Special section Biomedical applications of electroporation Guest Editors: Richard Heller, Maja Čemažar vol.50 no.1 march 2016 Prva in edina samostojna kemoterapija, ki v primerjavi z ostalimi možnostmi zdravljenja z enim zdravilom, pri bolnicah s predhodno že večkratno zdravljenim metastatskim rakom dojke, dokazano značilno podaljša celokupno preživetje.1,2 NOVA SMER DO PODALJŠANJA CELOKUPNEGA PREŽIVETJA Halaven (eribulin): ne-taksanski zaviralec dinamike mikrotubulov, prvo zdravilo iz nove skupine kemoterapevtikov, imenovanih halihondrini. Zdravilo HALAVEN je indicirano za zdravljenje bolnic z lokalno napredovalim ali metastatskim rakom dojke, ki je napredoval po vsaj enem režimu kemoterapije za napredovalo bolezen. Predhodna zdravljenja morajo vključevati antraciklin in taksan, bodisi kot adjuvantno zdravljenje ali za zdravljenje metastatskega raka dojke, razen če to zdravljenje za bolnice ni bilo primerno.1 Priporočeni odmerek 1,23 mg/m2, intravensko, v obliki 2-do 5-minutne infuzije, 1. in 8. dan vsakega 21-dnevnega cikla. Ena 2 ml viala vsebuje 0,88 mg eribulina. Raztopina, pripravljena za uporabo, redčenje ni potrebno. SKRAJŠAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA HALAVEN 0,44 mg/ml raztopina za injiciranje (eribulin) TERAPEV TSKE INDIKACIJE: Zdravljenje lokalno napredovalega ali metastatskega raka dojke, ki je napredoval po vsaj enem režimu kemoterapije za napredovalo bolezen vključno z antraciklinom in taksanom (adjuvantno zdravljenje ali zdravljenje metastatskega raka dojke), razen če to ni bilo primerno. ODMERJANJE IN NAČIN UPORABE: Halaven se daje v enotah, specializiranih za dajanje citotoksične kemoterapije, in le pod nadzorom usposobljenega zdravnika z izkušnjami v uporabi citotoksičnih zdravil. Odmerjanje: Priporočeni odmerek eribulina v obliki raztopine je 1,23 mg/m2 i.v. v obliki 2- do 5-minutne infuzije 1. in 8. dan vsakega 21-dnevnega cikla. Bolnikom je lahko slabo ali bruhajo. Treba je razmisliti o antiemetični profilaksi, vključno s kortikosteroidi. Preložitev odmerka med zdravljenjem: Dajanje Halavena je treba preložiti, če se pojavi kaj od naslednjega: absolutno število nevtrofilcev (ANC) < 1 x 109/l, trombociti < 75 x 109/l ali nehematološki neželeni učinki 3. ali 4. stopnje. Zmanjšanje odmerka med zdravljenjem: Za priporočila za zmanjšanje odmerka ob pojavu hematoloških ali nehematoloških neželenih učinkov glejte celoten povzetek glavnih značilnosti zdravila. Okvara jeter zaradi zasevkov: Priporočeni odmerek pri blagi okvari jeter (stopnje A po Child-Pughu) je 0,97 mg/m2 v obliki 2- do 5-minutne i.v. infuzije 1. in 8. dan 21-dnevnega cikla. Priporočeni odmerek pri zmerni okvari jeter (stopnje B po Child-Pughu) je 0,62 mg/m2 v obliki 2- do 5-minutne i.v. infuzije 1. in 8. dan 21-dnevnega cikla. Pri hudi okvari jeter (stopnje C po Child-Pughu) se pričakuje, da je treba dati še manjši odmerek eribulina. Okvara jeter zaradi ciroze: Zgornje odmerke se lahko uporabi za blago do zmerno okvaro, vendar se priporoča skrbno nadziranje, saj bo odmerke morda treba ponovno prilagoditi. Okvara ledvic: Pri hudi okvari ledvic (očistek kreatinina < 40 ml/min) bo morda treba odmerek zmanjšati. Priporoča se skrbno nadziranje varnosti. Način uporabe: Odmerek se lahko razredči z do 100 ml 0,9 % raztopine natrijevega klorida (9 mg/ml) za injiciranje. Ne sme se ga redčiti v 5 % infuzijski raztopini glukoze. Pred dajanjem glejte navodila glede redčenja zdravila v celotnem povzetku glavnih značilnosti zdravila ter se prepričajte, da obstaja dober periferni venski dostop ali prehodna centralna linija. Ni znakov, da bi eribulin povzročal mehurje ali dražil. V primeru ekstravazacije mora biti zdravljenje simptomatsko. KONTRAINDIKACIJE: Preobčutljivost na zdravilno učinkovino ali katerokoli pomožno snov. Dojenje. POSEBNA OPOZORILA IN PREVIDNOSTNI UKREPI: Mielosupresija je odvisna od odmerka in se kaže kot nevtropenija. Pred vsakim odmerkom eribulina je treba opraviti pregled celotne krvne slike. Zdravljenje z eribulinom se lahko uvede le pri bolnikih z vrednostmi ANC . 1,5 x 109/l in s trombociti > 100 x 109/l. Bolnike, pri katerih se pojavijo febrilna nevtropenija, huda nevtropenija ali trombocitopenija, je treba zdraviti v skladu s priporočili v celotnem povzetku glavnih značilnosti zdravila. Hudo nevtropenijo se lahko zdravi z uporabo G-CSF ali enakovrednim zdravilom v skladu s smernicami. Bolnike je treba skrbno nadzirati za znake periferne motorične in senzorične nevropatije. Pri razvoju hude periferne nevrotoksičnosti je treba odmerek prestaviti ali zmanjšati. Če začnemo zdravljenje pri bolnikih s kongestivnim srčnim popuščanjem, z bradiaritmijami ali sočasno z zdravili, za katera je znano, da podaljšujejo interval QT, vključno z antiaritmiki razreda Ia in III, in z elektrolitskimi motnjami, je priporočljivo spremljanje EKG. Pred začetkom zdravljenja s Halavenom je treba popraviti hipokaliemijo in hipomagneziemijo in te elektrolite je treba občasno kontrolirati med zdravljenjem. Eribulina ne smemo dajati bolnikom s prirojenim sindromom dolgega intervala QT. To zdravilo vsebuje majhne količine etanola (alkohola), manj kot 100 mg na odmerek. Eribulin je pri podganah embriotoksičen, fetotoksičen in teratogen. Halavena se ne sme uporabljati med nosečnostjo, razen kadar je to nujno potrebno. Ženske v rodni dobi naj ne zanosijo v času, ko same ali njihov moški partner dobivajo Halaven, in naj med zdravljenjem in še do 3 mesece po njem uporabljajo učinkovito kontracepcijo. Moški naj se pred zdravljenjem posvetujejo o shranjevanju sperme zaradi možnosti nepopravljive neplodnosti. INTERAKCIJE: Eribulin se izloča do 70 % prek žolča. Sočasna uporaba učinkovin, ki zavirajo jetrne transportne beljakovine, kot so beljakovine za prenos organskih anionov in beljakovine, odporne na številna zdravila, z eribulinom se ne priporoča (npr. ciklosporin, ritonavir, sakvinavir, lopinavir in nekateri drugi zaviralci proteaze, efavirenz, emtricitabin, verapamil, klaritromicin, kinin, kinidin, dizopiramid itd). Sočasno zdravljenje z indukcijskimi učinkovinami, kot so rifampicin, karbamazepin, fenitoin, šentjanževka, lahko povzroči znižanje koncentracij eribulina v plazmi, zato je ob sočasni uporabi induktorjev potrebna previdnost. Eribulin je blag inhibitor encima CYP3A4. Priporočljiva je previdnost in spremljanje glede neželenih učinkov pri sočasni uporabi snovi, ki imajo ozko terapevtsko okno in se odstranjujejo iz telesa predvsem preko CYP3A4 (npr. alfentanil, ciklosporin, ergotamin, fentanil, pimozid, kinidin, sirolimus, takrolimus). NEŽELENI UČINKI: Povzetek varnostnega profila Neželeni učinek, o katerem najpogosteje poročajo v zvezi s Halavenom, je supresija kostnega mozga, ki se kaže kot nevtropenija, levkopenija, anemija, trombocitopenija s pridruženimi okužbami. Poročali so tudi o novem začetku ali poslabšanju že obstoječe periferne nevropatije. Med neželenimi učinki, o katerih poročajo, je toksičnost za prebavila, ki se kaže kot anoreksija, navzea, bruhanje, driska, zaprtost in stomatitis. Med drugimi neželenimi učinki so utrujenost, alopecija, zvečani jetrni encimi, sepsa in mišičnoskeletni bolečinski sindrom. Seznam neželenih učinkov: Zelo pogosti (. 1/10): nevtropenija (57,0 %) (3./4. stopnje: 49,7 %), levkopenija (29,3 %) (3./4. stopnje: 17,3 %), anemija (20,6 %) (3./4. stopnje: 2,0 %), zmanjšan apetit (21,9 %) (3./4. stopnje: 0,7 %), periferna nevropatija (35,6 %) (3./4. stopnje: 7,6 %), glavobol (17,2 %) (3./4. stopnje: 0,8 %), dispnea (13,9 %) (3./4. stopnje: 3,1 %), kašelj (13,6 %) (3./4. stopnje: 0,6 %), navzea (33,8 %) (3./4. stopnje: 1,1 %), zaprtost (19,6 %) (3./4. stopnje: 0,6 %), driska (17,9 %) (3./4. stopnje: 0,8 %), bruhanje (17,6 %) (3./4. stopnje: 0,9 %), alopecija, artralgija in mialgija (19,4 %) (3./4. stopnje: 1,1 %), bolečina v hrbtu (13,0 %) (3./4. stopnje: 1,5 %), bolečina v udu (10,0 %) (3./4. stopnje: 0,7 %), utrujenost/astenija (47,9 %) (3./4. stopnje: 7,8 %), pireksija (20,4 %) (3./4. stopnje: 0,6 %), zmanjšanje telesne mase (11,3 %) (3./4. stopnje: 0,3 %). Pogosti (. 1/100 do < 1/10): okužba sečil (8 %) (3./4. stopnje: 0,5 %), pljučnica (1,2 %) (3./4. stopnje: 0,8 %), ustna kandidiaza, ustni herpes, okužba zgornjih dihal, nazofaringitis, rinitis, limfopenija (4,9 %) (3./4. stopnje: 1,4 %), febrilna nevtropenija (4,7 %) (3./4. stopnje: 4,5 %), trombocitopenija (4,3 %) (3./4. stopnje: 0,7 %), hipokaliemija (6,1 %) (3./4. stopnje: 1,7 %), hipomagneziemija (2,9 %) (3./4. stopnje: 0,2 %), dehidracija (2,8 %) (3./4. stopnje: 0,5 %), hiperglikemija, hipofosfatemija, nespečnost, depresija, disgevzija, omotičnost (7,9 %) (3./4. stopnje: 0,5 %), hipoestezija, letargija, nevrotoksičnost, obilnejše solzenje (6,0 %) (3./4. stopnje: 0,1 %), konjunktivitis, vrtoglavica, tahikardija, vročinski valovi, orofaringealna bolečina, epistaksa, rinoreja, bolečina v trebuhu, stomatitis (9,3 %) (3./4. stopnje: 0,8 %), suha usta, dispepsija (5,9 %) (3./4. stopnje: 0,2 %), gastroezofagealna refluksna bolezen, razjede v ustih, distenzija trebuha, zvišanje alanin-aminotransferaze (7,6 %) (3./4. stopnje: 2,1 %), zvišanje aspartat-aminotransferaze (7,4 %) (3./4. stopnje: 1,5 %), zvišanje gama-glutamiltransferaze (1,8 %) (3./4. stopnje: 0,9 %), hiperbilirubinemija (1,5 %) (3./4. stopnje: 0,3 %), izpuščaj, pruritus (3,9 %) (3./4. stopnje: 0,1 %), bolezni nohtov, nočno potenje, suha koža, eritem, hiperhidroza, bolečina v kosteh (9,6 %) (3./4. stopnje: 1,7 %), mišični spazmi (5,1 %) (3./4. stopnje: 0,1 %), mišično-skeletna bolečina in mišično­skeletna bolečina v prsih, mišična oslabelost, disurija, vnetje sluznice (8,3 %) (3./4. stopnje: 1,1 %), periferni edem, bolečina, mrzlica, bolečina v prsih, gripi podobna bolezen. Občasni (. 1/1.000 do < 1/100): sepsa (0,5 %) (3./4. stopnje: 0,2 %), nevtropenična sepsa (0,1 %) (3./4. stopnje: 0,1 %), herpes zoster, tinitus, globoka venska tromboza, pljučna embolija, hepatotoksičnost (1,0 %) (3./4. stopnje: 0.6 %), palmarno-plantarna eritrodisestezija, hematurija, proteinurija, odpoved ledvic. Redki (. 1/10.000 do < 1/1.000): diseminirana intravaskularna koagulacija, intersticijska pljučna bolezen, pankreatitis, angioedem. Za popoln opis neželenih učinkov glejte celoten povzetek glavnih značilnosti zdravila. Vrsta ovojnine in vsebina: viala z 2 ml raztopine. Režim izdaje: H Imetnik dovoljenja za promet: Eisai Europe Ltd, European Knowledge Centre, Mosquito Way, Hatfield, Hertfordshire, AL10 9SN, Velika Britanija HAL-270614, julij 2014 Pred predpisovanjem in uporabo zdravila prosimo preberite celoten povzetek glavnih značilnosti zdravila! Viri: (1) Povzetek glavnih značilnosti zdravila Halaven, junij 2014; (2) Cortes J et al. Lancet 2011; 377: 914–23. Odgovoren za trženje v Sloveniji: PharmaSwiss d.o.o., Brodišče 32, 1236 Trzin telefon: +386 1 236 47 00, faks: +386 1 283 38 10 HAL-0714-01, julij 2014 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 Radiology Societas Radiologorum Hungarorum Friuli-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 interventional radiology, computerized tomography, ultrasound, magnetic resonance, nuclear medicine, radiotherapy, clinical and experimental oncology, radiobiology, radiophysics and radiation protection. Editor-in-Chief Gregor Serša, Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia Executive Editor Viljem Kovač, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia Editorial Board Sotirios Bisdas, National Hospital for Neurology and Neurosurgery, University Collegge London Hospitals, London, UK Karl H. Bohuslavizki, Facharzt für Nuklearmedizin, Hamburg, Germany Serena Bonin, University of Trieste, Department of Medical Sciences, Trieste, Italy Boris Brkljačić, University Hospital “Dubrava”, Department of Diagnostic and Interventional Radiology, Zagreb, Croatia Luca Campana, Veneto Institute of Oncology (IOV-IRCCS), Padova, Italy Christian Dittrich, Kaiser Franz Josef - Spital, Vienna, Austria Metka Filipič, National Institute of Biology, Department of Genetic Toxicology and Cancer Biology, Ljubljana, Slovenia Maria Gődény, National Institute of Oncology, Budapest, Hungary Janko Kos, University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia Robert Jeraj, University of Wisconsin, Carbone Cancer Center, Madison, Wisconsin, USA Advisory Committee Tullio Giraldi, University of Trieste, Faculty of Medicine and Psychology, Trieste, Italy Vassil Hadjidekov, Medical University, Department of Diagnostic Imaging, Sofia, Bulgaria Deputy Editors Andrej Cör, University of Primorska, Faculty of Health Science, Izola, Slovenia Maja Čemažar, Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia Igor Kocijančič, University Medical Centre Ljubljana, Institute of Radiology, Ljubljana, Slovenia Karmen Stanič, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia Primož Strojan, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia Tamara Lah Turnšek, National Institute of Biology, Ljubljana, Slovenia Damijan Miklavčič, University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia Luka Milas, UT M. D. Anderson Cancer Center, Houston , USA Damir Miletić, Clinical Hospital Centre Rijeka, Department of Radiology, Rijeka, Croatia Häkan Nyström, Skandionkliniken, Uppsala, Sweden Maja Osmak, Ruder Bošković Institute, Department of Molecular Biology, Zagreb, Croatia Dušan Pavčnik, Dotter Interventional Institute, Oregon Health Science Universityte, Oregon, Portland, USA Geoffrey J. Pilkington, University of Portsmouth, School of Pharmacy and Biomedical Sciences, Portsmouth, UK Ervin B. Podgoršak, McGill University, Montreal, Canada Matthew Podgorsak, Roswell Park Cancer Institute, Departments of Biophysics and Radiation Medicine, Buffalo, NY ,USA Marko Hočevar, Institute of Oncology Ljubljana, Department of Surgical Oncology, Ljubljana, Slovenia Miklós Kásler, National Institute of Oncology, Budapest, Hungary Csaba Polgar, National Institute of Oncology, Budapest, Hungary Dirk Rades, University of Lubeck, Department of Radiation Oncology, Lubeck, Germany , Mirjana Rajer, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia Luis Souhami, McGill University, Montreal, Canada Borut Štabuc, University Medical Centre Ljubljana, Department of Gastroenterology, Ljubljana, Slovenia Katarina Šurlan Popovič, University Medical Center Ljubljana, Clinical Institute of Radiology, Ljubljana, Slovenia Justin Teissié, CNRS, IPBS, Toulouse, France Gillian M.Tozer, University of Sheffield, Academic Unit of Surgical Oncology, Royal Hallamshire Hospital, Sheffield, UK Andrea Veronesi, Centro di Riferimento Oncologico- Aviano, Division of Medical Oncology, Aviano, Italy Branko Zakotnik, Institute of Oncology Ljubljana, Department of Medical Oncology, Ljubljana, Slovenia Stojan Plesničar, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia Tomaž Benulič, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia Radiol Oncol 2016; 50(1): A. Editorial office Radiology and Oncology Zaloška cesta 2 P. O. Box 2217 SI-1000 Ljubljana Slovenia Phone: +386 1 5879 369 Phone/Fax: +386 1 5879 434 E-mail: gsersa@onko-i.si Copyright © Radiology and Oncology. All rights reserved. Reader for English Vida Kološa Secretary Mira Klemenčič Zvezdana Vukmirović Design Monika Fink-Serša, Samo Rovan, Ivana Ljubanović Layout Matjaž Lužar Printed by Tiskarna Ozimek, Slovenia Published quarterly in 400 copies Beneficiary name: DRUŠTVO RADIOLOGIJE IN ONKOLOGIJE Zaloška cesta 2 1000 Ljubljana Slovenia Beneficiary bank account number: SI56 02010-0090006751 IBAN: SI56 0201 0009 0006 751 Our bank name: Nova Ljubljanska banka, d.d., Ljubljana, Trg republike 2, 1520 Ljubljana; Slovenia SWIFT: 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: • Celdes • Chemical Abstracts Service (CAS) • Chemical Abstracts Service (CAS) - SciFinder • CNKI Scholar (China National Knowledge Infrastructure) • CNPIEC • DOAJ • EBSCO - Biomedical Reference Collection • EBSCO - Cinahl • EBSCO - TOC Premier • EBSCO Discovery Service • Elsevier - EMBASE • Elsevier - SCOPUS • Google Scholar • J-Gate • JournalTOCs • Naviga (Softweco) • Primo Central (ExLibris) • ProQuest - Advanced Technologies Database with Aerospace • ProQuest - Health & Medical Complete This journal is printed on acid- free paper On the web: ISSN 1581-3207 http://www.degruyter.com/view/j/raon http://www.radioloncol.com • ProQuest - Illustrata: Health Sciences • ProQuest - Illustrata: Technology • ProQuest - Medical Library • ProQuest - Nursing & Allied Health Source • ProQuest - Pharma Collection • ProQuest - Public Health • ProQuest - Science Journals • ProQuest - SciTech Journals • ProQuest - Technology Journals • PubMed • PubsHub • ReadCube • SCImago (SJR) • Summon (Serials Solutions/ProQuest) • TDOne (TDNet) • Thomson Reuters - Journal Citation Reports/Science Edition • Thomson Reuters - Science Citation Index Expanded • Ulrich's Periodicals Directory/ulrichsweb • WorldCat (OCLC) Radiol Oncol 2016; 50(1): B. editorial editorial How to increase the quality and visibility of Radiology and Oncology? Dear friends of Radiology and Oncology. First of all we would like to thank authors and reviewers for their valuable work that significantly contributes to the quality of our journal. Last year we published 58 articles in four issues. The internationality of our journal is evident through the participation of the authors from many European and other countries throughout the world. Furthermore, in 2015 our Impact factor increased to 1.912. We regret, but according to the limited place, we were unable to accept many manuscripts for publication. Currently we have been able to publish only part of submissions. The rejecting rate was about 78% (58/262). Thus, many otherwise worthwhile papers were not accepted relative to their publication priority assigned by the reviewers and editors. The published articles are well cited. Many authors from the United States and from China quoted papers from Radiology and Oncology. Furthermore, they were quoted also in some prestigious journals, such as Nature Reviews Cancer. This trend we would like to continue in the future. Our journal is general in the fields of oncology, radiology and nuclear medicine. We publish works from basic research to clinical studies. In relation to that we decided this year to devote part of the first issue to a theme of biomedical applications of electroporation. Our guest editors, Prof. Richard Heller from USA and Prof. Maja Čemažar from Slovenia have prepared several interesting manuscripts that are published in this issue of Radiology and Oncology. The results were in major part presented at the 1st World congress on electroporation that was held in Portorož in September 2015. Our intended next step in development of Radiology and Oncology is to apply for the MEDLINE. They adhere to specific requirements that journals have to follow. Among them are ethical issues that we have to comply with. Therefore we require that all clinical and animal data have clearance of the respectful bodies. We will also start to enforce that the prospective clinical studies - beside ethical consideration - are registered either in European or American clinical studies repositories. We would like to inform the authors and also the reviewers to be very strict in this respect. In the end we would like to thank you again for the support of our efforts for continuous development of Radiology and Oncology. With best regards, Prof. Gregor Serša, Ph.D. Editor in Chief Assoc. Prof. Viljem Kovač, M.D., Ph.D. Executive Editor Radiol Oncol 2016; 50(1): C. contents contents Biomedical applications of electroporation Guest editors: Richard Heller, Maja Čemažar 1 Recommendations for improving the quality of reporting clinical electrochemotherapy studies based on qualitative systematic review Luca G. Campana, A. James P. Clover, Sara Valpione,Pietro Quaglino, Julie Gehl, Christian Kunte, Marko Snoj, Maja Cemazar, Carlo R. Rossi, Damijan Miklavcic, Gregor Sersa 14 Electrochemotherapy in pancreatic adenocarcinoma treatment: pre-clinical and clinical studies Sabrina Bimonte, Maddalena Leongito, Vincenza Granata, Antonio Barbieri, Vitale del Vecchio, Michela Falco, Aurelio Nasto, Vittorio Albino, Mauro Piccirillo, Raffaele Palaia, Alfonso Amore, Raimondo di Giacomo, Secondo Lastoria, Sergio Venanzio Setola, Roberta Fusco, Antonella Petrillo, Francesco Izzo 21 Effectiveness of electrochemotherapy after IFN-. adjuvant therapy of melanoma patients Andrejc Hribernik, Maja Cemazar, Gregor Sersa, Masa Bosnjak, Marko Snoj 28 A statistical model describing combined irreversible electroporation and electroporation-induced blood-brain barrier disruption Shirley Sharabi, Bor Kos, David Last, David Guez, Dianne Daniels, Sagi Harnof, Yael Mardor, Damijan Miklavcic 39 Electrochemotherapy by pulsed electromagnetic field treatment (PEMF) in mouse melanoma B16F10 in vivo Simona Kranjc, Matej Kranjc, Janez Scancar, Jure Jelenc, Gregor Sersa, Damijan Miklavcic 49 A prototype of a flexible grid electrode to treat widespread superficial tumors by means of Electrochemotherapy Luca G. Campana, Fabrizio Dughiero, Michele Forzan, Carlo R. Rossi, Elisabetta Sieni 58 Combined local and systemic bleomycin administration in electrochemotherapy to reduce the number of treatment sessions Felipe Maglietti, Matias Tellado, Nahuel Olaiz, Sebastian Michinski, Guillermo Marshall Other articles review 64 Medical physics in Europe following recommendations of the International Atomic Energy Agency Bozidar Casar, Maria do Carmo Lopes, Advan Drljevic, Eduard Gershkevitsh, Csilla Pesznyak Radiol Oncol 2016; 50(1): E. contents radiology 73 Diagnostic accuracy of MRI to evaluate tumour response and residual tumour size after neoadjuvant chemotherapy in breast cancer patients Alberto Bouzón, Benigno Acea, Rafaela Soler, Ángela Iglesias, Paz Santiago, Joaquín Mosquera, Lourdes Calvo, Teresa Seoane-Pillado, Alejandra García clinical oncology 80 Antioxidant defence-related genetic variants are not associated with higher risk of secondary thyroid cancer after treatment of malignancy in childhood or adolescence Ana Lina Vodusek, Katja Goricar, Barbara Gazic, Vita Dolzan, Janez Jazbec 87 Cerebral toxoplasmosis in a diffuse large B cell lymphoma patient Lina Savsek, Tanja Ros Opaskar 94 Obstructive urination problems after high-dose-rate brachytherapy boost treatment for prostate cancer are avoidable Borut Kragelj 104 Prognostic factors of choroidal melanoma in Slovenia, 1986—2008 Boris Jancar, Marjan Budihna, Brigita Drnovsek-Olup, Katrina Novak Andrejcic, Irena Brovet Zupancic, Dusica Pahor 113 The impact of anaemia on treatment outcome in patients with squamous cell carcinoma of anal canal and anal margin Irena Oblak, Monika Cesnjevar, Mitja Anzic, Jasna But Hadzic, Ajra Secerov Ermenc, Franc Anderluh, Vaneja Velenik, Ana Jeromen,Peter Korosec radiophysics 121 Evaluation of dosimetric effect caused by slowing with multi-leaf collimator (MLC) leaves for volumetric modulated arc therapy (VMAT) Zhengzheng Xu, Iris Z. Wang, Lalith K. Kumaraswamy, Matthew B. Podgorsak I slovenian abstracts Radiol Oncol 2016; 50(1): F. review Recommendations for improving the quality of reporting clinical electrochemotherapy studies based on qualitative systematic review Luca G. Campana1,2, A. James P. Clover3, Sara Valpione2,4, Pietro Quaglino5, Julie Gehl6, Christian Kunte7, Marko Snoj8,9, Maja Cemazar10, Carlo R. Rossi1,2, Damijan Miklavcic11, Gregor Sersa10 1 Surgical Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, Padova, Italy 2 Department of Surgery Oncology and Gastroenterology, University of Padova, Padova, Italy 3 Department of Plastic Surgery, Cork University Hospital and Cork Cancer Research Centre, University College Cork, Cork, Ireland 4 Medical Oncology, Christie NHS Foundation Trust, Manchester, UK 5 Department of Medical Sciences, Dermatologic Clinic, University of Torino, Torino, Italy 6 Center for Experimental Drug and Gene Electro transfer, Department of Oncology, Copenhagen University Hospital Herlev, Herlev, Denmark 7 Department of Dermatology and Allergology, Ludwig-Maximilian University Munich, Munich, Germany 8 Department of Surgical Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia. 9 University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia 10 Department of Experimental Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 11 University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia Radiol Oncol 2016; 50(1): 1-13. Received 14 December 2015 Accepted 11 January 2016 Correspondence to: Prof. Gregor Serša, Ph.D., Institute of Oncology Ljubljana, Department of Experimental Oncology, Zaloška 2, SI-1000 Ljubljana, Slovenia. E-mail: gsersa@onko-i.si Disclosure: DM holds patents on electrochemotherapy that have been licensed to IGEA S.p.a. and is also a consultant to IGEA. The other co­authors have nothing to disclose. Background. Electrochemotherapy is becoming a well-established treatment for malignancies of skin and non-skin origin and its use is widening across Europe. The technique was developed and optimized from solid experimental and clinical evidence. A consensus document is now warranted to formalize reporting results, which should strengthen evidence-based practice recommendations. This consensus should be derived from high quality clinical data col­lection, clinical expertise and summarizing patient feedback. The first step, which is addressed in this paper, aims to critically analyze the quality of published studies and to provide the recommendations for reporting clinical trials on electrochemotherapy. Methods. The quality of reporting in published studies on electrochemotherapy was analyzed in order to produce procedure specific reporting recommendations. A comprehensive literature search of studies published from 2006 to 2015 was performed followed by qualitative analysis of manuscripts assessing for 47 quality criteria grouped into four major clusters: (1) trial design, (2) description of patient population, (3) description of treatment delivery and patient outcome, (4) analysis of results and their interpretation. The summary measure during literature assessment was the proportion of studies fulfilling each manuscript quality criteria. Results. A total of 56 studies were screened, from the period 2006 to 2015, of which 33 were included in the quali­tative analysis, with a total of 1215 patients. Overall, the quality of reporting was highly variable. Twenty-four reports (73%) were single-center, non-comparative studies, and only 15 (45%) were prospective in nature (only 2 of them were entered into a clinical trials registry). Electrochemotherapy technique was consistently reported, with most studies (31/33) adhering closely to published standard operating procedures. The quality of reporting the patient population was variable among the analyzed studies, with only between 45% and 100% achieving dedicated quality criteria. Reporting of treatment delivery and patient outcome was also highly variable with studies only fulfilling between 3% and 100%. Finally, reporting study results critically varied, fulfilling from 27% to 100% of the quality criteria. Based on the critical issues emerging from this analysis, recommendations and minimal requirements for reporting clinical data on electrochemotherapy were prepared and summarized into a checklist. Conclusions. There is an increasing body of published clinical data on electrochemotherapy, but more high quality clinical data are needed. Published papers often lack accurate description of study population, treatment delivery as well as patient outcome. Our recommendations, provided in the form of a summary checklist, are intended to ameliorate data reporting in future studies on electrochemotherapy and help researchers to provide a solid evidence basis for clinical practice. Key words: electrochemotherapy; clinical trials, recommendations Introduction Electrochemotherapy is becoming a well-estab­lished non-thermal ablative technique for malig­nancies of skin and non-skin origin.1,2 The medical applications of electrochemotherapy are based on the principle of electroporation, which dates back to 1982, when sequences of electric pulses were applied to deliver naked DNA molecules within mouse lyoma cells.3 Preclinical studies carried out by several research groups, coupled with technical developments, culminated in the clinical applica­tion of electroporation during the early 1990s.4-13 These initial data on electroporative uptake of mol­ecules are viewed as seminal for various biotechno­logical and medical applications.14,15 The principle of electrochemotherapy is the use of electropora­tion to enhance chemotherapeutic drug delivery. Two agents, bleomycin or cisplatin, can achieve a several fold increase in their intracellular avail­ability, and consequently cytotoxicity, when the tumor tissue is exposed to reversible electropora­tion and transient cell membrane permeabiliza­tion, thus achieving an optimal intratumor drug distribution.7,16-18 Electrochemotherapy has proven effective for the treatment of different tumor his­totypes, including both skin and non-skin cancers, as well as for the palliation of metastases involving cutaneous and subcutaneous tissues.19-22 The treat­ment of primary skin tumors is largely restricted to multifocal cutaneous tumors, most notably some selected cases of basal cell carcinoma, when tumor anatomical location and patient medical conditions contraindicate more aggressive treatments.23 The publication of the European Standard Operating Procedures of Electrochemotherapy (ESOPE) in 2006 facilitated a broad acceptance of electrochemotherapy for treatment of cutane­ous tumors and metastases.24 Over a number of years, several clinical reports have confirmed its effectiveness. Interestingly, the vast majority of studies used the Standard Operating Procedure (SOP) as a guideline for electrochemotherapy. The availability of SOP allowed for reproduc­ibility and improvement of results in the clinical practice. Several large follow up series confirmed the efficiency of electrochemotherapy. A recent meta-analysis of the use of electrochemotherapy in the treatment of cutaneous metastasis places it well amongst other, more established, treat­ment options.2 Recently, electrochemotherapy has also been recognized by the National Institute for Health and Care Excellence (NICE) as an integral part of the multidisciplinary treatment for pa­tients with skin metastases of non-skin origin and melanoma (NICE interventional procedure guid­ance IPG 446, http://www.nice.org.uk/guidance/ ipg446). More recently, electrochemotherapy has been introduced into the treatment of deep-seated and endoluminal tumors.25-28 The first clinical re­port on visceral metastases indicates its effective­ness, and suggests a possible role of electrochemo­therapy for the treatment of liver metastases, es­pecially when located close to major blood vessels and when not manageable with surgery or other ablative techniques.29 Overall, literature data from Web of Science da­tabase indicate a steady increase in number of pub­lications and their citations under the key word “electrochemotherapy” (Figure 1A,B) and “clini­cal electrochemotherapy” (Figure 1C,D). Despite a steady increase in the number of published reports, a higher quality and standardization of reported studies is needed to improve and support a truly evidence based practice. In our study we only in­cluded papers published after 2006, specifically on­ly to include reports published after the Standard Operating Procedures (SOP).24 The purpose of this recommendation paper is to provide practical recommendations in order to improve the precision of reported clinical stud­ies on electrochemotherapy (a summary checklist is provided as Supplementary file). This, in turn, we hope will stimulate the scientific community to report research using these guidelines to give comprehensive reports on areas including study design, definition of study endpoints, patient se­lection criteria, treatment plan and outcome assess­ment. The adoption of more precision in reporting will enable researchers and clinicians to perform more meaningful outcome comparisons with other ablative techniques, to clarify the direction for fu­ture research, and to produce more evidence-based practice. It is our hope that these advancements may improve patient selection, resource allocation, and ultimately patient outcome. This report was prepared based on initiative of the Steering Committee of the COST TD 1104 Action (www.electroporation.net) and in response to a general call for increased awareness and con­cern for low quality reporting practice30; moreover, it has been prepared by the committee within the Working group of Medical applications of elec­troporation, in COST action TD 1104 EP4Bio2Med, and is included in the series of publications ad­dressing the same topic in preclinical research in electroporation as well as in the pulsed electric fields for industrial purposes.31 Campana LG et al. / Recommendations for reporting electrochemotherapy clinical studies guidance for reporting the aim, methods, results and implications of randomized controlled trials; the PRISMA statement (www.prisma-statement. org) indicates preferred reporting for systematic reviews; finally, the REporting recommendations for tumor MARKer prognostic studies (REMARK, www.equator-network.org/reporting-guidelines/ reporting-recommendations-for-tumour-marker­prognostic-studies-remark/) suggest guidelines to provide relevant information about study design, preplanned hypotheses, patient and specimen char­acteristics, assay methods, and statistical analyses Several guidelines exist with the aim of assur­ing sound research practices, and improving the quality of clinical trials and, ultimately, allow for generalizable results. At a basic level, Good Clinical Practice (GCP) represents an international ethical and scientific quality standard for designing, con­ducting, recording and reporting trials that involve the participation of human subjects. At a higher level, dedicated guidelines and recommendations have been developed according to the specific type of study performed. For instance, the STROBE statement (www.strobe-statement.org) indicates a checklist for details that should be reported in ob­servational trials; the Consolidated Standards of Reporting Trials (CONSORT) statement (www.con- Published Items Each Year Citation in Each Year 100 3500 90 3000 80 70 2500 60 2000 50 40 1500 30 1000 10 20 500 0 0 C D FIguRe 1. Search in Web of Science demonstrates a steady increase in number of publications under the key word “electrochemotherapy” (a,B) as well under the “electrochemotherapy, clinical” (C,D). The Meta data indicate the expanding field. sort-statement.org/consort-statement/) provides trochemotherapy and identified possible pitfalls A Published Items Each Year Citation in Each Year 30 1200 25 1000 20 800 15 600 10 400 5 200 0 0 1996 1996 1997 1997 1998199819991999 2000 2000 200120012002200220032003 2004 2004 20052005 2006 20062007 2007 20082008 2009 20092010 20102011 2011 20122013 2012 20132014 2014 when evaluating tumor markers in oncology. In ad­dition, these guidelines provide helpful suggestions on how to present data and important elements to include in discussions. Although these guidelines provide a fundamental guidance for conducting a valid clinical trial and reporting generalizable find­ings, nonetheless it is recognized that there is a need for specialty-specific guidelines and that these guidelines will lead to improvement in the quality of reports and to higher impact publications.32,33 In the field of electrochemotherapy, comprehensive meta-analyses or Cochrane style reviews of effi­ciency are hampered by the lack of some relevant clinical data in published reports. Therefore, we evaluated the published papers on clinical elec- B 1997 1997 1998 1998 1999 1999 20002000200120012002200220032003200420042005200520062006200720072008200820092009 20102011201220132014 20102011201220132014 Radiol Oncol 2016; 50(1): 1-13. in data reporting. On this basis, we prepared rec­ommendations for improving the quality of future studies and fostering further rational development of electrochemotherapy. Systematic review and qualitative analysis of publications Methods The initial step was to identify and access all pub­lished trials evaluating the efficacy of electrochem­ TaBle 1. Manuscript quality criteria 1. Prospective trial 1. Setting (curative / palliative) 1. Type of anaesthesia 1. Summary of trial endpoints 2. Trial registration 2. Drug route and dosages 3. Comparative trial 2. Demographic data (in tabular form) 3. Pulse generator 2. Predictive factors 4. Mention of trial design 4. EP parameters 5. Multicenter study 3. No of tumors 5. Electrode description 3. Other patient outcome parameters 6. Mention of sponsor 6. Tumor safety margins indicated 7. Trail hypothesis and sample size 4. Tumor location 7. Deviation from SOPs 4. Results interpretation 8. Informed consent 5. Tumor histotype 9. Criteria for retreatment 5. Comparison to historical controls 9. EC approval 8. Tumor coverage with EP 10. Structured abstract 10. Total No of ECT sessions 11. Rationale of the trial 6. Tumor size 11. ECT sessions requireda 6. Future directions 12. A priori inclusion criteria 12. Toxicity criteria 13. Follow-up dates 7. Visceral mts indicated 13. Response criteria 7. COI statement 14. Statistical methods 14. Evaluation of tumor control 15. Software used 8. Concomitant treatments 15. ECT successb 16. C.I., p-values 16. Keep track of patients lost to follow-up C.I. = confidence intervals; COI = conflict of interest statement; EC = Ethic Committee; EP = electric pulses (including number, duration and amplitude); mts = metastases; SOPs = Standard Operating Procedures. a Number of electrochemotherapy (ECT) sessions required for achieving response (either complete or partial) on baseline tumors b Decision rule for determining ECT success otherapy in the treatment of tumors including skin cancers, cutaneous/subcutaneous metastases from other histotypes, deep-seated tumors or visceral metastases. From October 4 to 10, 2015, we conducted a comprehensive literature assessment that included searches of Medline (EBSCO), Pubmed (NLM), Web of Science and Embase. The search terms used were “electrochemotherapy”, “electrochemother­apy” AND “clinical trial”. We limited our search to humans. Articles published from January 2006 to September 30, 2015 were retrieved. We included studies on the clinical application of electrochemo­therapy regardless of study design (both prospec­tive and retrospective) patient population, tumors histotype and anatomical location or electrochem­otherapy treatment protocol. However, treatment outcome had to include tumor response and fol­low-up tumor control evaluation, procedural mor­bidity and toxicity or patient quality of life. Two of the authors (LGC and SV) and an external col­laborator with experience in clinical trials indepen­dently screened the retrieved studies based on the title, key words, and abstract to exclude non-rele­vant and non-English written studies. After com­pletion of all searches, duplicates were removed and only the most recent report from follow-up series was included in order to avoid overlapping series. Both retrospective and prospective studies were included, while case reports and small se­ries were excluded because of their intrinsic lower level of evidence (the minimum number of pa­tients was arbitrarily set at 9). Published reviews on electrochemotherapy were similarly excluded, but their reference list was reviewed in order to identify possible additional studies. Studies whose main purpose was unrelated to electrochemo­therapy efficacy and biological studies (i.e., those exploring immune effects of treatment) were also excluded, unless clear and standardized descrip­tion of patient outcome was retrievable from the manuscript. Studies that did not meet the inclusion criteria were discarded during the initial review. When uncertainty existed in the abstract evalu­ation, we retrieved and assessed the full text. A third author (GS) resolved differing opinions. Full text of the included articles was independently re­viewed by two of the authors using a predefined checklist quality criteria. These quality criteria were discussed and agreed among the authors in a series of operative meetings which were hold during the 1st World Congress on Electroporation in Portoroz, Slovenia, between September 6 to 10 2015 and were also based on deliberations at the Recommendation paper workshop organized by COST TD1104 on 28th March 2014 in Copenhagen, Denmark. The checklist was also adapted from similar reporting standard guidelines in the field of neuro-oncology, isolated limb perfusion and in phase II cancer trials.34-36 As a result, we had a fi­nal count of 47 quality criteria that were clustered into four domains: trial design, description of pa­tient population, treatment delivery and outcome assessment, and analysis of results and their inter­pretation (Table 1). The summary measure during literature assessment was the proportion of studies fulfilling each manuscript quality criteria. Results A total of 56 papers were initially identified. Of these, only 33 reports were finally retained in the qualitative synthesis; the reasons for exclusion of the remaining reports are listed in Figure 2. A summary of the studies included in the final analysis is presented in Table 2.20-22,29,37-65 The total number of patients across all studies was 1215. Electrochemotherapy protocol was following the SOP as defined in ESOPE study in all but two cas­ es.40,65 The majority (24/33) of reports were single-cent­er studies. There were 24 tumor-specific studies (melanoma, n=8; breast cancer, n=5; head and neck squamous cell carcinoma, n=4; Kaposi sarcoma, n=3; pancreatic cancer, n=1; colorectal cancer, n=1; soft tissue sarcomas, n=1; vaginal squamous cell cancer, n=1) and 9 studies including heterogene­ous histologies. Response assessment was based on clinical evaluation in all except 3 studies on pancreatic cancer41, liver metastases from colorec­tal cancer29, and chest wall recurrence from breast cancer57, where response assessment was radio­logical (ultrasound scan, magnetic resonance im­aging, computed tomography, or fluorine-18-de­oxyglucose PET-CT scan). Details of the quality criteria used to assess trial design are presented in Figure 3. Less than half (15/33, 45%) of studies FIguRe 2. PRISMA flow diagram of identification, screening, eligibility and inclusion of studies. Prospective trial Trial registration Comparative trial Mention of trial design Multicenter study Mention of sponsor Trial hypothesis and sample size Informed Consent Ethic Committee approval Structured abstract Rationale of the trial A prori de.nition of inclusion criteria Follow-up dates Statistical methods Software Con.dence Intervals, p-values 6 45 3 9 18 0 12 88 85 85 70 58 64 64 54 97 0 20 40 60 80 100 % of included studies (n=33) FIguRe 3. Assessment of published studies according to quality criteria concerning trial design. were prospective and only two of them (6%) were entered into a publicly accessible clinical trials reg­istry.29,57 Eighteen percent (6/33) of papers repre­sented the report of a multicenter study. There was a single comparative trial (an internally controlled TaBle 2. Trials identified included in the qualitative analysis Rotunno, 2015 37 Two-center, Italy 55 non-melanoma SC ESOPE Cabula, 2015 38 Multi-center, Italy 125 BC ESOPE Mozzillo, 2015 39 Single-center, Italy 15 melanoma ESOPE Landstrom, 2015 40 Single-center, Sweden 19 HNSCC Other a Granata, 2015 41 Single-center, Italy 13 pancreatic cancer ESOPE Kreuter, 2015 42 Multi-center, Germany 56 various ESOPE Quaglino, 2015 43 Multi-center, Europe 121 various ESOPE Mir-Bonafé, 2015 44 Single-center, Spain 31 melanoma ESOPE Campana, 2014 45 Single-center, Italy 39 HNSCC ESOPE Ricotti, 2014 46 Single-center, Italy 30 melanoma ESOPE Campana, 2014 47 Single-center, Italy 55 BC ESOPE Edhemovic, 2014 29 Single-center, Slovenia 16 CRC-liver mts ESOPE b Seccia, 2014 48 Single-center, Italy 9 HNSCC ESOPE Campana, 2014 50 Two-center, Italy 34 STS ESOPE Solari, 2014 51 Single-center, Italy 39 various ESOPE Di Monta, 2014 52 Single-center, Italy 19 KS ESOPE Caraco, 2013 49 Single-center, Italy 60 melanoma ESOPE Perrone, 2013 53 Single-center, Italy 9 V-SCC ESOPE Benevento, 2012 54 Single-center, Italy 12 BC ESOPE Mevio, 2012 55 Single-center, Italy 15 HNSCC ESOPE Campana, 2012 20 Single-center, Italy 35 BC ESOPE Latini, 2012 56 Single-center, Italy 18 KS ESOPE Matthiessen, 2012 57 Single-center, Denmark 12 BC ESOPE Gargiulo, 2012 58 Single-center, Italy 52 non-melanoma SC ESOPE Campana, 2012 21 Single-center, Italy 85 melanoma ESOPE Curatolo, 2012 59 Two-center, Italy 23 KS ESOPE Kis, 2011 60 Single-center, Hungary 9 melanoma ESOPE Matthiessen, 2011 22 Two-center, Denmark-UK 52 various ESOPE Skarlatos I, 2011 61 Multi-center, Greece 52 various ESOPE Campana, 2009 62 Single-center, Italy 52 various ESOPE Quaglino, 2008 63 Single-center, Italy 14 melanoma ESOPE Larkin, 2007 64 Single-center, Ireland 30 various ESOPE Gaudy, 2006 65 Single-center, France 12 melanoma Other c BC = breast cancer; ECT = electrochemotherapy; CRC-liver mts = colorectal cancer liver metastases; HNSCC = head and neck squamous cell cancer; KS = Kaposi’s sarcoma; SC = skin cancer; STS = soft tissue sarcomas; V-SCC = vaginal squamous cell cancer a Intratumoral BLM injection (1000 IU/cm3 and tumor electroporation by means of six 1100 V/cm square wave pulses with 0.1 ms duration b In this trial, the ESOPE protocol was integrated by the application of variable geometry electrodes for the treatment of deep visceral metastases. c Intratumoral BLM injection (concentration, 4 mg/mL; dose, 1 mg/cm3 of tumor volume was followed, after 10 minutes, by the application of electric pulses (six 100 µsec-long pulses, 4 pulses/sec, electric field >600V/cm study with intrapatient randomization of mela­noma metastases to intralesional bleomycin versus intralesional bleomycin followed by electric puls­es)65; a formal sample size calculation or analysis of “intent-to-treat” population was found in only 4/33 (12%) studies.20,50,57,65 Details of the quality criteria used to assess the description of patient population are presented in Figure 4. Treated tumors were described in detail in most reports: number of tumors, 94%; tumor loca­tion, 100%; tumor histotype, 100%; tumor size, 91%. On the other hand, additional clinical information was less frequently reported: study setting -pallia­tive/curative-, 54%; presence of visceral metastases, 54%; concomitant oncologic treatments, 45%. Details of the manuscript quality criteria used to assess the description of treatment delivery and response assessment are presented in Figure 5. Treatment details were accurately described in most reports: type of anaesthesia, 32/33 (97%); drugs, 33/33 (100%); pulse generator, 33/33 (100%); electrode types, 31/33 (93%); electric pulse param­eters, 32/33 (97%). The criteria for response assess­ment were clearly stated in 29/33 (88%) of studies, while toxicity criteria were indicated in only 14/33 (42%) of papers. Details of the quality criteria used to assess the analysis of results and their interpretation are presented in Figure 6. The majority of reports in­cluded a critical analysis: interpretation of results, 33/33 (100%); comparison to historical control, 25/33 (76%); indication of possible future direc­tions, 33/33 (100%); conflict of interest statement, 27/33 (82%). On the contrary, only a minority of them fulfilled other specific quality criteria: sum­mary of primary and secondary endpoints, 13/33 (39%); indication of predictive factors, 9/33 (27%); additional patient outcome parameters, 9/33 (27%). Based on the results of this analysis, the con­sensus between authors was to recommend some minimal requirements for reporting clinical data in future studies. Recommendations and minimal requirements for reporting clinical trial results on electrochemotherapy Trial design Any consolidation of the evidence base of electro­chemotherapy requires that reports adhere strictly to research reporting standards and are the re­sult of well-designed clinical trials. Much of these Setting, curative/palliative 54 Demographic data in tabular form 91 No. of tumors 94 Tumor location 100 Tumor histotype 100 Tumor size 91 Visceral metastases indicated 54 Indication of concomitant treatments 45 0 20 40 60 80 100 % of included studies (n=33) FIguRe 4. Assessment of published studies according to quality criteria concerning description of patient population. Type of anaesthesia 97 Drug, route and dosages 93 24 6 9 39 79 36 42 88 36 6 3 100 Pulse generator 100 EP parameters 97 Electrode type Safety margins Deviation from SOPs indicated Tumor coverage with EP Criteria for retreatment Total No of ECT sessions No of ECT required on baseline tumors Toxicity criteria Response criteria Evaluation of tumor control Decision rule for determining success Track of patients lost to follow-up 0 20 40 60 80 100 % of included studies (n=33) FIguRe 5. Assessment of published studies according to quality criteria concerning treatment delivery and outcome assessment. ECT = electrochemotherapy; EP = electric pulses. Summary of trial endpoints 39 Predictive factors 27 Other outcome parameters (QoL, PRO) 27 Results interpretation 100 Comparison to historical controls 76 Future direction 100 COI statement 82 0 20 40 60 80 100 % of included studies (n=33) FIguRe 6. Assessment of published studies according to quality criteria concerning analysis of results and interpretation. COI = conflict of interest statement; PRO = patient reported outcomes; QoL = quality of life. topics are covered by STROBE (STrengthening the Reporting of Observational studies in Epidemiology, http://www.strobe-statement. org/) checklist and CONSORT (CONsolidated Standards of Reporting Trials, http://www.consort­statement.org/checklists/view/32-consort/66-title) guidelines which should be adhered to as much as possible when reporting observational studies and randomized controlled trials, respectively. Incorporation of these electrochemotherapy guide­lines will further improve the quality of the reports. So far, only phase I-II single-arm trials have been reported, with the exception of a single small-sized study, which included an intra-patient randomi­zation of tumors to direct bleomycin injection or bleomycin injection followed by electroporation.65 It is likely that improving the evidence base will involve conducting properly designed, prospec­tive comparative - possibly randomized - clinical trials in order to perform accurate analyses of the advantages of electrochemotherapy against other ablative procedures or alternative local treatments. Of utmost importance, future trials should aim to be prospective and preferably multicentric, with clearly defined endpoints and inclusion criteria. It is also advisable that all trials should be registered at publicly accessible clinical trials registries, (e.g., clinicaltrials.gov, ISRCTN registry at http://www. isrctn.com, WHO registry at www.apps.who.int/ trialsearch, or similar ) and approved by institu­tional review boards or respective national bodies. Finally, according to the current requirements of most scientific journals – which refer to the rec­ommendation of the International Committee of the Medical Journal Editors (ICMJE, http://www. icmje.org/), manuscripts should conform to well-defined general principles and include, for exam­ple, a statement about patient informed consent, modalities of study conduct, as well as authors conflicts of interest. Key elements of trial design: • Explanation of the rationale of the study • Description of trial design and sponsorship • Indication of trial endpoints • Indication of inclusion and exclusion criteria • Trial approval and registration • Informed consent statement Description of patient population Electrochemotherapy was initially used with pal­liative intent. First trials demonstrated remark­able efficiency in the treatment of skin metastases from malignant melanoma.21,60,63 Subsequently, electrochemotherapy was also evaluated for the treatment of other tumor histotypes (e.g., non-melanoma skin cancers and cutaneous metastases from other tumor histotypes) with equally high success.20,37-38,47,57-58 Reports of small series indicate also its possible usefulness in the treatment of pri­mary basal cell carcinomas23 and a clinical trial is currently ongoing comparing the effectiveness of electrochemotherapy to standard surgical resec­tion and is due to report 5 year follow up data next year (EudraCT Number: 2010-019260-37). A particular advantage of electrochemotherapy is that it is a reliable alternative treatment option for patients who have exhausted more conven­tional oncological treatments or are judged unfit for or refuse repetitive surgical interventions.47 Therefore, future reports need to include detailed description of patient’s demographic and clinical data including detailed description of previous treatments. A detailed description of tumor loca­tion, histotype as well as number and size of the electrochemotherapy target and non-target lesions is paramount. Authors should also specify whether targeted lesions had previously received irradia­tion or not, whether visceral metastases are present and whether the treatment is intended as pallia­tive or curative. Additionally, since electrochemo­therapy is finding its place among other oncologic treatments, and will be increasingly used also in combination with them, an accurate record of con­comitant treatments is also advisable.66 Key elements of patient population: • Patient demographic data (in tabular form) • Setting - palliative or curative • Tumor histology • Disease stage (lymph node or visceral metastases) • Description of target lesions treated with electrochemotherapy (anatomical location, number and size ) • Previous local treatments • Concomitant oncological treatments • Adjuvant and / or following oncological treatments Treatment information The treatment is applied by performing a proce­dure conjugating the administration of a drug and local application of electric pulses. In one “session” developing satellite lesions such as in the case of malignant melanoma or soft tissue sarcomas. In order to improve reporting, the information about the safety margins and their extent should also be reported. In addition, electrochemotherapy can be repeated several times (however there is a ceiling for the total lifetime dose of bleomycin), according to tumor response and disease behavior.62,63 This fundamental aspect is not covered by the currently available SOP. For providing a more informative report, data regarding repetitive treatments should be included, along with the description on what basis the retreatment was performed and at what time interval. Key elements on treatment information: • Indication of electroporation protocol (adherence to SOP or other ) • Type of anesthesia • Drug (producer ) • Drug details (dose, concentration and route of administration • Time interval between drug administration and application of electric pulses • Technical details of the electric pulse generator, including type, manufacturer and version of software, if applicable • Information about the electrodes used, for respective tumor(s) • Number of electric pulses application per tumor • Inclusion of a report on electrical parameters (n, T, U, I, f )* • Adequacy of tumor treatment (treatment application success rate) • Extent of the safety margins treated • Number of treatment sessions (with interval between sessions) * Legend: n = number; T = duration of pulses; U = voltage amplitude applied; I = measured current; f = pulse repetition frequency Outcome assessment The early studies on electrochemotherapy antitu­mor activity have carefully evaluated the response of treated tumors. Response assessment was initial­ly performed by the bidimensional WHO criteria.71 According to these criteria, baseline and post-treat­ment tumor size is determined by bidimensional measurements e.g. the sum of the two longest di­ameters in the perpendicular dimensions. The tu­mor response to treatment is divided into four cat­egories (complete response, partial response, sta­ble disease, progressive disease, according to the change from baseline tumor measurement). Indeed, most past studies were focused on tu­mor response and on patient early outcomes. Nevertheless, a number of reports indicate that the disease locally relapsed or progressed elsewhere, but only few reports indicated the value of electro­chemotherapy in the local management of patient symptoms. Hence, the clinical benefit for patients, especially in the palliative setting, where preserva­tion of quality of life (QoL) and evaluation of pa­tient reported outcome (PRO) are crucial, should be based on dedicated assessments and described. The new RECIST (Response Evaluation Criteria in Solid Tumors) version 1.1 criteria, with some ad­aptations, have proven a suitable tool for response assessment of superficial tumors21,72, whereas for the setting of treatment of deep-seated tumors (i.e., electrochemotherapy application on liver metas­tases) the modified RECIST criteria represent the most appropriate and standardized method for the evaluation of tumor response.73 In general, for standard electrochemotherapy on superficial tu­mors, the RECIST 1.1 criteria, which are based on one-dimensional measurements, seem even more practical and offer highly concordant response as­sessment compared with the bidimensional WHO criteria.74 So far, most of the published papers do not report on any serious treatment related adverse event after electrochemotherapy. Nevertheless, the process surrounding the determination, recording and reporting of adverse events remains moderate­ly challenging especially for the clinician who may not be involved in drug or device-related research. Nevertheless, it is important to understand the ba­sic definitions of adverse events reporting in order to ensure that the proper information is collected in clinical protocols. Moreover, a comprehensive pa­tient observation and a detailed report of all types and grades of toxicities are essential for providing a comprehensive report of treatment outcome, not only in the early, but also in the long-term follow-up. In this way, only large cohorts of patients will enable in-deep view of long-term toxicity and more detailed analyses of treatment-related ad­verse events according to different patients sub­group, as demonstrated by a recently published report on electrochemotherapy-related pain.43 For this purpose, Common Terminology Criteria for Adverse Events (CTCAE v4.0) is widely accepted throughout the oncology community as the stand­ard classification and severity grading scale for adverse events. Unfortunately, most of the studies conducted so far do not report consistently on this crucial aspect. Key elements of treatment outcome assessment: • Time of response assessment • Standardized response evaluation criteria (e.g. WHO, RECIST 1.1, mRECIST) • Time to local and systemic disease progression • Standardized toxicity criteria (e.g. CTCAE v4.0) • Quality of Life (QoL), patient reported outcomes (PRO) • Track of patients lost to follow-up analysis and interpretation of the results A clear summary of the trial endpoints is essential. In fact, the field is moving beyond simply report­ing on tumor control, as treatment now includes, in some instances, also primary tumors. Here it is important to report and discuss other parameters, such as time to local/systemic progression and, if possible, also the patient survival time and QoL as well. Such data will increase the evidence level of electrochemotherapy effectiveness, and consoli­date a role for electrochemotherapy outside the palliative setting and into a confirmed primary treatment modality. It has been clearly demonstrated that tumor size is the most reliable predictive factor for re­sponse in patients who underwent electrochemo­therapy.21,22,69 In future, detailed reports including data on previous local therapies (e.g., radiation) as well as on local (within electrochemotherapy field) tissue status (e.g., presence of lymphedema or fi­brosis) and concomitant/adjuvant oncologic treat­ments would allow for the identification of other reliable predictive indicators for response. Key elements for analysis and interpretation of the results: • Summary of trial endpoints • Additional patient outcome parameters (e.g., QoL, PRO) • Predictive factors • Results interpretation • Future research directions Conclusions Electrochemotherapy represents an effective treat­ment option for an increasing number of cancer patients with superficial tumors. Nevertheless, to further improve its evidence basis, it will be crucial to raise the quality of future reports. In this study, we have highlighted some relevant aspects of clinical data reporting, with the aim of improving the quality of future studies in the field of electrochemotherapy. Although a large amount of data are published so far, clinical research needs to adopt detailed and accurate reporting as well as moving from small, non-comparative series to well-designed, possibly randomized, clinical trials. Despite the encouraging results indicated, the vast majority of included reports are case series from single institutions. Although there was a wide con­sensus to use previously published SOP for the treatment protocol, these studies often present a variety of designs and reporting methods, thus lim­iting the understanding of patient selection, treat­ment effect, toxicity and overall patient outcome. Of note, published studies often lack sufficient pro­cedural as well as patient data. These shortcomings represent a major hurdle to performing systematic reviews or meta-analysis, which may provide a more robust evaluation of treatment effectiveness and, ultimately, encourage wider acceptance of electrochemotherapy in the clinical practice. Our study has some limitations. We identified a set of manuscript quality criteria from available lit­erature and we have expanded this list by includ­ing additional, procedure-specific criteria that were discussed and agreed among the authors. The list of 47 quality criteria that were used for reviewing published reports represents an arbitrary selection of criteria performed by a relatively small number of authors. There is potential for selection bias in the inclusion of papers for analysis, as the initial screen was based on broad, non-selective inclusion criteria. However, we feel that these were widely inclusive and fitting in order to develop the pro­posed recommendations. Nevertheless, we believe that our suggestions largely cover the most crucial aspects, which are required to improve the quality of clinical practice and future research: trial design and conduction, definition of study endpoints, pa­tient selection, treatment delivery, patient manage­ment and follow-up, standardization of outcome assessment. Our recommendations are open to a broader discussion with the community users of electrochemotherapy and, possibly, to further improvements in line with other interventional oncology procedures.75,76 Electrochemotherapy re­quires standardization of terminology and report­ing criteria to facilitate effective communication among researchers and appropriate comparison between different treatment technologies. As such, investigators involved in this field should be fa­miliar with these recommendations and use them for future study design and conduction, treatment application as well as data reporting. We envision that the adoption of these recommendations will further improve the quality of future studies and allow more meaningful comparisons of outcome data of patients treated with electrochemotherapy (Supplementary file). acknowledgements The authors thank Roberto Marconato, Padova School of Medicine, for his help in literature search and screening of papers. The paper was discussed at the 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine, and Food & Environmental Technologies, September 6 to 10, 2015, Portoroz, Slovenia (wc2015.electropo­ration.net) organized by COST TD1104 Action (www.electroporation.net), supported by COST (European Cooperation in Science and Technology) and Slovenian Research Agency. References 1. Mali B, Jarm T, Snoj M, Sersa G, Miklavcic D. Antitumor effectiveness of electrochemotherapy: A systematic review and meta-analysis. EJSO 2013; 39: 4-16. 2. Spratt DE, Spratt EAG, Wu SH, DeRosa A, Lee NY, Lacouture ME, et al. 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J Vasc Interv Radiol 2009; 20: 1527-40. review Electrochemotherapy in pancreatic adenocarcinoma treatment: pre-clinical and clinical studies Sabrina Bimonte1*, Maddalena Leongito1*, Vincenza Granata2, Antonio Barbieri3, Vitale del Vecchio3, Michela Falco3, Aurelio Nasto1, Vittorio Albino1, Mauro Piccirillo1, Raffaele Palaia1, Alfonso Amore1, Raimondo di Giacomo1, Secondo Lastoria4, Sergio Venanzio Setola2, Roberta Fusco2, Antonella Petrillo2, Francesco Izzo1 1 Division of Abdominal Surgical Oncology, Hepatobiliary Unit, Istituto Nazionale per lo studio e la cura dei Tumori “Fondazione G. Pascale”, IRCCS, Naples, Italy 2 Division of Radiology, Istituto Nazionale per lo studio e la cura dei Tumori “Fondazione G. Pascale”, IRCCS, Naples, Italy 3 S.S.D Sperimentazione Animale, Istituto Nazionale per lo studio e la cura dei Tumori “Fondazione G. Pascale”, IRCCS, Naples, Italy 4 Division of Nuclear Medicine, Department of Diagnostic Imaging and Radiotherapy, Istituto Nazionale Tumori “Fondazione G.Pascale” IRCCS, Naples, Italy Radiol Oncol 2016; 50(1): 14-20. Received 17 September 2015 Accepted 13 December 2015 Correspondence to: Dr. Sabrina Bimonte, Division of Abdominal Surgical Oncology, Hepatobiliary Unit, Istituto Nazionale per lo studio e la cura dei Tumori “Fondazione G. Pascale”, IRCCS, Via Mariano Semmola, Naples, Italy. E-mail: s.bimonte@istitutotumori.na.it Disclosure: No potential conflicts of interest were disclosed. *The first two authors contributed equally to this manuscript Background. Pancreatic adenocarcinoma is currently one of the deadliest cancers with high mortality rate. This disease leads to an aggressive local invasion and early metastases, and is poorly responsive to treatment with chemo­therapy or chemo-radiotherapy. Radical resection is still the only curative treatment for pancreatic cancer, but it is generally accepted that a multimodality strategy is necessary for its management. Therefore, new alternative thera­pies have been considered for local treatment. Conclusions. Chemotherapeutic resistance in pancreatic cancer is associated to a low penetration of drugs into tumour cells due to the presence of fibrotic stroma surrounding cells. In order to increase the uptake of chemothera­peutic drugs into tumour cells, electrochemotherapy can be used for treatment of pancreatic adenocarcinoma leading to an increased tumour response rate. This review will summarize the published papers reported in literature on the efficacy and safety of electrochemotherapy in pre-clinical and clinical studies on pancreatic cancer. Key words: electrochemotherapy; pancreatic carcinoma; bleomycin; pre-clinical study; clinical study; safety; ef­ficacy Introduction Pancreatic adenocarcinoma, despite extensive re­search, remains one of the deadliest cancers with high mortality rate. Surgical resection represents the only curative treatment for this pathology, but the majority of the patients are incurable at initial presentation with metastatic (stage IV) or surgi­cally non-resectable disease (stage III disease).1-3 At present gemcitabine and paclitaxel (Abraxane) are the best chemotherapeutic agents used for treatment of pancreatic cancer, however, patients develop drug resistance over the time. Thus, new alternative strategies, involving less toxic agents have been considered for local treatment of pan­creatic cancer.4-6 It is of note that chemotherapeutic TABLE 1. Electrochemotherapy treatment in pancreatic cancer cell lines Girelli et al., 201517 PANC1 MiaPaCa2 Bleomycin Cisplatin < 0.0001 . 0.0001 MTS assay FACS 8 pulses, 100 µs of duration, 5 Hz Sundararajan, 201426 PANC1 PANC28 Gemcitabine < 0.0001 . 0.0001 MTS assay high intensity, low duration (microseconds) pulses; low intensity and long duration pulses (milliseconds). resistance in pancreatic cancer is associated with low penetration of drugs into tumour cells due to the presence of fibrotic stroma. Reversible electroporation (EP) is a physical method used to overcome the barrier of the cell membranes by applying short and intense electric field, depending by cell characteristics as shape, size, and cytoskeleton structure and membrane composition. Applications of these pulses create rapid voltage changes that usually reach values be­tween 0.5-1 V. This local polarization, deforms me­chanically the membrane by forming a hydrophilic pore with a radius of . 