ADIOLOGY 11111 NCOLOGY December 2006 ol. 40 No. 4 jubljana ISSN 1318-2099 Editorial office RadiologtJ and Oncologtj December 2006 Institute of Oncology Vol. 40 No. 4 Zaloška 2 Pages 205-288 SI-1000 Ljubljana ISSN 1318-2099 Slovenia UDC 616-006 Phone: +386 1 5879 369 CODEN: RONCEM Phone/Fax: +386 1 5879 434 E-mail: gsersa@onko-i.si Aims and scope Radiology and OncologrJ is a journal devoted to publication of original contributions in diagnostic and interventional radiology, computerized tomography, u/trasound, magnetic resonance, nuclear medicine, radiotherapy, clinical and experimental oncology, radiobiologrj, radiophysics and radiation protection. Editor-in-Chief Editor-in-Chief Emeritus Gregor Serša Tomaž Benulic Ljubljana, S/ovenia Ljubljana, Slovenia Executive Editor Editor Viljem Kovac Uroš Smrdel Ljubljana, Slovenia Ljubljana, S/ovenia Editorial board Marija Auersperg Valentin Fidler MajaOsmak Ljubljana, Slovenia Ljubljana, S/ovenia Zagreb, Croatia Nada Bešenski Be?a Fornet Branko Palcic Zagreb, Croatia Budapest, Hungary Vancouver, Canada Karl H. Bohuslavizki Tullio Giraldi ]urica Papa Hamburg, Germany Trieste, Italy Zagreb, Croatia Haris Boko Andrija Hebrang Dušan Pavcnik Zagreb, Croatia Zagreb, Croatia Portland, USA Nataša V. Budihna Ltiszl6 Horvtith Stojan Plesnicar Ljubljana, S/ovenia Pecs, Hungary Ljubljana, S/ovenia Marjan Budihna Berta Jereb Ervin B. Podgoršak Ljubljana, Slovenia Ljubljana, Slovenia Montreal, Canada Malte Clausen Vladimir Jevtic Jan C. Roos Hamburg, Germany Ljubljana, Slovenia Amsterdam, Netherlands Christoph Clemm H. Dieter Kogelnik Slavko Šimunic Miinchen, Gennany Salzburg, Austria ZagrebCroatia Mario Corsi Jurij Lindtner ,_, Lojze Smid Udine, Italy Ljubljana, Slovenia Ljubljm.a,Slovenia Ljubomir Diankov Ivan Lovasic Borut Stabuc Sofia, Bulgaria Rijeka, Croatia Ljubljana, S/ovenia Christian Dittrich Marijan Lovrencic Andrea Veronesi Vienna, Austria Zagreb, Croatia Aviano, Italy Ivan Drinkovic LukaMilas iiva Zupancic Zagreb, Croatia Houston, USA Ljubij'ana, S/ovenia Gillian Duchesne Metka Milcinski Melbourne, Australia Ljubljana, Slovenia Publisher Association of Radiology and OncologtJ Affiliated with Slovenian Medica/ Association -Slovenian Association of Radiology, Nuclear Medicine Society, Slovenian Society far Radiotherapy and Oncology, and Slovenian Cancer Society Croatian Medica/ Association -Croatian Society of Radiologij Societas Radiologorum Hzmgarorum Friuli-Venezia Giulia regional groups of S.I.R.M. (Italian Society of Medica/ Radiology) Copyright © Radiology and OncologtJ. Ali rights reserved. Reader for English Vida Kološa Key words Eva Klemencic Secretary Mira Klemencic Design Monika Fink-Serša Printed by Imprint d.o.o., Ljubljana, Slovenia Published quarterly in 700 copies Beneficiary name: DRUŠTVO RADIOLOGIJE IN ONKOLOGIJE Zaloška cesta 2, 1000 Ljubljana Slovenia Beneficiary bank account number: SI56 02010-0090006751 JEAN: SI56020100090006751 Our bank name: Nova Ljubljanska banka, d.d., Ljubljana, Trg republike 2, 1520 Ljubljana; Slovenia SWIFT: LJBASI2X Subscription fee far institutions EUR 100 (20.000 SIT), individuals EUR 50 (5000 SIT) The publication of this journal is subsidized by the Slovenian Research Agency. Indexed and abstracted by: BIOMEDICINA SLOVENICA CHEMICAL ABSTRACTS EMBASE / Excerpta Medica Sci Base Scopus This journal is printed on acid-free paper Radiology and Oncology is available on the internet at: http://www.onko-i.si/radioloncol ISSN 1581-3207 Ljubljana, Slovenia ISSN 1318-2099 December 2006 UDC 616-006 Vol. 40 No. 4 CODEN: RONCEM CONTENTS RADIOLOGY AND SONOGRAPHY Intracapsular and para-articular chondroma of knee: a report of four cases and review of the literature 205 Samardziski M, Foteva M, Adamov A, Zafiroski G Doppler ultrasound in the diagnosis and follow-up of the muscle rupture and an arteriovenous fistula of the thigh in 12 year boy 211 Pavcec Z, Žokalj I, Saghir H, Pal A, Roic G IMAGING IN CLINICAL MEDICINE Carcinoma of the lower lip Jancar B ONCOLOGY 217 Tumor vaccines 219 Frank M, Ihan A Preoperative concomitant chemoradiotherapy in esophageal cancer 231 Šeruga B, Sok M, Eržen J, Jerman], Jancar B, Zakotnik B Body mass index and lung cancer risk in never smokers Kagohashi K, Satoh H, Kurishima K, Ishikawa H, Ohtsuka M Schedule-dependency of Doxorubicin and Vinblastine in EAT tumours in mice 245 Auersperg M, Pogacnik A, Kloboves-Prevodnik V, Serša G, Cemažar M Cysteine cathepsins, stefins and extracellular matrix degradation during invasion of transformed human breast cell lines 259 Zajc I, Bervar A, Lah IT SLOVENIAN ABSTRACTS 273 NOTICES 281 AUTHORS INDEX 2006 SUBJECT INDEX 2006 Radiology and Oncology is covered in Biomedicina Slovenica, Chemical Abstracts, EMBASE / Excerpta Medica, Sci Base and Scopus Radiol Oncol 2006; 40(4): 205-9. Intracapsular and para- articular chondroma of knee: a report of four cases and review of the literature Milan Samardziski, Marta Foteva, Aleksandar Adamov, George Zafiroski University Clinic for Orthopaedic Surgery, Skopje, Macedonia Background. Intracapsular and para-articular chondroma is a rare variant of the extraskeletal chondromas. It arises from the capsule and/or the para-articular connective tissue of the large joints (mostly the knee) and is a result of cartilaginous metaplasia. In course of time these tumors ossify and this is where their second name comes from: Para-articular osteochondromas. According to Jaffe, not dependent on the degree of ossification of this tumor, there is one single entity in question. Cases report. We report four new cases of para-articular chondroma of the knee. On physical examination there was slow-growing solid mass in the knee and moderate pain, the radiological findings and CT scan show soft-tissue mass with variable amount of ossification, and on histological examination the presence of mature hyaline and connective cartilage was confirmed in all of the cases. Conclusions. The diagnosis of these benign tumors is made with correlation of clinical, radiological and histological features. The treatment of choice is surgical excision in toto. Key words: chondroma, osteochondroma, knee Introduction Extraskeletal chondromas are benign tu­mours which appear in three variants: synovial chondromatosis, para-articular chondroma and soft tissue chondroma. The first type is very common, but the last two variants are quite rare and they may show atypical features.1-3 Received 23 October 2006 Accepted 29 November 2006 Correspondence to: Milan Samardziski, MD, MSc, Clinic for Orthopaedic Surgery, Vodnjanska 17, 1000 Skopje, Macedonia; Phone +389 2 314 7626; Fax: +389 2 3165 137; E-mail: milan_ samardziski@yahoo.com or milansamardziski@gmail.com The fibrous coat of the capsule of a joint and/or the para-articular connective tissue, very rare, can suffer cartilaginous meta-plasia. As an end result of this metaplasia, intracapsular or para-articular chondromas are formed. In time, they usually ossify so they are also known as capsular and para- articular osteochondromas. Mostly seen in the large joints (the knee), they vary in size depending on the size of the joint.4,5 We have found only 30 cases of para-articular chondromas in the reviewed literature.3,6,7 We report four new cases of capsular and para-articular chondroma of the knee with their clinical, radiological and histological features. Case reports Case 1 A male patient, 24, reports with painful mass on the medial side of the right knee, with no record of trauma. He first noticed it one year prior to the examination. On physical examination the timorous forma­tion is movable, tender on palpation, pro-duces pain during active motion. The pro­file X-ray shows para- and supra-patellar soft tissue tumorous formation (Figure 1a). On CT- scan this soft-tissue mass is clearly seen, oval shaped and intracapsular (Figure 1b). The tumour was surgically excised. During the operation, the intracapsular but extrasynovial position of the tumour was confirmed. It was oval shaped, 8 x 5 x 2.5 cm. The histological examination showed mature hyaline cartilage with foci of mixomatous tissue with benign charac­teristics. The diagnosis was: intracapsular chondroma without ossification. Case 2 A female patient, age 41 complains of a solid mass on the lateral aspect of the left knee that has been slowly growing for the past two years. It caused limitation of joint movement and required surgical removal. The lateral radiograph of the left knee showed infrapatellar ossified mass (Figure 2a), whilst the CT- scan showed mostly ossified, encapsulated tumor just Radiol Oncol 2006; 40(4): 205-9. beneath the lateral border of the patella, but not attached to it (Figure 2b). The surgically excised mass was oval, 3 x 3.5 x 2.5 cm (Figure 2c), situated in the con­tinuity with the capsule of the joint, but extrasynovial. Macroscopically, on cross section there is a central zone of mature trabecular bone, surrounded by a hyaline cartilage cup (Figure 2c). On histological examination there was mature trabecular bone surrounded by hyaline cartilage with endochondral ossification. The diagnosis was: intracapsular chondroma with high rate of ossification. Case 3 A female patient aged 72 was admitted after a mild trauma of the left knee. Physical exami­nation showed painful mass under the patella and limited flexion and extension of the knee which were present for more than 10 years. The recent trauma of the left knee caused pain and swelling of the knee. The lateral radio-graph of the knee showed subpatellar, partly ossified mass (Figure 3a) and the transverse section on CT-Scan showed posttraumatic haematoma in the knee joint, as well as soft tissue tumour with ossification beneath the patella, situated in the para-articular connec­tive tissue (Figure 3b). The diagnosis was: para-articular chondroma of the knee with ossification. Case 4 A female patient aged 56 complained of intense pain and lack of extension in her right knee. There was no history of trauma, but she could remember heavy activities after which the progressive restriction of Radiol Oncol 2006; 40(4): 205-9. the extension and pain started. History of the patient showed that she had slightly painful, slowly growing lump seated be­neath and laterally of the patella for more than 30 years (Figure 4a). After the surgical extirpation of the para-articular chondroma (Figure 4b), full range of painless motion was regained. Discussion Para-articular and intracapsular chondro-mas are rare benign tumours mostly seen in the vicinity of the large joints. They were often named capsular osteoma, osteochon­droma or chondroma depending on the pro­portion of bone and cartilage.8 According to Jaffe, there is only one single lesion in question regardless of the ossification, and in 1958 he classified all these terms under one entity: intracapsular and para-articular chondroma.6 The Pathogenesis of these tumours is also controversial. They most likely originate from the connective tissue in the vicinity of the capsule of a joint or from the outer coat of the capsule as a result of cartilaginous metaplasia. Prior trauma is unlikely to play any significant role in the pathogenesis of these tumours. In the beginning comprising exclusively of cartilaginous tissue, in the course of time they usually ossify.3,4 This is where their “second name” used in the literature comes from: osteochondromas. From the relatively small number of re­ported cases we can conclude that, although Radiol Oncol 2006; 40(4): 205-9. there have been cases in the ankle, elbow and the hip joint,3,5 they are mostly seen in the knee joint.4,7,8 The location is para-articular and intracapsular, mostly infrapa­tellar or medial to the patella. The reported age varies from 12 to 75 years. The clinical complaints are of some months to several years of local discomfort, moderate pain, slow growing mass and some degree of limited motion in the joint. Radiologically, there is a soft tissue mass with a different degree of central radiodensity due to ossi­fication. Macroscopically their size varies, depending on the size of the involved joint, from 2 to 10 cm. The four cases we report have all the fea­tures of the previously reported chondro-mas found in the literature. Clinically they present with moderate pain and restricted range of motion in the involved knee joint. On plain radiographs, there was a soft tissue mass with a different degree of os­sification while the CT- scan has enabled us to make a more detailed analysis as to the exact position of the tumour (intracapsular or extracapsular), its relationship with the adjacent structures, its size and structure. In all of our cases, the tumours were int-racapsular, but with no direct contact with the joint. Grossly, they were large, and the pathological analysis confirmed the presence of hyaline cartilage with variable amounts of mature trabecular bone. Using the definition of Jaffe, the diagnosis in all of the four cases was: para-articular chon­droma. The treatment in all of the cases was surgical excision. The diagnosis of these benign tumours is made clinically and radiologically in correlation with the pathological features. Although rarely seen, they should be con­sidered in the differential diagnosis of soft tissue masses around the joints: haemath­oma, bursitis, periosteal chondroma, syno-vial sarcoma, synovial chondrosarcoma. The treatment of choice for these tumours is surgical excision, while being careful not to injure the joint integrity. Malignant transformation has never been reported. With correct diagnosis unnecessary aggres­sive surgical treatment will be avoided. References 1. Chung EB, Enzinger FM. Chondroma of soft parts. Cancer 1978; 41: 1414-24. 2. Hagan RF, Schoneker PL. Para-articular osteo­chondroma. Am J Orthop 1995; 24: 65-7. 3. Gayle EL, Morrison WB, Carino JA, Parsons TW, Liang CY, Stevenson A. Extrasceletal osteochon­droma of the foot. Skeletal Radiol 1999; 28: 594-8. 4. Gonzales-Lois C, Garcia-de-la-Torre JP, SantosBriz-Terron A, Vila J, Manrique-Chico J, Martinez-Tello FJ. Intracapsular and para-articular chondroma adjacent to large joints: report of three cases and review of the literature. Skeletal Radiol 2001; 30: 672-6. 5. Steiner GC, Meushar N, Norman A, Present D. Intracapsular and para-articular chondromas. Clin Orthop 1994; 303: 231-6. 6. Jaffe HL. Intracapsular and para-articular chondro-ma. In: Henry L, Jaffe, editors. Tumors and tumor­ous conditions of the bones and joints. Philadelphia: Lea & Febiger; 1958. p. 567-9. 7. Reith J.D, Bauer T.W, Joyce M.J. Para-articular osteochondromas of the knee: report of two cases and review of the Lliterature. Clin Orthop 1997; 334: 225-32. 8. Sakai H, Tamai K, Iwamoto A, Saotome K. Para-articular chondroma and osteochondroma of the infrapatellar fat pad: a report of three cases. Int Orthop 1999; 23: 114-7. Radiol Oncol 2006; 40(4): 205-9. Radiol Oncol 2006; 40(4): 211-5. case repor t Doppler ultrasound in the diagnosis and follow-up of the muscle rupture and an arteriovenous fistula of the thigh in 12 year boy Zlatko Pavcec1, Ivan Žokalj1, Hussein Saghir1, Andrej Pal1, Goran Roic2 1Department of Radiology and Ultrasound, Cakovec County Hospital, Cakovec 2Department of Pediatric Radiology, Children's Hospital, Zagreb, Croatia Background. With this case report the authors wish to present the accuracy of non-invasive vascular imag­ing methods, especially Doppler ultrasound, in the evaluation of the muscular trauma and periskeletal soft tissue vascular anomalies. Case report. Twelve year-old boy has been admitted with the right femoral quadriceps muscle traumatic rupture. Postoperative B-mod sonography (US) visualised recidivuous haematoma and Power Doppler de­picted hypervascularized area, suspected vascular malformation (angioma). Doppler findings obtained on the right thigh vasculature gave us reasons to think about posttraumatic arteriovenous fistula. Doppler has been repeated in the specialized paediatric institution with the same results. Digital subtraction angiography, 8 months after trauma, did not confirm suspicions reported in US findings. Spiral computed tomographic angiography (CTA) performed 11 months after trauma clearly depicted a lesion which had been repeatedly described in US findings. Fourteen months after trauma the vascular surgeon performed the deep femoral artery muscular branches ligation, but in the official report only arteriovenous fistula was mentioned. After the surgery the patient was clinically better. The aetiology of the right femoral arteriovenous fistula and hypervascularized structure remains unclear. Conclusions. Every inadequately behaving, recidivous posttraumatic haematoma should be evaluated with Doppler ultrasound. CTA can be performed if it is needed to clarify US findings. Key words: hematoma – ultrasonography; arteriovenous fistula; muscle, skeletal - injuries Received 24 November 2006 Accepted 2 December 2006 Correspondence to: Ivan Žokalj, MD, Department of Radiology and Ultrasound, Cakovec County Hospital, Ivana Gorana Kovacica 1e, 40 000 Cakovec, Croatia; Phone: + 385 40 375 297; Fax: + 385 40 313 325; E-mail: ivan.zokalj@ck.t-com.hr Introduction Soft tissue injury and its complications can be accurately evaluated with ultra-sonography (US) because this method has possibilities of multiplanar approach, the dynamic examination of muscle during the contraction and rest and assessment of the potential concomitant vascular injury with Doppler modalities.1-4 US doesn't carry danger of ionizing radiation and it is widely available; these facts make US the imaging method of choice for the diagnostic evalua­tion of muscular injuries and first method for vascular injury diagnosing. A muscle rupture of the lower limbs with consequent haematomas is often relat­ed with a sports injury. Haematoma is the most important sign of a muscle rupture, it is usually depicted as a hypo- or anechoic circumscribed lesion1-3 an arteriovenous (AV) fistula is an abnormal communication between the arterial and venous systems. AV fistulas of the extremities are the conse­quence of trauma or medical procedures in most of the cases. Post-traumatic AV fistula is usually the consequence of penetrating trauma, very rarely after blunt trauma.5-7 Non-invasive imaging diagnostic meth­ods, such as US, computed tomography (CT) and magnetic resonance imaging (MRI) have big potentials for the safe and even quick assessment of vascular anomalies and traumatic vascular lesions.8 Doppler ultrasound methods can give the majority of necessary information about traumatic vascular lesions and vascular anomalies, especially if US is combined with another vascular imaging method, such as computed tomographic angiogra­phy (CTA) and magnetic resonance angiog­raphy (MRA).3,4,9-12 Case report Twelve year-old boy was admitted with signs of the right femoral quadriceps mus­cle traumatic rupture caused by sudden extension during the football match, six months after trauma actually happened. B-mod US, performed before the surgi­cal intervention showed the right femoral quadriceps muscle rupture with haemato-ma. First postoperative US included B-mod and Doppler modalities (colour and power Doppler). B-mod US depicted recidivous haematoma. With Doppler methods high flow and high systolic peak values were revealed in the popliteal, superficial and deep femoral vein, the AV communication was suspected (Figure 1) Hypervascularized area with sonograph­ic characteristics of vascular malforma­tion (haemangioma) was depicted near the haematoma, ventrolateraly in the proximal third of the thigh (Figure 2). Recidivous haematoma was evacuated by punction after the US control. Clinically, the patient had bigger diameter of all parts of the right leg and oedema ventrolateraly in the right femoral region, but without a thrill and bruit over the site of the muscle injury. Radiol Oncol 2006; 40(4): 211-5. The patient has been sent into the paedi­atric hospital to clarify the suspicion of the post-traumatic AV communication (fistula) and the right thigh vascular malformation. Control US and axial CT scan showed again recidivous haematoma and right femoral hypervascularized structure. The digital subtraction angiography (DSA) indicated by the paediatric surgeon and performed 8 months after trauma, also depicted neither AV fistula nor vascular malformation. The repeated Doppler US, performed 10 and 11 months after trauma, showed a higher flow with high peak systolic values only in the deep femoral vein and the reduc­tion of the right femoral hypervascularized structure size. The spiral CT scan, per­formed 11 months after trauma, depicted a hypervascularized lesion supplied from the deep femoral artery muscular branches posi­tioned ventrolateraly in the right thigh proxi­mal third. The hypervascularized lesion was equally good opacified with contrast mate­rial in the arterial and venous phase, with one avascular zone ventromedially (Figures 3, 4). The patient still had swollen right leg but there was no palpable mass in the area of vascular malformation described in the CT report with thrill and bruit over them. MIP- right femoral region –axial plane (horizontal orientation). Hypervascularized lesion located ventrolaterally in right thigh proximal third, equally good opacified with contrast material in the arterial and venous phase, with one avascular zone ventromedially. A lesion receives arterial supply from the deep femoral artery muscular branches. The vascular surgeon performed the deep femoral artery muscular branches ligation, 14 months after trauma. In the surgery report only AV fistula was men­tioned. After the surgery clinical manifes­tations and AV fistula ultrasonographic signs disappeared. This was an indirect confirmation of posttraumatic AV com­munication, which had not been supported with the digital subtraction angiography. The control US, performed two years after the surgical intervention showed neither haematoma nor AV communications, and the patient was clinically better without right leg oedema. Discussion Ultrasonography is a standard diagnostic method for the evaluation of soft-tissue structures trauma. Doppler ultrasound vas- Radiol Oncol 2006; 40(4): 211-5. cular imaging is routinely included in the assessment of suspected vascular trauma. Duplex ultrasonography has sensitivity 95%, the specifity 99% and 98% accuracy in the assessment of peripheral vascular injuries, even 100% sensitivity and specifity compared with the conventional arteriogra­phy and operative exploration by Fry and colleagues 1994.4 Doppler vascular imaging can help to detect the origin and pattern of vascular supply and the degree of blood flow in periskeletal soft tissue masses.3,4 The combination of B-mod and Doppler sonography has 90% sensitivity and 91% specifitiy and 91% accuracy in the evalua­tion of musculoskeletal masses.9 Soft tissue vascular masses can be distinguished with these characteristics: morphostructural fea­tures, the presence of colour or power sig­nals, the site of vascular branches, their cal-ibre and course, the number of afferent vas­cular poles, resistance index, vessel density and peak flow velocities.10,11 Haemangioma and AV malformation have higher vessel density than other vascular malformations. There is no statistically significant differ­ence between haemangioma and vascular malformation in vessel density and mean peak velocity. Solid-tissue mass is the fac­tor for differentiation between haemangi­oma and vascular malformation.11,12 AV fistula clinical manifestations in the extremities usually are swelling of the in­jured limb, a thrill and bruit over the site of injury, but if the thrombus has occluded the AV communication the appearance of these signs will be delayed. The severity of AV fistula clinical manifestation can vary from local changes, as it was in this case, till the venous hypertension and congestive heart failure.5,6 In the case reported in this article the pa­tient had unrecognised AV fistula. The pen­etrating injury of the right thigh was denied by the patient. There were no characteristic clinical signs like bruit and thrill over the region where the AV fistula was situated, although the right leg was swollen. The ae­tiology of the AV fistula and hypervascular­ized structure near the femoral quadriceps muscle rupture remained unclear. Working hypothesis about vascular malformation injured by trauma was not confirmed with DSA and operative findings. To the authors' knowledge the differential diagnosis of posttraumatic bleeding of a congenital AV-malformation has not yet been reported. In this case of inadequately behaving posttraumatic haematoma, the correct diagnosis of an abnormal AV communi­cation, an AV fistula, was made on the non-invasive vascular imaging methods findings (Doppler and CT angiography) ground. DSA didn't depict a right thigh AV fistula the existence of which was indi­rectly confirmed with the disappearance of clinical signs after the deep femoral artery muscular branche ligation, an AV fistula feeding artery. The point is that every inad­equately behaving, recidivous posttrauma-tic haematoma should raise the suspicion of vascular injury, and must be evaluated with the vascular imaging methods. The facts presented in this case report support opinion that non-invasive vascular imaging methods like Doppler ultrasound and CT angiography can give enough information for diagnostic and therapeutic decisions and a follow-up after the treatment. Radiol Oncol 2006; 40(4): 211-5. References 1. Krolo I, Babic N, Marotti M, Klaric-Custovic R, Matejcic A, Hat J. Ultrasound in the evaluation of sports muscular injury. Acta Clin Croat 2000; 39: 15-9. 2. Peetrons P. Ultrasound of muscles. Eur Radiol 2002; 12: 35-43. 3. Bynoe RP, Miles WS, Bell RM, Greenwold DR, Sessions G, Haynes JL, et al. Noninvasive diagno­sis of vascular trauma by duplex ultrasonography. J Vasc Surg 1991; 14: 346-52. 4. Fry WR, Smith RS, Sayers DV, Henderson VJ, Morabito DJ, Tsoi EK, et al. The success of duplex ultrasonography scanning in diagnosis of extrem­ity vascular proximity trauma. Arch Surg 1994; 129: 669-70. 5. Khougeer G, Bayoumi Okda AH. Difficult femoral aretriovenous fistula in a child. Ann Saudi Med 2000; 20: 150-1. 6. Tayama K, Akashi H, Hiromatsu S, Okazaki T, Yokokura Y, Aoyagi S. Acquired arteriovenous fis­tula of the right forearm caused by repeated blunt trauma:a report of rare case. Ann Thorac Cardiovasc Surg 2005; 11: 59-62. 7. Bail HJ, Melcher I, Raschke MJ, Schroeder RJ, Schaser KD. Unknown AV-fistula as reason for post-traumatic hematoma of the thigh. Vasa 2003; 32: 108-10. 8. Bohndorf K, Kilcoyne RF. Traumatic injuries: im­aging of peripheral musculoskeletal injuries. Eur Radiol 2002; 12: 1605-16. 9. Belli P, Costantini M, Mirk P, Maresca G, Priolo F, Marano P. Role of color Doppler sonography in the assesment of musculoskeletal soft tissue masses. J Ultrasound Med 2001; 20: 587-95. 10. Taylor GA, Perlman EJ, Scherer LR, Gearhart JP, Leventhal BG, Wiley J. Vascularity of tumor sin children:evaluation with color Doppler imaging. AJR Am J Roentgenol 1991; 157: 1267-71. 11. Paltiel HJ, Burrows PE, Kozakewich HPW, Zurakowski D, Mulliken JB. Soft-Tissue Vascular Anomalies: Utility of US for Diagnosis. Radiology 2000; 214: 747-54. 