vol.49 no.3 september 2015 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 noveskupinekemoterapevtikov, imenovanih halihondrini. Zdravilo HALAVEN je indicirano za zdravljenje bolnic z lokalno napredovalim ali metastatskimrakomdojke,kije napredovalpovsaj enemrežimukemoterapije 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 odmerek1,23mg/m2, intravensko,v obliki2-do 5-minutne infuzije, 1.in8.danvsakega21-dnevnega cikla. Ena2ml vialavsebuje 0,88mg eribulina. Raztopina, pripravljena za uporabo, redčenje ni potrebno. SKRAJŠAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA HALAVEN 0,44 mg/ml raztopina za injiciranje (eribulin) TERAPEVTSKE 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, University Clinic Tübingen, Department of Neuroradiology, Tübingen, Germany 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 2015; 49(3): 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 2015; 49(3): B. contents contents review 209 The concept of radiation-enhanced stem cell differentiation Adam A. Mieloch, Wiktoria M. Suchorska 217 Gamma-enolase: a well-known tumour marker, with less known role in cancer Tjasa Vizin, Janko Kos nuclear medicine 227 The impact of reconstruction algorithms and time of flight information on PET/CT image quality Alen Suljic, Petra Tomse, Luka Jensterle, Damijan Skrk radiology 234 Careful treatment planning enables safe ablation of liver tumors adjacent to major blood vessels by percutaneous irreversible electroporation (IRE) Bor Kos, Peter Voigt, Damijan Miklavcic, Michael Moche 242 Central nervous system imaging in childhood Langerhans cell histiocytosis – a reference center analysis Luciana Porto, Stefan Schöning, Elke Hattingen, Jan Sörensen, Alina Jurcoane, Thomas Lehrnbecher 250 Correlation of diffusion MRI with the Ki-67 index in non-small cell lung cancer Adem Karaman, Irmak Durur-Subasi, Fatih Alper, Omer Araz, Mahmut Subasi, Elif Demirci, Mevlut Albayrak, Gökhan Polat, Metin Akgun, Nevzat Karabulut clinical oncology 256 The influence of cytokine gene polymorphisms on the risk of developing gastric cancer in patients with Helicobacter pylori infection David Stubljar, Samo Jeverica, Tomislav Jukic, Miha Skvarc, Tadeja Pintar, Bojan Tepes, Rajko Kavalar, Borut Stabuc, Alojz Ihan 265 Inflammatory myofibroblastic tumor of the pancreatic head - a case report of a 6 months old child and review of the literature Ales Tomazic, Diana Gvardijancic, Joze Maucec, Matjaz Homan Radiol Oncol 2015; 49(3): C. contents 271 Neoadjuvant chemotherapy in 13 patients with locally advanced poorly differentiated thyroid carcinoma based on Turin proposal - a single institution experience Nikola Besic, Marta Dremelj, Andreja Schwartzbartl-Pevec, Barbara Gazic 279 Fibulin-3 as a biomarker of response to treatment in malignant mesothelioma Viljem Kovac, Metoda Dodic-Fikfak, Niko Arneric, Vita Dolzan, Alenka Franko 286 Impact of tumour volume on prediction of progression free survival in sinonasal cancer Florian Hennersdorf, Paul-Stefan Mauz, Patrick Adam, Stefan Welz, Anne Sievert, Ulrike Ernemann, Sotirios Bisdas radiophysics 291 Comparison of hybrid volumetric modulated arc therapy (VMAT) technique and double arc VMAT technique in the treatment of prostate cancer Christopher Amaloo, Daryl P. Nazareth, Lalith K. Kumaraswamy 299 Rounded leaf end effect of multileaf collimator on penumbra width and radiation field offset: an analytical and numerical study Dong Zhou, Hui Zhang, Peiqing Ye 307 A comparison of the quality assurance of four dosimetric tools for intensity modulated radiation therapy Jaeman Son, Taesung Baek, Boram Lee, Dongho Shin, Sung Yong Park, Jeonghoon Park, , Young Kyung Lim, Se Byeong Lee, Jooyoung Kim, Myonggeun Yoon 314 erratum slovenian abstracts Radiol Oncol 2015; 49(3): D. 209 research article The concept of radiation-enhanced stem cell differentiation Adam A. Mieloch and Wiktoria M. Suchorska Radiobiology Laboratory, Department of Medical Physics, The Greater Poland Cancer Centre Radiol Oncol 2015; 49(3): 209-216. Received 9 March 2015 Accepted 5 June 2015 Correspondence to: Dr. Wiktoria M. Suchorska, Radiobiology Laboratory, Department of Medical Physics, The Greater Poland Cancer Centre, 15th Garbary Street, 61-866 Poznan, Poland. Phone: +48 618 850 477; wiktoria.suchorska@wco.pl Disclosure: No potential conflicts of interest were disclosed. Background. Efficient stem cell differentiation is considered to be the holy grail of regenerative medicine. Pursuing the most productive method of directed differentiation has been the subject of numerous studies, resulting in the de­velopment of many effective protocols. However, the necessity for further improvement in differentiation efficiency remains. This review contains a description of molecular processes underlying the response of stem cells to ionizing radiation, indicating its potential application in differentiation procedures. In the first part, the radiation-induced dam­age response in various types of stem cells is described. Second, the role of the p53 protein in embryonic and adult stem cells is highlighted. Last, the hypothesis on the mitochondrial involvement in stem cell development including its response to ionizing radiation is presented. Conclusions. In summary, despite the many threats of ionizing radiation concerning genomic instability, subjecting cells to the appropriate dosage of ionizing radiation may become a useful method for enhancing directed differen­tiation in certain stem cell types. Key words: stem cells; ionizing radiation; differentiation; regenerative medicine; tissue engineering Introduction Stem cells (SCs) possess a set of unique advantag­es, including the ability to replicate and the ability to differentiate into many different types of cells, called “pluripotency”. Due to the pluripotent char­acteristic of these cells, they play a pivotal role in tissue development and maintenance by replen­ishing the depletion of cells caused by damag­ing factors or that occurs physiologically during tissue turn-over.1 The majority of recent studies have mainly focused on two types of stem cells: Embryonic stem cells (ESCs) and adult stem cells (ASCs), also known as somatic tissue or mesenchy­mal stem cells.2 ESCs are derived from the inner cell mass of the blastocyst and are capable of dif­ferentiating into the three embryonic germ layers: ectoderm, mesoderm and endoderm, thus contrib­uting to the formation of almost every cell type. ASCs reside in tissue-specific niches in a quiescent state. Upon activation, they undergo asymmetric division, which simultaneously increases the num­ber of cells in the niche and the number differenti­ating into tissue specific lineages, providing cells required for tissue regeneration.3,4 Stem cells have had a significant impact on the progress of many fields of biotechnology, includ­ing cell-based regenerative therapies, drug testing and screening, disease modeling, side effects in radiotherapy and many more. In 2006, Yamanaka et al. announced a breakthrough finding in regen­erative medicine, describing the reprogramming of mouse adult fibroblasts into induced pluripo­tent stem cells (iPSs) by introducing four factors: Oct3/4, Sox2, c-Myc, and Klf4. iPSs in many aspects resemble ESCs.5 This discovery solved many of the ethical disputes concerning the procurement of ESCs from human embryos and began a new era in regenerative medicine. Since then, many attempts to harness the pluri­potency of stem cells into directed differentiation have been successful.6,7 Some of developed pro­ 210 tocols require the formation of embryoid bodies (EBs) prior to further differentiation. EBs are three-dimensional cellular aggregates obtained by spon­taneous differentiation of ESCs or IPSs. EBs con­sist of ESCs that are mostly differentiated into the embryonic germ layers: ectoderm, endoderm and mesoderm.8 The differentiation process in many aspects mimics early mammalian embryogenesis, including cell to cell interactions. Moreover, the most essential method of EBs formation is based on suspension culture deprived of antidifferentiation factors. Due to simple methodology and similarity to embryogenesis, EBs are widely utilized as an in­termediate stage during in vitro differentiation of both human and murine ESCs. 9 Currently, many stem cell-based therapies are un­dergoing clinical trials, for example, “Intravenous Stem Cells After Ischemic Stroke”, “Human Neural Stem Cell Transplantation in Amyotrophic Lateral Sclerosis (ALS)” and “Treatment of Knee Osteoarthritis by Intra-articular Injection of Bone Marrow Mesenchymal Stem Cells”.10–12 These trials are just a few from a still enlarging group of stud­ies investigating the potential applications of stem cells in regenerative medicine, which indicates a growing need for reliable methods of directed dif­ferentiation of SCs. Ionizing radiation (IR) has been used for many years as a basic tool in cancer treatment.13 The re­sponse of non-stem cells to irradiation has been extensively investigated by a number of studies, and to date, many molecular mechanisms of this phenomena have been thoroughly elucidated.14–16 However, based on the current understanding con­cerning non-SCs, the radio-response of SCs cannot be anticipated and it could result in unexpected outcomes. Radiation-induced differentiation has already been reported in multiple studies17,18; however, it has not been investigated as a potential tool in stem cell differentiation protocols. The main goal of this review is to present research based indications that radiation-enhanced differentiation is a promising technology for further development of stem cell engineering. Radiation-induced DNA damage response in stem cells Radiation-induced damage to genomic DNA trig­gers a cascade of biochemical reactions known as the DNA damage response (DDR), which includes cell cycle arrest, DNA repair and, in the case of un­manageable lesions, senescence or apoptosis. The functional mechanism of DNA damage repair is crucial for the maintenance of genomic stability. The most dangerous type of DNA lesions are double strand breaks (DSBs), which are usually caused by IR or free radical exposure. Repair of DSBs is driven by two major pathways: homolo­gous recombination (HR) and non-homologous end joining (NHEJ).19 In the process of HR, sister chromatids serve as a template; thus, the repair is considered error-free. NHEJ does not utilize sister chromatids as a template and is therefore signifi­cantly more prone to error introduction. Depending on the phase of the cell cycle, one of the pathways is used predominantly. The requirement for sister chromatids in HR restrains its activity to the S and G2 phases. The NHEJ response dominates through the rest of the cell cycle.20 There are also other types of DNA damage repair mechanisms: nucleotide ex­cision repair (NER), base excision repair (BER) and mismatch repair (MMR). However, their contribu­tion to radiation-enhanced differentiation seems to be negligible and will not be considered in this study. DNA damage repair in embryonic stem cells (ESCs) It has been proven that the mechanisms of DNA damage repair in ESCs are more efficient compared to other cell types.21 ESCs display a unique cell cy­cle structure. The G1 phase is significantly short­ened and the G1 to S transition is facilitated in or­der to promote rapid self-renewal. Consequently, the majority of the ESC population is in the phases of cell cycle where sister chromatids are available for use as a template. Due to this phenomena, ESCs predominantly utilize high-fidelity HR.22 ESCs serve as a pool of cells for the development of the whole organism. Therefore, DNA repair in these cells requires high efficiency and accuracy in order to provide genomic stability. In the case of in­sults in the genomic DNA that cannot be repaired, the cell undergoes apoptosis, which is significantly facilitated by a mechanism known as mitochon­drial priming in ESCs.23 Mitochondrial priming is determined by the equilibrium between levels of anti-apoptotic and pro-apoptotic proteins of the B-cell lymphoma 2 (Bcl-2) protein family. ESCs display elevated levels of pro-apoptotic proteins within the mitochondria. Consequently, the initia­tion of apoptosis requires a considerably weaker stimuli in order to cross the apoptotic threshold. This phenomenon ensures elimination of geneti­cally unstable cells and prevents further transmis­sion of mutations. 211 A previous study by Sokolov and Naumann re­vealed that human embryonic stem cells (hESCs) undergo apoptosis after relatively low-dose irra­diation. In the study, a 1.0 Gy dose of X-ray radia­tion triggered robust apoptosis. Conversely, doses of 0.5 Gy and 0.2 Gy did not increase the apoptotic response.24 A study by Lan et al. reported that a 2.0 Gy dose of X-ray radiation resulted in an almost 60% decrease in the survival rate of hESCs 5 days post-irradiation. The same study found that X-ray irradiation elevated metabolic activity (XTT assay) 1.5-fold after a 2.0 Gy dose and 2.5-fold after a 5.0 Gy dose. The same dosage of 2.0 Gy and 5.0 Gy resulted in elevated levels of reactive oxygen spe­cies (ROS) and nitrogen (RNS) species for 1 week following exposure.25 DNA damage repair in adult stem cells (ASCs) ASC sensitivity to irradiation varies greatly, de­pending on their type and developmental stage. However, it is postulated that the DNA repair mechanism becomes less efficient upon differenti­ation in general. Therefore, ASCs display reduced DNA damage repair (DDR) capabilities in compar­ison to ESCs, which has been shown previously.26 It is important to note that the mechanism of DDR in ASCs is distinctly different than the one observed in ESCs.27 ASCs reside in a quiescent state in the G0 phase of the cell cycle. Slower cell cycle progres­sion corresponds to a higher radioresistance.28,29 Therefore, despite a lower efficacy of DDR, ASCs exhibit a lower sensitivity to IR compared to the rapidly dividing ESCs. It has been shown that upon DNA damage, ASCs can exit quiescence and progress into the G1 phase, in which error-prone NHEJ repair is performed.30 Consequently, ASCs are more susceptible to DNA damage accumula­tion, which can be passed onto progeny. In 1996, Schwenke et al. 17 found that .