1 nm. It was hypothesized, that primary hydrophobic pores induced the transi­tion to hydrophilic based precisely on a strong and nonlinear local transmembrane voltage. This treat­ment enables access for extracellular agents into the cells. For this reason EP may be combined with chemotherapeutic agents to improve their uptake, in particular hydrophilic drugs that, differently from lipophilic, are poorly or not permeant; this new cancer treatment modality is named electro­chemotherapy (ECT).7-10 The use of ECT for tumour treatments leads to a local potentiation of chemo­therapy by reducing the doses of the drugs, mini­mizing the side effects, and increasing the efficacy of chemotherapy. In the perspective of pancreatic tumour treatment with ECT, several pre-clinical and clinical studies have been performed. This re­view will summarize the published papers reported in literature on the efficacy and safety of ECT in pre-clinical and clinical studies on pancreatic cancer.11-17 ECT in treatment of pancreatic cancer: in vitro studies Several studies have been conducted in order to identify the optimal electric field characteristics (amplitude, duration and number of electric pulses and repetition frequency) and the appropriate an­tineoplastic drugs whose cytotoxicity could be in­creased by combination with EP.18,19 MTT assay allowing to define cellular cytotoxic­ity, is the method commonly used to screen drugs for feasibility and safety on both electro-permea­bilized and not electro-permeabilized cells. It has been shown that, cell death due to the EP proce­dure was less than 4%, and that more than 90% of cells were permeabilized. Among several substanc­es evaluated in association to EP some of them, such as daunorubicin, doxorubicin, etoposide and paclitaxel, did no show increased cytotoxicity. For carboplatin and cisplatin (CDDP) the efficacy of EP was indexed with a factor 3 and 2.3, respectively, on the IC50 (inhibitory concentration 50%), while bleomycin (BLM) was indexed with a value of 300.20-22 These data suggest that BLM, by inducing apoptosis of several cancer cell lines, is considered the drug of choice for ECT, while the use of CDDP still remains to be fully explored.23, 24 Few studies have been performed on the use of ECT in pancreatic cancer treatment, but the emerg­ing results are very encouraging. In particular, in a recent publication, were reported the feasibility and the safety of ECT for the treatment of pancre­atic ductal adenocarcinoma (PDAC), a highly ag­gressive disease which normally is diagnosed in advanced stage. In this study it was demonstrated that EP represents a safe procedure in the treat­ment of PDAC and that can potentiate the effect on cytotoxicity of bleomycin and cisplatin in pan­creatic tumour cell lines, PANC-1 and Miapaca-2 (Table 1).17 New applications of ECT protocol on pancre­atic cancer cells have been developed. These tech­niques combined the use of natural compounds and chemotherapeutics drugs to ECT procedure, in order to reduce cytotoxic drug effects. In particu­lar, two different studies have demonstrated that nanocurcumin, a polymeric nanoparticle-encapsu­lated curcumin, has better efficacy on pancreatic or breast tumour cell lines respect to normal cur-cumin and it is able to activate the same molecu­lar pathways.25,26 Another natural compound that could be used in association with ECT is the epi­gallocatechin-3-gallate (EGCG), the most abundant catechin found in green tea, in particular combined with BLM. Recently, we showed the efficacy and synergism of EGCG and BLM on the inhibition of pancreatic cancer MiaPaCa-2 cell proliferation by inducing apoptosis.27 Several studies are ongoing in our laboratory, to demonstrate that ECT treatment can potentiate the efficacy and synergism of EGCG and BLM in pan­creatic cancer cells and in pancreatic cancer mouse model. Taken together, these studies suggest that ECT could be considered as a valid technique for treat­ment of pancreatic cancer, although more studies will be needed in order to refine the ECT protocols. Electrochemotherapy in pancreatic cancer: in vivo animal models It is has been reported that (EP), has been used for different types of clinical applications in treat­ment of cancer: irreversible (IRE) or reversible electroporation combined with chemotherapeutic agents (ECT). IRE is an ablative non-thermal tech­nique which uses a high voltage (maximum 3,000 V) small microsecond pulse lengths (70 to 90 µs) to induce cell membrane permeability which leads to slow/protracted cell death over time. Pre-clinical data supporting both the safety and effectiveness of IRE in treatment of pancreatic cancer have been published. In particular, Bower et al. performed an in vivo study demonstrating no adverse events of IRE around the portal veins in a large porcine ani­mal model. All pigs have been exposed to a pulse field generated with maximum 3,000 V for 70-90 µs and revealed only mild adhesions, no ascites, and no pancreatic necrosis. This study demonstrates that IRE protocol of the pancreas performed at an optimal voltage is well tolerated, with rapid reso­lution of pancreatic inflammation and preserva­tion of vascular structures.28 Similar results were confirmed by Charpentier et al. who generated an acute animal model (2 hours survival) and also demonstrated no vascular thrombosis as well as ef­fectiveness with complete ablation in pancreas and liver.29-31 Another group, showed the feasibility of IRE against pancreatic ductal adenocarcinoma (PDAC). This study demonstrated that IRE treat­ment had significant antitumour effects and pro­longed survival in mice with orthotopic xenografts. Extensive tumour necrosis, reduced tumour cell proliferation and disruption of microvessels, were observed at different days post-IRE.32 Recently, it has been demonstrated the efficacy of irrevers­ible electroporation in human pancreatic adeno­carcinoma by using heterotopic murine model. In this paper, authors optimized IRE parameters and evaluated the effects of IRE on surrounding tissues, recurrence, and biomarker expression changes in recurrent/incompletely electroporated mice tu­mours.33,34 In alternative to use of IRE in treatment of can­cer, some pre-clinical studies using different animal models have been performed to investigate the lo­cal and systemic effects of ECT in cancer. Sersa et al., in mice models of murine fibrosarcoma SA-1 treated with bleomycin-ECT, described mice tu­mour destruction due to the immune system activ­ity highly stimulated by ECT, an increased apop­tosis of endothelial cells surrounding the tumour, and a reduction of blood flow in the vessels sup­plying the lesion.35 Roux et al., by analyzing two tumour mouse models (sarcoma and melanoma) treated with bleomycin-ECT, have demonstrated an increase of local T-dependent response due to a massive recruitment of CD11c and CD11b positive cells in the tumours depending on tumour-associ­ated antigen (TAA) release.36 However, the param­eters and safety of ECT are well calibrated for the treatment of cutaneous and subcutaneous lesions, but not for deep-seated tumours as the pancreatic cancer. So, in order to perform a standardization of ECT protocol for pancreatic tumours, other studies on animal models will be needed. One of the first works on the use of ECT in pre-clinical pancreatic cancer treatment was published in 1998 by Nanda et al. In this study, human pancreatic tumours (Pan-4­JCK) implanted subcutaneously in nude mice, were treated with ECT using BLM, mitomycin C or car­boplatin. Tumours were monitored for a period of 89 days after the therapy and showed a significant regression (Table 2).12 Similar results were obtained in another study in which nude mice, xenografted with pancreatic adenocarcinoma cells, were sub­jected to a different scheme of EP with new elec­trodes for drugs (doxorubicin, fluorouracil or cispl­atin) delivery. Tumour growth analysis performed after 28 days of ECT treatment, revealed a signifi­cant regression (Table 2).37 Another study investi­gated the use of electrically mediated drug delivery for the treatment of pancreatic adenocarcinoma in a hamster model. Authors showed that treatment of subcutaneous tumours with bleomycin and electric fields resulted in a 100% complete response rate while treatment of tumours induced in the gland, resulted in a 25% complete response rate (Table 2).13 TABLE 2. Electrochemotherapy (ECT) in animal models of pancreatic cancer Nanda et al., 199812 Nude mice Pan-4JCK ECT Bleomycin Carboplatin Mitomicin C Tumour regression after 89 days Dev et al., 200037 Nude mice BxPc3 ECT Cisplatin Doxorubicin Tumour regression after 28 days Fluruoracil Jaroszeski et al., 199913 Golden Syrian hamster PC-1 ECT Bleomycin 100% complete response rate in subcutaneous tumours, 25% response rate in orthotopic tumours As reported previously, recently Girelli et al. have demonstrated the feasibility and the safety of ECT for the treatment of pancreatic ductal adenocarci­noma. In this study, New Zealand non pathologi­cal rabbits were subjected to open surgery EP of pancreas and duodenum, according to the ESOPE pulse protocol. Neither systemic nor local toxic ef­fects due to the electroporation procedure were ob­served, demonstrating the safety of the optimized electric parameters in the treatment of the pancreas in vivo.17 Taken together these studies suggest that ECT can be used for the local control of non-resectable pancreatic cancer adenocarcinoma (PDAC). Electrochemotherapy in clinical studies of pancreatic cancer Recently, different experiences showed the clinical approach of ECT for the treatment of deep-seated tumours as pancreatic cancer diseases15,38 and liver metastases from colorectal cancer.39 Specifically for pancreatic cancer, a clinical phase I/II study on patients with locally advanced disease, is on­going at the National Cancer Institute, “G. Pascale Foundation” of Naples.38 Patients are enrolled in this study by using the following inclusion criteria: age between 18 and 80 years; good mental health; life expectancy . 3 months; diagnosis of pancreatic adenocarcinoma or pancreatic neuroendocrine tu­mours, confirmed by histological analysis; locally advanced disease [stage III]. In this study, were not included patients with one or more of the fol­lowing conditions: pregnancy positive test for women, significant heart disease, coagulation dis­turbances, and allergy to bleomycin, lung and kid­ney dysfunction, concomitant presence of distant metastases. It is important to underline that all pa­tients received systemic chemotherapy (GEMOX or FOLFIRINOX). Subsequently, to choose the patients suitable to receive ECT treatment, were performed clinical and radiological examinations (CT, MRI and PET). By using functional MRI pa­rameters, it was observed a significant reduction of viable tumour tissue in ECT treated target area. Results from PET analysis, indicated that the up­take of 18FDG during post-operative PET examina­tion was lower in respect to pre-operative evalua­tions. No serious side effects for the patients were observed. In addition, pain reduction of patients (evaluated by VAS-score) was reported immedi­ately after the ECT treatment compared to pre­operative status. Preliminary data on feasibility and safety of the ECT treatment on patients with locally advanced cancer were reported by Granata et al.15 For a significant number of patients, a re­duced diameter and tumourigenicity of the lesions associated with good clinical parameters were re­ported. These data suggest that ECT can be safely per­formed in locally advanced pancreatic tumours. ECT vs IRE in treatment of patients with unresectable pancreatic cancer It is of note that multi-modality therapy, including chemotherapy, surgery and/or radiation therapy represent the optimal treatment option for patients with pancreatic adenocarcinoma especially stage II disease. Since the incidence of more advanced staged disease (stage III and stage IV) is becoming higher over the time, only a small percentage of pa­tients who are diagnosed with pancreatic adeno­carcinoma are eligible for definitive surgical resec­tion. Due to this high incidence, alternative tech­niques have been developed in order to improve quality-of-life especially in patients with stage III pancreatic adenocarcinoma. Radiofrequency ab­lation (RFA) has been studied as possible therapy centered on thermal techniques, but the reported morbidity rates were high in the majority of these TABLE 3. Clinical studies on irreversible electroporation (IRE) in pancreatic cancer Bagla et al., 201254 78 Stage III No residual disease and a decreasing cancer antigen 19-9 level. Mansson et al., 201455 5 No serious treatment-related adverse events were observed. Paiella et al.,201556 10 Stage III Overall survival of 7.5 months Martin et al, 201357 54 Stage III Improvement in local progression-free survival (14 vs. 6 months, p = 0.01), distant progression-free survival (15 vs. 9 months, p = 0.02), and overall survival (20 vs. 13 months, p = 0.03). Martin et al, 201458 48 Stage III No significant vascular complications were seen, and of the high-grade complications, bleeding (2), biliary complications (3) and DVT/PE (3) were the most common. DVT/PE = deep vein thrombosis and pulmonary embolism published studies.40-44 In addition, anatomy of pan­creas represents a significant obstacle to other ther­mal ablation techniques including cryoablation, high intensity focal ultrasonography, and MWA which to date have not been as well studied as RFA. To bypass the problems relative to thermal techniques, irreversible electroporation (IRE) has been introduced to treat pancreatic cancer, since it does not use thermal energy and does not dam­age blood vessels and bile ducts.45-49 Recent studies have demonstrated the safety and palliation with encouraging improvement in overall survival. It has also been demonstrated that for patients with LAPC (stage III), the addition of IRE to conven­tional chemotherapy and radiation therapy results in substantially prolonged survival compared with historical controls.50 Table 3 summarizes clinical studies on IRE in pancreatic cancer. It is important to underline, that recent studies have shown that a small area of thermal effect of IRE is likely present immediately adjacent to the probe.51 In addition, treating deep seated tumours either during open surgery or percutaneously in liver or other organs due to high voltage (up to 3000 V) and consequently high currents (up to 50 A) delivered pulses could potentially interfere with cardiac activity.52 Moreover, one limitation of IRE remains tissue heterogeneity and the unique settings based on tumour histology and prior in­duction therapy. For this reason, based on our knowledge, IRE could not be considered a stand-ard-of-care practice for treatment of locally ad­vanced pancreatic cancer. As previously described, preliminary studies indicate that ECT represents a feasible and safe treatment modality in patients with locally advanced pancreatic adenocarcinoma. Differently from IRE, ECT protocols for pancreatic cancer uses a lower voltage and lower currents of delivered pulses. In this way the risk of interfer­ence with cardiac activity of patients is lower than those induced by IRE protocols. A recent obser­vational study on the effects of ECT in colorectal liver metastases treatment, demonstrated that in patients after ECT treatment, were found in ECG signals recorded during early post-operative care, no major changes in functioning of the heart or pathological morphological changes.53 Conclusions IRE and ECT represent new non-thermal tech­niques with high interest in treatment of locally advanced pancreatic cancer. IRE applies a higher voltage leading to cell death by apoptosis rather than necrosis. Despite the exact mechanism by which IRE induces apoptosis is still unclear, it seems to induce permanent nanopore formation and consequent ion disruption. As previously re­ported, although IRE is known as non-thermal technique, studies provided evidence that induces a small area of thermal effect near the probe. One complication for patients treated with IRE is a sig­nificant musculature contraction as consequence of high voltage induced. On the other hand, ECT protocol for pancreatic cancer uses a lower voltage and lower currents of delivered pulses. No side ef­fects or major complications have been recorded for ECT treatment of patients with pancreatic can­cer, although clinical studies need to be improved. Taken together, these data suggest that IRE and ECT are promising techniques for treatment of pancreatic cancer, although both require more in­vestigation in the future. Authors’ contributions BS and LM performed preparation of the manu­script; BA, DV, FM performed experimental sup­port; AV, PM, AA, DR, NA, GV, PA, SL, SS, FR performed bibliographic research. AN and IF were responsible for coordination of this study. All au­thors read and approved the final manuscript. Acknowledgments The authors would like to specially thank Massimiliano Spinelli Data Manager of S.S.D. Animal Sperimentation, from Istituto Nazionale per lo Studio e la Cura dei Tumori “Fondazione Giovanni Pascale”, IRCCS, Italia, for kind help in providing informatics assistance. References 1. Ferlay J, Parkin DM, Steliarova-Foucher E. Estimates of cancer incidence and mortality in Europe in 2008. Eur J Cancer 2010; 46: 765-81. 2. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011; 61: 69-90. 3. Krejs GJ. Pancreatic cancer: epidemiology and risk factors. Dig Dis 2010; 28: 355-8. 4. Cunningham D, Chau I, Stocken DD, Valle JW, Smith D, Steward W, et al. Phase III randomized comparison of gemcitabine versus gemcitabine plus capecitabine in patients with advanced pancreatic cancer. J Clin Oncol 2009; 27: 5513-8. 5. 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Irreversible electroporation of the liver and liver hilum in swine. HPB (Oxford) 2011; 13: 168-73. 32. Jose A, Sobrevals L, Ivorra A, Fillat C. Irreversible electroporation shows efficacy against pancreatic carcinoma without systemic toxicity in mouse models. Cancer Lett 2012; 317: 16-23. 33. Philips P, Li Y, Li S, St Hill CR, Martin RC. Efficacy of irreversible electropora­tion in human pancreatic adenocarcinoma: advanced murine model. Mol Ther Methods Clin Dev 2015; 2: 15001. 34. Philips P, Li Y, Martin RC 2nd. Low-energy DC current ablation in a mouse tumor model. Meth Mol Biol 2014; 1121: 257-65. 35. Sersa G, Cemazar M, Snoj M. Electrochemotherapy of tumours. Curr Oncol 2009; 16: 34-5. 36. Roux S, Bernat C, Al-Sakere B, Ghiringhelli F, Opolon P, Carpentier AF, et al. Tumor destruction using electrochemotherapy followed by CpG oligode­oxynucleotide injection induces distant tumor responses. Cancer Immunol Immunother 2008; 57: 1291-300. 37. Dev SB, Hofmann GA, Nanda GS. Treatment of human pancreatic tumors xenografted in nude mice by chemotherapy combined with pulsed electric fields. Methods Mol Med 2000; 37: 277-83. 20 38. Tafuto S, von Arx C, De Divitiis C, Tracey Maura C, Palaia R, Albino V, et al. Electrochemotherapy as a new approach on pancreatic cancer and on liver metastases. Int J Surg 2015; 21(Suppl 1): S78-82. 39. Edhemovic I, Gadzijev EM, Brecelj E, Miklavcic D, Kos B, Zupanic A, et al. Electrochemotherapy: a new technological approach in treatment of metas­tases in the liver. Technol Cancer Res Treat 2011; 10: 475-85. 40. Girelli R, Frigerio I, Salvia R, Barbi E, Tinazzi Martini P, Bassi C. Feasibility and safety of radiofrequency ablation for locally advanced pancreatic cancer. Br J Surg. 2010; 97: 220-5. 41. Girelli R, Frigerio I, Giardino A, Regi P, Gobbo S, Malleo G, et al. Results of 100 pancreatic radiofrequency ablations in the context of a multimodal strategy for stage III ductal adenocarcinoma. Langenbecks Arch Surg 2013; 398: 63-9. 42. Giardino A, Girelli R, Frigerio I, Regi P, Cantore M, Alessandra A, et al. Triple approach strategy for patients with locally advanced pancreatic carcinoma. HPB (Oxford) 2013; 15: 623-7. 43. Cantore M, Girelli R, Mambrini A, Frigerio I, Boz G, Salvia R, et al. Combined modality treatment for patients with locally advanced pancreatic adenocar­cinoma. Br J Surg 2012; 99: 1083-8. 44. Matsui Y, Nakagawa A, Kamiyama Y, Yamamoto K, Kubo N, Nakase Y. Selective thermocoagulation of unresectable pancreatic cancers by using radiofrequency capacitive heating. Pancreas 2000; 20: 14-20. 45. Young SJ. Irreversible electroporation and the pancreas: What we know and where we are going? World J Gastrointest Surg 2015; 7: 138-44. 46. Scheffer HJ, Nielsen K, de Jong MC, van Tilborg AA, Vieveen JM, Bouwman AR, et al. Irreversible electroporation for nonthermal tumor ablation in the clinical setting: a systematic review of safety and efficacy. J Vasc Interv Radiol 2014; 25: 997-1011. 47. Trueba-Arguinarena FJ, de Prado-Otero DS, Poves-Alvarez R. Pancreatic adenocarcinoma treated with irreversible electroporation Case Report: first experience and outcome. Medicine (Baltimore) 2015; 94: e946. 48. Gall TM, Thompson Z, Dinneen EP, Sodergren M, Pai M, Frampton AE, et al. Surgical techniques for improving outcomes in pancreatic ductal adenocar­cinoma. Expert Rew Gastroenter Hepatol 2014; 8: 241-6. 49. Weiss MJ, Wolfgang CL. Irreversible electroporation: a novel pancreatic cancer therapy. Curr Probl Cancer 2013; 37: 262-5. 50. Martin RG, II, McFarland K, Ellis S, Velanovich V. Irreversible electroporation in locally advanced pancreatic cancer: potential improved overall survival. Ann Surg Oncol 2013; 20: 443-9. 51. Long G, Bakos G, Shires PK, Gritter L, Crissman JW, Harris JL, et al. Histological and finite element analysis of cell death due to irreversible electroporation. Technol Cancer Res Treat 2014; 13: 561-9. 52. Ball C, Thomson KR, Kavnoudias H. Irreversible electroporation: a new challenge in “out of operating theater” anesthesia. Anesth Analg 2010; 110: 1305-9. 53. Mali B, Gorjup V, Edhemovic I, Brecelj E, Cemazar M, Sersa G, et al. Electrochemotherapy of colorectal liver metastases - an observational study of its effects on the electrocardiogram. Biomed Eng Online 2015; 14(Suppl 3): S5. 54. Bagla S, Papadouris D. Percutaneous irreversible electroporation of surgi­cally unresectable pancreatic cancer: a case report. Journal of vascular and interventional radiology: J Vasc Interv Radiol 2012; 23: 142-5. 55. Mansson C, Bergenfeldt M, Brahmstaedt R, Karlson BM, Nygren P, Nilsson A. Safety and preliminary efficacy of ultrasound-guided percutaneous irrevers­ible electroporation for treatment of localized pancreatic cancer. Anticancer Res 2014; 34: 289-93. 56. Paiella S, Butturini G, Frigerio I, Salvia R, Armatura G, Bacchion M, et al. Safety and feasibility of irreversible electroporation (IRE) in patients with locally advanced pancreatic cancer: results of a prospective study. Digest Surg 2015; 32: 90-7. 57. Martin RC, McFarland K, Ellis S, Velanovich V. Irreversible electroporation in locally advanced pancreatic cancer: potential improved overall survival. Ann Surg Oncol 2013; 20(Suppl 3): S443-9. 58. Martin RC, Philips P, Ellis S, Hayes D, Bagla S. Irreversible electroporation of unresectable soft tissue tumors with vascular invasion: effective palliation. BMC Cancer 2014; 14: 540. research article Effectiveness of electrochemotherapy after IFN-. adjuvant therapy of melanoma patients Andrejc Hribernik1, Maja Cemazar2, Gregor Sersa2, Maša Bosnjak2, Marko Snoj1 1 Department of Surgical Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Department of Experimental Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia Radiol Oncol 2016; 50(1): 21-27. Received 20 October 2015 Accepted 30 November 2015 Correspondence to: Prof. Marko Snoj, M.D., Ph.D., Department of Surgical Oncology, Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. E-mail: msnoj@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. The combination of electrochemotherapy with immuno-modulatory treatments has already been explored and proven effective. However, the role of interferon alpha (IFN-.) adjuvant therapy of melanoma patients and implication on electrochemotherapy effectiveness has not been explored yet. Therefore, the aim of the study was to retrospectively evaluate the effectiveness and safety of electrochemotherapy after the previous adjuvant treatment with IFN-. in melanoma patients. Patients and methods. The study was a retrospective single-center observational analysis of the patients with ad­ vanced melanoma, treated with electrochemotherapy after previous IFN-. adjuvant therapy. Five patients, treated between January 2008 and December 2014, were included into the study, regardless of the time point of IFN-. ad­juvant therapy. Results. Electrochemotherapy of recurrent melanoma after the IFN-. adjuvant therapy proved to be a safe and effective treatment. Patients with one or two metastases responded completely. Among patients with multiple metas­tases, there was a variable response rate. In one patient all 23 metastases responded completely, in second patient more than 85% of all together 80 metastases responded completely and in third patient all 5 metastases had partial response. Taking into account all metastases from all patients together there was an 85% complete response rate. Conclusions. The study showed that electrochemotherapy of recurrent melanoma after the IFN-. adjuvant therapy is a safe and effective treatment modality, which results in a high complete response rate, not only in single metastasis, but also in multiple metastases. The high complete response rate might be due to an IFN-. immune-editing effect, however, further studies with a larger number of patients are needed to support this presumption. Key words: electrochemotherapy; melanoma; IFN-. Introduction Melanoma is, due to the high risk of metastases de­velopment and its resistance to different treatment strategies, still the most lethal skin cancer. The most effective treatment for primary melanoma of stage I and II is its removal by radical surgical exci­sion with the associated safety margin, usually fol­lowed by a sentinel lymph node biopsy. Although this approach is curative in many cases, relapse with disseminated disease occurs in some patients. Therefore, in patients with an increased risk for re­current disease adjuvant immunotherapy might be applied.1,2 The sole recognized postsurgical adjuvant therapy still is interferon alpha (IFN-.). In several clinical studies both interferon types, the high-dose interferon alpha-2b (IFN-.2b) and low-dose interferon alpha-2a (IFN-.2a) were shown to have significant effect on progression-free survival.3-5 Recent meta-analysis showed statistically signifi­cant improvement in disease free survival and overall survival in patients with high risk mela­noma (stage IIb-IIIc according to The American Joint Committee on Cancer [AJCC] TNM Cancer Staging Manual 7th edition6) treated with adjuvant IFN-. after surgery.7,8 When melanoma recurs, other treatment modal­ities are needed for local or systemic control of the disease. Most commonly used are systemic chem­otherapy with dacarbazine, irradiation, isolated limb perfusion or electrochemotherapy and in re­cent years also new targeted therapies with BRAF and/or MEK inhibitors and antibodies against CTLA-4.9,10 Electrochemotherapy is one of the treatment modalities for local treatment of malignant mela­noma, which is using electroporation as a delivery system for the chemotherapeutic drugs bleomycin or cisplatin into the tumor.11-14 Under the high ex­ternal electric field the plasma membrane becomes permeable, thus facilitating drug delivery into the tumors. Numerous clinical studies have demon­strated the effectiveness of electrochemotherapy on a variety of cutaneous and also deep seated tumors, such as liver metastases of colorectal can­cer.15 Among cutaneous tumors, electrochemo­therapy is very effective in treatment of melanoma, with a complete response rate after a single treat­ment of 74%.16 Some studies have recently reported on beneficial effect on melanoma treatment after combining electrochemotherapy with new target­ed therapies such as dabrafenib or ipilimumab.17,18 Furthermore, electrogene therapy with plasmids coding for interleukin-12 (IL-12) or antiangiogenic molecules, is also in clinical testing for melanoma treatment.19-22 In preclinical studies, it was shown that adju­vant therapy with TNF-., IL-2, IL-12 and CpG oligonucleotides might boost electrochemotherapy response.23-30 However, the role of adjuvant IFN-. has not been explored yet, neither on preclinical or clinical level. Therefore, the aim of the study was to evaluate the safety and effectiveness of electro­chemotherapy on recurrent melanoma after IFN-. adjuvant therapy of melanoma patients. Patients and methods Study design and patient selection The study was conducted as a retrospective single-center analysis of patients with advanced malig­nant melanoma treated with electrochemotherapy, who previously received IFN-. adjuvant therapy, after surgery of primary melanoma. All the patient files where electrochemotherapy was performed in the last 6 years, between January 2008 and December 2014, were reviewed regardless of the time point of IFN-. adjuvant therapy. Among all the (50) patients treated with electrochemotherapy in that time period only 5 of them met the require­ments and were further investigated. The trial was approved by an Institutional Review Board and National Medical Ethic Committee 97/06/02. The patients signed the informed consent before the treatment. Investigated entities First the patient’s general characteristics (gender, age), the site and TNM, pathological stage (AJCC TNM Cancer Staging Manual, 7th edition) and Breslow thickness of primary melanoma were re­corded. Than therapeutic dose and duration of IFN-. adjuvant therapy were recorded for each pa­tient. The treatment free-interval between the end of IFN-. adjuvant therapy and electrochemothera­py treatment was calculated. The site and number of melanoma nodules treated with electrochemo­therapy were further recorded and afterwards ef­fectiveness of electrochemotherapy was evaluated. IFN-. adjuvant therapy All five patients received IFN-. as a post-surgical adjuvant therapy. Patient 1 and 2 received low- dose IFN-. since the IFN-. adjuvant therapy was administered before 2010, when new guidelines for melanoma treatment at the Institute of Oncology Ljubljana were accepted. Patient 1 and 2 received Roferon-A® (interferon Alfa-2a) (Roche, Basel, Switzerland) subcutaneously at a dose of 3 or 6 million IU three times a week (Table 2) for 33 and 7 months, respectively, according to the instructions of an oncologist. Patient 3, 4 and 5 received high dose interferon Intron® A (interferon a- alpha 2b) (Merck, Kenilworth, New Jersey, USA) according to the Kirkwood scheme.31 The exact schedule and the dose was adjusted for each patient by his on­ cologist (Table 2). Electrochemotherapy Patients were treated according to the Standard Operating Procedure (SOP) for electrochemother­apy.16 Briefly, electrochemotherapy of cutaneous melanoma nodules was performed using either in­travenous bleomycin (Bleomedac, Medac, Wedel, Germany) in a dose of 15,000 IU/m2 or intratumoral cisplatin (Cisplatin, Medac) injection in a concen­tration of 2 mg/ml and dose is applied according TablE 1. Patients’ characteristics Gender Male Male Female Female Female Birth year 1958 1935 1937 1953 1941 TNM* T3aN0M0 T2aN0M0 T4bN1aM0 T3aN1aM0 T4bN1aM0 Patological stage* Stage IIa Stage Ib Stage IIIb Stage IIIa Stage IIIb Breslow 4 mm 1.5 mm 10 mm 2.8 mm 9.5 mm Ulceration / / 10 mm / 2.5 mm Localisation of primary tumor Right lower leg Left foot Right foot Right lower leg Back Localisation of metastases treated with ECT Right lower leg Left foot (dorsum) Left lower leg Right lower leg Breast, left side * according to AJCC TNM Cancer Staging Manual 7 th edition (2010)6; ECT = electrochemotherapy to ESOPE protocol.16 Standard pulse parameters for electrochemotherapy (voltage to distance ratio 1300 V/cm, 8 pulses, 100 µs, 5000 Hz) were used.16 Electric pulses were generated by Cliniporator pulse generator (IGEA, s.r.l., Carpi, Italy) and de­livered by parallel stainless steel plate electrodes with 6 or 8 mm distance in between. Electric pulses were applied to the tumors nodules in a way so as to cover the whole tumor area, including the safety margin. Response assessment Antitumor efficacy was evaluated 4 weeks after electrochemotherapy, patient were then monitored monthly. Treatment response was defined either as complete response (CR), when the tumor was not palpable, partial response (PR), when the tu­mor decreased more than 50% of the measurable lesions; no change (NC), when tumor decreased less than 50% or increased up to 25%, or progres­ sive disease (PD), when tumor increased for more than 25%. Determination was based on criteria of WHO Handbook for Reporting Results of Cancer Treatment where for all response definitions mini­mum 4-week duration was required for qualifying the response. Results Patients’ characteristics Only 5 patients fulfilled all the requirements for in­clusion into this retrospective study. Among them there were two male and three female patients with a median age of electrochemotherapy treatment 71 years (range from 50–76 years). TNM and patho­logical stage were recorded for all five patients, as well as Breslow thickness (Table 1). The localiza­tions of primary tumors were on the upper leg, foot or the back (Table 1). All the patients were iden­tified as patients with high risk of recurrence and were therefore assigned for IFN-. adjuvant ther­apy. The relapse of melanoma occurred in all five patients. Recurrence time was variable among pa­ tients; from a few months to a few years (Table 1). Other comorbidities were also recorded for all 5 patient; only patient 2 had arterial hypertension and no other comorbidities were recorded. IFN-. adjuvant therapy and disease progression Adjuvant therapy with IFN-. in our investigated patients can be divided into two subtypes; low-dose treatment for patients 1 and 2 and high dose treatment for another three patients (Table 2). After the completed adjuvant therapy with IFN-., in all five patients disease had progressed to a meta­static disease. Disease free interval, progression of the disease and treatment procedures vary for each patient (Table 2). Based on the decision of an institutional committee for melanoma treatment, electrochemotherapy was offered to the patients as another treatment option after several surgical ex­cisions and in patient 1 and 2 also irradiation. • In patient 1 inguinal lymph node metastasis oc­curred after a disease free interval and inguinal and retroperitoneal lymph node dissection was performed thereafter. The patient was irradi­ated as well. When progression of the disease occurred 3 years later, two metastases were excised within 2 months and the patient was irradiated again. In 2 months new metastases, TablE 2. Treatment regime INF-. dose 6 million IU 3 x weekly (s.c.) 3 million IU 3 x weekly (s.c.) 35 milion IU 20 x (i.v.) in 8 weeks/ 15 million IU 3x weekly (s.c.) for 13 weeks/ 10 milion IU 3 x weekly (s.c.) for 13 weeks* 30 milion IU 20 x (i.v.) in 4 weeks followed by 15 million IU 3x weekly (s.c.) for 35 weeks followd by 10 milion IU 3 x weekly (s.c.) for 12 weeks 30 milion IU 9 x (i.v.) in 2 weeks followed by 20 milion IU 11x (i.v.) in 3 weeks followed by 15 million IU 3x weekly (s.c.) for 13 weeks** INF-. treatment period 2 years 9 months 7 months 8 months 12 months 4 months Last date of INF-. treatment March 2004 August 1998 July 2012 January 2012 June 2011 Disease free interval 4 months 5 years 6 months 6 months 3 months 2 months Surgically excised metastases+ Yes Yes No Yes Yes Interval between INF-. and ECT treatment 4 years 8 months 12 years 11 months 7 months 6 months 7 months ECT treatment November 2008 July 2011 February 2013 July 2013 January 2012 ECT drug Cisplatin Cisplatin Bleomycin Bleomycin Bleomycin Number of lesions 2 1 80 23 5 Size of the nodules 1.0 x 1.5 cm 1.5 x 1.5 cm 3 x 3 cm 0.3–1 cm 0.1–0.8 cm 0.7–1.5 cm Effect after 4 weeks CR CR > 85% CR All CR 100% PR LRD*** or date of death (D) April 2010 (LRD) December 2014 (LRD) April 2014 (LRD) December 2013 (D) March 2013 (D) CR = complete response; ECT = electrochemotherapy; i.v. = intravenous; PR = partial response; s.c. = subcutaneously; s.c = subcutaneous; *prematurelly terminated treatment due to ineffective treatment; ** intravenous dose (i.v. dose) was decreased to 20 million IU due to pathological liver tests - prematurelly terminated treatment due to side effects, *** after last record date at the Institute of Oncology Ljubljana patients were given only paliative care at their regional centers, +details on localization and number of excised metastates in paragraph Pacients’ caracteristics. which were treated with electrochemotherapy, occurred. • In patient 2 after a disease free interval a metas­tasis on the limb occurred and was immediately excised. Four years later, another excision with following inguinal dissection and irradiation was performed. In the following 2 years three in-transit metastases were excised from the dor­sum of the left foot. A new metastasis occurred 1 year after the last in-transit metastasis excision, which was then treated with electrochemother­apy. • Disease free interval for the patient 3 was 6 months; thereafter multiple metastases occurred on the limb and were treated with electrochemo­therapy. • In patient 4 a metastasis occurred on the scar of a primary excised melanoma only 3 months after completing adjuvant therapy with IFN-.. It was immediately excised, although PET/CT scan and thin needle biopsy showed multiple metastases in the same area, which were then treated with electrochemotherapy. • In patient 5 the disease-free interval was 2 months, followed by two excisions of metasta­ ses and shortly afterwards electrochemotherapy of 5 metastases on the trunk. Due to partial re­sponse of all 5 metastases, these metastases and newly formed metastases on the trunk were again treated with electrochemotherapy 6 weeks after the first treatment. Electrochemotherapy following IFN-. adjuvant therapy At the time of electrochemotherapy patient 2 was presented with a single metastasis on the limb, whereas patients 1, 3, 4 and 5 were presented with multiple metastases on the limb (patient 1, 3 and 4) or trunk (patient 5). All metastases present at the time of electrochemotherapy were treated. Electrochemotherapy was effective in all five pa­ tients, with a variable response rate (Table 2). In patient 1 and 2 cisplatin was given intratu­moraly due to previous irradiation of the patients. In some studies, it was reported that previous irra­diation can cause lower effectiveness of i.v. electro­chemotherapy.32 Fibrosis can be one of the causes for lower effectiveness. Less fluid in the tissue re­sults in less lymphatic infiltration and also lower current in the nodule and can therefore contribute to the lower effectiveness of electrochemotherapy of pre-iradiated tissues. Intratumoral injection of chemotherapeutic drug can overcome those obsta­cles and can results in higher effectiveness. Electrochemotherapy following IFN-. adjuvant therapy was effective treatment modality, regard­less of drug used for electrochemotherapy, bleo­mycin or cisplatin. Single metastasis responded completely, while multiple metastases had a vari­able response rate. In patient 4 all 23 metastases responded completely, in patient 3 more than 85% of all together 80 metastases responded com­pletely and in patient 5 all 5 metastases had partial response. Taking into account all metastases from all patients together there was an 85% complete response rate. After electrochemotherapy no side effects, such as local erythema, bleeding, infec­tion on the site of electrochemotherapy, or mus­cle contractions, were reported. Nevertheless new metastases mostly occurred within 1 month (pa­tients 1, 3 and 5) or 2 months (patient 4) after the treatment. In patient 2, with a single metastasis at the time of electrochemotherapy, new metastases occurred after 1 year and 10 months. In patient 1 additional electrochemotherapy of 14 new lesions was performed thereafter, which also resulted in 100% complete response. Nevertheless the disease progressed and although systemic chemotherapy with dacarbazine was administered, new distant metastases in the head and neck region occurred. Electrochemotherapy was then used for palliative care. In patient 2 disease also progressed and due to several metastases, isolated limb perfusion was performed 4 years after electrochemotherapy. No new metastasis occurred yet. Dacarbazine and later also ipilimumab were prescribed for patient 3 with progressive metastatic disease, with metas­tases present also in liver and lungs. In patient 4 new subcutaneous metastases were effectively ir­radiated and year after brain metastasis occurred. Electrochemotherapy response in all those patients (patient 1–4) remains the same during the whole observational period. Due to partial response of all 5 metastases in patient 5, these metastases and 12 newly formed metastases on the trunk were again treated with electrochemotherapy, 6 weeks after the first treatment. After the second electro­chemotherapy treatment all 17 metastases (includ­ing 5 retreated metastases) responded completely. The patient was later treated with dacarbazine and vemurafenib due to soft tissue and lung metasta­ses, but disease progressed with new metastases in lungs and brain. Before ECT 6 weeks after ECT Discussion This is the first study to our knowledge which dis­cusses the effectiveness of electrochemotherapy af­ter IFN-. adjuvant therapy for treatment of mela­noma metastases. In recent years electrochemotherapy has been widely used in clinical studies for treatment of cu­taneous and also deep seated tumors. Among skin cancers electrochemotherapy was very effective in the treatment of malignant melanoma, with a complete response rate after single treatment 74%, according to ESOPE study.16 Although the electro­chemotherapy response rate is quite high, and ef­fective on most of tumor histologies, recently there is some evidence that there is a variability in the response rates of different tumor histologies. The meta-analysis by Mali et al. has shown that melano­ma tumors are less responsive than non-melanoma tumors. The effectiveness of electrochemotherapy was tumor type dependent, namely the complete re­sponse rate was 57% of melanoma tumors and 67% of other, non-melanoma tumors. Among carcino­mas, basal cell carcinoma was the most responsive tumor type, with up to 89% complete responses.33 Furthermore, the importance of the immune re­sponse elicited by electrochemotherapy locally was explored.34,35 Immunogenic cell death of cancer cells was proposed to contribute to the curability of the treated metastases. The concept of immunogenic cell death, which is triggered by some cancer thera­pies, is initiated by damage-associated molecular patterns, which can further trigger an adaptive im­mune response against tumors.34 Some pre-clinical studies have explored the possibility of adjuvant electrogene therapy with plasmid encoding IL-12, which greatly increase the response rate of the elec­trochemotherapy treated tumors.30 The recent clini­cal study, investigating the combined treatment of ipilimumab and electrochemotherapy has shown a better response than ipilimumab alone.17 In this report we show that electrochemothera­py was safe and effective also after IFN-. therapy. IFN-., although given to the patients in different periods before electrochemotherapy may also con­tribute to the response rate of the electrochemo­therapy treated melanoma metastases. Namely, re­sponse rate in patients with electrochemotherapy after adjuvant IFN-. was 100% partial response (patient 5) or from 85% to 100% complete response (patient 1, 3 and 4) in patients with multiple me­tastases, which is an equal or even higher percent­age than demonstrated in previous studies; 85% of metastases responded completely in the present study, while the results of meta-analysis showed that 57% of melanoma metastases responded com­pletely.16,33 We might speculate that the effectiveness may be increased by the previous immunostimulatory IFN-. adjuvant therapy, which would be reflected in high response rate of the treated tumors. IFN-. is one of the type I interferons, an important inter­feron family, involved in immune-editing process. Their main importance is the effect on the hemat­opoietic cells; induction of bystander T cell prolif­eration, long-term survival and expression of anti­apoptotic genes.36-37 Furthermore, interferons have also great impact on maturation and differentia­tion of dendritic cells, cells which are considered to be the most effective antigen presenting cells.34 Taking all this findings into account, IFN-. might have a significant role in a link between the innate and adaptive immune system. Similarly, new tar­geted therapy with ipilimumab acts on dendritic cells -cytotoxic T lymphocytes (CTLs) interac­tion. Dendritic cells are presenting tumor antigens to CTLs, which can then destroy cancer cells. But along with tumor antigens the dendritic cells pre­sent also an inhibitory signal, which can bind to a receptor on the CTLs; cytotoxic T lymphocyte-as­ sociated antigen 4 (CTLA-4) and thereby block the cytotoxicity of CTL. Ipilimumab binds to CTLA-4 and block the inhibitory signal.38,39 One of the possible reasons for the very good efficiacy of electrochemotherapy, following IFN-. adjuvant therapy is immune system activation by electrochemotherapy, which was previously mod­ulated by IFN-..40 Calvet et al. demonstrated in an in vivo preclinical study that electrochemothera­py not only has a cytotoxic effect towards cancer cells, but can also generate a systemic anticancer immune response, with imunogenic cell death.33,40 Dying cancer cells then behave as a therapeutic vaccine, leading to a cytotoxic immune response against remaining tumor cells.35,42 It was also dem­onstrated that electrochemotherapy is more effec­tive in immunocompetent mice, causing complete tumor regression, whereas in immunodeficient mice the complete response was not obtained.35 The drawback of our study is that we have no data on the immune status of the patients and that this is an observational study. Nevertheless, although this group of patients is small it might indicate on the potential of combining immu­nostimulatory treatments with electrochemothera­py, which can be explored in different ways. One of the recent ideas is that electrochemotherapy can serve as a vaccination to adjuvant peritumoral im­munostimulatory therapy that can boost the local effect as well it may have the systemic effect.42 Conclusions The report demonstrates that combining elec­ trochemotherapy with preceded IFN-. adjuvant therapy is a safe and effective treatment modal­ity, which results in high complete response rate, not only in single metastasis, but also in multiple metastases. The high complete response rate might be due to IFN-. immune-editing effect, however further controlled studies on a larger number of patients are needed to support this presumption. Acknowledgements We greatly appreciate the help of our research nurse Tjasa Pecnik, B. Sc. The research was financially supported from the Slovenian Research Agency (program no. P3-0003). This manuscript is a re­sult of the networking efforts of the COST Action TD1104 (www.electroporation.net). Research was conducted in the scope of EBAM European Associated Laboratory (LEA). References 1. Veronesi U, Adamus J, Aubert C, Bajetta E, Beretta G, Bonadonna G, et al. A randomized trial of adjuvant chemotherapy and immunotherapy in cutane­ous melanoma. N Engl J Med 1982; 307: 913-6. 2. Pflugfelder A, Kochs C, Blum A, Capellaro M, Czeschik C, Dettenborn T, et al. Malignant melanoma S3-guideline “diagnosis, therapy and follow-up of melanoma”. J Dtsch Dermatol Ges 2013; 11 (Suppl 6): 1-116, 1-126. 3. Hauschild A. Adjuvant interferon alfa for melanoma: new evidence-based treatment recommendations? Curr Oncol 2009; 16: 3-6. 4. Cole BF, Gelber RD, Kirkwood JM, Goldhirsch A, Barylak E, Borden E. Quality­of-life-adjusted survival analysis of interferon alfa-2b adjuvant treatment of high-risk resected cutaneous melanoma: an Eastern Cooperative Oncology Group study. J Clin Oncol 1996; 14: 2666-73. 5. Grob JJ, Dreno B, de la Salmoniere P, Delaunay M, Cupissol D, Guillot B, et al. Randomised trial of interferon alpha-2a as adjuvant therapy in resected primary melanoma thicker than 1.5 mm without clinically detectable node metastases. French Cooperative Group on Melanoma. Lancet 1998; 351: 1905-10. 6. Edge SB, Compton CC. The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol 2010; 17: 1471-4. 7. Mocellin S, Pasquali S, Rossi CR, Nitti D. Interferon alpha adjuvant therapy in patients with high-risk melanoma: a systematic review and meta-analysis. J Natl Cancer Inst 2010; 102: 493-501. 8. Mocellin S, Lens MB, Pasquali S, Pilati P, Chiarion Sileni V. 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Consolidation electro­chemotherapy with bleomycin in metastatic melanoma during treatment with dabrafenib. Radiol Oncol 2015; 49: 71-4. 19. Cemazar M, Jarm T, Sersa G. Cancer electrogene therapy with interleu­kin-12. Curr Gene Ther 2010; 10: 300-11. 20. Daud AI, DeConti RC, Andrews S, Urbas P, Riker AI, Sondak VK, et al. Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma. J Clin Oncol 2008; 26: 5896-903. 21. Spanggaard I, Snoj M, Cavalcanti A, Bouquet C, Sersa G, Robert C, et al. Gene electrotransfer of plasmid antiangiogenic metargidin peptide (AMEP) in dis­seminated melanoma: safety and efficacy results of a phase I first-in-man study. Hum Gene Ther Clin Dev 2013; 24: 99-107. 22. Heller LC, Heller R. Electroporation gene therapy preclinical and clinical trials for melanoma. Curr Gene Ther 2010; 10: 312-7. 23. Cemazar M, Todorovic V, Scancar J, Lampreht U, Stimac M, Kamensek U, et al. Adjuvant TNF-. therapy to electrochemotherapy with intravenous cis­platin in murine sarcoma exerts synergistic antitumor effectiveness. Radiol Oncol 2015; 49: 32-40. 24. Gerlini G, Di Gennaro P, Borgognoni L. Enhancing anti-melanoma im­munity by electrochemotherapy and in vivo dendritic-cell activation. Oncoimmunology 2012; 1: 1655-7. 25. Sersa G, Cemazar M, Menart V, Gaberc-Porekar V, Miklavcic D. Anti-tumor effectiveness of electrochemotherapy with bleomycin is increased by TNF-alpha on SA-1 tumors in mice. Cancer Lett 1997; 116: 85-92. 26. Heller L, Pottinger C, Jaroszeski MJ, Gilbert R, Heller R. In vivo electropora­tion of plasmids encoding GM-CSF or interleukin-2 into existing B16 mela­nomas combined with electrochemotherapy induces long-term antitumour immunity. Melanoma Res 2000; 10: 577-83. 27. Mir LM, Roth C, Orlowski S, Belehradek J, Fradelizi D, Paoletti C, et al. Potentiation of the antitumoral effect of electrochemotherapy by immuno­therapy with allogeneic cells producing interleukin 2. C R Acad Sci III 1992; 314: 539-44. 28. Mir LM, Orlowski S, Poddevin B, Belehradek J. Electrochemotherapy tumor treatment is improved by interleukin-2 stimulation of the host’s defenses. Eur Cytokine Netw 1992; 3: 331-4. 29. Roux S, Bernat C, Al-Sakere B, Ghiringhelli F, Opolon P, Carpentier AF, et al. Tumor destruction using electrochemotherapy followed by CpG oligode­oxynucleotide injection induces distant tumor responses. Cancer Immunol Immunother 2008; 57: 1291-300. 30. Sedlar A, Dolinsek T, Markelc B, Prosen L, Kranjc S, Bosnjak M, et al. Potentiation of electrochemotherapy by intramuscular IL-12 gene electro­transfer in murine sarcoma and carcinoma with different immunogenicity. Radiol Oncol 2012; 46: 302-11. 31. Moreno Nogueira JA, 1 Valero Arbizu M, Pérez Temprano R. Adjuvant treat­ment of melanoma. ISRN Dermatol. 2013; 2013: 545631. 32. Groselj A, Kos B, Cemazar M, Urbancic J, Kragelj G, Bosnjak M, Veberic B, Strojan P, Miklavcic D, Sersa G. Coupling treatment planning with naviga­tion system: a new technological approach in treatment of head and neck tumors by electrochemotherapy. Biomed Eng Online. 2015; 14 Suppl 3: S2. 33. Mali B, Jarm T, Snoj M, Sersa G, Miklavcic D. Antitumor effectiveness of electrochemotherapy: a systematic review and meta-analysis. EJSO 2013; 39: 4-16. 34. Calvet CY, Famin D, André FM, Mir LM. Electrochemotherapy with bleomy­cin induces hallmarks of immunogenic cell death in murine colon cancer cells. Oncoimmunology 2014; 3: e28131. 35. Sersa G, Miklavcic D, Cemazar M, Belehradek J, Jarm T, LM. Mir. Electrochemotherapy with CDDP on LPB sarcoma: comparison of the anti-tumor effectiveness in immunocompetent and immunodeficient mice. Bioelectrochem Bioenerg 1997; 43: 279-83. 36. Tough DF, Borrow P, Sprent J. Induction of bystander T cell proliferation by viruses and type I interferon in vivo. Science 1996; 272: 1947-50. 37. Paquette RL, Hsu NC, Kiertscher SM, Park AN, Tran L, Roth MD, et al. Interferon-alpha and granulocyte-macrophage colony-stimulating factor differentiate peripheral blood monocytes into potent antigen-presenting cells. J Leukoc Biol 1998; 64: 358-67. 38. Melero I, Hervas-Stubbs S, Glennie M, Pardoll DM, Chen L. Immunostimulatory monoclonal antibodies for cancer therapy. Nat Rev Cancer 2007; 7: 95-106. 39. Hodi FS, O’Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 2010; 363: 711-23. 40. O’Brien MA, Power DG, Clover AJ, Bird B, Soden DM, Forde PF. Local tumour ablative therapies: opportunities for maximising immune engagement and activation. Biochim Biophys Acta 2014; 1846: 510-23. 41. Sersa G, Kotnik V, Cemazar M, Miklavcic D, Kotnik A. Electrochemotherapy with bleomycin in SA-1 tumor-bearing mice--natural resistance and immune responsiveness. Anticancer Drugs 1996; 7: 785-91. 42. Sersa G, Teissie J, Signori E, Kamensek U, Marshall G, Cemazar M, et al. Electrochemotherapy of tumors as in situ vaccination boosted by immuno­gene electrotransfer. Cancer Immunol Immunother 2015; 64: 1315-27. research article A statistical model describing combined irreversible electroporation and electroporation-induced blood-brain barrier disruption Shirley Sharabi1,2, Bor Kos3, David Last1, David Guez1, Dianne Daniels1,2, Sagi Harnof2,4, Yael Mardor1,2, Damijan Miklavcic3 1 The Advanced Technology Center, Sheba Medical Center, Ramat-Gan, Israel 2 Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel 3 University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia 4 Neurosurgery Department, Sheba Medical Center, Ramat-Gan, Israel Radiol Oncol 2016; 50(1): 28-38. Received 23 October 2015 Accepted 3 January 2016 Correspondence to: Dr. Yael Mardor, Ph.D., The Advanced Technology Center, Sheba Medical Center Tel-Hashomer, 52621 Israel. Phone: +972 3 530 2993; Fax: 972 3 530 3146; E-mail: yael.mardor@sheba.health.gov.il Disclosure: We declare that Damijan Miclavcic holds patents in the area of electroporation and is consulting for various companies with financial interest in the area of electroporation use in medicine. The other authors declare no competing financial interests. Background. Electroporation-based therapies such as electrochemotherapy (ECT) and irreversible electroporation (IRE) are emerging as promising tools for treatment of tumors. When applied to the brain, electroporation can also induce transient blood-brain-barrier (BBB) disruption in volumes extending beyond IRE, thus enabling efficient drug penetration. The main objective of this study was to develop a statistical model predicting cell death and BBB disrup­tion induced by electroporation. This model can be used for individual treatment planning. Material and methods. Cell death and BBB disruption models were developed based on the Peleg-Fermi model in combination with numerical models of the electric field. The model calculates the electric field thresholds for cell kill and BBB disruption and describes the dependence on the number of treatment pulses. The model was validated using in vivo experimental data consisting of rats brains MRIs post electroporation treatments. Results. Linear regression analysis confirmed that the model described the IRE and BBB disruption volumes as a func­tion of treatment pulses number (r2 = 0.79; p < 0.008, r2 = 0.91; p < 0.001). The results presented a strong plateau effect as the pulse number increased. The ratio between complete cell death and no cell death thresholds was relatively narrow (between 0.88-0.91) even for small numbers of pulses and depended weakly on the number of pulses. For BBB disruption, the ratio increased with the number of pulses. BBB disruption radii were on average 67% ± 11% larger than IRE volumes. Conclusions. The statistical model can be used to describe the dependence of treatment-effects on the number of pulses independent of the experimental setup. Key words: electroporarion; blood brain barrier; Peleg-Fermi Introduction Electroporation (EP) is a physical phenomenon in which electric fields make cell membranes tran­siently permeable to ions and macromolecules which are otherwise deprived of or have limited trans-membrane transport mechanisms.1-3 Electric pulses applied to the tissue induce an electric field which in turn induces a change in cell membrane potential. This change depends on various tissue related parameters such as tissue type and cell size as well as pulse parameters including pulse ampli­tude, shape, duration, number of pulses, and pulse repetition frequency. As a function of the induced electrical field, electric pulses can either: reversibly permeabilize the cell membrane (reversible EP) or permeabilize the cell membrane in a manner that leads to cell death (irreversible EP).4 It was recent­ly demonstrated that when applying EP to brain tissue it also induces reversible disruption of the blood-brain barrier (BBB).5-7 Both irreversible EP (IRE), and reversible EP combined with chemotherapy, also known as electrochemotherapy (ECT), are emerging as new treatment techniques for solid tumors.3,8-16 ECT uses EP to allow increased uptake of chemothera­peutic drugs into tumor cells12 and IRE is a method aimed at inducing tumor ablation without thermal damage.17,18 Brain tumors are excellent candidates for local EP treatment. Glioblastoma multiforme (GBM) is the most frequent and most aggressive primary brain tumor with an average survival of 14 months from diagnosis. Existing treatments of­fer poor prognosis for GBM mainly due to tumor infiltration into the surrounding brain, high resist­ance to therapeutic apoptotic stimuli and poor BBB penetration of most therapeutic agents.19,20 A com­bined approach, consisting of inducing significant/ rapid necrosis in the tumor mass and simultaneous delivery of high chemotherapy doses to the tumor and surrounding infiltrating zone is suggested as a treatment strategy. EP-induced tissue necrosis within the massive region of the tumor and sur­rounding BBB disruption, enabling efficient local delivery of systemically administered chemothera­py was recently demonstrated.7,21 Individual treatment planning is an important key for EP-based treatment success.22 Treatment parameters should be chosen in such a manner that will induce maximal damage to the tumor while sparing surrounding healthy tissue. This is usu­ally done by numerical models. Several numerical models describing the electric filed distribution in the tissue have been introduced, and are ap­plied for predicting treatment outcome and plan­ning the electrodes placement to ensure full tumor coverage by electrical fields higher than the EP threshold.23-28 These models are usually based on experimental data. Treatment volumes calculated from MRI29 or histological data30,31 are incorporated into computerized models together with the organ characteristics and the electrodes configuration. These calculations traditionally use deterministic models, i.e all the cells exposed to electrical fields higher than a specific threshold, known in the lit­erature, will be irreversibly/reversibly electropo­rated. Nevertheless, live tissues are more complex, especially malignant tissues which are inherently inhomogeneous, and therefore assuming a statisti­cal effect of EP parameters maybe more appropri­ate.32,33 For this reason we chose to apply a statisti­cal model to describe reversible/irreversible effects in vivo. The Peleg-Fermi model is the most widely used mathematical model for describing cell death as a consequence of IRE in medicine.32-34 Although several other models have been proposed35 the Peleg-Fermi model seems the most adequate since it includes dependency on the number of pulses as well as in electrical field. For this reason we decid­ed to apply it on our experimental data and further extend it to irreversible and reversible EP effects in vivo. The Peleg-Fermi statistical model was first in­troduced as a model describing the survival of bacteria after exposure to pulsed electrical fields.36 Later on it was suggested that this model can be adapted to describe the effects of IRE.32,34 Goldberg and Rubinsky32 extrapolated experimental data ob­tained using prostate cancer cells and demonstrat­ed the feasibility of applying this model to describe the effects of IRE for up to 10 treatment pulses. Garcia et al. extended the model up to 90 pulses, by theoretical analysis that is yet to be confirmed with experimental data.34 Treatment parameters such as pulse shape, am­plitude, frequency, duration and number of puls­es37,38 affect treatment outcome. Here, we chose to study and model the effect of number of pulses while other pulse parameters remain fixed. A numerical model describing electric field dis­tribution in the brain tissue based on the applied voltage, tissue and electrodes electrical properties and electrodes configuration was constructed. The calculated electrical field was then implemented in the statistical model that was estimating the effect of the number of pulses on the outcome- irrevers­ible damage and BBB disruption. The first goal of our study presented below was to extend the Peleg-Fermi model to describe a wid­er range of the number of treatment pulses in vivo and to validate the extended model using experi­mental data obtained from naive rats treated with EP in the brain. The second goal was to adapt the statistical Peleg-Fermi model to describe the effects of pulse parameters on BBB disruption. BBB disruption is a vital key in treating brain tumors since it is impor­tant to disrupt a large enough volume surrounding the tumor mass to enable efficient drug penetration into the infiltrating zone. Once established, models describing both IRE and BBB disruption can be im­plemented to provide a complete treatment plan­ning for brain tumors with EP. Materials and methods Animal experiments The study was approved by and performed in ac­cordance with the guidelines of The Animal Care and Use Committee of the Sheba Medical Center, which is approved by the Israeli authorities for ani­mal experimentation. We have recently presented the results of an animal experiment designed to study both IRE and BBB disruption using the same experimental setup.6,7 Here we describe in detail the aspects rel­evant to our statistical model which are based on that experimental data. Our unique electrode setup employs a single insulated intracranial needle elec­trode with an exposed tip placed in the target tissue and an external surface electrode pressed against the skin. The electric field produced by this elec­trode configuration is highest at the exposed tip of the intracranial electrode tissue interface and then decays with the square of the distance. Therefore, the electric fields surrounding the needle electrode tip induce nearly spherical IRE effects at the target tissue and gradually decrease further away to re­versible EP effects which induce BBB disruption. Regions of interest (ROIs) plotted on MR images acquired post EP treatments with various pulse pa­rameters were used for calculating the tissue dam­age and BBB disruption radii. We then studied the correlation between the experimental radii and the extended statistical model. Animal model and procedure The study was performed by treating 46 male Spring Dawly rats with 50 µs monopolar electric pulses at 1 Hz and 600 V, as previously described.7 The rats were divided into seven groups of 5-7 rats each, treated with varying number of pulses (N = 10, 45, 90, 180, 270, 450 and 540). MR imaging Rats were scanned 30 minutes post treatment and periodically thereafter up to 2 weeks post treatment, using a 1.5 T GE Optima MR system (Optima MR450w, General Electric, Milwaukee). The MR sequences included contrast-enhanced T1­weighted MRI for depiction of BBB disruption and T2-weighted MRI for depiction of tissue response. Gradient echo (GE) MRI was acquired to assess possible procedure-related bleeding. The damage radius induced by IRE (rd) (in mm) for each rat was calculated from the hyper-intense regions on T2-weighted MR images acquired two weeks post treatment. This time point was previ­ously determined by histology as adequate to de­scribe IRE.7 BBB disruption radius (rb), referring to the maximal radius of tissue in which the BBB was breached, was calculated from enhancing regions on contrast-enhanced T1-weighted MR images ac­quired 30 minutes post EP treatment. In both cases the radii were calculated by de­lineating ROIs over the entire enhancing region in each slice (excluding the ventricles). The number of pixels in the ROIs was then counted and mul­tiplied by the volume of a single pixel to receive the ROI volume. The slice thickness was 2 mm and in-plane pixel size was 0.3 X 0.3 mm. Radii of each slice was then extracted by calculating the biggest radius based on the Euclidean distance transform of the corresponding slice. The biggest radii com­puted over all slices were chosen as IRE radius and BBB disruption radius. The radii rd and rb where then plotted as a func­tion of the number of treatment pulses (N) to deter­mine the dependence of the radii on the number of treatment pulses. Numerical modeling The mathematical models were based on a two-di­mensional finite element model (assuming spheri­cal symmetry of the produced IRE lesions and BBB disruption) (Figure 1) that was implemented in the COMSOL software package (Comsol Multiphysics, v.4.2a; Stockholm, Sweden) as previously de­scribed.7,34 The rat head and chest were modeled as a 30 x 15 mm ellipse (Figure 1C) with an initial conduc­tivity of 0.258 S/m to match the conductivity used by Sel et al.37 The electric field was described by the Laplace equation for electric potential distribution in a volume conductor: [1] where . is the electric conductivity of the tissue, E is the applied electric field and . is the potential. The .