cause pain, suffering or lasting harm must obtain the approval of the competent administrative authority, provide conditions in which such experiments may be carried out, restrict the use of domestic animals in experiments and for educational purposes, and keep a protocol of the experiment and a report on the experi-ment by the person in charge of it. A draw­back to these instructions was that the penal­ties they prescribed were low and the execu­tion of inspection and control ineffective. By adopting the Protection of Animals Act1, progress has been made in regulation of the protection of experimental animals. The act includes some of the basic provisions of EU Directive 86/609/EEC8 as follows: • Experiments on vertebrate animals may be performed only when it is anticipated that the suffering of the animal will be ethical­ly acceptable in the light of the envisaged results. • Experiments on animals may only be per­formed by organisations registered to car­ry out experiments on animals and duly authorised by the competent administra­tive authority. • Experimental animals may only originate from an organised and registered rearing establishment for the rearing of experi­mental animals; • The person carrying out the experiments shall have appropriate professional qualifi­cations. • The person carrying out the experiments shall keep records on the number and species of animals used and the type of ex­periment, and notify the competent ad­ministrative authority thereof. Slovenia has the Rules on the Protection of Experimental Animals, which transpose all other provisions of the EU Directive.8 The rules lay down the detailed conditions for granting permits to carry out experiments on animals, the procedure, documentation, records, reports and obligations of profes­sionals relating to the protection of animals, staff and other conditions for the execution of experiments, and the procedures relating to the use of animals in experiments. The rules lay down detailed conditions regarding the establishments involved in the rearing, sup­ply and use of animals in experiments, and the premises, equipment and staffing thereof. The rules include provisions concerning the planning of experiments under consideration of the 3R concept (1R – replacement, or re­placement of a planned method with another; 2R – reduction, or selection of an experiment that requires fewer animals; 3R – refinement, or the conducting of an experiment to perfec­tion so as to guarantee its success) and the use of alternative methods. The EU Directive8 and the draft rules include the 3R concept in the following provisions: • An experiment may not be repeated when any other scientifically satisfactory me­thod is available that enables attainment of the envisaged result and does not require the use of animals. • When an experiment is urgently neces­sary, the selection of animal species must be carefully studied. An experiment must be chosen which requires the minimum number of animals, and animals with the minimum neuro-physiological sensitivity, which causes the minimum pain, suffer­ing, stress and lasting harm, and which in all probability will give satisfactory results. • All experiments must be planned so as to avoid stress, avoidable pain and suffering on the part of experimental animals. • Experiments causing lasting pain to ani­mals are subject to a special application to the competent authority and their use must be justified. Efforts have been made in Slovenia to rati­fy the European Convention for the Protec­tion of Vertebrate Animals Used for Expe­rimental and Other Scientific Purposes (ETS 123).7 Data on the use of experimental animals in European countries (including Slovenia) shows a decline in the use of animals in ex­periments. The annual number of experimen­tal animals used in Slovenia in the past three years has totalled approximately 22,000 (in 1996 it was still 30,000, and between 1993 and 1995 it was 33,000). Most of the experi-ments are carried out on laboratory rodents by the chemical and pharmaceutical indus­tries for substance testing in compliance with applicable legislation, regulations and inter­national agreements. Institutes and laborato­ries of the faculties of medicine, veterinary medicine, biology, zootechnics and physiolo­gy use animals for basic research or scientific and research activities; these account for nearly one fifth of all experimental animals used. To a lesser extent, animals are also used in the diagnosis of disease and for education and training purposes. An important role in reducing the number of experimental animals is played by the applicable legislation, the re­placement of animals with alternative meth­ods (and a legal requirement to do so), the re­quired permits for conducting experiments, ongoing staff training, successful co-opera­tion between research institutions and re­searchers at national and international levels, active associations and societies, and in­creased responsibilities of the commissions for the protection of animals. References 1. Zakon o zašciti živali (Protection of Animals Act); (ZZZiv). Uradni List RS (Official Gazette of the Republic of Slovenia) 1999; 98 (9):14645-53. 2. Navodilo o pogojih za izdajo dovoljenja za poskuse na živalih v znanstveno-raziskovalne namene (Instructions on Conditions for the Issuing of Authorisations for Experiments on Animals for Scientific and Research Purposes). Uradni List RS 1985; 41 (40): 2030-1. 3. Pravilnik o eticni komisiji za poskuse na živalih (Rules on the Ethics Commission for Experiments on Animals). Uradni List RS 2000; 10 (84): 9958-9 4. Uredba o zavarovanju ogroženih živalskih vrst (Regulation on the Protection of Endangered Animal Species). Uradni List RS 1993; 3 (57): 2851­4. 5. Zakon o veterinarstvu (Veterinary Practice Act). Uradni List RS 2001; 11(33): 3673-93. 6. Kazenski zakonik (Penal Code). Uradni List RS 1994; 4(63): 3455-503. 7. European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (ETS 123). Strasbourg: Council of Europe, 1986. 8. EEC. Council Directive 86/609/EEC of 24 November 1986 on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of ani­mals used for experimental and other scientific purposes. Off J EEC 1986; 29(L 358): 1-28. Standardisation of laboratory animals for biomedical research in Poland Hanna Szymanska, Elzbieta Krysiak, Joanna Piskorowska, Marek Woszczynski, Alina Czarnomska The Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology – Warsaw, Poland Increasing demands for biomedical research require laboratory animals of good quality. Simultaneously, the scientists aim at limiting the number of sacrificed laboratory animals. Some international organisations like ICLAS, FELASA, SOLAS deal with the promotion of proper breeding and care of laboratory animals. In 1997’ The Animal Protection Act’ was adopted by Polish Parliament. The standardisation of laboratory an­imals for biomedical research in Poland is the main task of the Commission on Biology of Laboratory Animals P.A.Sci. and Polish National Committee for Collaboration with ICLAS. In 2000, the Department of Genetics and Laboratory Animal Breeding, M. Sklodowska-Curie Memorial Cancer Center and Institute of Oncology in Warsaw was designed as National Reference Centre for SPF Animal Breeding. The department consists of five integral parts: (1) bank of SPF (Specified Pathogen Free) inbred mouse and rat strains, (2) experimental animals laboratory, (3) health monitoring laboratory, (4) genetic laboratory, (5) anatomo-pathological laboratory. Trends in development of laboratory animal science in Poland and Europe are overlapping. The standardi­sation of laboratory animals according to international recommendation seems to be sufficient in leading centres. Key words: laboratory animals; SPF mice research; Poland Correspondence to: dr. Hanna Szymanska, Oslavijska 9, 1000 Ljubljana, Slovenia. Phone: +386 01 425 98 52; E-mail: hanszym@yahoo.com The paper was presented at the seminar “Laboratory Animals in Biomedical Research”, held in Ljubljana 27 October 2001. Received 23. October 2001 Accepted 29 October 2001 Introduction Permanently increasing demands GLP (Good Laboratory Practice) laboratory rules as well as Polish law regulations require laboratory animals of good quality for biomedical re­search.1 Simultaneously, the scientists aim at limiting the number of sacrificed laboratory animals. The use of animals in biomedical re­search provokes considerable conflicts and discussion. The international co-operation and collaboration is necessary to solve the problem. Some international organisations deal with the promotion of proper breeding and care for laboratory animals. The following international organisations are the best known within Europe and world­wide: 1. The International Council on Laboratory Animal Science, ICLAS 2. Federation of European Laboratory Animal Science Associations, FELASA 3. Gesellschaft für Versuchstierkunde, GV­SOLAS 4. Institute for Laboratory Animals Resour­ces, ILAR 5. Fund for Replacement of Animals in Medical Experiments (FRAME) founded as a scientific charity. In Europe, two intergovernmental organi­ sations have adopted regulations for protec­tion of laboratory animals: The Council of Europe, CE and The European Community, EC. Although the aims of these organisations are not the subject of this presentation, it should be mentioned that their statutory ac­tivities are based on the concept of “Three R-s”, namely: Reduction, Refinement and Replacement. The standardisation of laboratory animals for biomedical research in Poland is the main task of the Commission on Biology of Laboratory Animals P.A.Sci. and Polish National Committee for Collaboration with ICLAS. The breeding of high quality experimental animals is not possible without progress in the field of laboratory animal science and ap­propriate political decisions. Background of laws and policies that impact protection of animals in Poland (including laboratory animals) In 1928, the regulation referring to the prob­lem of protection of animals was issued by the President of