ADIOLOGY 11111 NCOLOGY March 2003 Vol. 37 No. 1 Ljubljana ISSN 1318-2099 SIEMENS SiemensMedical.com/oncology SEEK-FIND-ACT-FOLLOW -the Continuum of Oncology Care™ Siemens oncology portfolio comprises comprehensive maximized utilization potential, and patient-friendly design workflow solutions integrating the tuli spectrum ot care and teatures. tram screening/early detection and diagnosis through therapy and follow-up. AII tram one provider -with over Every day in the United States alone, 29,000 cancer 100 years history ot innovation in medical technology. patients receive radiation therapy delivered by Siemens linear accelerators. As clinical protocols transition to Siemens praven clinical methods can help you to achieve include IMRT and IGRT. Siemens seamlessly integrates more successful outcomes. How? Through industry­the diagnostic and treatment modalities. That's what we leading technology, increased productivity measures tor call Sest Practice Oncology Care. Siemens medical Solutions that help ADIOLOGY AND NCOLOGY Editorial office Radiologij and 011cology l11stit11/e oj Oncology Zaloška 2 Sl-1000 Ljubljmrn Plwne/Fax: +386 1 4337 410 Radiology mui Oncology is a jouma/ devoted la publicaliou oj original conlributions in diagnosfic and inlervelllional mdiology, compulerized lon10gmplzy, ultmsowu/, 111agnetic reson@ce, mtclear medici/le, mdiothempy, clinicnl nnd experimenla/ oncology, mdiobiology, mdiophysics and radia/ion pro/eclio11. Editor-in-Chief Gregor Serša Ljubljana, Slovenia Executive Editor Viljem Kovac Ljubljana, Slovenia Editorial board Marija Auersperg Ljubljana, Slovenia Nada Beše11ski Zagreb, Croatia Karl H. Bolwslavizki Hnmburg, Cer11111111; Haris Boko Zagreb, Croatia Nataša V. Budihna Ljubljmrn, S/ovenia Maijan Budilma Ljublja11a, Slovenia Malte Clause11 Hamburg, Cermnm; Christoph Clemm Miine/zen, Cemrnny Mario Corsi Udine, llaly Ljubomir Dia11kov Sofia, Bulgaria Christian Dittrich Vie1111a, Auslria Ivan Drinkovic Zagreb, Croatia Gillian Duclzes11e Melboume, Auslmlia Editor-in-Chief Emeritus Tomaž Benulic Ljubljmrn, Slovenia Editor Uroš Smrdel Ljubljana, Slove/lin Valenti11 Fidler Ljublja11a, Slove11ia Bela Fornet Budapesl, Hungnry Tullio Giraldi Trieste, ltnly Andrija Hebrang Zagreb, Croalin Lasz/6 Horvtitlz Pecs, H111zgary Berta Jereb Lj11blja11a, Slovenia Vladimir Jevtic Ljubljana, S/ovenia H. Dieter Kogelnik Salzbzug, Austrin Jurij Lindtner Ljubljaua, S/ovenia Iva11 Lovasic Rijekn, Croalia Marijan Lovrencic Zagreb, Croalin Luka Mi/as Houston, USA Metka Milcinski Ljubljana, S/ove11in 0.05). There was no difference in the average contrast res­olution (0-3 scale; 1.50 versus 1.51). Discussion Due to the great radiosensitivity of glandular breast tissue there is small but inevitable risk of inducing breast cancer during mammogra­phy (6.6 radiation induced breast cancers per million women per year per 0.01 Gy per all western women exposed after age of 20).7 The incidence of radiation induced breast cancer depends on the radiation dose and the age at the exposure. It progressively decreas­es after the age of 40 years because of the low­er proportion of glandular breast tissue and fatty substitution.8 The minimal latent period was estimated to 10 years from the radiation exposure until breast cancer develops and it was unaffected by dose.7,9 A linear dose-re­sponse curve without a threshold is generally accepted for the radiation induced breast can­ cer.8,10 It is obvious that a theoretical carcinogenic risk from mammography appears to be negli­gible compared to benefits of early cancer de­tection, even in women beginning annual screening at age of 35 and continuing until age 75 years the benefit widely outweigh the risk.4 Regardless of this Ètheoretical riskÇ of carcinogenesis, we consider that efforts made to reduce radiation dose during mammogra­phy are welcome, especially when we take in­to account rapidly increased number of women attending to the mammographic ex­amination. According to the prior statement, intention of this study was to indicate a way to reduce radiation dose during mammogra­phy, without impairing image quality. Routinely used 45° -films were proved to be suitable for the majority of patients con­sidering different body constitution and breast types. We were curious, what will hap­pen with the thickness of the compressed breast, time-current product, exposure and image quality if we choose another central beam angle? It is well known that the proper breast compression is a prerequisite for ob­taining mammograms of satisfying quality and for reducing radiation dose. Gentry and DeWerd state that exposure dose and com­pressed breast thickness were linearly corre­lated.9 It reinforces the importance of the firm breast compression during mammogra­phy in order not only to reduce the exposure but also to achieve some additional benefits affecting image quality: lower scatter, re­duced motion artefacts, reduced geometric unsharpness (shorter object-film distance), reduced breast tissue superimposition and equalised breast thickness.11,12 If we intend to obtain good image quality with as low as possible radiation dose a central beam angle, which allows a better breast compression, should be chosen. Considering the radiation dose measure­ment two approaches are available: recording the exposure parameters (tube voltage, focus-film distance, mAs, the thickness of the com­pressed breast) or the direct assessment us­ing TLDs, which was performed in our study.13 In a previous study14 the authors es- Radiol Oncol 2003; 37(1): 5-8. timated the breast irradiation indirectly recording exposure parameters and found differences in favour of 60° -films which agrees with the results of this study. In both studies Èfixed kVp protocolÇ was used: the tube voltage was constant and the variable breast thickness was compensated by mAs values. Mc Parland and Boyd investi­gated the patientÕs dose in Èfixed kVp proto­colÇ versus Èvariable kVp protocolÇ and found a lower radiation dose for thicker breast when Èvariable kVp protocolÇ was used, with a small reduction in image quality.15 In spite of this, we used Èfixed kVp protocolÇ because we consider that the patientÕs dose reduction should not interfere with the image quality. We are aware of the possible shortages of our study: We did not assess the mean glan­dular dose (MGD) which is of the greatest im­portance in assessing the carcinogenic risk. But, when we are aware of the linear correla­tion between MGD and the exposure, we can assume that by reducing exposures we will reduce MGD and the carcinogenic risk, as well. We also did not take into consideration the patientÕs body constitution and the con­stitution of the breasts. It was found in a pre­vious study that the breast compressibility with an angle of 60° was the best in thin women with the pendulous breast.14 We conclude that 60° -films were obtained with better breast compressibility comparing to 45° -films, which results in lower time-cur­rent product and exposure whereby the im­age quality was the same or even better. By introducing 60° -films instead of 45° -films the mammograms of at least the same quality can be obtained with a lower radiation dose and a lower carcinogenic risk. References 1. Bassett LW, Gold RH. Breast radiography using the oblique projection. Radiology l983; 149: 585-7. 2. Eklund GW, Cardenosa G. The art of mammo- graphic positioning. Radiol Clin North Am 1992; 30: 21-53. 3. Lundgren B. The oblique views at mammography. Br J Radiol 1976; 50: 626-8. 4. Mettler FA, Upton AC, Kelsey CA. Benefits versus risks from mammography: a critical reassessment. Cancer l996; 77: 903-9. 5. Feig SA. Radiation risk from mammography. Radiology 1977; 124: 1-6. 6. Ranogajec-Komor M, Muhiy Ed Din F, Milkovi“ ., Veki“ B. Thermoluminescence characteristics of various detectors for X-ray diagnostic measure­ments. Radiat Prot Dosim 1993; 47: 529-34. 7. Feig SA, Ehrlich SM. Estimation of radiation risk from screening mammography: recent trends and comparison with expected benefits. Radiology 1990; 174: 638-47. 8. Feig SA. A new method for assessment of radia­tion risk from screening mammography. Recent Results Cancer Res 1990; 119: 141-50. 9. Gentry JR, DeWerd LA. TLD measurements of in vivo mammographic exposures and calculated mean glandular dose across the United States. Med Phys 1996; 23: 899-903. 10. Hurley SF, Kaldor JM. The benefits and risks of mammographic screening for breast cancer. Epidemiol Rev 1992; 14: 101-30. 11. Tanner RL. Mammographic unit compression force: acceptance test and quality control proto­cols. Radiology 1992; 184: 45-8. 12. Eklund GW. Mammographic compression: sci­ence or art. Radiology 1991; 181: 339-41. 13. Dance DR, Skinner CL, Alm Carlsson G. Breast dosimetry. Appl Radiat Isot 1999; 50: 185-203. 14. Brni“ Z, Hebrang A. Breast compression and radi­ation dose in two different mammographic oblique projections: 45 and 60 degrees. Eur J Radiol 2001; 40: 10-5. 15. Mc Parland BJ, Boyd MM. A comparison of fixed and variable kVp technique protocols for film-screen mammography. Br J Radiol 2000; 73: 613­26. Scintigraphic detection of peptic lesions with the method of radiolabelled sucralfate John Naumovski1, Nina Simova2, Emilija Janevik-Ivanovska2, Elizabeta Kovkarova1, Sonja Georgievska- Kuzmanovska2 1Clinic of Toxicology and Urgent Internal Medicine, Clinical Center -Skopje, 2Institute of Pathologic Physiology and Radionuclear Medicine, Medical Faculty-Skopje, Macedonia Background. Sucralfate is an antiulcer agent that after peroral application strongly adheres to mucosal de­fects and in that way provides a protective barrier to further damage from acid and pepsin. If radiolabelled with a gamma isotope, it could be detected under a gamma camera pointing lesions to which it adhered. With the aim to confirm a suitable noninvasive method for investigation of caustic lesions of the upper gastroin­testinal tract we evaluated in a preliminary study the validity of the radiolabelled Sucralfate scintigraphy in detection of peptic disease. Patients and methods. With that purpose, 35 patients after an endoscopic examination underwent scintig­raphy with Tc-99m-DTPA sucralfate. Patients were divided in two groups: a group of 20 patients with en­doscopic confirmed peptic disease and a control group of 15 persons who had not any disease of the upper gastrointestinal tract. Results. Using the test for clinical evaluation of a new method, the scan showed sensitivity of 75 %, speci­ficity of 100 % and accuracy of 85.7 %. Conclusions. Scintigraphy with Tc-99m-DTPA Sucralfate promoting it as an additional method, comple­mentary to routine investigations in detecting mucosal lesions. Key words: peptic ulcer-radionuclide imaging; sucralfate; isotope labelling Received 7 February 2003 Accepted 21 February 2003 Correspondence to: John Naumovski, M.D., Clinic of Toxicology and Urgent Internal Medicine, Clinical Center Skopje, Vodjanska 17, 91000 Skopje, Macedonia; Phone: +389 91 375 133; Fax: +389 91 363 602; E-mail: naumovski@sonet.com.mk Introduction Sucralfate, a complex polyaluminium hydrox­ide salt of polysulphated sucrose, is used in medical treatment of peptic disease as a coat­ing agent that provides a protective barrier to further damage from acid and pepsin. Its ac­tions are principally local and at acid pH be­comes highly polar and binds by way of strong electrostatic interaction to ulcer tissue for up to 12 hours, while relatively little binds to intact gastric or duodenal mucosa.1 Utilizing its selective binding characteris­tics, Vasquez et al. first radiolabelled it with a gamma emitting isotope, developing a new method for detection of gastrointestinal ul­cerations.2 Since its original publication, this method has been used in different modifica­tions to detect and to evaluate other diseases in addition to peptic ulcers, such as oral mi-crolesions, oesophagitis, oesophageal transit time, gastric carcinoma, inflammatory bowel diseases and many other that have underly­ing mucosal defects.3-6 The presence of mucosal and submucosal lesions at peroral caustic ingestion allows the idea that the detection of these injuries is pos­sible with this method. With aim to evaluate the efficacy of the method of radiolabelled sucralfate and gain primary experience about its technique and interpretation of results, a preliminary study was undertaken on a group of patients with and without peptic disease. Patients and methods Subjects After a fiber-endoscopic investigation of the upper gastrointestinal tract, 35 patients (18 male, 17 female; medium age 38.5, range 15 ­72 years) were divided in two groups: an in­dex group, consisting of 20 patients, with en­doscopic verified peptic disease and a control group, consisting of 15 patients, who under­went endoscopy because of the suspicion of a peptic disease and the same excluded. Preparation of radiolabelled sucralfate After suspending 500 mg sucralfate (1/2 a tablet of Venter, Krka, Slovenia) in 5 ml of normal physiological saline in a test tube, 1 ml DTPA was added and incubated for 2 min­utes. 2 mCi of TcO4 was added, rotated and centrifuged for 10 minutes. After decanting the supernatant, pellet is resuspended in 20 ml water and applicated per os (modification of the method of Scopinaro et al.).7 The paper chromatography of the supernatant showed a consistently labeling efficiency of 87-91%. Patient imaging Isotope scan was carried out in the morning after an overnight fasting within 48 hours of endoscopy. Images of the upper gastrointesti­nal tract were obtained using a large field of view gamma camera with the patient in the supine position. Images were initially ob­tained in the anterior position and, if neces­sary, additional images were made in the left and right decubitus. Serial analog images on 30 minutes were taken for 2 hours and if gas­tric emptying was slow, we continued. To hurry up the gastric emptying we gave 100 ml of water by mouth and a intra- muscular in­jection of metoclopropamid. Positive images appeared as areas of accu­mulation of radiopharmaceutical and re­mained so with no changes of position, time and after drinking 100 ml of water. Negative results were interpreted if the first seen accu­mulation changed position with time or van­ished after drinking water. Results The number of detected peptic lesions visual­ized with both investigations, fiber-en­doscopy and radiolabelled sucralfate scintig­raphy, are given in Table 1. After comparing the results obtained from the radiolabelled sucralfate scans with the en­doscopic findings, they were estimated as Table 1. Number of detected peptic lesions with both methods peptic lesion FE Sc reflux esophagit 4 3 gastric ulcer 7 5 duodenal ulcer 9 7 control group* 0 0 total 20 15 FE=fiber-endoscopy; Sc =scans; * no lesions true positive (TP), true negative (TN), false positive (FP) and false negative (FN). This study gave 15 TP, 15 TN, no FP and 5 FN re­sults. Using the methods of clinical est evaluation (Bayesian analysis), the sensitivity (Sn=TP/ TP+FN), the specificity Sp=TN/TN+FP), posi­tive predictive value (PPV=TP/TP+FP), nega­tive predictive value (NPV=TN/TN+FN) and the accuracy (Ac=TP+TN/TP+TN+FP+FN) were calculated as Sn=75%, Sp=100%, PPV= 100%, NPV=75% and Ac= 85.7%. Discussion and conclusion Many authors found this method sensitive for detecting various mucosal defects in the up­per and lower gastrointestinal