vol.44 no.1 march 2010 pemetreksed Pravi Izbor Pravi Bolnik Pravi Cas Edina histološko usmerjena citostaticna terapija ALIMTA/cisplatin: Zdravljenje prvega reda pri bolnikih z nedrobnocelicnim pljucnim karcinomom, ki nimajo pretežno luskaste histologije Edina kombinirana terapija s signifikantno izboljšanim preživetjem: 12,6 meseca pri bolnikih z adenokarcinomom pljuc1 SKRAJŠAN POVZETEK GLAVNIH ZNACILNOSTI ZDRAVILA Ime zdravila ALIMTA 100 mg prašek za koncentrat za raztopino za infundiranje in ALIMTA 500 mg prašek za koncentrat za raztopino za infundiranje Kakovostna in kolicinska sestava ALIMTA 100 mg: vsaka viala vsebuje 100 mg pemetrekseda (v obliki dinatrijevega pemetrekseda). Po pripravi vsebuje vsaka viala 25 mg/ml pemetrekseda. Pomožne snovi: Vsaka viala vsebuje približno 11 mg natrija. Manitol, klorovodikova kislina, natrijev hidroksid. ALIMTA 500 mg: vsaka viala vsebuje 500 mg pemetrekseda (v obliki dinatrijevega pemetrekseda). Po pripravi vsebuje vsaka viala 25 mg/ml pemetrekseda. Pomožne snovi: Vsaka viala vsebuje približno 54 mg natrija. Manitol, klorovodikova kislina, natrijev hidroksid. Terapevtske indikacije: ALIMTA je v kombinaciji s cisplatinom indicirana za zdravljenje bolnikov z neresektabilnim malignim plevralnim mezoteliomom, ki jih še nismo zdravili s kemoterapijo. ALIMTA je v kombinaciji s cisplatinom indicirana kot zdravljenje prvega izbora za bolnike z lokalno napredovalim ali metastatskim nedrobnocelicnim pljucnim karcinomom, ki nima pretežno luskaste celicne histologije. ALIMTA je indicirana kot monoterapija za zdravljenje lokalno napredovalega ali metastatskega nedrobnocelicnega pljucnega karcinoma, ki nima pretežno luskaste celicne histologije pri bolnikih, pri katerih bolezen ni napredovala neposredno po ke­moterapiji na osnovi platine. Zdravljenje prvega izbora naj bo platinasta dubleta z gemcitabinom, paklitakselom ali docetakselom. ALIMTA je indicirana kot monoterapija za zdravljenje drugega izbora bolnikov z lokalno napredovalim ali metastatskim nedrobnocelicnim pljucnim karcinomom, ki nima pretežno luskaste celicne histologije. Odmerjanje in nacin uporabe: ALIMTO smemo dajati le pod nadzorom zdravnika, usposobljenega za uporabo kemoterapije za zdravljenje raka. ALIMTA v kombinaciji s cisplatinom Priporoceni odmerek ALIMTE je 500 mg/m2 telesne površine (TP), dan kot intravenska infuzija v 10 minutah prvi dan vsakega 21-dnevnega ciklusa. Priporoceni odmerek cisplatina je 75 mg/m2 TP, infundiran v dveh urah približno 30 minut po zakljucku infuzije pemetrekseda prvi dan vsakega 21 dnevnega ciklusa. Priporoceni odmerek cisplatina je 75 mg/m2 TP, infundiran v dveh urah približno 30 minut po zakljucku infuzije pemetrekseda prvi dan vsakega 21 dnevnega ciklusa. Bolniki morajo prejeti zadostno antiemeticno zdravljenje, pred in/ali po prejemanju cisplatina jih moramo tudi ustrezno hidrirati. ALIMTA kot samostojno zdravilo Priporoceni odmerek ALIMTE je 500 mg/m2 TP, dan kot intravenska infuzija v 10 minutah prvi dan vsakega 21 dnevnega ciklusa. Režim premedikacije Da zmanjšamo incidenco in resnost kožnih reakcij, dajemo kortikosteroid dan pred dajan­jem pemetrekseda, na dan dajanja pemetrekseda in naslednji dan. Kortikosteroid naj ustreza 4 mg deksametazona, danega peroralno dvakrat dnevno. Za zmanjšanje toksicnosti morajo bolniki dnevno jemati tudi peroralno folno kislino ali multivitaminski pripravek, ki jo vsebuje (350 do 1000 mikrogramov). V sedmih dneh pred prvim odmerkom pemetrek­seda morajo vzeti vsaj pet odmerkov folne kisline, odmerjanje pa morajo nadaljevati ves cas zdravljenja in še 21 dni po zadnjem odmerku pemetrekseda. Bolniki morajo prejeti tudi intramuskularno injekcijo vitamina B12 (1000 mikrogramov) v tednu pred prvim odmerkom pemetrekseda in enkrat vsake tri cikluse zatem. Kasnejše injekcije vitamina B12 lahko dajemo isti dan kot pemetreksed. Kontraindikacije: Preobcutljivost za zdravilno ucinkovino ali katerokoli pomožno snov. Dojenje. Socasno cepljenje proti rumeni mrzlici. Posebna opozorila in previdnostni ukrepi: Pemetreksed lahko zavre delovanje kostnega mozga, kar se kaže kot nevtropenija, trombocitopenija in anemija (ali pancitopenija). Pri bolnikih, ki pred zdravljenjem niso prejemali kortikosteroidov, so porocali o kožnih reakcijah. Uporabe pemetrekseda pri bolnikih z ocistkom kreatinina < 45 ml/min ne priporocamo. Bolniki z blagim do zmernim popušcanjem delovanja ledvic naj se izogibajo jemanju nesteroidnih protivnetnih zdravil (NSAID), denimo, ibuprofena in acetilsalicilne kisline 2 dni pred dajanjem pemetrekseda, na dan dajanja in še 2 dni po dajanju pemetrekseda. Vsi bolniki, ki jih lahko zdravimo s pemetreksedom, naj se izogibajo jemanju NSAID-ov z dolgimi razpolovnimi casi izlocanja vsaj 5 dni pred dajanjem pemetrekseda, na dan dajanja in še vsaj 2 dni po dajanju pemetrekseda. Porocali so o resnih ledvicnih primerih, vkljucno z akutno ledvicno odpovedjo, s pemetreksedom samim ali v povezavi z drugimi kemoterapevtiki. Pri bolnikih s klinicno pomembno tekocino tretjega prostora moramo razmisliti o drenaži izliva pred dajanjem pemetrekseda. Kot posledico toksicnosti pemetrekseda v kombinaciji s cisplatinom za prebavila so opažali hudo dehidracijo, zato moramo bolnike pred prejemanjem terapije in/ali po njej ustrezno hidrirati, prejeti morajo zadostno antiemeticno zdravljenje. Obcasno so v klinicnih študijah pemetrekseda, obicajno ob socasnem dajanju z drugo citotoksicno ucinkovino, porocali o resnih srcnožilnih dogodkih, vkljucno z miokardnim infarktom in možganskožilnimi dogodki. Odsvetujemo uporabo živih oslabljenih cepiv. Spolno zrelim moškim odsvetujemo zaploditev otroka v casu zdravljenja in še 6 mesecev zatem. Priporocamo ukrepe prosti zanositvi ali vzdržnost. Zaradi možnosti, da zdravljenje s pemetreksedom povzroci trajno neplodnost, naj se moški pred zacetkom zdravljenja posvetujejo o shranjevanju semena. Ženske v rodni dobi morajo v casu zdravljenja s pemetreksedom uporabljati ucinkovito kontracepcijo. Porocali so o primerih radiacijske pljucnice pri bolnikih, ki so jih zdravili z radiacijo pred, med ali po zdravljenju s pemetreksedom. Porocali so o radiacijskem izpušcaju pri bolnikih, ki so se zdravili z radioterapijo pred tedni ali leti. Zdravilo Alimta 500 mg vsebuje približno 54 mg natrija na vi-alo. Pomembno za bolnike, ki so na dieti z nadzorovanim vnosom natrija. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Socasno dajanje nefrotoksicnih zdravil (denimo, aminoglikozidov, diuretikov zanke, spojin platine, ciklosporina) lahko potencialno povzroci zakasnjeni ocistek pemetrekseda. Socasno dajanje snovi, ki se tudi izlocajo s tubulno sekrecijo (denimo, probenecid, penicilin), lahko potencialno povzroci zakasnjeni ocistek pemetrekseda. Pri bolnikih z normalnim delovanjem ledvic lahko visoki odmerki nesteroidnih protivnetnih zdravil (NSAID-i, denimo, ibuprofen) in acetilsalicilna kislina v visokih odmerkih zmanjšajo eliminacijo pemetrekseda in tako lahko povecajo pojavnost neželenih ucinkov pemetrekseda. Pri bolnikih z blagim do zmernim popušcanjem delovanja ledvic se moramo izogibati socasnemu dajanju pemetrekseda z NSAID-i (denimo, ibuprofenom) ali acetilsalicilne kisline v visokih odmerkih 2 dni pred dajanjem pemetrekseda, na dan dajanja in še 2 dni po dajanju pemetrekseda. Socasnemu dajanju NSAID-ov z daljšimi razpolovnimi casi s pemetreksedom se moramo izogibati vsaj 5 dni pred dajanjem pemetrekseda, na dan dajanja in še vsaj 2 dni po dajanju pemetrekseda.Velika razlicnost med posamezniki v koagulacijskem statusu v casu bolezni ter možnost medsebojnega delovanja med peroralnimi antikoagulacijskimi ucinkovinami ter kemoter­apijo proti raku zahtevata povecano pogostnost spremljanja INR. Kontraindicirana socasna uporaba: Cepivo proti rumeni mrzlici: tveganje za smrtno generalizirano bolezen po cepljenju. Odsvetovana socasna uporaba: Živa oslabljena cepiva (razen proti rumeni mrzlici): tveganje za sistemsko, potencialno smrtno bolezen. Neželeni ucinki Klinicne študije malignega plevralnega mezotelioma Zelo pogosto: znižani nevtrofilci/granulociti, znižani levkociti, znižan hemoglobin, znižani trombociti, nevropatija-senzoricna, diareja, bruhanje, stomatitis/faringitis, slabost, anoreksija, zaprtje, izpušcaj, alopecija, povišan kreatinin, znižan ocistek kreatinina, utrujenost. Pogosti: dehidracija, motnje okusa, konjuktivitis, dispepsija. Klinicne študije nedrobnocelicnega pljucnega karcinoma - ALIMTA monoterapija, zdravljenje 2. izbora: Zelo pogosti: znižan nevtrofilci/granulociti, znižani levkocitit, znižan hemoglobin, diareja, bruhanje, stomatitis/faringitis, slabost, anoreksija, izpušcaj/lušcenje, utrujenost. Pogosti: znižani trombociti, zaprtje, povišanje SGPT (ALT), povišanje SGOT (AST), srbenje, alopecija, povišana telesna temperatura. Klinicne študije nedrobnocelicnega pljucnega karcinoma - ALIMTA v kombinaciji s cisplatinom, zdravljenje 1. izbora: Zelo pogosti: znižan hemoglobin, znižani nevtrofilci/granulociti, znižani levkocitit, znižani trombociti, slabost, bruhanje, anoreksija, zaprtje, stomatitis/faringitis, diareja brez kolostomije, alopecija, izpušcaj/lušcenje, povišan kreatinin. Pogosti: nevropatija-senzoricna, motnje okusa, dispepsija/zgaga. Klinicne študije nedrobnocelicnega pljucnega karcinoma - ALIMTA monoterapija, vzdrževalno zdravljenje: Zelo pogosti: znižan hemoglobin, slabost, anoreksija, utrujenost, izpušcaj/lušcenje, utrujenost. Pogosti: infekcija, znižani levkociti, znižani nevtrofilci, nevropatija-senzoricna, bruhanje, mukozitis/stomatitis, diareja, povišanje ALT (SGPT), povišanje AST (SGOT). Obcasno so v klinicnih študijah pemetrekseda porocali o primerih resnih srcnožilnih in možganskožilnih dogodkov, vkljucno z miokardnim infarktom, angino pektoris, cerebrovaskularnim insultom in prehodnimi ishemicnimi atakami; primerih kolitisa ter o primerih intersticijske pljucnice z respiratorno insuficience, primerih edema in o ezofagitisu/radiacijskem ezofagitisu. Redkeje pa o primerih potencialno resnega hepatitisa in pancitopenije. Po uvedbi zdravila na trg so porocali o primerih akutne odpovedi ledvic s pemetreksedom samim ali v povezavi z drugimi kemoterapevtiki, primerih radiacijske pljucnice pri bolnikih, ki so jih zdravili z radiacijo pred, med ali po njihovem zdravljenju s pemetreksedom, primerih radiacijskega izpušcaja pri bolnikih, ki so se v preteklosti zdravili z radioterapijo in o primerih periferne ishemije, ki je vcasih vodila v nekrozo okoncin. Imetnik dovoljenja za promet Eli Lilly Nederland B.V., Grootslag 1 5, NL 3991 RA, Houten, Nizozemska. Datum zadnje revizije besedila 21.09.2009. Nacin izdaje zdravila: H Podrobnejše informacije o zdravilu Alimta, so na voljo na lokalnem predstavništvu SIALM00005 Eli Lilly Farmacevtska družba, d.o.o. Brnciceva 41G, 1231 Ljubljana - Crnuce, Slovenija Telefon: +386 (0)1 5800 010 Faks: +386 (0)1 5691 705 Publisher Association of Radiology and Oncology Affiliated with Slovenian Medical Association – Slovenian Association of Radiology, Nuclear Medicine Society,Slovenian Society for Radiotherapy and Oncology, and Slovenian Cancer Society Croatian Medical Association – Croatian Society of RadiologySocietas Radiologorum HungarorumFriuli-Venezia Giulia regional groups of S.I.R.M. Italian Society of Medical Radiology Aims and scope Radiology and Oncology is a journal devoted to publication of original contributions in diagnostic and interventionalradiology, computerized tomography, ultrasound, magnetic resonance, nuclear medicine, radiotherapy, clinical andexperimental oncology, radiobiology, radiophysics and radiation protection. Editor-in-Chief Gregor SeršaLjubljana, Slovenia Executive Editor Viljem KovacLjubljana, Slovenia Deputy Editors Andrej Cör Ljubljana, Slovenia Igor KocijancicLjubljana, Slovenia Editorial Board Karl H. Bohuslavizki Hamburg, GermanyMaja Cemažar Ljubljana, Slovenia Christian Dittrich Vienna, Austria Metka Filipic Ljubljana, SloveniaTullio Giraldi Trieste, ItalyMaria Godény Budapest, Hungary Vassil Hadjidekov Sofia, BulgariaMarko Hocevar Ljubljana, Slovenia Maksimilijan Kadivec Ljubljana, Slovenia Miklós Kásler Budapest, Hungary Michael Kirschfink Heidelberg, Germany Janko Kos Ljubljana, SloveniaTamara Lah Turnšek Ljubljana, SloveniaDamijan Miklavcic Ljubljana, SloveniaLuka Milas Houston, USADamir Miletic Rijeka, CroatiaMaja Osmak Zagreb, Croatia Branko Palcic Vancouver, CanadaDušan Pavcnik Portland, USAGeoffrey J. Pilkington Portsmouth, UKErvin B. Podgoršak Montreal, CanadaUroš Smrdel Ljubljana, Slovenia Primož Strojan Ljubljana, SloveniaBorut Štabuc Ljubljana, SloveniaRanka Štern-Padovan Zagreb, CroatiaJustin Teissié Tolouse, FranceSándor Tóth Orosháza, HungaryGillian M. Tozer Sheffield, UKAndrea Veronesi Aviano, ItalyBranko Zakotnik Ljubljana, Slovenia Advisory Committee Marija Auersperg Ljubljana, SloveniaTomaž Benulic Ljubljana, SloveniaJure Fettich Ljubljana, SloveniaValentin Fidler Ljubljana, SloveniaBerta Jereb Ljubljana, SloveniaVladimir Jevtic Ljubljana, SloveniaStojan Plesnicar Ljubljana, SloveniaMirjana Rajer Ljubljana, SloveniaŽiva Zupancic Ljubljana, Slovenia Radiol Oncol 2010; 44(1): A. Editorial office Radiology and Oncology Institute of Oncology Zaloška 2SI-1000 Ljubljana Slovenia Phone: +386 1 5879 369Phone/Fax: +386 1 5879 434E-mail: gsersa@onko-i.si Copyright © Radiology and Oncology. All rights reserved. Reader for English Vida Kološa Secretary Mira Klemencic Design Monika Fink-Serša, Samo Rovan, Ivana Ljubanovic Layout Matjaž Lužar Printed byImprint d.o.o., Ljubljana, Slovenia Published quarterly in 600 copies Beneficiary name: DRUŠTVO RADIOLOGIJE IN ONKOLOGIJEZaloška cesta 2,1000 LjubljanaSlovenia Beneficiary bank account number: SI56 02010-0090006751IBAN: SI56020100090006751Our bank name: Nova Ljubljanska banka, d.d., Ljubljana, Trg republike 2,1520 Ljubljana; SloveniaSWIFT: LJBASI2X Subscription fee for institutions EUR 100, individuals EUR 50 The publication of this journal is subsidized by the Slovenian Research Agency. Indexed and abstracted by: Science Citation Index Expanded (SciSearch®)Journal Citation Reports/Science EditionScopusEMBASE/Excerpta MedicaOpen J-gateChemical AbstractsBiomedicina Slovenica This journal is printed on acid- free paper On the web: ISSN 1581-3207 http://versita.com/science/medicine/ro/http://www.onko-i.si/radioloncol/ Radiol Oncol 2010; 44(1): B. editorial Progress of Radiology and Oncology In front of you is a new issue of Radiology and Oncology, an international journal devoted to publica­tions in the field of radiology, nuclear medicine and oncology. The journal’s scope is to publish review papers, scientific papers and case reports on these and related topics, including experimental oncology, radiophysics and radiation protection. It is my pleasure to inform you that Radiology and Oncology has grown over the past two years, thanks to inclusion into Web of Science and the Journal Citation Reports/Science Edition database from 2008. In oth­er words, we will gain an Impact Factor (IF), probably in 2010 or in 2011. The inclusion into Web of Science is already reflected in a higher submission rate of papers, which enabled us to increase the number of published articles. It is additionally reflected in higher average citations per item and in a higher h-index. Along with the printed version, Radiology and Oncology is also published electronically with open access on www.versita.com. This enables swift publication of the papers as E-ahead of print - early birds. In light of making our journal more attractive, we have prepared a new format of the journal with a more attractive appearance. We now publish the journal in full color text, so that the papers in the printed and electronic edition of the journal will be more appealing. At the end I would like to encourage you to submit papers to Radiology and Oncology and also encourage your colleagues and students to choose our journal for their publications. We in the editorial office will strive to be swift in processing the papers and to publish papers with a higher impact and influence in the scientific community. Best regards, Prof. Gregor Serša, Ph.D.Editor in Chief Viljem Kovac, M.D.Executive Editor Radiol Oncol 2010; 44(1): C. contents contents review 1 Role of radiotherapy in melanoma management Primoz Strojan 13 Genetic markers in oligodendroglial tumours Tomaz Velnar radiology 19 CT colonography in detection of colorectal carcinoma Amela Sofic, Serif Beslic, Igor Kocijancic, Nedzad Sehovic 24 Diffusion weighted MR imaging in the differential diagnosis of haemangiomas and metastases of the liver Nagihan Inan, Furkan Kilinc, Tahsin Sarisoy, Sevtap Gumustas, Gur Akansel, Ali Demirci 30 Percutaneous transcatheter arterial embolization in haemodynamically stable patients with blunt splenic injury Peter Popovic, Dragoje Stanisavljevic, Miran Jeromel experimental oncology 34 Numerical study of the electroporation pulse shape effect on molecular uptake of biological cells Damijan Miklavcic, Leila Towhidi 42 Development of human cell biosensor system for genotoxicity detection based on DNA damage-induced gene expression Valerija Zager, Maja Cemazar, Irena Hreljac, Tamara T. Lah, Gregor Sersa, Metka Filipic Radiol Oncol 2010; 44(1): E. contents clinical oncology 52 Attitudes of midwifery students towards teaching breast-self examination Andrej Plesnicar, Martina Golicnik1, Irena Kirar Fazarinc, Bozo Kralj, Viljem Kovac, Blanka Kores Plesnicar 57 Genetic testing for young-onset colorectal cancer: case report and evidence-based clinical guidelines Yaolin Zhou, Lisa A. Boardman, Robert C. Miller radiophysics 62 A neutron track etch detector for electron linear accelerators in radiotherapy Branko Vukovic, Dario Faj, Marina Poje, Maja Varga, Vanja Radolic, Igor Miklavcic, Ana Ivkovic, Josip Planinic I slovenian abstracts Radiology and oncology is covered in Science Citation Index Expanded (SciSearch®),Journal Citation Reports/Science Edition, Scopus, EMBASE/Excerpta Medica, Open J-gate, Chemical Abstracts, Biomedicina Slovenica Radiol Oncol 2010; 44(1): F. review Role of radiotherapy in melanoma management Primoz Strojan Department of Radiation Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia Received 28 January 2010 Accepted 18 February 2010 Correspondence to: Assoc. Prof. Primož Strojan, M.D., Ph.D., Department of Radiation Oncology, Institute of Oncology Ljubljana, Zaloska 2, Ljubljana, Slovenia. Phone: +386 1 5879 110; Fax: +386 1 400; E-mail: pstrojan@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. In melanoma, radiotherapy has generally been considered as a palliative treatment option indicated only for advanced cases or disseminated disease. In the 70s of the previous century, the technological advances in radiotherapy, linked to rapid development of computer sciences, resulted in restored interest for radiotherapy in melanoma management. Although a fundamental lack of well designed prospective and/or randomized clini­cal trials critically influenced the integration of radiotherapy into treatment strategies in melanoma, radiotherapy was recently recognized as an indispensable part in the multidisciplinary management of patients with melanoma. Altogether, approximately 23% of melanoma patients should receive at least one course of radiotherapy during the course of the disease. In this review, radiobiological properties of melanoma that govern the decisions for the frac­tionation patterns used in the treatment of this disease are described. Moreover, the indications for irradiation and the results of pertinent clinical studies from the literature, creating a rationale for the use of radiotherapy in the manage­ment of this disease, are reviewed and a brief description of radiotherapy techniques is given. Conclusions. Basic treatment modality in melanoma is surgery. However, whenever surgery is not radical or there are adverse prognostic factors identified on histopathological examination of resected tissue specimen, it needs to be supplemented. Also, in patients with unresectable disease or in those not being suitable for major surgery or who refuse proposed surgical intervention, other effective mode(s) of therapy need to be implemented. From this perspec­tive, supported by clinical experiences and literature results, radiotherapy is a valuable option: it is effective and safe, in curative and palliative setting. Key words: melanoma; radiobiology; radiotherapy; fractionation; indications; toxicity Introduction Changes in human behavior, particularly those re­lated to sun exposure and global environmental al­terations have contributed to an observed increase in the incidence of cutaneous melanoma in Europe since the 1950s.1In Slovenia with the population of two million, the melanoma incidence doubled during the last decade, being 10.2 per 100.000 in­habitants in 1997 and 19.6 in 2006.2,3As melanoma is a significant health burden, its management was continuously in focus of extensive laboratory and clinical research. Surgery is basic and the most effective treat­ment modality for melanoma, whereas radiother­apy, one of the corn stones of anti-cancer manage­ment, has been evolving steadily and, during the time, taking over greater role in the management of this disease. It has long been negatively marked by the lack of well designed prospective and/or randomized clinical trials which finally gave more credit to lucid observations of clinicians dealing with the disease.4 First experiences of radiation oncologists with melanoma were marked with technologically infe­rior irradiation devices and the label of tumor as radioresistant, which originated from categoriza­tion of tumor radiosensitivity by histological type introduced in 1930s.5Consequently, radiotherapy was generally considered as a palliative treatment option indicated only for advanced cases or dis­seminated disease. In the 70s of previous century, the interest for radiotherapy in melanoma management was re­stored. During the decades, new knowledge on radiobiological characteristics of melanoma cells Strojan P / Radiotherapy in melanoma management RADIOTHERAPY DOSE (Gy) FIguRe 1. Dose-response curve for melanoma cells. High in­trinsic capacity of melanoma cells for repair of sublethal DNA damages caused by irradiation is graphically presented by a distinctly broad shoulder in the low-dose portion of the logarith­mic cell survival curve. Accordingly, the ability of melanoma cell to overcome sublethal DNA injuries suggests increased sensitivity to large doses per fraction (hypofractionation). as well as more favorable clinical experiences were obtained. Furthermore, modern radiotherapy de­vices, including treatment planning systems, ap­peared on the market, allowing more sophisticated treatment planning and accurate targeting. These novelties contributed to a change in perception of clinicians confronted with this disease, which directly contradicted to long standing belief that melanoma is radioresistant tumor. Nowadays, RT is recognized as the most effec­tive non-surgical mode of locoregional therapy of melanoma and is an integral part of the multidisci­plinary management, thus providing a valuable in­put to the best treatment of melanoma. According to Delaney et al., the recommended proportion of all patients with melanoma who, according to the best available evidence, should receive at least one course of radiotherapy is 23%.6 Clinical radiobiology of melanoma and fractionation pattern Response of melanoma to irradiation depends on tumor volume, radiotherapy dose and fraction size. From preclinical studies as well as clinical ob­servations, an abundance of evidences emerge con­firming a positive relationship between response to irradiation and radiotherapy dose corrected for tumor volume, i.e.the number of clonogenic cells that need to be sterilized. Within the timeframe of the schedules used, the overall treatment time showed no effect on response rate.7,8 From radiobiological perspective, the most in­triguing is the observation that melanomas have a wide range of sensitivities to ionizing irradia­tion.9-13The results of in vitro studies on melanoma radiosensitivity suggest high intrinsic capacity of melanoma cells for repair of sublethal DNA dam­ages caused by photon beam radiotherapy.11-14This particular characteristic of melanoma cells is graphically presented by a distinctly broad shoul­der in the low-dose portion of the logarithmic cell survival curve (Figure 1).13However, variations in the cellular radiosensitivity recognized in vitro and in clinic imposed other factors to add to the observed heterogeneity among treated tumors.10,12There are several candidates, i.e.intra-tumor vari­ability (clonogenic subpopulations with different radiosensitivity, variations in tendency to apopto-sis); tumor physiological factors (the existence of hypoxic fraction and/or differences in reoxygena­tion capacity of tumor clonogens, the intracellular level of glutathione – scavenger of free radicals responsible for DNA damage); tumor cell kinet­ics (different propensity to cell cycle disruption); and host-related factors (immune competence of the patient).9-12According to the results of in vitrostudies, sublethal irradiation doses increase the risk of metastases, possibly due to increased hy­poxic fraction and hypoxia-induced up-regulation of urokinase-type plasminogen activator receptor in regrowing primary tumors.15 Theoretically, the ability of melanoma cell to overcome sublethal DNA injuries caused by irra­diation suggests that, clinically, melanoma should be more sensitive to large doses per fraction (hy­pofractionation) than to lower fraction doses (hy­perfractionation).14This concept is mirrored in a low value of the ratio of the parameters a and b in the linear-quadratic model, a determinant of the shape (or bendiness) of survival curve in the model and an indicator used to quantify the fractionation sensitivity of tissues. As derived from clinical data, the a/b ratio for cutaneous melanoma ranges from 0.6 Gy to 2.5 Gy and is characterized by wide con­fidence intervals, implying large variations in the sensitivity of individual tumors to radiotherapy.7,8 Also, wide range of values of the a/b ratio resulted from calculations in preclinical studies.16 Despite seemingly firm theoretical arguments, clinical data on optimal fractionation pattern are not equivocal and no consensus was accepted on the best radiation regimen. This issue is further complicated by an increased probability of morbid­ Strojan P / Radiotherapy in melanoma management ity from late reacting normal tissue injury when us­ing hypofractionated regimens.13,14Whereas good arguments for the use of fraction doses of =4 Gy were provided by several retrospective studies (for review see Bello andAng17), the results of the only prospective randomized trial addressing the issue of low versus high fraction doses neglected the ex­pected advantage of hypofractionation. In RTOG 83-05, 137 patients with measurable lesions were randomized between 20 x 2.5 Gy in 26-28 days and 4 x 8 Gy in 21 days.18No differences in local control, either complete or partial, were reported between the two arms; unfortunately, no data on the dura­tion of responses were provided from this trial.18On adjuvant setting, a retrospective comparison of conventional and hypofractionated regimens19-22and reported in-field relapse rates from rare ret­rospective23or prospective24series implementing more conventional fraction doses (i.e.1.7–2.4 Gy) support this observation. On the other hand, prospective randomized comparison of different hypofractionation sched­ules (3 x 9 Gy versus 5 x 8 Gy, 2 fractions per week) in recurrent or metastatic melanoma resulted in virtually identical durable complete response rates of 65% and 72%, respectively, in the two arms of the trial.25 In another randomized study, after add­ing hyperthermia as an adjuvant to radiotherapy (3 x 8-9 Gy in 8 days) for macroscopic lymph node and skin disease, multivariate analysis with either complete response or 2-year local control rates as an endpoint showed that tumor dose, on the top of additional hyperthermia and tumor size, was an independent prognostic variable.26 Indications for radiotherapy Considering the treatment intent and the time point at which radiotherapy is to be introduced into melanoma management, indications for irra­diation can be divided into four groups: upfront radiotherapy (as the main treatment modality, re­placing surgery); adjuvant radiotherapy (after sur­gery), elective and palliative radiotherapy. Radiotherapy as primary therapy Radiotherapy is rarely used as a primary treatment modality instead of surgery which is the curative treatment of choice for all types of primary melano­ma lesions. Poor performance status of the patient with severe comorbidities or refusal of proposed surgery are potential but less plausible motives in clinic for replacing surgery with radiotherapy. More frequent indication for upfront radio­therapy is lentigo maligna melanoma (LMM). Particularly when LMM is extensive and located on the face of elderly patient, radiotherapy is a good alternative to surgery. In three larger series with a total of 107 patients, 3 local recurrences were observed 13-44 months after radiotherapy (85 lesions) or combination of surgical excision of the nodular part followed by irradiation of the lentiginous part of the lesion (22 lesions).27-29Time to complete regression of the lesion after irradia­tion took up to 24 months. Regional node metas­tases developed in 3 patients 6, 8 and 18 months after therapy, respectively, whereas in one patient, pulmonary metastases occurred 44 months after treatment. All theses patients had their primaries controlled.27-29Thus, whenever surgery attempting to achieve clear margins would result in excessive mutilation, either cosmetic or functional, or in eld­erly patients, it should be replaced with radiother­apy, which is effective and has curative potential in LMM. Because the incidence of regional metastases is extremely low, no elective irradiation of regional lymphatics is required. Primary curative radiotherapy should be at­tempted also in localized inoperable mucosal melanoma (MM) where it is considered the most effective treatment modality. After the extensive literature review, Krengli et al.summarized their analyses in a way that high local control rates, over 70%, can be achieved with radiotherapy alone in MM, which could be – taking into account some preliminary results – further improved by utiliz­ing high-linear energy transfer (LET) radiation.30Primary tumor area only should be included into the irradiation field in clinically N0 disease as it is unlikely that elective nodal treatment affects the overall course of the disease.30,31The exception might be the oral cavity primaries with higher re­gional failure rates.32 Adjuvant radiotherapy – primary melanoma Afterexcisionofprimarylesion,thedecisionabout theuseofpostoperativeradiotherapyisdictatedby theriskestimateforrecurrence,treatmentrelated side-effectsandthepossibilityforsuccessfulsal­vagewhenrecurrenceoccurred.Becauseofsuperfi­cialnatureofthetargettissue(s),theriskforserious complicationsafterlocalradiotherapyislow. Strojan P / Radiotherapy in melanoma management TAble 1. Nodal field relapse rates (number of relapses/dissections) after therapeutic surgery according to adverse clinicopatho-logical features negatively impacted disease control in dissected nodal basin No. of involved nodes 1 9, 9, 25, 45 45,46,47,48 1-3 19, 14, 25a, 15, 24 47,49,50a,51,52 2-4 15, 10 45,46 =4 17, 22, 20, 60, 53, 46a, 8, 37 45,46,47,48,49,50a,51,52 >10 33, 26, 63a, 47 45,46,50a,52 Diameter of largest node <3 cm vs. 3-6 cm vs. >6 cm 25 vs. 42 vs. 80a 50a Extracapsular tumor spread No 15, 38, 23a, 9 45,48,50a,51 Yes 28, 54, 63a, 24, 43 45,48,50a,51,52 Matted nodes Yes 29, 44, 12 45,46,52 Nodal basin Parotid & neck 41,15, 19, 50, 43a, 14, 43, 50, 50 22,45,47,48,50a,51,52,53,54 Axilla 15, 60, 28a, 30, 14, 10 45,48,50a,52,53,55 Groin 17, 44, 18, 23, 19, 8, 34, 19, 34, 8 45,48,49,50a,52,53,56,57,58,59 All nodal sites 16, 52, 18, 30a, 28, 15, 34, 41 45,48,49,50a,52,53,60,61 aActuarial nodal basin control rates at 10 years are reported. Factors that adversely influence local control after wide excision alone are close or positive mar­gins, early and/or multiple recurrences, extensive satellitosis, desmoplasia or neurotropism, and MM primaries. The incidence of local recurrence when tumor satellites are noted histologically was reported to be 12-14%33,34, and in desmoplastic tu­mors, as high as 11-48%.35-38In the latter case, it ap­pears that local recurrence may be related to the presence of neutropism38and to inadequate surgi­cal margins36-39, which could be of importance for lesions arising in anatomically critical regions of the head and neck. In high-risk clinical situations, postoperative radiotherapy has a potential to re­duce the risk of local recurrence significantly.37,40-42In MM, a number of retrospective studies suggest that postoperative radiotherapy yields better out­come, although it has no influence on survival. Combined approach is currently recommended af­ter non-radical surgery, but seems to improve local control also after excision of large primary tumors, especially those in sinonasal localization, and those with perineural invasion.30 Adjuvant radiotherapy – regional lymphatic metastases After dissection of regional lymph nodes, radio­therapy adds significantly to an improved control in the operative bed. Only two randomized con­trolled trials were conducted to clarify this issue. The first was carried out in the 1970s with small sample size (56 patients) using an unusual regimen (split course, 50 Gy total and 1.78 Gy daily mid-plane dose, one field was treated daily) and was found inconclusive.43Only recently, the results of the intergroup multicenter randomized trial (ANZMTG 01.02/TROG 02.01) were published (in an abstract form, Henderson et al.44). After lym­phadenectomy for isolated regional recurrence of melanoma, 250 patients considered to be at high risk (>25%) of in-field recurrence were randomized into radiotherapy group (126 patients) and con­trol group (127 patients); 227 patient were avail­able for analysis. After a median follow up of 27 months, a statistically significant improvement in lymph node field control was observed with radio­therapy (hazard ratio 1.77, 95% confidence interval 1.02-3.08, P=0.041), but not also in median survival times (P=0.14).44 Thus far, identification of factors increasing the risk for regional recurrence after lymphadenecto-my and recommendations for adjuvant irradiation were based on retrospective analyses or rare non-randomized prospective studies. In high-risk set­ting, the rates of relapse in nodal basin could reach 50% or even more after surgery alone. The factors contributing to an increased recurrence in surgical field are the presence of residual disease after sur­ Strojan P / Radiotherapy in melanoma management TAble 2. Therapeutic lymph node dissection in melanoma patients with or without adjuvant radiotherapy: comparison of nodal basin recurrence rates Parotid & neck Parotid & neck Bayers, 198654 28 50 Ang et al., 199462 95 8 Calabro et al., 198945 287 15 O’Brian et al., 199747 45 7 O’Brian et al., 199747 107 19 Shen et al., 200051 21 14 Shen et al., 200051 196 14 Ballo et al., 200263 160 8 Pidhorecky et al., 200152 44 43 Strojan et al., 201022 45 18 Strojan et al., 201022 42 40 Total 366 10 Total 704 20 Axilla Axilla Bowsher et al., 198653 22 14 Ballo et al., 200264 89 10 Calabro et al., 198945 438 15 Beadle et al., 200965 200 10 Pidhorecky et al., 200152 116 30 Total 289 10 Kretschmer, et al., 200155 63 10 Total 639 17 Groin Groin Bowsher et al., 198653 36 8 Ballo et al., 200466 40 23 Kissin et al., 198756 44 34 Calabro et al., 198945 276 17 Hughes et al., 200057 132 19 Pidhorecky et al., 200152 93 19 Kretschmer et al., 200158 104 34 Allan et al., 200859 72 8 Total 757 20 All sites All sites Bowsher et al., 198653 66 15 Burmeister et al., 199567 26 12 Calabro et al., 198945 1001 16 Corry et al., 199923 42 21 Miller et al., 199249 55 18 Stevens et al., 200068 1741 11 Monsour et al., 199348 48 52 Cooper et al., 200141 401 8 Pidhorecky et al., 200152 253 28 Fuhrmann et al., 200169 58 16 Mayer et al., 200260 140 34 Chang et al., 200621 54 12 Henderson et al., 200944 108 31 Burmeister et al., 200624 234 7 Agrawal et al., 200961 106 41 Ballo et al., 200670 466 9 Total 1777 23 Henderson et al., 200944 123 18 Agrawal et al., 200961 509 10 Total 1726 11 gery, extracapsular tumor extension, nodes meas­uring =3 cm in the largest diameter, multiple nodal involvement or recurrence after previous lymph node dissection (and RT was not used at that time) (Table 1).22,45-61The criteria for using adjuvant irra­diation vary slightly among different nodal basins, reflecting various potential outcomes for distinc­tive anatomical body region. In recent ANZMTG/TROG randomized trial, the high-risk features (in addition to non-radical surgery and recurrent disease) were as follows: =1 parotid, =2 cervical or axillary or =3 groin nodes; extracapsular spread of tumor; maximum metastatic node diameter =3 cm in neck and axilla or =4 cm in the groin.44 Comparison of studies using surgery alone or surgery plus radiotherapy provides a strong argu­ment for the effectiveness of adjuvant irradiation when adverse prognostic factors are found at his-topathological examination of resected specimen. In-field tumor control is roughly 90% in adjuvantly irradiated patients using either conventional or hypofractionated schedules (Table 2).21-24,41,44-49,51-70However, in these studies, only the patients with less favorable disease characteristics were referred TAble 3. Nodal basin control after surgery with or without adjuvant radiotherapy O’Brian et al., 199747 Surgery 107 Parotid & neck 20 43 9 56 19 40 35 n.r. Surgery + XRT 45 Parotid & neck 49 67 24 38 7 17 40 n.r. Shen et al,. 200051 Surgery 196 Parotid & neck 23 n.r. 27 32a 14 17 n.r. 32 Surgery + XRT 21 Parotid & neck 43 n.r. 48 n.r. 14 25 n.r. n.r. Fuhrmann et al., 200169 Surgery 58 All sites n.r. 74 n.r. n.r. 21 26 n.r. 25 Surgery + XRT 58 All sites n.r. 74 n.r. n.r. 16 22 n.r. 23 Moncrieff et al., 200871 Surgery 587 Parotid & neck n.r. n.r. n.r. 35 n.r. 6b n.r. n.r. Surgery + XRT 129 Parotid & neck n.r. n.r. n.r. 35 n.r. 10b n.r. n.r. Henderson et al., 200944 Surgery 108 All sites 27 31 n.r. n.r. n.r. All patients at high risk for regional recurrence Surgery + XRT 109 All sites 27 18 n.r. n.r. n.r. Agrawal et al., 200961 Surgery 106 All sites 60 41 48 30 n.r. All patients at high risk for regional recurrence Surgery + XRT 509 All sites 60 10 13 51 n.r. Strojan et al., 201022 Surgery 42 Parotid & neck 21 38 n.r. 25 40 44a n.r. 58a Surgery + XRT 45 Parotid & neck 44 64 n.r. 25 18 22a n.r. 51a ECE – Extracapsular extension of tumor; N+ - Number of positive nodes; FUP – Follow-up; XRT – Radiotherapy; n.r. – Not reported.aAt 2 years. bAt 6 years. Strojan P / Radiotherapy in melanoma management Strojan P / Radiotherapy in melanoma management to radiotherapy; thus, the existing selection bias should be aware of when comparing the results be­tween these two groups of studies. Finally, while matching the results of studies simultaneously reporting on the outcome in surgically and post­operatively irradiated patients, it seems that adju­vant radiotherapy compensates effectively for the negative impact of adverse histopathological fea­tures to the disease control in the dissected nodal basin (Table 3).22,44,47,51,61,69,71No effect of postopera­tive irradiation on survival was observed in these studies. To the contrary, Agrawal et al.recently re­ported that adjuvant radiotherapy also could have a positive impact on melanoma specific survival.61 Owing to the increased probability of serious treatment-related side effects after adding radio­therapy to surgery, particularly lymphedema, and due to high likelihood of distant metastases in the patients with extensive lymph node involvement and no survival advantage for the adjuvantly ir­radiated patients, the question has been raised on the meaning of adjuvant use of radiotherapy. In view of these obstacles, the primary goal of post­operative irradiation must be emphasized, i.e.to prevent the uncontrolled regional recurrences with local destruction and associated infection (with secretion and stench), hemorrhages, edema, dis­figurement or pain, which produce considerable morbidity that significantly reduces the quality of patient’s life. The probability of systemic dissemination, which is the most powerful predictor of the risk of dying due to the disease, seems to be associ­ated with the number of involved nodes.22,70Thus, it sounds reasonable to use as cut point a certain number of involved nodes at which the risk of dis­tant failure is that high that regional radiotherapy should not be delivered, despite its proven effec­tiveness in controlling regional disease. A reason­able number might be between 10-15 nodes, at which point the risk of distant metastasis reaches 70%.22,70 Whenacomprehensivenodalresectionisnot doneandonlylocalexcisionofpalpablenode(s)is performedinstead,eitherduetosignificantmedical comorbiditiesorpatient’srefusalofmoreextensive surgicalprocedure,radiotherapyseemstohavea potentialtocompensateforthisdeficiency.Inase­riesof36patientswithparotidorcervicalnodeme­tastasesfrommelanomatreatedwithlocalexcision ofpalpablenodaldiseaseandpostoperativeradio­therapy(totheprimarysite–ifknown,thesiteof nodalexcisionandtheundissectedipsilateralneck), thedisease,afterthemedianfollowupof5.3years, recurredwithintheregionalbasinintwopatients onlyandatdistantsitesin14patients.72Inthisset­ting,itseemsunlikelythatacomprehensivesurgi­caldissectionwouldimprovetheregionalcontrol, butobservationonlywouldplacethepatientatun­necessaryriskofregionalrecurrence. elective radiotherapy – regional lymphatic metastases Elective neck irradiation is a viable treatment op­tion for the patients at risk for nodal micrometas­tases who are not candidates for sentinel lymph node biopsy.62,73In a retrospective series of 157 patients with high risk cutaneous melanoma of the head and neck for lymph node involvement (stages I or II), elective regional radiotherapy was found effective and safe treatment option. After a median follow up of 68 months, the disease recurred in the neck lymph nodes in 15 patients and distantly in 57 patients.73However, in the sentinel lymph node dissection era, this particular indication is less rel­evant. Palliative radiotherapy The primary goal of palliative radiotherapy is to reduce signs and symptoms related to the disease and improve quality of patients’ life; eventual pro­longation of her/his life is in the second plane. Palliative RT is to be introduced whenever sur­gery is not possible (i.e.technically unresectable tumors, poor general condition of the patient) or is deemed ineffective (i.e.multiple metastases, par­ticularly when occurring in different organs). In general, all types of metastases or metastatic sites can be irradiated, including cutaneous, lymphatic, brain, bone, and visceral lesions. The effectiveness of radiotherapy in palliative setting is primarily de­pendent on tumor burden and site. According to the results of in vitrostudies, cells from metastatic lesions are more radioresistant than those from pri­mary tumors.74 Whereas more than 85% complete response rate could be expected after irradiation of small-size (i.e.