the Republic of Poland. (The first comparable act has been in force in England since 1924.) In 1957, the regulation issued by the Ministry of Justice and the Ministry of Interior described the general requirements to receive a licence to experimentation on an­imals and possible penalties. In 1959, the Ministry of High Education is­sued a regulation concerning the problem of using animals for scientific purposes. Today’s legislation In 1986, in Strasbourg, 11 members of Council of Europe signed ‘The Convention for the Protection of Vertebrate Animals Used for Experimental and other Scientific Purposes’. In September 1997, the Polish Parliament adopted the ‘Animal Protection Act’ to har­monise Polish legislation with European Community. General requirements for ani­mal care and use were based on the above mentioned Convention. In September 1999, the State Committee for Scientific Research appointed National Ethics Committee on Animal Experimenta­tion. Its members are representatives of bio­logical, medical, veterinary sciences and hu­manities. Simultaneously, 17 Local Ethics Committees were approved. In November 1999, the Government is­sued an act that listed 85 scientific insti-tutes, medical and veterinary faculties, uni­versities as well as pharmaceutical firms that are allowed to perform experiments on ani­mals. Progress of laboratory animal sciences and alternatives Polish Commission on Biology of Laboratory Animals P.A.Sci. Since 1962, the Polish Commission on Biology of Laboratory Animals has been act­ing under the auspices of Polish Academy of Sciences. The organisation is an excellent place of contact between researchers and people dealing with the breeding of laborato­ry animals. In the period 1964-1993, the Polish journal “Laboratory Animals” was published under the supervision of the Commission on Biology of Laboratory Animals. In the last is­sue, the Convention signed in Strasbourg was published in Polish translation. The journals ‘Comparative Medicine’ for­merly ‘Laboratory Animal Science’, ‘Labo­ratory Animals’ as well as the Japanese jour­nal ‘Experimental Laboratory Animals’ are also available in Poland. Membership in ICLAS The long-lasting collaboration of P.A.Sci with ICLAS has resulted in the participation of Polish National Representative in the Governing Board of ICLAS. There are three laboratory animal breeding centres in Poland fulfilling the criteria to function as an ICLAS Reference or Monitoring Centre: ‘The ICLAS Reference and Monitoring Centre System is a regional, international and inter-institutional collaborative network in line with ICLAS pol­icy’.2,3 In 1978, the Reference Centre for Histo-compatibility Testing in Mice was designated at the Institute of Oncology in Warsaw. Ten years later (1988), ICLAS Regional Monito­ring Centres for Microbiology and Genetics were designated at the Institute of Oncology in Warsaw and at the Institute of Immu­nology & Experimental Therapy in Wroclaw (Poland). In 2000, the Department of Genetics and Laboratory Animal Breeding in the M. Sklodowska-Curie Memorial Cancer Center and the Institute of Oncology in Warsaw was designated as National Reference Centre for SPF Animal Breeding. Development of alternatives in Poland The end of 1980s was the period when Polish scientists were stimulated to develop alterna­tives mainly in pharmacology and toxicology testing. It has resulted in Poland’s participa­tion in EU Fifth Framework Programme in 1999 and numerous joint publications. INVI­TOX ON LINE service is available from Medical University of Warsaw server. The 13th ESTIV INVITOX (European Society for Toxicology in Vitro) Workshop will be held in Poland in 2004.4 Presentation of the National (Polish) Reference Centre for SPF Animal Breeding The Department of Genetics and Lab. Animal Breeding consists of five integral parts: 1. bank of SPF (Specified Pathogen Free) in­bred mouse and rat strains, 2. experimental animal division, 3. health monitoring laboratory (microbio­logy), 4. genetic laboratory, 5. anatomo-pathological laboratory, — bank of SPF inbred mouse and rat strains SPF animals are an essential tool in the in­vestigations in oncology, immunology, and transplantology. SPF mice became indispen­sable models in oncology because their longer life span allows to develop numerous, spontaneous tumours with rare different his­tological patterns. The incidence of chemical­ly or irradiation induced neoplastic tumours is significantly higher when SPF animals are used. Three inbred strains of rats and 18 of mice (7 inbred and 11 congenic resistant strains) are maintained in the barrier system in SPF condition.5 They are bacteriologically, viro-logically and parasitologically controlled. Breeding division consists of 12 breeding rooms, each equipped with lock-chamber. The doors of lock-chambers are blockaded. Room temperature around 22°C, higher pres­sure and 12 h light/dark are regulated auto­matically and controlled for each room sepa­rately. There are 15 exchanges of air per hour. The air is filtrated by three filters, the last one is the absolute filter. Pelleted food (LABO­FEED H), cages with bedding material and bottles with water are sterilised in double-door autoclaves and transferred through the barrier into the clean area. The entrance to the breeding part is allowed only to the em­ployed, specially trained personnel after tak­ing a shower and donning sterile medical suits.6 Pelleted food LABOFEED H is produced under the supervision of the Institute of Animal Physiology and Nutrition P.A.Sci. Animal breeding is carried out according to the single line system and breeding proto­cols are kept in Excel computer program. The animals are registered at the Committee on Standardised Nomenclature for Inbred Strains of Mice and at the National Academy of Science, NW Washington DC 20418. The symbol is W. Figure 1. A trained and properly dressed personnel takes care for animals. — experimental animal division Conditions in the division for experimental animals are the same as in the SPF breeding part, however animals are maintained in the “semi barrier” system. Experimental animals at this division are kept at the least MD (Minimal Disease) standard. The researchers are allowed to enter the animal and operating rooms after conforming the hygienic rules. — health monitoring The health monitoring includes parasitologi-cal, bacteriological and virusological control according to the recommended international rules.7,8,9 Bacterial colonies cultured from nasophar­ynx, eye, caecum, colon and vagina are iden­tified on standardized diagnostic media. Bacteriological identification is performed us­ing BioMerieux API kits (French production). Mycoplasma sp. and virusological infections are verified by ELISA – (Enzyme-linked im­munosorbent assay) using Perimmune MAT kits (American production). As a rule, three mature animals are sacrificed for examina­tion three times per month. The list of microorganisms (viruses, bacte­ria and mycoplasma, ecto- and endoparasites) tested has been published.10 — genetic control In 1981-1998, genetic monitoring including morphological, biochemical and immunologi­cal markers was being performed. Five mor- Figure 2. Irradiation-induced mammary gland tumour – adenoacanthoma. phological and 9 biochemical markers were used for genetic monitoring in rats, whereas 4 morphological, 15 biochemical and 9 im­munological markers in mice.10,11,13 Genetic profiles were constructed for all maintained animals. Since 1999 genetic control of mice from seven inbred strains and one congenic resistant line by random amplification of polymorphic DNA (RAPD) method has been performed.14 Ten arbitrary primers were test­ed. Moreover, genetic profiles (chromosome No 1 – 8) were constructed for these strains using microsatellite markers. — anatomo-pathological and histopathological control Emphasis is being placed on the incidence of spontaneous and radiation-induced tumo-urs.15,16 The data obtained from 7 inbred strains (AKR/W, BALB/cW, BN/aW, CBAT6/ W, C3H/W, C57BL/10PhW, DBA/2W) are col­lected. The observed tumours are histologi­cally classified. H&E staining as well as histo­chemical and immunohistochemical methods are routinely used. The incidence of mamma­ry gland, lung, liver and haematopoietic sys­tem tumours was analysed. Today’s trends and problems The mouse strains with defined genetic background are used in fundamental re­searches. The trends in the development of laboratory animal science in Poland and Europe are overlapping. In oncology, the in­bred strains, recombinant inbred strains and recombinant congenic strains are the most preferred. The high cost of mice and rats supplied from the breeding reference centres to other institutes reduces the number of animals per project, but parallely discourages the investi­gators to new experiments. Besides, in practice it is very difficult to prove researchers’ ignorance of law and labo­ratory animal science and punish them espe­cially when an experiment is failed. Conclusion Standardisation of laboratory animals accord­ing to international recommendations seems to be sufficient in leading centres. However, the general state of laboratory animals breed­ing in Poland still needs improvement and additional funds. The law regulation is respected by the in­stitutes conducting fundamental researches; however, it has rather resulted from the awareness of scientists than effectiveness of law regulations. In future, high priority for development of modern genetics, immunology, oncology and others may cause seeing laboratory animals more as research tools than as companions in scientific investigation. References 1. Öbrink K, Rehbinder C. Animal definition: A ne­cessity for the validity of animal experiments? Laboratory Animals 2000; 34: 121-8. 2. Guidelines for breeding and care of laboratory an­imals, Veterinary Public Health Reports, Rome ISS/WHO/FAO-CC/IZSTe/94.23, 1994. 3. Heine W, editor ICLAS Reference and Monitoring Centres, Hannover: Zentralinstitut für Versu­chstierzuch; 1991. 