tract. With variable successes in visualizing lesions, re­ported sensitivity was in the range from 67­75 % in the cases of gastroduodenal ulcers and up to 95% in the cases of detecting in­flammatory bowel disease.5,6,8 In all these studies the Sp was constantly high 97- 100 %. Lesions as small as 0.5-2 mm were detected after biopsy of gastric mucosa.9 Although of many optimistic reports, some authors re­ported unsatisfactory results and discontin­ued studies in the assessment of localization and extent of inflammatory bowel disease mostly because of the need of purgation in se­verely ill patients.10 Our preparing of the pa­tient was only an overnight fasting. Only se­vere oesophagitis was detected in other stud­ies explaining unfavorable conditions while drinking the radiopharmaceutic in erect posi­tion and giving short time of contact to the not enough proteinaceus excudate overlying lesser degrees of esophageal inflamma­tion.4,11 In our study we got similar results as the previously reported in the literature. The false negative results may be due to the Èinactivi­tyÇ of the peptic disease that means reepiteli­sation of the visualized ulcer craters. The pep­tic lesions in this study were not histological-ly verified and mostly depended on morpho­logical judgment and experience of the endo­scopist. However, acute and ÈactiveÇ peptic lesion with mucosal denudation seems to be detectable and not chronic ÈinactiveÇ struc­tural ulcers. How the aim of this study was to get experience with this method and later on to apply it in the investigation of caustic in­gestion that is acute and sometimes deeper than the mucosal and submucosal layer, these preliminary results give an opportunity to try. Most authors conclude that this method is insufficient in comparison with endoscopy, with equal reliability to contrast x-rays, but admits its advantage to be noninvasive, easy to perform, not needing active collaboration from the patients and gives an opportunity to evaluate any seriously ill patient not in condi­tion for endoscopythe or barium meal.5,12 Although the group is small, the results suggest that this method could be useful as noninvasive help in the clinical follow up and the detection of mucosal lesions. It seems to be a preferable option for patients after the ingestion of a caustic that would be an aim of a further clinical trial. Figure 1. Scintigraphy visualization of gastric region on 30 minute intervals after drinking a portion with radio labeled Sucralfate in a patient with previously endoscopic verified antral ulcer. (Arrow pointing to the radiotracer accumulation, matching endoscopic location of peptic ulcer; M-Marker , Int - Intestinal loops) Radiol Oncol 2003; 37(1): 9-12. Acknowledgment We are thanking to Dr Stefan Kostadinov, Direktor of KRKA -FARMA, Skopje, Mace­donia for helping us. References 1. James E, McGuigan ?. Peptic Ulcer. In: Braunwald E, editor. HarrisonÕs principles of internal medicine. 11th Edition. New York: McGraw-Hill Book Company; 1987. p. 1239-53. 2. Vasquez TE, Bridges RL, Braunstein P, Jansholt A, Meshkinpour H Gastrointestinal ulcerations: de­tection using a Technetium-99m-labeled ulcer-avid agent. Radiology 1983; 148: 227-31. 3. Bonazza A; Fila G; Gravili S. Scintigraphic demon­stration of the adherence of Technicium-99m-su­cralfate to oral microlesions (letter). J Nucl Med 1991; 32: 1465. 4. Goff JS, Adcock KA, Schmelter R. Detection of esophageal ulcerations with Technetium-99m al­bumin sucralfate. J Nucl Med 1986, 27: 1143-46. 5. Vasquez TE, Pretorius HT, Greenspan G. Radiolabeled sucralfate: a review of clinical efica­cy. Nucl Med Commun 1987; 8: 327-34. 6. Dawson DJ, Khan AN. Detection of inflammatory bowel disease in adults and children: Evaluation of a new isotopic technique. BMJ 1985; 291: 1227­30. 7. Scopinaro F, Linari G, Baldieri M, Corti E, Signori C, Liberatore M. 99mTc labelled llcer avid agents:sucrolphate(SF) and sucrose octasulfate (SO). J Nucl Med Allied Sci 1985; 29: 211. 8. Dawson DJ, Khan AN, Nuttall P, Shreeve DR. Technetium-99m-labelled-sucralphate isotope scanning in the detection of peptic ulceration. Nucl Med Commun 1985; 6: 319-25. 9. Braunstein TE, Vasquez TE, Bridges RL, Jansholt AL, Meshkinpour H. Tagged ulcer-avid material imaging: a potent new method for the evaluation of peptic ulcer disease (abstract). J Nucl Med 1987; 25: 78. 10. Crama-Bohbouth GE, Arndt JW, Pena AS, Blok D, Verspaget HW, Weterman IT et al. Is radiolabelled sucralphate scintigraphy of any use in the diagno­sis of inflammatory bowel disease. Nucl Med Commun 1988; 9: 591-5. 11. Mearns AJ, Hart GC, Cox JA. Dynamic radionu­clide imaging with 99m Tc-sucralfate in the detec­tion of oesophageal ulceration. Gut 1989; 30: 1256­9. 12. Pera A, Seevers RH, Meyer K, Hall C, Bekerman C, Anderson TM et al. Gastric ulcer localization by direct in vivo labeling of sucralfate. Radiology 1985; 156: 783-6. The accuracy of chest sonography in the diagnosis of small pleural effusion Igor Kocijan‰i‰ Department of Radiology, Institute of Oncology, Ljubljana, Slovenia Background. The aim of the study was to evaluate the accuracy of chest sonography in the radiological di­agnosis of small pleural effusions. Patients and methods. Patients referred for abdominal and/or chest sonographies for various reasons were examined for sonographic features of pleural effusion. From January 1997 till January 2000, 69 pa­tients were included into the study. Fifty-two patients were found to have pleural effusion not exceeding 15 mm in depth, the rest of them served as controls. Subsequently erect posteroanterior and expiratory lateral decubitus projections were done in all patients. Results. Compared to radiological examination chest sonography had a positive predictive value of 92% in the diagnosis of small pleural effusions in our study population. The mean thickness of fluid was 9.2 mm on ultrasonography and 7.6 mm on expiratory lateral decubitus views (P<0.01). Conclusions. Chest sonography showed a high degree of accuracy for demonstrating small pleural effusions and could replace lateral decubitus chest radiographs adequately. Key words: pleural effusion-ultrasonography; thoracic radiography Introduction A small amount of fluid (5-10 ml) is often present in the pleural space of healthy indi­viduals.1 Small pleural effusions are not read­ily identified on conventional radiographic views of the chest.2 Lateral decubitus radi­ographs or chest ultrasonography proved to Received 23 January 2003 Accepted 7 February 2003 Correspondence to: Igor Kocijan‰i‰, M.D., M.Sc., Department of Radiology, Institute of Oncology, Zaloäka 2, SI - 1000 Ljubljana, Slovenia; Phone: + 386 1 522 39 81; Fax: + 386 1 43 14 180; E-mail: ikocijancic @onko-i.si be more efficient methods for demonstrating small amounts of free pleural fluid.3-6 The da­ta on the smallest amount of pleural fluid de­tectable vary considerably, but they are es­sentially within the same broad range whether computed tomography, sonography or X-ray examination are used.1,3,6-12 Rigler used lateral decubitus chest radi­ographs for the detection of small pleural ef­fusions.13 Other investigators3,14 have devel­oped the technique and using cadaveric stud­ies15 have shown that volumes of pleural flu­id as little as 5 ml may be detected. Recent re­ports have proved that minute pleural effu­sions can be detected using chest ultrasonog-raphy.6,7,17 No formal comparison has been Kocijan‰i‰ I / Diagnosing small pleural effusions made between the thickness of the pleural ef­fusion as seen on sonography with X-ray and the amount of aspirated fluid. We have compared sonographically de­tected small pleural effusions with expiratory lateral decubitus radiographs. Patients and methods Patients referred for abdominal sonography for a variety of clinical conditions were also examined for unsuspected pleural effusion. Small control group was made up of 17 pa­tients, examined only for clinically suspected pleural effusion, which was not sonographi­cally confirmed. Between January 1997 and January 2000, 69 patients (51 males, 18 females, 28-80 years old, with the mean age of 57.1 years) were in­cluded into the study. Their condition was clinically diagnosed as lung cancer in 30, car­diac failure in 13, metastasis to the lung in 11, pneumonia and pulmonary tuberculosis in 6 and liver cirrhosis in 3 cases. Following abdominal sonography, the pa­tient was positioned in the lateral decubitus position for 5 minutes; sonography of the lower pleural space was performed with the patient leaning on the elbow.17 During the ex­amination maximal fluid thickness was meas­ured, with the position of the probe perpen­dicular to the thoracic wall.18 A Toshiba SSA-340A ultrasound unit was used with a 3.7 or 6 MHz convex transducer. Radiological examination followed if sonography showed a small pleural effusion. A 140 kV Siemens unit was used, with a 2 m film-focus distance for the erect views of the chest, and 1.5 m film-focus distance for later­al decubitus views. For these, the patient was put into lateral decubitus position with 100 hip elevations, for 5 minutes prior to expo­sure. Exposures were taken in expiration, with the central beam aimed at the lateral chest wall and the patient slightly rotated on­to the back. The films were evaluated inde­pendently by two experienced radiologists with no knowledge of the sonographic find­ings. On sonography, the criteria for determin­ing the presence of pleural fluid were: a non/hypo - echogenic zone between the pari­etal and the visceral pleura and/or changing between expiration and inspiration as well as changing with different positions of the pa­tient or fluttering of the pulmonary edge dur­ing respiration.6,9,19,20 On x - ray, the criteria were as follows: minimum 3 mm thick density with horizontal level on lateral decubitus view and costop­hrenic angle density with meniscus sign on erect views.3,21 Matching pairÕs t-test was used for analysis of differences between measurements of the fluid layer thickness on chest sonography and expiratory lateral decubitus projections. The study was approved by relevant ethic committee. Results On erect posteroanterior chest radiographs pleural fluid was demonstrated in only 17 of 52 (33%) patients. Lateral decubitus views were positive in 48 of 52 patients (ie, a positive predictive value of 92%) with sonographically visible fluid. In two cases pleural effusions detected sono-graphically were confirmed by thoracocente-sis. In one patient sonographically positive re­sult was not confirmed either way. In the last case radiography revealed diagnostic error occurred on sonography (Figure 1). The range of fluid thickness was 3-6 mm in these three patients. In a small control group of 17 patients pleural fluid was not confirmed sonographi­cally nor radiographically. The mean thickness of fluid was 9.2 mm (SD= +/- 3.3 mm) on sonography and 7.6 mm Kocijan‰i‰ I / Diagnosing small pleural effusions (SD= +/- 4.0 mm) on expiratory lateral decu­bitus views (P<0.01). The ranges of fluid thickness on gray-scale sonography and later­al decubitus radiography were 3-15 mm and 3-11 mm, respectively (Figures 1a, 1b). Discussion In the literature we could not find any exact definition of small pleural effusions. So, our term of small pleural effusions includes clini­cally silent effusions, which are usually unex­pected finding on x-ray or sonographic exam­inations undertaken for other reasons. Rigler13 was the first to use lateral decubi­tus views for pleural fluid demonstration. He did not use exposure in expiration, however, nor did he expose with central beam aimed at the lateral chest wall, parallel to the expected fluid level. The latter technical improvement was introduced by Hessen3 together with the elevation of the patientÕs hip, while obtaining radiograph during expiration was tested in the work of Kocijan‰i‰ et al.17 The amounts of pleural fluid detectable this way have been assessed in cadaveric experiments15 and has been shown to be as little as 5 ml in experi­mental conditions. This is probably less reli­able in practice, because the fluid may not al­ways be completely aspirated with thoraco­centesis. Radiol Oncol 2003; 37(1): 13-6. Kocijan‰i‰ I / Diagnosing small pleural effusions With the advent of sonography it was shown that very small amounts of pleural flu­id can be demonstrated this way.4-8 However no one precisely determined the sonographic criteria that should be fulfilled for reliable di­agnosis of small pleural effusions. In our study population all effusions were ane­chogenic, the only case with hypoechogenic ÈfluidÇ turned out to be pleural thickness. Interestingly, the main sign, allowing the demonstration of the smallest effusions on sonography as well as on radiography,17 was changing of the fluid layer during inspiration - expiration (Figures 2a, 2b). In the course of our study searching for small pleural effusions of about 200 ml or less,12,18 we have achieved comparable results using sonography and radiography, but sonography appears to assess the thickness of fluid layer more accurately. References 1. Felson B. Chest rentgenology. Philadelphia: W.B. Saunders, 1973. p. 352. 2. Collins JD, Burwell D, Furmanski S, Lorber P, Steckel R. Minimal detectable pleural effusions. A roentgen pathology model. Radiology 1972; 105: 51-3. 3. Hessen I. Roentgen examination of pleural fluid. A study of the localisation of free effusions, the potentialities of diagnosing minimal quantities of fluid and its existence under physiological condi­tions. Acta Radiol 1951; 86(Suppl 1): 1-80. 4. Gryminski J, Krakowa P, Lypacewicz G. The diag­nosis of pleural effusion by ultrasonic and radio­logic techniques. Chest 1976; 70: 33-7. 5. Lipscomb DJ, Flower CDR. Ultrasound in the di­agnosis and management of pleural disease. Br J Dis Chest 1980; 74: 353-61. 6. Mathis G. Thoraxsonography - part I.: chest wall and pleura. Ultrasound Med Biol 1997; 23: 1131-9. 7. Eibenberger KL, Dock WI, Ammann ME, Dorffner R, Hirmann MF, Grabenwirger F. Quantification of pleural effussions: sonography versus radiogra­phy. Radiology 1994; 191: 681-4. 8. Lorenz J, Birner N, Nikolaus HP. Sonographische volumetrie von pleuraerg.ssen. Ultraschall 1988; 9: 212-5. 9. Mc Loud TC, Flower CDR. Imaging of the pleura: Sonography, CT and MR imaging. Am J Roentgenol 1991; 156: 1145-53. 10. Mikloweit P, Zachgo W, Lircher U, Meier-Sydow J. Pleuranage lungenprozesse: diagnostische wer­tigkeit sonographie versus CT. Bildgebung 1991; 58: 127-31. 11. Leung AN, Muller NL, Miller RR. CT in the differ­ential diagnosis of pleural disease. Am J Roentgenol 1990; 154: 487-92. 12. Maffesanti M, Tommasi M, Pellegrini P. Computed tomography of free pleural effusions. Europ J Radiol 1987; 7: 87-90. 13. Rigler LG. Roentgen diagnosis of small pleural ef­fusions. JAMA 1931; 96: 104-108. 14. M.ller R, Lifstedt S. Reaction of pleura in primary tuberculosis of the lungs. Acta Med Scand 1945; 122: 105-33. 15. Moskowitz H, Platt RT, Schachar R, Mellus H. Roentgen visualization of minute pleural effusion. Radiology 1973; 109: 33-5. 16. Reu§ J. Sonographic imaging of the pleura: nearly 30 years experience. Eur J Ultrasound 1996; 3: 125­39. 17. Kocijan‰i‰ I, Ter‰elj M, Vidmar K, Jereb M. The value of inspiratory-expiratory lateral decubitus views in the diagnosis of small pleural effusions. Clin Radiol 1999; 54: 595-7. 