12. Latifi HR, Siegel MJ. Color Doppler flow imaging of pediatric soft tissues masses. J Ultrasound Med 1994; 13: 165-9. Radiol Oncol 2006; 40(4): 211-5. Radiol Oncol 2006; 40(4): 217. Images in clinical medicine Carcinoma of the lower lip Boris Jancar Department of Radiation Oncology, Institute of Oncology, Ljubljana, Slovenia In 71-year-old patient, the squamous cell carcinoma extended over 2/3 of the lower lip (Figures 1, 2). Surgical treatment would apparently be too mutilating; the patient was therefore referred to radiotherapy and received tmour dose (TD) 40 Gy in 10 fractions in 2 weeks, ortovoltage machine (Pantak). The tumour regressed completely. A photo of the patient was taken two years after the completed radio­therapy. On the last follow-up control ten years after the completed radiotherapy, no evidence of local or regional recurrence of the carcinoma was observed. Figure 1. Huge carcinoma of the lower lip. Figure 2. Exofitic huge carcinoma of the lower lip. Received 15 November 2006 Accepted 22 November 2006 Correspondence to: Prim. Boris Jancar, MD, MS, Department of Radiation Oncology, Institute of Oncology, Zaloška 2, Ljubljana, Slovenia; Phone; + 386 1 5879 295; Fax: + 386 1 5879 295; E-mail: bojan­car@onko-i.si Radiol Oncol 2006; 40(4): 219-29. review Tumor vaccines Mojca Frank, Alojz Ihan Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Tumor vaccines have several potential advantages over standard anticancer regiments. They represent highly specific anticancer therapy. Inducing tumor-specific memory T-lymphocytes, they have potential for long-lived antitumor effects. However, clinical trials, in which cancer patients were vaccinated with tumor vaccines, have been so far mainly disappointing. There are many reasons for the inefficiency of tumor vac­cines. Most cancer antigens are normal self-molecules to which immune tolerance exists. That is why the population of tumor-specific lymphocytes is represented by a small number of low-affinity T-lymphocytes that induce weak antitumor immune response. Simultaneously, tumors evolve many mechanisms to actively evade immune system, what makes them poorly immunogenic or even tolerogenic. Novel immunotherapeu-tic strategies are directed toward breaking immune tolerance to tumor antigens, enhancing immunogenicity of tumor vaccines and overcoming mechanisms of tumor escape. There are several approaches, unfortu­nately, all of them still far away from an ideal tumor vaccine that would reject a tumor. Difficulties in the activation of antitumor immune response by tumor vaccines have led to the development of alternative immunotherapeutic strategies that directly focus on effector mechanisms of immune system (adoptive tu­mor-specific T-lymphocyte transfer and tumor specific monoclonal antibodies). Key words: cancer vaccines; antigens, neoplasms; immunotherapy Introduction Development of tumor vaccines is based on the researches that have shown that many tumors express tumor antigens and are able Received 29 September 2006 Accepted 19 October 2006 Correspondence to: Mojca Frank, Institute of Microbiology and Immunology, Zaloška 4, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia. Phone: +386 1 5437493; Fax: +386 1 5437401; E-mail address: mojca_frank@yahoo.com to elicit tumor-specific B- and T-lymphocyte responses. Tumor vaccines have several potential advantages over standard anti­cancer regimens. They are directed against tumor antigens and represent highly spe­cific anticancer therapy. Inducing tumor-specific memory T-lymphocytes, they have potential for long-lived antitumor effects. Side effects of tumor vaccines are rare, in most cases limited to local reactions with minimal systemic toxicity (transient elevated body temperature, flu-like symp­toms). Autoimmune reactions are also rare (vitiligo with melanoma vaccines). Tumor antigens Genetic and epigenetic changes character­istic of carcinogenesis make cancer cells antigenically distinct from normal human cells.1 Cancer cells express tumor-specific antigens and tumor-associated antigens. Tumor-specific antigens are ideal targets for antitumor therapy. They are protein prod­ucts of mutated normal cell genes and are expressed only by cancer cells. They are foreign to immune system and therefore elicit high-affinity antitumor T-lymphocyte responses with low probability of intercur-rent autoimmune reactions.1-4 Their main disadvantages are that they are highly heter­ogenic and expressed only by certain types of tumors, therefore they cannot be used as a universal antigen in a cancer vaccine. A special subgroup of tumor-specific antigens are idiotypic sequences of B-cell membrane immunoglobulins or T-cell receptor.5,6 Tumor-associated antigens. Most antigens expressed by tumor cells are normal, non-mutated self-molecules to which immune tolerance exists.3,7 There are several classes of tumor-associated antigens: tissue-specific antigens (PSA, melanocyte antigens), on-cofetal proteins (normally expressed only during fetal development, foreign to im­mune system, reactivated in undifferenti­ated tumors), cancer testes antigens (normal testicular proteins, overexpressed on can­cer cells, foreign to immune system - sper­matocytes do not express MHC molecules), overexpressed normal cell proteins (HER2 Neu in breast carcinoma) and self-proteins with abnormal posttranslational modifica­tions (overglycosilated mucins, as MUC1 in breast carcinoma, changes in glycosilation can expose kryptotopes, foreign to immune system).1,9,10 Viral oncoproteins (human papilloma vi­rus proteins E6 and E7) are a special group of tumor antigens, playing a critical role in malignant transformation of infected cells. Being foreign to a body, they can induce high-affinity T-lymphocyte responses.11,12 Antitumor immune response Tumor-specific cytotoxic T-lymphocytes are central effector cells of antitumor immune response. They are the only cells capable of efficiently killing cancer cells, induc­ing their apoptosis or lysing them by ac­tion of perforines and granyzimes. They are produced in cross-priming of naive CD8+ T-lymphocytes, mediated by mature dendritic cells (DC). The process is es­sential for induction of antitumor immune response and involves cross-presentation of antigenic peptides originating from ex­tracellular proteins through MHC I mol­ecules on the surface of DC to naive CD8+ T-lymphocyte.9,13 The pathway involves en-dosome to cytosol shuffling of antigenic peptides from extracellular proteins medi­ated by TAP transporters, and is a major pathway of cross-presentation under physi­ological conditions. Efficient cross-activation of naive CD8+ T-lymphocytes requires 3 types of cells, mature DC, naive CD8+ T-lymphocyte and helper T-cell, and 2 signals. Signal 1 (spe­cific antigenic signal) arises from interaction between antigenic peptide, presenting on dendritic cell MHC I molecule, and antigen specific T-cell receptor (TCR) of naive CD8+ T-lymphocyte; signal 2 (costimulatory signal) is mediated by costimulatory molecule B7 on the surface of DC and its CD28 receptor on naive CD8+ T-lymphocyte (two signal hypothesis).9,13 For activation of naive CD8+ T-lymphocyte to be effective, licensing of DC is necessary. It is mediated by an in­teraction between CD40 ligand (CD40L) of DC and its receptor CD40 of helper T-cell, specific for antigenic peptide presented on MHC II molecule of DC. Interaction CD40 - CD40L results in an upregulation of cos- Radiol Oncol 2006; 40(4): 219-29. timulatory molecules B7 on the surface of DC - DC licensing. B7 interact with CD28 receptor, providing a dominant costimula­tory signal for the activation of naive CD8+ T-lymphocyte. Immune tolerance for tumor antigens Existence of tumor-specific lymphocytes and antitumor antibodies before and after vaccination has been found in many cancer patients; however, there has been no correla­tion with clinical improvement so far.1 There are many reasons for inefficiency of tumor vaccines. Tumor vaccines contain mainly weakly immunogenic tumor-associated (self) antigens and elicit weak antitumor immune responses. Frequency of tumor-specific lym­phocyte precursors, that arise during tumor vaccination, is small (=1%) compared to the frequency of lymphocyte precursors (=10%) against the infectious agents arising during classic vaccination.8 Even more importantly, the population of potentially tumor-reactive lymphocytes is represented by low-affinity T-lymphocytes, as high-affinity self-reactive T-lymphocytes have been deleted in a proc­ess of self-tolerance.8 Self-tolerance protects the body from autoimmune reactions and plays an essential role in inefficiency of tu­mor vaccines.1 Mechanisms of tumor escape Tumors evolve many mechanisms to evade actively or silence antitumor immune re­sponse, what makes them poorly immuno­genic or even tolerogenic.14 Tumor cells inefficiently present antigens to effector T-lymphocytes Genetic instability of tumors results in changing the tumor antigenic profile.15 Mutations of immunodominant tumor epitopes can prevent the recognition of a tumor cell by immune system.1 Level of tumor peptide presentation through MHC I molecules can be so low that the tu­mors remain undetected by specific T-lym­phocytes.8 Mechanisms of antigen pres­entation and processing are defective in many tumors. Low levels of surface MHC I molecules are characteristic of many tu­mors and correlate with worse prognosis. However, total absence of surface MHC I molecules makes a tumor cell more suscep­tible for lysis by natural killer cells.14 Induction of tolerance - anergy or deletion- of tumor specific lymphocytes Tumor cells actively participate in the in­duction of immune tolerance of tumor-specific lymphocytes. They interfere with maturation of DC, express surface Fas ligand inducing apoptosis of Fas positive tumor-specific lymphocytes, produce im­munosuppressive cytokines (IL-10, TGF-ß) and redirect immune response in the devel­opment of regulatory CD4+CD25+ T-lym­phocytes that inhibit the action of effector T-lymphocytes.1,13-17 Tumor interference with function of dendritic cell It has been found that DC are numerically and functionally defective in cancer pa­tients.9 Adoptively transferred tumor-spe­cific T-lymphocytes in mouse tumor model become anergic soon after their transfer to a mouse that has already developed the tumor.16 Anergy is caused by interaction of unmature DC, lacking a costimulatory signal, with tumor-specific T-lymphocytes. Level of DC maturation is essential in directing immune response either in anti-tumor immunity or in unresponsiveness to tumor antigens.9 Mechanisms of tumor Radiol Oncol 2006; 40(4): 219-29. cell interference with the function of DC involve an early and a late inhibition of DC maturation.13 The early inhibition of DC maturation is a result of cytokine mediated redirection of granulo/monocyte precursors from DC line to monocyte/macrophage line, decreasing the number of circulating DCs and increasing the number of circulating monocytes/macrophages which are ineffi­cient antigen-presenting cells in antitumor immunity.13 The late inhibition of DC mat­uration is a result of tumor mediated sup­pression of antigen cross-presentation and DC costimulatory molecules expression. Many tumors downregulate the expression of heat shock proteins that participate in endosome to cytosol shuffling pathway and provide maturation signals for DC. IL-10 limits availability of lysosomal proteases that are essential for the production of antigenic peptides.13 Phagocytosis of early apoptotic melanoma cells, rich in IL-10, inhibits the induction of DC costimulatory molecules.13 Peripheral deletion of tumor-infiltrating lymphocytes Peripheral deletion of tumor-infiltrating lymphocytes is mediated by Fas ligand on the tumor cells inducing apoptosis of Fas receptor positive tumor-specific T-lym­phocytes.8 Expression of Fas ligand on esophageal carcinoma cells is an early sign of disease progression.14 Immunoregulatory CD4+CD25+ T-lymphocytes Immunoregulatory CD4+CD25+ T-lym­phocytes are important negative regulators of immune response and represent 5 to 10% of peripheral T-lymphocytes. They induce anergy of high-affinity self-reactive T-lym­phocytes that have escaped central deletion process, and protect the body from au­toimmune diseases and overdriven normal immune responses against microbes.8,18 An increasing number of evidence show that they significantly suppress the anti-tumor immune response and participate in the induction of immune tolerance to tumor antigenes.5,19 Simultaneous use of anti-CD25 monoclonal antibodies and tu­mor vaccine in a mouse tumor model increases the efficiency of the vaccine and prolongs the survival of the experimen­tal animal. The ratio of peripheral immu­noregulatory CD4+CD25+ T-lymphocytes correlates negatively with the prognosis of gastrointestinal malignancies.23 Cancer pa­tients (23 ± 4%) have increased numbers of peripheral immunoregulatory CD4+CD25+ T-lymphocytes compared to healthy (6 ± 3%) volunteers.5,19 Immunoregulatory control points The high-affinity inhibitory CTLA-4 recep­tor expressed on activated T-lymphocytes competes with the lower-affinity stimula­tory receptor CD28 for binding the B7 costimulatory molecules on DC. It is im­plicated in the induction of self-tolerance and regulates the amplitude of normal T-lymphocyte responses. Similar actions are mediated by the B7-H1 and B7-H4 mole­cules that are frequently over-expressed in pancreatic carcinoma. Monoclonal antibo­dies directed against the B7-H1 and B7-H4 molecules are already in development.17 Production of immunosuppressive cytokines and cytokine immunostimulation of tumors Many cytokines (especially IL-10 and TGFß), produced by tumor cells or tumor-infiltrating lymphocytes, have several dif­ferent immunosuppressive actions.14 Cytokines can also accelerate tumor growth. The immunostimulatory actions of cytokines are seen mainly in hematologic Radiol Oncol 2006; 40(4): 219-29. malignancies. However, most solid tumors express the low-affinity IL-2 receptor ßµ (IL2R ßµ) which correlates with the increased therapeutic resistance of a tumor. Cytokines should therefore be used cautiously in can­cer patients as they could have detrimental effects on the survival of patients. Tumor microenvironment Infiltration of a tumor by tumor-specific lymphocytes is highly dependent on local tumor microenvironment.20 Tumor micro-vasculature represents significant barrier for lymphocytes. Although peritumor re­gions are rich in lymphocytes and tumors are usually well vascularized, lymphocytic infiltration of tumor remains poor.20 High-endothelial venules with activated endothe­lium that are important for entrance of lym­phocytes in an inflamed tissues are rarely present in intratumor regions.20 Tumor cells actively suppress expression of endothe­lial adhesion molecules by local secretion of angiogenic factors and cytokines. Poor lymphocyte infiltration is characteristic of many tumors and bears poor prognosis. Tumor vaccines Ideal tumor vaccine is a specific-tumor anti­gen expressed only by tumor cells (cannot induce autoimmune reactions) that par­ticipates in carcinogenesis and is crucial to tumor cell survival (preventing selection of immunoresistant clones during immu­notherapy). It must be expressed in high levels at all stages of the disease and must be common (universal) to different tumors. It is foreign to immune system and elicits high-affinity cellular and humoral immune responses with long-lived antitumor immu­nological memory.5,9 The real situation is far from being ideal. Most tumor antigens are self-molecules tol­erated by immune system.7,21 Tumor-specific lymphocytes isolated from cancer patients are rare and mainly anergic. Antitumor immune response imposes selective pres­sure over a genetically unstable tumor and accelerates the emergence of immunore­sistant clones. Simultaneously, the tumor develops many strategies to evade suc­cessfully antitumor immune response.16,21 Novel immunotherapeutic strategies are therefore directed toward breaking the im­mune tolerance to tumor antigens, enhanc­ing the immunogenicity of tumor vaccines and overcoming the mechanisms of tumor escape.16,22 There are several different im­munotherapeutic approaches, all of them unfortunately still far away from an ideal tumor vaccine that would reject a tumor. The present role of tumor vaccines in cancer therapy is minor. They are used mainly as adjuvant treatment in the pa­tients with advanced cancer. However, best results with tumor vaccines could be ex­pected in a state of minimal residual dis­ease after the majority of tumor burden has been removed by surgery or chemotherapy as the probability of immunoselection of resistant clones is the smallest and the im­munosuppressive effects of tumor are least pronounced. Cellular vaccines Cellular vaccines are either autologous or allogenic and contain tumor cells or their lysates. Classic autologous cellular vaccines con­tain attenuated patients' own tumor cells (requiring surgical resection of a sample of the patient tumor). Their primary ad­vantages are that they contain all antigens of the patient's tumor, can be specifically tailored for each patient and in every mo­ment match the tumor's changing antigenic profile.23,24 Previous identification of tumor antigens is not required and there are no Radiol Oncol 2006; 40(4): 219-29. limitations concerning the patient's HLA haplotype.15,23 They induce polyclonal anti-tumor immune response that more readily overcomes several tumor evading strate­gies and imposes smaller immunoselective pressure over the tumor.15 However, as the identity of tumor antigens is unknown, there are difficulties with the standardiza­tion of vaccine production protocol and measurement of postvaccination immune responses. Besides, variable immunogenic­ity of tumor antigens among different pa­tients influences the efficiency of immune response elicited by vaccine.15,24,25 Allogenic cellular vaccines are based on the idea that tumors of the same type from different patients share many com­mon antigens. They are prepared from cul­tured tumor cell lines, standardized, read­ily available and can be applied to many patients.15,25 Canvaxin and Melacin are al­logenic cellular vaccines, approved in adju­vant therapy of metastatic melanoma, and induce regression of melanoma lesions in 5 to 10% of treated patients.22,25 The main dis­advantage of cellular vaccines is their weak immunogenicity that can be improved by transfection of tumor cells with the genes that code for different immunostimulatory molecules (cytokines, chemokines, adhe­sion, MHC and costimulatory molecules) or by hybridizing tumor cells and DC.15,16,25 Genetic modifications have been shown to increase importantly immunogenicity of tumor vaccines, however clinical improve­ment has remained poor. 16 Peptide vaccines Peptide vaccines are intended to stimulate T-lymphocyte responses to tumor-specific antigenic peptides presented on the surface of tumor cells through MHC I molecules. Namely, most tumor antigens originate in tumor cell cytosol or cellular organelles and present themselves in the complex with the surface MHC I molecules. Peptide vaccines have several advantages; they are easily produced, inexpensive, safe, synthesized in big quantities and represent a standard­ized, well defined antigen, allowing post-vaccination immune response monitor­ing.2,23 Their main disadvantage is MHC I allotype restriction that makes them useful only in the patients matching MHC I al-lotype.2,23,27 Vaccination of cancer patients with one or two antigenic peptides has so far induced specific immune response in as many as 80% of patients; however, clinical improvement has been found only in 10 to 20% of patients.2 It has been proposed that a combination of many peptides would be necessary to achieve clinical results. Dendritic cell vaccines DCs are professional antigen-presenting cells essential in cross-presentation and dif­ferentiation of naive tumor-specific CD8+ T-lymphocytes in efficient cytotoxic cells. The basis for the development of DC vac­cines has been established with the proto­cols for ex vivo preparation of DC.28 DC can be prepared from CD34+ precursor cells isolated from bone marrow or peripheral blood after their incubation with different cytokine combinations, as are TNFa, GM­ 28 CSF, Flt3 ligand, CD40 ligand and TGFß. Mature low-phagocytic DC are produced, expressing high levels of membrane cos-timulatory molecules. Alternatively, DC are prepared from peripheral blood monocytes in culture with GM-CSF and IL-4. These DC are unmature, highly phagocytic and efficiently take up tumor antigens (tumor cells, peptides, proteins, tumor exosomes, heat shock proteins) they are incubated with.15,16,28 Another possibility of DC an­tigen loading is transfection of DC with cDNA or mRNA, coding for tumor anti­gens, mediated by viral vectors, electropo-ration or lipofection.16,28 Still better method Radiol Oncol 2006; 40(4): 219-29. is the transfection of DC with total tumor mRNA. After antigen loading of unmature DC is finished, it is necessary to induce DC maturation, mainly by TNFa, Toll-like receptor agonists (CpG oligonucleotides), IL-1ß or IL-6.16,21 Tumor heat shock protein vaccines An increasing number of evidence show that heat shock proteins (HSP), as are GP96 in HSP70 isolated from tumor cells, can induce a specific antitumor immune response. HSP are able to bind the anti­genic peptides arising in a tumor cell, to be actively taken up by DC in a process of receptor mediated endocytosis and to induce DC maturation through the interac­tion with DC Toll-like receptors.23,29 After HSP internalization, the antigenic peptides are released from HSP and enter antigen processing and cross-presentation proc­ess, finally emerging as a complex with MHC I on the surface of APC. There are many advantages of HSP vaccines. They contain many, if not all tumor antigenic peptides, induce polyclonal antitumor im­mune response, bring antigens to DC and induce their maturation.23,29,30 The disad­vantages of HSP vaccines are time consum­ing isolation of peptide-HSP complexes and unknown antigenic profile.23,29 Many animal models have confirmed in vivo im­munogenicity of HSP70 or GP96 vaccines. They have proven efficient in the induction of prophilactic and therapeutic antitumor immune responses in many preclinical tri­als and are now being tested in first- and second-phase clinical trials.33 In a study by Castelli et al., the vaccination of color-ectal carcinoma and melanoma patients by GP96 induced statistically significant antitumor T-cell immune response in 59% of melanoma patients and 47.8% colorectal carcinoma patients. A complete regression of melanoma lesions was achieved in 18% of patients and the survival of colorectal carcinoma patients was prolonged.29 Nanovesicular vaccines - tumor exosomes Exosomes (nanovesicles) are small mem­branous vesicles, originating from late en-dosome. They bud from the membrane of subcellular multivesicular bodies, fuse with plasmalema and are released extracellularly, where they refuse with the neighbor cells' membranes. Exosomes are composed of dif­ferent cytosolic and membrane proteins and have dual function. They represent a vehicle for removing redundant cellular proteins and are a pathway for trafficking proteins be­tween cells, thereby participating in a com­plex intercellular communication.31 The DC exosomes are enriched in adhesion proteins, costimulatory molecules, MHC I and MHC II molecules together with antigenic peptides ­they have immunomodulatory capacity. The tumor cell exosomes are enriched in native tumor proteins and are constitutively secret­ed by tumor cells. They bring tumor antigens to DC and, through action of surface HSP70, accelerate self-internalization in DC. The incubation of DC with the tumor exosomes in vitro and in vivo in mouse tumor models results in the activation of specific cytotoxic T-lymphocytes. Because of important role in antitumor immune response and proven preclinical antitumor efficiency, the DC and tumor cell exosomes are being tested in the first-phase clinical trials.31 Idiotypic vaccines Idiotypic vaccines use the variable region of B-lymphocyte membrane immunoglobulin as an antigen. The variable region contains epitopes unique to malignant B-lymphocyte clone and is therefore highly specific for the tumor.6 Idiotypic epitopes elicit polyclo­nal immune responses. Multiple myeloma patients have antiidiotypic antibodies and Radiol Oncol 2006; 40(4): 219-29. idiotype-specific T-lymphocytes in their blood. In vitro experiments and animal tu­mor models have shown that antiidiotypic immune response can destroy malignant myeloma cells.5 Polyclonal nature of antiid­iotypic immune response strongly reduces immunoselective pressure and the result­ant emergency of immunoresistant myelo-ma cells. Idiotypic vaccines could therefore represent a promising immunotherapeutic antitumor strategy.22 Their main disadvan­tage is their weak immunogenicity as idi­otype is a self-protein. It has been shown that the conjugation of an idiotype with a strongly immunogenic adjuvant is neces­sary for eliciting an efficient antiidiotypic immune response.23 Specific idiotypic pro­tein can be produced from hybridoma cells or can be synthetisized by methods of re­combinant gene technology. Total idiotypic protein can be produced, or better, only its single-chain variable fragment avoiding harmful reactions against the immunoglob­ulin constant region.23 Lately, idiotype fu­sion DNA vaccines have emerged. They are composed of cDNA coding for heavy or light chain variable region linked to bacte­rial DNA or cDNA coding for the tetanus toxoid C fragment.5 Viral vaccines Cervical carcinoma is caused by persistent infection of cervical epithelia with cancer-associated types of human papilloma virus (HPV). The genome of cancer-associated HPV is found in 99% of cervical malignant lesions.12 HPV infects the basal cells of cer­vical squamous epithelia. HPV proliphera­tion and assembly are intimately linked to epithelial cell differentiation program; infective virions are produced only in fully differentiated epithelial cells.12 As tissue damage with HPV infection is minimal and double helical RNA, an effective APC activator, is not produced during HPV cycle, the spontaneous anti-HPV immune response is weak.11 Despite that, most HPV infections spontaneously disappear in few years.11,12 As cervical carcinoma is caused by HPV16 or HPV18 in two thirds of patients, prophylactic and therapeutic vaccines are directed mainly against their antigens. Prophylactic vaccines contain re-combinantly produced HPV16/HPV18 cap-side antigens L1, forming virus-like parti­cles, and are entering in clinical use.12 Two important randomized placebo controlled studies that included young sexually active women have shown the vaccination with HPV16/HPV18-like particles to be safe and effective and protects against persistent HPV infection and development of precan­cerous cervical lesions.11 Therapeutic HPV vaccines are directed against HPV proteins E6 and E7 and are mostly experimental with limited clinical efficiency.11,12 Difficulties in activation of antitumor im­mune response by tumor vaccines have led lately to the development of alternative im­munotherapeutic strategies directly focus­ing on the effector mechanisms of immune system. Such approaches include adoptive tumor-specific T-lymphocyte transfer and tumor-specific monoclonal antibodies. Adoptive T cell transfer Autologous tumor-specific T-lymphocytes isolated from the patient's peripheral blood, tumor or tumor-infiltrated lymph nodes are activated and multiplied ex vivo in the pres­ence of specific T-cell epitopes and then re­turned to the patient.16,32 There are several advantages of the adoptive T-cell transfer. It provides large numbers of tumor specific T-lymphocytes which are activated in the absence of inhibitory and tolerogenic tu­mor actions.7 Compared to tumor vaccines, it is a better immunotherapeutic option for the patients with widespread disease and Radiol Oncol 2006; 40(4): 219-29. high tumor burden.32 However, the iden­tity of tumor antigens used in the ex vivo activation of antitumor T-lymphocytes has to be known and the process of antigen identification is difficult and time con­suming. Main disadvantages are decreased ability of transferred lymphocytes for tu­mor infiltration and their shorter survival that can be partially improved by adding IL-2.16,32 Lymphodepletion with the result­ant removal of regulatory T-lymphocytes, preceding adoptive T-cell transfer, is an im­portant factor in achieving an efficient an-titumor immune response mediated by the transferred tumor-specific lymphocytes.7,21 Adoptive transfer of Melan-A/MART1 epitope-specific T-lymphocytes or GP100­specific CD8+ T-lymphocytes induced re­gression of melanoma lesions in meta­static melanoma patients; however, target melanoma epitopes were eventually lost with the resultant overgrowth of immu­noresistant melanoma cells.21 Monoclonal antibodies Antitumor monoclonal antibodies (mAb) are an alternative form of effector im­munotherapy. Rituximab (anti-CD20 mAb) and Herceptin (anti HER-2 Neu mAb) are successfully used in the treatment of B-cell non-Hodkin’s lymphoma (NHL) and breast carcinoma patients, respectively.3 There are increasing numbers of novel an-titumor mAb that are tested in preclinical and first-phase clinical trials.33 Rituximab (MabThera®) is a chimeric IgG1 kappa mAb, produced by recombinant gene tech­nology methods. It is used in the treatment of III-/IV-stage chemoresistant follicular NHL and its relapses.34 CD20 antigen is expressed by healthy B-lymphocytes and more than 90% of B-cell NHLs, but not by plasma cells. Rituximab quickly depletes CD20+ B-lymphocytes with the restoration of their numbers only 9 to 12 months after treatment.34 Mechanisms involved in the depletion of B lymphocytes are antibody mediated cell cytotoxicity, complement de­pendent cytolysis and induction of apopto­sis.34 As the plasma cells are not affected, the production of immunoglobulins is prac­tically normal.34 Monoclonal antibody ef­ficiency can be improved by their conjuga­tion with toxins, radionuclides or cytotoxic drugs. Mylotrag, anti-CD33 immunotoxin, is used in the treatment of CD33 positive acute myeloic leukemia in older patients and shows comparable antileukemic ef­ficiency to chemotherapy with fewer side effects.3 Conclusion There are three main requirements for can­cer immunotherapy to be effective. First, there must be enough high-affinity tumor-specific lymphocytes; second, tumor-specific lymphocytes must successfully infiltrate the tumor and third, the tumor infiltrating lym­phocytes must effectively kill tumor cells. Real situation is totally different. First, po­tentially tumor-reactive T-lymphocyte pop­ulation is represented by a small number of low-affinity T-lymphocytes. Tumor vaccines can elicit only weak immune response against tumor antigens. Second, local tumor microenvironment is an important barrier for T-lymphocyte infiltration of the tumor. Third, tumor cells develop several strategies to evade successfully antitumor immune response. Although tumor vaccines arose as promising anticancer strategy with sev­eral potential advantages over standard anticancer regimens, the results of clinical trials on tumor vaccines have so far been disappointing. Considering all the barriers that the immune system must overcome to reject the tumor, disappointing results are all but surprising. Radiol Oncol 2006; 40(4): 219-29. References 1. Mosolits S, Ullenhag G, Mellstedt H. Therapeutic vaccination in patients with gastrointestinal ma­lignancies. A review of immunological and clinical results. Ann Oncol 2005; 16: 847-62. 