=1 cm in diameter) cutaneous lesions, the fre­quency of complete response is less than 30% in tumors of 5 cm in diameter or larger.7Single-shot or fractionated radiotherapy of bone metastases re­sults in complete or partial pain relief at one month after the completed therapy in more than 65% of cases.75In the case of impending or known patho­ Strojan P / Radiotherapy in melanoma management Strojan P / Radiotherapy in melanoma management logic fracture, a combination of adjuvant irradia­tion that follows upfront surgical intervention re­sulted in immediate pain relief and prolongation of disease-free interval. Surgical treatment of existing bone fracture is indicated when the expected sur­vival exceeds 6 weeks and the patient’s condition permits operation, when no greater benefit from nonoperative treatment is expected, when internal stability can be obtained and when early mobiliza­tion is possible. The criterion for choosing between radiotherapy and combined treatment approach when impending fracture is diagnosed is metasta­sis in weight-bearing bones of a diameter >2-3 cm or with cortical destruction of >50%.75 Patients with brain metastases are usually re­ferred to whole brain radiotherapy (WBRT), whenever their number or location excludes sur­gical intervention or stereotactic radiotherapy. In combination with corticosteroids, WBRT resulted in life prolongation for uninspiring 1-2 months76, whereas an improvement of performance status, at least temporary, could be expected in 60-70% of patients.17In a recent report on 686 melanoma patients with brain involvement, supportive car-ealone resulted in a median survival of only 2.1 months, WBRT 3.4 months, neurosurgery 9.7 months and combination of surgical resection and WBRT 8.9 months.77For patients with lower number of metastatic lesions (usually =3) and of maximal diameter between 2.5-3 cm, stereotactic radiosurgery, either with linear accelerator or gam­ma-knife-based, represents a comparative alterna­tive to surgery (Figure 2).78In this clinical scenario, local control in the range of 90% with sporadic long-term survivorships can be expected, whereas in the majority of patients treated with stereotac-tic irradiation the prevailing cause of death is pro­gression of extracranial disease.77,78Recently, as no difference in local control or survival was found when WBRT and stereotactic radiosurgery versus surgery plus WBRT and a boost were compared, the less invasive of the two combinations, WBRT and stereotactic irradiation, was recommended as a treatment of choice for the patients with one or two brain metastases.79 In metastases causing spinal cord compression, radiotherapy can be used as a single modality (in conjunction with high dose corticosteroids) or in combination with surgery to reverse neurologi­cal impairment or to prevent further loss of motor functions.80The decision on the use of upfront sur­gery versus radiotherapy alone depends on the as­sessment of neurological deficit, mechanical insta­bility, radioresponsiveness and extent of malignant disease, patient’s performance status and comor­bidities. Combined treatment offers good chance for pain relief and restoration of affected neurolog­ical functions as well as delay in tumor regrowth and prolongation of symptoms-free period.80,81 Radiotherapy regimens and techniques As the best radiotherapy regimen for melanoma re­mains undetermined, fractionation pattern should be in line with treatment intent and adapted to treated patient: i.e., anatomical localization and ex­tent of radiation volume/target, life expectancy and convenience for the patient, taking into account her/his performance status and preferences. All existing radiotherapy armamentarium can be used when irradiating melanoma, from simple kilovolt-age machines or telecobalts to sophisticated linear accelerators, tomotherapy units or cyber-knife. FIguRe 2. Stereotactic radiosurgery. This radiotherapy technique is characterized with maximal accuracy and is used for focal irradiation of small brain lesions (usually up to 3 tumors of 3.5 cm maximal diameter). After rigid fixation of the head with specific frame, several small beams coming from various directions are focused on one spot inside of the target, creating a steep dose gradient on periphery of the target. Tumor doses in the range of 16-25 Gy are prescribed on 80% isodose encompassing the lesion, whereas 1-2 mm from the edge of the target, the dose drops to 20-30% of its prescribed value. Local control is in the range of 90% and the prevailing cause of death is progression of extracranial disease. In January 2009, a 59-year-old male with melanoma, diagnosed 4 years earlier, presented with 4 metastatic lesions in the brain. On PET-CT, two additional metastases were identified elsewhere in the body, occupying the third lumbar vertebra and the mus­culature of the posterior abdominal wall. T1-weighted post-contrast MRIs revealed a lesion of 30x20 mm (long arrow) in the right temporoparietal region, a smaller one in the left half of the pons (short arrow), a 6 mm lesion in the left frontal lobe (thick arrow) and a 7 mm lesion in the left cerebellar hemisphere (arrowhead) (Figure 2A). The patient was treated with surgical resection of the large temporoparietal metastasis, whole brain irradiation (10 x 3 Gy), temozolamide and stereotactic radiosurgery of other three (smaller) brain metastases with the irradiation doses to 80% isodose of 20 Gy (the lesion in the frontal lobe) and 18 Gy (the lesions in the pons and cerebellum). Four months after the procedure, the size of all three irradiated tumors was reduced and no new lesion was identified in the brain (Figure 2B). In September 2009, disease progression was recorded after detecting a metastasis in the spinal cord which was treated with surgery, postoperative irradiation and chemotherapy. No progression of treated brain metastases occurred so far (January 2010, 11 months after stereotactic radiosurgery). 10 Strojan P / Radiotherapy in melanoma management At the Institute of Oncology Ljubljana, in melanoma patients irradiated with curative intent, the choice of fractionation pattern and total dose is governed mainly by the region to be irradiated. If there is no particular risk for lymphedema, e.g.targets on the trunk or neck region, higher fraction doses are used (4-6/fx Gy), although, owing to the risk of subcutaneous fibrosis particularly on the neck, lower fraction doses are sometimes preferred. In other clinical scenarios (axilla, groin), more con­ventionally fractionated radiotherapy regimens are implemented (1.8-2.5 Gy/fx). In palliative ra­diotherapy, smaller number of higher daily doses is usually employed (4-8 Gy/fx). The complexity of treatment plans, including the number of beams implemented, beam shaping and irradiation tech­niques (simple 2D, 3D-conformal, intensity modu­lated, image-guided) is also adjusted to the treated region and treatment intent. Formacroscopicdisease,curativedoseshould beinarangeof66-70Gy(equivalentdose,i.e.whenconventionallyfractionatedwith2Gy/day and5fractions/week,a/b =2Gy;Jonesetal.14). Radiotherapydoseprescribedpostoperativelyto theoperatedsideoftheneckshouldbeintherange of=60Gy22,41,47,61-63,68,70,71,althoughafavorableout­comewasalsoreportedwithlowerdoses.21-24For irradiationofaxillaryandinguinalnodalbasins, atotaldoseof50-55Gy,causingatolerablepro­fileofirradiationinducedside-effects,isusedas recommended.24,44Inpalliativesetting,theradio­therapydosesareusuallylower(equivalentdose 24-50Gy). The stereotactic technique is a valuable option for clearly defined subset of patients with brain metastases. It based on rigid fixation of the head with specific frame, allowing more accurate posi­tioning of the head (and tumor – target) in 3-di­mensional space compared to non-stereotactic con­ditions. Several small beams coming from various directions are focused on one spot inside of the target, creating a steep dose gradient on periphery of the target. Tumor doses in the range of 16-25 Gy are prescribed on 80% isodoseencompassing the lesion, whereas 1-2 mm from the edge of the tar­get the dose drops to 20-30% of its prescribed value (Figure 2). Conclusions Basic treatment modality in melanoma is sur­gery. However, whenever surgery is not radical or there are adverse prognostic factors identified on histopathological examination of resected tis­sue specimen, it needs to be supplemented. Also, in patients with unresectable disease or in those not being suitable for major surgery or who refuse proposed surgical intervention, other effective mode(s) of therapy need to be implemented. From this perspective, supported by clinical experiences and literature results, radiotherapy is a valuable option: it is effective and safe, in curative and pal­liative setting. However, the highest benefit in terms of best achievable disease control rates and, simultaneously, minimal treatment-related toxicity is obtainable when modern radiotherapy equip­ment and techniques are used and indications for irradiation are followed consistently, on patient-to patient basis. Acknowledgement The article resumes the lecture given by the au­thor at the International Melanoma Conference “From Prevention to Treatment«, held in Ljubljana, Slovenia on November 7-8, 2008. The author thanks to Uroš Smrdel, M.D., M.Sc. for providing the re­port on the case and images presented in Figure 2. References 1. de Vries E, Bray FI, Coebergh JW, Parkin DM. Changing epidemiology of malignant cutaneous melanoma in Europe 1953-1997: rising trends in incidence and mortality but recent stabilization in western Europe and decreases in Scandinavia. Int J Cancer 2003; 107: 119-26. 2. Cancer incidence in Slovenia 1997. Report no. 39. Ljubljana: Institute of Oncology Ljubljana, Cancer Registry of Republic of Slovenia; 2000. 3. Cancer incidence in Slovenia 2006. Report no. 48. Ljubljana: Institute of Oncology Ljubljana, Cancer Registry of Republic of Slovenia; 2009. 4. Kingham TP, Karakousis G, Ariyan C. Randomized clinical trials in melano­ma. Surg Oncol Clin N Am 2010; 19: 13-31. 5. Paterson R. Classification of tumours in relation to radiosensitivity. Br J Radiol 1933; 6: 218-33. 6. Delaney G, Barton M, Jacob S. Estimation of an optimal radiotherapy utilization rate for melanoma. A review of the evidence. Cancer 2004; 100: 1293-301. 7. Overgaard J, Overgaard M, Hansen V, von der Maase H. Some factors of importance in the radiation treatment of malignant melanoma. Radiother Oncol 1986; 5: 183-92. 8. Bentzen SM, Overgaard J, Thames HD, Overgaard M, Hansen PV, von der Maase H, et al. Clinical radiobiology of malignant melanoma. Radiother Oncol 1989; 16: 169-82. 9. Rofstad EK, Wahl A, Tveit KM, Monge OR, Brustad T. Survival curves after X-ray and heat treatments for melanoma cells derived directly from surgical specimens of tumours in man. Radiother Oncol 1985; 4: 33-44. 10. Bentzen SM, Thames HD, Overgaard J. Does variation in the in vitro cel­lular radiosensitivity explain the shallow clinical dose-control curve for malignant melanoma? Int J Radiat Biol 1990; 57: 117-26. 11. Barranco SC, Romsdahl MM, Humphrey RM. The radiation response of human malignant melanoma cells frown in vitro. Cancer Res 1971; 31: 830-3. 12. McKay MJ, Kefford RF. The spectrum of in vitro radiosensitivity in four human melanoma cell lines is not accounted for by differential induction or rejoining of DNA double strand breaks. Int J Radiat Oncol Biol Phys 1995; 31: 345-52. 13. Trott KR. The optimal radiation dose per fraction for the treatment of malignant melanoma. Int J Radiat Oncol Biol Phys 1991; 20: 905-7. 14. Jones B, Dale RG, Deehan C, Hopkins KI, Morgan DA. The role of bio­logically effective dose (BED) in clinical oncology. Clin Oncol (R Coll Radiol) 2001; 13: 71-81. 15. Rofstad EK, Mathiesen B, Galappathi K. Increased metastatic dissemina­tion in human melanoma xenografts after subcurative radiation treat­ment: radiation-induced increase in fraction of hypoxic cells and hypoxia-induced up-regulation of urokinase-type plasminogen activator receptor. Cancer Res 2004; 64: 13-8. 16. Rofstad EK. Fractionation sensitivity (a/b ratio) of human melanoma xenografts. Radiother Oncol 1994; 33: 133-8. 17. Bello MT, Ang KK. Radiation therapy for malignant melanoma. Surg Clin N Am 2003; 83: 323-42. 18. Sause WT, Cooper JS, Rush S, Ago CT, Cosmatos D, Coughlin CT, et al. Fraction size in external beam radiation therapy in the treatment of melanoma. Int J Radiat Oncol Biol Phys 1991; 20: 429-32. 19. Dvorák E, Haas RE, Liebner EJ. Contribution of radiotherapy to the man­agement of malignant melanoma. A ten year experience at the University of Illinois Hospital in Chicago. Neoplasma 1993; 40: 387-99. 20. Fenig E, Eidelevich E, Njuguna E, Katz A, Gutman H, Sulkes A, Schechter J. Role of radiation therapy in the management of cutaneous malignant melanoma. Am J Clin Oncol 1999; 22: 184-6. 21. Chang DT, Amdur RJ, Morris CG, Mendenhall WM. Adjuvant radiotherapy for cutaneous melanoma: comparing hypofractionation to conventional fractionation. Int J Radiat Oncol Biol Phys 2006; 66: 1051-5. 22. Strojan P, Jancar B, Cemažar M, Pohar Perme M, Hocevar M. Melanoma metastases to the neck: role of adjuvant irradiation. Int J Radiat Oncol Biol Phys. In press. 23. Corry J, Smith JG, Bishop M, Ainslie J. Nodal radiation therapy for meta­static melanoma. Int J Radiat Oncol Biol Phys 1999; 44: 1065-9. 24. Burmeister BH, Smithers BM, Burmeister E, Baumann K, Davis S, Krawitz, et al. A Prospective phase II study of adjuvant postoperative radiation therapy following nodal surgery in malignant melanoma – Trans Tasman Radiation Oncology Group (TROG) study 96.06. Radiother Oncol 2006; 81: 136-42. 25. Overgaard J, von der Maase H, Overgaard M. A randomized study comparing two high-dose per fraction radiation schedules in recurrent or metastatic malignant melanoma. Int J Radiat Oncol Biol Phys 1985; 11: 1837-9. 26. Overgaard J, Gonzalez Gonzalez D, Hulshof MC, Arcangeli G, Dahl O, Mella O, et al. Randomized trial of hyperthermia as adjuvant to radiotherapy for recurrent or metastatic malignant melanoma. Lancet 1995; 345: 795-8. 27. Harwood AR. Conventional fractionation radiotherapy for 51 patients with lentigo maligna and lentigo maligna melanoma. Int J Radiat Oncol Biol Phys 1983; 9: 1019-21. 28. Schmid-Wendtner MH, Brunner B, Konz B, Kaudewitz P, Wendtner CM, Peter RU, et al. Fractionated radiotherapy of lentigo maligna and lentigo maligna melanoma in 64 patients. J Am Acad Dermatol 2000; 43: 477-82. 29. Farshad A, Burg G, Panizzon R, Dummer R. A retrospective study of 150 patients with lentigo maligna and lentigo maligna melanoma and the efficacy of radiotherapy using Grenz or soft X-rays. Br J Dermatol 2002; 146: 1042-6. 30. Krengli M, Jereczek-Fossa BA, Kaanders JHAM, Masini L, Beldě D, Orecchia R. What is the role of radiotherapy in the treatment of mucosal melano­ma of the head and neck? Crit Rev Oncol Hematol 2008; 65: 121-8. 31. Trotty A, Peters LJ. Role of radiotherapy in the primary management of mucosal melanoma of the head and neck. Sem Surg Oncol 1993; 9: 246-50. 32. Krengli M, Masini L, Kaanders JHA, Maingon P, Oei SB, Zouhair A, et al. Radiotherapy in the treatment of mucosal melanoma of the upper aero-digestive tract: analysis of 74 cases. A Rare Cancer Network study. Int J Radiat Oncol Biol Phys 2006; 65: 751-9. 33. Leon P, Daly JM, Synnestvedt M, Schultz DJ, Elder DE, Clark WH. The prognostic implications of microscopic satellites in patients with clinical stage I melanoma. Arch Surg 1991; 126: 1461-8. 34. Kelly JW, Sagebiel RW, Calderon W, Murillo L, Dakin RL, Blois MS. The frequency of local recurrence and microsatellites as a guide to reexci­sion margins for cutaneous malignant melanoma. Ann Surg 1984; 200: 759-63. 35. Posther KE, Selim MA, Mosca PJ, Stanley WE, Johnson JL, Tyler DS, et al. Histopathologic characteristics, recurrence patterns, and survival of 129 patients with desmoplastic melanoma. Ann Surg Oncol 2006; 13: 728-39. 36. Jaroszewski DE, Pockaj BA, DiCaudo DJ, Bite U. The clinical behavior of desmoplastic melanoma. Am J Surg 2001; 182: 590-5. 37. Vongtama R, Safa A, Gallardo D, Calcaterra T, Juillard G. Efficacy of radia­tion therapy in the local control of desmoplastic malignant melanoma. Head Neck 2003; 25: 423-8. 38. Quinn MJ, Crotty KA, Thompson JF, Coates AS, O’Brien CJ, McCarthey WH. Desmoplastic and desmoplastic neutropic melanoma: experience with 280 patients. Cancer 1998; 83: 1128-35. 39. Arora A, Lowe L, Su L, Rees R, Bradford C, Cimmino VC, et al. Wide exci­sion without radiation for desmoplastic melanoma. Cancer 2005; 104: 1462-7. 40. Johanson CR, Harwood AR, Cummings BJ, Quirt I. 0-7-21 radiotherapy in nodular melanoma. Cancer 1983; 51: 226-32. 41. Cooper JS, Chang WS, Oratz R, Shapiro RL, Roses DF. Elective radiation therapy for high-risk malignant melanoma. Cancer J 2001; 7: 498-502. 42. Seegenschmiedt MH, Keilholz L, Altendorf-Hofmann A, Urban A, Schell H, Hohenberger W, et al. Palliative radiotherapy for recurrent and meta­static malignant melanoma: prognostic factors for tumor response and long-term outcome: a 20-year experience. Int J Radiat Oncol Biol Phys 1999; 44: 607-18. 43. Creagan ET, Cupps RE, Ivins JC, Pritchard DJ, Sim FH, Soule EH, et al. Adjuvant radiation therapy for regional nodal metastases from malignant melanoma: a randomized, prospective study. Cancer 1978; 42: 2206-10. 44. Henderson MA, Burmeister B, Thompson JF, Di Iulio J, Fisher R, Hong A, et al. Adjuvant radiotherapy and regional lymph node field control in melanoma patients after lymphadenectomy: results of an intergroup randomized trial (ANZMTG 01.02/TROG 02.01). [Abstract]. J Clin Oncol 2009; 27 (Suppl): LBA9084. 45. Calabro A, Singletary SE, Balch CM. Patterns of relapse in 1001 con­secutive patients with melanoma nodal metastases. Arch Surg 1989; 124: 1051-5. 46. Singletary SE, Byers RM, Shallenberger R, McBride CM, Guinee VF. Prognostic factors in patients with regional cervical nodal metastases from cutaneous malignant melanoma. Am J Surg 1986; 152: 371-5. 47. O’Brien CJ, Petersen-Schaefer K, Stevens GN, Stevens GN, Bass PC, Tew P, et al. Adjuvant radiotherapy following neck dissection and parotidectomy for metastatic malignant melanoma. Head Neck 1997; 19: 589-94. 48. Monsour PD, Sause WT, Avent JM, Noyes RD. Local control following therapeutic nodal dissection for melanoma. J Surg Oncol 1993; 54: 18-22. 49. Miller EJ, Daly JM, Synnestvedt M, Schultz D, Elder D, Guerry D. Loco-regional nodal relapse in melanoma. Surg Oncol 1992; 1: 333-40. 50. Lee RJ, Gibbs JF, Proulx GM, Kollmorgen DR, Jia C, Kraybill WG. Nodal basin recurrence following lymph node dissection for melanoma: im­plications for adjuvant radiotherapy. Int J Radiat Oncol Biol Phys 2000; 46: 467-74. 51. Shen P, Wanek LA, Morton DL. Is adjuvant radiotherapy necessary after positive lymph node dissection in head and neck melanomas? Ann Surg Oncol 2000; 7: 554-9. 52. Pidhorecky I, Lee RJ, Proulx G, Kollmorgen DR, Jia C, Driscoll DL, et al. Risk factors for nodal recurrence after lymphadenectomy for melanoma. Ann Surg Oncol 2001; 8: 109-15. 12 Strojan P / Radiotherapy in melanoma management 53. Bowsher WG, Taylor BA, Hughes LE. Morbidity, mortality and local recur­rence following regional node dissection for melanoma. Br J Surg 1986; 73: 906-8. 54. Bayers RM. The role of modified neck dissection in the treatment of cu­taneous melanoma of the head and neck. Arch Surg 1986; 121: 1338-41. 55. Kretschmer L, Preusser KP. Standardized axillary lymphadenectomy improves local control but not survival in patients with palpable lymph node metastases of cutaneous malignant melanoma. Langenbeck Arch Surg 2001; 386: 418-25. 56. Kissin MW, Simpson DA, Easton D, White H, Westbury G. Prognostic factors related to survival and groin recurrence following therapeutic lymph node dissection for lower limb malignant melanoma. Br J Surg 1987; 74: 1023-6. 57. Hughes TMD, A’Hern RP, Thomas JM. Prognosis and surgical manage­ment of patients with palpable inguinal lymph node metastases from melanoma. Br J Surg 2000; 87: 892-901. 58. Kretschmer L, Neumann C, Preußer KP, Marsch WC. Superficial inguinal and radical ilioinguinal lymph node dissection in patients with palpable melanoma metastases to the groin. Acta Oncol 2001; 40: 72-8. 59. Allan CP, Hayes AJ, Thomas JM. Ilioinguinal lymph node dissection for palpable metastatic melanoma to the groin. ANZ J Surg 2008; 78: 982-6. 60. Meyer T, Merkel S, Göhl J, Hohenberger W. Lymph node dissection for clinically evident lymph node metastases of malignant melanoma. Eur J Surg Oncol 2002; 28: 424-30. 61. Agrawal S, Kane JM, Guadagnolo BA, Kraybill WG, Ballo MT. The benefits of adjuvant radiation therapy after therapeutic lymphadenectomy for clinically advanced, high-risk, lymph node-metastatic melanoma. Cancer 2009; 115: 5836-44. 62. Ang KK, Peters LJ, Weber RS, Morrison WH, Frankenthaler RA, Garden AS, et al. Postoperative radiotherapy for cutaneous melanoma of the head and neck region. Int J Radiat Oncol Biol Phys 1994; 30: 795-8. 63. Ballo MT, Bonnen MD, Garden AS, Myers JN, Gershenwald JE, Zagars GK, et al. Adjuvant irradiation for cervical lymph node metastases from melanoma. Cancer 2003; 97: 1789-96. 64. Ballo MT, Strom EA, Zagars GK, Bedikian AY, Prieto VG, Mansfield PF, et al. Adjuvant irradiation for axillary metastases from malignant melanoma. Int J Radiat Oncol Biol Phys 2002; 52: 964-72. 65. Beadle BM, Guadagnolo BA, Ballo MT, Lee JE, Gershenwald JE, Cormier JN, et al. Radiation therapy field extent for adjuvant treatment of axillary metastases from malignant melanoma. Int J Radiat Oncol Biol Phys 2009; 73: 1376-82. 66. Ballo MT, Zagars GK, Gershenwald JE, Lee JE, Mansfield PF, Kim KB, et al. A critical assessment of adjuvant radiotherapy for inguinal lymph node metastases from melanoma. Ann Surg Oncol 2004; 11: 1079-84. 67. Burmeister BH, Smithers BM, Poulsen M, McLeod GR, Bryant G, Tripcony L, et al. Radiation therapy for nodal disease in malignant melanoma. World J Surg 1995; 19: 369-71. 68. Stevens G, Thompson JF, Firth I, O’Brien CJ, McCarthy WH, Quinn MJ. Locally advanced melanoma: results of postoperative hypofractionated radiation therapy. Cancer 2000; 88: 88-94. 69. Fuhrmann D, Lippold A, Borrosch F, Ellwanger U, Garbe C, Suter L. Should adjuvant radiotherapy be recommended following resection of regional lymph node metastases of malignant melanomas? Br J Dermatol 2001; 144: 66-70. 70. Ballo MT, Ross MI, Cormier JN, Myers JN, Lee JE, Gershenwald JE, et al. Combined-modality therapy for patients with regional nodal metastases from melanoma. Int J Radiat Oncol Biol Phys 2006; 64: 106-13. 71. Moncrieff MD, Martin R, O’Brien CJ, Shannon KF, Clark JR, Gao K, et al. Adjuvant postoperative radiotherapy to the cervical lymph nodes in cu­taneous melanoma: is there any benefit for high-risk patients? Ann Surg Oncol 2008; 15: 3022-7. 72. Ballo MT, Garden AS, Myers JN, Lee JE, Diaz Jr EM, Sturgis EM, et al. Melanoma metastatic to cervical lymph nodes: can radiotherapy replace formal dissection after local excision of nodal disease? Head Neck 2005; 27: 718-21. 73. Bonnen MD, Ballo MT, Myers JN, Garden AS, Diaz JREM, Gershenwald JE, et al. Elective radiotherapy provides regional control for patients with cutaneous melanoma of the head and neck. Cancer 2004; 100: 383-9. 74. Rofstad EK. Radiation sensitivity in vitro of primary tumors and metastatic lesions of malignant melanoma. Cancer Res 1992; 52: 4453-7. 75. GojkovicHorvatA,KovacV,StrojanP.Radiotherapyinpalliativetreatment of painful bone metastases. Radiol Oncol 2009; 43: 213-24. 76. Majer M, Samlowski W. Management of metastatic melanoma patients with brain metastases. Curr Oncol Rep 2007; 9: 411-6. 77. Fife KM, Colman MH, Stevens GN, Firth IC, Moon D, Shannon KF, et al. Determinants of outcome in melanoma patients with cerebral metas­tases. J Clin Oncol 2004; 22: 1293-300. 78. Gaudy-Marqueste C, Regis JM, Muracciole X, Laurans R, Richard MA, Bonerandi JJ, et al. Gamma-Knife radiosurgery in the management of melanoma patients with brain metastases: a series of 106 patients without whole-brain radiotherapy. Int J Radiat Oncol Biol Phys 2006; 65: 809-16. 79. Rades D, Kueter JD, Pluemer A, Veninga T, Schild SE. A matched-pair analysis comparing whole-brain radiotherapy plus stereotactic radiosur­gery versus surgery plus whole-brain radiotherapy and a boost to the metastatic site for one or two brain metastases. Int J Radiat Oncol Biol Phys 2009; 73: 1077-81. 80. Rajer M, Kovac V. Malignant spinal cord compression. Radiol Oncol 2008; 42: 23-31. 81. Klimo P, Kestle JRW, Schmidt MH. Clinical trials and evidence-based medi­cine for metastatic spinal disease. Neurosurg Clin N Am 2004; 14: 549-64. review Genetic markers in oligodendroglial tumours Tomaz Velnar1,2, Uros Smrdel2, Mara Popovic3, Gorazd Bunc1 1 Department of Neurosurgery, University Medical Centre Maribor, Maribor, Slovenia 2 Institute of Oncology Ljubljana, Ljubljana, Slovenia 3 Department of Pathology, Medical Faculty Ljubljana, Ljubljana, Slovenia Received 3 December 2009 Accepted 10 December 2009 Correspondence to: Tomaž Velnar, MD, Department of Neurosurgery, University Medical Centre Maribor, Ljubljanska ulica 5, 2000 Maribor, Slovenia. Phone: +386 2 332 4830; Fax: +386 2 321 1457; E-mail: tvelnar@hotmail.com Disclosure: No potential conflicts of interest were disclosed. Background. Oliogodendrogliomas are brain tumours composed of the cells resembling oligodendrocytes. They represent the third most common glial tumour, comprising 2.5% of all primary brain tumours and 5-20% of all gliomas. Conclusions. Oligodendroglial tumours with 1p and 19q loss demonstrate a better overall prognosis due to more indolent clinical behaviour and higher sensitivity to treatment. Additionally, 1p and 19q loss is a marker of clinical utility, helping to assess tumour sensitivity to chemotherapy and harbouring the potential for improving the diagnosis and survival of oligodendroglioma patients as well as future clinical practice. Key words: oligodendroglioma; genetic markers; chemotherapy; prognosis Introduction Over the past decade, a remarkable progress in the aspects of cancer treatment was made.1Advances in imaging methods, molecular biology, surgical and chemotherapeutic techniques and radiation delivery have all improved the prognosis of cancer patients more than ever before.1-6Despite optimal diagnostics and treatment, cancer burden in the so­ciety is still ranking too high. After cardiovascular disease, cancer is the sec­ond leading cause of morbidity and mortality worldwide.7The most frequent brain tumours are gliomas. According to cell morfology they may be divided into astrocytomas, oligodendrogliomas, ependymomas and oligoastrocytomas.4,8Due to a favourable response to chemotherapy, oligoden­drogliomas have gained much interest in the past decade. They may be additionally subdivided into prognostic subgroups according to the histopathol­ogy, molecular and biological characteristics. The genetic alterations in tumour cells, together with clinical and histolpathological properties, may all define the most appropriate therapy and predict the outcome of the treatment.4,9 Epidemiology and aetiology Oliogodendroglialtumoursarisefromoligodendro­glialcellsorimmatureglialprecursorsandrepre­sentthethirdmostfrequentlyencounteredtypeof glialtumours,rankingafterglioblastomaandastro­cytomas.Theyconstitute5-20%ofallgliomasand 2.5%ofallprimarybraintumours.4,9-11About1500 newcasesarediagnosedinEurope,andinSlovenia thereareabout20caseseachyear.12,13Theannual incidencerateofoligodendrogliomasistwotofour per1,000,000peopleandtheincidenceisincreasing everyyear.4,9Ithassignificantlyincreasedoverthe pastyears,butthismayprimarilybeduetotheuse ofadditionaldiagnosticcriteriainrecentyears.4,9,11,12 Although oligodendrogliomas may occur at any age, there is a peak incidence between 40 and 45 years.4,11In spite of, the brain tumours in children are very frequent14,oligodendrogliomas are rare and representing 2% of all brain tumours in pa­tient younger than 14 years.11Male-to-female ratio is 1.5 to 1. In males, peak incidence was described between 45 to 49 years and in females between 55 to 59 years.4,9In younger patients, low grade oli­godendrogliomas predominate. Familial clustering of tumours was found, with neither genetic factors nor special pattern of inheritance.15-17Moreover, 14 Velnar T et al. / Genetic markers in oligodendroglial tumours causes for oligodendroglioma evolvement are un­known, no lifestyle or environmental factors are discovered, even though rare individual cases of oligodendroglioma in patients previously irradia­ted for other reasons have been documented.9,11,15,18More than 90% of oligodendrogliomas arise su­pratentorially in the cerebral white matter, pre­dominantly in the frontal lobes, but patients have been reported with oligodendroglima in basal gan­glia, posterior fossa, or spinal cord. Supratentorial locations according to tumour frequency are as fol­lows: 55% in frontal lobes, 47% in temporal, 20% in parietal and 4% in occipital lobes.4,9,17 Pathology and neurooncology Oligodendrogliomas exhibit an infiltrative growth pattern, although not to such an extent as astrocyto-mas.4,19,20According to the growth pattern and his­tological characteristics, two grades of malignancy are distinguished by the WHO: well differentiated oligodendroglioma of grade II and anaplastic oli­godendroglioma of grade III.17,19,21The latter may evolve from a low-grade oligodendroglioma, be­coming gradually more anaplastic over time, or present de novo, without a preceding low-grade tumour. There are 77% of low-grade and 23% of anaplastic oligodendrogliomas.4,9,21According to the tumour cell morphology, two types have been described: pure oligodendrogliomas and mixed gliomas or oligoastrocytomas, containing neoplas­tic cells of oligodendroglial and astroglial pheno­ type.4,13,21-23 Oligodendrogliomas may sometimes invade meninges.4Very rarely, the tumours may metastasize to other locations, such as lung, liver, bone and cervical lymph nodes.24,25Although ex­tremely rare, metastatic disease is encountered more frequently due to the improved survival of oligodendroglioma patients.4,9 Because no specific immunohistological mark­ers for oligodendrogliomas exist, the histological diagnosis may be challenging for a pathologist.9However, a chromosomal alteration has been re­ported, which is the most common lesion found in oligodendroglial tumours and involves a deletion at chromosomal loci 1p and 19q. A combined loss of 1p and 19q identifies a group of good prognosis tumours and has been reported, depending on the literature, in the range of 50% to 90% or 60% to 70% of oligodendrogliomas of any grade. On the other hand, the incidence of either 1p or 19q deletions alone is 75%.4,9,17,26,27 Tumour signs and symptoms Thesymptomsofoligodendroglialtumoursaresim­ilartootherprimaryandsolitarysecondarybrain neoplasms28,29,withepilepticseizuresbeingthe mostcommonsymptom,presentingin35%to85% ofpatients.4Seizuresmaybegeneralised,simple orcomplexpartial,oracombinationofthese.They maybeexperiencedforanumberofyearsbefore thediagnosis.30Othersymptomsincludeheadaches, sensoryandmotordisturbancesintermsoflocalised limbweakness,suddenorinsidiouschangeinper­sonalityandmood,visualcomplaints,nauseaand dizziness.Symptomsusuallyprecedethedefinitive diagnosisfor2.9monthsto5years.4,31,32 Oligodendroglioma treatment There are three therapeutic modalities for the treatment of oligodendrogliomas that are con­nected and combinable: surgery, radiation thera­py and chemotherapy.4,9All three are often used successively. Surgery remains a most frequently employed method both in order to perform a tu­mour reduction or a gross resection where pos­sible and to obtain tissue samples for the definite diagnosis.9,33The resection decreases the tumour mass effect on the brain with concomitant neuro­logical consequences and reduces the tumour load during radiotherapy, which is the next and often the following form of the treatment in grade 3 tu­mours.24,35Radiotherapy is used due to an invasive nature of tumour growth where a deep infiltration of tumour cells cannot be determined during sur­gery and, therefore, prevents a complete removal. As a consequence, the disease relapses slowly but inevitably.35,36A tumour relapse after a removal, which was not possible to be detected by clinical means, is termed a recurrence. It takes place at the operative site in the form of a high grade tumour, an anaplastic oligodendroglioma or even glioblas­toma. While low-grade tumours may recur after many years, anaplastic ones tend to do so sooner.4 The third option of the treatment is chemother­apy, which is being widely used, again for grade 3 tumours. The most frequently employed agents are procarbazine, vincristine, and lomustine (CCNU) (PCV scheme).37-39It has been reported that 60% to 75% of patients respond to PCV chemotherapy with 10-32 months of median response duration.9Chemotherapy is used as a treatment option and with or without radiotherapy, the latter option in children, where radiation is usually withheld due to adverse effects on the developing nervous sys­tem. Chemotherapy application before radiother­apy is becoming a standard practice also in adults in order to spare the side effects of radiation and to have a second line of the treatment option in case of tumour progression with comparable time to progression and overall survival.4,37-40 Besides standard PCV chemotherapy, temo­zolomide is being widely used as an alternative or supplement treatment both in primary and in met­astatic disease.10,41-43In comparison to PCV chemo­therapy, there are fewer side effects reported and the therapy regimen is more convenient.38,40,44-46On the contrary to low-grade oligodendrogliomas, where radiation is delayed until tumour progres­sion, patents with anaplastic oligodendrogliomas receive both radiation and chemotherapy and this combination is superior to either treatment alone.4,9,34,35Other chemotherapeutic agents used are carboplatin, cisplatin, etoposide, melphalan, thiotepa and other nitrosourea drugs, as well as interferon-ß and recently bevacizumab.47A rea­son for an increase of chemotherapy comes from the observations that low-grade and anaplastic oli­godendrogliomas are chemosensitive tumours. Many genetic abnormalities are encountered in brain neoplasm and many of these identified may emphasize potential diagnostic, therapeutic and prognostic implications.9,40 Genetic markers in oligodendrogliomas Various genetic markers have been described in connection to oligdendroglial tumours and are briefly discussed below. Chromosomes 1 and 19 Asalreadystated,abnormalitiesinchromosomes1 and19arethemostsignificant.1pand19qlossesare encounteredin80%to90%ofgrade2andin50%to 70%ofgrade3oligodendrogliomas.17Onthecontra­ry,childhoodoligodendrogliomasonlyrarelyexhib­itchromosomalabnormalities.Inadults,inthema­jorityofcaseschromosomelossesinvolvetheentire longarmofchromosome19andarepresentincon­nectionwithlossesfromchromosome1p.Theywere observedtobemorecommoninfrontal,occipital andparietallobesthanintemporallobetumours.48,49 Methylation of MGMT genes Another common finding in oligodendrogliomas is methylation of DNA regions that code for MGMT genes, present in approximately 93% of cases. The end result is transcriptional silencing of genes re­sponsible for DNA repair enzyme, which may con­tribute to higher chemosensitivity.17,49 Mutations in p53 gene Mutations in p53 gene are described in 10% to 15% of tumours without 1p and 19q loss.17Such tumours arise most commonly in the temporal lobes; histologically they are anaplastic or mixed oligoastrocytomas and express poor chemosensi­tivity.50,51Response rate or efficacy of the chemo­therapy treatment was observed only in 33% of patients with p53 mutation and intact 1p and 19q chromosomes, as opposed to tumours with intact p53 gene and 1p and 19q or only 1p mutation, where the response rate was 100%.17,50 Growth factors and other genetic abnormalities Growth factors overexpression includes epidermal growth factor receptor (EGFR), vascular endothe­lial growth factor (VEGF) and platelet-derived growth factor (PDGF).17,52EGFR overexpression was observed in 50% of oligodendrogliomas; the percentage of other two overexpressed factors is somewhat lower. Other chromosomal abnormali­ties consist of genetical abnormalities or losses from chromosomes 10q and 9p. They are preferen­tially encountered in anaplastic oligiodendroglio-mas without of 1p and 19q loss.17,48Additionally, oligodendrocyte transcription factors, such as Olig 1 and Olig 2 that may be used as markers of oligodendrogliomas, are highly expressed in oli­godendrogliomas, as well as in astrocytomas.17,53,54 Prognostic and diagnostic value of 1p and 19q abnormalities Pureoligodendrogliomasshowabetterprognosis thanastrocytomasofthesamegradeandoligoastro­cytomasareprognosticallyinbetweentheformer two.9Forthemanagementoftumoursandforprog­nosticandtherapeuticdecisions,itisimportantto 16 Velnar T et al. / Genetic markers in oligodendroglial tumours identifythetumourtypecorrectly.23Microscopical appearance,whichformsthebasisforthedistinc­tionofgliomas,isnotalwaysasclearastosetthe diagnosisdirectly.Itissometimesparticularlydif­ficulttodistinguisholigodendrogliomasandoli­goastrocytomas.Inliteraturestudies,thediagnostic concordanceobservedinthesetumoursmayrange from52%to86%amongpathologists.48Thisfactne­cessitatedasearchforanadditionaldiagnostictool fortheoligodendrogliomas.Twofactorsinfluence thedifficultiesinhistopatologicaldiagnostics:(1) lackofaspecificimmunohistochemicalcellmarker foroligodendroglialtumoursand(2)avariationin tumourmicroscopicmorphology.Genetically,a combinedlossof1pand19qistypicalforoligoden­drogliomasandrareingliomasofothertype,while isolated19qlossoccursinmixedoligoastrocytomas andinastrocytomas.48,49,55Chromosome1pand19q statusmaybeassessedbyavarietyoftechniques, suchasmicrosatelliteanalysis,fluorescencein situhybridization(FISH),genomichybridization andquantitativepolymerasechainreactions.17,48,55Besidesbeingavaluablediagnosticmarkerdueto itsspecificity,itwasdiscoveredthat1pand19qloss alsoactsasapowerfulmarkerintheprognosisof thediseaseandasapredictorofchemotherapeutic responseandsurvival.42,48,56Thefollow-upperiod describedinthestudiesvariesfromtwotofive years.23,57Thisissomehowshort,whenonetakes intoaccountthesurvivalperiodinoligodendrog­lioma,whichvariesbetweenfourtosevenyears, dependingonthegrade.Oliogodendrogliomas harbouring1pand19qdeletionbehavemoreindo­lentlyandrespondfavourablytoPCVchemothera­pyandtemozolomideaswellastoradiotherapy.42,55Forexample,thereportedcorrelationbetween1p and19qlossandPCVregimeninthetreatmentre­sponserangedfrom93%to100%.Temozolomide asareplacementforPCVtherapy,duetoabetter toxicityprofile,showed46%to55%responserate tothetreatment.Also,timetoprogressionofthe diseasecorrelatedwith1pand19qloss.9,58,59Onthe otherhand,thetherapeuticsensitivityof1pand 19q-intacttumoursislessfavourableandthesur­vivalisthereforeshorter.58,60,61 Another factor reported to bear the prognostic significance is o6-methylguanine-DNAmethyl­transferase (MGMT), an enzyme involved in DNA repair.62,63In many tumours, including gliomas, alterations in DNA may be found, such as meth-ylation of the promoter region and their genes. Methylated DNA is less readily accessible to tran­scription factors and results in the loss of gene function. As MGMT is one of the key factors in resistance to chemotherapy, hypermethylation in­hibits the repair mechanism due to a lower level of the active enzyme.6,17,58,63MGMT methylation rates in oligodendrogliomas range from 25% to 85% and were reported to be strongly associated with 1p and 19q loss.58,59However, the response rate to chemotherapy and time to progression of oligodendrogliomas were not observed to be in correlation with the degree of MGMT methyla­tion, as is the case with glioblastoma, where pro­moter methylation correlated with response to the alkylating agent treatment and survival. The cause probably lies in different genes and pattern of pro­moter methylation, which is present in astrocytic cells.17,58,62-64 1p and 19q deletion is a predictive factor of tu­mour response principally to chemotherapy, and radiotherapy as well.48,59-61A number of centres em­ploy evaluation of 1p and 19q status as a laboratory test, which is used in conjunction to clinical status, imaging and patohistological diagnosis for pre­dicting the patient response to the treatment. This enables to tailor the most effective and appropriate therapy for the individual patient.23,55,58However, there are still unexplained issues in connection to 1p and 19q loss. To begin with, the genes and their exact functions in the pathogenesis of oligodendro­gliomas, located on the long arms of chromosomes 1 and 19, need to be identified for some patients with 1p and 19q intact tumours which respond well to the therapy and vice versa.9,58Despite the fact that 1p and 19q status helps in selecting pa­tients with respect to therapeutic regimen, there were no revolutionary improvements in the treat­ment outcomes.55A further investigation is re­quired in order to elucidate the unsolved questions in oligodendroglioma biology. Conclusions Oligodendroglial tumours with 1p and 19q loss demonstrate a better overall prognosis due to a more indolent clinical behaviour and higher sensi­tivity to treatment. The 1p and 19q status acts as a prognostic marker, since its loss is associated with an improved outcome compared to non-1p and 19q deleted oligodendrogliomas and astrocytomas of a same grade. 