(E) dependence of brain tissue was described by an smoothed Heaviside function using 500 V/ cm and 700 V/cm as reversible and irreversible TABLE 1. Material properties used for numerical model Brain . - basic conductivity 0.258[S/m] k - Thermal conductivity 0.0565[W/(m*K)] Cp - Heat capacity 3680 [J/(kg*K)] . - density 1039 [kg/m^3] Q’’’- metabolic heat generation 10437 [W/m^3] T - temperature 37°C Blood Cp-heat capacity 3840 [J/(kg*K)] . density 1060 [kg/m^3] Wb-Perfusion rate 7.15E-3 [1/s] copper . - basic conductivity 5.998E7 [S/m] k - thermal conductivity 400 [W/(m*K)] Cp heat capacity 385 [J/(kg*K)] . - Density 8700 [kg/m^3] Silver . - basic conductivity 6.273E7 [W/m^3] k - thermal conductivity 429 [W/(m*K)] Cp heat capacity 234 [J/(kg*K)] . - Density 10500 [kg/m^3] First, the model was adapted to predict tissue damage (cell death) probability induced by EP. In the Peleg-Fermi model the probability for cells sur­vival is given by: [7] where E is the electrical field, N is the number of pulses, Ec is the critical electric field in which 50% of the cells are killed and A is a kinetic constant which defines the slope of the curve. The electric field calculated using the nu­merical model was exported to Matlab (R2011a, Mathworks, USA) and was implemented in the Peleg-Fermi model. We have previously shown that the hyper­intense regions on T2-weighted MRI obtained 14 days post treatment were significantly correlated with rarified regions in histology, confirming that these regions represent damaged tissues.7 Based on this, rd was set as S(E,N) = 0, assuming over 99.99% of the cells were irreversibly electroporated. An optimization based on A Nelder-Mead sim­plex algorithm43 with added constrains was ap­plied to Equation [7] for each group treated with N pulses, calculating a map of S(E), until r(S = 0) matched rd. The coefficients Ec and A for the differ­ent number of pulses were extracted and behavior equations were fitted to Ec(N) and A(N). For each group treated with N pulses, Electric field distribution (E) was calculated using COMSOL Multiphisics and extracted to Matlab. The map E, along with the equation [7] allows to associate a map of S with any pair of the Fermi distribution (Ec,A). From the S map, the two iso­contours of S = 0.9999 and S = 0.0001 are fitted to circles. An optimization based on A Nelder-Mead simplex algorithm on Ec and A as variables is used to find the (Ec,A) pair of parameters best corre­sponding to rd/rb. The process of extracting r from S is nonlinear as it is based on fitting S = 1 iso-contour to a cir­cle. Therefor the global dependency between Ec, A and rb/rd is noisy. This noisiness could potentially cause problems with computation of derivatives. Additionally, since S is monotonous there is no risk of the simplex finding a local minimum. In order to assess whether the goodness of the (N) and Ad(N) (Ec and A for IRE) fits to the ex- Ecd perimental data, r(S = 0) for different number of treatment pulses was calculated and compared to rd. Although the Peleg-Fermi model was originally used to describe cell death, here we adapted it to describe BBB disruption as well and calculated the relevant coefficients. For this purpose the model was fitted to the radii calculated from contrast-en­hanced T1-weighted MR Images. This time rb was set as BBB(E,N) = 1, meaning less then 0.001% of the BBB was disrupted in radii larger than rb. After determining Ecb and Ab (Ec and A for BBB disruption) for each N and the behavior equations Ec(N) and A(N), the goodness of the fit to the ex­perimental data was evaluated by recalculating r(BBB = 1) and fitting it to rb. The electrical field threshold for cell kill, i.e. IRE extent and for BBB disruption, i.e. reversible EP extent, for different N values were then extracted from the results of the model and compared with thresholds reported in the literature. Results MR images of 46 rats that were previously treated with EP as described above were included in the current analysis. Treatment parameters were 600 V, 50 µs pulses at 1 Hz with varying number of treatment pulses from 10 to 540 pulses. The extent of tissue damage and BBB disruption, i.e. rd- the ir­reversible damage radius and rb – the BBB disrup­tion radius were calculated from the MR images acquired 30 minutes post treatment and 2 weeks post treatment as described in the Methods section. rd and rb can be seen in Figure 3. The average ra­tio between rb(N)and rd(N) was found to be 1.67 ± 0.11 (s.e.m), confirming the coverage of significant volumes surrounding the IRE with BBB disruption. The small error suggests that the ratio between rd(N) and rb(N) is not affected by the number of applied pulses. The ratio between rd(N) and rb(N) plotted as a function of the number of treatment pulses sup­ports this observation (Figure 3B). The coefficients of the empirical function for the BBB disruption are higher, because the BBB is disrupted by electric fields lower than those required for IRE ablation. Irreversible damage model The coefficients Ecd and Ad of equation [7] were cal­culated for each value of N as shown in Figure 4A­ B. In order to find Ecd and Ad we used rd values obtained from equation [8] rather than using the average values obtained from the experiments, as this equation describes the dependence of rd on the number of treatment pulses based on the experi­mental data. Although Ec(N) is traditionally de­scribed with an exponential function we chose to describe it here using a power function as it fitted the data considerably better (r2 was considerably larger: 0.89 for the power function versus 0.5 for the exponential function), especially in the high N range. Still, when fitting the optimization results of Ec(N) of only the first 90 pulses to an exponential function, r2 increased to 0.83 (Figure 4C). FIGURE 3. (A) radii of irreversible damage and BBB disruption calculated from the MRIs, as a function of the number of treatment pulses, and the logarithmic equations fits (B) ratio between rb(N) and rd(N) as a function of number of the number of treatment pulses. FIGURE 4. Dependence of Ecd (A) and Ad (B) on the number of treatment pulses. (C) Exponential dependence of Ecd on the number of treatment pulses with N limited to 90 pulses. (D) Correlation between radii obtained from experimental data and radii obtained from the statistical model for IRE. Error bars represent 95% confidence level. TABLE 2. Average radii of IRE and BBB disruption for each treatment group. Each group of 5-7 rats was treated with different number of pulses (10-540) at 600V, 50µs pulses at 1Hz # of pulses 10 45 90 180 270 450 540 IRE radius (mm) 0.62 ± 0.15 1.35 ± 0.18 0.89 ± 0.20 1.42 ± 0.15 1.37 ± 0.16 1.92 ± 0.07 1.80 ± 0.21 BBB disruption radius (mm) 1.25 ± 0.06 1.74 ± 0.04 1.84 ± 0.07 2.54 ± 0.15 2.19 ± 0.14 2.84 ± 0.04 2.69 ± 0.12 X 102 1,000 200 data with rb set to BBB(E,N) = 1, meaning that for 1000 0 0 radii larger than rb the BBB was not breeched(less 6000 BBB disruption model 5000 800 Ec(n) [V/m] 4000 600 The same optimization method that was used to cal­ 3000 400 culate Ecd and Ad was applied to the BBB disruption 2000 0 200 400 600 than 0.001% ). Ecb and Ab were calculated for each Number of pulses Number of pulses value of N as can be seen in Figure 5A-B. FIGURE 5. Dependence of Ecb (A) and Ab (B) on the number of treatment pulses for As for the IRE models, the goodness of the fit to BBB disruption. Error bars represent 95% confidence level. the experimental data was also evaluated. r(BBB = 1) and r(BBB = 0) were calculated from BBB(E,N) for each value of N using Ab(N) and Ecb(N). Next we evaluated the correlation between EF threshold (V/m) X10 2 EF threshold (V/m) X10 2 1200 1000 800 1200 1000 800 600 200 rb(N) obtained from the experimental data, and r(BBB = 0) linear regression analysis confirmed that r(BBB = 1), calculated from the extended Peleg Fermi model with Ecb(N) and Ab(N) described well the behavior of rb obtained from experimental data: F(1,5) = 45, p < 0.001, r2= 0.91. The regression equa­ 600 400 200 0 200 400 600 tion was: rb = 0.19 + 0.87 x (x = r(S = 0)). number of pulses number of pul ses Electric field thresholds The electrical field thresholds for E(S = 0) and E(S = 1) were calculated from the model for cell death. E(BBB = 0) and E(BBB = 1) were calcu­ lated for BBB disruption. In the cell death model, E(S = 0) represents the threshold needed for over 99.99% cells death whereas electric field lower then 0 200 400 600 number of pul 0 200 400 600 number of pul E(S = 1) will cause cell death lower than 0.001%. In ses ses FIGURE 6. Electrical field thresholds. (A) IRE thresholds. Dashed line represents published IRE thresholds for white matter for 80 50 µs pulses at 4 Hz. (B) BBB disruption thresholds. Dashed line represent previously published threshold for 90 50 µs pulses at 4 Hz.5 (C) Thresholds for E(S = 0) for the IRE and E(S = 1) for BBB disruption. (D) Ratio between E(S = 1) and E(S = 0) for IRE and E(BBB = 0) and E(BBB =1 ) for BBB disruption. Error bars are smaller than markers. Linear regression analysis confirmed that r(S = 0), calculated from the Peleg-Fermi equation with (N) and Ad(N)described well the rd obtained Ecd from the experimental data: F(1,5) = 45, p < 0.008, r2= 0.79. The resulting regression equation was: rd = 0.19 + 0.87 x, (x = r(S = 0)). the BBB disruption model E(BBB = 0) represents the threshold needed for over 99.99% of the BBB to be breeched while electric field lower then E(BBB = 1) will not disrupt the BBB(BBB disruption lower than 0.001%). Thresholds are presented in Figure 6. The ratio between S(E,N) = 0 and S(E,N) = 1 thresholds, representing the transition zone be­tween over 99.99% cell death and no cell death (S(E,N) = 0 / S(E,N) = 1) thresholds was calculated. The ratio is relatively high (between 0.88 and 0.91) even for small numbers of pulses and depends only weakly on the number of treatment pulses. This means that the transition between 99.99% cell death threshold and no cell death threshold is nar­row and gets even narrower for large numbers of treatment pulses. This is not the case with BBB dis­ruption, where the ratio between the thresholds in­creases with the number of treatment pulses even­tually converging to one (Figure 6). The average ra­tio between BBB disruption ratio and damage ratio is 1.67 ± 0.11 (s.e.m). Thermal model The initial temperature of the rats’ brain in the simulation was set to 37°C to show that the treat­ment should not induce thermal damage in clinical use. The maximum temperature reached in the tis­ sue after treatment at 600 V was 38.9°C (Figure 1B). This temperature was reached using 540 pulses. Although temperature was not measured during EP treatment, histological analysis of brains ex­tracted 60 min post treatment revealed no signs indicative of thermal damage such as coagulation, or extensive hemorrhages.45 Connective tissue and blood vessels were preserved in the treated area suggesting damage induced only by IRE.46 Discussion When treating tumors by EP, it is important to de­liver the electric pulses so that the entire tumor vol­ume will be treated to avoid recurrence. It is also vital to treat the infiltrating zone surrounding the tumor mass with high efficacy while preserving the healthy tissue. This is especially important in the case of brain tumors, where the infiltrative zone is relatively large47 and the preservation of healthy brain tissue is of critical importance. The electrode configuration in this experiment, which consists of a single intracranial insulated electrode with an exposed tip combined with an external surface ground electrode, provides an electric field distribution that is strongest at the in­tracranial needle tip tissue interface and decreases with the square of the radius. This setup provides a well-controlled region of permanent damage in­duced by IRE, further surrounded by significant BBB disruption zone. This combined response of­fers the potential of this configuration for the treat­ment of brain tumors combining IRE and chemo­therapy. This setup induces rapid tissue damage in the tumor mass surrounded by significant BBB disruption, thus potentially enabling efficient drug delivery of systemically administered drugs to the infiltration zone surrounding the tumor. Since GBM cells are highly resistant to therapeutic ap­optotic stimuli, however, they exhibit a paradoxi­cal propensity for extensive cellular necrosis19,20, IRE may be efficient for treating the tumor mass. Disrupting the BBB in the local vicinity of the tu­mor can also improve drug intake since peripheral administration of therapeutic agents is inefficient due to poor penetration of most drugs across the BBB. As clinical trials using IRE or ECT for the treatment of deep seated tumor are becoming more common8-16, a model that can predict treatment outcome and enable individual treatment planning is increasingly being recognized as a need. The statistical model of EP-induced cell death used in this manuscript was originally suggested by Golberg et al.32 who validated the model using experimental data in vitro. Here, we present for the first time an experimentally validated statistical model for tissue IRE where the Peleg-Fermi model was extended to a wide range of number of treat­ment pulses r and to BBB disruption. The results of this study demonstrate the fea­sibility of applying the Peleg-Fermi model for de­scribing irreversible EP in the brain and for treat­ment planning. Furthermore, since our model is based on experiments with up to 540 pulses we were able to extend the model beyond the up to 90 traditional pulses used for IRE. This is important since protocols outside the traditional 100 pulses are being evaluated48,49 and a tool to evaluate proto­cols with higher number of pulses is needed. The results of the model indicate, as expected, that with increasing number of treatment pulses it is possible to treat larger volumes of tissue and that the IRE threshold decreases with the number of pulses. This is however true up to a limited ex­tent since both Ec(N) and the thresholds eventually plateau. This suggests that although increasing the number of pulses while lowering the treat­ment voltage may represent a safe way to avoid thermal damage while still achieving large enough treatment volumes, there is an upper limit for this effect. In addition, when using higher voltages, raising the number of pulses will eventually lead to increased damage induced by Joule heating but will not increase the damage induced by IRE. This plateau phenomenon does not only result from the logarithmic behavior of r(N),as can be seen in Equations [8], [9] (Figure 6) but also in Ecd(N) (Figure 4B) and Ecb(N) (Figure 5B). Although the behavior of the equations de­scribing Ec(N) was previously described as expo­nential32,36, which supports the claim that larger number of pulses increases lethality, we found that Ec(N) is better described by a power function. As power functions plateau faster than exponential functions it further supports the limited effect of increasing the number of treatment pulses. One ex­planation for the difference might be that previous­ly the model was limited to 10-90 pulses32,36, and therefore the plateau effect was not yet reached. In a paper published about evaluation of the Fermi equation as a model of dose-response curves on dose response the author described Ec as a Weibull function suggesting exponential function is just a simplification for limited range of pulses.50 Once we found that the Peleg-Fermi model can be used to describe IRE, we continued to further extend the model to describe BBB disruption in­duced by EP. For the model, we correlated radii calculated from contrast-enhanced T1-weighted MRI with BBB(E,N) = 1 since even a relatively small BBB disruption can be visible. We found that the extended Peleg-Fermi model describes well not only the behavior of IRE radii but also that of BBB disruption induced by EP with high statistical sig­nificance. This also indicates that there are possi­bly similar underlying mechanisms at play, which cause the effects. The combination of the two models can be used for efficient treatment planning for brain tumors where IRE is used for ablating the tumor mass while BBB is disturbed in the rims and infiltrating zone thus allowing efficient access of therapeutic agents. The rims in this setup are on average 1.67 ÷ 0.11 mm wider than the damage, with no corre­lation to number of pulses. This suggests that the volume of BBB disruption is over 4 times larger than the volume of IRE. Although both the IRE and the BBB disruption models were constructed separately, when plan­ning a treatment protocol for brain, both should be used since BBB disruption with no irreversible damage only occurs in relatively low electric fields and when higher voltages or higher number of pulses are used, irreversible damage is difficult to avoid. The electric field thresholds for IRE and revers­ible EP are mostly limited to the traditional treat­ment protocol, i.e. 90 pulses, but when using pro­tocols that include a different number of pulses, different thresholds should be used.51 In this study we calculated the thresholds needed for the differ­ent number of pulses and fitted them to a power function. The thresholds we found for 90 pulses fit well within the thresholds previously reported in the literature for IRE in white matter52 and BBB dis­ruption5 as can be seen in Figure 6. The ratio between the thresholds of BBB(E,N) = 0 and BBB(E,N) = 1 were found to increase with the number of pulses suggesting that the window between 99.99% of BBB disruption and no BBB dis­ruption narrows with the number of pulses. This suggests that while increasing the number of puls­es will eventually not lead to bigger radius of BBB disruption, larger percentage of the BBB will be disrupted thus improving drug penetration to the tissue. This is not the case for IRE where the ratio between the thresholds for S(E,N) = 0 and S(E,N) = 1 seems to be nearly independent on the number of treatment pulses. This is somewhat surprising but could be explained by the fact that the ratio is relatively high to begin with (between 0.88-0.91) and that our dataset starts with 10 pulses, however the range of pulse numbers in this study covers the most commonly used IRE protocols in clinical practice. This is also consistent with previous pub­lications saying there is a sharp delineation of IRE treated and healthy tissue53 and demonstrates that the sharp delineation is maintained even for high number of pulses. The ratio between rb and rd was found to be nearly independent on the number of pulses. This is further supported by the relatively constant ra­tio between the thresholds that where calculated from the model. Thus, during treatment planning it might be sufficient to calculate one radius. It also indicates that BBB disruption may be used as a safety limit for irreversible EP. Though when using other electrode configurations, caution is needed. If thermal damage occurs, typically at high volt­ages or high number of pulses, it may influence the ratio between cell death and BBB disruption. The thermal model showed only a mild increase in brain temperature. The maximal temperature at the end of 540 pulses reached 38.9°C. Since 42°C is often considered the thermal damage threshold if sustained for long durations42, it is safe to assume that the tissue damage found in our experiments was induced solely by EP and not by thermal ef­fects. This was also confirmed by histology7 show­ing no signs of thermal damage, although a tem­perature assessment in real time is advisable. Despite our understanding that this model may be used by physicians and researchers for the se­lection of treatment protocols, a model that also incorporates dependence on additional treatment parameters such as frequencies and pulse dura­tions should be developed.28 Such all-inclusive model would enable physicians to choose the saf­est and most efficient protocol on a per-patient bas­es. Another point to bear in mind is that although the electrode configuration suggested in this paper produces very low Joule heating, using other elec­trode configurations with high number of pulses might induce thermal damage in addition to IRE.54 Although this study indicates that the combination of IRE and BBB disruption may be applied for the treatment of brain tumors, experimental validation using animals bearing intracranial tumors is yet to be done. In conclusion, the results of our study indicate that it is possible to apply high voltage electric pulses in a manner that induces localized focused irreversible damage in the brain surrounded by a larger volume of BBB disruption while using a single minimally invasive intracranial electrode. We used existing statistical models of cell kill by electric pulses that were based on theoretical cas­es and validated them using in vivo experimental data and extended the knowledge of EP thresholds beyond the traditional 90 pulses protocol used in IRE. We further extended the model to describe BBB disruption induced by EP. These models can assist physicians and researchers in selecting opti­mal treatment protocols allowing them to achieve the desired outcome in treating brain tumors. Although validation of the model in tumors is yet to be done, the results confirm that treatments outside the most commonly used protocols can achieve expected outcome. Acknowledgements This work was performed in partial fulfillment of the requirements for a Ph.D. degree of Shirley Sharabi, Sackler Faculty of medicine, Tel Aviv University, Israel and was supported by COST TD1104 STSM number 010315-057446. 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A statistical model for multidimensional irreversible electroporation cell death in tissue. Biomed Eng Online 2010; 9: 13. 33. Dermol J, Miklavčič D. Predicting electroporation of cells in an inho­mogeneous electric field based on mathematical modeling and experi­mental CHO-cell permeabilization to propidium iodide determination. Bioelectrochemistry 2014; 100: 52-61. 34. Garcia PA, Davalos RV, Miklavcic D. A numerical investigation of the electric and thermal cell kill distributions in electroporation-based therapies in tis­sue. PloS One 2014; 9: e103083. 35. Dermol J, Miklavcic D. Mathematical models describing Chinese hamster ovary cell death due to electroporation in vitro. J Membr Biol 2015; 248: 865-81. 36. Peleg M. A model of microbial survival after exposure to pulsed electric fields. J Sci Food Agric 1995; 67: 93-9. 37. Sel D, Lebar AM, Miklavcic D. Feasibility of employing model-based op­timization of pulse amplitude and electrode distance for effective tumor electropermeabilization. IEEE Trans Biomed Eng 2007; 54: 773-81. 38. Miklavcic D, Towhidi L. Numerical study of the electroporation pulse shape effect on molecular uptake of biological cells. Radiol Oncol 2010; 44: 34-41. 39. Corovic S, Lackovic I, Sustaric P, Sustar T, Rodic T, Miklavcic D. Modeling of electric field distribution in tissues during electroporation. Biomed Eng Online 2013; 12: 16. 40. Elwassif MM, Kong Q , Vazquez M, Bikson M. Bio-heat transfer model of deep brain stimulation-induced temperature changes. J Neural Eng 2006; 3: 306-15. 41. Garcia PA, Rossmeisl JH, Jr., Neal RE, 2nd, Ellis TL, Olson JD, Henao-Guerrero N, et al. Intracranial nonthermal irreversible electroporation: in vivo analy­sis. J Membr Biol 2010; 236: 127-36. 42. Garcia PA, Rossmeisl JH, Jr., Neal RE, 2nd, Ellis TL, Davalos RV. A parametric study delineating irreversible electroporation from thermal damage based on a minimally invasive intracranial procedure. Biomed Eng Online 2011; 10: 34. 43. Lagarias JC, Reeds JA, Wright MH, Wright PE. Convergence properties of the Nelder--Mead simplex method in low dimensions. SIAM J Optim 1998; 9: 112-47. 44. Pucihar G, Krmelj J, Rebersek M, Napotnik TB, Miklavcic D. Equivalent pulse parameters for electroporation. IEEE Trans Biomed Eng 2011; 58: 3279-88. 45. Sherar M, Moriarty J, Kolios M, Chen JC, Peters RD, Ang LC, et al. Comparison of thermal damage calculated using magnetic resonance ther­mometry, with magnetic resonance imaging post-treatment and histology, after interstitial microwave thermal therapy of rabbit brain. Phys Med Bio 2000; 45: 3563-76. 46. Maor E, Ivorra A, Leor J, Rubinsky B. The effect of irreversible electropora­tion on blood vessels. Technol Cancer Res Treat 2007; 6: 307-12. 47. Nieto-Sampedro M, Valle-Argos B, Gomez-Nicola D, Fernandez-Mayoralas A, Nieto-Diaz M. Inhibitors of glioma growth that reveal the tumour to the immune system. Clin Med Onco 2011; 5: 265-314. 48. Faroja M, Ahmed M, Appelbaum L, Ben-David E, Moussa M, Sosna J, et al. Irreversible electroporation ablation: Is all the damage nonthermal? Radiology 2013; 266: 462-70. 49. Olweny EO, Kapur P, Tan YK, Park SK, Adibi M, Cadeddu JA. Irreversible electroporation: evaluation of nonthermal and thermal ablative capabilities in the porcine kidney. Urology 2013; 81: 679-84. 50. Peleg M. Evaluation of the Fermi equation as a model of dose-response curves. Appl Microbiol Biotechnol 1996; 46: 303-6. 51. Pucihar G, Krmelj J, Rebersek M, Napotnik T, Miklavcic D. Equivalent pulse parameters for electroporation. IEEE Trans Biomed Eng 2011; 58: 3279-88. 52. Garcia PA, Neal RE, Rossmeisl JH, Davalos RV. Non-thermal irreversible electroporation for deep intracranial disorders. Conf Proc IEEE Eng Med Biol Soc 2010; 2010: 2743-6. 53. Ellis TL, Garcia PA, Rossmeisl JH, Jr., Henao-Guerrero N, Robertson J, Davalos RV. Nonthermal irreversible electroporation for intracranial surgical applica­tions. Laboratory investigation. J Neurol 2011; 114: 681-8. 54. Kos B, Voigt P, Miklavcic D, Moche M. Careful treatment planning enables safe ablation of liver tumors adjacent to major blood vessels by percutane­ous irreversible electroporation (IRE). Radiol Oncol 2015; 49: 234-41. research article Electrochemotherapy by pulsed electromagnetic field treatment (PEMF) in mouse melanoma B16F10 in vivo Simona Kranjc1, Matej Kranjc2, Janez Scancar3, Jure Jelenc4, Gregor Sersa1, Damijan Miklavcic2 1 Department of Experimental Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 University of Ljubljana, Faculty of Electrical Engineering 3 Jozef Stefan Institute, Ljubljana, Slovenia 4 Iskra Medical LLC, Ljubljana, Slovenia Radiol Oncol 2016; 50(1): 39-48. Received 30 October 2015 Accepted 20 January 2016 Correspondence to: Prof. Damijan Miklavčič, Ph.D., University of Ljubljana, Faculty of Electrical Engineering, Tržaška 25, SI-1000 Ljubljana, Slovenia. E-mail: damijan.miklavcic@fe.uni-lj.si Disclosure: DM holds a patent on electrochemotherapy that have been licensed to IGEA S.p.a. and is also consultant to various companies having commercial interests in electroporation based treatments and therapies. Other co-authors have nothing to disclose. Introduction. Pulsed electromagnetic field (PEMF) induces pulsed electric field, which presumably increases mem­brane permeabilization of the exposed cells, similar to the conventional electroporation. Thus, contactless PEMF could represent a promising approach for drug delivery. Materials and methods. Noninvasive electroporation was performed by magnetic field pulse generator con­nected to an applicator consisting of round coil. Subcutaneous mouse B16F10 melanoma tumors were treated with intravenously injection of cisplatin (CDDP) (4 mg/kg), PEMF (480 bipolar pulses, at frequency of 80 Hz, pulse duration of 340 µs) or with the combination of both therapies (electrochemotherapy – PEMF + CDDP). Antitumor effectiveness of treatments was evaluated by tumor growth delay assay. In addition, the platinum (Pt) uptake in tumors and serum, as well as Pt bound to the DNA in the cells and Pt in the extracellular fraction were measured by inductively coupled plasma mass spectrometry. Results. The antitumor effectiveness of electrochemotherapy with CDDP mediated by PEMF was comparable to the conventional electrochemotherapy with CDDP, with the induction of 2.3 days and 3.0 days tumor growth delay, respectively. The exposure of tumors to PEMF only, had no effect on tumor growth, as well as the injection of CDDP only. The antitumor effect in combined treatment was related to increased drug uptake into the electroporated tumor cells, demonstrated by increased amount of Pt bound to the DNA. Approximately 2-fold increase in cellular uptake of Pt was measured. Conclusions. The obtained results in mouse melanoma model in vivo demonstrate the possible use of PEMF induced electroporation for biomedical applications, such as electrochemotherapy. The main advantages of electropora­tion mediated by PEMF are contactless and painless application, as well as effective electroporation compared to conventional electroporation. Key words: pulsed electromagnetic field; bipolar pulses; contactless electroporation; CDDP; electrochemotherapy; platinum determination; mouse melanoma Introduction proteins and genetic material (plasmid DNA, siR- NA, miRNA) into cells.1 Electroporation is related Electroporation is a physical method enabling to the induced transmembrane voltage which if delivery of impermeable drugs, macromolecules, sufficiently high, enables the formation of tempo­rary structural changes in the plasma membrane and increases its permeability for molecules other­wise deprived of transmembrane transport mecha­nisms.2-5 Electroporation of cells is predominantly induced by pulsed electric fields, which are gener­ated with the train of square wave electric pulses of sufficient amplitude establishing local electric field (hundreds of V/cm).1,3,6,7 The electric field intensity and duration of the pulses determine whether the structural changes in the plasma membrane are re­versible, allowing cells to survive, or irreversible, leading to cell death, due to the loss of homeo­stasis.8-11 Nowadays, reversible electroporation is used as a platform technology12 and among others for drug delivery to various tissues, with therapeu­tic purposes for the treatment of cancer, known as electrochemotherapy.7,13-18 Electrochemotherapy is used in treatment of hu­man cutaneous tumors of different histology, and has been translated also in treatment of deep seat­ed tumors.15,18-22 In parallel, electrochemotherapy is being used for treatment of tumors in veterinary oncology.23-26 The main chemotherapeutics used in electrochemotherapy are nonpermeable bleo­mycin and poorly permeable cisplatin (CDDP), via systemic or intratumoral administration route. Electric pulses can be delivered to the tumors via noninvasive plate electrodes, which embrace the tissue, or invasive needle electrodes, which are in­serted into the tumor.7,27 In the past the effects of externally applied pulsed electromagnetic fields (PEMF) on the cells were studied extensively. It was demonstrated that externally applied PEMF can influence intracellu­lar signal transduction, affect the cytoskeletal pro­teins involved in cell shape modification, induce changes in mitochondrial membrane potential, and besides that increase transmembrane molecu­lar transport (electroporation).28-34 Since then, a few studies actually defined the PEMF parameters that enabled successful electropermeabilization of cells; i.e. large number of 25 up to 800 the µs long magnetic field pulses applied at frequencies from 25 Hz up to 40 Hz and strength from 725 V/m up to 160 kV/m.30,35,36 Furthermore, its use as electropo­ration tool was shown in an approach for drug as well as for plasmid DNA delivery.36,37 Thus, such electromagnetic induction with alternating cur­rents has the potential for simple contactless tissue electroporation, used for electrochemotherapy and gene electrotransfer. The majority of studies with PEMF induced elec­troporation were using bipolar pulses. Generally, shorter and larger number of pulses resulted in better membrane permeabilization.30,34 In a recent study, time varying magnetic field of 6.1 T was shown as an interesting tool in drug delivery for antifungal treatment, as well as for irreversible electroporation.36,38 Furthermore, the bipolar puls­es generated by magnetic field (4 T) were used for gene electrotransfer of plasmid DNA into the skin.37 Thus, as simple, noninvasive and contactless appli­cation of PEMF, which could enable electropora­tion of cells in the tissue, this approach showed the potential use for the clinical applications. Due to only few studies in the field of electropo­ration induced by pulsed electromagnetic field we designed experiments and considered the use of such physical delivery technique in treatment of cancer, as a model for electroporation of tissues in vivo induced by PEMF. If feasible and effective, electroporation induced by PEMF would have the advantage over “conventional” electroporation, since it is noninvasive, contactless and does not in­duce pain during electroporation. We assessed the feasibility and antitumor effectiveness of electropo­ration induced with PEMF as drug delivery system for CDDP to murine melanoma B16F10 subcutane­ous tumors. To prove the underlying mechanism of electroporation we measured the platinum (Pt) bound to DNA in tumors. Electroporation induced by PEMF proved to facilitate drug uptake in tu­mors, such as CDDP, thus providing evidence of its feasibility and effectiveness. Materials and methods Drug CDDP, a chemotherapeutic drug used in electro­chemotherapy protocol in human and veterinary clinic, was chosen in the study to test the appli­cation of induced electroporation mediated with magnetic field. The stock solution of the chemother­apeutic drug used in the study, CDDP (5 mg/mL, Cysplatyl, Aventis Laboratory, Paris, France) was dissolved in aqua pro injection and frozen in ali­quots of 1 mL. In order that each animal received a dose of 80 µg of CDDP, a fresh solution at ap­propriate concentration of CDDP (1 mg/mL) was prepared in 0.9% sodium chloride solution daily before each experiment. Mouse tumor model Female C57Bl/6 mice were purchased from Charles River Laboratories Italy s.r.l. (Calco, Italy) and were maintained in an adaptation period for 14 days. They were kept at a constant room tempera­ture with a 12 hours light cycle in a conventional animal facility. Eight-week old animals weighing 20–22 g were used in the experiments. Tumors in C57Bl/6 mice were implanted subcutaneously in the right flank of the mice by inoculation of sus­pension 1 × 106 B16F10 melanoma cells prepared in 100 µL of phosphate-buffered saline (PBS) for electrochemotherapy experiments. All animal ex­perimental manipulations were conducted in ac­cordance with the principles and procedures out­lined with the guidelines for animal experiments of the EU directives and the permission from The administration of the Republic of Slovenia for food safety, veterinary and plant protection (permission No.: 34401–4/2012/2). In vivo electrochemotherapy protocol using noninvasive electroporation induced by PEMF or conventional electroporation Seven days after subcutaneously induction of B16F10 melanoma tumors (40 mm3) mice were randomly divided into the experimental groups as follows: intravenously injection of saline solution alone (Control) or combined with electroporation induced pulsed electromagnetic field (PEMF), in­travenously injection of CDDP (CDDP) or com­bined with electroporation induced PEMF (PEMF + CDDP). Noninvasive electroporation was per­formed 3 minutes after intravenous injection of chemotherapeutic drugs by magnetic field pulse generator (TESLA Stym, Iskramedical, Slovenia) connected to an applicator consisted of round coil with 72 turns. The generator supplied the applica­tor with pulses of electric current that generated time-varying magnetic field around the coil, which in turn induced an electric field in the treated tissue (Figure 1). In order to obtain precise application of elec­troporation mice were initially anesthetized with inhalation anaesthesia in the induction chamber with 2% (v/v) of isoflurane (Isoflurane; Piramal Healthcare UK Limited, London, UK) and after­wards the mouse muzzle was placed under inha­lation tube to remain anesthetized during experi­ment. The applicator for electroporation was posi­tioned over the tumor so that the tumor was in the middle of the applicator (Figure 1A). Based on the preliminary experiments, where four different sequences of bipolar pulses of elec­tric current alone or in the combination with bleomycin were tested (Supplementary Table 1, FIGURE 1. (A) Illustrated lateral view of multi-turn coil (colored orange) and the treated tumor (colored blue). Tumor was located in the center of the coil, i.e. 31 mm and 42 mm from the inner (r1) and outer boundary (r2) of the coil, respectively. The number of turns (N) in coil was 72. Due to the casing of the applicator the coil was placed 23 mm above the tumor (h). (B) Illustrated view from above. FIGURE 2. Sequence of bipolar electric pulses where tp is a duration of the pulse, Ip is a pulse amplitude, tint is an interval between pulses and fp is a repetition frequency. Supplementary Figure 1), the most promising se­quence of bipolar pulses (Supplementary Figure 1) was used in the combination with CDDP. Briefly, the sequence had 480 bipolar pulses with dura­tion of tp = 340 µs, with a peak of Ip = 400 A, repeti­tion frequency (fp) of 80 Hz and duration of each sequence (ts) of 6 s (Figure 2). Electric pulses were measured using an oscilloscope (WavePro 7300A, LeCroy, Chestnut Ridge, NY) and current probe CWT Rogowski Current Transducer (Powertek, UK). In previous experiments, groups of positive controls, such as conventional electroporation (EP) and the combination of EP with CDDP (ECT), were obtained. The growth of untreated tumors (tumor doubling time (the time in which tumor reaches twice of the initial volume, DT) in control was 1.4 ± 0.2, n = 6) and of CDDP treated tumors alone (2.3 ± 0.1, n = 4) in that independent experiment (data previously not published) were comparable to the experiment performed with PEMF treat­ment). In the conventional electrochemotherapy protocol three minutes after intravenously injec­tion of CDDP eight square wave electric pulses at 1300 V/cm voltage to distance ratio, 100 µs long and 1 Hz (Cliniporator™, IGEA s.r.l., Carpi, Italy) were applied by plate electrodes (d = 8 mm) to the tumors. Electric pulses were delivered in perpen­dicular orientation (4 + 4) and good contact be­tween the electrodes and tumor was assured using conductive gel. Determination of magnetic and electric field in the tumor Time-varying magnetic field and induced electric field of PEMF in the tumor was determined by means of numerical modelling. Numerical mod­el of the applicator was modelled as multi-turn coil node which is a lumped model for tightly wound 72 wires separated by electrical insulator. Numerical model of the tumor was represented by an ellipsoid (Figure 1). Since volume of mice tu­mors varied from 30 to 40 mm3 an average volume, i.e. 35 mm3, was used in the numerical model of the tumor. Bipolar pulse (Figure 2) was used as electric current in the numerical model of the applicator. Calculations of time-varying magnetic field and induced electric field were performed using finite element method on a desktop PC (Windows 8.1, 3.50 GHz, 32 GB RAM) using commercial finite ele­ment software package COMSOL Multiphysics 5.1 (COMSOL AB, Stockholm, Sweden). Treatment evaluation The muscle contraction during PEMF treatment and conventional EP, tumor growth after therapy, skin area above the tumor and 2 cm in diameter around the tumor exposed to PEMF or conven­tional EP and the general well-being of animals (consumption of water and food, weight loss) were monitored during the experiment. Tumor growth was followed by measuring three mutually orthog­onal tumor diameters (a, b, and c) with a Vernier caliper, every day. The tumor volumes were calcu­lated by the formula: . The arithmetic mean of the tumor volumes and the standard error of the mean (SE) were calculated for each experimental group for each measurement day. The tumor growth delay was determined for each individual tumor by subtracting the average DT of the control group from the DT of each indi­vidual tumor. Platinum determination in the serum and tumors The measurements of platinum accumulation in the serum, tumors, platinum bound to the DNA in the cells and in extracellular fraction were per­formed by inductively coupled plasma mass spec­trometry (ICP-MS, Agilent Technologies, model 7700x, Tokyo, Japan). 195Pt isotope was monitored. At optimized instrumental parameters, instrumen­ tal limit of detection (LOD) was 0.005 ng Pt/mL (3. of the blanks). The linearity of the signal was con­firmed from LOD to 10 µg Pt/mL. Repeatability of the measurements was better than 3%. The platinum uptake in tumors and its total con­centration in the serum were measured 1 hour after the treatment of mice with intravenously injection of CDDP, electroporation induced by PEMF or the combination of those therapies (PEMF + CDDP). The blood was collected with glass capillary from intraorbital sinus (3–8 samples per group) and was coagulated at room temperature for two hours. Thereafter the blood was centrifuged at 3000 rpm for 10 minutes and serum was collected and stored at the temperature -20°C. On the day of measure­ments total amount of serum samples were di­gested in 1 mL of 1 : 1 mixture of 65% nitric acid (MERCK KgaA, Dermstadt, Germany) and 30% hydrogen peroxide (MERCK KgaA, Dermstadt, Germany) by incubation at 90°C for 48 hours. Obtained clear solutions were diluted with Milli-Q water before analysis. For platinum determination in tumors, animals were sacrificed after the blood collection. The tu­mors (3–8 tumors per group) were excised and removed from the overlying skin. Each tumor was weighed, and placed into a 15 mL graduated polyethylene tube. For tumors digestion, the same procedure as for serum was applied, with the ex­ception that 2 mL instead of 1 mL of 1 : 1 mixture of 65% nitric and 30% hydrogen peroxide was used. Before analysis samples were diluted with Milli-Q water. Determination of platinum bound to the DNA in the tumor cells and the extracellular fraction (fluid) The tumors were obtained as described in the chapter above, weighed and immediately mechan­ically disintegrated. The sample was washed with 3 mL of freshly prepared PBS and filtered through the cell strainer with pore size of 40 µm (Corning Incorporated, Life Sciences, Durham, USA). The obtained cells in suspension were centrifuged at 1500 rpm for 10 minutes. Collected cells were used for the fast DNA isolation by salting-out protocol. Briefly, cells were lysed with lysis buffer (10 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid [EDTA], 1% sodium dodecyl sulfate [SDS]) with proteinase K (20 µg) for 30 minutes at 55°C by con­stant shaking. After the samples were cooled down proteins were precipitated by adding of 120 µL 4 M NaCl and shaken for 15 seconds. Precipitated pro­teins were centrifuged at 13000 rpm for 6 minutes. Supernatant was collected and centrifuged one times more at 13000 rpm for 6 minutes. In addition, DNA was precipitated with 1 mL of ethanol (70%) for 2 min by gentle mixing of tube and centrifuged at 13000 rpm for 2 minutes. Precipitated DNA was washed with additional 1 mL of ethanol (70%) and centrifuged at 13000 rpm for 2 minutes. The pellet of DNA was dried out, resuspended in 100 µL of distilled water, digested under the same procedure as serum and the concentration determined in di­luted samples by ICP-MS. The rest of two fractions, supernatant and the interstitial fraction on the top of the cell strainer, named as extracellular fraction, were collected and stored at -20°C till the digestion with the mixture of nitric acid and hydrogen peroxide (see the section above). Statistical analysis All data were tested for normal distribution with the Shapiro–Wilk test. A t-test and one-way analy­sis of variance followed by a Holm–Sidak test were used for evaluation of the differences between the experimental groups. A p value less than 0.05 was considered significant. SigmaPlot Software (Systat Software, Chicago, IL, USA) was used for statistical analysis and graphical representation. Results Antitumor effectives of electrochemotherapy mediated by PEMF Exposure of tumors to PEMF or conventional EP, performed 3 minutes after intravenous injection of CDDP, resulted in significant tumor growth de­lay, up to 3 days compared to untreated tumors, as well as compared to monotherapies. Nevertheless TABLE 1 Tumor doubling times of melanoma B16F10 tumors after treatment with CDDP or combined with electroporation induced by PEMF. Control* 12 1.5 ± 0.1 CDDP* 4 mg/kg 12 2.2 ± 0.2 0.7 PEMF 9 1.9 ± 0.1 0.4 PEMF + CDDP 10 3.8 ± 0.1 2.3 <0.001 (to PEMF) EP* 12 2.2 ± 0.3 0.7 ECT CDDP* 8 4.5 ± 0.2 3.0 <0.009 (to PEMF + CDDP) CDDP = intravenously injection of cisplatin (4 mg/kg); PEMF = pulsed electromagnetic field treatment; PEMF + CDDP = PEMF after intravenously injection of CDDP; EP = electric pulses treatment; ECT = electrochemotherapy, EP after intravenously injection of CDDP; DT = tumor doubling time; GD = tumor growth delay; p < 0.05 statistically significant difference; *Data pooled from separate experiments after checking that DT in control and CDDP treatment alone were comparable. FIGURE 3. Antitumor effectiveness of electrochemotherapy with CDDP mediated by PEMF in mouse melanoma B16F10. Data were collected from two individual experiments and each point on graph represents mean and standard error of the mean (AM ± SE). Each group consisted at least of 8 animals. CDDP = intravenously injection of cisplatin (4 mg/kg); ECT = electrochemotherapy, EP after intravenously injection of CDDP; EP = electric pulses treatment; PEMF = pulsed electromagnetic field treatment; PEMF + CDDP = PEMF after intravenously injection of CDDP significantly higher antitumor effect was obtained after conventional electrochemotherapy com­pared to electrochemotherapy mediated by PEMF. Treatment of tumors with CDDP alone or exposure to PEMF or conventional EP had no significant ef­fect on tumor growth (Table 1, Figure 3). The application of PEMF did not exert mus­cle contraction, indicating on painless treatment procedure. Additionally, all treatments were well tolerated by animals, since no body weight loss or any detectable changes in skin, exposed to PEMF treatment during observation period was obtained, indicating that there was no systemic toxicity after treatments. FIGURE 4. Platinum (Pt) accumulation in FIGURE 5. Platinum (Pt) bound to tumor and serum after electroporation in-the DNA in tumor cells representing duced by PEMF. Each group consisted intracellular fraction and Pt content from 3–8 animals. Data represent mean in extracellular fractions after elec­and standard error of the mean (AM ± SE). troporation induced by PEMF. Data represent mean and standard errorCDDP = intravenously injection of cisplatin (4 mg/ of the mean (AM ± SE). Each group kg); PEMF = pulsed electromagnetic field treat­ment; PEMF + CDDP = PEMF after intravenously consisted from 3–8 animals. injection of CDDP. * = p < 0.05 statistically signifi­cant difference; ** = p < 0.05 statistically significant CDDP = intravenously injection of cisplatin difference to measured Patinum (Pt) content in (4 mg/kg); PEMF = pulsed electromagnetic the whole tumor field treatment; PEMF + CDDP = PEMF after intravenously injection of CDDP. *, ** = p < 0.05 statistically significant difference A B C D FIGURE 6. (A) Evaluation surfaces (., ., .) in three different planes (xy, yz, xyyzzx zx) where electric field distribution and magnetic flux density were simulated by means of numerical modelling. (B) Distribution of induced electric field in evaluation surfaces when it reached its peak at tE. (C) Time course of magnetic flux density and induced electric field at evaluation point Pev. Time points when magnetic flux density and induced electric field reached its maximum are marked with tE and tB, respectively. (D) Distribution of magnetic flux density in evaluation surfaces when it reached its peak at tB. Overall, the antitumor effect CDDP, i.e. the chemotherapeutic drug used, in the electrochem­otherapy performed by PEMF was significantly increased in comparison to monotherapies, pre­sumably due to facilitated transport of CDDP. However, electrochemotherapy after conventional EP was more effective than PEMF mediated elec­troporation. Determination of platinum in the serum and tumor after electroporation induced by PEMF In order to determine whether electroporation in­duced by PEMF facilitates drug delivery into the cells, as the underlying antitumor mechanism, Pt accumulation in the serum and tumors with plas­ma mass spectrometry was determined. First, Pt was measured as indicator of CDDP39 in whole tu­mors and plasma of the blood in mice. Intravenous CDDP injection demonstrated the drug accumu­lation in tumors, and electroporation induced by PEMF as successful method for increasing Pt accu­mulation in whole tumors, one hour after the drug administration (Figure 4). A statistically significant increase in platinum content in the tumors treat­ed by electroporation induced by PEMF was ob­served, however this measurement does not indi­cate whether the electroporation induced by PEMF in fact facilitated drug delivery into the cells. To prove that electroporation induced by PEMF facilitates transmembrane transport of CDDP, the extracellular and intracellular Pt amounts were measured. After mechanically disintegration of tumors, the suspension of tumor cells and extra-cellular fractions were obtained, and Pt was meas­ured in both fractions. The concentration of the Pt detected in extracellular fraction statistically sig­nificantly decreased after electroporation induced by PEMF (Figure 5). Moreover, the Pt bound to DNA as indicator of the drug bound to the intra­cellular target was significantly increased after the electroporation induced by PEMF (Figure 5). Approximately 2 times higher values of Pt bound to DNA were obtained. Estimation of PEMF in the tumor Simulation results of PEMF in the tumor are pre­sented in Figure 6. Electric field distribution in the tumor was linearly decreasing from the boundary of the tumor towards the center with a peak value of 8.6 V/m on the tumor boundary. Magnetic flux density had a peak value of 0.3 T and its distribu­tion in evaluation surfaces remained homogeneous through the whole surface of the tumor. Discussion This study demonstrated the use of contactless pulsed electromagnetic field (PEMF) treatment as an approach to achieve electroporation of mela­noma tumor tissue, which increases drug uptake in vivo. We evaluated for the first time the antitumor effectiveness of electrochemotherapy obtained by PEMF after systemic injection of chemotherapeu­tic drug, CDDP, which is used in conventional electroporation protocol. Furthermore, we proved that the antitumor effect was related to increased drug uptake into the electroporated tumor cells, demonstrated by increased amount of Pt bound to the DNA. Thus PEMF treatment can be used (once optimized) for noninvasive drug delivery in vivo, which may be important for research where deli­cate tissues and organs needs to be avoided and for clinical applications, since it is noninvasive, contactless and painless compared to the classical electroporation using different electrodes. Potential use of strong time-varying magnetic field which induced electric field to increase trans-membrane molecular transport (i.e. electropora­tion), was already suggested before.30,34-36,38 In or­der to expand the use of contactless PEMF induced electroporation as drug delivery method the mela­noma B16F10 tumor model in vivo was chosen, as it represents a great challenge in the treatment of human melanoma.1,14,40-43 We have shown that mag­netic field generated by round coil which induced the 480 bipolar pulses, at frequency of 80 Hz, pulse duration of 340 µs, significantly improved the antitumor effectiveness of electrochemotherapy with CDDP. Our results are in accordance with in vitro study, where much stronger PEMF (6.1 T) was indicated as delivery method for therapeutic molecules in human pathogenic fungi. Namely, the synergistic effect of simultaneos treatment of 200 applied magnetic field pulses at frequency of 35 Hz and drug was observed.36 However, the ap­plication of stronger magnetic field (up to 16.4 T) did not result in better membrane permeabiliza­tion.35 The membrane permeabilization seems to be dependent more on the shape, number and fre­quency of generated pulses34-36 in addition to the amplitude of induced electric field, similar to the conventionally generated bipolar pulses.44 In addi­tion, bipolar pulses were demonstrated two times more effective as monopolar and almost equally effective as conventional square wave pulses.37 In fact, for the almost equal membrane permeabili­zation larger number of short duration induced bipolar electric pulses at higher frequency has to be delivered.37 Similarly, in our study the obtained antitumor effectiveness of cisplatin by using short and larger number of induced electric pulses at higher frequency in comparison to conventional electric pulses was comparable. Furthermore, it is known that simple round coils induce less focused and lower peak electric field than figure-of-eight coils.45,46 It was also demonstrated in vitro that by using figure-of-eight coils the increased transmem­brane molecular transport could be obtained by pulses of lower frequencies and larger number.34 However, we and others34 have shown that by in­creasing the number of pulses at the same frequen­cy the effect of electroporation can be improved (supplementary data). Presently, in conventional electrochemotherapy protocols mainly square wave or monotonically de­creasing electric pulses are delivered through plate or needle electrodes to the cells or tissues.15,18,23,25 On the contrary, only a few studies were performed with bipolar electric pulses for the electroporation of cells in vitro and tissues in vivo.44,47-55 In general, the lower pulse amplitudes were needed for effec­tive electroporation of cells in vitro with respect to unipolar pulses.48,51 Besides that, the pulse shape played important role in electroporation of cells as well.34,51,56 It was shown that electroporation, cell death and the uptake of Lucifer Yellow occurred by using the rectangular bipolar pulses at lowest, the sine bipolar pulses at medium and the triangular bipolar pulses at the highest pulses amplitudes.44 Bipolar pulses were already applied successfully in electrochemotherapy for human and veterinary clinic.49,57,58 In fact, the combination of PEMF induced elec­troporation with CDDP had significant antitumor effectiveness, whereas the application of PEMF or the drug alone had none. The antitumor effective­ness of electrochemotherapy of applied PEMF (480 bipolar pulses, at frequency of 80 Hz, pulse dura­tion of 340 µs) was presumably due to improved membrane permeabilization of cells in the tissue, since the monotherapies alone had very little but no significant effect on tumor growth in comparison to control. Similarly, preclinical and clinical studies performed in conventional scheme of electrochem­otherapy with CDDP have shown great antitumor effectiveness of electrochemotherapy on different tumor types15,42,59-62, mostly due to direct cytotoxic effect on tumor cells.60-62 It has been demonstrated that after conventional electroporation the cytotox­icity of CDDP could be improved by 70-times.61-63 Nevertheless, in our study sufficient antitumor effectiveness on melanoma B16F10 was obtained with electrochemotherapy after PEMF induced electroporation, despite the effect was significant­ly lower compared to that obtained after conven­tional electrochemotherapy with CDDP. However, calculations suggest that levels of electric field are 4 orders of magnitude lower than those associated to classical electroporation64, but tend to be high enough to induce electroporation. On the other side, while using electric field bellow 0.09 V/cm at the position of tumor site, we suspect there was no possible occurrence of irreversible electropora­tion or thermal effect, as obtained at much higher electric field induced by PEMF (up to 40 V/cm).38 Even more, we speculate that PEMF could be im­proved by positioning of tumor towards the edge of the coil, where based on calculations the high­est strength of magnetic field could be obtained, which consequently could induce higher electric field strength and thus, even more cells could be successfully electroporated at deeper parts of tu­mor tissue. Therefore, further studies to optimize the PEMF are warranted. Even though it was previously known that the time varying magnetic field could induce electropo­ration34-37,65, affect the cytoskeleton and intracellular signal transduction28,32,66,67, the mechanisms of its action in the combination with CDDP have not been studied yet. Therefore, to clarify the antitumor effectiveness of electrochemotherapy the measure­ments of platinum amount after electroporation induced by PEMF were performed. Observed sig­nificant 1.6-fold increased platinum uptake into melanoma B16F10 tumors after electroporation induced by PEMF indirectly confirmed membrane permeabilization of the tumor cells and thus, its correlation with the antitumor effectiveness of elec­trochemotherapy. Our results are in accordance with results reported in other studies, where up to two times higher platinum uptake was obtained in sarcoma SA-1 and fibrosarcoma LPB tumors after conventional electrochemotherapy with CDDP.60,61 Even though lower increase of platinum amount in tumors was obtained after PEMF in comparison to conventional electrochemotherapy60,61, the final amount of platinum in the tumors was comparable. The difference of platinum amount in tumors treat­ed only with CDDP, might be tumor type depend­ent, since melanoma tumors are well vascularized and contained large spherical cells, with less sur­rounding extracellular matrix component in com­parison to stiff SA-1 and LPB tumors with small spindle-shaped cells and high content of extracel­lular matrix component.68,69 On the other hand, the amount of platinum in the serum was significant­ly up to 6-times lower compared to tumor tissue. Therefore we could assume that excess of the drug which was not entrapped in the tumors after elec­troporation was washed out with similar kinetic as in nonelectroporated tumors. Moreover, our results indicated that CDDP in the cells reached its main intracellular target DNA, in fact significantly two times higher amount of Pt was bounded to the DNA in PEMF and CDDP treated tumors than in CDDP only treated tumors. At the same time, as expected the pool of Pt amount in the extracellular fraction of these tumors was lowered, up to 1.4- times. Thus, the increased Pt uptake in the cells and its binding to the DNA could indicate the main reason for anti­tumor effectiveness of electrochemotherapy medi­ated by PEMF. Presently, it is not clear if the membrane permea­bilization obtained after application of time varying magnetic field occurs only due to induced electric field as in conventional electroporation or due to direct effect of magnetic field with the plasma membrane and surrounding ions. Thus, the precise mechanism of cisplatin uptake after electroporation mediated by PEMF into the cells remains unclear. Obtained Pt amount in the tumor cells after treat­ment with CDDP only could be ascribed to passive diffusion and active transport mechanisms of cispl­atin through the membrane, which are carrier-me­diated through formed pores or via endocytosis.70,71 In addition, it has been demonstrated that expo­sure of cells to train of unipolar pulsed low electric fields at strength from 1.2 up to 20 V/cm can induce electro-endocytosis.72,73 Thus, we suspect that gen­erated bipolar electric field of just below 0.09 V/cm by PEMF might also trigger endocytosis besides membrane permeabilization which enables the in­ternalization of cisplatin in the cell and contributes partially to the increase of platinum amount. In conclusion, our results show that PEMF at magnetic field below of 1 T was sufficient to achieve membrane permeabilization of tumor cells, thus, small molecules such as drug (CDDP) improved delivery and cellular uptake in solid tumors was enabled. Due to simple, contactless, painless, fo­cused local application of PEMF, better field distri­bution irrespective of tissue type and thus, achiev­ing electric field strength for membrane permeabi­lization can be established in deeper parts of tissue. However this approach has the limitation that the strength of the magnetic field decreases rapidly with distance from the coil which has to be taken into account by designing coils74 in order to achieve successful permeabilization at a greater tissue depth. PEMF might thus represent an alternative to conventional electroporation with electric fields in electrochemotherapy. However further studies are needed to improve the equipment, to optimize and establish precise protocols of drug application and PEMF parameters, as well as to reveal the effects of PEMF on variety of normal and tumor tissues. Acknowledgements The work was performed in the scope of The European Associated Laboratory entitled Pulsed Electric Fields Applications in Biology and Medicine (LEA-EBAM). This research was sup­ported by the Slovenian Research Agency un­der program grants (P2-0249 and P3-0003) and by Slovenian Ministry of Education, Science and Sport under program grant M-1330E. The authors acknowledge Tanja Dolinšek and Lara Prosen for technical help in preparation of tumor samples sub­jected to platinum measurements. The research has been achieved due to the networking efforts of the COST TD1104 Action (www.electroporation.net). The paper was presented at the 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine, and Food & Environmental Technologies, September 6 to 10, 2015, Portorož, Slovenia (wc2015.electroporation.net) organized by COST TD1104 Action (www.electroporation. net), supported by COST (European Cooperation in Science and Technology). References 1. 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Antov Y, Barbul A, Mantsur H, Korenstein R. Electroendocytosis: exposure of cells to pulsed low electric fields enhances adsorption and uptake of macromolecules. Biophys J 2005; 88: 2206-23. 74. Deng Z, De Lisanby SH, Peterchev AV. Electric field depth-focality trade off in transcranial magnetic stimulation: Simulation comparison of 50 coil designs. Brain Stimul 2013; 6: 1-13. research article A prototype of a flexible grid electrode to treat widespread superficial tumors by means of Electrochemotherapy Luca G. Campana1,2, Fabrizio Dughiero3, Michele Forzan3, Carlo R. Rossi1,2, Elisabetta Sieni3 1 Surgical Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, Padova, Italy 2 Department of Surgery Oncology and Gastroenterology, University of Padova, Italy 3 Department of Industrial Engineering, University of Padova, Italy Radiol Oncol 2016; 50(1): 49-57. Received 3 November 2015 Accepted 20 January 2016 Correspondence to: Dr. Elisabetta Sieni, University of Padova, Department of Industrial Engineering, Via Gradenigo 6/a, 35131 Padova, Italy. Phone: +39 049 8277514; E-mail: elisabetta.sieni@unipd.it Disclosure: No potential conflicts of interest were disclosed. Background. In recent years, superficial chest wall recurrence from breast cancer can be effectively treated by means of electrochemotherapy, with the majority of patients achieving response to treatment. Nevertheless, tumor spread along superficial lymphatic vessels makes this peculiar type of tumor recurrence prone to involve large skin ar­eas and difficult to treat. In these cases, electroporation with standard, small size needle electrodes can be time-con­suming and produce an inhomogeneous coverage of the target area, ultimately resulting in patient under treatment. Materials and methods. Authors designed and developed a prototype of a flexible grid electrode aimed at the treatment of large skin surfaces and manufactured a connection box to link the pulse applicator to a voltage pulse generator. Laboratory tests on potato tissue were performed in order to evaluate the electroporation effect, which was evaluated by observing color change of treated tissue. Results. A device has been designed in order to treat chest wall recurrences from breast cancer. According to pre­liminary tests, the new flexible support of the electrode allows the adaptability to the surface to be treated. Moreover, the designed devices can be useful to treat a larger surface in 2–5 minutes. Conclusions. Authors developed the prototype of a new pulse applicator aimed at the treatment of widespread superficial tumors. This flexible grid needle electrode was successfully tested on potato tissue and produced an elec­troporation effect. From a clinical point of view, the development of this device may shorten electrochemotherapy procedure thus allowing clinicians to administer electric pulses at the time of maximum tumor exposure to drugs. Moreover, since the treatment time is 2–5 min long, it could also reduce the time of anesthesia, thus improving patient recovery. Key words: electrochemotherapy; electrode; flexible support; breast cancer recurrence Introduction Electrochemotherapy (ECT) is an effective local therapy in use for unresectable skin cancers as well as cutaneous metastases from different tumor his­totypes such as melanoma, head and neck cancer, soft tissue sarcomas and breast cancer.1-3 During the procedure, high voltage pulses are applied to a needle pair implanted into tumor tissue in order to generate an electric field aimed at increasing cell membrane permeability and the uptake of chemo­therapeutic drugs (bleomycin or cisplatin). In fact, electric fields over a suitable threshold allow the opening of transient aqueous pores on the cell membrane, thus inducing a temporary permeabi­lization (reversible electroporation).1,4-8 In recent years, ECT has shown efficacy in sev­eral tumors types and has been adopted by several centers for the treatment of skin tumors and, most of all, superficial metastases with the aim to im­prove local tumor control without discontinuation of concomitant systemic treatments.4,8-13 Among ECT indications, breast cancer (BC) represents a promising, but challenging field. Chest wall recur­rence (i.e. the occurrence of skin / soft tissue metas­tases on the chest wall after previous mastectomy) is an uncommon, but not negligible, pattern of recurrence observed in BC patients. It may occur also after optimal multidisciplinary management (e.g. mastectomy, radiation and systemic therapy). Its occurrence is more frequent (up to 45%) if the primary tumor was advanced in stage, while drops to 2–15% if adjuvant radiotherapy was applied af­ter mastectomy. About 40–50% of chest wall recur­rences occur around the mastectomy scar.14-18 ECT represents a promising treatment option in these patients and its efficacy in achieving an effective tumor control is particularly high when disease is limited in size.10,19,20 Nevertheless, when chest wall recurrence is multifocal and widespread, it poses a therapeutic challenge to the treating on­cologist. Generally, during ECT procedure a 7-needle electrode, arranged in hexagonal geometry, is used to apply the electric fields to tumor tissue.8,11 This type of electrode covers a surface close to 3 cm2 at each single application. Since it is well known that bleomycin main­tains a sufficiently high concentration in tissue for a limited time interval (i.e., 20 minutes according to Standard Operative Procedures8,21), the volt­age pulses have to be applied within this interval. Consequently, large tumor-involved areas can be managed only by applying the 7-needle electrode several times during the 20-minute interval which follows the infusion of bleomycin. In theory, the standard hexagonal array needle electrode can be applied indicatively 100–120 times during a single ECT procedure, thus allowing for the coverage of approximately an area of 200–360 cm2. In the clini­cal practice, this surface area may prove to be in­sufficient for effective treatment of patients with widespread skin tumor infiltration of the chest wall (Figure 1A). As a consequence, a larger area can be managed only planning an additional ECT cycle, which in­evitably will require a new anesthesia and an addi­tional administration of drugs. As a consequence, many patients need to undergo repetitive ECT cycles in order to completely treat their chest wall recurrences or to treat newly occurred metastases outside treatment field (Figure 1A). To avoid this problem, some electrodes designed to treat surfaces up to tens of cm2 have been pro­posed.22-24 Among these solutions, a composition of triangular configurations or arrays of parallel nee­dles mounted on rigid support has been indicated25-28. An alternative strategy is represented by the use of planar antenna technology, thus avoiding needles insertion and developed in Nenzi et al.29 In this paper we present the prototype of a grid electrode suitable for not-plane surfaces such as chest wall and the treatment of recurrences from BC. The concept is based on a grid device includ­ing several electrodes arranged in a regular mesh (Figure 1). The new device is mounted on a flex­ible support that can be adapted to the skin surface and is equipped with removable needles that can be positioned one by one on the treating area.44 As a result, the proposed device is a grid composed by a several needles that can be positioned before elec­tric field application. For instance, the flexible ver­sion of the prototype tested in Ongaro et al. 30 has 13 electrodes and can cover an area of 50 cm2. The treatment time in this case can be equivalent to one application of the 96 voltage pulses sequence pre­scribed by ECT Standard Operative Procedures8,21 for hexagonal electrodes. Then, by using such de­vice, the electric field could be applied in a shorter time interval compared with the currently used procedure which requires multiple, juxtaposed electrode placements (e.g. at least 16 applications for an area of 50 cm2 considering the area covered by the standard electrode of 3 cm2). In this paper, a prototype with 67 needles mounted in a flexible support covering an area of 225 cm2 is proposed. The aim of this device is to apply the electric field more homogeneously in the treatment field and to respect the suggested time interval of 20 minutes after bleomycin infusion. Also the electric con­nection to the voltage pulse generator is here de­scribed. The prototype of the new flexible device consists of a grid of needles distant 2 cm apart, i.e. the needle configuration use hexagonal geometry of needle as in standard ECT needles, but their distance has been increased in order to reduce the number of needles per cm2. The amplitude of the electric field generated by this needle configuration has been verified by simulation and compared with the one obtained with lower distance needles.30 Moreover, the effect of the field in term of electroporated cells has been also verified using potato tissue tests and in vitro tests.28,30,31 Finally, in order to verify the electroporation, the results of simple preliminary tests carried on vegetable tissue are presented. Materials and methods The prototype of the grid electrode (Figure 2) is composed by a flexible support and 67 (5 or 10 mm-long) stainless steel needles that can be insert­ed one by one and linked to the electrical connec­tion of the support. The flexible support is equipped with some elec­trical conductive strips with some holes where the needles can be inserted. Each hole is provided with an insertion guide. The guides allow maintaining the perpendicularity of the needle with the surface where they are inserted. The guides center is posi­tioned in points belonging to the vertex of adjacent equilateral triangles like in28,30 with side 2 cm long. The connection guides are arranged in hexagons as shown in Figure 2A in order to reproduce the standard hexagonal electrode geometry.8,21,28,30 The guides allow both the insertion and electrical con­nection of the needle electrodes. The flexible plastic support is also provided with electric connections to the voltage pulses generator. The flexible elec­trode in Figure 2A has the electrical connections formed by copper strip, whereas in the electrode of Figure 2C the electrical connections are made, for sake of simplicity, with copper wires. Figure 2B shows the insertion of the needles in the guides. The connection guides are realized in order to eas­ily place and remove the needles. The needles are provided with an insulant cap (Figure 2B) and the flexible support by needle guides in order to allow the normal penetration of the tip. Moreover, for ensuring a user friendly, safe and effective appli­cation on chest wall BC recurrences, needle length was limited to 5–10 mm. In practice, it is expected that, when the flexible plastic support is fixed on a curved surface (e.g. the thoracic wall), the short length of the needles and the 2 cm distance will limit the approach of their tips. Finally, the device is connected to the pulse generator by means of fast connectors. #1 1–2 #14 A-3 #2 1–3 #15 A-4 #3 1–4 #16 A-5 The prototype of the grid electrode has been re­alized in two different sizes. The first has a diam­eter of 8 cm with 13 needles and the second one is a square of side 15 cm with 67 needles that covers an area of 225 cm2. Electrode supply The new grid electrodes are supplied by a volt­age pulse generator manufactured by Igea S.p.A. (Carpi (MO), Italy).32,33 In each test performed the voltage generator delivers sequences of 10 rectan­gular pulses between 0 and 2000 V with pulse du­ration of 100 µs at 100 Hz (total time of 10 msec). In this work the voltage has been fixed to 2000 V for all test performed. Figure 3A shows the connection device. It is a box equipped with 16 plug clamps that can be con­nected two by two to the voltage pulse generator. The 16 clamps are arranged in groups of 8 each one identified by a number except the eighth that is identified by the letter ‘A’ like in Figure 3B. The needles in the flexible electrode are ar­ranged in hexagon and numbered consequently following the scheme in Figure 3B. This schema is formed by a hexagonal structure of numbered points and a point named ‘A’ (corresponding to the clamping numbers). Continuous gray hexagons in Figure 3C highlight the hexagonal scheme in Figure 3B in which the central