-irradiation of murine erythroid progenitor cells resulted in enhanced differentiation. This observed enhance­ment was determined to be due to the omission of mitotic cell cycling, which is necessary for pro­genitor cells to undergo terminal differentiation. Moreover, Zheng et al. 31 found that DSB suppress­es the self-renewal and promotes the further dif­ferentiation of neuronal stem cells (NSCs) in a p53­dependent manner. Role of p53 in stem cells The p53 protein has been widely studied for many years, and a number of its properties have been elucidated.32 However, the complexity of its inter­actions and associations with various molecular processes has left many novel functions of this pro­tein remaining to be discovered. p53 is a tumor suppressor protein responsible for the induction of reversible cell cycle arrest, which enables DNA repairs to be conducted, and the initiation of apoptosis in the case of irreversible DNA damage. p53 is a transcription factor that, upon activation, binds to the promoters of target genes, either inducing or repressing their tran­scription depending on the gene.33 p53 can trigger apoptosis via two pathways: the transcriptional (intrinsic) pathway, as described above, or the non-transcriptional (mitochondrial) pathway by direct interactions with pro- and anti-apoptotic proteins. The main target genes for its proapoptotic activ­ity include p53 upregulated modulator of apopto­sis (Puma) and Bcl-2-associated X (Bax) proteins, which belong to the Bcl-2 family.34 DNA damage results in ataxia telangiectasia mutated (ATM) pro­tein activation, which drives mouse double minute 2 homolog (Mdm2) polyubiquitination and further degradation. Mdm2 is an oncoprotein that medi­ates p53 polyubiquitination and further degrada­tion by the 26S proteasome. Therefore, Mdm2 deg­radation contributes to the increased stability of p53. It is worth noting that other mechanisms of p53 regulation also exist. Furthermore, p53 performs a regulatory function over cell proliferation by con­trolling the expression of the p21 protein, known as cyclin-dependent kinase inhibitor.35 Silencing of p53 expression has also been shown to increase the efficiency of reprogramming in iPSs genera­tion, indicating its contribution to the maintenance of a differentiated state.36 Nonetheless, p53 activ­ity during reprogramming ensures elimination of cells bearing genomic aberrations. Therefore, disruption of p53 pathway increases the efficacy of reprogramming and the risk of mutations con­comitantly.37,38 Down regulation of p53 activity has been shown to induce normal SCs transformation towards neoplastic, tumor cells.39 This may in turn result in cancer stem cells (CSs) formation. CSs share the fundamental properties of SCs, but their activity contributes to the cancer grow and main­tenance instead of replenishing normal cell pool.40 Moreover, teratomas generated from p53 knockout iPSs showed the presence of double-strand DNA breaks and DDR activation, leading to the conclu­sion that p53 inhibition decreases genomic stabil­ity.41 Due to the high risk of tumor generation after transplantation, methods utilizing p53 inhibition in iPSs generation seem to be unsuitable for thera­peutic use. 212 TAble 1. The examples of adult stem cell (ASC) types and their corresponding tissue of origin, progenitors and fully differentiated cells Bone marrow Hematopoietic stem cells Myeloid progenitor cells, Lymphoid progenitor cells Intestine Intestinal stem cells Enterocytes, Goblet cells, Entero-endocrine cells, Paneth cells Brain Neural stem cells Neurons, Astrocytes, Oligodendrocytes Mammary gland Mammary stem cells Luminal cells, Myoepithelial cells Muscle Myosatellite cells Mioblasts p53 in embryonic stem cells (ESCs) It has been shown that p53 accumulates at low levels in the nucleus of hESCs, although in a deacetylated, inactive state. Apart from its canonical activity, p53 also performs a regulatory function over cell prolif­eration by controlling the expression of p21, known as cyclin-dependent kinase inhibitor. p21 inhibits the activity of cyclin/cdk2 complexes and restrains cell cycle progression. Dolezalova et al. revealed that after UVC-irradiation of hESCs, p21 mRNA is pre­sent, although its translation is inhibited by various microRNAs.42 However, a study by Maimets et al. contradicts these findings, revealing that the small molecule Nutlin, functioning as a p53 activator, el­evates p21 protein levels in hESCs.43 Therefore, the role of p21 in the p53 pathway remains elusive. p53 plays an important role in ESC differentia­tion. It has been shown that spontaneous differ­entiation occurs at significantly lower rates when the p53 level is reduced.44 However, one of the most crucial mechanisms supporting the theory of radiation-enhanced differentiation is the re­duced expression of pluripotency factors driven by p53 activity. p53 binds directly to the promot­ers of NANOG and octamer-binding transcription factor 3/4 (Oct3/4), inhibiting their transcription. Moreover, elevated levels of p53 induce expression of differentiation markers GATA4 and GATA6.43 Furthermore, upon stabilization, in addition to its canonical function, p53 triggers the expression of miR-34a and miR-145, which subsequently repress the pluripotency factors Oct3/4, Kruppel-like fac­tor 4 (Klf4), protein lin-28 homolog A (Lin-28A) and sex determining region Y-box 2 (Sox2), which supports differentiation.45 Retinoic acid (RA) is a commonly used differ­entiation factor utilized in various differentiation protocols, including those inducing the genera­tion of neural cells, cardiomyocytes or chondro­cytes.46–48 RA treatment results in the suppression of NANOG expression. However, this effect was not observed after p53 gene deletion, suggesting that p53 is required for RA-mediated NANOG suppression.49 Therefore, synergistic cooperation between these two proteins may be hypothesized. It is important to mention that p53 also performs anti-differentiation stimulation through the Wnt canonical signaling pathway, which is responsible for the maintenance and self-renewal of human and murine ESCs.50,51 p53 in adult stem cells (ASCs) Adult stem cells comprise endothelial progenitors cells (ESC) and hematopoietic stem cells (HSC) and tissue cells, called mesenchymal stem cells (MSC), found in many different organs of the human body and the one discussed in this review are listed in Table 1. Every ASC type contributes to a different cell lineage; therefore, any indications concerning radiation-enhanced differentiation may be true for some ASC types and completely false for others. To clarify the reasoning behind this statement, the properties of p53 activity in three different types of ASCs will be described: neural stem cells (NSCs), hematopoietic stem cells (HSCs) and mammary stem cells (MaSCs). Neural stem cells (NSCs) Neural stem cells have the potential to differentiate into neurons, astrocytes and oligodendrocytes. In adults, neurogenesis of the central nervous system begins within the subventricular zone (SVZ) and the subgranular zone of the dentate gyrus of the hippocampus, which serves as a niche for NSCs. The SVZ is a narrow zone of tissue in the wall of the lateral ventricle in the forebrain and is the most active neurogenic region in the adult brain.52 Neurons generated within SVZ migrate through a path called rostral migratory stream and reach their final destination within the olfactory bulb. A complete turn-over of resident cells within SVZ occurs every 2 to 4 weeks. Nearly 30 000 neuronal precursors are produced daily.53 It has been demonstrated that the neuronal pro­genitors of p53-/-mice display a significantly higher 213 proliferation rate compared to wild-type mice. NSCs can be maintained in culture as aggregates or neurospheres. p53-/--derived NSCs formed sub­stantially larger neurospheres than wild type cells, which was due to an increased number of cells per sphere, rather than larger cells. This finding indi­cates that one of the functions of p53 in NSCs is to restrain excessive proliferation.54 Monje et al. found that gamma irradiation of neural progenitor cells resulted in a higher effi­ciency of differentiation. Cultures irradiated with a 10.0 Gy dose showed increased differentiation compared to cells irradiated with a 2.0 Gy dose and control cells. However, the ratio between neurons and astrocytes/oligodendrocytes remained undis­turbed, which is an important factor to consider in the context of radioenhancement.55 As previously mentioned, p53 also stimulates the Wnt signaling pathway. Data obtained by Wei et al. indicated that the Wnt/ß-catenin signaling pathway plays a crucial role in the proliferation and differentiation of NSCs in the hippocampus. In this study, a low dose of ionizing radiation (0.3 Gy) was shown to activate the Wnt/ß-catenin pathway. As a result, NSCs subjected to irradiation showed increased proliferation and differentiation with a concomitant decrease in apoptosis. Moreover, a water-maze test performed on mice indicated an improvement in the behavioral learning of these mice after low-dose irradiation compared to non-irradiated mice.56 Mammary stem cells (MaSCs) Mammary stem cells are located in the mammary glands. They can differentiate into all lineages of mammary epithelial cells. MaSCs are also respon­sible for mammary gland development during puberty and pregnancy.57 MaSCs can be cultured in vitro as floating aggregates called mammos­pheres. A mammosphere is a spherical colony de­rived from a single MaSC by clonal proliferation.58 However, the division of MaSCs occurs predomi­nantly by asymmetric division. Therefore, mam­mospheres usually contain a single stem cell sur­rounded by more differentiated progeny.59 Despite their self-renewal capabilities, they were shown to have a limited life span in culture conditions. MaSCs derived from p53-/- mice displayed an in­creased self-renewing potential, resulting in an in­creased number of MaSCs per mammosphere and an unlimited life span in culture conditions. This finding suggests that p53 is involved in the pre­vention of pathological proliferation by promoting asymmetric division, thus contributing to increased differentiation.60 It is also consistent with many sci­entific data regarding the role of p53 mutation in breast cancer development.61 Interestingly, MaSCs subjected to 4.0 Gy irradiation showed 2.7 fold in­crease in mammosphere reconstitution capacity, confirming that X-ray increases MaSCs prolifera­tion.62 Hematopoietic stem cells (HSCs) Hematopoietic stem cells (HSCs) are one of the best characterized human stem cells. For many years, they have been used in clinical applications, includ­ing leukemia treatment. HSCs differentiate into all of the blood cell lineages. They can be found in the red bone marrow. HSCs differentiate into myeloid and lymphoid progenitors, which may differentiate further giving rise to monocytes, erythrocytes, neu­trophils and macrophages (myeloid progenitors) or T-lymphocytes, B-lymphocytes and NK-cells (lymphoid progenitors). The majority of HSCs re­side in a quiescent state, while only a small fraction remains active and replenishes the blood cell pool.63 During steady-state hematopoiesis, p53 regu­lates HSC self-renewal and quiescence. It is also responsible for cell competition in the HSC niche. Cells expressing higher than average level of p53 undergo cell cycle arrest and senescence. This mechanism contributes to the maintenance of tis­sue homeostasis by the eradication of less func­tional cells. Milyavsky et al. found that HSCs subjected to a 3.0 Gy irradiation dose exhibited a delayed DSB repair and an increased apoptotic response via the p53/antiphagocytic protein 1 (APP1) pathway compared to progenitor cells, which indicated a high sensitivity of HSCs to ionizing radiation.64 This finding is in agreement with the common no­tion that HSCs are one of the cell types most vul­nerable to ionizing radiation (IR). However, de­spite its deteriorating effects, X-ray radiation has induced almost twofold increase in absolute num­ber of murine HSCs. Increased number of murine HSCs in bone marrow was still detectable 2 months after irradiation. This effect was not observable in p21-/-mice, suggesting p21 as a key factor of X-ray induced proliferation.62 Mitochondria in the context of enhanced differentiation Mitochondria are double-layered organelles that conduct the metabolic activities associated with energy production through oxidative phospho­rylation. Their morphology varies between tissues TAble 2. Examples of differences between human and murine cells affecting IR response Murine cells are deficient in p53 global genomic repair 72 Human ESC rejoin X-ray induced DSB faster than murine ESC 71 Murine cells repair DNA base damage more efficiently 73 Murine cells are more sensitive to oxidative stress 74,75 Murine cells are more prone to oncogenic transformation 76,77 DSB = double strand breaks; ESC = embryonic stem cells and is strictly connected to the metabolic state of a given cell. In addition to tissue specific differences, mitochondria may undergo fusion or fission, giv­ing rise to tubular or fragmented mitochondria, re­spectively. The fusion/fission mechanism is strictly connected with proliferation and differentiation.65 However, the outcome of tubular or fragmented mitochondria generation differs between cell types. In ESCs, the mitochondria reside in a frag­mented state, and an increase in mitochondrial fu­sion precedes differentiation. Ionizing radiation affects mitochondria in vari­ous ways. Mitochondrial DNA (mtDNA) is signifi­cantly more susceptible to IR compared to genomic DNA because it does not possess repair mecha­nisms as efficient as those found in the nucleus. Furthermore, mtDNA does not contain histones, which results in decreased resistance to various in­sults and a higher mutation rate.66 IR has also been found to induce both intracellular and mitochon­drial oxidative stress.67 However, IR-induced mi­tochondrial production of reactive oxygen species (ROS) has been proven to be the most influential in mediating cellular damage compared to ROS gen­erated in other compartments.68 Damage to mitochondria may trigger apoptosis, autophagy or, in the case of less severe lesions, fu­sion. This mechanism provides cross-complemen­tation between impaired mitochondria, supporting their functionality by alleviation of IR-induced defi­ciencies.69 A 0.005 to 5.0 Gy dose of X-ray radiation has been shown to prompt a 1.5- to 3.8- fold increase in mitochondrial mass, which supports a theory of increased mitochondrial fusion after IR exposure.70 Lan et al. have shown that ESCs subjected to IR display a significantly increased level of ROS gen­eration and metabolic activity.25 Both of these phe­nomena contribute to the induction of mitochon­drial fusion, which in turn is a stimulus for differ­entiation. Therefore, it may be speculated that the radio-enhancement of differentiation could also in­volve changes in the mitochondrial fission/fusion machinery. Summary A growing amount of evidence indicates that ra­diation-enhanced stem cell differentiation may be­come a potent tool for use in stem cell engineering FIguRe 1. The pathway of radiation-enhanced differentiation. 215 (Figure 1). Ionizing radiation triggers an excessive amount of side effects and does not enable the use of directed differentiation as a sole method in stem cell applications. However, a proper dosage may increase its efficacy while concomitantly reduc­ing its disadvantages. It is important to highlight that despite very efficient mechanisms of DDR in the majority of stem cells, IR bears the risk of in­troducing genomic instability. Therefore, it is of great importance to define the radiation dose that maximizes the stimulation of differentiation and minimizes the genotoxic effects. The response to ir­radiation varies between different stem cell types; thus, each type of stem cell requires an independ­ent evaluation of dosage. The deteriorating effects of irradiation could also be partially overcome by the formation of embryoid bodies, which display a significant increase in radioresistance compared to human embryonic stem cells (hESCs). It is also important to note that there are significant discrep­ancies between murine and human cell models in response to IR (Table 2.); thus, any assumptions based on murine models should be confirmed in human cells.71 Nonetheless, despite the presence of molecular evidence indicating the probable appli­cation to stem cell differentiation methodologies, the concept of radiation-enhanced stem cell differ­entiation remains to be scientifically proven. Acknowledgement This work was supported by grant no. 2/2014(61), project no. 30/06/2014/PRB/WCO/03. References 1. Jones DL, Wagers AJ. No place like home: anatomy and function of the stem cell niche. Nat Rev Mol Cell Biol 2008; 9: 11-21. 