1p and 19q testing proved to be particularly useful for determining the tumour type in morphologically ambiguous cases, as it acts as a valid marker of classical oligodendroglial tumours, when present. Additionally, 1p and 19q loss is a marker of clinical utility, helping to assess tumour sensitivity to chemotherapy and harbour­ing the potential for improving the diagnosis and survival of oligodendroglioma patients as well as future clinical practice. References 1. Gilbert MR, Lang FF. Management of patients with low-grade gliomas. Neurol Clin 2007; 25: 1073-88. 2. Strojnik T, Gornik-Kramberger K. [Stereotactic biopsy of brain tumours – Maribor experience with MHT stereotaxy system]. [Slovenian]. Zdrav Vestn 2008; 77: 601-8. 3. Asthagiri AR, Pouratian N, Sherman J, Ahmed G, Shaffrey ME. Advances in brain tumorsurgery. Neurol Clin 2007;25:975-1003.Engelhard HH, Stelea A, Mundt A. Oligodendroglioma and anaplastic oligodendroglioma: clinical features, treatment and prognosis. Surg Neurol 2003; 60: 443-56. 4. StrojnikA.Searchoftheshortestregimen:fractionationofafullyisoeffec­tive combination of hyperfractionated and hypofractionated treatment. Radiol Oncol 2008; 42: 170-2. 5. HeikalAA,WachowiczK,ThomasSD,FalloneBG.Aphantomtoassessthe accuracy of tumor delineation using MRSI. Radiol Oncol 2008; 42: 232-9. 6. Giampaoli S. Epidemiology of major age-related diseases in women com­pared to men. Aging (Milano) 2000; 12: 93-105. 7. Pytel P, Lukas RV. Update on diagnosic practice: tumors of the nervous system. Arch Pathol Lab Med 2009; 133: 1062-77. 8. van den Bent MJ, Reni M, Gatta G, Vecht C. Oligodendroglioma. Crit Rev Oncol Hematol 2008; 66: 262-72. 9. Siker ML, Chakravarti A, Mehta MP. Should concomitant and adjuvant treatment with temozolomide be used as standard therapy in patients with anaplastic glioma? Crit Rev Oncol Hematol 2006; 60: 99-111. 10. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, editors. WHO classifica­tion of tumours of the central nervous system. Lyon 2007: WHO, IARC; 2007. p. 53-67. 11. Ohgaki H Epidemiology of brain tumors. Methods Mol Biol 2009; 472: 323-42. 12. Cancer incidence in Slovenia 2005. Ljubljana: Institute of Oncology Ljubljana, Cancer Registry of Slovenia; 2008. 13. Kachanov DY, Dobrenkov KV, Shamanskaya TV, Abdullaev RT, Inushkina EV, Savkova RF, et al. Solid tumors in young children in Moscow Region of Russian Federation. Radiol Oncol 2008; 42: 39-44. 14. Wrensch M, Fisher JL, Schwartzbaum JA, Bondy M, Berger M, Aldape KD. The molecular epidemiology of gliomas in adults. Neurosurg Focus 2005; 19: E5. 15. Wrensch M, Minn Y, Chew T, Bondy M, Berger MS. Epidemiology of pri­mary brain tumors: current concepts and review of the literature. Neuro Oncol 2002; 4: 278-99. 16. Hilton DA, Melling C. Genetic markers in teh assesment of intrinsic brain tumours. Current Diagnostic Pathology 2004; 10: 83-92. 17. Scelsi R. Epidemiology of cerebral gliomas. Minerva Med 1984; 75: 1259-63. 18. Louis DN. Molecular pathology of malignant gliomas. Annu Rev Pathol 2006; 1: 97-117. 19. Wong ET, Hess KR, Gleason MJ, Jaeckle KA, Kyritsis AP, Prados MD, et al. Outcomes and prognostic factors in recurrent glioma patients enrolled onto phase II clinical trials. J Clin Oncol 1999; 17: 2572-8. 20. Engelhard HH, Stelea A, Cochran EJ. Oligodendroglioma: pathology and molecular biology. Surg Neurol 2002; 58: 111-7. 21. Engelhard HH, Stelea A, Mundt A. Oligodendroglioma and anaplastic oligodendroglioma: clinical features, treatment, and prognosis. Surg Neurol 2003; 60: 443-56. 22. Figarella-Branger D, Colin C, Coulibaly B, Quilichini B, Maues De Paula A, Fernandez C, et al. Histological and molecular classification of gliomas. Rev Neurol (Paris) 2008; 164: 505-15. 23. Zustovich F, Della Puppa A, Scienza R, Anselmi P, Furlan C, Cartei G. Metastatic oligodendrogliomas: a review of the literature and case re­port. Acta Neurochir (Wien) 2008; 150: 699-703. 24. Volavsek M, Lamovec J, Popovic M. Extraneural metastases of anaplastic oligodendroglial tumors. Pathol Res Pract 2009; 205: 502-7. 25. Bromberg JE, van den Bent MJ. Oligodendrogliomas: molecular biology and treatment. Oncologist 2009; 14: 155-63. 26. van den Bent MJ. Diagnosis and management of oligodendroglioma. Semin Oncol 2004; 31: 645-52. 27. Franko A, Holjar-Erlic I, Miletic D. Lateral ventricle epidermoid. Radiol Oncol 2008; 42: 66-8. 28. Bisof V, Juretic A, Saric N, Melada A, Perkovic Z, Rados M, et al. Pituitary metastasis of renal cell carcinoma: a case report. Radiol Oncol 2008; 42: 225-31. 29. Liigant A, Haldre S, Oun A, Linnamägi U, Saar A, Asser T, et al. Seizure disorders in patients with brain tumors. Eur Neurol 2001; 45: 46-51. 30. Engelhard HH. Current diagnosis and treatment of oligodendroglioma. Neurosurg Focus 2002; 12: E2. 31. Celli P, Nofrone I, Palma L, Cantore G, Fortuna A. Cerebral oligodendrogli­oma: prognostic factors and life history. Neurosurgery 1994; 35: 1018-35. 32. Burton EC, Prados MD. Malignant gliomas. Curr Treat Options Oncol 2000; 1: 459-68. 33. Gannett DE, Wisbeck WM, Silbergeld DL, Berger MS. The role of post­operative irradiation in the treatment of oligodendroglioma. Int J Radiat Oncol Biol Phys 1994; 30: 567-73. 34. Paleologos NA, Cairncross JG. Treatment of oligodendroglioma: an up­date. Neuro Oncol 1999; 1: 61-8. 35. Bullard DE, Rawlings CE 3rd, Phillips B, Cox EB, Schold SC Jr, Burger P, et al. Oligodendroglioma. An analysis of the value of radiation therapy. Cancer 1987; 60: 2179-88. 36. van den Bent MJ. Chemotherapy of oligodendroglial tumours: current developments. Forum (Genova) 2000; 10: 108-18. 37. van den Bent MJ. New perspectives for the diagnosis and treatment of oligodendrogliomav. Expert Rev Anticancer Ther 2001; 1: 348-56. 38. Medical Research Council Brain Tumor Working Party. Randomized trial of procarbazine, lomustine, and vincristine in the adjuvant treatment of high-grade astrocytoma: a Medical Research Council trial. J Clin Oncol 2001; 19: 509-18. 39. Soffietti R. Chemotherapy of anaplastic oligodendroglial tumours. Expert Opin Pharmacother 2004; 5: 295-306. 40. Yung WK, Prados MD, Yaya-Tur R, Rosenfeld SS, Brada M, Friedman HS, et al. Multicenter phase II trial of temozolomide in patients with anaplastic astrocytoma or anaplastic oligoastrocytoma at first relapse. Temodal Brain Tumor Group. J Clin Oncol 1999; 17: 2762-71. 41. Michotte A, Chaskis C, Sadones J, Veld PI, Neyns B. Primary leptomenin­geal anaplastic oligodendroglioma with a 1p36-19q13 deletion: Report of a unique case successfully treated with Temozolomide. J Neurol Sci 2009; 287: 267-70. 42. Morrison T, Bilbao JM, Yang G, Perry JR. Bony metastases of anaplastic oligodendroglioma respond to temozolomide. Can J Neurol Sci 2004; 31: 102-8. 43. van den Bent MJ. Anaplastic oligodendroglioma and oligoastrocytoma. Neurol Clin 2007; 25: 1089-109. 44. Buckner JC, Reid JM, Wright K, Kaufmann SH, Erlichman C, Ames M, et al. Irinotecan in the treatment of glioma patients: current and future studies of the North Central Cancer Treatment Group. Cancer 2003; 97: 2352-8. 18 Velnar T et al. / Genetic markers in oligodendroglial tumours 45. Soffietti R, Nobile M, Rudŕ R, Borgognone M, Costanza A, Laguzzi E, et al. Second-line treatment with carboplatin for recurrent or progressive oli­godendroglial tumors after PCV (procarbazine, lomustine, and vincristine) chemotherapy: a phase II study. Cancer 2004; 100: 807-13. 46. Chamberlain MC, Johnston S. Bevacizumab for recurrent alkylator-refrac­tory anaplastic oligodendroglioma. Cancer 2009; 115: 1734-43. 47. Gadji M, Fortin D, Tsanaclis AM, Drouin R. Is the 1p/19q deletion a di­agnostic marker of oligodendrogliomas? Cancer Genet Cytogenet 2009; 194: 12-22. 48. Stupp R, Hegi ME. Neuro-oncology: oligodendroglioma and molecular markers. Lancet Neurol 2007; 6: 10-2. 49. Ino Y, Betensky RA, Zlatescu MC, Sasaki H, Macdonald DR, Stemmer-Rachamimov AO, et al. Molecular subtypes of anaplastic oligodendro­glioma: implications for patient management at diagnosis. Clin Cancer Res 2001; 7: 839-45. 50. Mueller W, Hartmann C, Hoffmann A, Lanksch W, Kiwit J, Tonn J, et al. Genetic signature of oligoastrocytomas correlates with tumor location and denotes distinct molecular subsets. Am J Pathol 2002; 161: 313-9. 51. Ducray F, Idbaih A, de Reyničs A, Bičche I, Thillet J, Mokhtari K, et al. Anaplastic oligodendrogliomas with 1p19q codeletion have a proneural gene expression profile. Mol Cancer 2008; 7: 41. 52. Riemenschneider MJ, Koy TH, Reifenberger G. Expression of oligodendro­cyte lineage genes in oligodendroglial and astrocytic gliomas. Acta Neuropathol 2004; 107: 277-82. 53. Li H, Lu Y, Smith HK, Richardson WD. Olig1 and Sox10 interact synergisti­cally to drive myelin basic protein transcription in oligodendrocytes. J Neurosci 2007; 27: 14375-82. 54. Aldape K, Burger PC, Perry A. Clinicopathologic aspects of 1p/19q loss and the diagnosis of oligodendroglioma. Arch Pathol Lab Med 2007; 131: 242-51. 55. Glioma meta-analysis trialists group. Chemotherapy in adult high-grade glioma: a systematic review and meta-analysis of individual patient data from 12 randomised trials. Lancet 2002; 359: 1011-8. 56. van den Bent MJ, Carpentier AF, Brandes AA, Sanson M, Taphoorn MJ, Bernsen HJ, et al. Adjuvant procarbazine, lomustine, and vincristine improves progression-free survival but not overall survival in newly diagnosed anaplastic oligodendrogliomas and oligoastrocytomas: a ran­domized European Organisation for Research and Treatment of Cancer phase III trial. J Clin Oncol 2006; 24: 2715-22. 57. Brandes AA, Tosoni A, Cavallo G, Reni M, Franceschi E, Bonaldi L, et al. Correlations between O6-methylguanine DNA methyltransferase pro­moter methylation status, 1p and 19q deletions, and response to temo­zolomide in anaplastic and recurrent oligodendroglioma: a prospective GICNO study. J Clin Oncol 2006; 24: 4746-53. 58. Idbaih A, Omuro A, Ducray F, Hoang-Xuan K. Molecular genetic markers as predictors of response to chemotherapy in gliomas. Curr Opin Oncol 2007; 19: 606-11. 59. Fontaine D, Vandenbos F, Lebrun C, Paquis V, Frenay M. Diagnostic and prognostic values of 1p and 19q deletions in adult gliomas: critical review of the literature and implications in daily clinical practice. Rev Neurol (Paris) 2008; 164: 595-604. 60. Sonabend AM, Lesniak MS. Oligodendrogliomas: clinical significance of 1p and 19q chromosomal deletions. Expert Rev Neurother 2005; 5: S25-32. 61. Cankovic M, Mikkelsen T, Rosenblum ML, Zarbo RJ. A simplified labora­tory validated assay for MGMT promoter hypermethylation analysis of glioma specimens from formalin-fixed paraffin-embedded tissue. Lab Invest 2007; 87: 392-7. 62. Mueller WC, von Deimling A. Gene regulation by methylation. Recent Results Cancer Res 2009; 171: 217-39. 63. Norden AD, Wen PY. Glioma therapy in adults. Neurologist 2006; 12: 279-92. research article CT colonography in detection of colorectal carcinoma Amela Sofic1, Serif Beslic1, Igor Kocijancic2, Nedzad Sehovic1 1 Institute of Radiology, Clinical Centre of University of Sarajevo, Sarajevo, Bosnia and Herzegovina 2 Clinical Institute of Radiology, University Medical Centre Ljubljana, Ljubljana, Slovenia Received 9 November 2009 Accepted 6 December 2009 Correspondence to: Amela Sofic, MD, MSc, Institute of Radiology, Clinical Centre of University of Sarajevo, Bolnicka 25, 71000 Sarajevo, Bosnia and Hercegovina. Phone/Fax:+387 33 444 553, E-mail: amelasofic@yahoo.com Disclosure: The authors have no conflicts of interest to disclose. Background. Diagnostic methods used in screening and detecting colorectal carcinoma are digitorectal examina­tion, faecal occult blood testing, sigmoidoscopy, DNA stool analysis, barium enema, colonoscopy, and as of recently CT colonography. The aim of this study was to establish diagnostic accuracy and comfort of CT colonography com­pared to colonoscopy and barium enema. Patients and methods. We included 231 patients in the prospective study. For all patients CT colonography and barium enema followed by colonoscopy were performed. After the procedures a comfort assessment was done in all patients. Diagnostic positive results were verified by the pato-histological examination. Sensitivity, specificity, positive predicative value (PPV) and negative predicative value (NPV) were calculated for each procedure. Results. With CT colonography, barium enema and colonoscopy 95 lesions were found, 56 (59%) of them were tumours and 39 (41%) were polyps. Among polyps pato-histology revealed 34 adenomas, 3 tubulovillous adenomas and 2 lipomas, among tumours there were 55 adenocarcinomas and 1 lymphoma. Results showed CT colonography sensitivity to polyps to be 89.7%, barium enema 48.7%, and colonoscopy 94.9%. Sensitivity to tumours of CT colonogra­phy and colonoscopy was 100% and of barium enema 94.6%. Specificities and PPV were 100% in all procedures. The comfort assessment showed CT colonography as the far most comfortable out of three procedures. Key words: CT colonography; barium enema; colonoscopy; colorectal polyp; colorectal carcinoma Introduction Colorectal carcinoma (CRC) is the second leading cause of illness and the third leading cause of death in Western countries.1Pato-histologically (PH) CRC is most commonly adenocarcinoma in 98% of cases. CRC starts as a polyp, representing precur­sor of CRC. Consumption of meat and animal fats, physical inactivity, smoking and consumption of alcohol increase the risk for CRC. Prevention and screening of CRC are very com­plex and depend on financial and organizational capacities of health institutions where they are per­formed. There are several basic tests applied in the screening of CRC: digitorectal examination, faecal occult blood testing (FBOT), sigmoidoscopy, colon­oscopy, barium enema, DNA stool analysis and re­cently CT colonography (CTC).2,3 We conducted this comparative study to estab­lish the diagnostic accuracy and comfort of CTC comparing with C and barium enema. Patients and methods Of 231 patients included in the study 106 (47%) were males and 125 (53%) were females. The aver­age age of patients was 57.9 years (SD ± 11.3y, range 23-83y). Only patients with suspected symptoms of CRC were included with the history of blood in the stool, anaemia, constipation, and changes in the stool or positive FBOT test. In all patients CT colonography, barium enema and colonoscopy were performed. Positive diag­nostic findings were correlated with PH results of biopsies taken during colonoscopy. Two hundred 20 Sofic A et al. / CT colonography in detection of colorectal carcinoma FIguRe 1. Polypous adenoma of cecal region in a 55 years old female patient, obtained by our CTC evaluation. TABle 1. Age distribution of patients with positive hystologic findings 20-30 4 10.26% 0 0.00% 31-40 5 12.82% 1 1.79% 41-50 10 25.64% 4 7.14% 51-60 10 25.64% 19 33.93% 61-70 9 23.08% 11 19.64% 71-... 1 2.56% 21 37.50% Total 39 100.00% 56 100.00% and twenty-seven patients were included in the statistical analysis; four patients were excluded due to undetermined PH results. Anidenticalprotocolforcleansingthebowels (Dulcolax®tabletsandsuppositories,aswellas Coloclens®syrup)wasperformedbeforethecom­mencementofeachofthreeprocedures.TheCTC procedurewasperformedaftertheairhadbeenin-sufflatedinthecleansedcolorectalregionuntilan optimalextension,withanintravenousapplication ofspasmolytics.Patientswithintraluminalresidu­alcontentorsuboptimaldistensionofthebowels wereexcludedfromthestudysothatreliableimag­escouldbeachieved.CTscanningwasperformed on4sliceMDCT(VolumezoomSiemens,Erlangen, Germany)equipmentintheproneandsupineposi­tionofthepatient.2Dand3Dreconstructionswere performedonthe«Syngo»softwareworkstation. Double contrast barium enema was performed on an X-ray diascopic equipment (Practix 100, Philips, Aidhoven The Netherlands). The colon­oscopy procedure was performed by a gastroen­terologist on Videocolonoscopic device (CF Q-165 L Olympus» Tokyo Japan). PH examination was done on the tissue obtained by polypectomy or tu­mour sample that was taken either during an endo­scopic examination or a surgical procedure. In relation to PH findings sensitivity and spe­cificity as well as PPV and NPV, using Kappa sta­tistical method for all three procedures were calcu­lated. All hypotheses were tested for the statistical significance of p <0.05 value. Confidence intervals (CI) were also presented. Patients self evaluated comfort of all three procedures as being comfort, less comfort or discomfort. Results The histological examination was conclusive in 227 patients. There were 39 benign lesions in 31 patients and 56 malignant lesions in 56 patients. Benign lesions were present among females in 22 cases (56%), and in males in 17 cases (44%). In male patients tumours were found in 30 cases (54%), and in the females in 26 (46%). Age distribution of the patients regarding benign lesions and tumours is presented in Table 1 (Table 1). The most common symptoms in the case of polyps were: bowel distur­bances in 14 cases, constipation in 14 cases, blood in the stool in 7 cases, followed by anaemia and abdominal pain each in 1 case. In case of tumours, most commonly reported symptoms were: blood in the stool in 35 cases, anaemia in 11 cases and constipation in 10 cases. Most polyps were detected in colon descedens, followed by rectum, colon transversum, caecum and colon ascendens. The most frequent localiza­tion of carcinoma was rectum in 27 cases followed by sigmoid part of colon in 13 cases while the de­scedent part of colon in 5 cases. In the remaining nine cases, carcinoma was found in colon ascedens, transversum and caecum, three cases in each of FIguRe 2. Adenocarcinoma in the middle third part of the rectum with infiltration of mesorectal fat tissue and reactive lymph nodes, obtained by our CTC evaluation. TABle 2. Comparison of results regarding all three methods Sensitivity 89.7% 48.7% 94.9% 100.0% 94.6% 100.0% 95.8% 75.8% 97.9% Specificitiy 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% and PPV these localisations. In one case carcinoma was lo­cated on hepatic flexure whilst a single case of lym­phoma was located on Valvula Bauchini. With the CTC procedure the size of benign le­sions (polyps) detected was: less than 6 mm in 2 cases, 6 to 10 mm in 15 cases and larger than 10 mm in 18 cases. The size of carcinomas detected by CTC was more than 10 mm in all 56 cases (Figure 1). Barium enema detected benign lesions between 6 and 10 mm in 2 cases and in 17 cases larger than 10 mm. This procedure did not detect any polyps smaller than 6 mm. With BE 53 carcinomas were found, all of them were larger than 10 mm. Colonoscopy detected benign lesions smaller than 6 mm in 6 cases, 6 to 10 mm in 9 cases and larger than 10 mm in 22 cases. The size of carci­nomas detected by colonoscopy was larger than 10 mm in all 56 cases. Amongst polyps, there were 34 adenomas, fol­lowed by tubuloviluous adenomas in three cases and lipomas in three cases. According to PH analy­sis adenocarcinoma was far most common (in 98% of cases, n=55), since there was only a single case of lymphoma. (2%). (Figure 2). In all 231 patients CTC side findings were found in 25 cases and extracolic extension in 36 cases. Sensitivity, specificity and PPV for all three pro­cedures are presented in Table 2. We obtained sta­tistically significant results validating CTC proce­dure regarding sensitivity and specificity on polyps and tumours, which are approximately identical in comparison with colonoscopy, and significantly above the method of barium enema. It is important to point out that the CTC method missed to locate 4 polyps which were found by colonoscopy, but did not miss any tumours. Out of 22 cases which were missed by barium enema, all 22 were located by colonoscopy, and 19 by CTC. 3 of those cases were carcinoma and all were diagnosed by both colon­oscopy and CTC. Colonoscopy did not miss any carcinoma; however it missed two polyps located by CTC. Both were later confirmed by colonoscopy and PH; however, it did not miss any tumours. To evaluate staging of carcinoma, we used Dukes method of clinical staging and achieved conform­ity in 96.4% of cases in comparison with the post-surgical oncology staging. Inasurveyofallexamineesinourresearch,the CTCprocedurewasassessedasthemostcomfort­ableincomparisonwiththebariumenemaand colonoscopy;all231patientsassessedtheCTCpro­cedureascomfort.Bariumenemawasassessedasa lesscomfortprocedureby224ofpatients,andasdis­comfortby7patients.CCwasassessedastheleast comfortprocedureby224patients,assesseditasdis­comfort,whilst7patientsassesseditaslesscomfort. 22 Sofic A et al. / CT colonography in detection of colorectal carcinoma Discussion In recent years there has been an extremely rapid development of CT due to the development of CT multislice technology. Its more frequent use in de­tecting CRC is also due to the fact that it has not yet been established an optimal procedure regarding comfort and high reliability in detection of color-ectal lesions.1 The CTC could become an important method in CRC and polyps screening due to its efficacy, cost-effectiveness and because it is an ultra-low dose radiation technique.4,5The more recent method is MR imaging. However, it is usually used in the di­agnostic of colorectal lesions and not in the screen­ing proceeding.6The most significant advantage of CTC is that it can detect the extraluminal tumour extension, which is not possible by other proce­dures.7It is extremely important for discovering the extent of the disease and enabling the proper choice of the treatment. On that way we can influ­ence on better surviving and quality of life of our patients.8,9Regarding comfort, the CTC procedure is undoubtedly in advance compared to other two procedures. It is also much safer, although the colorectal injuries during barium enema are very rare.10In our research the comfort of the procedure was assessed as being 100%. Gluecker published that 72% and Svenson stated that 82% of patients would rather have a CTC than any of the other two procedures.11,12 There are many reports regarding detecting benign and malignant colorectal lesions in the lit­erature. Winawer published the lowest result re­garding sensitivity of barium enema in detecting polyps to be 48%.13Smith reported sensitivity of barium enema in detecting tumours as 83% and of colonoscopy as 97.5%.14Hara stated that sensitiv­ity of CTC for polyps larger than 1 cm was 75%, or 85% in a follow up study.15 Fletcher reported that sensitivity of CTC for polyps larger than 1 cm was 85%.16Gennen published that sensitivity of barium enema regarding carcinoma is in the range of 85-95%, and that sensitivity in detecting polyps smaller than 1 cm is between 50-80%.17Johnson published that sensitivity of CTC to polyps larger than 1 cm was 81%, and of barium enema of 45%. For those smaller than 1 cm, sensitivity of CTC was 72%, compared to barium enema which was 44%. Specificities of CTC were 96-99%, compared to 99-100% of barium enema.18Cotton’s multicen­tric study included 600 participants and showed that sensitivity of CTC to lesions smaller than 6 mm was 39%, to those larger than 1 cm was 96%.19Macari reported 100% sensitivity of CTC regard­ing polyps larger than 1 cm, and 52.9% regarding those between 6-9 mm.20Iacanconne found 100% sensitivity of CTC regarding polyps of 1 cm and larger, and 86% for those smaller than 6 mm, which is slightly more compared to colonoscopy, which was 84%.21Mulhal reported 48% sensitivity of CTC regarding polyps smaller than 6 mm; 70% for those between 6-9 mm; and 85% for those larger than 9 mm.22Ramjii reported sensitivity of 71-93% for pol­yps larger than 1 cm, 55-71% for those between 5 and 9 mm, and 39% for those smaller than 6 mm.23In 2008, Johnson acquired 90%sensitivity of CTC for polyps larger than 9 mm.24 In our research CTC was equally sensitive (100%) in detection of CRC lesions as colonoscopyand much better than barium enema (94.6%). The CTC demonstrated similar sensitivity in detecting polyps larger than 1 cm (89.7%) compared to the colonoscopy (94.9%), and better sensitivity com­pared to barium enema (sensitivity of 48.7%). The CTC is very efficient in pain-intolerant patients and in cases of tumours causing obstruction, doli­chocolons, spasms, and other reasons preventing the colonoscopeto reach the caecum. The CTC is suitable for screening and staging of tumours, as well as for obtaining unexpected findings on other abdominal or pelvic organs. In the detection of le­sions smaller than 5 mm, colonoscopy showed to be better in regard to other two methods. Having considered all results of our study and having compared all three procedures, we have ob­tained statistically significant differences regard­ing sensitivity and specificity of CTC regarding polyps and tumours. These results are quite similar to those compared to colonoscopy, but much more advanced compared to the barium enema. We could state here that our results regarding sensitiv­ity of CT colonography are much better compared with the results of initial studies published in the early nineties in the world, and are quite close to the results of the latest studies published at the be­ginning of this century. References 1. Perše M, Cerar A. Physical activity and colorectal cancer. [Slovenian]. Zdrav Vestn 2009; 78: 399-405. 2. Brkic T, Grgic M. Colorectal Carcinoma. Medicus 2006; 15: 89-97. 3. Colorectal cancer. http.//www.wikimedia.org/wik/colorectal-cancer 4. Kim DH, Pickhardt PJ, Taylor AJ, Leung WK, Winter TC, Hinshaw JL, et al. CT colonography versus colonoscopy for the detection of advanced neoplasia. N Engl J Med 2007; 357: 1403-12. 5. Summers RM, Yao J, Pickhardt PJ, Franaszek M, Bitter I, Brickman D, et al. Computed tomographic virtual colonoscopy computer aided polyp detection in screening population. Gastroenterology 2005; 129: 1832-44. 6. Sofic A, Šehovic N, Bešlic Š, Prnjavorac B, Bilalovic N, Caluk J, et al. MR rectum imaging with ultra sound gel as instrumental contrast media in tubulovillous adenoma. Radiol Oncol 2008; 42: 136-42. 7. Zalis ME, Perumpillichira JJ, Kim JY, Del Frate C, Magee C, Hahn PF. Polyp size at CT colonography after electronic subtraction cleansing in an an­thropomorphic colon phantom. Radiology 2005; 236: 118-24. 8. Ocvirk J. Advances in the treatment of metastatic colorectal carcinoma. Radiol Oncol 2009; 43: 1-8. 9. Velenik V, Oblak I, Anderluh F. Quality of life in patients after combined modality treatment of rectal cancer: Report of a prospective phase II study. Radiol Oncol 2008; 42: 207-14. 10. Yildirim M, Oztekin O, M. Bayam E, Yagli E, Yakan S. Retroperitoneal perfo­ration of the rectum during double-contrast barium-enema examination: a life-threatening complication. Radiol Oncol 2009; 43: 26-9. 11. Glucker TM, Johnson DC, Harmsen WS, Offord KP, Harris AM, Wilson LA, et al. Colorectal cancer screening with CT colonography, colonoscopy, and double-contrast barium enema examination; prospective assessment of patient perceptions and preferences. Radiology 2003; 227: 378-84. 12. Svensson MH, Svensson I, Lasson A, Hellstrom M. Patient acceptance of CT colonography and conventional colonoscopy: prospective com­parative study in patients with or suspected of having colorectal disease. Radiology 2002; 222: 337-45. 13. Winawer SJ, Stewart ET, Zauber AG, Bond JH, Howard A, Waye JD, et al. A comparison of colonoscopy and double-contrast barium enema for surveillance after polypectomy. N Engl J Med 2000; 342: 1766-72. 14. Smith GA, O’Dwyer PJ. Sensitivity of double contrast barium enema and colonoscopy for the detection of colorectal neoplasms. Surg Endosc 2001; 15: 649-52. 15. Hara AK, Johnson CD, McCarty RL, Welch TJ, McCollough CH, Harmsen WS. CT colonography: single versus multi detector row imaging. Radiology 2001; 219: 461–5. 16. Fletcher JG, Johnsom CD, Krueger WR, Ahlquist DA, Nelson H, Ilstrup D, et al. Contrast Enhanced CT colonography in recurrent colorectal carcin­oma: Feasibility of simultaneous evaluation for metastatic disease, local recurrence, and metachronous neoplasia in colorectal carcinoma. AJR 2002; 178: 283-90. 17. Geenen RWF, Hussain SM, Cadamartini F, Poly JW, Sierseno PD, Krestin GP. CT and MRI colonography scanning techniques postprocessing and emphasis on polyp detection. Radiographics 2003; 154: 18-24. 18. Johnson CD, Dachman AH. CT colonography the next colon screening examination. Radiology 2004; 216: 331–41. 19. Cotton P, Durkalski VD, Pineau BC, Dvining Y, Small WC, Affronti JA, et al. Computed tomographic colonography (Virtual colonoscopy). JAMA 2004; 291: 1713-19. 20. Macari M, Bini EJ, Jacobs SL, Naik S, Lui YW, Milano A. Colorectal polyps and cancers in asymptomatic average risk patients: evaluation with CT colonography. Radiology 2004; 230: 629-36. 21. Iannaccone R, Catalano C, Mangiapane F, Murakami T, Lamazza A, Fiori A, et al. Colorectal polyps detection with low dose multi detector row; helical CT colonography versus two sequential colonoscopy. Radiology 2005; 237: 927-37. 22. Mulhall BP, Verrappan GR, Jackson JL. Meta analysis: Computed tomo­graphic colonography. Annals of Internal Medicine 2005; 142: 635-50. 23. Alnoor R.Villous Adenoma. http//www.emedicine.com 24. Johnson CD, Chen MH, Toledano AY. CTC screening (Virtual Colonoscopy) is it virtually ready to replace optical colonoscopy? N Engl J Med 2008; 359: 1207-17. research article Diffusion weighted MR imaging in the differential diagnosis of haemangiomas and metastases of the liver Nagihan Inan, Furkan Kilinc, Tahsin Sarisoy, Sevtap Gumustas, Gur Akansel, Ali Demirci Department of Radiology, University of Kocaeli, Kocaeli, Turkey Received 5 August.2009 Accepted 5 September 2009 Correspondence to: Assist. Prof. Nagihan Inan, MD, PhD, Department of Radiology, University of Kocaeli, Tip Fak, Radyoloji AD, 41380 Umuttepe, Kocaeli, Turkey. Phone: +90 262 3037242; Fax: +90 26203038003; E-mail: inannagihan@ekolay.net Disclosure: No potential conflicts of interest were disclosed. Background. The purpose of the study was to evaluate the value of diffusion-weighted imaging in the differential diagnosis of haemangiomas from metastases of the liver. Patients and methods. We analyzed 69 lesions in 38 patients (33 haemangiomas; 36 metastases) in the retrospec­tive study. Diffusion-weighted imaging was performed using a breath-hold single-shot echo-planar spin echo sequence with three b factors (0, 500 and 1000 sec/mm2), and apparent diffusion coefficients (ADCs) were calculated. For the quantitative evaluation, signal intensity of the lesions, lesion-to-liver signal intensity ratios, ADC of the lesions, and lesion-to-liver ADC ratios were compared between the groups. The statistical significance was determined by student’s-t test. Results. With the b factor 500 sec/mm2, no statistical significance was achieved (p>0.05). With the b factor of 1000 sec/mm2, both the signal intensity and lesion-to-liver signal intensity ratio of the metastases were significantly higher than those for haemangiomas (p<0.001). The cut-off value at 2.6 yielded a sensitivity of 86% and a specificity of 82% for the lesion-to-liver signal intensity ratio. The ADC, and lesion-to-liver ADC ratio of the metastases were significantly lower than those of haemangiomas (p<0.001). With cut-off value of 1.7, ADC ratio had a sensitivity of 88% and a specificity of 72% for ADC lesion/liver. Conclusions. Diffusion-weighted imaging with high b value may help in the differential diagnosis of metastases from haemangiomas of the liver. Key words: liver; haemangioma; metastasis; magnetic resonance imaging; diffusion-weighted imaging, apparent diffusion coefficient. Introduction A liver lesion detected in a patient with the known malignant disease requires a further assessment, as the liver is common site for metastatic spread and haemangiomas are encountered in about 7-20% of the population.1The radiologic imaging plays a cri­tical role in the differential diagnosis of these lesi­ons. On postcontrast computed tomography (CT) and magnetic resonance (MR) images, most hae­mangiomas have a typical enhancement. However, atypical haemangiomas may imitate metastases. The differential diagnosis of these lesions is essen­tial to determine the therapy.2A variety of radio­logic imaging is currently available for the clinical use in these cases.3CT arterioportography has been widely used in the differential diagnosis, however, this technique is invasive and the results are not always reliable.4Over the years, the success rates have increased with the development of new MR contrast agents such as superparamagnetic iron oxide (SPIO).3However, we may encounter some problems in interpreting SPIO-enhanced MR ima­ges because of the difficulty in differentiating thin vessels, small cysts, haemangiomas, and metasta­ses.5Therefore, a non-invasive method is required in the diagnosis of such lesions. Inthisstudy,weevaluatedthecontributionof diffusion-weighted(DW)imaginginthedifferen­tiationofmetastasesfromhaemangiomas,particu­larlyinapatientwiththeknownmalignantdisease, whichposesachallengeinthedifferentialdiagnosis. Patients and methods Patients Our retrospective data were obtained in a 14-month period (September 2007 to October 2008). During this period 63 patients were referred for MR im­aging of the liver for the following indications: suspected haemangioma or metastatic liver mass based on the findings of other imaging modalities and the evaluation for metastases in patients with known primary cancer. However, 25 patients were excluded from the study because of size (< 1cm) (n = 11), low image quality of DW images (n = 8), and incomplete characterization of lesions on the follow-up imaging or the histopathologic examina­tion (n = 6). As a consequence, a total of 69 solidle­sions with a diameter of at least 1 cm in 38 patients (20 women, 18 men) were included in this study. Of these lesions, 36 (in 15 patients) were metastases and 33 (in 23 patients) were haemangiomas. Fifteen patients had multiple lesions (two metastases in 1 patient, three metastases in 3 patients, five metas­tases in 1 patient, six metastases in 2 patients, two haemangiomas in 6 patients, three haemangiomas in 2 patients). For subjects with more than six le­sions only six largest lesions and one region of hepatic parenchyma were analyzed. Imaging was performed prior to the administration of the neo­adjuvant treatment or biopsy. The diagnosis of all metastases was confirmed histopathologically after MR imaging. For subjects with multiple metastases only one lesion was ana­lyzed histopathologically. Remaining similar ra­diologic appearing lesions were accepted as metas­tases because all of them increased in size during the radiologic follow-up (4-10 months). The pri­mary cancer sites in each patient were as follows: 3 colorectal cancers, 2 pancreatic cancers, 1 common bile duct cancer, 3 lung cancers, 2 breast cancers, 1 non-Hodgkin’s lymphoma, 1 neuroblastoma, 1 en-dometrial cancer, 1 adenoid cystic carcinoma of the appendix. All patients with a tentative radiologi­cal diagnosis of haemangiomas showed no change during the clinical and radiological follow up (US every 3 months for 9-14 months). Five patients with haemangiomas had a primary cancer as following sites: 3 colorectal cancers, 2 breast cancers. MR imaging The study was approved by the institutional re­view board and the protocol review committee. 26 Inan N et al. / Diffusion weighted MR imaging in the differential diagnosis Since the tests employed were a part of the routine clinical work-up of these patients, the informed consent was not required by the review board. Allpatientswereexaminedwitha1.5TeslaMR scanner(GyroscanIntera;PhilipsMedicalSystems, Eindhoven,TheNetherlands)usingafourelement phased-arraybodycoil.Thissystemhasamaximal gradientstrengthof30mT/mandaslewrateof150 mT/m/msec.Allpatientswereexaminedinitially withtheroutineMRimagingprotocolfortheup­perabdomenthatincluded:precontrastaxialT1­weightedbreath-holdspoiledgradientecho(fast fieldecho:FFE)withandwithoutfatsuppression (TR/TE/FA/NEX:169/4.6/80/1),coronalandaxial T2-weightedsingleshotturbospinecho(SS-TSE) (TR/TE/NEX/TSEfactor:700/80/1/72),andaxialT2­weightedSS-TSEwithfatsuppression(TR/TE/NEX/TSEfactor:700/80/1/72).Subsequently,3seriesof axialsingle-shotspin-echoecho-planar(SS-SE-EP) DWimages(TR/TE/echo-planarimagingfactor: 1000/81/77;sensitizinggradientsinx,y,zdirec­tions)wereacquiredusingthefollowingbvalues: 0,500and1000sec/mm2.ADCmapswererecon­structedfromtheseimages.Thefatsuppressionwas performedbyusingaspectralsaturationinversion recovery(SPIR)technique.Subsequently,0.1mmol/kgGd-DTPA(Magnevist,Schering,Germany)was administeredasahand-injectedbolusin5seconds followedbyarapidflushwith10-20mlofsaline. Fivedynamicseriesandanadditionallatephase (5thmin)imagewereacquiredwithaT1-weighted breath-holdFFE(TR/TE/FA:169/4.6/80)sequence. MRimaging,includingtheDWI,consistedofa multisectionacquisitionwithaslicethicknessof6 mm,anintersectiongapof1mm,andanacquisition matrixof128x256.Thefieldofviewvariedbetween 455and500mm.Allsequenceswereacquiredusing apartially-parallelimagingacquisitionandSENSE reconstructionwithareductionfactor(R)of2.The scantimeoftheacquisitionofeachDWimagingse­riesduringasinglebreath-holdwas26seconds. Image analysis Quantitativemeasurementsweremadeusinga dedicatedwork-station(DellWorkstationprecision 650,ViewForumrelease3.4system).SIofthele­sionsandliverweremeasuredbyoneoftheradi­ologists(N.I)foreachbfactor(0,500and1,000sec/mm2)usingaregionofinterest(ROI).TheROIwas placedcentrallyandthesizeoftheROIwaskept aslargeaspossible,coveringatleasttwo-thirdsof thelesion,yetavoidingtheinterferencefromthe surroundinglivertissueandmajorbloodvessels. Table 1. Quantitative analysis of DW imaging Inaddition,theADCmapswerecreatedautomati­callyandthemeanADCvaluesoflesionsandliver weredeterminedonimageswithbfactor0and1000 sec/mm2.Theaverageofthreemeasurementswas SIR 3.4 ± 0.9 2.2 ± 0.5 < 0.001 (b=1000 sec/mm2 ) recordedasthefinalSIorADC.SIofthelesions, lesion-to-liverSIratio(SIR),ADCofthelesions,and ADC 1.9 ± 0.4 2.5 ± 0.3 < 0.001 (x10-3 mm2/sec) lesion-to-liverADCratio(ADCR)werecalculated. ADCR 1.6 ± 0.3 1.9 ± 0.3 < 0.001 Statistical analysis SI, SIR, ADC, and ADCR were compared between the groups. The fitness of numeric data set to nor­mal distribution was determined by Kolmogorov-Smirnov test. The data were normally distribut­ed;hencethe differences in SIs, SIRs, ADCs, and ADCRs were analyzed by the student-t test. A p value of less than 0.05 was considered statistically significant. To evaluate the diagnostic performance of the quantitative tests (SIR and ADCR) for dif­ferentiating metastases from haemangiomas and to describe the sensitivity and specificity of the tests, the receiver operating characteristic (ROC) analy­sis was performed. The areas and standard errors for each ROC curve were calculated by the method described by Metz.6The area under the ROC curve reflects the performance of the tests. The optimum cut-off point was determined as the value that best discriminates between the two groups in terms of maximum sensitivity and minimum number of false-positive results. All statistical analyses were performed using SPSS (Statistical Package for Social Science) software. Results The mean age was 66.9 ± 9.3 years and 45.5 ± 12.5 years for patients with metastases and haemangi­omas, respectively. The mean size for metastases and haemangiomas were 44.7 ± 28.4 mm and 38.1 ± 23.2 mm, respectively. 80% of the metastases were found in the right lobe (segment 5 to 8) with the remaining in the left lobe (segments 1 to 4). 