electrode is named ‘1’. Considering the dotted hexagons, the central point is named ‘A’, whereas the other points of the hexagon correspond to the points identified by numbers. Figure 3C shows also the connection scheme of the flexible grid electrode with 67 nee­dles to the voltage pulse generator. Some exam­ples of supplied electrode pairs are presented in the same Figure. For instance, Table 1 reports the supply sequence where the needle pairs selected involve needle ‘1’ or needle ‘A’. The clamps are subdivided in two groups that supply a half of the needle pairs. In fact, since the average resistance at electrode ends, Re, is 130–200 W (value measured at needle extremities during pulses application) considering a current of 40 A (maximum value deliverable by the voltage pulse generator, Imax) and an applied voltage, VA, of 2000 V the maximum number of electrode pairs that can be parallel connected, Nm, is between 3 and 5.28 [1] For instance, like shown in Figure 3C for the pair 3–6, the number of electrode pairs between needles with the same number is up to 8. Consequently, the larger grid electrode is divided into two areas where there are up to 4 electrode pairs between needles with the same number. Each group of eight clamps in the box in Figure 3A supplies half of the grid electrode, and the electrode pairs on the boundary are supplied by connecting two clamps belonging to different group as it has been highlighted for pair ‘1–5’ in Figure 3C. All the electrodes, both the activated and not-supplied ones, are inserted in the treating area before pulse delivery. They remain inserted during the entire treatment. In fact the influence of the not-supplied electrodes is irrelevant, as report­ed in a previous work, since they act as an ‘open circuit’30 and any current can flow. Potato test procedure The two models of the grid electrode have been tested on a phantom made of potato tissue. In fact, it is well known that after few hours of application of voltage pulses, the potato tissue appears dark if cell membranes have been electroporated.28,30,34,35 For instance in Castiello et al.28 and Ongaro et al.30 have observed potato color after 24 h even if the darkening started after few hour after pulses ap­plication. Voltage pulses have been applied to the potato tissue using the pulse generator and applying 2000V at each electrode pair following the pair-sequence described in Standard Operative Procedure.8,21 A sequence of 10 pulses has been applied to each pos­sible electrode pair. Potatoes tissue was preserved at room temperature for 24 hours after application of voltage pulses and then observed looking for the electroporation effect; pictures of the investigated tissue were also taken (Figure 4A,B). The electropo­rated potatoes appear dark only in the areas where the voltage pulses have been applied (Figure 4A), whereas any color change did not occur for the potato cut and maintained at room temperature (Figure 4B) as described in Ongaro et al.30. The in­tense dark coloration is due to the electroporation and not to the cut. Nevertheless, this technique cannot discriminate between reversible or irrevers­ible electroporation and is a simple qualitative test to visualize the area where the electroporation was occurred. The two prototypes of the device have been tested on two type of phantoms: a single potato tuber for the 13 needles electrode, whereas the larger device with 67 needles has been tested using more pota­toes piece immerged in Meat Liver Agar gel (46379 Fluka Analytica) dissolved in hot water at 5% and cooled at room temperature. Agar gel allows elec­trical conduction between adjacent potato pieces. Potatoes are arranged as shown in Figure 5A. The flexible electrode is positioned over the phantom, a single half of potato tuber for the 13 needle device or the arrangement in Figure 5A for the large electrode. The needles are posi­tioned manually one by one in each contact as in Figure 5B. Figure 5C shows the flexible electrode with 67 needles ready to apply the voltage puls­es to all possible needles pairs. Finally, Figure 5D shows the connection of larger flexible electrode to the connection box. FIGURE 6. (A) Potato tuber surface appears dark after 24 h from voltage pulse application and (B) effect of voltage pulses inside the potato. Results Figure 6 shows the electroporation effect observed 24 hours after the application of voltage pulses by means of the 13-needles flexible electrode present­ed in Figure 2A. The 15 cm2 area of potato surface appears dark (Figure 6). After 24 h the potato has been cut vertically in order to evaluate the effect of electroporation inside the phantom. The tissue appears dark for a depth larger than the electrode length as shown in Figure 6B. In this case, the nee­dle is 10 cm long and the dark area has a depth double than the needle length (close to 20 mm). This effect has been described also in Castiello et al.28 and in Ongaro et al.30 In fact, the electrode dis­tance affects the electric field distribution in the treated volume: using electrode with a larger dis­tance the electric field can penetrate more deeply in the tissue as it has been demonstrated in Ongaro et al.30 by means of numerical modeling by means of finite element method.30,36-39 Figure 7A shows the surface of phantom in Figure 5A used to test large electrode, captured 24 hours after voltage pulse application. It is evi­dent the electroporation effect produced by the device from the dark color of the tissue surface. Nevertheless, in some areas the electroporation did not occur. Considering the pairs underlined with continuous or dotted line in Figure 7A, it appears that in these cases almost one needle pair is entirely immersed in Agar gel that is more conductive than potato tissue. In these cases, the pulse generator has not delivered the current to the load because the in­ternal control of the device has measured a current greater than the maximum allowed (Imax = 40 A). Finally, pieces of potato in the phantom have been vertically cut 24 h after the voltage pulse ap­plication showing the appearance of dark areas in­side the tissue. The electrode has 67 needles and can treat an area of 225 cm2 (a square with side of 15 cm). Also in this case the electroporation depth is larger than the electrode length as shown in Figure 7B, where the needle length is 10 mm and the electroporation depth is 20 mm. Tests on potato tissue are very easy and cheap. Nevertheless, they are not sufficient to explore if the electroporation is reversible or irreversible even though some authors have tried to couple simulation results and dark color gradation40-42 and searched for the dark intensity for which the elec­tric field overcame the irreversible electroporation threshold. The evidence whether electropoation effect is reversible or irreversible can be obtained by means of in-vitro tests using cell culture as re­ported in previous experiences.30,31 Discussion Chest wall recurrence from BC represents a pecu­liar type of tumor recurrence, and, when superficial and widespread (due to lymphangitic diffusion), it is crucial, during ECT, to apply electric fields on a wide and thin surface instead of a target volume. The preliminary experimental results show that the flexible device is able to electroporate larger tis­sue surfaces with respect to the standard hexago­nal electrode. In fact, the designed device covers an area from 50 cm2 (by using 13 needles) to 225 cm2 (by using 67 needles). From a practical point of view, the plastic flexible support allows the adapt­ability to non-planar surfaces as the chest wall and can be applied to the skin as a plaster. Its main drawback is represented by the loss of parallelism between adjacent needles because of the curved surface. Nevertheless, this kind of device has been primarily conceived to treat superficial tumors of the chest wall, which has a limited radius of curva­ture; moreover, this an anatomical area, especially in mastectomy patients, is characterized by the presence of a rigid, underlying plane represented by the rib cage, which limits the penetration of needle electrodes. Moreover, and importantly, tu­mor growth in these patients follows a superficial pattern of spread and tumor thickness is limited to 4–5 mm from the superficial skin layer; conse­quently, needle electrode should be inserted only few millimeters, thus limiting the convergence of their tips. With the proposed device, needle elec­trodes can penetrate only few millimeters (maxi­mum 10 mm), and the distance between two points of insertion was fixed at 20 mm, so that the effect of parallelism loss is limited. On the other hand, single needle insertion may be more user friendly for the clinician in presence of fibrous tissue. In fact, in this case, it is simpler to insert one needle instead of 7 needles at the same time. Moreover, each needle is inserted using a rigid guide provided by the insertion mask in or­der to allow parallel penetration of tips. Moreover, the device has been designed to use needles with a length between 5 and 10 mm, so that the effect on electric field intensity due to tip convergence should be limited. During the procedure, when the needle tips are too close (e.g. by visual inspec­tion or by resistance evaluation before pulse ap­plication), applied voltage can be accordingly re­duced. The device and its positioning have been de­signed in order to contain the time of ECT proce­dure and to permeabilise tumor cell membrane when drug concentration is higher. To make elec­trode application time-sparing, the needles of the new device can be pre-positioned on the flexible support, before pulse delivery. In this way, the re­petitive, operator-dependent, placement and dis­placement of standard electrodes could be avoid­ed and voltage pulse delivery may be performed within the optimal time interval from the infusion of bleomycin. Authors are aware that the present study has several drawbacks. In fact, the phantom used to test the electrode shows areas with large difference in conductivity, likely due to Agar gel between potato pieces. These areas have caused the failure of the electroporation procedure since the pulse generator in some case has not delivered voltage pulses for its proper current limitation (internal control detected low impedance and blocked pulse delivering). Nevertheless, in previous works a low­er scale device with 52 needles 1 cm apart has been already tested on a single potato tissue and experi­mental results are in Ongaro et al.28. These tests on single potato piece did not show any drawback due to high conductivity area. Since, the designed electrode can be considered an extension of existing devices for clinical ECT treatments (e.g. hexagonal array electrodes), then current and electric field thresholds are borrowed from standard protocol for ECT.4,8,20,21,43 In future experiments, the new grid electrode will be tested in animal models in order to verify its efficacy. Moreover, it will be tested also a pro­totype with more distant needles (2 cm apart) with respect to standard electrode as well as the incre­ment of electric field depth, up to two times the electrode length shown in Figure 7B. The flexible device has the potential to reduce the time required for the application of electric pulses. For instance, the 13 needles device which, can treat an area of 50 cm2, is supplied by means of two 96-pulse sequences for hexagonal electrode described in Standard Operative Procedures.8,21 Therefore, the 50 cm2 area can be treated in less than 2 min (one 96-pulse sequence has a time dura­tion approximately of 20 ms) considering the time to arm the pulse generator. Moreover, the 67 nee­dles electrode, which covers an area of 225 cm2, can be supplied by means of 5 sequences of 96 pulses as described in the Electrode supply paragraph. Considering the time to change connections and arm the pulse generator, the treatment can be per­formed in approximately 5 min. This short time may assure a higher drug availability in tumor tissue during tumor electroporation. The new de­vice is advantageous since the clinician can repeat the 96-pulse sequence up to 150 times moving the standard electrode several times to cover all the chest wall surface. Considering, for instance, an ap­plication time of 20 ms per sequence (i.e. 96 pulses) and the time required to move the electrode and arm the generator of approximately 15s it gives a treatment time of approximately 40 min. Conclusions A flexible device has been designed in order to man­age chest wall recurrence from BC, which usually involves large skin areas in mastectomy patients. A prototype of a grid electrode aimed to treat wide­spread superficial tumors has been designed and tested in preclinical preliminary experiments. This report presents the results obtained with a flex­ible device that can be used to treat by means ECT skin areas between 50 cm2 and 225 cm2. The tip ap­proach effect is limited by needle distance, which was fixed to 2 cm, and by needle lenght, which was set between 5 and 10 mm. Moreover, the possibil­ity to pre-insert all the needles before pulse deliv­ery has several advantages: it may allow to reduce the duration of the procedure and anesthesia, to expose tumors to higher drug concentration and, hopefully, to increase the antitumor effectiveness of ECT in a challenging subgroup of BC patients. Acknowledgments Authors are gratefull to Dr. Federico Bertoldi, Dr. Roberto Bordin and Dr. Mose Castiello for the re­alization of the prototypes. Authors thank Igea S.p.A. (Carpi, Modena Italy), for the loan of the pulse generators. Project granted by CPDA138001 (Padua University). The paper was presented at the 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine, and Food & Environmental Technologies, September 6 to 10, 2015, Portoroz, Slovenia (wc2015.electroporation. net) organized by COST TD1104 Action (www.elec­troporation.net), supported by COST (European Cooperation in Science and Technology)”. References 1. 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Campana, Patent No. VR2013A000184 “APPLICATORE PER ELETTROPORAZIONE”, 01/08/2013. research article Combined local and systemic bleomycin administration in electrochemotherapy to reduce the number of treatment sessions Felipe Maglietti1,2, Matias Tellado1,3, Nahuel Olaiz1,2, Sebastian Michinski1,2, Guillermo Marshall1,2 1 Laboratorio de Sistemas Complejos, Departamento de Computación e Instituto de Física del Plasma, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina 2 Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina 3 Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Buenos Aires, Argentina Radiol Oncol 2016; 50(1): 58-63. Received: 20 October 2015 Accepted: 18 January 2016 Correspondence to: Felipe Maglietti, Laboratorio de Sistemas Complejos, Departamento de Computación e Instituto de Física del Plasma, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Intendente Güiraldes 2160, Buenos Aires, Argentina. E-mail: felipemaglietti@gmail.com Disclosure: No potential conflicts of interest were disclosed. Background. Electrochemotherapy (ECT), a medical treatment widely used in human patients for tumor treatment, increases bleomycin toxicity by 1000 fold in the treated area with an objective response rate of around 80%. Despite its high response rate, there are still 20% of cases in which the patients are not responding. This could be ascribed to the fact that bleomycin, when administered systemically, is not reaching the whole tumor mass properly because of the characteristics of tumor vascularization, in which case local administration could cover areas that are unreach­able by systemic administration. Patients and methods. We propose combined bleomycin administration, both systemic and local, using compan­ion animals as models. We selected 22 canine patients which failed to achieve a complete response after an ECT treatment session. Eleven underwent another standard ECT session (control group), while 11 received a combined local and systemic administration of bleomycin in the second treatment session. Results. According to the WHO criteria, the response rates in the combined administration group were: complete response (CR) 54% (6), partial response (PR) 36% (4), stable disease (SD) 10% (1). In the control group, these were: CR 0% (0), PR 19% (2), SD 63% (7), progressive disease (PD) 18% (2). In the combined group 91% objective responses (CR+PR) were obtained. In the control group 19% objective responses were obtained. The difference in the response rate between the treatment groups was significant (p < 0.01). Conclusions. Combined local and systemic bleomycin administration was effective in previously to ECT non re­sponding canine patients. The results indicate that this approach could be useful and effective in specific population of patients and reduce the number of treatment sessions needed to obtain an objective response. Key words: electrochemotherapy; combined treatment; systemic and local; bleomycin; resistant Introduction Electrochemotherapy (ECT) is an ablative approach that is rapidly growing, both in human and veteri­nary medicine. ECT is based on administration of bleomycin followed by application of an electric field on the tumor that enhances cell permeability to the drug. This technique can increase bleomy­cin cytotoxicity by 1000 fold. The effectiveness of ECT is approximately 80% objective response (OR) rate.1,2 A meta-analysis of ECT clinical studies in hu­man oncology showed that the overall OR rates vary from 62.6% and 82.2% OR rate depending also on the route of the drug administration, be­ing either intravenous or intratumoral.3 Despite its success related to its low cost and minimum side effects, ECT still has room for improvement. Even with such a high response rate there are 20% of cases on which attention must be focused in order to improve the outcome of the treatment. The application of ECT in companion animals showed the same pattern of success as in humans, with many studies demonstrating its high efficien­cy, with a very similar response rate to that of hu­man patients.4,5 The use of companion animals with spontane­ous tumors as models for tumor treatment therapy became a generalized practice due to its many ad­vantages. The most important is that these tumors behave similarly to human ones and are thus bet­ter preclinical models for testing new therapies. As these animals were exposed to environmental carcinogens, they developed the tumors in the con­text of an intact immune system that has the same tumor-host interactions.6,7 A study on melanomas in dogs conducted by Spugnini et al. reported 80% effectiveness.8 Another study by Tamzali et al. on spontaneouslyoccurring tumors showed very high effectiveness when treat­ing sarcoid tumors in equines using ECT with local cisplatin in up to 6 sessions of ECT.9 A ganglioneu­roblastoma case was published in which a cat with a very small tumor was treated with up to 3 ses­sions of ECT in order to obtain an OR.10 In large tumors, however, it is often the case that no OR is possible with a single treatment session.11,12 Systemic bleomycin administration consists of injecting the drug into a vein, thus allowing the drug to reach the tumor through the bloodstream and diffuse from the vessels into the tumor.13 On the other hand, local bleomycin administration consists of directly injecting the drug into the tu­mor tissue, thus allowing it to diffuse from the in­jecting point to the target. Multiple injections into the tumor can provide an adequate coverage in small tumors13, but the case of large tumors is dif­ferent where it is very difficult to homogeneously cover them. Tumor vasculature is structurally and functionally abnormal; blood vessels leak and are tortuous, dilated, and saccular and have a random pattern of interconnection.14 In solid tumors, these aberrant vessels determine an increase in the liquid outlet out of these, together with the contribution of the compression caused by the proliferation of can­cer cells, leading to an increase in interstitial hydro­static pressure.15 The heterogeneous flow of blood and interstitial hypertension pose a serious obstacle to the antineoplastic agents, especially in the case of large tumors with a broader vascular system that are more likely to have areas of tumor that cannot be reached by the systemic route.16,17 This character­istic of tumor vessels could lead to an insufficient bleomycin distribution when administered system­ically. Repeated ECT sessions could lead to modifi­cations in the characteristics of the tumors, such as its size reduction and changes in its vasculature that improve treatment response after each session. For these reasons, performing many treatment sessions can improve the results obtained in the first session, increasing, however, the cost of the treatment and its risks related to multiple anesthetic procedures. To address this problem, here, we propose com­bined bleomycin administration, both systemic and local, using companion animals as models for ECT tumor treating. The aim of this study was to determine whether it is possible to reduce the number of treatment ses­sions using a combined administration of bleomy­cin (both systemic and local) vs. systemic bleomy­cin administration alone in ECT. Accordingly, for the purpose of this work, we selected companion animals with spontaneous tumors. Patients and methods Patients Consent was obtained from the dog’s owner to use the dog’s image in this scientific work and for the treatment of the other patients. In all cases, all recommendations from the Consejo Profesional de Medicos Veterinarios de Buenos Aires (Buenos Aires Veterinary Council) were observed, as well as the relevant local legislation in Argentina, Act No. 14072 which governs veterinary medicine practice. Twenty-two patients from the oncology ser­vice from the Centro de Epecialidades Medicas Veterinarias (CEMV), Buenos Aires, Argentina, were selected. These patients had tumors of a varied histology and had failed to achieve a com­plete response after an ECT treatment session. We divided them into two groups: eleven received combined bleomycin administration in a second treatment session, and 11 underwent another standard ECT session (control group). The first ECT session in both groups and the second ECT session in the control group were performed in ac­cordance with the Standard Operating Procedure for Electrochemotherapy.13 The patients were al­located on a ‘first come, first served’ basis to the control group first, and from the eleventh patient onwards, they were allocated to the combined ad­ministration group. The size of their tumors was calculated by multiplying their two diameters and their height. The patients underwent a full clinical examina­tion, blood samples were taken, and a biopsy for his­tological confirmation of the tumor was performed. The histological analysis of the biopsies was per­formed with hematoxilin-eosin staining. Most pa­tients treated in the first session were expected to require further ECT sessions in order to obtain an objective response because of their tumor size. Treatment procedure General anesthesia procedure consisted of pre-medication with 0.5 mg/kg of xylazine (Xilacina 100®, Richmond, Buenos Aires, Argentina), 2 mg/ kg of tramadol (Tramadol®, John Martin, Buenos Aires, Argentina) and induction with 3 mg/kg of propofol (Propofol Gemepe®, Gemepe, Buenos Aires, Argentina). Then maintenance was assured with 2–3% of isofluorane (Zuflax®, Richmond, Buenos Aires, Argentina) and 2 mcg/kg of fenta­nyl (Fentanilo Gemepe®, Gemepe, Buenos Aires, Argentina). Meloxicam (Meloxicam Denver Farma®, Denver Farma, Munro, Argentina) 0.2 mg/kg was administered for analgesia after the treatment. This scheme of anesthesia provided ad­equate comfort during the treatment. Prophylactic antibiotic amoxicillin/clavulanic acid (Clavamox® Zoetis®, San Isidro, Argentina) 12.5mg/kg/bid was administered. ECT with systemic bleomycin administration alone was performed as follows: the patient was anesthetized using general anesthesia, after an intravenous bolus of bleomycin (Blocamicina®, Gador, Buenos Aires, Argentina) at a dose of 15 000 IU/m2 BSA in 30–45 seconds was adminis­tered. Eight minutes after the intravenous injec­tion, to allow drug distribution, the pulses were delivered covering the whole tumor surface. ECT with systemic and local bleomycin admin­istration was performed as follows: the patient was anesthetized using general anesthesia. An intravenous bolus of bleomycin (Blocamicina®) at a dose of 15 000 IU/m2 BSA in 30–45 seconds was administered, after a local injection of bleomycin (Blocamicina®) at a dose of 125 IU/cm3 of tumor was administered.13 The drug was injected into the tumor using a 27G 2.5 cm needle (Terumo, Tokyo, Japan) in a 3 ml syringe (Darling, Korea), and for an even distribution of the drug, the injections were placed 5 mm apart in one plane and 2 or 3 planes of injections were placed 1 cm apart accord­ing to the size of the tumor. The injections started at the center of the tumor and continued at its pe­riphery.9 Healthy margins were not injected with bleomycin since they are covered by the systemic administration of the drug; there are no vascular abnormalities in healthy tissue to justify the addi­tional administration. The pulses were administered using a six needle electrode, consisting of three rows of two needles 2 cm long and 1 mm diameter, each row separated by 4 mm and each column separated by 8 mm. The pulse generator used was a BTX ECM 830 (Harvard Apparatus, Holliston, MA, USA). A train of 8 elec­tric pulses (1000 V/cm, 100 microseconds, 10 Hz) was applied, covering the whole tumor13, begin­ning at the periphery of the tumor in a circular fashion in order to have maximum drug concen­tration at the margins and prevent the spreading of tumor cells. The superposition of electric fields was avoided in order to prevent overtreatment of the lesions. The response to each treatment was evaluated according to the WHO criteria for tumor response18, 30 days after the treatment. A complete response (CR) is obtained when there is a complete disap­pearance of all known disease, a partial response (PR) when there is a 50% reduction of the tumor or more, a stable disease (SD) when PR or PD criteria are not met, and a progressive disease (PD) when there is a 25% or more increase in the size of the tumor, and no CR, PR or SD is documented before the increase of the disease or new lesions appear. All of this must be confirmed within 4 weeks after the treatment. After the treatment, the patients returned to the veterinary clinic within 7, 15, 21, 30 and 60 days in order to evaluate response, toxicity and side effects by means of a full clinical examination and ques­tions to their owners. Results were compared and statistical signifi­cance was evaluated using the chi square test. Results The total dose of bleomycin in combined treatment was slightly higher than that of systemic adminis­tration alone; in both cases, no toxicity or side ef­fects were reported. Table 1 shows the response of the patients in which combined treatment was performed in the second session. Table 2 shows the control group, for which patients the second ses­sion was a repetition of the first procedure. The responses obtained with combined bleo­mycin administration were significantly different TABLE 1. List of group 1 patients treated using combined systemic and local bleomycin administration in the second treatment session 1 Labrador retriever Oral 32 Mastocytoma II 10.6 PR CR 2 Cross-breed Oral 21 Squamous cell carcinoma II 36.2 SD PR 3 Labrador retriever Nasal 32 Squamous cell carcinoma II 43.5 PR CR 4 Yorkshire Perianal 5 Solid differentiated carcinoma IV 173.8 SD SD 5 Cross-breed Elbow 12 Schwannoma I 67.6 SD PR 6 Rottweiler Oral 37 Fibrosarcoma I 109.5 SD CR 7 Labrador retriever Nasal 38 Squamous cell carcinoma III 42.4 SD PR 8 Boxer Oral 37 Fibrosarcoma III 112.2 SD PR 9 Cocker spaniel Oral 15 Melanoma II 8.7 PR CR 10 Beagle Oral 16 Melanoma III 12.4 PR CR 11 Cocker spaniel Oral 16 Melanoma III 26.64 PR CR CR = complete response; ECT = electrochemotherapy; PR = partial response; SD = stable disease; S+L = systemic + local from those of systemic administration alone in se­lected cases (p < 0.01). In the combined administra­tion group the following response were obtained: CR 54% (6), PR 36% (4), SD 10% (1). In the control group the obtained response were: CR 0% (0), PR 19% (2), SD 63% (7), PD 18% (2). Figure 1 shows a case treated using combined intravenous and in­tratumoral bleomycin administration in which a CR was obtained. The OR rates obtained were significantly better when using combined treatment compared with the standard ECT treatment (p < 0.01). As seen in Figure 2, in the combined group, 91% (10) of OR (CR+PR) were obtained, and 19% (2) were obtained in the control group. It is worth noting that no complete responses were obtained in the control group with two ses­sions of ECT, as opposed to 54% of CR obtained when applying combined treatment in the second session. The average tumor size in the control group was 99.9 cm3, while it was 58.5 cm3 in the combined group. In general, the patients were at a lower stage of the disease in the control group compared with the combined group. Discussion ECT is based on a physical phenomenon, elec­troporation, which acts directly on cell membranes, which accounts for its effectiveness in practically all histological types of tumors. In our experience with veterinary patients, we found that large tu- FIGURE 1. Case number 6. (A) before combined treatment, a fibrosarcoma which failed to respond to the first ECT treatment. (B) CR was obtained after combined treatment. FIGURE 2. Graph shows the objective response rate obtained in the second session, in a comparison between combined bleomycin administration, both systemic and local (S+L), and systemic alone (S Alone). ECT = electrochemotherapy TABLE 2. List of group 2 patients (control) treated using a repetition of the first session 12 Cross-breed Oral 30 Melanoma I 158.2 PR SD 13 Cross-breed Oral 21 Sarcoma III 79.76 PR SD 14 Cross-breed Oral 20 Carcinoma III 96.5 PR SD 15 Toy Poodle Oral 5 Fibrosarcoma II 23.23 PD PD 16 Cross-breed Oral 11 Melanoma II 73.8 PR SD 17 Cross-breed Oral 16 Schwannoma II 467.02 PD SD 18 Cross-breed Oral 6 Squamous cell carcinoma II 12.32 SD SD 19 Labrador retriever Oral 32 Fibrosarcoma II 40 PR SD 20 Rottweiler Oral 34 Melanoma II 33 PR PR 21 German Shepherd Oral 39 Fibrosarcoma II 101.18 PR PD 22 Cross-breed Oral 14 Melanoma II 14.4 PR PR CR = complete response; ECT = electrochemotherapy; PD = progressive disease; PR = partial response; SD = stable disease; S+L = systemic + local mors have poorer responses and require further sessions to obtain an objective response. Our hy­pothesis was that the abnormal vasculature of large tumors impedes proper drug distribution when it is administered intravenously, even though this route of drug administration is prescribed for tu­mors of this size in standard operating procedure (SOP).13 Based on this hypothesis, we decided to make an approach by combining both systemic and lo­cal bleomycin administration to improve drug dis­tribution in the tumor. In this way, local adminis­tration can cover areas where vasculature proves insufficient. There are many reasons against con­sidering using a local injection alone to improve results. According to literature, in tumors above 2 cm in diameter, intravenous administration is rec­ommended.13 It is highly challenging to provide proper drug distribution in the tumor by using local administration only, because during its local application, it is easy to leave sections without the adequate drug concentration, and sometimes it is even impossible to reach the base of the lesion. It is worth mentioning that some authors ob­tained good response rates with several treatment repetitions. These repetitions lead to changes in the tumor that can improve drug distribution in later applications.12,19-22 Here, we obtained good results with only one repetition. Tamzali et al. obtained very good results with a local injection of cisplatin in multiple applications treating sarcoids. It is important to take into ac­count that this kind of tumors behave like benign tumors, thus giving a veterinarian time to perform multiple treatments. Our scenario is different since these kinds of tumors are significantly large, and the survival of the patients is compromised, so we need to reduce the tumor as fast as possible in order to improve their quality of life. Frequently, patients with large tumors are in bad clinical shape, so it is important to reduce the number of treatment ses­sions in order to reduce the risk of anesthetic pro­cedures. On the other hand, costs are also a very important issue, as performing many sessions of treatment increases the cost of the procedure, and makes it rather impossible with our resources. The fact that the tumors in the combined group were smaller could contribute to better responses achieved, but we also have to consider that the stages were higher. Tumor size rather than disease stage is likely to be a better prognostic factor in ECT, but this speculation is yet to be confirmed. Further study is needed in order to determine in difficult cases whether practitioners should firstly try ECT with systemic bleomycin alone, or directly apply ECT with its combined systemic and local administration. Since the dose of bleomycin used is very low, the greatest risk of the ECT procedure lies in the application of anesthesia. Reducing an­esthesia procedures outweighs the risk of adverse reactions related to the accumulated dose of bleo­mycin.23,24 Acknowledgment F. Maglietti holds a fellowship from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), S. Michinski is CPA-CONICET, N. Olaiz and G. Marshall are researchers at CONICET. This work was supported by grants from CONICET (PIP 2012), Universidad de Buenos Aires (UBACyT 2014) and the International European Cooperation in Science and Technology (COST Action TD 1104). The funders had no role in the study, design, da­ta collection and analysis, decision to publish, or preparation of the manuscript. This article was proof read by YasminTranslations.com The paper was presented at the 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine, and Food & Environmental Technologies, September 6 to 10, 2015, Portoroz, Slovenia (wc2015.electroporation. net) organized by COST TD1104 Action (www.elec­troporation.net), supported by COST (European Cooperation in Science and Technology)”. References 1. Yarmush ML, Golberg A, Serša G, Kotnik T, Miklavcic D. Electroporation­based technologies for medicine: principles, applications, and challenges. Annu Rev Biomed Eng 2014; 16: 295-320. 2. Marty M, Sersa G, Garbay J, Gehl J, Collins C, Snoj M, et al. Electrochemotherapy - a simple, highly effective and safe treatment of cutaneous and subcutaneous metastases: results of ESOPE (European Standard Operating Procedures for Electrochemotherapy) study. EJC Suppl 2006; 4: 3-13. 3. Mali B, Jarm T, Snoj M, Sersa G, Miklavcic D. Antitumor effectiveness of electrochemotherapy: a systematic review and meta-analysis. Eur J Surg Oncol 2013; 39: 4-16. 4. Spugnini EP, Baldi F, Mellone P, Feroce F, D’Avino A, Bonetto F, et al. Patterns of tumor response in canine and feline cancer patients treated with electro­chemotherapy: preclinical data for the standardization of this treatment in pets and humans. J Transl Med 2007; 5: 48. 5. Cemazar M, Tamzali Y, Sersa G, Tozon N, Mir LM, Miklavcic D, et al. Electrochemotherapy in veterinary oncology. J Vet Intern Med 2008; 22: 826-31. 6. Spugnini EP, Fanciulli M, Citro G, Baldi A. Preclinical models in electrochemo­therapy: the role of veterinary patients. Future Oncol 2012; 8: 829-37. 7. London CA. Abstract SY28-01: Spontaneous cancer in dogs: Opportunities for preclinical evaluation of novel therapies. Cancer Res 2011; 71: SY28-01. 8. Spugnini EP, Dragonetti E, Vincenzi B, Onori N, Citro G, Baldi A. Pulse-mediated chemotherapy enhances local control and survival in a spontane­ous canine model of primary mucosal melanoma. Melanoma Res 2006; 16: 23-7. 9. Tamzali Y, Borde L, Rols M, Golzio M, Lyazrhi F, Teissie J. Successful treatment of equine sarcoids with cisplatin electrochemotherapy: a retrospective study of 48 cases. Equine Vet J 2012; 44: 214-20. 10. Spugnini EP, Citro G, Dotsinsky I, Mudrov N, Mellone P, Baldi A. Ganglioneuroblastoma in a cat: a rare neoplasm treated with electrochemo­therapy. Vet J 2008; 178: 291-3. 11. Valpione S, Campana LG, Pigozzo J, Chiarion-Sileni V. Consolidation electro­chemotherapy with bleomycin in metastatic melanoma during treatment with dabrafenib. Radiol Oncol 2015; 49: 71-4. 12. Campana LG, Mocellin S, Basso M, Puccetti O, De Salvo GL, Chiarion-Sileni V, et al. Bleomycin-based electrochemotherapy: clinical outcome from a single institutional experience with 52 patients. Ann Surg Oncol 2009; 16: 191-9. 13. Mir LM, Gehl J, Sersa G, Collins CG, Garbay JR, Billard V, et al. Standard operating procedures of the electrochemotherapy: instructions for the use of bleomycin or cisplatin administered either systemically or locally and electric pulses delivered by the Cliniporator TM by means of invasive or non-invasive electrodes. EJC Suppl 2006; 4: 14-25. 14. Jain RK. Normalization of tumor vasculature: an emerging concept in antian­giogenic therapy. Science 2005; 307: 58-62. 15. Padera TP, Stoll BR, Tooredman JB, Capen D, di Tomaso E, Jain RK. Pathology: cancer cells compress intratumour vessels. Nature 2004; 427: 695. 16. Kumar V, Abbas AK, Aster JC. Robbins basic pathology. Elsevier Health Sciences; 2012. 17. Warren BA. The vascular morphology of tumors. In: Peterson HI, editor. Tumor blood circulation: angiogenesis, vascular morphology and blood flow of experimental and human tumors. Boca Raton FL: CRC Press Inc.; 1979. p. 1-47. 18. WHO handbook for reporting results of cancer treatment. Geneva, Switzerland: WHO Offset Publications; 1979; 48: 22-7. 19. Jaroszeski M, Gilbert R, Perrott R, Heller R. Enhanced effects of multiple treatment electrochemotherapy. Melanoma Res 1996; 6: 427-33. 20. Testori A, Tosti G, Martinoli C, Spadola G, Cataldo F, Verrecchia F, et al. Electrochemotherapy for cutaneous and subcutaneous tumor lesions: a novel therapeutic approach. Dermatol Ther 2010; 23: 651-61. 21. Matthiessen LW, Johannesen HH, Hendel HW, Moss T, Kamby C, Gehl J. Electrochemotherapy for large cutaneous recurrence of breast cancer: a phase II clinical trial. Acta Oncol 2012; 51: 713-21. 22. Sersa G, Cufer T, Paulin SM, Cemazar M, Snoj M. Electrochemotherapy of chest wall breast cancer recurrence. Cancer Treat Rev 2012; 38: 379-86. 23. Jules-Elysee K, White D. Bleomycin-induced pulmonary toxicity. Clin Chest Med 1990; 11: 1-20. 24. Cohen IS, Mosher MB, O’Keefe EJ, Klaus SN, De Conti RC. Cutaneous toxicity of bleomycin therapy. Arch Dermatol 1973; 107: 553-5. review Medical physics in Europe following recommendations of the International Atomic Energy Agency Bozidar Casar1, Maria do Carmo Lopes2, Advan Drljević3, Eduard Gershkevitsh4, Csilla Pesznyak5 1 Institute of Oncology Ljubljana, Slovenia 2 Portuguese Institute of Oncology Coimbra, Portugal 3 University Clinical Centre Sarajevo, Bosnia and Herzegovina 4 North Estonia Medical Centre, Tallinn, Estonia 5 BME, Institute of Nuclear Techniques, Budapest, Hungary Radiol Oncol 2016; 50(1): 64-72. Received 2 October 2015 Accepted 17 October 2015 Disclosure: No potential conflicts of interested were disclosed. Correspondence to: Bozidar Casar, MPE, Head of Radiophysics Department, Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. Phone: +386 1 5879 516; Fax: +386 1 5879 416; E-mail: bcasar@onko-i.si Background. Medical physics is a health profession where principles of applied physics are mostly directed towards the application of ionizing radiation in medicine. The key role of the medical physics expert in safe and effective use of ionizing radiation in medicine was widely recognized in recent European reference documents like the European Union Council Directive 2013/59/EURATOM (2014), and European Commission Radiation Protection No. 174, European Guidelines on Medical Physics Expert (2014). Also the International Atomic Energy Agency (IAEA) has been outspoken in supporting and fostering the status of medical physics in radiation medicine through multiple initiatives as technical and cooperation projects and important documents like IAEA Human Health Series No. 25, Roles and Responsibilities, and Education and Training Requirements for Clinically Qualified Medical Physicists (2013) and the International Basic Safety Standards, General Safety Requirements Part 3 (2014). The significance of these documents and the recogni­tion of the present insufficient fulfilment of the requirements and recommendations in many European countries have led the IAEA to organize in 2015 the Regional Meeting on Medical Physics in Europe, where major issues in medical physics in Europe were discussed. Most important outcomes of the meeting were the recommendations addressed to European member states and the survey on medical physics status in Europe conducted by the IAEA and European Federation of Organizations for Medical Physics. Conclusions. Published recommendations of IAEA Regional Meeting on Medical Physics in Europe shall be followed and enforced in all European states. Appropriate qualification framework including education, clinical specialization, certification and registration of medical physicists shall be established and international recommendation regarding staffing levels in the field of medical physics shall be fulfilled in particular. European states have clear legal and moral responsibility to effectively transpose Basic Safety Standards into national legislation in order to ensure high quality and safety in patient healthcare. Key words: medical physics; Europe; International Atomic Energy Agency; recommendations; basic safety standards Introduction Medical physics is a dynamic and constantly growing field of applied physics mainly directed towards the application of physics principle to health care in order to ensure safety and quality in diagnostic and therapeutic procedures involv­ing the application of ionizing radiation. Medical physics traditionally covers four main areas of ap­plied physics in medicine: 1. Diagnostic and interventional radiology physics 2. Radiation oncology/radiotherapy physics 3. Nuclear medicine physics 4. Radiation protection physics sometimes referred also as health physics Within these four subspecialties, medical physi­cists are involved in four basic activities: clinical service, research and development, teaching and management/administration. Although mentioned specialties of medical physics cover almost com­pletely the area of medical physics profession, med­ical physicists are and where appropriate should be involved in other applications of physics in medi­cine as well, such as ultrasound imaging, magnetic resonance imaging, bioelectrical investigation of the brain and heart (electroencephalography and elec­trocardiography), bio magnetic investigation of the brain (magneto encephalography) applications of lasers in medicine and medical informatics.1 Within the present discussion we limit ourselves to the ap­plication of ionizing radiation to medicine. Over hundred years ago three major events opened doors of medicine to applied radiation physics: discovery of x rays by Wilhelm Conrad Roentgen in 1895, discovery of natural radioactiv­ity by Henry Becquerel in 1896 and discovery of ra­dium by Pierre and Marie Curie in 1898, followed in 1934 by the discovery of artificial radioactivity by Irene Curie and Frederic Joliot, resulting from the creation of short-lived radioisotopes from the bombardment of stable nuclides and the advances in radar and radiofrequency technology during World War II that made linear accelerators devel­opment possible. Since then physics has started to play an important role in medicine for routine use of ionizing radiation in medical diagnostic and therapy. Over the last few decades we were witnessed of enormous development of radiation medicine, mainly through the technological devel­opment of the equipment which is used for accu­rate diagnostic or therapeutic procedures: optimi­zation of image quality for computed tomography and magnetic resonance imaging, development of radiation therapy equipment (high energy linear accelerators with sophisticated options for dose de­livery, computerized treatment planning systems, record and verify systems, etc.) and overall inte­gration of computers into the routine clinical work. This is reflected in the huge increase of medical ra­diological procedures in the world; presently there are around 4 billion x-ray examinations, 35 million nuclear medicine examinations and 5 million ra­diotherapy courses undertaken annually. Such tremendous development has triggered demands and need for more highly educated and well trained medical physicists. Introduction of formal systems for education and clinical training became crucial and many universities in Europe offer academic programmes in medical physics. However, there is still a lack of accredited clinical training programs in the majority of countries in Europe. Although international and European pro­fessional medical physics organizations together with European Commission (EC) and International Atomic Energy Agency (IAEA) have undertook ef­forts to raise awareness of national authorities across Europe regarding the role and the importance of medical physics in radiation medicine it seems, that these efforts have not been fully successful. There is still no harmonization and full recognition of medi­cal physics profession in Europe, there is still short­age of well-educated and clinically trained medical physicists, there are still lack of educational frame­works and structured clinical training programmes in several European countries, there are difficulties to implement continuous professional develop­ment (CPD) systems and unfortunately, there are still reports and news about incidents and accidents in the field of radiation medicine.2-8 This review has no intention to cover current status of medical physics in Europe, neither has the ambition to discuss medical physics history or its future perspectives and importance in radiation medicine. The subject is far too broad and complex and it is described and discussed in depth in gen­eral medical physics textbooks and international literature.9-11 The main purpose of this paper is to present com­ments on most recent recommendations from the IAEA after the “Regional Meeting on Medical Physics in Europe: Current Status and Future Perspectives” held in Vienna from 7th to 8th May 2015. Invited rep­resentatives - over 60 from more than 30 European countries - from World Health Organization (WHO), international professional organiza­tions and societies (International Organisation for Medical Physics – IOMP, European federation of Organisations for Medical Physics – EFOMP and European Society for Radiotherapy and Oncology – ESTRO) , national regulatory bodies and Health Ministries and representatives of medical physi­cists, were discussing the current status and future perspectives of medical physics in Europe.12 The recommendations of the IAEA Regional Meeting, serving as an outcome of the meeting, are presented in original form and are the bases of the paper, while notes and observation from the same document were omitted due to the journal space considerations.13 Issues related to medical physics in the European countries Di.culties to .nd funding to attend CPD activities Lack of structured clinical training Shortage of Medical Physicists Inability to participate in the management/decision making process Lack of recognition at national level Lack of educational framework Unattractiveness of the career Inability of professional growth Di.culties in implementing new Basic Safety Standards Other Problems with inter -professional communication 0 10 20 30 40 50 60 70 80 90 % of responses FIGURE 1. Issues/difficulties in medical physics identified in IAEA/EFOMP survey in 2015 (Damilakis J, Lopes M. C. Overview of medical physics status and future prospects: Results of survey in Europe. “Regional meeting on Medical Physics in Europe: Current Status and Future Perspectives”, IAEA 7th -8th May 2015). Survey has revealed pronounced problems in several issues: difficulties to find funding to attend continuous professional development (CPD) activities, lack of structured clinical training, shortage of medical physicists, inability to participate in the management/decision making process, lack of recognition and lack of educational framework, were appointed by more than 50% of the 32 respondent countries to be felt problems concerning medical physics. For each of seven IAEA recommendations, it has been tried to find justifications for and rationales behind the recommendations as well as to limited extent also legislative backgrounds, mostly within recently published international documents and basic safety standards (BSS) – European Union Council Directive 2013/59/EURATOM (EU BSS Directive), European Commission RP 174 European Guidelines for Medical Physics Experts (EC RP 174), IAEA Radiation Protection and Safety of Radiation Sources: International Basic Safety Standards (IAEA IBSS) and IAEA Human Health Series No. 25, Roles and Responsibilities, and Education and Training Requirements for Clinically Qualified Medical Physicists (IAEA HHS 25).14-17 Although the cita­tions from various documents are presented only fragmentally, they provide sufficient information about the solid background of presented recom­mendations of the IAEA Regional Meeting regard­ing the medical physics profession in the Europe region. Recommendations have additional basis in the convincing and unambiguous results of the survey on medical physics status in Europe conducted by the IAEA and EFOMP in 2015 (Figure 1).18 Recommendations of the IAEA Regional Meeting on medical physics in Europe “Recalling the provisions of “Radiation Protection and Safety of Radiation Sources: International Basic Safety Standards” (General Safety Requirements Part 3, IAEA 2014) regarding the role of medical physicists in ensuring safety in diagnostic and therapeutic procedures involving application of ionizing radiation, the Meeting recommended that Member States of the Europe Region should fully recognize Clinically Qualified Medical PhysicistA (CQMP) as a health professional with specialist education and training in the concepts and tech­niques of applying physics in medicine and com­petent to practice independently in one or more of the subfields (specialties) of medical physics The Meeting also recommended that Member States of the Europe Region should, in particular: 1. Recognize medical physics as an independent profession in health care with radiation protec­tion responsibilities, as given in the “Joint posi­tion statement by the IAEA and WHO – Bonn call for action”; 2. Ensure that medical physics aspects of thera­peutic and diagnostic procedures, including patient and equipment related tasks and activi­ties are performed by CQMPs or under their supervision; 3. Establish the appropriate qualification frame­work for CQMPs including education, special­ized clinical training, certification, registration A The term “clinically qualified medical physicsts” was defined in Roles and responsibilities and Education and training Requirements for Clinically Qualified Medical Physicists, IAEA Human Health Series No. 25, IAEA 2013 corresponds to “qualified expert in medical physics” defined in the IAEA International Basic Safety Standards and the “medical physics expert” defined in the European Council Directive 2013/59/EURATOM and continuing professional development in the specializations of medical physics, i.e. diag­nostic and interventional radiology, radiation oncology and nuclear medicine; 4. Follow and fulfil international recommenda­tions regarding the staffing levels in the field of medical physics; 5. Establish mechanisms for medical physics ser­vices integration in all centres practicing radia­tion medicine, and establish, where appropri­ate, independent medical physics departments in which accredited clinical training can take place; 6. Promote involvement of CQMPs in hospital governance boards and relevant national health committees; 7. Establish and enforce the legislative and regu­latory requirements related to radiation safety in medical imaging and therapy where medi­cal physics is concerned, in accordance with the international and, where applicable, European basic safety standards.” Recognition of medical physics as independent health profession In 2012 the IAEA, co-sponsored by WHO, held the “International Conference on Radiation Protection in Medicine: Setting the Scene for the Next Decade” in Bonn, Germany. The specific outcome of this conference was the published document “Joint po­sition statement by the IAEA and WHO – Bonn call for action”19, where some actions were identified as being essential for the strengthening of radia­tion protection in medicine over the next decade. Regarding the strengthening of radiation safety culture in health care, Action 8f: says the follow­ing: “Work towards recognition of medical physics as an independent profession in health care, with radiation protection responsibilities”. Furthermore in the IAEA IBSS16, medical physi­cist is defined as “A health professional with specialist education and training in the concepts and techniques of applying physics in medicine and competent to practice independently in one or more of the subfields (special­ties) of medical physics”. Through the committed efforts of the IOMP and other organizations, medical physicists have been included for the first time, in 2008 in “The inter­national Standard Classification of Occupations (ISCO.08)”.20,21 Medical physicists are classified un­der the group 2111, “Physicists and Astronomers” but 5 out of 11 enumerated tasks concern explicitly medical physicists.B There is also an explicit note of 2111 group stating “… medical physicists are considered to be an integral part of the health work force alongside those occupations classified in sub-major group 22, Health professionals”. On the other hand, under the group 22 of “Health professionals” also a specific note is included say­ing that “it should be noted that a number of professions considered to be a part of the health work force are clas­sified in groups other than sub-major group 22, Health professionals. Such occupations include but are not re­stricted to: addictions counsellors, biomedical engineers, clinical psychologists and medical physicists.” Mentioned statements and definitions from quoted documents give unambiguous justification for the first recommendation of the IAEA Regional Meeting. The recognition of medical physicists as a health profession is of paramount importance and should be reflected at the national level (list of rec­ognized professions, legal and fiscal environment, involvement in hospital governance etc.). Roles and responsibilities of medical physics experts Recommendation No. 2 clearly emphasizes the role and responsibilities of medical physics expert (MPE) in the fields of medical diagnostic and thera­peutic procedures. In the new EU BSS Directive14 from 2013, MPE is mentioned in 9 articles, while in the former EU BSS Directive22 from 1997, MPE was mentioned only in 2 articles. New EU BSS Directive14 thus recognizes the importance and growing role of medical physics profession in Europe. In Article 83 of the directive definitions are found of roles and responsibilities of MPE which are required to be implemented by the EU Member states: “Member States shall ensure that depending on the medical radiological practice, the medical physics expert takes responsibility for dosimetry, including physical measurements for evaluation of the dose delivered to the patient and other individuals subject to medical expo­sure, give advice on medical radiological equipment, and contribute in particular to the following: B “(e) ensuring the safe and effective delivery of radiation (ionising and non­ ionising) to patients to achieve a diagnostic or therapeutic result as pre­ scribed by a medical practitioner; (f) ensuring the accurate measurement and characterization of physical quantities used in medical applications; (g) testing, commissioning and evaluating equipment used in applications such as imaging, medical treatment and dosimetry; (h) advising and con­sulting with medical practitioners and other health care professionals in optimizing the balance between the beneficial and deleterious effects of radiation; … (j) developing, implementing and maintaining standards and protocols for the measurement of physical phenomena and for the use of nuclear technology in industrial and medical applications;”… (a) optimisation of the radiation protection of pa­tients and other individuals subject to medical exposure, including the application and use of diagnostic reference levels; (b) the definition and performance of quality assur­ance of the medical radiological equipment; (c) acceptance testing of medical radiological equip­ment; (d) the preparation of technical specifications for medical radiological equipment and installation design; (e) the surveillance of the medical radiological instal­lations; (f) the analysis of events involving, or potentially involving, accidental or unintended medical exposures; (g) the selection of equipment required to perform ra­diation protection measurements; (h) the training of practitioners and other staff in rel­evant aspects of radiation protection;” It is evident that the tasks described in the EU BSS Directive14 impose indispensable role and re­sponsibility of medical physics experts and can on­ly be performed by experienced medical physicists with high level of competence. Article 79 of the di­rective specifically requires from member states to ensure arrangements for the recognition of medical physics experts. One of the most important requirements from the new EU BSS Directive14 is that MPE shall be in­volved in all three major clinical fields of radiation medicine: radiotherapy, nuclear medicine and di­agnostic and interventional radiology. The document IAEA HHS 2517, published by IAEA in 2013 and endorsed by IOMP and American Association of Physicists in Medicine (AAPM), de­fines appropriately and unequivocally the roles and responsibilities of CQMP in the different specialties of medical physics and recommends minimum re­quirements for their academic education and clini­cal training, including recommendations for their accreditation, certification and registration, along with continuing professional development. The main goal of all these documents and rec­ommendations is to establish criteria that support the harmonization of education and clinical train­ing, as well as to promote the recognition of medi­cal physics as a health profession. Establishment of the appropriate qualification framework European commission has recently published guidelines for medical physics expert – EC RP 174.15 In this document detailed qualification framework (QF) for MPE in Europe is presented and discussed. QF for medical physicists in Europe should be referred to the European Qualification Framework (EQF) for lifelong learning, laid down by the European parliament and council of the European Union with learning outcomes ex­pressed as inventories of Knowledge, Skills and Competences (KSC).23 Education and clinical train­ing requirements for medical physicists are dis­cussed comprehensively in the IAEA HHS 25.17 In depth description and guidance on clinical train­ing of medical physicists specializing in radiation oncology, diagnostic and interventional radiology and nuclear medicine can be found in the IAEA Training Course Series.24-26 Education and training of medical physicist in Europe is also covered in the EFOMP Policy Statement No. 12.27 According to these documents, appropriate QF for medical physicists should consist of adequate education, accredited clinical training in hospitals and CPD programmes in place. In the IAEA IBSS16 similar accent is given al­ready within the definition: “medical physicists is a health professional with specialist education and train­ing in the concepts and techniques of applying physics in medicine and competent to practise independently in one or more of the subfields (specialties) of medical physics.” and further defines that “qualified expert is an individual who, by virtue of certification by ap­propriate boards or societies, professional licence or aca­demic qualifications and experience, is duly recognized as having expertise in a relevant field of specialization, e.g. medical physics, radiation protection, occupational health, fire safety, quality management or any relevant engineering or safety specialty.” From the definitions in IAEA IBSS16 it can be deducted that qualified expert in medical physics (i.e. MPE) is a health professional having officially recognized specialization in one or more fields of medical physics. Regarding the subject discussed in this section, new EU BSS Directive14 requires from European Union member states in Article 14, point 2 the fol­lowing: “Member States shall ensure that arrangements are made for the establishment of education, training and retraining to allow the recognition of radiation protec­tion experts and medical physics experts, as well as oc­cupational health services and dosimetry services, in relation to the type of practice.” Also EC RP 17415 pre­sents as the first of the seven final recommendations that “Each Member State should consider designating, through a legal instrument, a Competent Authority spe­cifically for the recognition of the MPE”. And recom­mendation No. 3 of EC RP 17415 clearly links recog­nition to a proper qualification framework as stated: “The Competent Authority designated for the recognition of the MPE, should use the Qualifications Framework and KSC of the MPE specified in the present document, for the recognition of the MPE to Level 8 of the EQF.” Within this frame, recognition of MPE encom­passes also certification and registration of an in­dividual professional. Certification is the formal process by which an authorized body evaluates and recognizes the knowledge and proficiency of an individual, which must satisfy pre-determined requirements or criteria. The process must thus always be based on a proper qualification frame­work involving both education and clinical train­ing. Professional certification of medical physi­cists should be formally conducted by competent national boards - designated governmental body or alternatively national medical physics organi­zation authorized by the government. In either case, members of such boards shall be predomi­nantly senior MPEs in order to ensure competency in assessment and decision making procedures. The process of certification should be followed by formal registration of medical physics profession­als and the register should be operated at the na­tional level by an official authority (e.g. Ministries of health) or professional medical physics society/ organization if an official authorization is given by the government. Re-certification system should be established as well in order to maintain high level of proficiency of medical physics experts (EQF lev­el 8). This is usually achieved via formal CPD pro­gramme which should ensure up to date KSC of an individual professional. It is evident that without appropriate education, clinical training and CPD system, it cannot be expected medical physics ser­vice to play effective role in radiation medicine. However, in order to have a transparent system of certification and re-certification for MPEs, it needs to be consistent with the certification and recogni­tion system of other health professionals/special­ists (physicians, dentists) and Ministries of health have to play a key role in this process. Situations where a formal QF system is not estab­lished yet, are mentioned in IAEA IBSS16 (footnote under definition of medical physicist): “Competence of persons is normally assessed by the State by having a formal mechanism for registration, accreditation or certification of medical physicists in the various spe­cialties (e.g. diagnostic radiology, radiation therapy, nuclear medicine). States that have yet to develop such a mechanism would need to assess the education, train­ing and competence of any individual proposed by the licensee to act as a medical physicist and to decide, on the basis of either international accreditation standards or standards of a State where such an accreditation sys­tem exists, whether such an individual could undertake the functions of a medical physicist, within the required specialty.” In countries where the desirable qualification system is not (completely) implemented yet, ad­equate mechanisms for transition period should be established in order to recognize and certify expe­rienced professionals who have been already con­tinuously employed in the field of medical physics for a specific period.13 In such cases, the certifica­tion through an international or European instance may be a solution. In this concern EFOMP has given recognized steps to fostering education and train­ing on a European level, encouraging the establish­ment of national training centres, networking and cooperative actions within European projects (e.g. EUTEMPE.RX) that may be taken as facilitators to­wards European certification process.28 Moreover, senior professionals who have been working for a longer period on active duty as medical physicists and are in possession of the core KSC of medical physics should be deemed to sat­isfy the requirements for recognition as an MPE. For these professionals a “grandparenting clause” might and shall be applied and they should be recognized/certified by competent authorization board as MPEs and not required to meet new leg­islative, educational or training (specialization) re­quirements. Staffing levels in the field of medical physics Fulfilment of the recommendation regarding the staffing levels in the field of medical physics is of major importance if high quality radiation health care service is to be ensured and the risk of radio­logical incidents and accidents reduced. Among many reports about incidents/accidents published within the last two decades, several of them can be attributed to shortage of experienced medi­cal physicists.3,4 Many national and international recommendations and other publications regard­ing the staffing levels in medical physics were published in the past.29-35 Most recent documents about staffing levels for all subspecialties of medi­cal physics have been published as Annex 2 of EC RP 17415 and Staffing in Radiotherapy: An Activity Based Approach IAEA Human Health Reports No. 13 (2015).36 Despite all recommendations, there is still un­acceptable understaffing in the field of medical physics in many European countries.18 Call for ac­tion is addressed to the national authorities (e.g. Ministries of health) and hospital’s management to incorporate recommendations regarding medi­cal physics staffing levels into national legislations and standards in close cooperation with profes­sional societies and organizations. Insufficient number of qualified and competent medical physi­cists – MPEs - will result in lower level of health care, even if requests, recommendations and stand­ards from EU BSS Directive14 and IAEA IBSS16 will be formally transposed into national legislations. Independent medical physics departments EU BSS Directive14 defines MPE as: “medical phys­ics expert means an individual or, if provided for in national legislation, a group of individuals, having the knowledge, training and experience to act or give ad­vice on matters relating to radiation physics applied to medical exposure, whose competence in this respect is recognised by the competent authority.” In this con­text “group of individuals” clearly means group of medical physics professionals (e.g. medical physics departments) with appropriate knowledge, skills and competencies in relevant medical physics spe­cialization fields. It seems reasonable that medical physics service is governed by the size, type and specific needs of the medical facility. In large hospitals medical physicists are often organized into an autonomous medical physics department which provides ser­vices to the various clinical departments e.g. di­agnostic and interventional radiology, radiation oncology/radiotherapy and nuclear medicine.17 If at least two major medical physics subspecial­ties are required for clinical work in hospitals, autonomous and independent medical physics departments shall be established as appropriate with well-defined safety and quality management system.37 In many large European hospitals inde­pendent medical physics departments have been already established. Examples from developed countries are Institute Gustave Roussy in Paris and Royal Marsden Hospital in London and from less developed countries University Clinical Centre in Sarajevo, which offer services to various clinical departments. Such medical physics departments should competently cover also the field of radia­tion protection as the fourth major specialty where medical physicists have clear responsibilities, roles and competency. The added value of a medical physics de­partments is multiple folded and can be shown through clinical and economic indicators in terms of efficiency and profitability, services quality, improved patient safety and patient satisfaction, increased patient throughput, improved commu­nication and moral of professionals and reduce costs and liabilities. Accredited clinical training for medical physicists and other health professionals (clinicians, technologists, and nurses) is also pro­moted through such organizational structures that may be constituted as accredited clinical training centres by competent authorities. Importance of in­tegrated medical physics departments was recog­nized by EFOMP already more than two decades ago in EFOMP Policy No. 5.38 Involvement of MPEs in hospital governance boards EFOMP has recently published Policy statement no. 15, where guidelines on the role of the medi­cal physicist within the hospital governance board are laid down.39 Explicit recommendation is given regarding the involvement of medical physicists in hospital governance board: “EFOMP recommends that National Member Organisations encourage their Medical Physicists to be closely involved in hospital governance and, where this has not already happened, to seek membership of their hospital’s governance boards and its committees, emphasising the importance of such membership for the good of the patients and the hospital as a whole.” Involvement of medical physicists in the hos­pital governance is presently very limited across Europe and often they are not officially included in management and decision making processes (Figure 1). We have entered the era of fragile and sensi­tive economy with constantly growing demands for higher quality and safer health care system es­pecially in the field of radiation medicine, where medical physicists are and should be strongly in­volved. The work of medical physicists in hospitals goes far beyond routine clinical and research tasks and reach demanding fields from radiation protec­tion of patients, personnel and general public to the selection of expensive and complex equipment used in radiation medicine. Recalling the roles and responsibilities of MPE as defined and requested in the EU BSS Directive14, it is clear that all mentioned tasks cannot possibly be fulfilled, if MPE is not of­ficially involved in the policy and decision making processes in the hospitals. Legislative and regulativerequirements Throughout this paper the two most important recently published documents were quoted sev­eral times: EU BSS Directive14 and IAEA IBSS.16 In the foreword of the second document, the IAEA Director General Yukiya Amano among other said: “Standards are only effective if they are properly applied in practice.” And continued: “Regulating safety is a national responsibility, and many States have decided to adopt the IAEA’s standards for use in their national regulations. For parties to the various international safety conventions, IAEA standards provide a consist­ent, reliable means of ensuring the effective fulfilment of obligations under the conventions. The standards are also applied by regulatory bodies and operators around the world to enhance safety in nuclear power generation and in nuclear applications in medicine, industry, agri­culture and research.” Any standard, if it is not implemented into na­tional legislations and regulations, followed by a committed introduction into the clinical work, have a limited value. IAEA IBSS are important and extremely well prepared official recommendations from distinguished authority; however, adoption of these standards is, as said by Director General, a national responsibility. It is even binding for those IAEA Member States who are involved in Technical Cooperation (TC) activities with the IAEA. EU BSS Directive14 on the other hand is legally binding. In Article 106 the obligations for European Union member states are clearly stated: “Member States shall bring into force the laws, regulations and administrative provisions necessary to com­ply with this Directive by 6 February 2018.” The two above mentioned documents require from national authorities to transpose written standards and recommendations into local legisla­tion. Concerning IAEA IBSS16 national authorities have at least moral obligation to follow and imple­ment recommended safety standards in order to optimize medical diagnosis and treatment of hu­man diseases and to improve human health and well-being. Regarding the EU BSS Directive14, there is a clear and firm legal obligation and responsi­bility for all European Union countries to adopt national legislation in order to comply with the re­quirements of the directive. Conclusions Work and devotion of medical physicists was nicely described by the esteemed medical physicist Prof. Ervin B. Podgorsak in his speech after accept­ance of Coolidge award in 2006: “A healthy man has a thousand wishes, a sick man has only one. Most of the work of medical physicists is indirectly related to people who have only one wish. We must not forget that, despite our scientific and technical training, our strong­est guiding attributes must be compassion for pa­tients and discipline toward our work.” Call for action is addressed to the national au­thorities, ministries of health and hospitals, to implement the latest international recommenda­tions discussed in this paper without hesitation, completely, with great care and empathy in close cooperation with professional bodies, societies and organizations; it is their moral and legal responsi­bility. National authorities shall follow this road, above all for the benefit of millions of patients throughout the Europe and all over the world, oth­erwise “compassion for patients and discipline toward our work” might soon become an insufficient driv­ing force. Acknowledgments Authors were members of the IAEA working core group for preparation of the “Regional meeting on Medical Physics in Europe: Current Status and Future Perspectives” held in Vienna from 7th to 8th May 2015. The work of this group was supported by the IAEA technical cooperation project “RER/6/031 Strengthening Medical Physics in Radiation Medicine” and authors express sincere thanks to the IAEA. Thanks go to the IAEA staff Mr. Ivan Videnović and Tomislav Bokulić for valuable comments dur­ing the preparation of the meeting and above all to Ms. Joanna Izewska for leading our working group and for her constant support and devotion to the medical physics profession over many years. References 1. Podgorsak EB. 