2. Tropepe V, Turksen K. The ontogeny of somatic stem cells. 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Armesilla-Diaz A, Bragado P, Del Valle I, Cuevas E, Lazaro I, Martin C, et al. p53 regulates the self-renewal and differentiation of neural precursors. Neuroscience 2009; 158: 1378-89. 55. Monje ML, Mizumatsu S, Fike JR, Palmer TD. Irradiation induces neural precursor-cell dysfunction. Nat Med 2002; 8: 955-62. 56. Wei L-C, Ding Y-X, Liu Y-H, Duan L, Bai Y, Shi M, et al. Low-dose radiation stimulates Wnt/ß-catenin signaling, neural stem cell proliferation and neu­rogenesis of the mouse hippocampus in vitro and in vivo. Curr Alzheimer Res 2012; 9: 278-89. 57. Visvader JE, Stingl J, Genes D. Mammary stem cells and the differentiation hierarchy: current status and perspectives. Genes Dev 2014; 28: 1143-58. 58. Shackleton M, Vaillant F, Simpson KJ, Stingl J, Smyth GK, Asselin-Labat ML, et al. Generation of a functional mammary gland from a single stem cell. Nature 2006; 439: 84-8. 59. Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ, et al. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 2003; 17: 1253-70. 60. Cicalese A, Bonizzi G, Pasi CE, Faretta M, Ronzoni S, Giulini B, et al. The tumor suppressor p53 regulates polarity of self-renewing divisions in mam­mary stem cells. Cell 2014; 138: 1083-95. 61. Ziyaie D, Hupp TR, Thompson AM. P53 and breast cancer. Breast 2000; 9: 239-46. 62. Insinga a, Cicalese a, Faretta M, Gallo B, Albano L, Ronzoni S, et al. DNA damage in stem cells activates p21, inhibits p53, and induces symmetric self-renewing divisions. Proc Natl Acad Sci U S A 2013; 110: 3931-6. 63. Kondo M, Wagers AJ, Manz MG, Prohaska SS, Scherer DC, Beilhack GF, et al. Biology of hematopoietic stem cells and progenitors: implications for clinical application. Annu Rev Immunol 2003; 21: 759-806. 64. Milyavsky M, Gan OI, Trottier M, Komosa M, Tabach O, Notta F, et al. A dis­tinctive DNA damage response in human hematopoietic stem cells reveals an apoptosis-independent role for p53 in self-renewal. Cell Stem Cell 2014; 7: 186-97. 65. Mitra K. Mitochondrial fission-fusion as an emerging key regulator of cell proliferation and differentiation. Bioessays 2013; 35: 955-64. 66. Yakes FM, Van Houten B. Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proc Natl Acad Sci 1997; 94: 514-9. 67. Kam WW-Y, Banati RB. Effects of ionizing radiation on mitochondria. Free Radic Biol Med 2013; 65: 607-19. 68. Azzam EI, Jay-Gerin JP, Pain D. Ionizing radiation-induced metabolic oxida­tive stress and prolonged cell injury. Cancer Lett 2012; 327: 48-60. 69. Youle RJ, van der Bliek AM. Mitochondrial fission, fusion, and stress. Science 2012; 337: 1062-5. 70. Nugent SME, Mothersill CE, Seymour C, McClean B, Lyng FM, Murphy JEJ. Increased mitochondrial mass in cells with functionally compromised mi­ tochondria after exposure to both direct . radiation and bystander factors. Radiat Res 2007; 168: 134-42. 71. Banuelos CA, Banáth JP, MacPhail SH, Zhao J, Eaves CA, O’Connor MD, et al. Mouse but not human embryonic stem cells are deficient in rejoining of ionizing radiation-induced DNA double-strand breaks. DNA Repair (Amst) 2008; 7: 1471-83. 72. Hanawalt P. Functional characterization of global genomic DNA repair and its implications for cancer. Mutat Res Mutat Res 2003; 544: 107-14. 73. Purschke M, Kasten-Pisula U, Brammer I, Dikomey E. Human and rodent cell lines showing no differences in the induction but differing in the repair kinetics of radiation-induced DNA base damage. Int J Radiat Biol 2004; 80: 29-38. 74. Parrinello S, Samper E, Krtolica A, Goldstein J, Melov S, Campisi J. Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nat Cell Biol 2003; 5: 741-7. 75. Hornsby PJ. Mouse and human cells versus oxygen. Sci Aging Knowl Environ 2003; 2003: PE21. 76. Wright WE, Shay JW. Telomere dynamics in cancer progression and preven­tion: fundamental differences in human and mouse telomere biology. Nat Med 2000; 6: 849-51. 77. Hornsby PJ. Replicative senescence of human and mouse cells in culture: significance for aging research. Mech Ageing Dev 2003; 124: 853-5. 217 review Gamma-enolase: a well-known tumour marker, with a less-known role in cancer Tjasa Vizin, Janko Kos Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2015; 49(3): 217-226. Received 9 May 2015 Accepted 13 July 2015 Correspondence to: Prof. Janko Kos, Ph.D., Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, SI-1000 Ljubljana, Slovenia. E-mail: Janko.kos@ffa.uni-lj.si Disclosure: No potential conflicts of interest were disclosed. Background. Gamma-enolase, known also as neuron-specific enolase (NSE), is an enzyme of the glycolytic path­way, which is expressed predominantly in neurons and cells of the neuroendocrine system. As a tumour marker it is used in diagnosis and prognosis of cancer; however, the mechanisms enrolling it in malignant progression remain elu­sive. As a cytoplasmic enzyme gamma-enolase is involved in increased aerobic glycolysis, the main source of energy in cancer cells, supporting cell proliferation. However, different cellular localisation at pathophysiological conditions, proposes other cellular engagements. Conclusions. The C-terminal part of the molecule, which is not related to glycolytic pathway, was shown to promote survival of neuronal cells by regulating neuronal growth factor receptor dependent signalling pathways, resulting also in extensive actin cytoskeleton remodelling. This additional function could be important also in cancer cells either to protect cells from stressful conditions and therapeutic agents or to promote tumour cell migration and invasion. Gamma-enolase might therefore have a multifunctional role in cancer progression: it supports increased tumour cell metabolic demands, protects tumour cells from stressful conditions and promotes their invasion and migration. Key words: gamma-enolase; cancer; glycolysis; cell survival; tumour marker Introduction Enolases (EC 4.2.1.11) are intracellular enzymes that catalyse the dehydration of 2-phospho-D­glycerate to phosphoenolpyruvate in the catabolic direction of the glycolytic pathway, a process con­verting glucose into pyruvate, which enables the formation of high-energy compounds of ATP and NADH. In the anabolic direction during gluconeo­genesis, they catalyse the reverse reaction of hy­dration of phosphoenolpyruvate to 2-phospho-D­glycerate. The glycolytic pathway and its enzymes are one of the most conserved and important meta­bolic networks in living organisms and therefore, enolases are among the most ubiquitously and abundantly expressed proteins.1-4 Despite being expressed in most cells, the gene that encodes for enolase is not a housekeeping gene since its expres­sion varies during several developmental, metabol­ic or pathophysiological conditions.5 In addition to their innate glycolytic function, many enzymes of the glycolytic pathway, including enolase, were shown to possess various specific regulatory func­tions and to play a pleiotropic role in physiological and pathological processes, including cancer.1,2,6,7 In this paper we review the properties, distribu­tion and function of gamma-enolase and its role in enhanced glycolysis and proliferation of tumour cells. Additionally, we expose new mechanisms through which gamma-enolase may promote cancer progression: aiding adaptation of tumour cells to stressful conditions by activating survival promoting signalling pathways and promoting migration of tumour cells. Finally, we discuss the role of gamma-enolase as a marker of exposure to carcinogenic pollutants and review the diagnostic and prognostic utility of gamma-enolase in cancer patients. 218 Properties and distribution of enolase Enolases are functionally active as dimers, com­ posed of non-covalently linked subunits alpha- (.), beta- (ß) and gamma- (.), facing each other in an antiparallel fashion, which may form five homodi­meric or heterodimeric isoenzymes, expressed in a development and tissue-specific manner. The iso­enzyme .. (alpha-enolase) is localized in all foe­tal and in the majority of adult mammal tissues. During tissue development, it is replaced by other isoforms: in skeletal and heart muscles by .ß and ßß (beta-enolase), and in neuronal cells and cells of the diffuse neuroendocrine system by isoenzymes .. and .. (gamma-enolase). In mammals, each of the three isoenzymes is encoded by an independent loci.8,9 All enolase isoforms have a molecular range between 82 and 100 kDa and share high sequence identity and kinetic properties.1,6,10-12 However, each isoform possesses characteristic short vari­able regions, which are situated predominantly on the surface of the molecule and might be the sites of contact with different cytoskeleton elements or other cell components.13 Besides the peptide molecule, enolase requires a divalent metal ion for its stabilisation and catalytic activity. Six divalent metals have been demonstrat­ed to activate enolase: Mg2+, Zn2+, Cd2+, Co2+, Mn2+ and Ni2+. The most abundant is Mg2+, which pro­vides the highest activation strength.1,14,15 The met­al ion is not firmly bound into the protein part of the molecule; therefore enolase is not a typical met­alloenzyme, but defined as a “metal-ion-activated enzyme complex”.16 Enolase has two binding sites for Mg2+, both contributing to catalysis: binding to the first site, Mg2+ induces conformational changes of the active site enabling the binding of the sub­strates, whereas the binding of a second Mg2+ is an essential part of the catalytic apparatus.1,17-19 Enolase localizes predominantly in the cytosol however, variations in cellular localisation were observed for all three enolase isoforms. Alpha­enolase was observed in the nucleus, on the cell surface and in extracellular space. It may interact with different cytoplasmic, nuclear and membrane molecules and exhibits several other functions besides catalysis.1,20 The nuclear form of alpha­enolase was recognized as Myc promoter-binding protein-1 (MBP-1), an alternative splicing form in­volved in regulation of transcription by repressing the function of Myc and acting as a tumour sup­pressor.6,21-23 Alpha-enolase localizes also on cell surface of neuronal, endothelial and hematopoi­etic cells as well on pancreatic, breast and lung cancer cells. Its surface expression was shown to depend on the pathophysiological conditions of the cells and its C-terminal lysine residue acts as a plasminogen-binding receptor modulating peri­cellular fibrinolytic activity and promoting migra­tion and metastasis of cancer cells. The cell surface alpha-enolase is catalytically active, maintaining its active dimeric form. Alpha-enolase was shown also to be secreted from cells by exosomes, cell de­rived vesicles, proposed to play an important role in intercellular communication.23-25 However, the mechanisms of surface translocation, membrane attachment, cell surface expression or secretion re­main unknown.6,26-32 The properties and function of alpha-enolase in malignant disease have been ex­tensively studied and reviewed.1,2,6,20,23 Different subcellular localisation and interac­tions with other proteins were observed also for beta-enolase during maturation, normal function and regeneration of muscles. Specific interactions with macromolecules may address beta-enolase to the subcellular site where ATP, produced through glycolysis, is most needed for muscular contraction or regeneration.33-35 Increased expression of beta­enolase was detected in rhabdomyosarcoma tissue, which is, to our knowledge, the only evidence that this isoform might be involved in cancer.36,37 Gamma-enolase Gamma-enolase, is a 433 amino acid long acidic dimeric protein, which includes two enolase isoen­ zymes, .. and .., and is also referred as neuron- specific enolase (NSE). The subunit molecular mass is approximately 39 kDa, whereas Mr of the native form is 78 kDa which might vary on the subunit combination. Gamma-enolase localizes predomi­nantly in neuronal cells and in neuroendocrine cells, particularly in those of the amine precursor uptake and decarboxylation (APUD) lineage, for example in the intestine, lung, thyroid and pituary gland and pancreas.8,38 It is found in lower amounts also in non-neuronal and non-neuroendocrine tis­sues or cells, such as erythrocytes, platelets, breast tissue, prostate and uterus.39-41 The .. isoform is found predominantly in mature neurons and is also used as marker of neuronal maturation and differentiation, while the .. isoenzyme localizes in higher amounts in non-neuronal cells.8,9 The C-terminal end of gamma-enolase contains a PDZ-binding motif (431S-433L: SVL) (Figure 1), which might enable an interaction with several 219 proteins that contain a PDZ-domain and are in­volved in intracellular redistribution of molecules and signalling pathway events. Different gamma­enolase cellular localisation, which depends on the pathophysiological conditions of the cells, propose other cellular engagement besides glycolysis. In neuronal, glial and astrocytic cells, gamma-enolase was shown to associate with the plasma membrane, or even appear on the surface of cells42-45, which might occur through its hydrophobic domain in the N-terminal region (32A-43Y: AAVPSGASTGIY). Also, on the cell surface alpha-enolase may bind to plasminogen by C-terminal lysine.46 In contrast to alpha-enolase, gamma-enolase has no C-terminal lysine and does not bind plasminogen; therefore it might exert other functions on cell surface.43,46-48 Gamma-enolase was detected also in the nucleus of malignantly transformed urothelial and epithelial breast cells and in glioblastoma cells; however its role remains unknown.40,49-51 Significantly higher increase of gamma-enolase antigen levels than its catalytical activity was observed during exponen­tial growth of small-cell lung cancer cells, propos­ing that cellular gamma-enolase exists also as an enzymatically inactive compound, that might pos­sess other functions.52 The function of gamma-enolase in increased glycolysis in cancer It is generally known that glycolysis is drasti­cally enhanced in tumour cells and is a hallmark of cancer progression.53,54 In tumours that out­grow its feeding circulation, cells are exposed to an environment with poor oxygen and nutrients supply50, which leads to a prevalence of aerobic glycolysis over mitochondrial oxidative phospho­rylation.55-57 This metabolic switch referred also to as the Warburg effect, enables tumour cells to produce energy to survive and eventually prolifer­ate regardless the presence of oxygen. Glycolysis alone, however, is energetically less efficient than oxidative phosphorylation. Therefore, reactions of the glycolytic pathway have to be drastically ac­celerated to satisfy the higher metabolic needs of proliferating tumour cells, which is evident from a net increase in glucose consumption and higher expression of glycolytic enzymes.55,58-60 Gamma-enolase is overly-expressed in tumours39 and its major contribution to tumour progression is, no doubt, the participation to accelerated glycolysis of cancer cells. For instance, malignant transforma­tion of astrocytic61, breast40 and urothelial cells49 FIGURE 1. Position of gamma-enolase catalytical active site and the PDZ-binding motif containing C-terminal end. Subunits of the ..