69% of the haemangiomas were found in the right lobe (segment 5 to 8) with the remaining in the left lobe (segments 1 to 4). The results of the quantitative analysis of the DW imaging were reviewed in Table 1. With b fac­tors of 0 and 500 sec/mm2, no difference of statisti­cal significance was achieved (p > 0.05). With the b factor of 1000 sec/mm2, the SIs and SIRs of the metastases were significantly higher than those of Note. Data are mean ± SD. *SIR: lesion-to-liver SI ratio; ADC: apparent diffusion coefficients; ADCR: lesion-to-liver ADC ratio. the haemangiomas (p < 0.001) (Figures 1B, 2B). The area under the ROC curve was 0.891 ± 0.04 for SIR (p < 0.001). With a cut-off value of 2.6, SIR had a sensitivity of 86% and a specificity of 82% (Figure 3A). The ADCs and ADCRs of metastases were sig­nificantly lower than that of the haemangiomas (p < 0.001) (Figures 1C, 2C). The area under the ROC curve was 0.893 ± 0.04 for ADCR (p < 0.001). Setting the cut-off value at 1.7, we found a sensitivity of 88% and a specificity of 72% for ADCR. Discussion Forthedifferentialdiagnosisofhaemangiomas frommetastasesoftheliver,thesensitivityand specificityaregenerallysuperiorwithcontrast enhancedMRIwhencomparedtootherimaging modalities.2MRI-basedtechniquesarealsouseful toassesstheotherhepaticpathology.7However, thegreatestclinicalexperienceinthedifferen­tialdiagnosiswaswithnon-specificextracellular gadoliniumchelatescontrastsbecausetheyare safe,relativelyinexpensiveandtheyalsopro­videthecharacterizationofmostoftheselesions.3However,sometimesnon-specificextracellular gadoliniumchelatesmaynotallowustorecognize theselesionswell.Inthesepatients,newcontrast agents(SPIO-enhancedMRI)ornewMRItech­niques(DWI)mustbeused,especiallyinpatients withtheknownprimarymalignancy.Inareport publishedbyNasuetal.8,theauthorscompared accuracyofDWIwithofSPIO-enhancedMRIin theevaluationofhepaticmetastases.Inthatre­portitwasshownthatDWIhasmoresensitiv­itythanSPIO-enhancedMRI.However,intheir studyADCmeasurementwasnotperformed.In twootherreports,theauthorscomparedaccuracy ofDWIwithofSShT2-WTSEsequencesinthe evaluationofhepaticmetastases.Inthosereports, 28 Inan N et al. / Diffusion weighted MR imaging in the differential diagnosis ADCR Lesion 3 2,5 2 1,5 1 0,5 0 hemangiomasmetastases Lesion FIguRe 3. Scattergram distribution of lesion-to-liver SI ratios on DW images with b factor 1000 sec/mm2 (A) and ADC ratios (B) of haemangiomas and metastases. althoughimageartifactswerelowerwithT2-W TSEthanSSh-EPI,itisshownthatDWIwasmore usefulthanSShT2-WTSEsequencesforthedetec­tionoflesions.9,10However,indailypracticethele­sioncharacterizationisasimportantasthelesion detection. Recent reports have suggested that DWI with SS EPI may be helpful in the characterization of fo­cal and diffuse liver lesions, with high specificity and sensitivity.2,8,11-20Those studies reported that the ADC values in benign lesions (such as hae­mangiomas and cysts) were significantly higher than those of the malignant lesions (hepatocellular carcinomas, metastases). This difference was at­tributed to the difference in cellular density. Since malignant tumors often have higher cellularity than benign lesions, the ADCsof most malignant tumors are lower than benign masses. In these previous studies, different imaging parameters were applied to evaluate a wide range of hepatic lesions, including metastases and haemangiomas.The ADC values of both the normal liver and the liver lesions were differed significantly at different b values.18Namimoto et al.16reported a low ADC of the liver with low and high b values (30 and 1200 sec/mm2). On the other hand, high ADCs were re­ported by Taouli et al.15using low and intermediate b values (0 and 500 sec/mm2). In a study of Yamada et al.18in which the b values of 30, 300, 900 or 1100 sec/mm2 were used, high ADCs were obtained at low b values.In conclusion, when only a high b value is used, the ADC values reflect the true diffu­sion of the tissue. On the contrary, when only a low b value is used, the ADC may be influenced by the intravoxel perfusion.21 In our study, significant differences between the SIs and SIRs of haemangiomas and metas­tases were found only on images with a b factor of 1000 sec/mm2. At higher b values, the contribu­tion of the T2 shine-through to the signal intensity decreases, while tissue cellularity makes a greater contribution.22Hence, the hyperintensity of metas­tases on b 1000 sec/mm2images can not be totally attributed to the T2 shine-through effect. Diffusion can be quantitatively evaluated by ADC, which is free of the T2 shine-through effect.23In our series, the mean ADC of the metastases was significantly lower than that of the haemangiomas. Hence, at least a part of the increase in signal on DW images must have been caused by the reduced diffusion in metastases. Since the cavernous haemangiomas are mainly composed of liquid component which consists of fiber septation, scar, and hemorrhage the ADC of the haemangiomas is increased. On the contrary, the metastases have higher cellularity, hence the lower ADC.1 This study has several technical limitations. The main limitation was that the SSh-EPI sequence employed with a higher b value had a lower SNR, resulting in greater image distortion. In addition, the EPI sequence caused anatomic distortion due to susceptibility effects.22Although the best lesion conspicuity is achieved with low b value for detect­ing small focal liver lesions, the best lesion specifi­city is achieved with a high b value.22Because of that reason, we used a high b value for the char­acterization of lesions. Another important limita­tion was that there were not any atypical haeman­giomas (such as calcified, hyalinised or sclerosed) and cystic metastases in our study. The necrotic metastasis may exhibit the pronounced hyperin-tensity on T2-W image and less restricted diffusion. The differential diagnosis of most of the hae­mangiomas from metastases is usually possible with the combined use of specific radiologic fea­tures. However, sometimes the differential diagno­sis of these lesions may still be difficult. Our pre­liminary data suggest that DWI with a high b value may be helpful in this setting and it can be easily added to routine liver imaging protocols. References 1. Bartolozzi C, Cioni D, Donati F, Lencioni R. Focal liver lesions: MR imaging-pathologic correlation. Eur Radiol 2001; 11: 1374-88. 2. Vossen JA, Buijs M, Liapi E, Eng J, Bluemke DA, Kamel IR. Receiver operating characteristic analysis of diffusion-weighted magnetic resonance imaging in differentiating hepatic hemangioma from other hypervascular liver lesions. J Comput Assist Tomogr 2008; 32: 750-6. 3. Rappeport ED, Loft A, Berthelsen AK, von der Recke P, Larsen PN, Mogensen AM, et al. Contrast-enhanced FDG-PET/CT vs. SPIO-enhanced MRI vs. FDG-PET vs. CT in patients with liver metastases from colorectal cancer: a prospective study with intraoperative confirmation. Acta Radiol 2007; 48: 369-78. 4. Vogl TJ, Schwarz W, Blume S, Pietsch M, Shamsi K, Franz M, et al. Preoperative evaluation of malignant liver tumors: comparison of unen­hanced and SPIO (Resovist)-enhanced MR imaging with biphasic CTAP and intraoperative US. Eur Radiol 2003; 13: 262-72. 5. Coenegrachts K, Orlent H, ter Beek L, Haspeslagh M, Bipat S, Stoker J, et al. Improved focal liver lesion detection: comparison of single-shot spin-echo echo-planar and super paramagnetic iron oxide (SPIO)-enhanced MRI. J Magn Reson Imaging 2008; 27: 117-24. 6. Metz CE. ROC methodology in radiologic imaging. Invest Radiol 1986; 21: 720-33. 7. Mahmoud-Ghoneim D, Amin A, Corr P. MRI-based texture analysis: a po­tential technique to assess protectors against induced-liver fibrosis in rats. Radiol Oncol 2009: 43: 30-40 8. Nasu K, Kuroki Y, Nawano S, Kuroki S, Tsukamoto T, Yamamoto S, et al. Hepatic metastases: diffusion-weighted sensitivity-encoding versus SPIO-enhanced MR imaging. Radiology 2006; 239: 122-30. 9. Bruegel M, Gaa J, Waldt S, Woertler K, Holzapfel K, Kiefer B, et al. Diagnosis of hepatic metastasis: comparison of respiration-triggered dif­fusion-weighted echo-planar MRI and five T2-weighed turbo spin-echo sequences. AJR Am J Roentgenol 2008; 19: 1421-9. 10. Coenegrachts K, Delanote J, Ter Beek L, Haspeslagh M, Bipat S, Stoker J, et al. Improved focal liver lesion detection: comparison of single-shot diffusion-weighted echoplanar and single-shot T2-weighed turbo spin-echo techniques. Br J Radiol 2007; 80: 524-31. 11. Koh DM, Scurr E, Collins DJ, Pirgon A, Kanber B, Karanjia N, et al. Colorectal hepatic metastases: quantitative measurements using single-shot echo-planar diffusion-weighted MR imaging. Eur Radiol 2006; 16: 1898-905. 12. Inan N, Arslan A, Akansel G, Anik Y, Sarisoy HT, Ciftci E, Demirci A. Diffusion-weighted imaging in the differential diagnosis of simple and hydatid cysts of the liver. AJR Am J Roentgenol 2007; 189: 1031-6. 13. Chan JH, Tsui EY, Luk SH, Fung AS, Yuen MK, Szeto ML, et al. Diffusion-weighted MR imaging of the liver: distinguishing hepatic abscess from cystic or necrotic tumor. Abdom Imaging 2001; 26: 161-5. 14. Ichikawa T, Haradome H, Hachiya J, Nitatori T, Araki T. Diffusion-weighted MR imaging with single-shot echo-planar imaging in the upper abdomen: preliminary clinical experience in 61 patients. Abdom Imaging 1999; 24: 456-61. 15. Taouli B, Vilgrain V, Dumont E, Daire JL, Fan B, Menu Y. Evaluation of liver diffusion isotropy and characterization of focal hepatic lesions with two single-shot echo-planar MR imaging sequences: prospective study in 66 patients. Radiology 2003; 226: 71-8. 16. Namimoto T, Yamashita Y, Sumi S, Tang Y, Takahashi M. Focal liver mass­es: characterization with diffusion-weighted echo-planar MR imaging. Radiology 1997; 204: 739-44. 17. Muller MF, Prasad P, Siewert B, Nissenbaum MA, Raptopoulos V, Edelman RR. Abdominal diffusion mapping with use of a whole-body echo-planar system. Radiology 1994; 190: 475-8. 18. Yamada I, Aung W, Himeno Y, Nakagawa T, Shibuya H. Diffusion coefficients in abdominal organs and hepatic lesions: evaluation with intravoxel inco­herent motion echo-planar MR imaging. Radiology 1999; 210: 617-23. 19. Kim T, Murakami T, Takahashi S, Hori M, Tsuda K, Nakamura H. Diffusion-weighted single-shot echoplanar MR imaging for liver disease. AJR Am J Roentgenol 1999; 173: 393-8. 20. Ichikawa T, Haradome H, Hachiya J, Nitatori T, Araki T. Characterization of hepatic lesions by perfusion-weighted MR imaging with an echoplanar sequence. AJR Am J Roentgenol 1998; 170: 1029-34. 21. Goshima S, Kanematsu M, Kondo H, Yokoyama R, Kajita K, Tsuge Y, et al. Diffusion-weighted imaging of the liver: optimizing b value for the detec­tion and characterization of benign and malign hepatic lesions. J Magn Reson Imaging 2008; 28: 691-7. 22. Naganawa S, Kawai H, Fukatsu H, Sakurai Y, Aoki I, Miura S, et al. Diffusion-weighted imaging of the liver: technical challenges and prospects for the future. Magn Reson Med Sci 2005; 4: 175-86. 23. Ozsunar Y, Sorensen AG. Diffusion-and perfusion-weighted magnetic reso­nance imaging in human acute ischemic stroke: technical considerations. Top Magn Reson Imaging 2000; 11: 259-72. case report Percutaneous transcatheter arterial embolization in haemodynamically stable patients with blunt splenic injury Peter Popovicą, Dragoje Stanisavljevic˛, Miran Jeromelą ą Institute of Radiology, University Medical Centre Ljubljana, Ljubljana, Slovenia ˛ Clinical Department for Surgery, University Medical Centre Ljubljana, Ljubljana, Slovenia Received 28 December 2009 Accepted 10 January 2010 Correspondence to: Peter Popovic, MD, MSc, University Medical Centre Ljubljana, Clinical Institute of Radiology, Zaloška cesta 7, 1525 Ljubljana, Slovenia. E-mail: peter.popovic@kclj.si Disclosure: No potential conflicts of interest were disclosed. Background. The nonoperative management of the blunt splenic injury in haemodynamically stable patients has become an accepted treatment in recent years. We present a case of the blunt splenic injury successfully treated by supraselective embolization with microspheres. Case report. A young hockey player was brought to the Emergency Department with the history of blunt abdomi­nal trauma 2 h earlier. A Grade III splenic injury with haemoperitoneum was diagnosed on sonographic evaluation and the patient was treated with the selective distal splenic artery embolization with microspheres. Postprocedural ultrasound and computed tomography follow-up a year later revealed only a small area of parenchymal irregularity. Conclusions. The percutaneous splenic arterial embolization has a major role in the management of traumatic splenic injuries. Embolization is particularly beneficial in injuries of grade III or higher. Key words: splenic trauma; treatment; angiography; percutaneous transcatheter embolization Introduction Spleen injuries are most commonly associated with blunt abdominal trauma and represent a po­tentially life-threatening condition. The manage­ment of splenic trauma is still controversial, but there have been major changes over the last three decades. In the past, any damaged spleen was sur­gically removed to avoid a delayed rupture. The increased susceptibility of the patient to infec­tion after splenectomy – in particular, the risk of overwhelming, potentially fatal postsplenectomy sepsis – motivated physicians to favour splenic preservation procedures.1Nonsurgical manage­ment (NOM) with bed rest and observation has traditionally been the treatment of choice for the splenic injury in paediatric patients. Although the nonsurgical management of stable blunt splenic injuries in adults has gained popularity in recent years, the initial choice of surgical versus nonsurgi­cal management remains controversial. However, embolization is also wildly used for another indi­cations with much less controversial results.2,3The controversy of the splenic arterial embolization has been attributed to the relatively high failure rate of such a treatment (10–31%), with a result­ant need for secondary splenectomy, and to the potential of missing other intra-abdominal injures that require laparotomy. The splenic transcatheter arterial embolization (TAE) has been proposed to reduce the risk of nonsurgical management failure in adults and children. Sclafani et al. reported a se­ries of cases in which NOM by means of the tran­scatheter arterial embolization was successful in 91% of hemodynamically stable patients, and the splenic function was preserved in all patients who underwent TAE.1The most widely accepted indi­cation for TAE is evidence of the arterial injury on a computed tomography (CT) scan. In cases of the arterial injury, embolization is performed with mi-crocoils or gelfoam particles as distally as possible, in a small arterial branch that supplies the segment in which the extravasation is detected, to preserve perfusion to the remaining splenic parenchyma.1,2We report on a patient with blunt splenic trauma who was successfully treated by the supraselective embolization with microspheres. Case report A 20-year-old hockey player presented with blunt trauma to the left upper abdomen. The abdomi­nal ultrasound revealed a small amount of free fluid around the spleen (haemoperitoneum). A small intraparenchimal splenic haematoma and a laceration 4 cm in depth were seen in the lower pole of the spleen. The repeated abdominal ultra­sound (performed 4 h later) revealed an increased amount of free abdominal fluid (around 1000 cm3). The splenic haematoma had also increased in size (measuring 5.5 cm in diameter). The patient was haemodynamically stable. His blood pressure was 110/85 mmHg with a heart rate of 90 beats/min. A multidisciplinary decision for the nonopera­tive treatment – percutaneous embolization – was reached. An urgent angiogram was performed to identify and possibly also treat the source of bleed­ing. The informed, written consent of the patient was obtained before the procedure for both diag­nostic angiography and possible embolization. The selective catheterisation of the splenic artery with a 5F Sidewinder catheter (Cordis, Miami, FL, USA) demonstrated the extravasation of contrast media from the distal branch of the splenic artery (Figure 1A,B). A decision to perform the distal splenic ar­tery embolization was made. Supraselective cath­eterisation and embolization with 500–700 µm Bead Block (Biocompatibles, Farnham, Surrey, UK) microspheres via a Progreat 2.8 Fr microcath­eter (Terumo, Leuven, Belgium) were performed. A postprocedural splenic arteriogram showed the successful embolization (Figure 2). Follow-up ultrasound and CT examination revealed a small area of infarction at the site of the embolization (Figure 3). The patient was stable and discharged from hospital two weeks later. A postprocedural follow-up a year after the procedure revealed only a small area of parenchymal irregularity. Discussion The nonoperative management of blunt splenic in­juries is the treatment modality of choice in haemo-dynamically stable adults and paediatric patients regardless of the severity of the injury. The em-bolization is a useful adjunct in the nonoperative management of patients who continue to bleed (Eastern Association for the Surgery of Trauma, Trauma Practice Management Guidelines, 2003). These guidelines are now accepted in most mod­ern trauma centres. Contrast-enhanced CT has been shown to be highly accurate in diagnosing 32 Popovic P et al. / Percutaneous transcatheter embolization in splenic injury acute splenic injuries. It enables the classification of the splenic injury according to severity, for which the Organ Injury Scale for the spleen (American Association for the Surgery of Trauma-AAST) is a widely accepted grading system.4-6The scale is as follows: Grade I – subcapsular haematoma of less than 10% of surface area or capsular tear of less than 1 cm in depth; Grade II – subcapsular he-matoma of 10–50% of surface area or intraparen-chymal haematoma of less than 5 cm in diameter or laceration of 1–3 cm in depth and not involv­ing trabecular vessels; Grade III – subcapsular haematoma of greater than 50% of surface area (or expanding and ruptured subcapsular or parenchy­mal haematoma) or intraparenchymal hematoma of greater than 5 cm (or expanding) or laceration of greater than 3 cm in depth (or involving trabecular vessels); Grade IV – laceration involving segmental or hilar vessels with the devascularisation of more than 25% of the spleen; Grade V – shattered spleen or hilar vascular injury. Patients with AAST grade I or II splenic injuries and no associated splenic vas­cular injuries can be managed with just a simple observation. Those who are found to have one of the previously mentioned CT findings indicative of angioembolization — including AAST grade III–V splenic injury, active contrast extravasation or vascular injury of the spleen (pseudoaneurysm or A-V fistula) — should proceed to angiography and splenic embolization.4,6 The objective of the splenic arterial emboliza­tion is to improve the results of the nonoperative management.7The embolization is performed via percutaneous access (usually via the common femoral artery). There are two methods regarding the splenic artery embolization. The decision about which method to use depends on angiographic findings. The distal splenic artery embolization is the method of choice for the management of a haemorrhage which originates from a distal branch of the splenic artery. This type of embolization is usually performed with microcoils and/or gelatin sponge pledgets that are injected through a micro­catheter.2,8-11 This technique achieves haemostasis to the injured parts while preserving the perfusion to the remainder of the spleen. When a haemorrhage persists in spite of the dis­tal embolization or the patient is at high risk of sec­ondary spleen rupture (injury Grade III or higher), a more proximal splenic artery embolization is per­formed which reduces the pressure in the splenic parenchyma.5,9-13 This type of embolization is usu­ally performed with microcoils inserted in the mid­dle segment of the splenic artery. Coils inserted at this site allow the reconstitution of blood supply through collateral vessels (short gastric and gastro­epiploic, transgastric and transpancreatic arteries). The proximal embolization has been shown to be associated with less frequent and smaller infarcts than the distal embolization. It does not affect spleen anatomy or immune function.14The success of the splenic arterial embolization is defined by the splenic salvage rate. The Quality Improvement Guidelines of the Society of Interventional Radiology reports a suc­cess rate between 87–100%.4,11,13 Major postprocedural complications are splenic abscess and infarct. Postembolization CT shows splenic infarcts in two thirds of patients after the proximal embolization and in all cases after the distal embolization.14The reported rate of the splenic abscess after the proximal or distal emboli­zation is 3%.10,13 Other relatively rare complications include coil migration, iatrogenic vascular injury and missed injuries to the diaphragm or pancreas. Grading of the spleen injury in our case was not based on CT but on the sonographic evaluation. It was estimated as a Grade III injury (intraparen­chymal haematoma greater than 5 cm and lacera­tion greater than 3 cm in depth). The patient was haemodinamically stable but continued to bleed, and, therefore, the decision to perform emboliza­tion was made. Arteriography revealed bleeding from a small distal branch of the splenic artery. The successful distal splenic artery embolization with microspheres was performed. An early postproce­dural follow up with CT and ultrasound was per­formed, revealing only a small area of parenchymal infarct (less than 2 cm in diameter). A postproce­dural follow-up a year after the procedure revealed only a small area of parenchymal irregularity. Conclusions The percutaneous splenic arterial embolization has a major role in the management of traumatic splenic injuries. The embolization is particularly beneficial in injuries of AAST grade III or higher. Microspheres can be used as an alternative to mi-crocoil or gelfoam particles for the distal splenic artery embolization. references 1. Sclafani SJ, Shaftan GW, Scalea T, Patterson LA, Kohl L, Kantor A, et al. Nonoperative salvage of computed tomography–diagnosed splenic inju­ries: utilization of angiography for triage and embolization for hemostasis. J Trauma 1995; 39: 818-25. 2. Vidjak V, Novacic K, Hebrang A, Mazuranic I, Samarzija M, Ljubic S, et al. Transcatheter embolization of bronchial arteries in the treatment of haemoptysis. Radiol Oncol 2009; 43: 152-61. 3. Kutlu R, Soylu A. Deep dorsal vein embolization with N-butyl-2­cyanoacrylate and lipiodol mixture in venogenic erectile dysfunction: early and late results. Radiol Oncol 2009; 43: 17-25. 4. Shanmuganathan K, Mirvis SE, Boyd-Kranis R, Takada T, Scalea T. Nonsurgical management of blunt splenic injury: Use of CT criteria to select patients for splenic arteriography and potential endovascular therapy. Radiology 2000; 217: 75-82. 5. Thompson BE, Munera F, Cohn SM, MacLean AA, Cameron J, Rivas L, et al. Novel computed tomography scan scoring system predicts the need for intervention after splenic injury. J Trauma 2006; 60: 1083-6. 6. Anderson SW, Varghese JC, Lucey BC, Burke PA, Hirsch EF, Soto JA. Blunt splenic trauma: delayed-phase CT for differentiation of active hemor­rhage from contained vascular injury in patients. Radiology 2007; 243: 88-95. 7. Wahl WL, Ahrns KS, Chen S, Hemmila MR, Rowe SA, Arbabi S. Blunt splenic injury: operation versus angiographic embolization. Surgery 2004; 136: 891-9. 8. Bessoud B, Denys A, Calmes JM, Madoff D, Qanadli, Schnyder P, et al. Nonoperative management of traumatic splenic injuries: Is there a role for proximal splenic artery embolization? Am J Roentgenol 2006; 186: 779-85. 9. Haan J, Biffl W, Knudson M, David KA, Oka T, Majericik S, et al. Splenic embolization revisited: a multicenter review. J Trauma 2004; 56: 542-7. 10. Madoff DC, Denys A, Wallace MJ, Murthy R, Gupta S, Pillsbury EP, et al. Splenic arterial interventions: anatomy, indications, technical consid­erations, and potential complications. Radiographics 2005; 25(Suppl 1): S191-211. 11. LiuPP,LeeWC,ChengYF,HsiehPM,HsiehYM,TanBL,etal.Useofsplenic artery embolization as an adjunct to nonsurgical management of blunt splenic injury. J Trauma 2004; 56: 768-72. 12. Tessier DJ, Stone WM, Fowl RJ, Abbus MA, Andrews JC, Bower TC, et al. Clinical features and management of splenic artery pseudoaneurysm: case series and cumulative review of litarature. J Vasc Surg 2003; 28: 969-74. 13. Velmahos G, Chan L, Kamel E. Nonoperative management of splenic injuries: have we gone too far? Arch Surg 2000; 135: 674-9. 14. Killeen K, Shanmuganathan K, Boyd-Kranis R, Scalea T, Mirvis S. CT find­ings after embolization for blunt splenic trauma. J Vasc Interv Radiol 2004; 12: 209-14. research article Numerical study of the electroporation pulse shape effect on molecular uptake of biological cells Damijan Miklavcic and Leila Towhidi Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia Received 16 November 2009 Accepted 2 December 2009 Correspondence to: Prof. Damijan Miklavcic, PhD, Faculty of Electrical Engineering, University of Ljubljana, Trzaska 25, 1000 Ljubljana, Slovenia. Phone: +386 1 4768 456; Fax: +386 1 4264 658; E-mail: damijan.miklavcic@fe.uni-lj.si Disclosure: No potential conflicts of interest were disclosed. Background. In order to reduce the side-effects of chemotherapy, combined chemotherapy-electroporation (elec-trochemotherapy) has been suggested. Electroporation, application of appropriate electric pulses to biological cells, can significantly enhance molecular uptake of cells due to formation of transient pores in the cell membrane. It was experimentally demonstrated that the efficiency of electroporation is under the control of electric pulse parameters. However, the theoretical basis for these experimental results is not fully explained. In order to predict the outcome of experiments and optimize the efficiency of electroporation before each treatment, we developed a model to inves­tigate the effect of pulse shape on efficiency of electroporation. Results. Our model is based on a developed chemical-kinetics scheme and trapezium barrier model, while self-consistency was taken into account. This model is further supplemented with a molecular transport model to acquire the molecular uptake of cells. The investigated pulse shapes in this study were unipolar rectangular pulses with dif­ferent rise and fall times, triangular, sinusoidal and bipolar rectangular pulses and also sinusoidal modulated unipolar pulses with different percentages of modulation. The obtained results from our modelling and simulations are in good agreement with previously published experimental results. Conclusions. We therefore conclude that this model can be used to predict the effects of arbitrarily shaped electro-poration pulses on cell membrane conductivity and molecular transport across the cell membrane. Key words: electrochemotherapy, optimization; membrane permeability; membrane conductivity Introduction Cancerisaleadingcauseofdeatharoundthe worldandforthisreasonithasdrawntheat­tentionofmanyresearchers.Chemotherapyhas beenusedformanyyearsandisoneofthemost commontreatmentsforcancer.Cytotoxicchemo­therapeuticdrugsareusuallyhydrophilicwith verylowtransportthroughthecellmembrane andthushighdosesofthesedrugsareneededfor treatment.Therefore,whilechemotherapycanbe quiteeffectiveintreatingcertaincancersbyinter­feringwiththecancercell’sabilitytogroworre­produce,chemotherapeuticdrugsreachallparts ofthebody,notjustthecancercells.Becauseof this,theremaybemanyside-effectssuchasnau­sea,bloodcelldeficiency,fatigueandlossofhair duringtreatment.1 Two decades ago, electrochemotherapy was suggested for its use in clinical treatment of local­ized tumors.2Electrochemotherapy consists of elec­troporation and chemotherapy. Electroporation is a technique in which permeability of the plasma membrane increases transiently and reversibly with appropriate pulse parameters3-6and is nowa­days widely used not only in electrochemothera­py,7-9but also in biotechnology10,11and in medical applications such as gene electrotransfer12,13and transdermal drug delivery.14Electroporation in combination with chemotherapy can increase drug delivery into the cells and consequently drug doses and thus side-effects of chemotherapy can be re­ duced.10, 11,15,16 It was experimentally demonstrated in a number of studies that the efficiency of electroporation is under the control of electric pulse parameters such as pulse amplitude, duration, and shape.17-22 and, therefore, electrochemotherapy using mod-Optimization of electric field parameters for suc-elling and simulation. Our model was based on a cessful electroporation requires time-consuming chemical-kinetics scheme with two types of pores29and costly experiments for different experimental which have been recently confirmed.30We used de-criteria unless an appropriate model for this phe-veloped equations with field-dependent rate coef­nomenon can be suggested. Although the known ficients in order to obtain the pore distribution on models were proposed in previous studies23-28, they the membrane. Besides, the conductivity of pores are still unable to explain the effect of some param-was defined based on a trapezium barrier model eters such as pulse shape, pulse repetition frequen-for the image forces.31A self-consistent set of equa­cy and number of pulses on the molecular uptake tions was used to consider all simultaneous chang-enhancement of the cells under exposure. es. This model was supplemented with a molecu- The suggested mechanism for electroporation lar transport model for a single cell to acquire the consists of structural changes resulting in forma-molecular uptake of cells. The investigated pulse tion of transient aqueous pores in the cell mem-shapes in this study were unipolar rectangular brane. In order to reveal the exact mechanisms and pulses with different rise and fall times, triangular, dynamics of pore formation and closure and more sinusoidal and bipolar rectangular pulses and also importantly resealing of the membrane, theoretical sinusoidal modulated unipolar pulses with dif-models have drawn a great deal of attention.ferent percentages of modulation – all previously In our present study, we investigated the effect used in experimental studies.of pulse shape on the efficiency of electroporation Material and methods Model description Modified chemical-kinetics model for electroporation When a cell is exposed to an external electric field, the induced transmembrane voltage (ITV) starts to increase based on the Laplace equation which leads to structural changes of the cell membrane. Based on a previously suggested29, and recently confirmed30kinetic model, in the first step the intact closed lipids (C) transform to tilted lipid headgroups (C1). In the second step, the prepores (P1) are formed and finally, in the last step the final pores (P2) are formed. The sequential reaction can be described by: [1] The permeability of the P1 state is negligibly small and P2 is predominantly responsible for molecular uptake. Pore formation and closure are denoted by kiand k-i(i=1,2,3) rate coefficients, respectively. For simplicity, the rate coefficients k1, k2and k3are considered equal (k1=k2=k3=kp).29The governed rate laws of constituting steps for the scheme [1] are: [2] wheretand denotetimeandposition,respectively.[C],[C1],[P1]and[P2]shownormalizeddistribution ofeachmembranelipidstaterelativetotheinitialvalueoftheclosedstate Regarding the Van’t Hoff relationship in electro-thermodynamics, the rate coefficient of pore formation can be obtained from:29,31 36 Miklavcic D and Towhidi L / Dependence of electroporation on pulse shape [3] where ITV is the potential difference between the outer and inner layer of the membrane, is the mean volume change due to pore formation, is the permittivity of the vacuum and and are dielectric constants of water and lipids, respectively. kB is the Boltzmann constant, dm is the thickness of the mem­brane and T is temperature. While the pore formation rate coefficient kp is electric field-dependent, the closure rate coefficients (k-1, k-2 and k-3) are constant and independent of electric field strength.29 Whenever electroporation occurs, an increase in conductivity during the pulse is observed32which can be explained by the formation of pores in the cell membrane. Based on the trapezium barrier model for the image forces,the intrinsic pore conductivities (i=1 and 2 represents P1 and P2 pores, respectively) are expressed as follows:31,33 [4] where [5] In the above equations, and are the extracellular and intracellular conductivities respectively, n is the geometrical parameter of the trapezium model for energy barrier, F is the Faraday constant and is the intrinsic pore barrier potential. Therefore, conductivity of the membrane ( ) can be obtained by: [6] where is the physiological/baseline conductivity of the membrane. Thus conductivity at each point on the membrane changes with time during and after the pulse, depending on pore distribution variations which affect ITV and in turn the distribution of pores. Transmembrane molecular transport model Based on previous studies34-36, we defined two distinct phases for the electroporated membrane and two related transport mechanisms: the first one is the porated phase [P2] with relatively fast relaxation due to pore closure according to Eq. [1]. The second phase is the memory phase [M] due to enhanced membrane perturbation and ruffling with quite slow relaxation34-36which returns to its baseline value with a dual exponential decay function:29 [7] where is the normalized distribution of [P2] pores at the end of the pulse, kfand ksare decay rate coefficients for this second phase and B is a constant. The considered transport mechanisms for these two phases were interactive diffusion through the pores and endocytotic-like transport through the permeabilized area of the membrane.34-37 Thus, the per­meability of the membrane can be written as the sum of two distinct contributions: [8] where Dpand Drare the attributed diffusion coefficients for interactive transport and endocytotic-like transport, respectively. While the membrane is being permeabilized due to the electric field, the molecules pass through the membrane due to a concentration gradient. A quantitative description of diffusion is contained in Fick’s first law. The total flux can be approximated by , where and are the outer and inner concentrations adjacent to the membrane. The total number of molecules transported through the mem­ Table 1. Values of parameters used in simulations Membrane thickness dm 5e-9 m 29 Extracellular conductivity 0.14 S/m a 29, 38 Intracellular conductivity 0.3 S/m b 29, 39 Initial conductivity of membrane 5e-7 S/m 40 Extracellular permittivity 7.1e-10 As/Vm 29 Intracellular permittivity 7.1e-10 As/Vm 29 Membrane permittivity 4.4e-11 As/Vm c 29 Water relative dielectric constant 80 As/Vm 29 Lipid relative dielectric constant 2 As/Vm 29 Free diffusion coefficient D0 5e-10 m2/s 29 Zero-field equilibrium constant K0 2e-2 29 Mean average aqueous pore volume 9e-27 m3 29 Intrinsic barrier potential of P1 state 0.13 V 31 Intrinsic barrier potential of P2 state 0.084 V 31 A geometrical parameter n 0.12 33 Decay rate coefficient for C1 k-1 105 s-1 41-43 Decay rate coefficient for P1 pores k-2 2000 s-1 41-43 Decay rate coefficient for P2 pores k-3 2 s-1 41 Decay rate coefficients for endocytotic-like process kf , ks 0.044, 0.003 s-1 29 a This is for SMEM. The range of extracellular medium is quite large. b Reported between (0.2-0.55) S/m c Reported between (4.4-5)*10-11 As/Vm brane (N) was computed with integration of transported molecules through the cell membrane over time and the cell surface: [9] where S is the surface of the cell membrane, is the time at which the quantity of transported molecules is to be determined and is Avogadro’s number. Construction of the model The simulations in this study were performed using the COMSOL 3.3 package (COMSOL Inc., Burlington, MA) based on the finite element meth­od. To construct the geometrical model, a spheri­cal cell with radius of 5.6 µm was located between two virtual electrodes. Since incorporating an ex­tremely thin membrane is problematic in meshing and solving the problem, we assigned the bound­ary condition to the membrane.38We neglected the resting transmembrane voltage. The initial intra­cellular and extracellular concentrations of probe were set to 0 and 10 mM, respectively. The diffu­sion coefficients for interactive diffusion and for an induced endocytotic-like process are considered as D0/5 and D0/10000. These two values, however, depend considerably on the type and size of the transported molecules. The necessary parameters used in our simulations are given in Table 1. Our simulation was designed to solve the Laplace equa­tion considering all related equations in this model (Eq. [2], [3], [6]) taking into account self-consistency of parameters to find the distribution of pores on the cell membrane, spatially and temporally, and all related parameters such as ITV, cell membrane conductivity and permeability. Afterwards, the up­take of the cells for each different pulse shape was obtained. All simulations were performed on a PC (2.8 GHz Pentium IV processor, 3 GB RAM) and 38 Miklavcic D and Towhidi L / Dependence of electroporation on pulse shape FIgure 3. The sine-modulated 50 kHz unipolar pulses with (A) 10% and (B) 90% modulation investigated in this paper. FIgure 4. Temporal evolution of the overall membrane conductivity during the pulse for (A) unipolar and bipolar rectangular, triangular and sinusoidal pulses and (B) 10% and 90% sine-modulated unipolar pulses. each simulation lasted 3-25 minutes depending on the considered pulse shape and number of pulses in each train of pulse. The investigated pulse shapes in this study were unipolar rectangular pulses with different rise and fall times of 2, 10 and 100 µs (Figure 1); triangular, sinusoidal and bipolar rectangular pulses (Figure 2); and also sinusoidal modulated unipolar pulses with different percentages of modulation of 10% and 90% with 50 kHz frequency (Figure 3). results Immediately after the smoothed step pulse is switched on, ITV starts to increase based on the Laplace equation and causes membrane structur­al changes initiation, which in turn results in the membrane conductivity increase according to Eq. [6]. The temporal behaviour of average conductivi­ties over the cell membrane due to application of the considered pulse shapes (Figures 1, 2 and 3) are shown in Figure 4. All pulses were considered to have a peak of 1 kV/cm and total duration of 1 ms. It can be observed in Figure 4 that the overall conductivity changes for unipolar and bipolar pulses have negligible differences. The reason for this fact is a very quick switch between positive and negative voltage, as well as ignoring the rest­ing voltage in this model. Besides, a comparison between conductivity increases due to rectangular, triangular and sinusoidal pulses was performed. Figure 4A shows that the largest and smallest changes were due to rectangular and triangular pulse shapes, respectively. Figure 4B shows that a conductivity change due to 10% modulation is higher than the 90% one but both are still lower than the rectangular pulse. The temporal behaviour of averaged cell mem­brane permeability for pulses in Figures 1 to 3 is illustrated in Figure 5. Permeability changes occur slowly. Therefore, for bipolar pulses and modu­lated pulses in which the fall and rise is very fast, there is not enough time for resealing of permeabil­ity which causes different behaviour for membrane permeability related to membrane conductivity. Based on Figure 5, we expect the order of efficien­cy of pulses of the same peak as follows: unipolar FIgure 5. Temporal evolution of the overall membrane permeability during the pulse for (A) unipolar and bipolar rectangular, triangular and sinusoidal pulses and (B) 10% and 90% sine-modulated unipolar pulses. 40 Miklavcic D and Towhidi L / Dependence of electroporation on pulse shape and bipolar rectangular, 10% modulated, sine, 90% modulated and finally bipolar triangular pulses. Note that unlike membrane conductivity in Figure 4, membrane permeability does not recover as fast after the pulse ceases. To be able to check the validity of our simulation results, the uptake enhancement of the cell was calculated for the same pulse parameters of previ­ously obtained experimental results.19The chosen parameters were 8 pulses of 100 µs duration and 1 Hz pulse repetition frequency with different pulse strengths for each pulse shape. Figure 6A shows the results of simulation for 8 pulses of bipolar rectangular, sine and triangu­lar pulses. It shows that the rectangular pulses are more efficient than sinusoidal pulses which in turn are more efficient than triangular pulses. These re­sults are in good agreement with experimentally obtained results.19 As can be seen in Figure 2A for the bipolar pulses, the pulse switch from positive to negative takes place very fast. During the switch time, the pore creation rate and, therefore, membrane con­ductivity decrease. But due to very short time of switching related to pulse duration, these changes are negligible in comparison to the conductivity change related to the unipolar pulse (Figure 4A). Consequently, the uptake due to unipolar pulses is larger than bipolar pulses but this difference is negligible and not observable (data not shown). While our simulation shows no significant differ­ence between these two pulse types, in experimen­tal results bipolar pulses are significantly more efficient than unipolar pulses. The reason for this inconsistency is most probably due to neglecting resting voltage in the simulations. In addition, Figure 6B demonstrates the com­parison between unipolar pulses of 0, 10 and 90% modulation. The results are also in good qualita­tive agreement with previously obtained experi­mental results.19The uptake enhancement results for 8 pulses of unipolar trapezoidal pulses of 1 ms duration with 2, 10 and 100 µs rise and fall times are shown in Figure 6C. It can be seen from the fig­ure that there is no significant difference between these pulses which is again in good agreement with previously published experimental results.19 Conclusions Thedescribedmodelenablesdeterminationand predictionofallelectricalanddiffusionparameters fordifferentpulseshapes.Thus,knowingelectrical anddiffusionpropertiesofthecellsandthespecific dye,optimizationoftheelectroporationprotocol canbeperformedbeforethetreatment.Ourresults showthatrectangularpulsesaremoreeffective thanthesinusoidalandtriangularpulses.Besides, ourresultsindicatethatthehigherthepercentageof unipolarpulsesmodulationwithsineshapepulses of50kHz,thelowertheuptakeenhancementofthe cells.Moreover,theriseandfalltimesofunipolar rectangularpulsesdonotsignificantlyaffectthe uptakeofmoleculesbythecells.Oursimulationre­sultsareconsistentwithexperimentalobservations. acknowledgements This research was supported by the Research Agency of the Republic of Slovenia. references 1. Airley R. Cancer Chemotherapy: Basic Science to the Clinic, West Sussex: Wiely-Blackwell; 2009. 2. Mir LM, Belehradek M, Domenge C, Orlowski S, Poddevin B, Belehradek J Jr, et al. Electrochemotherapy, a new antitumor treatment: first clinical trial. CR Acad Sci III 1991; 313: 613-8. 3. Tsong TY. Electroporation of cell membranes. Biophys J 1991; 60: 297-306. 4. Neumann E, Kakorin S, Toensing K. Fundamentals of electroporative deliv­ery of drugs and genes. Bioelectrochem Bioenergy 1999; 48: 3-16. 5. Teissie J., Eynard N, Gabriel B, Rols MP. Electropermeabilization of cell membranes. Adv Drug Del Rev 1999; 35: 3-19. 6. Neumann E, Kakorin S, Tönsing K. Fundamentals of electroporative delivery of drugs and genes. Bioelectrochem Bioenerg 1999; 48: 3-16. 7. Mir LM. Bases and rationale of the electrochemotherapy. EJC Suppl 2006; 4: 38-44. 8. Sersa G, Miklavcic D, Cemazar M, Rudolf Z, Pucihar G, Snoj M. Electrochemotherapy in treatment of tumours. EJSO 2008; 34: 232-40. 9. Zupanic A, Corovic S, Miklavcic D. Optimization of electrode position and electric pulse amplitude in electrochemotherapy. Radiol Oncol 2008; 42: 93-101. 10. Faurie C, Golzio M, Phez E, Teissie J, Rols MP. Electric field induced cell membrane permeabilization and gene transfer: theory and experiments. Eng Life Sci 2005; 5: 179-86. 11. Teissie J, Eynard N, Vernhes MC, Bénichou A, Ganeva V, Galutzov B, et al. Recent biotechnological developments of electropulsation. A prospective review. Bioelectrochem 2002; 55: 107-12. 12. Golzio M, Mazzolini L, Moller P, Rols MP, Teissie J. Inhibition of gene expres­sion in mice muscle by in vivo electrically mediated siRNA delivery. Gene Ther 2005; 12: 246-51. 13. Mesojednik S, Kamensek U, Cemazar M. Evaluation of shRNA-mediated gene silencing by electroporation in LPB fibrosarcoma cells. Radiol Oncol 2008; 42: 82-92. 14. Pliquett U, Weaver JC. Feasibility of an electrode-reservoir device for transdermal drug delivery by noninvasive skin electroporation. IEEE Trans Biomed Eng 2007; 54: 536-8. 15. Gothelf A, Mir LM, Gehl J. Electrochemotherapy: results of cancer treat­ment using enhanced delivery of bleomycin by electroporation, Cancer Treat Rev 2003; 29: 371-8. 16. Pavselj N, Miklavcic D. Numerical modeling in electroporation-based bio­medical applications. Radiol Oncol 2008; 42: 159-68. 