4. Sladowski D. Regulation of the use of animals in research in Poland, and the development of alter­natives. ATLA 2000; 28: 869-70. 5. Czarnomska A, Sitarz M, Woszczynski M. Inbred strains of rats maintained at the Institute of Oncology, Tumour Biology Department. Rat News Letter 1991; 25: 30. 6. FELASA Recommendations for the education and training of persons carrying out animal experiments (Category B). Laboratory Animals 2000; 34: 229-35. 7. Allen AM, Nomura T. Anual of microbiologic monitoring of laboratory animals, U.S.Dept Health Human Serv., Natl. Inst.Health, Div. Res. Serv., Bethesda: NIH Publication; 1986. 8. Heine WOP. Environmental management in labo­ratory animal units – basic technology and hy­giene – methods and practice. Lengerich, Berlin, Düsseldorf, Leipzig, Riga, Scottsdale, Wien, Zagreb Pabst Science Publishers, 1998. 9. List of pathogens for specification in SPF labora­tory animals. Biberach, Basel: GV SOLAS, 1989. 10. Woszczynski M, Czarnomska A. ZwierzÍta labora­toryjne SPF – niezbÍdne modele w badaniach bio-medycznych. Medycyna Weterynaryjna 2000; 56: 222-5. 11. Hedrich HJ. Genetic monitoring of inbred strains of rats, a manual on colony management, basic monitoring techniques, and genetic variants of the laboratory rat. For International Council for Laboratory Animal Science (ICLAS), Stuttgart and New York: Gustav Fischer Verlag; 1990. 12. Sitarz M, Woszczynski M, Czarnomska A. Charakterstyka wsobnych szczepów myszy pod wzgledem genetycznych markerów biochemicz­nych Mod-1, Gpd-1, Idh-1, Pgm-1 i Gpi-1s. Zwierzeta Laboratoryjne 1990; 27: 45-52 (abstract in English). 13. Nomura T, Esaki K, Tomita T. ICLAS Manual for ge­netic monitoring. University of Tokyo; 1984. 14. Gajewska M, Wirth-DziÍciolowska E. Application of the random amplification of polymorphic DNA (RAPD) method in genetic monitoring of inbred mouse strains. Animal Science Papers and Reports 2000; 18: 269 -75. 15. Szymanska H et al. Genetics of susceptibility to radiation-induced lymphomas, leukemias and lung tumors studied in recombinant congenic strains. Int J Cancer 1999; 83: 674-8. 16. Sitarz M, Wirth-Dzieciolowska E, Demant P. Loss of heterozygosity on chromosome 5 in vicinity of the telomere in gamma – radiation inducesd thymic lymphomas in mice. Neoplasma 2000; 47: 149. Radio/ Oncol 2001; 35(4): 237-42. Diagnosticna vrednost pneumoperitoneja pri rentgenskem slikanju abdomna na prazno Frkovic M, Klapan T, Moscatello I, Frkovic M Izhodišca. Pneumoperitonej imenujemo prisotnost zraka izven širine lumna crevesa in je naj­važnejši dokaz perforacije crevesa pri rentgenskem slikanju abdomna na prazno. Perforacija je lahko spontana ali pa posledica poškodbe. Najpogostejši vzrok spontane perforacije je razjeda želodca ali dvanajsternika. Zato je bil namen naše raziskave preveriti diagnosticno vrednost pneumoperitoneja pri rentgenskem slikanju abdomna na prazno. Bolniki in metode. V retrospektivni raziskavi smo analizirali 79 bolnikov, ki so bili v dvoletnem obdobju (1998-1999) napoteni v našo bolnico in operirani zaradi perforirane razjede želodca ali dvanajsternika. Preverjali smo diagnosticno vrednost rentgenske slike abdomna na prazno, predhodno narejene na bolnikih v stojecem polažju, leže na hrbtu in leže ne levem boku. Rezultati. Deset (12,66 %) od 79 bolnikov je bilo operiranih brez predhodne radiološke diagnos­tike. Pri 16 (87,34 %) bolnikih smo naredili radiološke preiskave, 53 (76,81 %) pa jih je imelo za­cetne znake pneumoperitoneja pri slikanju abdomna na prazno. Zakljucki. Najpogostejši vzrok pneumoperitoneja je bila perforirana razjeda dvanajsternika pri starostnikih. Najpogostejši rentgenološki znak pneumoperitoneja pa je bila srpasta kolekcija zraka pod trebušno prepono. Rndiol Oncol 2001; 35(4): 243-7. So podatki o umrljivosti za rakom verodostojni -analiza podatkov o umrljivosti zaradi raka maternicnega vratu Primic Žakelj M, Pompe Kirn V, Škrlec F, Šelb J Izhodišce. Uradni podatki o umrljivosti za rakom maternicnega vratu so manj zanesljivi, saj v zdravniških porocilih o vzroku smrti mesto izvora raka v maternici ni vedno dovolj natancno opredeljeno. Želeli smo oceniti, v kolikšni meri objavljeni podatki o umrljivosti za rakom mater­nicnega vratu v Sloveniji odražajo dejansko umrljivost za to boleznijo. Gradivo in metode. 2245 smrti, pri katerih je bil v podatkovni zbirki Inštituta za varovanje zdrav­ja v letih 1985-1999 kot vzrok smrti naveden rak maternicnega vratu, telesa ali maternice, smo racunalniško povezali s podatkovno zbirko Registra raka za Slovenijo. Izsledki. V obdobju 1985-1999 je po uradnih podatkih za rakom maternicnega vratu umrlo 878 žensk, v Registru raka je bilo od njih prijavljenih s to boleznijo le 87,7%. Med ženskami, ki naj bi umrle zaradi raka maternicnega telesa, jih je 17,1 % imelo v resnici raka maternicnega vratu, od 717, pri katerih je bil kot vzrok smrti naveden le rak maternice, pa je 31,4 % dejansko imelo raka maternicnega vratu. Ce med vzroki smrti upoštevamo pravilno opredeljen rak maternicne­ga vratu, dodatno pa še napacno ali premalo natancno opredeljene primere, je zaradi raka ma­ternicnega vratu umrlo 1106 žensk, kar pomeni, da so uradni podatki o umrljivosti za rakom ma­ternicnega vratu podcenjeni za 26 %. Zakljucek. Uradni podatki o umrljivosti za rakom maternicnega vratu podcenijo dejansko stanje, kar je treba upoštevati pri vrednotenju bremena te bolezni. To pa ne velja za vse rakave bolezni. S/ove11ia11 abstrncts Radio/ Onco/ 2001; 35(4): 249-54. Sporazumevanje po laringektomiji Hocevar-Boltežar I, Žargi M Izhodišca. Pri bolnikih z napredovalim rakom grla in spodnjega žrela je kirurška odstranitev gr­la najbolj primeren nacin zdravljenja. To zdravljenje pa negativno vpliva na številne pomembne bolnikove funkcije in tudi na govor. Bolniki in metode dela. Po odstranitvi grla je možnih vec razlicnih nacinov tvorbe glasu, tako da se vsak laringektomirani bolnik lahko nauci katerega od teh nacinov govornega sporazumeva­nja. Rezultati. Znanih je vec razlicnih, tudi elektronskih naprav, ki s pomocjo lastnih baterij proiz­vajajo zvok. Drugo možnost predstavlja ezofagalni govor, pri katerem se bolnik nauci uporabl­jati mišicni segment na prehodu v zgornji požiralnik kot generator glasu. Kirurško možnost gov­orne rehabilitacije po laringektomiji pa predstavlja vstavitev proteze v kirurško narejeno fistulo med zgornjim požiralnikom in sapnikom. Proteza usmeri zrak iz pljuc v zgornji požiralnik, glas pa nastane pri prehodu zraka skozi že omenjeni mišicni segment. Zakljucki. Številni dejavniki vplivajo na izbiro najprimernejšega nacina nadomestnega govora pri posameznem bolniku. V Sloveniji najvec laringektomiranih bolnikov uporablja ezofagalni govor. Radio/ 011col 2001; 35(4): 255-8. Kombinirano zdravljenje plošcatocelicnega karcinoma ustne votline in ustnega žrela. Globina tumorske invazije kot napovedni dejavnik Cizmarevic B, Lanišnik B, Didanovic V, Kramberger K Izhodišca. V raziskavi smo nameravali ugotoviti pomembnost kirurškega zdravljenja plošcato­celicnega karcinoma glave in vratu ter najti najpomembnejši napovedni dejavnik za širjenje kar­cinoma v vratne bezgavke in dolociti, kateri dejavnik v najvecji meri vpliva na preživetje bol­nikov. Bolniki in metode. Od 1. 6. 1992 do 31. 5. 1998 smo na Oddelku za otorinolaringologijo in cervikofacialno kirurgijo Ucne bolnišnice v Mariboru obravnavali 154 bolnikov s plošcatocelicn­im karcinomom ustne votline in ustnega žrela. Kriterijem naše retrospektivne študije je us­trezalo 142 bolnikov, a le 62 je bilo primernih za kombinirano zdravljenje s kirurgijo in pooper­ativno radioterapijo. Od 62/142 operairanih bolnikov je bilo 149 tudi pooperativno obsevanih, 13/62 bolnikov pa je odklonilo nadaljnje zdravljenje. Na kirurškem resektatu smo ugotavljali, ali tumor zaseva v vratne bezgavke, ali je bil izrezan v zdravo in kakšna je bila globinska invazija tumorja. Prav tako smo na tumorskih celicah dolocali proliferativni faktor Ki67. Rezultati. V multivariantni analizi, ki je vkljucevala tudi stopnjo diferenciacije tumorja, klinicni in patološki stadij tumorja (T in pT), je bila globina tumorske invazije najvažnejši napovedni de­javnik za širjenje karcinoma v vratne bezgavke. Ob uporabi Coxovega regresijskega modela pa je bila patohistološka opredelitev prizadetosti bezgavk (pN) pomemben dejavnik za preživetje bolnikov (p<0,05). Našli smo statisticno pomembno razliko med klinicno in patološko opre­delitvijo prizadetosti vratnih bezgavk (N in pN). V 23 primerih so bile bezgavke napacno ocen­jene kot prizadete s tumorskimi celicami, v 3 primerih pa so bile spregledane. Celotno 5-letno preživetje bolnikov je bilo 55 %. Izražanje Ki67 je bil odvisno od stopnje diferenciacije tumorja in statisticno znacilne povezave s širjenje karcinoma v vratne bezgavke nismo ugotovili. Zakljucki. Pri bolnikih s karcinomom ustne votline in ustnega žrela je bila globina tumorske in­ vazije najvažnejši napovedni dejavnik za zasevanje karcinoma v vratne bezgavke, preživetje bol­ nikov pa je bilo odvisno od prizadetosti vratnih bezgavk s tumorskimi celicami. Radio/ 011co/ 2001; 35(4): 259-66. Spremenjeni nacini zdravljenja rabdomiosarkoma pri otrocih, slovenske izkušnje Pohar-Marinšek Ž, Anžic J, Jereb B Izhodišca. Neoadjuvantna kemoterapija (KT) je spremenila nacin zdravljenja rabdomiosarkoma (RMS) otrok. Namen naše študije je bil analizirati rezultate zdravljenja pri otrocih, ki so bili zdravljeni v letih med 1974 in 1996. Bolniki in metode. V študijo smo vkljucili 51 otrok med enim in 15 letom starosti. Primarni tu­morji so bili v podrocju glave in vratu pri 15, v orbiti pri 6, v genitourinskem podrocju pri 12, na udih pri 9, na trupu pri 5 in paratestikularno pri 4 bolnikih. Dvanajst bolnikov je imelo bolezen s stadijem I, 10 s stadijem II, 26 s stadijem III in 3 s stadijem IV. Med 43 histološko potrjenimi primeri RMS je imelo 25 bolnikov embrionalni podtip, 13 alveolarni, 1 botrioidni, 1 vretenasto­celicni in 3 sarkom brez nadalnje opredelitve. Pri 8 bolnikih je bila morfološka diagnoza opre­deljena le iz vzorca aspiracijske biopsije s tanko iglo (ABTI). Vsi bolniki so prejeli KT, 29 neoad­juvantno, 20 je bilo