-dimer are represented by separate colours (wheat and violet). The orange part represents the catalytical active site, yellow balls represent Mg2+ ions and the magenta part represents the C-terminal end of the molecule (the last 6 amino acids). For better representation, active site and C-terminal end are shown only in one subunit. The image was created using PyMOL (DeLano LLC Scientific). Gamma-enolase crystal structure (1TE6) was obtained from Protein Data Bank (PDB). The image was prepared by authors and has not been published elsewhere. led to occurrence of gamma-enolase in originally gamma-enolase-negative cells and to colony forma­tion and proliferation, which strongly suggests that transformed cells might obtain the ability to express gamma-enolase in order to adapt to increased met­abolic needs of a neoplastic state.61,62 Further, ma­lignantly transformed urothelial cells, which were able to proliferate and form tumours when inocu­lated into immune compromised mice, were shown to express higher levels of gamma-enolase, com­pared to less active and differentiated cells. Authors proposed that cells, which express gamma-enolase at higher rates, might have an advantage in tumour initiation and subsequent growth.49 Gamma-enolase was significantly up-regulated also in glioblastoma cells exposed to hypoxia and serum starvation, and additionally, its knock-down significantly dimin­ished cell growth50, supporting the findings that the dependence of tumour cell growth on glycolysis is even more emphasized in stressful conditions.55,60,63 Finally, in non-small cell lung cancer cells, an alter­native splicing form of c-H-ras, p19ras, was shown to specifically bind gamma-enolase and inhibit its en­zymatic activity, resulting in diminished cell prolif­eration.58 The glycolytic function of gamma-enolase and its impact on promoting tumour cell growth represents a promising target for cancer therapy.64 220 The pro-survival function of gamma-enolase in cancer Gamma-enolase was shown to act as a neurotropic factor in neuronal cells.7,65,66 This function is mani­fested through an additional active site, which is not a part of the catalytical apparatus involved in glycolysis, but localized at the C-terminal end of the molecule. For instance, a 30 amino acid long pep-tide, mimicking the C-terminal part of gamma-eno­lase, was shown to promote survival, differentiation and regeneration of neurons by activating signal transduction pathways which are normally trig­gered by the activation of Trk receptor: phosphati­dylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. Additionally, the C-terminal peptide of gamma-enolase was dem­onstrated to impair apoptosis and to interact with p75 neurotrophin receptor (p75NTR) and suppress the activation of its downstream effectors in apop­totic signalling. Despite having similar amino acid sequence in the C-terminal part, other enolase iso­forms do not show a neurotropic function.7,43,46,67-69 Gamma-enolase neurotrophic effect is regulated by cathepsin X, a cysteine carboxymonopeptidase, which is frequently expressed in neuronal and glial cells.70,71 Cathepsin X was shown to sequentially cleave the final two amino acids (433L and 432V) at the C-terminal end of gamma-enolase and to dis­rupt the PDZ motif, through which gamma-enolase binds to the scaffold protein gamma-1-syntrophin. The latter mediates the translocation of gamma­enolase and its association with plasma membrane, which is a prerequisite for neurotrophic activity.43,46 Therefore, only C-terminally uncleaved gamma­enolase has a pro-survival activity. The protec­tive function of gamma-enolase was observed also in brains of a mouse model of Alzheimer disease (Tg2576): C-terminally truncated gamma-enolase localized in immediate plaque vicinity and strong­ly colocalized with cathepsin X, while uncleaved gamma-enolase exhibiting neuroprotective activity, localized in microglia cells in close proximity of se­nile plaques. Additionally, using a mouse microglial cell model, gamma-enolase was shown to protect neuronal cells from amyloid-ß peptide toxicity and cathepsin X reversed its function.66 Gamma-enolase has been proposed to act as a pro-survival factor also in cancer cells. It was shown to support glioblastoma cell adaptation to cellular stress, such as serum starvation, hypoxia, chemo­therapy and radiotherapy; however, no specific mechanism has yet been proposed.50 Both, starva­tion and hypoxia have been linked to progression of FIGURE 2. Co-localization of gamma-enolase and cathepsin X in human glioblastoma cells U87-MG grown in serum-free medium for 72 h. U87-MG cells were grown in Eagle´s Minimum Essential Medium (EMEM, Sigma), supplemented with 10% (v/v) foetal bovine serum (HyClone), 1% L-glutamine (Sigma) and 1% penicillin/streptomycin (Sigma) at 37oC and humidified atmosphere with 5% CO2. For protein visualization, cells were seeded on glass coverslips at a concentration of 1 x 104 cells/ml in 24 well plates. After 24 h, complete growth medium was replaced with serum-free medium and cells were left to grow for additional 72 h. After treatment cells were fixed with 10% formalin for 30 min at room temperature and then permeabilized by 0.5% Tween®20 in phosphate buffered saline (PBS), pH 7.4 for 10 min. Non-specific binding was blocked with 3% bovine serum albumin (BSA) in PBS, pH 7.4 for 1.5 h at room temperature. Cells were then incubated with primary antibody against N-terminal end of gamma-enolase (10 µg/ml, goat polyclonal, Santa Cruz Biotechnology) and active cathepsin X (10 µg/ml, mouse monoclonal, 2F12) in 3% BSA in PBS pH 7.4 for 2 h at room temperature. After three washes with PBS, pH 7.4, cells were incubated with Alexa Fluor 555 donkey anti-goat (Molecular Probes™) and Alexa Fluor 488 donkey anti-mouse (Molecular Probes™) secondary antibody in 3% BSA in PBS, pH 7.4. After washing with PBS, ProLong® Gold Antifade Mountant with 4’,6-diamidino-2-phenylindole, dilactate (DAPI, Molecular Probes™) was used to mount coverslips on glass slides. Fluorescence microscopy was performed by Carl Zeiss LSM 710 confocal microscope (Carl Zeiss Oberkochen) with ZEN 2012 image software. Gamma-enolase (red) and cathepsin X (green) staining showed co-localisation in the perimembrane region. The blue staining with DAPI represents the nucleus. The image was prepared by authors and has not been published elsewhere. cancer and resistance to treatment by inducing bio­logical changes in tumour cells, one of them being increased glycolysis.55,60,72 However, C-terminally uncleaved gamma-enolase might additionally sup­port tumour cell adaptation to stressful conditions 221 by activating survival promoting signalling path­ways as it does in neuronal cells, and cathepsin X, which is present also in tumour cells71, might reg­ulate its function (Figure 2). For instance, in glio­blastoma cell lines, exposed to serum starvation or hypoxia, gamma-enolase expression was signifi­cantly increased50; moreover, significant increases in protein and phosphoprotein levels were observed also in PI3K/Akt and MAPK/ERK and anti-apop­totic signalling pathways73,74, which are triggered by gamma-enolase in neuronal cells. Separate analysis of expression and role of C-terminally uncleaved and truncated gamma-enolase in cancer cells and tumour tissue might provide new information on its involvement in tumour progression. The role of gamma-enolase inmigration of tumour cells Recently, a study on glioma cells showed that gamma-enolase knockdown significantly reduced migration of cells; however, no specific mechanism has been proposed.75 An important prerequisite for cell migration is a dynamic remodelling of actin cytoskeleton. Remodelling is stimulated by several molecules that link migratory signals to the actin filaments and are upregulated in invasive and metastatic cancer cells.76 In neuroblastoma cells, gamma-enolase was shown to co-localize with ac­tin filaments, an interaction that depends on the presence of gamma-1-syntrophin.43 Additionally, gamma-enolase C-terminal peptide was shown to regulate RhoA kinase, a regulator of actin cytoskel­eton organization. Consequently, gamma-enolase induced actin polymerisation and its redistribution to growth cones of neurites.68 Similarly, alpha-eno­lase was shown to bind to actin and tubulin77 and to mediate invasiveness of tumour cells78 and sen­sitivity to microtubule targeted drugs.79 These re­sults provide evidence, that gamma-enolase might be involved in migration of tumour cells through interactions with actin filaments and regulation of RhoA kinase function. Gamma-enolase as a marker of exposure to environmental carcinogenic pollutants arsenic and cadmium Arsenic and cadmium exposure is linked to breast and bladder cancer occurrence. Exposure of breast epithelial and urothelial cells to As3+ or Cd2+ was shown to induce malignant transformation of cells and an increase of mRNA and protein levels of gamma-enolase in the cytoplasm and nucleus of cells, while expression of alpha-enolase did not change. Authors proposed that gamma-enolase might be translated as a possible biomarker for chronic environmental exposure to As3+ or Cd2+. Its expression in non-malignant cells was influenced also by methylation and histone modifications, in­duced by a histone deacetylase inhibitor (MS-275) and a methylation inhibitor (5-AZC), which pro­posed that gamma-enolase gene expression is con­trolled by methylation and histone modifications. The later provides evidence that environmental carcinogenic pollutants, such as cadmium and ar­senic, might cause changes in epigenetic regulation of genes, which specifically affect the expression and function of gamma-enolase in breast epithelial cells and urothelial cells.40,49 Gamma-enolase in tumour tissues Gamma-enolase is typically overexpressed in tu­mours of neurogenic and neuroendocrine origin and has been used as a marker for detection of neuroendocrine differentiation of tumour cells. It is considered the most important tumour marker for poorly differentiated neuroendocrine tumours, since a tumour is classified as a neuroendocrine tumour only when it expresses at least two neu­roendocrine markers of which one is gamma­enolase.80,81 Immunohistochemistry of gamma­enolase is regularly used for differential diagnosis of small-cell lung cancer (SCLC) from other lung cancer histological subtypes (Table 1).82,83 Gamma­enolase increased expression was observed also in other tumours, including breast cancer, with increased staining in lymph node metastases com­pared to primary breast tumours84 or in glioblasto­mas, with higher levels in advanced stage tumours, which were related to shorter patient survival.50 Nevertheless, immunostaining of gamma-enolase in tumour tissue has limited diagnostic or prog­nostic utility, since many clinical studies provided contradictory results.80,85-87 Gamma-enolase in extracellular fluids of cancer patients In general, gamma-enolase serum levels are bet­ter indicators than its tissue expression (Table 1).80 222 TABLE 1. Use of gamma-enolase as a tumour marker Tumour tissues Serum SCLC Other neuroendocrine tumours (neuroblastoma, endocrine pancreatic tumours, seminoma, medullary thyroid carcinoma, phaeochromocytoma, ect.) SCLC NSCLC Testicular cancer (seminoma) Carcinoids Medullary thyroid carcinomas Phaeochromocytoma Endocrine pancreatic tumours Paraganglioma Neuroblastoma Differential diagnosis from other lung cancer subtypes Diagnosis or detection of neuroendocrine differentiation of tumour Differential diagnosis from other lung cancer subtypes when biopsy is not possible Prognosis Post-operative surveillance Monitoring efficacy of therapy Detection of recurrent disease after primary surgery Monitoring therapy in advanced disease Prognosis Diagnosis Diagnosis Monitoring efficacy of therapy Detection of early relapse Monitoring efficacy of therapy Detection of early relapse Monitoring efficacy of therapy Detection of early relapse Diagnosis Monitoring efficacy of therapy Detection of early relapse Diagnosis Differential diagnosis Prognosis Monitoring efficacy of therapy Detection of recurrent disease Yes Yes Yes Unknown Yes Yes Yes No Unknown Experimental Unknown Yes Yes Yes Yes Yes Yes Yes Yes Unknown Unknown Unknown Yes Yes Yes [82, 83] EGTM, NACB [80, 81, 95, 96] [82, 83] EGTM, NACB [82, 83, 97] [82, 83] EGTM, NACB [82, 83] EGTM, NACB [82, 83] NACB [83] [83] [98] EGTM [96, 99] [8, 39] EGTM [8, 39, 96] [8, 39] EGTM [8, 39] [8, 39] EGTM [8, 39] [95, 96] [8, 39] EGTM [8, 39, 99] [99] [8] [100] ACS [8, 100] EGTM [97] ACS = American Cancer Society; EGTM = European Group for Tumour Markers; NACB = National Academy of Clinical Biochemistry; NSCLC = non-small-cell lung cancer; SCLC = small-cell lung cancer Levels of gamma-enolase are elevated in sera from injury or cardiac arrest, gamma-enolase is released patients with various cancers, however, its appear-into the cerebrospinal fluid and eventually into the ance in extracellular fluids without any apparent bloodstream due to damage or death of neuronal cellular damage is not clear.1,88 After stroke, brain cells or impairment of the blood-brain barrier in­ 223 tegrity. For instance, levels of gamma-enolase in cerebrospinal fluid and serum have been used as a biomarker of cerebral injury and for the assessment of neurological disorders.38,89,90 Gamma-enolase is the only neuroendocrine tumour marker, which is used as a serum marker for follow up and moni­toring of therapy effectiveness. Increased gamma­enolase levels in extracellular fluids are related to cancer progression and are typical for cancer in advances stages with distant metastases.8,39,80,84-87 The levels of gamma-enolase in non-treated cancer increase proportionally to the tumour mass, stage and number of metastases and are related to worse prognosis, however, the levels are not related to the location of metastases.39 Gamma-enolase is used in clinical practice in patients with SCLC and neuroblastoma. Its lev­els are significantly elevated compared to healthy subjects; however, specificity and sensitivity are too low to be used in screening.39,91 According to the recommendations of expert groups for the use of markers in lung cancer, gamma-enolase is rec­ommended as an auxiliary marker in SCLC for differential diagnosis when biopsy is not possible and when other neuroendocrine tumours are ex­cluded. Further, it is recommended for SCLC post­operative surveillance, for monitoring of therapy in advanced disease and for detection of recurrent disease.83,91 During chemotherapy, a transient rise of gamma-enolase serum levels occurs due to cy­tolysis of tumour cells, which disappears in case of successful treatment. However, persistently el­evated levels show unsuccessful therapy. Gamma­enolase is not a recommended tumour marker in neuroblastoma; however, it is frequently used for differential diagnosis of neuroblastoma from ne­froblastoma and for disease monitoring.8,91 Gamma-enolase is used as an auxiliary serum marker for follow-up and monitoring of therapy ef­fectiveness in patients with carcinoids, melanoma, seminoma, feocromocitoma, medullary thyroid carcinoma, and endocrine pancreatic tumours. In patients with brain tumours, the levels of gamma­enolase in sera are not elevated, however, increased levels were reported in cerebrospinal fluid.39,91 Increased serum levels of gamma-enolase were reported also in patients with cancers of non­neuroendocrine origin, such as T-cell leukaemia92, B-cell lymphoma93 and malignant melanoma.94 In general, higher serum levels of gamma-enolase are related to worse prognosis and are the highest in patients with advanced metastatic stage.39 Gamma-enolase is usually measured in serum samples and less frequently in cerebrospinal fluid, pleural exudate or ascites. Its half-life in serum is estimated to be approximately 