2. Singh-Jasuja H, Emmerich NP, Rammensee HG. The Tubingen approach: identification, selection, and validation of tumor-associated HLA peptides for cancer therapy. Cancer Immunol Immunother 2004; 53: 187-95. 3. Huber CH, Wolfel T. Immunotherapy of cancer: from vision to standard clinical practice. J Cancer Res Clin Oncol 2004; 130: 367-74. 4. Wong KK Jr, Chatterjee S. Vaccine development for chronic myelogenous leukaemia. Lancet 2005; 365: 631-2. 5. Harrison SJ, Cook G. Immunotherapy in multiple myeloma-possibility or probability? Br J Haematol 2005 Aug; 130: 344-62. 6. Kunkel LA. Idiotype vaccines in the treatment of B-cell non-Hodgkin's lymphoma. Cancer Invest 2004; 22: 97-105. 7. Rosenberg SA. Shedding light on immunotherapy for cancer. N Engl J Med 2004; 350: 1461-3. 8. Emens LA, Jaffee EM. Leveraging the activity of tumor vaccines with cytotoxic chemotherapy. Cancer Res 2005; 65: 8059-64. 9. Turtle CJ, Hart DN. Dendritic cells in tumor im­munology and immunotherapy. Curr Drug Targets 2004; 5: 17-39. 10. Adam JK, Odhav B, Bhoola KD. Immune responses in cancer. Pharmacol Ther 2003; 99: 113-32. 11. Frazer IH. Prevention of cervical cancer through papillomavirus vaccination. Nat Rev Immunol 2004; 4: 46-54. 12. Mahdavi A, Monk BJ. Vaccines against human papillomavirus and cervical cancer: promises and challenges. Oncologist 2005; 10: 528-38. 13. Cho HJ, Bhardwaj N. Against the self: dendritic cells versus cancer. APMIS 2003; 111: 805-17. 14. Pawelec G. Tumour escape: antitumour effec­tors too much of a good thing? Cancer Immunol Immunother 2004; 53: 262-74. 15. Rousseau RF, Brenner MK. Vaccine therapies for pediatric malignancies. Cancer J 2005; 11: 331-9. 16. Mapara MY, Sykes M. Tolerance and cancer: mechanisms of tumor evasion and strategies for breaking tolerance. J Clin Oncol 2004; 22: 1136-51. 17. Laheru D, Jaffee EM. Immunotherapy for pancre­atic cancer - science driving clinical progress. Nat Rev Cancer 2005; 5: 459-67. 18. Velders MP, Markiewicz MA, Eiben GL, Kast WM. CD4+ T cell matters in tumor immunity. Int Rev Immunol 2003; 22: 113-40. 19. Terabe M, Berzofsky JA. Immunoregulatory T cells in tumor immunity. Curr Opin Immunol 2004; 16: 157-62. 20. Chen Q, Wang WC, Evans SS. Tumor microvascu­lature as a barrier to antitumor immunity. Cancer Immunol Immunother 2003; 52: 670-9. 21. Mahnke YD, Speiser D, Luescher IF, Cerottini JC, Romero P. Recent advances in tumour antigen-specific therapy: in vivo veritas. Int J Cancer 2005; 113: 173-8. 22. Antonia S, Mule JJ, Weber JS. Current develop­ments of immunotherapy in the clinic. Curr Opin Immunol 2004; 16: 130-6. 23. Campoli M, Ferrone S. T-cell-based immunothera­py of melanoma: what have we learned and how can we improve? Expert Rev Vaccines 2004; 3: 171­87. 24. Frankenberger B, Regn S, Geiger C, Noessner E, Falk CS, Pohla H, Javorovic M, Silberzahn T, Wilde S, Buchner A, Siebels M, Oberneder R, Willimsky G, Pezzutto A, Blankenstein T, Schendel DJ. Cell-based vaccines for renal cell carcinoma: genetical-ly-engineered tumor cells and monocyte-derived dendritic cells. World J Urol 2005; 23: 166-74. 25. Demierre MF, Allten S, Brown R. New treatments for melanoma. Dermatol Nurs 2005; 17: 287-95. 26. Cheuk AT, Chan L, Czepulkowski B, Berger SA, Yagita H, Okumura K, Farzaneh F, Mufti GJ, Guinn BA. Development of a whole cell vac­cine for acute myeloid leukaemia. Cancer Immunol Immunother 2006; 55: 68-75. 27. Baxevanis CN, Sotiriadou NN, Gritzapis AD, Sotiropoulou PA, Perez SA, Cacoullos NT, Papamichail M. Immunogenic HER-2/neu peptides as tumor vaccines. Cancer Immunol Immunother 2006; 55: 85-95. 28. Grunebach F, Muller MR, Brossart P. New devel­opments in dendritic cell-based vaccinations: RNA translated into clinics. Cancer Immunol Immunother 2005; 54: 17-25. Radiol Oncol 2006; 40(4): 219-29. 29. Castelli C, Rivoltini L, Rini F, Belli F, Testori A, Maio M, Mazzaferro V, Coppa J, Srivastava PK, Parmiani G. Heat shock proteins: biological functions and clinical application as personal­ized vaccines for human cancer. Cancer Immunol Immunother 2004; 53: 227-33. 30. Stebbing J, Bower M, Srivastava P. Kaposi's sarco­ma as a model for cancer immunotherapy. Trends Mol Med 2004; 10: 187-93. 31. Li X, Zhang Z, Beiter T, Schluesener HJ. Nanovesicular vaccines: exosomes. Arch Immunol Ther Exp (Warsz) 2005; 53: 329-35. 32. Knutson KL, Wagner W, Disis ML. Adoptive T cell therapy of solid cancers. Cancer Immunol Immunother 2006; 55: 96-103. 33. Finley RS. Overview of targeted therapies for can­cer. Am J Health Syst Pharm 2003; 60 (24 Suppl 9): S4-10. 34. White CA. Rituxan immunotherapy and zevalin radioimmunotherapy in the treatment of non-Hodgkin's lymphoma. Curr Pharm Biotechnol 2003; 4: 221-38. 35. Villinger F. Cytokines as clinical adjuvants: how far are we? Expert Rev Vaccines 2003; 2: 317-26. Radiol Oncol 2006; 40(4): 219-29. Radiol Oncol 2006; 40(4): 231-7. Preoperative concomitant chemoradiotherapy in esophageal cancer Boštjan Šeruga1, Miha Sok2, Janez Eržen2, Jože Jerman2, Boris Jancar1, Branko Zakotnik1 1Institute of Oncology Ljubljana, 2Department of Thoracic Surgery, Clinical Center Ljubljana, Slovenia Background. Currently primary treatment options for esophageal cancer are surgery only or concomitant chemoradiotherapy (CRT) and the long-term survival of patients with locally advanced disease is rare. Preoperative concomitant CRT seems to be beneficial, mostly in patients who achieve a complete pathologic response (pCR) after CRT. In this retrospective analysis the efficiency and toxicity of preoperative CRT in patients with locally advanced esophageal cancer was analysed as well as the influence of pCR on the survival. Patients and methods From 1996 to 2002 41 patients with locoregionally confined esophageal cancer were treated with cisplatin 75 mg/m2 and 5-FU 1000 mg/m2 as 4 day contonuous infusion starting on days 1. and 22. with concomitant radiotherapy 4500 cGy, 200-300 cGy/day. Esophagectomy followed 4-5 weeks after radiotherapy. After the surgery patients were followed-up regularly at 3-6 months intervals. Results. The pCR was achieved in 26.8% of patients. The overall median survival time was 18 months for all patients, 21.2 months for patients who achieved pCR and 16 months in those with residual disease (p= 0,79). Postoperative mortality rate was 22%. The median dose intensity for cisplatin was 92% and for 5-FU 71.5% of the planned dose. Disease recurred most often locoregionally (31.7%) and the overall recurrence rate was 43.9%. Conclusion. Modern radiation techniques and the adequate dose intensity could further improve the loco-regional control. The selection of patients without comorbid conditions and without already present distant metastases is essential for this combined treatment approach. Key words: esophageal neoplasms – drug therapy - radiotherapy Received 23 October 2006 Accepted 29 November 2006 Correspondence to: Assist. Prof. Branko Zakotnik, MD, PhD, Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia; Phone: + 386 1 5879 280; Faks: + 386 1 5879 400; E-mail: bzakotnik@onko-i.si Introduction In the last few decades the incidence of esophageal cancer is constantly growing in Western Europe and USA where adenocar­cinoma represents 60% of all esophageal carcinomas.1,2 According to data of the Cancer registry of Slovenia the incidence of esophageal cancer has risen in recent years (76 patients in 1998, 100 patients in 2002) but adenocarcinoma still represents only 14% of all histological confirmed can­cers and most of them are squamous cell carcinomas.3,4 Primary treatment modalities include surgery alone or concomitant chemora­diotherapy. The surgical treatment is a standard treatment for stage I, II and III-T3 (www.cancernet.nci.nih.gov) and is feasi­ble in 40-60% of patients.5,6 In 75% of patients esophageal cancer is diagnosed when already locally advanced (stage IIB, III). The postoperative mortality rate is 10-15%, in experienced centers less than 5%.7 Postoperatively locoregional recur­rence occurs in 30-60%.8-10 After the only primary surgical treatment, 5-year survival for stage I disease and locally advanced dis­ease is 50-80% and 5-10%, respectively.11-13 Concomitant chemoradiotherapy was supe­rior when compared to radiotherapy alone in the primary treatment of locally ad­vanced esophageal cancer.14,15 Preoperative radiotherapy does not improve outcome in comparison to surgery alone.16-21 Combined modality therapies (preopera­tive chemotherapy, preoperative concomi­tant chemoradiotherapy) are still under the clinical evaluation. According to the results from randomized trials, the role of preoperative chemotherapy is still incon­clusive.9,22-24 In nonrandomized clinical tri­als with preoperative concomitant chemo-radiotherapy (CRT) a pathologic complete response (pCR) was achieved in average in 32% (11%-76%) of patients and predicted a better survival. The survival of patients with pCR at 3-years and 5-years was 29­92% and 20-71%, respectively. The survival of patients who did not achieve pCR at 3­years was 23-33%. The disease recurred in 46% of all patients. In patients with a pCR the disease recurred in 20%, mostly (80%) as distant metastases.25 In one randomized clinical trial the concomitant preoperative CRT showed some modest survival ben­efit over surgery alone26 but there was no benefit in other randomized trials. 10,20,27-29 The inconsistency of these results might be due to heterogeneous patients’ population, tumours characteristics and different treat­ment protocols. In this retrospective study we analyzed the efficacy and toxicity of preoperative concomitant CRT in our patients with lo­cally advanced esophageal cancer. Patients and methods Patients Medical records of patients with esophageal cancer treated with preoperative concomi­tant CRT from 1996 to 2002 at the Institute of Oncology Ljubljana and Department of Thoracic Surgery Ljubljana were reviewed. Patients with histological confirmed loco-regionally confined esophageal carcinoma (stage II and III), performance status < 2 ac­cording to WHO, adequate function of bone marrow, liver and kidney and absence of other malignances in their medical history with the exception of skin carcinoma were eligible. Staging of the tumour was based on the results of physical examination, blood tests, chest radiography, ultrasonog­raphy of abdomen, computed tomography of chest and upper abdomen, esophagogas­troscopy with biopsy, liquid oral contrast examination of esophagus, endoscopic ul­trasound of esophagus and bronchoscopy in patients with tumours in the middle and the upper third of esophagus. Treatment Patients were treated with concomitant preoperative chemotherapy (cisplatin 75 mg/m2 days 1, 22 and 5-FU 1000 mg/m2 days 1 - 4 and 22 – 25) and concomitant ra- Radiol Oncol 2006; 40(4): 231-7. diotherapy (4500 cGy, 200 – 300 cGy /day, field designs were either two or three-field plans on the linear accelerator, the radia­tion field included primary tumour with 5­cm superior and inferior margins and 2 cm lateral margins) (Figure 1). Esophagectomy followed 4 to 5 weeks after radiotherapy. After the surgery patients were followed– up regularly at 3-6 months intervals. Statistical analysis A statistical analysis with descriptive sta­tistics and survival times and curves was performed using SPSS 10.0 for Windows statistical software. The survival was cal­culated according to Kaplan-Meier method and compared by a log-rank test between groups. The overall survival was estimated from the date of diagnostic biopsy to death and the relapse-free survival from the date of diagnostic biopsy to the first event of recurrence (local, regional, distant or their combinations). Results Forty-one patients (38 men, 3 women) with locoregionally confined esophageal cancer and without other comorbid conditions were treated with preoperative concomi­tant CRT in the period 1996 to 2002. The Table 1. Patients and tumors characteristics CHARACTERISTIC No. of patients TOTAL 41 MALES 38 (92,5%) FEMALES 3 (7,5%) MEDIAN AGE (min - max) 60 (41-74) HISTOLOGY squamous cell 39 (95,2%) adenocarcinoma 1 (2,4%) poorly differentiated carci­ 1 (2,4%) noma TUMOR LOCALIZATION upper third 2 (4,9%) middle third 22 (53,6%) lower third 17 (41,5%) median age was 60 years (range 41-74). Squamous cell carcinoma was present in 39 (95.2%) patients and adenocarcinoma and poorly differentiated carcinoma in 2 (4.8%) patients. Tumour was located in the upper third of esophagus in 2 (4.9%) patients, in the middle third in 22 (53.6%) patients and in the lower third in 17 (41.5%) patients (Table 1). Median dose intensity of received cispla-tin was 23,2 mg/m2/week (range, 16-34.2 mg/m2/week) and of 5-FU 954.1 mg/m2/ week (range, 231.5-1394 mg/m2/week) com­prising 92.8% and 71.5% of the planned dose intensity, respectively. Patients who received in average less than 80% of planned dose intensity of both cisplatin and 5-FU had higher recurrence rate than patients who received 80% or more of the planned dose intensity (50% vs 30.8%; difference not statistically significant). Both, distant and locoregional recurrences were more com­mon in patients with lower dose intensity, 28.6% vs. 7.7% and 35.7% vs. 23.1%, respec­tively (Table2). Transthoracic esophagectomy (Lewis) and transhiatal esophagectomy were per­formed in 38 patients and 3 patients, re­spectively. R0 resection (microscopically free margins) was achieved in 39 patients and R1 resection (microscopically residual Radiol Oncol 2006; 40(4): 231-7. Table 2. Chemotherapy (ChT) dose intensity and recurrence rates No. of patients (%)RECURRENCE DOSE INTENSITY OF ChT < 80% LOCOREGIONAL 6/28 (21,4%) DISTANT 4/28 (14,3%) LOCOREGIONAL+DISTANT 4/28 (14,3%) TOTAL 14/28 (50%) disease) in 2 patients. Postoperatively 9 patients (22%) died in 30 days. All patients died in a septic shock with multiorgan failure. Another 10 patient had nonfatal postoperative complications: pneumonia, empyema, necrosis of the stomach wall (fundus), fistula of the anastomosis and hylothorax. In patients with carcinoma lo­cated in the upper and middle third of the esophagus both fatal and nonfatal postop­erative complications were more common than in patients with their carcinoma in the lower third, 25% vs. 17.6% and 29.2 vs. 17.6%, respectively (Table 3). In 4 out of 5 patients with necrosis of the stomach wall and in 3 out of 4 patients with fistula of anastomosis the tumour was present in the middle third of the esophagus. A pathologic complete response (pCR) was achieved in 11/41 (26.8%) patients, in the upper two thirds of esophagus in 7/24 patients (29.1%) and in the lower third in 4/17 (23.5%) patients. Postoperatively 2 patients with pCR died. After a thorough lymph nodes examination by the patholo­gist metastatic disease was found in 9 patients (3 patients M1a, 6 patients M1b dis­ =80% 3/13 (23,1%) 1/13 (7,7%) 0 4/13 (30,8%) ease) and during the follow-up only 5 of these patients relapsed. The median follow up was 40 months (6-52 months). The overall risk for recur­rence was 43.9% (9 recurred locoregionally, 5 distant and 4 locoregionally and distant). Patients with and without pCR had a simi­lar risk for recurrence (45.5% vs. 43.3%). The median time to relapse was 21.5 months (95% CI: 7.3 – 35.7 months) and the median overall survival time was 18 months (95% CI: 10.8 – 25.1 months). Overall 2-year and 3-year survival was 36% and 28% respectively (Figures 2, 3). Patients with pCR had the median survival time of 21.2, months (95% CI: 2.4-40 months) and patients without a pCR 16 months (95% CI: 7.6-24.4 months, p=0.79). Discussion In this retrospective analysis of our pa­tients with locally advanced esophageal cancer treated with preoperative concomi­tant CRT the pathologic complete response rate (26.8%), recurrence rate (43.9%) and Radiol Oncol 2006; 40(4): 231-7. median overall survival time (18 months) are comparable with the results of pub­lished trials.25 In the majority of published clinical tri­als, the survival of patients who achieved a pCR was significantly better than of those without a pCR. In our study the median survival of patients with a pCR (21.2 months) was also better than in those without a pCR (16 months) but the differ­ence was not statistically significant. The main reason could due to small number of patients included in our study. We also observe a high postoperative mortality rate and efforts to reduced post­operative mortality could further improve the overall survival. Necrosis of stomach wall occurred in 5 patients and led to death in 2 patients. This treatment compli­cation is not listed among the common in the literature. The postoperative mortality rate was higher in patients with tumours located in the upper two thirds of es­ophagus in comparison to the lower third (27.3% vs. 17.6%) but the difference was not statistically significant. Altogether, 80% of all stomach wall necrosis and 75% of all fistulas on the anastomoses occurred in patients with tumours in the upper two thirds. Therefore, concomitant CRT with­out surgery might be a reasonable option for patients with cancer localized in the up­per two thirds of the esophagus. Although there are no randomized studies compar­ing surgery versus concomitant CRT alone, the survival of patients in the concomitant CRT arms (in some randomized clinical trials comparing concomitant CRT versus radiotherapy) is similar to the survival of patients treated in the trials compar­ing surgery and preoperative concomitant CRT plus surgery. This comparison is speculative and not evidence based, but it might be reasonable to adopt it for the subgroup of patients with high mortality rate after the surgery as are in our case the patients with their cancers in the upper two thirds. Improved surgical techniques and more intense postoperative care are also important options for these patients since the surgery was beneficial in 5 out of 9 our patients with residual carcinoma in the lymphnodes (M1a and M1b disease) after concomitant CRT who are still free of recurrence after the median time of fol­low up of 40 months. Currently it is also hard to predict who is going to achieve a pCR after concurrent chemoradiotherapy and the achieved pCR rates are relatively low. For these reasons the role of surgery remains an important part of this multi-modality treatment approach. The median dose intensity for cispla-tin and 5-FU was 92.8% and 71.5% of the Radiol Oncol 2006; 40(4): 231-7. planned dose intensity, respectively. The main reason for the lower dose inten­sity for 5-FU might be due to well known higher incidence of mucositis in case of concurrent chemoirradiaton. Patients with median dose intensity of less than 80% for both cisplatin and 5-FU had a higher locore­gional recurrence rate (35.7% vs 21.4%) and increased incidence of distant failure by almost four-fold (28.6% vs 7.7,%). Therefore it seems important that the dose intensity is delivered as planned in the schedule. It seems that this is feasible, since in our study the postoperative complication rates (including fatal) were similar regardless of the dose intensity received. Modern three dimensional conformal radiotherapy plan­ning could be of additional benefit for the locoregional control. An extremely important issue is the selection of patients for this combined modality treatment. We should exclude patients in poor performance status with distant metastases who will not benefit with this kind of treatment. Conclusion Preoperative concomitant CRT might be beneficial at least in a subset of patients with locally advanced esophageal cancer in good performance status and without important comorbidity. For tumours origi­nating in the upper two thirds of esopha­gus the role of surgery should be used in highly selected cases. A multidisciplinary approach of surgeons, radiation oncolo­gists and medical oncologist is essential. References 1. Blot WJ, Mclaughlin JK. The changing epidemiol­ogy of esophageal cancer. Semin Oncol 1999; 26(5 Suppl 15): 2-8. 2. Devesa SS, Blot WJ, Fraumeni JF Jr. Changing pat­terns in the incidence of esophageal and gastric carcinoma in the United States. Cancer 1998; 83: 2049-53. 3. Cancer incidence in Slovenia 2002. Ljubljana: Institute of Oncology, Cancer registry of Slovenia, 2005. 4. Survival of patients with cancer in Slovenia 1983-1997. Ljubljana: Institute of Oncology, Cancer registry of Slovenia, 2003. 5. Macfarlane SD, Hill LD, Jolly PC, Kozarek RA, Anderson RP. Improved results of surgical treat­ment for esophageal and gastroesophageal junc­tion carcinomas after preoperative combined chemotherapy and radiation. J Thorac Cardiovasc Surg 1988; 95: 415-22. 6. Saagar PM, Gauperaa T, Sue-Ling H, Mcmahon MJ, Johnston D. An audit of the treatment of can­cer of the oesophagus. Gut 1994; 35: 941-5. 7. Lozac'h P, Topart P, Perramant M. Ivor Lewis procedure for epidermoid carcinoma of the es-ophageus. A series of 264 patients. Semin Surg Oncol 1997; 13: 238-44. 8. Barbier PA, Luder PJ, Schupfer G, Becker CD, Wagner HE. Quality of life and patterns of recur­rence following transhiatal esophagectomy for cancer: results of prospective follow-up in 50 pa­tients. World J Surg 1988; 2: 270-6. 9. Kelsen DP, Ginsberg R, Pajak TF, Sheahan DG, Gunderson L, Mortiner J, et al. Chemotherapy followed by surgery compared with surgery alone for localized esophageal cancer. N Engl J Med 1998; 339: 1979-84. 10. Urba S, Orringer M, Turrisi A, Iannettoni M, Forastiere A, Strawderman M. Randomized trial of preoperative chemoradiation versus surgery alone in locoregional esophageal cancer. J Clin Oncol 2001; 19: 305-13. 11. Ellis FH Jr. Standard resection for cancer of the esophagus and cardia. Surg Oncol Clin North Am 1999; 8: 279-94. 12. Orringer MB, Marshal B, Iannettoni MD. Transhiatal esophagectomy: clinical experience and refinements. Ann Surg 1999; 230: 392-400. Radiol Oncol 2006; 40(4): 231-7. 13. Vigneswaran WT, Trastek VF, Pairolero PC, Deschamps C, Daly RC, Allen MS. Transhiatal esophagectomy for carcinoma of the esophagus. Ann Thorac Surg 1993; 56: 838-44. 14. Cooper JS, Guo MD, Herskovic A, Macdonald JS, Martensen JA Jr, Al-Sarraf M, et al. Chemoradiotherapy of locally advanced esopha­geal cancer: long term of follow-up of a prospective randomized trial (RTOG 85-01). Radiation Therapy Oncology Group. JAMA 1999; 281: 1623-7. 15. Smith RJ, Ryan ML, Douglass HO Jr, Haller DG, Dayal Y. Combined chemoradiotherapy vs. radiotherapy alone for early stage squamous cell carcinoma of the esophagus: a study of the Eastern Cooperative Oncology Group. Int J Radiat Oncol Biol Phys 1998; 42: 269-76. 16. Arnott SJ, Duncan W, Gignoux M, Girling DJ, Hansen HS, Launois B, et al. Preoperative radio­therapy in esophageal carcinoma: a meta-analysis using individual patient data (Oesophageal Cancer Collaborative Group). Int J Radiat Oncol Biol Phys 1998; 41: 579-83. 17. Arnott SJ, Duncan W, Kerr GR, Walbaum PR, Cameron E, Jack WJ, et al. Low-dose preoperative radiotherapy for carcinoma of the esophagus: re­sults of a randomized clinical trial. Radiother Oncol 1992; 24: 108-13. 18. Gignoux M, Roussel A, Paillot B, Gillet M, Schlag P, Favre JP, et al. The value of preoperative radio­therapy in esophageal cancer: results of a study of the EORTC. World J Surg 1987; 11: 426-32. 19. Launois B, Delarue D, Campion JP, Kerboal M. Preoperative radiotherapy for carcinoma of the esophagus. Surg Gynecol Obstet 1981; 153: 690-92. 20. Nygaard K, Hagen S, Hansen HS, Hatlevoll R, Hultborn R, Jakobsen A, et al. Pre-operative radio­therapy prolongs survival in operable esophageal carcinoma: a randomized, multicenter study of pre-operative radiotherapy and chemotherapy. The second Scandinavian trial in esophageal cancer. World J Surg 1992; 16: 1104-10. 21. Wang M, Gu XZ, Yin WB, Huang GJ, Wang LJ. Randomized clinical trial on the combination of preoperative irradiation and surgery in the treat­ment of esophageal carcinoma: report on 206 patients. Int J Radiat Oncol Biol Phys 1989; 325-7. 22. Kok TC, Van Lancshot J, Siersema PD, Van Overhagen H, Tilanus HW, for the Rotterdam Esophageal Tumor Study Group. Neoadjuvant chemotherapy in operable esophageal squamous cell cancer: final report of a phase III multicenter randomised controlled study. Proc Am Soc Clin Oncol 1997; 16: A984. 23. Law S, Fok M, Chow S, Chu KM, Wong J. Preoperative chemotherapy versus surgical thera­py alone for squamous cell carcinoma of the es­ophagus: a prospective randomised trial. J Thorac Cardiovasc Surg 1997; 114: 210-17. 24. Medical Research Council Oesophageal Cancer Working Group. Surgical resection with or with­out preoperative chemotherapy in oesophageal cancer: a randomized controlled trial. Lancet 2002; 359 (9319): 1727-33. 25. Geh JI, Crellin M, Glynne-Jones R. Preoperative (neoadjuvant) chemoradiotherapy in esophageal cancer. Br J Surg 2001; 88: 338-56. 26. Walsh TN, Noonan N, Holywood D, Kelly A, Keling N. A comparison of multimodal therapy and surgery for esophageal adenocarcinoma. N Engl J Med 1996; 335: 462-7. 27. Apinop C, Puttisak P, Preecha N. A prospective study of combined therapy in esophageal cancer. Hepatogastroenterology 1994; 41: 391-3. 28. Bosset JF, Gignoux M, Triboulet JP, Tiret E, Mantion G, Elias D, et al. Chemoradiotherapy fol­lowed by surgery compared with surgery alone in squamous-cell carcinoma of the esophagus. N Engl J Med 1997; 337: 161-7. 