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Unintended exposure in radiotherapy: identification of prominent causes. Radiother Oncol 2009; 93: 609-17. 8. Derreumaux S, Etard C, Huet C, Trompier F, Clairand I, Bottollier-Depois JF, et al. Lessons from recent accidents in radiation therapy in France. Radiat Prot Dosimetry 2008; 131: 130-5. 9. Webb S. The contribution, history, impact and future physics in medicine. Acta Oncol 2009; 48: 169-77. 10. Keevil SF. Physics and medicine: a historical perspective. Lancet 2012; 379: 1517-24. 11. Jeraj R. Future of physics in medicine and biology. Acta Oncol 2009; 48: 178-84. 12. Izewska J. Summary of the IAEA “Regional Meeting on Medical Physics in Europe: Current Status and Future Perspectives”. Med Phys Internat J 2015; 3: 33-4. 13. IAEA. Recommendation of the Regional Meeting on Medical Physics in Europe: Current Status and Future Perspectives. 7-8 May 2015, IAEA, Vienna, Austria. [Citated 15 Sept 2015)]. Available at: https://rpop.iaea. org/RPOP/RPoP/Content/Documents/Whitepapers/Recommendations_ RER6031_7-8May2015.pdfhttps://rpop.iaea.org /RPOP/RPoP/Content/ Documents/Whitepapers/Recommendations_RER6031_7-8May2015.pdf 14. Council of the European Union. (2013). Council Directive 2013/59/Euratom laying down basic safety standards for protection against the dangers aris­ing from exposure to ionising radiation, and repealing Directives 89/618/ Euratom, 90/641/Euratom, 96/29/Euratom, 97/43/Euratom and 2003/122/ Euratom. Official Journal L-13 of 17.01.2014. 15. European Commission. European Guidelines on Medical Physics Experts. Radiation Protection No 174; 2014. 16. International Atomic Energy Agency. Radiation Protection and Safety of Radiation Sources: International Basic Safety Standards, General Safety Requirements Part 3. Wienna: IAEA; 2014. 17. IAEA. Roles and Responsibilities and Education and Training Requirements for Clinically Qualified Medical Physicsts. IAEA Human Health Series No. 25. Vienna: IAEA; 2013. 18. Damilakis J, Do Carmo Lopes M. Overview of medical physics status and fu­ture prospects: results of survey in Europe. In: Regional meeting on Medical Physics in Europe: Current Status and Future Perspectives”. Wienna, 7th–8th May 2015. Wienna: IAEA; 2015. 19. IAEA , WHO. Bonn call-for action. Joint position statement by the IAEA, WHO. [Citated 16 Sept 2015)]. Available at: http://www.who.int/ioniz­ing_radiation/medical_exposure/Bonn_call_action.pdf 20. International Standard Classification of Occupations. ISCO O8, Vol I. Geneva: International Labour Organization; 2012. 21. Smith PHS, Nusslin F. Benefits to medical physics from the recent inclusion of medical physicists in the international classification of standard occupa­tions. ISCO 08. Med Phys Internat J 2014; 1: 10-14. 22. Council of the European Union. Council Directive 97/43/Euratom of 30 June 1997 on health protection of individuals against the dangers of ionizing radiation in relation to medical exposure, and repealing Directive 84/466/ EURATOM. Official Journal L-180 of 09. 07. 1997. 23. European Parliament and Council of the European Union. Recommendation 2008/C 111/01 on the establishment of the European Qualifications Framework for Lifelong Learning. Official Journal of the European Union 6. 5. 2008. 24. International Atomic Energy Agency. Clinical training of medical physicists specializing in radiation oncology. Training Course Series 37. Vienna: IAEA; 2010. 25. International Atomic Energy Agency. Clinical Training of Medical Physicists Specializing in Diagnostic Radiology. Training Course Series 47. Vienna: IAEA; 2010. 26. International Atomic Energy Agency. Clinical Training of Medical Physicists Specializing in Nuclear Medicine. Training Course Series 50. Vienna: IAEA; 2010. 27. Eudaldo T, Olsen K. European Federation of Organisations for Medical Physics policy statement no.12: The present status of medical physics edu­cation and training in Europe. New perspectives and EFOMP recommenda­tions. Phys Med 2010; 26: 1-5. 28. EUTEMPE.RX, European training and education for medical physics experts in radiology. [Citated 18 Sept 2015)]. Available at: http://www.eutempe-rx. eu/ 29. European Federation of Organisations for Medical Physics. Policy statement no. 7: criteria for staffing levels in a medical physics department. Phys Med 1997; 13: 187-94. 30. International Atomic Energy Agency. Setting up a radiotherapy programme: clinical, medical physics, radiation protection and safety aspects. Vienna: IAEA; 2008. 31. Institute of Physics and Engineering in Medicine. Recommendations for the Provision of a Physics Service to Radiotherapy. IPEM 2009; York, UK. 32. SSRMP. Medical physicist staffing for nuclear medicine and dose-intensive X-ray procedures. Schweizerische Gesellschaft für Strahlenbiologie und Medizinische Physik. Report No. 20. 2009. 33. Klein EE. A grid to facilitate physics staffing justification. J Appl Clin Med Phys 2010; 11: 263-73. 34. Battista JJ, Clark BG, Patterson MS, Beaulieu L, Sharpe MB, Schreiner LJ, et al. Medical physics staffing for radiation oncology: a decade of experience in Ontario, Canada. Can J Appl Clin Med Phys 2012; 13: 93-110. 35. Lievens Y, Defourny N, Coffey M, Borras JM, Dunscombe P, Slotman B, et al. Radiotherapy staffing in the European countries: final results from the ESTRO-HERO survey. Radioth Oncol 2014; 112: 178-86. 36. International Atomic Energy Agency. Staffing in radiotherapy: an activity based approach IAEA. Human Health Reports No. 13; Vienna: IAEA; 2015. [Citated 19 Sept 2015]. Available at: http://www-pub.iaea.org/books/ IAEABooks/10800/Staffing-in-Radiotherapy-An-Activity-Based-Approach 37. Christofides S, European Federation of Organisations for Medical Physics. 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Phys Med 2015; 31: 201-3. research article Diagnostic accuracy of MRI to evaluate tumour response and residual tumour size after neoadjuvant chemotherapy in breast cancer patients Alberto Bouzón1, Benigno Acea1, Rafaela Soler2, Ángela Iglesias2, Paz Santiago3, Joaquín Mosquera2, Lourdes Calvo4, Teresa Seoane-Pillado5, Alejandra García1 1 Department of Surgery; Breast Unit. Complexo Hospitalario Universitario de A Coruna Sergas, Spain 2 Department of Radiology, Breast Unit. Complexo Hospitalario Universitario de A Coruna Sergas, Spain 3 Department of Anatomic Pathology, Breast Unit. Complexo Hospitalario Universitario de A Coruna Sergas, Spain 4 Department of Clinical Oncology, Breast Unit. Complexo Hospitalario Universitario de A Coruna Sergas, Spain 5 Clinical Epidemiology and Biostatistics Unit, Breast Unit. Complexo Hospitalario Universitario de A Coruna Sergas, Spain Radiol Oncol 2016; 50(1): 73-79. Received 30 September 2015 Accepted 30 December 2015 Correspondence to: Alberto Bouzón, M.D., Department of Surgery, Breast Unit, Hospital Abente y Lago, Rd: Gral Sir John Moore, no1, A Coruna, Spain. Phone: +34 690103810; Email: dr.alberto@aecirujanos.es Disclosure: No potential conflicts of interest were disclosed. Background. The aim, of the study was to estimate the accuracy of magnetic resonance imaging (MRI) in assessing residual disease in breast cancer patients receiving neoadjuvant chemotherapy (NAC) and to identify the clinico­pathological factors that affect the diagnostic accuracy of breast MRI to determine residual tumour size following NAC. Patients and methods. 91 breast cancer patients undergoing NAC (92 breast lesions) were included in the study. Breast MRI was performed at baseline and after completion of NAC. Treatment response was evaluated by MRI and histopathological examination to investigate the ability of MRI to predict tumour response. Residual tumour size was measured on post-treatment MRI and compared with pathology in 89 lesions. Clinicopathological factors were ana­lyzed to compare MRI-pathologic size differences. Results. The overall sensitivity, specificity, positive predictive value, negative predictive value, and accuracy for diagnosing invasive residual disease by using MRI were 75.00%, 78.57%, 88.89%, 57.89%, and 76.09% respectively. The Pearson´s correlation coefficient (r) between tumour sizes determined by MRI and pathology was r = 0.648 (p < 0.001). The size discrepancy was significantly lower in cancers with initial MRI size . 5 cm (p = 0.050), in cancers with high tu­mour grade (p < 0.001), and in patients with hormonal receptor-negative cancer (p = 0.033). Conclusions. MRI is an accurate tool for evaluating tumour response after NAC. The accuracy of MRI in estimating residual tumour size varies with the baseline MRI tumour size, the tumour grade and the hormonal receptor status. Key words: breast cancer; MRI; residual tumour; neoadjuvant chemotherapy Introduction Neoadjuvant chemotherapy (NAC) has been used in the management of large operable breast tu­mours with the purpose of modifying the surgical planning and increase the rate of breast conserva­tive surgery (BCS).1-6 Currently, NAC has been ex­tended to selected patients with early-stage disease to improve the cosmetic outcome of BCS, espe­cially in women with small breast size.7-9 NAC has proved to be equivalent to postoperative chemo­therapy in terms of disease-free and overall surviv­al.10-12 However, in the neoadjuvant setting, there is evidence that patients who achieve a pathologic complete response (pCR) in the breast after NAC have a better prognosis than patients with a partial response or non-responders.6,11,13-15 Based on the different behaviour of each tumour subtype, a mo­lecular classification system identifies subgroups of breast invasive carcinoma patients who are most likely to achieve a pCR.16 An accurate imaging assessment of tumour re­sponse to NAC may facilitate the surgical plan­ning. Magnetic resonance imaging (MRI) is more accurate and sensitive than conventional methods in assessing residual tumour extent after NAC.17-23 In addition; there has been a positive correlation between MRI-determined and pathologic residual tumour size.24-29 However, it is necessary to know the factors affecting the diagnostic accuracy of MRI in breast cancer treated with NAC. The aims of the present study are to evaluate the diagnostic accuracy of MRI to detect residual dis­ease and to predict the tumour extent in patients with breast cancer receiving NAC, and to identify the factors that influence the accuracy of MRI in predicting residual tumour size. Patients and methods Patients and tumour molecular characteristics A total of 91 patients with invasive breast cancer (92 carcinomas) were included in this institutional retrospective study. All patients were diagnosed by core needle biopsy between October 2006 and June 2013. All patients were treated with NAC followed by surgical treatment and underwent MRI before and after NAC for monitoring tumour response to treatment. Considering the Helsinki Declaration principles, the Institutional Research Ethics Committee approved this study (No. 2015/059). According to the results of the initial diagnostic biopsy, tumours were classified into 5 molecular subtypes based on immunohistochemical charac­teristics of breast cancer30 (hormone receptor status, human epidermal growth factor receptor 2 (HER2) status and level of expression of ki-67). Estrogen receptor (ER) and progesterone receptor (PR) were considered positive if . 10% of tumour cell nuclei stained positive. Hormone receptors (HR) were considered positive when the ER, PR, or both were positive. HER2 tumours scoring 3+ (intense homo­geneous membranous staining in . 10% of tumour cells) were considered positive. In case of 2+ scores (moderate complete membranous staining in . 10% of tumour cells), the technique of fluorescent in situ hybridization was used to determine HER2 gene amplification. Samples with scores 0 to 1+ were considered negative. The cut-off of ki-67 expression level was set at 14%, to determine whether the cell proliferation index was high (> 14%) or low (. 14%). The five categories of molecular subtypes were: lu­minal A-like subtype (HR positive, HER2 negative, ki-67 . 14%), luminal B/HER2-negative-like subtype (HR positive, HER2 negative, ki-67 > 14%), luminal B/HER2-positive-like subtype (HR positive, HER2 positive), HER2-positive-like subtype (HR negative, HER2 positive) and triple negative subtype (HR neg­ative, HER2 negative). Chemotherapeutic and MRI protocols The treatment plan was chosen by oncologists and explained to every patient. The initial evaluation of patients before NAC included a complete medi­cal history, physical examination, complete blood work, chest X-ray, CT scan and bone scan. Clip tita­nium was placed in the tumour bed in all patients before starting chemotherapy, to identify the area of the primary tumour at the time of surgery. All patients with HER2-positive cancers received tras­tuzumab-based regimen as neoadjuvant therapy. Tumour size was measured on the last MRI performed after completing NAC. The stud­ies were done on a 1.5 T MRI scanner (Best, The Netherlands). The patient was placed in a prone position. Protocol in space-occupying lesions in the breast includes an axial T1-weighted sequence (TR: 494 msec, TE: 8 msec, number of acquired signals: 2, slice thickness: 3 mm, interval: 0.3 mm) and T2-weighted sequence (TR: 5000 msec, TE: 120 msec, number of acquired signals: 2, slice thick­ness: 3 mm, interval: 0.3 mm), followed by 3D T1-weighted fast spoiled gradient-echo dynamic sequence, selective excitation of water, (TR: 23 msec, TE: 5.7 msec, angle: 20o, slice thickness: 2 mm) acquiring 6 series, one pre-contrast series and five consecutive post-contrast series at 90-second intervals. Contrast agent (Gd-DOTA, DOTAREM, Guerbet) was administered using a bolus injection (2 mL/s) at a dose of 0.1 mmol/kg followed by a bolus of saline solution (20 mL). All images are analysed at a workstation. Subtraction images be­tween the without contrast stage and the 2nd-3rd-4th­5th post-contrast phase were obtained, which were interpreted with the help of specific programs for the analysis of contrast enhancement and time-sig­nal intensity curves. TABLE 1. Clinical and tumour characteristics MRI assessment Assessment of response was based on changes in tumour size in the MRI contrast sequences. Tumour size was calculated by summing the maxi­mum diameters of tumour enhancement on axial slices of MRI, as the Response Evaluation Criteria in Solid Tumours (RECIST). The absence of a clear enhancement indicates no residual cancer. The fi­nal response was defined as the change in size be­tween the pre-treatment and post-treatment MRI. Response categories, based on radiological exami­nation with contrast MRI, were classified as: (1) im­aging complete response on MRI (iCR: no evidence of residual disease on posttreatment MRI); and (2) non-iCR: residual disease on posttreatment MRI. Histopathologic analysis Pathologic measurement of the tumour size was used as the “gold standard” and compared to the MRI-measured residual tumour size. Samples for histopathological examination were cut into 5 mm slices, fixed in 10 % neutral-buffered formalin, try­ing to identify any lesion that corresponded with invasive carcinoma. If the tumour lesion was evi­dent, it was included in its entirety for morphologi­cal study with hematoxylin and eosin (H&E). If no evident tumour was found, the clip marker was identified, and slides from the block containing the marker as well as the adjacent blocks were exam­ined. Tumour response after NAC was classified as (1) pCR: no residual invasive tumour in the breast on final pathology; and (2) non-pCR: presence of residual invasive cancer on final pathology. If any residual invasive disease, pathologic tumour size was determined by measuring the longest dimen­sion of a sample stained with H&E and the number of blocks in which invasive tumour was detected. Statistical analysis A descriptive analysis of the variables included in the study was performed. Continuous variables were expressed as mean and standard deviation, and categorical variables were expressed as abso­lute values and percentages with their estimated 95% confidence interval. Comparison of means was performed using Student´s t test or Mann-Whitney test and analysis of variance or Kruskal-Wallis test, as appropriate after checking normality with the Kolmogorov-Smirnov test. Association of qualita­tive variables was estimated using the Chi-square test. Pearson correlation analysis was used to com- AGE (years) 47.22 10.10 42.0 31.0-75.0 BASAL TUMOR SIZE (cm) 3.99 1.97 3.40 1.60-13.0 CLINICAL TUMOR STAGE HISTOLOGICAL TYPE HISTOLÓGICAL GRADE HORMONAL RECEPTOR STATUS HER2 STATUS MOLECULAR SUBTYPE PRE-NAC AXILLARY STATUS T1 7 7.6 1.6-13.6 T2 69 75.0 65.6-84.4 T3 13 14.1 6.5-21.8 T4 3 3.3 0.7-9.2 Ductal 85 92.4 86.4-98.4 Lobular 7 7.6 1.6-13.6 G1 9 10.0 3.2-16.8 G2 32 35.6 25.1-46.0 G3 49 54.4 43.6-65.3 NA 2 Positive 60 65.2 54.9-75.5 Negative 32 34.8 24.5-45.1 Positive 25 27.2 17.5-36.8 Negative 67 72.8 63.2-82.5 Luminal A 11 12.0 4.8-19.1 Luminal B/HER2-35 38.0 27.6-48.5 Luminal B/HER2+ 16 17.4 9.1-25.7 HER2+ 9 9.8 3.2-16.4 Triple negative 21 22.8 13.7-31.9 Positive 76 82.6 74.3-90.9 Negative 16 17.4 9.1-25.7 CI = confidence interval; NA = not available; SD = standard deviation pare the MRI-measured and pathological tumour sizes. Linear regression model was used to analyse the diagnostic accuracy of MRI. P . 0.05 was con­sidered significant. Basic statistical indicators to assess the accuracy of MRI in detecting residual disease after NAC were calculated. The efficacy of MRI was measured by the predictive values. True negative (TN) was defined as negative in both MRI and pathology; true positive (TP) was defined as positive in both MRI and pathology; false negative (FN) was defined as negative on MRI and positive on pathology; and false positive (FP) was defined as positive on MRI and negative on pathology. To assess the accuracy of MRI in detecting residual disease, sensitivity: TP / (TP + FN), specificity: TN / (TN + FP), positive predictive value (PPV): TP / (TP + FP), negative predictive value (NPV): TN / (TN + FN), and overall accuracy: (VN + VP) / (TP + FIGURE 1. Dispersion graph of the correlation between residual tumour sizes (cm) calculated at preoperative MRI (on the X-axis) and at pathological examination (on the Y-axis). Each point represents a tumour; if the pairs of data coincide, the points overlap. The bisector corresponds to the equivalence line. On the graph, the closer the points are to the equivalence line, the greater the correlation between MRI and pathology. TN + FP + FN) were calculated. Analyses were per­formed using IBM SPSS Statistics version 19. Results 92 invasive breast tumours were analysed (1 pa­tient with bilateral breast cancer was recorded). Initial clinicopathologic characteristics of patients and tumours of the study are shown in Table 1. Patients age ranged between 31 and 75 years (mean 47.22 years). Mean baseline tumour size de­termined by MRI was 3.99 cm. Most tumours were diagnosed as T2 stage (75%) and grade 3 (53.3%). The initial biopsy of the lesions revealed 85 cases of invasive ductal carcinoma and 7 cases of inva­sive lobular carcinoma. 11 tumours were classified as luminal A, 35 as luminal B/HER2-, 16 as lumi­nal B/HER2+, 9 as HER2+ and 21 as triple negative. Most patients (65.9%) received taxane-anthracy­cline based chemotherapy; HER2-positive patients (27.5%) were treated with a trastuzumab-based regimen. After NAC, mastectomy was performed in 21 cases (21.8%) and BCS was attempted in 71 cases (77.2%). Of the latter group, 16 cases were re­operated because tumour involvement or proxim­ity to the resection margins. Accuracy of MRI to detect residual disease after NAC Tumour responses to NAC were compared based on the results obtained by MRI and pathological examination. MRI showed complete remission in TABLE 2. MRI diagnostic performance in predicting pathologic response MRI No iCR TP = 48 FP = 6 54 (58.70%) iCR FN =16 TN = 22 38 (41.30%) Total 64 (69.60%) 28 (30.40%) 92 (100%) FN = false negative; FP = false positive; iCR = imaging complete response; pCR = pathologic complete response; TN = true negative; TP = true positive 38 cases (41.3%) and residual disease in 54 cases (58.7%). The pathological study showed a pCR in 28 cases (30.4%) and invasive residual tumour was found in 64 samples (69.6%).The diagnostic performance of MRI for detecting residual tumour is summarized in Table 2. The sensitivity of MRI for detecting residual disease after NAC was 75% (48/64) and the specificity was 78.57% (22/28). The PPV (accuracy of MRI for detecting residual dis­ ease) was 88.89% (48/54). The NPV (accuracy of MRI in predicting pCR) was 57.89% (22/38). The overall accuracy of MRI was 76.09% (70/92). MRI showed FN diagnoses in 25% cases (16/64). Accuracy of MRI to predict the residual tumour size after NAC It was possible to compare the residual tumour size determined by MRI and pathological examination in 89 of the 92 cases of the study. In 3 cases it was not possible to determine the pathologic tumour size due to the presence of scattered residual mul­tifocal disease. The mean residual tumour size de­termined by MRI after NAC was 1.44 cm. The final pathologic tumour size was 1.53 cm. The two meas­urements are correlated forwardly significantly (r = 0.648, p < 0.001) (Figure 1). The mean discrepancy between the two measures was 0.96 cm. The dis­ crepancy was less than 1 cm in 57 cases (64.04%). Analysis of factors influencing the accuracy of MRI for predicting residual tumour size. Linear regression models were performed to find clinicopathological predictors of the diagnostic accuracy of MRI based on the absolute difference between the MRI-measured and pathologic resid­ual tumour size (Table 3). The strongest predictor was tumour grade (p < 0.001). The mean absolute TABLE 3. Factors affecting the MRI diagnostic accuracy based on the discrepancy between MRI and pathologic residual tumour size Age (years) .45 43 1.09 ±1.14 0.281 >45 46 0.84 ±1.01 Baseline tumour size (cm) .5 74 0.85 ±0.99 0.050 >5 15 1.53 ±1.33 Histological type 0.818 ductal 83 0.97 ±1.09 lobular 6 0.87 ±0.82 Histological grade <0.001 1 or 2 40 1.44 ±1.24 3 47 0.56 ±0.71 Hormonal receptor status 0.033 positive 59 1.14 ±1.13 negative 30 0.63 ±0.87 HER2 status 0.906 positive 24 0.99 ±1.12 negative 65 0.96 ±1.07 FIGURE 2. Discrepancy between MRI-measured and pathologic residual tumour size, based on molecular subtypes. discrepancy was significantly lower in the group of high-grade tumours. In addition, the HR status was associated significantly with the diagnostic accuracy of MRI, observing a lower discrepancy in the group of non-luminal tumours (p = 0.033). Baseline tumour size was kept in the limit of signif­icance, with a lower discrepancy in the group with baseline tumour size . 5 cm. The age, histological type and HER2 status were not associated with the diagnostic accuracy of MRI. Triple negative subtype showed the smallest difference between the two measurements (Figure 2), although no statistical­ly significant difference regarding the molecular phenotype of the tumour (p = 0.055). After a mul­tivariate linear regression analysis, tumour grade (p = 0.001) and baseline tumour size (p = 0.030) re­mained significant independent predictors of MRI accuracy (Table 4). Discussion Several researchers have previously studied the diagnostic accuracy of MRI in detecting invasive breast carcinoma in patients undergoing NAC31­34; however, an accurate determination of residual tumour size is necessary to perform an optimal surgery and achieve negative margins. We con­ducted a comparative analysis of post-NAC MRI and pathological findings to describe the diagnos­tic accuracy of MRI to detect residual invasive dis­ease and to estimate the residual tumour size after NAC. In the current study, the overall diagnostic Molecular subtype Luminal A 10 1.59 ±1.34 Luminal B-HER2-34 1.05 ±1.06 Luminal B-HER2+ 15 1.02 ±1.14 HER2+ 9 0.92 ±1.14 Triple negative 21 0.50 ±0.70 0.055 SD = standard deviation TABLE 4. Results from the Multivariate Regression Analysis Tumour grade 0.807 0.236 0.001 0.338-1.276 HR status 0.086 0.249 0.729 -0.408-0.581 BTS (MRI) 0.610 0.277 0.030 0.060-1.161 BTS = baseline tumour size; CI = confidence interval; se = standard error accuracy of MRI for detecting residual invasive carcinoma in the breast was 76.09%. The PPV and NPV were 88.89% and 57.89%, respectively. These data suggest that breast MRI is an accurate tool for assessing tumour response after NAC, although it is more limited in predicting pCR, which may be due to the NAC antiangiogenic effect in the tu­mour bed. Correlation coefficients of residual tumour size assessed by MRI and pathology were considered good. Lobbes et al.35 reviewed 17 studies compar­ing MRI-measured and pathologic residual tumour size. In this review, the mean correlation coefficient was 0.698. In our series, the value of the correlation coefficient was 0.648, reflecting a moderate corre­lation. If we remove the luminal A-like cases from the calculation, in which it has been observed that NAC is not the optimal therapeutic strategy, the value of global correlation coefficient rises to 0.706 (p < 0.001). Although correlation coefficients pro­vide important information about the MRI´s abil­ity to assess response to NAC, the determination of the absolute difference between MRI-measured and pathologic residual tumour size is necessary to evaluate the diagnostic accuracy of MRI. MRI over­estimation of residual tumour size can increase the number of unnecessary mastectomies and alter the cosmetic outcome of BCS with wide resection mar­gins, while MRI underestimation can increase the number of reoperations. In the current study, the mean discrepancy was 0.96 cm. Less than 1 cm dif­ference in the calculation of residual tumour size between MRI and pathology was observed in 57 tumours (64.04%). Identifying factors that may af­fect the accuracy of MRI for predicting residual tu­mour size could help to interpret breast MRI find­ings. Three recent studies evaluated the accuracy of MRI to predict residual tumour size after NAC and investigated the factors that influence the ac­curacy of MRI. In a study conducted by Ko et al.36, the Pearson´s correlation coefficient between the tumour sizes measured using MRI and pathology was 0.749 (p < 0.001) and the mean of size discrep­ancy was 1.26 cm. According to the molecular sub­type, tumour grade and tumour morphology on initial MRI, statistically significant differences of size discrepancy between both measurements were observed. Triple negative subtypes were measured more accurately (mean, size discrepancy = 0.8 cm). In the study by Chen et al.37, the mean discrepancy was 1.0 cm, and predictive factors found in the univariate analysis were histological type, tumour morphology, HR status, HER2 status and type of MRI. Multivariate analysis identified as independ­ent predictors histological type, tumour morphol­ogy and the combination of HER2-HR status. Finally, in a study by Moon et al.38, the Pearson´s correlation coefficient and the mean difference be­tween MRI-measured and pathologic tumour size was 0.664 (p < 0.001) and 1.39 cm, respectively. The clinicopathological factors associated with MRI ac­curacy were the initial T stage, the age at the time of the diagnosis and the ER expression status. In addition, Moon et al observed increased accuracy of MRI in predicting the residual tumour extent after NAC in triple negative breast cancer. In our study, a statistically significant association was ob­served between the absolute discrepancy and each of covariates: baseline tumour size, tumour grade and HR status. A minor discrepancy was observed in tumours with an initial size . 5 cm, in high-grade tumours and in non-luminal tumours. Despite a sta­tistically significant association between molecular subtypes and the diagnostic accuracy of MRI was not observed (p = 0.055), a tendency to find bet­ter accuracy in triple negative tumours was found. The mean discrepancy in residual tumour size was lower in the group of triple negative tumours (0.50 cm). Multivariate analysis identified only as independent predictors baseline tumour size and tumour grade. There are two important limitations to note in our study. The absence of ductal carcinoma in situ (DCIS) was not included in the definition of pCR, which can affect the accuracy of MRI. In addition, the current molecular classification includes a cut­off value of ki-67 expression level at 20% to define low or high level.39 Conclusions In conclusion, MRI can accurately measure tumour response and residual tumour size in breast cancer patients treated with NAC. Both overestimation and underestimation of MRI-measured residual tumour size may cause an incorrect surgical plan­ning so it´s important to consider the clinicopatho­logical factors that can affect the diagnostic ac­curacy of breast MRI. In our series, evaluation of residual tumour size was more accurate in baseline tumour size . 5 cm lesions, in high tumour grade lesions and in non-luminal breast cancer. References 1. Hortobagy GN, Ames FC, Buzdar AU, Kau SW, McNeese MD, Paulus D, et al. Management of stage III primary breast cancer with primary chemotherapy, surgery, and radiation therapy. Cancer 1988; 62: 2507-16. 2. Mauriac L, Durand M, Avril A, Dilhuydy JM. 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Ann Oncol 2013; 24: 2206-23. research article Antioxidant defence-related genetic variants are not associated with higher risk of secondary thyroid cancer after treatment of malignancy in childhood or adolescence Ana Lina Vodusek,1 Katja Goricar,2 Barbara Gazic,3 Vita Dolzan,2 Janez Jazbec4 1 Department of Radiation Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Pharmacogenetics Laboratory, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Department of Pathology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 4 Department of Hematology and Oncology, University Children’s Hospital, Ljubljana, Slovenia Radiol Oncol 2016; 50(1): 80-86. Received 2 February 2015 Accepted 23 February 2015 Correspondence to: Prof. Janez Jazbec, Ph.D., M.D., Department of Hematology and Oncology, University Children’s Hospital, Bohoričeva 20, 1525 Ljubljana, Slovenia. Phone: +386 1 522 8653; Fax: +386 1 522 4036; E-mail: janez.jazbec@mf.uni-lj.si Disclosure: No potential conflicts of interest were disclosed. Background. Thyroid cancer is one of the most common secondary cancers after treatment of malignancy in child­hood or adolescence. Thyroid gland is very sensitive to the carcinogenic effect of ionizing radiation, especially in chil­dren. Imbalance between pro- and anti-oxidant factors may play a role in thyroid carcinogenesis. Our study aimed to assess the relationship between genetic variability of antioxidant defence-related genes and the risk of secondary thyroid cancer after treatment of malignancy in childhood or adolescence. Patients and methods. In a retrospective study, we compared patients with childhood or adolescence primary malignancy between 1960 and 2006 that developed a secondary thyroid cancer (cases) with patients (controls), with the same primary malignancy but did not develop any secondary cancer. They were matched for age, gender, primary diagnosis and treatment (especially radiotherapy) of primary malignancy. They were all genotyped for SOD2 p.Ala16Val, CAT c.-262C>T, GPX1 p.Pro200Leu, GSTP1 p.Ile105Val, GSTP1 p.Ala114Val and GSTM1 and GSTT1 dele­tions. The influence of polymorphisms on occurrence of secondary cancer was examined by McNemar test and Cox proportional hazards model. Results. Between 1960 and 2006 a total of 2641 patients were diagnosed with primary malignancy before the age of 21 years in Slovenia. Among them 155 developed a secondary cancer, 28 of which were secondary thyroid can­cers. No significant differences in the genotype frequency distribution were observed between cases and controls. Additionally we observed no significant influence of investigated polymorphisms on time to the development of secondary thyroid cancer. Conclusions. We observed no association of polymorphisms in antioxidant genes with the risk for secondary thyroid cancer after treatment of malignancy in childhood or adolescence. However, thyroid cancer is one of the most com­mon secondary cancers in patients treated for malignancy in childhood or adolescence and the lifelong follow up of these patients is of utmost importance. Key words: secondary thyroid cancer; antioxidant genes; genetic polymorphism Introduction dren and adolescents with malignancies.1,2 With increasing number of survivors and years of follow Modern treatment modalities and better diagnostic up late effects of treatment are encountered more techniques greatly improved the survival of chil-frequently.3,4 The most detrimental late effects are second­ary cancers. Several studies report on increased risk of subsequent secondary cancers even several decades after treatment of primary malignancy.5 Increased incidence of secondary thyroid cancer was reported even up to 40 years after radiother­apy.6-9 The thyroid gland is very sensitive to the car­cinogenetic effect of ionizing radiation, especially in children.7 Ionizing radiation damages DNA directly or indirectly through production of free radicals and reactive oxygen species (ROS). It has been shown that gamma radiation and hydrogen peroxide (H2O2), which is one of the ROS, induce similar DNA damages in the thyroid.10 Oxidative DNA damage involves single- or double DNA strand breaks, purine and pyrimidine or deoxy­ribose modifications as well as DNA cross links. ROS can also damage the cell through lipid perox­idation, protein modification, membrane disrup­tion and mitochondrial damage.11,12 The thyroid cell is constantly exposed to ROS and an imbalance between pro- and anti-oxidative factors has been suggested as an important mecha­nism in thyroid carcinogenesis. The accumulation of oxidative DNA damage may drive genomic instability events and lead to somatic mutations. Many studies have shown that oxidants are in­creased and antioxidants are decreased in patients with thyroid cancer.13-19 The most important antioxidants in the thyroid are antioxidant enzymes such as superoxide dis­mutase (SOD), glutathione peroxidase (GPX) and catalase (CAT). Manganese superoxide dismutase (SOD2) is the major antioxidant in mitochondria, catalysing the dismutation of superoxide anion to H2O2, which is then reduced to water by CAT or GPX.20,21 Many studies have investigated genetic variability in genes coding for antioxidant en­zymes and their relationship to cancer risk, how­ever the results were inconclusive22 and the data on thyroid cancer risk are lacking.23 The most common polymorphism in the gene coding for SOD2 (SOD2) leads to substitution of alanine (Ala) with valine (Val) at codon 16 (p.Ala16Val) and affects transport of the enzyme into the mitochondria.24 According to several stud­ies the 16Ala allele of SOD2 polymorphism is as­sociated with an increased risk of prostate and oe­sophageal cancer.21,25 CAT activity is affected by functional single nucleotide polymorphism (SNP) CAT c.-262C>T in the promoter region of CAT gene, which leads to lower catalase activity. This polymorphism was implicated in increased susceptibility to breast and cervical cancer.26,27 The most common GPX1 polymorphism re­sults in the amino acid substitution of leucine with proline at codon 200 (p.Leu200Pro) and re­sults in lower enzyme activation. As it may in­fluence the balance between oxidative stress and antioxidant defence, it may therefore increase can­cer risk.28 Indeed several studies associate GPX1 p.Pro200Leu polymorphism with increased sus­ceptibility to prostate and breast cancer.29,30 Glutathione S-transferase (GSTs) enzymes, en­coded by GST genes, are implicated in detoxifica­tion of xenobiotics and reactive products of ROS, so they may have a crucial role in protecting tissue from oxidative damage.31 Deletions of the GSTM1 and GSTT1 genes result in null genotypes and lead to impaired enzyme activity.32 In GSTP1 two frequent SNPs resulting in an amino acid substi­tution have been reported. The GSTP1 p.Ile105Val SNP results in Ile to Val substitution near the active site and leads to decreased enzyme activity. The functional role of the GSTP1 p.Ala114Val polymor­phism is not clear, however, reduced conjugation capacity was reported in the enzyme with both polymorphisms present.30 Variants of these loci have been implicated in the aetiology of numerous cancers.29,31-34 There is some inconclusive data on GST poly­morphisms in association to thyroid cancer risk. According to some studies individuals with ho­mozygous deletions of GSTM1 or GSTT1 have an increased risk of thyroid cancer, whereas Lemos et al. found the opposite in his study.33-35 Mertens et al. studied radiotherapy related malignancies in survivors of Hodgkin disease and found that individuals lacking GSTM1 but not GSTT1 were at increased risk of any subsequent SMN.36 To our knowledge data on genetic variability of an­tioxidant enzymes in primary thyroid cancer are scarce, while no data have been published regard­ing the secondary thyroid cancer. The aim of the present study was to investigate the relationship between genetic variability in anti­oxidant defence-related genes (SOD2 p.Ala16Val, CAT c.-262C>T, GPX1 p.Pro200Leu, GSTM1 de­letion, GSTT1 deletion, GSTP1 p.Ile105Val and GSTP1 p.Ala114Val) and the risk of secondary thyroid cancer after treatment of malignancy in childhood or adolescence. Patients and methods Patients A population based study of all patients known to have developed a secondary thyroid cancer af­ter treatment of malignancy in childhood or ado­lescence was performed. A retrospective matched case-control study was designed. Individuals were eligible for inclusion in the study patients group (cases) if they were diagnosed with any kind of pri­mary malignancy between 1960 and 2006 and before the age of 21 and were treated at the Department of Hematology and Oncology, University Children’s Hospital, Ljubljana, or at the Institute of Oncology, Ljubljana and had a secondary thyroid cancer di­agnosed 5 years or later after the primary malig­nancy. Study controls were patients with a primary malignancy in childhood or adolescence but free of secondary thyroid cancer. They were selected with a ratio of 1 control to 1 case matched for: type of the primary malignancy, treatment of primary ma­lignancy, especially regarding the radiotherapy to the neck, head or mediastinal region, sex and age at the time of primary malignancy diagnosis (if pos­sible not more than 2 years younger or older then the case). Study patients and study controls were identified from a search from the Cancer Registry of Slovenia.37 All patients with primary childhood or ado­lescent malignancy were followed up at the Department of Hematology and Oncology, University Children’s Hospital, Ljubljana, or at the out-patient Clinic for Late Effects at the Institute of Oncology, Ljubljana.38 Thyroid follow-up included yearly thyroid stimulating hormone (TSH) and thyroglobulin level evaluation and occasional neck ultrasound. All patients with palpable nodules and/or elevated thyroglobulin levels underwent a neck ultrasound as a method commonly used in the work-up of thy­roid diseases.39 If malignancy was suspected, fine needle aspiration biopsy (FNAB) was performed. When papillary/follicular lesions were detected or were just suspected by cytology, thyroidec­tomy was performed at the Institute of Oncology, Ljubljana. The follow up interval was defined as the time between primary malignancy and secondary thy­roid cancer in the study group or between primary malignancy and the last appointment at the Out-Patient Clinic for Late Effects in the control group. All patient’s data (demographic, clinical and treat­ment data) were collected from the patient’s medi­cal records. Single experienced pathologist reviewed all the primary malignancies and corresponding thyroid cancers. The study was approved by the Slovenian Ethics Committee for Research in Medicine (No.138/04/10) and was carried out according to the Declaration of Helsinki. DNA isolation and genotyping According to the presence of tumour or normal tis­sue on hematoxylin and eosin (HE) slides the pa­thologist chose one representative paraffin block from each biopsy and marked the tumour and nor­mal tissue area on the block. From the marked area (if possible we chose normal tissue) two to three cores of 1 mm in diameter were obtained for DNA extraction using a QIAamp DNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.40 For two control patients genomic DNA was isolated from archived cytological smears of bone marrow specimens using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany).41 For all other pa­tients DNA was obtained from paraffin blocks con­tained tumour or normal tissue as described above. Genotypes of GPX1 p.Pro200Leu (rs1050450) and SOD2 p.Val16Ala (rs4880) were determined by TaqMan genotyping method (Applied Biosystems, Foster City, CA, USA) as described previously.42 Genotyping of CAT c.-262C>T (rs1001179), GSTP1 p.Ile105Val (rs1695) and GSTP1 p.Ala114Val (rs1138272) was carried out using a fluorescence-based competitive allele-specific (KASPar ) assay (KBiosciences, Herts, UK).43 Multiplex polymerase chain reaction (PCR) was used for detection of GSTM1 and GSTT1 gene dele­tions. GSTM1, GSTT1, and BGLO genes were simul­taneously amplified in a multiplex PCR reaction as previously described.41 This approach allowed us to identify homozygous GSTM1 or GSTT1 gene de­letion, but we could not distinguish between carri­ers of one or two copies of each gene. Statistical analysis Frequencies were used to describe the distribution of categorical variables and median and interquar­tile ranges were used for continuous variables. Standard chi-square test was used to assess devia­tion from Hardy-Weinberg equilibrium (HWE), comparing the distribution of genotype frequen­cies in the control group with the expected distri­bution within the population.44 To compare the genotype distribution, McNemar test for the analysis of matched samples based on binomial distribution was used. The influence of polymorphisms on the time to the occurrence of second cancer was examined by Cox proportional hazards model with stratification on the matched pairs to calculate relative risks (RRs) and their 95% confidence intervals (CIs). All statistical analyses were carried out by IBM SPSS Statistics, version 19.0 (IBM Corporation, Armonk, NY, USA), except for odds ratios (OR) and 95% CIs in McNemar test that were calculated us­ing GraphPad Software. A dominant genetic model was used in all statistical analyses and p values be­low 0.05 were considered statistically significant. Results Patients’ characteristics Based on data from The Cancer Registry of Slovenia, in the period between 1960 and 2006 a to­tal of 2641 patients were diagnosed with primary cancer before the age of 21 years.40 Among them 155 developed secondary cancer (5.9%), out of which 28 (18.1%) were secondary thyroid cancers. Only 24 (85.7%) eligible cases were included in the study because we could not get the histopatho­logical material for 4 controls. Therefore the study group included 8 (33.3%) males and 16 (66.7%) fe­males with a median age of 12.7 years at diagnosis of primary cancer (range 7.1.6.7 years) and 29.2 years at diagnosis of secondary thyroid cancer (range 23.5.35.4 years). Six out of 24 patients (25.0%) were under 5 years old at the time of primary diagnosis. The control group included 8 (33.3%) males and 16 (66.7%) females with a median age of 12.9 years at diagnosis of primary cancer (range 5.0.15.4 years). The most frequent primary cancer was Hodgkin’s disease (HD) (15 pairs, 62.5%), then acute lympho­blastic leukemia (ALL) (2 pairs, 8.3%) and central nervous system (CNS) tumours (2 pairs, 8.3%). Non-Hodgkin lymphoma (NHL), neuroblastoma, rhabdomyosarcoma, nasopharyngeal carcinoma and ovarian tumours were observed in 1 pair each (4.2%). Most of the patients with secondary thyroid cancer received radiation therapy to the head, neck or mediastinum during the treatment for primary cancer (23 patients, 95.8%): 15 (62.5%) to the neck, 6 (25.0%) to the head and 2 (8.3%) to the mediasti­num. The same distribution of irradiated sites was observed also in the control group. The most frequent histology of secondary thy­roid cancer was papillary carcinoma (23, 95.8%). Only 1 tumour (4.2%) was follicular neoplasm of undefined malignant potential. Using TNM clas­sification for staging most of thyroid cancer were stage 1 with tumour localised to the thyroid and/ or lymph nodes (23, 95.8%), 1 (4.2%) was stage 2 with lung metastasis (M1). Among 12 (50.0%) T1 tumours (tumour diameter . 2cm), there were 9 (37.5%) microcarcinoma (T1a: tumour . 10 mm). There were 2 (8.3%) T2 tumours (tumour > 2 cm but . 4 cm in greatest dimension, limited to the thyroid); 6 (25.0%) were T3 (minimal extrathyroid extension) and 3 (12.5%) were T4 (extending be­yond the thyroid capsule to invade subcutaneous soft tissues, larynx, trachea, oesophagus, or recur­rent laryngeal nerve) and 11 (45.8%) had regional lymph node metastasis (N1). A total or near total thyroidectomy was carried out in all cases. Additional radioiodine treatment was applied to 19 (79.2%) patients. In 6 (25.0%) cas­es lymph node metastases were excised. The follow up interval was comparable in both groups and was 19.6 (range 9.0.23.6) years in the primary group and 18.8 (range 12.80.27.6) years in the control group. Both groups did not differ sig­nificantly regarding the demographic data. Genotype frequencies of the antioxidant de­fence-related genes are presented in the Table 1. All the investigated polymorphisms were in HWE in the control group. To assess if the investigated polymorphisms influence the risk of secondary thyroid cancer, we performed a matched analysis. When all the cases were compared to controls, no significant differ­ences in the genotype frequency distribution were observed (Table 2). There were also no differences in genotype distribution between microcarcinoma and other secondary thyroid cancers. We also assessed the influence of investigated polymorphisms on time to development of second­ary thyroid cancer using Cox regression with strat­ification on matched pairs. We have not observed any association between studied polymorphisms and the time pattern of occurrence of secondary thyroid cancers after treatment of malignancy in childhood or adolescence. Discussion In the present study we investigated if SOD2 p.Ala16Val, CAT c.-262C>T, GPX1 p.Pro200Leu, GSTM1, GSTT1, GSTP1 p.Ile105Val and GSTP1 p.Ala114Val polymorphisms influence the risk of secondary thyroid cancer after treatment of malig­ TABLE 1. Genotype frequencies of the antioxidant defence-related genes GPX1 CC 28 (58.3) 16 (66.7) 12 (50) 0.967 rs1050450 p. Pro200Leu CT TT 17 (35.4) 3 (6.3) 7 (29.2) 1 (4.2) 10 (41.7) 2 (8.3) SOD2a GG 10 (21.7) 4 (16.7) 6 (27.3) 0.338 rs4880 p.Val16Ala GA AA 31 (67.4) 5 (10.9) 18 (75) 2 (8.3) 13 (59.1) 3 (13.6) CAT GG 32 (66.7) 16 (66.7) 16 (66.7) 0.834 rs1001179 c.-262G>A GA AA 14 (29.2) 2 (4.2) 7 (29.2) 1 (4.2) 7 (29.2) 1 (4.2) GSTP1 AA 22 (45.8) 10 (41.7) 12 (50) 0.432 rs1695 p.Ile105Val AG GG 23 (47.9) 3 (6.3) 12 (50) 2 (8.3) 11 (45.8) 1 (4.2) GSTP1 CC 39 (81.3) 19 (79.2) 20 (83.3) 0.106 rs1138272 p.Ala114Val CT TT 8 (16.7) 1 (2.1) 5 (20.8) / 3 (12.5) 1 (4.2) GSTM1b non-null 23 (48.9) 14 (58.3) 9 (39.1) gene deletion null 24 (51.1) 10 (41.7) 14 (60.9) GSTT1b non-null 39 (83) 19 (79.2) 20 (87) gene deletion null 8 (17) 5 (20.8) 3 (13) CAT = catalase; GPX = glutathione peroxidase; GSTM1 = glutathione S-transferase Mu 1; GSTP1= glutathione S-transferase pi gene; GSTT1 = glutathione S-transferase theta 1; HWE= Hardy-Weinberg equilibrium; N = number; SNP = single nucleotide polymorphism; SOD2 = manganese superoxide dismutase; adata missing for 2 controls; bdata missing for 1 control nancy in childhood or adolescence. To the best of our knowledge no such study was yet performed in the secondary thyroid cancer, while data on the role of common functional polymorphisms in anti­oxidant defence-related genes in the primary thy­roid cancer are scarce.29 In total 5.9% of patients diagnosed between 1960 and 2006 with primary malignancy before the age of 21 years has developed a secondary cancer. Secondary thyroid cancers represented 18% of sec­ondary cancers. Our data are comparable to other studies and show that thyroid cancer is one of the most common secondary cancers after treatment of malignancy in childhood or adolescence. Similar to other studies the most frequent primary malig­nancy was Hodgkin’s lymphoma and the median time to develop a secondary thyroid cancer was 19.6 years.2-6 According to several studies the SOD2 16Ala al­lele was associated with an increased risk of pros­tate and oesophageal cancer, but with decreased risk of lung cancer.21,24 Never the less, the meta­analysis showed no significant effect of SOD2 TABLE 2. Influence of selected polymorphisms on the risk for secondary thyroid cancer GPX1 (rs1050450) 0.43 (0.07-1.88) 0.344 SOD2a (rs4880) 1.50 (0.36-7.23) 0.754 CAT (rs1001179) 1.00 (0.27-3.74) 1.000 GSTP1 (rs1695) 1.40 (0.38-5.59) 0.774 GSTP1 (rs1138272) 1.25 (0.27-6.30) 1.000 GSTM1 (gene deletion)b 0.43 (0.07-1.88) 0.344 GSTT1 (gene deletion)b 2.00 (0.29-22.11) 0.687 CAT = catalase; CI = confident interval; GPX = glutathione peroxidase; GSTM1 = glutathione S-transferase Mu 1; GSTP1= glutathione S-transferase pi gene; GSTT1 = glutathione S-transferase theta 1; OR = odd ratio; SNP = single nucleotide polymorphism; SOD2 = manganese superoxide dismutase; adata missing for 2 controls; bdata missing for 1 control p.Val16Ala polymorphism on overall cancer risk.21 This is also in concordance with our data that show no association between the SOD2 polymorphism and the risk of secondary thyroid cancer. Recent studies suggested that the GPX1 p.Pro200Leu polymorphism increased the suscep­tibility to bladder cancer27, whereas a meta-anal­ysis showed no significant association of GPX1 p.Pro200Leu polymorphism with cancer risk in general.45 Similar to the meta-analysis we observed no association between GPX1 p.Pro200Leu poly­morphism and the risk of secondary thyroid cancer. CAT polymorphism was implicated in cancero­genesis of several tumours, including breast and cervical cancer24, but again, meta-analysis has not confirmed these observations for the breast cancer risk.46 Our results also showed no association be­tween CAT polymorphism and risk of secondary thyroid cancer. Genetic variability at the GSTM1, GSTT1 and GSTP1 loci has been linked to increased suscep­tibility to several cancers, including thyroid can­cer.28,29,31 Our study was the first to analyse the association between the GST polymorphisms (GSTM1, GSTT1, GSTP1 p.Ile105Val and GSTP1 p.Ala114Val) and the risk of developing second­ary thyroid cancer after treatment of malignancy in childhood or adolescence, however, we were not able to detect any significant association. Mertens et al. found only a non-significantly increased risk of thyroid cancer in GSTM1 or GSTT1 homozygous patients that had Hodgkin lymphoma as a primary cancer36, but a meta-analysis concluded that GST polymorphisms are unlikely to be major determi­nants of susceptibility to primary thyroid cancer.29 Our results are comparable to the results of rel­evant studies on antioxidant defence-related poly­morphisms and the risk of cancer in general. While the sample size is small and therefore in some in­stances lacks statistical power, its prime advantage is the homogeneity of data because we designed a population-based study, which included all pa­tients diagnosed and treated for secondary thyroid cancer in Slovenia after treatment of malignancy in childhood or adolescence between 1960 and 2006. In conclusion, we observed no association of common functional polymorphisms in antioxidant defence related genes with the risk for secondary thyroid cancer after treatment of malignancy in childhood or adolescence. However, thyroid can­cer is one of the most common secondary cancers after treatment of malignancy in childhood or ado­lescence and it can develop several decades after the treatment. Hence the lifelong follow up of pa­tients with childhood or adolescent malignancy is of utmost importance and further studies on genetic factors associated with thyroid cancer risk should be performed. Acknowledgements The authors thank Matej Kastelic, Ph.D. for the help with the laboratory analyses, Maruša Debeljak, Ph.D. for help with DNA isolation from archived cytological smears of bone marrow specimens and prof. Berta Jereb, Ph.D., M.D. for all the support. This work was financially supported by the Infrastructure program for the Lifelong follow-up of the survivals from childhood or adolescent cancer (Grant No. I0 - 0010) and by the Slovenian Research Agency (ARRS Grant No.P1-0170). References 1. Perme MP, Jereb B. Trends in survival after childhood cancer in Slovenia between 1957 and 2007. Pediatr Hematol Oncol 2009; 26: 240-51. 2. Kachanov DY, Dobrenkov KV, Shamanskaya TV, Abdullaev RT, Inushkina EV, Savkova1 RF, et al. Solid tumors in young children in Moscow Region of Russian Federation. Radiol Oncol 2008; 42: 39-44. 3. Jazbec J, Ećimović P, Jereb B. Second neoplasms after treatment of child­hood cancer in Slovenia. Pediatr Blood Cancer 2004; 42: 574-81. 4. 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Jereb B. Model for long-term follow-up of survivors of childhood cancer. Med Pediatr Oncol 2000; 34: 256-8. 39. Tatar IG, Kurt A, Yilmaz KB, Dogan M, Hekimoglu B, Hucumenoglu S. The role of elastosonography, gray-scale and colour flow Doppler sonography in prediction of malignancy in thyroid nodules. Radiol Oncol 2014; 48: 348-53. 40. Goricar K, Kovac V, Jazbec J, Lamovec J, Dolzan V. Homologous recombina­ tion repair polymorphisms and the risk for osteosarcoma. J Med Biochem 2014; 33: 1-8. 41. Jazbec J, Aplenc R, Dolzan V, Debeljak M, Jereb B. GST polymorphisms and occurrence of second neoplasms after treatment of childhood leukemia. Leukemia 2003; 17: 2540-2. 42. Erculj N, Zadel M, Dolzan V. Genetic polymorphisms in base excision repair in healthy slovenian population and their influence on DNA damage. Acta Chim Slov 2010; 57: 182-8. 43. Goricar K, Erculj N, Zadel M, Dolzan V. Genetic polymorphisms in homolo­gous recombination repair genes in healthy Slovenian population and their influence on DNA damage. Radiol Oncol 2012; 46:46-53. 44. Guo S, Thompson E. Performing the exact test of Hardy–Weinberg propor­tion for multiple alleles. Biometrics 1992; 48: 361-72. 45. Cao M, Mu X, Jiang C, Yang G, Chen H, Xue W. Single-nucleotide polymor­phisms of GPX1 and MnSOD and susceptibility to bladder cancer: a system­atic review and meta-analysis. Tumour Biol 2014; 35: 759-64. 46. Saadat M, Saadat S. Genetic polymorphism of CAT C-262 T and susceptibility to breast cancer, a case-control study and meta-analysis of the literature. Pathol Oncol Res 2015; 21: 433-7. case report Cerebral toxoplasmosis in a diffuse large B cell lymphoma patient Lina Savsek1, Tanja Ros Opaskar2 1 Department of Neurology, General Hospital Celje, Celje, Slovenia 2 Unit of Neurology, Institute of Oncology Ljubljana, Ljubljana, Slovenia Radiol Oncol 2016; 50(1): 87-93. Received 27 May 2014 Accepted 21 August 2014 Correspondence to: Tanja Ros Opaskar, M.D., M.Sc., Unit of Neurology, Institute of Oncology Ljubljana, Ljubljana, Slovenia. E-mail: tros@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. Toxoplasmosis is an opportunistic protozoal infection that has, until now, probably been an under­estimated cause of encephalitis in patients with hematological malignancies, independent of stem cell or bone marrow transplant. T and B cell depleting regimens are probably an important risk factor for reactivation of a latent toxoplasma infection in these patients. Case report. We describe a 62-year-old HIV-negative right-handed Caucasian female with systemic diffuse large B cell lymphoma who presented with sudden onset of high fever, headache, altered mental status, ataxia and findings of pancytopenia, a few days after receiving her final, 8th cycle of rituximab, cyclophosphamide, vincristine, doxo­rubicin, prednisolone (R-CHOP) chemotherapy regimen. A progression of lymphoma to the central nervous system was suspected. MRI of the head revealed multiple on T2 and fluid attenuated inversion recovery (FLAIR) hyperintense parenchymal lesions with mild surrounding edema, located in both cerebral and cerebellar hemispheres that dem­onstrated moderate gadolinium enhancement. The polymerase chain reaction on cerebrospinal fluid (CSF PCR) was positive for Toxoplasma gondii. The patient was diagnosed with toxoplasmic encephalitis and successfully treated with sulfadiazine, pyrimethamine and folic acid. Due to the need for maintenance therapy with rituximab for lymphoma remission, the patient now continues with secondary prophylaxis of toxoplasmosis. Conclusions. With this case report, we wish to emphasize the need to consider cerebral toxoplasmosis in patients with hematological malignancies on immunosuppressive therapy when presenting with new neurologic deficits. In such patients, there are numerous differential diagnoses for cerebral toxoplasmosis, and the CNS lymphoma is the most difficult among all to distinguish it from. If left untreated, cerebral toxoplasmosis has a high mortality rate; there­fore early recognition and treatment are of essential importance. Key words: toxoplasmosis;,cerebral; lymphoma, B-cell; rituximab; hosts, immunocompromised; magnetic resonance imaging; treatment Introduction Toxoplasmosis is caused by an infection with the obligate intracellular parasite Toxoplasma gondii. The general assumption is that approximately 25 to 30% of the world’s human population is in­fected by Toxoplasma1, which makes it one of the most common human infections in the world. The prevalences vary widely between countries, with the lowest seroprevalences observed in the coun­tries of North America, South East Asia, Northern Europe and the Sahelian countries of Africa (be­ tween 10.30%). Toxoplasmosis is highly prevalent in Latin America and tropical African countries, while in the countries of Central and Southern Europe, including Slovenia, moderate seropreva­ lences have been found (30.50%).1 Toxoplasma gondii has a complex lifecycle in­volving felines, in which the sexual phase is com­pleted, as it’s definite host. Oocysts shed in feline FIGURE 1. (A) MRI at time of diagnosis demonstrating multiple T2 and (B) fluid attenuated inversion recovery (FLAIR) hyperintense parenchymal lesions, located in both cerebral and cerebellar hemispheres, with mild surrounding edema. (C) On T1 sequences, these lesions were hypointense. (D) After contrast administration, only moderate rim enhancement was seen. feces can infect a wide range of animals, including birds, rodents, grazing domestic animals, and hu­mans.2 Humans usually get infected either by the ingestion of tissue cysts in infected meat or by in­gestion of soil, water, or food contaminated with sporulated oocysts derived from the environment or, less frequently, directly from feline feces.1 In addition to oral transmission, direct transmission of the parasite by blood or organ products dur­ing transplantation takes place at a low rate. Apart from horizontal transmission, a vertical route of transmission is also recognized; parasite transmis­sion to the fetus occurs in about one-third of preg­nant women with primary toxoplasmic infection.1.2 In the immunocompetent host, the infection is usually rapidly cleared and only about 10-20% of infected individuals present with a self-limited and nonspecific illness, that only rarely requires therapy.1-3 In the immunocompromised host, the immune factors necessary to control the spread of the infection are lacking and the disease can be life-threatening. In these individuals, toxoplasmosis al­most always happens as a result of reactivation of a latent infection. The most common site affected by toxoplasmosis is the central nervous system (CNS). Besides CNS, multiple other organs may be in­volved, including the lungs, gastrointestinal tract, pancreas, skin, eyes, heart, and liver. With the ad­vent of the human immunodeficiency virus (HIV) pandemic, toxoplasmic encephalitis has become one of the most frequent opportunistic infections. Other heavily immunocompromised patients like those after allogeneic stem cell transplantation (SCT) or previous T cell depleting treatment regi­mens (e.g. with fludarabine or alemtuzumab) are also at high risk for opportunistic infections. If left untreated, the infection with Toxoplasma gondii in such individuals is usually fatal. Case report A 62-year-old HIV-negative right-handed Caucasian female with systemic diffuse large B cell lymphoma (DLBCL) presented to our oncology clinic in the beginning of September 2013 with high fever, headache and altered mental status. Her past medical history included arterial hy­pertension and type 2 diabetes mellitus, both well controlled with medications. Since 2010, she was treated for marginal zone B cell lymphoma stage IV A, involving the spleen, bone marrow and lymph nodes. A splenectomy was performed in July 2011. At that time, no other treatment was administered due to clinical remission. In August 2012, the disease progressed and she was started on chemotherapy with rituximab and chlorambu­cil (R-LP), but was switched after only two cycles of R-LP to fludarabine and cyclophosphamide in combination with rituximab (R-FC) due to further disease progression. During this time, she suffered an episode of cutaneous herpes zoster, so valacy­clovir prophylaxis was initiated. In January 2013, a transformation into DLBCL was confirmed and rituximab, cyclophosphamide, vincristine, doxoru­bicin and prednisolone (R-CHOP) chemotherapy regimen introduced. After 4 cycles of R-CHOP, disease remission was achieved and chemothera­py continued until the beginning of August 2013, when a neutropenia was noted and prophylactic therapy with ciprofloxacin and fluconazole initiat­ed. Only two days later she had to be hospitalized in a regional hospital because of febrile neutrope­nia due to urinary infection with E. Coli, which was successfully treated with amoxicilline/clavulanic acid. At the same time, a left posterior tibial vein thrombosis was discovered and therapy with low molecular weight heparin (LMWH) initiated. By the end of August 2013 she had received her 8th, final cycle of R-CHOP. Only five days after, the patient started complaining of headache with pho­tophobia and was admitted to a regional hospital a day later with high fever, confusion and pancyto­penia. A head CT scan revealed diffuse hypodensi­ties involving the right parieto-temporo-occipital region, spreading into the frontal lobe and across the corpus callosum into the left parieto-occipital lobe. A hypodensity in the central region of the cer­ebellum was present as well. There were no mid­line brain shifts or herniations and the ventricular system was normal. Progression of the lymphoma to the CNS was suspected and the patient trans­ferred to our oncology clinic for further evaluation and management. At the time of our first neurological consulta­tion, the patient was conscious but apathetic, with elements of dysexecutive syndrome. She had poor orientation in time and situation, seemed to fill in the memory gaps with confabulations and exhib­ited finger agnosia. Her speech was normal. A mini mental state examination (MMSE) test could not be applied at that time due to the lack of attention needed to complete the task. A left homonymous hemianopsia, denser in the lower quadrant, was clinically suspected. A slight mask-like appearance of the face was noted. The rest of the cranial nerve examination revealed no abnormalities. Meningeal signs were absent. A mild pyramidal weakness of the right upper extremity was present and she ex­hibited a moderate symmetrical ataxia along with dysmetria of all four extremities. The plantar re­sponses were flexor. There were no marked sen­sory deficits. The clinical picture indicated bilateral multifocal cerebral cortico-subcortical involvement along with damage to the cerebellum. She was started on antiedematous therapy with 20% mannitol solution. A diagnostic lumbar puncture was performed and the cerebrospinal fluid (CSF) analysis revealed an elevated white cell count of 27 cells (1 neutrophil, 20 lymphocytes, 6 monocytes), with increased lactate, LDH and protein concentration, but normal glucose level. Cytological analysis of the CSF with Giemsa stain­ing and flow cytometric immunophenotypisation revealed no malignant cells, but there were signs of reactive pleocytosis with T cell predominance. Magnetic resonance imaging (MRI) of the head revealed multiple T2 and fluid attenuated inver­ sion recovery (FLAIR) hyperintense parenchymal lesions with mild surrounding edema, located in both cerebral and cerebellar hemispheres, with the largest lesion located in the right occipital lobe. The lesions were hypointense on T1 sequences and demonstrated moderate rim enhancement after contrast administration (Figure 1). The polymerase chain reaction on cerebrospi­nal fluid (CSF PCR) was positive for Toxoplasma gondii and serological analysis revealed border­line positive IgG and negative IgM antibodies. Microbiological assays for other bacteria, fungus or viruses, such as Borrelia burgdorferi, Mycobacterium tuberculosis, Cryptococcus neoformans, herpes sim­plex virus type 1 and 2, varicella zoster virus, en­teroviruses, polioviruses, tick-borne encephalitis virus, JC virus, Epstein-Barr virus, and cytomeg­alovirus were negative. There was no apparent evidence of toxoplasmo­sis in other organs. A full body 18F-FDG PET/CT showed remission of lymphoma and local hypo-metabolism in the right occipital lobe, correspond­ing to the location of the largest toxoplasma lesion (Figure 2). Based on these findings, the patient was diag­nosed with toxoplasmic encephalitis and therapy with sulfadiazine, pyrimethamine and folic acid was administered. Due to an underlying mild car­diomyopathy, caused by anthracycline therapy in the past, the patient suffered an acute heart failure episode in the beginning of treatment, which was managed conservatively. This condition was not directly associated with toxoplasma infection and was caused by fluid volume overload. After a few days of antibiotic therapy the patient slowly began improving. She became more alert and her cogni­tive status improved. A follow-up MRI of the head FIGURE 3. Lesion size and edema reduction after 6 weeks of intensive antibiotic therapy, as demonstrated by (A) fluid attenuated inversion recovery (FLAIR) and (B) T1 sequence. 