30 h.101 The .. iso­form is expressed in large amounts also in eryth­rocytes and in platelets, therefore it is important to separate blood cells from plasma or serum within 60 minutes from sample collection to prevent hae­molysis of blood samples, which could lead to falsely elevated levels of gamma-enolase.80,102,103 Falsely elevated serum levels of gamma-enolase might be also due to various noncancerous patho­logical causes104, such as benign pulmonary dis­eases105, renal failure106, brain injuries, seizures, stroke38,107, severe hypoglycaemia108, benign liver diseases109 or systemic sclerosis.110 Concluding remarks Glycolytic enzymes were shown to exert various specific regulatory functions and to play a pleio­tropic role in physiological and pathological pro­cesses. Therefore, their participation to accelerated glycolysis could not be the only contribution to tumour progression.2 Alpha-enolase, the most ex­haustively studied enolase isoform, was found to be one of the most frequently altered proteins in human pathologies and suggested as a universal cellular sensor that responds to multiple stimu­li and reacts through multiple mechanisms.6,111 Gamma-enolase, sharing high-sequence identity with alpha-enolase, is also emerging as a multi­functional molecule. Different cellular localisation and interactions with other molecules strongly suggest its multiple cellular engagements. Gamma-enolase primary role in cancer is the participation to the accelerated glycolysis, which supports increased tumour cell metabolic demands and enables their proliferation. Its C-terminal end might protect tumour cells from stressful condi­tions and action of therapeutic agents by activating survival-promoting signalling pathways and reg­ulating apoptosis. An additional role of gamma­enolase in cancer progression is its involvement in actin remodelling and consequently in promotion of migration and invasion of tumour cells. These findings suggest that the role of this well-known tumour marker, whose expression is altered dur­ing development and progression of a variety of cancers, is pleiotropic and still has to be defined. Future work should be focused on elucidation of gamma-enolase cellular redistribution, interac­tions with other molecules and involvement in cell signalling. Understanding these processes, togeth­er with the tools enabling effective inhibition of 224 gamma-enolase glycolytic activity, might provide new opportunities for cancer treatment. Acknowledgements We thank Dr. Bojan Doljak for constructing Figure 1. This project was supported by Research Agency of the Republic of Slovenia (grants P4-0127 and J4-4123 to JK). References 1. Pancholi V. Multifunctional alpha-enolase: its role in diseases. 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Phone: +386 40 202812; E-mail: alen.alterego@gmail.com Disclosure: No potential conflicts of interest were disclosed. Background. The aim of the study was to explore the influence of various time-of-flight (TOF) and non-TOF recon­struction algorithms on positron emission tomography/computer tomography (PET/CT) image quality. Materials and methods. Measurements were performed with a triple line source phantom, consisting of capil­laries with internal diameter of ~ 1 mm and standard Jaszczak phantom. Each of the data sets was reconstructed using analytical filtered back projection (FBP) algorithm, iterative ordered subsets expectation maximization (OSEM) algorithm (4 iterations, 24 subsets) and iterative True-X algorithm incorporating a specific point spread function (PSF) correction (4 iterations, 21 subsets). Baseline OSEM (2 iterations, 8 subsets) was included for comparison. Procedures were undertaken following the National Electrical Manufacturers Association (NEMA) NU-2-2001 protocol. Results. Measurement of spatial resolution in full width at half maximum (FWHM) was 5.2 mm, 4.5 mm and 2.9 mm for FBP, OSEM and True-X; and 5.1 mm, 4.5 mm and 2.9 mm for FBP+TOF, OSEM+TOF and True-X+TOF respectively. Assessment of reconstructed Jaszczak images at different concentration ratios showed that incorporation of TOF information improves cold contrast, while hot contrast only slightly, however the most prominent improvement could be seen in background variability - noise reduction. Conclusions. On the basis of the results of investigation we concluded, that incorporation of TOF information in re­construction algorithm mostly affects reduction of the background variability (levels of noise in the image), while the improvement of spatial resolution due to incorporation of TOF information is negligible. Comparison of traditional and modern reconstruction algorithms showed that analytical FBP yields comparable results in some parameter measure­ments, such as cold contrast and relative count error. Iterative methods show highest levels of hot contrast, when TOF and PSF corrections were applied simultaneously. Key words: time of flight; PET/CT; point spread function; reconstruction algorithm; image quality Introduction The first advantages of time-of-flight (TOF) tech­nique for positron emission tomography (PET) were presented in the early 1980s. The idea of using the TOF information in PET was implemented in the first generation of the TOF PET scanners using crystal materials with relatively low time resolu­tion.1,2 TOF PET is characterized by a better trade-off between contrast and noise in the image.3-7 This property is used in more challenging clinical con­ditions, allowing shorter examinations at lower count rates, successful scanning of larger patients, clearer characterization of low uptake areas and visualization of smaller lesions.8-12 Accompanied with the specific point spread function (PSF) cor­rection it produces images with high image qual­ity.13,14 Current endeavours in research are mainly 228 oriented towards improving the time resolution. Recent study of TOF PET using Cherenkov light reached coincidence resolution of 71 ps full width at half maximum (FWHM).15 Karp et al. investigated the benefits of TOF cor­rection in experimental phantoms and concluded that TOF correction leads to a better contrast-to­noise trade-off than non-TOF. They pointed out that complete impact of TOF should not be investi­gated in terms of a simple sensitivity gain improve­ment.10 Akamatsu et al. investigated the effect of PSF and TOF corrections on PET/CT image qual­ity with different reconstruction parameters and count rates. They determined that PSF and TOF corrections slightly improve contrast and back­ground variability.16 Review of the literature indicates that image quality improvement is expected with incorporat­ing TOF correction in reconstruction algorithm.16,17 The aim of present research was to evaluate image quality parameters using different reconstruction algorithms, altering phantoms, activity concentra­tion ratios and regions of interest with special fo­cus on TOF information impact. Materials and methods All measurements were performed at the Department of Nuclear Medicine, University Medical Centre Ljubljana on Biograph mCT PET/ CT scanner, manufactured by Siemens. Scanner combines a 128-slice CT and patented lutetium oxyorthosilicate (LSO) PET system for whole body imaging with included TOF technique. The gantry aperture is 78 cm wide and the tunnel length is 136 cm. This model of PET/CT scanner has incorporat­ed PET Syngo VG30 software. The study was per­formed on a triple line source phantom and on the Jaszczak phantom. To insure adequate comparison with presented values in literature, the measure­ments in both phases were performed according to National Electrical Manufacturers Association (NEMA) NU-2-2001 standard.18 Measurement of spatial resolution Spatial resolution was evaluated following the NEMA NU-2-2001 standard using a triple line source of 18F (activity concentration 7 MBq/ml). Triple line insert phantom (Triple Line Insert, Data Spectrum Co.) was used to obtain three 1 mm diam­eter parallel lines of tracer material spaced 7.5 cm apart (Figure 1). The total activity was low enough to keep dead time losses and the ratio of randoms to total events below 5%, as suggested by the pro­tocol.18-20 The acquisition of data was performed with 4.1 ns coincidence window and 12% energy window. The measurements were performed with phantom centre positioned at three locations within PET ring; (1) x = 0 and y = 1 cm (to avoid the exact centre of the scanner where the sampling density of lines of response may be very high), (2) x = 0 and y = 10 cm, and (3) x = 10 and y = 0 cm. The acquired data was reconstructed using analyti­cal filtered back projection (FBP), iterative ordered subsets expectation maximization (OSEM) (4 itera­tions, 24 subsets) and iterative True-X (4 iterations, 21 subsets) which incorporates PSF correction. All images were reconstructed into a matrix of 400×400 with a 1 mm pixel size. All reconstructions includ­ed a Gaussian post-filter of 4 mm FWHM. Values of intrinsic spatial resolution – FWHMint were cal­culated according to equation in Skreting et al.21, in which FWHMeff is the FWHM of profile measured on the reconstructed image and FWHMfilter is the width of the Gaussian reconstruction filter. Measurements of image quality parameters Due to the complex interplay of different aspects of imaging system, it is desirable to be able to com­pare the image quality of different systems using a standardized imaging situation that simulates a clinical imaging condition. In order to evaluate the quality of the image simulating a clinical whole body acquisition, Jaszczak phantom PET/FL-X2/P (Data Spectrum Co.) was used (Figure 1). The phantom consists of the lid, the body of phantom 229 and the cold spheres insert. Lid has seven little cyl­inders, six of which are hollow with external diam­eters of 8 mm, 12 mm, 16 mm and 3 cylinders with diameters of 25 mm. The seventh cylinder is solid and simulates bone on reconstructed image (tef­lon). The body of the phantom holds the volume of ~ 6 L. The cold insert holds spheres with diam­eters of 9.5 mm, 12.7 mm, 15.9 mm, 19.1 mm, 25.4 mm and 31.8 mm.18 The four smallest cylinders (8 mm, 12 mm, 16 mm and 25 mm) and the body of the phantom were filled with a radioactive solution with three different cylinder-to-background activ­ity concentrations of 2:1 (48 kBq/ml:24 kBq/ml), 4:1 (88 kBq/ml: 22 kBq/ml) and 8:1 (144 kBq/ml:18 kBq/ ml) in 3 sequential acquisitions. The coincidence and energy window settings remained the same as in spatial resolution measurements. The two larger cylinders were filled with water and air, respec­tively. The phantom was placed so that the spheres were in the same transversal plane, coinciding with the central plane of the scanner. Corrections (in­tensity normalization, scatter and random events, dead time losses and attenuation with the CT) were applied in the reconstruction into a matrix of 512 × 512 with 1.6 mm pixel size and Gaussian post-filter of 4 mm of FWHM. We used different image reconstruction algorithms - analytical filtered back projection (FBP), iterative OSEM (4.24) and itera­tive True-X with PSF correction (4.21). TOF infor­mation was alternately incorporated in each recon­struction algorithm. Baseline iterative OSEM (2.8) reconstruction method was added for comparison. Evaluation of image quality was performed by cal­culation and observation of the following image parameters: percentage of contrast of hot cylin­ders and cold spheres, percentages of background variability (in the vicinity of hot cylinders and cold spheres) and percentage of relative count error. Percentage of the contrast of the hot cylinders and cold spheres was determined from the average counts in the cylinders and spheres, as well as in the background which were measured in regions of interest (ROI) with the same size as the cylinders or spheres. Contrast QH,j for cylinder j was calcu­lated by: where CH,j is the average counts in ROI for the cyl­inder j, CB,j is the average of background ROI, aH is the activity concentration in cylinders and aB is the activity concentration in the background (both aH and aB were measured in dose calibrator before PET acquisition). Contrast of spheres QC,j for each cold sphere j was calculated by: where CC,j is the average counts in the ROI for sphere j and CB,j is the average of the background ROI counts for sphere j. Percentage of background variability was calcu­lated as the ratio between the standard deviation and the mean value in 12 randomly placed con­centric ROI in the background that were at least 15 mm away from any cylinder, sphere or the edge of the phantom. The sizes of ROI corresponded to the diameters of the spheres. The percent background for sphere j is calculated as: where SDj is the standard deviation of the back­ground ROI counts for sphere j and CB,j is the aver­age of the background ROI counts for sphere j. The relative count error that evaluates the accu­racy of the scatter and attenuation corrections was determined as the average of the relative count er­rors in 2 planes. This was obtained as the ratio be­tween the mean value of counts in a circular region (of 22 mm or 25 mm in diameter, positioned in the air filled cylinder) and the mean background value (evaluated in 12 regions of the same size). We ex­pected the contribution of scatter and attenuation error that was evaluated for air to be most promi­nent in the voxels closest to the background which also includes 1.5 mm plastic cylinder wall. Besides estimating the value for purely air medium, we found that it was important to take into account the cylinder wall for comparison. Therefore 2 diame­ters of ROI were used, including and excluding the cylinder wall (22 mm and 25 mm). The residual er­ror in scatter and attenuation corrections .Cair,i for each slice i was calculated as: where Cair,i is the average counts in the air filled cyl­inder ROI and CB,i is the average count of the back­ground ROI for slice i. Results Results are presented in the same order as they were presented theoretically in the previous chap­ter. Spatial resolution results, measured on triple line insert, are followed by contrast, background variability and relative count error results, meas­ured on Jaszczak phantom. 