18. Eibenberger KL, Dock W, Metz V, Weinstabl C, Haslinger B. Grabenwiger F. Wertigkeit der tho­rax bettaufnahmen zur diagnostik und quan­tifizierung von pleuraerg.ssen - .berpr.fung mit-tels sonographie. RiFo 1991; 155: 323-6. 19. Targhetta R, Bourgeois JM, Marty-Double C, Chavagneux R, Proust A, Coste E, et al. Vers une autre approche du diagnostic des masses pul­monaires périphériques. [Towards another diag­nostic approach of peripheral pulmonary masses. Ultrasound-guided puncture] J Radiol 1992; 73: 159-64. 20. Marks WM, Filly RA, Callen PW. Real - time eval­uation of pleural lesions: New observations re­garding the probability of obtaining free fluid. Radiology 1982; 142: 163-4. 21. Raasch BN, Carsky EW, Lane EJ, OÕCallaghan JP, Heitzman ER. Pleural effusion: explanation of some typical appearances. AJR Am J Roentgenol 1982; 139: 889-904. Endosonographic and manometric assessment of the anal sphincters after ileal pouch-anal anastomosis Iwona Sudo¸-SzopiÄska1, Adam Ciesielski2, Krzysztof Bielecki2, Lech Baczuk2, Wies¸aw Jakubowski1,Wies¸aw Tarnowski2 1Imaging Diagnostic Department, Second Medical Faculty, Warsaw, Poland 2Postgraduate Education Medical Centre, Department of General Surgery, Warsaw, Poland Background. The aim of this study was to compare endosonography and manometry of the anal sphincters in patients after ileal pouch-anal anastomosis (IPAA). Patients and methods. Ten patients aged between 23 and 50 years with IPAA performed for ulcerative co­litis were examined with anal endosonography (AES) and manometry. Results. AES visualised abnormal image of the internal anal sphincter (IAS) in 9 patients (90%). Defects of the external anal sphincter (EAS) and puborectalis muscle (PR) were shown in 4 patients (40%). In 5 pa­tients (50%) correlation between endosonographic and manometric assessment for the all analysed muscles: IAS, EAS and PR was found. In 4 cases (40%) both methods correlated with the evaluation of the EAS on­ly and in 1 patient (10%) no correlation was found. Correlation between both methods for the IAS was found in half of the patients (50%) while in the evaluation of the EAS and PR dynamic activity, it was found in 9 cases (90%). Conclusions. Anal endosonography and manometry allow us to assess the morphology as well as the func­tion of the anal sphincters in patients with IPAA. The methods mentioned above show high correlation in the assessment of the EAS function (9 cases; 90%) whereas in the case of IAS, manometry frequently (5 pa­tients; 50%) does not confirm endosonografically detected defects. Key words: colitis, ulcerative; anus-ultrasonography; manometry; proctocolectomy, restorative Received 4 September 2002 Accepted 11 September 2002 Correspondence to: Iwona Sudo¸-SzopiÄska, M.D., Ph.D., Zak¸ad Diagnostyki Ultrasonograficznej,WojewŠdzki Szpital BrŠdnowski, 03 285 Warszawa, ul. Kondratowicza 8: Phone +48 5017 16407; E-mail: mdyvonne@wp.pl Introduction Ileal pouch-anal anastomosis (IPAA) has be­come the operation of choice for most pa­tients with ulcerative colitis. Patients prefer this form of therapy to formation of the sto-my although it is accompanied by a notice­able percentage of anal incontinence which affects even 84% of patients.1-4 Anal sphinc­ters defects can be partially responsible for this high incidence of incontinence. The aim Sudo¸-SzopiÄska I et al. / Endosonography and manometry of the anal sphincters of this study was to visualise the suspected defects of the anal sphincters on anal en-dosonography (AES) and to compare them with the results of the ano-rectal manometry. Patients and methods Ten patients (8 women and 2 men) aged be­tween 23 and 50 years (median age 33.2 years) with J - pouch and stapled IPAA per­formed without mucosal dissection for the ul­cerative colitis were examined with the use of anal endosonography and ano-rectal manom­etry. Examinations were performed after 3-11 years after IPAA formation (mean 5,5 years). None of them had any operation on the anal canal prior to IPAA formation and none of the women had a complicated delivery. In or­der to assess the severity of anal inconti­nence, the Jorge- WexnerÕs grading system was used.5 Anal endosonography was per­formed with the use of Bruel and Kjaer scan­ner type 1846 with a 7.0 MHz rotating endo-probe that provides a 360° image. The probe was covered with a plastic cone with an ex­ternal diameter of 17 mm, which was filled with degassed water for acoustic coupling. The cone was covered with a condom. Patients were examined in the prone posi­tion, and no preparation was required prior to AES. As the probe was withdrawn from the anal canal, images of the puborectalis muscle (PR), external anal sphincter (EAS) and inter­nal anal sphincter (IAS) were documented. The thickness, echogenicity and outlines of the IAS and echogenicity of the EAS were as­sessed on each level of the anal canal. The thickness of the IAS was measured at 3 and 9 oÕclock position of the coronal plane of imag­ing, using electronic calipers on the monitor. The normal IAS was defined as a homoge-nous, hypoechoic ring with thickness greater than 1mm.6 Increased and nonhomogenous echogenicity and ill-defined margins of the IAS were diagnosed as abnormal. The EAS was identified as non-homogenous muscle with striated echogenicity and was defined as abnormal if hypoechoic areas were visible within it.6 Dynamic activity of EAS and PR was assessed as good (++), poor (+) or lack (0) of contraction using a subjective scale which depends on comparing their image at rest and during maximal voluntary contraction. Anorectal manometry was performed with the patients in the left lateral position. No en­ema was given. A lower gastrointestinal manometry system (PC Polygraf HR; Synec-tics Medical Stockholm, Sweden) with four ­lumen polyvinyl chloride catheter with rectal distending balloon (AMC4-B; Zinectics Medi­cal, Stockholm, Sweden) was used. Perfusion ports were located in 1 cm intervals arranged circumferentially. After positioning at the depth of 6 cm from the anal verge the cat­heter was allowed to accommodate for sever­al minutes. Maximum resting anal pressure (MRP), maximum voluntary pressure (MVP) and maximal duration of squeeze (D) were recorded. Pouch capacity was also recorded by distending air-filled, thin- walled balloon positioned 6 cm within the pouch to assess maximal tolerable volume (MTV). Results The results of anal endosonography and ano-rectal manometry are presented in Tables 1 and 2. In anal endosonography, thinning of the IAS was visible in all but one patient (9 cases; 90%). Increased echogenicity of the IAS in 6 (60%) and ill-defined borders was detected in 3 patients (30%). Echogenicity defect of the EAS was visible in 3 cases (30%). Dynamic examination revealed good EAS and PR contraction in 6 patients (60%), poor in 3 (30%) and lack in 1 patient (10%). Manometry revealed decreased maximum resting anal pressure suggesting dysfunction of the IAS in 3 cases (30%), decreased maxi- Sudo¸-SzopiÄska I et al. / Endosonography and manometry of the anal sphincters Table 1. Anal endosonography in patients with IPAA No IAS Echogenicity Thickness Increased Il-defined defect of the Dynamic exam [mm] echogenicity borders EAS 1. BA <1 + ++ 2. EB <1 + + ++ 3. GP <1 ++ 4. ME <1 + + 5. NM 2.5 ++ 6. SK <1 + + 7. ST <1 + + + + 8. WE <1 + + ++ 9. WM <1 ++ 10. Wm 0 + + + 0 Table 2. Ano-rectal manometry in patients with IPAA (sequence of patients as in Table 1) No MRP* MVP D MTV I 1. BA 30 180 16 150 8 2. EB 60 180 48 100 0 3. GP 60 200 40 190 0 4. ME 60 81 40 150 0 5. NM 80 110 30 200 6 6. SK 45 125 64 120 12 7. ST 60 200 40 350 0 8. WE 60 257 80 /-/ /-/ 9. WM 75 148 50 260 0 10. Wm 30 45 /-/ 160 9 Normal values 60-80 100-250 >40 >150 0 *MRP - Maximum resting anal pressure [mmHg], MVP -Maximum voluntary pressure [mmHg], D - Maximal duration of squeeze (sec) MTV- Maximal tolerable volume [ml], I - Jorge-WexnerÕs fecal incontinence severity score (points) /-/ - not assessed: in one patient - MTV and I - because of pouch-vaginal fistula and in one patient - D - be­cause of low MVP mal voluntary anal pressure in 3 patients muscles (IAS, EAS and PR), and in 4 patients (30%), implying dysfunction of the EAS and (40%) for the EAS and PR only. No correlation PR, and in another 2 patients (cases 1 and 5 between the methods was found in 1 patient from the Table 2) the shortage of the maximal (10%). Although in this case AES showed duration of squeeze indicating dysfunction of thin, hyperechoic, with ill-defined marginat-the EAS and PR was revealed as well. ed IAS, and also poor contraction and scars In all cases manometry correlated with within EAS, manometry revealed preserved clinical examination (Table 2). Correlation be-function of the anal sphincters. tween endosonography and manometry was The analysis of the each assessed element found in 5 patients (50%) for all analysed of the IAS (thickness, echogenicity and bor- Radiol Oncol 2003; 37(1): 17-22. Sudo¸-SzopiÄska I et al. / Endosonography and manometry of the anal sphincters ders) showed that normal image of this sphincter, which was observed in only 1 pa­tient correlated with its preserved function in manometry. However, the abnormal image, which was visible in the remaining 9 patients correlated with its dysfunction in manometry in 4 cases only (44.4%). This included 2 out of 3 patients with thin IAS (66.6% correlation) and 2 out of 6 patients who had thin and hy­perechoic IAS (33.4%). Correlation between AES and manometry in the assessment of the EAS and PR function was found in the majority of the patients (9 cases; 90%). In the remaining one case, en-dosonographic image of the PR and EAS showing their poor contraction and scars did not corresponded with their preserved func­tion (case 7 from the tables 1 and 2). Discussion Anal endosonography, apart from magnetic resonance imaging using endorectal coil, is the most appropriate method to assess the morphology of the anal sphincters. Ileal pauch-anal anastomosis has become an operation of choice for most patients with ulcerative colitis.2,4,7-9 Patients prefer the pelvic reservoir to an ileostomy, although the results of the IPAA formation are not fully satisfactory, its greatest problem being the loss of continence after treatment.2,4,7-9 The images of the anal sphincters and their func­tion after IPAA have not been precisely in­vestigated so far. Individual reports in the lit­erature concentrated on the results of anal endosonography and ano-rectal manometry after IPAA and show the thinning of the IAS and the reduction of the maximum anal rest­ing pressure in most of the operated pa­tients.2,7,9 These disturbances are present af­ter endoanal manipulation (handsewn trans-anal anastomosis with or without mucosecto-my) as well as after stapled anastomosis.4,7 Nevertheless, avoidance of endoanal proce­dures and transabdominal anal pursestring placement and stapled IPAA without muco­sectomy provides higher anal resting pres­sure comparing to endoanal manipulations.4,7 The thinning of the IAS was also the most fre­quent abnormality we observed in our study in all but one patient (9 patients; 90%). There are several reasons leading to the thinning of the IAS, such as denervation, ischemia or a direct trauma to the IAS as a result of transanal mucosectomy, and also, as men­tioned above, hand sewn anastomosis.2,7,9 During the IPAA, the formation dissection and mobilisation of the anorectum is respon­sible for the IAS trauma as a result of damage to the extrinsic autonomic nerve supply, which plays an important role in the IAS function.3 Additionally, the transsection of Table 3. Jorge-WexnerÕs grading system of anal incontinence Type Frequency of Incontinence Never* Rarely Sometimes Usually Always Solid 0 1 2 3 4 Liquid 0 1 2 3 4 Gas 0 1 2 3 4 Wears pad 0 1 2 3 4 Lifestyle alteration 0 1 2 3 4 *Never = 0 Rarely £ 1/month Sometimes £ 1/week, ³ 1/month Usually ³ 1/day Sudo¸-SzopiÄska I et al. / Endosonography and manometry of the anal sphincters the rectal wall at the level of the levator ani muscles may cut through the layer of spe­cialised circular muscle which forms the IAS. This could cause damage of the intramural nerve plexus and blood supply.2 The sphinc­ter trauma at this level is presumably in­evitable.2 In our study only one patient had a normal image and pressure of the IAS. In the remain­ing 9 cases, endosonography suggested its de­generation in 6 out of 9 patients. Correlation with manometry was found in less than half of these patients (4 out of 9; 44.4%). Gene­rally, manometry revealed preserved function of the sphincters in the majority of the pa­tients. Our results were as in other studies, for instance in Stryker et al.,7 who found no differences in anal canal resting and squeeze pressures between patients with IPAA and controls as well as no correlation between them regarding clinical data. Although our small study does not lead to definite conclu­sions, such a high incidence of patients with abnormal image of the IAS, but without func­tional disturbances is striking. Undoubtedly, our group of patients with aged 33.2 years on the average is young, and it is well known that the thickness of the IAS normally in­creases with age.10 So in the young popula­tion thickness is the smallest. It is expected to be over 1.9 mm at the age of 19-65 years.10 Norms of the thickness of the sphincter may need to be verified. Increased echogenicity of the IAS was the most likely consequence of surgery and represented fibrosis of the sphincter. The possibility of the IAS degener­ation related to age, which manifests typical­ly as thinning, increased echogenicity, and ill-defined borders of the IAS, is excluded be­cause of the young age of our patients. On the other hand, there were predominantly women in our group of patients (8 versus 2) and it has been shown in the literature11 that a relevant number of women, who have had uncomplicated deliveries, endosonographi­cally show sphincter defects. The results of the findings would be more reliable if pa­tients had been examined before and after the pauch procedure, which was not the case in our study. Dynamic anal endosonography appeared a valuable adjunct to the examina­tion at rest. Dynamic endosonography is es­pecially valuable in diagnosing anal sphinc­ters trauma, and shows high correlation with electromyography.10 In our study, correlation between dynamic anal endosonography and manometry was found in 9 cases (90%). The assessment of the anal sphincters in both ano-rectal manometry and anal en-dosonography in patients with IPAA enables structural and functional evaluation of the sphincters. The lack of high correlation be­tween these methods in our group of patients emphasizes the complexity of the many mechanisms that contribute to normal conti­nence. One of the examinations of anal sphincter function, besides clinical investiga­tion, is anal manometry, which showed nor­mal function of the anal sphincters in most of our patients. Anal endosonography visu­alised defects of the IAS in 90% of the pa­tients. They might reflect the presence of sub­tle (not disturbing the function) changes of the sphincter, as a consequence of surgery, which in the future might predispose to fur­ther trauma (for instance obstetric), with the risk of anal incontinence development. Conclusions Ano-rectal monometry and anal endosonog­raphy are complementary methods in the as­sessment of the anal sphincters after IPAA. Although in our study manometry showed preserved function of the IAS in most of the cases, the abnormal image of this sphincter might be indicative of its subtle or imminent dysfunction. Dynamic anal endosonography supplements manometric evaluation of anal sphincters and enables prognosis of the sphincter function. Radiol Oncol 2003; 37(1): 17-22. Sudo¸-SzopiÄska I et al. / Endosonography and manometry of the anal sphincters References 1. Choen S, Tsunoda A, Nicholls RJ. Prospective ran­domized trial comparing anal function after hand sewn ileoanal anastomosis with mucosectomy ver­sus stapled ileoanal anastomosis without muco­sectomy in restorative proctocolectomy. Br J Surg 1991; 78: 430-4. 