6 weeks later showed reduction of edema and le­sion size, with hemorrhagic inclusions in some of the lesions (Figure 3.) After 6 weeks of intensive antibiotic treatment we started with maintenance therapy with rituximab for lymphoma remission and continued with prophylactic doses of sulfadia­zine, pyrimethamine and folic acid. Three months after the first presentation, the patient was seen again in the neurooncology out­patient clinic for a follow-up. There were only mild cognitive deficits still present and she scored 28/30 points on MMSE. In January 2014, follow-up MRI of the head showed further reduction of brain le­sions, this time without any visible hemorrhagic inclusions (Figure 4.) Patient’s written consent to publish this case re­port has been obtained. Discussion In patients with malignancies, especially those af­ter previous allogeneic bone marrow or stem cell FIGURE 4. Follow-up MRI after 4 months reveals further reduction of lesion size. (A) T1 sequence + gadolinium, (B) T2 sequence. transplantation, the incidence of various CNS in­fections may be up to 15%.4 There are several risk factors for CNS infection in these individuals, de­pending on the underlying malignancy, its treat­ment and various other factors. Among causative organisms, Toxoplasma gondii is the most prevalent.4 The majority of clinical experience with cerebral toxoplasmosis was acquired in HIV/AIDS patients, in whom the risk of reactivation of latent infec­tion with T. gondii is greatest when the CD4+ T-cell count decreases below 100 cells/uL.5 Cerebral toxo­plasmosis is also a rare, but well known opportun­istic infection in bone marrow transplant (BMT) recipients6, as well as in solid organ transplant re­cipients.7 Clinical presentation of cerebral toxoplasmosis is unspecific, ranging from altered mental status, fever, seizures and headache to focal neurologic deficits, including motor deficits, cranial nerve pal­sies, visual field defects, aphasia, movement disor­ders and cerebellar dysfunction. The gray-white matter junction, basal ganglia and thalamus are the areas predisposed to cerebral toxoplasmosis; however, the brainstem and the cor­pus callosum may also be involved. The lesions are usually characterized by a central zone of necrosis containing only few organisms. The central zone is surrounded by a hypervascular intermediate zone, comprised of numerous inflammatory cells mixed with tachyzoites and encysted organisms, the lat­ter being the predominating ingredient of the final, peripheral zone.8 On nonenhanced CT scans, the lesions are usually hypo- to isodense to grey mat­ter with surrounding vasogenic edema and mass effect, while solitary or multiple solid, nodular or ring enhancing lesions are demonstrated on con­trast-enhanced CT scans.8,9 The method of choice for evaluation of mass lesions in the brain remains MRI. On MRI scans, a so-called “target sign” on T2 and FLAIR images, consisting of at least three alter­nating zones, is usually found. The classic constel­lation consists of a hypointense core surrounded by an intermediate hyperintense region and a pe­ripheral hypointense rim, delineated by surround­ing edema. Contrast T1-weighted images show an inverse appearance with ring enhancement of the inflammatory zone.8,9 A highly specific, but rela­tively insensitive finding for cerebral toxoplasmo­sis, seen in less than 30 % of cases, is an asymmetric target sign, exhibiting an off-centre nodular lesion along the wall of the enhancing rim.8,10 Our patient had multiple parenchymal lesions on MRI with only moderate enhancement and surrounding edema. In patients on immunosup­pressant therapy, such as those after bone mar­ row transplant (BMT), cerebral toxoplasmosis may manifest differently than in patients with HIV/ AIDS. Due to a more global loss of immune cells, these patients are usually unable to build a suffi­cient immune inflammatory response at the blood-brain barrier, therefore allowing for passage of gadolinium and vasogenic edema. However, due to a more global loss of the immune system, most of the parenchymal lesions are non-enhancing, al­though mass effect and edema are usually present, and there is only subtle enhancement along the meninges.8,11 In post-BMT patients, parenchymal lesions may undergo hemorrhagic transforma­tion8, as had our patient’s lesions. There are numerous differential diagnoses for cerebral toxoplasmosis in immunocompromised patients, among which CNS lymphoma can often be the most difficult to distinguish it from. Features suggestive of cerebral toxoplasmosis are subcorti­cal location, multiplicity, and asymmetric target sign.8,10 In addition to standard MR imaging, MR spectroscopy, perfusion and diffusion MRI, thal­lium-201 SPECT or 18F-FDG PET/CT8,12-14 can be of further aid in distinguishing cerebral toxoplas­mosis from lymphoma.8,12-14 On 18F-FDG/PET CT scan, which provides remarkable accuracy for detection, treatment monitoring and follow-up of patients with systemic lymphoma as well as of patients with primary central nervous system lym­phoma15, the standardised uptake for cerebral le­sions is much higher than in toxoplasma lesions8. Cerebral toxoplasmosis in HIV-negative patients has been most commonly described in patients af­ter BMT due to underlying lymphoma or leuke­mia. There are only rare reports of toxoplasmosis in patients with hematologic malignancies, inde­pendent of BMT.16,17 Despite clinical advancements and good safety profiles, chemotherapeutics, espe­cially T and B cell depleting regimens, present an important risk factor for infectious complications. Rituximab is a chimeric human-murine mono­clonal antibody, designed to specifically target the transmembrane protein CD20 of B cells. The mechanism of rituximab-induced B cell depletion includes antibody-dependent cell-mediated cyto­toxicity (ADCC), complement-dependent cytotox­icity (CDC) and the direct induction of apoptosis, in addition to enhancing the sensitivity of B cells to chemotherapy and inducing cell cycle arrest.18 Rituximab can deplete peripheral B cells while B cell precursors and mature plasma cells remain rel­atively unaffected, allowing for a new population of B cells to develop from lymphoid stem cells.19 This remarkable activity allowed for its approval by the US Food and Drug Administration (FDA) in 1997 and European Medical Agency (EMA) in 1998 for treatment of CD20 positive cancers, including DLBCL, follicular lymphoma and chronic lym­phocytic leukemia.19,20 In addition, it is also widely used in therapy of autoimmune diseases, including FDA approved therapy of rheumatoid arthritis and various off-label uses.21 In the pre-rituximab era, CHOP regimen was considered the best therapy for DLBCL patients. The addition of rituximab to such chemotherapy (R-CHOP) resulted in signifi­cant improvement of outcomes in DLBCL patients, therefore becoming the preferred treatment regi­men for DLBCL patients.18 Rituximab shows a good safety profile with its main adverse event being infusion reactions, how­ever concerns of increasing the risk of infections are being raised. In addition to a rapid depletion of B cells, which can remain at low or undetectable levels for 2-6 months before returning to baseline levels, rituximab may also cause immunosuppres­sion through several other mechanisms. Growing evidence, supported by an increased incidence of viral infections with the use of rituximab, now suggests rituximab also influences T cell immu­nity and predisposes patients to opportunistic in­fections.19,22 When administered for long periods, such as in maintenance therapy, it also causes delayed-onset neutropenia and hypogamma­globulinemia.22 Several studies have implicated B cells and antibodies (Abs) in host survival and protozoan parasite clearance, especially the B1 cell subpopulation, which appears to be evolutionary selected and maintained to facilitate prompt Ab responses. The B1 cells also appear to modulate T cell response and are implicated in the pathogen­esis of toxoplasmosis through fine tune regulation of the exacerbated Th1 response by secretion of IL­10, and production of Abs against heat shock pro­tein 70 of T. Gondii.23 Parasitic infections have rarely been described in association with rituximab use22, although recently a case of reactivation of cerebral toxoplasmosis in a patient with cutaneous vascu­litis was attributed to rituximab therapy.24 On the other hand, a recent study by Lanini et al. found several factors influencing the risk for infections, independent of rituximab use, among them HIV sero-status, presence of graft-versus-host-disease, type of malignancy and lymphocyte count at na­dir.19 In BMT patients, the risk of latent toxoplasmo­ sis reactivation is highest within 2.4 months post- BMT, but can be prolonged to 6 months, especially in cases of prolonged immune reconstitution.25 In cases of toxoplasmosis in patients with hemato­logical malignancies, the exact time relationship between immunosuppresive therapy and reacti­vation of latent toxoplasma infection is not clearly established. In addition to rituximab, other immu­ nosuppressants (e.g. fludarabine) also influence T cell function and this impact may persist for 1-2 years after discontinuation of therapy.16 Therefore, the emergence of febrile neutropenia in an immu­nosuppressed patient should be an important clini­cal sign, which prompts the physician to look for opportunistic infections. Diagnosis of cerebral toxoplasmosis in immu­nocompromised patients is considered a medi­cal emergency, since undiagnosed and untreated infection can rapidly be lethal. PCR testing has revolutionized the diagnosis of toxoplasmosis al­lowing for early detection of the parasite DNA in the CSF and blood, thereby reducing the need of direct demonstration of tachyzoites in body flu­ids or tissues by conventional methods, such as Giemsa staining.1,3 Serologic testing has only a limited value in immunocompromised patients, nowadays being used mostly as (i) an exclusion criterion when negative for patients, except for haematopoietic stem cell transplant patients, with symptoms consistent of acute toxoplasmosis or (ii) as a monitoring indicator, mainly for solid-organ transplant patients, prompting further investiga­tions in cases of strong increases of IgG titers.1 An IgG avidity ratio can be helpful in establishing the diagnosis of recent toxoplasmic infection in immu­nocompetent patients1,3, with questionable results in immunocompromised patients due to their lack of ability to mount a sufficient immune response. Treatment of cerebral toxoplasmosis comprises either (i) a combination of pyrimethamine plus sul­fadiazine plus leucovorin, (ii) a combination of py­rimethamine plus clindamycin plus leucovorin or (iii) trimethoprim-sulfametoxazole.4,26 Prophylactic therapy is an important factor in toxoplasmosis prevention. In immunocompromised patients with high risk of reactivation of a latent T. gondii infec­tion (eg. HIV patients with CD4+ T cell count less than 100 cells/uL or transplant patients), primary prophylaxis with trimethoprim-sulfametoxazole is usually initiated.5,25,27 Secondary prophylaxis guidelines for patients with HIV/AIDS who have completed initial therapy for cerebral toxoplasmo­sis clearly state that such patients should be admin­istered lifelong suppressive therapy (eg. secondary prophylaxis) unless immune reconstitution occurs as a consequence of antiretroviral therapy, in which case discontinuation of treatment is indicated.26 To our knowledge, there are no such guidelines for other immunocompromised patients. Considering the fact that a combination of pyrimethamine plus sulfadiazine plus leucovorin is highly effective in HIV/AIDS patients26, we decided to proceed with secondary prophylaxis for the duration of mainte­nance therapy with rituximab. If rituximab was to be discontinued in the future, we planned for an additional prolongation of prophylactic therapy until rituximab-mediated depletion of B cell popu­lation would be reversed. Within 10 days of therapy initiation, the effect of medications can already be detected with MRI; there is a decrease in the number and size of the lesions, with a reduction of edema and mass effect. Complete resolution may take as long as 6 months, and healed foci may calcify or show changes con­sistent with leukomalacia.8 Sometimes, a paradoxi­cal worsening of the clinical and radiological pic­ture is seen due to immune reconstitution inflam­matory syndrome (IRIS) after initiation of therapy.8 Our patient showed a good clinical and radiologi­cal response to therapy, but further follow-up will be needed. With this case report, we wish to emphasize the need to consider cerebral toxoplasmosis in patients with malignancies on immunosuppressive therapy when presenting with new cognitive or neurologic deficits. Although to our knowledge, no guidelines for screening of patients for latent toxoplasmosis prior to administration of potent immunosuppres­sants, such as rituximab, exist, we would strongly recommend it. On the basis of serology results, the need for primary prophylaxis against toxoplasmo­sis should then be determined for every patient. Conclusions Despite the widespread prevalence of latent toxo­plasmosis, cerebral toxoplasmosis is an opportun­istic infection that has until now only rarely been reported in patients with hematological malig­nancies, independent of stem cell or bone marrow transplant. Chemotherapeutics, such as rituximab and fludarabine, probably present an important risk factor for reactivation of a latent toxoplasma infec­tion in patients with hematological malignancies. There are numerous differential diagnoses for cerebral toxoplasmosis, among which CNS lym­phoma is the most difficult to distinguish it from. Early recognition and confirmation of cerebral toxoplasmosis is of essential importance, since an undiagnosed and untreated infection presents a life-threatening condition. Management of cerebral toxoplasmosis in immunocompromised patients with malignancies is currently based on guidelines for prevention and treatment of cerebral toxoplas­mosis in patients with HIV/AIDS or BMT patients, for whom abundant epidemiological data exist. With the advent of new chemotherapeutic drugs there is an evolving need for recommendations for prevention and management of toxoplasmosis and other opportunistic infections in patients receiving these agents. References 1. Robert-Gangneux F, Dardé ML. Epidemiology of and diagnostic strategies for toxoplasmosis. Clin Microbiol Rev 2012; 25: 264-96. 2. Kasper LH. Toxoplasma infections. In: Fauci AS, Kasper DL, Longo DL, Braunwald E, Hauser SL, Jameson JL, et al., editors. Harrison’s principles of internal medicine. 17th Edition. New York: McGraw Hill Medical; 2008. p. 1305–11. 3. Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet 2004; 12; 363(9425): 1965-76. 4. Schmidt-Hieber M, Zweigner J, Uharek L, Blau IW, Thiel E. Central nervous system infections in immunocompromised patients: update on diagnostics and therapy. Leuk Lymphoma 2009; 50: 24-36. 5. Yan J, Huang B, Liu G, Wu B, Huang S, Zheng H, et al. Meta-analysis of pre­vention and treatment of toxoplasmic encephalitis in HIV-infected patients. Acta Trop 2013; 127: 236-44. 6. Mele A, Paterson P, Prentice H, Leoni P, Kibbler C. Toxoplasmosis in bone marrow transplantation: a report of two cases and systematic review of the literature. Bone Marrow Transplant 2002; 29: 691-8. 7. Da Cunha S, Ferreira E, Ramos I, Martins R. Cerebral toxoplasmosis after re­nal transplantation. Case report and review. Acta Medica Port 1994; 7: 61-6. 8. Abdel Razek AA, Watcharakorn A, Castillo M. Parasitic diseases of the cen­tral nervous system. Neuroimaging Clin N Am 2011; 21: 815-41. 9. Offiah CE, Turnbull IW. The imaging appearances of intracranial CNS infec­tions in adult HIV and AIDS patients. Clin Radiol 2006; 61: 393-401. 10. Masamed R, Meleis A, Lee EW, Hathout GM. Cerebral toxoplasmosis: case review and description of a new imaging sign. Clin Radiol 2009; 64: 560-3. 11. Ionita C, Wasay M, Balos L, Bakshi R. MR imaging in toxoplasmosis encepha­litis after bone marrow transplantation: paucity of enhancement despite fulminant disease. Am J Neuroradiol 2004; 25: 270-3. 12. Miller RF, Hall-Craggs MA, Costa DC, Brink NS, Scaravilli F, Lucas SB, et al. Magnetic resonance imaging, thallium-201 SPET scanning, and laboratory analyses for discrimination of cerebral lymphoma and toxoplasmosis in AIDS. Sex Transm Infect 1998; 74: 258-64. 13. Camacho DL a, Smith JK, Castillo M. Differentiation of toxoplasmosis and lymphoma in AIDS patients by using apparent diffusion coefficients. Am J Neuroradiol 2003; 24: 633-7. 14. Sarrazin JL, Bonneville F, Martin-Blondel G. Brain infections. Diagn Interv Imaging 2012; 93: 473-90. 15. Maza S, Buchert R, Brenner W, Munz DL, Thiel E, Korfel A, et al. Brain and whole-body FDG-PET in diagnosis, treatment monitoring and long-term follow-up of primary CNS lymphoma. Radiol Oncol 2013; 47: 103-110. 16. Abedalthagafi M, Rushing EJ, Garvin D, Cheson B, Ozdemirli M. Asymptomatic diffuse “encephalitic” cerebral toxoplasmosis in a patient with chronic lymphocytic leukemia: case report and review of the literature. Int J Clin Exp Pathol 2009; 3: 106-9. 17. Touahri T, Pulik M, Fezoui H, Genet P, Lionnet F, Louvel D. Toxoplasmic encephalitis in a non-HIV patient with follicular lymphoma. Int J Hematol 2002; 75: 111-2. 18. Marcus R, Hagenbeek A. The therapeutic use of rituximab in non-Hodgkin’s lymphoma. Eur J Haematol Suppl 2007; 67: 5-14. 19. Lanini S, Molloy AC, Prentice AG, Ippolito G, Kibbler CC. Infections in patients taking rituximab for hematologic malignancies: two-year cohort study. BMC Infect Dis 2013; 13: 317. 20. Eisenberg R. Update on rituximab. Ann Rheum Dis 2005; 64(Suppl 4): iv55-7. 21. Gürcan HM, Keskin DB, Stern JNH, Nitzberg MA, Shekhani H, Ahmed AR. A review of the current use of rituximab in autoimmune diseases. Int Immunopharmacol 2009; 9: 10-25. 22. Kelesidis T, Daikos G, Boumpas D, Tsiodras S. Does rituximab increase the incidence of infectious complications? A narrative review. Int J Infect Dis 2011; 15: e2–16. 23. Amezcua Vesely MC, Bermejo DA, Montes CL, Acosta-Rodríguez EV, Gruppi A. B-Cell response during protozoan parasite infections. J Parasitol Res 2012; 2012: 362131. 24. Safa G, Darrieux L. Cerebral toxoplasmosis after rituximab therapy. JAMA Intern Med 2013; 173: 924-6. 25. Derouin F, Pelloux H. Prevention of toxoplasmosis in transplant patients. Clin Microbiol Infect 2008; 14: 1089-101. 26. Kaplan JE, Benson C, Holmes KK, Brooks JT, Pau A, Masur H. Guidelines for prevention and treatment of opportunistic infections in HIV-infected adults and adolescents: recommendations from CDC, the National Institutes of Health, and the HIV Medicine Association of the Infectious Diseases Society of America. MMWR Recomm Rep 2009; 58(RR-4): 1-207. 27. Soave R. Prophylaxis strategies for solid-organ transplantation. Clin Infect Dis 2001; 33(Suppl 1): S26-31. research article Obstructive urination problems after high-dose-rate brachytherapy boost treatment for prostate cancer are avoidable Borut Kragelj Institut of Oncology Ljubljana, Zaloska 2, Ljubljana, Slovenia Radiol Oncol 2016; 50(1): 94-103. Received 15 October 2014 Accepted 20 January 2015 Correspondence to: Assist. Prof. Borut Kragelj, M.D., Ph.D., Institute of Oncology Ljubljana, Zaloska 2, Ljubljana, Slovenia. Phone: +386 1 5879 489; E-mail: bkragelj@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. Aiming at improving treatment individualization in patients with prostate cancer treated with combina­tion of external beam radiotherapy and high-dose-rate brachytherapy to boost the dose to prostate (HDRB-B), the objective was to evaluate factors that have potential impact on obstructive urination problems (OUP) after HDRB-B. Patients and methods. In the follow-up study 88 patients consecutively treated with HDRB-B at the Institute of Oncology Ljubljana in the period 2006-2011 were included. The observed outcome was deterioration of OUP (DOUP) during the follow-up period longer than 1 year. Univariate and multivariate relationship analysis between DOUP and potential risk factors (treatment factors, patients’ characteristics) was carried out by using binary logistic regression. ROC curve was constructed on predicted values and the area under the curve (AUC) calculated to assess the per­formance of the multivariate model. Results. Analysis was carried out on 71 patients who completed 3 years of follow-up. DOUP was noted in 13/71 (18.3%) of them. The results of multivariate analysis showed statistically significant relationship between DOUP and anti-coagulation treatment (OR 4.86, 95% C.I. limits: 1.21-19.61, p = 0.026). Also minimal dose received by 90% of the urethra volume was close to statistical significance (OR = 1.23; 95% C.I. limits: 0.98-1.07, p = 0.099). The value of AUC was 0.755. Conclusions. The study emphasized the relationship between DOUP and anticoagulation treatment, and suggested the multivariate model with fair predictive performance. This model potentially enables a reduction of DOUP after HDRB-B. It supports the belief that further research should be focused on urethral sphincter as a critical structure for OUP. Key words: prostate cancer; high-dose-rate brachytherapy boost; late effects; urinary stricture; obstructive urination problems. Introduction Several modes of radical local treatment are on disposal for patients with localized or locally ad­vanced prostate cancer. Beside radical prostatecto­my, radical irradiation in the form of external beam radiotherapy (EBRT), and permanent brachythera­py (PB) or high-dose-rate brachytherapy (HDRB) are established ways of treatment. Both treatment modalities should be considered as equally effec­tive in the absence of randomized trials. Similar consideration should also be given to different ways of radiation treatment.1,2 When radiation therapy is applied, EBRT could be combined with either form of brachytherapy. The combination of EBRT and HDRB to boost the dose to prostate (HDRB-B) is effective treatment, according to some reports more effective than EBRT alone.3-5 Any of above mentioned treatments could ex­pose patients to late side effects. Especially long-term consequences could be decisive for patients’ determination for one or other treatment. Well known long-term complications are bladder neck contractures after radical prostatectomy6, and ure­thral strictures after PB7,8 or HDRB-B.9-11 In HDRB-B obstructive urination problems (OUP), including urethral strictures, are the most frequent urinary severe late effect. According to results of studies in the past the frequency of only strictures was 1.5.9%.9-12 Some recent stud­ies report even considerably higher frequency.4,13 Previous transurethral resection of the prostate (TURP)14-16, fractionation schedule of HDRB-B with increased risk with higher fractional dose13, older age14, and hypertension15, were considered as po­tential patient-related risk factors but their role was not clearly defined.17 Furthermore, there were found no reliable normal tissue absolute dose con­straints for urinary toxicity.17 This high stricture risk with problems that evolve with stricture formation should be factored into counselling all men who are considering HDRB-B and could curtail patient’s decision for HDRB-B, since with refinement of radical prostatectomy, or PB, the expected frequency of these marked lower urinary tract OUP could be considerably lower.18-20 At the Ljubljana Institute of Oncology HDRB-B was started in October 2006, and up to now, late ef­fects, including OUP and stricture formation, have not been evaluated yet. Aiming at improving treatment individualiza­tion in patients with prostate cancer treated with HDRB-B, the objective of the present study was to evaluate factors that have potential impact on OUP after HDRB-B. Patients and methods Patients In the follow-up study 88 patients, consecutively treated by the author with HDRB-B at the Institute of Oncology Ljubljana in the period 2006.2011, were included. HDRB-B treatment was primarily offered to patients with intermediate- or high-risk clinically localized or locally advanced prostate cancer (ac­cording to D’Amico risk stratification of prostate cancer patients)21 and to low risk patients refused to get radical prostatectomy, if feasible for brachy­therapy. In general, patients were considered eligi­ble for HDRB-B if (i) ultrasound showed no pubic arch interference, (ii) were eligible to undergo re­gional anaesthesia with spinal block, and (iii) were eligible to perform CT/MRI scan. TURP was con­sidered as a contraindication for the procedure. Study protocol was approved by the Protocol and Ethical Committee of the Institute of Oncology Ljubljana. Treatment characteristics Brachytherapy consisted of ultrasound guided transperineal insertion of 20 or 30 cm long close-end plastic needles (Varian, California, USA) into the prostate, and in selected patients also into the initial part of seminal vesicles. An in-house made template allowing needle fixation was used. Needles were typically placed into prostate pe­riphery. Cystoscopy was performed to control for urinary bladder or urethral puncture. CT or MRI scan was used for planning purposes. Brachyvision planning system (Varian, California, USA) was used for image registration, contouring and do­simetry. Two planning target volumes (PTVs) were routinely defined: PTV1 and PTV2. PTV1 encircled the prostate with additional 3 mm margin around the zone of suspected capsular invasion, while PTV2 encircled peripheral part of the prostate. If visible on the MRI images, also gross tumour vol­ume (GTV) was defined and included in the PTV2. Initially, prescribed dose was 21 Gy to PTV1 and 22.5 Gy to PTV2, with 7 Gy and 7.5 Gy per frac­tion, respectively. Later, the dose was reduced to 18 Gy to PTV1 and 19.5 Gy to PTV2, also given in 3 fractions in 2 consecutive days. Urethra was in­dentified with the urinary catheter. The contour also enclosed additional 1-2 mm margin around the catheter. Contouring of urethra started at bladder base and extended to genitourinary dia­phragm inferiorly (always at least 0.5 cm caudally from the last slice of contoured apex of the pros­ tate). Minimal dose received by 90% of the urethra ) was planned to be below volume (D90urethra volume 110%, while minimal dose received by 1% of the urethra volume (D1urethra volume) was planned to be below 130% of the prescribed dose. Treatment was delivered with Gammamed plus device (Varian, California, USA) using 192Iridium with the activity of 0.7-1.4 Ci. EBRT was delivered as 3-dimensional conformal radiation. The details of technique have been de­scribed elsewhere.22 The patients were simulated in supine position with knee and feet fixation device and urethrogram, to define prostatic apex. Clinical target volume (CTV) included prostate and distal 2/3 of seminal vesicles with lymph nodes along external, internal and common iliac vessels in pa­tients with Gleason Score (GS) 8.10 or locally ad­vanced tumours, or if the risk of positive lymph TABLE 1. Questions addressing obstructive urination problems in Ljubljana Institute of Oncology questionnaire about adverse health effects of radiation therapy Did you have a sensation of not emptying your bladder in the previous month Yes/No Did you find stopping and starting again several times when you urinated in the previous month Yes/No Did you have weak urinary stream in the previous month Yes/No Did you have to strain to start urination in the previous month Yes/No Occasionally If you had any of the problems, how often did they occur At least once a week Daily At every urination No problem Very small problem How big were these problems for you Small problem Moderate problem Big problem Did you have to get urinary catheter in the last half of the year Yes/No Were you operated because of the mentioned problems Yes/No Did you still have urinary catheter Yes/No nodes (RiskN+) exceeded 15% according to the equation of Roach et al. (Equation 1).23 [Equation 1] PSA = the pre-treatment prostate-specific antigen GS = the pre-treatment Gleason score Uniform 1 cm margin was added to CTV to form PTV. Prior to treatment planning three gold­en markers were implanted into the prostate. If treatment started with HDRB treatment, markers were implanted together with needle insertion. Prescribed dose was 50.4 Gy. The PTVs were re­quired to be enclosed in 95% isodose relative to the prescribed dose. Basically, box technique was used, with additional small fields to homogenize the dose delivery. All patients were treated using 15 MV photons. During treatment prostate posi­tion was determined using MV portal imaging and implanted markers. Daily off-line position correc­tion was used. A constitutive part of treatment was also the an­drogen deprivation therapy. In principle 3 years of androgen deprivation was advised to high-risk pa­tients and also to some intermediate-risk patients with cancer overgrowth in the majority of biopsy cores or with MRI evidenced infiltration of peripro­static tissue or seminal vesicles. One year of andro­gen deprivation was advised to the rest of interme­diate-risk patients. In low-risk patients androgen deprivation therapy was given either to reduce the prostate volume, or was initiated by the referring urologist after prostate biopsy and continued after­wards until the end of radiation treatment. Patients on androgen deprivation were followed-up every 6 months. After the discontinuation of androgen deprivation, patients were seen yearly, with PSA testing every six months. After the first follow-up visit 3.6 months after treatment, the same way of annual check-ups was used in patients without ad­juvant androgen deprivation. Study instrument for assessment of obstructive urination problems An in-house made questionnaire, used already for several years, was used as the study instrument. The questionnaire was discussed with patients at follow-up visits. The aim of the questionnaire was to detect late effects of treatment more pre­cisely as would be by open questions, and to al­low to grade late toxicity according to Radiation Therapy Oncology Group (RTOG)24 subjective part of RTOG/EORTC Soma Scales25 and Common Terminology Criteria for Adverse Events Ver. 3.0 (CTCAE). Late urinary toxicity was addressed with regard to dysuria, frequency, haematuria, inconti­nence and obstruction. In Table 1 the questions ad­dressing OUP are presented. Patients were asked to complete the questionnaire just before the start of the HDRB-B treatment, at first follow-up visit and yearly thereafter. Observed outcome The observed outcome was deterioration of OUP (DOUP) during the follow-up period longer than 1 year, supposing to be a manifestation of late radia­tion urethral injury. It was defined in several steps. Firstly, the presence of OUP just before the start of the HDRB-B treatment was established. The problems were graded according to the following scale: 1-occasional (less than weekly), 2-regular (about daily), 3-regular (daily) with at least one episode of urgent urethral catheter placement, 4-regular (daily) with at least one episode of ure­thral dilatation or endoscopic intervention, 5-re­fractory obstruction (permanent urinary catheter, supravesical urine derivation). During the follow-up period the grade of OUP was checked-up in the 2nd, the 3rd, the 4th and the 5th year after the beginning of the HDRB-B treatment. Finally, the difference in grade of OUP between OUP at the start of the HDRB-B treatment and lat­ter follow-up visits was assessed. The following scale was used: 1-major improvement (decrease in OUP for two or more grades), 2-minor im­provement (decrease in OUP for one grade), 3-no change, 4-minor deterioration (increase in OUP for one grade), 4-major deterioration (increase in OUP for two or more grades). Since minor and major deterioration were the categories of interest, these two categories were combined in the observed out­come - DOUP (0 = no, 1 = yes). In order to achieve a sufficiently large number of observed persons, analysis of association be­tween observed outcome and potential risk factors was carried out only in patients who completed 3 years of follow-up. The occurrence of DOUP in the 2nd or in the 3rd year of follow-up was considered. Risk factors for deterioration of obstructive urination problems Two groups of risk factors were observed. The first group consisted of HDRB-B and supportive treat­ment factors, while the second group consisted of patients’ characteristics. In the group of HDRB-B and supportive treat­ment factors following factors were observed: num­ber of implanted needles (Nimplanted needles), planning imaging (1 = CT, 2 = MRI), number of interventions ) (0 = 1, 1 = 2+), and dosimetric factors, (Ninterventions being PTV1, minimal dose received by 100% of the PTV1 (D100PTV1), minimal dose received by 90% of the PTV1 (D90PTV1), minimal dose received by 100% of the PTV2 (D100PTV2), urethral volume (Vurethra), mean urethral dose (D-MEANurethra ), D90urethra vol­ ume, minimal dose received by 10% of the urethra . All of volume (D10urethra volume), and D1urethra volume dosimetric factors were retrospectively extracted from Dose-Volume Histograms stored in electronic patients’ files. As supportive treatment factor the duration of androgen deprivation therapy was in­ cluded (0 = < 12 months, 1 = . 12 months). In the group of patients’ characteristics the fol­lowing factors were observed: age, co-morbidity (hypertension: 0 = no, 1 = yes, diabetes: 0 = no, 1 = yes, coronary insufficiency: 0 = no, 1 = yes, hyper­lipidemia: 0 = no, 1 = yes, history of cerebrovascular insult or peripheral deep venous thrombosis: 0 = no, 1 = yes), and anticoagulant treatment (vitamin K antagonist or antiplatelet drug) (0 = no, 1 = yes). All of them were extracted from patients’ files. Statistical analysis All data were first statistically described. Parametric (mean ± standard deviation, minimum and maxi­mum) or nonparametric methods (median, range) for numerical data, or percentages for attributable data were used. Afterwards the univariate and multivariate rela­tionship analysis between DOUP and potential risk factors (treatment factors, patients’ characteristics) was carried out. Univariate analysis was carried out by using binary logistic regression or Fisher’s exact test (in one variable logistic regression analy­sis could not be performed due to no observed out­come in one category). In multivariate analysis bi­nary logistic regression was used. All variables that were meaningful for the observed outcome and univariately at least marginally statistically signifi­cantly associated with DOUP (p < 0.250) were in­cluded in the multivariate model.26 On the basis of logistic regression model, the risk-score (logit) for each participant was calculated (Equation 2) and afterwards converted to the risk estimates (p(x)) for the observed outcome (Equation 3).26 [Equation 2] [Equation 3] Finally, the risk estimate values were put in an ordered series from the lowest to the highest value. TABLE 2. Characteristics of dosimetric parameters of high-dose-rate brachytherapy in Ljubljana Institute of Oncology study of late toxicity after high-dose-rate brachytherapy boost treatment for prostate cancer PTV1 18 ml 95 ml 37.6 ± 14.8 ml D100PTV1 8.3 Gy 17.1 Gy 11.8 ± 1.9 Gy D90PTV1 13.4 Gy 24.6 Gy 19.2 ± 2.0 Gy D100PTV2 12.8 Gy 23.7 Gy 17.3 ± 2.1 Gy Vurethra 1.2 ml 4.0 ml 1.9 ± 0.8 ml D-MEANurethra 14.4 Gy 26.1 Gy 19.0 ± 2.6 Gy D90urethra volume 7.5 Gy 18.9 Gy 13.2 ± 2.9 Gy D10urethra volume 17.8 Gy 31.1 Gy 23.1 ± 2.7 Gy D1urethra volume 17.3 Gy 36.0 Gy 24.5 ± 3.4 Gy = minimal dose received by 100% of the PTV1; D90PTV1 = minimal dose received by 90% of the PTV1; D100PTV2 = minimal dose received by 100% of the PTV2; PTV1 = planning target volume 1; PTV2 = planning target volume 2; D-MEANurethra = mean urethral dose; D90urethra volume = minimal dose received by 90% of the urethra volume; D10urethra volume = minimal dose received by 10% of the urethra volume; D1urethra volume = minimal dose received by 1% of the urethra volume; Vurethra = urethral volume D100PTV1 At every value the cut-point was placed, a decision matrix defined taking into account the actual status of presence/absence of observed outcome, and no­sological (true-positive, false-positive, true-nega­tive and false-positive rates) as well diagnostic test validity measures (positive and negative predic­tive values) calculated.27-29 Also the receiver oper­ating characteristic (ROC) analysis was performed (ROC curve constructed and the area under the curve calculated).29 Decision about the best possi­ble cut-point (the cut-point with the highest true positive rate at the highest acceptable false positive rate) was supported by calculating Youden index (the maximum vertical distance between the ROC curve and the diagonal/chance line).30 In all statistical tests p-value 0.05 or less was considered significant. SPSS statistical package for Windows Ver. 21.0 was used for analysis. Results Description of the study group Patients in the study group were aged 67.6 ± 6.1 years, with following tumour characteristics: Gleason score: L 6 22/88 (25.0%), 7 37/88 (42.0%), . 8 29/88 (33.0%); percent of positive cores: median 50 (range 10.100); PSA: median 10 ng/ml (range 4.60 ng/ml); stage: T1 18/88 (20.5%), T2 40/88 (45.5%), T3 30/88 (34.0%); risk category: low 13/88 (14.8%), intermediate 27/88 (30.7%), high 48/88 (54.5%). Androgen deprivation treatment was received by 81/88 (92.0%) patients (median duration 24 months (1.60 months); duration less than 12 months: 10/81 (12.3%) patients). In most patients the comorbidities were present. The most frequent was hypertension (46/86, 53.5%), followed by hyperlipidemia (20/85, 23.5%), coro­nary insufficiency (17/86, 19.8%), history of cerebro­vascular insult or peripheral deep venous thrombo­sis (11/86, 12.8%) and diabetes (10/85, 11.8%). During the HDRB-B treatment 22/86 (25.6%) of patients were receiving anticoagulation therapy. Description of the treatment Regarding the dose to PTV1, 21 Gy was delivered to 27/88 (30.7%) patients, while 18 Gy to 61/88 (69.3%) patients. Target volume was restricted to prostate in 79/88 (89.8%), while in 9/88 (10.2%) of patients it was enlarged to enclose infiltrated parts of semi­nal vesicles. Dosimetric parameters of HDRB-B ap­plied in the study are summarized in Table 2. Mean Nimplanted needles was 15±3, while Ninterventions was one in 80/88 (90.9%), and two or more in 8/88 (9.1%) patients. CT based planning was used in 30/88 (34.1%), while MRI was used in 58/88 (65.9%) patients. Obstructive urination problems at the beginning of the study At the start of treatment OUP were declared in 52/82 (63.4%) patients that had complete entry da­ta. In majority of them problems were not signifi­ cant and were assessed as grade 1 in 46/52 (88.5%). In the rest of patients, problems were more pro­ nounced and assessed as grade 2 in 5/52 (9.6%) and as grade 3 in 1/52 (1.9%) patients. Analysis of deterioration of obstructive urination problems during the follow-up period The course of OUP after treatment in relation to initial problems is presented as a prevalence rates during the 2nd, the 3rd, the 4th and the 5th year of observation (Table 3). In this time frame the pro­portion of patients with DOUP after initial increase remained stable. On the other hand it seemed that the proportion of patients that experienced improvement of initial obstructive problems in­creased (Table 3). TABLE 3. Prevalence rates of alteration of obstructive urination problems in Ljubljana Institute of Oncology study of late toxicity after high-dose-rate brachytherapy boost treatment for prostate cancer 2nd year 80 1 (1.3%) 10 (12.5%) 57 (71.3%) 12 (15.0%) 0 (0%) 3rd year 71 1 (1.4%) 11 (15.5%) 51 (71.8%) 8 (11.3%) 0 (0%) 4th year 45 2 (4.4%) 7 (15.6%) 31 (68.9%) 4 (8.9%) 1 (2.2%) 5th year 25 0 (0%) 6 (24.0%) 16 (64.0%) 3 (12.0%) 0 (0%) Analysis of association between deterioration of obstructive urination problems and potential risk factors Analysis of association between observed outcome and potential risk factors was carried out in 71 pa­tients who completed 3 years of follow-up. DOUP occurred in the 2nd or in the 3rd year in 13/71 (18.3%) of patients. In the group of HDRB-B and supportive treat­ment factors none of them was statistically sig­nificantly associated with the DOUP (Table 4). However, according to predefined criterion PTV1 were candidates for entering the and D90urethra volume multivariate analysis. In the group of patients’ characteristics also the vast majority of factors did not show statistically significant association with DOUP (Table 5). The only exception was anticoagulation treatment in which association with observed outcome was sta­tistically significant, and thus was a candidate for entering the multivariate analysis. In the majority of patients receiving this treatment, it consisted of acetyl salicylic acid either alone (11/17 patients) or in the combination with warfarin (2/17 patients). Tidapidine was given to 2/17, warfarin to 1/17, TABLE 4. Results of univariate logistic regression analysis of association between deterioration of obstructive urination problems and treatment factors in Ljubljana Institute of Oncology study of late toxicity after high-dose-rate brachytherapy boost treatment for prostate cancer Nimplanted needles 71 0.89 0.71 1.11 0.305 Planning imaging CT 71 3/25 (12.0%) 1.00 MRI 10/46 (21.7%) 2.04 0.51 8.22 0.317 Ninterventions 1 71 10/63 (15.9%) 1.00 2+ 3/8 (37.5%) 3.18 0.65 15.48 0.152 PTV1 (ml) 70 1.03 0.99 1.07 0.149 D100PTV1 (Gy) 71 1.08 0.76 1.53 0.656 D90PTV1 (Gy) 71 0.93 0.68 1.28 0.673 D100PTV2 (Gy) 70 0.99 0.75 1.31 0.941 Vurethra (ml) 70 1.22 0.55 2.74 0.626 D-MEANurethra (Gy) 70 1.06 0.84 1.35 0.629 D90urethra volume (Gy) 71 1.18 0.95 1.47 0.145 D10urethra volume (Gy) 70 1.07 0.87 1.31 0.521 D1urethra volume (Gy) 71 1.03 0.86 1.22 0.780 Androgen deprivation < 12 months 69 2/9 (22.2%) 1.00 . 12 months 11/60 (18.3%) 0.79 0.14 4.31 0.781 Ntot=total number of observations, Ndet= number of patients with deterioration; Ncat= number of patients within the category; Nimplanted needles = number of implanted needles; Ninterventions = number of interventions; PTV1 = planning target volume 1; PTV2 = planning target volume 2; D100PTV 1 = minimal dose received by 100% of the PTV1; D90PTV1 = minimal dose received by 90% of the PTV1; D100PTV2 = minimal dose received by 100% of the PTV2; Vurethra = urethral volume; D-MEANurethra = mean urethral dose; D90urethra volume = minimal dose received by 90% of the urethra volume; = minimal dose received by 10% of the urethra volume; D1urethra volume = minimal dose received by 1% of the urethra volume D10urethra volume 100 TABLE 5. Results of univariate logistic regression analysis of association between deterioration of obstructive urination problems and patients’ characteristics in Ljubljana Institute of Oncology study of late toxicity after high-dose-rate brachytherapy boost treatment for prostate cancer Age 70 1.05 0.94 1.17 0.372 Hypertension No 69 5/32 (15.6%) 1.00 Yes 7/37 (18.9%) 1.26 0.36 4.44 0.719 Diabetes No 68 12/62 (19.4%) NA Yes 0/6 (0.0%) NA NA NA 0.581* Hyperlipidemia No 68 8/52 (15.4%) 1.00 Yes 4/16 (25.0%) 1.83 0.47 7.14 0.382 CVI No 69 10/62 (16.1%) 1.00 Yes 2/7 (28.6%) 2.08 0.35 12.26 0.418 Coronary insufficiency No 69 8/54 (14.8%) 1.00 Yes 4/15 (26.7) 2.09 0.53 8.22 0.291 Anticoagulation treatment No 69 6/52 (11.5%) 1.00 Yes 6/17 (35.5%) 4.18 1.13 15.48 0.032 Ntot=total number of observations, Ndet= number of patients with deterioration; Ncat= number of patients within the category; NA = not applicable; * = Fisher exact test results; C.I. = confidence interval; CVI = history of cerebrovascular insult or peripheral deep venous thrombosis; OR = odds ratio TABLE 6. Results of multivariate logistic regression analysis of association between deterioration of obstructive urination problems and selected treatment factors and patients’ characteristics in Ljubljana Institute of Oncology study of late toxicity after high-dose-rate brachytherapy boost treatment for prostate cancer (N = 68) PTV1 (ml) 1.02 0.98 1.07 0.292 D90urethra volume (Gy) 1.23 0.96 1.57 0.099 No 1.00 Anticoagulation treatment Yes 4.86 1.21 19.61 0.026 = minimal dose received by 90% of the urethra volume; OR = odds ratio; PTV1 = planning target volume 1; OR = odds ratio D90urethra volume while acenocoumarol to 1/17 patients. In the group of 6 patients with DOUP, anticoagulation treat­ment consisted of acetyl salicylic acid in 4 patients, a combination of acetyl salicylic acid and warfarin in one, and warfarin alone in one patient. All data necessary to perform multivariate anal­ ysis were present in 68/71 patients (95.8%). The results of the logistic regression model showed that anticoagulation treatment not only remained statistically significantly associated with observed outcome but even increased (the odds for DOUP were about 4.9-times higher in patients on anti-coagulation treatment). In addition, D90urethra volume came closer to the border of statistical significance (for a one-Gy increase in D90urethra volume, about 23% increase in odds of experiencing observed outcome could be expected). All other results are presented in Table 6. Finally, the risk of DOUP was estimated for each patient. The values varied between 0.02509 and 0.62421, with the median value 0.10973. The value of area under ROC curve was 0.755, indicating fair predictive performance of the model. The best cut-point was placed at value 0.16441 (true positive rate: 9/12 or 0.750; false positive rate: 16/56 or 0.286; true negative rate: 40/56 or 0.714; false negative rate: 3/12 or 0.250; positive predictive value: 9/25 or 0.360; negative predictive value: 40/43 or 0.930; Youden index: 0.464). Discussion The main results of the study The most prominent result of present study was strong association of DOUP after HDRB-B with anticoagulation treatment. Based on the available literature this association has not been reported yet up to now. Generally, patient-related risk factors, with exception of age and prior TURP, were only 101 exceptionally considered in the studies of OUP after HDRB-B. In the available literature only one study that considered patients’ characteristics could be found. In this study hypertension was identified as an independent predictor of urinary tract obstruc­tion grade 2 or more according to CTCAE after HDRB-B, but anticoagulation treatment was not considered.15 Anticoagulation treatment, however, was found to be, together with total dose, the most important predictive factor for 5-year risk of global urinary toxicity in a large study of 965 patients who received definitive EBRT. One of the conclusions of the study was that urinary toxicity might be more related to patients’ risk factors than dose parame­ters.31 A similar conclusion can be drawn on the ba­sis of results of present study. They pointed out that perhaps higher dose sensitivity of urethral sphinc­ter region in comparison to the urethra, as already suggested by Hindson13, is further increased with anticoagulation treatment (with almost 5-times higher odds of DOUP in patients receiving either vitamin K antagonists or antiplatelet agents in pre­sent study). One can only speculate about exact ae­tiology of this increased radiosensitivity. Since anti-coagulation treatment is as a rule considered in pa­tients with vasculopathy, poor circulation could be the underlying cause. However, other parameters that implicate this mechanism and were considered in present study (hypertension, diabetes, hyperlipi­demia, history of cerebrovascular insult and deep venous thrombosis) did not show significant asso­ciation with the observed outcome. Other important results of the study Additionally, study offered other results that could be interesting. Urethra is, as suggested by Hsu32, with regard to late urinary toxicity, a dose limiting structure. In present study D90urethra volume expressed the strongest, although statistically only marginal, association with observed outcome. This relation of dose applied to the major part of urethra to OUP seems to be reasonable as the location of stricture formation is at or beyond the prostatic apex, which is at the margin or beyond the contoured ure­thra.13,15,33 Apparently, this is in the area of external urethral sphincter. This way it is more likely that the dose applied to the major part of urethra is a better representative of the actual sphincter dose than are dose parameters that represent high doses to small parts of (contoured) urethra. Some similarity can be found between the significance of the D90urethra volume and the minimum dose to bulbomembranous ure­thra which was found to be (with regard to maxi­mum prostatic urethral dose, the mean, maximum and minimum bulbomembranous urethral doses, and bulbomembranous urethral doses 10 and 15 mm from apex) the stongest predictor of stricture formation in a large study of patients treated with PB.18 However, in two studies that addressed whole range of dose-volume histogram urethral data, and related them to late urinary toxicity grade 2 or higher according to CTCAE32, or late urinary tox­icity grade 3 or higher according to RTOG34, small urethral volumes and high doses were emphasized. In the study of Ischiyama this was the volume that received minimal 13 Gy per fraction with the pre­scribed dose of 5×7.5 Gy, and in the study of Hsu multiple dose levels above 110% of the prescribed 19 Gy in 2 fractions. Although in 8/12 patients with grade 3 toxicity in the study of Ischiyama was due to stricture formation, studies that focused only on strictures, failed to prove the value of high-dose urethral volumes. In the study of Hindson this was the minimal dose received by the »hottest« 10% of the urethral volume13, and in the study of Ghadjar the minimal dose to the urethral volumes that re­ ceived at least 100%, 120%, 125% of the prescribed target dose and the minimal dose received by the »hottest« 1% urethral volume.35 Both were negative as no significant association was found with 5-year stricture-free survival in the study of Ghadjar, and no correlation was recorded within dose groups of 18 Gy in 3 fractions, 20 Gy in 4 fractions, 19 Gy in 2 fractions, or 16 Gy in 2 fractions between D10urethra and stricture risk in the study of Hindson. volume Nevertheless, low-dose urethral volumes were decisive for obstructive or any other urinary toxic­ity after HDRB-B. We can only speculate that with analogy to EBRT data, different dose-volume his­togram parameters should be emphasized for dif­ferent grades of toxicity. In all of patients that were included in the present study, OUP would either be missed if RTOG criteria were used, or graded as grade 1 morbidity according to CTCAE. As it is suggested by the results of present study, low-dose parameters may be crucial for this low-grade uri­nary toxicity. Another parameter that was included in pre­dictive model in present study was PTV1. It was also only marginally significantly associated with observed outcome and was included in the final model primarily due to results of other studies that reported positive correlation between late genitou­rinary toxicity and PTV.32,36 Additionally, the study of Pinkawa et al. showed that PTV may relate to the length of prostatic urethra that is also predictive for late genitourinary toxicity.37 102 Limitations of the study The presented study has some potential limita­tions. Firstly, one could argue that in the study the internationally validated International Prostate Symptom Score (I-PSS) Questionnaire was not used as a study instrument instead of in-house made questionnaire. This is certainly a limitation, how­ever, the in-house made questionnaire was used already for several years at Ljubljana Institute of Oncology and replacing the questionnaire would affect the comparability of responses in time. Secondly, a shortcoming of the study was that pa­tients with OUP did not undergo cystoscopic evalu­ation so it was unclear weather OUP were actually a consequence of urethral obstruction. The deduction from OUP to urethral stricture, at least if assessed by I-PSS, may not be so straightforward, as it has been already shown.13 Thirdly, some caution may be posed also to two different prescribed target doses. However, as the technique and the fractiona­tion remained the same and not prescribed doses but dose volume parameters obtained at planning were considered, this concern may be redundant. Perhaps more plausible may be the effect of either CT or MRI based planning. It may be expected that the dose that urethra was actually exposed is dif­ferent when CT or MRI based planning is used. Delineated volume of urethra is different with dif­ferent imaging technique -urethra with inserted urinary catheter is more clearly visible on MRI and impression is that urethral volumes are larger on MRI even if urethral sphincter is not considered, in contrast to other organs involved in the contouring of prostate cancer.38 Finally, according to merely statistical criterion also Ninterventions could be consid­ered in the model. However, as this factor primarily reflects excessive needle movement during treat­ment that could not be predicted in the phase of treatment planning, it was not treated as important in the present study. Actually, only one interven­tion was planned in all patients. Exact evaluation of needle movement with regard to apical fiducial marker using orthogonal x-ray or CT images was done only before third fraction was applied in the morning next day after needle placement. Separate intervention was considered only if needle move­ment could not be compensated with additional planning. This primarily caudal needle transloca­tion after HDRB-B is well documented but poten­tial detrimental effect, as perhaps suggested by the results of presented study, is less obvious. The importance of the study results for clinical practice The study stresses the importance of clinical pa­tients’ data in the evaluation of late toxicity after HDRB-B. Considering clinical patients’ data to­gether with treatment and dosimetric parameters it is possible to estimate toxicity of HDRB-B more precisely and also give better opportunity to allevi­ate side effects. The implementation of results of presented study can hopefully reduce OUP after HDRB-B, and can perhaps also reduce stricture formation that requires surgical intervention. Possibilities for further research in the field Further research in the field should be focused in improvement of safety of HDRB-B treatment. For improvement of safety in terms of late adverse ef­fects it is vital to recognize structures which could be at risk for certain type of toxicity. It seems that the critical structure for OUP may be urethral sphincter. However, it is needed to confirm a re­lation between urethral sphincter, potential risk factors and OUP. Among potential risk factors also anticoagulation treatment should be considered. Conclusions Treatment factors as well as patients’ characteris­tics that are associated with OUP, and can predict it, and eventually prevent overt stricture forma­tion after HDRB-B, are not sufficiently known. The study emphasizes the relationship between DOUP and anticoagulation treatment and suggests a fair predictive performance of the model which includes its high negative predictive value. It sup­ports the belief that further research should be fo­cused on urethral sphincter as a critical structure for OUP. References 1. 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Urethral strictures following high-dose-rate brachytherapy for prostate cancer: analysis of risk factors. Brachytherapy 2013; 12: 50-5. 14. Martínez-Monge R, Moreno M, Ciérvide R, Cambeiro M, Pérez-Gracia JL, Gil-Bazo I, et al. External-beam radiation therapy and high-dose-rate brachy­therapy combined with long-term androgen deprivation therapy in high and very high prostate cancer: preliminary data on clinical outcome. Int J Radiat Biol Phys 2012; 82: 469-76. 15. Sullivan L, Williams SG, Tai KH, Foroudi F, Cleeve L, Duchesne GM. Urethral stricture following high dose rate brachytherapy for prostate cancer. Radiother Oncol 2009; 91: 232-6. 16. Galalae RM, Kovács G, Schultze J, Loch T, Rzehak P, Wilhelm R, et al. Long-term outcome after elective irradiation of the pelvic lymphatics and local dose escalation using high-dose-rate brachytherapy for locally advanced prostate cancer. Int J Radiat Biol Phys 2002; 52: 81-90. 17. Yamada Y, Rogers L, Demanes DJ, Morton G, Prestidge BR, Pouliot J, et al. American brachytherapy society consensus guidlines for high-dose-rate prostate brachytherapy. Brachytheapy 2012; 11: 20-32. 18. Merrick GS, Butler WM, Wallner KE, Galbreath RW, Anderson RL, Allen ZA, et al. Risk factors for the development of prostate brachytherapy related urethral strictures. J Urol 2006; 175: 1376-81. 19. Parihar JS, Ha YS, Kim IY. Bladder neck contracture-incidence and man­agement following contemporary robot assisted radical prostatectomy. Prostate Int 2014; 1: 12-8. 20. Breyer BN, Davis CB, Cowan JE, Kane CJ, Carroll PR. Incidence of bladder neck contracture after robot-assisted laparoscopic and open radical prosta­tectomy. BJU Int 2010; 106: 1734-8. 21. D’Amico AV, Whittington R, Malkowicz SB, Schultz D, Blank K, Broderick GA, et al. A. Biochemical outcome after radical prostatectomy, external beam radiation therapy, or interstitial radiation therapy for clinically localized prostate cancer. JAMA 1998; 280: 969-74. 22. Grabec D, Kragelj B. The sigmoid colon and bladder shielding in whole pelvic irradiation at prostate cancer (forward planned IMRT from Institute of on­cology Ljubljana). Radiol Oncol 2009; 43: 56-64. 23. Roach M 3rd, Marquez C, Yuo HS, Narayan P, Coleman L, Nseyo UO, et al. Predicting the risk of lymph node involvement using the pre-treatment prostate specific antigen and Gleason score in men with clinically localized prostate cancer. Int J Radiat Oncol Biol Phys 1994; 28: 33-7. 24. Cox JD, Stetz JA, Pajak TF. Toxicity criteria of the Radiation Therapy Oncology Group (RTOG) and the European Organization for Research and Treatment of Cancer (EORTC). Int J Radiat Biol Phys 1995; 31: 1341-6. 25. Marks LB, Carroll PR, Dugan TC, Anscher MS. 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Pract Radiat Oncol 2013; 3: e1-9. 104 research article Prognostic factors of choroidal melanoma in Slovenia, 1986—2008 Boris Jancar1, Marjan Budihna1, Brigita Drnovsek-Olup2, Katrina Novak Andrejcic2, Irena Brovet Zupancic2, Dusica Pahor3,4 1 Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Eye Hospital, University Clinical Centre Ljubljana, Ljubljana, Slovenia 3 Department of Ophthalmology, University Medical Centre Maribor, Maribor, Slovenia 4 Faculty of Medicine, University of Maribor, Maribor, Slovenia Radiol Oncol 2015; 50(1): 104-112. Received 17 November 2014 Accepted 9 December 2014 Correspondence to: Boris Jančar M.D., M.Sc., Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia. Phone +386 1 500 96 90; E-mail: bojancar@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Introduction. Choroidal melanoma is the most common primary malignancy of the eye, which frequently metasta­sizes. The Cancer Registry of Slovenia reported the incidence of choroid melanoma from 1983 to 2009 as stable, at 7.8 cases/million for men and 7.4/million for women. The aim of the retrospective study was to determinate the prognostic factors of survival for choroidal melanoma patients in Slovenia. Patients and methods. From January1986 to December 2008 we treated 288 patients with malignant choroidal melanoma; 127 patients were treated by brachytherapy with beta rays emitting ruthenium-106 applicators; 161 pa­tients were treated by enucleation. Results. Patients with tumours thickness < 7.2 mm and base diameter < 16 mm were treated by brachytherapy and had 5- and 10-year overall mortality 13% and 32%, respectively. In enucleated patients, 5- and 10-year mortality was higher, 46% and 69%, respectively, because their tumours were larger. Thirty patients treated by brachytherapy de­veloped local recurrence. Twenty five of 127 patients treated by brachytherapy and 86 of 161 enucleated patients developed distant metastases. Patients of age . 60 years had significantly lower survival in both treatment modali­ties. For patients treated by brachytherapy the diameter of the tumour base and treatment time were independent prognostic factors for overall survival, for patients treated by enucleation age and histological type of tumour were independent prognosticators. In first few years after either of treatments, the melanoma specific annual mortality rate increased, especially in older patients, and then slowly decreased. Conclusions. It seems that particularly younger patients with early tumours can be cured, whereby preference should be given to eyesight preserving brachytherapy over enucleation. Key words: choroid melanoma; therapy; brachytherapy; prognostic factors Introduction Uveal melanoma is the most common primary malignancy of the eye.1 Approximately 90% of all uveal melanoma develop in the choroid, 7% in the ciliary body and 3% in the iris.2 The disease is more common in older age, with the highest incidence at about 60 years of age.1,2 For the period 1983.1994, the incidence of uveal melanoma in 16 European countries was analysed by the European Cancer Registry (EUROCARE).3 The incidence in Europe was found ascend from South to North, being 2/ million inhabitants in Spain and southern Italy and more than 8/million in Denmark and Norway. In Slovenia, the incidence of choroid melanoma be­tween 1983-2009 was stable, at 7.8 cases/million for men and 7.4/million for women.4 In the majority of patients, the biopsy of tumour is not indicated because the accuracy of clinical diagno­sis is reaching 99%.5 However, there is no agreement 105 about the optimal therapy.6-10 Until development of eye conserving therapies in 1960’s, for more than 100 years, enucleation was the only mode of treat­ment. The first among eye conserving approaches was the plaque brachytherapy9,11-14, followed by proton beam15-17 and helium ion radiotherapy18-20, stereotactic radiotherapy, transscleral or transreti­nal local resection10,21,22, and phototherapy brachy­therapy23, several types of radioactive plaques with photon emitting isotopes were used, including co­balt-60, iodine-125, and iridium-192. Beta emitting plaques with ruthenium (106Ru/106Ro), however, were introduced in 1964 by Lommatzsch.24-26 In Slovenia, treatment of choroidal melanoma by brachytherapy with ruthenium plaques using the Lommatzsch method was introduced in 1985 by the Eye Clinic at the University Clinical Centre Ljubljana in collaboration with the Institute of Oncology Ljubljana.27,28 Before that time, the only available treatment was enucleation of the dis­eased eye. The aim of this retrospective study was to evaluate these two modalities in the treatment of choroidal melanoma in Slovenia during the period from 1986 to 2008 and to determinate the prognos­tic factors of survival for choroidal melanoma pa­tients in Slovenia. Patients and methods Patients The database of the Cancer Registry of Slovenia was used for identification of patients with the diagnosis of choroidal melanoma in Slovenia in the years 1986.2008.4 The medical records of identified patients from the Eye Hospital of the University Clinical Centre Ljubljana and from the Department of Ophthalmology of the University Medical Centre Maribor were reviewed for rele­vant information on clinical characteristics, treat­ment and outcome. The diagnosis of choroidal melanoma was based on clinical features and full ophthalmologic examination, indirect ophthal­moscopy, fundus photography, ultrasonography and fluorescein angiography. At the time of diag­nosis, the patients were evaluated by chest radiog­raphy, lymph gland and liver ultrasonography29 and routine blood tests. Genetic testing was not done because it was not available at the time of the study. The study was approved by the institutional review board and was carried out according the Helsinki Declaration. Treatment Applicators manufactured by Bebig (Eckert& Ziegler BEBIG Gmbh, Berlin; later Amersham, GB) were used. The applicators were concave, shell-shaped, with Ru-106/Ro-106 isotope covering the concave surface as a thin, insoluble film and emit­ ting beta rays with the energy of 3.54 MeV and half-life of 373 days. The tumours were localized by transillumination and indirect ophthalmosco­py, and the applicators were sutured to the sclera. The dose at the tumour apex was aimed to be about 120 Gy. The applicator was removed after expira­tion of appropriate time. Treatment was selected according to the tumour size: patients with tumours . 16 mm in diameter and . 7.2 mm thick, with useful vision preserved, were treated by brachytherapy, patients with larg­er tumours had enucleation. The enucleation was performed in general anaesthesia. First follow-up visits took place one month af­ter the procedure, in 3-month intervals during the first year and once a year thereafter. At each follow up visit, patients underwent ophthalmologic ex­aminations with indirect ophthalmoscopy, fundus photography and ultrasonography. Statistical methods For comparative analyses, the Fisher exact test for two proportions as well as t-test and Mann-Whitney test for data of two independent groups were used. Survival estimates were carried out using the Kaplan-Meier method and reported at 5 and 10 years follow up. The difference between the survival curves was evaluated by means of a log-rank comparison. Multivariate survival analy­sis for study of an independent effect of various parameters that appeared statistically significant on univariate analysis to treatment outcome and survival was performed according to Cox’s pro­ portional hazard models with backward stepwise selection. The end points of survival analysis were locoregional control (LRC, persistent disease or lo­coregional recurrence considered as an event), dis-ease-free survival (DFS, appearance of loco-region­al recurrence or systemic metastases considered as event), disease-specific survival (DSS, melanoma related death considered as event) and overall survival (OS, death from any cause considered as event) which were measured from the first day of therapy. These statistical analyses were performed by using SPSS version 18.0 (SPSS Inc., Chicago, IL) 106 TABLE 1. The characteristics of patients and tumours by treatment modality 2008 at the Eye Hospital of the University Clinical All patients 130 161 291 Excluded 3 palliations -3 Treated 127 161 288 Gender Man 58 84 142 Women 69 77 146 Age (median) Men 58 (29-74) 58 (19-86) Women 60 (22-89) 61 (23-92) T-stage (AJCC) 1 38 2 69 3 8 No data 12 Thickness < 3 mm 11 3.1-5.0 mm 64 5.1-7.2 mm 49 > 7.8 mm 3 No data 0 Basal diameter . 