230 TABLE 1. Measured values of intrinsic spatial resolution in FWHM for various line source positions and reconstruction methods 1 cm offset (x=0, y=1 cm) Transverse 5.2 mm 5.1 mm 4.5 mm 4.5 mm 2.9 mm 2.9 mm 10 cm offset (x = 10 cm, y = 10 cm) Transverse radial 5.9 mm 5.9 mm 4.8 mm 4.8 mm 2.7 mm 2.8 mm Transverse tangential 5.9 mm 5.8 mm 5.3 mm 5.8 mm 3.7 mm 3.9 mm FBP = filtered back projection; FBP+TOF = filtered back projection with incorporated time of flight information; OSEM = ordered subsets expectation maximization; OSEM+TOF = ordered subsets expectation maximization with incorporated time of flight information; True-X = iterative reconstruction method which incorporates point spread function (PSF) correction; True-X + TOF = iterative reconstruction method which incorporates point spread function (PSF) correction with incorporated time of flight information age reconstruction. The measurement of spatial resolution characterizes the shape of the recon­structed point spread function at the FWHM level. Such measurement allows a reliable evaluation of scanners, taking into account the variation in spa­tial resolution with radial distance. The data are taken at low counting rates, so that potential event pileup is not encountered. Table 1 summarizes the results of the spatial resolution measured in air on PET/CT scanner. Hot and cold contrast The measured parameters of image quality de­pend on reconstruction algorithm used. Figures 2 and 3 show the response of observed reconstruc­tion methods in relation to cylinder or sphere di­ameter and activity concentration ratio between cylinders or spheres and background. Presented activity concentration ratios were chosen for best representation of the results. The iterative algo­rithm True-X with TOF correction displayed the best results of hot contrast. Slightly lower levels of contrast were shown (with smallest spheres) with iterative OSEM (4.24), followed closely by analyti­cal FBP. Algorithms with incorporated TOF cor­rection displayed slightly better results as their non TOF counterparts. Iterative algorithm True-X with TOF correction displayed the best result of cold contrast, followed closely by analytical FBP. Baseline iterative OSEM (2.8) showed the low­est hot and cold contrast. TOF information had higher impact with cold contrast performance in comparison with hot contrast performance. For all sizes of cylinders and spheres, the hot contrast increased with iterative reconstruction methods, however in cold contrast traditional FBP showed slightly better results, especially for larger spheres. Background variability Figures 4 and 5 show variability of background for all reconstruction methods and all (cylinders or spheres) diameters. The TOF correction sig­nificantly reduced background variability – up to 50% for all reconstruction algorithms especially with the smallest diameter spheres. The measure­ment of background variability in the vicinity of cold spheres is not foreseen in the NEMA proto­col; however our research shows that the values of background variability in the vicinity of hot cylin­ders and cold spheres differ by a factor of three. The impact was more prominent for cylinders and spheres of smaller diameters. Baseline OSEM (2.8) produced images with lowest values of back­ground variability, or in other words, highest uni­formity and lowest noise levels. Relative count error Relative count errors for various reconstruction methods and activity concentration ratios are pre­sented in Table 2. We found some difficulties with positioning ROI in areas with low concentration ra­tio, since there is a cylinder wall around observed air medium in the background with higher specific activity, which has to be taken into account. This is­sue was not addressed in standard protocol but as we found it important, we chose to compare meas­urements with and without 1.5 mm thick cylinder wall accounted in ROI measurements (22 mm and 25 mm). The measurements were made in cylinder filled with air as opposed to the measurements made in lung insert with fixed density 0.3 g/cm3, cited by NEMA protocol and other authors.5,17,18,22 Discussion The spatial resolution measurements show that PSF modelling successfully counteracts the paral­lax error and is responsible for spatial resolution improvement throughout field of view. The results are in line with results of other authors and con­firmed the accuracy of used methods. Slight mis­alignments of a line source with the scanner axis leads to degraded resolution compared with that measured with a point source. The spatial resolu­tion measured with a point source, therefore, can be expected to be slightly better than that determined with a line source (approximately few tenths of a millimetre).23 The objective of the image quality 231 TABLE 2. Relative count error for various reconstruction methods, performed with regions of interest (ROI) with diameter equal to external diameter of air insert and diameter equal to internal diameter of air insert (in brackets) FBP 11.1 (9.8) 16.2 (14.5) 9.7 (8.4) FBP+TOF 11.1 (12.1) 12.6 (10.2) 9.8 (8.8) OSEM 15.0 (15.3) 25.0 (23.6) 21.0 (19.8) OSEM+TOF 15.3 (15.4) 17.7 (15.3) 14.7 (13.8) True-X 20.4 (20.5) 25.0 (23.5) 20.3 (18.9) True-X+TOF 14.1 (14.1) 17.2 (14.7) 14.9 (12.3) OSEM (2,8) 48.0 (46.9) 45.9 (47.5) 49.1 (47.6) FBP = filtered back projection; FBP+TOF = filtered back projection with incorporated time of flight information; OSEM = ordered subsets expectation maximization; OSEM+TOF = ordered subsets expectation maximization with incorporated time of flight information; True-X = iterative reconstruction method which incorporates point spread function (PSF) correction; True-X+TOF = iterative reconstruction method which incorporates point spread function (PSF) correction with incorporated time of flight information 232 FBP FBP+TOF OSEM OSEM+TOF True-X True-X+TOF 2:1 4:1 8:1 FIGURE 6. Visual assessment of image quality according to the reconstruction method and activity concentration ratio. FBP = filtered back projection; FBP+TOF = filtered back projection with incorporated time of flight information; OSEM = ordered subsets expectation maximization; OSEM+TOF = ordered subsets expectation maximization with incorporated time of flight information; True-X = iterative reconstruction method which incorporates point spread function (PSF) correction; True-X+TOF = iterative reconstruction method which incorporates point spread function (PSF) correction with incorporated time of flight information test was to produce images simulating whole body scans with hot and cold lesions. The measurements were extended to include the contrast ratios 2:1, 4:1 and 8:1 between the hot cylinders and background, in addition to evaluation of different modern and especially traditional reconstruction algorithms, not contemplated by NEMA protocol. Results of hot and cold contrast show that incorporation of TOF information only marginally improves con­trast recovery. Best results were achieved with it­erative reconstruction algorithm incorporating PSF modelling-True-X with TOF information. Baseline OSEM (2.8) produced images with lowest con­trast ratio in comparison with other reconstruction methods. The most important improvement of con­trast was obtained with the incorporation of PSF in the reconstruction, while TOF having lower impact. Results of background variability showed that TOF information has the most profound impact. Incorporation of TOF information resulted in up to 50% reduction of background variability with all observed reconstruction algorithms. In clini­cal application the improvement of background variability means lower patient dose or reduction of the imaging time at the same level of image noise. The background variability in the vicinity of hot inserts was higher up to three times com­pared to background variability in the vicinity of cold inserts. Best results were achieved with base­line reconstruction algorithm OSEM (2.8) where we reconstructed images with the lowest levels of noise. This algorithm was included in this research because it was the usual method of reconstruction in the previous generation of PET tomographs.16 It is important to understand that the background variability parameter presents not only statistical noise but also non uniformities in the image which arise from inaccurate attenuation correction or poor convergence during iterative reconstruction. The background variability does not reflect noise correlations or streak artefacts in the image.23 The results of relative count error which pro­vides information of accuracy of attenuation and scatter corrections show, that incorporation of TOF reconstruction in most cases improved (decreased) relative count error, especially at higher activity concentration ratios. Best results were surprisingly obtained with FBP with incorporated TOF correc­tion. The use of PSF correction does not show the improvement of the results, already obtained with TOF correction. The results were similar in evalua­tion of the cold contrast and the error in the air, since the radioactivity is measured in an image segment in which there is no activity and only the medium varies. The different measurements of relative count error show that the differences between measure­ments with internal diameter sized ROI and exter­nal diameter sized ROI can be as high as 10%. It is important that the images are also examined visually for inconsistencies and artefacts (Figure 6). Visual assessment of reconstructed Jaszczak images at different activity concentrations showed that in­corporation of TOF information in reconstruction algorithm substantially improves contrast levels and lowers noise with analytical FBP. FBP showed the lowest levels of contrast and the highest levels of background variability. Iterative reconstruction al­gorithm (OSEM) and iterative reconstruction algo­rithm with PSF modelling-True-X produced images with clearly shaped cylinders and spheres with high contrast and low image noise. TOF information had lower impact on improvement of the images recon­structed with iterative reconstruction methods. TOF information showed best results with low activity concentration ratios and less advanced reconstruc­tion methods, where more noise was present. Conclusions The performance characteristics of Siemens Biograph mCT PET/CT scanner were evaluated fol­lowing the NEMA NU-2-2001 standard, adjusted NEMA NU-2-2001 standard and some additional tests using different methods of topographic re­construction.. While other studies present either 233 results with NEMA phantoms, or results with in­house-made phantoms, we found it interesting to compare and present both types of the results, which might be applicable in the institutions where NEMA equipment is not available. All algorithms offered by the Biograph mCT software were included and applied to the wide range of activity concentration ratios. Thus ana­lytical FBP method as traditional reconstruction method was also included into study in order to compare it with modern iterative reconstruction algorithms, which is novelty compared to results performed by other authors. Our most important interest was in observing the impact of TOF information. On the basis of measurements evaluation we concluded that incor­poration of TOF information in the reconstruction algorithm had the greatest impact on background variability reduction, while improvement of spatial resolution is negligible. The comparison of levels of background variability in the vicinity of hot cylinders revealed that they can be higher up to three times compared to background variability in the vicinity of cold inserts for smallest diameters. Lower levels of background variability in the area of spheres could be obtained using separate phan­toms for cylinders and spheres. Measurements of relative count error or accuracy of attenuation and scatter corrections showed that TOF correction im­proved relative count error, especially with higher activity concentration ratios. We observed substan­tial difference in relative count error for the cases excluding/including the plastic wall. Relative count error measurements should be performed with the same diameter of ROI as the internal diameter of cylinder. When comparing traditional and modern reconstruction algorithms we found out that ana­lytical FBP yields comparable or even better results in some parameter measurements, such as cold contrast and relative count error. Iterative meth­ods show the highest levels of hot contrast, when PSF and TOF correction were applied simultane­ously. However, iterative method with PSF model­ling produced higher values of relative count error, which can be decreased with implementing TOF corrections. The impact is especially prominent at higher activity concentration ratios. Baseline itera­tive OSEM (2.8) showed substantially lower levels of background variability than any other recon­struction algorithm, on the other hand, it was infe­rior in all other parameter measurements. References 1. Budinger TF. 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Karp JS, Suleman S, Daube-Witherspoon ME, Muehllehner G. Benefit of time-of-flight in PET: experimental and clinical results. J Nucl Med 2008; 49: 462-70. 11. Lois C, Jakoby BW, Long MJ, Hubner KF, Barker DW, Townsend DW. An as­sessment of the impact of incorporating Time-of-Flight (TOF) information into clinical PET/CT imaging. J Nucl Med 2010; 51: 1-20. 12. Kadrmas DJ, Casey ME, Conti M, Jakoby BW, Lois C, Towsend DW. Impact of time of-flight on PET tumor detection. J Nucl Med 2009; 50: 1315-23. 13. Casey ME. Point spread function reconstruction in PET. Knoxville, USA: Siemens Medical Solutions, Inc; 2007. p. 1-7. 14. Chang JK, Laforest R. Evaluation of the HD and HD+TOF reconstructions for Siemens’ Biograph-mCT TOF PET scanner. Nuclear Science Symposium and Medical Imaging Conference (NSS/MIC), 2011 IEEE. Valencia; 23-29 October 2011. p. 4131-4. DOI: 10.1109/NSSMIC.2011.6153787 15. Korpar S, Dolenec R, Križan P, Pestotnik R, Stanovnik A. Study of TOF PET using Cherenkov light. Nucl Instrum Methods Phys Res A 2012; 654: 532-8. 16. Akamatsu G, Ishikawa K, Mitsumoto K, Taniguchi T, Ohya N, Baba S, et al. Improvement in PET/CT Image Quality with a Combination of Point-Spread Function and Time-of-Flight in Relation to Reconstruction Parameters. J Nucl Med 2012; 53: 1-7. 17. Martí-Climent JM, Prieto E, Domínguez-Prado I, García-Velloso MJ, Rodríguez-Fraile M, Arbizu J, et al. Contribution of time of flight and point spread function modeling to the performance characteristics of the PET/CT Biograph mCT scanner. Rev Esp Med Nucl Imagen Mol 2012; 32: 1-9. 18. National Electrical Manufacturers Association. NEMA standards publication NU-2-2001. Performance measurements of positron emission tomographs. Rosslyn, VA: National Electrical Manufacturers Association; 2001. p. 1-39. 19. Prieto E, Martí-Climent JM, Arbizu J, Garrastachu P, Domínguez I, Quincoces G, et al. Evaluation of spatial resolution of a PET scanner through the simu­lation and experimental measurement of the recovery coefficient. Comput Biol Med 2010; 40: 75-80. 20. Lodge MA, Rahmin A, Wahl RL. A practical, automated quality assurance method for measuring spatial resolution in pet. J Nucl Med 2009; 50: 1307-14. 21. Skretting A. A method for on-site measurement of the effective statial resolution in PET image volumes reconstructed with OSEM and gaussian post-filters. Radiat Prot Dosimetry 2010; 139: 195-8. 22. Jakoby BW, Bercier Y, Conti M, Casey ME, Bendriem B, Towsend DW. Physical and clinical performance of the mCT time-of-flight PET/CT scanner. Phys Med Biol 2011; 56: 2375-89. 23. Daube-Witherspoon ME, Karp JS, Casey ME, DiFilippo FP, Hines H, Muehllehner G, et al. PET Performance Measurements Using the NEMA NU 2-2001 Standard. J Nucl Med 2002; 43: 1398-409. 