2. Lavery IC, Tuckson WB, Easley KA. Internal anal sphincter function after total abdominal colecto-my and stapled ileal pouch-anal anastomosis with­out mucosal proctectomy. Dis Colon Rectum 1989; 32: 950-3. 3. Williams NS, Marzouk DE, Hallan RI, Waldron DJ. Function after ileal pouch and stapled pouch-anal anastomosis for ulcerative colitis. Br J Surg 1989; 76: 1168-71. 4. Johnston D, Holdsworth PJ, Nasmyth DG, Neal DE, Primrose JN, Womack N, et al. Preservation of the entire anal canal in conservative proctocolec­tomy for ulcerative colitis: a pilot study comparing end-to-end ileo-anal anastomosis without mucosal resection with mucosal proctectomy and endoanal anastomosis. Br J Surg 1987; 74: 940-4. 5. Jorge JM, Wexner SD. Etiology and management of fecal incontinence. Dis Colon Rectum 1993; 36: 77-97. 6. Bartram CI, Frudinger A. Handbook of anal en-dosonography. Petersfield: Wrightson Biomedical Publishing LTD; 1997. 7. Tuckson W, Lavery I, Fazio V, Oakley J, Church J, Milsom J. Manometric and functional comparison of ileal pouch anal anastomosis with and without anal manipulation. Am J Surg 1991; 161: 90-95. 8. Stryker SJ, Kelly KA, Phillips SF, Dozois RR, Beart RW Jr. Anal and neorectal function after ileal pouch-anal anastomosis. Ann Surg 1986; 203: 55­61. 9. Silvis R, Eekelen JW, Delemarre JB, Gooszen HG. Endosonography of the anal sphincter after ileal pouch-anal anastomosis. Dis Colon Rectum 1995; 38: 383-8. 10. 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: 665-7. 11. Sultan AH, Kamm MA, Hudson CN, Thomas JM, Bartram CI. Anal-sphincter disruption during vagi­nal delivery. N Engl J Med 1993; 329(26): 1956-7. Malignant lymphomas of the testis Ferhat Berkmen Department of Uro-Oncology, Ankara Oncology Education and Research Hospital, Ankara, Turkey Background. The aim of the study was to analyse 10 patients with malignant lymphomas of the testis, and to discuss the necessity of immunocytochemical staining to confirm the histologic diagnosis and an effective treatment policy. Patients and methods. Ten patients with malignant lymphomas of the testis were reviewed in order to iden­tify and study the incidence, histologic findings, the type of treatment administered and the overall outcome. Results. Testicular malignant lymphomas were identified ten times between 1984 and 1999. Bilateral tu­mours occurred simultaneously in 4 patients, and a metachronous malignancy and testicular relapses de­veloped in 2 patients. Of the remaining patients 4 had unilateral testicular involvement. None had elevated AFP and b -HCG or a history of undecided testis. Eight of patients were younger than 50 years. Five of the lymphomas were high grade, 3 were intermediate and 2 were low grade diffuse non-HodgkinÕs lymphoma. All patients were initially treated with radical orchiectomy and were, according to their clinical stage, treat­ed with chemotherapy and/or radiotherapy. Five of 10 patients were alive with no evidence of disease with follow-up ranging from 9 to 62 months. The remaining 5 patients died between 3 and 42 months respec­tively. Conclusions. Testicular lymphomas are similar to those of testicular germ cell tumours and account for ap­proximately 5% of all testis tumours and represents 1% of all lymphomas. Testicular lymphomas differ from germ cell tumours of the testis by following points: (1) Testicular lymphomas tend to occur in the middle ages, (2) tumour markers AFP and b -HCG are in normal limits, (3) a development in cryptorchid testis is extremely rare, (4) an early systemic therapy is indicated and watchful waiting policy can not be performed, (5) the prognosis is poor. The recognition of histologic diagnosis with immunocytochemical staining for leukocyte common antigen (LCA) is essential and should help the future treatment policy. Key words: testicular neoplasms, lymphoma; germinoma; lymphoma, non-Hodgkin Received 23 August 2000 Accepted 8 August 2002 Correspondence to: Assoc. Prof. Ferhat Berkmen, M.D., Ankara Oncology Education and Research Hospital, Demetevler, Ankara 6200, Turkey; Phone: +312 336 0909 ext. 333; Fax: +312 345 49 79; E-mail: fberkmen@yahoo.com Introduction The average incidence of testis tumours is in the range of 2.1 to 2.3 per 100 000 males and remains the most common solid cancer in men between the ages of 20 and 34 years.1-3 Berkmen F / Malignant lymphomas of the testis Testicular lymphomas account for approx­imately 5% of testis tumours and constitute the most frequent of all testicular tumours in patients over 50 years of age. The median age of occurrence is about 60 years. Primary lym­phoma of the testis rarely occurs in children.4-7 Testicular involvement by lymphoma may be a manifestation of primary extranodal dis­ease, an initial manifestation of clinically no­dal disease or a later manifestation of di­sseminated nodal lymphoma. The most com­mon histologic pattern is a diffuse histiocytic lymphoma and testicular lymphomas are sometimes misdiagnosed as spermatocytic or anaplastic seminomas. The prognosis is poor within one year after the diagnosis if a dis­seminated disease is evident.5,6,9 The aim of this paper was to analyse a group of patients with malignant lymphomas of the testis and to discuss the necessity of performing leukocyte common antigen (LCA) in pathologically reported anaplastic or sper­matocystic seminomas in order to confirm the histologic diagnosis. Patients and methods From 1984 to 1996, 1201 patients, 17 to 73 years of age were treated for a testicular tu­mours in Ankara Oncology Education and Research Hospital. We reviewed medical records of these patients to identify and study lymphomas in the testis with respect to their incidence, histologic findings, the type of treatment administered and the overall out­come. Results Malignant lymphomas of the testis were iden­tified in 10 patients. The information record­ed for each patients included age, date of di­agnosis, initial symptoms and physical find­ings, initial haematologic data, clinical stage, histology, mode of therapy, response to ther­apy, survival time in months and the patientÕs condition to the last date of follow-up. The data are summarised in Table 1. The common clinical presentation was a painless enlargement of the testis. Of 10 pa­tients 7 had generalized constitutional symp­toms including anaemia (3 patients), anorexia (6 patients), weakness and weight loss (2 pa­tients). The investigation included a complete blood count, peripheral smears, chest radi­ographs, ultrasonography and/or comput­erised tomography of abdomen, ultrasound of testes, tumour markers AFP and b -HCG. None of patients had elevated AFP and b ­HCG. Bilateral tumours were identified in six patients (60 %). The tumours occurred simul­taneously in 4 patients, and metachronous tu­mours plus testicular relapses developed in 2 patients after one and 6 months of the diag­nosis. Initially, all patients were treated with rad­ical orchiectomy. Five of the testicular lym­phomas were high grade, 3 were intermediate and 2 were low grade diffuse non-HodgkinÕs lymphoma. Discussion Cancer of the testis accounts for less than 3 % of all malignant tumours in males. Most of these malignancies are of germinal cell ori­gin.1-3 To date, approximately 100 % of germ cell tumours can be cured. The involvement of the testis by lym­phomas accounts for almost 5 % of testicular tumours and 50% of patients with bilateral tu­mours have lymphoma.3,7,8,10 Testicular lym­phomas differ from germ cell tumours in re­gard to age, incidence, relation to cryp­torchidism, frequency of bilateral involve­ment, normal tumour marker levels of AFP and b -HCG, and prognosis. Despite reports that the median age of occurrence is about 60 years, in our series the age ranged from 32 to Berkmen F / Malignant lymphomas of the testis 73 years with a median of 49. In contrast, 8 of 10 patients were younger than 50 years at di­agnosis. Risk factors, like cryptorchidism, have been reported to range between 7 and 35 % of the total number of patients with tes­ticular germ cell cancer.11-13 In the medical lit­erature, to our knowledge, testicular lym­phoma arising in cryptorchid testis was re­ported only once.5 None of our patients had a history of cryptorchidism. In principle, bilateral tumours can occur synchronously or metachronously and bilat­eral testicular involvement is reported to be a more common feature of malignant lym­phomas of the testis than of germ cell tu­mours.14-16 The relative incidence of bilateral testis tumours, reported in the literature from 1981 to 1995, can be calculated as 2.38 %.17-22 Our study doesnÕt confirm this, bilateral testis tumours comprise 1.17 %, and lym­phoma of the testis comprises 60 % of the to­tal incidence of bilateral testicular carcino­mas. Four of 10 patients had radical inguinal or-chiectomy in different hospitals. Tissue spec­imens of all patients were reviewed in our pathology department to confirm the patho­logic diagnosis, because testicular lym­phomas are sometimes pathologically misdi­agnosed as spermatocytic or anaplastic semi-nomas. Our study confirms this in 2 of 10 pa­tients (20 %). There were 2 patients initially misdiagnosed as spermatocytic seminoma. In order not only to prevent misdiagnosis but al­so for further evaluation and treatment, we recommended to perform immunocytochem­ical staining with LCA if the levels of b -HCG and AFP are normal in the sera, especially in the clinical stages to IIC to IV seminomas. Until several years ago, the traditional treatment for testicular lymphoma was radia­tion therapy for the localized and regional spread and chemotherapy for distant metas­tasis. To date multiagent chemotherapy, the combined modality treatment has become more frequently used as the initial one.23 Irradiation of contra lateral testicle as a pro­phylactic treatment for patients with testicu­lar lymphoma is controversial.6,24,25 Even though, prophylaxis for the normal opposite testis can be performed safely to the elder pa­tients, the need to preserve testicular func­tion may be vital for young cases. Most previ­ous reports have also indicated a poor prog­nosis for patients with testicular lymphoma, especially in patients with bilateral disease and there were no 5-year survivors. In centres cumulating a sufficient number of cases, even Table 1. PatientsÕ data presenting with testicular lymphomas Patients Age Laterality Clinical Treatment Disease Status (months) stage free interval (months) 1 32 Right III Chemo 24 Alive with NED 2 38 Right I XRT 11 DD 3 38 Left IV Chemo 42 DD 4 43 Bilateral, M I Chemo+XRT 52 Alive with NED 5 36 Bilateral, M IV Chemo 10 DD 6 48 Bilateral, S I XRT 62 Alive with NED 7 48 Bilateral, S I XRT 18 Alive with NED 8 58 Left IV Chemo 9 Alive with NED 9 70 Bilateral, S IV Chemo 6 DD 10 73 Bilateral, S IV Chemo 3 DD S = simultaneously; M = metachronous; Chemo = chemotherapy; XRT = radiotherapy; NED = no evidence of disease; DD = died of disease Radiol Oncol 2003; 37(1): 23-7. Berkmen F / Malignant lymphomas of the testis in patients with low-stage tumours, a 5-year survival rate has been documented as 30 %.6,12,13,26-29 Thus, even patients whose dis­ease seems to be limited to the testis may have a relatively short survival time. In pa­tients with lymphoma at other sites and who later experience testicular relapses a poor prognostic factor exisists.5,6,9 The reported in­cidence of relapse in the contra lateral testicle is 0 to 35 % for patients with testicular lym­phoma.13,24,26,27,30 Testicular relapse was oc­curred in our 2 cases (20 %). Disease free mean survival times in reported series ranges between 16 months to 30 months.4,6 Five of our cases were alive with no evidence of dis­ease with follow-up ranging from 9 to 62 months. The remaining 5 patients died of dis­seminated disease, with survival ranging 3 ­42 months. Even though we treated our pa­tients with combine modality, we did not ob­tain good results. Conclusions Testicular tumours of germ cell origin reach their peak incidence in the age group 20 and 34 years. Malignant lymphomas of the testis account for almost 5% of all testis tumours. The common clinical presentation, initial treatment and the pattern of dissemination of testicular lymphomas are similar to those of testicular germ cell tumours. In contrast, by the following points testicular lymphomas differs from germ cell tumours: (1) Lymphoma of the testis tends to occur in the middle ages; (2) The levels of AFP and b -HCG are in normal limits; (3) Development of tes­ticular lymphoma in cryptorchid testis is ex­tremely rare; (4) In view of aggressive behav­iour, an early systemic chemotherapy is indi­cated and a watchful waiting policy is con­traindicated; (5) Patients with disease appar­ently confined to the testis and who have no clinical evidence of generalised disease 1 year after therapy may or may not have a high probability of cure; (6) Survival is poor espe­cially with bilateral disease, and later experi­encing a testicular relapse. Since testicular lymphomas are sometimes misdiagnosed as spermatocytic and anaplas-tic seminomas, immunocytochemical stain­ing for LCA should be performed in order to confirm the histologic diagnosis. The recogni­tion of histologic diagnosis in testicular lym­phoma is essential and should help the future treatment policy. References 1. Morse MJ, Whitmore WF Jr. Neoplasms of the testis. In: Walsh PC, Gittes RF, Perlmutter AD, Stamey TA, eds. CampbellÕs urology. Philadelphia: WB Saunders; 1986. p. 1539-44. 2. Davies JM. Testicular cancer in England and Wales: some epidemiological aspects. Lancet 1981; 1(8226): 928-32. 3. Silverberg E, Lubera JA. Cancer statistics, 1989. CA Cancer J Clin 1989; 39(1): 3-20. 4. Richie JP. Neoplasms of the testis. In: Walsh PC, Gittes RF, Perlmutter AD, Stamey TA, eds.CampbellÕs urology. 6th Edition. Philadelphia: WB Saunders; 1986. p. 1222-68. 5. Kiely JM, Massey BD Jr, Harrison EG Jr, Utz DC. Lymphoma of the testis. Cancer 1970; 26(4): 847­52. 6. Turner RR, Colby TV, McIntosh FR. Testicular lymphomas: a clinicopathologic study of 35 cases. Cancer 1981; 48(9): 2095-102. 7. Abell MR, Holtz F. Testicular and paratesticular neoplasms in patients 60 years of age and older. Cancer 1968; 21(5): 852-70. 8. Mostofi FK, Price EB Jr. Tumors of the male geni­tal system. In: Atlas of tumor pathology. Fasc 8, se­ries 2. Washington, D.C.: Armed Forces Institute of Pathology; 1973. 9. Paladugu RR, Bearman RM, Rappaport H. Malignant lymphoma with primary manifestation in the gonad: a clinicopathologic study of 38 pa­tients. Cancer 1980; 45(3): 561-71. 