10 mm 52 10,1-12,0 mm 38 > 12 mm 25 No data 12 Histology 161 Spindle cell 33 Epithelioid 38 Mixed 23 No data 37 AJCC = American Joint Committee on Cancer and nonlinear regression Gaussian curve fitting was performed by GraphPad Prism version 5. All tests were two-sided and a P-value of 0.05 was con­sidered statistically significant. Results Clinical records of 288 patients with choroidal melanoma treated from January 1986 to December Centre Ljubljana and from the Department of Ophthalmology of the University Medical Centre Maribor were reviewed. The follow-up close-out date was December 31, 2013. Median follow-up of all patients was 15 years (range, 4.27 years). In December 2013, 130 patients were alive. The cause of death was melanoma in 107 patients and 51 patients died of melanoma unrelated disease; 20 among them died of other malignant diseases. The characteristics of patients and tumours are shown in Table 1. Survival In univariate analysis of all patients, the LRC and DFS were better in enucleation than in brachy­therapy group and better in females than in males. Patients < 60 years had better DFS, DSS and OS than older patients. In brachytherapy group, fe­ males had statistically better LRC and DFS than males; younger patients had better DSS and OS than older patients. Tumour thickness < 6 mm was associated with better LRC and DFS than thicker tumours, while the base diameter < 11 mm was a good prognostic sign for LRC; DFS, DSS and OS. The treatment time influenced LRC and DFS, while the dose-rate had no influence of the outcome of the treatment. In the enucleation group, age and histology influenced DFS, DSS and OS, while sex had no effect on survival. The detailed data of sur­ vival are presented in Tables 2–4. In multivariate analysis for all patients, gender was independent prognostic factor for LRC, while first treatment and age were independent prognos­tic factors for DFS, DSS and OS. In the brachythera­py group, gender was independent prognostic fac­ tors for LRC; treatment time for LRC and DFS; base diameter for DFS and OS. The age was independ­ent prognostic factor for DFS and OS. In enuclea­tion group, age and histology were independent prognostic factors for DFS and DSS, while on OS influenced only age (Table 5). Second treatment In 30 patients treated by brachytherapy, a local re­currence of the tumour occurred. The second ap­plication of ruthenium plaque was performed in 13 of these patients, and in 17 patients had enuclea­ tion: 12 patients - because of extent of the recurrent tumour and 5 patients - because of the treatment-related side effects (glaucoma, cataract). The eyes were enucleated from 7 months to 18 years (median 24 months) after the first brachytherapy (Figure 1). 107 TABLE 2. Univariate analysis of survival: all patients (N = 288) 5 yrs 10 yrs p 5 yrs 10 yrs p 5 yrs 10 yrs p 5 yrs 10 yrs p All 288 90 88 - 65 50 - 76 58 68 46 - Ruthenium Enucleation 127 161 78 100 75 100 0.000 71 60 60 42 0.014 92 64 79 42 0.000 87 54 68 31 0.000 Men Women 142 146 85 95 82 93 0.026 61 69 51 49 0.673 74 78 61 55 0.647 66 70 47 46 0.952 < 60 years . 60 years 150 138 89 90 86 90 0.648 74 56 58 40 0.002 86 65 68 47 0.000 84 52 64 28 0.000 DFS = disease free survival; DSS = disease specific survival; LRC = loco-regional control; n = number of patients; OS = overall survival; yrs = years TABLE 3. Univariate analysis of survival: patients treated by brachytherapy (N = 127) n 5 yrs 10 yrs p 5 yrs 10 yrs p 5 yrs 10 yrs p 5 yrs 10 yrs p Men Women 58 69 66 89 60 87 0.003 60 81 49 69 0.039 90 93 76 81 0.703 87 88 67 70 0.859 < 60 years . 60 years 68 59 76 80 71 80 0.305 76 65 66 51 0.156 98 84 89 65 0.002 98 75 83 52 0.000 T-stage 1 38 79 79 73 67 97 84 94 71 2 69 79 74 0.451 72 57 0.354 90 75 0.378 86 72 0.508 3 8 60 40 45 45 86 86 0 50 Tumour thickness 2-5.9 mm 6-.2 mm 97 29 83 64 82 54 0.003 74 64 66 39 0.021 92 96 79 80 0.489 86 96 68 70 0.724 Base < 11 mm . 11mm 61 54 83 70 83 64 0.043 80 60 72 45 0.002 96 87 84 72 0.024 96 78 77 64 0.002 Dose rate Top (Gy/h) . 108 Gy < 108 Gy 53 52 81 74 78 68 0.302 74 68 66 46 0.099 95 89 84 72 0.280 87 87 72 62 0.690 Dose- rate base (Gy/h) . 532 < 532 53 52 82 74 74 71 0.708 74 68 57 55 0.804 95 89 81 75 0.665 87 87 69 65 0.862 Treatment time . 96 hours > 96 hours 52 53 87 68 84 62 0.015 80 62 72 40 0.004 95 89 84 71 0.400 89 85 74 60 0.565 DFS = disease free survival; DSS = disease specific survival; LRC = loco-regional control; n = number of patients; OS = overall survival; yrs = years 108 Overall survival (%) 100 Enucleation, n=17 Ru106, n=13 80 No recurrence, n=97 60 40 20 all metastases were localized in the liver. The ac­tuarial rates of metastases by treatment modality are depicted in Figure 2. At 5 and 10 years, the inci­dences were 39% and 57%, respectively, for enucle­ated patients, and 11% and 21%, respectively, for irradiated patients (P < 0.001). 0 Years after therapy FIGURE 1. Overall survival of patients treated by brachytherapy after treatment of recurrence. FIGURE 2. Incidence of distant metastases according to the type of treatment. FIGURE 3. Percentage of annual post-treatment melanoma specific mortality according to the type of treatment. *There was no peak in ruthenium treated patients < 60 years. FIGURE 4. Linear regression analysis of melanoma related mortality per age decades, according to the type of treatment. Points represent percent mortality rate for the elapsed decade. No patient less than 40 years who was treated with Ru-106 died of melanoma. Distant metastases Twenty-five of 127 patients treated by brachyther­apy and 86 of 161 those treated by enucleation de­veloped systemic metastases. Seventy per cent of In patients treated by brachytherapy, half of the metastases developed in 5 years, and in those treat­ed by enucleation in 2.6 years. Annual melanoma specific mortality rate The mortality of patients was increased in the first few years after treatment and then slowly returned to pre-treatment values. Melanoma specific mortal­ity rate is displayed in Figure 3. The peak percentage of annual melanoma spe­cific mortality after treatment was achieved at 3.6 years for patients older than 60 years treated by enucleation and at approximately 6 years for younger enucleated patients and for all patients treated with brachytherapy. The irradiated pa­tients below 60 years contributed little to the peak because of low mortality rate. No patient from brachytherapy group aged be­ low 40 years died of melanoma. In brachytherapy treated patients the mortality began to increase af­ ter the age of 40 and reached 40 % in 70.80 year’s age group. In patients treated by enucleation, the mortality started to increase one decade earlier: the rise started with about 40% and reached about 70 % in patient’s 80.90 years of age (Figure 4). Complications Because of retrospective character of the study, acute complications were not systematically re­corded. For chronic complications patients were reviewed annually. Post-radiation retinopathy started to appear after two years and was docu­mented in 18 patients (12 mild, 6 severe), neovas­cular glaucoma in 5 patients and cataract in 6 pa­tients. None of the patients had optic neuropathy. Vision after treatment After brachytherapy, the eye was retained in all pa­tients and the vision was assessed in 112 patients. Compared to pre-treatment status, 22 (19.6%) pa­tients had better visual acuity; in 12 (10.7%) pa­tients the vision was unchanged while in 78 (69.6%) patients the acuity of vision was worse. The major­ity of brachyradiotherapy patients retained vision which was better than counting fingers. 109 Discussion Our retrospective study reports results of the treatment of patients with choroidal melanoma in Slovenia from 1986 to 2008. In our study, the over­all and specific mortality rate in patients treated by enucleation was higher mainly because larger tu­mours were selected for enucleation as compared to those treated by brachytherapy. Brachytherapy could be used only for selected tumours, depend­ ing on their size, location and shape of applicators, for which a satisfactory dose distribution of dose can be achieved. Because no data about the dimen­sions of the enucleated tumours was available, comparison of results between the two treatment modalities by tumour stage could not be made. The randomized as well as nonrandomized studies reported no difference in survival rates in patients treated either by enucleation or brachy­therapy when matched by the stage, age and other prognostic parameters.6-8,11,12,30-33 The largest of these studies was the COMS, which included 1317 patients and prospectively compared on ran­domized fashion enucleation and brachytherapy. There was no statistical difference in 5- and 10-year OS between the two treatment groups.30 In the matched pairs study of Guthoff et al. melanoma specific survival at 12 years of follow-up was 77.9% in irradiated patients and 78.6% in enucleated pa­tients (P > 0.05).31 When the OS at 10 years of our patients treated by brachytherapy was compared with that from COMS study, no difference could be observed: 32% vs. 35%; similarly, the DSS at 10 years in our series was 79% and was comparable with DSS reported by Guthoff. There are several prognostic factors for out­come of the choroidal melanoma, including age30-33, gender33, basal tumour diameter34, tumour thickness33-37, T-stage35, cell morphology1,7,33,38 and various genetic changes of the tumour, especially monosomy of chromosome 3.33,39-41 Some of them appeared statistically significant also in the present study, although the strength of our results should be interpreted with caution. Namely, we only had complete information on age and gender of the patients, histology of the enucleated tumours, and data of tumour diameter, thickness, irradiation dose on the base and top of the tumour and the treatment time for brachytherapy patients, but not also on some other highly relevant prognosticators (e.g. genetic alterations), which limits the strength of statistical analysis. In both treatment groups, the post-treatment annual mortality related to melanoma at first in- TABLE 4. Univariate analysis of survival: patients treated by enucleation (N = 161)* n 5 yrs 10 yrs p 5y 10 yrs p 5 yrs 10 yrs p Men Women 84 77 62 59 51 33 0.154 63 65 51 34 0,275 53 56 34 27 0.775 < 60 years . 60 years 82 79 73 49 52 30 0.001 76 50 52 30 0.000 74 34 51 10 0.000 Spindle cell epithelioid Mixed cell 33 38 23 74 56 62 70 36 28 0.050 81 61 67 72 33 24 0.029 66 55 52 49 24 15 0.026 *None of enucleated patients had local recurrence; DFS = disease free survival; DSS = disease specific survival; n = number of patients; OS = overall survival; yrs = years creased, as expected due to systemic metastases, but few years later it decreased to a few or zero percent. In patients of 60 years or more who were treated by enucleation, mortality reached its peak of 18% at 3.7 years after treatment, while in patients younger than 60 years the peak was reached at six years after treatment and was 7%. Patients treated by brachytherapy fared better: regardless of age, six years after treatment completion the peak mor­tality was 3%. However, the mortality of irradiated patients aged . 60 years reached the peak of 7% at 6 years post-treatment, while no increase in mortal­ity was noticed among younger patients, probably due to the small number of deaths. The increase in annual mortality following enu­cleation was first observed by Zimmermann.42,43 He re-analysed the data of Paul et al.38 who moni­tored 2652 patients for 40 years and found a steep increase in mortality following enucleation. In this study, the peak of 8% was reached at 2 years after enucleation, slowly diminishing during the next few years to the “normal” 1%.43,44 Similarly, Seddon et al. reported the increase in mortality to 6.5 % in the first 2.3 years after treatment and slowly return to »normal« 1% during the next 7 years.45 The post-treatment increase in melanoma re­lated mortality can be attributed to the loss of antiangiogenic activity of the primary tumour after its removal or destruction. Uveal melanoma cells produce angiostatin, growth inhibitor of metastatic foci46,47, which was found to be present in the circu­lation only up to five days after the removal of the primary tumour.48,49 Damato et al.33 found that the probability of metastases was greater in older patients as their 110 TABLE 5. Multivariate analysis of survival of all patients (N = 288) All patients LRC First treatment 40.842 5.565 299.717 0.000 Gender 2.658 1.245 5.678 0.012 DFS First treatment 1.628 1.144 2.316 0.007 Age < 60 years vs. . 60 years 1.800 1.275 2.540 0.001 DSS First treatment 3.937 2.509 6.178 0.000 Age < 60 years vs. . 60 years 2.534 1.714 3.747 0.000 OS First treatment 3.153 2.218 4.480 0.000 Age < 60 years vs. . 60 years 3.818 2.710 5.377 0.000 Gender 2.306 1.013 5.251 0.047 Ruthenium LRC Treatment time (. 96 h vs. > 96 h) 2.841 1.220 6.623 0.015 Treatment time (. 96 h vs. > 96 h) 2.674 1.276 5.587 0.009 DFS Base (< 11 mm vs. . 11 mm) 2.519 1.015 6.250 0.046 T-stage 2.320 1.002 5.376 0.050 DSS Age (< 60 years vs. . 60 years) 4.762 1.709 13.333 0.003 OS Base (< 11 mm vs v11 mm) 3.610 1.391 9.434 0.008 Age (< 60 years vs. . 60 years) 5.650 2.538 12.658 0.000 Nucleation LRC - - - - - DFS Age (< 60 years vs. . 60 years) 2.132 1.149 3.968 0.016 Histology S VS E VS M 1.467 1.000 2.151 0.050 DSS Age (< 60 years vs. . 60 years) 2.326 1.229 4.403 0.009 Histology S vs. E vs. M 1.555 1.052 2.298 0.027 OS Age (< 60 years vs. . 60 years) 3.876 2.222 6.757 0.000 Histology (S vs. E vs. M) 1.444 1.051 1.983 0.023 CI = confident interval; DFS = disease free survival; DSS = disease specific survival; E = epitheloid; HR = hazard ratio; LRC = loco-regional control; M = mixed cell; n = number of patients; OS = overall survival; S = spindle cell tumours grew longer and had more time for ac­cumulation of chromosome instabilities, making the tumour more malignant and more prone to metastasize. Accordingly, the younger patients should have smaller and perhaps less malignant tumours, and the appearance of metastases is less likely. It is assumed that following primary tumour removal, metastases in younger patients reach the lethal tumour mass at a later time. The peak in melanoma-related mortality in younger enucleated patients from our series appeared 2.5 years later than in older counterpart, confirming this assumption. However, not all patients from advanced age group have advanced primary tu­ mour and metastases. In our series, 59 patients . 60 years had primary tumours small enough to be treated by brachytherapy. The annual melanoma related mortality curve suggests that the burden of their metastases was also smaller, and reached the lethal mass at a later time. The synchronous peaks of enucleated patients < 60 years and of irradiated patients . 6o years suggests that the burden of me­tastases in enucleated group, was similar in these two groups (Figure 3). There is no good scientific evidence that treat­ment can prolong patients’ life.33 The increase in annual post-treatment mortality rate implies that the life of some patients might be shortened due to 111 the therapy, particularly of older ones. This obser­vation and the fact that some tumours and their me­tastases grow very slowly raise the question when the treatment of uveal melanoma can be withheld. The COMS study showed that the estimated risk of death at 5 years of follow-up in 42 untreated pa­tients was 50%, and risk of 1317 patients treated by a standard method, was 18%.50 It seems that treat­ment in older patients without eyesight problems, in spite of evident metastases, could be postponed until the problems eventually ensue. On the other hand, it may be assumed that some of the younger patients are without micrometastases at the time of therapy and can be cured by the early treatment. Indeed, in our study, none of the patients younger than 40 years from brachytherapy group died of metastases, while death of metastases in older pa­ tients steeply increased with age (Figure 4). To conclude, treatment-specific and age-de­pendent pattern of -related mortality was con­firmed in our study, confirming observation of other authors. For quality of life reasons we be­lieve that preference should be given to eyesight preserving brachytherapy or other eye preserv­ing treatments of choroidal melanoma over enu­cleation, if the size and location are suitable even though the definite opinion on the best treatment differed in the literature.51,52 Acknowledgments The authors thank to Dr. Primoz Logar, ophthal­mologist of Eye Hospital of the University Clinical Centre Ljubljana for his enthusiasms while he treated the patients with choroidal melanoma. References 1. Singh AD, Bergman L, Seregard S. Uveal melanoma: epidemiological as­pects. Ophthalmol Clin North Am 2005; 18: 75-84. 2. Singh AD, Turell ME, Topham AK. Uveal melanoma: trends in incidence, treatment and survival. Ophthalmology. 2011; 118: 1881-5. 3. Virgili D, Gatta G, Ciccolallo L, Capoccacia R, Biggeri A. Incidence of uveal melanoma in Europe. Ophthalmology 2007; 114: 2309-15. 4. Cancer incidence in Slovenia 1983-2009. Ljubljana: Institute of Oncology Ljubljana, Cancer Registry of Republic of Slovenia, 1987–2012. 5. Accuracy of diagnosis of choroidal melanomas in the Collaborative Ocular Melanoma Study. COMS report No1. Arch Ophthalmol 1990; 108: 1268-73. 6. Shields JA, Shields CL. Current management of posterior uveal melanoma. Mayo Clin Proc 1993; 68: 1196-200. 7. Shields JA, Shields CL, De Potter P, Singh AD. Diagnosis and treatment of uveal melanoma. Semin Oncol 1996; 23: 763-7. 8. Hungerford JL. Management of ocular melanoma. British Medical Bulletin 1995; 51: 694-716. 9. Albert DM. The ocular melanoma story, Edward Jackson memorial lecture: Part II. Am J Ophthalmol 1997; 123: 729-41. 10. Kertes PJ, Johnson JC, Peyman GA. Internal resection of posterior uveal melanomas. B J Ophthalmol 1998; 82: 1147-53. 11. Packer S, Stoller S, Lesser ML, Mandel FS, Finger PT. Long-term results of io­dine 125 irradiation of uveal melanoma. Ophthalmology 1992; 99: 767-73. 12. Vrabec TR, Augsburger JJ, Gamel JW, Brady LW, Hernandez C, Woodleigh R. Impact of local tumor relapse on patient survival after cobalt 60 plaque radiotherapy. Ophthalmology 1991; 89: 984-8. 13. Augsburger JJ, Mullen D, Kleineidam M. Planned combined I 125 plaque irradiation and indirect ophthalmoscope laser therapy for choroidal malig­nant melanoma. Ophthalmic Surgery 1993; 24: 76-81. 14. Papageorgiou KI, Cohen VML, Bunce C, Kinsella M, Hungerford JL. Predicting local control of choroidal melanomas following 106 Ru plaque brachy­therapy. Br J Ophthalmol 2011; 95: 166-70. 15. Bercher L, Zografos L, Egger E, Chamot L, Uffer S, Gaillaud C. [Treatment of exterior extension of choroid melanomas by accelerated proton beams]. [French]. Klin Monbl Augenheilkd 1992; 200: 440-3. 16. Zografos L, Bercher L, Egger E. [Treatment of eye tumors by accelerated proton beams. 7 years experience]. [French]. Klin Monbl Augenheilkd 1992; 200: 431-5. 17. Saornil MJ, Egan KM, Gragoudas ES, Seddon JM, Walsh SM, Albert DM. Histopathology of proton beam-irradiated vs. enucleated uveal melanomas. Arch Ophthalmol 1992; 110: 1112-8. 18. Char DH, Castro JR, Kroll SM, Irvine AR, Quivery JM, Stone RD. Five-year follow-up of helium ion therapy for uveal melanoma. Arch Ophthalmol 1990; 108: 209-14. 19. Char DH, Quivery JM, Castro JR, Kroll SK, Phillips T. Helium ions versus iodine 125 brachytherapy in the management of uveal melanoma. Ophthalmology 1993; 100: 1547-54. 20. Char CH, Kroll SM, Castro J. Ten-year follow-up of helium ion therapy for uveal mela-noma. Am J Ophthalmol 1998; 25: 81-9. 21. Damato BE, Paul J, Foulds WS. Risk factors for residual and recurrent uveal melanoma after trans-scleral local resection. Br J Ophthalmol 1996; 80: 102-8. 22. Char DH. Clinical ocular oncology. 2nd edition. Philadelphia: Lippincott-Raven Publishers; 1997. p. 114-60. 23. Oosterhuis JA, Journee-de Korver HG, Kakebeeke-Kemme HM, Bleeker JC. Transpupillary thermotherapy in choroidal melanomas. Arch Ophthalmol 1995; 113: 315-21. 24. Lommatzsch PK. Results after beta-irradiation (106Ru/106Rh) of choroidal melanomas: 20 years experience. Br J Ophthalmol 1986; 70: 844-51. 25. Lommatzsch PK. Radiotherapie der intraokularen Tumoren, insbesondere bei Aderhautmelanom. [Experience in treatment of retinoblastoma in the German Democratic Republic]. [German]. Klin Monbl Augenheilkd 1979; 174: 948-58. 26. Lommatzsch PK, Werschnik C, Schuster E. Long-term follow-up of Ru-106/ Rh-106 brachytherapy for posterior uveal melanoma. Graefe’s Arch Clin Exp Ophthalmol 2000; 238: 129-37. 27. Jančar B. [Choroidal melanoma]. [Slovenian]. Zdrav Vestn 1992; 61: 439-41. 28. Novak-Andrejčič K, Logar P, Brovet-Zupančič I, Jančar B. [Treatment of choroidal melanoma with Ru-106 brachytherapy - 14-year experience]. [Slovenian]. Zdrav Vestn 2002; 71(Suppl II): 67-70. 29. Solivetti FM, Elia F, Santaguida MG, Guerrisi A, Visca P, Cercato MC, et al. The role of ultrasound and ultrasound-guided fine needle aspiration biopsy of lymph nodes in patients with skin tumours. Radiol Oncol 2014; 48: 29-34. 30. The COMS randomized trial of Iodine125 brachytherapy for choroidal mela­noma. COMS report No. 28. Arch Ophthalmol 2006; 124: 1684-93. 31. Guthoff R, Frischmuth J, Jensen OA. [Choroid melanoma. A retrospective randomized comparative study of ruthenium irradiation vs enucleation]. [German]. Klin Monbl Augenheilkd 1992; 200: 257-61. 32. Rouberol F, Roy P, Kodjikian L, Gerard JP, Jean-Louis B. Survival, anatomic and functional long-term results in choroidal and ciliary body melanoma after ruthenium brachytherapy. Am J Ophthalmol 2004; 137: 893-900. 112 33. Damato BE, Heimann H, Kalirai H, Coupland SE. Age, survival predictors, and metastatic death in patients with choroidal melanoma: tentative evidence of a therapeutic effect on survival. JAMA Ophthalmol 2014; 132: 605-13. 34. Damato B, Coupland SE. A reappraisal of the significance of largest basal diameter of posterior uveal melanoma. Eye (Lond) 2009; 23: 2152-60. 35. Kujala E, Damato B, Coupland SE, Desjardins L, Bechrakis NE, Kivela T. Staging of ciliary body and choroidal melanomas based on anatomic extent. J Clin Oncol 2013: 31: 2825-31. 36. Shields CL, Furuta M, Thangappan A, Nagori S. Metastasis of uveal mela­noma milimeter by milimeter in 8033 consecutive eyes. Arch Ophthalmol 2009; 127: 898-98. 37. Damato B. Progress in the management of patients with uveal melanoma. Eye (Lond) 2012; 26: 1157-72. 38. Paul EU, Paunell BL, Braker M. Prognostic factors in malignant melanoma of the choroid and ciliary body. Int J Ophthalmol Clin 1962; 2: 387-402. 39. Prescher G, Bornfeld N, Hirche H, Horsthemke B, Jöckel KH, Becher R. Prognostic implications of monosomy in uveal melanoma. Lancet 1996; 347: 1222-5. 40. Onken MD, Worley LA, Person E, Char DH, Bowcock AM, Harbour JW. Loss of heterozygosity of chromosome 3 detected with single nucleotide poly­morphisms is superior to monosomy 3 for predicting metastasis in uveal melanoma. Clin Cancer Res 2007; 13: 2923-7. 41. Tschentscher F, Prescher G, Zeschnigk M, Horsthemke B, Lohmann DR. Identification of chromosomes 3, 6, and 8 aberrrations in uveal melanoma by microsatellite analysis in comparison to comparative genomic hybridiza­tion. Cancer Genet Cytogenet 2000; 122: 13-7. 42. Mossbock G, Rauscher T, Winkler P, Kapp KS, Langman G. Impact of dose rate on clinical course in uveal melanoma after brachytherapy with ruthe­nium-106. Strahlenther Onkol 2007; 10: 571-5. 43. Zimmerman L, McLean IW, Foster WD. Does enucleation of the eye con­taining malignant a melanoma prevent or accelerate the dissemination of malignant cells. Br J Ophthalmol 1978; 62: 420-5. 44. Zimmerman L, McLean IW. An evaluation of the enucleation in the manage­ment of uveal melanoma. Am J Ophthalmol 1979; 87: 741-60. 45. Seddon JM, Gragoudas ES, Egan KM, Polivogianis L. Relative survival rates after alternative therapies for uveal melanoma. Ophthalmology 1990; 97: 769-77. 46. Westphal JR, Hullenaar RV, Geurts-Moespot A, Sweep FC, Verheijen JH, Bussemakers MM. Angiostatin generation by human tumor cell lines. Int J Cancer 2000; 86: 760-7. 47. Apte RS, Niederkorn JY, Mayhew E, Alizadeh H. Angiostatin produced by certain primary uveal melanoma cell lines impedes the development of liver metastases. Arch Ophthalmol 2001; 119: 1805-9. 48. Grossniklaus HE. Progression of ocular melanoma metastasis to the liver JAMA Ophthalmol 2013; 131: 462-9. 49. Kauffman EC, Robinson VL, Stadler WM, Sokoloff MH, Rinker-Schaeffer CW. Metastasis suppression: the evolving role of metastasis suppressor genes for regulating cancer cell growth at the secondary site. J Urol 2003; 169: 1122-33. 50. Diener-West M, Reynolds SM, Agugliaro DJ, Caldwell R, Cumming K, Earle JD, et al. Development of metastatic disease after enrollment in the COMS trials for treatment of choroidal melanoma: Collaborative Ocular Melanoma Study Group Report No.26. Arch Ophthalmol 2005; 123: 1639-43. 51. Straatsma BR, Diener-West M, Caldwell R, Engstrom RE. Mortality after deferral of treatment or no treatment for choroidal melanoma. Am J Ophthalmol 2003; 136: 47-54. 52. Damato B. Does ocular treatment of uveal melanoma influence survival. Br J Cancer 2010; 103: 285-90. 113 research article The impact of anaemia on treatment outcome in patients with squamous cell carcinoma of anal canal and anal margin Irena Oblak1,2, Monika Cesnjevar2, Mitja Anzic2, Jasna But Hadzic1, Ajra Secerov Ermenc1, Franc Anderluh1, Vaneja Velenik1,2, Ana Jeromen1, Peter Korosec1 1 Department of Radiotherapy, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2016; 50(1): 113-120. Received 26 November 2014 Accepted 22 December 2014 Correspondence to: Assist. Prof. Irena Oblak, M.D., Ph.D., Department of Radiotherapy, Institute of Oncology Ljubljana, Ljubljana, Slovenia. Phone: +386 1 5879 515; Fax: +386 1 5878 304; E-mail: ioblak@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. Radiochemotherapy is the main treatment for patients with squamous cell carcinoma of the anal ca­nal. Anaemia is reported to have adverse effect on survival in cancer patients. The aim of the study was to evaluate the influence of anaemia on radiochemotherapy treatment outcome in patients with squamous cell carcinoma of the anal canal. Patients and methods. One hundred consecutive patients with histologically confirmed squamous cell carcinoma of the anal canal were treated radically with 3-dimensional conformal or intensity-modulated radiation therapy fol­lowed by brachytherapy or external beam radiotherapy boost and with concurrent mitomycin C and 5-fluorouracil. The influence on survival of pre-treatment, mean on-treatment and end-of-treatment haemoglobin (Hb) concentra­tions was studied. Results. The 5-year locoregional control, disease free survival, disease specific survival and overall survival rates for all patients were 72%, 71%, 77% and 62%, respectively. In univariate analysis, patients with pre-treatment and end-of­ treatment Hb > 120 g/L survived statistically significantly better compared to patients with Hb . 120 g/L. Patients with mean on-treatment Hb > 120 g/L only had statistically significant better locoregional control and overall survival than patients with Hb . 120 g/L. In multivariate analysis, independent prognostic factors were pre-treatment Hb (> 120 g/L vs. . 120 g/L) for overall survival (hazard ratio [HR] = 0.419, 95% confidence interval [CI] = 0.190–0.927, p = 0.032) and stage (I & II vs. III) for disease specific (HR = 3.523, 95% CI = 1.375–9.026, p = 0.009) and overall survival (HR = 2.230, 95% CI = 1.167–4.264, p = 0.015). Conclusions. The pre-treatment, mean on-treatment and end-of-treatment Hb concentration > 120 g/L carried better prognosis for patients for with squamous cell carcinoma of the anal canal treated with radiochemotherapy. The pre-treatment Hb > 120 g/L was an independent prognostic factor for overall survival of patients with anal canal cancer. Key words: anaemia; anal canal squamous cell carcinoma; radiochemotherapy Introduction Squamous cell anal cancer is a rare tumour which represents 1.5% of gastrointestinal cancers, but in Slovenia only 0.5%.1-5 Despite its infrequent occur­rence its incidence is increasing.4 Women are more commonly affected than men.3-6 Causal factors in the anal canal cancer are usually associated with human papilloma virus (HPV) infection (being the most important risk factor ), human immuno­ 114 deficiency virus (HIV) infection, anal intercourse, higher lifetime number of sexual partners, genital warts and cigarette smoking.3,6-8 Anal canal cancer is predominantly a loco-re­gional disease, because it metastasizes in less than 10% of patients, mainly to lungs and liver.6 The management of anal canal cancer has un­dergone an interesting transformation over the course of the past three decades. With the report by Nigro et al. in 1974 it shifted from abdomino­perineal resection with or without inguinal lymph node dissection to radical radiochemotherapy.9,10 Radiochemotherapy with 5-fluorouracil and mito­mycin C, nowadays being the main treatment, re­sults in complete tumour response in 70–90% and has a 5-year survival rate of 60–70%, leaving sur­gery only as a salvage treatment for tumours that do not respond to radiochemotherapy or recur.4,7 Anal margin cancers are classified as skin tumours and small tumours can be treated by surgery, while tumours T2 or larger should be treated with defini­tive radiochemotherapy.11 Radiotherapy as well as chemotherapy is known to be more efficacious in the presence of oxygen than in hypoxic conditions.12-15 Tumours are more hypoxic than the surrounding normal tis­sue.13 Anaemia, present in 75% of cancer patients, could increase the proportion of hypoxic tumour cells.13 Hypoxia is widely recognized as a major factor leading to the resistance of tumour cells to radiotherapy, but several mechanisms may also cause cells in the hypoxic region to be resistant to anticancer drugs.16 The influence of anaemia on the outcome of treatment was first recognized in 1940s in cervical cancer patients and later in pa­tients with other tumours such as head and neck squamous cell carcinoma, carcinoma of the lungs, bladder, prostate and anus.7,17,18 The purpose of present study was to evaluate the influence of anaemia on radiochemotherapy treatment out­come in patients with squamous cell carcinoma of the anal canal. Patients and methods One hundred consecutive patients (60 females and 40 males) with histologically confirmed squamous cell carcinoma of the anal canal were included in the retrospective study. They were treated at the Institute of Oncology Ljubljana from January 2003 till June 2013. For performance status (PS) the scoring system of the World Health Organization (WHO) was used19, and for TNM staging the criteria of the Union for International Cancer Control (UICC).20 Pre-treatment evaluation Pre-treatment evaluation consisted of physical and digital rectal examination, rectoscopy with biopsy and fine needle aspiration biopsy of enlarged in­guinal lymph nodes, also ultrasound-guided, like in other cancer patients.21 Imaging included chest X-ray or computer tomography (CT) of chest, ab­dominal ultrasound (US) or CT and magnetic reso­nance imaging (MRI) of the pelvis. Laboratory tests included serum chemistry and complete blood count in all patients, and testing for HIV infection in high-risk patients. A multidisciplinary team con­sisting of a surgeon, a radiation oncologist and a medical oncologist decided the treatment for each patient. Radiotherapy Clinical target volume (CTV) consisted of the tu­mour volume with a safety margin of 2–2.5 cm and the regional lymph node areas. An additional margin of 1 cm was applied to the CTV for the planning target volume (PTV). Initial tumour bor­ders were marked with tattoo. Positron emission tomography with computed tomography (PET­CT) was used as an aid in treatment planning. The treatment schedule for external beam radio­therapy (EBRT) consisted of 3-dimensional (3-D) conformal photon beam radiotherapy or intensity modulated radiotherapy (IMRT) with individual field arrangement. The total dose was 45 Gy in 25 fractions, 5-times weekly with 15 MV photon beam linear accelerator, plus a boost 10–15 Gy with interstitial pulsed-dose rate brachytherapy if tumour size was less than 5 cm. Metal needles were homogeneously implanted through a per­ineal template according to the rules of the Paris system. In tumours larger than 5 cm or in N2–3 disease, the boost was delivered with EBRT. CTV (brachytherapy/EBRT) of the boost correspond­ed to the initial gross tumour extension. In cases with positive inguinal lymph nodes, inguinal ar­eas were boosted with electrons to a total dose of 59.4 Gy. When IMRT technique was used, ingui­nal lymph nodes were involved in CTV and PTV and irradiated to the same total dose of 59.4 Gy. If the tumour involved or crossed the external anal sphincter, this area was covered with a 1 cm thick gelatinous bolus to raise the dose at the surface to at least 95% of the planned dose. 115 Chemotherapy Chemotherapy protocol consisted of 2 cycles of 96-hour continuous infusion of 5-fluorouracil with a daily dose of 1000 mg/m2 of body surface in the first and fifth week of radiotherapy. On day 1 the patients also received a bolus of mitomycin C in a dose of 10 mg/m2. Since 2006, we administered peroral cytostatic capecitabine in a dose of 825 mg/m2, twice daily, to cooperative patients with good performance status and without important comorbidities. First dose of capecitabine was ad­ministered one hour before the irradiation and the second dose 12 hours after. In cases of severe treatment toxicity according to common toxicity criteria22 radiotherapy and/or chemotherapy was modified according to the patient’s general condi­tion and laboratory findings or was even temporar­ily interrupted. Follow-up During treatment, the patients were examined weekly to assess acute toxicity and compliance with radiochemotherapy, and complete blood count and serum biochemistry were performed as well. The first post treatment examination was per­formed six weeks after the completion of radio-chemotherapy, and then every 2–3 months for the first 2 years and every 6 months in the following 3 years. When tumour response was incomplete, pa­tients were examined every 6 weeks over a period of 4 months after the end of the treatment. In this period we performed all necessary investigations to prove tumour viability or its progression and in such cases surgery (abdomino-perineal resection) was recommended. Tumour response was evaluated according to the WHO criteria.19 Statistical analysis The survival estimates were carried out by using the Kaplan-Meier method23 and a log rank test24 was used to test the differences in survival between subgroups. The end points of survival analysis were defined as follows: loco-regional control (LRC) as the time interval from the beginning of the treatment to the appearance of local and/or regional progression; disease-free survival (DFS) as the time interval from the beginning of the treatment to the appear­ance of local and/or regional progression and/or TABLE 1. Patients’ and tumours’ characteristics Gender female 60 male 40 Mean age (range) 63 (34–87) Performance status (WHO) 0 76 1 20 2 3 3 1 Tumour type Carcinoma of the anal canal 72 Carcinoma of the anal margin 28 Tumour histology Basaloid 12 Squamous 88 TNM N0 N1 N2 N3 T1 9 0 1 0 T2 36 6 1 0 T3 19 10 3 1 T4 1 1 7 5 Tumour stage I 9 II 55 IIIA 17 IIIB 19 WHO = World Health Organization appearance of distant metastases; disease-specific survival (DSS) as the time interval from the begin­ning of the treatment to the death because of can­cer; and overall survival (OS) as the time interval from the beginning of the treatment to the death due to any cause. For multivariate analysis, Cox proportional haz­ard model (with “Enter method”) was used.25 All statistical tests were two-sided and a P-value of p . 0.05 was considered statistically significant. Statistical analyses were performed by using SPSS version 22 (Chicago, IL). 116 TABLE 2. Haemoglobin (Hb) values in subgroups of patients Pre-treatment Hb 128 86–169 > 120 g/L 69 136 122–169 . 120 g/L 31 107 86–120 Mean on-treatment Hb 127 96–157 > 120 g/L 67 134 121–157 . 120 g/L 33 113 96–119 End-of-treatment Hb 121 77–159 > 120 g/L 46 134 121–159 . 120 g/L 54 114 77–120 Ethical consideration The study was carried out according to the Helsinki Declaration (1964, with later amend­ments) and according to the European Council Convention on Protection of Human Rights in Bio-Medicine (Oviedo, 1997). It was approved by the Institutional Review Board Committee and by the National Committee for Medical Ethics, Ministry of Health, of the Republic of Slovenia. Results The study was closed on February 15, 2014. Median follow-up time of all patients was 52 months (range: 1–129 months) and 72 months (range: 6–129 months ) for the survivors. On the day of analysis, 59 patients were alive, 22 patients died of anal ca­nal cancer, 15 patients died of other causes and in 4 patients the cause of death was unknown. Characteristics of patients and tumours are shown in Table 1. Characteristics of Hb values in subgroup of pa­tients are shown in Table 2. Ninety-two patients (92%) completed their treatment according to the protocol. In 8 patients the treatment was modified: three did not receive chemotherapy due to significant comorbidities (is­chemic heart disease or significant hepatopathy); in 1 patient chemotherapy was terminated due to acute side effects (chest pain due to a suspected is­chemic event) and in 1 patient due to febrile neu­tropenia. One patient refused further treatment after 45 Gy and 1 patient refused chemotherapy. One patient received concurrent chemotherapy with cisplatin due to simultaneous treatment of the synchronous oropharyngeal cancer. Median duration of radiochemotherapy was 1.9 months (range: 1–3.7 months). Fifty-six pa­tients received brachytherapy boost with medial dose of 18.5 Gy (range: 10–25 Gy) or EBRT boost with medial dose of 14.4 Gy (range: 9–14.4 Gy). Capecitabine was used instead of 5-fluorouracil in 25 patients. Tumour response to treatment Complete clinical remission of the disease was achieved in 80 patients. The tumour disappeared within six weeks after the treatment completion in 73 patients, and within 4 months in 7 patients. One of them was operated on because of presumed persistent disease, yet the pathologist did not find disease residues. Of the remaining 20 patients, in 1 patient the disease progressed during treatment, 9 patients had APR performed and 2 patients had inguinal lymphadenectomy due to recurrence in inguinal lymph nodes; 8 patients had inoperable residual disease. Survival The 5-year LRC, DFS, DSS and OS rates for all pa­tients were 72%, 71%, 77% and 62%, respectively. Univariate analysis for survival according to the Hb level and other parameters is shown in Table 3. In multivariate analysis, pre-treatment Hb (> 120 g/L vs. . 120 g/L) was an independent prognostic factor only for OS (hazard ratio [HR]= 0.419, 95% confidence interval [CI] = 0.190–0.927, p = 0.032) and stage (I & II vs. III) for DSS (HR = 3.523, 95% CI = 1.375–9.026, p = 0.009) and OS (HR = 2.230, 95% CI = 1.167–4.264, p = 0.015). Patients’ age, gender, tumour site, type of radio­therapy boost (tele- or brachytherapy) and type of chemotherapy (5-fluorouracil or capecitabine) did not have an influence on survival. Haemoglobin concentration during treatment In the group of patients with Hb > 120 g/L the mean Hb concentration during the treatment slightly but not significantly decreased (mean pre-treatment Hb = 139 g/L, mean end-of-treatment Hb = 125 g/L). However in the group of patients with Hb . 120 g/L it slightly increased (mean pre-treatment Hb = 106 g/L, mean end-of-treatment Hb = 113 g/L). One third of patients had low iron levels and received iron preparations. Nine patients received blood transfusion due to a drop in their Hb concentration below 100 g/L. 117 Acute side effects TABLE 3. Univariate analysis of survival of patients at 5 years by Hb level, tumour-, None of the patients died because of acute side patients-, and treatment characteristics effects. Most grade 3 side effects were caused by radiodermatitis. Serious, life-threatening infec­ tions were observed in 3 patients: 2 patients expe­rienced severe pneumonia that requested transfer to the intensive care unit and 1 patient developed Pre-treatment Hb > 120 g/L . 120 g/L 69 31 79% 57% P = 0.011 77% 57% P = 0.017 85% 56% P = 0.003 73% 39% P = 0.000 febrile neutropenia which required termination of radiochemotherapy. One patient developed severe stomatitis and needed parenteral nutrition. In 1 pa­tient, serious diarrhoea developed, which required Mean on-treatment Hb > 120 g/L . 120 g/L 67 33 78% 60% P = 0.037 76% 60% P = 0.054 82% 67% P = 0.081 68% 50% P = 0.007 hospitalization. Frequency and intensity of acute side effects are shown in Table 4. End-of-treatment Hb > 120 g/L . 120 g/L 46 54 82% 63% P = 0.022 80% 63% P = 0.037 89% 65% P = 0.011 75% 49% P = 0.003 Discussion Survival rates of our patients and the profile and frequency of acute side effects are similar to the Performance status PS 0 PS 1–3 76 24 73% 69% P = 0.480 73% 64% P = 0.283 80% 66% P = 0.231 72% 34% P = 0.000 results of other researchers.2,7,26-29 There was no dif­ference in survival of anal canal and anal margin cancer patients. The survival rate of patients with higher pre-treatment and end-of treatment Hb concentrations was generally better, compared to those patients with lower Hb concentrations, yet only pre-treatment Hb concentration was an in- Tumour stage T1–3 T4 Lymph node involvement no yes 86 14 65 35 75% 50% P = 0.054 79% 59% P = 0.032 75% 44% P = 0.015 79% 56% P = 0.017 84% 38% P = 0.000 87% 60% P = 0.000 68% 25% P = 0.001 70% 48% P = 0.000 dependent prognostic factor for OS. Patients with mean on-treatment Hb > 120 g/L only had sta­tistically significant better LRC and OS than pa­tients with Hb . 120 g/L. Many authors found that Overall disease stage I / II IIIA / IIIB 64 36 79% 59% P = 0.044 79% 57% P = 0.025 87% 61% P = 0.000 70% 49% P = 0.000 anaemic patients respond worse to radiotherapy and/or chemotherapy and have worse survival Histologic tumour type basaloid 12 100% 100% 100% 100% rates.2,8,12,13,15-18,30-41 There is convincing evidence of a correlation between Hb concentration and tu­ squamous 88 68% P = 0.030 67% P = 0.026 74% P = 0.051 57% P = 0.016 mour oxygenation in various kinds of tumours.42 Nordsmark’s et al. comparison of pre-treatment Hb with pre-treatment tumour pO2 measurements in head and neck cancer showed a quadratic regres- Tumour site anal canal anal margin 72 28 69% 81% P = 0.250 68% 81% P = 0.212 78% 73% P = 0.994 62% 61% P = 0.738 sion correlation between Hb concentration and Blood transfusion median pO2.43 Tumours of anaemic patients are consequently more hypoxic and more resistant to radiotherapy (and chemotherapy).16 The National no yes 91 9 72% 0% P = 0.993 71% 0% P = 0.950 78% 0% P = 0.333 64% 0% P = 0.044 Comprehensive Cancer Network (NCCN) guidelines recommend the use of blood transfusion in symp­tomatic patients with Hb concentration <100 g/L Overall radiation time . 1,08 months > 1,08 months 29 71 89% 64% P = 0.015 89% 63% P = 0.011 93% 69% P = 0.012 83% 51% P = 0.012 to improve oxygen delivery to the tumour.44 Nine patients in our study received blood transfusion. Operation no 73 89% 88% 88% 69% They had statistically significant worse OS than yes 27 29% 29% 52% 45% other patients. The conclusions about beneficial ef- P = 0.000 P < 0.000 P = 0.001 P = 0.018 fect of transfusion in our study cannot be made be- DFS = disease-free survival; DSS = disease-specific survival; Hb = haemoglobin; LRC = loco-regional cause the patients who received transfusion were control; N = number of patients; OS = overall survival few. The contribution to low survival of other un­ 118 TABLE 4. Acute treatment toxicities 0 1 2 3 4 Total Stomatitis 68 12 10 9 1 100 Nausea, vomiting 79 9 9 3 0 100 Diarrhoea 57 17 12 13 1 100 Hand-foot syndrome* 22 0 1 2 0 25 Radiodermatitis 10 12 13 64 1 100 Infection 51 14 23 9 3 100 Leucocyte count 37 31 20 10 2 100 Haemoglobin level 43 44 11 2 0 100 Platelet count 58 36 3 3 0 100 * Only in patients treated with capecitabine favourable factors, which are often combined with anaemia, was not possible to assess. The reports in the literature of the influence of transfusions on the outcome are not consist­ent. Some authors found favourable effect45, some found none46,47 and some found unfavourable ef­fect.2,32 It is possible that a better oxygen delivery is not sufficient to improve oxygenation of a tumour with high oxygen consumption.30,35 Moreover, anaemic patients are assumed to have a more ag­gressive disease from the start.35,46 Immune sup­pression in patients could also play a part (7, 35).7,35 The use of erythropoetin is controversial due to the possible effect on tumour growth14,33,48, how­ever, only in the subpopulation of patients whose tumours expressed erythropoetin receptors.49 Another potential mechanism by which erythro­poetin therapy may result in negative outcomes in cancer patients is through promotion of thrombo­vascular events.50 Therefore, it was not used in our patients. De Los Santos et al. believe the connection between anaemia and hypoxia is complex; there­fore, it is not clear whether transfusion or erythro­poetin do patients any favour.51 The Hb concentration during treatment pro­gressively decreased, which is in agreement with other reports.2,7,17,18,30-33,46 At the beginning of treat­ment, 31% of our patients were anaemic, and at the end 54%. That should cause more hypoxia in the tumour. It is possible that a decreased delivery of oxygen to the tumour due to of Hb drop dur­ing the treatment is partially counterbalanced by the reoxygenation due to shrinkage of the tumour and does not influence very much the outcome. In some patients with Hb . 120 g/L it was possible to raise the mean Hb level by the blood transfusion or by iron preparations. The significance of mean on-treatment Hb con­centration and end-of-treatment Hb concentration is less clear. Some authors found a positive effect of higher mean on-treatment Hb concentration on treatment outcome2,15,18,32,33,35 and some found a positive effect of higher end-of-treatment Hb con­centration on treatment outcome35,36, while others found no influence on outcome of either mean- or end- of-treatment Hb level.31 In our patients, the mean- or end- of treatment Hb levels had less in­fluence on survival compared to the pre-treatment values of Hb concentration. Our study showed that pre-treatment Hb was an important independent prognostic factor for over­all survival in patients with squamous cell carcino­ma of the anal canal and anal margin treated with radiochemotherapy, which is in agreement with findings of most other authors. Mean on-treatment Hb and end-of-treatment Hb do not seem to have much influence on survival. Because of a small number of patients who needed blood transfusion its influence on survival could not be assessed in our study. References 1. 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Prevalence and significance of anaemia in patients receiving long-course neoadjuvant chemoradiotherapy for rectal carcinoma. Colorectal Dis 2013; 15: 52-6. 34. Hoff CM, Hansen HS, Overgaard M, Grau C, Johansen J, Bentzen J, et al. The importance of haemoglobin level and effect of transfusion in HNSCC pa­tients treated with radiotherapy – Results from the randomized DAHANCA 5 study. Radiother Oncol 2011; 98: 28-33. 35. Lee SD, Park JW, Park KS, Lim SB, Chang HJ, Kim DY, et al. Influence of anemia on tumor response to preoperative chemoradiotherapy for locally advanced rectal cancer. Int J Colorectal Dis 2009; 24: 1451-8. 36. Hoff CM. Importance of hemoglobin concentration and its modification for the outcome of head and neck cancer patients treated with radiotherapy. Acta Oncol 2012; 51: 419-32. 37. van Acht MJ, Hermans J, Boks DE, Leer JW. The prognostic value of hemo­globin and a decrease in hemoglobin during radiotherapy in laryngeal carcinoma. Radiother Oncol 1992; 23: 229-35. 38. 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J Clin Oncol. 2006; 10; 24: 4708-13. 50. Hadland BK, Longmore GD. Erythroid-stimulating agents in cancer therapy: potential dangers and biologic mechanisms. J Clin Oncol 2009; 27: 4217-26. 51. De Los Santos JF, Thomas GM. Anemia correction in malignancy manage­ment: threat or opportunity? Gynecol Oncol 2007; 105: 517-29. 121 research article Evaluation of dosimetric effect caused by slowing with multi-leaf collimator (MLC) leaves for volumetric modulated arc therapy (VMAT) Zhengzheng Xu1,2, Iris Z. Wang1,2, Lalith K. Kumaraswamy1, Matthew B. Podgorsak1,2 1 Department of Radiation Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263 2 Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, NY 14214 Radiol Oncol 2016; 50(1): 121-128. Received: 16 June 2015 Accepted: 19 October 2015 Correspondence to: Zhengzheng Xu, Department of Radiation Medicine, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY, USA, 14263. Phone: + 1 919 724 7764; Email: zhengzheng.xu@roswellpark.org Disclosure: The authors declare no conflict of interest. Background. This study is to report 1) the sensitivity of intensity modulated radiation therapy (IMRT) QA method for clinical volumetric modulated arc therapy (VMAT) plans with multi-leaf collimator (MLC) leaf errors that will not trig­ger MLC interlock during beam delivery; 2) the effect of non-beam-hold MLC leaf errors on the quality of VMAT plan dose delivery. Materials and methods. Eleven VMAT plans were selected and modified using an in-house developed software. For each control point of a VMAT arc, MLC leaves with the highest speed (1.87-1.95 cm/s) were set to move at the maximal allowable speed (2.3 cm/s), which resulted in a leaf position difference of less than 2 mm. The modified plans were considered as ‘standard’ plans, and the original plans were treated as the ‘slowing MLC’ plans for simulating ‘standard’ plans with leaves moving at relatively lower speed. The measurement of each ‘slowing MLC’ plan using MapCHECK®2 was compared with calculated planar dose of the ‘standard’ plan with respect to absolute dose Van Dyk distance-to-agreement (DTA) comparisons using 3%/3 mm and 2%/2 mm criteria. Results. All ‘slowing MLC’ plans passed the 90% pass rate threshold using 3%/3 mm criteria while one brain and three anal VMAT cases were below 90% with 2%/2 mm criteria. For ten out of eleven cases, DVH comparisons between ‘standard’ and ‘slowing MLC’ plans demonstrated minimal dosimetric changes in targets and organs-at-risk. Conclusions. For highly modulated VMAT plans, pass rate threshold (90%) using 3%/3mm criteria is not sensitive in detecting MLC leaf errors that will not trigger the MLC leaf interlock. However, the consequential effects of non-beam hold MLC errors on target and OAR doses are negligible, which supports the reliability of current patient-specific IMRT quality assurance (QA) method for VMAT plans. Key words: VMAT; MLC; MapCHECK2; quality assurance; DVH Introduction Volumetric modulated arc therapy (VMAT) de­mands high level of precision and reliability from the linear accelerator (LINAC) control system be­cause the gantry rotation is synchronized with multi-leaf collimator (MLC) movement for accu­rate dose delivery.1,2 Highly modulated dose dis­tribution commonly requires MLC leaves of high speed moving along the whole arc.3-5 However, fast leaf motion during gantry rotation may be affected by interleaf friction or MLC motor problems that result in leaf position errors.6 Wijesooriya et al and Ling et al reported an increase in MLC leaf position errors due to fast moving leaves.3,7 Detecting dosimetric variation caused by MLC leaf errors is one important concern in patient-specific quality assurance (QA). Currently, several dose measuring systems are available for patient-specific QA.8-11 Fredh et al. evaluated the dosimet­ric effect of MLC position error on four single arc plans using Delta4®, OCTAVIUS®, COMPASS® and 122 TABLE 1. Volumetric modulated arc therapy (VMAT) plan parameters (dose, gantry speed, Leaf travel and modulation complexity score [LTMCS] and arcs), ranges of the maximal leaf speed, multi-leaf collimator (MLC) leaf position changes along 178 control points (CPs), and total modified leaves percentage of each VMAT arc P1 2.0 x 33 4.8 0.164 1 1.87-1.92 -1.90-1.88 1.5 P2 2.0 x 33 4.8 0.262 1 1.87-1.90 -1.90-2.00 1.6 2 1.87-1.90 -1.90-2.00 1.6 P3 2.0 x 33 4.8 0.163 1 1.87-1.90 -1.90-2.00 1.5 2 1.87-1.90 -1.90-2.00 1.6 B1 1.8 x 33 4.8 0.204 1 1.87-1.92 -1.88-1.90 1.6 B2 1.8 x 33 4.8 0.199 1 1.87-1.92 -1.88-1.90 1.5 2 1.87-1.92 -1.88-1.90 1.7 B3 1.8 x 33 4.8 0.217 1 1.85-1.90 -1.88-2.00 1.5 2 1.85-1.90 -1.88-2.00 1.6 A1 1.8 x 33 4.8 0.081 1 1.90-1.92 -1.80-1.80 1.7 2 1.90-1.92 -1.80-1.80 1.7 3 1.90-1.92 -1.80-1.80 1.7 4 1.90-1.92 -1.80-1.80 1.8 A2 1.8 x 33 4.8 0.083 1 1.92-1.95 -1.87-1.87 1.5 2 1.92-1.95 -1.87-1.87 1.6 3 1.92-1.95 -1.87-1.87 1.6 4 1.92-1.95 -1.87-1.87 1.5 A3 1.8 x 33 4.8 0.105 1 1.92-1.95 -1.87-1.87 1.7 2 1.92-1.95 -1.87-1.87 1.7 3 1.92-1.95 -1.87-1.87 1.7 A4 1.8 x 33 4.8 0.076 1 1.87-1.90 -1.80-1.93 2.2 2 1.87-1.90 -1.80-1.93 2.2 3 1.87-1.90 -1.80-1.93 2.3 4 1.87-1.90 -1.80-1.93 2.3 A5 1.8 x 33 4.8 0.084 1 1.90-1.92 -1.80-1.80 1.7 2 1.90-1.92 -1.80-1.80 1.7 3 1.90-1.92 -1.80-1.80 1.8 a P: Prostate VMAT cases; B: Brain VMAT cases; A: Anal VMAT cases. b Leaf travel and modulation complexity score (LTMCS) for a VMAT plan. LTMCS ranges from 0 to 1. Low LTMCS indicates high modulation complexity. EpiqaTM.11 All detectors demonstrated low pass rate failure to the 2 mm widening of both MLC banks, and this error has the least dosimetric impact on the plans. In addition, several studies have stated poor correlation between the gamma index pass rates of QA procedure and DVH deviations.12, 13 Although previous studies have studied both systematic and random MLC leaf errors in VMAT plans, in actual beam delivery, if the difference between actual leaf position and planned one is larger than 2 mm, the LINAC will trigger an MLC interlock which invokes a “beam hold-off”.14 Therefore, the purpose of this study is to evaluate the dosimetric error of clinical VMAT plans caused by MLC leaf position errors that will not trigger MLC interlock (i.e. beam hold-off ). In addition, we evaluated the sensitivity of patient-specific IMRT QA method using MapCHECK®2 for VMAT plans with non-beam-hold MLC errors. Methods A. Patient selection and plan complexity evaluation We selected 11 VMAT plans (Table 1) on three types of targets: anus, brain and prostate. Leaf trav­el and modulation complexity score (LTMCS) was used to characterize the modulation complexity of each VMAT plan.15 LTMCS ranges from 0 to 1 and 123 it approaches 0 for increasing degree of modula­tion and increasing total leaf travel distance. In this study, anal VMAT plans had low LTMCS while brain and prostate VMAT plans had moderate to high LTMCS (Table 1). LTMCS is derived by the product of leaf trav­el index (LTi) and modulation complexity score (MCSv).15,16 MCSv was derived by Masi et al to characterize the modulation degree of the MLC leaves of a VMAT plan. MCSv value of 1 indicates no modulation by MLC leaves (i.e. a plan of the least complexity), and the value decreases as modula­tion complexity increases. Total travel distance of all in-field moving MLC leaves was calculated and normalized to acquire LTi. When LTi approaches 1, it indicates short travel distance of all in-field mov­ing MLC leaves. LTi decreases to zero as total leaf travel distance increases. B. MLC leaf speed modifications All original treatment plan DICOM files were ex­ported from the EclipseTM (version 10.0, Varian Medical Systems, Inc., Palo Alto, USA) treatment planning system (TPS). The DICOM plans were modified through an in-house developed software. For each arc in the VMAT plan, in-field moving MLC leaves of 178 control points (CPs) on both banks were selected for leaf speed modifications. Speed of each moving MLC leaf per CP was calcu­lated based on MLC leaf position, gantry rotation angle and gantry speed as shown in Equation [1]. [1] and Here .t(n) is the gantry rotation time between two adjacent CPs, .(n) is the gantry angle of CP ‘n’, u(n) is the gantry speed of CP ‘n’, Vleaf (m, n) is the speed of mth leaf of CP ‘n’ and LP(m, n) is the position of mth leaf of CP ‘n’. MLC leaves on bank ‘A’ were marked from 1 to 60 while those on bank ‘B’ were marked from 61 to 120. For each CP of the arc, leaves on both banks (MLC leaf: 1-120) with the highest speed were set to move at 2.3 cm/s, resulting in a leaf position dif­ference at a maximum of 2 mm (Table 1: Range of leaf position chages along 178 CPs). New leaf position of CP ‘n’ is: [2] If one leaf was moving with the highest speed at two consecutive CPs, leaf motion direction of each CP was further considered as shown below: FIGURE 1. Illustration of multi-leaf collimator (MLC) leaf position modifications of one leaf when it is moving with the highest speed at two consecutive control points (CPs) (i.e. CP2 and CP3). (A) The leaf is moving in the same direction. (B) The leaf is moving back and forth. Red arrow (Vmax1 or 2.3cm/s) represents leaf speed from CP1 to CP2; Black arrow (Vmax2 or 2.3cm/s) represents leaf speed from CP2 to CP3. Black bars represent original leaf positions. Blue bars represent new leaf positions after modification. 1. If the motion directions of the next two CPs remained the same, then both leaf positions of corresponding CP were subject to modification (Figure 1A). 2. If the motion directions of the next two CPs were different, we only modified the leaf position of the middle CP so that both leaf speed values would be increased (Figure 1B). The total modified MLC leaves percentage (Equation 3 and Table 1) was an indicator of the amount of MLC leaves that had been changed to the maximal speed in each arc; [3] where modified in field leaves on both banks of CPi is the total modified MLC leaves that moving with the highest speed of current CP. Leaf speed modi­fication would not be applied if it caused any leaf pair collision (Gap between leaf pair should be no less than 0.5 mm in actual delivery). In this study, modified plans were considered as ‘standard’ plans where MLC leaves were allowed to move at the maximal speed (2.3 cm/s). The origi­nal plans were considered as ‘slowing MLC’ plans where the highest MLC speed was lower than 2.3 cm/s. There were no changes in monitor unit (MU) and gantry speed per CP in all modified VMAT plans. C. MLC leaf speed change evaluation Having increased the leaf speed of one CP to the maximal limit without triggering the MLC error interlock (i.e. MLC leaf position difference was less than 2 mm), leaf speed of the next CP would be 124 TABLE 2. Demonstration of the impact on leaf speed of adjacent control points (CPs) due to leaf modifications A (ori) 4.6 5.4 5.9 1.8 1.1 A (mod) 4.6 5.6 5.9 2.3 0.7 B (ori) 4.6 5.4 4.9 1.8 -1.1 B (mod) 4.6 5.6 4.9 2.3 -1.6 a Scenario A: Leaf moved forward from LP1 to LP2, then moved forward from LP2 to LP3. Scenario B: Leaf moved forward from LP1 to LP2, then moved backward from LP2 to LP3. In the table, ‘LPn’: leaf position at CP ‘n’ =1,2,3; ‘ori’: original leaf positions; ‘mod’: leaf positions after modification; positive speed: leaf moved forward; negative speed: leaf moved backward. The speed was the distance between LP1,2,3 divided by .t =0.435s. For both scenarios A and B, we only modified LP2 from 5.4 to 5.6 to increase LP1-2 speed from 1.8 cm/s to 2.3 cm/s. affected by this modification. With .t(n) of each CP remained unchanged, increasing leaf speed by modifying leaf position of current CP while keep­ing the leaf position of the next CP unchanged resulted in consequential change of leaf speed of the next CP. As a result, total number of leaf speed changes in one arc is twice as many as the total number of MLC leaves that were set to the maximal leaf speed (see Equation [4] below). This accompa­nied effect caused by the MLC leaf modification either increases or decreases the MLC leaf speed of the next CP according to the leaf motion direc­tion (Table 2). We have taken this accompanied leaf speed modification into account when evaluating dosimetric changes. Because of the high complexity (i.e. low LTMCS) of the anal VMAT plans, we further analyzed MLC leaf changes in these VMAT plans. The average percentage of modified MLC leaves (Table 3) was the summation of total percentage of modified MLC leaves for all arcs (Table 1: Total Modified MLC leaves %) divided by number of total arcs in the plan. The average percentage of faster moving leaves ( ) depends on MLC leaves that were set to the maximal speed and total faster moving leaves after modifications including those modified leaves of current CP and affected leaves of the next CP (Equation [4]); [4] where n is the number of total arcs. D. Planar dose measuring system In this study, we used MapCHECK®2 2D di­ode array system (Model 1177, Sun Nuclear Co., Melbourne, FL) for evaluating the effect of slow­ing MLC leaves on planar dose delivery accuracy. MapCHECK®2 along with its software have been widely used as the clinical implementation for patient-specific verification of VMAT plans due to its compact diode size (0.8 mm×0.8 mm), dose lin­earity, real-time measurement, reproducibility and sensitivity.17-21 E. Dosimetric evaluation E.1 Measurement and uncertainty evaluation All the VMAT plans (‘standard’ and ‘slowing MLC’ plans) were delivered using a Varian Trilogy® LINAC on the same day. Measurement of each arc was then compared with the corresponding calculated planar dose from the TPS with respect to absolute dose Van Dyk distance-to-agreement (DTA) comparison (dose difference is normalized to global maximum) using 3%/3 mm criteria.22 All measurements were repeated on two consecutive days. The uncertainty was then obtained by evalu­ating the variation in repeated measurements. E.2 Dosimetric evaluation of ‘slowing MLC’ plans According to current pre-treatment IMRT QA method for VMAT plans with MapCHECK®2, measurement of each arc in the ‘standard’ plan was compared with calculated planar dose of the ‘standard’ plan with respect to absolute dose Van Dyk DTA comparison using 3%/3 mm and 2%/2 mm criteria. Pass rate (PSstandard) of the comparison was demonstrated in percentage. PSstandard of each arc using 3%/3 mm criteria was used as a baseline to verify that all the plan parameters had been cor­rectly transferred from control console computer to LINAC for delivery. Each ‘slowing MLC’ plan was considered as a ‘standard’ plan with MLC leaf errors that would not trigger any MLC interlock to interrupt the beam delivery. In order to evaluate the sensitivity of the IMRT QA method for VMAT plans with non-beam-hold leaf errors, we delivered each ‘slowing MLC’ plan and compared the measurement with calcuated planar dose of the ‘standard’ plan with respect to absolute dose Van Dyk DTA comparison using 3%/3 mm and 2%/2 mm criteria to acquire the pass rate (PSslowing MLC) in percentage. Because of the MLC leaf errors in each ‘slowing MLC’ plan, there was a decrease in pass rate of each arc (Equation [5]). [5] 125 The correlations between the decreases in pass rates using 3%/3mm and 2%/2mm criteria and LTMCS were analyzed through Spearman’s corre­lation coefficient.23 Finally, the 3D dose distribution of each plan was calculated in the TPS and dose-volume histo­gram (DVH) for targets and organs-at-risk (OAR) were obtained. For clinical dosimetric evaluation, mean target dose (Dmean), dose that covers 95% (D95) of the planning target volume, and Normal Tissue Complication Probability (NTCP) using Lyman Kutcher Burman (LKB) model23 were calcu­lated for all the plans. Clinical dosimetric param­eters of ‘standard’ and ‘slowing MLC’ plans were compared using the Wilcoxon signed-rank test.24 Results A. Pass rate and uncertainty evaluation Figure 2 demonstrated pass rate of each arc and variation of measurements based on repeated measurements on two consecutive days. Among all the arcs in both ‘standard’ and ‘slowing MLC’ plans, the maximal variation found was 0.3% with respect to the 91.5% pass rate. B. Prostate cases For all three prostate cases, PSstandard and PSslowing MLC using 3%/ 3 mm and 2%/2 mm criteria were all higher than 90% (Figure 3 and 4: Prostate). Dosimetric differences of Dmean, D95, NTCP (bladder, rectum, leaf and right femoral heads) be­tween ‘slowing MLC’ plans and ‘standard’ plans were: 0.47 ± 0.17 Gy (p > 0.05), 0.33 ± 0.13 Gy (p TABLE 3. Target dose differences between ‘standard’ and ‘slowing multi-leaf collimator (MLC)’ anal volumetric modulated arc therapy (VMAT) plans, total leave states, and average percentages of modified leaves and faster moving leaves of anal cases A1 -0.8 -0.2 120 × 178 1.7 56.5 A2 -0.9 -0.3 120 × 178 1.6 53.6 A3 -1.2 -0.5 120 × 178 1.7 51.3 A4 -2.2 -1.0 120 × 178 2.3 69.0 A5 -1.1 -0.3 120 × 178 1.7 52.7 a Negative sign means dose of ‘standard’ plan is lower than that of ‘slowing MLC’ plan CP = control point > 0.05), 1% ± 1%bladder (p > 0.05), 3% ± 2%(p > rectum 0.05), 2% ± 1%left fem (p > 0.05), 2% ± 1%right fem (p > 0.05), respectively. C. Brain cases For all three brain cases, PSstandard and PSslowing MLCusing 3%/ 3 mm and 2%/2 mm criteria were all higher than 90% (Figure 3 and 4: Brain) except for arc 2 of brain case B2. Dosimetric differences of Dmean, D95, NTCP (brain stem, cerebellum, spi­nal cord, left and right cochlea) between ‘slowing MLC’ plans and ‘standard’ plans were: 0.13 ± 0.05 Gy (p > 0.05), 0.17 ± 0.09 Gy (p > 0.05), 1% ± 1%brain (p > 0.05), 1% ± 1%cerebellum (p > 0.05), 0% ± 1%spinal stem (p > 0.05), 1% ± 2%left cochlea (p > 0.05), 1% ± 1%right cord (p > 0.05), respectively. cochlea 126 D. Anal cases The LTMCS scores of Anal VMAT plans were smaller than both brain and prostate VMAT plans (Table 1: LTMCS) indicating higher modulation by MLC leaves. For anal VMAT cases, PSstandard and PSslowing MLC using 3%/3 mm criteria were all higher than 90% while ‘slowing MLC’ plans of cases A3, A4 and A5 demonstrated less than 90% pass rates using 2%/2 mm criteria (Figure 4: Anus). Dosimetric differences of NTCP (bladder, rectum, large bowel and femoral heads) between ‘slowing MLC’ plans and ‘standard’ plans were: 2% ± 2%blad­(p > 0.05), 3% ± 1%(p > 0.05), 2% ± 1%large der rectum (p > 0.05), 1% ± 1%femheads (p > 0.05), respective- bowel ly. Compared with anal case A3 and A5, case A4 demonstrated substantial dosimetric differences between the ‘standard’ and ‘slowing MLC’ plans where .Dmean and .D95 were 2.2 Gy and 1.0 Gy re­spectively (Figure 5 and Table 3). E. Correlation between LTMCS and dosimetric parameters The correlation between decreases in pass rates of VMAT arcs using 2%/2 mm criteria and LTMCS is moderate to strong (rs = 0.597, Figure 6A). When using 3%/3 mm criteria, the correlation is weak to moderate (rs = 0.453, Figure 6B). Discussion A. Measurement uncertainty By using lasers and front pointer for device posi­tioning, the measurement setup was of high consist­ency. Absolute dose calibration for MapCHECK®2 was performed every day before dose measure­ment.25,26 Therefore, the source of the uncertainty is mainly due to variability of MLC leaf motion. The small error bars in Figure 2 indicate that the meas­urement variability is very small. B. Anal case A4 results For anal case A4, since the MU of each control point remained unchanged, and ‘slowing MLC’ plan had more slowly moving MLC leaves, more area were being irradiated that resulted in higher dose. The average percentage of faster moving leaves indicates the amount of MLC leaves moving back-and-forth. The average percentages of modified MLC leaves (2.3%) and average percentage of faster moving leaves (69%) of anal case A4 are higher compared with other anal 127 cases (Table 3), indicating that more high speed MLC leaves were moving back and forth to create a highly modulated VMAT plan. Accordingly, the anal case A4 has the minimal LTMCS among all anal cases studied. Moreover, all four arcs of anal case A4 have large fields (e.g. 14 cm ×30 cm, 14 cm ×29 cm, 30 cm ×14 cm, 30 cm ×14 cm for arc 1, 2, 3, 4 respectively). Wijesooriya et al reported the accuracy of RapidArc delivery holds for leaf velocities with small dosi­metric uncertainties for 5 mm width MLC leaves which are in the central 20 cm of field.7 They found that three VMAT plans with large MLC leaves with 1cm width at high speed (2.1–2.4 cm/s) demon­strated higher leaf position inaccuracy. Therefore, large MLC leaves in the VMAT plan of anal case A4 have more effect on dose delivery inaccuracy. C. Pass rates and dosimetric parameters When using 3%/3 mm criteria, all 11 cases includ­ing ‘standard’ and ‘slowing MLC’ plans passed the institutional 90% acceptance threshold of absolute dose DTA comparison. Dosimetric differences (e.g. .Dmean , .D95 and NTCP) between ‘standard’ and ‘slowing MLC’ plans in targets and normal tissues were minimal indicating that VMAT plans with non-beam-hold MLC leaf errors (leaf position dif­ ference . 2 mm) remain the planned dose coverage except for anal case A4. Using 2%/2 mm criteria, decrease in pass rates of VMAT arcs demonstrat­ed stronger correlation with VMAT modulation complexity which is characterized by LTMCS (Figure 6A). Some arcs in ‘slowing MLC’ plans of anal cases A3 and A5 showed less than 90% pass rates us­ing 2%/2mm criteria although differences in do­simetric parameters are small (e.g. .Dmean and ). However, anal case A4 showed a consist­ .D95 ent decrease in pass rates and dose conformity. Compared with the ‘standard’ plan, ‘slowing MLC’ plan of anal case A4 delivered higher planar dose (Figure 7) which is consistent with the changes in DVH curves in Figure 5B. To further ensure the do­simetric quality of VMAT plans like anal case A4 that have the following features: 1. Highly modu­lated multiple arc VMAT plan (e.g. LTMCS < 0.1); and 2. Arc has large field size that involves more thick MLC leaves, we recommend 2%/2mm criteria for absolute dose Van Dyk DTA comparison which is more sensitive to non-beam-hold leaf position er­rors. Conclusions For ten out of eleven cases, DVH comparisons be­tween ‘standard’ and ‘slowing MLC’ VMAT plans 128 demonstrated minimal dosimetric changes in tar­gets and OAR. Pass rate threshold (90%) using 3%/3 mm criteria is not sensitive in detecting MLC leaf errors that will not trigger the MLC leaf inter­lock. However, the consequential effects on target and OAR are negligible, which supports the reli­ability of current IMRT QA method for VMAT plan verification. References 1. Clivio A, Fogliata A, Franzett PA, Nicolini G, Vanetti E, Wyttenbach R, et al. Volumetric-modulated arc radiotherapy for carcinomas of the anal canal: A treatment planning comparison with fixed field IMRT. Radiother Oncol 2009; 92: 118-24. 