17. Teissie J, Escoffre JM, Rols MP, Golzio M. Time dependence of electric field effects on cell membranes. A review for a critical selection of pulse duration for therapeutical applications. Radiol Oncol 2008; 42: 196-206. 18. Rols MP, Teissie J. Electropermeabilization of mammalian cells to macro­molecules: control by pulse duration. Biophys J 1998; 75: 1415-23. 19. Kotnik T, Pucihar G, Rebersek M, Mir LM, Miklavcic D. Role of pulse shape in cell membrane electropermeabilization. Biochim Biophys Acta 2003; 1614: 193-200. 20. Pucihar G, Mir LM, Miklavcic D. The effect of pulse repetition frequency on the uptake into electropermeabilized cells in vitro with possible applica­tions in electrochemotherapy. Bioelectrochem 2002; 57: 167-72. 21. Macek-Lebar A, Sersa G, Kranjc S, Groselj A, Miklavcic D. Optimisation of pulse parameters in vitro for in vivo electrochemotherapy. Anticancer Res 2002; 22: 1731-6. 22. Macek-Lebar A, Miklavcic D. Cell electropermeabilization to small mole­cules in vitro: control by pulse parameters. Radiol Oncol 2001; 35: 193-202. 23. DeBruin KA, KrassowskaW. Modeling Electroporation in a Single Cell. II. Effects of Ionic Concentrations. Biophys J 1999; 77: 1225-33. 24. Gowrishankar TR, Weaver JC. An approach to electrical modeling of single and multiple cells. Proc Natl Acad Sci USA 2003; 100: 3203-8. 25. Joshi RP, Hu Q, Schoenbach KH, Bebe SJ. Simulations of electroporation dy­namics and shape deformations in biological cells subjected to high voltage pulses. IEEE Trans Plasma Sci 2002; 30: 1536-46. 26. Joshi RP, Hu Q, Schoenbach KH. Modeling studies of cell response to ultrashort, high-intensity electric fields—implications for intracellular ma­nipulation. IEEE Trans Plasma Sci 2004; 32: 1677-86. 27. Neu JC, Krassowska W. Asymptotic model of electroporation. Phys Rev E 1999; 59: 3471-82. 28. Kotnik T, Bobanovic F, Miklavcic D. Sensitivity of transmembrane voltage induced by applied electric fields – a theoretical analysis. Bioelectrochem Bioenerg 1997; 43: 285-91. 29. Neumann E, Toensing K, Kakorin S, Budde P, Frey J. Mechanism of electro-porative dye uptake by mouse B cells. Biophys J 1998; 74: 98-108. 30. Böckmann RA, Groot BL, Kakorin S, Neumann E, Grubmüller H. Kinetics, statistics, and energetics of lipid membrane electroporation studied by molecular dynamics simulations. Biophys J 2008; 95: 1837-50. 31. Kakorin S, Neumann E. Ionic conductivity of electroporated lipid bilayer membranes. Bioelectrochem 2002; 56: 163-6. 32. Pavlin M, Leben V, Miklavcic D. Electroporation in dense cell suspension – Theoretical and experimental analysis of ion diffusion and cell permeabi­lization. Biochim Biophys Acta 2007; 1770: 12-23. 33. Schmeer M, Seipp T, Pliquett U, Kakorin S, Neumann E. Mechanism for the conductivity changes caused by membrane electroporation of CHO cell – pellets. Phy Chem Chem Phys 2004; 6: 5564-74. 34. Glogauer M, Lee W, McCulloch CA. Induced endocytosis in human fibrob­lasts by electrical fields. Exp Cell Res 1993; 208: 232-40. 35. Rols MP, Femenia P, Teissié J. Long-lived macropinocytosis takes place in electropermeabilized mammalian cells. Biochem Biophys Res Commun 1995; 208: 26-38. 36. Zimmermann U, Schnettler R, Klöck G, Watzka H, Donath E, Glaser RW. Mechanisms of electrostimulated uptake of macromolecules into living cells. Naturwissenschaften 1990; 77: 543-5. 37. Puc M, Kotnik T, Mir LM, Miklavcic D. Quantitative model of small mol­ecules uptake after in vitro cell electropermeabilization. Bioelectrochem 2003; 60: 1-10. 38. Pucihar G, Kotnik T, Valic B, Miklavcic D. Numerical determination of trans-membrane voltage induced on irregularly shaped cells. Annals Biomed Eng 2006; 34: 642-52. 39. Kotnik T, Miklavcic D, Slivnik T. Time course of transmembrane voltage induced by time-varying electric fields – a method for theoretical analysis and its application. Bioelectrochem Bioenerg 1998; 45: 3-16. 40. Pucihar G, Kotnik T, Miklavcic D, Teissie J. Kinetics of transmembrane trans­port of small molecules into electropermeabilized cells. Biophys J 2008; 95: 2837-48. 41. Gowrishankar TR, Pliquett U, Lee RC. Dynamics of membrane sealing in transient electropermeabilization of skeletal muscle membranes. Ann NY Acad Sci 1999; 888: 195-210. 42. Hibino M, Itoh H, Kinosita K. Time courses of cell electroporation as re­vealed by submicrosecond imaging of transmembrane potential. Biophys J 1993; 64:1789-800. 43. Chernomordik LV, Sukharev SI, Popov SV, Pastushenko VF, Sokirko AV, Abidor IG, et al. The electrical breakdown of cell and lipid membranes: the similarity of phenomenologies. Biochim Biophys Acta 1987; 902: 360-73. research article Development of human cell biosensor system for genotoxicity detection based on DNA damage-induced gene expression Valerija Zager1, Maja Cemazar1, Irena Hreljac2, Tamara T. Lah2, Gregor Sersa1, Metka Filipic2 1 Institute of Oncology Ljubljana, Department of Radiotherapy and Department of Experimental Oncology, Ljubljana, Slovenia 2 National Institute of Biology, Department for Genetic Toxicology and Cancer Biology, Ljubljana, Slovenia Received 4 February 2010 Accepted 28 February 2010 Correspondence to: Prof. Metka Filipic, Ph.D., National Institute of Biology, Department for Genetic Toxicology and Cancer Biology, Vecna pot 101, SI-1000 Ljubljana, Slovenia. E-mail: metka.filipic@nib.si Disclosure: No potential conflicts of interest were disclosed. Background. Human exposure to genotoxic agents in the environment and everyday life represents a serious health threat. Fast and reliable assessment of genotoxicity of chemicals is of main importance in the fields of new chemicals and drug development as well as in environmental monitoring. The tumor suppressor gene p21, the major down­stream target gene of activated p53 which is responsible for cell cycle arrest following DNA damage, has been shown to be specifically up-regulated by genotoxic carcinogens. The aim of our study was to develop a human cell-based biosensor system for simple and fast detection of genotoxic agents. Methods. Metabolically active HepG2 human hepatoma cells were transfected with plasmid encoding Enhanced Green Fluorescent Protein (EGFP) under the control of the p21 promoter (p21HepG2GFP). DNA damage was induced by genotoxic agents with known mechanisms of action. The increase in fluorescence intensity, due to p21 mediated EGFP expression, was measured with a fluorescence microplate reader. The viability of treated cells was determined by the colorimetric MTS assay. Results. The directly acting alkylating agent methylmethane sulphonate (MMS) showed significant increase in EGFP production after 48 h at 20 µg/mL. The indirectly acting carcinogen benzo(a)pyren (BaP) and the cross-linking agent cisplatin (CisPt) induced a dose-dependent increase in EGFP fluorescence, which was already significant at concen­trations 0.13 µg/mL and 0.41 µg/mL, respectively. Vinblastine (VLB), a spindle poison that does not induce direct DNA damage, induced only a small increase in EGFP fluorescence intensity after 24 h at the lowest concentration (0.1 µg/ mL), while exposure to higher concentrations was associated with significantly reduced cell viability. Conclusions. The results of our study demonstrated that this novel assay based on the stably transformed cell line p21HepG2GFP can be used as a fast and simple biosensor system for detection of genetic damage caused by chemical agents. Key words: HepG2 cells; biosensor system; green fluorescent protein; reporter gene assay; genotoxicity; p21 promoter Introduction Genotoxicity data play an important role in eval­uating health hazards associated with exposure of humans and living organisms to chemical substances. Genotoxicity assays are needed for screening compounds that are candidate drugs, food additives, or cosmetics to assess whether the compound of interest induces DNA damage. The methods for detecting genotoxic agents are also needed to monitor contamination of water sup­plies with genotoxic pollutants. In addition, geno-toxicity screening should be introduced to monitor environmental pollution through industrial and municipal waste disposal. Regulatory require­ments for genotoxicity testing of chemicals and products such as pharmaceuticals, pesticides, food additives, and cosmetics rely on a battery of geno-toxicity tests, which generally consist of an in vitrotest for gene mutations in bacteria and mammalian cells, an in vitrotest for chromosomal damage and an in vivotest for chromosomal damage in rodent hematopoetic cells.1However these same methods are unsatisfactory for rapid screening for several reasons: testing can take many weeks, when it is desirable to obtain genotoxic data in a shorter time frame, or large quantities of a tested compound are needed, when only limited quantities are available, such as during drug development or in environ­mental monitoring when concentrated samples are tested. Here we have developed a method suitable for primary genotoxicity screening. Genotoxic agents cause different types of dam­age to the DNA molecule. To counteract the con­sequences of DNA damage, cells have evolved complex defense mechanisms resulting in cell cycle arrest, DNA damage repair and apoptosis, which positively contribute to genomic stability. In bac­teria, DNA damage or inhibition of its replication invokes a well-characterized SOS response with the induction of about 20 different genes.2An even larger number of genes are involved in the cel­lular response to DNA damage in yeasts3, and in mammalian cells.4Alteration in expression of these genes can be used as a surrogate for early detec­tion and quantification of DNA damage caused by genotoxic agents. Reporter gene expression sys­tems that measure changes in expression of DNA damage response-associated genes as the markers of DNA damage have been shown to be suitable as high-throughput screens for genotoxicity. The most widely used are bacterial systems in which genotoxic effects are identified based on the chang­es in expression of SOS response genes.5-6Recently, yeast Saccharomyces cerevisiaeDNA reporter assays in which the RAD54promoter is fused to green fluorescent protein (GFP)7and RAD51 promoter fused to Renilla luciferase8, have been developed. In mammalian cells, the most prominent path­way of cellular response to DNA damage is acti­vation of the tumor suppressor and transcription factor p53 through phosphorylation by DNA damage-responsive kinases.9Activated p53 then induces the expression of genes involved in DNA repair, cell cycle arrest, or apoptosis.10The cyclin-dependent kinase 1A (CDKN1A) inhibitor p21(Waf1/Cip1) is the major downstream target gene of activated p53 and is responsible for causing cell cycle arrest following DNA damage.11The acti­vated p53 protein directly stimulates expression of p21which, through its negative effect on vari­ous CDKs, inhibits both the G1 to S and the G2 to mitosis transition.12In addition, by binding to the proliferating cell nuclear antigen (PCNA), p21 in­terferes with PCNA-dependent DNA polymerase activity, thereby inhibiting DNA replication and modulating various PCNA-dependent DNA repair processes.13Up-regulation of p21expression upon exposure to irradiation or genotoxic chemicals has been reported in several in vitroand in vivostud­ ies.14-17 Here we describe a new genotoxicity test sys­tem based on a p21-dependent GFP reporter gene assay with stably transformed human hepatoma HepG2 cells. The HepG2 cells were chosen because of their human origin and their retained activities of xenobiotic-metabolizing enzymes, which make them a better model for reflecting the processes in an intact liver than other in vitrotest systems.18In addition, HepG2 cells express wild-type tumor suppressor p5319, making them an appropriate model for development of the test system based on the p53-mediated DNA damage response. The re­sults showed that this test could be used for a high throughput screening for genotoxic agents. Materials and methods Chemicals and reagents Methyl methane sulphonate (MMS), benzo[a]pyrene (BaP) and dimethyl sulphoxide (DMSO) were purchased from Sigma, St. Louis, USA). Cisplatin (CisPt) was obtained from Medac, Hamburg, Germany, and vinblastine sulphate (VLB) from Lilly France S.A., Fagersheim, France. Cell line The human hepatoma HepG2 cell line was ob­tained from ECACC (Wiltshire, UK), and was grown in minimum essential medium (MEM, ad­vanced, GIBCO, Invitrogen, Paisley, UK) without phenol red supplemented with 10% heat inacti­vated fetal calf serum (FCS, SIGMA, St. Louis, MO, USA). Cells were routinely subcultured twice per week and were maintained in a humidified atmos­phere with 5% CO2 at 37°C. Construction of plasmids The plasmid pEGFP-N1, encoding Enhanced Green Fluorescent Protein (EGFP) controlled by the CMV promoter (Clontech, Basingstoke, UK)was used as a source of the coding sequence of the EGFP gene. 44 Zager V et al. / Biosensor system for genotoxicity FIGURE 1. pp21-EGFP plasmid (A) and confirmation of successful construc­tion of the plasmid (B). The identity of the plasmid was confirmed with SaII and EcoRI restriction. Sample 1 is WWP-Luc from which the p21 promoter (marked yellow) was isolated. Sample 2 is linearized pEGFP-N1 without CMV plasmid from which CMV was cut out with the same restriction en­zymes to form blunt ends. Sample 3 is pp21-EGFP plasmid resulting from ligation of a 2.4 kB p21 promoter from WWP-Luc and sample 2 restricted with SaII and EcoRI. The source of the coding sequence of the p21pro­moter was the WWP-LUC plasmid, which was a gift from Prof. Bert Vogelstein (Johns Hopkins Oncology Center, Baltimore, Maryland, USA). The construction of a recombinant vector containing the p21promoter reporter cassette and EGFP was done in several steps using the Clontech pEGFP­N1 plasmid as a backbone and standard molecu­lar biology techniques of restriction and ligation. In addition, the gene for neomycin resistance was included into the plasmid, which enabled the isola­tion of HepG2 cells with stable expression of the re­porter gene under pressure of Geneticin® (neomy­cin, GIBCO). The constructed plasmid pp21-EGFP was cloned into E. coli(strain DH5a, Invitrogen, UK), andisolated using the Qiagen Maxi Endo-Free kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Purified plasmid DNA was subjected to quality control and quantity determinations, performed by agarose gel electro­phoresis and by means of spectrophotometry. Transfection of HepG2 cell line The HepG2 cells were transfected with the pp21­EGFP plasmid using electroporation as described.2040 µl of cell suspension (2.5 x107cells/ml) were mixed with 10 µg of plasmid DNA and placed between two flat parallel stainless steel electrodes with a 2-mm gap and subjected to 8 square-wave shaped electric pulses of 5 ms duration, repetition frequency 1 Hz. Different electric field intensities were tested: 400 V/cm, 600 V/cm, 700 V/cm, 800 V/cm and 1000 V/cm. The electric pulses were gen­erated by an electroporator (GT-1, electropora-tor, Faculty of Electrical Engineering, University of Ljubljana, Slovenia). After exposure to electric pulses, the cells were incubated for 5 min at room temperature. Thereafter, cells were maintained in non-selective medium for 1-2 days after trans-fection. The selection of stably transfected clones was performed by culturing the cells in medium containing 1 mg/ml Geneticin®. Cultivation in the selective medium was continued for 2-3 weeks. During this period, the cells without plasmid died while the cells containing stably incorporated plas­mid were able to replicate and form colonies. Separate colonies were picked and transferred into wells of 96-well microtiter plates and cultivat­ed under pressure of 0.5 mg/ml Geneticin®. After reaching a sufficient number, the cells were trans­ferred to larger plates for further propagation to obtain a sufficient number of cells for further selec­tion of the most responsive clones. The clones with visible morphological and/or replication changes were discharged. Cell treatment with model genotoxic agents and EGFP measurement Model genotoxic agents with known mecha­nisms of action were used to test and validate the cell biosensor system. Stock solutions were pre­pared prior to testing: MMS, and CisPt were dis­solved in distilled water at concentrations 50 mg/mL (454 mM) and 2 mg/mL (6.7 mM), respectively. BaP was dissolved in DMSO at a concentration 2.52 mg/mL (10 mM) and VLB in 0.9% NaCl at a concentration 1 mg/mL (1.1 mM). Further dilutions were made in cell culture media. A suspension of exponentially growing p21­HepG2GFP cells (3x105cells/mL) in minimum es­sential medium without phenol red with 10% fetal calf serum was distributed in 3 mL aliquots to plas­tic test tubes. 30 µL of test chemical of appropriate concentration (100-fold higher concentrations than final treatment concentrations) or 30 µL of vehicle for controls were added to each tube . The follow­ing final concentrations were used: MMS: 5, 10, 20, 40, 50 mg/mL; CisPt: 0.4125, 0.825, 1.65, 3.3, 6.6 mg/mL; BaP: 0.05, 0.13, 0.25, 0.5, 1.26 µg/mL, and VBL 0.1, 0.5, 1.0, 2.5, 5.0 mg/mL. For the EGFP fluores­cence measurements, 100 µL aliquots from each tube were distributed to 6 wells of 96-well black microtiter plates with a clear bottom (Greiner BIO­ONE, Nuernberg, Germany). The plates were in­cubated at 37oC, 5% CO2for 7 days, and the EGFP fluorescence was determined after 24, 48, 72, 120 and 168 h. The intensity of EGFP fluorescence was measured at 485 nm excitation and 535 nm emis­sion wavelengths with a fluorescence microplate reader (Tecan Infinite 200). The experiments were repeated three times. From fluorescence intensity measurements, a relative EGFP induction ratio was calculated. Fluorescence intensity of the treated cells was di­vided by the fluorescence intensity of control cells and normalized to the relative cells viability deter­mined with the MTS assay. Determination of cell viability (MTS assay) The cell viability was determined by the colometric (3-(4.5-dimethylthiazol-2-yl)-5-(3-car­boxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetra-MTS solution were added to each well of 96-well zolium, inner salt) MTS assay with the CellTiter microtiter plates and incubated for 2 h in a humidi­96 Aqueous One Solution Cell Proliferation Assay fied atmosphere with 5% CO2at 37°C. After the (Promega, Madison, USA) according to the manu-incubation with MTS, the microtiter plates were facturer’s instructions. The 100 µL aliquots from shaken for 30 s and the absorbance of the resulting each test tube of treated or control cells were dis-solution was measured at 492 nm using a Labtec tributed into 4 wells of normal 96 well microtiter HT2 microplate reader (Anthos, Wals, Austria). plates and incubated for 24, 48, 72 120 or 168 h. For Relative survival of cells was calculated by divid-each of the 5 time point measurements, a separate ing the absorbance of the treated cells with the ab-microtiter plate was prepared. At the end of the sorbance of the control cells. The experiments were incubation period with chemical agents, 20 µL of performed in quadruplets and repeated 3-times. 46 Zager V et al. / Biosensor system for genotoxicity Statistical analysis Statistical analysis was performed using SigmaStat software (Systat Software, Inc., Richmond CA). All data were first tested for normality with the Kolmorogov-Smirnov normality test. Significance tests were carried out using analysis of variance (ANOVA) and two-tailed Student’s t-test. Values of p<0.05 were considered significant. Data were presented as the arithmetic mean (AM) ± standard deviation of the mean (SD). Results Construction of reporter gene plasmid and stably transformed HepG2 cells For this genotoxicity screening system, a plasmid pp21-EGFP with the p21promoter inserted in front of the EGFP reporter gene was constructed (Figure 1A). Successful construction and isolation of the pp21-EGFP plasmid was confirmed with restric­tion analysis (Figure 1B). The pp21-EGFP plasmid was then transfected to HepG2 cells. In the final step, HepG2 cell clones expressing low basal and high inducible EGFP expression were isolated. For the isolation of DNA damage-responsive clones we used MMS. After measuring the basal and MMS induced EGFP levels in 36 independent clones the one with the highest inducible and the lowest basal level of EGFP expression was selected for further propagation and characterization and for the experiments with the known model geno-toxic compounds. The clone was named p21HepG­2GFP. Microscopic observations of p21HepG2GFP cells demonstrated a clear increase of EGFP fluo­rescence intensity induced by 50 µg/mL MMS after 48 h exposure (Figure 2). Cell viability as an internal standard Sinceitisknownthatgenotoxicchemicalsare toxicatcertainconcentrationsandthussuppress cellgrowthduringexposurewhichwascontin­uedforupto7days,itwasnecessarytonormal­izetheobservedlevelofEGFPtothenumberof FIGURE 4. Dose- and time-dependent induction of EGFP expression in viablecells.TheinductionofEGFPfluorescence p21HepG2GFP cells treated with graded doses of MMS (A), BaP (B), wasmeasuredafter24,48,72,120and168hon CisPt (C) and VBL (D) after 24, 48 and 72 h of exposure. The response is thesamepopulation,whilethiswasnotpossible presented as the relative EGFP induction ratio which is the ratio between fordeterminationofcellviability,sincenoappro- EGFP fluorescence of the treated cells and the background fluorescence of control cells normalized to the relative cell viability. The values represent priatemethodthatwouldallowfordetermination means of four independent experiments ± SD; *p<0.05 ofcellviabilitywithoutterminationofcellcultur­ ingisavailable.TheMTSassaythatmeasuresthe conversionofMTStotheformazanproductby dehydrogenaseenzymesoftheintactmitochon­driaoflivingcellscorrelateswiththenumberof viablecells.Wethereforeusedthisassaytoindi­rectlydeterminetherelativechangesincellnum­bersduringtheexposuretotestedchemicals.For eachtreatment,wepreparedfiveplatesforthe measurementofcellviability(oneplateforeach timepoint)inparalleltotheplateforEGFPfluo­rescencemeasurements.Thecorrelationanalysis oftheproliferationofp21HepG2GFPcellsshowed thatabsorbanceoftheformedformazanproduct correlatedtocellproliferation(r=0.94)(Figure3). Thedataalsoindicatethatduringtheexponential growthphasethedoublingtimeofthep21HepG­2GFPcellsisabout48h.Ateachtimepoint,the relativecellviabilitycomparedtonon-treatedcon­trolcellswascalculatedandthefactorwasused fornormalizationoftherelativeEGFPinduction ratiotothenumberofviablecells.Areductionof relativecellviabilitybymorethan30%(reduction factor0.7)wasconsideredascytotoxic. Responses of p21HepG2GFP cells to exposure to model genotoxic agents To demonstrate the sensitivity of this bioassay for detection of genotoxic agents, we tested several ge­notoxic agents with known mechanisms of action. To determine the optimal exposure conditions, a time and dose dependence of p21-dependent EGFP fluorescence induced by model genotoxic agents was investigated. Methyl methane sulphonate (MMS), a direct acting genotoxic agent that induces alkylation of DNA bases, induced a statistically significant increase in EGFP fluorescence after 24 h to 50 mg/mL and after 48 h exposure to 20, 40 and 50 µg/mL (Figure 4A, Table 1). The MMS-induced increase of EGFP fluorescence was time- and dose-dependent, which is clearly reflected in the increasing values of rela­tive EGFP induction ratio (Figure 4A, Table 1). After 120 and 168 h exposure, a significant increase in EGFP fluorescence associated with the increase in relative EGFP induction ratio was observed at all concentrations (Table 1). The parallel measure­ment of cell viability during the exposure to MMS showed that it was not significantly affected dur­ing the initial 72 h of exposure, while after 120 and 168 h it was reduced by more than 30% compared to non-treated control cells (Table 1). Benzo[a]pyrene(BaP) is a mutagenic and carci­nogenic indirectly-acting genotoxic agent which forms BaP diolepoxide (BPDE)-DNA adducts af­ter metabolic activation. BaP induced a significant dose-dependent increase in EGFP fluorescence at all exposure times and all concentrations except the lowest one (0.05 µg/mL). However, the relative EGFP induction ratio did not increase with pro­longed exposure indicating that the EGFP induc­tion reached a plateau (Figure 4B, Table 1). BaP did not significantly reduce the cell viability during the exposure up to 72 h (Table 1), while with further exposure the viability was reduced by more than 30% at all doses of BaP (Table 1). Cisplatin (CisPt),a well known chemotherapeu­tic, is a directly-acting genotoxic agent that induces alkylation of DNA and DNA cross-links. CisPt in­duced significant increase of EGFP fluorescence already after 24 h exposure at all concentrations. With further exposure, the relative EGFP induction ratio tended to increase with the time of exposure (Figure 4C, Table 1). In cells exposed to 3.3 µg/mL CisPt, the relative EGFP induction ratio increased from 1.40, determined after 24 h, to 2.83 deter­mined after 72 h of exposure (Figure 4C, Table 1). CisPt did not reduce cell viability after 24 h of ex­posure. After 48 and 72 h exposure, the viability of the cells was significantly reduced at the two high­est concentrations (3.3 and 6.6 mg/mL) while after 120 and 168 h exposure, CisPt reduced cell viabil­ity by more than 30% at all tested concentrations (Table 1) Vinblastine (VLB)is a chemotherapeutic that does not induce DNA damage but induces dis­turbances in cell replication due to its interference with mitotic spindle formation. This compound induced significant increase of EGFP fluorescence after 24 h exposure to all concentrations, except the highest (5.0 mg/mL). After 48 h exposure, a signifi­cant increase of EGFP fluorescence was detected at the lowest three concentrations (0.1, 0.5 and 1.0 µg/mL), while at higher concentrations and with prolonged exposure the EGFP fluorescence intensity was reduced (Figure 4D, Table 1). The vi­ability measurements showed that VBL was highly cytotoxic. Although after 24 and 48 h exposure cell viability was not reduced by more than 30%, except at the highest concentration, after prolonged expo­sure it rapidly decreased. After 72 h exposure the viability was reduced by more than 40% at all con­centrations and after 168 h exposure it decreased by more than 90% compared to the viability of non-treated control cells (Table 1). TABLE 1. Cell viability and induction of EGFP fluorescence in p21 HepG2 GFP cells exposed to Methylmethane sulphonate (MMS), Benzo[a]pyrene (BaP), Cisplatin (CisPt) and Vinblastine (VLB) for 24, 48, 72, 120 and 168 h. MMS 24 hours 48hours 72hours 120hours 168hours Conc. µg/ml Viab. (% )± SD a GFP int. ± SD b GFP ind.± SD c Viab. (% )± SD a GFP int. ± SD b GFP ind.± SDc Viab. (% )± SD a GFP int. ± SD b GFP ind.± SDc Viab. (% ) ± SD a GFP int. ± SDb GFP ind.± SD c Viab. (% )± SD a GFP int. ± SDb GFP ind.± SD c 0 100 ± 0,05 8,8 ± 0,87 1,00 ± 0,10 100 ± 0,02 12,8 ± 1,29 1,00 ± 0,10 100 ± 0,03 15,4 ± 1,58 1,00 ± 0,10 100 ± 0,06 10,5 ± 1,42 1,00 ± 0,14 100 ± 0,06 13,4 ± 1,94 1,00 ± 0,14 5,00 102 ± 0,04 9,4 ± 0,98 1,04 ± 0,10 117 ± 0,04 13,8 ± 1,79 0,92 ± 0,12 91 ± 0,04 16,1 ± 1,34 1,14 ± 0,09 12,9 ± 1,03 16,7 ± 1,11 1,85 ± 0,11 10,00 108 ± 0,03 10,6 ± 1,17 1,11 ± 0,12 106 ± 0,02 15,8 ± 1,48 1,16 ± 0,11 104 ± 0,03 18,7 ± 2,08 1,16 ± 0,12 18,5 ± 2,16 20,6 ± 1,13 2,43 ± 0,11 20,00 104 ± 0,04 10,8 ± 1,06 1,17 ± 0,11 109 ± 0,02 18,6 ± 1,47 1,33 ± 0,11 107 ± 0,03 22,6 ± 1,58 23,3 ± 0,87 25,1 ± 2,47 3,13 ± 0,16 40,00 110 ± 0,04 10,6 ± 1,13 1,09 ± 0,11 110 ± 0,02 20,5 ± 1,07 1,45 ± 0,09 102 ± 0,04 26,3 ± 1,58 24,8 ± 1,56 25,3 ± 2,17 5,03 ± 0,15 50,00 88 ± 0,04 10,4 ± 0,75 1,33 ± 0,09 104 ± 0,02 20,1 ± 1,35 1,51 ± 0,10 94 ± 0,03 25,5 ± 1,78 23,4 ± 0,81 24,5 ± 0,81 6,74 ± 0,10 CisPt 24 hours 48hours 72hours 120hours 168hours Conc. µg/ml Viab. (% ) ± SD a GFP int. ± SD b GFP ind.± SD c Viab. (% )± SD a GFP int. ± SD b GFP ind.± SD c Viab. (% ) ± SD a GFP ind.± SD c Viab. (% ) ± SD a GFP int. ± SD b GFP ind.± SD c Viab. (% )± SD a GFP int. ± SD b GFP ind.± SD c 0 100 ± 0,01 13,0 ± 0,45 1,00 ± 0,03 100 ± 0,00 18,2 ± 0,78 1,00 ± 0,04 100 1,00 ± 0,06 100 ± 0,03 18,5 ± 1,06 1,00 ± 0,06 100 ± 0,10 20,8 ± 1,17 1,00 ± 0,06 0,10 94 ± 0,01 26,3 ± 1,89 2,15 ± 0,09 81 ± 0,01 25,9 ± 2,01 1,76 ± 0,08 57 ± 0,01 2,41 ± 0,07 18 ± 0,02 18,2 ± 1,12 5,46 ± 0,06 5 ± 0,01 17,2 ± 1,22 16,55 ± 0,05 0,50 95 ± 0,01 24,3 ± 2,87 1,96 ± 0,13 80 ± 0,01 22,3 ± 2,27 1,54 ± 0,08 58 ± 0,01 1,71 ± 0,07 14 ± 0,00 12,2 ± 0,92 4,70 ± 0,05 4 ± 0,00 11,2 ± 1,22 13,45 ± 0,05 1,00 90 ± 0,00 19,6 ± 0,49 1,67 ± 0,04 85 ± 0,00 18,0 ± 0,75 1,17 ± 0,04 56 ± 0,01 1,28 ± 0,04 16 ± 0,02 9,6 ± 1,01 3,24 ± 0,06 5 ± 0,01 8,3 ± 0,57 8,03 ± 0,04 2,50 85 ± 0,01 17,2 ± 0,82 1,55 ± 0,05 77 ± 0,01 15,0 ± 1,08 1,07 ± 0,05 52 ± 0,00 1,08 ± 0,04 16 ± 0,01 7,3 ± 0,78 2,45 ± 0,05 5 ± 0,01 7,3 ± 0,80 7,07 ± 0,05 5,00 78 ± 0,04 10,4 ± 0,76 1,03 ± 0,05 70 ± 0,01 7,9 ± 1,07 0,62 ± 0,05 42 ± 0,01 1,10 ± 0,05 15 ± 0,00 3,3 ± 0,56 1,17 ± 0,04 6 ± 0,01 3,7 ± 0,67 2,95 ± 0,04 GFP int. ± SD b ± 0,04 20,8 ± 1,27 28,7 ± 1,50 20,7 ± 1,50 14,9 ± 0,58 11,7 ± 0,47 9,6 ± 0,75 a Cell viability was measured with the MTS assay and is expressed as % of viable p21 HepG2 GFP cells treated with MMS, BaP, CisPt and VLB compared to control, non-treated cells. b Intensity of EGFP fluorescence measured at 485 nm excitation and 535 nm emission wavelengths.Relative EGFP induction expressed as the ratio between the EGFP fluorescence intensity of the treated cells and non-treted control cells, normalized to cell viability.Light grey areas represent significantly different values compared to control (P<0.001)Dark grey areas represent values of cell viability below 70% of control. Zager V et al. / Biosensor system for genotoxicity Discussion We developed a novel microplate genotoxicity as­say test system using EGFP as the reporter that enables simple and rapid detection of genotoxic agents. The assay is based on a p21HepG2GFP cell line that contain the EGFP reporter under the con­trol of the p21promoter. In response to DNA dam­age, the transcription of the p21promoter is acti­vated leading to concurrent accumulation of EGFP that is detected in intact cells with the fluorescence microplate reader. Several reporter genotoxicity assays using mam­malian cells and DNA damage responsive genes as the biomarkers of genotoxic injury have been de­scribed. Todd et al.21were the first who exploited DNA damage responsive genes: p53R2, GADD45aand GADD153for construction of a chlorampheni-col acetyl transferase (CAT) reporter that was sta­bly integrated into HepG2 cells. However, there is very little data published from this assay. The p53R2, one of the p53 target genes that encode a subunit of ribonucleotide reductase, which is ex­pressed mainly in response to DNA damage22, 23, has been used more recently for construction of a reporter assay with MCF7 and HepG2 cells us­ing luciferase as the reporter gene.24,25The growth arrest and DNA damage (GADD)-inducible gene family is another group of target genes regulated by p53 that are expressed in response to various environmental stresses including DNA damage. In response to DNA-damage GADDgenes induce arrest in cell cycle progression at G1/S or G2/M checkpoints.26Hastwell et al.27developed an assay that exploits a reporter system in which the expres­sion of EGFP is controlled by regulatory elements of the GADD45agene hosted in the p53-competent human lymphoblastoid TK6 cell line. A thorough validation of this assay showed its high sensitiv­ity and specificity.28The assay is commercially available as GreenScreen HC assay provided by Gentronics Ltd (UK). Recently Zhang et al.29devel­oped a stably transfected HepG2 cell line contain­ing GADD153promoter regions coupled to the lu­ciferase reporter gene. p21belongs to p53mediated DNA damage re­sponsive genes that has not been previously used as an indicator of genotoxic injury. For the con­struction of our reporter system, we selected the p21promoter to drive EGFP expression since re­cently Ellinger-Ziegelbaure et al.17reported that p21was up-regulated only by genotoxic carcino­gens in the liver of rats exposed to genotoxic and non-genotoxic carcinogens. The GADD45agene was up-regulated by both, genotoxic and non-ge­notoxic carcinogens. Therefore, it could be that our test system will allow for discrimination of the two types of carcinogens. We evaluated the sensitivity of the assay and es­tablished optimal exposure conditions for induced EGFP fluorescence data collection using four model genotoxic agents with known mechanisms of action. The results showed that the optimal ex­posure time for detection of EGFP expression is 48 h. Although the EGFP fluorescence in cells ex­posed to MMS, BaP and CisPt increased with the time of exposure, the lowest effective concentration (LOEC) at which a significant increase in EGFP fluorescence was observed did not change. Longer exposures lead to reduced cell viability, resulting either from cytotoxicity or inhibition of cell divi­sion that may interfere with the reliability of EGFP fluorescence detection and calculation of the rela­tive EGFP induction ratio as a quantitative meas­ure of genotoxic activity. When measurements of EGFP fluorescence are performed in wells with a very different number of control vs. treated cells, interference with the optical measurements due to changes in the background reflectance and absorb-ance of the microplate is possible. The half-life of EGFP in mammalian cells has been reported to be in the range of 24 – 48 h.30,31As the relative EGFP induction ratio is normalized to the cell viability, which was significantly reduced after prolonged exposure, normalization might give unreliable high values of the EGFP induction ratio due to the accumulated EGFP. The reason for unreliable results can also be cytotoxicity per se. The break­down of cell integrity can lead to non-specific DNA damage and thus to p21 activation, which does not lead to genetic consequence if cells are dying or dead. Therefore, only the EGFP measurements at which cell viability was not reduced by more than 20% were considered as relevant for genotoxicity evaluation while reduction of cell viability by more than 30% was considered as cytotoxic. The alkylating agent MMS is a known mutagen and rodent carcinogen.32,33Recently, it has been re­ported that MMS induces phosphorylation of the p53 protein and increases its DNA-binding proper­ties to cause an increased expression of p21.34MMS induced a dose- dependent increase of EGFP fluo­rescence with a LOEC of 20 µg/mL. The sensitivity of our system for MMS genotoxicity detection is similar to that of the GreenScreen HC assay with the GADD45apromoter fused to an EGFP gene, in which the LOEC was 25 µg/mL27, and to that with the p53R2promoter fused to the luciferase reporter 50 Zager V et al. / Biosensor system for genotoxicity in MCF-7 cells in which the LOEC was around 10 µg/mL.25 BaPisanindirectly-actinggenotoxiccarcinogen thatismetabolizedbycytochromeP450enzymes todiolepoxideBPDE,whichbindscovalentlyto guaninebases.35ExposuretoBaPisknowntoin­duceactivationofthep53proteinanditsdown­ 36,37 streamregulatedgenesincludingp21.The LOECforBaPwasat0.13µg/mL(0.5µM),and atthehighesttestedconcentration1.26µg/mL(5 µM)therelativeEGFPinductionratiowas8.54 after24hexposure.HepG2cellstransfectedwith GADD153fusedtoluciferaseweresignificantly moresensitiveforBaPgenotoxicitydetection;the LOECwas0.0025µg/mL(10nM).29Theauthors ascribedhighsensitivityoftheirassaycompared tootherreportersystemstothesensitivityoflu­ciferase,whichseemstobehigherthanthatof EGFP.29InMCF-7cellstransfectedwithp532Rcoupledtotheluciferasereportergene,theLOEC forBaPwas0.26µg/mLwhentestedwithoutmet­abolicactivationand0.12µg/mLinthepresence ofmetabolicactivation.24Thelowersensitivityof MCF-7cellsintheabsenceofmetabolicactivation comparedtoHepG2cellscanbeascribedtotheir lowerexpressionofmetabolicenzymes.Whenus­ingmetabolicallyincompetentcells,theindirect-ly-actinggenotoxicagentshavetobetestedinthe presenceofexogenousmetabolicactivation,usu­allyS9liverextracts.However,S9islight-absorb­ingandfluorescentthatcanconfoundspectropho­tometricmeasurementsoffluorescence,whichis themainlimitationofreportersystemsbasedon EGFP.FortheGreenScreenHCtestsystem,apro­tocolbasedonflowcytometry(FCM)hasbeen developedforthedetectionofindirectly-acting genotoxicchemicals,andtheLOECforBaPwas 1.25µg/mL.38Thus,ourtestsystemwithHepG2 cellsrepresentsgreatpotentialfordirectdetection oftheindirectly-actinggenotoxicagents. ADNAcross-linkerCisPtinducesbulkyle­sions,whichblockDNAtranscriptioninvitro.39TheresponsetoCisPt-inducedDNAdamage activatesp53throughtheATR-Chk2pathway.40ThebulkyDNAdamageinducedbydifferent genotoxicchemicalssuchasDNAcross-linkers orBaParerepairedbynucleotideexcisionrepair (NER).Thestudiesshowedthattriggeringofthe signaltransductioncascadethatleadstophospho­rylationofp53orChk1requiresrecognitionand processingofthelesionsbyNER.41Inp21HepG­2GFP,CisPtinducedadose-dependentinduction ofEGFPfluorescence.TheLOECwas0.41µg/mL, whichismoresensitivecomparedtotheresponse observedwiththeGreenScreenHCassayinwhich theLOECwas1µg/mL.27TheMCF-7cellscarry­ing thep53R2promoter linked to the luciferase re­porterwerelesssensitive;theLOECwasaround 10µg/mL.25 VBL belongs to spindle poisons that block po­lymerization of tubulin into microtubules and inhibit cell division without directly damaging DNA.42These chemicals induce activation of p53 and cell cycle arrest mediated by p2143, although the details of this process are not clear. VBL in­duced a significant increase of EGFP fluorescence at the lowest tested concentration of 0.1 µg/mL, which decreased at higher concentrations. VBL showed a cytostatic effect, which is reflected in rapid decrease of relative cell viability during pro­longed exposure. At all tested concentrations, the relative cell viability was reduced by 20% or more already after 48 h exposure. Therefore, only the ef­fect observed after 24 h exposure was considered. Lower induction of p21-mediated EGFP expression at higher concentrations may be explained by its toxicity. In MCF-7 cells with the p53R2-mediated luciferase reporter, VBL induced comparable cyto-toxicity and induction of the reporter gene25as we observed in our test system. VBL was highly cyto­toxic also in the GreenScreen HC test with LOEC for growth inhibition and GFP induction at 0.02 µg/mL.27 In conclusion, our study showed that the new biosensor system with the human hepatoma cell line p21HepG2GFP efficiently detects different types of genotoxic agents. Its main advantages are the use of metabolically competent human cells that allow for direct detection of indirectly-acting genotoxic chemicals and spectrofluorimetric meas­urement of reporter genes on a microplate format ensuring easy handling and rapid data acquisition. After further validation of the test system, which is currently in progress, this genotoxicity assay based on p21gene expression can become a valuable tool with potential applications in the fields of chemical and drug safety evaluation as well as for environ­mental and occupational monitoring of exposure to chemical agents. Acknowledgement The study was supported by the Slovenian Ministry of Defense projects M1-0031 and M1-0151 and the Slovenian Research Agency, Project J3-9580. References 1 Elespuru RK, Agarwal R, Atrakchi AH, Bigger CAH, Heflich RH, Jagannath DR, et al. Current and Future Application of Genetic Toxicity Assays: The Role and Value of In Vitro Mammalian Assays. Toxicol Sci 2009; 109: 172-9. 2 Sutton MD, Smith BT, Godoy VG, Walker GC. The SOS response: Recent insights into umuDC-dependent mutagenesis and DNA damage tolerance. Ann Rev Genet 2000; 34: 479-97. 3 Putnam CD, Jaehnig EJ, Kolodner RD. Perspectives on the DNA damage and replication checkpoint responses in Saccharomyces cerevisiae. DNA Repair 2009; 8: 974-82. 4 Holbrook NJ, Fornace AJ. Response to adversity -molecular control of gene activation following genotoxic stress. New Biologist 1991; 3: 825-33. 5 Quillardet P, Huisman O, Dari R, Hofnung M. SOS chromotest, a direct assay of induction of an sos function in escherichia-coli k-12 to measure genoto­xicity. P Natl Acad Sci USA 1982; 79: 5971-5. 6 Oda Y, Nakamura S, Oki I, Kato T, Shinagawa H. Evaluation of the new system (umu-test) for the detection of environmental mutagens and car­cinogens. Mutat Res 1985; 147: 219-29. 7 Walmsley RM, Billinton N, Walsh L, Barker MG, Knight AW, Cahill PA. A yeast RAD54-GFP genotoxicity assay, is effective in identifying direct acting mutagens in addition to clastogens not detected by bacterial tests. Toxicol Sci 2003; 72: 1106. 8 Liu X, Kramer JA, Swaffield JC, Hu Y, Chai G, Wilson AGE. Development of a highthroughput yeast-based assay for detection of metabolically activated genotoxins. Mutat Res-Gen Tox En 2008; 653: 63-9. 9 Zhou B-BS, Elledge SJ. The DNA damage response: putting checkpoints in perspective. Nature 2000; 408: 433-9. 10 Sionov RV, Haupt Y. The cellular response to p53: the decision between life and death. Oncogene 1999; 18: 6145-57. 11 WaldmanT,KinzlerKW,VogelsteinB.P21isnecessaryfortheP53-mediated G1 arrest in human cancer cells. Cancer Res 1995; 55: 5187-90. 12 Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature 2000; 408: 307-10. 13 Moldovan G-L, Pfander B, Jentsch S. PCNA, the maestro of the replication fork. Cell 2007; 129: 665-79. 14 Park SY, Lee SM, Ye SK, Yoon SH, Chung MH, Choi J. Benzo[a]pyrene-induced DNA damage and p53 modulation in human hepatoma HepG2 cells for the identification of potential biomarkers for PAH monitoring and risk assess­ment. Toxicol Lett 2006; 167: 27-33. 15 Zegura B, Zajc I, Lah TT, Filipic M. Patterns of microcystin-LR induced altera­tion of the expression of genes involved in response to DNA damage and apoptosis. Toxicon 2008; 51: 615-23. 16 Hreljac I, Zajc I, Lah T, Filipic M. Effects of model organophosphorous pesticides on DNA damage and proliferation of HepG2 cells. Environ Mol Mutagen 2008; 49: 360-7. 17 Ellinger-Ziegelbauer H, Stuart B, Wahle B, Bomann W, Ahr HJ. Comparison of the expression profiles induced by genotoxic and nongenotoxic carcino­gens in rat liver. Mutat Res-Gen Tox En 2005; 575: 61-84. 18 Knasmuller S, Mersch-Sundermann V, Kevekordes S, Darroudi F, Huber WW, Hoelzl C, et al. Use of human-derived liver cell lines for the detection of environmental and dietary genotoxicants; current state of knowledge. Toxicology 2004; 198: 315-28. 