29. Le Prise E, Etienne PL, Meunier B, Maddern G, Ben Hassel M. A randomized study of chemo­therapy, radiation therapy and surgery versus surgery for localized squamous cell carcinoma of the esophagus. Cancer 1994; 73: 1779-84. Radiol Oncol 2006; 40(4): 231-7. Radiol Oncol 2006; 40(4): 239-44. Body mass index and lung cancer risk in never smokers Katsunori Kagohashi, Hiroaki Satoh, Koichi Kurishima, Hiroichi Ishikawa, Morio Ohtsuka Division of Respiratory Medicine, Institute of Clinical Medicine, University of Tsukuba, Japan Background. A relationship between body mass index (BMI) and lung cancer risk in never smokers has not been reported precisely. To evaluate the risk of lung cancer associated with BMI in never smokers, we conducted a case-control study. Methods. The relationship between BMI and the risk of lung cancer in never smokers was investigated in a study of 204 lung cancer cases and 398 controls admitted between 1987 and 2005. Controls were selected from hospitalized age-matched never-smoking patients with non-malignant respiratory disease. Results. When compared with BMI of the leanest group (BMI<20.8) in men, no inverse association between BMI and lung cancer was observed after the adjustment for age (the second BMI group: BMI= 20.8 to < 22.9; p=0.683, the third BMI group: BMI= 22.9 to < 24.9; p=0.745, and the highest BMI group: BMI= 25.0; p=0.327). Similarly, no association in women was found between BMI and lung cancer in these three BMI groups (the second group, p=0.639; the third group, p=0.667; the highest group, p=0.978) when compared with that of the leanest BMI group. Conclusions. Our present study indicated that the association between leanness and the risk of lung cancer might be influenced by other factors such as smoking. Key words: lung neoplasms – epidemiology; risk factors; adenocarcinoma; body mass index; smoking Introduction An elevated risk of lung cancer associated with lower levels of body mass index (BMI) has been reported in previous studies.1-9 However, the interpretation of the associa- Received 3 December 2006 Accepted 14 December 2006 Correspondence to: Hiroaki Satoh, MD, Division of Respiratory Medicine, Institute of Clinical Medicine, University of Tsukuba, Tsukuba-city, Ibaraki, 305­8575, Japan; Phone: + 81 29 853 3210; Fax: + 81 29 853 3320; E-mail: hirosato@md.tsukuba.ac.jp tion between low BMI and lung cancer is complicated by the fact that weight loss may be a sign of smoking. In general, smokers tend to be lighter than non-smok-ers,1, 10-15 and it is believed that nicotine is responsible for the effect of smoking on body weight because nicotine appears to increase the metabolic rate. We have recently performed a case-control study of subjects participating in a mass-screening program and found an increased risk of lung cancer for lower BMI in male patients.16 Most of them were current smokers.16 However, the inverse association between the risk of lung cancer and BMI was not found in female patients. This was inconsistent with the results of previous studies.1-9 The difference between the findings of previous studies and our own was most probably due to difference in study population. The subjects in previ­ous studies were symptomatic, but all the subjects in our recent study were asymp­tomatic preclinical patients.16 In addition, there were higher proportions of never-smoking women and lung adenocarcinoma in our study, and the different results might also be influenced by them. Whereas, it remains an unsettled ques­tion whether there is an inverse association between the risk of lung cancer and BMI among never smokers, especially in those with lung adenocarcinoma. In order to evaluate the association between BMI and the risk of lung cancer in patients who never smoked, we conducted a hospital-based case-control study. In this study, we also estimated the association between BMI and the risk of lung adenocarcinoma in never-smokers. Methods Study Design A hospital-based case-control study was conducted from January 1987 to September 2005 in Respiratory Division of Tsukuba University Hospital, to explore the risk of lung cancer associated with lower levels of BMI at the time of initial diagnosis. Patients All cases and controls were recruited at Tsukuba University Hospital and were identified from the medical record. All patients who were histopatologically con­firmed to have lung cancer were included in this analysis. Pathological diagnoses were based on the 1999 WHO classification of lung neoplasms. Only lung cancer pa­tients as well as control subjects, who had never smoked and without occupational or domestic exposure to other recognized car­cinogens, were included in this study. The hospital-based controls matched by age (±5 yr), gender, and time of hospitalization were recruited from our division, including inpatients with non-malignant respiratory diseases. Patients with other cancer at any site were excluded. This study was ap­proved by the institutional ethics commit­tee of University of Tsukuba. Statistical analysis BMI was calculated from body height and weight, which were measured and reported by nurses at the time of admission, using the formula for Quetelet’s index (expressed in kg/m2). BMI was categorized into four levels on the basis of the distribution in the total study population (BMI < 20.8, leanest; 20.8 = BMI < 22.9, second; 22.9 = BMI < 25.0, third; 25.0 = BMI, highest), which was the same category as our previous study based on the results of community mass screening.16 We used the leanest category (BMI < 20.8) as the reference group for analyses. Logistic regression was used to exam­ine the effect of BMI on lung cancer risk. Results with a p value less than 0.05 were regarded as significant. The software pack­age SSPE (SSPE Inc., Chicago, IL, USA) and Microsoft Excel (Microsoft, Redmond, WA, USA) were used to perform the analyses stated above. Results During the study period, 919 patients with primary lung cancer were diagnosed in our division. Among them, a total of 204 Radiol Oncol 2006; 40(4): 239-44. (22.2 %) documented never-smokers with primary lung cancer were registered. Table 1 shows the characteristics of 204 lung cancer cases. Three-fourths of all cas­es were women. Although the age range for this study was between 22 and 85 years, median age of the male and female patients was 64 and 67 years, respectively. Twenty-four (64.9 %) of 37 male patients, and 151 (90.4 %) of 167 female patients had lung ad-enocarcinoma. There was no patient with small cell lung cancer in both genders (Table 1). Among the 204 patients, 81 had stage IA-IIIA, 35 had stage IIIB, and 88 had stage IV disease. Table 2 shows odd ratios and 95 percent confidence intervals for the association between the risk of lung cancer and lower levels of BMI at the time of diagnosis with lung cancer. When compared with BMI of the leanest group (BMI<20.8) in men, no inverse association between BMI and lung cancer was observed after the adjustment for age (the second BMI group: BMI= 20.8 to < 22.9; p = 0.683, the third BMI group: BMI= 22.9 to < 24.9; p = 0.745, and the high­est BMI group: BMI= 25.0; p = 0.327). In women, no association was found between Table 2. Association of BMI with lung cancer Table 1. Characteristics of 204 lung cancer patients Characteristics Men Women Number of patients 37 167 Age (median, range), Yr 64, 22 – 80 67, 35 – 85 Histologic types Adenocarcinoma 24 151 Squamous cell carcinoma 10 9 Large cell carcinoma 1 4 Others 2 3 Stage IA – IIIA 11 70 IIIB 11 24 IV 15 73 BMI and lung cancer in these three BMI group (the second BMI group, p = 0.639; the third BMI group, p = 0.667; the high­est BMI group, p = 0.978) when compared with that of the leanest BMI group. In male adenocarcinoma, the second BMI group (p=0.967), the third BMI group (p=0.310), and the highest BMI group (p = 0.378) did not exhibit higher odd ratio than that of the leanest BMI group after the adjustment for age. As above mentioned, 175 of 204 patients were adenocarcinomas. There were too-few BMI Case Patients, No. ControlSubjects No. OR 95% CI p-value Men < 20.8 20.8 – 22.8 22.9 – 24.9 = 25.0 13 10 5 9 32 20 10 13 1.0 0.8 0.8 0.6 0.3 – 2.2 0.2 – 2.8 0.2 – 1.7 0.683 0.745 0.327 Women < 20.8 20.8 – 22.8 22.9 – 24.9 = 25.0 67 43 23 34 131 75 51 66 1.0 0.9 1.1 1.0 0.6 – 1.4 0.6 – 2.0 0.6 – 1.7 0.639 0.667 0.978 OR: odd ratio; CI: confidence interval Radiol Oncol 2006; 40(4): 239-44. Table 3. Association of BMI with lung adenocarcinoma BMI Case Patients, No. ControlSubjects No. OR 95%CI p-value Men < 20.8 20.8 – 22.8 22.9 – 24.9 = 25.0 8 6 4 6 26 19 6 11 1.0 1.0 2.2 1.7 0.3 – 3.5 0.5 – 9.6 0.5 – 6.3 0.967 0.310 0.378 Women < 20.8 20.8 – 22.8 22.9 – 24.9 = 25.0 61 36 22 32 120 67 46 58 1.0 1.1 0.9 1.1 0.6 – 1.8 0.5 – 1.7 0.6 – 1.8 0.831 0.841 0.762 OR: odd ratio; CI: confidence interval patients to stratify the histologic subtypes in both men and women, therefore, we examined the association only in adenoca­cinoma cases (Table 3). In men, the second BMI group (p = 0.967), the third BMI group (p = 0.310), and the highest BMI group (p = 0.378) did not exhibit higher odd ratio than that of the leanest BMI group after the ad­justment for age. In women, no association was found between BMI and lung cancer in these three BMI groups (the second BMI group, p = 0.831; the third BMI group, p = 0.841; the highest BMI group, p = 0.762) when compared with that of leanest BMI group. For adenocarcinoma, therefore, the inverse association was not observed in both genders. Discussion An inverse gradient between BMI and the incidence of lung cancer has been reported in several case control and cohort studies.1­9 However, the interpretation of the asso­ciation between low BMI and lung cancer is complicated by the fact that low BMI may be influenced by other factors such Radiol Oncol 2006; 40(4): 239-44. as smoking. It is possible that smokers tend to have lighter body weight, possibly a consequence of the metabolic effects of nicotine. In order to investigate the association between lower levels of BMI and the risk of lung cancer in never-smoking patients, we, therefore, conducted a hospital-based case-control study. The results of the present study indicate two important points. The first point of importance is that we found the absence of an inverse gradient between BMI and the risk of lung cancer in never-smoking female patients, which was consistent with our recent study of subjects participating in a mass screening program.16 Interestingly, Rauscher et al reported an elevated risk of lung cancer associated with not “low” but “high” levels of BMI in non-smoking female patients.17 The difference between the find­ings of Rauscher and our own was prob­ably due to different study populations. All patients and controls in our study were never-smokers. On the other hand, howev­er, not all subjects in the study by Rauscher et al were never-smokers.17 They included 188 patients who haven’t smoked more than 100 cigarettes in their lifetime and 224 patients who haven’t smoked more than 100 cigarettes during the last 10 years.17 Therefore, there was a possibility that the results were influenced by the residual ef­fects of smoking. The majority of published results inves­tigating the association between lower BMI and lung cancer risk were based on stud­ies conducted in Western countries where the prevalence of obesity is high.2,8,17 The difference in categorized BMI levels might also influence the difference between the findings of previous authors and our own. The second important point is that both thin male and female never smokers did not have an increased risk of lung adeno-carcinoma. The results of the present study indicate that lower BMI is not significantly associated with the risk of lung adenocarci­noma. Some lung adenocarcinomas in never smokers can arise without the growth pro­moting effects of the carcinogens present in cigarette smoke,18 one can postulate that the mechanism of carcinogenesis of adenocarcinomas arising in smokers and never-smokers may be different, and these distinct tumorigenic mechanisms can imply differences in tumor biology, demographic characteristics as Brownson and colleague suggested.19 Although we showed the above-men­tioned two findings of importance, we must acknowledge the limitation of this study. First, it was a hospital-based case-control study. As with any hospital-based case-control study, it has been suggested that such case-control studies may reflect the presence of disease other than lung cancer in control subjects.2 The second limitation of the present study is that it included only small number of patients and controls in a single institute. The third, 123 (60.3%) of 204 patients had locally advanced or meta­static lung cancer and stage of the disease would be important in that more advanced disease might itself be associated with weight loss. It is interesting to know the as­sociation between BMI and the risk of lung cancer among patients with early disease, but we could not evaluate the association because of small number of study popula­tion. The last, we could not examine the im­pact of environmental tobacco smoke (ETS) exposure on this association. An accurate assessment of the amount of ETS exposure in never smokers is necessary for determin­ing the lung cancer risks associated with ETS exposure. It is well known that smokers tend to be leaner than non-smokers.1,10-15 Several previous studies have reported an asso­ciation between leanness and risk of lung cancer, mainly among smokers,12,14 and among men with smoking-related disease.14 Inconsistence with previous studies,1-9 our results indicated the possibility that the previously reported association between leanness and the risk of lung cancer might be influenced by other factors such as smoking. A large cohort study will be needed to confirm the current results. References 1. Nomura A, Heilbrun LK, Stemmermann GN. Body mass index as a predictor of cancer in men. J Natl Cancer Inst 1985; 74: 319-23. 2. Kabat GC, Wynder EL. Body mass index and lung cancer risk. Am J Epidemiol 1992; 135: 769-74. 3. Knekt P, Heliovaara M, Rissanen A, Aromaa A, Seppanen R, Teppo L, et al. Leanness and lung-cancer risk. Int J Cancer 1991; 49: 208-13. 4. Chyou PH, Nomura A, Stemmermann GN. A prospective study of weight, body mass index and other anthropometric measurements in relation to site-specific cancers. Int J Cancer 1994; 57: 313-7. 5. Drinkard CR, Sellers TA, Potter JD, Zheng W, Bostick RM, Nelson CL, et al. Association of body mass index and body fat distribution with risk of lung cancer in older women. Am J Epidemiol 1995; 142: 600-7. Radiol Oncol 2006; 40(4): 239-44. 6. Kark JD, Yaari S, Rasooly I, Goldbourt U. Are lean smokers at increased risk of lung cancer? The Israel Civil Servant Cancer Study. Arch Intern Med 1995; 155: 2409-16. 7. Goodman MT, Wilkens LR. Relation of body size and risk of lung cancer. Nutr Cancer 1993; 20: 179­86. 8. Olson JE, Yang P, Schmitz K, Vierkant RA, Cerhan JR, Sellers TA. Differential association of body mass index and fat distribution with three major histologic types of lung cancer: evidence from a cohort of older women. Am J Epidemiol 2002; 156: 606-15. 9. Albanes D, Jones DY, Schatzkin A, Micozzi MS, Taylor PR. Adult stature and risk of cancer. Cancer Res 1988; 48: 1658-62. 10. Garrison RJ, Feinleib M, Castelli WP, McNamara PM. Cigarette smoking as a confounder of the re­lationship between relative weight and long-term mortality: the Fremingham Heart Study. JAMA 1983; 249: 2199-203. 11. Vandenbroucke JP, Mauritz BJ, de Bruin A, Verheesen JH, van der Heide-Wessel C, van der Heide RM. Weight, smoking, and mortality. JAMA 1984; 252: 2859-60. 12. Sidney S, Friedman GD, Siegelaub AB. Thinness and mortality. Am J Public Health 1987; 77: 317-22. 13. Marti B, Tuomilehto J, Korhonen HJ, Kartovaara L, Vartiainen E, Pietinen P, et al. Smoking and lean­ness: evidence for change in Finland. BMJ 1989; 298: 1287-90. 14. Wannamethee G, Shaper AG. Body weight and mortality in middle aged British men: impact of smoking. BMJ 1989; 299: 1497-502. 15. Henley SJ, Flanders WD, Manatunga A, Thun MJ. Leanness and lung cancer risk: fact or artifact? Epidemiology 2002; 13: 268-76. 16. Kanashiki M, Sairenchi T, Saito Y, Ishikawa H, Satoh H, Sekizawa K. Body mass index and lung cancer. A case-control study of subjects participat­ing in a mass-screening program. Chest 2005; 128: 1490-6. 17. Rauscher GH, Mayne ST, Janerich DT. Relation between body mass index and lung cancer risk in men and women never and former smokers. Am J Epidemiol 2000; 152: 506-13. 18. Nordquist LT, Simon GR, Cantor A, Alberts WM, Bepler G. Improved survival in never smokers vs current smokers with primary adenocarcinoma of the lung. Chest 2004; 126: 347-51. 19. Brownson RC, Loy TS, Ingram E, Myers JL, Alavanja MC, Sharp DJ, et al. Lung cancer in non­smoking women. Histology and survival patterns. Cancer 1995; 75: 29-33. Radiol Oncol 2006; 40(4): 239-44. Radiol Oncol 2006; 40(4): 245-57. Schedule-dependency of doxorubicin and vinblastine in EAT tumours in mice Marija Auersperg, Ana Pogacnik, Veronika Kloboves-Prevodnik, Gregor Serša, Maja Cemažar Institute of Oncology Ljubljana, Ljubljana, Slovenia Background. Antitumour schedule-dependency of the doxorubicin and vinblastine combination was ex­plored. Materials and methods. Intraperitoneal Ehrlich ascites tumours (EAT) syngeneic to CBA mice were treated with vinblastine or doxorubicin alone, or in combined treatment schedules. Results. Combinations of doxorubicin and vinblastine administered at 48-h, but not at 24-h interval, regard­less of the sequence of drugs, significantly reduced the number of tumour cells in the ascites in comparison with all other treatments. In the combined treatment schedules, the predominant morphological changes as well as DNA distribution pattern were dependent on the first drug applied. Regardless of the sequence of the drugs, median survival times of animals did not significantly differ between the treatment groups. Conclusions. The effect of combination of vinblastine and doxorubicin is schedule-dependent. The time interval, but not the sequence of drugs seems to be crucial for the observed effect. The data from preclinical studies are important for planning combined treatment schedules in clinical setting. Key words: carcinoma Ehrlich tumor – drug therapy; doxorubicin; vinblastine; drug administration schedule Introduction Doxorubicin (Doxo) and vinblastine (VLB) or their analogues are used in combined treatment schedules for a variety of ma­lignant tumours, i.e. breast, ovarian, lung, urothelial cancer and Hodgkin disease.1-6 The combination chemotherapy is used Received 28 November 2006 Accepted 10 December 2006 Correspondence to: Prof. Maja Cemažar, PhD, Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. Phone: +386 (0)1 5879 544; Fax: +386 (0)1 5879 434; E-mail: mcemazar@onko-i.si with the aim to enhance antitumour effi­cacy. In planning chemotherapy protocols in patients, drugs with different mecha­nisms of action, non–cross-resistant and with non-overlapping toxicities are used. In planning the intervals between sequential cycles of chemotherapy mostly the toler­ance of bone marrow is taken into consid­eration. However, in clinical setting, atten­tion is rarely paid to possible drug interfer­ence and schedule dependency of the drug combinations. There are usually no data on the sequence and exact timing of par­ticular drugs in combined chemotherapy protocols. Schedule dependency and drug interference has been extensively studied in tumour models.7-11 The implementation of results from preclinical studies could make planning of treatment schedules more rational and thus improve the effect of chemotherapy in patients. In our clinical research on the combined treatment of anaplastic thyroid carcinoma, VLB or Doxo combined with radiation was a promising treatment for achieving local control of the primary tumour.12 A logic fur­ther development of treatment of this very aggressive tumour would be a schedule com­bining both Doxo and VLB with radiation. In our previous preclinical and clinical studies, we demonstrated that, after pretreatment of tumour cells with VLB, the accumula­tion of bleomycin or cisplatin in the tumour cells was increased.13,14 The increased cell membrane permeability and consequently a better penetration of the drug into the cell was the proposed mechanism of action. It would be clinically relevant to explore if pretreatment with VLB also influences the accumulation of Doxo in the tumour cells. The primary objective of the current work was to explore in a preclinical study whether there is a schedule-dependency of the combination of VLB and Doxo. The second objective was to find out whether, after pretreatment with VLB, the accumula­tion of Doxo within the tumour cells could be increased. Materials and methods Drug formulation VLB (Vinblastine sulphate, Lilly France S.A., Fagersheim, France) was dissolved in 0.9% NaCl solution at a concentration 2.5 µg/ml. Doxorubicin (Doxo; Doxorubin, Pharmachemie B.V., Haarlem, The Netherlands) stock solution (2 mg/ml) was further diluted in 0.9% NaCl solution to achieve doses of 0.9 to 3.6 mg/kg. Each ani­mal was injected i.p. with adjusted volume (approx 0.5 ml) of drug solution to achieve VLB dose of 62.5 µg/kg and different Doxo doses ranging from 0.9 to 3.6 mg/kg. This low VLB dose was selected according to our previous studies where we demonstrated that this dose significantly affected cell membrane fluidity with a minimal effect on cell survival.15,16 All doses used were far below the maximal tolerated dose level. Animals Inbred CBA mice were purchased from the Institute of Pathology, Medical Faculty Ljubljana (Slovenia). Mice were maintained at a constant room temperature (22°C) and natural day/night light cycle in a conven­tional animal colony. Before experiments, mice were subjected to an adaptation peri­od of at least 10 days. Mice of both sexes, in good condition, weighing 22-30 g, without signs of infection, 10-15 weeks old, were in­cluded in the experiments. Animal studies were carried out according to the guidelines of the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia, permis­sion No. 323-02-200/2004 and in compli­ance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Bethesda MD). Tumour model Intraperitoneal (i.p.) Ehrlich ascites carci­noma (EAT) syngeneic to CBA mice was used in the study. The tumour was main­tained i.p. as ascites by serial transplanta­tions once a week. For the transplantation of i.p. tumours, the tumour cells from the donor mouse were harvested by peritoneal lavage with 4 ml of 0.9% NaCl solution, washed and resuspended at a concentra­tion of 3 x 106 cells/ml. The tumours were transplanted by i.p. injection of 1.5 x 106 Radiol Oncol 2006; 40(4): 245-57. viable EAT cells in 0.5 ml 0.9% NaCl solu­tion. Cell viability, determined by Trypan dye exclusion test, was over 95%. Treatment protocol In the first part of the study, different doses of Doxo (0.9, 1.8 and 3.6 mg/kg) were test­ed in order to determine the cell survival, accumulation of Doxo in the tumour cells and effects of Doxo on DNA distribution. Three days after tumour transplantation, the animals were treated with Doxo and thereafter sacrificed at different post treat­ment intervals (24, 48 and 72 h) to evaluate the treatment effectiveness. In the combined treatment schedule, the animals were randomly allocated three days after tumour transplantation into the following groups: control (i.p. treated with 0.9% NaCl solution), VLB alone, Doxo alone, VLB followed by Doxo, Doxo followed by VLB and both drugs given simultaneously. The time interval between i.p. injection of the first and second drug was 24 or 48 h. When the chemotherapy with VLB or Doxo was tested alone, 0.9% NaCl was injected 24 h or 48 h afterwards, as a sham interven­tion (Table 1). The mice were sacrificed 24 h after the completion of therapy by cervical dislocation. Each experimental group con­sisted of at least 3 mice and the data were pooled from 2-3 independent experiments. Cell number, flow cytometric analysis of DNA content, Doxo fluorescence and cell morphology Twenty-four hours after the completion of therapy, the mice were sacrificed and tu­mour cells were harvested by peritoneal lavage with phosphate-buffered saline sup­plemented with 20% bovine serum albumin. The tumour cells harvested from individual animals were used for the measurement of cell number, flow cytometric DNA measure­ments and cell cycle analysis, Doxo accu­mulation and study of cell morphology. The effect of different treatments on cell survival Table 1. Treatment protocol. A – 24 h interval between treatments; B. 48 h –interval between treatments. A B Group Day 0 Day 3 Day 5 Day 6 Radiol Oncol 2006; 40(4): 245-57. was determined by counting the tumour cells in the peritoneal lavage of the animals by means of haemocytometer. The results of cell number were presented as the percent of cells compared to the number of cells in the control, saline treated animals. For the flow cytometric DNA measurements, the cells were prepared according to a modi­fied Otto method.17 In brief, the cells were treated for 20 min with a solution consist­ing of 0.2 M citric acid and 0.5% Tween 20, and then fixed with 70% ethanol for at least 24 h. After the treatment with 0.5% pepsin (Serva, Heilderberg, Germany) for 5 min, the cells were stained with 4’6-diamidino-2 phenylindole (DAPI, Serva) for DNA. The measurements of DNA content of cells were performed using a PAS III (Partec, Münster, Germany) flow cytometer. The results were presented in the histograms of cell number against fluorescence. The data were analyzed with Multicycle AV (Phoenix Floe Systems, San Diego, CA) program. For flow cytomet­ric measurement of Doxo accumulation in the tumour cells, the cells were centrifuged and resuspended in phosphate buffered saline at a concentration of 5x105 cells/ml. The samples were analysed on FacsCalibur flow cytometer (BD PharMingen, San Jose, California, USA) using 585/42 bandpass filter and the results were presented in the histograms of cell number against fluores­cence. In addition, the cell samples counter-stained with DAPI to distinguish between dead cells (DAPI positive) and the cells with Doxo accumulation were studied on fluorescence microscope. Cell morphology was studied on the cell smears stained with Giemsa (Merck, Darmstadt, Germany). Statistical analysis The data are presented as arithmetic means ± SE (standard error of the mean). The significance of the effect was determined using post-hoc Tukey’s t-test after One-way analysis of variance was performed; the lev­els of less than 0.05 were taken as indicative of significant differences. Survival curves were plotted by the Kaplan-Meier method. The differences between the survival curves were determined using Dunn’s method af­ter Kruskal-Wallis One way analysis of vari­ance on ranks was performed. Statistical analysis was carried out using SigmaStat statistical software (SPSS, Chicago, USA). Results Effect of different Doxo doses in EAT tumour cells The growth curves of the cells in the ascites of CBA mice treated with different Doxo doses did not differ up to 48 h post treat­ment. Only at 72 h post treatment, already the lowest dose of Doxo (0.9 mg/kg) induced a significant reduction of the number of cells in the ascites (Figure 1). The increase in Doxo dose resulted in an increased cyto-toxicity at this time interval. However, there was no statistical difference between the two higher doses tested (1.8 and 3.6 mg/kg). Based on these results, the lowest, rela­tively non-cytotoxic Doxo dose (0.9 mg/kg) was chosen for the subsequent experiments combining VLB and Doxo. In order to detect a possible potentiation of Doxo or VLB cyto-toxicity in combined therapy schedules, an excessive cell kill caused by higher dose of Doxo alone would not be desirable. Beside cytotoxicity of different doses of Doxo, intracellular accumulation of Doxo, morphological changes and DNA distribu­tion in EAT tumour cells were also studied. From the flow cytometric measurements of number of cells with internalized Doxo it was evident that the number of fluorescent cells did not differ between the doses tested at 24 and 48 h post treatment, whereas at 72 h post treatment, significantly less fluorescent cells were observed at two Radiol Oncol 2006; 40(4): 245-57. Figure 1. Cytotoxic effect of Doxo to EAT tumour cells in ascites. EAT tumour cell number as a function of time in the ascites of mice treated with different doses of Doxo injected i.p. Points are mean of 9 mice per group. *p<0.05 compared to control group. higher doses because of significant cell kill induced by these doses (Figure 2). However, the amount of Doxo in the tumour cells increased in a dose-dependent manner, as evident from the median value of the peak of fluorescence intensity of cells and fluorescence microscopy (Figure 3). Median value of the peak of fluores­cence intensity at the lowest dose was 167 and increased to 320 at 3.6 mg/kg Doxo 24 h post treatment (data not shown).The cell cycle phase distribution in EAT tumour cells 48 h post treatment demonstrated that Doxo greatly reduced the number of cells in G1 phase of cell cycle and caused a block in G2M compartment (see Figure 6). The Figure 2. Percentage of EAT tumour cells with Doxo accumulation as determined from fluorescent histograms obtained by flow cytometer. Points are mean of 9 mice per group .