234 research article Careful treatment planning enables safe ablation of liver tumors adjacent to major blood vessels by percutaneous irreversible electroporation (IRE) Bor Kos1, Peter Voigt2, Damijan Miklavcic1, Michael Moche2 1 University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia 2 Leipzig University Hospital, Department of Diagnostic and Interventional Radiology, Leipzig, Germany Radiol Oncol 2015; 49(3): 234-241. Received 4 June 2015 Accepted 7 July 2015 Correspondence to: Michael Moche, M.D., Leipzig University Hospital, Department of Diagnostic and Interventional Radiology, Liebigstraße 20, D-04103 Leipzig, Germany. E-mail: michael.moche@medizin.uni-leipzig.de Disclosure: DM holds patents on electrochemotherapy that have been licensed to IGEA S.p.a. and is also a consultant to IGEA. The other co-authors have nothing to disclose. Background. Irreversible electroporation (IRE) is a tissue ablation method, which relies on the phenomenon of electroporation. When cells are exposed to a sufficiently electric field, the plasma membrane is disrupted and cells undergo an apoptotic or necrotic cell death. Although heating effects are known IRE is considered as non-thermal ablation technique and is currently applied to treat tumors in locations where thermal ablation techniques are con­traindicated. Materials and methods. The manufacturer of the only commercially available pulse generator for IRE recommends a voltage-to-distance ratio of 1500 to 1700 V/cm for treating tumors in the liver. However, major blood vessels can influence the electric field distribution. We present a method for treatment planning of IRE which takes the influence of blood vessels on the electric field into account; this is illustrated on a treatment of 48-year-old patient with a metastasis near the remaining hepatic vein after a right side hemi-hepatectomy. Results. Output of the numerical treatment planning method shows that a 19.9 cm3 irreversible electroporation lesion was generated and the whole tumor was covered with at least 900 V/cm. This compares well with the volume of the hypodense lesion seen in contrast enhanced CT images taken after the IRE treatment. A significant temperature raise occurs near the electrodes. However, the hepatic vein remains open after the treatment without evidence of tumor recurrence after 6 months. Conclusions. Treatment planning using accurate computer models was recognized as important for electrochemo­therapy and irreversible electroporation. An important finding of this study was, that the surface of the electrodes heat up significantly. Therefore the clinical user should generally avoid placing the electrodes less than 4 mm away from risk structures when following recommendations of the manufacturer. Key words: irreversible electroporation; liver tumors; colorectal carcinoma; finite element method Introduction Irreversible electroporation (IRE) is a tissue abla­tion method which relies on the phenomenon of electroporation.1 Electroporation occurs, when cells are exposed to sufficiently strong electric fields. These fields disrupt the plasma membrane and cause increased permeability of the plasma membrane to ions, larger molecules, and even DNA. When the electric field is sufficiently strong, the cells cannot recover from the disruption of the membrane and consequently undergo apoptotic or necrotic cell death.2,3 When this method is used for curative tumor ablation, it requires the whole tu­ 235 TABLE 1. Parameters of the electrical and thermal model .L — Liver initial conductivity 0.091 S/m Haemmerich et al.10 .L — Liver final conductivity 0.45 S/m Cukjati17 .T — Tumor initial conductivity 0.4 S/m Haemmerich et al.16 .T — Tumor final conductivity 1.6 S/m Extrapolated from Cukjati17 .Vfinal — Vessel initial conductivity 0.7 S/m Marčan et al.15 .Vfinal — Vessel final conductivity 1.05 S/m Marčan et al.15 .T — Tissue conductivity thermal coefficient 1.5 %/K Haemmerich et al.16 CT — Tissue thermal capacity 3540 J/(kg K) Hasgall et al.19 .T — Tissue density 1079 kg/m3 Hasgall et al.19 kT — Tissue thermal conductivity 0.52 W/(m K) Hasgall et al.19 .b —Blood perfusion 1.8 mL /s /100 mL Hasgall et al.19 Cb —Blood thermal capacity 3840 J/(kg K) Hasgall et al.19 .B — Blood density 1060 kg/m3 Garcia et al.9 T — Initial tissue temperature 310 K q’’’ — Tissue metabolic heat generation 10740 W/m3 Hasgall et al.19 Ea — Activation energy 5.06×105 J/mol Henriques et al.20 . — Frequency factor 2.984×1080 s-1 Henriques et al.20 R — Universal gas constant 8.314 J/(mol*K) .L — Electrode conductivity 106 S/m kE — Electrode thermal conductivity 15 W/(m K) Garcia et al.9 .E — Electrode density 6000 kg/m3 Garcia et al.9 CE — Electrode thermal capacity 500 J/(kg K) Garcia et al.9 mor to be covered with sufficiently strong electric fields, which requires placement of at least two, but typically four or more electrodes around and/or in­side the tumor. The electrodes can be positioned percutaneously under ultrasound or CT guidance, or intra-operatively.4,5 IRE relies on applying electric fields in excess of 600 V/cm in the target volume.6,7 Since tissues are rather good conductors, and tissue conductiv­ity even increases during pulse application, elec­tric fields in tissue are accompanied by significant currents, which can be up to 50 A (maximum cur­rent limit of the Nanoknife® device). This leads to large power dissipation in the tissue during the pulses, which can be up to 150 kW during the pulse. However, the pulses are typically equal or less than 100 µs long and always delivered syn­chronized with the heart rate, which usually is lower than 100 beats per minute. This results in duty cycles of less than 0.1 % and consequently the actual delivered power is less than 15 W, which is between a factor of 5 to 10 less than in thermal ablation methods. Nevertheless, this power dis­sipation causes a non-negligible temperature rise which can be found most prominently around the tissue-electrode boundary.8 However, tempera­ture itself is not the primary, nor the desired cell-killing mechanism.9 In fact, one of the most prom­ising uses of IRE is to treat tumors, which are very close to major blood vessels, bile ducts (in liver), or nerves (in prostate), which limit thermal abla­tion methods like radiofrequency, laser or micro­wave ablation.10–12 The reason for this limitation is on one hand the risk of leaving residual vital tu­mor due to the heat sink effect induced from the cooling of the vessel. On the other hand, there is a significant risk of heat caused coagulation of sensi­tive structures like nerves and bile ducts with se­rious complications for the patient. However, the local electric field distribution, which is the most important factor for successful treatment with IRE is affected by the higher conductivity of blood and blood vessels.13–15 This means that additional care has to be taken when IRE is performed near blood 236 vessels, where it is most preferred over the other thermal ablation techniques. Currently, the manufacturer of the only certi­fied medical device available for IRE treatments (NanoKnife, Angiodynamics, Latham, NY) recom­mends electrodes for ablation of liver tumors to be no more than 2.2 cm apart, positioned in parallel around the target volume. A total of 90 pulses with voltage to distance ratio of 1500-1700 V/cm, and 90 µs duration are recommended per electrode pair according to System Procedure Guide Software Version 2.2.0. The graphical user interface of the software provides a simple treatment planning op­tion in two dimensions (2D), while not taking into account differences in electric conductivities of tis­sue between normal and tumor tissue.16 In this study we present a method for numerical patient-specific treatment planning for IRE, which takes into account the influence of blood vessels on electric field distribution. The method is illustrated on a 48-years-old. female patient with a recurrent metastasis directly adjacent to the last remaining hepatic vein after previous right-side hemi-hepa­tectomy and the successful treatment with IRE. Materials and methods Electric field computation and temperature distribution computation Since IRE relies on applying local fields in excess of 600 V/cm in the whole target volume, electrodes need to be introduced in the target volume itself, but preferably minimizing the number of elec­trodes inserted in the tumor to preclude needle track metastasis seeding. The electric field is how­ever affected by the local conductivity of tissue, which generally varies between different tissues, especially at frequencies, which are present in elec­troporation pulses (Table 1). Additionally, conduc­tivity of tissue was shown to increase due to the electric field during the pulse delivery due to mem­brane electroporation.17,18 Together with the com­plex geometry of the target location (blood vessels, tumors and liver) this generally requires numerical computation of the electric fields. In order to differentiate the tissues of different conductivities and separate the target volume from the healthy tissue a segmentation step is required in the treatment planning procedure. We use a treatment planning procedure based on optimization in Matlab, while electric fields are solved in Comsol Multiphysics. The simulations consist of solving the Laplace equation for electric potential, given boundary conditions for electric potential on the electrodes. A stationary solver is used for the simulations, but iterated 6 times, in­creasing the conductivity of the tissues above elec­troporation thresholds between each iteration.18,21–23 From the electric field simulations, we obtained the electric field distribution and final volumes of tumor and liver covered with fields above the IRE threshold. Since more than one electrode pair is generally required to obtain clinically relevant treatment volumes in IRE, the electric field from each electrode pair are compared and the maxi­mum value at each location is considered when evaluating the total coverage of the target volume. For verifying the tissue heating during the treat­ment, the thermal dissipation of the electric field computation step can be used to set a heat source for a Pennes’ bioheat equation for a transient solver of temperature fields. To shorten simulation times, a duty-cycle approach is used, wherein we use the thermal dissipation multiplied by the duty cycle of the pulse delivery to model heating. This provides a comparable temperature increase in the bulk tissue9, but is numerically more stable and much faster. The reason for this is that it does not have to account for the very fast temperature rise dur­ing the 90 µs that the pulse is turned on in com­parison to the 10000 times longer interval between pulses. All parameters of the electrical and thermal model were taken from the literature and are listed in Table 1. Patient data The patient was a 48-years-old female who had previously undergone right hemi-hepatectomy for treatment of metastases of cholangiocellular carcinoma. During follow-up imaging a small (14 × 9 × 15 mm, i.e. 0.96 cm3) focal recurrent metasta­sis was detected in the remaining left liver. Since the metastasis was adjacent to the only remaining left hepatic vein it was not surgically resectable. Percutaneous CT guided IRE ablation was selected as the best treatment option to preserve this last liver vein since primary thermal ablations like ra­diofrequency ablation would have been contrain­dicated in this constellation. IRE can achieve com­plete ablation of tumors even nearby major blood vessels, since it is not negatively affected by their cooling effect such as thermal therapies24 where this so called heat sink effect may lead to incom­plete tumor ablation. Furthermore, it is also com­monly reported, that it is sparing for larger blood vessels25 which could have been damaged during 237 TABLE 2. Reconstructed distances and angles between the electrodes 1 — 2 18 4.1 1 — 3 14 1.2 1 — 4 12 1.8 2 — 3 15 3.2 2 — 4 12 5.2 3 — 4 17 2.0 thermal ablations. The patient was treated in the scope of the research project GoSmart (funded by the European Commission – grant agreement no. 600641). Ethical approval was obtained from Leipzig University Hospital Institutional Review Board under code AZ206 -13 – 15072013. Informed consent to use their personal data for scientific pur­poses was obtained from the patient. Treatment was performed using the above-mentioned pulse generator and the ablation protocol recommended from the manufacturer. Electrodes were positioned using CT guidance (Figure 1). Electrode exposure length for the treatment was 2 cm. All data was collected from clinical records or from the genera­tor, where electric pulse data it is saved by default. The data is routinely recorded for improvement of quality, practice and performance of this novel treatment. For the illustration case, we used MRI images of the patient acquired 3 weeks prior to the treat­ment. The images were anonymized and upload­ed into the web based electric fields visualization tool Visifield (www.visifield.com, University of Ljubljana, Slovenia). Liver was segmented using automatic segmentation method for liver segmen­tation26 and the tumor and blood vessel were seg­mented manually. Interventional CT images ob­tained during the procedure were used to exactly reconstruct the electrode positions during treat­ment and to have these available for the numerical simulations. The reconstructed distances between the tips of the electrodes and angles between the tips of the electrodes are given in Table 2. It is also demonstrated, that the radiologist performing the procedure has managed to place the electrodes al­most completely in parallel. The pulse generator measures the pulse volt­age and current during electric pulse delivery and stores the results in an xml document, which was parsed into Matlab. The same voltages, as were actually delivered during the actual treatment for each electrode pair were also used in a finite ele­ment computational model. The electric field dis­tribution was computed only for the first pulse of each pulse train using a stationary solver, but taking into account increase of conductivity due to electroporation. Results A total of 660 pulses were delivered in three se­quences to six electrode pairs (pulses are always delivered to pairs of electrodes; sequentially, pairs of two electrodes are selected from available elec­trodes), with the electrode positioning and num­ 238 TABLE 3. List of delivered pulses and comparison with computed currents 1 [3,4] 2720 20 26.6 27.4 3 2 [1,2] 2550 20 21.5 23.9 11 3 [1,3] 2380 20 23.6 25.8 9 Test pulses 4 [2,3] 2210 20 21.3 21.0 -2 5 [4,1] 2200 20 22.5 25.7 14 6 [2,4] 1650 20 17.4 16.2 -7 7 [1,2] 3000 70 30.5 28.4 -7 8 [3,4] 2720 70 30.5 27.4 -10 9 [2,3] 2405 70 30.3 22.8 -25 Treatment pulses 10 [1,3] 2380 70 30.7 25.9 -16 11 [2,4] 2200 70 31.1 22.3 -28 12 [4,1] 2200 70 29.3 25.6 -13 13 [1,2] 2380 20 24.2 22.1 -9 14 [3,4] 2380 20 28.5 23.6 -17 15 [1,3] 1960 20 22.1 20.6 -7 Additional pulses 16 [2,3] 1820 20 20.5 16.8 -18 17 [2,4] 1540 20 18.9 14.9 -21 18 [4,1] 1540 20 17.2 17.1 0 1 3 -4 1 -2 0.9 1 -3 2 -3 0.8 4 -1 2 -4 1 -2 0.7 0.6 3 -4 2 -3 0.5 1 -3 2 -4 0.4 4 -1 1 -2 0.3 3 -4 1 -3 0.2 2 -3 2 -4 0.1 4 -1 0 FIGURE 2. Coverage progression after delivery of pulses to each electrode pair. The graph shows the combined maximum fields in the tumor following each electrode pair. Electrode pair progression is the same as in Table 3. Arrow indicates the direction of increase of coverage with delivery of successive electric pulses. The graph shows that the first electrode pair already covers the whole tumor with electric fields above the irreversible electroporation threshold. bering as shown in Figure 1. Initially trains of 20 pulses were delivered (test pulses), then trains of 70 pulses were delivered, with some voltage ad­justments (treatment pulses), and finally trains of 20 pulses were delivered (additional pulses) with Volume fraction of tumor tissue lower voltages. Table 3 lists all delivered pulses and pulse parameters. The root mean square error (RMSE) of the com­puted currents versus measured currents from the pulse generator is 3.8 A. The cumulative coverage of tumor with electric fields after each electrode pair is shown in Figure 2, while the coverage of liver tissue is shown in Figure 3. While the IRE threshold for tumors has not yet been rigorously determined, we are using a value of 800 V/cm in the following graphs, consistent with previous work.6,23,27 For liver we used IRE threshold deter­mined from experiments on rabbit livers – 700 V/ cm.21 To be