19 Bressac B, Galvin KM, Liang TJ, Isselbacher KJ, Wands JR, Ozturk M. Abnormal structure and expression of p53 gene in human hepatocellular-carcinoma. P Natl Acad Sci USA 1990; 87: 1973-7. 20 Mesojednik S, Kamensek U, Cemazar M. Evaluation of shRNA-mediated gene silencing by electroporation in LPB fibrosarcoma cells. Radiol Oncol 2008; 42: 82-92. 21 Todd MD, Lee MJ, Williams JL, Nalezny JM, Gee P, Benjamin MB, et al. The cat-tox (l) assay -a sensitive and specific measure of stress-induced transcription in transformed human liver-cells. Fund Appl Toxicol 1995; 28: 118-28. 22 Tanaka H, Arakawa H, Yamaguchi T, Shiraishi K, Fukuda S, Matsui K, et al. A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage. Nature 2000; 404: 42-9. 23 Guittet O, Hakansson P, Voevodskaya N, Fridd S, Graslund A, Arakawa H, et al. Mammalian p53R2 protein forms an active ribonucleotide reductase in vitro with the R1 protein, which is expressed both in resting cells in response to DNA damage and in proliferating cells. J Biol Chem 2001; 276: 40647-51. 24 Ohno K, Tanaka-Azuma Y, Yoneda Y, Yamada T. Genotoxicity test system based on p53R2 gene expression in human cells: Examination with 80 chemicals. Mutat Res-Gen Tox En 2005; 588: 47-57. 25 Ohno K, Ishihata K, Tanaka-Azuma Y, Yamada T. A genotoxicity test system based on p53R2 gene expression in human cells: Assessment of its reactiv­ity to various classes of genotoxic chemicals. Mutat Res-Gen Tox En 2008; 656: 27-35. 26 Siafakas RA, Richardson DR. Growth arrest and DNA damage-45 alpha (GADD45a). Int J BiochemCell B 2009; 41: 986-9. 27 Hastwell PW, Chai LL, Roberts KJ, Webster TW, Harvey JS, Rees RW, et al. High-specificity and high-sensitivity genotoxicity assessment in a human cell line: Validation of the GreenScreen HC GADD45a-GFP genotoxicity as­say. Mutat Res-Gen Tox En 2006; 607: 160-75. 28 Birrell L, Cahill P, Hughes C, Tate M, Walmsley RM. GADD45a-GFP GreenScreen HC assay results for the ECVAM recommended lists of ge­notoxic and non-genotoxic chemicals for assessment of new genotoxicity tests. Mutat Res-Gen Tox En 2010; 695: 87-95. 29 Zhang R, Niu YJ, Do HR, Cao XW, Shi D, Hao QL, et al. A stable and sensitive testing system for potential carcinogens based on DNA damage-induced gene expression in human HepG2 cell. Toxicol In Vitro 2009; 23: 158-65. 30 Yang TT, Cheng LZ, Kain SR. Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic Acids Res 1996; 24: 4592-3. 31 Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 1996; 173: 33-8. 32 Lawley PD. Mutagens as carcinogens -development of current concepts. Mutat Res 1989; 213: 3-25. 33 Beranek DT. Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating-agents. Mutat Res 1990; 231: 11-30. 34 Jaiswal AS, Narayan S. S(N)2 DNA-alkylating agent-induced phosphorylation of p53 and activation of p21 gene expression. Mutat Res-Fund Mol M 2002; 500: 17-30. 35 PerlowRA,KolbanovskiiA,HingertyBE,GeacintovNE,BroydeS,Scicchitano DA. DNA adducts from a tumorigenic metabolite of benzo[a]pyrene block human RNA polymerase II elongation in a sequence-and stereochemistry-dependent manner. J Mol Biol 2002; 321: 29-47. 36 Wang A, Gu J, Judson-Kremer K, Powell KL, Mistry H, Simhambhatla P, et al. Response of human mammary epithelial cells to DNA damage induced by BPDE: involvement of novel regulatory pathways. Carcinogenesis 2003; 24: 225-34. 37 Sadikovic B, Rodenhiser DI. Benzopyrene exposure disrupts DNA methyla­tion and growth dynamics in breast cancer cells. Toxicol Appl Pharm 2006; 216: 458-68. 38 Jagger C, Tate M, Cahill PA, Hughes C, Knight AW, Billinton N, et al. Assessment of the genotoxicity of S9-generated metabolites using the GreenScreen HC GADD45a-GFP assay. Mutagenesis 2009; 24: 35-50. 39 Corda Y, Job C, Anin MF, Leng M, Job D. Spectrum of DNA platinum adduct recognition by prokaryotic and eukaryotic DNA-dependent RNA-polymerases. Biochemistry 1993; 32: 8582-8. 40 Pabla N, Huang S, Mi QS, Daniel R, Dong Z. ATR-Chk2 signaling in p53 activa­tion and DNA damage response during cisplatin-induced apoptosis. J Biol Chem 2008; 283: 6572-83. 41 Marini F, Nardo T, Giannattasio M, Minuzzo M, Stefanini M, Plevani P, et al. DNA nucleotide excision repair-dependent signaling to checkpoint activa­tion. P Natl Acad Sci USA 2006; 103: 17325-30. 42 Owellen RJ, Hartke CA, Dickerson RM, Hains FO. Inhibition of tubulin­microtubule polymerization by drugs of vinca alkaloid class. Cancer Res 1976; 36: 1499-502. 43 Tishler RB, Lamppu DM, Park S, Price BD. Microtubule-active drugs taxol, vinblastine, and nocodazole increase the levels of transcriptionally active P53. Cancer Res 1995; 55: 6021-5. research article Attitudes of midwifery students towards teaching breast-self examination Andrej Plesnicar1, Martina Golicnik1, Irena Kirar Fazarinc2, Bozo Kralj3, Viljem Kovac4, Blanka Kores Plesnicar5 1 Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia, 2 Student Health Center, Ljubljana, Slovenia, 3 School of Health Science, Novo Mesto, Slovenia, 4 Department of Radiotherapy, Institute of Oncology, Ljubljana, Slovenia, 5 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Received 20 December 2009 Accepted 29 December 2009 Correspondence to: Andrej Plesnicar, MD, MSc, Faculty of Health Sciences, University of Ljubljana, Zdravstvena pot 5, 1000 Ljubljana, Slovenia. Phone: +386 1 300 11 67; E-mail: andrej.plesnicar@zf.uni-lj.si Disclosure: No potential conflicts of interest were disclosed. Background. The purpose of this study was to asses the attitude of undergraduate midwifery students towards teach­ing other women in methods of breast self-examination (BSE). Participants and methods. The study was performed at the beginning and at the end of students’ study at the Faculty of Health Sciences in Ljubljana, Slovenia. It was carried out during the academic year 2002/2003 and involved 28 first and 25 third year undergraduate midwifery students. The data were gathered from questionnaires and proc­essed with the use of descriptive and inferential statistics. Results. All study participants were of the opinion that teaching other women in methods of BSE is of great impor­tance for an early detection of breast cancer (BC) and that this task ought to be one of their duties. There were no significant differences between the two groups when the readiness to upgrade their own knowledge of BSE or when the optimism regarding the progress in breast cancer detection and therapy in the future were concerned. Conclusions. The readiness of midwifery students to pass the knowledge of BSE to other women could help to increase their breast health awareness and thus improve their willingness and ability to detect early changes, associated with BC. Key words: midwifery students; breast cancer, teaching breast self-examination; breast health awareness; specific education Introduction Breast cancer is the most common form of cancer and the cause of death from cancer in women in Slovenia (population two million), where it was di­agnosed in 1112 women in the year 2006 and has shown a fivefold increase in its incidence in the last five decades.1-3Breast cancer incidence may be low in less developed countries, but survival rates of those suffering from this disease are also low, thus making it an important health care problem all over the world.4,5 The efficacy of BC screening with mammogra­phy has been confirmed in a number of randomized and observation studies.6,7However, small palpa­ble tumours can also be detected with the clinical breast examination (CBE), performed in clinical surroundings, and with BSE, which can be done by properly trained women themselves.6-8Despite many controversies, it has been accepted that BSE increases the awareness of normal appearance and structure of breasts and the consequent ability to detect subtle changes that don’t correspond to the normal features of healthy breasts. Breast health awareness may, therefore, increase the ability of women to find small palpable tumours early, lead­ing them to consult their physician immediately af­ter discovering undue changes in their breasts.6,9,10Unfortunately, few women perform BSE and there is little information on the attitude of individual TAble 1. The attitudes of midwifery students (Faculty of Health Sciences, Ljubljana, Slovenia, academic year 2002/2003) towards teaching BSE, receiving additional BSE education and breast towards BC detection and treatment in the future Teaching BSE helps early BC detection Yes No 28 (100%) 0 (0%) 25 (100%) 0 (0%) / / Teaching BSE should be one of midwives’ duties Yes No 28 (100%) 0 (0%) 25 (100%) 0 (0%) / / I am willing to receive further BSE education Yes No 26 (92.86%) 2 (7.14%) 22 (88%) 3 (12%) 0.362 0.5469 I favour practical demonstration of BSE Yes No 22 (78.56%) 6 (21.44%) 16 (64%) 9 (36%) 0.9327 0.3341 I am optimistic about BC detection and treatment in the future Yes No 24 (85.71%) 4 (14.29%) 23 (92%) 2 (8%) 0.5311 0.4664 n = number; BSE = breast self-examination; BC = breast cancer women and specific groups of women towards this way of breast change detection.3,6 In this study, we, thus, assessed the attitude of midwifery students towards the teaching other women in methods of BSE. We also assessed how the specific education of midwifery students, gained during the course of their three year study, affects their attitude towards BSE. Finally, we ex­amined their attitude towards the progress in BC detection and treatment in the future. Participants and methods Twenty eight first year and 25 third year midwife­ry students were included in the study carried out in the academic year 2002/2003 at the Faculty of Health Sciencesin Ljubljana, Slovenia. There were no males among the midwifery students in both study groups in that academic year. The part of the study involving the first-year students was carried out at the end of their first term and the part in­volving third-year students at the end of their sixth term. A written consent was obtained from all the involved participants before enrolling them in the study and all were asked to complete the question­naires with data about their personal characteris­tics. Questionnaires also included five statements concerning their attitudes whether teaching other women in methods of BSE helps in BC detection, whether teaching other women about BSE ought to be one of their duties, about their attitude regard­ing additional BSE training and the form of such training. Finally, they were asked to express their attitude about the development and successfulness of the BC treatment in the future. The acquired data were sorted and analyzed with the help of Microsoft Excel 97 and SPSS 11.0 for Windows programs. Most answers were as­sessed with the use of Chi-square test for 2X2 contingency tables with which the differences in frequencies were verified. The results were consid­ered as statistically significant if p<0.05.11 Results The mean age of the first-year midwifery students in the study was 19.86 ± 1.18 (SD) years (range 18­23 years), the mean age of the third-year students was 21.76 ± 2.26 (SD) years (range 20-32 years). None of them had undergone a mammography or ultrasound breast examination prior to participat­ing in the study. All the participants, regardless of the year of study, were of the opinion that teaching BSE to other women helps in the early detection of BC. All were of the opinion that teaching and inform­ing women of the advantages and disadvantages of BSE ought to be one of their duties. Most of the participants from both groups would be willing to receive additional training in how to perform BSE. The number of the participants willing to receive this training was slightly smaller in the group of the third-year students, but the difference was not statistically significant. In the group of the first-year students more participants were in favour of practical BSE lessons given live by experts and demonstrators as compared to a brochure on BSE obtained by mail (Table 1). The majority of the par­ticipants from both groups were optimistic in their opinion regarding the progress in the develop­ 54 Plesnicar A et al. / Midwifery students and teaching breast self-examination ment and successfulness of the BC detection and the treatment in the future. The number of partici­pants holding such opinion was slightly larger in the group of the third-year students, the difference between both groups was not statistically signifi­cant (Table 1). Discussion In this study, we found that midwifery students could be regarded as having a positive attitude to­wards teaching other women in methods of BSE. They were of opinion that this type of teaching helps in the BC detection and that it ought to be one of their duties. Most of the participants were also willing to receive the additional training in BSE themselves. The opinion that they should play an active part in educating other women and that they should additionally educate themselves prob­ably makes them optimistic in their views of the development and successfulness of the BC detec­tion and the treatment in the future. A favourable attitude of midwifery students to­wards BSE may come from an inherent motivation to become involved in problems associated with BC and from the specific education they gain during the course of their study. This knowledge makes them more informed than other women5,12-18, giv­ing them a profound insight into the anatomy, his­tology and physiology of healthy breasts, as well as into the significance of the incidence, mortality and pathogenic characteristics of BC.19Midwifery stu­dents are at the end of their study in possession of an important amount of information about BC and know that it is an important public health problem. In this context, it is safe to conclude that their spe­cific education is of utmost importance. A special emphasis should be given to the atti­tude of all the participants in the study that BSE is important in the early detection of BC. All of them would fully participate in teaching other women about BSE that certainly must include its advan­tages and disadvantages.6,15Their willingness to teach BSE should receive the additional support by giving the results of the studies investigating BSE some further consideration. According to some of the studies, BSE does not decrease the mortal­ity caused by BC.20-22However,the results of one of such studies also show that the compliance of one of the study groups to BSE program (BSE every month or every two months) helped to achieve a higher rate of 15 year survival.22Some of the studies also show that BSE reduces the size of newly dis­covered primary breast tumours20,23,24, which may lead to a prompter use of modern diagnostic proce­dures and enables carrying out more conservative surgical procedures.10,25Even when examining the negative results from some of these studies, a care­ful consideration should be given to local cultural values, level of readiness of participants to perform BSE and the likelihood that all of them were prop­erly informed about BC issues.20,26-29 Despite some of the doubts mentioned above, the possibility that BSE is used for achieving the in­creased breast health awareness in women should be examined carefully.6,30-32The breast health awareness means accepting health responsibili­ties to a greater extent by being able to recognize a normal appearance and structure of breasts dur­ing different cycle periods and with regard to age, by being able to recognize undue changes and to inform the physician immediately.6,23,30,31,33If the breast health awareness in women is to increase, the easiest way to achieve this would probably be by teaching them how to perform BSE. In some opinions the breast health awareness should take the role of BSE30, with BSE itself being not much different from what the concept of the breast health awareness includes.23,34 The majority of midwifery students in our study were willing to receive the additional education in relation to BSE. Most of them favoured the practi­cal demonstration of the BSE procedures. A small­er interest in the additional training and practical demonstrations is understandable with the stu­dents finishing their study and, on the other hand, this smaller interest again proves the importance of knowledge gained during the study of midwife­ry.19A slightly bigger number of those who hold an optimistic view of the future of the BC treatment in the group of the third-year students probably also points to the importance of this knowledge.18,35,36 In this study we found that female midwifery students included in the study have a favourable attitude towards teaching BSE, although it is un­doubtedly hard to learn and teach.37-39All consid­ered BSE to be of great importance for an early breast cancer detection and all were willing to teach it to other women, including those with previously treated malignant disease.40As a consequence of their attitude towards BSE and specific contents of their study, midwifery students could towards the end of their study probably also perform CBE quite efficiently and relieve other women of difficult en-deavour of learning all the steps of the appropriate and effective BSE.41,42In Slovenia and elsewhere, this application of their knowledge would be es­pecially important when younger women are in question, because in their cases mammography screening is not so efficient.9,23On the other hand, the participants in this study could, due to their knowledge and other characteristics, represent a specific group continuing with quality and regular BSE. Their attitude towards and knowledge of BSE could, in due course, be evaluated again. However, in some of the less developed parts of the world, the knowledge of BSE could enable similarly edu­cated midwives to have an important impact on the early detection of breast cancer and, along with other nurses, an important role in health promo­tion, prevention and education activities associ­ated with breast cancer.43,44 In conclusion, in this study we show that mid­wifery students, a highly motivated and specifical­ly educated group, are ready to pass the knowledge of BSE to other women, to receive the further BSE education themselves and are generally optimistic about the BC detection and the treatment in the fu­ture. In this way, they could have an important role in increasing the breast health awareness of other women and thus improve their willingness and ability to detect early changes associated with BC. References 1. Pisani P, Parkin DM, Ferlay J. Estimates of worldwide mortality from eight­een major cancers in1985. Implications for prevention and projections of future burden. Int J Cancer 1993; 55: 891-903. 2. Parkin DM, Whelan SL, Ferlay J, Raymond L, Young J. Cancer incidence in five continents, Volume VII. Lyon: International Agency for Research on Cancer; 1997. 3. Cancer Registry of Slovenia. Cancer Incidence in Slovenia: 2006. Report No. 48. Ljubljana: Institute of Oncology; 2009. 4. Lantz PM, Weisman CS, Itani Z. A disease-specific Medicaid expansion for women. The Breast and Cervical Cancer Prevention and Treatment Act of 2000. Womens Health Issues 2003; 13: 79-92. 5. Anderson BO, Braun S, Carlson RW, Gralow JR, Lagios MD, Lehman C, et al. Overview for breast health guidelines for countries with limited resources. Breast J 2003; 9(Suppl. 2): S42-50. 6. Smith RA, Saslow D, Sawyer KA, Burke W, Costanza ME, Evans WP 3rd, et al. American Cancer Society guidelines for breast cancer screening: update 2003. CA Cancer J Clin 2003; 53: 141-69. 7. Olsen O, Gřtzsche PC. Cochrane review on breast cancer screening with mammography. Lancet 2001; 358: 1340-2. 8. Tabar L, Duffy SW, Vitak B, Chen HH, Prevost TC. The natural history of breast carcinoma: what have we learned from screening? Cancer 1999; 86: 449-62. 9. Hertl K. Diagnostic methods in breast cancer detection. [Slovenian]. Zdrav Vest 2009; 78(Suppl 1): 191-5. 10. Podkrajšek M, Žgajnar J, Hocevar M. What is the most common mammo-graphic appearance of T1a and T1b invasive breast cancer? Radiol Oncol 2008; 42: 173-80. 11. Jekel JF, Elmore JG, Katz DL. Epidemiology, Biostatistics and Preventive Medicine. 1st edition. Philadelphia: W. B. Saunders Company; 1996. 12. Fitzgibbon ML, Gapstur SM, Knight SJ. Mujeres felices por ser saludables: a breast cancer risk reduction program for Latino women. Prev Med 2003; 36: 536-46. 13. Jirojwong S MacLennan R. Health beliefs, perceived self-efficacy, and breast self-examination among Thai migrants in Brisbane. J Adv Nurs 2003; 41: 241-9. 14. Sadler GR, Dhanjal SK, Shah NB, Shah RB, Ko C, Anghel M, et al. Asian Indian women: knowledge, attitudes and behaviors toward breast cancer early detection. Public Health Nurs 2001; 18: 357-63. 15. Frank E, Rimer B, Brogan D, Elon L. US women physicians’ personal and clinical breast cancer screening practices. J Women’s Health Gender Based Med 2000; 9: 791-801. 16. Fischer V, Pabst R, Nave H. Seminar in breast self-examination for female medical students integrated into a human gross anatomy course. Clin Anat 2003; 16: 160-4. 17. Budden L. Student nurses’ breast self-examination health beliefs, atti­tudes, knowledge, and performance during the first year of a preregistra­tion degree program. Cancer Nurs 1999; 22: 430-7. 18. Devine SL, Frank DL. Nurses self-performing and teaching others breast self-examination: implications for advanced practice nurses. Clin Excell Nurse Pract 2000; 4: 216-23. 19. Skoberne M, Mihelic A, eds. Zdravstvena nega, porodniško ginekološka smer. Visoko strokovni študijski program. Ljubljana: University of Ljubljana, University College of Health Studies; 1996. 20. Thomas DB, Gao DL, Ray RM, Wang WW, Allison CJ, Chen FL, et al. Randomized trial of breast self-examination in Shangai: final results. J Natl Cancer Inst 2002; 94: 1445-57. 21. Semiglazov VF, Moiseyenko VM, Bavli JL, Migmanova NSh, Seleznyov NK, Popova RT, et al. The role of breast self-examination in early breast cancer detection (results of the 5-years USSR/WHO randomized study in Leningrad). Eur J Epiedmiol 1992; 8: 498-502. 22. Semiglazov VF, Manikhas AG, Moiseenko VM, Protsenko SA, Kharikova RS, Seleznev IK, et al. Results of a prospective randomized investigation [Russia (St. Petersburg)/WHO] to evaluate the significance of self-exam­ination for the early detection of breast cancer. [Russian]. Vopr Onkol 2003; 49: 434-41. 23. Philip J, Harris WG, Flaherty C, Joslin CA, Rustage JH, Wijesinghe DP. Breast self-examination: clinical results from a population-based prospec­tive study. Br J Cancer 1984; 50: 7-12. 24. Dowle CS, Mitchell A, Elston CW, Roebuck EJ, Hinton CP, Holliday H, et al. Preliminary results of the Nottingham breast self-examination education programme. Br J Surg 1987; 74: 217-9. 25. Koibuchi I, Iino Y, Takei H, Maemura M, Horiguchi J, Yokoe T, et al. The effect of mass screening by physical examination combined with regular breast self-examination on clinical stage and course of Japanese women with breast cancer. Oncol Rep 1998; 5: 151-5. 26. Crossing S, Manaszewics R. Breast self examination: be alert but not alarmed? Med J Aust 2003; 178: 646-7. 27. Sadler GR, Wang K, Wang M, Ko CM. Chinese women: behaviors and attitudes toward breast cancer education and screening. Womens Health Issues 2000; 10: 20-6. 28. Tang TS, Solomon LJ, Mc Cracken LM. Cultural barriers to mammography, clinical breast exam, and breast self exam among Chinese-American women 60 and older. Prev Med 2000; 31: 575-83. 29. Smith RA. Commentary: Breast self examination. Do we really know what we think we know? Br Med J USA 2003; 3: 168-9. 30. Austoker J. Breast self examination. Br Med J 2003; 326: 1-2. 31. Brett J, Austoker J. Evaluation of breast awareness training resource pack for primary care nurses: final report. Oxford: Cancer Research Campaign; 1999. 32. Mitchell A. Breast health awareness. In: Perkins ER, Simnett, Wright L, editors. Evidence-based health promotion. 4th edition. Chichester: John Wiley & Sons Ltd; 2002. p. 266-74. 33. Plesnicar A, Kovac V, Kralj B. Breast cancer and breast health awareness as an evolving health promotion concept. Radiol Oncol 2004; 38: 27-34. 56 Plesnicar A et al. / Midwifery students and teaching breast self-examination 34. Epstein RJ. Breast self examination provides entry strategy. Br Med J 2003; 326: 710. 35. Baum M. The changing face of breast cancer-past, present and future perspectives. Breast Cancer Res Treat 2002; 75(Suppl. 1):S1-5; Discussion S33-5. 36. Walter FM, Kinmoth AL, Hyland F, Murrell P, Marteau TM, Todd C. Experiences and expectations of the new genetics in relation to familial risk of breast cancer: a comparison of the views of GPs and practice nurses. Fam Pract 2001; 18: 491-4. 37. Harvey BJ, Miller AB, Baines CJ, Corey PN. Effect of breast self-examina­tion techniques on the risk of death from breast cancer. Can Med Assoc J 1997; 157: 1205-12. 38. Pilgrim CA, Lannon C, Harris RP, Cogburn W, Fletcher SW. Improving clinical breast examination training in a medical school: a randomized controlled trial. J Gen Intern Med 1993; 8: 685-8. 39. Campbell HS, Fletcher SW, Pilgrim CA, Morgan TM, Lin S. Improving physi­cians’ and nurses’ clinical breast examination: a randomized controlled trial. Am J Prev Med 1991: 7: 1-8. 40. Plesnicar A, Kovac V. Breast metastases from cutaneous melanoma: a report of three cases. Tumori 2000; 86: 170-3. 41. Miller AB, To T, Baines CJ, Wall C. Canadian National Breast Screening Study-2: 13-year results of a randomized trial in women aged 50-59 years. J Natl Cancer Inst 2000; 92: 1490-9. 42. Phillip J, Harris WG, Flaherty C, Joslin CA. Clinical measures to assess the practice and efficiency of breast self-examination. Cancer 1986; 58: 973-7. 43. Plesnicar S, Plesnicar A. Cancer: a reality in the emerging world. Semin Oncol 2001; 28: 210-16. 44. Henke Yarbro C. International nursing and breast cancer. Breast J 2003; 9(Suppl 2): S98-100. case report Genetic testing for young-onset colorectal cancer: case report and evidence-based clinical guidelines Yaolin Zhou1, Lisa A. Boardman2, Robert C. Miller3 1 Mayo Medical School, College of Medicine, Mayo Clinic, Rochester, Minnesota 2 Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota 3 Department of Radiation Oncology, Mayo Clinic, Rochester, Minnesota Received 29 December 2009 Accepted 10 January 2010 Correspondence to: Robert C. Miller, MD, MS, Department of Radiation Oncology, Mayo Clinic, 200 First St SW, Rochester, MN 55905. E-mail: miller.robert@mayo.edu Disclosure: No potential conflicts of interest were disclosed. Background. Young-onset colorectal cancer is clinicopathologically different from older-onset colorectal cancer and tends to occur in patients with hereditary germline conditions such as Lynch syndrome and familial adenomatous polyposis. Case report. We describe the case of a 44-year-old man with a paternal history of colon polyps, a personal 2-year history of hematochezia, and a diagnosis of rectal cancer. Further clinical evaluation of the patient at our institution determined the cancer to be stage IIIA. The patient underwent genetic counseling and testing, which indicated he was negative for the most common familial cancer syndromes. After treatment with neoadjuvant chemoradiother­apy, surgery, and adjuvant chemotherapy, the patient has done well. We review the hereditary cancer syndromes and genetic tests to consider for patients with early-onset colorectal cancer. Conclusions. This case underscores the importance of following cancer-screening guidelines. Key words: adenomatous polyposis coli; attenuated familial adenomatous polyposis; colorectal cancer; familial ad-enomatous polyposis; microsatellite instability; MYH-associated polyposis Introduction Colorectal cancer (CRC) is a common malignancy in North America and Europe; most patients have sporadic disease without a known genetic predis­position to the illness. With the exception of the hereditary germline conditions Lynch syndrome, familial adenomatous polyposis (FAP), and attenu­ated FAP, which are marked by an early age of on­set and familial CRC clustering, most cases of CRC do not develop until age 65 years or older. Still, up to 20% of all cases of CRC arise in persons aged 50 years or younger who do not have Lynch syn­drome or FAP.1-3 Lynch syndrome, formerly known as heredi­tary nonpolyposis colorectal cancer, is caused by a germline mutation in 1 of several DNA mismatch repair (MMR) genes and is the most common sin-gle-gene–related cause of hereditary colon cancer in North American and European populations.4,5CRC in Lynch syndrome has unique histopatho-logic and clinical findings. These cancers tend to be more responsive to treatment despite being poorly differentiated.2 One report based on the experience from 2 can­cer registries in the United States, the National Program of Cancer Registries and the Surveillance, Epidemiology and End Results studies, empha­sized that CRC is of concern for young adults: it is among the top 10 cancers in persons aged 20 to 49 years of all races.6Young-onset and older-onset CRC are clinicopathologically different in that young-onset CRC usually presents at a later stage and is more poorly differentiated.6Here, we present the case of a young middle-aged man with rectal cancer. 58 Zhou Y et al. / Young-onset colorectal cancer Case report A 44-year-old man sought medical care in 2001 for a 2-year history of hematochezia. Colonoscopy per­formed at an outside institution in September 2001 showed a lesion within the rectum. Biopsy per­formed at that time confirmeda poorly differen­tiated, grade 3 adenocarcinoma, which measured 2×1.3×1 cm, with invasion through the muscularis mucosa. He came to our institution in October 2001 for further evaluation. The patient’s family history of cancer was pri­marily limited to a brother who received a diag­nosis of chronic myelogenous leukemia at age 47 years. His father had colon polyps removed in his mid 50s, and a maternal aunt had colon polyps at age 35 years. Distant paternal relatives may have had CRC in old age. The patient had been a smoker for the previous 16 years and had a history of gastroesophageal re­flux disease. He had a history of sebaceous cysts on the left scapula and the back of the thighs and a lipoma on the inferior border of the right scapula. He did not have supernumerary teeth. Evaluation of the patient included genetics counseling and testing because of his young age at diagnosis (Figure 1). Immunohistochemical testing (IHC) for MLH1, MSH2, and MSH6 showed intact MMR. The patient was not tested for the APC gene mutation because he was not suspected to have FAP (Figure 2). He was also not tested for MYH, which was not discovered until later.7 Endorectal ultrasonography with guided fine-needle aspiration biopsy of suspicious lymph nodes confirmed an ulcerated lesion extending from the anal verge to 3.5 cm proximally along the anterior wall of the rectum. Fine-needle aspirates from 2 enlarged perirectal lymph nodes were posi­tive for adenocarcinoma. In the ascending colon, a 5-mm hyperplastic polyp was removed with cold snare. Abdominal and pelvic computed tomography detected a 0.6-cm sclerotic lesion within the left ili­ac bone, which was considered benign after a bone scan demonstrated no bony metastases. Results of upper endoscopy were normal. On the basis of the clinical evaluations, the patient’s cancer was deter­mined to be stage IIIA (T2N1M0) by the American Joint Committee on Cancer 6th edition criteria. Neoadjuvant chemoradiotherapy was rec­ommended on the basis of the tumor stage and the proximity of the tumor to the prostate. From November through December 2001, the patient was given a continuous infusion of radiosensitizing 5-fluorouracil (5-FU) (225 mg/m2). He was treated with 180 cGy in 28 fractions, for a total of 5040 cGy. He tolerated the therapy well, with some mild di­arrhea, weight loss, decreased energy, and perianal irritation. The cancer in the rectum showed com­plete response to therapy. In January 2002, the patient underwent surgery to have an abdominoperineal resection and perma­nent colostomy. He tolerated the procedure well, and no residual tumor was identified. Two of 31 re­gional lymph nodes were positive for grade 3 mu-cinous adenocarcinoma. In the following month, the patient had several perineal drain site infec­tions and was treated for depression. In April 2002 the patient began his first of 4 cycles of adjuvant 5-FU and leucovorin systemic chemotherapy. Prolonged neutropenia and gas­trointestinal tract bleeding occurred in the middle of the first cycle of chemotherapy; therefore, the dosage of 5-FU (425 mg/m2) was decreased by 10% (to 380 mg/m2). He completed chemotherapy in August 2002. Surveillance colonoscopies in the following years have been negative. The patient was last seen at our institution in January 2007. He has had no new gastrointestinal tract or genitourinary symp­toms or problems. His appetite has returned to normal, and he feels strong. He has returned to work as a carpenter and is now being monitored with colonoscopy every 3years. Discussion According to the Fearon and Vogelstein model of carcinogenesis, the accumulation of multiple mu­tations is required for the transformation of nor­mal colonic mucosa into dysplastic adenomas and then into invasive carcinomas.4CRC can be clas­sified broadly as exhibiting either chromosomal instability through gain-of-function mutations (APC/ß-catenin pathway) or microsatellite insta­bility (MSI) (defects in DNA MMR). In sporadic CRC, mutations are acquired in a stepwise fash­ion. In the case of single-gene hereditary colorectal cancer syndromes, all DNA-containing cells have a germline mutation in one allele of the involved gene and thus are a step closer to the accumulation of additional acquired mutations necessary to lead to CRC. In human CRC, 80% of the tumors are micro-satellite stable, which means they have intact DNA MMR and can correct single-base and small-loop base-pair mismatches present throughout the non­ FIGure 1. Scheme describing the recommended initial evaluation of a patient aged 50 years or younger with a diagnosis of colorectal cancer (CRC). AFAP indicates attenuated familial adenomatous polyposis; FAP, familial adenomatous polyposis; IHC, immunohistochemistry; JPS, juvenile polyposis syndrome; MAP, MYH-associated adenomatous polyposis; MSI, microsatellite instability; PJS, Peutz-Jeghers syndrome. coding and coding regions of the genome. The re­maining 20% of CRC tumors exhibit MSI due to defects in this DNA MMR pathway that corrects small base-pair mistakes in mononucleotide, dinu­cleotide, and trinucleotide repeat regions through­out the genome and are classified as having high or low MSI (MSI-H or MSI-L, respectively).5A small fraction of MSI-H tumors result from germline mu­tations in 1 of 4 DNA MMR genes—MLH1, MSH2, MSH6, and PMS2—and result in the hereditary CRC condition called Lynch syndrome. However, the greater proportion of MSI-H tumors arises via impairment of DNA MMR through hypermethyla­tion of the MLH1 gene. Although only 15% to 20% of sporadic can­cers are MSI-H, 90% of patients who meet the Amsterdam criteria for Lynch syndrome have MSI-H CRC.8Tumor DNA can be evaluated for MSI using polymerase chain reaction to amplify a panel of DNA sequences with nucleotide repeats. Lynch syndrome, an autosomal dominant disor­der, is the most common hereditary colon cancer syndrome. Mutations in 1 of the MMR genes usual­ly result in truncated or lost protein product. Thus, tumors can be screened for defective DNA MMR by using polymerase chain reaction to test for MSI or IHC to test for loss of MMR protein expres­sion. The results of IHC may then be used to direct germline sequencing toward a specific DNA MMR gene in young-onset cases or in persons with clini­cal or family history criteria suggestive of Lynch syndrome. Hypermethylation assays and BRAF V600E mutation testing of tumor DNA can be used to distinguish an MSI-H tumor with absent MLH1 expression caused by hypermethylation of the MLH1 promoter from a tumor caused by a germ-line MLH1 mutation. Tumors with hypermethyla­tion of MLH1 and with the BRAF V600E mutation nearly always represent sporadic CRC not caused by a germline MLH1 mutation and not associated with Lynch syndrome. In the case presented here, the patient was tested for a familial syndrome because of his relatively young age at presentation (44 years); however, he lacked many of the features of either FAP or Lynch syndrome. The genetic diagnosis of Lynch 60 Zhou Y et al. / Young-onset colorectal cancer FIGure 2. Scheme describing the recommended genetic testing for a patient with a diagnosis of colorectal cancer. AFAP indicates attenuated familial adenomatous polyposis; APC, adenomatous polyposis coli; BRAF, v-raf murine sarcoma viral oncogene homolog B1; FAP, familial adenomatous polyposis; IHC, immunohistochemistry; MAP, MYH-associated adenomatous polyposis; MLH1, MutL homolog 1; MMR, mismatch repair; MSI, microsatellite instability; MYH, MutY homolog. syndrome requires a germline mutation in 1 of the MMR genes. The patient’s tumor showed normal expression of the MMR genes MLH1, MSH2, and MSH6 by IHC. MSI testing was not performed. A previous study from our institution showed that a normal IHC test for MLH1 and MSH2 has a 96.7% positive predictive value for a microsatellite stable/MSI-L phenotype.9On the basis of the IHC data alone, it is highly unlikely that the patient has a germline mutation in an MMR gene, which would lead to Lynch syndrome with MSI.9 Another hereditary CRC syndrome that can be considered is MYH-associated polyposis (MAP). MAP has a phenotypic overlap with FAP, attenu­ated FAP, and Lynch syndrome; biallelic carriers have an 80% cumulative lifetime risk of CRC by age 70 years.10In several studies, among patients with early-onset CRC (diagnosed before age 50 years) who tested negative for Lynch syndrome, 1% to 2% were biallelic carriers of the MYH muta­ tion.11-13 CrC screening and testing recommendations Although CRC does not usually develop until age 65 years or older, up to 20% of CRC cases will arise in persons 50 years or younger who do not have ei­ther of the known hereditary CRC conditions.1-3,6,14The American Gastroenterological Association has published guidelines for CRC screening for aver­ age-risk and higher-risk patients.15,16Persons with references a family history of CRC or adenomatous polyps (a first-degree relative with CRC or adenomatous polyps diagnosed before age 60 years, or 2 first-degree relatives with CRC diagnosed at any age) should have screening colonoscopy starting at age 40 years, or 10 years younger than the earliest diag­nosis, whichever comes first, with repeat colonos­copy every 5 years. If testing for MMR is negative, patients with ear-ly-onset CRC may be tested for MYH mutations, regardless of their family history or the number of colon polyps. IHC can be used in clinical practice to test for MAP regardless of the specific MYH mu­tations.17 Conclusions The case presented here highlights that CRC can occur at an age younger than the cancer-screening guidelines suggest for average-risk patients and also shows the importance of using family history to determine the timing of the first CRC screen­ing. Had our patient undergone his first screening colonoscopy at age 40 years as recommended by the American Gastroenterological Association—given that the patient’s father had colon polyps re­moved before age 60 years—his CRC might have been diagnosed at an earlier stage. Similarly, the patient’s 2-year history of hematochezia warranted a colon examination. We stress the importance of acknowledging and pursuing these symptoms, even in younger patients. 1. Adloff M, Arnaud JP, Schloegel M, Thibaud D, Bergamaschi R. Colorectal cancer in patients under 40 years of age. Dis Colon Rectum 1986; 29: 322-5. 2. Gryfe R, Kim H, Hsieh ET, Aronson MD, Holowaty EJ, Bull SB, et al. Tumor microsatellite instability and clinical outcome in young patients with colorectal cancer. N Engl J Med 2000; 342: 69-77. 3. Moore PA, Dilawari RA, Fidler WJ. Adenocarcinoma of the colon and rectum in patients less than 40 years of age. Am Surg 1984; 50: 10-4. 4. Lynch HT, Smyrk TC, Watson P, Lanspa SJ, Lynch JF, Lynch PM, et al. Genetics, natural history, tumor spectrum, and pathology of hereditary nonpolyposis colorectal cancer: an updated review. Gastroenterology 1993; 104: 1535-49. 5. Peltomaki P, Vasen HF; the International Collaborative Group on Hereditary Nonpolyposis Colorectal Cancer. Mutations predisposing to hereditary nonpolyposis colorectal cancer: database and results of a col­laborative study. Gastroenterology 1997; 113: 1146-58. 6. Fairley TL, Cardinez CJ, Martin J, Alley L, Friedman C, Edwards B, et al. Colorectal cancer in U.S. adults younger than 50 years of age, 1998-2001. Cancer 2006; 107(5 Suppl): 1153-61. 7. Al-Tassan N, Chmiel NH, Maynard J, Fleming N, Livingston AL, Williams GT, et al. Inherited variants of MYH associated with somatic G:C.T:A muta­tions in colorectal tumors. Nat Genet 2002; 30: 227-32. 8. Jenkins MA, Hayashi S, O’Shea AM, Burgart LJ, Smyrk TC, Shimizu D, et al; Colon Cancer Family Registry. Pathology features in Bethesda guidelines predict colorectal cancer microsatellite instability: a population-based study. Gastroenterology 2007; 133: 48-56. 9. Lindor NM, Burgart LJ, Leontovich O, Goldberg RM, Cunningham JM, Sargent DJ, et al. Immunohistochemistry versus microsatellite instability testing in phenotyping colorectal tumors. J Clin Oncol 2002; 20: 1043-8. 10. Jenkins MA, Croitoru ME, Monga N, Cleary SP, Cotterchio M, Hopper JL, et al. Risk of colorectal cancer in monoallelic and biallelic carriers of MYH mutations: a population-based case-family study. Cancer Epidemiol Biomarkers Prev 2006; 15: 312-4. 11. Eliason K, Hendrickson BC, Judkins T, Norton M, Leclair B, Lyon E, et al. The potential for increased clinical sensitivity in genetic testing for poly­posis colorectal cancer through the analysis of MYH mutations in North American patients. J Med Genet 2005; 42: 95-6. 12. Riegert-Johnson DL, Johnson RA, Rabe KG, Wang L, Thomas B, Baudhuin LM, et al. The value of MUTYH testing in patients with early onset mic-rosatellite stable colorectal cancer referred for hereditary nonpolyposis colon cancer syndrome testing. Genet Test 2007; 11: 361-5. 13. Wang L, Baudhuin LM, Boardman LA, Steenblock KJ, Petersen GM, Halling KC, et al. MYH mutations in patients with attenuated and clas­sic polyposis and with young-onset colorectal cancer without polyps. Gastroenterology 2004; 127: 9-16. Erratum in: Gastroenterology 2004; 127: 1651. 