10. Ferguson JD. Tumours of the testis. Brit J Urol 1962; 34: 407-21. Berkmen F / Malignant lymphomas of the testis 11. Rosai J. AckermanÕs surgical pathology. 8th Edition. Mosby; 1996. p. 1287-97. 12. Root M, Wang TY, Hescock H, Parker M, HudsonP, Balducci L. BurkittÕs lymphoma of the testicle: report of 2 cases occurring in elderly patients. J Urol 1990; 144(5): 1239-41. 13. Sussman EB, Hajdu SI, Lieberman PH, Whitmore WF. Malignant lymphoma of the testis: a clinico-pathologic study of 37 cases. J Urol 1977; 118(6): 1004-7. 14. Abeshouse BS, Trongson A, Goldfarb M. Bilateral tumors of the testicles. Review of literature and re­port of case of bilateral simultaneous lymphosar-coma. J Urol 1955; 74: 522-32. 15. Tellem M, Faulk A, Meranze DR. Bilateral malig­nant lymphoma of the testes. Arch Patol 1961; 71: 151-5. 16. Collins DH, Pugh RCB. Classification and fre­quency of testicular tumors. Br J Urol 1964; 36: 1­11. 17. Dieckmann KP, Boeckmann W, Brosig W, Jonas D, Bauer HW. Bilateral testicular germ cell tumors. Report of nine cases and review of the literature. Cancer 1986; 57(6): 1254-8. 18. Montie JE. Carcinoma in situ of the testis and bi­lateral carcinoma. Urol Clin North Am 1993; 20(1): 127-32. 19. Hoekstra HJ, Wobbes T, Sleyfer DT, Schraffordt Koops H. Bilateral primary germ cell tumors of testis. Urology 1982; 19(2): 152-4. 20. Ware SM, Heyman J, Al-Askari S, Morales P. Bilateral testicular germ cell malignancy. Urology 1982; 19(4): 366-72. 21. Cockburn AG, Vugrin D, Batata M, Hajdu S, Whitmore WF. Second primary germ cell tumors in patients with seminoma of the testis. J Urol 1983; 130(2): 357-9. 22. Strohymeyer T, Hartmann M. Doppelseitige ho-dentumoren: Fallprasentation und thera­piekonzept. Akt Urol 1984; 15: 186-9. 23. Connors JM, Klimo P, Voss N, Fairey RN, Jackson S. Testicular lymphoma: improved outcome with early brief chemotherapy. J Clin Oncol 1988; 6(5): 776-81. 24. Doll DC, Weiss RB. Malignant lymphoma of the testis. Am J Med 1986; 81(3): 515-24. 25. Read G. Lymphomas of the testis-results of treat­ment 1960-77. Clin Radiol 1981; 32(6): 687-92. 26. Duncan PR, Checa F, Gowing NF, McElwain TJ,Peckham MJ. Extranodal non-HodgkinÕs lym­phoma presenting in the testicle: a clinical and pathologic study of 24 cases. Cancer 1980; 45(7): 1578-84. 27. Crellin AM, Hudson BV, Bennett MH, Harland S, Hudson GV. Non-HodgkinÕs lymphoma of the testis. Radiother Oncol 1993; 27(2): 99-106. 28. Selli C, Amorosi A, Nesi G, Bartoletti R, De Benedetto A, Cionini L. Asynchronous bilateralnon-HodgkinÕs lymphoma of the testis: report of three cases. Urology 1994; 44(6): 930-2. 29. Sasai K, Yamabe H, Tsutsui K, Dodo Y, Ishigaki T, Shibamoto Y, et al. Primary testicular non-HodgkinÕs lymphoma: a clinical study and review of the literature. Am J Clin Oncol 1997; 20(1): 59-62. 30. Jackson SM, Montessori GA. Malignant lym­phoma of the testis: review of 17 cases in British Columbia with survival related to pathological subclassification. J Urol 1980; 123(6): 881-3. Radiol Oncol 2003; 37(1): 23-7. The urokinase plasminogen activator and its inhibitors PAI-1 and PAI-2 in primary cutaneous melanoma Jasmina Markovi‰1, Borut átabuc2 1Clinical Department of Anaesthesiology and Intensive Therapy, 2Clinical Department of Gastroenterology, University Clinical Centre Ljubljana, Ljubljana, Slovenia Background. We investigated the differences in urokinase plasminogen activator (uPA) and its inhibitors type1 and 2 (PAI-1/2) concentrations in clinically suspected nevi, primary cutaneous melanoma and nor­mal skin and correlations with histopathological prognostic factors of primary melanoma. Patients and methods. Fifty-one patients were enrolled. The tissue concentrations of uPA, PAI-1 and PAI­2 were quantified by enzyme-linked immunosorbent assay (ELISA). Results. Mean uPA and PAI-1 concentrations in melanomas were higher than in normal surrounding skin (uPA: 1.08; vs. 0.48 ng/mgp; PAI-1: 14.07 vs. 2.07 ng/mgp; p < 0.001). uPA and PAI-1 concentrations were higher in melanomas than in nevi, and higher in nevi than in normal surrounding skin (uPA: p > 0.05; PAI-1: p = 0.02). PAI-2 concentration was higher in normal surrounding skin than in nevi and melanomas (p > 0.05). Melanoma uPA, PAI-1 and PAI-2 concentrations correlated significantly with normal skin (r = 0.73, 0.54, 0.38 respectively). PAI-1 was significantly lower in melanomas of Breslow thickness £ 0.75 mm, Clark invasion of 0+I, without microscopic ulceration, without vascular invasion (p < 0.01) than in melanomas of Breslow thickness > 0.75 mm, Clark invasion > II, with ulceration and vascular invasion. Conclusions. Determination of uPA and PAI-1 can provide significant additional prognostic information for melanoma patients. Key words: skin neoplasms - melanoma; urokinase plasminogen activator; plasminogen activator in­hibitor 1; plasminogen activator inhibitor 2; prognosis Received 24 January 2003 Accepted 14 February 2003 Correspondence: Jasmina Markovi‰, M.D., M.Sc., University Clinical Centre Ljubljana, Clinical Department of Anaesthesiology and Intensive Therapy, Zaloäka 7, 1000 Ljubljana, Slovenia; Phone: +386 (0) 31626577; E-mail: jasmina.markovic1@kclj.si Introduction Malignant melanoma is one of the most ag­gressive human tumours. The prognosis of melanoma as well as other cancers is mainly dependent on its ability to invade and metas­tasise. Prognostic markers such as Breslow tumor thickness, Clark level of invasion, ul­ceration and vascular invasion are used for defining melanoma patients at risk and fol­lowing adjuvant therapy. The clinical impor­tance of other factors (clinical, biochemical-molecular) has not been established yet.1 Cancer invasion and metastasis are multi-step events involving local invasion of the ex­tra cellular matrix, angiogenesis, invasion of the blood vessel wall, survival of malignant cells in the vascular system, extravasation, and establishment of a secondary growth. During most of these steps, extra cellular ma­trix and basement membrane have to be de­graded.2 The breakdown of these barriers is catalysed by the proteolytic enzymes, which are released from the invading tumour. Four different proteases are mainly involved: met-alloproteinases (collagenases, stromelysin, gelatinases), cysteine proteinases (cathepsin B, H, L), aspartyl proteases (cathepsin D), ser­ine proteases (plasminogen activation sys­tem).3 The two known plasminogen activators (PAs) are tissue type (tPA) and urokinase type plasminogen activator (uPA). Proteolytic activity of tPA is important for the degrada­tion of intravascular blood clots, while uPA contributes to extracellular proteolysis in a wide variety of physiological and pathological processes.2-4 uPA has many activities. It con­verts inactive plasminogen to plasmin, which degrades most substrates in the extracellular matrix (proteoglycans, laminin, fibronectin, vitronectin), and activates other proteases (procollagenases, uPA). uPA is activated also by kallikrein, trypsin, cathepsin B, L, ther­molysin and nerve growth factor.3,4 Beside these degradative functions uPA exerts other activities that may enable it to play a role in invasion and metastasis. These include stim­ulation of cellular proliferation, enhancement of cellular migration, alteration of cellular ad­hesive properties and activation of specific growth factors.3-5 Active uPA binds to a mem-brane-bound receptor known as u-PAR. uPA activity is controlled by two inhibitors, plas­minogen activator inhibitor type - 1 (PAI-1) and type - 2 (PAI-2). In addition to inhibiting uPA, PAI-1 modulates cellular adhesion and migration by its attachment to extracellular protein vitronectin. PAI-2 is important in in­hibition of apoptosis.3,4 A strong impact of these proteolytic factors on prognosis of the cancer disease has been observed in a variety of malignancies. The strongest and most con­sistent evidence of a prognostic role exists with breast cancer. Elevated concentrations of uPA, PAI-1 and uPAR are associated with poor prognosis, high levels of PAI-2, on the other hand, correlate with good outcome.2,5 uPA is prognostic also in gastric, colorectal, oesophageal, renal, endometrial, and ovarian cancer.2,4,5 It is well known, that uPA, PAI-1 and PAI-2 expression in human melanoma cell lines correlated with a high metastatic ca­pacity in nude mice.6 However in human cu­taneous and uveal melanoma uPA, PAI-1 and PAI-2 had not been detected in early stage of melanoma but appeared frequently in ad­vanced primary melanoma and melanoma metastatic lesions.7,8 Our study was aimed to find out the dif­ferences of uPA, PAI-1 and PAI-2 concentra­tions in clinically suspected nevi, primary melanoma and normal skin concentrations and their correlation to the most important histopathological prognostic factors: Breslow thickness, Clark invasion, ulceration and vas­cular invasion. Patients and methods Patients Fifty-one patients with clinical confirmed pri­mary cutaneous melanoma (27 women: mean age 49.7; range 21-84 years and 24 men: mean age 56.5; range 17-83 years) were enrolled in­to a prospective study between 1998-2000. Inclusion criteria were: absence of metastases and macroscopically and histological com­plete surgical removal of the primary cuta­neous melanoma (UICC pT1or T2N0M0, AJCC stage I and II).9 The Medical Ethics Committee at the Ministry of Health of the Republic of Slovenia approved the study pro­tocol. We totally excised melanoma lesions with safety edge of 1-2 cm. We excised 2 x 2 x 2 mm tissue specimens of the lesions and of the normal skin (at least 2 cm far away from the edge of tumours) for the quantification of uPA and PAI-1/2. They were snap-frozen in liquid nitrogen and stored at - 80° C. The remaining tissue was fixed in 10% for­malin and embedded in paraffin for histolog­ical examination. The histological diagnosis in 8 patients was dysplastic nevus and in 43 primary cutaneous malignant melanoma with clinical stage I (T1-2 N0 M0) less than 1.5 mm thick. The clinical and histopathological char­acteristics of primary tumours are shown in Table 1. Tissue extraction and ELISA for uPA, PAI-1 and PAI-2 The uPA concentrations were determined in 41 pairs of triton extracts, and the PAI-1 and PAI-2 concentrations in 51 pairs of cytosols prepared from tumour and adjacent normal tissue samples (matched pairs) weighing 50 mg, obtained at surgery. The still frozen cut sections were dipped into liquid nitrogen and then pulverized in a microdismembrator (Braun - Melsungen, Melsungen, Germany). For the triton extracts the still frozen pul­ver was dispensed with Tris buffered saline (TBS) (0.02 M Tris-HCl, 0.125 M NaCl, pH 8.5) containing 1% non-ionic detergent Triton X-100 (Sigma, St. Louis, Missouri, U.S.A.). The suspension was gently shaken for 3 hours at 4° C. For the cytosol the still frozen pulver was dispensed in a phosphate buffer (5 mM Na2HPO4, 1.7 mM KH2PO4, 1 mM monothioglycerol, 10% (vv-) glycerol, pH 7.4). Both, the tissue extracts and cytosol suspen­sion were subjected to ultracentrifugation (100 000 g/45 min at 4° C) to separate tissue debris. Supernatants were collected, divided into aliquots and stored at - 70° C until use. uPA, PAI-1 and PAI-2 concentrations were determined by commercially available ELISA kits (American Diagnostica, Inc., Greenwich, U.S.A.) for uPA, PAI-1 and PAI-2. Details of the kits are described elsewhere.10 Levels of the uPA, PAI-1 and PAI-2 are expressed in ng/mg proteins. Protein content was deter­mined with Bio Rad method. Statistical analysis Statistical analysis was performed using the SPSS for Windows program. Differences of uPA, PAI-1 and PAI-2 concentrations in melanomas, nevi and normal skin were analysed by the Wilcoxon test. SpearmanÕs Table 1. Clinical and histopathological characteristics of the tumours Characteristics Number Dysplastic nevus 8 Melanoma 43 Localization of primary lesion Head 4 Trunk 34 Extremities 13 Histopathological characteristics of N = 43 melanomas Breslow £ 0,75 28 > 0,75 15 Clark 0+I 12 II + III 31 Ulceration Yes 7 No 36 Vascular invasion Yes 3 No 20 Undetermined 20 Histopathological type Lentigo maligna 2 Superficial spreading 35 Nodular 2 Unclassified 4 Radiol Oncol 2003; 37(1): 29-35. rank correlations were evaluated for the rela­tions between uPA, PAI-1 and PAI-2 concen­trations in melanomas and normal skin. Differences of uPA, PAI-1 and PAI-2 concen­trations in melanomas and histomorphologi-cal variables among various groups of pa­tients were analysed by the two-tailed t-test and the analysis of variance (ANOVA). The p-values £ 0.05 were considered sig­nificant. Results uPA, PAI-1 and PAI-2 concentrations in melanomas, nevi and normal skin Among 51 tumour specimens (43 melanomas, 8 dysplastic nevi) and normal skin, the con­centration of u-PA was determined in 41 pairs of triton extracts (36 melanomas, 5 nevi), and of PAI-1 and PAI-2 in 51 pairs of cytosols (43 melanomas, 8 nevi) (Table 2). The mean uPA and PAI-1 concentrations in melanomas were significantly higher than in normal skin (p < 0.0001). The mean uPA con­centration was higher in melanomas than in dysplastic nevi and higher in dysplastic nevi than in normal skin, however statistically in­significant (p > 0.05). Mean PAI-1 concentra­tion was higher in melanomas than in dys-plastic nevi, and higher in dysplastic nevi than in normal skin with significant results (p = 0.02). Mean PAI-2 concentration was not significantly higher in normal skin than in dysplastic nevi and melanomas (p > 0.05). There was a significant positive correlation (r= 0.45) between melanoma uPA and PAI-1 s concentrations. Significant correlations be­tween melanoma and normal skin concentra­tions for uPA, PAI-1 and PAI-2 were found (r= 0.73; 0.54; 0.38) (Figure 1). s Table 2. Differences of uPA, PAI-1 and PAI-2 concentrations between melanomas, nevi and normal skin uPA (ng/mgp) PAI-1 (ng/mgp) PAI-2 (ng/mgp) Melanoma 1,08 ± 0,58 14,07 ± 16,55*** 9,21 ± 18,32 Dysplastic nevi 0,99 ± 0,58 4,84 ± 8,32**** 13,44 ± 17,32 Normal skin around 0,48 ± 0,21* 2.07 ± 1,83** 13,05 ± 19,72 Melanoma Normal skin around 0,51 ± 0,36 0,89 ± 0,91 28,8 ± 39,55 Dysplastic nevi Levels are mean ± sd *, ** p < 0,0001, statistically significant lower uPA and PAI-1 normal skin levels according to melanoma levels ***, **** p = 0,021 statistically significant higher PAI-1 melanoma levels according to nevi and higher nevi levels according to normal skin levels Correlation between melanoma uPA, PAI-1 and PAI-2 concentrations and relevant prognostic factors Melanoma uPA, PAI-1 and PAI-2 concentra­tions were compared to established histomor­phological factors (Table 3). In contrast to uPA and PAI-2, PAI-1 concentrations were significantly lower in melanomas of Breslow tumour thickness £ 0.75 mm, Clark level of invasion 0+I, without microscopic ulceration on the tumour surface and absents vascular invasion of the tumours. Much higher positive correlations (r= s 0.67, 0.62 and 0.63 respectively; p £ 0.02) be­tween u-PA and PAI-1 concentrations were found in the group of the melanomas of Breslow thickness > 0.75 mm, with the level of Clark invasion III and present vascular in­vasion. Discussion In the present study using specific ELISA per­formed on tumour extracts of primary melanomas less than 1.5 mm thick, higher uPA and PAI-1 and lower PAI-2 concentra­tions than in nevi and normal skin were found. The correlation between uPA, PAI-1 and PAI-2 concentrations in melanomas and normal skin was found