* p<0.05 compared to treatment with Doxo dose of 0.9 mg/kg. Radiol Oncol 2006; 40(4): 245-57. morphological changes observed in the cell smears prepared from the same samples corresponded to the changes in cell cycle phase distribution. Enlarged cells with en­larged nuclei and nucleoli were observed at all doses tested compared to the untreated control cells. In addition, mitoses were rare and degenerative changes, such as poto- Figure 4. Survival of EAT tumour cells after treatment with 24-h (black bars) or 48-h (grey bars) interval be­tween the treatments with Doxo and VLB. Pertinent control groups, i.e. treatment with either of the drugs alone and VLB + Doxo given simultaneously, are included. For protocol see Table 1. Cells from ascites were harvested 24 h after completion of treatment. Bars are mean of at least 6 mice per group. * p<0.05 compared to all treatment groups; ** p<0.05 com­pared to treatment with VLB, but not to treatment with Doxo; *** p<0.05 compared to control. Figure 5. Accumulation of Doxo in EAT tumour cells. Percentage of Doxo positive EAT tumour cells after treatment with 24-h (black bars) or 48-h (grey bars) time interval between Doxo and VLB administration. Pertinent control groups, i.e. treatment with either of the drugs alone and VLB+Doxo given simultane­ously, are included. For protocol see Table 1. Cells from ascites were harvested 24 h after completion of therapy. Bars are mean of at least 6 mice per group.* p<0.05 compared to treatment with Doxo and VLB + Doxo. cytosis and fragments of cytoplasm, were observed at all doses tested (see Figure 7). Effects of different VLB and Doxo treatment combinations delivered with a 24-h interval between the administration of the two drugs in EAT tumour cells To determine the effect of VLB and Doxo combinations delivered with a 24-h inter­val between the two drug administrations on the survival of EAT tumour cells, the cell number was determined in the ascites. All the treatments significantly reduced the EAT tumour cell number in the ascites compared to the untreated control animals. However, there was no significant reduc­tion in cell number following the treatment with different treatment combinations com­pared to the treatment with Doxo alone. The treatment combinations in either of the schedules reduced the survival only to the level of the survival induced by Doxo alone (Figure 4). In contrast, compared to the treatment with VLB alone, the treat­ment combinations with two drugs, inject­ed simultaneously or with Doxo preceeding Radiol Oncol 2006; 40(4): 245-57. VLB resulted in significantly reduced cell number (Figure 4). The percentages of fluorescent (Doxo positive) cells measured 24 h after the com­pletion of treatment were the same in all groups regardless of the treatment sched­ule (Figure 5). The DNA distribution measurement of tumour cells from the animals treated with Doxo alone showed an increased G2M com­partment of the cell cycle. In the cytological smears (Doxo 24 h) prepared from the same samples as for the DNA measurements, en­larged cells with enlarged nuclei and nucle­oli were observed. Mitoses were very rare. The same effects on the cells were seen also in the samples when Doxo preceded VLB for 24 h as well as when the drugs were given simultaneously. Rare mitoses in cyto-logical smears together with enlarged G2M compartment in DNA histograms speaks for a block in G2 phase of the cell cycle. The DNA distribution measurements of the cells treated with VLB alone (VLB 24 h) or when VLB preceded Doxo for 24 h showed the cells with very high DNA values, but with no distinctive peaks (data not shown). The cells taken from the same samples were enlarged, but to the lesser degree than the cells treated with Doxo. Multinucleated cells and cells with irregular mitoses were also observed (data not shown). In cytological smears of all treatment groups, a high percentage of inflammatory cells was observed, which was also shown in DNA histograms. Effects of different VLB and Doxo treatment combinations delivered with a 48-h interval between the administration of the two drugs in EAT tumour cells The cytotoxic effects of VLB and Doxo combinations delivered with a 48-h interval Radiol Oncol 2006; 40(4): 245-57. between the applications of the two drugs were also tested. In contrast to the 24-h interval between the drugs, at the 48-h in­terval, both treatment combinations (VLB preceding Doxo or VLB following Doxo) resulted in a significantly reduced cell sur­vival in comparison to all other treatment groups (Figure 4). Measurement of percentage of fluores­cent cells using flow cytometer demon­strated that, in the treatment combinations with the 48-h interval, the number of Doxo positive cells was significantly lower than in the treatment with Doxo alone or with both drugs injected simultaneously (VLB + Doxo). The lower percentage of fluorescent cells observed after the treatments can be ascribed to a significant cell kill induced by these treatments (Figure 5). This is in agree­ment with the results observed when the animals were treated with increasing doses of Doxo alone. In the group treated with Doxo and VLB applied with the 48-h interval between the two drug administrations, the DNA distribution measurements of the samples showed an increased G2M compartment. The same phenomenon was observed when the animals were treated with Doxo alone or when Doxo and VLB were applied si­multaneously. The DNA histograms of the samples from VLB treated animals or when VLB preceded Doxo showed less distinctive peaks and cells with very high DNA values (Figure 6). Morphological changes corre­sponded to the measured DNA content in the cells (Figure 7). In the experiments when VLB preceded Doxo, the effect of VLB was dominant (multinucleated cells), while in the case when Doxo preceded VLB, the dominant effect on the cell morphology was due to Doxo (enlarged cells). However, in both cases, extremely enlarged cells with potocytosis were also observed. When the drugs were given simultaneously, a certain percentage of unaffected tumour cells were observed, the rest of them displayed mor­phological changes that could be ascribed predominantly to Doxo action. In other treatment groups (VLB 48 h and Doxo 48 h alone), the cells displayed typical changes pertinent to the action of the drug. The cells treated with VLB alone were enlarged. In addition, irregular mitoses and multi-nucleated cells with potocytosis were also observed. The cells treated with Doxo alone were also enlarged having enlarged nuclei and nucleoli. In cytological smears of all treatment groups, a high percentage of inflammatory cells was observed, which was also shown in DNA histograms, as a diploid peak. Effect of different VLB and Doxo treatment combinations on animal survival Survival of animals was also determined after various schedules of Doxo and VLB treatments delivered with 24-h and 48-h in­tervals between the injections of the drugs. Despite the significant difference in reduc­tion of EAT tumour cell number in the ascites after the treatment with Doxo and VLB delivered with the 48-h interval be­tween the injections, this reduction did not translate into statistically significantly pro­longed animal survival. Regardless of the schedule of the treatment, median survival times did not significantly differ between the treatment groups either delivered with the 24-h interval (data not shown) or 48-h interval between the drug administration (Figure 8). However, there was a trend to prolonged survival in the group treated with Doxo and 48h later with VLB. Discussion The results of our study show that the combination of VLB and Doxo is schedule-dependent. The time interval between the Radiol Oncol 2006; 40(4): 245-57. drug administrations, but not the sequence, seems to be crucial for the obtained effects. The interval of 48 h between the drug ad­ministrations resulted in more pronounced antitumour effectiveness compared to the 24-h interval. In planning multidrug (combined) chem­otherapy in clinical setting, attention is focused mainly on defining an optimal dosage of the drugs in a protocol with ac­ceptable toxicity. However, for the effect of a protocol, beside dosage and toxicity, an interaction of drugs with consequent schedule-dependency could be decisive. A combination of drugs can result in a synergistic, additive or antagonistic effect. In the literature on multidrug schedules in clinical chemotherapy, there are usually only the data on the dosage of drugs and their distribution according to days in a particular cycle of chemotherapy. There are mostly no data on the sequence and exact timing of drugs.18-22 In the reports in which authors define the sequence of drugs, there are usually no data on their exact timing and intervals between drugs 1,4,6, i.e. factors which could have an important influence on the effect of chemotherapy. In clinical stud­ies comparing multidrug chemotherapy to single agent chemotherapy, a clear advan­tage of multidrug chemotherapy over single agents was not always shown. In a meta analysis of randomized trials in metastatic breast cancer, Fossati23 found an advantage of multidrug chemotherapy in the response rate, but only a very modest benefit in survival of patients. Similarly, Ejlertsen19 described a higher response rate with no benefit in survival of patients, whereas Norris and Joensuu found no advantage of combined schedules in metastatic breast cancer.22,24 Among other factors for these conflicting results, schedule-dependency could also play a role. Recently, there have been many reports on schedule-dependency between antra­cyclins and microtubule active drugs.7-11,25 In our work, we explored the schedule-dependency of VLB combined with Doxo in Ehrlich ascites tumour cells in mice. We found that the combination was schedule-dependent. The most cytotoxic combina­tions were VLB injected 48 h before Doxo and the reverse order of the drug injections, Doxo injected 48 h before VLB. Both orders of sequence were equally effective and showed a statistically significant reduction of tumour cell survival compared to all oth­er combinations (Figure 4). The combina­tion of VLB and Doxo was not better than Doxo alone when given simultaneously or with the 24-h interval between the drugs. In contrast to our results, Zeng 10 found an an­tagonistic effect of Doxo and a mitotic poi­son Docetaxel if the two drugs were applied simultaneously or in the sequence Doxo-Docetaxel. The reverse sequence Docetaxel- Doxo delivered with an interval of 12 h between the drugs resulted in an enhanced cytotoxicity. The explanation for the an­tagonism of the sequence Doxo-Docetaxel backed up by flow cytometry was that Doxo produced a block in G2 phase of the cell cycle and thus prevented the mitotic arrest by Docetaxel.10 Similarly, the best inhibition of the tumour by the schedule Docetaxel interval 12 h Doxo was reported by To et. al. in Ehrlich ascites cell tumours bearing mice.9 In contrast to these results, Zoli found a synergistic and not antagonis­tic effect of the sequence Doxo and another microtubule stabilizing agent Paclitaxel.11 The results of schedule-dependency stud­ies are influenced by many factors, such as the dosage, time of exposure to the drugs and the intervals between the injections of drugs. In addition, the results could de­pend on the tumour model as different cell lines can respond with different cell kinetic changes to the same drug.26 Flow cytometric study showed two dis­tinctive patterns of DNA distribution after Radiol Oncol 2006; 40(4): 245-57. the treatment with VLB or Doxo. The latter drug produced a block of cells in the G2M compartment as shown previously27,28, whereas after VLB, a wide scatter of DNA values with less distinctive peaks in com­parison with Doxo were observed. When both drugs were given simultaneously, the effect of Doxo prevailed, whereas in combi­nations delivered with an interval of 24 or 48 h between the drugs, the pattern of the first drug prevailed (Figure 6). Similarly, cytomorphologic studies showed two dis­tinctive patterns of changes after VLB or Doxo. After Doxo, the tumour cells were enlarged with enlarged both nuclei and nu­cleoli. Mitoses in the smears were very rare, which means that the signals appearing in the DNA histograms in the G2M compart­ment represent nuclei blocked in the G2 phase of the cell cycle. In contrast, after VLB, cells with irregular mitoses as well as multinucleated cells as a consequence of unaccomplished cell division were ob­served. In the DNA histograms, scattered, very high DNA values depassing the G2M peak of the tumour were found, corre­spondingly. VLB exerts its action at least in part by binding to tubulin and disturbs the function of microtubules, necessary for the formation of mitotic spindle. After discov­ery of VLB, it was believed that its primary action is depolymerization of microtubules. Only recently it was discovered that, at low doses, VLB stabilizes microtubule dynam­ics and blocks or slows down the mitosis by acting on microtubule dynamics and not by depolymerization as at high doses.29,30 The consequence of impaired function of mitotic spindle is the mitotic arrest and inhibition of cell proliferation. However, as antiproliferative activity of VLB does not correlate well with binding to tubulin, there must be other targets for VLB such as RNA, DNA and lipid biosynthesis.29 Better results of the combinations of VLB and Doxo applied with the 48-h inter­vals over the drugs applied simultaneously can only partly be explained by DNA meas­urements and cell morphology studies. The block in G2 could prevent cells entering mitosis, which is the part of the cell cycle where VLB can exert its maximal effect. We could speculate that the sequence of Doxo and VLB could therefore be self-lim­iting. Such an explanation could be valid for the simultaneous application of both drugs or when the time interval was 24 h where the effect was not better than that of Doxo alone. On the other hand, such an as­sumption does not explain the best results obtained after administering the drugs in 48-h intervals between them irrespective of their sequence. Our DNA measurements in this experiment showed that the block of the cells in G2 was still present 48 h and even 72 h after the application of Doxo, yet the tumour cell toxicity was enhanced with the combination of Doxo and VLB provided the drugs were delivered with the 48-h in­terval between the drug administrations. Moreover, not the sequence of drugs, but the time interval between them was crucial for obtaining better effect. According to our results, the explanation could be that the action of either of the drugs, Doxo or VLB, needs 48 h to make the tumour cells either prone to the action of the other drug or to trigger a cell death pathway. Our second objective was to explore whether pretreatment with VLB will in­crease the accumulation of Doxo in tumour cells. In our previous preclinical study, we showed that VLB increased cell membrane fluidity.16 Pretreatment with VLB increased the uptake of cisplatin into the tumour cells, which led to an increased antitumour effectiveness of cisplatin.14 An increase antitumour effectiveness was also demon­strated for bleomycin applied after VLB.15 In the present study, the pretreatment with VLB did not result either in an increased accumulation of Doxo or in the increased Radiol Oncol 2006; 40(4): 245-57. antitumour effectiveness compared to the treatment with reverse order of the drugs. There are reports that the effect of Doxo is not dependent solely on entering the cell and binding to DNA, but also on binding to the cell membrane, which is very important for inducing the cell death.31-33 Therefore, we assumed that the cytotoxic effect of the combination of Doxo and VLB applied with the 48-h interval between the drug administration might be the result of the effects on DNA, cell membrane events and transmembrane signalling.31-34 Another ex­planation for better results of the schedule with 48-h interval could be a repopulation of tumour cells which might occur after that interval and the elimination of the re­populated cells by the second drug. The fact that no drug combination re­sulted in a prolonged survival of animals (Figure 9) is not surprising. We deliberately used low doses of VLB and Doxo with the aim to demonstrate a possible interaction of the combinations. Even after the best combi­nations with 48-h interval between the drug injections, there were still some unaffected tumour cells in the specimens. In view of rapid repopulation of tumour cells in this fast growing tumour model, the difference in animal survival could not be expected after only one cycle of chemotherapy. In conclusion, the combination of VLB and Doxo is schedule dependent. It seems that for the effect of treatment the time interval between the drug administrations, but not the sequence of drugs, is crucial. Pretreatment with VLB does not increase the accumulation of Doxo in EAT tumour cells. When translating the results of pre-clinical studies to clinical setting, we need to be cautious, since different tumour mod­els used can yield controversial results. Nevertheless, the data from preclinical studies should be taken into consideration when planning combined treatment sched­ules in clinical situation. Acknowledgement The authors acknowledge the financial sup­port of the state budget by the Slovenian Research Agency. References 1. Palmeri S, Leonardi V, Tamburo De Bella M, Morabito A, Vaglica M, Accurso V, et al. Doxorubicin-docetaxel sequential schedule: re­sults of front-line treatment in advanced breast cancer. Oncology 2002; 63: 205-12. 2. Canellos GP, Gollub J, Neuberg D, Mauch P, Shulman LN. Primary systemic treatment of ad­vanced Hodgkin's disease with EVA (etoposide, vinblastine, doxorubicin): 10-year follow-up. Ann Oncol 2003; 14: 268-72. 3. Diehl V. Advanced Hodgkin's disease: ABVD is better, yet is not good enough! J Clin Oncol 2003; 21: 583-5. 4. Dreicer R, Manola J, Roth BJ, See WA, Kuross S, Edelman MJ, et al. Phase III trial of methotrexate, vinblastine, doxorubicin, and cisplatin versus carbo­platin and paclitaxel in patients with advanced carci­noma of the urothelium. Cancer 2004; 100: 1639-45. 5. Johnson E, Lake D, Herndon JE, Box JW, Lynch TJ, Green MR. Phase II trial of vinorelbine plus doxo­rubicin in relapsed small-cell lung cancer: CALGB 9332. Am J Clin Oncol 2004; 27: 19-23. 6. Tambaro R, Greggi S, Iaffaioli RV, Rossi A, Pisano C, Manzione L, et al. An escalating dose finding study of liposomal doxorubicin and vinorelbine for the treatment of refractory or resistant epithe­lial ovarian cancer. Ann Oncol 2003; 14: 1406-11. 7. Akutsu M, Kano Y, Tsunoda S, Suzuki K, Yazawa Y, Miura Y. Schedule-dependent interaction be­tween paclitaxel and doxorubicin in human cancer cell lines in vitro. Eur J Cancer 1995; 31A: 2341-6. 8. Serrano MJ, Sanchez-Rovira P, Algarra I, Jaen A, Lozano A, Gaforio JJ. Evaluation of a gemcitab­ine-doxorubicin-paclitaxel combination schedule through flow cytometry assessment of apoptosis extent induced in human breast cancer cell lines. Jpn J Cancer Res 2002; 93: 559-66. 9. To H, Shin M, Tabuchi M, Sakaguchi H, Takeuchi A, Matsunaga N, et al. Influence of dosing sched­ule on toxicity and antitumor effects of a combi­nation of adriamycin and docetaxel in mice. Clin Cancer Res 2004; 10: 762-9. Radiol Oncol 2006; 40(4): 245-57. 10. Zeng S, Chen YZ, Fu L, Johnson KR, Fan W. In vitro evaluation of schedule-dependent interac­tions between docetaxel and doxorubicin against human breast and ovarian cancer cells. Clin Cancer Res 2000; 6: 3766-73. 11. Zoli W, Ricotti L, Barzanti F, Dal Susino M, Frassineti GL, Milandri C, et al. Schedule-depend­ent interaction of doxorubicin, paclitaxel and gemcitabine in human breast cancer cell lines. Int J Cancer 1999; 80: 413-6. 12. Auersperg M, Us-Krasovec M, Petric G, Pogacnik A, Besic N. Results of combined modality treat­ment in poorly differentiated and anaplastic thy­roid carcinoma. Wien Klin Wochenschr 1990; 102: 267-70. 13. Auersperg A, Erjavec M, Us-Kraševec M. Accumulation of 99mTc-Bleomycin in human squa­mous cell carcinoma in vivo after synchronization by vinblastin. IRCS (Int Res Commun Sys) Med Sci 1975; 3: 560. 14. Cemazar M, Auersperg M, Scancar J, Kirbis IS, Pogacnik A, Sersa G. Schedule-dependent interac­tion between vinblastine and cisplatin in Ehrlich ascites tumors in mice. J Pharmacol Exp Ther 2002; 302: 337-43. 15. Cemazar M, Auersperg M, Sersa G. Antitumor effectiveness of bleomycin on SA-1 tumor after pretreatment with vinblastine. Radiol Oncol 2000; 34: 49-57. 16. Sersa G, Cemazar M, Sentjurc M, Us-Krasovec M, Kalebic S, Draslar K, Auersperg M. Effects of vinblastine on cell membrane fluidity and the growth of SA-1 tumor in mice. Cancer Lett 1994; 79: 53-60. 17. Otto F. DAPI staining of fixed cells for high-resolu­tion flow cytometry of nuclear DNA. Methods Cell Biol 1990; 33: 105-10. 18. Bonadonna G, Bonfante V, Viviani S, Di Russo A, Villani F, Valagussa P. ABVD plus subtotal nodal versus involved-field radiotherapy in early-stage Hodgkin's disease: long-term results. J Clin Oncol 2004; 22: 2835-41. 19. Ejlertsen B, Mouridsen HT, Langkjer ST, Andersen J, Sjostrom J, Kjaer M, Scandinavian Breast Group. Phase III study of intravenous vinorelbine in combination with epirubicin versus epirubicin alone in patients with advanced breast cancer: a Scandinavian Breast Group Trial (SBG9403). J Clin Oncol 2004; 22: 2313-20. 20. Keller AM, Mennel RG, Georgoulias VA, Nabholtz JM, Erazo A, Lluch A, et al. Randomized phase III trial of pegylated liposomal doxorubicin versus vinorelbine or mitomycin C plus vinblastine in women with taxane-refractory advanced breast cancer. J Clin Oncol 2004;22: 3893-901. 21. Nistico C, De Matteis A, Rossi E, Carnino F, Valenza R, Agostara B, et al. Primary chemo­therapy with epirubicin and vinorelbine in women with locally advanced breast cancer. Anticancer Res 2005; 25: 1343-8. 22. Norris B, Pritchard KI, James K, Myles J, Bennett K, Marlin S, et al. Phase III comparative study of vinorelbine combined with doxorubicin versus doxorubicin alone in disseminated metastatic/re­current breast cancer: National Cancer Institute of Canada Clinical Trials Group Study MA8. J Clin Oncol 2000; 18: 2385-94. 23. Fossati R, Confalonieri C, Torri V, Ghislandi E, Penna A, Pistotti V, et al. Cytotoxic and hormonal treatment for metastatic breast cancer: a systemat­ic review of published randomized trials involving 31,510 women. J Clin Oncol 1998; 16: 3439-60. 24. Joensuu H, Holli K, Heikkinen M, Suonio E, Aro AR, Hietanen P, Huovinen R. Combination chem­otherapy versus single-agent therapy as first- and second-line treatment in metastatic breast cancer: a prospective randomized trial. J Clin Oncol 1998; 16: 3720-30. 25. Blagosklonny MV, Robey R, Bates S, Fojo T. Pretreatment with DNA-damaging agents permits selective killing of checkpoint-deficient cells by microtubule-active drugs. J Clin Invest 2000; 105: 533-9. 26. Sciola L, Spano A, Monaco G, Bottone MG, Barni S. Different apoptotic responses and patterns in adhering and floating neoplastic cell cultures: effects of microtubule antagonists. Histochem Cell Biol 2003; 119: 77-90. 27. Gohde W, Meistrich M, Meyn R, Schumann J, Johnston D, Barlogie B. Cell-cycle phase-depend­ence of drug-induced cycle progression delay. J Histochem Cytochem 1979; 27: 470-3. 28. O'Loughlin C, Heenan M, Coyle S, Clynes M. Altered cell cycle response of drug-resistant lung carcinoma cells to doxorubicin. Eur J Cancer 2000; 36: 1149-60. 29. Jordan MA, Thrower D, Wilson L. Mechanism of inhibition of cell proliferation by Vinca alkaloids. Cancer Res 1991; 51: 2212-22. Radiol Oncol 2006; 40(4): 245-57. 30. Jordan MA. Mechanism of action of antitumor drugs that interact with microtubules and tubulin. Curr Med Chem 2002; 2: 1-17. 31. Dalmark M, Hoffmann EK. Doxorubicin (Adriamycin) transport in Ehrlich ascites tumour cells: comparison with transport in human red blood cells. Scand J Clin Lab Inv 1983; 43: 241-8. 32. Larrivee B, Averill DA. Modulation of adriamy­cin cytotoxicity and transport in drug-sensitive and multidrug-resistant Chinese hamster ovary cells by hyperthermia and cyclosporin A. Cancer Chemoth Pharm 2000; 45: 219-30. 33. Triton TR, Yee G. The anticancer agent adriamycin can be actively cytotoxic without entering cells. Science 1982; 217: 248-50. 