14. O’Connell JB, Maggard MA, Liu JH, Etzioni DA, Livingston EH, Ko CY. Rates of colon and rectal cancers are increasing in young adults. Am Surg 2003; 69: 866-72. 15. American Gastroenterological Association. American Gastroenterological Association medical position statement: hereditary colorectal cancer and genetic testing. Gastroenterology 2001; 121: 195-7. 16. Winawer S, Fletcher R, Rex D, Bond J, Burt R, Ferrucci J, et al; Gastrointestinal Consortium Panel. Colorectal cancer screening and surveillance: clinical guidelines and rationale: update based on new evi­dence. Gastroenterology 2003; 124: 544-60. 17. Di Gregorio C, Frattini M, Maffei S, Ponti G, Losi L, Pedroni M, et al. Immunohistochemical expression of MYH protein can be used to identify patients with MYH-associated polyposis. Gastroenterology 2006; 131: 439-44. research article A neutron track etch detector for electron linear accelerators in radiotherapy Branko Vukovic1, Dario Faj2, Marina Poje1, Maja Varga1, Vanja Radolic1, Igor Miklavcic1, Ana Ivkovic2, Josip Planinic1 1 Department of Physics, University of Osijek; Osijek, Croatia 2 Clinical Hospital Osijek, Osijek, Croatia Received 16 September 2009 Accepted 16 November 2009 Correspondence to: Branko Vukovic, PhD, Department of Physics, University of Osijek, P.O. Box 125, HR-31000 Osijek, Croatia. Phone: +385 31 232 700; Fax: +385 31 232 701. E-mail: branko@fizika.unios.hr Disclosure: No potential conflicts of interest were disclosed. Background. Electron linear accelerators in medical radiotherapy have replaced cobalt and caesium sources of radiation. However, medical accelerators with photon energies over 10 MeV generate undesired fast neutron contami­nation in a therapeutic X-ray photon beam. Photons with energies above 10 MeV can interact with the atomic nucleus of a high-Z material, of which the target and the head of an accelerator consist, and lead to the neutron ejection. Results and conclusions. Our neutron dosimeter, composed of the LR-115 track etch detector and boron foil BN-1 converter, was calibrated on thermal neutrons generated in the nuclear reactor of the Josef Stefan Institute (Slovenia), and applied to dosimetry of undesirable neutrons in photon radiotherapy by the linear accelerator 15 MV Siemens Mevatron. Having considered a high dependence of a cross-section between neutron and boron on neutron energy, and broad neutron spectrum in a photon beam, as well as outside the entrance door to maze of the Mevatron, we developed a method for determining the effective neutron detector response. A neutron dose rate in the photon beam was measured to be 1.96 Sv/h. Outside the Mevatron room the neutron dose rate was 0.62 mSv/h. PACS: 87.52. Ga; 87.53.St; 29.40.Wk. Key words: electron linear accelerator; photoneutron; track etch detector; neutron dose equivalent Introduction Nowadays,cobaltandcaesiumteletherapymachines inmedicalradiotherapyarebeingreplacedbylinear accelerators.1Thegreatadvantageofthisnewequip­mentisthatithasnoattachedradioactivesource, whichmakesthemsaferfromtheradiologicalpoint ofview.However,medicalacceleratorswithphoton energiesover10MeVgenerateundesiredfastneu­troncontaminationinatherapeuticbeam.Photons withenergiesabove10MeVcaninteractwiththe atomicnucleusofahigh-Zmaterial,ofwhichthetar­getandtheheadoftheacceleratorconsist,andlead totheneutronejection.Consequently,thiscanin­creasethepatientdoseandposeaprobleminroom shieldingdosimetry,whichisessentialforthequal­ityassuranceinradiotherapy.2,3 Neutrons are principally produced through giant dipole resonance in a nuclear reaction (g, n) between photons and target nuclei.4The giant resonance process produces two groups of neutrons; the first and the largest group has a spectrum, which can be described by a Maxwellian distribution, with the most probable energy, typically between 0.4 and 1 MeV; the second group of neutrons is produced by direct emission and is somewhat higher in energy (up to 10-20% of the total neutrons in general).5The mean energy of the neutron spectrum generated by the (g,n) reaction is around a few MeV, but, at the patient plane, neutrons have a more complex dis­tribution and a less mean energy. As beam energies increase (>10 MeV), an undesirable photoneutron dose also increases. Otherwise one can expect, that inside the area treated by linear accelerator, the neu­tron dose in a tissue will not exceed 1% of the pho­ton dose.6 Material and methods The neutron dosimeter consisted of the LR-115 track detector and boron foil BN-1 (Kodak-Pathe, France) or 10B converter for reaction (n, a); it was calibrated on neutrons generated in the nuclear re­actor of the J. Stefan Institute (Slovenia).7Neutron irradiation was carried out in the thermal column of the TRIGA Mark II reactor where the neutron flux was F = 3.33x105cm-2s-1; for irradiation time, t = 240 s, we got the fluence, f = Ft = 7.99x107 cm-2. The LR-115 detectors, 2x3 cm2, were etched in a 10% NaOH aqueous solution at 60 °C for 120 min and afterwards counted visually using a micro­scope of (10x16) magnification. The irradiated neutron track etch detector had a net track density Dt= (63394– B) @ 63370 cm-2, where the background, B, was 24 cm-2; the standard deviation of the Dtwas sDt= 570. The response, rt, of the neutron dosimeter for thermal neutrons was: rt = Dt/f = (7.930 ± 0.071) x 10-4,[1] where the error was given as a standard devia­tion, srt = sDt/f = 7.1x10-6. TheelectronlinearacceleratorSiemensMevatron 15MVhasbeenusedasanX-rayradiotherapy sourceintheClinicalHospitalOsijek.Thesame acceleratorwasusedtoperformexperimentsfor determiningadoseequivalentofundesirablepho­toneutronsbyusingtheneutrontracketchdetector. Results and discussion Linear accelerator neutrons – track detector in the beam In order to determine a dose equivalent of pho­toneutrons produced by linear accelerator, operat­ing in a photon production mode at an acceleration potential of 15 MV, we used the LR-114 track etch detector, which was positioned in the beam at 1 m from the accelerator head. For fast neutrons with higher energy, a detector response should be lower; having considered a to­tal cross-section of neutrons on boron, 10B (n, a)7Li, depending on neutron energy (s(E), Figure 1)8, as well as a neutron fluence spectrum on energies by the linear accelerator (Figure 2)2, we divided neu­tron energy Ea(MeV) in the two areas as follows: the first area , 0 < Ea1< 0.5, and the second one, 0.5 = Ea2 = 7.5. Afterwards, we estimated the respective mean cross-sections st = 1000 b (for thermal neutrons), sa1 = 1.33 b and sa2= 0.25 b (for energies Ea1 and Ea2, respectively) from the curve s(E) in Figure 1, and 64 Vukovic B et al. / Neutron track etch detector for electrons we determined the neutron detector responses for the tow energy areas as follows: sr t/sa1= t/ra1, st/sa2= r, where the rwas the response that had al­ t/ra2t ready been calculated by using equation [1]. Thus we got ra1= 1.06 x 10-6and ra2= 2.00 x 10-7, with the standard deviations of 9.1x10-9and 1.8x10-9, respec­tively, and we were able to determine an average or effective value of the neutron detector response rae, but as a weighted or pondered mean.9 Therefore, we took the surfaces under the neu­tron fluence spectrum curve (Figure 2; f(E)) for the two energy areas as above and we got the rela­tive surfaces: sa1= 0.58 and sa2= 0.42, those had the meaning of the relative frequencies in the calcula­tion of the pondered mean (for instance, s1 is the ra­tio of the surface under the curve between 0 and 0.5 MeV to the total surface under the curve between 0 and 7.5 MeV). The mean or effective detector re­sponse was: rae = ra1sa1 + ra2 sa2 =(6.97 ± 0.07) x10-7. For a measureddetector density Da, the respec­tive fluence was (like in equation [1]): f= D/r. aeaae When the neutron fluence is known, a conversion coefficient (k) from neutron fluence to doseequiv­alent, depending on neutron energy, gives a per­sonal dose equivalent (H), as follows:10 H= k f. aaaeae Having considered a great dependence of kon neutron energy (Figure 3), we calculated the aver­age or effective kaefor the two energy areas, like above, and we used the same relative frequencies (i = 1, 2); taking the average values of kfor the saianeutron energies Ea1 and Ea2 as ka1= 200 and ka2= 430 pSv cm2, respectively, we calculated the effec­tive conversion factor as follows: kae = ka1sa1 + ka2sa2 = 296.4 pSv cm2. Thus, the measured detector net density Da= (383.1± 0.04) cm-2corresponded to the following dose equivalent: H = kf = k/r D= 425.5D (mSv) ,[2] aaeaeaeaeaa Ha = (0.163 ± 0.002) Sv The dose rate was calculated as a ratio of the dose equivalent and exposure time, t, or: .H= H/t , and for t = 5 min, we got the dose rate .H= (1.96 ± 0.02) Sv/h. The measurement errors were determined as variances or standard deviations for track densi­ties in the following way (according to the Poisson distribution): sD2 = sDb2 + sB2 = Db + B ; The dose equivalent variance was calculated as total differential of the function of the form like in equitation [2], which led to the following expres­sion: sH2 = (kD/r2)2sr2 + (k/r)2sD2 . Linear accelerator neutrons – track detector behind the wall The neutron track etch detector was positioned outside the entrance door to the accelerator maze. The neutron spectrum in the same position was measured by Schraube et al.11, hereby presented in Figure 4. We used the given neutron spec­trum in the procedure for determining a neutron dose equivalent by the track detector, as above. Otherwise, neutron spectrum can vary depending on the wall construction of a room. We divided neutron energy, Eb, in two areas as follows: the first area, 0 < Eb1< 100 eV, and the sec­ond one, 100 eV = Eb2= 3750 eV. Because the Eb1was the area of thermal neutrons, we estimated the respective mean cross-sections st = sb1 = 120 b and = 14.75 b (for energies Eb1 and Eb2, respectively) sb2from the curve s(E) in Figure 1, and we determined the neutron detector responsesfor the low energy areas as follows: sb1/sb2= rb1, where rb1= rwas /rb2talready assessed response by using equation [1]. Thus we got rb1= 7.94x10-4and rb2= 9.75x10-5, and were able to determine the average or effective va­lue of the neutron detector response rbefor neu­trons in the energy area Eb.As in the previous case, we took the surfaces un­der the neutron fluence spectrum curve (Figure 4; f(E)) for the two energy areas and we got the rela­tive surfaces: sb1= 0.18 and sb2= 0.82, those had the meaning of the relative frequencies in the calcula­tion of the pondered mean. The mean or effective detector response was: rbe = rb1sb1 + rb2 sb2 = 2.2x10-4.For the measured detector density Db, the re­spective fluence is (like in equation [1]): fbe= Db/rbe. When the neutron fluence is known, a conversion coefficient (k) from neutron fluence to dose equiva­lent, depending on neutron energy, gives a person­al dose equivalent (H), as follows: Hb= kbe a fbe. Having considered a great dependingkon neu­tron energy (Figure 3), we calculated the average or effective kbefor the two energy regions, like above, and we used the same relative frequencies sbi(i = 1, 2); taking the average values of kbfor the neutron energies Eb1 and Eb2 as kb1= 11.58 and kb2= 9 pSv cm2, respectively, we calculated the effective con­version factor as follows: = kb1sb1 + kb2sb2 = 9.45 pSv cm2. kbe Thus, the measured detector net density Db= 1.2± 0.01 cm-2corresponded to the following dose equivalent: = kbe = kbe Db= 42.9x10-3 Db (µSv), Hbfbe/rbe Hb = (0.050 ± 0.0006) µSv. Thedoseratewascalculatedasaratioofthedose equivalentandexposuretimeof5min,andwegot theneutrondoserateoutsidetheentrancedoorto .themazeofthe15MVMevatron,Hb=(0.62±0.007) µSv/h.Thelinacroom1.7mwallswereconstructed ofbariteconcrete,withdensityof3200kg/m3. Although the obtained neutron dose rate out­side the accelerator room was 3165 times smaller than the neutron dose rate in the photon beam, the .measured dose rate Hbwas not negligible from the aspect of personal dosimetry. Some considerations of neutron energy attenu­ation after crossing the treatment room walls were performed for different concrete barrier thickness and materials. A neutron spectrum attenuation from the 15 MV linear accelerator, after passing a conventional 1 m concrete barrier, with density of 2260 kg/m3, was measuredby Facure et al., hereby presented in Figure 5.2Observing the neutron spectra in Figure 5 and Figure 4, one can notice a broad neutron energy area in Figure 5 (from 0.1 eV to 10 MeV), that con­tributes to the neutron dose outside the Mevatron treatment room; neutrons that crossed the 1 m con­crete barrier had higher energies than those behind the 1.7 m concrete wall (with energy below 1 keV). In order to compare the neutron detection pa­rameters reand ke, we dividedneutron energy Ec(MeV) of the spectrum (Figure 5) in the two areas as follows: first area Ec1< 2 MeV, and the second one 2 MeV = Ec2 = 7.5 MeV.Afterwards, we estimated the respective mean cross-sections st = sc1= 0.619 b (for thermal neu­trons, like above), sc2 = 0.203 b (for energies Ec1 , , respectively) from the curve s(E) in Figure1, Ec2 and we determined the neutron detector responses for the three energy areas as follows: sc1/sc2= rc1/, where rc1 = r= 7.94x10-4was the response that rc2t had already been calculated by using the equation [1]. Thus we got rc2= 2.6 x 10-4and we were able to determine the average or effective value of the neutron detector response rce, but as a weighted or pondered mean. Therefore, we took the surfaces under the neu­tron fluence spectrum curve (Figure 5; f(E)) for the two energy regions, like above, and we got the relative surfaces: sc1= 0.22, sc2= 0.78, which had the meaning of the relative frequencies in the calcula­tion of the pondered mean. The mean or effective detector response was: rce= rc1sc1+ rc2 sc2 = (3.78 ± 0.03) x10-4. Having considered a great depending kon neu­tron energy (Figure 3), we calculated the average or effective kcefor the three energy areas, and we used the same relative frequencies sci(i = 1, 2, 3); taking the average values of kcfor the neutron energies Ec1and Ec2 as kc1= 366 , kc2= 433 pSv cm2, respectively, 66 Vukovic B et al. / Neutron track etch detector for electrons we calculated the effective conversion factor as fol­lows: kce = kc1sc1 + kc2sc2 = 418 pSv cm2. Thus, since kc/rc= 25,75 kb/rb , and according to equation [2], one can see that neutrons of the given distribution in Figure 5 (behind the 1 m concrete barrier) contribute to a neutron dose 26 times more than neutrons from the distribution in Figure 4 (be­hind the 1.7 m concrete wall). Conclusions The neutron dosimeter, consisting of the LR-115 track etch detector and boron foil BN-1, was cali­brated on thermal neutrons generated in the nucle­ar reactor of the J. Stefan Institute (Slovenia), and was applied to dosimetry of undesirable neutrons in photon radiotherapy with the Siemens Mevatron 15 MV electron linear accelerator. Having considered a broad neutron spectrum of energies in the photon beam and high dependence of the track detector response on neutron energy, we divided the spectrum in the two energy areas, below and over 2 MeV. Afterwards we determined the detector responses (r) for the energy areas us­ing corresponding cross-sections for neutron and boron, and then we calculated the pondered or ef­fective response depending on surfaces under the respective neutron spectrum areas. Using the em­pirical curve k(E), we performed the similar proce­dure for determining an effective conversion coef­ficient (k) from neutron fluence to dose equivalent, depending on neutron energy (E). The relative measurement errors made by track etching method with the LR-115 detector were about 1%. It is to mention that we took the data from the empirical curves (e.g. s(E), k(E) ) as aver­age values without respective experimental errors. The measurement of the neutron dose equiva­lent by the track etch detector, positioned outside the Siemens Mevatron 15 MV room (room wall of 1.7 m, density of 3200 kg/m3), gave the dose rate of 0.62 mSv/h. Observing the neutron spectrum attenuation from 15 MV Mevatron behind the 1m concrete barrier, one was able to notice that the neutrons (Figure 5) had higher energies and contributed to a neutron dose 26 factor times more than neutrons from the distribution in Figure 4 (behind the 1.7 concrete wall). References 1. Strojnik A. In search of the shortest regimen: fractionation of a fully isoef­fective combination of hyperfractionated and hypofractionated treatment. Radiol Oncol 2008; 42: 170-2. 2. Facure A, Falcăo RC, Silva AX, Crispim VR, Vitorelli JC. A study of neutron spectra from medical linear accelerators. Appl Radiat Isotop 2005; 62: 69­72. 3. D Ryan C Rivest, Terence A Riauka, Albert D Murtha, B Gino Fallone. Dosimetric implications of two registration based patient positioning me­thods 203 in prostate image guided radiation therapy (IGRT). Radiol Oncol 2009; 43: 203-12. 4. Ongaro C, Zanini A, Nastasi U, Rodenas J, Ottaviano G, Manfredotti C. Analysis of photoneutron spectra produced in medical accelerators. Phys Med Biol 2005; 45: 55-61. 5. Králík M, Turek K. Characterisation of neutron fields around high-energy x-ray radiotherapy. Radiat Prot Dosimetry 2004; 110: 503-7. 6. Rivera JC, Falcăo RC, deAlmeida CE. The measurement of photoneutron dose in the vicinity of clinical linear accelerators. Radiat Prot Dosimetry 2008; 130: 403-9. 7. Izerrouken M, Skvarc J, Ilic R. A wide range personnel neutron dosimeter. Radiat Meas 2003; 37: 21-4. 8. Lilley J. Nuclear Physics. Chichester: John Wiley & Sons; 2001. p. 196. 9. Vukovic B, Radolic V, Lisjak I, Vekic B, Poje M, Planinic J. Some cosmic ra­diation dose measurements aboard flights connecting Zagreb Airport. Appl Radiat Isotop 2008; 66: 247-51. 10. Lopez Ponte MA, Castellani CM, Currivan L, et al. A catalogue of dosimeters and dosimetric services within Europe—an update. Radiat Protec Dosim 2004; 112: 45-68. 11. Schraube H, Kneschaurek P, Schraube G, Wagner FM, Weitzenegger E. Neutron spectra around medical treatment facilities. Nucl Instrum Meth A 2002; 476: 463-7. Radiol Oncol 2010; 44(1): 1-12. doi:10.2478/v10019-010-0008-x Vloga radioterapije pri zdravljenju melanoma Strojan P Izhodišca. V primeru melanoma je bila radioterapija dolgo casa obravnavana kot paliativna možnost zdravljenja in kot taka indicirana le za zdravljenje napredovalih ali sistemsko razširjenih primerov bolezni. V 70-ih letih preteklega sto­letja je tehnološki napredek v radioterapiji, ki je bil tesno povezan z napredkom racunalništva, prispeval k obujenemu zanimanju za radioterapijo tudi pri zdravljenju melanoma. Ceprav je pomanjkanje dobro zasnovanih prospektivnih in/ ali randomiziranih klinicnih raziskav pomembno zaznamovalo vkljucevanje radioterapije v obstojece nacine zdravlje­nja melanoma, je v zadnjem obdobju radioterapija prepoznana kot nepogrešljiv del multidisciplinarne obravnave bolnikov s to boleznijo. Kar približno 23% bolnikov z melanomom naj bi bilo med svojo boleznijo obsevanih vsaj enkrat. V tem pregledu so opisane radiobiološke znacilnosti melanoma, ki narekujejo nacine frakcionacije, ki so v rabi pri tej bolezni. Poleg tega je podan pregled indikacij za obsevanje in pregled rezultatov klinicnih raziskav iz literature, ki so dale osnovo za uporabo radioterapije pri melanomu, ter kratek opis radioterapevtskih tehnik. Zakljucki. Temeljno zdravljenje pri melanomu je kirurško. Vendar takšno zdravljenje potrebuje dopolnilo, ko kirurški poseg ni radikalen ali je pri histopatološkem pregledu odstranjenega tkiva ugotovljena prisotnost neugodnih napove­dnih dejavnikov. Tudi pri bolnikih z neresektabilno boleznijo ali pri takih, ki niso sposobni za vecji kirurški poseg oziroma ga odklanjajo, obstaja potreba po drugih ucinkovitih nacinih zdravljenja. Iz tega gledišca predstavlja radioterapija pomembno možnost: je ucinkovita in varna, tako pri zdravljenju z namenom ozdravitve kot v paliativne namene, kar potrjujejo klinicne izkušnje, kot tudi podatki iz strokovne literature. Radiol Oncol 2010; 44(1): 13-18. dol:10.2478/v10019-010-0007-y Genetski oznacevalci pri oligodendrogliomih Velnar T, Smrdel U, Popovic M, Bunc G Izhodišca. Oligodendrogliomi so po pogostosti pojavljanja med možganskimi tumorji na tretjem mestu. Zajemajo 2,5% primarnih možganskih tumorjev in 5-20% vseh gliomov. Sestavljajo jih celice, podobne oligodendrocitom. Zakljucki. Mutacije lokusov 1p in 19q se v oligodendrogliomih pogosto pojavljajo in so v histološko nejasnih primerih pomembne za tocno diagnozo. So neodvisen ugodni napovedni dejavnik poteka bolezni in hkrati napovedujejo vecjo obcutljivost na kemoterapevtsko zdravljenje. Ugotavljanje omenjenih mutacij je izjemnega pomena pri nacrto­vanju zdravljenja, ker imajo bolniki z mutacijami lokusov 1p in 19q znatno daljše preživetje. Radiol Oncol 2010; 44(1): I-V. Radiol Oncol 2010; 44(1): 19-23. doli:10.2478/v10019-010-0012-1 Odkrivanje raka debelega crevesja in danke z racunalniško tomografsko kolonografijo Sofic A, Bešlic Š, Kocijancic I, Šehovic N Izhodišca. Diagnosticne metode pri odkrivanju in presejanju kolorektalnega raka so: rektalni klinicni pregled, test bla­ta na okultno krvavitev, rektoskopija, analiza DNA blata, irigografija, kolonoskopija in zadnjih nekaj let CT kolonografija. Namen raziskave je bil ugotoviti diagnosticno zanesljivost CT kolonografije in pocutje bolnikov ob preiskavi v primerjavi s klasicno kolonoskopijo in irigografijo. Bolniki in metode. V prospektivno raziskavo smo vkljucili 231 bolnikov, pri vseh smo naredili omenjene tri preiskave, ki smo jih želeli primerjati. Po opravljenih preiskavah so bolniki izpolnili vprašalnik, kako so se pocutili med preiskavami. Diagnosticno pozitivne rezultate smo potrdili s patohistološkimi vzorci, odvzetimi pri kolonoskopiji. Za vsako preiskavo smo izracunali obcutljivost in specificnost ter dolocili njihovo pozitivno in negativno napovedno vrednost. Rezultati. Z vsemi tremi preiskavami smo odkrili 95 patoloških sprememb, 56 (59%) tumorjev in 39 (41%) polipov. Izmed polipov je patohistološka preiskava pokazala 34 adenomov, 3 tubuloviloznih adenomov in 2 lipoma. Med malignimi tumorji je bilo 55 adenokarcinomov in 1 limfom. Za odkrivanje polipov je CT kolonografija pokazala 89,7% obcutljivost, irigografija 48,7%, in kolonoskopija 94,9% obcutljivost. Za tumorje sta bili CT kolonografija in irigografija 100% obcutljivi, irigografija pa 94.6%. Specificnost in pozitivna napovedna vrednost sta bili za vse preiskave 100%. Vprašalnik o pocutju med preiskavami je pokazal, da je CT kolonografija za bolnike izrazito najbolj udobna preiskava. Zakljucki. CT kolonografija je statisticno znacilno za bolnike najbolj udobna preiskava debelega crevesja in je zna-cilno bolj obcutljiva za odkrivanje polipov kot irigografija. Ob intraluminalnih spremembah lahko s CT kolonografijo pri­kažemo tudi spremembe ob lumnu debelega crevesja in nekatere druge nakljucno odkrite spremembe v abdomnu. Metoda je zelo uporabna za presejanje raka debelega crevesja in za oceno stadija pred zdravljenjem. Radiol Oncol 2010; 44(1): 24-29. doi:10.2478/v10019-010-0001-4 Razlikovanje hemangiomov od zasevkov v jetrih z magnetnoresonancnim difuzijskim slikanjem Inan N, Kilinc F, Sarisoy T, Gumustas S, Akansel G, Demirci A Izhodišca. Namen raziskave je bil ugotoviti pomen magnetnoresonancnega difuzijskega slikanja pri razlikovanju hemangiomov od zasevkov v jetrih. Bolniki in metode. V retrospektivni raziskavi smo pri 38 bolnikih analizirali 69 žarišcnih sprememb v jetrih (33 heman­giomov in 36 zasevkov). Slikanje z difuzijo je bilo narejeno med zadržanim vdihom s t.i. tehniko single-shot-echo-shot echo-planar spin eho sekvenco s tremi razlicnimi b vrednostmi (0, 500 in 1000 sek/mm2). Izracunali smo pripadajoce difuzijske koeficiente. Za kvantitativno oceno smo med skupinama primerjali intenzitete signala v žarišcnih spremebah, razmerja v intenziteti signala med žarišcnimi spremembami in jetri, difuzijske koeficiente žarišcnih sprememb ter razmerja difuzijskih koeficientov med žarišcnimi spremembami in jetri. Za statisticno analizo smo uporabili Studentov test. Rezultati. Pri b vrednosti 500 sec/mm2 nismo dokazali statisticno znacilne razlike (p>0,05). Pri b vrednosti 1000 sek/ mm2 sta bili obe – intenziteta signala ter razmerje med intenziteto signala med žarišcnimi spremembami in jetri – pri zasevkih znacilno višji kot pri hemangiomih (p<0,001). Pri mejni vrednosti 2,6 za razmerje intenzitet med žarišcnimi spre­membami in jetri sta bili obcutljivost 86% in specificnost 82%. Difuzijski koeficienti ter razmerje difuzijskih koeficientov med žarišcnimi spremembami in jetri so bili za zasevke znacilno nižje kot za hemangiome (p<0,001). Pri mejni vrednosti 1,7 za razmerje difuzijskih koeficientov med žarišcnimi spremembami in jetri je bila obcutljivost 88% in specificnost 72%. Zakljucki. Magnetnoresonancno difuzijsko slikanje z visoko b vrednostjo pomaga razlikovati zasevek od hemangi­oma v jetrih. Radiol Oncol 2010; 44(1): I-V. Radiol Oncol 2010; 44(1): 30-33. doi:10.2478/v10019-010-0011-2 Perkutana transkateterska arterijska embolizacija tope poškodbe vranice pri hemodinamsko stabilnem pacientu Popovic P, Stanisavljevic D, Jeromel M Izhodišca. Neoperativno zdravljenje tope poškodbe vranice pri hemodinamsko stabilnem bolniku je v zadnjih letih postalo sprejemljiv nacin obravnave bolnika. Predstavljamo primer tope poškodbe vranice uspešno zdravljene s su­praselektivno embolizacijo z majhnimi delci. Prikaz primera. Na Urgentni oddelek je bil pripeljan mlad igralec hokeja z anamnezo tope poškodbe trebuha, ki je nastala pred dvema urama. Z ultrazvocno preiskavo smo ugotovili poškodbo vranice III. stopnje s hematoperitonejem. Bolnika smo zdravili s selektivno embolizacijo distalne vranicne arterije z majhnimi delci. Kontrolna ultrazvocna in racunal­niško tomografska preiskava po posegu in po enem letu sta pokazali zgolj majhno podrocje nepravilnosti v parenhimu. Zakljucki. Perkutana arterijska embolizacija vranice ima pomembno vlogo pri zdravljenju mehanskih poškodb vra-nice. Embolizacija je zlasti koristna pri poškodbah III. ali višje stopnje. Radiol Oncol 2010; 44(1): 34-41. doi: 10.2478/v10019-010-0002-3 Numericno modeliranje vpliva oblike elektroporacijskega elektricnega pulza na vnos molekul v celico Miklavcic D, Towhidi L Izhodišce. Elektrokemoterapijo – kombinacijo elektroporacije in kemoterapije – so uvedli zaradi želje, da bi povecali lokalno ucinkovitost nekaterih kemoterapevtikov in zmanjšali stranske ucinke. Elektroporacija, ki jo izzovemo z uporabo ustreznih elektricnih pulzov, povzroci znantno povecan vnos nekaterih molekul, ki sicer ne prehajajo plazmaleme, v celico. Elektroporacija in ucinkovitost transmembranskega molekulskega transporta sta odvisni od parametrov elek­tricnih pulzov. To odvisnost so že pred casom eksperimentalno pokazali razlicni avtorji, pri cemer pa je treba poudariti, da teoreticne osnove še niso v celoti znane in potrjene. V pricujoci študiji smo razvili model, s katerim smo raziskali vpliv oblike elektroporacijskega pulza na vnos molekul v celico z namenom napovedovanja izida elektroporacije celic. Rezultati. Predstavljeni model temelji na že prej znanem kemicno-kineticnem opisu elektroporacije ob upoštevanju trapezne prepreke pore in samokonsistentnosti. Model smo nadgradili s transportom molekul prek plazmaleme, da smo tako lahko pridobili podatek o kolicini vnosa molekul v celico. Raziskali smo elektroporacijo celic z razlicnimi obli­kami elektroporacijskih pulzov – monopolarnih pravokotnih pulzov z razlicnimi casi vzpona in upada pulza, bipolarnimi pulzi trikotne, pravokotne in sinusne oblike, ter uniporalne pulze z amplitudno modulacijo razlicnih stopenj s sinusnim signalom. Rezultati, ki smo jih dobili z uporabo opisanega modela in elektroporacijskih pulzov razlicnih oblik, se dobro ujemajo z že pred casom objavljenimi ekperimentalnimi rezultati dobljenimi v in vitro pogojih z enakimi oblikami elek­troporacijskih pulzov. Zakljucki. Predstavljeni model je uporaben za opis in napovedovanje ucinka elektroporacijskega pulza poljubne oblike na plazmalemo, spremembo njene prevodnosti in transporta molekul preko plazmaleme ter posledicnim vno­som molekul v celico. Radiol Oncol 2010; 44(1): I-V. Radiol Oncol 2010; 44(1): 42-51. doi:10.2478/v10019-010-0010-3 Razvoj celicnega biosenzorskega sistema za zaznavanje genotoksicnosti na osnovi izražanja gena, ki se odziva na poškodbe DNA Žager V, Cemažar M, Hreljac I, Lah TT, Serša G, Filipic M Izhodišca. Izpostavljenost cloveka genotoksicnim snovem v okolju in vsakodnevnem življenju resno groža zdravje lju­di. Hitra in zanesljiva ocena genotoksicnosti kemikalij je kljucnega pomena pri razvoju novih kemikalij in zdravil, kot tudi pri opazovanju okolja. Tumorski supresorski gen p21 je glavni ciljni gen aktiviranega proteina p53, ki je odgovoren za ustavitev celicnega ciklusa po poškodbi DNK. Ugotovili so, da genotoksicni karcinogeni specificno povecajo njegovo izražanje. Cilj naše raziskave je bil razviti celicni biosenzorski sistem za enostavno in hitro odkrivanje genotoksicnih snovi. Metode. Metabolno aktivne cloveške jetrne celice (HepG2) so bile genetsko modificirane (transfecirane) s plazmi­dom. Plazmid pa pod kontrolo promotorja za gen p21 (p21HepG2 EGFP) nosi zapis za zeleno fluorescirajoci protein -Enhanced Green Fluorescent Protein (EGFP). Poškodbe DNA smo povzrocili z genotoksicnimi snovmi z znanimi meha­nizmi delovanja. Povecano intenzivnost fluorescence zaradi povecanega izražanja EGFP pod vplivom p21 smo merili s spektrofluorimetrom za mikrotitrske plošce. Živost tretiranih celic smo dolocili s kalorimetricnim MTS testom. Rezultati. Neposredno delujoca alkilirajoca snov methylmethane sulphonate (MMS) je povzrocila statisticno znacil-no povecanje tvorbe EGFP po 48 urah pri 20 µg/mL. Posredno delujoc rakotvoren benzo(a)pyren (BaP) in navzkrižno povezovalna snov cisplatin (CisPt) sta povzrocila dozno odvisno povecanje EGFP fluorescence. To povecanje je bilo statisticno znacilno že pri koncentracijah 0,13 µg/mL oziroma 0,41 µg/mL. Vinblastin (VLB), inhibitor delitvenega vre­tena, ki ne inducira direktnih poškodb DNK, je povzrocil le majhen porast intenzivosti EGFP fluorescence po 24 urah pri najmanjši koncentraciji (0,1 µg/mL), medtem ko je bila izpostavljenost višjim koncentracijam povezana z znacilno zmanjšanim preživetjem celic. Zakljucki. Rezultati naše raziskave so pokazali, da je nov test, ki temelji na stabilno preoblikovani celicni liniji p21He­pG2EGFP, primeren za hitro in enostavno odkrivanje poškodb genetskega materiala, ki jih povzrocajo kemicne snovi. Radiol Oncol 2010; 44(1): 52-56. doi:10.2478/v10019-010-0009-9 Odnos študentk babištva do poucevanja samopregledovanja dojk Plesnicar A, Golicnik M, Kirar Fazarinc I, Kralj B, Kovac V, Kores Plesnicar B Izhodišca. Namen raziskave je bil ugotoviti, kakšen je odnos študentk dodiplomskega študija babištva do pouceva­ nja samopregledovanja dojk. Udeleženke in metode. Raziskava je potekala ob zacetku in na koncu študija na Fakulteti za zdravstvene vede v Ljubljani, v akademskem letu 2002/2003. Vanjo je bilo vkljucenih 28 študentk prvega in 25 študentk tretjega letnika do-diplomskega študija. Podatki z izpolnjenih vprašalnikov so bili zbrani in analizirani s pomocjo deskriptvinih in inferencnih statisticnih metod. Rezultati. Vse udeleženke raziskave so bile mnenja, da je poucevanje drugih žensk o samopregledovanju dojk zelo pomembno za zgodnje odkrivanje raka dojk in da bi tako poucevanje moralo biti ena od njihovih dolžnosti. Med skupinama študentk ni bilo statisticno znacilnih razlik glede pripravljenosti za izpopolnjevanje njihovega znanja samopregledovanja dojk ali glede optimizma v zvezi z napredkom odkrivanja in zdravljenja raka dojke v prihodnosti. Zakljucki. Pripravljenost študentk babištva, da svoje znanje samopregledovanja dojk posredujejo drugim ženskam, je povezano z njihovo ozavešcenostjo o zdravju dojk. S tem bodo lahko prispevale k zgodnjemu odkrivanju sprememb v dojkah ter omogocile njihovo zgodnjo diagnostiko in ucinkovito zdravljenje. Radiol Oncol 2010; 44(1): I-V. Radiol Oncol 2010; 44(1): 57-61. doi:10.2478/v10019-010-0005-0 Genetsko testiranje za ugotavljanje raka debelega crevesa in danke pri mladih: prikaz primera in na izsledkih temeljece smernice Zhou Y, Boardman LA, Miller RC Izhodišca. Rak debelega crevesa in danke, ki nastane pri mlajših, se klinicnopatološko razlikuje od takšnega raka pri starejših. Odkrivamo ga pri bolnikih z dednimi boleznimi, kot sta sindrom Lynch in familiarna adenomatoidna polipoza. Prikaz primera. Opisujemo primer 44-letnega bolnika. Njegov oce je imel polipe debelega crevesa, bolnik pa je 2 leti odvajal svetlordec krvav feces. Diagnosticirali smo rak debelega crevesa, stadij IIIA. Ob genetskem svetovanju smo bolnika tudi genetsko testirali, vendar nismo ugotovili najpogostejših familiarnih sindromov povezanih z rakom. Bolnika smo zdravili z neoadjuvantno kemoradioterapijo, operacijo in adjuvantno kemoterapijo. Po šestih letih nismo ugotovili ponovitve bolezni. Zakljucki. Pri mlajših bolnikih z rakom debelega crevesa in danke svetujemo ugotavljanje morebitnih familiarnih sin-dromov povezanih z rakom in genetsko testiranje. Opisani primer kaže na pomembnost presejalnih testov, ki morajo temeljiti na izsledkih. Radiol Oncol 2010; 44(1): 62-66. doi: 10.2478/v10019-010-0003-2 Detektor sledi za dozimetrijo nevtronov iz linearnih elektronskih pospeševalnikov v radioterapiji Vukovic B, Faj D, Poje M, Varga M, Radolic V, Miklavcic I, Ivkovic A, Planinic J Izhodišca. Linearni elektronski pospeševalniki so v radioterapiji zamenjali kobaltove in cezijeve izvore sevanja. Vendar pospeševalniki s fotonskimi energijami nad 10 MeV proizvajajo neželeno kontaminacijo fotonskega snopa s hitrimi nev­troni. Med fotoni z energijami nad 10 MeV lahko prihaja do interakcije z atomskimi jedri snovi z visokim vrstnim številom. Ti sestavljajo tarco in glavo pospeševalnika, kar vodi do izsevanja nevtronov. Rezultati in zakljucki. Nevtronski dozimeter sestavljata detektor sledi LR-115 in pretvornik BN-1 in so ga umerili s termalnimi nevtroni iz jedrskega reaktorja v Inštitutu Jožef Stefan (Slovenija). Uporabili smo ga za dozimetrijo neželenih nevtronov v fotonskem snopu iz linearnega pospeševalnika 15 MV Siemens Mevatron. Upoštevali smo visoko odvisnost preseka reakcije med nevtronom in borom od energije nevtrona ter širok spekter nevtronov tako v fotonskem žarku kot tudi pred vhodnimi vrati labirinta k Mevatronu. Na ta nacin smo razvili metodo dolocanja efektivnega odziva nev­tronskega detektorja. Izmerjena nevtronska hitrost doze v fotonskem žarku je bila 1.96 Sv/h. Zunaj sobe z Mevatronom je bila nevtronska hitrost doze 0.62 mSv/h. PACS: 87.52. Ga; 87.53.St; 29.40.Wk Radiol Oncol 2010; 44(1): I-V. Fundacija "Docent dr. J. Cholewa" je neprofitno, neinstitucionalno in nestrankarsko zdrućenje posameznikov, ustanov in organizacij, ki ćelijo materialno spodbujati in poglabljati raziskovalno dejavnost v onkologiji. Dunajska 106 1000 Ljubljana ĆR: 02033-0017879431 Activity of "Dr. J. Cholewa" Foundation for Cancer Research and Education – a report for the first quarter of 2010 One of the main activities of Dr. J. Cholewa Foundation for Cancer Reasearch and Education is The Dr. J. Cholewa Foundation for Cancer Research and Education is of the opinion that the spread of knowledge about cancer among health professionals and population in general through cancer research, information about new approaches in cancer therapy and cancer education should be regarded as its main activity. Among the activities mentioned above, the Foundation continues to support the publication of "Radiology and Oncology” international medical scientific journal that is edited, published and printed in Ljubljana, Slovenia. “Radiology and Oncology” is an open access journal, available free of charge on its own website. In this way, the Foundation indirectly complements the approaches mentioned previously, with the spread of information supporting the ever increasing number of patients with various types of cancer in Slovenia. In ad­dition, the Foundation is therefore also active in increasing the amount of scientific information about various aspects of cancer among the scientists in different fields with a budding interest in cancer research. One of the main activities of Dr. J. Cholewa Foundation for Cancer Research and Education is to carefully asses the requests for research grants and scholarships submitted by experts in on­cology and other scientific activities in Slovenia. The Foundation’s opinion is that the advances in cancer research, therapy and education must not be hindered for the simple lack of financial support. Within its possibilities, the Foundation supports the implementation of all advances in cancer therapy and education into everyday hospital, ambulatory and health promotion practice. Hopefully, the results of cancer research will thus find their way into the practical application across Slovenia and elsewhere as quickly as possible. The Foundation hopes to successfully continue with its activities in 2010, leading to greater appli­cation of the latest cancer diagnostic, therapy and education methods and knowledge to everyday research, clinical and public environment in Slovenia. Borut Štabuc, MD, PhDAndrej Plesnicar, MD Tomaž Benulic, MD Zadeli smo pravo tar;o Izredno u;inkovito zdravljenje prvega reda pri nedrobnoceli;nem plju;nem raku z mutacijo EGFR Iressa je prva in edina tar;na monoterapija, ki dokazano podaljša preživetje brez napredovanja bolezni v primerjavi z dvojno kemoterapijo kot zdravljenje prvega reda pri bolnikih z napredovalim nedrobnoceli;nim plju;nim rakom z mutacijo EGFR. 1 IRESSA® (GEFITINIB) 1. Povzetek glavih zna;ilnosti zdravila Iressa (gefitinib). Junij 2009. SKRAJŠAN POVZETEK GLAVNIH ZNA:ILNOSTI ZDRAVILA Sestava' Filmsko obložene tablete vsebujejo 250 mg gefitiniba. Indikacije' zdravljenje odraslih bolnikov z lokalno napredovalim ali metastatskim nedrobnoceli;nim plju;nim rakom z aktivacijskimi mutacijami EGFR-TK Odmerjanje in na;in uporabe' Zdravljenje z gefitinibom mora uvesti in nadzorovati zdravnik, ki ima izkušnje z uporabo zdravil proti raku. Priporo;eno odmerjanje zdravila IRESSA je ena 250-mg tableta enkrat na dan. Tableto je mogo;e vzeti s hrano ali brez nje, vsak dan ob približno istem ;asu. Kontraindikacije' preob;utljivost za zdravilno u;inkovino ali katerokoli pomožno snov, dojenje Opozorila in previdnostni ukrepi' Pri 1,3 % bolnikov, ki so dobivali gefitinib, so opažali intersticijsko bolezen plju; (IBP). Ta se lahko pojavi akutno in je bila v nekaterih primerih smrtna. :e se bolniku poslabšajo dihalni simptomi, npr. dispneja, kašelj in zvišana telesna temperatura, morate zdravljenje z zdravilom IRESSA prekiniti in bolnika takoj preiskati. ;e je potrjena IBP, morate terapijo z zdravilom IRESSA kon;ati in bolnika ustrezno zdraviti. ;eprav so bile nepravilnosti testov jetrnih funkcij pogoste, so jih redko zabeležili kot hepatitis. Zato so priporo;ljive redne kontrole delovanja jeter. V primeru blagih do zmernih sprememb v delovanju jeter je treba zdravilo IRESSA uporabljati previdno. :e so spremembe hude, pride v poštev prekinitev zdravljenja. Zdravilo IRESSA vsebuje laktozo. Bolniki z redko dedno intoleranco za galaktozo, laponsko obliko zmanjšane aktivnosti laktaze ali malabsorpcijo glukoze/galaktoze ne smejo jemati tega zdravila. Bolnikom naro;ite, da morajo takoj poiskati zdravniško pomo;, ;e se jim pojavijo kakršnikoli o;esni simptomi, huda ali dolgotrajna driska, navzea, bruhanje ali anoreksija, ker lahko vse te posredno povzro;ijo dehidracijo. Medsebojno delovanje zdravile' Induktorji CYP3A4 lahko pove;ajo presnovo gefitiniba in zmanjšajo njegovo koncentracijo v plazmi. Zato lahko so;asna uporaba induktorjev CYP3A4 (npr. fenitoina, karbamazepina, rifampicina, barbituratov ali zeliš;nih pripravkov, ki vsebujejo šentjanževko\Hypericum perforatum) zmanjša u;inkovitost zdravljenja in se ji je treba izogniti. Pri posameznih bolnikih, ki imajo genotip slabih metabolizatorjev s CYP2D6, lahko zdravljenje z mo;nim zaviralcem CYP3A4 pove;a koncentracijo gefitiniba v plazmi. Na za;etku zdravljenja z zaviralcem CYP3A4 je treba bolnike natan;no kontrolirati glede neželenih u;inkov gefitiniba. Pri nekaterih bolnikih, ki so jemali varfarin skupaj z gefitinibom, so se pojavili zvišanje internacionalnega normaliziranega razmerja (INR) in\ali krvavitve. Bolnike, ki so;asno jemljejo varfarin in gefitinib, morate redno kontrolirati glede sprememb protrombinskega ;asa (P:) ali INR. Zdravila, ki ob;utno in dolgotrajno zvišajo pH v želodcu npr. zaviralci protonske ;rpalke in antagonisti H2, lahko zmanjšajo biološko uporabnost gefitiniba in njegovo koncentracijo v plazmi in tako zmanjšajo u;inkovitost. Redno jemanje antacidov, uporabljenih blizu ;asa jemanja zdravila IRESSA, ima lahko podoben u;inek. Neželeni u;inki' V kumulativnem naboru podatkov klini;nih preskušanj III. faze so bili najpogosteje opisani neželeni u;inki, ki so se pojavili pri ve; kot 20 % bolnikov, driska in kožne reakcije (vklju;no z izpuš;ajem, aknami, suho kožo in srbenjem). Neželeni u;inki se ponavadi pojavijo prvi mesec zdravljenja in so praviloma reverzibilni. Ostali pogostejši neželeni u;inki so' anoreksija, konjunktivitis, blefaritis in suho oko, krvavitev, npr. epistaksa in hematurija, intersticijska bolezen plju; (1,3 %), navzea, bruhanje, stomatitis, dehidracija, suha usta, nepravilnosti testov jetrnih funkcij, bolezni nohtov, alopecija, asimptomati;no laboratorijsko zvišanje kreatinina v krvi, proteinurija, astenija, pireksija. Vrsta in vsebina ovojnine' škatla s 30 tabletami po 250 mg gefitiniba Na;in izdajanja zdravila' samo na recept Datum priprave besedila' junij 2009 Imetnik dovoljenja za promet' AstraZeneca AB, S-151 85, Sodertalje, Švedska Pred predpisovanjem, prosimo, preberite celoten povzetek glavnih zna;ilnosti zdravila. Dodatne informacije so na voljo pri' AstraZeneca UK Limited, Podružnica v Sloveniji, Verovškova 55, 1000 Ljubljana, telefon' 01\51 35 600. Erbitux 5 mg/ml raztopina za infundiranje (skrajšana navodila za uporabo) Cetuksimab je monoklonsko IgG1 protitelo, usmerjeno proti receptorju za epidermalni rastni faktor (EGFR). Terapevtske indikacije: Zdravilo Erbitux je indicirano za zdravljenje bolnikov z metastatskim kolorektalnim rakom in nemutiranim tipom KRAS; v kombinaciji s kemoterapijo in kot samostojno zdravilo pri bolnikih, pri katerih zdravljenje z oksaliplatinom in irinotekanom ni bilo uspešno. Zdravilo Erbitux je indicirano za zdravljenje bolnikov z rakom skvamoznih celic glave in vratu; v kombinaciji z radioterapijo za lokalno napredovalo bolezen in v kombinaciji s kemoterapijo na osnovi platine za ponavljajoco se in/ali metastatsko bolezen. Odmerjanje in nacin uporabe: Zdravilo Erbitux pri vseh indikacijah infundirajte enkrat na teden. Zacetni odmerek je 400 mg cetuksimaba na m2 telesne površine. Vsi naslednji tedenski odmerki so vsak po 250 mg/m2. Kontraindikacije: Zdravilo Erbitux je kontraindicirano pri bolnikih z znano hudo preobcutljivostno reakcijo (3. ali 4. stopnje) na cetuksimab. 