najprej operiranih, 40 je bilo obsevanih (RT), 2 bonika z boleznijo v stadiju IV sta imela presaditev kostnega mozga. Kombinirana KT se je v razlicnih obdobjih razlikovala: z VRC, AMD in ciklofosfamidom (V AC) smo zdravili v 70-tih letih, kombinirano z Adriablastinom (T2), MTX in/ali drugimi kemoterapevtiki (T6, Tll) v 80-tih letih, ciklofosfamid smo zamenjali z ifosfamidom v 90-tih letih (VAIA). S kemoterapijo smo priceli pri tumorjih or­bite, glave in vratu in pri vecini tumorjev genitourinskega podrocja. Operacija je bila na prvem mestu pri paratestikularnih tumorjih in pri vecini tumorjev na udih. Rezultati. Vsi trije bolniki z boleznijo v stadiju IV so umrli. Med bolniki z lokaliziranimi tumor­ji je bilo 34 (70 %) živih in brez znakov bolezni 5 let po pricetku zdravljenja: 80% bolnikov s stadi­jem I, 75 % s stadujem II in 61 % s stadijem III. En bolnik je umrl zaradi odpovedi srca, 3 zaradi posledic KT in eden zaradi spremljajoce bolezni. Zakljucki. Preživetje otrok z RMS se je v Sloveniji izboljšalo v zadnjih dveh desetletjih iz 57 % na 70 % po letu 1985 in je sedaj primerljivo s preživetjem v centrih po svetu. Z uvedbo neoadju­vantne KT sta postali kirurgija in RT bolj ohranitveni (konzervirajoci) in se jima vcasih lahko ce­lo izognemo, s cimer zmanjšamo možnost neželenih posledic. Embrionalni RMS, ki zraste v or­biti, genitourinskem ali paratestikularnem podrocju ima v nizkem stadiju bolezni dobro prognozo. Tumorji, ki zrastejo na udih in v podrocju glave in vratu, vecinoma alveolarnega tipa, imajo slabo prognozo. Za alveolarni tip RMS in IV stadij bolezni današnji nacin zdravljenja ni ucinkovit, vkljucno s presaditvijo kostnega mozga. Radio/ Onco/ 2001; 35(4): 267-72. Obsevanje kraniospinalnega predela pri otrocih, ki ležijo na hrbtu Šlampa P, Seneklova Z, Simicek J, Soumarova R, Burkon P, Burianova L Izhodišca. S pooperativnim obsevanjem lahko veckrat preprecimo lokalno ponovitev bolezni in podaljšamo preživetje otrok z možganskimo tumorji. V clanku smo želeli predstaviti nov nacin pooperativnega obsevanja kraniospinalnega predela pri otrocih, ki so bili operirani zaradi možganskega tumorja; želeli pa smo tudi opisati morebitne stranske ucinke takšnega zdravlje­nja. Bolniki in metode. V štirih letih smo pooperativno obsevali kraniospinalno podrocje pri 17 otro­cih, starih manj kot 15 let. 8 jih je imelo meduloblastom, 6 ependimom in 3 glioblastom. Obsevali smo jih z novim nacinom, ki srno ga razvili na našem oddelku. Bolniki so ležali na hrb­tu in ne na trebuhu, kakor je sicer obicajno. Pri obsevanju srno uporabili nesimetricne celjusti linearnega pospeševalnika. Rezultati. Bolniki so radioterapijo dobro prenašali, pri nobenem ni bilo potrebno zmanjševati obsevalne doze. Pri vseh bolnikih so bile kožne reakcije blage. Prav tako so bili gastrointestinal­ni in hematološki ucinki blagi ali zmerni (stopnje I ali II po klasifikaciji SZO). Zakljucki. Nov nacin obsevanja kraniospinalnega podrocja bolnika ležecega na hrbtu povzroca malo stranskih ucinkov, zato ga priporocamo. Je alternativna metoda glede na obicajno obsevan­je, ko leži bolnik na trebuhu. Ker je opazovana doba naših bolnikov kratka, je oceana ucinkovi­tosti takšnega obsevanja (vpliv na lokalno ponovitev bolezni in preživetje) še nezanesljiva. Radio/ Oncol 2001; 35(4): 273-6. Diagnosticna ultrazvocna preiskava anusa za ugotavljanje analnega karcinoma. Prikaz primera Sudol-Szopiriska I, Szczepkowski M, Jakubowski W Izhodišca. Opisujemo primer bolnika z adenokarcinomom, s katerim želimo prikazati uporab­nost ultrazvocne preiskave v diagnostiki analnega karcinoma. Prikaz primera. Ultrazvocno preiskavo smo opravili z odcitalcem Bruel&Kjaer, tip 3535 z en­doskopsko sondo s frekvcenco 70 MHz. Preiskava je bila opravljena na bolniku, ki je ležal na levem boku. Z ultrazvocno preiskavo anusa smo natancno izmerili globino infiltracije tumorja v steno anusa ter ugotovili lokacijo in ultrazvocne znacilnosti tkiva. Z ultrazvocno preiskavo smo ocenili tudi prizadetost perianalnih limfnih bezgavk in razširjenost tumorja v sosednja tkiva. Zakljucki. Ultrazvocna preiskava anusa je zelo primerna metoda za ugotavljanje globine infil­tracije analnega karcinoma in za zbiranje podatkov, ki so pomembni pri odlocitvi o nacinu zdravljenja. S/ovenian abstracts Radio/ Onco/ 2001; 35(4): 277-91. Prve izkušnje z novo luminescencno opticno metodo za merjenje oksigenacije v tumorjih Jann T, Lešnicar H, Serša G, Miklavcic D Izhodišca. S to uvodno študijo smo želeli predvsem ugotoviti uporabnost nove luminescencne opticne metode (sistem OxyLite) za merjenje delnega tlaka kisika v tumorjih (pO2) in za casovno spremljanje sprememb pO2. Hkrati s to novo metodo smo uporabili opticno metodo laser Doppler, s katero lahko merimo relativni pretok krvi v tkivu. Materija! in metode. Meritve smo opravili na tumorjih SA-1 pri miših A/J, ki so bile med poskusi anestetizirane. Za casovno zvezno zajemanje meritev pO2 in pretoka krvi smo uporabili senzor­je v obliki opticnih vlaken, ki smo jih vstavili v tumor. S spreminjanjem koncentracije vdihane­ga anestetika in z uporabo vazoaktivne ucinkovine hidralazin smo izzvali spremembe v pretoku krvi in oksigenaciji. Rezultati. Rezultati so pokazali, da sta v splošnem obe merilni metodi uspešno zaznali spre­membe v pretoku krvi oziroma pO2 . Izmerjene spremembe pO2 in spremembe v mikrocirkulaciji so bile v tesni korelaciji. V vecini meritev pO2 smo naleteli na nepricakovan potek signala med umirjanjem izmerjene vrednosti takoj po vstavitvi merilne sonde. Tak potek signala je najver­jetneje posledica lokalnih poškodb tkiva, ki nastanejo med vstavljanjem sonde v tumor. Zakljucek. Nadaljne reziskave bodo potrebne za podrobnejšo osvetlitev tega vidika meritev z novo luminescencno opticno metodo za merjenje oksigenacije. Radio/ Onco/ 2001; 35(4): 293-302. Prisotnost kromosomskih sprememb v somatskih celicah pri bolnikih s tumorji mod po razlicnih nacinih zdravljenja Bilban Jakopin C in Bilban M Izhodišca. Raziskave v zadnjih 35 letih so pokazale, da so spremembe v genomu posameznih cel­ic povzrocene tako zaradi zdravljenja s citostatiki, kot zaradi obsevanja z ionizirajocimi žarki. Te spremembe v celici nam lahko služijo kot biološki dozimeter. Namen raziskave je bil najti pris­otnost kromosomskih sprememb v somatskih celicah pri bolnikih s tumorji mod po razlicnih nacinih zdravljenja. Pacienti in metode. V prospektivno študijo smo vkljucili 60 bolnikov s tumorji mod, ki srno jih glede na histološke izvide in nacine zdravljenja razdelili v štiri skupine. Pred zdravljenjem ni bi­lo odstopanj v genomski sliki v primerjavi z enako kontrolno skupino pregledovancev brez ma­lignoma. Vpliv zdravljenja na genom bolnikov smo ugotavljali s pregledom genornske slike pred zdravljenjem, po zdravljenju in šest mesecev kasneje s priznanimi citogenetskimi testi, s kater­imi smo pogledali strukturne spremembe kromosomov -SCA, izmenjavo med sestrskimi kro­matidami -SCE in mikronukleus -MN pri dvojedrnih limfocitih. Rezultati. Takoj po koncanem obsevanju smo ugotovili mocno inhibicijo mitotske aktivnosti limfocitov in zvecano število nestabilnih kromosomskih sprememb (dicentrikov). Kemoterapija je vplivala na genomsko sliko v manjšem odstotku, tip kromosomskih sprememb je bil dru­gacen. S citološko mutagenetskega vidika se je pokazalo, da je samo obsevanje bolj agresivno od kemoterapije. Šest mesecev po zakljucenem zdravljenju je mitoticna aktivnost vecinoma normalna, vendar pa je še vedno zelo velik odstotek sprememb glede na zacetne vrednosti in v primerjavi z bolniki, ki so bili le operirani. Zakljucek. Tako po obsevanju kot po zdravljenju s citostatiki pride do normalizacije genoma, ka­jti poškodovane celice odmrejo. Glede na to, da je pri obsevanih bolnikih le majhen tkivni celicni volumen izpostavljen obsevanju, pri kemoterapiji pa je celotni organizem izpostavljen kemi­jskemu agensu, upraviceno pricakujemo hitrejšo popravo genoma pri obsevanih bolnikih. Slovenia11 abstracls Radio! 011col 2001; 35(4): 303-8. Zakonodaja o zašciti poskusnih živali Ornik D, Pogacnik M Namen pricujocega dela je bil ugotoviti stanje na podrocju zakonodaje o zašciti poskusnih živali v Republiki Sloveniji. Zašcita poskusnih živali je urejena z dolocili Zakona o zašciti živali. Na njegovi podlagi se uporablja Navodilo o pogojih za izdajo dovoljenja za poskuse na živalih v znanstveno-razisko­valne namene in Pravilnik o eticni komisiji za poskuse na živalih. Osnovna dolocila zašcite poskusnih živali so v sistemu izdaje dovoljenj za poskuse. Dovoljenje za poskuse na živalih izdaja upravni organ, pristojen za veterinarstvo, ce so poskusi nujno potrebni in nujni iz medicinskih, veterinarsko-medicinskih ali znanstveno-raziskovalnih razlo­gov in se pricakuje, da bodo rezultati prinesli pomembna nova spoznanja, oziroma je trpljenje živali eticno sprejemljivo v primerjavi s pricakovanim dosežkom; nadalje tudi ce gre za temeljne raziskave, ce se poskusnih ciljev ne da doseci z drugimi metodami in postopki, in ce se poskus izvede z najmanjšim možnim številom živali z najnižjo nevrofiziološko obcutljivostjo in metodo, ki povzroca najmanj trpljenja, bolecin in trajnih poškodb. Osebje za vodenje in izvajanje poskusa ter za nego in oskrbo živali, prostori za bivanje oziroma rejo in oskrbovanje živali ter naprave in priprave morajo izpolnjevati predpisane pogoje. Z sprejetjem zakona, ki je usklajen z evropskimi predpisi, se je zacelo urejati tudi podrocje zašcite poskusnih živali. Kot doloca zakon bodo izvršilni predpisi dolocali natancnejše pogoje za izdajo dovoljenj za opravljanje poskusov na živalih, o postopku, dokumentaciji, evidenci, porocilih in o obveznostih strokovne osebe za zašcito živali, kakor tudi o kadrovskih in drugih pogojih za izvajanje poskusov ter o postopku z živalmi po koncanem poskusu. Radia/ 011cal 2001; 35(4): 309-15. Standardizacija laboratorijskih živali za biomedicinske raziskave na poljskem Szymariska H, Krysiak E, Piskorowska J, Woszczyriski M, Czarnomska A Vedno vecje potrebe po biomedicinskih raziskavah zahtevajo kakovostne laboratorijske živali. Hkrati pa si znanstveniki prizadevajo