2. Sale C, Moloney P. Dose comparisons for conformal, IMRT and VMAT pros­tate plans. J Med Imaging Radiat Oncol 2011; 55: 611-21. 3. Ling CC, Zhang P, Archambault Y, Bocanek J, Tang G, Losasso T. Commissioning and quality assurance of RapidArc radiotherapy delivery system. Int J Radiat Oncol Biol Phys 2008; 72: 575-81. 4. Tatsumi D, Hosono MN, Nakada N, Ishii K, Tsutsumi S, Inoue M, et al. Direct impact analysis of multi-leaf collimator leaf position errors on dose distribu­tions in volumetric modulated arc therapy: a pass rate calculation between measured planar doses with and without the position errors. Phys Med Biol 2011; 56: N237-46. 5. Peng J, Zhang Z, Zhou L, Zhao J, Wang J, Kong L, et al. A study on investigating the delivery parameter error effect on the variation of patient quality assur­ance during RapidArc treatment. Med Phys 2013; 40: 031703. 6. Oliver M, Gagne I, Bush K, Zavgorodni S, Ansbacher W, Beckham W. Clinical significance of multi-leaf collimator positional errors for volumetric modu­lated arc therapy. Radiother Oncol 2010; 97: 554-60. 7. Wijesooriya K, Aliotta E, Benedict S, Read P, Rich T, Larner J. RapidArc patient specific mechanical delivery accuracy under extreme mechanical limits us­ing LINAC log files. Med Phys 2012; 39: 1846-53. 8. Arumugam S, Xing A, Goozee G, Holloway L. Detecting VMAT delivery er­rors: a study on the sensitivity of the ArcCHECK-3D electronic dosimeter. Journal of Physics: Conference Serire 444(1) 2013; 1-4. 9. Schreibmann E, Dhabaan A, Elder E, Fox T. Patient-specific quality assurance method for VMAT treatment delivery. Med Phys 2009; 36: 4530. 10. Bakhtiari M, Kumaraswamy L, Bailey DW, deBoer S, Malhotra HK, Podgorsak MB. Using an EPID for patient-specific VMAT quality assurance. Med Phys 2011; 38: 1366-73. 11. Fredh A, Scherman JB, Fog LS, Munck af RP. Patient QA systems for rota­tional radiation therapy: a comparative experimental study with intentional errors. Med Phys 2013; 40: 031716. 12. Zhen H, Nelms BE, Tome WA. Moving from gamma pass rates to patient DVH-based QA metrics in pretreatment dose QA. Med Phys 2011; 38: 5477-89. 13. Oliver M, Bush K, Zavgorodni S, Ansbacher W, Beckham WA. Understanding the impact of RapidArc therapy delivery errors for prostate cancer. J Appl Clin Med Phys 2011; 12: 32-43. 14. LoSasso T, Chui CS, Ling CC. Comprehensive quality assurance for the deliv­ery of intensity modulated radiotherapy with a multileaf collimator used in the dynamic mode. Med Phys 2001; 11: 2209-19. 15. McNiven AL, Sharpe MB, Purdie TG. A new metric for assessing IMRT modu­lation complexity and plan deliverability. Med Phys 2010; 37: 505. 16. Masi L, Doro R, Favuzzaet V, Cipressi S, Livi L. Impact of plan parameters on the dosimetric accuracy of volumetric modulated arc therapy. Med Phys 2013; 40: 071718. 17. Iftimia I, Cirino ET, Xiong L, Mower HW. Quality assurance for methodology for Varian RapidArc treatment plans. J Appl Clin Med Phys 2010; 11: 130-43. 18. Gloi AM, Buchanan RE, Zuge CL, Goettler AM. RapidArc quality assurance through MapCHECK. J Appl Clin Med Phys 2011; 12: 39-47. 19. Jursinic PA, Sharma R, Reuter J. MapCHECK used for rotational IMRT measurements: step-and-shoot, TomoTherapy, RapidArc. Med Phys 2010; 37: 2837-46. 20. Rinaldin G, Perna L, Agnello G, Pallazzi G, Cattaneo GM, Fiorino C, et al. Quality assurance of RapidArc treatments: performances and pre-clinical verifications of a planar detector (MapCHECK2). Phys Medica 2014; 30:184­ 90. 21. Shim SJ, Shim JB, Lee SH. Quality assurance of volumetric modulated arc therapy for Elekta Synergy. Korean J Med Phys 2012; 23: 33-41. 22. Van Dyk J, Barnett RB, Cygler JE, Shragge PC. 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Radiol Oncol 2016; 50(1): 1-13. doi:10.1515/raon-2016-0006 Priporočila za izboljšanje kvalitete poročanja pri kliničnih raziskavah o elektrokemoterapiji, ki so utemeljena na kvantitativnem sistematičnem pregledu Campana LG, Clover AJP, Valpone S, Quaglino P, Gehl J, Kunte C, Snoj M, Čemažar M, Rossi CR, Miklavčič D, Serša G Izhodišča. Elektrokemoterapija postaja uveljavljena metoda za zdravljenje malignomov kože in ostalih lokalizacij. Njena uporaba v Evropi narašča. Izvedba je razvita iz čvrstih eksperimentalnih in kliničnih dokazov. S predlaganim konsenzom želimo formalizirati poročanje in okrepiti z dokazi utemeljena priporočila za prakso. Konsenz naj nastane na podlagi visoko kvalitetnih kliničnih podatkov, kliničnega znanja in odgovora bolnikov. Prvi korak je objavljen v pričujočem članku, katerega namen je kritično ovrednotiti kvaliteto poročanja o elektrokemoterapiji in podati priporočila za nadaljne poročanje. Metode. Analizirali smo kvaliteto poročanja v objavljenih raziskavah o elektrokemoterapiji, da bi ustvarili posebna, za meto­do specifična priporočila za poročanje. Opravili smo vseobsežen pregled literature, ki je bila objavljena med leti 2006 in 2015. Nato smo kvantitativno analizirali tekste s 47 kriteriji za kvaliteto, med katere so sodili zasnova raziskave, opis obravnavanih bolnikov, opis izvedbe zdravljenja in izida zdravljenja ter tudi analiza rezultatov. Končna ocena je temeljila na deležu raziskav, ki so izpolnjevale posamezen kriterij za kvaliteto. Rezultati. Pregledali smo 56 raziskav, ki so jih objavili v obdobju med leti 2006 in 2015, in jih 33 analizirali. Skupaj smo analizirali 1215 bolnikov. Kvaliteta poročanja je bila zelo raznolika. 24 (73 %) raziskav so izvedli v enem centru, brez primerjave z drugimi centri, in samo 15 (45 %) raziskav je bilo prospektivnih, samo dve od teh pa sta bili uvrščeni v register raziskav. Tehnični način izvedbe elektrokemoterapije so vedno opisali, pri večini raziskav (31/33) glede na standardne pogoje izvedbe. Kakovost po­ročanja o značilnostih bolnikov je bila raznolika, med 45 % in 100 % izpolnjevanja kakovostnih meril za opis populacije bolnikov. Poročanje o načinu in izidu zdravljenja je bilo prav tako zelo raznoliko; izpolnjenih je bilo med 3 % in 10 0% meril. Na koncu lahko ugotovimo, da poročanje o rezutatih raziskav variira, med izpolnitvijo 27 % do 100 % meril za kakovost. Na osnovi teh rezultatov smo pripravili priporočila za izboljšanje kvalitete poročanja pri kliničnih raziskavah o elektrokemoterapiji. Zaključki. Obseg kliničnih podatkov o elektrokemoterapiji narašča, vendar potrebujemo kakovostneješe podatke. Objavljeni članki velikokrat ne navajajo primernega opisa preučevane populacije, načina zdravljenja in izida zdravljenja. Naša priporočila so namenjena izboljšanju kakovosti podatkov v prihodnjih raziskavah o elektrokemoterapiji, želimo, da bi bila v pomoč raziskovalcem, da bi zagotovili večjo enovitost publikacij, ki bi omogočila utemeljitve na dokazih. Radiol Oncol 2016; 50(1): 14-20. doi:10.1515/raon-2016-0003 Elektrokemoterapija žleznega raka trebušne slinavke Bimonte S, Leongito M, Granata V, Barbieri A, del Vecchio V, Falco M, Nasto A, Albino V, Piccirillo M, Palaia R, Amore A, di Giacomo R, Lastoria S, Setola SV, Fusco R, Petrillo A, Izzo F Izhodišča. Žlezni rak trebušne slinavke je eden od najbolj agresivnih rakov z visoko umrljivostjo. Bolezen se širi agresivno infiltrativno lokalno in zgodaj metastazira. Hkrati ni odzivna na kemoterapijo ali kemo-radioterapijo. Radikalna resekcija je še vedno edino kurativno zdravljenje raka trebušne slinavke, vendar smernice priporočajo multimodalno zdravljenje. Zato išče­mo druge terapevtske modalitete za lokalno zdravljenje. Zaključki. Kemorezistenca raka trebušne slinavke je pogojena s slabim privzemom citostatikov v tumorske celice zaradi prostega fibrotičnega tkiva. Če bi povečati privzem citostatikov v tumorske celice z elektrokemoterapijo, bi lahko povečali terapevtski odgovor. Povzemamo do sedaj objavljene raziskave o učinkovitosti in varnosti elektrokemoterapije na predklinič­nem nivoju kot tudi v objavljenih kliničnih raziskavah. Radiol Oncol 2016; 50(1): 21-27. doi:10.1515/raon-2015-0048 Učinkovitost elektrokemoterapije pri bolnikih z malignim melanomom po predhodnem zdravljenju z IFN-. Hribernik A, Čemažar M, Serša G, Bošnjak M, Snoj M Izhodišča. Kombinacija elektokemoterapije z imunomodulatorji je do sedaj že povečala uspešnost zdravljenja malignega melanoma. Vendar pa učinkovitost elektrokemoterapije pri bolnikih z malignim melanomom po predhodnem zdravljenju z interferonom alfa (IFN-.) še ni bila ovrednotena. Cilj naše raziskave je bil retrospektivno ovrednotiti varnost in učinkovitost elekrokemoterapije po predhodnem zdravljenju z IFN-.. Bolniki in metode. Z retrospektivno opazovalno raziskavo smo analizirali bolnike z napredovalim malignim melanomom, ki smo jih po predhodnem pooperativnem dopolnilnem zdravljenju z IFN-. zdravili z elektokemoterapijo. V raziskavo smo lah­ko vključili pet bolnikov, ki smo jih zdravili z elektrokemoterapijo med januarjem 2008 in decembrom 2014, ne glede na čas predhodnega zdravljenja z IFN-.. Rezultati. Elektrokemoterapija se je pri bolnikih z malignim melanomom, po predhodnem zdravljenju z IFN-., pokazala kot varna in učinkovita metoda zdravljenja. Pri bolnikih z eno ali dvema metastazama je bil odgovor na zdravljenje popoln, bolniki z multiplimi metastazami pa so na zdravljenje odgovorili različno. Pri prvem bolniku z 23 metastazami so vse metastaze na zdravljenje odgovorile popolnoma, pri drugem bolniku je več kot 85 % izmed 80 zdravljenih metastaz odgovorilo popolnoma, pri tretjem bolniku z multiplimi metastazami pa je vseh 5 zdravljenih metastaz na zdravljenje odgovorilo delno. Če upoštevamo vse metastaze vseh pacientov, je kar 85 % metastaz na zdravljenje odgovorilo popolnoma. Zaključki. Elektrokemoterapija po predhodnem zdravljenju z IFN-. se je pokazala kot varna in učinkovita metoda zdra­vljenja, z visoko stopnjo popolnega odgovora tako pri posameznih, kot tudi pri multiplih metastazah. Razlog za visoko stopnjo popolnega odgovora je morda imunomodulatorni učinek IFN-., vendar so za potrditev te domneve potrebne nadaljnje klinične raziskave. Radiol Oncol 2016; 50(1): 28-38. doi:10.1515/raon-2016-0009 Statistični model za opis istočasne ireverzibilne elektroporacije in poškodb krvno-možganske pregrade z elektroporacijo Sharabi A, Kos B, Last D, Guez D, Daniels D, Harnol S, Mardor Y, Miklavčič D Izhodišča. Na elektroporaciji temelječe terapije kot sta elektrokemoterapija in ireverzibilna elektroporacija predstavljajo obetajoča orodja za zdravljenje tumorjev. Kadar jih uporabljamo v možganih, lahko elektroporacija povzroči začasno po­škodbo krvno-možganske pregrade, ki je večja od prostornine ireverzibilno elektroporiranega tkiva in s tem lahko omogoči učinkovito prehajanje zdravil. Glavni namen raziskave je bil razvoj statističnega modela, ki bi napovedal celično smrt in poškodbo krvno-možganske pregrade po elektroporaciji. Model bi lahko uporabljali za individualno načrtovanje zdravljenja. Materiali in metode. Modele celične smrti in poškodbe krvno-možganske pregrade smo razvili na osnovi Peleg-Fermijevega modela v kombinaciji z numeričnimi modeli električnega polja. Model lahko računa pražne vrednosti električne­ga polja za celično smrt in poškodbo krvno-možganske pregrade ter razloži odvisnost od števila električnih pulzov. Validirali smo ga z uporabo posnetkov MRI možganov podgan po elektroporaciji. Rezultati. Analiza z uporabo linearne regresije je potrdila, da model dobro razloži prostornine ireverzibilno elektropori­ranega tkiva in poškodbi krvno-možganske pregrade kot funkcijo števila električnih pulzov (r2 = 0,79; p < 0,008, r2 = 0.91; p < 0.001). Rezultati so pokazali, da je naraščanje učinka z naraščanjem števila pulzov omejeno. Razmerje med pražnimi vrednostmi električnega polja za celično smrt in preživetje celic je bilo relativno ozko (med 0,88-0,91) tudi za majhno število električnih pulzov in je bilo šibko odvisno od števila električnih pulzov. Za poškodbo krvno-možganske pregrade je razmerje med volumnom poškodovane in nepoškodovane krvno-možganske pregrade naraščalo s številom električnih pulzov. Premeri poškodbe krvno-možganske pregrade so bili v povprečju večji za 67 % ± 11 % kot premeri ireverzibilno elektroporiranega tkiva. Zaključki. Statistični model se lahko uporablja za razlago odvisnosti učinkov zdravljenja od števila električnih pulzov, ne glede na zasnovo poskusa. Radiol Oncol 2016; 50(1): 39-48. doi:10.1515/raon-2016-0015 Zdravljenje mišjega melanoma B16F10 z elektrokemoterapijo s pulznim magnetnim poljem (PEMF) in vivo Kranjc S, Kranjc M, Ščančar J, Jelenc J, Serša G, Miklavčič D Izhodišča. S pulznim magnetnim poljem (PEMF) inducirano električno polje domnevno poveča prepustnost membrane ma­gnetnemu polju izpostavljenih celic, podobno kot pri običajni elektroporaciji. Takšno nekontaktno PEMF predstavlja obetaven pristop vnosa zdravilnih učinkovin v celico. Materiali in metode. Neinvazivno elektroporacijo smo izvedli s pulznim generatorjem magnetnega polja, ki smo ga do­vedli preko aplikatorja s tuljavo. Podkožne mišje tumorje melanoma B16F10 smo zdravili z intravenoznim vbrizganjem cisplatina (CDDP) (4 mg/kg), lokalno aplikacijo PEMF (480 bipolarnih pulzov pri frekvenci 80 Hz, posamezni pulz je trajal 340 µs) in kom­binacijo obeh zdravljenj (elektrokemoterapija = PEMF + CDDP). Protitumorsko delovanje zdravljenja smo vrednotili s testom zaostanka rasti tumorjev. Nadalje smo določili vnos platine (Pt) v tumorje, Pt v serumu kot tudi vezavo Pt na DNK v celicah in vsebino Pt v medceličnini z induktivno sklopljeno plazmo in masno spektrometrijo. Rezultati. Protitumorsko delovanje elektrokemoterapije s CDDP posredovano s PEMF je bilo primerljivo z običajno elektroke­moterapijo s CDDP. Zaostanek rasti tumorjev je bil 2,3 dneva in pri slednji 3,0 dni. Zdravljenje tumorjev samo s PEMF ali samo s CDDP ni zakasnilo rasti tumorjev. Učinek kombiniranega zdravljenja tumorjev je posledica povečanega vnosa Pt v tumorje po izpostavitvi PEMF, kot tudi njene vezave na DNK. Dokazali smo približno dvakratno povečanje vnosa Pt v celice. Zaključki. Rezultati zdravljenja mišjega melanoma in vivo nakazujejo možno uporabo elektroporacije s pulznim magnetnim poljem v biomedicini, npr. pri elektrokemoterapiji. Prednosti takšne elektroporacije so nekontaktna, neboleča aplikacija ob primerljivi elektroporaciji z običajno elektroporacijo. Radiol Oncol 2016; 50(1): 49-57. doi:10.1515/raon-2016-0013 Prototip fleksibilnih in prilegajočih elektrod za zdravljenje velikih površinskih tumorjev z elektrokemoterapijo Campana LG, Dughiero F, Forzan M, Rossi CR, Sieni E Izhodišča. Kožne recidive raka dojke na prsnem košu lahko učinkovito zdravimo z elektrokemoterapijo. Zaradi limfogenega razsoja pa so te lezije večkrat velike in jih je težko zdravimo z ustaljeno obliko elektrokemoterapije. Metoda zdravljenja je s komercialno dostopnimi elektrodami lahko zamudna in je možno, da nehomogeno pokrije zdravljeno področje. Materiali in metode. Namen raziskave je bil izdelati prototip elektrod s fleksibilnim vzorcem elektrod za izvajanje elektro­kemoterapije velikih kožnih tumorjev. Naredili smo konektor za priključitev na generator električnih pulzov. Izvedli smo labora­torijske teste na krompirju, da bi ocenili učinkovitost elektroporacije. Rezultati. Razvili smo nov tip elektrod za elektrokemoterapijo, ki je primeren za zdravljenje kožnih recidivov raka dojke na prsnem košu. Fleksibilnost vzorca namestitve elektrod smo preizkusili na krompirju, kjer se spremeni barva ob učinkoviti elek­troporaciji. Preliminarni rezultati nakazujejo da fleksibilna podlaga elektrod omogoča prilagajanje površini prsnega koša in omogoča zdravljenje večjih površinskih tumorjev v kratkem času 2–5 minut. Zaključki. Naredili smo prototip novih elektrod za zdravljenje večjih površinskih tumorjev. Nove elektrode so klinično upo­rabne, saj je njihova prednost v kratkem času dovajanje večjega števila električnih pulzov, kar tudi omogoča izvedbo elekt­rokemoterapije v dovoljenem časovnem oknu. Tako smo tudi skrajšali čas anestezije bolnika. Radiol Oncol 2016; 50(1): 58-63. doi:10.1515/raon-2016-0015 Kombinacija lokalne in sistemske aplikacije bleomicina pri elektrokemoterapiji za zmanjšanje ponovitev zdravljenja Maglietti F, Tellado M, Olaiz N, Michinski S, Marshall G Izhodišča. Elektrokemoterapija, ki je pogost način zdravljenja tumorjev v humani in veterinarski medicini, poveča toksičnost bleomicina 1000-krat in dosega 80 % objektivnih odgovorov tumorjev. Kljub temu visokemu deležu objektivnih odgovor pa pri 20 % bolnikov zdravljenje ni uspešno. Razlog za to je morda, ker se pri sistemski aplikaciji bleomicina ta ne razporedi po celotnem tumorju zaradi nezadostnega tumorskega krvožilja. Lokalna aplikacija bleomicina bi lahko pokrila dele tumorja, ki jih sistemska ne doseže. Bolniki in metode. Predlagamo kombinacijo sistemske in lokalne aplikacije bleomicina na modelu tumorjev domačih živali. V raziskavo smo vključili 22 pasjih bolnikov, pri katerih nismo dosegli popolni odgovor na elektrokemoterapijo. Enajst psov je prejelo še eno standardno elektrokemoterapijo (kontrolna skupina), drugih enajst pa smo zdravili s kombinacijo sistemske in lokalne aplikacije bleomicina pri ponovitvi zdravljenja z elektrokemoterapijo. Rezultati. Po kriterijih Svetovne zdravstvene organizacije smo pri skupini, ki smo jo zdravili s kombinirano elektrokemoterapijo, dosegli popoln odgovor (CR) pri 54 % (6), delni odgovor (PR) pri 36 % in stabilno bolezen (SD) pri 10 % (1) bolnikov. Pri kontrolni skupini smo CR dosegli pri 0 %, PR pri 19 % (2), SD pri 63 % (7) in pri 18 % (2) bolnikov je bolezen napredovala. Objektivni odgovor na zdravljenje smo dosegli pri 91 % (CR+PR) bolnikov, ki smo jih zdravili s kombinacijo sistemske in lokalne aplikacije bleomicina, medtem ko je bil pri kontrolni skupini ta odstotek nižji, 19 %. Razlika med obema skupinama je bila statistično značilna (p < 0,01). Zaključki. Kombinirana sistemska in lokalna aplikacija bleomicina je bila učinkovita pri pasjih bolnikih, ki smo jih predhodno neučinkovito zdravili z elektrokemoterapijo. Ti rezultati nakazujejo, da bi lahko bil tak pristop uporaben in učinkovit pri specifični populaciji bolnikov in da bi se lahko zmanjšalo število ponovitev zdravljenja z elektrokemoterapijo, ki so potrebni za dosego objektivnih odgovorov. Radiol Oncol 2016; 50(1): 64-72. doi:10.1515/raon-2016-0004 Medicinska fizika v Evropi po priporočilih Mednarodne agencije za atomsko energijo Casar B, Lopes MC, Drljević A, Gershkevitsh E, Pesznyak C Izhodišča. Medicinska fizika je zdravstveni poklic, kjer fizikalna načela uporabne fizike služijo pretežno uporabi ionizirajočega sevanja v medicini. Ključno vlogo specialista medicinske fizike pri varni in učinkoviti uporabi ionizirajočega sevanja v medicini so določili v nedavnih uradnih evropskih smernicah, imenovanih "Direktiva Sveta Evropske unije 2013/59/EURATOM (2014)" in v "Smernicah za specialista medicinske fizike Evropske komisije (2014)". Tudi Mednarodna agencija za atomsko energijo (International Atomic Energy Agency, IAEA) je jasno izrazila svoja stališča glede podpore in spodbujanja pri ureditvi statusa medicinske fizike na področju medicine v okviru tehničnih projektov in objavljenih dokumentov kot sta "IAEA Human Heath Series No. 25: Vloga, odgovornost in zahteve po izobrazbi in usposabljanju klinično kvalificiranih medicinskih fizikov (2013)" ter "Mednarodni temeljni varnostni standardi (2014)". Pomembnost omenjenih dokumentov ter ugotovitev o neizpolnjevanju zahtev in priporočil v več evropskih državah je vodilo IAEA do organizacije Regionalnega srečanja o medicinski fiziki v Evropi, kjer so udeleženci razpravljali o ključnih vprašanjih glede medicinske fizike v Evropi. Najpomembnejši rezultat srečanja so pri­poročila naslovljena na evropske države in raziskava o statusu medinske fizike v Evropi, ki sta jo izvedli IAEA in Evropska zveza organizacij za medicinsko fiziko (European Federation of Organizations for Medical Physics). Zaključki. Objavljena priporočila IAEA z regionalnega srečanja o medicinski fiziki v Evropi morajo biti upoštevana in uvelja­vljena v vseh evropskih državah. Vzpostaviti moramo primerne okvirje kvalifikacij, ki vključujejo izobrazbo, klinično specializa­cijo, certifikacijo in registracijo medicinskih fizikov, s posebnim poudarkom na izpolnjevanju mednarodnih priporočil po kadro­vskih normativih. Evropske države imajo jasno pravno in moralno odgovornost, da učinkovito prenesejo temeljne varnostne standarde v lokalno zakonodajo in s tem zagotavljajo visoko kakovost in varnost pri zdravljenju bolnikov. Radiol Oncol 2016; 50(1): 73-79. doi:10.1515/raon-2016-0007 Natančnost slikanja z magnetno resonanco za opredelitev odgovora raka dojk na neoadjuvantno zdravljenje in za ugotavljanje velikosti rezidualnega tumorja Bouzón A, Acea B, Soler R, Iglesias Á, Santiago P, Mosquera J, Calvo L, Seoane-Pillado T, García A Izhodišča. Namen raziskave je bil oceniti natančnost slikanja z magnetno resonanco (MRI) za ugotavljanje rezidualnega tumorja pri bolnicah z rakom dojk, ki so dobivale neoadjuvantno kemoterapijo. Poleg tega smo želeli opredeliti kliničnopato­loške dejavnike, ki vplivajo na diagnostično natančnost MRI, za določanje velikosti rezidualnega tumorja po neoadjuvantnem zdravljenju. Bolnice in metode. V raziskavo smo vključili 91 bolnic z rakom dojk (92 tumorjev dojk), ki so dobivale neoadjuvantno ke­moterapijo. MRI dojke smo izvedli na začetku in po končani neoadjuvantni kemoterapiji. Odziv na zdravljenje smo ovrednotili s pomočjo MRI in s pomočjo histopatološke analize. Ovrednotili smo možnost MRI, da opredeli odgovor tumorja na zdravljenje. Pri 89 tumorjih smo s pomočjo MRI določili velikost rezidualnega tumorja po koncu neoadjuvantne kemoterapije ter jo primer­jali z velikostjo rezidulanega tumorja, ugotovljeno s patološko analizo. Analizirali smo kliničnopatološke dejavnike, ki vplivajo na velikost razlike v velikosti rezidualnega tumorja med MRI in patološko analizo. Rezultati. Občutljivost za diagnosticiranje rezidualnega tumorja s pomočjo MRI je bilo 75,00 %, specifičnost 78,57 %, po­zitivna napovedna vrednost 88,89 %, negativna napovedna vrednost 57,89 % in natančnost 76,09 %. Pearsonov korelacijski koeficient (r) med velikostjo rezidualnega tumorja, ugotovljeno s pomočjo MRI, in velikostjo, izmerjeno s patološko analizo, je bil 0,648 (p < 0,001). Razlika v velikosti rezidualnega tumorja je bila značilno manjša pri tumorjih, pri katerih je bila z MRI ugo­tovljena velikost . 5 cm (p = 0,050), pri tumorjih visokega gradusa (p < 0,001), in pri tumorjih, ki so bili negativni na hormonske receptorje (p = 0,033). Zaključki. MRI je natančno orodje za opredelitev odziva tumorja po neoadjuvantni kemoterapiji. Natančnost MRI pri oceni velikosti rezidualnega tumorja se razlikuje glede na velikosti tumorjev pred zdravljenjem, glede na gradus ter glede na njihov status hormonskih receptorjev. Radiol Oncol 2016; 50(1): 80-86. doi:10.1515/raon-2015-0026 Polimorfizmi antioksidantnih genov niso povezani s povišanim tveganjem za nastanek sekundarnega raka ščitnice po zdravljenju zaradi raka v otroštvu ali mladostništvu Vodušek AL, Goričar K, Gazić B, Dolžan V, Jazbec J Izhodišča. Sekundarni rak ščitnice je eden izmed najpogostejših rakov, ki nastanejo kot posledica zdravljenja raka v otroštvu ali mladostništvu. Ščitnica je predvsem v otroštvu zelo občutljiva na kancerogene učinke ionizirajočega sevanja. Nesorazmerje med pro- in antioksidantnimi dejavniki je eden izmed možnih mehanizmov za nastanek raka ščitnice. Z raziskavo smo želeli oceniti vpliv polimorfizmov antioksidantnih genov na nastanek sekundarnega raka ščitnice po zdravljenju raka v otroštvu ali mladostništvu. Bolniki in metode. V retrospektivno raziskavo smo vključili bolnike, ki smo jih med letoma 1960 in 2006 zdravili zaradi pri­marnega raka v starosti manj ali enako 21 let, in ki so nato zboleli zaradi sekundarnega raka ščitnice. Naredili smo raziskavo s kontrolnimi primeri in zato dodatno poiskali primerljive bolnike po starosti, spolu, diagnozi in zdravljenju primarnega raka (predvsem obsevanju), ki pa niso zboleli zaradi sekundarnega raka. Vsem smo določili polimorfizme SOD2 p.Ala16Val, CAT c.-262C>T, GPX1 p.Pro200Leu, GSTP1 p.Ile105Val, GSTP1 p.Ala114Val in GSTM1 in GSTT1. Njihov vpliv na pojav sekundarnega raka smo opredelili z McNemarovim testom in Coxovo regresijsko analizo. Rezultati. Med letoma 1960 in 2006 je v Sloveniji zbolelo zaradi raka 2641 bolnikov mlajših od 21 let. Sekundarni rak smo ugo­tovili pri 155 bolnikih, od katerih je 28 zbolelo zaradi ščitničnega raka. Med primeri in kontrolami ni bilo statistično pomembnih razlik v porazdelitvi genotipov preiskovanih polimorfizmov, kakor tudi ne statistično pomembnega vpliva polimorfizmov na čas nastanka sekundarnega raka ščitnice. Zaključki. Nismo ugotovili vpliva polimorfiizmov antioksidantnih genov na tveganje za nastanek sekundarnega raka šči­tnice pri bolnikih, ki smo jih v otroštvu ali mladostništvu zdravili zaradi raka. Ker je rak ščitnice eden izmed najbolj pogostih sekundarnih rakov po zdravljenju zaradi primarnega raka v otroštvu ali mladostništvu in lahko nastane tudi več deset let po zdravljenju, je potrebno doživljenjsko spremljanje teh bolnikov. Radiol Oncol 2016; 50(1): 87-93. doi:10.2478/raon-2014-0042 Toksoplazmoza osrednjega živčevja pri bolniku z limfomom B Savšek L, Roš Opaškar T Izhodišča. Oportunistična okužba s protozojem Toxoplasmo gondii je doslej najverjetneje slabše prepoznana pri bolnikih s hematološkimi malignimi obolenji, ki niso bili zdravljeni s presaditvijo zarodnih celic ali kostnega mozga. Sodobne metode zdravljenja rakavih bolezni vključujejo načine, ki povzročijo zmanjšanje števila limfocitov B in T ter najverjetneje predstavljajo pomemben dejavnik tveganja za reaktivacijo latentne okužbe s Toxoplasmo gondii. Prikaz primera. Opisujemo 62-letno HIV-negativno desnoročno bolnico z difuznim velikoceličnim limfomom B, ki je nekaj dni po zadnjem (osmem) krogu zdravljenja z rituksimabom, ciklofosfamidom, vinkristinom, doksrubicinom in prednisolonom (R-CHOP) akutno zbolela s povišano telesno temperaturo, glavobolom, motnjo zavesti, ataksijo in pancitopenijo. Postavili smo sum na napredovanje limfoma v osrednje živčevje. MR glave je na sekvencah T2 in sekvencah z izločanjem signalov tekočin (FLAIR) pokazala številne hiperintenzivne spremembe obojestransko v možganovini malih in velikih možgan z okolnim ede­mom. Zmerno so se obarvale z gadolinijem. Polimerazna verižna reakcija (PCR) likvorja je bila pozitivna za DNK Toxoplasme gondii. Postavili smo diagnozo toksoplazmoze osrednjega živčevja. Bolnico smo uspešno zdravili s kombinacijo sulfadiazina, pirimetamina in folne kisline. Zaradi vzdrževalne terapije z rituksimabom ob remisiji limfoma je bolnica nato nadaljevala s se­kundarno profilakso toksoplazmoze. Zaključki. S prikazom tega primera želimo poudariti pomembnost vključitve toksoplazmoze osrednjega živčevja v dife­rencialno diagnostiko bolnikov s hematološkimi malignimi obolenji, ki zbolijo z novo nevrološko simptomatiko. Izmed števil­nih diferencialno diagnostičnih možnosti je namreč najtežje razločevanje limfoma in toksoplazmoze osrednjega živčevja. Nezdravljena toksoplazmoza osrednjega živčevja ima visoko smrtnost, zato je zgodnje prepoznavanje in zdravljenje tovrstnih bolnikov ključnega pomena. Radiol Oncol 2016; 50(1): 94-103. doi:10.1515/raon-2015-0010 Obstruktivne težave z uriniranjem po kombinaciji zunanjega obsevanja in brahiradioterapije raka prostate Kragelj B Izhodišča. Cilj raziskave je bil opredeliti dejavnike, ki vplivajo na nastanek obstruktivnih težav z uriniranjem po kombiniranem zdravljenja z zunanjim obsevanjem in brahiradioterapijo pri bolnikih z rakom prostate. Ob upoštevanju teh dejavnikov bi lahko zdravljenje prilagajali posameznemu bolniku. Bolniki in metode. V raziskavo smo vključili 88 bolnikov, ki smo jih na Onkološkem inštitutu v Ljubljani zdravili z zunanjim obsevanjem in brahiradioterapijo v letih 2006-2011. Opazovani izid je bilo poslabšanje obstruktivnih težav z uriniranjem eno ali več let po zaključenem zdravljenju. Univariatno in multivariatno analizo povezanosti poslabšanja uriniranja z morebitnimi dejavniki tveganja smo naredili z metodo binarne logistične regresije. Pri tem smo upoštevali značilnosti bolnikov ter značilnosti zunanjega obsevanja in brahiradioterapije. S pomočjo analize ROC, pri kateri smo ocenili površino pod krivuljo ROC (AUC), smo nato ocenili še učinkovitost multivariatnega modela pri napovedovanju poslabšanja obstruktivnih težav z uriniranjem. Rezultati. V končno analizo smo zajeli 71 bolnikov, ki smo jih sledili vsaj 3-leta po zdravljenju. Napovedovanju poslabšanja obstruktivnih težav z uriniranjem smo ugotovili pri 13/71 (18,3 %) bolnikov. Rezultati multivariatne analize so pokazali statistično značilno povezanost poslabšanja obstruktivnih težav z uriniranjem z antikoagulantnim zdravljenjem (razmerje obetov [OR] = 4,86; 95 % interval zaupanja [C.I.]: 1,21-19,61; p = 0,026) in mejno značilno povezanost z dozo ki jo prejme 90 % volumna uretre (OR = 1,23; 95 % C.I.: 0,98-1,07; p = 0,099). Vrednost AUC je bila 0,755 . Zaključki. Raziskava je izpostavila vpliv antikoagulantnega zdravljenja na napovedovanje poslabšanja obstruktivnih težav z uriniranjem. Izdelali smo model s sprejemljivo napovedno vrednostjo, ki bi lahko omogočil zmanjšanje obstruktivnih težav z uriniranjem. Izsledki raziskave podpirajo tudi pomen uretralnega sfinktra kot rizične strukture za poslabšanja obstruktivnih težav z uriniranjem. Radiol Oncol 2016; 50(1): 104-112. doi:10.1515/raon-2015-0009 Napovedni dejavniki pri zdravljenju malignih melanomov žilnice v Sloveniji, 1986--2008 Jančar B, Budihna M, Drnovšek-Olup B, Novak Andrejčič K, Brovet Zupančič I, Pahor D Izhodišča. Melanom žilnice je najpogostejši primarni tumor v očesu. Incidenca bolezni je bila v Sloveniji stabilna od 1983 do 2009 s 7,8 bolnikov/milijon za moške in 7,4 bolnikov/milijon za ženske. V retrospektivni raziskavi smo želeli ugotoviti napovedne dejavnike preživetja pri bolnikih z malignim melanomom žilnice, ki smo jih zdravili v Sloveniji. Bolniki in metode. Od januarja 1986 do decembra 2008 smo zdravili 288 bolnikov z malanomom žilnice. 127 bolnikov smo zdravili z brahiterapijo z rutenijevimi (Ru-106) aplikatorji in 161 bolnikov z enukleacijo. Rezultati. Pri bolnikih s tumorji višine < 7 mm in premera < 16 mm, ki smo jih zdravili z brahiterapijo, je bila 5- in 10-letna splošna smrtnost 13% in 32%, pri bolnikih zdravljenih z enukleacijo pa 46% in 69%. Razlika v preživetju je bila posledica izbora: bolnike z večjimi tumorji smo zdravili z enukleacijo. Pri 30 bolnikih smo ugotovili ponovno rast tumorja. Pri 25 bolnikih od 127, ki smo jih zdravili z brahiterapijo ter pri 86 od 161, ki smo jih zdravili z enukleacijo, pa smo po zdravljenju ugotovili oddaljene zasevke. Preživetje bolnikov starih . 60 let, ki smo jih zdravili tako z brahiterapijo kot z enuklacijo, je bilo krajše. V multivariatni analizi sta bila neodvisna dejavnika za preživetje bolnikov zdravljenih z brahiterapijo premer tumorja in čas implantacije, za bolnike zdravljene z enukleacijo pa samo histološki tip tumorja. V prvih letih po zdravljenju smo opazili začasen porast speci­fične smrtnosti, zlasti pri starejših bolnikih. Zaključki. Verjetnost ozdravitve bolnikov z malignim melanom žilnice je večja pri mlajših bolnikih in pri tistih z manjšimi tumor­ji. Brahiterapija je prednostna metoda zdravljenja glede na enukleacijo zaradi ohranitve očesa. Radiol Oncol 2016; 50(1): 113-120 doi:10.1515/raon-2015-0015 Vpliv anemije na izid zdravljenja bolnikov s ploščatoceličnim rakom analnega kanala Oblak I, Češnjevar M, Anžič M, But Hadžič J, Šečerov Ermenc A, Anderluh F, Velenik V, Jeromen A, Peter Korošec Izhodišča. Temeljno zdravljenje bolnikov s ploščatoceličnim rakom analnega kanala je radiokemoterapija. Anemija pa je lahko eden od dejavnikov, ki poslabša učinkovitost zdravljenja. Namen raziskave je bil ugotoviti, kakšen je vpliv anemije na izid zdravljenja bolnikov z rakom analnega kanala. Bolniki in metode. V retrospektivno raziskavo smo vključili 100 bolnikov s histološko potrjenim ploščatoceličnim rakom, ki smo jih zdravili s 3-dimenzionalno (3-D) konformalno planiranim obsevanjem ali intenzitetno modulirano radioterapijo (IMRT). Sočasno z obsevanjem so bolniki prejemali kemoterapijo z derivati fluoropirimidinov in mitomicina C. Kasneje so prejeli še do­datno obsevanje na tumor z brahi- ali teleradioterapijo. Analizirali smo vpliv koncentracij hemoglobina (Hb) pred, med in ob zaključku zdravljenja na izid poteka bolezni. Rezultati. 5-letno preživetje brez ponovitve bolezni lokalno in/ali področno je bilo 72 %, preživetje brez ponovitve bolezni 71 %, bolezensko specifično preživetje 77 % in celokupno preživetje 62 %. V univariatni analizi so bolniki s koncentracijo Hb >120 g/L pred začetkom zdravljenja in ob zaključku zdravljenja imeli statistično pomembno boljše preživetje kot bolniki, ki so imeli Hb . 120 g/L. Bolniki, ki smo jim med zdravljenjem ugotovili Hb > 120 g/L, so imeli statistično pomemben vpliv le na boljše preživetje brez ponovitve bolezni lokalno in/ali področno in celokupno preživetje. V multivariatni analizi se je koncentracija Hb pred zdravljenjem (> 120 g/L vs. . 120 g/L) pokazala kot neodvisni napovedni dejavnik za celokupno preživetje (razmerje ogroženosti [HR] = 0,419; 95 % interval zaupanja [CI] = 0,190—0,927; p = 0,032). Prav tako se je pokazal stadij bolezni (stadij I in II vs. III) kot neodvisni napovedni dejavnik za bolezensko specifično preživetje (HR = 3,523; 95 % CI = 1,375—9,026; p = 0,009) in celokupno preživetje (HR = 2,230; 95% CI = 1,1675—4,264; p = 0,015). Zaključki. Koncentracija Hb > 120 g/L pred, med in po zdravljenju z radiokemoterapijo je bila ugoden napovedni dejavnik preživetja pri bolnikih z rakom analnega kanala. Najpomembnejša je bila koncentracija Hb > 120 g/L pred začetkom zdravlje­nja, ki se je pokazala kot neodvisni napovedni dejavnik za celokupno preživetje. Radiol Oncol 2016; 50(1): 121-128 doi:10.1515/raon-2016-0008 Ocena dozimetričnega učinka povzročenega z upočasnitvijo lističev večlistnega kolimatarja na volumetrično modulirano ločno terapijo (VMAT) Xu ZZ, Wang IZ, Kumaraswamy LK, Podgorsak MB Izhodišča. Poročamo o občutljivosti metode zagotavljanja kakovosti načrtov intenzitetnega moduliranega obsevanja (IMRT) za napake lamel večlistnega kolimatorja (MLC) pri volumetrično moduliranem ločnem obsevanju (VMAT), ki ne sproži blokade MLC med postopkom obsevanja. Prav tako poročamo o učinku napak na kakovost obsevanja z VMAT zaradi neu­stavitve žarka z lističi. Materiali in metode. S pomočjo internega računalniškega programa smo izbrali in spremenili 11 planov VMAT. Za vsako kontrolno točko na loku VMAT so bili lističi MLC največje hitrosti (1,87–1,95 cm/s) nastavljeni tako, da so se premikali z največjo dovoljeno hitrostjo (2,3 cm/s), kar je povzročilo razliko položaja lističa za manj kot 2 mm. Spremenjene načrte smo obravnavali kot ‚standardne‘ načrte in prvotne načrte kot ‚upočasnjene‘ načrte MLC za simulacijo ‚standardnih‘ načrtov s premikanjem lističev pri relativno nižji hitrosti. Meritve vsakega ‚upočasnjenega‘ načrta MLC z uporabo MapCHECK®2 smo primerjali z izračunano planirano dozo ‚standardnega‘ načrta ob upoštevanju primerjave absolutne doze dogovorne razdalje Van Dyk (DTA) z meriloma 3 % / 3 mm in 2 % / 2 mm. Rezultati. Vsi ‚upočasnjeni‘ načrti MLC so uspešno prešli prag 90 % ob uporabi merila 3 % / 3 mm, medtem ko je bil en primer načrta VMAT za možgane in trije primeri za analni kanal pod pragom 90 % ob uporabi merila 2 % / 2 mm. Primerjava dozno-volumnih histogramov (DHV) ‚standardnih‘ načrtov in ‚upočasnjenih‘ načrtov MLC je pri desetih od enajstih primerov pokazala minimalne dozimetrične spremembe na tarčnih in rizičnih organih. Zaključki. Za visoko modulirane načrte VMAT prag merila 3 % / 3 mm ni dovolj občutljiv za odkrivanje napake lističev MLC, ki ne sprožijo njihove blokade. Vendar pa so učinki doze na tarčne in kritične organe kot posledica napak MLC zaradi ne ustavitve žarka zanemarljive, kar podpira zanesljivost trenutne metode bolniku specifičnega prilagojenega zagotavljanja kakovosti IMRT za načrte VMAT. Fundacija Doc. dr. Josip Cholewa razpisuje denarno pomoč za sofinanciranje materialnih stroškov pri znanstveno-raziskovalnih delih s področja onkologije. Prijava naj vsebuje: 1. kratko obrazložitev znanstveno-raziskovalnega dela s finančno konstrukcijo 2. kratko biografijo in bibliografijo prosilca/prosilcev Prijave, prosimo, pošljite do 30. 4. 2016 na naslov Združenje Fundacija Doc.dr. Josip Cholewa, Dunajska cesta 106, 1000 Ljubljana Fundacija "Docent dr. J. Cholewa" je neprofitno, neinstitucionalno in nestrankarsko združenje posameznikov, ustanov in organizacij, ki želijo materialno spodbujati in poglabljati raziskovalno dejavnost v onkologiji. Uporabljena moška slovnična oblika se enakovredno nanaša na oba spola. Activity of "Dr. J. Cholewa" Foundation for Cancer Research and Education – a report for the first quarter of 2016 The “Docent Dr. J. Cholewa Foundation for Cancer Research and Education” is named after Dr. Josip Cholewa, one of the first researchers in cancer in Slovenia and the founder of the “Banovinski Inštitut za raziskovanje in zdravljenje novotvorb” in 1937, that later became the Institute of Oncology in Ljubljana, Slovenia. His laboratory and clinical research work was based on an innovative and far-reaching multidis­ciplinary approach that included studies on prevention, detection and treatment of cancer. This pioneering approach facilitated the understanding of the complexities of all the problems and troubles experienced by cancer patients, their doctors and other medical staff when facing this disease. It could also be regarded as a harbinger of the progress observed in a large part of the world in the last half of the previous century. Therefore, the Foundation is a non-profit, non-political and non-government organisation that helps pro­fessionals, institutions and individuals obtaining financial help for cancer research and education in the Republic of Slovenia with the goal of continuing and expanding the great work and efforts of Dr. Josip Cholewa. The “Docent Dr. J. Cholewa Foundation for Cancer Research and Education” hopes and strives to provide at least part of the financial support needed by qualified individuals and organisations interested in cancer research in the Republic of Slovenia. One of the objectives of the Foundation is to facilitate the transmission of the latest diagnostic and therapy procedures to the clinical environment in Slovenia, thus benefiting the ever increasing number of patients with various types of cancer in Slovenia. With this in mind, it is impor­tant to note that the incidence rates of many cancer, like colon, prostate and breast cancer have kept rising in recent decades in Slovenia. The Foundation continues to provide financial support to "Radiology and Oncology”, an international sci­entific journal that is edited and published in Ljubljana, Slovenia. It publishes scientific research articles, re­views, and letters to the editor about research and studies in experimental and clinical oncology, supportive therapy, radiology, radiophyics, prevention and early diagnostics of different types of cancer. It is an open access journal freely available in pdf format and with a respectable Science Citation Index Impact factor. All the abstracts in “Radiology and Oncology” are available in Slovenian and the journal can thus provide suf­ficient scientific information from various fields of high quality cancer research to interested lay public in Slovenia. The “Docent Dr. J. Cholewa Foundation for Cancer Research and Education” has thus an important role in support of cancer research, cancer education and many of the related fields in the Republic of Slovenia. Tomaž Benulič, M.D. Viljem Kovač, M.D., Ph.D. A Borut Štabuc, M.D., Ph.D. Andrej Plesničar, M.D., M.Sc. OVA INDIKACIJA1 Vectibix® + FOLFIRI v 1. liniji zdravljenja bolnikov z mKRR in nemutiranim genom RAS Zdravilo Vectibix® je sedaj indicirano za zdravljenje odraslih bolnikov z metastatskim kolorektalnim rakom (mKRR) in nemutiranim genom RAS: v 1. liniji zdravljenja v kombinaciji s FOLFOX ali FOLFIRI v 2. liniji zdravljenja v kombinaciji s FOLFIRI pri bolnikih, ki so v prvi liniji zdravljenja prejemali kemoterapijo, ki je vkljucevala fluoropirimidin ˇ (vendar ni vkljucevala irinotekana) ˇ kot monoterapija po neuspehu shem kemoterapije, ki so vkljucevale fluoropirimidin, oksaliplatin in irinotekan. ˇ VECTIBIX® 20 mg/ml koncentrat za raztopino za infundiranje (sterilni koncentrat) (panitumumab) – SKRAJŠAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA Samo za strokovno javnost. Pred predpisovanjem si preberite celoten Povzetek glavnih značilnosti zdravila (SmPC). SESTAVA ZDRAVILA: 1 ml koncentrata vsebuje 20 mg panitumumaba. 1 viala vsebuje 100 mg panitumumaba v 5 ml, 200 mg panitumumaba v 10 ml ali 400 mg panitumumaba v 20 ml koncentrata. TERAPEVTSKE INDIKACIJE: Zdravljenje odraslih bolnikov z metastatskim kolorektalnim rakom (mKRR) z divjim tipom RAS in sicer v prvi liniji zdravljenja v kombinaciji s FOLFOX ali FOLFIRI, v drugi liniji zdravljenja v kombinaciji s FOLFIRI pri bolnikih, ki so v prvi liniji zdravljenja prejemali kemoterapijo, ki je vključevala fluoropirimidin (vendar ni vključevala irinotekana), ter kot monoterapija po neuspehu shem kemoterapije, ki so vključevale fluoropirimidin, oksaliplatin in irinotekan. ODMERJANJE IN NAČIN UPORABE: Zdravljenje z zdravilom Vectibix® mora nadzirati zdravnik z izkušnjami pri uporabi terapije proti raku. Pred začetkom zdravljenja z zdravilom Vectibix® mora biti potrjeno, da gre za stanje divjega tipa RAS (KRAS in NRAS) . Mutacijsko stanje mora ugotoviti izkušen laboratorij z uporabo validiranih testnih metod za detekcijo mutacij KRAS (eksoni 2, 3 in 4) in NRAS (eksoni 2, 3 in 4). Priporočeni odmerek zdravila Vectibix® je 6 mg/kg telesne mase enkrat na dva tedna. V primeru hudih (. 3. stopnja) dermatoloških reakcij je lahko potrebna prilagoditev odmerka zdravila Vectibix® . Bolniki z okvaro ledvic ali jeter: Varnost in učinkovitost zdravila Vectibix® nista raziskani. Starejši bolniki: Ni kliničnih podatkov, ki bi podprli prilagoditev odmerka. Pediatrična populacija: Zdravilo Vectibix® nima relevantne uporabe za indikacijo zdravljenja kolorektalnega raka. Zdravilo Vectibix® morate aplicirati v intravenski (i.v.) infuziji z infuzijsko črpalko. Če se pojavijo z infundiranjem povezane reakcije, je lahko potrebna upočasnitev infundiranja zdravila Vectibix® . Zdravila Vectibix® ne smete injicirati z intravenskim vbrizganjem ali v bolusu. KONTRAINDIKACIJE: Anamneza hude ali smrtno nevarne preobčutljivosti na zdravilno učinkovino ali katero koli pomožno snov, intersticijski pnevmonitis, pljučna fibroza. Pri bolnikih z mKRR z mutantnim RAS in tistih bolnikih z mKRR, pri katerih stanje RAS ni znano, je kontraindicirana kombinacija zdravila Vectibix® in kemoterapije, ki vključuje oksaliplatin. POSEBNA OPOZORILA IN PREVIDNOSTNI UKREPI: Dermatološke reakcije in toksičnost mehkih tkiv: Skoraj pri vseh bolnikih (približno 90%), zdravljenih z zdravilom Vectibix®, se pojavijo dermatološke reakcije, ki so farmakološki učinek, opažen pri zaviralcih EGFR. Če se pri bolniku pojavijo dermatološke reakcije 3. ali višje stopnje (po CTCAE verzija 4.0) ali dermatološke reakcije, ocenjene kot neznosne, je priporočljiva naslednja prilagoditev odmerka: Prvi pojav kožnih simptomov . 3. stopnje: Zadržite 1 ali 2 odmerka zdravila Vectibix® . Če se po tem simptomi izboljšajo (< 3. stopnja), nadaljujte infundiranje s 100 % odmerka, ki ste ga aplicirali pred pojavom kožnih simptomov; če izboljšanja ni, prekinite uporabo zdravila Vectibix® . Ob drugem pojavu kožnih simptomov . 3. stopnje: Zadržite 1 ali 2 odmerka zdravila Vectibix® . Če se simptomi izboljšajo (< 3. stopnja), nadaljujte infundiranje z 80 % odmerka, ki ste ga aplicirali pred pojavom kožnih simptomov; če izboljšanja ni, prekinite uporabo zdravila Vectibix® . Ob tretjem pojavu kožnih simptomov . 3. stopnje: Zadržite 1 ali 2 odmerka zdravila Vectibix® . Če se simptomi izboljšajo (< 3. stopnja), nadaljujte infundiranje s 60 % odmerka, ki ste ga aplicirali pred pojavom kožnih simptomov; če izboljšanja ni, prekinite uporabo zdravila Vectibix® . Ob četrtem pojavu kožnih simptomov . 3. stopnje: Prekinite uporabo. Bolnike s hudimi dermatološkimi reakcijami ali toksičnostjo mehkih tkiv ali poslabšanjem reakcij med uporabo zdravila Vectibix® morate nadzirati zaradi možnih vnetnih ali infekcijskih posledic (vključno s celulitisom in nekrotizirajočim fasciitisom) ter jim nemudoma uvesti ustrezno zdravljenje, če se pojavijo. Če se pojavi dermatološka toksičnost ali toksičnost mehkih tkiv, povezana s hudimi ali življenjsko ogrožujočimi vnetnimi ali infekcijskimi zapleti, zadržite ali prekinite zdravljenje z zdravilom Vectibix® . Zapleti na pljučih: V primeru akutnega nastanka ali poslabšanja pljučnih simptomov morate zdravljenje z zdravilom Vectibix® prekiniti in simptome takoj raziskati. Če diagnosticirate intersticijsko pljučno bolezen, morate zdravljenje z zdravilom Vectibix® za stalno prekiniti in bolnika ustrezno zdraviti. Pri bolnikih z anamnezo intersticijskega pnevmonitisa ali pljučne fibroze je potrebno skrbno razmisliti o koristih zdravljenja s panitumumabom v primerjavi s tveganjem za zaplete na pljučih. Elektrolitske motnje: Bolnike je treba pred uvedbo zdravljenja, med zdravljenjem in še 8 tednov po končanem zdravljenju z zdravilom Vectibix®, redno spremljati glede hipomagneziemije in spremljajoče hipokalciemije. Priporočljivo je dodajanje magnezija, kot je ustrezno. Opažali so tudi druge elektrolitske motnje, vključno s hipokaliemijo. Priporočljivo je spremljanje, kot opisano zgoraj in dodajanje teh elektrolitov, kot je ustrezno. Z infundiranjem povezane reakcije: Če se kadarkoli med ali po infundiranju pojavi huda ali smrtno nevarna reakcija (npr. z bronhospazmom, angioedemom, hipotenzijo, potrebo po parenteralnem zdravljenju ali anafilaksijo), je treba uporabo zdravila Vectibix® za stalno prekiniti. Bolnikom, ki se jim pojavi blaga ali zmerna (1. in 2. stopnja po CTCAE verzija 4.0) z infundiranjem povezana reakcija, je treba hitrost infundiranja za čas infuzije zmanjšati. To manjšo hitrost infundiranja je priporočljivo ohraniti tudi pri vseh nadaljnjih infuzijah. Opisane so preobčutljivostne reakcije, ki so se pojavile več kot 24 ur po infundiranju, vključno s smrtnimi primeri angioedema, ki so se pojavili več kot 24 ur po infundiranju. Bolnike je potrebno opozoriti na možnost reakcij s poznim nastankom in jim naročiti, naj se obrnejo na svojega zdravnika, če se jim pojavijo simptomi preobčutljivostne reakcije. Akutna odpoved ledvic: Opisana je akutna odpoved ledvic pri bolnikih, ki se jim pojavi huda driska in dehidracija. Bolnikom, ki se jim pojavi huda driska, je treba naročiti, da se takoj posvetujejo z zdravnikom. Zdravilo Vectibix®® v kombinaciji s kemoterapijo na podlagi oksaliplatina pri bolnikih z mKRR z mutantnim RAS oz. bolnikih z mKRR, pri katerih stanje RAS tumorja ni znano: Kombinacija zdravila Vectibix® s kemoterapijo, ki vključuje oksaliplatin, je kontraindicirana pri bolnikih z mKRR z mutantnim RAS ali pri katerih stanje RAS ni znano. Če je predvidena uporaba zdravila Vectibix® v kombinaciji s FOLFOX, je priporočljivo, da mutacijsko stanje določi laboratorij, ki sodeluje v programu Eksterno zagotavljanje kakovosti RAS, ali da se stanje divjega tipa potrdi z dupliciranjem preiskave. Očesni toksični učinki: Bolnike, ki se jim med prejemanjem zdravila Vectibix® pojavijo znaki in simptomi, ki kažejo na keratitis, kot so akutni pojav ali poslabšanje: vnetja očesa, solzenja, občutljivosti na svetlobo, zamegljenega vida, bolečine v očesu in/ali rdečih oči, je priporočljivo takoj napotiti k specialistu oftalmologu. Če je potrjena diagnoza ulcerativnega keratitisa, je treba zdravljenje z zdravilom Vectibix® začasno ali trajno prekiniti. Če je diagnosticiran keratitis, je treba skrbno pretehtati koristi in tveganja nadaljevanja zdravljenja. Zdravilo Vectibix® morate uporabljati previdno pri bolnikih z anamnezo keratitisa, ulcerativnega keratitisa ali zelo suhih oči. Bolniki z zmogljivostnim stanjem 2 po ECOG, zdravljeni z zdravilom Vectibix® v kombinaciji s kemoterapijo: Pri teh bolnikih je pred uvedbo zdravila Vectibix® v kombinaciji s kemoterapijo za zdravljenje mKRR priporočljivo oceniti koristi in tveganje, saj pri njih pozitivno razmerje med koristnostjo in tveganjem ni bilo zabeleženo. Starejši bolniki: V celoti niso ugotovili razlik v varnosti ali učinkovitosti med starejšimi bolniki (starimi . 65 let), ki so prejemali monoterapijo z zdravilom Vectibix® . Drugi previdnostni ukrepi: Zdravilo vsebuje manj kot 0,150 mmol natrija (kar je 3,45 mg natrija) na mililiter koncentrata. To je treba upoštevati pri bolnikih, ki potrebujejo prehrano z nadzorovano količino natrija. MEDSEBOJNO DELOVANJE Z DRUGIMI ZDRAVILI IN DRUGE OBLIKE INTERAKCIJ: Podatki študije o medsebojnem delovanju zdravil, ki je vključevala zdravili Vectibix® in irinotekan, pri bolnikih z mKRR kažejo, da se med sočasno uporabo zdravil farmakokinetika irinotekana in njegovega aktivnega metabolita SN-38 ne spremeni. Rezultati primerjave v navzkrižni študiji so pokazali, da sheme z irinotekanom (IFL ali FOLFIRI) ne vplivajo na farmakokinetiko panitumumaba. Zdravila Vectibix® se ne sme uporabljati v kombinaciji s kemoterapijo IFL ali kemoterapijo, ki vključuje bevacizumab. Kombinacija zdravila Vectibix® s kemoterapijo, ki vključuje oksaliplatin, je kontraindicirana pri bolnikih z mKRK z mutantnim RAS ali pri katerih stanje RAS ni znano. NEŽELENI UČINKI: Neželeni učinki, zabeleženi v kliničnih študijah in v spontanih poročilih v obdobju trženja zdravila pri bolnikih z mKRR, ki so dobivali panitumumab kot edino zdravilo ali v kombinaciji s kemoterapijo, so: Zelo pogosti (. 1/10): paronihija, anemija, hipokaliemija, anoreksija, hipomagneziemija, nespečnost, konjunktivitis, dispneja, kašelj, driska, navzea, bruhanje, bolečine v trebuhu, stomatitis, zaprtost, akneiformni dermatitis, izpuščaj (vključuje splošne termine kožne toksičnosti, eksfoliacije kože, eksfoliativnega izpuščaja, papuloznega izpuščaja, pruritičnega izpuščaja, eritematoznega izpuščaja, generaliziranega izpuščaja, makularnega izpuščaja, makulo papuloznega izpuščaja, kožne lezije), eritem, srbenje, suha koža, fisure na koži, akne, alopecija, bolečine v hrbtu, utrujenost, pireksija, astenija, vnetje sluznice, periferni edemi, paronihija, zmanjšanje telesne mase; Pogosti ogosti (. 1/100 do < 1/10): pustulozen izpuščaj, celulitis, okužba sečil, folikulitis, lokalizirana okužba, levkopenija, preobčutljivost, hipokalciemija, dehidracija, hiperglikemija, hipofosfatemija, anksioznost, glavobol, omotica, blefaritis, rast trepalnic, močnejše solzenje, očesna hiperemija, suho oko, srbenje oči, draženje oči, tahikardija, globoka venska tromboza, hipotenzija, hipertenzija, zardevanje, pljučna embolija, epistaksa, krvavitev iz danke, suha usta, dispepsia, aftozni stomatitis, heilitis, gastroezofagealna refluksna bolezen, sindrom palmarno plantarne eritrodizestezije, kožni ulkus, krasta, hipertrihoza, lomljenje nohtov, bolezni nohtov, hiperhidroza, dermatitis, bolečine v okončini, bolečine v prsih, bolečina, mrzlica, znižanje magnezija v krvi; Občasni (. 1/1.000 do < 1/100): okužba oči, okužba vek, draženje vek, keratitis, cianoza, bronhospazem, suhost nosu, razpokane ustnice, suhe ustnice, angioedem, hirzutizem, vraščanje nohta, oniholiza, z infundiranjem povezane reakcije; Redki (. 1/10.000 do < 1/1.000): anafilaktična reakcija, ulcerativni keratitis, nekroza kože, Stevens-Johnsonov sindrom, toksična epidermalna nekroliza; Pogostnost neznana ogostnost neznana (pogostnosti ni mogoče oceniti iz razpoložljivih podatkov): intersticijska pljučna bolezen. FARMACEVTSKI PODATKI: Shranjujte v hladilniku (2 °C 8 °C). Ne zamrzujte. Shranjujte v originalni ovojnini za zagotovitev zaščite pred svetlobo. Zdravilo Vectibix® ne vsebuje nobenih antimikrobnih konzervansov ali bakteriostatikov. Zdravilo je treba uporabiti takoj po razredčenju. Če ni uporabljeno takoj, so čas shranjevanja med uporabo in pogoji pred uporabo odgovornost uporabnika; čas običajno ne sme presegati 24 ur pri temperaturi od 2 °C do 8 °C. Razredčene raztopine ne smete zamrzniti. Zdravilo Vectibix® je namenjeno za enkratno uporabo. Inkompatibilnosti med zdravilom Vectibix® in natrijevim kloridom 9 mg/ml (0,9 %) raztopino za injiciranje v polivinilkloridnih ali poliolefinskih vrečkah niso ugotovili. NAČIN IN REŽIM PREDPISOVANJA TER IZDAJE ZDRAVILA: Predpisovanje in izdaja zdravila je le na recept s posebnim režimom – H. IMETNIK DOVOLJENJA ZA PROMET: Amgen Europe B.V., Minervum 7061, NLvum 7061, NL 4817 ZK Breda, Nizozemska. Dodatna pojasnila lahko dobite v lokalni pisarni: Amgen zdravila d.o.o., Šmartinska 140, SI 1000 Ljubljana. DATUM ZADNJE REVIZIJE BESEDILA: Marec 2015. DATUM PRPRAVE INFORMACIJE: Avgust 2015. Podrobne informacije o zdravilu so objavljene na spletni strani Evropske agencije za zdravila http://www.ema.europa.eu. Literatura: 1. Povzetek glavnih značilnosti zdravila Vectibix®, Amgen. SLHR-SL-P-954-0815-112854 (1) Kakovostna in količinska sestava 1 ml raztopine vsebuje 1,5 mg benzidaminijevega klorida, kar ustreza 1,34 mg benzidamina. V enem razpršku je 0,17 ml raztopine. En razpršek vsebuje 0,255 mg benzidaminijevega klorida, kar ustreza 0,2278 mg benzidamina. En razpršek vsebuje 13,6 mg 96 odstotnega etanola, kar ustreza 12,728 mg 100 odstotnega etanola, in 0,17 mg metilparahidroksibenzoata (E218). Terapevtske indikacije Samozdravljenje: lajšanje bolečine in oteklin pri vnetju v ustni votlini in žrelu, ki so lahko posledica okužb in stanj po operaciji. Po nasvetu in navodilu zdravnika: lajšanje bolečine in oteklin v ustni votlini in žrelu, ki so posledica radiomukozitisa. Odmerjanje in način uporabe Uporaba 2- do 6-krat na dan (vsake 1,5 do 3 ure). Odrasli: 4 do 8 razprškov 2- do 6-krat na dan. Otroci od 6 do 12 let: 4 razprški 2- do 6-krat na dan. Otroci, mlajši od 6 let: 1 razpršek na 4 kg telesne mase; do največ 4 razprške 2 do 6-krat na dan. Kontraindikacije Znana preobčutljivost za zdravilno učinkovino ali katerokoli pomožno snov. Posebna opozorila in previdnostni ukrepi Pri manjšini bolnikov lahko resne bolezni povzročijo ustne/žrelne ulceracije. Če se simptomi v treh dneh ne izboljšajo, se mora bolnik posvetovati z zdravnikom ali zobozdravnikom, kot je primerno. Zdravilo vsebuje aspartam (E951) (vir fenilalanina), ki je lahko škodljiv za bolnike s fenilketonurijo. Zdravilo vsebuje izomalt (E953) (sinonim: izomaltitol (E953)). Bolniki z redko dedno intoleranco za fruktozo ne smejo jemati tega zdravila. Uporaba benzidamina ni priporočljiva za bolnike s preobčutljivostjo za salicilno kislino ali druga nesteroidna protivnetna zdravila. Pri bolnikih, ki imajo ali so imeli bronhialno astmo, lahko pride do bronhospazma. Pri takih bolnikih je potrebna previdnost. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij Pri ljudeh raziskav o interakcijah niso opravljali. Nosečnost in dojenje Tantum Verde z okusom mentola 3 mg pastile se med nosečnostjo in dojenjem ne smejo uporabljati. Vpliv na sposobnost vožnje in upravljanja s stroji Uporaba benzidamina lokalno v priporočenem odmerku ne vpliva na sposobnost vožnje in upravljanja s stroji. Neželeni učinki Bolezni prebavil Redki: pekoč občutek v ustih, suha usta. Bolezni imunskega sistema Redki: preobčutljivostna reakcija. Bolezni dihal, prsnega koša in mediastinalnega prostora Zelo redki: laringospazem. Bolezni kože in podkožja Občasni: fotosenzitivnost. Zelo redki: angioedem. Rok uporabnosti 4 leta. Zdravila ne smete uporabljati po datumu izteka roka uporabnosti, ki je naveden na ovojnini. Posebna navodila za shranjevanje Za shranjevanje pastil niso potrebna posebna navodila. Plastenko z raztopino shranjujte v zunanji ovojnini za zagotovitev zaščite pred svetlobo. Shranjujte pri temperaturi do 25°C. Shranjujte v originalni ovojnini in nedosegljivo otrokom. Erbitux 5 mg/ml raztopina za infundiranje Skrajšan povzetek glavnih znaËilnosti zdravila Sestava: En ml raztopine za infundiranje vsebuje 5 mg cetuksimaba in pomožne snovi. Cetuksimab je himerno monoklonsko IgG1 protitelo. Terapevtske indikacije: Zdravilo Erbitux je indicirano za zdravljenje bolnikov z metastatskim kolorektalnim rakom z ekspresijo receptorjev EGFR in nemutiranim tipom RAS v kombinaciji s kemoterapijo na osnovi irinotekana, kot primarno zdravljenje v kombinaciji s FOLFOX in kot samostojno zdravilo pri bolnikih, pri katerih zdravljenje z oksaliplatinom in zdravljenje na osnovi irinotekana ni bilo uspešno in pri bolnikih, ki ne prenašajo irinotekana. 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 ponavljajoËo se in/ali metastatsko bolezen. Odmerjanje in naËin uporabe: Zdravilo Erbitux pri vseh indikacijah infundirajte enkrat na teden. Pred prvo infuzijo mora bolnik prejeti premedikacijo z antihistaminikom in kortikosteroidom najmanj 1 uro pred uporabo cetuksimaba. ZaËetni 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 preobËutljivostno reakcijo (3. ali 4. stopnje) na cetuksimab. Kombinacija zdravila Erbitux s kemoterapijo, ki vsebuje oksaliplatin, je kontraindicirana pri bolnikih z metastatskim kolorektalnim rakom z mutiranim tipom RAS ali kadar status RAS ni znan. Posebna opozorila in previdnostni ukrepi: Pojav hude reakcije, povezane z infundiranjem, zahteva takojšnjo in stalno ukinitev terapije s cetuksimabom. »e pri bolniku nastopi blaga ali zmerna reakcija, povezana z infundiranjem, lahko zmanjšate hitrost infundiranja. PriporoËljivo je, da ostane hitrost infundiranja na nižji vrednosti tudi pri vseh naslednjih infuzijah. »e se pri bolniku pojavi kožna reakcija, ki je ne more prenašati, ali huda kožna reakcija (. 3. stopnje po kriterijih CTCAE), morate prekiniti terapijo s cetuksimabom. Z zdravljenjem smete nadaljevati le, Ëe se je reakcija izboljšala do 2. stopnje. »e ugotovite intersticijsko bolezen pljuË, morate zdravljenje s cetuksimabom prekiniti, in bolnika ustrezno zdraviti. Zaradi možnosti pojava znižanja nivoja elektrolitov v serumu se pred in periodiËno med zdravljenjem s cetuksimabom priporoËa doloËanje koncentracije elektrolitov v serumu. Pri bolnikih, ki prejemajo cetuksimab v kombinaciji s kemoterapijo na osnovi platine, obstaja veËje tveganje za pojav hude nevtropenije. Takšne bolnike je potrebno skrbno nadzorovati. Pri predpisovanju cetuksimaba je treba upoštevati kardiovaskularno stanje in indeks zmogljivosti bolnika in soËasno dajanje kardiotoksiËnih uËinkovin kot so fluoropirimidini. »e je diagnoza ulcerativnega keratitisa potrjena, je treba zdravljenje s cetuksimabom prekiniti ali ukiniti. Cetuksimab je treba uporabljati previdno pri bolnikih z anamnezo keratitisa, ulcerativnega keratitisa ali zelo suhih oËi. Cetuksimaba ne uporabljajte za zdravljenje bolnikov s kolorektalnim rakom, Ëe imajo tumorje z mutacijo RAS ali pri katerih je tumorski status RAS neznan. Interakcije: Pri kombinaciji s fluoropirimidini se je v primerjavi z uporabo fluoropirimidinov, kot monoterapije, poveËala pogostnost srËne ishemije, vkljuËno z miokardnim infarktom in kongestivno srËno odpovedjo ter pogostnost sindroma dlani in stopal. V kombinaciji s kemoterapijo na osnovi platine se lahko poveËa pogostnost hude levkopenije ali hude nevtropenije. V kombinaciji s kapecitabinom in oksaliplatinom (XELOX) se lahko poveËa pogostnost hude driske. Neželeni uËinki: Zelo pogosti (. 1/10): hipomagneziemija, poveËanje ravni jetrnih encimov, kožne reakcije, blage ali zmerne reakcije povezane z infundiranjem, mukozitis, v nekaterih primerih resen. Pogosti (. 1/100 do < 1/10): dehidracija, hipokalciemija, anoreksija, glavobol, konjunktivitis, driska, navzeja, bruhanje, hude reakcije povezane z infundiranjem, utrujenost. Posebna navodila za shranjevanje: Shranjujte v hladilniku (2 °C -8 °C). Pakiranje: 1 viala z 20 ml ali 100 ml raztopine. NaËin in režim izdaje: Izdaja zdravila je le na recept-H. Imetnik dovoljenja za promet: Merck KGaA, 64271 Darmstadt, NemËija. Datum zadnje revizije besedila: november 2014. Pred predpisovanjem zdravila natanËno preberite celoten Povzetek glavnih znaËilnosti zdravila. Samo za strokovno javnost. Podrobnejše informacije so na voljo pri predstavniku imetnika dovoljenja za promet z zdravilom: Merck d.o.o., Ameriška ulica 8, 1000 Ljubljana, tel.: 01 560 3810, faks: 01 560 3830, el. pošta: info@merck.si www.merckserono.net www.Erbitux-international.com PM-ONC-05/2015 /13.10.2015 instructions Instructions for authors The editorial policy Radiology and Oncology is a multidisciplinary journal devoted to the publishing original and high quality scientific papers and review articles, pertinent to diagnostic and interventional radiology, computerized tomography, magnetic resonance, ultrasound, nuclear medicine, radiotherapy, clinical and experimental oncology, radiobiology, radiophysics and radiation protection. Therefore, the scope of the journal is to cover beside radiology the diagnostic and therapeutic aspects in oncology, which distinguishes it from other journals in the field. 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The statement of disclosure must be in the Cover letter accompany­ing the manuscript or submitted on the form available on www.icmje.org/coi_disclosure.pdf Page proofs Page proofs will be sent by E-mail 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 three days of receipt. Only grammatical corrections are ac­ceptable at that time. Open access Papers are published electronically as open access on www.degruyter.com/view/j/raon, also papers accepted for publication as E-ahead of print. BISTVENI PODATKI IZ POVZETKA GLAVNIH ZNAČILNOSTI ZDRAVILA SUTENT 12,5 mg, 25 mg, 37,5 mg, 50 mg trde kapsule Sestava in oblika zdravila: Ena 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) pri odraslih, če zdravljenje z imatinibom zaradi odpornosti ali neprenašanja ni bilo uspešno. Zdravljenje napredovalega/metastatskega karcinoma ledvičnih celic (MRCC) pri odraslih. Zdravljenje neizrezljivih ali metastatskih, dobro diferenciranih nevroendokrinih tumorjev trebušne slinavke (pNET), kadar gre za napredovanje bolezni pri odraslih (izkušnje z zdravilom Sutent kot zdravilom prve izbire so omejene). Odmerjanje in način uporabe: Terapijo mora uvesti zdravnik, ki ima izkušnje z uporabo zdravil za zdravljenje rakavih bolezni. GIST in MRCC: Priporočeni odmerek je 50 mg peroralno enkrat na dan, 4 tedne zapored; temu sledi 2-tedenski premor (Shema 4/2), tako da celotni ciklus traja 6 tednov. pNET: Priporočeni odmerek je 37,5 mg peroralno enkrat na dan, brez načrtovanega premora. Prilagajanje odmerka: Odmerek je mogoče prilagajati v povečanjih po 12,5 mg, upoštevaje individualno varnost in prenašanje. Pri GIST in MRCC dnevni odmerek ne sme preseči 75 mg in ne sme biti manjši od 25 mg; pri pNET je največji odmerek 50 mg na dan, z možnimi prekinitvami zdravljenja. Pri sočasni uporabi z močnimi zaviralci ali induktorji CYP3A4 je treba odmerek ustrezno prilagoditi. Pediatrična populacija: Uporaba sunitiniba ni priporočljiva. Starejši bolniki (. 65 let): Med starejšimi in mlajšimi bolniki niso opazili pomembnih razlik v varnosti in učinkovitosti. Okvara jeter: Pri bolnikih z jetrno okvaro razreda A in B po Child-Pughu prilagoditev odmerka ni potrebna; pri bolnikih z okvaro razreda C sunitinib ni bil preizkušen, zato njegova uporaba ni priporočljiva. Okvara ledvic: Prilagajanje začetnega odmerka ni potrebno, nadaljnje prilagajanje odmerka naj temelji na varnosti in prenašanju pri posameznem bolniku. Način uporabe: Zdravilo Sutent se uporablja peroralno, bolnik ga lahko vzame s hrano ali brez nje. Če pozabi vzeti odmerek, ne sme dobiti dodatnega, temveč naj vzame običajni predpisani odmerek naslednji dan. Kontraindikacije: Preobčutljivost na zdravilno učinkovino ali katerokoli pomožno snov. Posebna opozorila in previdnostni ukrepi: Bolezni kože in tkiv: obarvanje kože, gangrenozna pioderma (običajno izgine po prekinitvi zdravljenja), hude kožne reakcije (multiformni eritem (EM), Stevens-Johnsonov sindrom (SJS) in toksična epidermalna nekroliza (TEN)). Če so prisotni znaki EM, SJS ali TEN, je treba zdravljenje prekiniti. Krvavitve v prebavilih, dihalih, sečilih, možganih; najpogosteje epistaksa; krvavitve tumorja, včasih s smrtnim izidom. Pri bolnikih, ki se sočasno zdravijo z antikoagulanti, se lahko redno spremlja celotna krvna slika (trombociti), koagulacijski faktorji (PT / INR) in opravi telesni pregled. Bolezni prebavil: polegdiareje, navzee/bruhanja, bolečine v trebuhu, dispepsije, stomatitisa/bolečine v ustih in ezofagitisa tudi hudi zapleti (včasih s smrtnim izidom), vključno z gastrointestinalno perforacijo. Hipertenzija: pri bolnikih s hudo hipertenzijo, ki je ni mogoče urediti z zdravili, je priporočljivo začasno prenehanje zdravljenja. Hematološke bolezni: zmanjšanje števila nevtro.ilcev, trombocitov, anemija. Bolezni srca in ožilja: srčno-žilni dogodki, vključno s srčnim popuščanjem, kardiomiopatijo, miokardno ishemijo in miokardnim infarktom, v nekaterih primerih s smrtnim izidom; sunitinib povečuje tveganje za pojav kardiomiopatije; previdna uporaba pri bolnikih s tveganjem za te dogodke, ali ki so te dogodke imeli v preteklosti. Podaljšanje intervala QT: previdna uporaba pri bolnikih z znano anamnezo podaljšanja intervala QT, tistih, ki jemljejo antiaritmike ali zdravila, ki lahko podaljšajo interval QT, in tistih z relevantno, že obstoječo srčno boleznijo, bradikardijo ali elektrolitskimi motnjami. Venski in arterijski trombembolični dogodki; arterijski včasih s smrtnim izidom. Trombotična mikroangiopatija (TMA): TMA, vključno s trombotično trombocitopenično purpuro in hemolitično-uremičnim sindromom, v nekaterih primerih z odpovedjo ledvic ali smrtnim izidom. Dogodki na dihalih: dispneja, plevralni izliv, pljučna embolija ali pljučni edem; redki primeri s smrtnim izidom. Moteno delovanje ščitnice: bolnike je treba med zdravljenjem rutinsko spremljati glede delovanja ščitnice vsake 3 mesece. Pankreatitis, tudi resni primeri s smrtnim izidom. Hepatotoksičnost, nekateri primeri s smrtnim izidom. Holecistitis, vključno z akalkuloznim in em.izemskim holecistitisom. Delovanje ledvic: primeri zmanjšanega delovanja ledvic, odpovedi ledvic in/ali akutne odpovedi ledvic, v nekaterih primerih s smrtnim izidom. Fistula: če nastane .istula, je treba zdravljenje s sunitinibom prekiniti. Oteženo celjenje ran: pribolnikih, pri katerih naj bi bil opravljen večji kirurški poseg, je priporočljiva začasna prekinitev zdravljenja s sunitinibom. Osteonekroza čeljustnic: pri sočasnem ali zaporednem dajanju zdravila Sutent in intravenskih bisfosfonatov je potrebna previdnost; invazivni zobozdravstveni posegi predstavljajo dodatni dejavnik tveganja. Preobčutljivost/angioedem. Motnje okušanja. Konvulzije: obstajajo poročila, nekatera s smrtnim izidom, o preiskovancih s konvulzijami in radiološkimi znaki sindroma reverzibilne posteriorne levkoencefalopatije. Sindrom lize tumorja, v nekaterih primerih s smrtnim izidom. Okužbe: hude okužbe z ali brez nevtropenije (okužbe dihal, sečil, kože in sepsa), vključno z nekaterimi s smrtnim izidom; redki primeri nekrotizitajočega fasciitisa, vključno s prizadetostjo presredka, ki so bili včasih smrtni. Hipoglikemija: če se pojavi simptomatska hipoglikemija, je treba zdravljenje s sunitinibom začasno prekiniti. Pri sladkornih bolnikih je treba redno preverjati raven glukoze v krvi in, če je treba, prilagoditi odmerek antidiabetika. Medsebojno delovanje z drugimi zdravili: (Študije so izvedli le pri odraslih.) Zdravila, ki lahko zvečajo koncentracijo sunitiniba v plazmi (ketokonazol, ritonavir, itrakonazol, eritromicin, klaritromicin ali sok grenivke). Zdravila, ki lahko zmanjšajo koncentracijo sunitiniba v plazmi (deksametazon, fenitoin, karbamazepin, rifampin, fenobarbital, Hypericum perforatum oz. šentjanževka). Plodnost, nosečnost in dojenje: Zdravila Sutent ne smemo uporabljati med nosečnostjo in tudi ne pri ženskah, ki ne uporabljajo ustrezne kontracepcije, razen če možna korist odtehta možno tveganje za plod. Ženske v rodni dobi naj med zdravljenjem z zdravilom Sutent ne zanosijo. Ženske, ki jemljejo zdravilo Sutent, ne smejo dojiti. Neklinični izsledki kažejo, da lahko zdravljenje s sunitinibom poslabša plodnost samcev in samic. Vpliv na sposobnost vožnje in upravljanja s stroji: Sutent lahko povzroči omotico. Neželeni učinki: Najbolj resni neželeni učinki (nekateri s smrtnim izidom) so: odpoved ledvic, srčno popuščanje, pljučna embolija, gastrointestinalna perforacija in krvavitve (npr. v dihalih, prebavilih, tumorju, sečilih in možganih). Najpogostejši neželeni učinki (ki so se pojavili pri vsaj 20 % bolnikov v registracijskih preskušanjih) so: zmanjšan tek, motnje okušanja, hipertenzija, utrujenost, prebavne motnje (npr. driska, navzea, stomatitis, dispepsija in bruhanje), sprememba barve kože in sindrom palmarno-plantarne eritrodisestezije. Med najbolj pogostimi neželenimi učinki so tudi hematološke motnje (nevtropenija, trombocitopenija, anemija in levkopenija). Ostali zelo pogosti (. 1/10) neželeni učinki so: hipotiroidizem, nespečnost, omotica, glavobol, dispneja, epistaksa, kašelj, bolečina v trebuhu, zaprtje, obarvanje kože, izpuščaj, spremembe barve las, suha koža, bolečine v udih, artralgija, bolečine v hrbtu, vnetje sluznice, edem, pireksija. Način in režim izdaje: Predpisovanje in izdaja zdravila je le na recept, zdravilo pa se uporablja samo v bolnišnicah. Izjemoma se lahko uporablja pri nadaljevanju zdravljenja na domu ob odpustu iz bolnišnice in nadaljnjem zdravljenju. Imetnik dovoljenja za promet:P.izer Limited, Ramsgate Road, Sandwich, Kent, CT13 9NJ, Velika Britanija. Datum zadnje revizije besedila: 25.06.2015 Pred predpisovanjem se seznanite s celotnim povzetkom glavnih značilnosti zdravila. SUT-03-15 Samo za strokovno javnost