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 (= 3. stopnje po kriterijih US National Cancer Institute, Common Toxicity Criteria; NCI-CTC), morate prekiniti terapijo s cetuksimabom. Z zdravljenjem smete nadaljevati le, ce se je reakcija pomirila do 2. stopnje. Priporoca se dolocanje koncentracije elektrolitov v serumu pred zdravljenjem in periodicno med zdravljenjem s cetuksimabom. Po potrebi se priporoca nadomešcanje elektrolitov. 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 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: Opazili so progresivno zniževanje nivoja magnezija v serumu, ki pri nekaterih bolnikih povzroca hudo hipomagneziemijo. Glede na resnost so opazili tudi druge elektrolitske motnje, vecinoma hipokalciemijo ali hipokaliemijo. Posebna navodila za shranjevanje: Shranjujte v hladilniku (2 °C -8 °C). Ne zamrzujte. Vrsta ovojnine in vsebina: 1 viala po 20 ml ali 100 ml. Imetnik dovoljenja za promet: Merck KGaA, 64271 Darmstadt, Nemcija. Podrobne informacije o zdravilu so objavljene na spletni strani Evropske agencije za zdravila PM-ONC-02/09/10.04.2009 (EMEA) http://www.emea.europa.eu. Dodatne informacije so vam na voljo pri: Merck d.o.o., Dunajska cesta 119, 1000 Ljubljana, tel.: 01 560 3810, faks: 01 560 3831, el. pošta: info@merck.si www.oncology.merck.de Povzetek glavnih znacilnosti zdravila Ime zdravila: Temodal 20 mg, 100 mg, 140mg, 180 mg, 250 mg, Temodal 2,5 mg/ml prašek za raztopino za infundiranje Kakovostna in kolicinska sestava: Vsaka kapsula zdravila Temodal vsebuje 20 mg, 100 mg, 140 mg, 180 mg ali 250 mg temozolomida. Ena viala vsebuje 100 mg temozolomida Po rekonstituciji 1 ml raztopine za infundiranje vsebuje 2,5 mg temozolomida. Pomožna snov: Ena viala vsebuje 2,4 mmol natrija. Terapevtske indikacije: Zdravilo Temodal 2,5 mg/ml je indicirano za zdravljenje: odraslih bolnikov z novo diagnosticiranim multiformnim glioblastomom, socasno z radioterapijo (RT) in pozneje kot monoterapija in otrok, starih 3 leta in vec, mladostnikov in odraslih bolnikov z malignimi gliomi, npr. multiformnimi glioblastomi ali anaplasticnimi astrocitomi, ki se po standardnem zdravljenju ponovijo ali napredujejo. Odmerjanje in nacin uporabe: Zdravilo Temodal 2,5 mg/ml smejo predpisati le zdravniki, ki imajo izkušnje z zdravljenjem možganskih tumorjev. Odrasli bolniki z novo diagnosticiranim multiformnim glioblastomom Zdravilo Temodal 2,5 mg/ml se uporablja v kombinaciji z žarišcno radioterapijo (faza socasne terapije), temu pa sledi do 6 ciklov monoterapije (monoterapijska faza) z temozolomidom (TMZ). Faza socasne terapije TMZ naj bolnik jemlje v odmerku 75 mg/m2 na dan 42 dni, socasno z žarišcno radioterapijo (60 Gy, danih v 30 delnih odmerkih). Zmanjševanje odmerka ni priporoceno, vendar se boste vsak teden odlocili o morebitni odložitvi jemanja TMZ ali njegovi ukinitvi na podlagi kriterijev hematološke in nehematološke toksicnosti. TMZ lahko bolnik jemlje ves cas 42-dnevnega obdobja socasne terapije (do 49 dni), ce so izpolnjeni vsi od naslednjih pogojev: • absolutno število nevtrofilcev (ANC – Absolute Neutrophil Count) = 1,5 x 109/l; • število trombocitov = 100 x 109/l; • skupna merila toksicnosti (SMT) za nehematološko toksicnost = 1. stopnje (z izjemo alopecije, navzee in bruhanja). Med zdravljenjem morate pri bolniku enkrat na teden pregledati celotno krvno sliko. Faza monoterapije Štiri tedne po zakljucku faze socasnega zdravljenja s TMZ in RT naj bolnik jemlje TMZ do 6 ciklov monoterapije. V 1. ciklu (monoterapije) je odmerek zdravila 150 mg/m2 enkrat na dan 5 dni, temu pa naj sledi 23 dni brez terapije. Na zacetku 2. cikla odmerek povecajte na 200 mg/m2, ce je SMT za nehematološko toksicnost za 1. cikel stopnje = 2 (z izjemo alopecije, slabosti in bruhanja), absolutno število nevtrofilcev (ANC) = 1,5 x 109/l in število trombocitov = 100 x 109/l. Ce odmerka niste povecali v 2. ciklu, ga v naslednjih ciklih ne smete povecevati. Ko pa odmerek enkrat povecate, naj ostane na ravni 200 mg/m2 na dan v prvih 5 dneh vsakega naslednjega cikla, razen ce nastopi toksicnost. Zmanjšanje odmerka in ukinitev zdravila med fazo monoterapije opravite, kot je opisano v preglednicah 2 in 3. Med zdravljenjem morate 22. dan pregledati celotno krvno sliko (21 dni po prvem odmerku TMZ). Odrasli in pediatricni bolniki, stari 3 leta ali vec, s ponavljajocim se ali napredujocim malignim gliomom:Posamezen cikel zdravljenja traja 28 dni. Bolniki, ki še niso bili zdravljeni s kemoterapijo, naj jemljejo TMZ v odmerku 200 mg/m2 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 zacetni odmerek 150 mg/m2 enkrat na dan, v drugem ciklu pa se poveca na 200 mg/m2 enkrat na dan 5 dni, ce ni bilo hematoloških toksicnih ucinkov. Kontraindikacije: Preobcutljivost za zdravilno ucinkovino ali katerokoli pomožno snov. Preobcutljivost za dakarbazin (DTIC). Posebna opozorila in previdnostni ukrepi: Pljucnica, ki jo povzroca Pneumocystis carinii Pilotno preskušanje podaljšane 42-dnevne sheme zdravljenja je pokazalo, da pri bolnikih, ki so socasno prejemali TMZ in RT, obstaja še posebej veliko tveganje za nastanek pljucnice zaradi okužbe s Pneumocystis carinii (PCP). Malignosti Zelo redko so porocali tudi o primerih mielodisplasticnega sindroma in sekundarnih malignostih, vkljucno z mieloidno levkemijo. Antiemeticno zdravljenje Navzea in bruhanje sta pogosto povezana z zdravljenjem s TMZ. Antiemeticno zdravljenje se lahko da pred uporabo TMZ ali po njej. Odrasli bolniki z novo diagnosticiranim multiformnim glioblastomom Antiemeticna profilaksa je priporocljiva pred zacetnim odmerkom socasne faze in je mocno priporocljiva med fazo monoterapije. Ponavljajoci se ali napredujoci maligni gliom Pri bolnikih, ki so mocno bruhali (stopnja 3 ali 4) v prejšnjih ciklih zdravljenja, je potrebno antiemeticno zdravljenje. Laboratorijske vrednosti Pred jemanjem zdravila morata biti izpolnjena naslednja pogoja za laboratorijske izvide: ANC = 1,5 x 109/l in število trombocitov = 100 x 109/l. Na 22. dan (21 dni po prvem odmerku) ali v roku 48 ur od navedenega dne, morate pregledati celotno krvno sliko in jo nato spremljati vsak teden, dokler ni ANC > 1,5 x 109/l in število trombocitov > 100 x 109/l. Ce med katerimkoli ciklom ANC pade na < 1,0 x 109/l ali število trombocitov na < 50 x 109/l, morate odmerek zdravila v naslednjem ciklu zmanjšati za eno stopnjo (glejte poglavje 4.2). Stopnje odmerka so 100 mg/m2, 150 mg/m2 in 200 mg/m2. Najmanjši priporoceni odmerek je 100 mg/m2. Pediatricna uporaba Klinicnih izkušenj z uporabo TMZ pri otrocih, mlajših od 3 let, ni. Izkušnje z uporabo tega zdravila pri starejših otrocih in mladostnikih so zelo omejene. Starejši bolniki (stari > 70 let) Videti je, da je pri starejših bolnikih tveganje za nevtropenijo ali trombocitopenijo vecje, kot pri mlajših. Zato je pri uporabi zdravila TMZ pri starejših bolnikih potrebna posebna previdnost. Moški bolniki Moškim, ki se zdravijo s TMZ je treba svetovati, naj ne zaplodijo otroka še šest mesecev po prejetem zadnjem odmerku in naj se pred zdravljenjem posvetujejo o možnostih za shranitev zmrznjene sperme. Natrij To zdravilo vsebuje 2,4 mmol natrija na vialo. To je treba upoštevati pri bolnikih na nadzorovani dieti z malo natrija. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Študije medsebojnega delovanja so izvedli le pri odraslih. V loceni študiji 1. faze, socasna uporaba TMZ in ranitidina ni povzrocila spremembe obsega absorpcije temozolomida ali izpostavljenosti njegovem aktivnem presnovku monometiltriazenoimidazol karboksamidu (MTIK). Analiza populacijske farmakokinetike v preskušanjih 2. faze je pokazala, da socasna uporaba deksametazona, proklorperazina, fenitoina, karbamazepina, ondansetrona, antagonistov receptorjev H2 ali fenobarbitala ne spremeni ocistka TMZ. Socasno jemanje z valprojsko kislino je bilo povezano z majhnim, a statisticno pomembnim zmanjšanjem ocistka TMZ. Študij za dolocitev ucinka TMZ na presnovo ali izlocanje drugih zdravil niso izvedli. Ker pa se TMZ ne presnavlja v jetrih in se na beljakovine veže le v majhni meri, je malo verjetno, da bi vplival na farmakokinetiko drugih zdravil. Uporaba TMZ v kombinaciji z drugimi mielosupresivnimi ucinkovinami lahko poveca verjetnost mielosupresije. Neželeni ucinki: Pri bolnikih, ki se zdravijo s TMZ v kombinaciji z RT ali monoterapijo po RT zaradi novo diagnosticiranega multiformnega glioblastoma ali z monoterapijo pri bolnikih s ponavljajocim se ali napredujocim gliomom, so bili zelo pogosti neželeni ucinki podobni; slabost, bruhanje, zaprtje, neješcnost, glavobol in utrujenost. Pri bolnikih z novo diagnosticiranim glioblastomom multiforme na monoterapiji so zelo pogosto porocali o konvulzijah, medtem ko je bil izpušcaj opisan zelo pogosto pri bolnikih z novo diagnosticiranim multiformnim glioblastomom, ki so prejemali TMZ socasno z RT, ter pri tistih, ki so zdravilo prejemali v obliki monoterapije, pogosto pa pri tistih s ponavljajocim se gliomom. Pri obeh indikacijah so o vecini hematoloških neželenih reakcij porocali pogosto ali zelo pogosto. Imetnik dovoljenja za promet: Schering-Plough Europe, Rue de Stalle 73, Bruselj Belgija Nacin in režim izdaje zdravila: Zdravilo Temodal 20 mg, 100 mg, 140mg, 180 mg, 250 mg se izdaja na recept (Rp/Spec), Temodal 2,5 mg/ml prašek za raztopino za infundiranje pa je namenjeno uporabi samo v bolnišnicah (H). Datum priprave informacije: februar 2010 Literatura: 1 Povzetek temeljnih znacilnosti zdravila Temodal 2 Stupp R, et. al. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a 0233 Nova, intravenska oblika zdravila Temodal, za zdravljenje bolnikov z novo diagnosticiranim glioblastomom multiforme in bolnikov s ponavljajocim se ali napredujocim malignim gliomom.1 Resnicni napredek Pomembno izboljšanje preživetja potrjeno tudi ob daljšem spremljanju bolnikov.2 Dunajska 22, 1000 Ljubljana tel: 01 300 10 70 fax: 01 300 10 80 SKRAJ[AN POVZETEK GLAVNIH ZNA^ILNOSTI ZDRAVILA Samozastrokovnojavnost. Ime zdravila: Tarceva25mg/100mg/150mgfilmskoobloženetableteKakovostna in koli~inska sestava: Enafilmskoobloženatabletavsebuje25mg,100mgali150mgerlotiniba(voblikierlotinibijevegaklorida).Terapevtske indikacije:Nedrobnoceli~nirakplju~:ZdraviloTarcevajeindiciranozazdravljenjebolnikovzlokalnonapredovalimalimetastatskimnedrobnoceli~nimrakomplju~poneuspehuvsajenepredhodnekemoterapije.PripredpisovanjuzdravilaTarcevajetrebaupo{tevatidejavnike,povezanespodalj{animpreživetjem.Koristnegavplivanapodalj{anjepreživetjaalidrugihklini~nopomembnihu~inkovzdravljenjanisodokazalipribolnikihzEGFR-negativnimitumorji.Raktrebu{neslinavke:ZdraviloTarcevajevkombinacijizgemcitabinomindiciranozazdravljenjebolnikovzmetastatskimrakomtrebu{neslinavke.PripredpisovanjuzdravilaTarcevajetrebaupo{tevatidejavnike,povezanespodalj{animpreživetjem.Koristnegavplivanapodalj{anjepreživetjanisodokazalizabolnikezlokalnonapredovaloboleznijo.Odmerjanje in na~in uporabe: ZdravljenjezzdravilomTarcevamoranadzorovatizdravnikzizku{njamiprizdravljenjuraka.ZdraviloTarcevavzamemonajmanjenouropredzaužitjemhranealidveuripotem.Kadarjepotrebnoodmerekprilagoditi,gazmanj{ujemovkorakihpo50mg.Priso~asnemjemanjusubstratovinmodulatorjevCYP3A4bomordapotrebnaprilagoditevodmerka.PridajanjuzdravilaTarcevabolnikomzjetrnookvarojepotrebnaprevidnost.^esepojavijohudineželeniu~inkipridevpo{tevzmanj{anjeodmerkaaliprekinitevzdravljenjazzdravilomTarceva.UporabazdravilaTarcevapribolnikihshudojetrnoaliledvi~nookvaroterpriotrocihnipriporo~ljiva.Bolnikomkadilcemjetrebasvetovati,najprenehajokaditi,sajsoplazemskekoncentracijeerlotinibaprikadilcihmanj{ekotprinekadilcih.Nedrobnoceli~nirakplju~:Priporo~enidnevniodmerekzdravilaTarcevaje150mg.Raktrebu{neslinavke:Priporo~enidnevniodmerekzdravilaTarcevaje100mg,vkombinacijizgemcitabinom.Pribolnikih,prikaterihsekožniizpu{~ajvprvih4do8tednihzdravljenjanepojavi,jetrebaponovnopretehtatinadaljnjezdravljenjezzdravilomTarceva.Kontraindikacije: Hudapreob~utljivostzaerlotinibalikaterokolipomožnosnov.Posebna opozorila in previdnostni ukrepi: Mo~niinduktorjiCYP3A4lahkozmanj{ajou~inkovitosterlotiniba,mo~nizaviralciCYP3A4palahkopove~ajotoksi~nost.So~asnemuzdravljenjustemizdravilisejetrebaizogibati.Bolnikom,kikadijo,jetrebasvetovati,najprenehajokaditi,sajsoplazemskekoncentracijeerlotinibaprikadilcihzmanj{anevprimerjavisplazemskimikoncentracijamiprinekadilcih.Verjetnoje,dajevelikostzmanj{anjaklini~nopomembna.Pribolnikih,prikaterihseakutnopojavijonoviin/aliposlab{ajonepojasnjeniplju~nisimptomi,kotsodispneja,ka{eljinvro~ina,jezdravljenjezzdravilomTarcevatrebaprekiniti,doklerniznanadiagnoza.Bolnike,kiseso~asnozdravijozerlotinibomingemcitabinom,jetrebaskrbnospremljatizaradimožnostipojavatoksi~nosti,podobniintersticijskiplju~nibolezni.^ejeugotovljenaintersticijskaplju~nabolezen,zdraviloTarcevaukinemoinuvedemoustreznozdravljenje.Pripribližnopolovicibolnikov,kisosezdravilizzdravilomTarceva,sejepojaviladriska.Zmernodohudodriskozdravimozloperamidom.Vnekaterihprimerihbomordapotrebnozmanj{anjeodmerka.Vprimeruhudealidolgotrajnedriske,navzeje,anoreksijealibruhanja,povezanihzdehidracijo,jezdravljenjezzdravilomTarcevatrebaprekinitiindehidracijoustreznozdraviti.Ohipokaliemijiinledvi~niodpovedisoporo~aliredko.Posebnopribolnikihzdejavnikitveganja(so~asnojemanjedrugihzdravil,simptomi,boleznialidrugidejavniki,vklju~nozvisokostarostjo)moramo,~ejedriskahudaalidolgotrajnaoziromavodivdehidracijo,zdravljenjezzdravilomTarcevaprekinitiinbolnikomzagotovitiintenzivnointravenskorehidracijo.Dodatnojetrebapribolnikihsprisotnimtveganjemzarazvojdehidracijespremljatiledvi~nodelovanjeinserumskeelektrolite,vklju~noskalijem.PriuporabizdravilaTarcevasoporo~alioredkihprimerihjetrneodpovedi.Knjenemunastankujelahkopripomoglapredhodnoobstoje~ajetrnabolezenaliso~asnojemanjehepatotoksi~nihzdravil.Pritehbolnikihjetrebazatopremislitiorednemspremljanjujetrnegadelovanja.DajanjezdravilaTarcevajetrebaprekiniti,~esospremembejetrnegadelovanjahude.Bolniki,kiprejemajozdraviloTarceva,imajove~jetveganjezarazvojperforacijvprebavilih,kisojihopaziliob~asno.Pribolnikih,kiso~asnoprejemajozdravila,kizavirajoangiogenezo,kortikosteroide,nesteroidnaprotivnetnazdravila(NSAID)in/alikemoterapijonaosnovitaksanov,alisovpreteklostiimelipepti~niulkusalibolezendivertiklov,jetveganjeve~je.^epridedotega,jetrebazdravljenjezzdravilomTarcevadokon~noukiniti.Poro~aliso oprimerihkožnihboleznizmehurjiinlu{~enjemkože,vklju~nozzeloredkimiprimeri,kisonakazovalinaStevens-Johnsonovsindrom/toksi~noepidermalnonekrolizoinsobilivnekaterihprimerihsmrtni.ZdravljenjezzdravilomTarcevajetrebaprekinitialiukiniti,~esepribolnikupojavijohudeoblikemehurjevalilu{~enjakože.Zeloredkosoporo~alioprimerihperforacijealiulceracijeroženice;opazilisotudidrugeo~esnebolezni.ZdravljenjezzdravilomTarcevajetrebaprekinitialiukiniti,~esepribolnikihpojavijoakutneo~esnebolezni,kotjebole~inavo~eh,alisele-teposlab{ajo.Tabletevsebujejolaktozoinjihnesmemodajatibolnikomzredkimidednimistanji:intolerancozagalaktozo,laponskooblikozmanj{aneaktivnostilaktazealimalabsorpcijoglukoze/galaktoze. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Erlotinibsepriljudehpresnavljavjetrihzjetrnimicitokromi,primarnosCYP3A4invmanj{imerisCYP1A2.PresnovaerlotinibazunajjeterpotekasCYP3A4v~revesju,CYP1A1vplju~ihinCYP1B1vtumorskihtkivih.Zzdravilnimiu~inkovinami,kisepresnavljajostemiencimi,jihzavirajoalipasonjihoviinduktorji,lahkopridedointerakcij.Erlotinibjesrednjemo~anzaviralecCYP3A4inCYP2C8,kottudimo~anzaviralecglukuronidacijezUGT1A1in vitro.Prikombinacijiciprofloksacinaalimo~negazaviralca CYP1A2 (npr.fluvoksamina)zerlotinibomjepotrebnaprevidnost.Vprimerupojavaneželenihdogodkov,povezanihzerlotinibom,lahkoodmerekerlotinibazmanj{amo.Predhodnoaliso~asnozdravljenjezzdravilomTarcevanispremeniloo~istkaprototipovsubstratov CYP3A4,midazolamaineritromicina.Inhibicijaglukoronidacijelahkopovzro~iinterakcijezzdravili,kisosubstrati UGT1A1 inseizlo~ajosamopotejpoti.Mo~nizaviralci aktivnosti CYP3A4 zmanj{ajopresnovoerlotinibainzve~ajokoncentracijeerlotinibavplazmi.Priso~asnemjemanjuerlotinibainmo~nihzaviralcevCYP3A4jezatopotrebnaprevidnost.^ejetreba,odmerekerlotinibazmanj{amo,{eposebnopripojavutoksi~nosti.Mo~nispodbujevalci aktivnosti CYP3A4 zve~ajopresnovoerlotinibainpomembnozmanj{ajoplazemskekoncentracijeerlotiniba.So~asnemudajanjuzdravilaTarcevaininduktorjevCYP3A4sejetrebaizogibati.Pribolnikih,kipotrebujejoso~asnozdravljenjezzdravilomTarcevainmo~niminduktorjemCYP3A4jetrebapremislitiopove~anjuodmerkado300mgobskrbnemspremljanjunjihovevarnosti.Zmanj{anaizpostavljenostselahkopojavitudizdrugimiinduktorji,kotsofenitoin,karbamazepin,barbituratiali{entjanževka.^etezdravilneu~inkovinekombiniramozerlotinibom,jepotrebnaprevidnost.Kadarjemogo~e,jetrebarazmislitiodrugihna~inihzdravljenja,kinevklju~ujejomo~negaspodbujanjaaktivnostiCYP3A4.Bolnikom,kijemljejovarfarinalidrugekumarinske antikoagulante,jetrebarednokontroliratiprotrombinski~asaliINR.So~asnauporabazaviralcev P-glikoproteina,kotstaciklosporininverapamil,lahkovodivspremenjenoporazdelitevin/alispremenjenoizlo~anjeerlotiniba.Zaerlotinibjezna~ilnozmanj{anjetopnostipripHnad5.Zdravila, ki spremenijo pH v zgornjem delu prebavil,lahkospremenijotopnosterlotinibainposledi~nonjegovobiolo{kouporabnost.U~inkaantacidovnaabsorpcijoerlotinibanisoprou~evali,vendarjetalahkozmanj{ana,karvodivnižjeplazemskekoncentracije.Kombinacijierlotinibainzaviralcaprotonske~rpalkesejetrebaizogibati.^emenimo,dajeuporabaantacidovmedzdravljenjemzzdravilomTarcevapotrebna,jihjetrebajematinajmanj4urepredali2uripodnevnemodmerkuzdravilaTarceva.^erazmi{ljamoouporabiranitidina,moramozdravilijematilo~eno:zdraviloTarcevajetrebavzetinajmanj2uripredali10urpoodmerkuranitidina.V{tudijifazeIbnibilopomembnihu~inkovgemcitabina nafarmakokinetikoerlotiniba,pravtakonibilopomembnihu~inkoverlotinibanafarmakokinetikogemcitabina.Erlotinibpove~akoncentracijoplatine.Pomembnihu~inkovkarboplatina alipaklitakselanafarmakokinetikoerlotinibanibilo.Kapecitabin lahkopove~akoncentracijoerlotiniba.Pomembnihu~inkoverlotinibanafarmakokinetikokapecitabinanibilo.Neželeni u~inki: Zelo pogosti neželeni u~inki sokožniizpu{~ajindriska,kottudiutrujenost,anoreksija,dispneja,ka{elj,okužba,navzeja,bruhanje,stomatitis,bole~inavtrebuhu,pruritus,suhakoža,suhikeratokonjunktivitis,konjunktivitis,zmanj{anjetelesnemase,depresija,glavobol,nevropatija,dispepsija,flatulenca,alopecija,okorelost,pireksija.Pogosti neželeni u~inki sogastrointestinalnekrvavitve,krvaviteviznosu,nenormalnostitestovjetrnefunkcije,keratitis,zanohtnica.Redko soporo~aliojetrniodpovedi.Ob~asno pa operforacijahvprebavilih,pora{~enostimo{kegatipapriženskah,spremembahobrvi,krhkihnohtih,odstopanjunohtovodkože,blagihreakcijahnakoži(npr.hiperpigmentacija),spremembahtrepalnic,resniintersticijskiplju~nibolezni,vklju~nossmrtnimiprimeri.Zelo redko soporo~alioprimerih,kisonakazovalinaStevens-Johnsonovsindrom/toksi~noepidermalnonekrolizoinsobilivnekaterihprimerihsmrtni,teroulceracijahinperforacijahroženice.Režim izdaje zdravila: H/Rp.Imetnik dovoljenja za promet: RocheRegistrationLimited,6FalconWay,ShirePark,WelwynGardenCity,AL71TW,VelikaBritanija.Verzija: 2.0/09.Informacija pripravljena:januar2010. DODATNEINFORMACIJESONAVOLJOPRI:Rochefarmacevtskadružbad.o.o.Vodovodnacesta109,1000Ljubljana.Povzetekglavnihzna~ilnostizdravilajedosegljivnawww.roche.si. 002-10-TAR Lek farmacevtska družba d.d., Verovškova 57, 1526 Ljubljana, Slovenija, www.lek.si SKRAJŠAN POVZETEK GLAVNIH ZNACILNOSTI ZDRAVILA Epufen 12,5, 25, 50, 100 in 150 mikrogramov/uro transdermalni obliži SESTAVA: 1 transdermal­ni obliž vsebuje 2,89 mg, 5,78 mg 11,56 mg, 23,12 mg ali 34,65 mg fentanila. TERAPEVTSKE INDIKACIJE: Huda kronicna bolecina, ki se lahko ustrezno zdravi le z opioidnimi analgetiki. ODMERJANJE IN NACIN UPORABE: Odmerjanje je tre­ba individualno prilagoditi ter ga po vsaki uporabi redno oceniti. Izbira zacetnega odmerka: velikost odmerka fentanila je odvisna od predhodne uporabe opioidov, kjer se upošteva možnost pojava tolerance, socasnega zdravljenja, bolnikovega splošnega zdravstvenega stanja in stopnje resnosti obolenja. Pri bolnikih, ki pred tem niso dobivali mocnih opioidov, zacetni odmerek ne sme preseci 12,5-25 mi-krogramov na uro. Zamenjava opioidnega zdravljenja: pri zamenjavi peroralnih ali parenteralnih opioidov s fentanilom je treba zacetni odmerek izracunati na osnovi kolicine analgetika, ki je bila potrebna v zadnjih 24 urah, jo pretvoriti v odgovarjajoci odmerek morfina s pomocjo razpredelnice in nato preracunati ustrezen odmerek fentanila, spet s pomocjo razpredelnice (glejte SmPC). Prvih 12 ur po prehodu na transdermalni obliž Epufen bolnik še vedno dobiva predhodni analgetik v enakem odmerku kot prej; v naslednjih 12 urah se ta analgetik daje po potrebi. Titracija odmerka in vzdrževalno zdravljenje: obliž je treba zamenjati vsakih 72 ur. Odme­rek je treba titrirati individualno, dokler ni dosežen analgeticni ucinek. Odmerek 12,5 mikrogramov/uro je primeren za titriranje odmerka v manjšem odmernem ob-mocju. Ce analgezija na koncu zacetnega obdobja nošenja obliža ni zadostna, se lahko odmerek po 3 dneh zveca. Možno je, da bodo bolniki potrebovali obcasne dodatne odmerke kratko delujocih analgetikov (npr. morfina) za prekinitev bolecine. Sprememba ali prekinitev zdravljenja: vsaka zamenjava z drugim opioidom mora potekati postopoma, z majhnim zacetnim odmerkom in pocasnim zvecevanjem. Splošno veljavno pravilo je postopna ustavitev opioidne analgezije, da bi preprecili odtegnitvene simptome, kot so navzeja, bruhanje, diareja, anksioznost in mišicni tremor. Uporaba pri starejših bolnikih: starejše in oslabljene bolnike je treba skrb-no opazovati zaradi simptomov prevelikega odmerjanja ter odmerek po potrebi zmanjšati. Uporaba pri otrocih: transdermalni obliži Epufen se lahko uporabljajo le pri pediatricnih bolnikih (starih od 2 do 16 let), ki tolerirajo opioide in peroralno že dobivajo opioide v odmerku, enakovrednemu najmanj 30 mg morfina na dan. Bolnik mora prvih 12 ur po prehodu na Epufen še vedno dobivati predhodni anal-getik v enakem odmerku kot prej. V naslednjih 12 urah je treba ta analgetik dajati odvisno od klinicnih potreb. Titracija odmerka in vzdrževalno zdravljenje: ce je anal­geticni ucinek Epufena prešibak, je treba bolniku dodati morfin ali drugi opioid s kratkim delovanjem. Odvisno od dodatnih potreb po analgeziji in jakosti bolecine pri otroku se lahko uporabi vec obližev. Odmerek je treba prilagajati korakoma, po 12,5 mikrogramov/uro. Uporaba pri bolnikih z jetrno ali ledvicno okvaro: Zara-di možnosti pojava simptomov prevelikega odmerjanja je treba te bolnike skrbno spremljati in odmerek ustrezno zmanjšati. Uporaba pri bolnikih s povecano telesno temperaturo: Pri teh bolnikih bo morda treba prilagoditi odmerek. Nacin uporabe: transdermalni obliž Epufen je treba takoj po odprtju vrecke nalepiti na nerazdraženo, neobsevano kožo, na ravno površino prsnega koša, zgornjega dela hrbta ali nadlakti. Po odstranitvi zašcitne plasti je treba obliž trdno pritrditi na izbrano mesto in z dlanjo pritiskati približno 30 sekund, da se obliž popolnoma nalepi, še zlasti na robovih. Uporaba pri otrocih: pri mlajših otrocih je obliž priporocljivo nalepiti na zgornji del hrbta, ker je manjša verjetnost, da bi otrok odstranil obliž. Transdermalnega obliža se ne sme deliti, ker podatkov o tem ni na voljo. KONTRAINDIKACIJE: Preobcu­tljivost za zdravilno ucinkovino, hidrogenirano kolofonijo, sojo, arašide ali katerokoli pomožno snov. Akutna ali pooperativna bolecina, ko v kratkem casovnem obdobju ni možno titriranje odmerka in obstaja verjetnost za življenjsko ogrožajoco respirator-no depresijo. Huda okvara osrednjega živcnega sistema. POSEBNA OPOZORILA IN PREVIDNOSTNI UKREPI: Zaradi razpolovne dobe fentanila je treba bolnika v primeru pojava neželenega ucinka opazovati še 24 ur po odstranitvi obliža. Pri nekaterih bol­nikih, ki uporabljajo transdermalni obliž Epufen, se lahko pojavi respiratorna depre­sija. Epufen je treba previdno dajati: bolnikom s kronicno pljucno boleznijo, zvišanim intrakranialnim tlakom, možganskim tumorjem, boleznimi srca, jeter in ledvic, tistim z zvišano telesno temperaturo, pri starejših bolnikih in otrocih, bolnikih z miaste­nijo gravis. Odvisnost od zdravila: kot posledica ponavljajoce se uporabe se lahko razvijeta toleranca na ucinkovino ter psihicna in/ali fizicna odvisnost od nje. Ostali: lahko se pojavijo neepilepticne (mio)klonicne reakcije. MEDSEBOJNO DELOVANJE Z DRUGIMI ZDRAVILI IN DRUGE OBLIKE INTERAKCIJ: Derivati barbiturne kisline, opioidi, anksiolitiki in pomirjevala, hipnotiki, splošni anestetiki, fenotiazini, mišicni relaksanti, sedativni antihistaminiki in alkoholne pijace, zaviralci MAO, itrakonazol, ritonavir, ketokonazol, nekateri makrolidni antibiotiki, pentazocin, buprenorfin. VPLIV NA SPOSOBNOST VOŽNJE IN UPRAVLJANJA S STROJI: Zdravilo ima mocan vpliv na sposobnost vožnje in upravljanja s stroji. NEŽELENI UCINKI: Najbolj resen neželen ucinek fentanila je respiratorna depresija. Zelo pogosti (= 1/10): drema­vost, glavobol, navzeja, bruhanje, zaprtje, znojenje, srbenje, somnolenca. Pogosti (= 1/100 do < 1/10): kserostomija, dispepsija, reakcije na koži na mestu aplikacije, sedacija, zmedenost, depresija, tesnoba, živcna napetost, halucinacije, zmanjšan apetit. Obcasni (= 1/1000 do < 1/100): tahikardija, bradikardija, tremor, parestezija, motnje govora, dispneja, hipoventilacija, diareja, zastajanje urina, izpušcaj, rdecina, hipertenzija, hipotenzija, evforija, amnezija, nespecnost, vznemirljivost. Nekateri od naštetih neželenih ucinkov so lahko posledica osnovne bolezni ali drugih zdravljenj. Drugi neželeni ucinki: odpornost, fizicna in psihicna odvisnost se lahko razvijejo med dolgotrajno uporabo fentanila. Pri nekaterih bolnikih se lahko pojavijo odtegnitveni simptomi, ko zamenjajo prejšnje opiodne analgetike s transdermalnim obilžem s fentanilom ali po nenadni prekinitvi zdravljenja. NACIN IZDAJE: Samo na zdravni­ški recept. OPREMA: Škatle s 5 transdermalnimi obliži. IMETNIK DOVOLJENJA ZA PROMET: Lek farmacevtska družba, d.d., Verovškova 57, Ljubljana, Slovenija INFOR­MACIJA PRIPRAVLJENA: avgust 2009 Novartis Oncology prinaša spekter inovativnih zdravil, s katerimi poskuša spremeniti življenje bolnikov z rakavimi in hematološkimi obolenji. Ta vkljucuje zdravila kot so Glivec® (imatinib), Tasigna® (nilotinib), A. nitor® (everolimus), Zometa® (zoledronska kislina), Femara® (letrozol), Sandostatin® LAR® (oktreotid/i.m. injekcije) in Exjade® (deferasiroks). Novartis Oncology ima tudi obširen razvojni program, ki izkorišca najnovejša spoznanja molekularne genomike, razumskega nacrtovanja in tehnologij za odkrivnje novih ucinkovin. Novartis Pharma Services Inc. • Podružnica v Sloveniji • Tivolska cesta 30 • 1000 Ljubljana Samo za strokovno javnost. NV-JA-02/09-SI ucinkovita in preizkušena možnost zdravljenja anoreksije-kaheksije ... še vedno EDINO ZDRAVILO, ki je v Sloveniji registrirano za zdravljenje anoreksije-kaheksije pri bolnikih z napredovalim rakom 1,2 - predpisovanje na zeleni recept v breme ZZZS 6 izboljša apetit 1,5 pomaga ohraniti in pridobiti telesno težo 3,4,5 izboljša splošno pocutje bolnikov 3,4 SKRAJŠAN POVZETEK GLAVNIH ZNACILNOSTI ZDRAVILA: MEGACE 40 mg/ml peroralna suspenzija Sestava: 1 ml peroralne suspenzije vsebuje 40 mg megestrolacetata. TERAPEVTSKE INDIKACIJE: Zdravljenje anoreksije-kaheksije ali nepojasnjene, pomembne izgube telesne mase pri bolnikih z AIDS-om. Zdravljenje anorekticno-kahekticnega sindroma pri napredovalem raku. ODMERJANJE IN NACIN UPORABE: Pri aidsu je priporoceni zacetni odmerek Megace za odrasle 800 mg (20 ml peroralne suspenzije) enkrat na dan eno uro pred jedjo ali dve uri po jedi in se lahko med zdravljenjem prilagodi glede na bolnikov odziv. V raziskavah bolnikov z aidsom so bili klinicno ucinkoviti dnevni odmerki od 400 do 800 mg/dan (10 do 20 ml), uporabljani štiri mesece. Pri anorekticno-kahekticnem sindromu zaradi napredovalega raka je priporocljiv zacetni odmerek 200 mg (5 ml) na dan; glede na bolnikov odziv ga je mogoce povecati do 800 mg na dan (20 ml). Obicajni odmerek je med 400 in 800 mg na dan (10–20 ml). V raziskavah bolnikov z napredovalim rakom so bili klinicno ucinkoviti dnevni odmerki od 200 do 800 mg/dan (5 do 20 ml), uporabljani najmanj osem tednov. Pred uporabo je potrebno plastenko s suspenzijo dobro pretresti. Uporaba pri otrocih: Varnosti in ucinkovitosti pri otrocih niso dokazali. Uporaba pri starostnikih: Zaradi pogostejših okvar jeter, ledvic in srcne funkcije, pogostejših socasnih obolenj ali socasnega zdravljenja z drugimi zdravili je odmerek za starejšega bolnika treba dolociti previdno in obicajno zaceti z najnižjim odmerkom znotraj odmernega intervala. KONTRAINDIKACIJE: Preobcutljivost za megestrolacetat ali katerokoli pomožno snov. POSEBNA OPOZORILA IN PREVIDNOSTNI UKREPI: Uporaba gestagenov med prvimi štirimi meseci nosecnosti ni priporocljiva. Pri bolnikih s tromboflebitisom v anamnezi je treba zdravilo Megace uporabljati previdno. Zdravljenje z zdravilom Megace se lahko zacne šele, ko so bili vzroki hujšanja, ki jih je mogoce zdraviti, ugotovljeni in obravnavani. Megestrolacetat ni namenjen za profilakticno uporabo za preprecitev hujšanja. Ucinki na razmnoževanje virusa HIV niso ugotovljeni. Med zdravljenjem z megestrolacetatom in po prekinitvi kronicnega zdravljenja je treba upoštevati možnost pojava zavore nadledvicne žleze. Morda bo potrebno nadomestno zdravljenje s stresnimi odmerki glukokortikoidov. Megestrolacetat se v veliki meri izloci prek ledvic. Ker je verjetnost zmanjšanega delovanja ledvic pri starostnikih vecja, je pri dolocitvi odmerka potrebna previdnost, prav tako je koristno spremljanje ledvicne funkcije. Peroralna suspenzija vsebuje saharozo. Bolniki z redko dedno intoleranco za fruktozo, malabsorpcijo glukoze/galaktoze ali pomanjkanjem saharoza-izomaltaze ne smejo jemati tega zdravila. Peroralna suspenzija vsebuje tudi majhne kolicine etanola (alkohola), in sicer manj kot 100 mg na odmerek. INTERAKCIJE: Aminoglutetimid: porocali so o zmanjšanju koncentracije progestogena v plazmi z možno izgubo terapevtskega delovanja zaradi inducirane presnove. Socasno jemanje megestrolacetata (v obliki peroralne suspenzije) in zidovudina ali rifabutina ne povzroca sprememb farmakokineticnih parametrov. NEŽELENI UCINKI: Pogosti (= 1/100, < 1/10): navzea, bruhanje, driska, flatulenca, izpušcaj, metroragija, impotenca, astenija, bolecina, edem. Neznana pogostnost (pogostnosti ni mogoce oceniti iz razpoložljivih podatkov): poslabšanje osnovne bolezni (širjenje tumorja), adrenalna insuficienca, kušingoidni izgled, Cushingov sindrom, diabetes mellitus, motena toleranca za glukozo, hiperglikemija, spremembe razpoloženja, sindrom karpalnega kanala, letargija, srcno popušcanje, tromboflebitis, pljucna embolija (v nekaterih primerih usodna), hipertenzija, navali vrocine, dispneja, zaprtje, alopecija, pogosto uriniranje. Vrsta ovojnine in vsebina: Plastenka z 240 ml suspenzije. Režim izdaje: Rp/Spec. Imetnik dovoljenja za promet: Bristol-Myers Squibb spol. s r.o., Olivova 4, Praga 1, Ceška; Odgovoren za trženje v Sloveniji: PharmaSwiss d.o.o., Ljubljana, tel: 01 236 4 700, faks: 01 236 4 705; MGS-120609. Pred predpisovanjem preberite celoten povzetek glavnih znacilnosti zdravila! Reference: 1. Povzetek glavnih znacilnosti zdravila Megace – 12. junij 2009; 2. Register zdravil Republike Slovenije XI – leto 2008; 3. Beller, E., 1997. Ann Oncol 8: 277-283; 4. Cufer, T, 2002. Onkologija 9(2): 73-75; 5. Yavuzsen, T., 2005. J Clin Oncol 23(33): 8500-8511; 6. Bilten Recept 7(1), 22.5.2009 MEG1109-07; november, 2009 zdravnikom ali sfarmacevtom. Za Slovenijo kontakt: MEDIAS International d.o.o. Leskoškova cesta 9D, 1000 Ljubljana tel.: 01/5202302 info@medias int.si, www.medias int.si Prvi in edini obliž z aplikacijo dvakrat na teden Skrajšan povzetek glavnih znacilnosti zdravila TRANSTEC® 35 µg/h; 52,5 µg/h;70 µg/h transdermalni obliž Kakovostna in kolicinska sestava: TRANSTEC® 35µg/h:1 transdermalni obliž vsebuje 20mg buprenorfina (površina obliža:25cm2), hitrost sprošcanja:35µg buprenorfina na uro (v skupnem obdobju 96 ur); TRANSTEC® 52,5µg/h:1 transdermalni obliž vsebuje 30mg buprenorfina (površina obliža:37,5cm2), hitrost sprošcanja:52,5µg buprenorfina na uro (v skupnem obdobju 96 ur); TRANSTEC® 70µg/h:1 transdermalni obliž vsebuje 40mg buprenorfina (površina obliža:50cm2), hitrost sprošcanja:70µg buprenorfina na uro (v skupnem obdobju 96 ur). Pakiranje: Škatla s 4 transdermalnimi obliži. Indikacije: Za srednje hude do hude rakave bolecine in hude bolecine, ki se jih ne da obvladati z neopioidnimi analgetiki. Ni primeren za zdravljenje akutne bolecine. Odmerjanje: Bolniki, starejši od 18 let: Pri bolnikih, ki še niso prejemali zdravil za zdravljenje bolecine in pri bolnikih, ki so prejemali neopioidne analgetike iz prve stopnje lestvice SZO, zacnemo zdravljenje z najnižjo jakostjo (Transtec 35µg/h). Po namestitvi prvega obliža, vrednosti buprenorfina v serumu pocasi narašcajo (pri bolnikih, ki so že prejemali analgetike in pri tistih, ki jih niso). Zato naredimo prvo oceno analgeticnega zdravljenja šele po 24 urah. Titracija odmerka in vzdrževalno zdravljenje: Transtec obliž moramo zamenjati najkasneje po 96 urah (4 dneh). Zaradi poenostavitve uporabe naj bolnik zamenja obliž 2x tedensko v rednih casovnih razmikih, npr. vedno ob ponedeljkih zjutraj in cetrtkih zvecer. Odmerek titriramo za vsakega bolnika posebej, dokler ne dosežemo analgeticnega ucinka. Bolniku socasno ne predpišemo vec kot dveh obližev, ne glede na jakost. Bolniki, mlajši od 18 let: Pri teh uporabe Transtec obliža do sedaj niso proucevali, zato njegove uporabe ne priporocamo. Kontraindikacije: preobcutljivost za buprenorfin ali pomožne snovi; odvisnost od opioidov; nadomestno zdravljenje odvisnosti od mamil; oslabljeno delovanje in funkcija dihalnega centra ali ce do tega lahko pride; jemanje zaviralcev MAO v zadnjih dveh tednih; miastenija gravis; alkoholni delirij; nosecnost. Posebna opozorila in previdnostni ukrepi pri: akutni zastrupitvi z alkoholom; konvulzivnih motnjah; poškodbah glave; bolnikih v šoku; motnjah iz nejasnega vzroka; znakih zvišanega znotrajlobanjskega tlaka brez možnosti predihavanja; oslabljeni dihalni funkciji; socasnem jemanju zdravila, ki lahko povzrocijo zavoro dihanja; sumu na zlorabo zdravila; okvari jetrne funkcije; bolnikih mlajših od 18 let uporaba ni priporocena; pri zvišani telesni temperaturi ali zvišani temperaturi kože iz drugih razlogov (verjetnost opioidnih ucinkov je vecja, potrebno pozorno spremljanje). Neželeni ucinki: Zelo pogosti: slabost, pordela koža, srbenje. Pogosti: omoticnost, glavobol, obcutek težkega dihanja, bruhanje, zaprtje, kožne spremembe (eksantem) (predvsem ob ponavljajoci rabi), potenje, edem, utrujenost. Izdajanje zdravila: Le na recept, uporablja se po navodilu in pod posebnim nadzorom zdravnika specialista ali od njega pooblašcenega zdravnika. Datum priprave informacije: januar 2010 Podrobnejše informacije so na voljo pri: Grünenthal d.o.o., Dunajska 56, 000 Ljubljana* www.grunenthal.si * E-pošta: siinfo@grunenthal.com * tel. 0 589 67 0 Instructions Instructions for authors The editorial policyof the journal Radiology and Oncologyis topublish original scientific papers, professional papers, review articles case reports and varia (editorials, 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. 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This should be followed by the abstract page, summarizing in less than 250 words the reasons for the study, experimental approach, the major findings (with specific data if possible), and the principal conclusions, and providing 3-6 key words for indexing purposes. Structured abstracts are preferred. The text of the report should then proceed as follows: Introductionshould state the purpose of the article and summarize the rationale for the study or observation, citing only the essential references and stating the aim of the study.Materials and methodsshould provide enough information to enable experiments to be repeated. New methods should be described in detail. Reports on human and animal subjects should include a statement that ethical approval of the study was obtained. Resultsshould be presented clearly and concisely without repeating the data in the figures and tables. Emphasis should be on clear and precise presentation of results and their significance in relation to the aim of the investigation. Discussion should explain the results rather than simply repeating them and interpret their significance and draw conclu­sions. It should review the results of the study in the light of previously published work. Radiol Oncol 2010; 44(1) Instructions Illustrations and tablesmust be numbered and referred to in the text, with the appropriate location indicated. Graphs and photographs, provided electronically, should be of appropriate quality for good reproduction. Colour graphs and photo­graphs are encouraged. In photographs, mask the identities of the patients. Tables should be typed double-spaced, with a descriptive title and, if appropriate, units of numerical measurements included in the column heading. Referencesmust be numbered in the order in which they appear in the text and their corresponding numbers quoted in the text. 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In: Nelson DS, editor. Immunobiology of macrophage. New York: Academic Press; 1976. p. 45-74.Page proofswill be sent by E-mail or faxed to the corresponding author. It is their responsibility to check the proofs carefully and return a list of essential corrections to the editorial office within 48 hours of receipt. If corrections are not received by the stated deadline, proof-reading will be carried out by the editors. Reprints: The electronic version of the published papers will be available on www.versita.com free of charge. Radiol Oncol 2010; 44(1) BISTVENE INFORMACIJE IZ POVZETKA GLAVNIH ZNACILNOSTI ZDRAVILA SUTENT 12,5 mg, 25 mg, 37,5 mg, 50 mg trde kapsule Sestava in oblika zdravila: Vsaka trda kapsula vsebuje 12,5 mg, 25 mg, 37,5 mg ali 50 mg sunitiniba v obliki sunitinibijevega malata. Indikacije: Zdravljenje neizrezljivega in/ali metastatskega malignega gastrointestinalnega stromalnega tumorja (GIST), ce zdravljenje z imatinibijevim mesilatom zaradi odpornosti ali neprenašanja ni bilo uspešno. Zdravljenje napredovalega in/ali metastatskega karcinoma ledvicnih celic (MRCC). Odmerjanje in nacin uporabe: Terapijo mora uvesti zdravnik, ki ima izkušnje z zdravljenjem MRCC ali GIST. Priporoceni odmerek je 50 mg enkrat dnevno, peroralno vsak dan 4 tedne zapored; temu sledi 2-tedenski premor (Shema 4/2), tako da celotni ciklus traja 6 tednov. Odmerek je mogoce prilagajati v povecanjih po 12,5 mg, upoštevaje individualno varnost in prenašanje. Dnevni odmerek ne sme preseci 75 mg in ne sme biti manjši od 25 mg. Pri socasni uporabi z mocnimi zaviralci ali induktorji CYP3A4 je potrebno odmerek ustrezno prilagoditi. Uporaba pri otrocih in mladostnikih (< 18 let): Sutenta ne smemo uporabljati, dokler ne bo na voljo dodatnih podatkov. Uporaba pri starejših bolnikih (= 65 let): med starejšimi in mlajšimi bolniki niso opazili pomembnih razlik v varnosti in ucinkovitosti. Insuficienca jeter: pri bolnikih z jetrno okvaro razreda A in B po Child-Pughu prilagoditev odmerka ni potrebna; pri bolniki z okvaro razreda C Sutent ni bil preizkušen. Insuficienca ledvic: klinicnih študij niso izvedli. Sutent se uporablja peroralno, bolnik ga lahko vzame z ali brez hrane. Ce pozabi vzeti odmerek, ne sme dobiti dodatnega, temvec naj vzame obicajni predpisani odmerek naslednji dan. Kontraindikacije: Preobcutljivost za zdravilno ucinkovino ali katerokoli pomožno snov. Posebna opozorila in previdnostni ukrepi:Koža in tkiva. Krvavitve v prebavila, dihala, secila, v možganih ter krvavitve tumorja. Ucinki na prebavila: poleg navzee in driske tudi resni zapleti. Hipertenzija. Hematološke bolezni. Bolezni srca in ožilja: zmanjšanje LVEF in srcno popušcanje. Podaljšanje intervala QT. Venski trombembolicni dogodki. Dogodki na dihalih: dispneja, plevralni izliv, pljucna embolija ali pljucni edem. Moteno delovanje šcitnice. Pankreatitis. Delovanje jeter. Delovanje ledvic. Fistula. Preobcutljivost/angioedem. Motnje okušanja. Konvulzije. Pri krvavitvah, ucinkih na prebavila, hematoloških boleznih, dogodkih na dihalih, venskih trombembolicnih dogodkih, pankreatitisu in ucinkih na jetra so opisani tudi smrtni izidi. Medsebojno delovanje z drugimi zdravili: Zdravila, ki lahko zvišajo koncentracijo sunitiniba v plazmi (ketokonazol, ritonavir, itrakonazol, eritromicin, klaritromicin ali sok grenivke). Zdravila, ki lahko znižajo koncentracijo sunitiniba v plazmi (deksametazon, fenitoin, karbamazepin, rifampin, fenobarbital, Hypericum perforatum oz. šentjanževka). Antikoagulanti. Nosecnost in dojenje: Sutenta se ne sme uporabljati med nosecnostjo in tudi ne pri ženskah, ki ne uporabljajo ustrezne kontracepcije, razen ce možna korist odtehta možno tveganje za plod. Ženske v rodni dobi naj med zdravljenjem s Sutentom ne zanosijo. Ženske, ki jemljejo Sutent, ne smejo dojiti. Vpliv na sposobnost vožnje in upravljanja s stroji: Sutent lahko povzroci omotico. Neželeni ucinki: Najpogostejši neželeni ucinki: pljucna embolija, trombocitopenija, krvavitev tumorja, febrilna nevtropenija, hipertenzija, utrujenost, diareja, navzea, stomatitis, dispepsija, bruhanje, obarvanje kože, disgevzija, anoreksija, zvišanje ravni lipaze. Zelo pogosti: anemija, nevtropenija, hipotiroidizem, zmanjšanje teka, motnje okušanja, glavobol, bolecina v trebuhu / napihnjenost, flatulenca, bolecine v ustih, sindrom palmarno plantarne eritrodizestezije, spremembe barve las, astenija, vnetje sluznice, edemi. Nacin in režim izdajanja: Izdaja zdravila je le na recept, uporablja pa se samo v bolnišnicah. Izjemoma se lahko uporablja pri nadaljevanju zdravljenja na domu ob odpustu iz bolnišnice in nadaljnjem zdravljenju. Imetnik dovoljenja za promet: Pfizer Limited, Ramsgate Road, Sandwich, Kent, CT13 9NJ, Velika Britanija. Datum zadnje revizije besedila: 28.10.2009 Pred predpisovanjem se seznanite s celotnim povzetkom glavnih znacilnosti zdravila. SUT-01-10 Pfizer Luxembourg SARL, Grand Duchy of Luxembourg, 51, Avenue J.F. Kennedy, L-1855, PFIZER, Podružnica za svetovanje s podrocja farmacevtske dejavnosti, Ljubljana, Letališka 3c, 1000 Ljubljana, SLOVENIJA