34. Manche-Boitrel MT, Meurette O, Rebillard A, Lacour S. Role of early plasma membrane events in chemotherapy-induced cell death. Drug Resist Updat 2005; 8: 5-14. Radiol Oncol 2006; 40(4): 245-57. Radiol Oncol 2006; 40(4): 259-71. Cysteine cathepsins, stefins and extracellular matrix degradation during invasion of transformed human breast cell lines Irena Zajc, Aleš Bervar, Tamara T. Lah Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia Background. Human breast cellular model, comprising four cell lines originating from spontaneously im­mortalized human breast epithelial MCF10A cell line, its c-Ha-ras transfectant, MCF10AT, and two tumouri­genic derivatives, cultured from two sequential mouse xenographs, MCF10AT-Ca1a and MCF10AT-Ca1d, were used to compare the relative protein concentration of cathepsins and stefins in single cells. Methods. The relative protein concentration of cathepsins and stefins in single cells was analysed by confo­cal microscopy, and compared to their protein expression in cell homogenates. Results. The most invasive, MCF10AT cell line contained several fold higher protein concentration of cathepsin B and increased levels of stefins, but similar levels of cathepsin L, compared with the parental MCF10A cells. This was associated with five fold higher endocytosis of Matrigel-DQ-collagen IV (DQC) and a simultaneous increase in signal overlap between DQC and cathepsin L as well as DQC and stefin B, but a decrease in that of DQC and cathepsin B overlap in the MCF10AT cells. Simultaneously, increased sig­nal overlaps between both cathepsins and between cathepsins-stefins pairs, were observed in this cell line. Conclusions. These results suggest that the increased collagen endocytosis and degradation in the invasive phenotype significantly affect also the subcellular localization of cysteine cathepsins and stefins. Based on these and the reports of other authors, we hypothesize that the intracellular degradation may also be as­sociated with cathepsin L, whereas cathepsin B in the ras transformed breast cells is involved in both, the intracellular and pericellular degradation of extracellular matrix during cell migration and invasion. Key words: breast neoplasms; tumor cells, cultured; neoplasms invasiveness; cathepsins; extracellular matrix Received 9 October 2006 Accepted 29. November 2006 Correspondence to: Tamara T. Lah, Ph. D., Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Vecna pot 111, 1000 Ljubljana, Slovenia; Phone: +386 1 423 5017; Fax: +386 1 423 5038; E-mail: Tamara.Lah@nib.si ; http://www.nib.si Introduction Human genome is known to contain 11 related, but distinct cysteine proteases be­longing to papain family C1A, cathepsins (Cats) B, L, H, S, K, F, V, X, W, O and C.1 Early studies of CatB and CatL have shown their wide tissue distribution. They are mostly localized to lysosomes, where they can reach up to 1 mM concentration.2 In the tumour cell models, the increased se­cretion of the pro-enzyme and the mature enzyme forms of CatB3,4 and CatL5 after the oncogenic transformation has been re­ported. Besides other proteases, CatB6 and CatL7 can directly degrade components of the extracellular matrix, although under slightly different conditions. It has been shown that CatL activation may be acceler­ated by certain ECM components8, such as proteoclycans.9 Cathepsins may also be involved in the proteolytic cascade10, in which they activate other proteases. In breast carcinoma, altered expression of lys­osomal CatB and CatD and altered subcel­lular trafficking were initially demonstrat­ed by Sameni et al.11 and later confirmed by these and other authors (reviewed in12). The interest in the mechanisms of CatB and CatL regulation and activation increased when several clinical studies have shown elevated mRNA, protein and activity lev­els of CatB and CatL in malignant breast tumour tissues and demonstrated their potential for the prognosis of the disease.13­16 The activities of cysteine cathepsins are regulated by their endogenous inhibitors, a large superfamily of cystatins. The ste-fins (St) family comprise the intracellular inhibitors, of which StA and StB were also found to be altered in tumours and sera of cancer patients.17-20 The structural features of inhibitors and their tight complexes with cathepsins were revisited by Turk and Guncar.21 Presumably, their cytosolic location should guard the subcellular struc­tures from the accidental release of active lysosomal cysteine cathepsins, but their intracellular interactions with their target proteases are still not well understood. To establish whether CatB and CatL are associated with an invasive cell phenotype and with the ability to form tumours after the injection at a secondary site (tumouri­genicity), we have used a cell model of four breast cell lines, originating from parental MCF10A cell, which is described in details in Materials and methods below. In this model, we found that the in vitro invasive­ness did not correlate with cell ability to form malignant tumours in mice, the most invasive cell line being the MCF10AT and selective synthetic inhibitors of CatB and CatL impaired the in vitro invasion of these cells.22 In the present study, we focus on the expression and the localization of CatB and CatL and their inhibitors StA and StB in the transformed breast cells during the invasion through Matrigel-collagen matrix. The aims of this study were: (a) to deter­mine the relative abundance of cathepsins and stefins in different breast cells during their invasion into Matrigel-collagen matrix using confocal microscopy in comparison with their expression levels in cell homoge­nates, (b) to develop the software tools for faster, cost-effective, automatic and more objective single-cell image stack analysis, including analysis of signal overlapping of different antigens and (c) to examine the overlapping signals of cathepsins and stefins inside the tumour cells, along with the degraded endocytosed collagen type IV (DQ-collagen IV) in the parental MCF10A, and invasive MCF10AT cell lines. Materials and methods Cell lines We used a model of four epithelial breast cell lines derived from spontaneously immor­talized cells of a fibrocystic breast patient. The parental cell line was the immortalized diploid cell line MCF10A.23 The MCF10AT line is MCF10A transfected with c-Ha-ras oncogene24,25 and has an acquired ability to grow in immunodeficient mice. MCF10AT­Ca1a and MCF10AT-Ca1d, obtained by Radiol Oncol 2006; 40(4): 257-71. multiple passages of MCF10AT cells in nude mice, are fully malignant. MCF10AT­Ca1a cells mostly produce undifferentiated carcinoma and MCF10AT-Ca1d cells form heterogeneous carcinomas.26 These lines were originated at Barbara Ann Karamanos Cancer Institute (Detroit), and kindly pro­vided by Prof. Bonnie Sloane, Department of Pharmacology, WSU, Detroit MI, USA. Cell lines were grown as described previ­ously.22 Preparation of cell lysates and enzyme-linked immunosorbent assay (ELISA) The cells were scraped and pelleted by cen­trifugation at 150g for 5min. They were ho­mogenized by sequential freezing in liquid nitrogen and thawing at 37oC (3 X) in 50mM Tris buffer, pH 6.9, containing 0.05% (v/v) Brij 35, 0.5mM DTT (dithiothreitol), 5mM EDTA, 0.5mM PMSF (paramethylsulphonyl fluoride) and 10mM pepstatin A. The lysates were centrifuged at 12,000g for 15min and the supernatants stored at –20°C. ELISA kits for human CatB, CatL, StA and StB were obtained from Krka d.d., Slovenia and performed as suggested by the manufacturer. Purified human CatB, CatL, StA and StB were used as the stand­ards. For CatB, rabbit polyclonal anti-CatB antibodies (IgG) and sheep anti-CatB anti­bodies (horseradish peroxidase-conjugat­ed) were used as the capture and detection antibodies, respectively. For CatL, polyclo­nal sheep anti-CatL, for StA, monoclonal mouse anti-StA, and for StB, monoclonal mouse anti-StB antibodies were used as the capture and the detection antibodies. Total protein concentrations were determined using Bradford assay (Bio-Rad, USA). Mean values of at least three independent meas­urements and standard errors of the mean (SEM) were calculated. Statistical signifi­cance was determined with t-test and p < 0.05 was considered significant. Confocal microscopy Each of 8 wells on a Chamber SlideTM (Lab Tec®, Nunc Inc., USA) was pre-coated with 150 µl of fibronectin (16.7 µg/ml, 2.5 µg per well, Sigma) and coated with 200 µl of Matrigel (1 mg/ml, Becton Dickinson, USA), with added 0.2%, or 1% fluorescently labelled DQ-collagen IV, DQC (Molecular Probes, USA). 200,000 cells in 100 µl of me­dium were seeded and grown for another 24 h. Then the cells were washed with PBS and fixed with 200 µl of 3.7% formaldehyde for 30 min at 37 C in 5% CO2. After wash­ing with PBS, cells were permeabilized for 5 min with 150 µl of 0.2% Triton X-100 (Sigma). Polyclonal and monoclonal anti­bodies were tested with comparable inten­sity of labelling and herein mouse clones of the primary antibodies for cathepsins and stefins were used (all by Krka d.d., Slovenia) at 2 mg/ml final concentration. Secondary antibodies, excited at various wavelengths, AlexaFlour 488 rabbit anti-mouse IgG, AlexaFlour 546 goat anti-mouse IgG, and AlexaFlour 633 rabbit anti-mouse IgG, were then applied according to the instructions of the supplier (Molecular Probes, USA). Samples were imaged using laser scanning confocal microscope Leica TCS SP2 at the three channels with excitation wavelengths 488 nm, 546 nm and 633 nm. Images were recorded at 1000× enlargement in series of fifty to eighty 512×512 pixel images (size of a pixel in x and y directions app. 50-300 nm) at 150-1000 nm distance in the z-axis. Each cell (cell group), e.g. each stack of images, was then analyzed with Image software (available on the internet and developed by Wayne Rasband, wayne@codon.nih.gov). Software analysis, customised plug-ins, were developed in our laboratory specifi­cally for this purpose. Due to the space limi­tations these plug-ins are not explained in detail but are available upon request. The first plug-in was developed to detect edges of the cells. The selected regions of Radiol Oncol 2006; 40(4): 257-71. interest were then transformed by another plug-in into a mask, used to separate pos­sible multiple cells. These masks were then used for quantification, with pixels outside the mask taken as zero signals. The algorithm applied to the mask calculates the volume of the cell (in actual units, e.g. µm3), absolute and average values of the signal in each of the three channels (488 nm, 546 nm and 633 nm) and allows signal-overlap measurements of the three chan­nels using a specifically derived formula.27 Another plug-in detects background (noise) structures of non-specific shape and deletes them. Although some noise still has to be omitted manually by checking each slice, our method is much faster, and more pre­cise than manual selection of cell edges. Overlapping signals are the closest this method can get to actually monitor the antigens’ subcellular co-localizations. As the size of the cell in pixels (radius between about 50 and 200 pixels) is not large enough for precise separation and localization of the organelles, the signal overlap relates to the localization of the antigens to the same area of the cell, but not to the co-localiza­tion to the same subcellular organelle. This signal-overlap was determined by calculat­ing “overlap index” for each pixel of the image (500 × 500 × 50-80 pixels). The index was calculated for each pair of the channels separately (488 nm – 546 nm, 488 nm – 633 nm, 546 nm – 633 nm). The index took into account the relative signal intensity (0-256) of each pixel compared to the background of the individual slice (generally between 10 and 20). These relative intensities were therefore generally larger than one but not higher than about 20. Finally, relative signal intensities of the two signals (e.g. at 488 nm and 546 nm) were multiplied so that only in the cases where signals in both pixels were strong, the index could reach high enough values to be taken into consideration for calculation. The average Figure 1. Protein expression of CatB and CatL, StA and StB in the lysates of human breast epithelial cell lines, MCF10A, MCF10AT, MCF10AT-Ca1a and MCF10AT-Ca1d. Cells were grown on Matrigel, homogenized and protein expressions of cathepsins and stefins were determined by ELISAs as described in Materials and methods. Mean values from three independent ex­periments are presented. Error bars depict SEM. The statistical significance was determined by t-test and p < 0.05 was considered significant. The cell lines are listed according to their increased tumourigenicity. (a) CatB protein concentration was the highest in MCF10AT cells (p = 0.003) and significantly lower in MCF10AT-Ca1a (p = 0.002) and MCF10AT-Ca1d cell lines (p = 0.004), compared to MCF10A cells. CatL expression was similar in all four cell lines. (b) StA and StB were both significantly increased in MCF10AT, MCF10AT-Ca1a, and MCF10AT-Ca1d cells (all p<0.005) compared to the parental line. overlap index across the cell and at the wavelength pairs was calculated by the ap­propriate statistical analyses, using up to 50 single-cell measurements for each category, e.g. images with different combinations of labelled Cats B and L, Sts A and B and de­graded DQC, respectively.27 Radiol Oncol 2006; 40(4): 257-71. Results Cathepsins and stefins protein expression in cell cultures by ELISA As previously demonstrated, the MCF10AT, obtained after ras transfection of the paren­tal MCF10A cell line, was the most invasive, whereas the two cell lines, MCF10AT-Ca1a and MCF10AT-Ca1d, which were obtained from xenographts of the MCF10AT cell line, although having higher tumourigenicity than the parental cell line, are less invasive in in vitro Matrigel assays.22-27 CatB protein expression was the highest in MCF10AT (p = 0.003), and significantly lower in MCF10AT-Ca1a (p = 0.002) and MCF10AT­Ca1d cell lines (p = 0.004), compared to MCF10A cells (Figure 1a). CatL expressions were similar in all four cell lines. StA and StB were both significantly increased in MCF10AT, MCF10AT-Ca1a, and MCF10AT-Ca1d cells (all p<0.005) com­pared to the parental line (Figure 1b). Noteworthy, StB expression was the highest in MCF10AT, similar as was that of CatB. The relative expression of these antigens was similar as observed previously,22 when these cells were grown on the plastic sur­face, although on the Matrigel, all protein levels are about two fold lower compared to the plastic surface. Noteworthy, the mo­lar ratios calculations (between cathepsins and stefins) showed that there was about 60-90 fold overexpression of cathepsins in MCF10A, and slightly decreased in the malignant cells lines. Analysis of cathepsins, stefins and the Matrigel-collagen by confocal microscopy A representative example of immunohis­tochemical labelling in a model of the four lines is shown on Figure 2. The quantitative fluorescence analysis of single cell images by confocal microscopy parallels the data in cell lysates. Quantification of CatB in MCF10A and MCF10AT cells by confocal microscopy showed its significantly higher expression in the MCF10AT than in the pa­rental cells. The increased pericellular distri­bution of CatB was observed in MCF10AT, confirming the previous findings.11,28 In MCF10AT-Ca1a and MCF10AT-Ca1d cells, spreading of CatB immunostaining over the whole cell was observed. CatL distribution was not significantly changed in these cells compared to MCF10A cells. The expression of stefins was markedly elevated, particu­larly in tumourigenic cell lines, compared to MCF10A cells. The last panel represents degraded DQC (green) in the four cell lines, grown on Matrigel, enriched with DQC. Slight, peri-cellular degradation of DQC was observed in the parental cells. In contrast, the intra­cellular degradation of DQC in MCF10AT was about 5-times higher than in MCF10A cells and both, extracellular and pericellu­lar degradation of DQC was observed. The intracellular degradation of DQC was also abundant in the tumourigenic cells in spite of the fact that the latter are less invasive in vitro.22 Taken together, these results show that the parental cell line contained the least of endocytosed matrix, least disper­sion of cathepsins staining and low levels of both stefins. Subcellular distribution and co-localization of antigens The 3-D analysis was performed on a sample size a few hundred single cells. These were imaged in over 100 stacks of 50-80 slices with the size of 512 × 512 pixels at three different wavelengths of the excitation la­ser, as described in Material and methods. For reasons of clarity, we will present the process of the confocal image analysis us­ing one slice of a single stack. Figure 3a presents a raw image of the MCF10AT cell labelled with DQC (488nm channel – left), Radiol Oncol 2006; 40(4): 257-71. Figure 2. Immunolabeling of CatB, CatL, StA, StB and degraded DQ-collagen IV (DQC) in MCF10A, MCF10AT, MCF10AT-Ca1a and MCF10AT-Ca1d breast cells. Cells were grown on Matrigel and prepared for confo­cal microscopy as described in Material and methods. All images were taken at the same resolution. Size bar indicates 10µm. The cells were labelled with specific mouse antibodies and the fluorescence signals were coloured arbitrarily red for CatB, yellow for CatL, dark blue for StA and turquoise for StB. The degraded DQC (green) was also monitored. CatB spreading over the whole cell and its’ more pericellular distribu­tion was observed in MCF10AT, MCF10AT-Ca1a and MCF10AT-Ca1d cells. CatL distribution was similar in all cells (upper panel). The expression of StA and StB was elevated in the MCF10AT and both tumourigenic lines (MCF10AT-Ca1a and MCF10AT-Ca1d) compared to MCF10A cells (middle panel). The concentration of DQC in Matrigel for MCF10AT cells has to be 5-times lower than in other cell lines, otherwise the signal was too strong for imaging. Intracellularly degraded DQC was expressed at a higher level in MCF10AT, MCF10AT-Ca1a and MCF10AT-Ca1d cells compared to the parental cells. Radiol Oncol 2006; 40(4): 257-71. Figure 4. Signal-overlap of cathepsins, stefins and DQ-col­lagen in MCF10A and MCF10AT cells. The overlap index was calculated as described in Materials and methods. Error bars depict SEM. Note the different scales of panels A and B. (a) Overlap in­dexes between cathepsins and stefins were measured in all pairs. The signal overlap was higher in MCF10AT cells than in the MCF10A cells. This increase in the signal overlap was highly significant between CatB and both stefins, and between CatL and StB (p = 0.0001), less so between CatB and CatL (p = 0.0065), and barely significant between CatL and StA (p = 0.049). In invasive MCF10AT cells, CatB-CatL overlap­ping was relatively low compared with the overlap indexes between CatB and stefins. (b) Overlap indexes between DQC and CatB, CatL, StA, and StB showed a significant increase in the signal overlap observed in MCF10AT compared to MCF10A cells between DQC and CatL (p = 0.0002) and DQC and StB (p = 0.0155). In contrast, the overlap index between DQC and CatB was significantly lower (p = 0.0018) in MCF10AT cells. That between DQC and StA was similar in both cell lines. the antibodies against CatL (546nm – mid­dle) and the antibodies against StB (633nm – right). To these raw images we applied the first and most extensive plug-in, that used an algorithm designed to detect whether or not there is a significant gradient of signal strength, which dubbed an edge at each individual point. The plug-in also detected the angle at which this edge was going through the pixel in question. To achieve this, a roster of 9 × 9 pixels surrounding the pixel in question was analysed. If adjacent pixels had edges running in the same direc­tion (angles not differing by more than a preset number, e.g. 5° or 10°), an edge was drawn on the resulting image (Figure 3b). In Figure 3c, the results of the second plug­in are shown. It was designed to check for the size and shape of the resulting closed shapes and to fill the shapes that were meeting the criteria of size and circularity. The next plug-in (Figure 3d) combined all three channels into one (Figure 3d mid­dle). The red line was manually drawn after reviewing single slices (Figure 3d left) and comparing it to a composite image throughout the depth of all 55 slices (Figure 3d right). Figure 4a shows the signal overlap be­tween both cathepsins and between cathe­psins and stefins. In MCF10AT cells, CatB overlapped better with either of the stefins than CatL did. Consistently a higher signal overlap observed in MCF10AT cells than in the paternal MCF10A cells, may be also due to the increased immunostaining of both cathepsins and stefins in the former cell line. Figure 4b presents the signal over­lap indexes between the degraded DQC (Matrigel) and cathepsins and stefins in the invasive MCF10AT compared with the benign parental cell line. A significant in­crease in the signal overlap was observed in MCF10AT compared to MCF10A cells between DQC and CatL (p = 0.0002) and DQC and StB (p = 0.0155). In contrast, the Radiol Oncol 2006; 40(4): 257-71. Figure 3. The development of software tools for the quantitative image analysis of confocal images. A single slice of a MCF10AT cell is presented. The software plug-ins are described in Materials and methods. The stack of images can be browsed using a slide bar on the bottom to view any slice of the stack at any chosen time. (a) The un-edited images of a single slice of MCF10AT cell at three different wavelengths: left – 488nm, DQ-collagen IV; middle – 546nm, CatL, right – 633nm StB, as derived from the confocal microscope. The green color was chosen arbitrarily. (b) The same slice after the plug-in for edge detection. The plug-in automatically detects cell borders and intracellular margins based on the changes in the intensity of the signal that meet predefined criteria. (c) The slice after the plug-in that fills the encircled areas and removes the areas that are too small or do not meet the defined shape criteria. The removing process can be best observed when panels (b) and (c) of the left image are compared. (d) Final (manual) processing of the slice. Left: The three areas calculated in (c) were merged into one that repre­sents the total area of interest in the slice. Middle: The area representing a single cell was selected manually (red line). Right: The view through the whole stack (50-80 slices) allowed the easier overview of the cell size and posi­tion. Some structures shown on this view clearly do not represent cells and these were removed by the plug-ins. Radiol Oncol 2006; 40(4): 257-71. overlap index between DQC and CatB was significantly lower (p = 0.0018) in MCF10AT cells. That between DQC and StA was simi­lar in both cell lines. Similarly, the over­lap index between the DQC and StB was significantly increased in MCF10AT cells, whereas that of stefin A was similar as in MCF10A cells. Discussion The present work is the continuation of the study on lysosomal cathepsins and their inhibitors in a panel of four human breast epithelial cell lines, which represents a cellular model for the development of ma­lignant phenotypes.22 We examined the expression of cysteine cathepsins and their inhibitors at protein levels in cell lysates on one hand, and on the other hand by confo­cal microscopic imaging, which may give an alternative insight into the proteolysis associated with the invasion into collagen matrix. For the latter we developed a novel software programme for the partially auto­matic analysis of confocal images by using multiple imaging channels to monitor and quantitate the proteins along with the deg­radation of DQC. Imaging data confirmed the measurements in cell lysates, showing increased levels of CatB, but not CatL pro­tein in the most invasive, ras transfected cells. CatB was lower in the two less inva­sive, but more tumourigenic cell lines and in the paternal cells, whereas CatL expres­sion was similar in all four cell lines. These results indicated that the ras transfection of the breast epithelial cells affected spe­cifically CatB expression and mostly its trafficking,11,28 but not that of CatL. This is different as recently reported by Collette et al. for mouse fibroblasts and rat ovarian ep­ithelial cells, where ras signalling pathways resulted in markedly up-regulated CatL, but not CatB.5 The authors also emphasized that ras signalling is cell type specific and may affect lysosomal cathepsins in differ­ent ways. Indeed, in our transformed breast cells, CatL and CatB were differentially af­fected by the ras oncogene transfection and were obviously independently regulated. A functional role for ras in cathepsin B traf­ficking and redistribution to cell surface has also been observed in melanoma, os­teoclasts and human colorectal carcinoma (sumarized in ref 12), similar as observed in this study. We also reported on the in­dependent regulation of both cathepsins in another panel of breast cancer cell lines29-31 and same notion was deduced from the clinical studies.13,15,16,32 It has been suggested that a misbalance between cathepsins and their inhibitors contributes to the progression of tumors.16­18 In this study, both stefins were upregulat-ed in the more invasive as well as in the tu­mourigenic cells. From the calculated molar ratios between individual cathepsins and stefins, being much higher than equimolar, we may conclude that the concentration of these two inhibitors may not be sufficient to inhibit these two cysteine cathepsins. However, in another panel of breast cancer cells lines, obtained from breast cancer patients with different genetic alterations, we found an inverse correlation between the invasiveness and stefins’ expression.30 In this and other studies on expression of stefins in cancer progression, the findings are not conclusive: lower, similar or higher levels of stefins were found in tumour tis­sue homogenates in the clinical studies of breast and prostate carcinoma,32,33 sug­gesting the complex regulation of stefins in tumours progression. In this study, we observed a partial sig­nal overlap between cathepsins and stefins, indicating that they are to some extent co-localized to the same cellular area, but not necessarily to the same cellular organelles. This supports the data from the first at- Radiol Oncol 2006; 40(4): 257-71. tempt to localize the cathepsins and stefins by confocal microscopy, revealing their dif­ferential localization in human embryonic liver and hepatoma cells.34 Noteworthy, CatB and StB co-localization seems to be the highest in the invasive cells, whereas CatL co-localization with stefins was rela­tively lower in the invasive cells. This may result in higher CatL efficacy in the intra­cellular collagen degradation, which cer­tainly also depends on the local access of stefins to bind to CatL. Collagen turnover is critical to tumour expansion and several previous studies addressed the questions of the subcellu­lar sites of its degradation. Sameni et al.35 showed that collagen degradation was pre­dominantly pericellular in BT20, and most­ly intracellular, localized to lysosomes, in BT549, both breast cancer cell lines. They have also demonstrated that proteolysis is pericellular in breast carcinoma spheroids and pericellular, as well as intracellular, in the colon carcinoma spheroids, with the stroma and inflammatory cells contributing to the degradation of collagen.36 In prostate carcinoma cells, the DQ-collagen I and DQ-collagen IV degradation was reduced by inhibitors of matrix metallo, serine and cysteine proteases,37 suggesting a complex interplay among these proteases in matrix degradation. In the MCF10A-neoT cells, Premzl et al.28 clearly defined a co-localiza­tion of extracellular and intracellular frac­tion of CatB, presumably responsible for both, the pericellular and intralysosomal DQ-collagen IV degradation, respectively. The authors also reported on the major role of cathepsin B activity in the intracellular substratum degradation during capillary– like tube formation of endothelial cells grown on Matrigel by differential inhibi­tion of Z-Arg-Arg-cresyl violet degradation by the inhibitor CA-074Me and not by the non-methylated form CA-074, under the conditions permisive for cell penetration.38 However, Montaser et al.39 demonstrat­ed that CA-074Me inactivates both CatB and CatL within living murine fibroblasts, whereas CA-074 is really inhibiting CatB selectively and therefore all previous data do not exclude the involvement of CatL in the substratum degradation. However, to our knowledge, there is no direct evidence for CatL involvement in the intracellular substratum degradation, for example by using highly selective inhibitors and/or se­lective substrates for CatL that would easily penetrate the living cells. The intracellular localizations of colla­gen may be explained by findings of Kjller et al.,40 who reported that a glycoprotein, urokinase plasminogen activator receptor-associated protein (uPARAP/Endo180) was responsible for import and lysosomal deliv­ery of extracellular collagen IV and that its degradation was impaired in the presence of the cysteine protease inhibitors. Genetic ablation of uPARAP/Endo180 impaired collagen turnover during mammary carci­noma progression.41 Montcourrier et al.42 found that another lysosomal cathepsins, CatD, is co-localized to and responsible for the degradation of the extracellular matrix in large acidic vesicles in the breast cancer cells, and speculated that increased intrac­ellular substratum degradation is not only assisting tumour cell invasion, but is also important to provide tumour cells with amino acid reservoir, necessary for their the increased metabolic activity and pro­tein synthesis. In line with this hypothesis is our finding that the intracellular degra­dation of collagen was not only elevated in the invasive cell type, but was actually elevated in all cancer cells lines, compared to the parental cell line, regardless of their invasive potential. The presented confocal microscopy data are an attempt to quantitate the signal over­lap of the key players of the intracellular cysteine dependent proteolysis and their Radiol Oncol 2006; 40(4): 257-71. functions. A high signal overlap of CatL (but not that of CatB) with the intracellular-ly degraded collagen (despite being present in about five times lower concentrations than CatB) indicates that CatL may be more important for the intracellular degradation of collagen type IV. A high signal overlap in­dex between the stefins and degraded DQC suggests that stefins are in the proximity of the cathepsins-collagen complexes and may affect cathepsins activities. However, to confirm their functionality, in situ ac­tivity and the co-localization of enzymes and inhibitors, additional experiments by simultaneous organelle-specific labelling are needed. In conclusion, we were able to show that the semi-quantitative analysis of cellular levels of cathepsins and stefins by confocal images in single cell gives complementary information to the measurements in the cell lysates. Confocal microscopy and the developed software for the single-cell im­age stack analysis supported the biochemi­cal analysis, demonstrating an increased labelling of CatB, StA and StB in the inva­sive MCF10AT compared with the parental MCF10A cells, whereas CatL was expressed equally. Much higher levels of endocytosed, degraded DQC in the invasive than in the parental line, and a simultaneous increase in the signal overlap of collagen and CatL, suggests possible involvement of CatL in the intracellular degradation of the substra­tum, complementary to that of CatB, what has to be confirmed in future experiments. Acknowledgments This study was supported by the Ministry of Higher Education, Science and Technology of the Republic of Slovenia (Programme # 0105-509, T.T Lah) and by 6FP Cancerdegradome, # 503297 (to T.T.Lah, #17). We thank Prof. Dr. Bonnie Sloane for providing us with the cell lines thate have been obtaind via the collaboration on International & Cooperative Projects Foggarty International, NIH, USA,. 