za omejitev števila žrtvovanih živali. Nekatere mednaro­dne organizacije, kot so ICLAS, FELASA ali SOLAS, se ukvarjajo z propagiranjem primerne re­je in oskrbe laboratorijskih živali. Leta 1997 poljska vlada je sprejela Zakon o zašciti živali. Standarizacija laboratorijskih živali za biomedicinske raziskave na Poljskem je poglavitna nalo­ga Komisije za laboratorijske živali Poljske akademije znanosti in Poljskega narodnega komite­ja za sodelovanje z ICLAS-om. Leta 2000 je bil Oddelek za genetiko in rejo laboratorijskih živali Centra za zdravljenje raka Maria Sklodowska Curie in Onkološkega inštituta v Varšavi obliko­van kot Narodni referencni center za rejo laboratorijskih živali SPF. Oddelek je sestavljen iz petih delov: l. Banka SPF; 2. Laboratorij poskusnih živali; 3. Laboratorij za nadzor zdravja; 4. Genetski laboratorij; 5. Anatomsko-patološki laboratorij. Trendi v razvoju znanosti o laboratorijskih živalih na Poljskem in v Evropi so si podobni. Standarizacija labora­torijskih živali se zdi po mednarodnih priporocilih zadostna v vodecih centrih. Notices Notices submitted for publication s/10uld contain a mailing address, phone and/or fax number and/or e-mail oj a Contact person or department. Chemotherapy December 12-14, 2001. "5th The lnternational Symposium on Clinical Febrile Neutropenia" will be offered in Brussels, Belgium. Contact Mrs. Martine Hazard and call +32 2 541 3201; or fax +32 2 541 3202; or e-mail martine.haz­ard@bordet.be Radiation therapy Felmtary 6-8, 2002 The symposiurn "Cardiovascular Revascularization Therapy" and satellite meetings will take placc in Washington, DC, USA. Contact Cardiovascular Rcsearch Institute, CRT 2002 Registration, 110 lrving Street, NW, Suite 6D, Washington, DC 20010, USA; or cal +1 202 877 7947; or fax +1 202 877 8141; or see http://www.radiation­line.com Lung cancer March 7-9, 2002. The "2"'1 World Confcrence on Clinical Cooperative Research for Lung Cancer" will be offered in Brussels, Belgium. Contact European Lung Cancer Working Party, c/o Prof. J.-P. Sculier, Institute Jules Bordet, 1, rue Heger­ Bordet, B-1000 Brussels; or call +32 25 39 04 96; or fax +32 25 34 37 56; or e-rnail 101473.1044t"com­ puserve.com; or see http://www.elcwp.org Radiotherapy Marc/1 10-14, 2002 The ESTRO teaching course "Radiothernpy Treatrnent Planning: Principles and Practice" will take place in Dublin, Ireland. Contact ESTRO office, Av. E. Mounier, 83/ 4, B-1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail info«,>estro.be; or see http://www.estro.be Lung cancer March 14-15, 2002 The !ASLC international workshop "Early lnvasive Lung Cancer. New Diagnostic Tools & Trcatrnent Strategies will be offercd in Turin, ltaly. Contact Organising Secrctariat, CCI Centra Congressi lnternazionale srl, Via Cervino 60, 10155 Turin, ltaly; or call +39 011 244 69 16; or fax +39 011 244 69 00; or e-mail info«i>congressiefiere.com Gastroenterology and hepatology Mnrch 14-16, 2002 The teaching course "Gastroenterology and He­patology'' will take place in Las Croabas, Puerto Rico. Contact Program Coordinator, John Hopkins University, Turner 20/720 Rutland Avenue, Baltimore, Maryland 21205, USA; or call +1 410 955 2959; or fax +1 410 955 0807; or e-mail cmeneW'jhmi.edu; or sec http://www.med.jhu.edu/cme Brachyherapy Mnrc/1 24-26, 2002 The ESTRO teaching course "Endovascular Brachytherapy" will take place in Vienna, Austria. Contact ESTRO office, Av. E. Mounier, 83/4, B-'1 200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail info«oestro.bc; or see http://www.estro.be Breast cancer April 5-6, 2002 The teaching course "Current Concepts in the Multidisciplinary Management of Early-Stage Breast Cancer" will take place in Baltimore; Maryland, USA. Contact Program Coordinator, John Hopkins University, Turner 20/720 Rutland Avenue, Baltimore, Maryland 21205, USA; or call +1 410 955 2959; or fax +1 410 955 0807; or e-mail cmenet@jhrni.edu; or see http://www.med.jhu.edu/cme Thoracic surgery April 11-12, 2002. The "5°1 International Meeting on General Thoracic Surgery" will be offered in Barcelona, Spain. Contact RCT, C/ Aulestia i Pijoan, 12 Baixos 98012, Barcelona, Spain, or call +34 93 415 69 38; or fax +34 415 69 04; or e-mail rct@rct-congresos.com Radiation physics April 14-18, 2002 The ESTRO teaching course "Physics for Clinical Radiotherapy" (Extra edition) will take place in lzmir, Turkey. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Bclgium; or call +32 7759340; or fax +32 2 7795494; or e-rnail info@estro.be; or see http://www.estra.be Radiation oncology April 21-25, 2002 The ESTRO teaching course "Radiation Oncology: A Molecular Approach" will take place in Santorini, Greece. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-rnail info«vestro.be; or see http://www.estro.be Radiotherapy April 21-25, 2002 The ESTRO teaching course "Dose Determination in Modem Radiotherapy: Beam Characterisation, Calculation and Verification" will take place in Perugia, ltaly. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-rnail info@estro.be; or see http://www.estra.be Radiotherapy May 9-11, 2002 The Annual Brachytherapy Meeting GEC/ESTRO will take place in Antalya, Turkey. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail info@estro.be; or see http://www.estro.be Nuclear medicine May 12-15, 2002. The "41h International Congress of the Croatian Society of Nuclear Medicine" will be offered in Opatija, Croatia. Contact Prof. Damir Dodig or Mr. Božidar Kasal, Nuclear Medicine Congress Secretariat, KBC Rebro, Kišpaticeva 12, 10000 Zagreb, Croatia, or call +385 1 24 21 851; or fax +385 1 24 21 874; or e-mail bkasal«i>public.srce.hr; or see http://jygor.srce.hr/nu­clrnedzg-rebro/ Radiation therapy May 15-19, 2002 The 7th lnternational Meeting on Progress in Radio­Oncology ICRO/OGRO 7 will take place in Salzburg, Austria. Contact Prof. D.H. Kogelnik, Salzburg, Austria; call +43 662 44823900; or fax +43 662 4482887; or e-mail d.kogelnik@lkasbg.gv.at Radiotherapy Ju11e 2-6, 2002 The ESTRO teaching course "!MRI and Other Conformal Techniques in Practice" will take place in Amsterdam, the Netherlands. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail info@estro.be; or see http://www.estro.be Breast cancer june 5-7, 2002 The "4th Milan Breast Cancer Conference" will take place in Milan, ltaly. Contact ESO Office, Viale Beatrice d'Este 37, 20122 Milan, ltaly; or call +39 0258317850; or fax +39 0258321266; or e-mail esomi@tin.it; or see http://www.cancerworld.org Notices 327 Radiology June 9-11, 2002 The "UK Radiological Congress 2002 UKRC 2002" will take place in London, UK. Contact UKRC Secretariat, PO Box 2895, London, WlA SRS, UK; or call +44 (0)20 7307 1410; or fax +44 (0)20 7307 1414; or e-rnail conference@lukrc.org.uk Bronchology and bronchoesophagology June 16-19, 2002. The "12th World Congress for Bronchology" and the "12th World Congress for Bronchoesophagology" will be offered in Boston, USA. Contact Congress Secretariat. Tufts University School of Medicine. Office of Continuing Education, 136 Harrison Avenue, Boston, MA 02111, USA, or call +1 617 636 6509; or fax +l 617 636 0472; or see http://www.aabronchology.org Brachytherapy June 16-20, 2002 The ESTRO teaching course "Modem Brachy­therapy Techniques" will take place in Lisbon, Portugal. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail infoQDestro.be; or see http://www.estro.be Clinical oncology June 20-22, 2002. The "3rd Jnternational Anglo-Croatian Symposium on Clinical Oncology" in collaboration with "51 Radiotherapy Club" (UK) meeting will be offered in Dubrovnik Cavtat, Croatia. Contact Dr. Fedor Šantek, Executive Secretary; Medica! school, Clinic of Oncology and Radiotherapy, University Hospital Centre Rebro, Kišpati)eva 12, Zagreb, Croatia; or call +385 l 4552 333. Radiotherapy June 23-27, 2002 The ESTRO teaching course "IMRT and Other Confonnal Techniques in Practice" will take place in Amsterdam, The Netherlands. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail info@lestro.be; or see http://www.estro.be Radiotherapy June 23-27, 2002 The ESTRO teaching course "Jmaging for Target Volurne Determination in Radiotherapy" will take place in Coimbra, Portugal. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgiurn; or call +32 7759340; or fax +32 2 7795494; or e-mail infolIDestro.be; or see http://www.estro.be Oncology Ju11e 30 -July 5, 2002. The "18th UICC lnternational Cancer Congress" will be offered in Oslo, Norway. Contact Norwegian Cancer Society, P.O. Box 5327 Majorstua, N-0304 Oslo, Norway, or call +47 22 59 30 00; or fax +47 22 60 69 80; or e-mail cancer@oslo2002.org Radiology July 1-5, 2002 The "22nd Jnternational Congress of Radiology (JCR 2002)" will take place in Cancun, Mexico. Contact B.P. Servimed, S.A. de C.V., at Insergentes Sur No. 1188 50 piso, Col. Del Valle, 03210 Mexico DF, Belgium; or call +525 575 9931; or fax +525 559 9407; or e-mail fmricr«•'servimed.com.rnx Clinical Oncology August 4-9, 2002 The "Masterclass in Clinical Oncology" will take place in Montecatini Terme, ltaly. Contact Dr. Wolfgang Gatzermeier, ESO Office, Viale Beatrice d'Este 37, 20122 Milan, ltaly; or call +39 02583.17850; or fax +39 0258321266; or e-rnail es­ oweb«Dtin.it Radiotion physics August 25-29, 2002 The ESTRO teaching course "Physics for Clinical Radiotherapy" will take place in Leuven, Belgiurn. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgiurn; or call +32 7759340; or fax +32 2 7795494; or e-mail info«ilestro.be; or see http://www.estro.be Radiobiology August 25-29, 2002 The ESTRO teaching course "Basic Clinical Radiobiology" will take place in St. Petersburg, Russia. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail info@>estro.be; or see http://www.estro.be Prostate cancer Septe111ber 1-3, 2002 The ESTRO teaching course "Brachytherapy for Prostate Cancer" will take place in Utrecht, the Netherlands. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail info[i>estro. be; or see http://www.estro.be Lung cancer Septe111ber 1-4, 2002 The "8th Central European Lung Cancer Conferen­ce" will be offered in Vienna, Austria. Contact Conference Secretariat, Mondial Congress, Faulmanngasse 4, A-1040 Vienna, Austria; or call +43 1 588 04 O; or fax +43 1 586 91 85; or e-mail con­gress[!lmondial.at Lung cancer Seple111ber 8-12, 2002 The "IASLC Workshop on Progress and Guidelines in the Management of Non Small Celi Cancer" will be offered in Bruges, Belgium. Contact Secretariat, P. van 1-loutte, Dept. Radiotherapy, Institute Jules Bordet, Rue Heger­Bordet 1, B-1000 Brussels, Belgium; or call +32 2 541 3830; or fax +32 2 538 7542; or e-mail paul.van­houtte@bordet.be Medica! physics September 9-13, 2002 The "10'h lnternational Congress on Boron Neutron Capture Therapy" will take place in Essen, Germany. Contact Dr. Ray Moss with e-rnail moss«lljrc.nl Radiation therapy September 17-21, 2002 The 21 st Annual ESTRO Meeting will take place in Prague, Czech Republic. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgiurn; or call +32 7759340; or fax +32 2 7795494; or e-rnail info@estro.be; or see http://www.estro.be Oncology Seple111ber 29 October 3, 2002. The "2nd World Assembly on Tobacco Counters J-lealth" will be offered in New Delhi, India. Contact Convenor, W ATCH 2002, 509-B, Sari ta Vihar, New Delhi 110 044, lndia; or call +91 11 694 4551; or fax +91 11 694 4472; or e-mail cancerak@Jdel6.vsnl.net.in; or see http://www.watch­2000.org Radiation therapy October 6-9, 2002 ASTRO Annual meeting will bc held in New Orleans, Louisiana, USA. Contact American Society for Therapeutic Radiology and Oncology Office, 1891 Preston White Drive, Reston, VA 20191, USA; or see http://www.as­tro.org Radiation oncology Nove111ber 10-16, 2002 The ESTRO teaching course "Evidence-Based Radiation Oncology: Methodological Basis and Clinical Application" will take place in Tenerife, Spain. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail info«Destro.be; or see http://www.estro.be Radiation oncology Marc/1 15-19, 2003. The "211d International Conference on Translation Research and Pre-Clinical Strategies in Radiation Oncology, ICTR 2003" will be offered in Lugano, Switzerland. Fax +41 91 820 9044, or e-mail jbernier«Dpop.eu­net.ch, or see http://www.osg.ch/ictr2003.html Radio/ 011col 2001; 35(4): 325-9. Notices 329 Lung cancer Aug11s/ 10-14, 2003. The "10th World Confercnce of the lntcrnational Association for the Study of Lung Cancer" will be of­fered in Vancouver, Canada. Contact 10th World Conferencc of Lung Cancer, c/o [nternational Confcrence Services, 604-850 West Hastings, Vancouver BC Canada V6C 1E'J, or call +1 604 681 2] 53; or fax +1 604 681 1049; or c-mail con­fercnce."2003worldlungcancer.org Radiation therapy Seple111ber 21-25, 2003 Thc ESTRO 22 / ECCO ·12 Meeting will take place in Copenhagen, Denmark. Contact FECS office, Av. E. Mounier, 83/4, B-1200 Brnssels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail info«1>estro.be; or sec http://www.fccs.be Radiation therapy Octoher J 9-23, 2003 ASTRO Annual meeting will be held in Salt Lake City, Utah, USA. Contact American Society for Thernpcutic Radiology and Oncology Office, 1891 Preston White Drive, Reston, VA 20191, USA; or see http://www.as­tro.org Radiation therapy September 12-16, 2004 The 23rd Annual ESTRO Meeting will be held. Contact ESTRO office, Av. E. Mounier, 83/4, B-1200 Brnssels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-rnail info«!'cstro.bc; or see http://www.estro.be Radiation therapy Octo/Jer 3-7, 2004 ASTRO Annual mecting will bc held in Atlanta, USA. Contact Arnerican Society for Thernpeutic Radiology and Oncology Office, 1891 Preston White Drive, Reston, VA 20191, USA; or sce http:// www.as­tro.org As i:l service to our readcrs, notices of 111eetings or courses will be inscrtcd free of charge. Please scnt information to the Editorial office, Radiology and Oncology, Zaloška 2, Sl-1000 Ljubljana, Slovenia. Radio/ 011col 2001; 35(4): 325-9. Radio/ O/lcol 2001; 35(4): 330-4. Reviewers in 2001 Anžic J, Ljubljana, Slovenia -Berden P, Ljubljana, Slovenia -Bilban -Jakopin C, Ljubljana, Slove­nia Budja M, Slovenj Gradec, Slovenia -Burger J Ljubljana, Slovenia -Casar B, Ljubljana, Slo­venia -Cerar A, Ljubljana, Slovenia -Cemažar M, Ljubljana, Slovenia -Cufer T, Ljubljana, Slo­venia -Grošelj C, Ljubljana, Slovenia -Hertl K, Ljubljana, Slovenia -Horvat Dj, Zagreb, Croatia Kadivec M, Ljubljana, Slovenia -Kocijancic I, Ljubljana, Slovenia -Kocijancic K, Ljubljana, Slo­venia -Kolonic V, Ljubljana, Slovenia -Kotnik V, Ljubljana, Slovenia -Kovac V, Ljubljana, Slo­venia -Lešnicar H, Ljubljana, Slovenia -Lindtner J, Ljubljana, Slovenia -Majdic E, Ljubljana, Slovenia -Marolt F, Ljubljana, Slovenia -Miloševic Z, Ljubljana, Slovenia -Pavlin-Košir S, Lju­bljana, Slovenia -Princic J, Ljubljana, Slovenia -Rainer S, Slovenj Gradec, Slovenia -Repše S, Ljubljana, Slovenia -Robar V, Ljubljana, Slovenia -Serša G, Ljubljana, Slovenia -Snoj M, Lju­bljana, Slovenia -Škrk J, Ljubljana, Slovenia -Šmid L, Ljubljana, Slovenia -Šuštaršic J, Ljublja­na, Slovenia -Tomšic-Demšar R, Ljubljana, Slovenia -Velenik V, Ljubljana, Slovenia -Zaletel L, Ljubljana, Slovenia -Zebic A, Ljubljana, Slovenia Supplement 1/2001 Kadivec M, Ljubljana, Slovenia -Miklavcic L, Valdoltra, Slovenia -Pirc S, Kamnik, Slovenia -Rener M, Ljubljana, Slovenia -Vargazon T, Ljubljana, Slovenia -Zdešar U, Ljubljana, Slovenia Editors greatly appreciate the work of the reviewers who significantly contributed to the impro­ved quality of our journal. Author Index 2001 Altenhoff J: 2/95-103 Corles Cf1L: 1/1-10 Anžic J: 4/259-66 Czarnomska A: 4/309-15 Babic Z: 2/105-10 Cemažar M: 2/139-47 Bafaloukos D: 3/209-14 Cižmarevic B: 4/255-8 Baitchev G: 1/43-6; 2/133-7 Barbaric-Babic V: 1/11-6 Dabo N: 2/105-10 Beravs K: 2/139-47 Deliisky T: 2/133-7 Bilban-Jakopin C: 4/293-302 Demšar F: 2/139-47 Bilban M: 4/293-302 de Wit M: 1/21-30 Bohuslawizki KH: 1/21-30; 2/95-103; 3/185-91 Didanovic V: 4/255-8 Bracko M: 1/47-52 Brkljacic B: 2/83-8 Ebert M: 3/215-24 Brountzos E: 3/209-14 Edge SB: 1/35-41 Buchert R: 2/95-103 Ekl O: 1/31-4 Burger J: 2/117-26 Burianova L: 4/267-72 Fallone BG: 1/63-71 Burkon P: 4/267-72 Fidler V: 1/53-61 Flisar K: 3/203-7 Cerovic R: 3/175-7 Frkovic Marija: 1/11-6; 4/237-42 Clausen M: 2/95-103 Frkovic Marijan: 4/237-42 Fuckm· Ž: 3/167-73 Genser B: 2/127-31 Glavaš R: 3/167-73 Golouh R: 1/47-52 Gortchev G: 1/43-6; 2/133-7 Grga A: 2/83-8 Groell R: 3/161-5 Guna F: 2/117-26 Hackl A: 2/]27-31 Hebrang A: 2/83-8 Hocevar-Boltežar 1: 4/249-54 Hurd Th: 1/35-41 lvaniš N: 1/31-4 Ivanuša T: 2/139-47 Jakubowski W: 4/273-6 Jarrn T: 4/277-91 Jenicke L: 2/95-103; 3/185-91 Jereb B: 4/259-66 Jevtic V: 2/139-47 Jureti<' M: 3/175-7 Kelekis D: 3/209-14 Keller FS: 1/1-10 Klapan T: 4/237-42 Klutrnann S: 2/95-103 Koren A: 3/179-83 Kovac D: 1/31-4 Kragelj B: 2/117-26 Kramberger K: 4/255-8 Krysiak E: 4/309-15 Lachaine M: 1/63-71 Lanišnik B: 4/255-8 Lee RJ: 1/35-41 Lešnicar H: 4/277-91 Macek-Lebar A: 3/193-202 Mayer R: 2/127-31 Medved 1: 1/31-4 Miklavcic D: 3/193-202; 3/203-7; 4/277-91 Miletic D: 3/167-73 Molnar M. 1/11-6 M6r6 Zs: 2/89-93 Moscatello !: 4/237-42 Miiller V: 1/21-30; 3/185-91 Naumovski-Mihalic S: 2/105-10 Nebeling B: 2/111-5 Neuber K: 2/95-103 Nikolov S: 2/111-5 Niyyati M: 2/89-93 Ornik D: 4/303-8 Palcic!: 2/105-10 Panagiotou I: 3/209-14 Park JW: 1/1-10 Patrlj L: 2/83-8 Pavcnik D: 1/1-10 Perhoc Ž: 1/11-6 Piskorowska J: 4/309-15 Podgoršak MB: ·1/35-41 Pogacnik M: 4/303-8 Pohar-Marinšek Ž: 4/259-66 Pompe Kirn V: 4/243-7 Prepadnik M: 1/53-61 Prettenhofer U: 2/127-31 Primic Žakelj M: 4/243-7 Prkacin I: 2/105-10 Proulx GM: 1/35-41 Puc M: 3/203-7 Reberšek S: 3/203-7 Rosch J: 1/1-10 Schaffler G: 3/161-5 Schloffer S: 3/161-5 Seneklova Z: 4/267-72 Sergieva S: 1/43-6 Serša G: 2/139-47; 4/277-91 Simicek J: 4/267-72 Smrke D: 1/17-9 Soumarova R: 4/267-72 Stankovski V: 1/17-9 Steinhagen J: 1/21-30 Stomper PC: 1/35-41 Stranzl H: 2/127-31 Sudo;;c-Szopinska 1: 4/273-6 Szczepkowski M: 4/273-6 Szymanska H: 4/309-15 Šebecic B: 2/83-8 Šelb J: 4/243-7 Šeronja-Kuhar M: 1/11-6 Šimic O: 1/31-4 Škrlec F: 4/243-7 Šlampa P: 4/267-72 Šustic A: 1/31-4; 3/175-7 Timmermans H: 1/1-10 Topljak-Polic D: 3/167-73 Uchida BT: 1/1-10 Uravic M: 3/167-73 Varga G: 2/89-93 Velkova A: 2/133-7 Vujanic S: 2/105-10 Vukovic J: 1/11-6 W edler J: 2/95-103 Weiner GM: 3/185-91 Autlwr lndex 2001 Willfurth P: 2/127-31 de Wit M: 1/21-30 Woszczynski M: 4/309-15 Žargi M: 4/249-54 Xie Y: 1753-61 Yamada K: 1/1-10 Yamakado K: 1/1-10 Yovtchev Y: 2/111-5 Supplement 1/2001 Coer A: Suppl 1/44-50 Dahmane R: Suppl 1/44-50 Guna F: Suppl 1/7-18 Hertl K: Suppl 1/7-18, Suppl 1/51-9 Hudales B: Suppl 1/74-80, Suppl 1/81-6, Suppl 1/87-91 Jancar B: Suppl 1/60-7 Jokan B: Suppl 92-101 Kadivec M: Suppl 1/51-9 Kocijancic I: Suppl 1/60-7 Kofjac N: Suppl 1/I (Uvodnik) Kokot A: Suppl 1/74-80, Suppl 1/81-6, Suppl 1/87-91 Korat S: Suppl 1/26-9, Suppl 1/30-6 Krajnc Z: Suppl 1/26-9, Suppl 1/30-6 Miklavcic L: Suppl 1/1-6, Suppl 1/19-25 Novak G: Suppl 1/26-9, Suppl 1/92-101 Podkrajšek M: Suppl 1/51-9 Rener M: Suppl 1/7-18, Suppl 1/51-9 Škrk D: Suppl 1/37-43 Špeh P: Suppl 1/74-80, Suppl 1/81-6, Suppl 1/87-91 Vargazon T: Suppl 1/92-101 Vegelj-Pirc M: Suppl 1/68-73 Zdešar U: Suppl 1/30-6 Subject Index 2001 abdomen: 3/167-73 acguisition card: 1/53-61 acute ultrasonography, abdominal ultrasound: 2/89-93 amorphous selenium: 1/63-71 animal husbandry: 4/303-8 animal rights: 4/303-8 animals, laboratory: 4/303-8 anus neoplasms ultrasonography: 4/273-6 artifacts: 3/185-91 axilla: 1 / 43-6 benign bone diseases: 3/185-91 bile ducts, stents: 1/1-10 biomechanics: 1/17-9 biopsy: 1/ 43-6 bone: 3/185-91 -bone diseases -radionuclide imaging: 1/21-30 -bone neoplasms: 1/21-30; 3/209-14 bone scintigraphy, 99mTc-diphosphonates, indi­ cations: 1/21-30 brachytherapy: 2/1'17-26 brain neoplasms -radiotherapy: 4/267-72 brain neoplasms-secondary-radiotherapy, brain me- tastses: 2/127-31 breast neoplasms: 1/43-6 -breast neoplasms -radiotherapy: 1/35-41 -breast neoplasms, stage I breast carcinoma: 2/133-7 breath holding: 3/161-5 carcinoma, renal celi: 2/105-10 carcinoma, sguamous celi: 4/255-8 catheterization, central venous: 3/175-7 celi membrane