to be positive. The prognostic value of uPA and PAI-1 in early stages primary malignant melanoma has not yet been investigated. However, a strong correlation between the metastases of the human melanoma cells in the nude mouse model as well as in the human cuta­neous melanoma and expression of uPA and PAI-1 was found.6 Dysplastic nevi are sup­posed to be an important risk factor for the development of the cutaneous melanoma. In 1979 Fr-ki et al. found out higher concentra­tions of plasminogen activators in primary melanomas and melanoma metastases com­paring to extracts of nevi.11 Recently De Vries et al reported that uPA, PAI-1, PAI-2 and uPAR appeared frequently in advanced pri­mary cutaneous and uveal melanoma and melanoma metastasis lesions.7,8,12 However uPA and PAI-1 accumulation was observed also in atypical nevocytes, whereas uPA pro­teolytic activity was detected only in melanomas.13 Table 3. Differences of mean concentrations of uPA, PAI-1 and PAI-2 in primary cutaneous melanomas with his-tomorphological prognostic factors Variable N uPA± SD pa N PAI-1± SD pa N PAI-2± SD pa (ng/mgp) (ng/mgp) (ng/mgp) Breslow £ 0,75 mm 22 1,01± 0,54 n.s. 28 9,68± 11,92 28 10,89± 22,34 n.s. > 0,75 mm 14 1,2± 0,65 15 22,25± 20,92 0,016 15 6,07± 5,35 Clark 0+I 9 0,89± 0,55 12 4,74± 6,45 12 15,97± 32,36 II+III+IV 27 1,15± 0,59 n.s. 31 17,67± 17,89 0,001 31 6,59± 7,78 n.s. Ulceration Yes 7 1,38± 0,74 7 27,76± 23,27 7 3,2± 2,68 n.s. No 29 1,01± 0,53 n.s. 36 11,41± 13,82 0,015 36 10,38± 19,82 Vascular invasion Yes 2 1,99± 1,04 n.s. 3 37,77± 23,01 3 5,13 ± 6,74 n.s. No 15 1,02± 0,55 20 9,5± 12,74 20 11,48± 25,62 Undetermined 19 1,04± 0,52 20 15,1± 16,6 0,017 20 7,55± 8,33 aAnalysis of variance, two-tailed t-test, p £ 0,05; n.s. = not significant Radiol Oncol 2003; 37(1): 29-35. Previously, uPA and PAI-1 have been claimed to be of independent prognostic val­ue for disease free and overall survival in breast cancer patients.14,15 In addition to breast cancer, components of plasminogen activation system also have a prognostic val­ue in colorectal, gastric, oesophageal, blad­der, endometrian and ovarian cancer.2,5 Similarly Nekarda et al have found a stronger prognostic impact of PAI-1 than that of uPA in completely resected gastric cancer.16 The presence of the components of the PAs in malignant melanoma has earlier been studied using immunohistochemistry (IHC), in situ zymography and in situ hybridisation. Both, ELISA and IHC have their specific ad­vantages. ELISA methods give an objective quantification of analyte levels, whereas IHC yields at best semi-quantitative information. ELISA and IHC may detect fractions of PA components with different efficiencies.17,18 At present, ELISAs measuring the levels of uPA and PAI-1 performed consistently well, at least by their more extensively proven clin­ical value and unequivocal interpretation as demonstrated by the Quality Assurance Center in Nijmegen and from the European Organisation for Research and Treatment of Cancer (EORTC).10 Breslow tumour thickness, Clark level of invasion, ulceration and vascular invasion are the most important histological variables pre­dicting melanoma outcome. The prognostic role of other possible markers like molecular and biochemical is not known yet. Our re­sults prove the presence of the correlation of uPA and PAI-1 concentrations with prognos­tic value of Breslow thickness, Clark inva­sion, ulceration and vascular invasion also in early melanoma stage I. We observed that higher PAI-1 concentrations were associated with melanomas of Breslow > 0.75 mm, Clark II+III+IV, present ulceration and vascular in­vasion, which are prognostic adversely. Significant positive correlation between uPA and PAI-1 in melanomas with poor prognosis proves that also uPA has a prognostic impact. It indicates that their role in melanoma growth is co-dependent. There are various speculative explanations of the role of excessive PAI -1 production in the tumour:14,16,19 a) PAI-1 is important for reimplantation of circulating tumour cells at distant loci; b) since PAI-1 is present in en­dothelial cells and platelets, increased PAI-1 levels may reflect a high degree of angiogene-sis, thus favouring tumour spread and metas­tasis; c) PAI-1 binds to adhesive glycoprotein vitronectin and influences cell adhesion and migration. The prognostic role of PAI-2 is not well known yet. In breast cancer patients with high uPA concentrations, PAI-2 correlated with good prognosis.20 In colorectal cancer, however, high PAI-2 concentrations were as­sociated with aggressive disease.21 Our results prove that using ELISA it is possible to quantify the uPA, PAI-1 and PAI­2 concentration in very small specimens of early melanoma lesions. Analogous to breast cancer, uPA and PAI­1 could become an additional prognostic fac­tor for the progression of melanoma next to the established histological criteria. Further­more, the fundamental role of uPA and PAI-1 in tumour invasion and metastasising indi­cates that these factors should be explored as targets for tumour biology - oriented thera­pies.22 To conclude, our results indicate that us­ing ELISA uPA, PAI-1 and PAI-2 concentra­tions can be measured also in small samples of primary melanoma and that both uPA and PAI-1 might also be of prognostic importance in primary malignant melanoma. These find­ings need to be confirmed in further studies where relationship between uPA, PAI-1 and patient survival will be investigated. References 1. Slominski A, Ross J, Mihm MC. Cutaneous melanoma: pathology, relevant prognostic indica­tors and progression. Br Med Bull 1995; 51: 548-69. 2. Andreasen PA, Kjøller L, Christensen L, Duffy MJ. The urokinase-type plasminogen activator system in cancer metastasis: a review. Int J Cancer 1997; 72: 1-22. 3. Schmitt M, Harbeck N, Thomssen C, Wilhelm O, Magdolen V, Reuning U, et al. Clinical impact of the plasminogen activation system in tumor inva­sion and metastasis: prognostic relevance and tar­get for therapy. Thromb Haemost 1997; 78: 285-96. 4. Reuning U, Magdolen V, Wilhelm O, Fischer K, Lutz V, Graeff H, Schmitt M. Multifunctional po­tential of the plasminogen activation system in tu­mor invasion and metastasis. Int J Oncol 1998; 13: 893-906. 5. Duffy MJ, Maguire TM, McDermott EW, OÕHiggins N. Urokinase plasminogen activator: a prognostic marker in multiple types of cancer. J Surg Oncol 1999; 71: 130-5. 6. Quax PHA, Van Muijen GNP, Weening - Verhoeff EJD, Lund LR, Danø K, Ruiter DJ, et al. Metastatic behaviour of human melanoma cell lines in nude mice correlates with urokinase-type plasminogen activator, its type-1 inhibitor and urokinase medi­ated matrix degradation. J Cell Biol 1992; 115: 191­9. 7. De Vries TJ, Quax PHA, Denijn M, Verrijp KN, Verheijen JH, Verspaget HW, et al. Plasminogen activators, their inhibitors, and urokinase receptor emerge in late stages of melanocytic tumor pro­gression. Am J Path 1994; 144: 70-81. 8. De Vries TJ, Mooy CM, Van Balken MR, Luyten GPM, Quax PHA, Verspaget HW, et al. Components of the plasminogen activation sys­tem in uveal melanoma: a clinico - pathological study. J Pathol 1995; 175: 59-67. 9. American Joint Committee on Cancer: Manual for staging of cancer. Philadelphia: JB Lippincott Co.; 1997. 10. Benraad TJ, Geurts-Moespot J, Gro/ndahl-Hansen J, Schmitt M, Heuvel JJTM, de Witte JH, et al. Immunoassays (ELISA) of urokinase-type plas­minogen activator (uPA): report of an EORTC/ BIOMED-1 workshop. Eur J Cancer 1996; 32A (8): 1371-81. 11. Fr-ki JE, Nieminen S, Hopsu-Havu VK. Proteolytic enzymes and plasminogen activators in melanoma. J Cutan Pathol 1979; 6: 195-200. 12. De Vries TJ, Van Muijen GNP, Ruiter DJ. The plas­minogen activation system in melanoma cell lines and in melanocytic lesions. Mel Res 1996; 6: 79-88. 13. Delbaldo C, Masouye I, Saurat JH, Vassalli JD, Sappino AP. Plasminogen activation in melanocyt­ic neoplasia. Cancer Res 1994; 54: 4547-52. 14. J-nicke F, Schmitt M, Pache L, Ulm K, Harbeck N, Hofler H, Graeff H. Urokinase plasminogen acti­vator (u-PA) and its inhibitor PAI-1 are strong and independent prognostic factors in node-negative breast cancer. Breast Cancer Res Treat 1993; 24: 195­208. 15. Gro/ndahl-Hansen J, Christensen IJ, Rosenquist C, Br.nner N, Mouridsen HT, Dano/ K, et al. High levels of urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 in cytosolic extracts of breast carcinomas are associated with poor prognosis. Cancer Res 1993; 53: 2513-21. 16. Nekarda H, Schmitt M, Ulm K, Wenninger A, Vogelsang H, Becker K, et al. Prognostic impact of urokinase-type plasminogen activator and its in­hibitor PAI-1 in completely resected gastric can­cer. Cancer Res 1994; 54: 2900-7. 17. Ferrier CM, De Witte HH, Straatman H, van Tienoven DH, van Geloof WL, Rietveld FJR, et al. Comparison of immunohistochemistry with im­munoassay (ELISA) for the detection of compo­nents of the plasminogen activation system in hu­man tumour tissue. Br J Cancer 1999; 79: 1534-41. 18. Ferrier CM, Suciu S, vanGeloof WL, Straatman H, Eggermont AM, Koops HS, et al. High tPA-expres­sion in primary melanoma of the limb correlates with good prognosis. Br.J.Cancer 2000; 83: 1351-9. 19. Br.nner N, Nielsen HJ, Hamers M, Christensen IJ, Thorlacius-Ussing O, Stephens RW. The uroki­nase plasminogen activator receptor in blood from healthy individuals and patients with cancer. AP­MIS 1999; 107: 160-7. 20. Foekens JA, Buessecker F, Peters HA, Krainick U, van Putten WLJ, Look MP, et al. Plasminogen ac­tivator inhibitor-2: prognostic relevance in 1012 patients with primary breast cancer. Cancer Res 1995; 55: 1423-7. 21. Ganesh S, Sier CFM, Griffioen, Vloedgraven HJM, De Boer A, Welvaart K, et al. Prognostic relevance of plasminogen activators and their inhibitors in colorectal cancer. Cancer Res 1994; 54: 4065-71. 22. Schmitt M, Wilhelm OG, Reuning U, et al. The urokinase plasminogen activator system as a nov­el target tumor therapy. Fibrinol Proteol 2000; 14: 114-32. Radiol Oncol 2003; 37(1): 29-35. Breast cancer in the Czech Republic Lenka Hoda‰ov/ Department of Social Medicine, Charles University in Prague, Faculty of Medicine in Hradec Kr/lov” Background. Malignant neoplasms present one of the most serious chapters of morbidity, mortality, and the overall perspective of the health status of Czech population. Malignant neoplasms have been registered in the Czech Republic since the end of the 1950s. Guarantor of the all-state registry is the Institute of Health Information and Statistics of the Czech Republic (IHIS CR), and conceptual and methodological steering is performed by the Council of the Czech Cancer Registry. The five most frequently diagnoses in Czech males and seven most frequently diagnoses in Czech females were followed during the last 20 years. The most fre­quent malignant neoplasm in Czech women is breast cancer. Conclusions. The incidence of this cancer has increased by 75% during the studied period. During the year 2001, three pilot studies of preventive mammography screening were done in the country. One case of asymptomatic breast cancer in the study costs 80,000 K‰ (in reality it was 120,000 K‰). These costs are markedly lower than the combined therapy of advanced stages of breast cancer. Key words: breast neoplasms; mammography; Czech Republic Introduction Malignant neoplasms (MN) present one of the most serious chapters of morbidity, mor­tality, and the overall perspective of the health status of Czech population. They can Received 23 January 2003 Accepted 7 February 2003 The theme of the article was presented as an invitedlecture at the È1st Open Meeting on Public Health and Oncology: Breast CancerÇ, held in Ljubljana June 14th, 2002. Correspondence to: Lenka Hoda‰ov/, M.D., Department of Social Medicine, Charles University in Prague, Faculty of Medicine in Hradec Kr/lov”, áimkova 870, 500 01 Hradec Kr/lov”, Czech Republic; Phone: +420 495 816 417; Fax: +420 495 513 597; E­mail: hodacoval@lfhk.cuni.cz also be seen as an important measure of the health status of our population. Due to the ex­tent of their occurrence and lethality, fatality rate, the incomplete knowledge of their etiopathogenesis, therapy, and prevention, they must be considered as one of the pri­mary questions and tasks for the coming years. Neoplasms represent a challenge for health care and the whole society as well. MN are the second most common cause of death and constitute about 25% of all deaths in the Czech Republic.1 Methodology MN have been registered in the Czech Republic since the end of the 1950s. In 1976, Hoda‰ov/ L / Breast cancer in the Czech Republic the cancer registry was established. Since 1991, the Cancer Registry of the Czech Republic has been a member of the International Association of Cancer Registries (IACR). It collaborates with the European Network of Cancer Registries and keeps in contact with registries in many countries. In the early 1990s, the collection, supple­menting, checking, and completion of data were gradually taken over by the 85 District Units of the Czech Cancer Registry, which are aggregated into 8 Regional Units. Guarantor of the all-state registry is the Institute of Health Information and Statistics of the Czech Republic (IHIS CR), and conceptual and methodological steering is performed by the Council of the Czech Cancer Registry. The basic source of information contained in the Registry is the mandatory form ÈReport on neoplasmÇ (NZIS 022 2) filled in by the physician who diagnoses the disease. The no­tification is returned within 3 months to the District Unit of the Registry where it is checked, supplemented by additional data, and included in the database. The Registry is also complemented on the basis of check-up reports and by comparison with the database of deaths in the Czech Statistical Office (CSO). Registration is performed according to the Decree of the Ministry of Health and Social Affairs (MSHA) of the CSR no.3/1989 in Bulletin of MHSA CSR (reg. in Law Collection, part 19/1988) - ÈDispensary care for patients with precanceroses and cancer and mandatory notification of neoplasmsÇ, applying methodology NZIS (the National Health Information System) no.515/1987 -ÈCancer RegistryÇ, following the ÈInstruction on data entries and returnsÇ 56/1997 in the NZIS methodology ÈHandbooks for regional and district unitsÇ and other appropriate methodological instructions. Data for the publication ÈNeoplasmsÇ are usually processed with a two-year delay, which is necessary for the completion of the Registry with missing patients and missing data.2 Trends of registered cases of cancer Trends of incidence of registered cancer cas­es (ICD - 10, dg. C00 - C097 and dg. D00 ­D09) are shown in Figure 1. The relative indi­cators (cases per 100 000 males or females) are computed with reference to the mid-year population and also to world standard and European standard populations, the same for both genders, as published in the WHO Yearbook. The incidence of all registered cancer cas­es in males in the Czech Republic grew by 40.1% during the period 1985 - 1998 (1985 ­ 414.8 cases per 100 000 males; 1998 - 583.6 cases per 100 000 males), whereas in females, it grew by 50.1% during the same period (1985 - 363.7 cases per 100 000 females; 1998 - 548.7 cases per 100 000 females). In the pe­riod 1985 - 1998, the growing incidence trend is seen in both sexes also at the world and European levels: - by 27.8% - males world rates (1985 - 324.8 per 100 000 males; 1998 - 415.1 per 100 000 males) - by 38.7% - females world rates (229.4 ­318.2) - by 42.8% - males European rates (427.5 ­610.7) - by 39.2% - females European rates (319.7 ­444.9) In Czech females compared to the world and European rates, the highest incidence was observed in the followed period. The in­cidence of all registered cancer cases in Czech males is lower compared to the European rates, but higher than the incidence in the world standard. But the incidence growth is steeper in both sexes in the Czech Republic compared to the world and European rates.2,3 Trends of registered cancer cases in the Czech Republic - selected diagnosis (per 100 000 inhabitants) Trends of registered cases of selected diagno­sis for males and females separately during the studied period of the last 20 years (1980 ­1999) in Czech Republic are shown in Figures 2 and 3. The incidence of all registered cancer cases in males in the Czech Republic grew from 378.4 cases per 100 000 males (1980) to 570.5 cases per 100 000 males. The growth in­dex 1999/1980 is 150.8. The growth was faster in the nineties than in the eighties (in­dex 1990/1980 is 120.5; index 1999/1990 is 125.1). The incidence in Czech females was permanently lower than in males (1980 ­322.3; 1999 - 537.9), but the difference was systematically falling down, with the index M/F of 117.4 in 1980; 111.7 in 1990; and 106.1 in 1999. The incidence in females was grow­ing faster than in males. According to the selected diagnosis in Czech males, MN of the lung (C 33 + C 34) with 97.6 cases per 100 000 males was the most frequent cancer in the year 1980. MN of the stomach (C16) ranked second, with 32.4 cases per 100 000 males. The incidence of MN of the lung slightly increased in the eighties (99.6) and decreased during the nineties (88.9). The second most frequent cancer for males in 1999 was MN of the prostate (53.8 cases per 100 000 males). The incidence in­creased by 128% during the followed period. A considerable increase (120%) is seen in colon cancer (from 19.8 to 43.6). The inci­dence of MN of the rectum has grown by 49% (24.9 to 37.1). A remarkable decrease from 32.4 to 20.4 (37%) is seen in stomach cancer. The above-mentioned five selected diagnoses included 52.4% of all notified diagnoses in 1980, while in 1999, it was only 42.9%.3 The selected seven most frequent diag­noses in females in the Czech Republic were followed during the period 1980 - 1999. In 1980 and in 1999, the proportion of these di­agnoses to all registered cancer cases was 54.6%, and 43.8%, respectively. The most fre­quent cancer in 1980 was breast cancer (C 50) (51.5 cases per 100 000 females) and it kept that position also in 1999 (89.9 per 100 000 fe­males). During the followed period of 20 years, its incidence grew by 75%. The cancer of the colon ranked second in 1999 and its in­cidence grew by 76.4% during the last 20 years. A remarkable increase in Czech fe­males represents MN of the lung; it grew by 111.3% (from 10.6 to 22.4). In this same peri­od, the incidence of MN of the lung decreased in Czech males by 10%. In females, a system­atic decrease of 34% in stomach cancer inci­dence was observed in the followed period. A stable incidence (about 20 per 100 000) dur­ing the 20-year period was seen in MN of the cervix uteri. The incidence of the cancer of the rectum increased by 34% and the inci­dence of the cancer of the corpus uteri by 35%.3 Breast cancer The most frequent and most serious MN in women is breast cancer. MN of the breast has been registered since 1953 in the Czech Republic (in absolute numbers, there were 1,700 cases per year). In 1980, the incidence of breast cancer was 51.5 per 100 000 women (2,739 cases), and in 1999, it was already 89.8 per 100 000 women (4,740 cases in absolute numbers), which is 2.5% more cases than the year before.2,7 In 1999, the breast cancer incidence in fe­males was for the first time higher than that of the lung cancer in males (breast cancer in­cidence - 89.8; lung cancer incidence - 88.9). The incidence of breast cancer increased by 75% during last 20 years (as mentioned above).3 During the last ten years, the absolute number of deaths from breast cancer gradu­ally increased by 3.4%, while the fatality rate Radiol Oncol 2003; 37(1): 37-42. Hoda‰ov/ L / Breast cancer in the Czech Republic decreased. Compared to 1989, it decreased by more than 10% and its present value is ap­proximately 41%. The number of MN in an unspecified stage, or more properly, with no stage identi­fication in the notification, increased in cases of MN of the breast. This phenomenon was mostly seen in the 1990s. In cases where the stage was notified, the proportion of stages I and II was definitely growing, while the pro­portion of stages III and moderately also IV had a decreasing tendency. The cumulative 5­year survival rate in cases diagnosed in the years 1989-1993 increased in women by al­most 10% compared to the cases diagnosed in the period 1980-1984. Breast cancer incidence grows with age. During the last 5 years, the specific incidence of breast cancer increased more rapidly in the Czech Republic in the age groups of 55-59 years and 65-69 years (55-59: 1995 - 164.7 per 100 000; 1999 - 189.5; 65-69: 1995 - 218.7; 1999 - 248.7). It is important to mention that the specific incidence was markedly higher in all age groups over 50 years compared with the situation at the beginning of the 1990s. On the basis of mandatory form ÈReport on neoplasmsÇ it is possible to follow the changes in treatment modalities. Since the 1980s, the number of surgically treated breast cancer within 3 months from the diagnosis has been stabilized, ranging from 86 to 89 %. The proportion of radiotherapy is decreasing. In 1980, 76% of MN cases were treated by ra­diotherapy, in 1990, about 60%, and in 1999, 45%. On the other hand, the proportion of chemotherapy and hormonal therapy is in­creasing. In 1980 and 1999, respectively 11% and 46% of cases were treated by chemother­apy. As to the hormonal therapy, the propor­tion increased even more - from 8% of diag­nosed breast cancer cases in 1980 to 49% of these cases in 1999. The absolute number of deaths from breast cancer is under 2000 per year. Since the mid 1980s, the mortality rate has been ranging from 34 - 39 deaths per 100 000 women. The proportions of the mortality rate have been relatively stable in all age groups during the last 10 years. Breast cancer mor­tality increased only in the oldest age group (over 85 years old). This age group also had the highest specific mortality.2,7 Breast cancer incidence in the Czech Republic (even with its relatively steep grow­ing tendency) is still lower in comparison with Western European countries. But it is higher compared to Slovakia, Hungary and Poland and similar to the incidence in Slovenia (see Table 1). Table 1. Female breast cancer incidence per 100 000 in some selected European countries. Source: Database HFA 2000. 1996 1997 1998 1999 Czech Republic 83,8 85,1 87,5 94,9 Slovenia 82,2 87,9 . . Slovakia 62,1 61,2 63,8 . Hungary 35,0 41,7 43,0 97,5 Poland 48,8 . . . Austria 110,1 110,8 109,3 . Denmark 130,7 132,1 . . Finland 127,0 126,7 129,8 134,6 Norway 114,4 116,1 114,1 . Netherlands 126,6 125,9 . . Sweden 130,6 130,1 138,2 140,9 United Kingdom 122,0 125,6 . . Mammography - pilot study in the Czech Republic The question of preventive mammography screening for all women of age 45 - 70 was discussed in the Czech Republic by the pro­fessionals several times. The main reason why it wasnÕt introduced was financial. For mammography examination women needed a recommendation either from a GP or a gy­naecologist. During the year 2001, three pilot studies were done in the country with the aim to make a detailed audit in a relatively short time and answer the questions by General Health Insurance Company before the intro­duction of payments for preventive screen­ing. Two of the studies took place in Mamma Centrum in Prague and one in University Hospital in Hradec Kr/lov”. Each study in­cluded 1,500 women and the duration of each was 3 months. The results of the first (that took place in Mamma Centrum in Prague from November 2000 to January 2001) are the following: Eleven cases of breast cancer were found among 1,500 examined women (9 of the women were without any symptom). Medical diagnoses were made within 2 hours from the examination in 98.5% of women. The 9 women who were diagnosed, but showed no symptoms, obviously benefited from the screening in the pilot study. No doubt that preventive mammography is not a cheap method. Mamma Centrum obtain­ed altogether 750,000 K‰ to cover the pilot stu­dy; it means 500 K‰ per one examined woman (1 EURO was approximately 31 K‰ at that time). The real costs also included the clinical examination of a patient, registration, necessa­ry ultrasonography for 1/4 of women, audit, taking of anamnesis, and the work of health professionals were at least 1.5 times higher. One case of asymptomatic breast cancer in the study cost 80,000 K‰ (in reality it was 120,000 K‰). These costs are markedly lower than the combined therapy of advanced stages of breast cancer. The economical prof­itability of the screening was proved. Preventive mammography screening was rat­ified in spring 2002 by the Decree of the Ministry of Health of the Czech Republic. The screening should start in November 2002 and is targeted to women aged 44 - 70 years once every 2 years.8 Radiol Oncol 2003; 37(1): 37-42. Hoda‰ov/ L / Breast cancer in the Czech Republic References 1. Drbal C. Zdravotn™ stav populace ›R a jeho prognŠza jako dominantn™ determinanta poìadavku na syst”m p”‰e o zdrav™. Studie N/rodohospod/Þsk”ho œstavuJosefa Hl/vky. Praha: N/rodohospod/ÞskyÕ œstav Josefa Hl/vky; 1997. 2. tav zdravotnick” statistiky a informatiky ›R. Novotvary 1998 ›R. Cancer incidence in the Czech Republic. Praha: IS, NOR ›R; 1998. 3. Hoda‰ov/ L.: K veÞejn”mu zdrav™ na pÞelomu 20. a 21. stolet™ v kontextu trvale udrìiteln”ho ìivota. Diserta‰ni pr/ce, Hradec Kr/lov”: LF UK; 2002. 4. tav zdravotnick” statistiky a informatiky ›R. Zdravotnick/ ro‰enka vychodn™ch ›ech 1995-1999. Hradec Kr/lov”: IS ›R, Region/ln™ odbor vyÕ­chodn™ch ›ech. 1996-2000; periodical - annually. 5. tav zdravotnick” statistiky a informatiky ›R. Zdravotnictv™ ›R 1997 - 1999 ve statistickyÕch œdaj™ch. Praha: IS ›R, 1998 - 2000; periodical - annual­ly. 6. tav zdravotnick” statistiky a informatiky ›R. Zdravotnick/ ro‰enka ›R 1980, 1985, 1990, 1995­1999. Praha: IS ›R; 1981, 1986, 1991, 1996­2000; periodical - anually. 7. Jechov/ M. ZhoubnyÕ novotvar prsu u ìen - Aktualn™ informace; tav zdravotnickyÕch informac™ a sta­tistiky ›eske republiky, Narodn™ onkologickyÕ reg-istr - ZN prsu u ìen, Zdravotnick/ data novotvary 1998, Archiv aktualn™ch informac™; available on in-ternet at: http:// www.uzis.cz. 8. Skovajsov/ M. Preventivn™ vyäetÞovan™ prsu -prvn™ zkuäenosti podle novyÕch pravidel ma-marn™ho screeningu. Zdravotnick” noviny 2002; 51(18): 22-3. Tumor blood flow modifying effects of electrochemotherapy: a potential vascular targeted mechanism Gregor Seräa1, Maja ›emaìar1 and Damijan Miklav‰i‰2 1Institute of Oncology Ljubljana, 2University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia Background. The aim of this study was to determine the tumor blood flow modifying, and potential vas­cular targeted effect of electrochemotherapy with bleomycin or cisplatin. Materials and methods. Electrochemotherapy was performed by application of short intense electric puls­es to the tumors after systemic administration of bleomycin or cisplatin. Evaluated were antitumor effec­tiveness of electrochemotherapy by tumor measurement, tumor blood flow modifying effect by Patent blue staining technique, and sensitivity of endothelial and tumor cells to the drugs and electrochemotherapy by clonogenicity assay. Results. Electrochemotherapy was effective in treatment of SA-1 tumors in A/J mice resulting in substan­tial tumor growth delay and also tumor cures. Tumor blood flow reduction following electrochemotherapy correlated well with its antitumor effectiveness. Virtually complete shut down of the tumor blood flow was observed already at 24 h after electrochemotherapy with bleomycin whereas only 50% reduction was ob­served after electrochemotherapy with cisplatin. Sensitivity of human endothelial HMEC-1 cells to elec­trochemotherapy suggests a vascular targeted effect for electrochemotherapy in vivo with bleomycin as well as with cisplatin. Conclusion. These results show that, in addition to direct electroporation of tumor cells, other vascular tar­geted mechanisms are involved in electrochemotherapy with bleomycin or cisplatin, potentially mediated by tumor blood flow reduction, and enhanced tumor cell death as a result of endothelial damage by elec­trochemotherapy. Key words: sarcoma experimental - drug therapy - blood supply; bleomycin; cisplatin; electroporation; drug delivery systems Received 1 October 2002 Accepted 15 November 2002 Correspondence to: Gregor Seräa, Ph.D., Institute of Oncology Ljubljana, Zaloäka 2, SI-1000 Ljubljana, Slovenia; Phone/Fax: +386 1 433 74 10; E-mail: gser­sa@onko-i.si Introduction Enhanced delivery of chemotherapeutic drugs into tumor cells by electroporation is termed electrochemotherapy.1 A local in­crease in plasma membrane permeability, af­ter exposure of tumor nodules to electric pulses (electroporation), results in increased Seräa G et al. / Tumor blood flow modifying effects of electrochemotherapy uptake of chemotherapeutic drugs into the tumor cells. Electrochemotherapy has been shown to be successful for drugs such as bleomycin and cisplatin, which normally ex­hibit impeded transport through the plasma membrane. The increased antitumor effec­tiveness of bleomycin and cisplatin combined with electroporation has already been demonstrated in experimental and clinical studies although the underlying mechanisms remain to be clarified. 