1RO3 TW00952-01, 1999-2001. References 1. Berdowska I. Cysteine proteases as disease mark­ers. Clin Chim Acta 2004; 342: 41-69. 2. Xing R, Addington AK, Mason RW. Quantification of cathepsins B and L in cells. Biochem J 1998; 332: 499-505. 3. Linebauch BE, Sameni M, Day NA, Sloane BF, Keppler D. Exocytosis of active cathepsin B. Eur J Biochem 1999; 264: 100-9. 4. Frosch BA, Berquin I, Emmert.Buck M, Moin K, Sloane BF. Molecular recognition, membrane as­sociation and secretion of tumour cathepsin B. APMIS 1999; 107: 28-37. 5. Collette J, Ulku AS, Der CJ, Jones AS, Erickson AH. Enhanced Cathepsin L expression is mediat­ed by different ras effector pathways in fibroblasts and epithelial cells. Int J Cancer 2004; 112: 190-9. 6. Buck MR, Karustis DG, Day NA, Honn KV, Sloane BF. Degradation of extracellular-matrix proteins by human cathepsin B from normal and tumour tis­sues. Biochem J 1992; 282: 273-78. 7. Felbor U, Dreier L, Bryant RAR, Ploegh HL, Olsen BR, Mothes W. Secreted cathepsin L generates endostatin from collagen XVIII. EMBO J 2000; 19: 1187-94. 8. Ishidoh K, Kominami E. Procathepsin L degrades extracellular matrix proteins in the presence of glycosaminoglycans in vitro. 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Cysteine proteinases and their inhibitorsin extracellualr fluids: Markers for diagnosis and prognosis in cancer. Int J Biol Markers 2000; 15: 84-9. 20. Kos J, Lah TT. Cystatins in cancer. In: Kopitar Jerala N, Žerovnik E, editors. Human stefins and cystatins, molecular anatomy and physiology of pro­teins series. New York: NOVA Science Publishers Inc; 2006. 21. Turk D, Guncar G. Lysosomal cysteine proteases (cathepsins): promising targets. Acta Crystallogr 2003; D 59: 203-13. 22. Bervar A, Zajc I, Sever N, Katunuma N, Sloane BF, Lah TT. Invasiveness of transformed human breast epithelial cell lines is related to cathepsin B and inhibited by cysteine proteinase inhibitors. Biol Chem 2003; 384: 447-55. 23. Soule HD, Maloney TM, Wolman SR, Peterson WD Jr, Brenz R, McGrath CM, et al. Isolation and characterization of a spontaneously immortalized human breast epithelial cell line MCF10. Cancer Res 1990; 50: 6075-86. 24. Basolo F, Elliott J, Tait L, Chen XQ, Maloney T, Russo IH, et al. Transformation of human breast epithelial cells by c-Ha-ras oncogene. Mol Carcinogenesis 1991; 4: 25-35. 25. Dawson PJ, Wolman SR, Tait L, Heppner G, Miller FR. MCF10AT: a model for the evolution of cancer from proliferative breast disease. Am J Pathol 1996; 148: 313-9. 26. Santner SJ, Dawson PJ, Tait L, Soule H, Eliason J, Mohamed AN, et al. Malignant MCF10CA1 cell lines derived from premalignant breast epithelial MCF10AT cells. Breast Cancer Res Treat 2001; 65: 101-10. 27. Bervar A. Localization of cathepsins B and L and their inhibitors, stefins A and B in the invasiveness of breast cancer cell lines. PhD thesis. Ljubljana: Ljubljana University; 2002. 28. Premzl A, Zavašnik-Bergant V, Turk V, Kos J. Intracellular and extracellular cathepsin B fa­cilitate invasion of MCF-10A neoT cells through reconstituted extracellular matrix in vitro. Exp Cell Res 2003; 283: 206-14. 29. Lah TT, Calaf G, Kalman E, Shinde BG, Russo J, Jarosz D, et al. Cathepsins D, B and L in breast car­cinoma and transformed human breast epithelial cells (HBEC). Biol Chem 1995; 376: 357-63. 30. Zajc I, Sever N, Bervar A, Lah TT. Expression of cysteine peptidase cathepsin L and its inhibitors stefins A and B in relation to tumourigenicity of breast cancer cell lines. Cancer Lett 2002; 187: 185­90. 31. Zajc I, Frangež L, Lah TT. Expression of cathepsin B is related to tumourigenicity of breast cancer cell lines. Radiol Oncol 2003; 37: 233-40. 32. Levicar N, Kos J, Blejec A, Golouh R, Vrhovec I, Frkovic-Grazio S, et al. Comparison of potential biological markers cathepsin B, cathepsin L, stefin A and stefin B with urokinase and plasminogen activator inhibitor-1 and clinicopathological data of breast carcinoma patients. Cancer Det Prev 2002; 26: 42-9. 33. Sinha AA, Jamuar MP, Wilson MJ, Rozhin J, Sloane BF. Plasma membrane association of cathe­psin B in human prostate cancer: biochemical and immunogold electron microscopic analysis. 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CA-074, but not its methyl ester CA-074Me is a selective in­hibitor of cathepsin B witin living cells. Biol Chem 2002; 383: 1306-8. 40. Kjller L, Engelholm LH, Hyer-Hansen M, Danř K, Bugge TH, Behrendt N. uPARAP/endo180 di­rects lysosomal delivery and degradation of col­lagen IV. Exp Cell Res 2004; 293: 106-16. 41. Curino AC, Engelholm LH, Yamada SS, Holmbeck K, Lund LR, Molinolo AA, et al. Intracellular col­lagen degradation mediated by uPARAP/Endo180 is a major pathway of extracellular matrix turnover during malignancy. J Cell Biol 2005; 169: 977-85. 42. Montcourrier P, Mangeat PH, Valembois C, Salazar G, Sahuquet A, Duperray C, et al. Characterization of very acidic phagosomes in breast cancer cells and their association with invasion. J Cell Sci 1994; 107: 2381-91. Radiol Oncol 2006; 40(4): 257-71. Radio/ Oncol 2006; 40(4): 205-9. Intrakapsularni in paraartikularni hondrom kolena: Prikaz štirih primerov in pregled literature Samardziski M, Foteva M, Adamov A, Zafiroski G Izhodišca. Intrakapsularni in paraartikularni hondrom sta redka oblika hondroma, ki se pojavlja izven skeletnega tkiva. Zraste iz ovojnice in/ali obsklepnega vezivnega tkiva velikih sklepov (najveckrat kolena) zaradi hrustancne metaplazije. S casom tumorji osificirajo in od tod tudi njihovo drugo poimenovanje paraartikularni osteohondrom. Po mnenju Jaffeja gre za isto entiteto ne glede na stopnjo osifikacije. Prikaz primerov. Porocamo o štirih primerih paraartikularnega hondroma kolena. Pri klinicnem pregledu smo ugotovili pocasi rastoco zatrdlino na kolenu in zmerno bolecino. Pri vseh primerih sta rentgensko slikanje in racunalniška tomografija pokazali mehkotkivni tumor z razlicno stopnjo osifikacije; histološka preiskava pa prisotnost zrelega hialinega in vezivnega hrustanca. Zakljucki. Te benigne tumorje smo diagnosticirali s primerjavo klinicnih, radioloških in histoloških znacilnosti. Vse bolnike smo radikalno operirali, kar je najprimernejši nacin zdravljenja. Radio/ Oncol 2006; 40(4): 273-8. Radio/ Oncol 2006; 40(4): 211-5. Uporaba Dopplerskega ultrazvoka pri ugotavljanju in sledenju mišicne rupture in arteriovenozne fistule na stegnu 12-letnega decka Pavcec Z, Žokalj I, Saghir H, Pal A, Roic G Izhodišca. V clanku avtorji na klinicnem primeru prikazujejo natancnost neinvazivnih slikovnih metod, zlasti Dopplerskega ultrazvoka pri ocenjevanju mišicne poškodbe in ob­skeletnih žilnih nepravilnostih v mehkih tkivih. Prikaz primera. Obravnavali so 12-letnega decka s poškodbo desne štiriglave stegenjske mišice, kjer je nasta mišicna ruptura. Po operaciji so z obicajnim 2-dimenzionalnim ultraz­vokom videli ponavljajoci hematom. Z ultrazvocno preiskavo so pravtako ugotovili hiper­vaskularizirano podrocje in posumili na žilno nepravilnost (angiom). Ko so prekrvavljenost bolnikovega stegna pregledali z Dopplerskim ultrazvokom, so menili, da ima lahko bolnik arterijovenozno fistulo, ki je nastala po poškodbi. Podobnega mnenja so bili preiskovalci v pediatricni bolnici, kjer so decka ponovno pregledali z Dopplerskim ultrazvokom. Vendar digitalna subtrakcijska angiografija, ki so jo naredili 8 mesecev po poškodbi, ni potrdila izvidov ultrazvocnih preiskav. Zato so 11 mesecev po poškodbi naredili racunalniško tomografsko angiografijo, ki je jasno pokazala spremembe, ki so bile že veckrat opisane ob predhodnih ultrazvocnih preiskavah. 14 mesecev po poškodbi je bolnika operiral žilni kirurg in klinicno stanje se je znatno popravilo. Kljub preiskavam in kirurškemu posegu vzrok nastanka fistule pri decku ni popolnoma pojasnjen. Zakljucki. Ob neobicajnih ponavljajocih hematomih, ki nastanejo po poškodbi, je indicirana Dopplerska ultrazvocna priskava. Z racunalniško tomografsko angiografijo laho natancneje opredelimo ultrazvocno vidne spremembe. Radio/ Oncol 2006; 40(4): 273-8. Radio/ Onco/ 2006; 40(4): 219-29. Tumorska cepiva Frank M, Ihan A Tumorska cepiva imajo številne prednosti v primerjavi z drugimi oblikami zdravljenja raka. Predstavljajo visokospecificno protitumorsko terapijo in jih lahko usmerimo proti antig­enom, kljucnim za proces maligne preobrazbe. Imajo edinstven potencial za trajni proti­tumorski ucinek zaradi nastanka dolgoživega, za tumor specificnega imunskega spomina. Kljub velikim pricakovanjem so dosedanji klinicni poskusi cepljenja bolnikov z rakom s tumorskimi cepivi v glavnem prinesli razocaranje. Vzroki za neuspešnost tumorskih cepiv so številni. Potencialno protitumorsko populacijo limfocitov T predstavljajo nizkoafinitetni in maloštevilni periferni limfociti T. Vecina tumorskih antigenov namrec predstavlja lastne antigene, za katere je imunski sistem toleranten. Vzporedno tumorji razvijajo razlicne mehanizme, s katerimi se izogibajo imunskemu sistemu in so kot taki slabo imunogeni ali celo tolerogeni. Novejše imunoterapevtske strategije so usmerjene v premagovanje imunske tolerance na tumorske antigene, povecevanje imunogenosti tumorskih cepiv in naspro­tovanje mehanizmom tumorskega izogibanja imunskemu sistemu. Pristopi so številni, a še vedno dalec stran od idealnega tumorskega cepiva, ki bi uspešno zavrnilo tumor. Težave pri aktivaciji protitumorskega imunskega odziva s tumorskimi cepivi so privedle do razvoja alternativnih imunoterapevtskih strategij, ki neposredno vkljucujejo efektorske mehanizme imunskega odziva (adoptivni prenos limfocitov Tin monoklonska protitelesa). Radio/ Oncol 2006; 40(4): 231-7. Preoperativna socasna kemoradioterapija pri raku požiralnika Šeruga B, Sok M, Eržen J, Jerman J, Jancar B, Zakotnik B Izhodišca. Kirurško zdravljenje in zdravljenje s socasno kem.oradioterapijo (KRT) sta danes možna nacina primarnega zdravljenja raka požiralnika. Dolgotrajno preživetje bolnikov z lokalno napredovalo boleznijo je redko. Zdravljenje teh bolnikov s preoperativno KRT bi lahko bilo koristno, zlasti pri dosegu patološkega popolnega odgovora (pPO) po KRT. V retrospektivni analizi smo analizirali ucinkovitost in toksicnost preoperativne KRT pri bol­nikih z lokalno napredovalim. rakom požiralnika ter vpliv pPO na preživetje. Bolniki in metode. Od leta 1996 do 2002 smo zdravili 41 bolnikov z lokalno napredovalim. rakom požiralnika s cisplatinom. 75 m.glm.2 in 5-FU 1000 m.g/m.2 v štiridnevni kontinu­irani infuziji s pricetkom. l. in 22. dan. Socasno so prejeli 4500 cGy, 200-300 cGy/dan. Ezofagektom.ija je sledila 4-5 tednov po zakljuceni radioterapiji. Po operaciji smo jih redno sledili na 3-6 mesecev. Rezultati. Pri 26,8% bolnikov je bil dosežen pPO. Cas srednjega preživetja je bil 18 mesecev za vse bolnike, 21,2 meseca za bolnike s pPO in 16 mesecev za bolnike z rezidualno bolezn­ijo (p = 0,79). Postoperativna smrtnost je bila 22%. Srednja intenziteta odmerka za cisplatin je bila 92% in za 5-FU 71,5% predvidenega celokupnega odmerka. Bolezen se je najpogosteje ponovila lokoregionalno (31,7%), celokupno se je bolezen ponovila v 43,9%. Zakljucek. Sodobnejši nacini radioterapije in zadostna intenziteta odmerka bi lahko prispe­vali k izboljšanju lokoregionalne kontrole. Za ta kombiniran nacin zdravljenja je potrebna skrbna izbira bolnikov brez pridruženih socasnih obolenj in oddaljenih zasevkov. Radio/ Oncol 2006; 40(4): 273-8. Radio/ On col 2006; 40( 4): 239-44. Indeks telesne mase in tveganje za pljucnega raka pri nekadilcih Kagohashi K, Satoh H, Kurishima K, Ishikawa H, Ohtsuka M Izhodišca. Razmerje med indeksom telesne mase (BMI) in tveganjem za pljucnega raka pri nekadilcih do sedaj še ni bilo natancno opredeljeno. Da bi to tveganje ocenili, smo naredili študijo primerov. Metode. V letih 1987 do 2005 smo v raziskavi obravnavali 204 bolnikov nekadilcev s pljucnim rakom in 398 bolnikov v kontrolni skupini. Starostno primerljive bolnike v kon­trolni skupini smo izbrali med nekadilci, ki so se zdravili v naši bolnišnici zaradi nemaligne pljucne bolezni. Rezultati. Ko smo primerjali starostno primerljivo skupino bolnikov z najmanjšim BMI (BMI < 20.8), nismo ugotovili manjše povezave s pljucnim rakom kot v skupinah z višjim BMI (v drugi skupini z BMI;;,: 20,8 do< 22,9, p = 0,683; v tretji skupini z BMI;;,: 22,9 do< 24,9, p = 0,745; in v skupini z najvišjim BMI, BMI;;,: 25,0, p = 0,327). Prav tako primerjava omenjenih skupin pri ženskah ni pokazala povezave med BMI in po­javnostjo pljucnega raka (pri drugi skupini je bil p = 0,639; pri tretji p = 0,667; in pri skupini z najvišjim BMI p = 0,978). Zakljucki. Naša raziskava ni pokazala, da bi pri nekadilcih BMI bil povezan s stopnjo tveganja za pljucnega raka. Na obolevanje za pljucni rak še vedno najbolj vplivajo drugi dejavniki, najvec kajenje. Radio/ Oncol 2006; 40(4): 245-57. Pomen razlicnih shem zdravljenja z doksorubicinom in vinblastinom na EAT tumorjih pri miših Auersperg M, Pogacnik A, Kloboves-Prevodnik V, Serša G, Cemažar M Izhodišca. Dolocevali smo pomen razlicnih shem zdravljenja z doksorubicinom in vinlasti­nom na uspešnost zdravljenja tumorjev. Materiali in metode. CBA miši z intraperitonealno nasajenimi Ehrlich ascitesnimi tumorji smo zdravili z doksorubicinom, vinblastinom ali kombinacijo obeh v razlicnih zaporedjih in casovnih intervalih. Rezultati. Kombinacija doksorubicina in vinblastina z 48-h intervalom med aplikacijo ke­moterapevtikov, ne pa tudi s 24-h, je statisticno znacilno zmanjšala število tumorskih celic v ascitesu v primerjavi z drugimi terapijami, ne glede na zaporedje aplikacije kemoterape­vtikov. Pri kombiniranih terapijah je bil ucinek na morfološke spremembe in porazdelitev DNA dolocen s kemoterapevtikom, ki smo ga aplicirali najprej. Ne glede na zaporedje ap­likacije kemoterapevtikov se mediana casov preživetja živali ni statisticno razlikovala med posameznimi skupinami. Zakljucki. Ucinek kombinacije doksorubicina in vinblastina je odvisen od casovnega in­tervala med aplikacijo kemoterapevtikov, ne pa tudi od zaporedja. Rezultati predklinicnih študij so pomembni za nacrtovanje kombiniranih kemoterapevtskih shem v klinicni praksi. Radio/ Onco/ 2006; 40(4): 273-8. Radio/ Onco/ 2006; 40(4): 259-71. Cisteinski katepsini, stefini in razgradnja izvencelicnega matriksa med invazijo cloveških transformiranih celicnih linij raka dojke Zajc I, Bervar A, Lah TI Izhodišca. Celicni model cloveškega raka dojke, ki je vseboval štiri celicne linije, osnovno MCFlOA, spontano imortalizirano linijo epitelija cloveške dojke, MCFlOAT, ki je nastala s c-Ha-ras transfekcijo osnovne linije ter dve tumorigeni liniji, MCFlOAT-Cala in MCFlOAT­ Cald, pridobljeni iz zaporednih vsadkov MCFlOAT v miškah, smo uporabili za primerjavo relativne kolicine katepsinov in stefinov v posameznih celicah. Metode. Relativne kolicine katepsinov in stefinov v posameznih celicah smo ocenili s kon­fokalno mikroskopijo in z merjenjem proteinske koncentracije v celicnih homogenatih. Rezultati. Najbolj invazivne MCFlOAT celice so izražale nekajkrat vec katepsina B in vec stefinov, vsebovale pa so podobne koncentacije katepsina L kot osnovna, MCFlOA celicna linija. To je bilo povezano s petkrat višjo endocitozo substrata -Matrigela z DQ kolagenom IV (DQC) -in s hkratnim povišanjem prekrivanja signalov med DQC in katepsinom L ter DQC in stefinom B, a z manjšim prekrivanjem signalov med DQC in katepsinom B v MCFlOAT celicah. Pri teh celicah smo opazili tudi znatno vecje prekrivanje signalov med obema katepsinoma in med pari katepsinov in stefinov. Zakljucki. Ti rezultati kažejo, da pride pri invazivnem celicnem fenotipu do vecje endoci­toze in razgradnje kolagena ter istocasne spremembe v lokalizaciji katepsinov in stefinov v celici. Glede na te in podatke drugih avtorjev se zdi, da je znotraj celicna razgradnja bolj povezana s katepsinom L, medtem ko katepsin B v vecji meri sodeluje pri pericelularni razgradnji medcelicnega matriksa tekom celicne migracije in invazije. Radio/ Onco/ 2006; 40(4): 273-8. Notices Notices submitted far publication should contain a mailing address, phone and/ or fax number and/ or e-mail oj a Contact person or department. Prostate cancer ]anuary 14-18, 2007 The ESTRO multidisciplinary teaching course on pros­tate cancer will be offered in Ghent, Belgium. Contact ESTRO office, Avenue E. Mounierlaan, 83/12, B-1200 Brussels, Belgium; or call +32 2 775 93 40; or fax +32 2 779 54 94; or e-mail info@estro.be; or see http://www.estro.be Head and neck oncology February 22-24, 2007 The ESTRO international meeting on innovative ap­proaches in head and neck oncology will take place in Barcelona, Spain. Contact ESTRO office, Avenue E. Mounierlaan, 83/12, B-1200 Brussels, Belgium; or call +32 2 775 93 40; or fax +32 2 779 54 94; or e-mail info@estro.be; or see http://www.estro.be LungCancer March 2-3, 2007 The 8th European Congress: "Perspectives in Lung Cancer" will take place in Seville, Spain. Contact Congress Secretariat, 4325 Alexander Drive, Alpharetta, GA 30022-3740 USA; or call +1 770 751 7332; or fax +1 (770) 751 7334; or e-mail meetings@ imedex.com; or see http://www.imedex.com Oncology March 7-10, 2007 The "EORTC Groups Annual Meeting" will take place in Brussels, Belgium. Contact Mr. Danielle Zimmermann; EORTC Education Office, Avenue E. Mounier, 83, bte 11, B-1200 Brussels, Belgium; or call +32 2 774 16 02; or fax +32 2 772 61 33; or e-mail Danielle.zimmerrnann@eortc.be; or see http://www.eortc.be Radiotherapy March 25-29, 2007 The ESTRO course "Radiotherapy Treatrnent Planning: Principles and Practice" will be offered in Dublin, Ireland. Contact ESTRO office, Avenue E. Mounierlaan, 83/12, B-1200 Brussels, Belgiurn; or call +32 2 775 93 40; or fax +32 2 779 54 94; or e-rnail info@estro.be; or see http://www.estro.be Brachytherapy March 25-29, 2007 The ESTRO course "Modern Brachytherapy Techniques" will be offered in Como, Italy. Contact ESTRO office, Avenue E. Mounierlaan, 83/12, B-1200 Brussels, Belgiurn; or call +32 2 775 93 40; or fax +32 2 779 54 94; or e-rnail info@estro.be; or see http://www.estro.be Radio/ Oncol 2006; 40(4): 281-3. 282 Notices Chest tumours March 30 -April 1, 2007 The "ESMO International Symposium" will take place in Geneva, Switzerland. Contact ESMO Head Office, Congress Department, Via La Santa 7, CH-6962 Viganello-Lugano, Switzerland; or +41 (0)91 973 19 19; or fax +41 (0)91 973 19 18; or e­mail congress@esmo.org; or see http://www.esmo.org Brachytherapy May 10-12, 2007 The GEC-ESTRO brachytherapy meeting will take place in Wolfsberga, Germany. Contact ESTRO office, Avenue E. Mounierlaan, 83/12, B-1200 Brussels, Belgium; or call +32 2 775 93 40; or fax +32 2 779 54 94; or e-mail info@estro.be; or see http://www.estro.be Oncology June 12-15, 2007 The EORTC annual course "Clinical Trials Statistics for Non Statisticians" will take place in Brussels, Belgium. Contact Mr. Danielle Zimmermann; EORTC Education Office, Avenue E. Mounier, 83, bte 11, B-1200 Brussels, Belgium; or call +32 2 774 16 02; or fax +32 2 772 61 33; or e-mail Danielle.zimmermann@eortc.be; or see http://www.eortc.be Oncology July 5-8, 2007 The "ESMO Conference Lugano" will take place in Lugano, Switzerland. Contact ESMO Head Office, Congress Department, Via La Santa 7, CH-6962 Viganello-Lugano, Switzerland; or +41 (0)91 973 19 19; or fax +41 (0)91 973 19 18; or e-mail congress@esmo.org; or see http://www.esmo. org/ activi ties/ ecluconference/ Toxicology July 15-19, 2007 The "11 th International Congress of Toxicology" will be offered in Montreal, Canada. Contact Congress Secretariat, e-mail: ict2007@nrc­cnrc.gc.ca; or see http://www.ict2007.org Lung cancer September 2-6, 2007 The "12th World Conference on Lung Cancer" will be offered in Seoul, Korea. Contact Conference Secretariat; e-mail WCLC2007@ ncc.re.kr; or see http://www.iaslc.org!umages/ 12worldconfannounce.pdf Oncology September 7, 2007 The EORTC annual course "One-Day Introduction to EORTC Trials" will take place in Brussels, Belgium. Contact Mr. Danielle Zimmermann; EORTC Education Office, Avenue E. Mounier, 83, bte 11, B-1200 Brussels, Belgium; or call +32 2 774 16 02; or fax +32 2 772 61 33; or e-mail Danielle.zimmermann@eortc.be; or see http://www.eortc.be Radiotherapy September 8-13, 2007 "9th The Biennial ESTRO Meeting on physics and Radiation Technology for Clinical Radiotherapy will take place in Barcelona, Spain. Contact ESTRO office, Avenue E. Mounierlaan, 83/12, B-1200 Brussels, Belgium; or call +32 2 775 93 40; or fax +32 2 779 54 94; or e-mail info@estro.be; or see http://www.estro.be Oncology September 23-27, 2007 The "14th European Cancer Conference ECCO 15/ ESTRO 26" will take place in Barcelona, Spain. Contact Conference Secretariat, ECCO 14, The European Cancer Conference, European Cancer Societies (FECS), Avenue E. Mounier, 83, B-1200 Brussels, Belgium; or call +32 2 775 02 01; or fax +32 2 775 02 00; or e-mail ECC014@fecs.be; or see http:// www.fecs.be Prostate cancer September 15-17, 2007 The ESTRO multidisciplinary prostate cancer meeting will be offered. Contact ESTRO office, Avenue E. Mounierlaan, 83/12, B-1200 Brussels, Belgium; or call +32 2 775 93 40; or fax +32 2 779 54 94; or e-mail info@estro.be; or see http://www.estro.be Radio/ Oncol 2006; 40(1): 281-3. Notices 283 Lungcancer August 21-24, 2009 The "13th World Conference on Lung Cancer" will be offered in San Francisco, USA. Contact Conference Secretariat; e-mail WCLC2007@ ncc.re.kr; or see http://www.iaslc.orgiumages/ 12worldconfannounce.pdf Oncology September4-8, 2009 The "34th ESMO Congress" will take place in Vienna, Austria. Contact ESMO Head Office, Congress Department, Via La Santa 7, CH-6962 Viganello-Lugano, Switzerland; or +41 (0)91 973 19 19; or fax +41 (0)91 973 19 18; or e­mail congress@esmo.org; or see http://www.esmo.org As a service to our readers, notices of meetings ar courses will be inserted free of charge. Please send infonnation to the Editorial office, Radiologij and Oncology, Zaloška 2, 51-1000 Ljubljana, Slovenia Radio/ Onco/ 2006; 40(4): 281-3. Adamov A: 4/205-209 Akinwunmi J: 2/73-85 Amar S: 2/73-85 Auersperg M: 4/245-257 Bergant M: 3/183-188 Bervar A: 4/259-271 Bešlic š: 2/67-72 Bhatia S: 1/29-33 Bochenek A: 1/7-15 Božic M: 1/35-38 Campagnutta E: 3/175-181 Cemažar M: 3/163-174; 4/245-257 Da Ronch L: 3/149-161 De Piero G: 3/175-181 Del Pup L: 3/175-181 Dolenšek M: 3/147-148 Durinec M: 1/43-49 Eržen J: 4/231-237 Fallone BG: 2/125-132 Foteva M: 4/205-209 Frank M: 4/219-229 Franko A: 1/17-21 Garaj-Vrhovac V: 1/43-49 Geis N: 2/95-105 Giorda G: 3/175-181 Griffiths G: 3/183-188 Grochowicz M: 1/7-15 Hans-Sonke J: 2/125-132 Holjar-Erlic I: 1/17-21 Ihan A: 4/219-229 Ihan Hren N: 1/35-38 Ishikawa H: 4/239-244 Jakubowski W: 4/231-237 Jamar B: 3/147-148 Jancar B: 4/217-217; 4/231-237 Authors Index 2006 Jeras M: 3/183-188 Jerman J: 4/231-237 Kagohashi K: 4/239-244 Kirschfink M: 2/95-105 Kloboves-Prevodnik V: 4/245-257 Kocijancic I: 1/1-5 Kolodziejczak M: 1/7-15 Konatschnig T: 2/95-105 Kovac V: 1/39-42 Krnic A: 3/143-146 Krolo I: 3/143-146 Kurishima K: 4/239-244 La Mura N: 3/149-161 Lachance D: 1/29-33 Lah Turnšek T: 4/259-271 Lenassi M: 1/51-56 Lestuzzi C: 3/149-161 Lincender L: 2/67-72 Majdic E: 1/23-28 Miklavcic D: 3/163-174 Miletic D: 1/17-21 Miller R: 1/29-33 Miolo G: 3/149-161 Murrone A: 3/149-161 Nigri P: 3/149-161 Ohtsuka M: 4/239-244 Pal A: 4/211-215 Parker K: 2/87-93 Pavcec Z: 4/211-215 Petrovic O: 1/17-21 Pilkington GJ: 2/73-85; 2/87-93 Plemenitaš A: 1/51-56 Pogacnik A: 4/245-257 Pohar M: 2/115-124 Primic Žakelj M: 2/115-124 Rajer M: 1/23-28 Roic G: 4/211-215 Rudolf Z: 3/163-174 Saghir H: 4/211-215 Samardziski M: 4/205-209 Satoh H: 4/239-244 Schultz S: 2/95-105 Serša G: 3/163-174; 4/245-257 Sisto R: 3/175-181 Smrdel U: 1/39-42 Sofic A: 2/67-72 Sok M: 4/231-237 Sopracordevole F: 3/175-181 Stanescu T: 2/125-132 Stavrev P: 2/125-132 Strojan P: 2/107-113 Sucic Z: 3/143-146 Sudol-Szopir'iska I: 1/7-15 Šeruga B: 4/231-237 Škrk D: 3/189-195 Šubic T: 3/147-148 Swiatlowska M: 1/7-15 Veronesi A: 3/149-161 Viel E: 3/149-161 Vrcic D: 2/67-72 Vucic N: 3/143-146 Zadnik V: 2/115-124 Zafiroski G: 4/205-209 Zajc I: 4/259-271 Zakotnik B: 4/231-237 Zavašnik-Bergant T: 3/183-188 Zdešar U: 3/189-195 Žagar T: 2/115-124 Željezic D: 1/43-49 Žokalj I: 4/211-215 Žontar D: 3/189-195 Supplement 1/2006 Bartenjev I: S153-S157; S159-S161; S163-S170 Bavcar Vodovnik T: S53-S58 Bebar S: S137-S141 Dolenšek M: S53-S58 Dovšak D: S9-S17 Eržen J: S67-S76 Fischinger J: S19-S24 Gale N: S1-S8 Jerše M: S67-S76; S77-S85 Krajc M: S25-S30; S31-S43 Luzar B: S159-S161; S163-S170 Maucec Zakotnik J: S25-S30; S31-S43; S95-S105 Mikuž G: S121-S126 Možina A: S137-S141 Novak Mlakar D: S95-S105 Oblak C: S127-S136 Ovcak Z: S115-S120 Pogacnik A: S149-S151 Primic Žakelj M: S143-S148; S25-S30; S31-S43 Rakar S: S137-S141 Rott T: S67-S76 Skok P: S87-S94 Stržinar V: S137-S141 Štabuc B: S107-S114 Takac I: S137-S141 Tercelj M: S59-S66; S67-S76 Uršic Vršcaj M: S137-S141 Vakselj A: S137-S141 Žgajnar J: S45-S52 Subject Index 2006 adenocarcinoma: 4/239-244 angiography: 3/143-146 annexin V: 2/87-93 antidepressive agents, tricyclic: 2/73-85 antigens, neoplasms: 4/219-229 antineoplastic agents: 1/39-42; 1/51-56 antineoplastic agents -adverse -effects -toxic­ ity: 3/149-161 apoptosis: 1/51-56; 2/87-93; 2/73-85 arteriovenous fistula: 4/211-215 bleomycin: 3/163-174 body mass index: 4/239-244 brain neoplasms -drug therapy: 2/73-85; 2/87-93 brain neoplasms -radiotherapy: 2/125-132 breast neoplasms: 4/259-271 breast neoplasms -surgery: 1/23-28 cancer vaccines: 4/219-229 carcinoma Ehrlich tumor -drug therapy: 4/245-257 carcinoma, non-small-cell lung: 1/39-42 cathepsins: 4/259-271 cerebellar neoplasms: 1/17-21 chondroma, osteochondroma, knee: 4/205-209 chromosome aberrations: 1/43-49 cisplatin: 3/163-174 clomipramine: 2/73-85; 2/87-93 complement inactivators: 2/95-105 constipation: 1/7-15 cryopreservation: 3/175-181 cryoultramicrotomy: 3/183-188 CT scan: 2/67-72 cysteine endopeptidases: 2/107-113 cysteine proteinase inhibitors: 2/107-113 defecography: 1/7-15 dendritic cells: 3/183-188 diagnostic reference leve!: 3/189-195 DNA damage: 1/43-49 DNA repair: 1/43-49 Doppler duplex: 3/143-146 doxorubicin: 4/245-257 drug administration schedule: 4/245-257 electrochemotherapy, drug delivery systems: 3/163-174 electroporation: 3/163-174 embryo: 3/175-181 esophageal neoplasms -drug therapy -radio­ therapy: 4/231-237 extracellular matrix: 4/259-271 fertility: 3/175-181 fertilization in vitro: 3/175-181 fibroma: 1/35-38 foecal incontinence: 1/7-15 ganglioneuroma: 1/17-21 glioma: 2/87-93; 2/73-85 head: 1/29-33 head and neck neoplasms: 2/107-113 heart -drug effects: 3/149-161 hematoma -ultrasonography: 4/211-215 Hodgkin disease -radiotherapy: 1/29-33 immunohistochemistry: 3/183-188 immunology: 2/95-105 immunotherapy: 4/219-229 intestinal perforation-radiography-ultrasonogra­ phy: 2/67-72 ionizing radiation: 1/43-49 life tables: 2/115-124 lung neoplasms -epidemiology: 4/239-244 mandibula neoplasms: 1/35-38 MAP kinase: 1/51-56 micronucleus test: 1/43-49 microscopy, electron: 3/183-188 muscle weakness: 1/29-33 muscle, skeleta! -injuries: 4/211-215 muscular athrophy: 1/29-33 neoplasms: 3/175-181 neoplasms -drug therapy: 3/163-174; 2/95-105 neoplasms invasiveness: 4/259-271 odontoma: 1/35-38 oocytes: 3/175-181 ovary: 3/175-181 phytohaemagglutinins: 1/43-49 pleura: 1/1-5 pleural effusion: 1/1-5 public health: 2/115-124 radiation exposure: 3/189-195 radiography: 3/189-195 radiography, thoracic: 1/1-5 radiotherapy planning, computer-assisted: 2/125-132 risk factors: 4/239-244 smoking: 4/239-244 stomach-injuries-radiography: 2/67-72 subclavian artera -abnormalities -radiography -ultrasonography: 3/143-146 subclavian steal syndrome: 3/143-146 survival analysis: survival analysis: 1/23-28; 2/115-124 tumor cells cultured: tumor cells, cultured: 1/51-56; 4/259-271 tumour cells, cultured: 2/87-93 vertebral artery: 3/143-146 vinblastine: 4/245-257 Supplement 1/2006 biopsija igelna, Gleasonova ocena: S115-S120 biopsy: S67-S76; S77-S85 carcinoma in situ: S67-S76 cepljenje: S137-S141 debelo crevo in danka, novotvorbe-preprecevanje in nadzor: S87-S94; S95-S105; S107-S114 debelo crevo, polipi: S107-S114 dejavniki tveganja: S87-S94 dermoskopija: S159-S161 diagnostika, slikovna: S53-S58 dojka, bolezni, novotvorbe -diagnostika - zdravljenje: S45-S52 dojka, novotvorba -preprecevanje in nadzor: S31-S43 dojka, novotvorbe -diagnostika: S25-S30 državni program ZORA: S143-S148 eritroplakija: S9-S17 grlo, novotvorbe -diagnostika -zdravljenje: S19-S24 grlo, novotvorbe -patologija: Sl-S8 histološki napovedni dejavniki: S163-S170 hyperplasia: S67-S76 incidenca: S143-S148 karcinom plošcatocelicni: Sl-S8 karcinom, bazalnocelicni: karcinom, plošcatocelicni: S163-S170 kolonoskopija: S107-S114 kontrola kakovosti: S149-S151 kontrolirane raziskave: S87-S94 koža, novotvorbe -diagnostika: S159-S161; S153-S157 koža, novotvorbe -patologija: S163-S170 levkoplakija: S9-S17 light-microscopy: S67-S76 lung neoplasms -cytology -pathology: S77-S85 lung neoplasms -pathology: S67-S76 maligni epitelni tumorji, klinicna slika: S153­ S157 mamografija: S25-S30 maternica, vrat, novotvorbe -diagnostika: S143­ S148 Radio/ Oncol 2006; 40(4): 287-8. maternica, vrat, novotvorbe -preprecevanje in nadzor: S137-S141; S149-S151 melanom: S153-S157; S163-S170 metaplasia: S67-S76 nevusi -pigmentirani: S159-S161 novotvorba, stadij, TNM klasifikacija: S9-S17 papiloma virus cloveški: S137-S141 pathogenesis: S121-S126 pljuca, novotvorbe -diagnostika: S53-S58 pljucna novotvorba -preživetje: S59-S66 pojavnost: S87-S94 predrakava stanja: S115-S120; S19-S24; S1-S8 preinvasive epithelial lesions: S67-S76 presejanje: S143-S148; S95-S105; S149-S151; S25-S30; S31-S43; S137-S141 preventivni centri za dojke: S31-S43 prostata, novotvorba -diagnostika -zdravljenje: S127-S136 prostata, novotvorbe -patologija: S115-S120 prostatic neoplasms -diagnosis -therapy: S121­ S126 rekonstrukcije: S9-S17 simptomi in znaki bolezni: S59-S66 spremljanje: S127-S136 usta, novotvorbe: S9-S17 usta, novotvorbe -patologija: S1-S8 vaginalni brisi, test PAP: S149-S151 zagotavljanje in nadziranje kakovosti: S25-S30 zdravstvene službe -organizacija: S45-S52 zgodnje odkrivanje: S59-S66 zgodnje odkrivanje, presejanje: S53-S58 žrelo, novotvorbe -diagnostika -zdravljenje: S19-S24 žrelo, novotvorbe -patologija: S1-S8 FUNDACIJA "DOCENT DR. J. CHOLEWA" JE NEPROFITNO, NEINSTITUCIONALNO IN NESTRANKARSKO ZDRUŽENJE POSAMEZNIKOV, USTANOV IN ORGANIZACIJ, KI ŽELIJO MATERIALNO SPODBUJATI IN POGLABLJATI RAZISKOVALNO DEJAVNOST V ONKOLOGIJI. DUNAJSKA 106 1000 LJUBLJANA ŽR: 02033-001 7879431 Activity of "dr. J. Cholewa" Foundation for Cancer Research and Education -a report for the final quarter of 2006 The Dr. J. Cholewa Foundation for Cancer Research and Education is preparing plans to continue to support activities associated with cancer research and education in Slovenia in the coming 2007. The Foundation remains active in promoting any form of cancer educa­ tion in general population, among medica! and nursing students and among ali the others with a particular interest in cancer research and education. A number of requests for re­search grants and other forms of financial support were thus received from experts in vari­ous disciplines of cancer research and education in Slovenia in the year 2006 and needless to say, ali have been dealt with great attention and responsibly in respect to their proposals and contents. The role of the Foundation members with clinical and research experience in cancer and by members with important experience in finance has been instrumenta! in this activity and their contribution is gratefully acknowledged. The Dr. J. Cholewa Foundation for Cancer Research and Education also continues to sup­port the regular publication of "Radiology and Oncology" international scientific journal, which is edited, published and printed in Ljubljana, Slovenia. In line with the philosophy of the Foundation, the spread and expansion of the information and knowledge of cancer research and problems associated with cancer in general have thus found the way to many interested professionals, lay public and others in Slovenia and elsewhere. The support for cancer research and education in various forms, financial and otherwise, remains to be considered as one of the more important commitments of the The Dr. J. Cholewa Foundation. It is important to note that a number research and study grants have been bestowed and allocated by the Foundation to researchers from various scientific spheres of acticvity associated with oncology in Slovenia in the year 2006. Other forms of support were also allotted to attend scientific meetings, conferences and symposia dealing with oncology worldwide to a number of interested and qualified applicants. The Foundation will therefore also support the publication of the results from cancer research in Slovenia in any respectable international scientific journal and in any other form of dis­semination of scientific information. The Dr. J. Cholewa Foundation for Cancer Research and Education has since its inception every reason to respectfully acknowledge the importance of various forms of support from various public companies and private individuals to its cause. With best wishes for the year 2007, the Foundation extends its thanks and gratitude to all of them. Tomaž Benulic, MD Andrei Plesnicar, MD Borut Stabuc, MD, PhD SIEMENS SiemensMed,cal.com oncology SEEK-FIND-ACT-FOLLOW -the Continuum of Oncology Care™ Siemens oncology porttoho compnses comprehens1ve max1m1zed u111tza11on potenual. and pattent-tnendly des1gn workflow solut1ons mtegratmg the tuli spectrum ot care and teatures. tram screemng/early detectton and d1agnos1s through therapy and tollow-up AII from one prov1der -w1th over Every day m the Un,ted States alone. 29.000 cancer 100 years h1story ot mnovat,on ,n medica! technology pat,ents rece,ve rad,auon therapy deltvered by S,emens ltnear accelerators. As chn,cal protocols trans,1100 to Siemens praven chmcal methods can help you to ach1eve ,nclude IMRT and IGRT. Siemens seamlessly ,ntegrates more successful outcomes. How} Through 1ndustry­the d1agnost,c and treatment modaltt,es. That's what we lead1ng technology, mcreased productiv1ty measures tor call Best Practice Oncology Care Siemens medica! S o I uti on s that help Sanolabor •••.• kottermann . . Kcttermann (Nemcija): laboratorijsko pohištvo, varnostne omare za kisline, luge, topila, pline in strupe, ventilacijska tehnika in digestorji Angelantoni scientifica (Italija): hladilna tehnika in aparati za laboratorije, transfuzijo, patologijo in sodno medicino CORNING Corning (Amerika): specialna laboratorijska plastika za aplikacijo v imunologiji, mikrobiologiji, virologiji, ipd., mehanske eno-in veckanalne pipete in nastavki mi •• MICRONIC Micronic (Nizozemska): sistemi za shranjevanje vzorcev, pipete, nastavki za pipete Tmplanttech· The.re's No Reason to Ope.rate with Anyonc Elsc Implantech (Amerika): obrazni in glutealni vsadki Biomerica (Amerika): hitri testi za diagnostiko, EIA /RIA testi LABORMED d.o.o. Bežigrajski dvor Periceva 29, Ljubljana Tel.: (0)1 436 49 01 Fax: (0)1 436 49 05 Ehret (Nemcija): Laminar flow tehnika, inkubatorji, sušilniki, suhi sterilizatorji in oprema za laboratorijsko vzrejo živali -kletke Dako (Danska): testi za aplikacijo v imunohistokemiji, patologiji, mikrobiologiji, virologiji, mano-in poliklonalna protitelesa Eli Sakura finetek (Evropa): aparati za pripravo histoloških preparatov: mikro­inkriotomi, zalivalci, tkivni procesorji, barvalci, pokrivalci IBS INTEGRA WWt11WWtl Integra Biosciens (Švica}: laboratorijska oprema za mikrobiologijo, biologijo celic, molekularno biologijo in biotehnologijo SpectrumDesigns MEDICAL (Amerika): moški pektoralni vsadki Byron (Amerika): liposuktorji in kanile za liposukcijo lnfo@labormed.si www.labormed .. s ERBITUXS CETUKSIMAB Zavira EGFR -odpira nove možnosti Lokalno napredovali rak glave in vratu 1 Erbitux® in radioterapija sig n ifi ka ntno podaljšujeta preživetje2 Erbitux v kombinaciji z radioterapijo podaljša srednje preživetje za 20 mesecev.2·3 Erbitux skupaj z radioterapijo ne potencira stranskih ucinkov znacilnih za radioterapijo.3 Erbitux 2 mg/ml raztopina za infundiranje (skrajšana navodila za uporabo) Cetuksimab je monoklonsko lgGl protitelo, usmerjeno proti receptorju za epidermalni rastni faktor (EGFR). Terapevtske indikacije: Zdravilo Erbitux je v kombinirani terapiji z irinotekanom indicirano za zdravljenje bolnikov z metastatskim rakom debelega crevesa in danke, in sicer po neuspešni citotoksicni terapiji, ki je vkljucevala tudi irinotekan. Zdravilo Erbitux je v kombinaciji z radioterapijo indicirano za zdravljenje bolnikov z lokalno napredovalim rakom skvamoznih celic glave in vratu. Odmerjanje in nacin uporabe: Zdravilo Erbitux 2 mg/ml se daje z intravensko infuzijo prek linijskega filtra. Zdravilo Erbitux pri vseh indikacijah infundirajte enkrat na teden. Zacetni odmerek je 400 mg cetuksimaba/m2 telesne površine, vsi naslednji tedenski odmerki so vsak po 250 mg/m'. Pred prvo infuzijo mora bolnik prejeti premedikacijo z antihistaminikom. Ta premedikacija je priporocljiva tudi pred vsemi naslednjimi infuzijami. Kontraindikacije: Zdravilo Erbitux je kontraindicirano pri bolnikih z znano hudo preobcutljivostno reakcijo (3. ali 4. stopnje) na cetuksimab. Pred zacetkom kombiniranega zdravljenja morate upoštevati kontraindikacije za irinotekan ali radioterapijo. Posebna opozorila in previdnostni ukrepi: ce pri bolniku nastopi blaga ali zmerna reakcija, povezana z infundiranjem, lahko zmanjšate hitrost infundiranja. Priporocljivo je, da ostane hitrost infundiranja na nižji vrednosti tudi pri vseh naslednjih infuzijah. Ce se pri bolniku pojavi huda kožna reakcija (:c, 3. stopnje po kriterijih NCI-CTC), morate prekiniti terapijo s cetuksimabom. Z zdravljenjem smete nadaljevati le, ce se je reakcija pomirila do 2. stopnje. Posebna previdnost je potrebna pri oslabljenih bolnikih in pri tistih z obstojeco srcno-pljucno boleznijo. Neželeni ucinki: Zelo pogosti(?_ 1/10): dispneja, blago do zmerno povecanje ravni jetrnih encimov, kožne reakcije, blage ali zmerne reakcije, povezane z infundiranjem, blag do zmeren mukozitis. Pogosti(?_ 1/100, < 1/10): konjunktivitis, hude reakcije, povezane z infundiranjem. Pogostost ni znana: hipomagneziemija. Pakiranje: 1 viala po 50 ml. Imetnik dovoljenja za promet: Merek KGaA, 64271 Darmstadt, Nemcija. Podrobne informacije o zdravilu so objavljene na spletni strani Evropske agencije za zdravila (EMEA) http://www.emea.eu.int/ Dodatne informacije so vam na voljo pri: Merek, d.o.o„ Dunajska cesta 119, 1000 Ljubljana, tel.: 01 560 3810, faks: 01 560 3831, el. pošta:info@merck.si 1 plošcatocelicni rak glave in vratu 2 1 v primerjavi z radioterapijo II 3 Bonner et al. Radiotherapy plus Cetuximab far Squamous -Celi Carcinoma 1 II rimidex vodilni zaviralec aromataze anastrozol Kratka informacija o zdravilu Arimidex 1 mg .stava: Filmsko obložena tableta vsebuje mg anastrozola. dikacije: Adjuvantno zdravljenje žensk po enopavzi, ki imajo zgodnji invazivni rak >jke s pozitivnimi estrogenskimi receptorji in , ne morejo zdraviti s tamoksifenom zaradi ivecanega tveganja za tromboembolizem ali mormalnosti endometrija. Zdravljenje ipredovalega raka dojke pri ženskah po enopavzi. Ucinkovitost pri bolnicah z igativnimi estrogenskimi receptorji ni bila ikazana razen pri tistih, ki so imele edhodno pozitiven klinicni odgovor na moksifen. .merjanje In nacin uporabe: 1 tableta po 1 g peroralno, enkrat na dan. Pri zgodnjem ku je priporocljivo trajanje zdravljenja 5 let. lntraindikaclje: Arimidex je kontraindiciran i: ženskah pred menopavzo, nosecnicah in ijecih materah, bolnicah s hujšo ledvicno !povedjo (ocistek kreatinina manj kot 20 ljmin (oziroma 0,33 ml/s)), bolnicah z nemim do hudim jetrnim obolenjem in >lnicah, ki imajo znano preobcutljivost za 1astrozol ali za katerokoli drugo sestavino lravila. Zdravila, ki vsebujejo estrogen, ne nete dajati socasno z Arimidexom, ker bi se egovo farmakološko delovanje iznicilo. ,moksifena se ne sme uporabljati skupaj z imidexom, ker lahko pride do zmanjšanja njegovega delovanja. Posebna opozorila In previdnostni ukrepi: Uporabe Arimidexa ne priporocamo pri otrocih, ker njegova varnost in ucinkovitost pri njih še nista raziskani. Menopavzo je potrebno biokemicno dolociti pri vseh bolnicah, kjer obstaja dvom o hormonskem statusu. Ni podatkov o varni uporabi Arimidexa pri bolnicah z zmerno ali hudo jetrno okvaro ali hujšo ledvicno odpovedjo (ocistek kreatinina manj kakor 20 ml/min (oziroma 0,33 ml/s)). Ni podatkov o uporabi anastrozola z analogi LHRH. Te kombinacije zdravil se ne sme uporabljati zunaj klinicnih preskušanj. Pri ženskah z osteoporozo ali pri ženskah s povecanim tveganjem za razvoj osteoporoze je treba dolociti njihovo mineralno gostoto kosti z denzitometrijo, na primer s slikanjem DEXA na zacetku zdravljenja, pozneje pa v rednih intervalih. Po potrebi je treba zaceti z zdravljenjem ali preprecevanjem osteoporoze in to skrbno nadzorovati. Ni verjetno, da bi Arimidex zmanjšal bolnicino sposobnost za vožnjo ali upravljanje s stroji. Ker pa so med uporabo Arimidexa porocali o splošni oslabelosti in zaspanosti, je potrebna previdnost pri vožnji in upravljanju strojev, dokler simptoma trajata. Nosecnost in dojenje: Arimidex je med nosecnostjo in dojenjem kontraindiciran. Neželeni ucinki: Najpogostejši neželeni ucinki so navali vrocine, suhost vagine in redcenje las. Ostali neželeni ucinki vkljucujejo gastrointestinalne motnje (anoreksija, slabost, bruhanje, diareja), astenijo, bolecine/okorelost v sklepih, zaspanost, glavobol in izpušcaje. Obcasna porocila navajajo krvavitev iz nožnice, ki se pretežno pojavlja pri bolnicah z napredovalim obolenjem raka na dojki v prvih tednih po prehodu z dotedanjega hormonskega zdravljenja na zdravljenje z Arimidexom. ce krvavitev traja dlje casa, so potrebne dodatne preiskave. Hiperholesterolemija, obicajno blaga do zmerna. O povišanih nivojih gama-GT in alkalne fosfataze so porocali le obcasno. Vzrocna povezanost omenjenih sprememb ni bila ugotovljena. Medsebojno delovanje z drugimi zdravili: Zdravila, ki vsebujejo estrogen, ne smete dajati socasno z Arimidexom, ker bi se njegovo farmakološko delovanje iznicilo. Tamoksifena se ne sme uporabljati skupaj z Arimidexom, ker lahko pride do zmanjšanja njegovega delovanja. Vrsta ovojnine in vsebina: Pretisni omoti iz PVC in aluminija, ki vsebujejo 28 tablet v škatlici. Režim izdaje zdravila: Rp/Spec Datum priprave informacije: september 2006 Pred predpisovanjem, prosimo, preberite celoten povzetek temeljnih znacilnosti zdravila. odatne informacije in literatura so na voljo pri: ,traZeneca UK Limited, Podružnica v Sloveniji, Verovškova 55, Ljubljana ww.breastcancersource.com ww .rimirlAY nPt AstraZeneca i ©Ji"JtXgLQ)fuJJA PE: Stritarjeva 5, 4000 Kranj, Slovenija tel.: (0)4/ 2015 050, fax: (0)4/ 2015 055 e-mail: kemomed@siol.net, KEMOMED www.kemomed.si o agmrm1m11 Promega IZDELKI ZA MOLEKULARNO BIOLOGIJO DOKUMENTACIJA IN ANALIZA GELOV S.AIMYO CISTA VODA ZA LABORATORIJ SKRINJE IN HLADILNIKI m-11,-e-rv_a ___ ,<9 .,.c,. biolab, Biosciences ELEKTRONSKE IN MEHANSKE AVTOMATSKE PIPETE DIAGNOSTIKA SEKVENATORJI MIKOPLAZEM IN LEGIONEL remodal 20 mg; 100 mg, 250 mg. Sestava zdravila Vsaka kapsula zdravila Temodal vsebuje 20 mg, 100 mg ali 250 mg temozolamida.Terapevtske indikacije Temodal 1,5 x 109/1, število trombocitov > 100 x 109/1, skupni kriteriji toksicnosti (SKT) za nehematološko toksicnost < 1. stopnje (z izjemo llopecije, slabosti in bruhanja). Med zdravljenjem morate pri boiiiiku enkrat na teden pregledati celotno krvno sliko. Faza monoterapije Štiri tedne po zakljucku faze ,ocasnega zdravljenja z zdravilom Temodal in radioterapijo naj bolnik jemlje zdravilo Temodal do 6 ciklov monoterapije. V 1. ciklu (monoterapija) je odmerek zdravila 150 ng/m' enkrat na dan 5 dni, temu pa naj sledi 23 dni brez terapije. Na zacetku 2. cikla odmerek poveca.e na 200 mg/m2 , ce je SKT za nehematološko toksicnost za 1. cikel ,topnje s 2 (z izjemo alopecije, slabosti in bruhanja), absolutno število nevtrofi/cev (AŠN)? 1,5 x 109/1 in število trombocitov? 100 x 109/1. ce odmerka niste pov­:cali v 2. ciklusu, ga v naslednjih ciklusih ne smete povecevati. Ko pa odmerek enkrat povecate, naj ostane na ravni 200 mg/m2 na dan v prvih 5 dneh vsakega nasled­1jega ciklusa, razen ce nastopi toksicnost. Med zdravljenjem morate pregledati celotno krvno sliko na 22. dan (21 dni po prvem odmerku zdravila Temodal). Ponavljajoci :e ali napredujoci maligni gliom: Odrasli bolniki Posamezen ciklus zdravljenja traja 28 dni. Bolniki, ki še niso bili zdravljeni s kemoterapijo, naj jemljejo Temodal pero­alno v odmerku 200 mg/m' enkrat na dan prvih 5 dni, temu pa naj sledi 23-dnevni premor (skupaj 28 dni). Pri bolnikih, ki so že bili zdravljeni s kemoterapijo, je acetni odmerek 150 mg/m2 enkrat na dan, v drugem ciklusu pa se poveca na 200 mg/m2 enkrat na dan 5 dni, ce ni bilo hematoloških toksicnih ucinkov. Pediatricni 1olniki Pri bolnikih, starih 3 leta ali starejših, posamezen ciklus zdravljenja traja 28 dni. Temodal naj jemljejo peroralno v odmerku 200 mg/m2 enkrat na dan prvih 5 dni, 1otem pa naj sledi 23-dnevni premor (skupaj 28 dni). Otroci, ki so že bili zdravljeni s kemoterapijo, naj prejmejo zacetni odmerek 150 mg/m2 enkrat na dan 5 dni, s ,ovecanjem na 200 mg/m2 enkrat na dan 5 dni v naslednjem ciklusu, ce ni bilo hematoloških toksicnih ucinkov. Bolniki z motnjami v delovanju jeter ali ledvic Pri ,olnikih z blagimi ali zmernimi motnjami v delovanju jeter je farmakokinetika temozolomida podobna kot pri tistih z normalnim delovanjem jeter. Podatki o uporabi dravila Temodal pri bolnikih s hudimi motnjami v delovanju jeter (razred lil po Child-u) ali motnjami v delovanju ledvic niso na voljo. Na podlagi farmakokineticnih last­1osti temozolomida obstaja majhna verjetnost, da bo pri bolnikih s hudimi motnjami v delovanju jeter ali ledvic potrebno zmanjšanje odmerka zdravila. Kljub temu je ,otrebna previdnost pri uporabi zdravila Temodal pri teh bolnikih. Starejši bolniki Analiza farmakokinetike je pokazala, da starost ne vpliva na ocistek temozolomida. Kljub imu je potrebna posebna previdnost pri uporabi zdravila Temodal pri starejših bolnikih. Nacin uporabe Temodal mora bolnik jemati na tešce. Temodal kapsule mora olnik pogoltniti cele s kozarcem vode in jih ne sme odpirati ali žveciti. Predpisani odmerek mora vzeti v obliki najmanjšega možnega števila kapsul. Pred jemanjem dravila Temodal ali po njem lahko bolnik vzame antiemetik. ce po zaužitju odmerka bruha, ne sme še isti dan vzeti drugega odmerka. Kontraindikacije Temodal je ontraindiciran pri bolnikih, ki imajo v anamnezi preobcutljivostne reakcije na sestavine zdravila ali na dakarbazin (DTIC). Temodal je kontraindiciran tudi pri bolnikih s hudo 1ielosupresijo. Temodal je kontraindiciran pri ženskah, ki so nosece ali dojijo. Posebna opozorila in previdnostni ukrepi Pilotno preskušanje podaljšane 42-dnevne sheme dravljenja je pokazalo, da imajo bolniki, ki so socasno prejemali zdravilo Temodal in radioterapijo, še posebej veliko tveganje za nastanek pljucnice zaradi okužbe s 'neumocystis carinii (PCP). Profilaksa proti tovrstni pljucnici je torej potrebna pri vseh bolnikih, ki socasno prejemajo zdravilo Temodal in radioterapijo v okviru 42-dnevne heme zdravljenja (do najvec 49 dni), ne glede na število limfocitov. ce nastopi limfopenija, mora bolnik nadaljevati s profilakso, dokler se limfopenija ne povrne na topnjo < 1. Anliemeticna terapija: Z jemanjem zdravila Temodal sta zelo pogosto povezana slabost in bruhanje. Laboratorijske vrednosti Pred jemanjem zdravila 10rata biti izpolnjena naslednja pogoja za laboratorijske izvide: ANC mora biti ? 1,5 x 109/1 in število trombocitov ? 100 x 109/1. Na 22. dan (21 dni po prvem odmerku) li v roku 48 ur od navedenega dne, morate pregledati celotno krvno sliko in jo nato spremljati vsak teden, dokler ni ANC nad 1,5 x 109/1 in število trombocitov nad 100 109/1. ce med katerimkoli ciklusom ANC pade na < 1,0 x 109/1 ali število trombocitov na < 50 x 10'/1, morate odmerek zdravila v naslednjem ciklusu zmanjšati za eno dmerno stopnjo. Odmerne stopnje so 100 mg/m2 , 150 mg/m2 in 200 mg/m2 • Najmanjši priporoceni odmerek je 100 mg/m2 . Moški bolniki Temozolomid lahko deluje enotoksicno, zato morate moškim, ki se zdravijo z temozolomidom svetovati, da naj ne zaplodijo otroka še šest mesecev po zdravljenju. Interakcije Socasna uporaba jravila Temodal in ranitidina ni povzrocila spremembe obsega absorpcije temozolomida ali monometiltriazenoimidazol karboksamida (MTIC). Jemanje zdravila Temodal hrano je povzrocilo 33 % zmanjšanje Cmax in 9 % zmanjšanje površino pod krivuljo (AUC). Ker ne moremo izkljuciti možnosti, da bi bila sprememba Cmax lahko 'inicno pomembna, naj bolniki jemljejo zdravilo Temodal brez hrane. Analiza populacijske farmakokinetike v preskušanjih druge faze je pokazala, da socasna uporaba eksametazona, proklorperazina, fenitoina, karbamazepina, ondansetrona, antagonistov receptorjev H2 ali fenobarbitala ne spremeni ocistka temozolomida. Socasno ,manje z valprojsko kislino je bilo povezano z majhnim, a statisticno znacilnim zmanjšanjem ocistka temozolomida. Uporaba zdravila Temodal v kombinaciji z drugimi 1ielosupresivnimi ucinkovinami lahko poveca verjetnost mielosupresije. Nosecnost študij na nosecih ženskah ni bilo. Predklinicne študije na podganah in kuncih z dmerkom 150 mg/m2 so pokazale teratogenost in/ali toksicnost za plod. Zato naj nosece ženske naceloma ne bi jemale zdravila Temodal. Ce pa je uporaba v casu Jsecnosti nujna, morate bolnico opozoriti na možne nevarnosti zdravila za plod. Zenskam v rodni dobi svetujte, naj med zdravljenjem z zdravilom Temodal preprecijo rnositev. Dojenje Ni znano, ali se temozolomid izloca v materino mleko, zato ženske, ki dojijo, ne smejo jemati zdravila Temodal. Neželeni ucinki V klinicnih preskušanjih ) bili najpogostnejši neželeni ucinki, povezani z zdravljenjem, prebavne motnje, natancneje slabost (43 %) in bruhanje (36 %). Oba ucinka sta bila ponavadi 1. ali 2. opnje (od O do 5 epizod bruhanja v 24 urah) in sta prenehala sama ali pa ju je bilo mogoce hitro obvladali s standardnim antiemeticnim zdravljenjem. lncidenca hude sla­)Sti in bruhanja je bila 4 %. Laboratorijski izvidi: Trombocitopenija in nevtropenija 3. in. 4. stopnje sta se pojavili pri 19 % in 17 % bolnikov, zdravljenih zaradi malignega ioma. Zaradi njiju je bila potrebna hospitalizacija in/ali prekinitev zdravljenja z zdravilom Temodal pri 8 % in. 4 % bolnikov. Mielosupresija je bila predvidljiva (ponavadi 1 je pojavila v prvih nekaj ciklusih in je bila najizrazitejša med 21. in 28. dnem), okrevanje pa je bilo hitro, ponavadi v 1 do 2 tednih. Opazili niso nobenih dokazov Jmulativne mielosupresije. Trombocitopenija lahko poveca tveganje za pojav krvavitev, nevtropenija ali levkopenija pa tveganje za okužbe.Imetnik dovoljenja za pro­1et SP Europe 73, rue de Stalle B-1180, Bruselj, Belgija. Nacin in režim izdaje Zdravilo se izdaja samo na recept, uporablja pa se pod posebnim nadzorom zdravnika iecialista ali od njega pooblašcenega zdravnika. Datum priprave informacije januar 2006 Podrobnejše informacije o zdravilu Temodal dobite na sedežu podjetja. f, Schering-Plough resnicni napredek Pri na novo odkritem glioblastomu multiforme in malignih gliomih, ki se ponovijo ali napredujejo. lfMOOAl ffMOM IIMODAL ;10om9 1 250mg Dunajska 22, 1000 Ljubljana tel: 01 300 1 O 70 tax: 01 30010 80 Te111odal® . Schering-Plough temozolomid A therapeutic advance in second-line NSCLC Tarceva is indicated far the treatment of patients with locally advanced or metastatic Non-Small Celi Lung Cancer (NSCLC) after failure of at least one prior chemotherapy regimen. Far additional information please consult your local Roche office. <.D Refer e nce: o o 1-Tarceva (erlo tinib) summary of product r:,i N o characteristics, F.Hoffmann-La Roche LTD., 2005. (f'Tarceva® erlotinib HCI UKRC 2007 -Advances in Technology UKRC 2007 should prove to be one of the most stimulating congresses to date in terms of the scientific coverage of new and developing fields within medica! imaging. Key sessions will build on the !atest technology being presented within the technical exhibition, providing a unique multi-dis­ciplinary forum to exchange ideas about the !atest developments in technology which will impact on radiology and clinical applications. Monday sees a revisit to a very popular session from 2006 on image perception, featuring an in­ternational cast from Europe and the USA, a key update on radiation protection and x-ray equipment performance issues, including Dr. Walter Huda from the USA presenting a new paradigm for CT dosimetry. This first day also involves a look at new emerging imaging techniques such as PET/MR systems and what promises to be a very poignant debate, hosted by Prof. Mike Smith and Dr. Giles Maskell, on whether advances in technology will render the radiologists and radiographer redun­dant. Tuesday morning has a session on the !atest advances in cartilage imaging and two sessions on developments in MR imaging, incorporating presentations on blood pool agents by Dr. Giles Roditi, molecular MRI by Dr. Arne Hengerer from Erlangen and MR elasticity imaging by Dr. Ralph Sinkus from Paris. The afternoon covers quantitative imaging applications in medicine and a key session on CT with Prof. Mathias Prokop from Utrecht addressing the future role of multi slice CT, dual source CT described by Prof. Thomas Flohr from Erlangen, and 256 slice CT systems from Toshiba. The pace fails to slow down for the fina! day on Wednesday, so it's best not to have too late a night at Tiger-Tiger on the Tuesday night! The scientific sessions involve a look at interactive imaging applications for surgery, including interventional MR, stereotaxy in neurosurgery and surgical robot­ics along with a constantly topical look at the changing face of cardiac CT. The afternoon sees a CT teaching course for radiographers, clinicians, and scientists with everything you need to know about physics, technology, image quality and patient dose. The advances in technology sessions provide a unique opportunity to view and appreciate devel­oping areas of radiology from a truly multi-disciplinary standpoint. Where else could you meet col­leagues of all disciplines and enjoy stimulating presentations from clinicians, scientists and radiogra­phers; not to mention surgeons and cardiologists? Be there in Manchester or be disappointed! Best wishes, Mr Andrew Jones Vice President, UKRC Advances in Technology UKRC 2007 Organisers PO Box 2895 London W1A 5RS Tel: +44(0) 20 7307 1410/20 Fax: +44(0) 20 7307 1414 Email: conference@ukrc.org.uk exhibition@ukrc.org. uk Proffer work by 15 January 2007 and present your paper at the largest Radiological Congress in the UK. Take advantage of our 30% reduction in fees and register now! Catch our early deadline of 16 April 2007 and make a further saving up to f:60! View the Advance Programme, proffer work and register now at www.ukrc.org.uk Radiology a11d Oncology Instructions for authors Editorial policy of the journal Radiology and Oncology is to publish original scientific papers, professional papers, review articles, case reports and varia (editorials, reviews, short communications, professional informa­tion, book reviews, letters, etc.) pertinent to diagnostic and interventional radiology, com­puterized tomography, magnetic resonance, ultrasound, nuclear medicine, radiotherapy, clinical and experimental oncology, radiobiol­ogy, radiophysics and radiation protection. The Editorial Board requires that the paper has not been published or submitted for pub­lication elsewhere: the authors are responsible for all statements in their papers. Accepted articles become the property of the journal and therefore cannot be published elsewhere without written permission from the editorial board. 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