permeabilization: 3/203-7 celi survival: 3/193-202 cervix neoplasms -mortality: 4/243-7 child: 1/11-6; 4/267-72 chromosomal aberations: 4/293-302 colorectal neoplasrns -therapy: 2/111-5 cornbined modality therapy: 4/255-8 cornputed tornography: 3/179-83 computer systerns: 1/17-9; 1/53-61 contrast media: 2/139-47 death certificates: 4/243-7 diphosphonates: 1/21-30 diverticulitis; diverticulosis, colonic: 2/89-93 DQE, AMFPI, EPID: 1/63-71 drainage: 3/167-73 early breast cancer: 1/43-6 electromagnetic fields: 3/193-202 electroporation: 2/139-47; 3/193-202 -electroporation -instrurnentation -rnethods: 3/203-7 garnrna cameras: 1/53-61 germinoma, intracranial: 3/179-83 hip joint: 1/17-9 hyperthrophy: 1/11-6 imaging: 3/209-14 intestinal mucosa: 1/1-'J O intraoperative radiation, breast cancer, breast con­ servation therapy: 1/35-41 Karnofsky performance status: 2/127-31 kidney neoplasms -secondary: 2/105-10 laboratory animals: 4/309-15 laryngeal neoplasms: 4/249-54 laryngectomy: 4/249-54 laser -doppler flowmetry: 4/277-98 legislation: 4/303-8 luminiscence: 4/277-91 lymphatic metastasis: 1/43-6 lymph nodes pathology: 1/43-6 macromolecular systems: 2/139-47 magnetic resonance: 3/179-83 -rnagnetic resonance imaging: 2/139-47; 3/11'1-5 malignant bone diseases: 3/185-91 malignant melanoma, bone metastases: 3/209-14 melanoma: 2/95-103 micronuclei: 4/293-302 morphological imaging: 2/95-103 mouth neoplasms -therapy: 4/255-8 neoplasms invasiveness: 4/255-8 neoplasms staging: 1/47-52; 2/95-103 99mTc_diphosphonates: 3/185-91 oropharyngeal neoplasms therapy: 4/255-8 oxygen: 4/277-91 peptic ulcer perforation: 4/237-42 peripheral vascular diseases ultrasonography, ve­ ins: 2/83-8 PIP-GAMMA-PF system: 1/53-61 pleural effusion -ultrasonography, guantification: 1/31-4 pneumoperitoneum -radiography: 4/237-42 Poland: 4/309-15 portal imaging, Mante Carla method: 1/63-71 prognosis :1/ 4 7-52 -prognosis, prognostic factors: 2/133-71 propidium iodide: 3/193-202 prostatic neoplasms -radiotherapy: 2/117-26 pyloric stenosis -radiotherapy -surgery: 1/11-6 radiobiology: 3/215-24 radionuclide imaging: 3/185-91 radiotherapy: 3/209-14 -radiotherapy adverse effects: 2/117-26 -radiotherapy -dosage: 2/117-26 -radiotherapy -methods: 4/267-72 -radiotherapy planning, computer assisted: 3/215-24 recurrence: 2/133-7 recursive partitioning analysis, prognostic groups: 2/127-31 registries: 4/243-7 research: 4/309-15 rhabdomyosarcoma -therapy, child, survival analy­ sis: 4/259-66 sarcoma experimantal -blood supply: 4/277-91 sarcoma experimental -therapy -blood supply: 2/139-47 sarcoma pathology: 1/47-52 sentinel lymph node biopsy: 1/43-6 sigmoid diseases: 2/89-93 Slovenia: 4/243-7 soft tissue neoplasms: 1/47-52 sonography: 3/167-73; 3/175-7 speech, alaryngeal: 4/249-54 speech, esophageal: 4/249-54 SPF mice: 4/309-15 supine position: 4/267-72 survival analysis: 2/127-31; 4/255-8 technetium: 1/21-30 testicular neoplasms -therapy: 4/293-302 tomography, emission computed, 18"-FDG-PET: 2/95-103 tomography, X-ray computer: 2/105-10; 3/161-5 treatment strategy: 2/95-103 ultrasonography: 3/161-5 -ultrasonography, Doppler, duplex: 2/83-8 upgrading of analogue gamma cameras: 1/53-61 vinblastine: 2/139-47 weight bearing: 1/17-9 Note Supplement 1/2001 biopsija: Suppl 1/92-101 dojka: Suppl 1/51-9 -dojka -anatomija in histologija: Suppl 1/44-50 -dojka -bolezni: Suppl 1/51-9, Suppl 1/60-7 -dojka, bolezni -psihologija: Suppl 1/68-73 -dojka, novotvorbe -diagnostika: Suppl 1/92-10 kontrola: Suppl 1/26-9 kvaliteta: Suppl 1/26-9 -kvaliteta, kontrola: Suppl 1/19-25, Suppl 1/30-6 mamografija: Suppl 1/1-6, Suppl 1/19-25, Suppl 1/30-6, Suppl 1/37-43, Suppl 1/51-9, Suppl 1/60-7, Suppl 1/68-73, Suppl 1/74-80, Suppl 1/81-6, Suppl 1/87-91, Suppl 1/92-101 mamografija, kvaliteta: Suppl 1/7-18 radiografija, rentgenski film: Suppl 1/26-9 radiokirurgija: Suppl 1/92-101 rentgenski film: Suppl 1/19-25 tehnologija radiološka: Suppl 1/1-6, Suppl 1/74-80, Suppl 1/81-6, Suppl 1/87-91 -tehnologija radiološka -zakonodaja: Suppl 1/37-43 Združene države Amerike: Suppl 1/37-43 One issue of Radiolo !š}' & Oncology, Volume 35, 2001, was published only in Slovenian as Supplement 1 and was mten.ed exclus1vely to the students of School for Health Professionals at the University of Ljubljana. Hence, ali m.exes for the above Supplement (reviewers', author's and subject's) are quoted separately and are not mcluded mto the mdexes of regular issues. FONDACIJA "DOCENT DR. J. CHOLEWA" JE NEPROFITNO, NEINSTITUCIONALNO IN NESTRANKARSKO ZDRUŽENJE POSAMEZNIKOV, USTANOV IN ORGANIZACIJ, KI ŽELIJO MATERIALNO SPODBUJATI IN POGLABLJATI RAZISKOVALNO DEJAVNOST V ONKOLOGIJI. MESESNELOVA 9 1000 LJUBLJANA TEL 0 1 51 9 1 2 77 FAKS 01 251 81 13 ŽR: 50 1 00-620-1 33-05-1 033 1 1 5-2 1 4 779 FONDACIJA DR.].CHOLEWA Activity of "Dr. J. Cholewa" Foundation for Cancer Research and Education -A Repo rt f or the Final Quarter of 2001 The activity of the "Dr. J Cholewa" Foundation for cancer research and education contin­ued its activity throughout the remaining weeks and months of the year 2001 as it was out­lined during the last meeting of its Executive council. The change in the donors' attitude towards the Foundation in the last months prior to this report caused some concern and the ways how to adapt to the new circurnstances were seriously discussed. Some of the new initiatives were taken into consideration and it is hoped that it will be possible to re­view some tangible results of the activity under discussion in the near future. The Foundation is planning to intensify its contacts with some of the established donors and it also plans to attract new ones to its cause. It is believed that considerable interest ex­ists in collaboration with the Foundation arnong the executive officers and rnanagers of var­ious companies, especially those with the interests in pharmacology development and pro­duction and in banking. The activity of the Foundation is by now well established as a result of a relatively long and substantial experience, and through the hard work of its members it offers a certain guarantee that the rneans frorn the possible donations will be spent in an efficient and irnpartial way. The Foundation continues to support the regular publication of "Radiology and Oncology" international scientific journal that is edited, published and printed in Ljubljana, Slovenia. The support for the publication of the "Challenge Newsletter" is stili to be re-evaluated and the decision should be taken in due tirne. The Foundation has received a number of grant applirntions and it has considered the mer­its of the proposed research work thoroughly. The support is to be given to research work in the field of oncology originating in Slovenia with regard to the quality and to severa! oth­er important aspects presented in proposals for the grants. A dccision was also taken that a number of grants is to be awarded to experts from various parts of Slovenia in order to attend various conferences and meetings in the field of oncology in Slovenia and around the world. The Foundation is considering to invite new members to its Executive council in order to better represent and understand the present urgencies and advances in the continuos de­velopment of cancer research and education in Slovenia. It will thus continue to pursue its stated objectives with new vigour and objectivity. Andrej Plesnicar, MD, MSc Tomaž Benulic, MD, MSc Borut Štabuc, MD, PhD SENSE is rhe new acquisition method in MRI. It achieves imaging speeds far beyond those possible by merely boosting gradient fields alone. Oesigned to improve patient throughput and comforr, SENSE can be applied to virrually ali sequences and rherefore ali clinical applicarions. Moreovet, it also allows users to choose either increased spatial or remporal resolution without increasing scan tirne. Simply imagine performing in one breathhold, a large FOV srudy such as rhe liver, pancreas and kidneys and you'II see rhat SENSE is revolutionizing time-sensitive clinical studies. To discover how SENSE can boost your efficiency and why Philips is changing how rhe world looks ar MR visir us ar www.medical.philips.com today. PE: Stritarjeva 5, 4000 Kranj, Slovenija tel.: (0)4/ 2015 050, fax: (0)4/ 2015 055 e-mail: kemomed@siol.net KEMOMED o Promega SKRINJE IN HLADILNIKI BIOHIT ELEKTRONSKE IN MEHANSKE AVTOMATSKE PIPETE SIE ENS Rešitve po meri Mammomat 3000 modular Mammomat 3000 modular o univerzalni sistem za vse vrste mamografije o optimizacija doze in kompresije z OPDOSE in OPCOMP sistema o modularna zgradba zagotavlja posodabljanje sistema o servis v Sloveniji z zagotovljenimi rezervnimi deli in garancijo o izobraževanje za uporabnike SIEMENS d.o.o. Dunajsk a 22 1511 Ljubljana Telefon 01/47461 00 Telefaks 01 / 474 61 35 Sanolabor 50 let zastopa naslednja podjetja 1 . Kottermann (Nemcija): INTEGRA BIOSCIENCES (Svica): laboratorijsko pohištvo, laboratorijska oprema za !mikrobiologijo, varnostne omare za kisline, biologijo celic, molekularno biologijo luge, topila, pline in strupe, in1 biotehnologijo ventilacijska tehnika in digestorji 1 CO'}NING (ZDA): DAKO (Danska): specialna laboratorijska plastika testi za aplikacijo v imunohistokemiji, za aplikacijo v imupologiji, mikro­patologiji, mikrobiologiji, virologiji, biologiji-virologiji, ipd., fehanske eno­mono-in poliklonalna protitelesa in veckanalne pip ete in nastavki l SVANOVA Biotech (Švedska): EVL (Nizozemska): Elisa testi za diagnostiko v v.eterini diagnosticni tes.1 i za uporabo v veteri arski medicini NOVODIRECT BIOBLOCK (Francija): kompletna oprema in pripomocki HURNER (Nemcija): za delo v laboratoriju ventilrcijska tehnika GFL (Nemcija): CSL i Biosciences: laboratorijski aparati, omare in diagnosticni testi za uporabo skrinje za globoko zamrzovanje v veteriharski medicini 1 ANGELANTONI SCIENTIFICA (Italija): BIOMERICA (ZDA): hladilna tehnika in aparati za laboratorije, hitri testi b diagnostiko, transfuzijo, patologijo in sodno medicino j EIA /R!A testi EHRET (Nemcija): CHARLES ISCHI (Svica): laminar flow tehnika, inkubatorji, specialna oprema za testiranje izdelkov sušilniki, suhi sterilizatorji in oprema v farmacevtski induhriji;aparati za za laboratorijsko vzrejo živali -kletke procesno kontrolo in ko1 trolo kvalitete ROSYS -ANTHOS (Avstrija): fotometri, avtomatski pralni sistem za mikrotitrine plošce LABORMED d.o.o. LABORMED, razstavni salon Zg. Pirnice 96/c Bežigrajski dvor SI -1215 Medvode Periceva 29, Ljubljana Tel.: (0)1 362 14 14 Tel.: (0)1 436 49 01 Fax: (0)1362 14 15 in f o @ 1 a bo r m e d . si Fax: (0)1 436 49 05 www.labormed.s • MEDITRADE¦ d.o.o. Vodovodna 100 1000 Ljubljana, Slovenija Tel.: 01 5894 600 Fax: 01 5684 340 www.meditrade.si Zastopamo: Radiološki program firme Kodak Medicinsko opremo firme Marconi CT, MRI, IM Negatoskope firme Ella Roloskopi fit"me Planilux Mamografska oprema firme Metaltronica - - - - = aAnna•::.