1-4 In addition to increased drug delivery into the cells, application of electric pulses to the tumors was found to exert tumor blood flow modifying effect.5,6 Electric pulses, as used in preclinical and clinical studies were found to reduce tumor blood flow. Transient reduction in tumor blood flow down to 30% of control was found, but recovered to almost pre-treat­ment level within 24 hours.5 Application of electric pulses to solid tu­mors would not be expected to selectively electroporate tumor cells alone. All cells in all areas where electric field exceeds the critical threshold level would be electroporated.1,7 Therefore endothelial cells are also potential targets for electroporation. Since the initial concentration of the drug is the highest in tu­mor blood vessels, during electroporation, electrochemotherapy is probably effective on endothelial cells in the tumor blood vessels. This may lead to severe damage to the vascu­lature of the tumors and consequently induce a secondary cascade of tumor cell death, e.g. by abrogating oxygen supply to the cells. This phenomenon, described as vascular targeted therapy, has been exploited in several stud­ies.8 The aim of this study was to elucidate tu­mor blood flow modifying and vascular tar­geted effects of electrochemotherapy with bleomycin or cisplatin by measuring tumor perfusion, and cells survival of endothelial cells in relation to their antitumor effective­ness. Materials and methods Animals, tumors and cell lines A/J mice of both sexes, purchased from the Institute Rudjer Boäkovi“, Zagreb, Croatia, were used. Subcutaneous murine fibrosarco-ma SA-1 tumors (The Jackson Laboratory, Bar Harbour, ME) were implanted, by injecting 0.1 ml NaCl (0.9%) containing 5 x 105 viable tumor cells under the skin on the rear dor-sum. Six to 8 days after implantation, when tumors reached approximately 40 mm3 in vol­ume (6 mm in diameter) mice were randomly divided into experimental groups, consisting of at least 6 mice. Treatment protocols were approved by the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia No. 323-02-237/01. Human dermal microvascular endothelial cells (HMEC-1) cells were generously provid­ed by Dr. F.J. Candal (Center for Disease Control, Atlanta, USA). Cells were grown as monolayer in D-MEM supplemented with 10% fetal calf serum (FCS, Sigma, USA) in a humidified incubator at atmospheric oxygen supplemented with 5% CO2 at 37° C. They were routinely subcultured twice per week. Electrochemotherapy protocol Bleomycin (Bleomycin, Mack, Germany) was dissolved in phosphate buffered saline and the dose of 5mg/kg in 0.2 ml volume was in­jected intravenously. Bleomycin solution was prepared freshly for daily injections. cis-Diamminedichloroplatinum (II) (Cispla-tin) was obtained from Bristol Myers Squibb (Austria) as a crystalline powder and a stock solution prepared in sterile H2O at a concen­tration of 1 mg/ml. The final cisplatin solu­tion was freshly prepared in 0.9% NaCl each day. Cisplatin at a dose of 4 mg/kg in 0.2 ml volume was injected intravenously. Electric pulses were delivered by two flat, parallel stainless steel electrodes 8 mm apart (two stainless steel strips: length 35 mm, width 7 mm with rounded corners), which Seräa G et al. / Tumor blood flow modifying effects of electrochemotherapy were placed percutaneously at the opposite margins of the tumor. Good contact between the electrodes and the skin was assured by means of conductive gel (Parker Laboratories, New York, USA). Eight square-wave pulses of 1040 V amplitude (amplitude to distance ratio 1300 V/cm), with a pulse width of 100 m s and repetition frequency of 1 Hz were generated by electroporator Jouan GHT 1287 (Saint Herblaine, France). In the electrochemothera­py protocol, tumors were exposed to electric pulses 3 minutes after bleomycin or cisplatin injection. Treatments were performed with­out anesthesia and were well tolerated by the animals. Tumor growth was followed by measuring three mutually orthogonal tumor diameters (e1, e2 and e3) using a vernier caliper on each consecutive day following treatment. Tumor volumes were calculated by the formula P x e1 x e2 x e3 /6. From the calculated volumes the arithmetic mean and SE were calculated for each experimental group. Tumor growth delay was calculated for each individual tu­mor by subtracting the doubling time of each tumor from the mean doubling time of the control group and then averaged for each ex­perimental group. Assessment of tumor staining by Patent blue Patent blue (Byk Gulden, Switzerland) was used to estimate tumor perfusion. Patent blue (1.25%), diluted in 0.2 ml 0.9% NaCl, was in­jected into tail vein of animals after tumor treatment. The dye was distributed evenly through the blood at approximately 1 minute, thereafter animals were sacrificed and tu­mors were carefully dissected. Tumors were cut along their largest diameter and the stained versus non-stained tissue per cross-section was immediately estimated visually by two persons. The mean of both estima­tions was used as an indicator of tumor per­fusion. The results of individual experiments were pooled and presented as arithmetic mean and SE for each experimental group. Cytotoxicity assay for SA-1 and HMEC-1 cells treated by electrochemotherapy The sensitivity of the SA-1 and HMEC-1 cells to combined treatment with bleomycin or cis­platin and electric pulses (electrochemothera­py) was determined by in vitro colony forming assay. The cells (2.2 x 107 cells/ml) were mixed with bleomycin or cisplatin. One half of the mixture was exposed to 8 electric puls­es (electric field intensity 1400 V/cm, pulse duration 100 m s, frequency 1 Hz) and the oth­er half served as a control for bleomycin or cisplatin treatment alone. The bleomycin con­centrations used ranged from 0.1 nM to 100 m M and the cisplatin concentrations from 16.7 to 670 m M. The cells were incubated with each drug for 5 min. The survival of cells treated with electrochemotherapy was nor­malized to electric pulses treatment alone. The IC50 values (drug concentration that re­duced cell survival to 50% of control) were de­termined for each treatment group. Statistical analysis The significance of differences between the mean values of the groups was evaluated by modified t-test (Newman Keuls test) after a one way analysis of variance was performed and fulfilled. Sigma Stat statistical package (SPSS, USA) was used for statistical analysis. P levels less than 0.05 were taken as statisti­cally significant. Results Antitumor effectiveness Electrochemotherapy with either bleomycin or cisplatin was effective in inducing cytotox­icity in subcutaneous SA-1 tumors (Table 1). Treatment of tumors with electric pulses alone had a minor effect on tumor growth, re­sulting in only 1.3 days tumor growth delay. Treatment of mice with bleomycin or cis­platin alone had also minor effects on tumor Radiol Oncol 2003; 37(1): 43-8. Seräa G et al. / Tumor blood flow modifying effects of electrochemotherapy Table 1. Antitumor effectiveness of electrochemotherapy on SA-1 tumors in mice (* P<0.05) Therapy n Tumor doubling time Tumor growth delay Cures (Days, AM± SE) (Days, AM± SE) Control 20 1.8 ± 0.05 0 % Electric pulses 17 3.1 ± 0.2* 1.3 ± 0.2 0 % Bleomycin (5 mg/kg) 20 1.9 ± 0.1 0.1 ± 0.1 0 % Electrochemotherapy with bleomycin 17 34.5 ± 2.9* 32.7 ± 2.9 70 % Cisplatin (4 mg/kg) 10 3.7 ± 0.4* 1.9 ± 0.4 0 % Electrochemotherapy with cisplatin 10 12.1 ± 1.6* 10.3 ± 1.6 0 % growth; bleomycin having none, whereas cis­platin inducing 1.9 days tumor growth delay. When using bleomycin in electrochemothera­py, a highly significant growth delay of 32.7 days was achieved and 70% of the animals were cured (tumor free 100 days after the treatment). The animals tolerated the treat­ment well without scaring of the treatment area. Electrochemotherapy with cisplatin also resulted in good antitumor effect with reduc­tion in tumor size at three days after the treat­ment, regrowth after 8 days, however no tu­mor cures were achieved. Tumor growth de­lay was 10.3 days, which was highly signifi­cant compared to the antitumor effectiveness of either single modality. Figure 1. Changes in tumor blood flow at 1, 8 or 24 h after electrochemotherapy (ECT) with bleomycin (BLM) or cisplatin (CDDP), measured by Patent blue staining. Eight electric pulses (EP) were applied to the tumor (amplitude to distance ratio 1300 V/cm, pulse width 100 µs, repetition frequency 1 Hz) 3 minutes af­ter intravenous injection of 5 mg/kg of bleomycin or 4 mg/kg of cisplatin. Mean values ± SE of the mean of at least 6 mice per point. Tumor blood flow changes Electrochemotherapy, either with bleomy­cin or cisplatin, induced substantial reduc­tion of tumor blood flow. Untreated SA-1 tu­mors showed very low incidence of necrosis with approx. 90% of the tumor area stained with Patent blue. When electric pulses were applied to a tumor reduction in tumor stain­ing was observed (Figure 1). By 1 hour after the application of electric pulses the percent­age of unstained tumor section had increased to 45% and after 8 hours further increased to 65%, however tumor blood flow recovered al­most completely within 24 hours after this treatment. Treatment with bleomycin alone did not induce changes in tumor blood flow. However, electrochemotherapy with bleomy­cin demonstrated substantial increase in un­stained tumor area at 8 hours after treatment, and virtually complete shut down of tumor perfusion at 24 hours after therapy compared to electric pulses alone (Figure 1). Treatment with cisplatin alone had mini­mal tumor blood flow modifying effect. However, electrochemotherapy with cisplatin demonstrated greatly increased unstained tu­mor area at 8 hours after treatment which re­mained significantly higher at 24 hours after the treatment compared to treatment with electric pulses alone (Figure 1). Cytotoxicity of electrochemotherapy to tumor and endothelial cells The sensitivity of SA-1tumor cells and human endothelial cells HMEC-1 to bleomycin and Seräa G et al. / Tumor blood flow modifying effects of electrochemotherapy cisplatin as well as to electrochemotherapy was evaluated by in vitro colony forming as­say (Table 2). Endothelial cells were more sensitive to bleomycin than tumor cells. The potentiation of bleomycin cytotoxicity by electroporation was ~5000-fold for endothe­lial cells and ~2400-fold for tumor cells. Electrochemotherapy with cisplatin was less effective on endothelial as on tumor cells, but potentiation of cisplatin cytotoxicity by elec­troporation was bigger for endothelial cells (~10-fold), as for tumor cells (~8-fold). Discussion This study shows tumor blood flow modify­ing and vascular targeted effect of elec­trochemotherapy with bleomycin as well as with cisplatin. The sensitivity of endothelial cells to electrochemotherapy with either, bleomycin or cisplatin correlates well with the enhanced reduction of tumor blood flow induced by electrochemotherapy in vivo and its antitumor effectiveness. As many preclinical and clinical studies have shown, electrochemotherapy either with bleomycin or cisplatin leads to high percent­age of tumor cures, on many tumor types test­ed so far.1,3,4,9,10 Electroporation was shown to significantly increase drug accumulation in the tumor cells.1,11 In electrochemotherapy treated tumors more than twice the amount of platinum was determined in whole tumors, Table 2. Cytotoxicity of electrochemotherapy to hu­man endothelial HMEC-1 and mouse tumor SA-1 cells in vitro Cell line / Group HMEC-1 SA-1 (IC50; m M) (IC50; m M) Bleomycin 20.0 60.0 Electrochemotherapy with bleomycin 0.004 0.025 Cisplatin 380.0 166.0 Electrochemotherapy with cisplatin 40.0 20.0 as well as bound to DNA compared with cis­platin treatment alone.11 In view of our previ­ous study observing that electrochemothera­py with cisplatin induced more than 20-fold increase in cell kill compared with cisplatin treatment alone, we proposed that, in addi­tion to direct electroporation of tumor cells, other mechanisms may be involved in antitu­mor effectiveness of electrochemotherapy.11 The direct blood flow modifying effect of electric pulses applied to the tumors has now been established. Application of electric puls­es reduces blood flow selectively at the site of its application, i.e. within the tumor site, with­out modifying flow in normal tissues.5 Recently, a new method by staining of tumors with Patent blue was evaluated, giving data on tumor blood flow, in support of that found using the 86RbCl extraction technique.12 Sin­ce the two methods correlated well, Patent blue staining technique was preferred in this study, because of its simplicity. The present study confirms the results of our previous study that application of electric pulses to the tumors induces transient reduction in tumor blood flow. Tumor blood flow modifying effect of elec­trochemotherapy was greater than after ap­plication of electric pulses alone. This effect was especially dramatic in electrochemother­apy with bleomycin, but in lesser extent after electrochemotherapy with cisplatin. Tumor blood flow after electrochemotherapy with bleomycin was completely shut down already at 24 hours after therapy, indicating that tu­mor vasculature was irreversibly damaged.12 Since HMEC-1 endothelial cells were more sensitive to electrochemotherapy with bleo­mycin in vitro than SA-1 tumor cells, this vas­cular shut down may be ascribed in large part to the death of endothelial cells. In contrast, endothelial cells were less sensitive to elec­trochemotherapy with cisplatin in vitro, which was also reflected in less severe tumor blood flow changes induced by this therapy with flow partly restored after 24 hours.13 Radiol Oncol 2003; 37(1): 43-8. Seräa G et al. / Tumor blood flow modifying effects of electrochemotherapy In summary, several mechanisms are in­volved in antitumor effectiveness of elec­trochemotherapy. Electroporation of the tu­mors increases delivery of cytotoxic drugs in­to the tumor cells, potentiating their cytotoxi-city. Additionally, the current study demon­strates that nonselective electroporation of solid tumors enables cytotoxic action of elec­trochemotherapy to endothelial cells and en­hanced tumor cytotoxicity by a vascular tar­geted mechanism. Acknowledgment This work was supported by the Ministry of Education, Science and Sport of the Republic of Slovenia. References 1. Mir LM, Orlowski S. Mechanisms of elec­trochemotherapy. Adv Drug Deliver Rev 1999; 35: 107-18. 2. Seräa G, ›emaìar M, Miklav‰i‰ D. Antitumor ef­fectiveness of electrochemotherapy with cis-di­amminedichloroplatinum(II) in mice. Cancer Res 1995; 55: 3450-5. 3. Heller R, Gilbert R, Jaroszeski MJ. Clinical appli­cation of electrochemotherapy. Adv Drug Deliver 1999; 35: 119-29. 4. Seräa G, átabuc B, ›emaìar M, Miklav‰i‰ D, Rudolf Z. Electrochemotherapy with cisplatin: clinical experience in malignant melanoma pa­tients. Clin Cancer Res 2000; 6: 863-7. 5. Seräa G, ›emaìar M, Parkins CS, Chaplin DJ. Tumour blood flow changes induced by applica­tion of electric pulses. Eur J Cancer 1999; 35: 672-7. 6. Gehl J, Skovsgaard T, Mir LM. Vascular reactions to in vivo electroporation: characterization and consequences for drug and gene delivery. Biochim Biophys Acta 2002; 1569: 51-8. 7. Neumann E, Kakorin S. Digression on membrane electroporation and electroporative delivery of drugs and genes. Radiol Oncol 1998; 32: 7-17. 8. Chaplin DJ, Hill SA, Bell KM, Tozer GM. Modification of tumor blood flow: Current status and future direction. Sem Radiat Oncol 1998; 8: 151-63. 9. Mir LM, Glass LF, Seräa G, Teissie J, Domenge C, Miklav‰i‰ D, Jaroszeski MJ, Orlowski S, Reintgen DS, Rudolf Z, Belehradek M, Gilbert R, Rols MP, Belehradek JJr, Bachaud JM, DeConti R, átabuc B, ›emaìar M, Coninx P, Heller R. Effective treat­ment of cutaneous and subcutaneous malignant tumours by electrochemotherapy. Br J Cancer 1998; 77: 2336-42. 10. Seräa G. Electrochemotherapy: Animal work re­view. In: Jaroszeski MJ, Heller R, Gilbert R, edi­tors. Electrochemotherapy, electrogenetherapy and transdermal drug delivery. Electrically mediated deliv­ery of molecules to cells. Totowa: Humana Press; 2000. p. 119-36. 11. ›emaìar M, Miklav‰i‰ D, á‰an‰ar J, Dolìan V, Golouh R, Seräa G. Increased platinum accumula­tion in SA-1 tumour cells after in vivo elec­trochemotherapy with cisplatin. Br J Cancer 1999; 79: 1386-91. 12. Seräa G, ›emaìar M, Miklav‰i‰ D, Chaplin DJ. Tumor blood flow modifying effect of elec­trochemotherapy with bleomycin. Anticancer Res 1999; 19 (5B): 4017-22. 13. ›emaìar M, Parkins CS, Holder AL, Chaplin DJ, Tozer GM, Seräa G. Electroporation of human mi-crovascular endothelial cells: evidence for an anti-vascular mechanism of electrochemotherapy. Br J Cancer 2001; 84: 565-70.   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