¦:::11" kapsule v svetu najvec predpisovani sistemski( antimikotik edini peroralni sistemski antimikotik za zdravljenje vaginalne kandidoze, ki ga je odobril FDA Skrajšano navodilo Flukonazof je sistemski antimikotik iz skupine triazofov. Odmerjanje pri razlicnih indikacijah: vaginalna kandidoza 150 mg v enkratnem odmerku mukozna kandidoza 50 do 100 mg na dan dermatomikoze 50 mg na dan ali 150 mg na teden sistemska kandidoza prvi dan 400 mg, nato od 200 do 400 mg na dan Najvecji dnevni odmerek je 800 mg. preprecevanje kandidoze 50 do 400 mg na dan kriptokokni meningitis prvi dan 400 mg, nato od 200 do 400 mg na dan vzdrževalno zdravljenje 200 mg na dan Kontraindikacije: Preobcutljivost za zdravilo ali sestavine zdravila. Interakcije: Pri enkratnem'. odmerku flukonazola za zdravljenje vaginalne kandidoze klinicno pomembnih interal{oij ni.. Pri veckratnih in vecjih odmerkih so možne interakcije s terfenadinom, cisapridom, astemizolom, varfarinom, derivati sulfonilureje, hidroklorotiazidom, fenitoinom, rilampicincim, ciklosporil')or)l,. teofilinom, indinavirom in midazolamom. Nosecnost in dojenje: Nosecnica lahko jemlje zdravi\o . le, ce je korist zdravljenja za mater vecja od tveganja za plod. Dojece matere naj ined zdravlje/ijE!i]J.' s flukonazolom ne dojijo. Stranski ucinki: Povezani so predvsem s prebavnim traktom:·.sl!jl;!0.s.t,:< napenjanje, bolecine v trebuhu, driska, zelo redko se pojavijo preobcutljivostn.e kol!ne reakc!Je;.,. anafilaksija in angioedem v tem primeru takoj prenehamo jemati zdravilo. Pri bpl glivicnimi obolenji lahko pride do levkopenije in trombocitopenije in do povecana akt! enc·,mov. Oprema in nacin izdajanja: 7 kapsul po 50 mg, 28 kapsul pb 100 l'(ig,' 1 · 150 mg. Na zdravniški recept. 1/99. Podrobnejše informacije so na voljo pri proizvajalcu. epoetin a/ " Dodatne informacije o zdravilu lahko dobite pri imetniku dovoljenja za promet: JANSSEN-CILAG JOHNSON & JOHNSON S. E. Podružnica Ljubljana, Šmartinska cesta 140, 1000 Ljubljana, E.mail:jac_sto@jnjsi.jnj.com Instructions f or authors Editorial policy of the journal Radiology and Oncology is to publish original scientific pa­pers, professional papers, review articles, case reports and varia (editorials, reviews, short communications, professional infarmation, book reviews, letters, etc.) pertinent to diag­nostic and interventional radiology, computer­ ized tomography, magnetic resonance, ultra­sound, nuclear medicine, radiotherapy, clinical and experimental oncology, radiobiol­ogy, radiophysics and radiation protection. The Editorial Board requires that the paper has not been published or subrnitted far publica­tion elsewhere: the authors are responsible far ali statements in their papers. Accepted arti­cles become the property of the journal and therefare cannot be published elsewhere with­out written permission from the editorial board. Papers concerning the work on hu­mans, must comply with the principles of the declaration of Helsinki (1964). The approval of the ethical committee rnust then be stated on the manuscript. Papers with questionable jus­tification will be rejected. Manuscript written in English should be submitted to the Editorial Office in triplicate (the original and two copies), including the il­lustrations: Radiology and Oncology, Institute of Oncology, Zaloška 2, SI-1000 Ljubljana, Slovenia; (Phone: +386 1 432 00 68, Tel./Fax: +386 1 433 74 10, E-mail: gsersa@onko-i.si). Authors are also asked to submit their manu­scripts on a 3.5" 1.44 Mb farmatted diskette. The type of cornputer and word-processing package should be specified (W ord far Windows is preferred). Ali articles are subjected to editorial review and review by independent referee selected by the editorial board. Manuscripts which do not comply with the technical requirements stated herein will be returned to the authors far cor­rection befare peer-review. Rejected manu­scripts are generally returned to authors, how­ever, the journal cannot be held responsible far their loss. The editorial board reserves the right to ask authors to make appropriate changes in the contents as well as grammatical and stylistic corrections when necessary. The expenses of additional editorial work and re­quests far reprints will be charged to the au­thors. General instructions• Radiology and Onco­logy will consider manuscripts prepared accor­ding to the Vancouver Agreement (N Engl J Med 1991; 324: 424-8, BM] 1991; 302: 6772; JA­MA 1997; 277: 927-34.). Type the manuscript double spaced on one side with a 4 cm margin at the top and left hand side of the sheet. Write the paper in grammatically and stylisti­cally correct language. Avoid abbreviations unless previously explained. The technical da­ta should confarm to the SI system. The man­uscript, including the references may not ex­ceed 15 typewritten pages, and the number of figures and tables is limited to 4. If appropri­ate, organize the text so that it includes: Introduction, Material and methods, Results and Discussion. Exceptionally, the results and discussion can be combined in a single sec­tion. Start each section on a new page, and number each page consecutively with Arabic numerals. Title page should include a concise and in­formative title, followed by the full name(s) of the author(s); the institutional affiliation of each author; the name and address of the cor­responding author (including telephone, fax and e-mail), and an abbreviated title. This should be followed by the abstract page, sum­marising in less than 200 words the reasons for the study, experimental approach, the major findings (with specific data if possible), and the principal conclusions, and providing 3-6 key words for indexing purposes. Structured ab­stracts are preferred. If possible, the authors are requested to submit also slovenian version of the title and abstract. The text of the report should theri proceed as follows: Introduction should state the purpose of the article and summarize the rationale for the study or observation, citing only the essential references and stating the aim of the study. Material and methods should provide enough information to enable experiments to be re­peated. New methods should be described in detail. Reports on human and animal subjccts should include a statement that ethical ap­proval of the study was obtained. Results should be presented clearly and concisely without repeating the data in the ta­bles and figures. Emphasis should be on clear and precise presentation of results and their significance in relation to the aim of the inves­tigation. Discussion should explain the results rather than simply repeating them and interpret their significance and draw conclusions. It should review the results of the study in the light of previously published work. Illustrations and tables must be numbered and referred to in the text, with appropriate location indicated in the text margin. Illu­strations must be labelled on the back with the author's name, figure number and orien­tation, and should be accompanied by a de­scriptive legend on a separate page. Line drawings should be supplied in a form suit­able for high.quality reproduction. Photo­graphs should be glossy prints of high guality with as much contrast as the subject allows. They should be cropped as close as possible to the area of interest. In photographs mask the identities of the patients. Tables should be typed double spaced, with descriptive title and, if appropriate, units of numerical meas­urements included in column heading. References must be numbered in the order in which they appear in the text and their cor­responding numbers quoted in the text. Authors are responsible for the accuracy of their references. References to the Abstracts and Letters to the Editor must be identified as such. Citation of papers in preparation, or sub­mitted for publication, unpublished observa­tions, and personal communications should not be included in the reference list. lf essen­tial, such material may be incorporated in the appropriate place in the text. References fol­low the style of lndex Medicus. Ali authors should be listed when their number does not exceed six; when there are seven or more au­thors, the first six listed are followed by "et al.". The following are some examples of refer­ences from articles, books and book chapters: Dent RAG, Cole P. In vitro maturation of monocytes in sguamous carcinoma of the Jung. Br J Cancer 1981; 43: 486-95. Chapman S, Nakielny R. A guide to mdiolog­ical procedures. London: Bailliere Tindall; 1986. Evans R, Alexander P. Mechanisms of ex­tracellular killing of nucleated mammalian cells by macrophages. In: Nelson DS, editor. l111111unobiology oj macrophage. New York: Academic Press; 1976. p. 45-74. Page proofs will be faxed to the correspon­ding author whenever possible. It is their re­sponsibility to check the proofs carefully and fax a list of essential corrections to the editori­al office within 48 hours of rcceipt. If correc­tions are not received by the stated deadline, proof-reading will be carried out by the edi­tors. Reprints: Fifty reprints are free of charge, for more contact editorial board. For reprint information contact: Intemational Reprint Corpomtio11, 287 East "H" Street, Benicia, CA 94510, USA. Tei: (707) 746-8740; Fax: (707) 746-1643; E-mail: reprints