noted however, this value is for pulse trains of 8 pulses. It is very likely that actual IRE thresholds are much lower.7,28 In the post-IRE contrast enhanced CT images, a hypodense region can be seen in the area where the treatment was performed. We used this hypodense region to estimate the total IRE lesion as was previ­ously suggested.29 The volume of this hypodense lesion was 20.03 cm3. From Figure 3, the volume of the IRE lesion in the liver is 18.4 cm3. Together with the tumor (0.57 cm3) in the simulations, the to­tal volume of the computed lesion equals 19.9 cm3, which is comparable to the lesion size determined by CT. 239 Figure 4 shows a single slice of temperature data after the 7th pulse treatment set (first train of the treatment pulses, and the train with high­est pulse amplitude). The tumor is heated slightly more than the surrounding tissue, as its perfusion is lower, and also the conductivity is higher than that of the liver tissue, which both contributes to a higher Joule heating. Temperatures above 50 C are typically used for indication of thermal tissue dam­age.1 Therefore, we also show the volume of tissue above this threshold in Figure 5. Interesting to note is, that temperatures above 70°C are located less than 4 mm from the electrodes. If we approximate this volume with four cylinders, each with a radi­us of 4 mm and height of 28 mm (length of active electrode region with 4 mm added on either side), the total volume of high temperature caused by the electrodes is 5.6 cm3, which is consistent with the volume of tissue above 50°C shown in Figure 5. The curve in Figure 5 shows, that the 63% prob­ability of thermal damage, determined by the Arrhenius rate equation, increases strongly in the first 250 s while later the slope levels off. This is caused by the fast increase in temperature around the electrodes, and then a slower increase in tem­perature in the more distant areas. Finally, the slope gets flatter, since the heating does not extend further from the electroporated area, the pulse am­plitudes start to decrease, and diffusion moves the heat into tissue further away. Discussion The presented numerical results and clinical fol­low-up show that IRE is efficient at treating tumors in the immediate vicinity of major blood vessels. Since IRE is unaffected by the cooling of blood ves­sels, it is supposed to be not limited by their vicin­ity. The results of our computational model show a good correlation between the modelled IRE, electri­cal measurements during treatment, and imaging results. The tumor treatment has been classified as a complete ablation, and has shown no recurrence in the 6 months follow-up. With the number, amplitude, and duration of pulses in the presented treatment, it is therefore clear, that a non-negligible temperature rise occurs. Since IRE has also been classified as a non-thermal tissue ablation technique in the literature24,30, it needs to be clarified, that non-thermal does not in­dicate that there is no temperature rise, but rather, that the temperature is not the main mechanism which induces cell death. Our model clearly shows 140 120 100 80 60 40 20 0 FIGURE 3. Electric field coverage in the liver tissue. The tumor tissue is not included in the volume on this graph. The graph shows the combined maximum fields in the liver following each electrode pair. Electrode pair progression is the same as in Table 3. Arrow indicates the direction of increase of coverage with delivery of successive electric pulses. Volume of liver tissue [cm3] FIGURE 4. One slice showing computed temperature distribution after all pulses from the 7st pulse train (electrode pair 1 – 2 at 3000 V) superimposed on the corresponding MRI slice of the model. that some areas of the lesion do heat up signifi­cantly (Figure 5), and the temperature rise is also consistent with experimental results from the lit­erature.31 Although our results show, that IRE is not an exclusively nonthermal treatment, i.e. there is significant heating present in the vicinity of elec­trodes, the total treatment volume is significantly higher than the volume based on thermal effects would be expected. A limitation of the model is, that we assumed that pulses were delivered con­stantly at 1 Hz, while in reality, the pulses were delivered in synchronization with the patient’s ECG, which can realistically be up to 100 beats per minute, and would consequently result in a 241 due to the networking efforts of the COST TD1104 Action (www.electroporation.net). In particular, BK received a grant under reference COST-STSM­TD1104-21010. Co-funded by the European Commission in the scope of the research project GoSmart (grant agreement No. 600641). References 1 Davalos R, Mir L, Rubinsky B. Tissue ablation with irreversible electropora­tion. Ann Biomed Eng 2005; 33: 223-31. 2 Yarmush ML, Golberg A, Serša G, Kotnik T, Miklavčič D. Electroporation­based technologies for medicine: principles, applications, and challenges. Annu Rev Biomed Eng 2014; 16: 295-320. 3 Jiang C, Davalos R, Bischof J. A review of basic to clinical studies of irrevers­ible electroporation therapy. IEEE Trans Biomed Eng 2015; 62: 4-20. 4 Martin RCG. Irreversible electroporation of locally advanced pancreatic head adenocarcinoma. J Gastrointest Surg 2013; 17: 1850-6. 5 Scheffer HJ, Melenhorst MCAM, Vogel JA, van Tilborg AAJM, Nielsen K, Kazemier G, et al. Percutaneous irreversible electroporation of locally advanced pancreatic carcinoma using the dorsal approach: a case report. Cardiovasc Intervent Radiol 2015; 38: 760–5. 6 Qin Z, Jiang J, Long G, Lindgren B, Bischof JC. Irreversible electroporation: an in vivo study with dorsal skin fold chamber. Ann Biomed Eng 2013; 41: 619-29. 7 Neal RE, Garcia PA, Kavnoudias H, Rosenfeldt F, Mclean CA, Earl V, et al. In vivo irreversible electroporation kidney ablation: experimentally correlated numerical models. IEEE Trans Biomed Eng 2015; 62: 561-9. 8 Arena CB, Mahajan RL, Nichole Rylander M, Davalos RV. An experimental and numerical investigation of phase change electrodes for therapeutic irreversible electroporation. J Biomech Eng 2013; 135: 111009. 9 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. 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Segmentation of hepatic vessels from MRI images for planning of electropo­ration-based treatments in the liver. Radiol Oncol 2014; 48: 267-81. 15 Marčan M, Kos B, Miklavčič D. Effect of blood vessel segmentation on the outcome of electroporation-based treatments of liver tumors. PloS One 2015; 10: e0125591. 16 Haemmerich D, Schutt D, Wright A, Webster J, Mahvi D. Electrical conduc­tivity measurement of excised human metastatic liver tumours before and after thermal ablation. Physiol Meas 2009; 30: 459-66. 17 Cukjati D, Batiuskaite D, Andre F, Miklavcic D, Mir L. Real time elec­troporation control for accurate and safe in vivo non-viral gene therapy. Bioelectrochemistry 2007; 70: 501-7. 18 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. 19 Hasgall P, Neufeld E, Gosselin M, Klingenböck A, Kuster N. IT’IS Database for thermal and electromagnetic parameters of biological tissues. 2011. Available at http://www.itis.ethz.ch/database. Accessed 15 March 2015. 20 Henriques FC. Studies of thermal injury; the predictability and the signifi­cance of thermally induced rate processes leading to irreversible epidermal injury. Arch Pathol 1947; 43: 489-502. 21 Sel D, Cukjati D, Batiuskaite D, Slivnik T, Mir LM, Miklavcic D. Sequential finite element model of tissue electropermeabilization. IEEE Trans Biomed Eng 2005; 52: 816-27. 22 Aström M, Zrinzo LU, Tisch S, Tripoliti E, Hariz MI, Wardell K. Method for patient-specific finite element modeling and simulation of deep brain stimu­lation. Med Biol Eng Comput 2009; 47: 21-8. 23 Županič A, Kos B, Miklavcic D. Treatment planning of electroporation-based medical interventions: electrochemotherapy, gene electrotransfer and ir­reversible electroporation. Phys Med Biol 2012; 57: 5425-40. 24 Daniels C, Rubinsky B. Electrical field and temperature model of nonthermal irreversible electroporation in heterogeneous tissues. J Biomech Eng-Trans ASME 2009; 131: 071006. 25 Lee YJ, Lu DSK, Osuagwu F, Lassman C. Irreversible electroporation in por­cine liver: short- and long-term effect on the hepatic veins and adjacent tissue by CT with pathological correlation. Invest Radiol 2012; 47: 671-5. 26 Pavliha D, Mušič MM, Serša G, Miklavčič D. Electroporation-based treat­ment planning for deep-seated tumors based on automatic liver segmenta­tion of MRI images. PloS One 2013; 8: e69068. 27 Miklavcic D, Snoj M, Zupanic A, Kos B, Cemazar M, Kropivnik M, et al. Towards treatment planning and treatment of deep-seated solid tumors by electrochemotherapy. Biomed Eng Online 2010; 9: 10. 28 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. 29 Zhang Y, White SB, Nicolai JR, Zhang Z, West DL, Kim D, et al. Multimodality imaging to assess immediate response to irreversible electroporation in a rat liver tumor model. Radiology 2014; 271: 721-9. 30 Scheffer HJ, Nielsen K, de Jong MC, van Tilborg AAJM, Vieveen JM, Bouwman A (R. A), 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. 31 Wagstaff PGK, de Bruin DM, van den Bos W, Ingels A, van Gemert MJC, Zondervan PJ, et al. Irreversible electroporation of the porcine kidney: Temperature development and distribution. Urol Oncol 2015; 33: 168. e1–168.e7. 32 Dollinger M, Jung E-M, Beyer L, Niessen C, Scheer F, Müller-Wille R, et al. Irreversible electroporation ablation of malignant hepatic tumors: subacute and follow-up CT appearance of ablation zones. J Vasc Interv Radiol 2014; 25: 1589-94. 33 Meir A, Hjouj M, Rubinsky L, Rubinsky B. Magnetic resonance imaging of electrolysis. Sci Rep 2015; 5: 8095. 34 Pucihar G, Krmelj J, Reberšek M, Napotnik TB, Miklavčič D. Equivalent pulse parameters for electroporation. IEEE Trans Biomed Eng 2011; 58: 3279-88. 35 Garcia PA, Pancotto T, Rossmeisl JH, Henao-Guerrero N, Gustafson NR, Daniel GB, et al. Non-thermal irreversible electroporation (N-TIRE) and adjuvant fractionated radiotherapeutic multimodal therapy for intracranial malignant glioma in a canine patient. Technol Cancer Res Treat 2011; 10: 73-83. 36 Miklavčič D, Serša G, Brecelj E, Gehl J, Soden D, Bianchi G, et al. Electrochemotherapy: technological advancements for efficient electropo­ration-based treatment of internal tumors. Med Biol Eng Comput 2012; 50: 1213-25. 37 Kranjc M, Markelc B, Bajd F, Čemažar M, Serša I, Blagus T, et al. In Situ Monitoring of electric field distribution in mouse tumor during electropora­tion. Radiology 2015; 274: 115-23. 38 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. 242 research article Central nervous system imaging in childhood Langerhans cell histiocytosis – a reference center analysis Luciana Porto1, Stefan Schöning2, Elke Hattingen1, Jan Sörensen2, Alina Jurcoane1, Thomas Lehrnbecher2 1 Neuroradiology Department, Johann Wolfgang Goethe University, Frankfurt/Main, Germany 2 Pediatric Hematology and Oncology, Hospital for Children and Adolescents, Johann Wolfgang Goethe University, Frankfurt/Main, Germany Radiol Oncol 2015; 49(3): 242-249. Received 3 March 2015 Accepted 1 June 2015 Correspondence to: Thomas Lehrnbecher, Pediatric Hematology and Oncology, Hospital for Children and Adolescents, Johann Wolfgang Goethe University, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany. Phone: +49 69 6301 83481; Fax: +49 69 6301 6700; E-mail: Thomas.Lehrnbecher@kgu.de Disclosure: No potential conflicts of interest were disclosed. This work is dedicated to Robert Arceci. Background. The aim of our study was (1) to describe central nervous system (CNS) manifestations in children with Langerhans cell histiocytosis (LCH) based on images sent to a reference center and meeting minimum requirements and (2) to assess the inter-rater agreement of CNS-MRI results, which represents the overall reproducibility of this in­vestigation. Methods. We retrospectively reviewed brain MRI examinations in children with LCH, for which MRI minimum require­ments were met. Abnormalities were rated by two experienced neuroradiologists, and the inter-rater agreement was assessed. Results. Out of a total of 94 imaging studies, only 31 MRIs met the minimum criteria, which included T2w, FLAIR, T1w images before/after contrast in at least two different section planes, and thin post contrast sagittal slices T1w through the sella. The most common changes were osseous abnormalities, followed by solid enlargement of the pineal gland, thickened enhancing stalk and signal changes of the dentate nucleus. Whereas inter-rater agreement in assessing most of the CNS lesions was relatively high (. > 0.61), the application of minimum criteria often did not allow to evalu­ate the posterior pituitary. Conclusions. The diversity of radiological protocols from different institutions leads to difficulties in the diagnosis of CNS abnormalities in children with LCH. Although the inter-rater agreement between neuroradiologists was high, not all the LCH manifestations could be completely ruled out when using the minimum criteria. Brain MRIs should therefore follow LCH guideline protocols and include T1 pre-gadolinium sagittal images, and be centrally reviewed in order to improve the comparison of clinical trials. Key words: Langerhans cell histiocytosis; child; central nervous system; magnetic resonance imaging Introduction Langerhans cell histiocytosis (LCH) is a rare dis­ease of the monocyte-macrophage system, seen mainly in children, but which can occur in any age group. The clinical presentation of the disease may range from a self-healing bone-lesion to multi-sys­tem life-threatening disease. The choice of appro­priate therapy is therefore a significant challenge, with treatment options varying from watch-and­wait to intensive chemotherapy.1,2 The pathogene­sis of LCH remains unresolved, with data support­ing both malignant transformation and immune dysregulation.1,3 243 Whereas skin and bone lesions are the most frequent manifestations of LCH, central nerv­ous system (CNS) lesions are less common, and a wide variety of CNS findings have been de­scribed by magnetic resonance imaging (MRI).1,3,4 Unfortunately, many centers often do not follow a standardized protocol when evaluating these pa­tients, or are unaware of potential complications such as degenerative CNS changes.5 It is thus im­portant to evaluate the reliability of MRI in diag­nosing CNS changes. The aim of our study was (1) to describe CNS manifestations of LCH in chil­dren based on images sent to a reference center and meeting minimum requirements and, more importantly, (2) to assess the inter-rater agreement of CNS-MRI results, which represents the overall reproducibility of this investigation in this patient population. Patients and methods Patients and MR imaging As the national reference center for German child­hood LCH, we regularly receive clinical data and imaging studies from children and adolescents with LCH. We included in our analysis all chil­dren with biopsy proven LCH from whom we had received MRI studies during the 2-year period between 2012 to 2014; notably, children could have had also MRI studies performed prior to 2012. Since the centers where the patient was treated often used different MR protocols, we included only patients with the following MR image se­quences in the analysis: (1) Sagittal T1-weighted (w) images post contrast, permitting evaluation of the infundibulum (the majority of the images did not include sagittal T1-w pre contrast, with the result that this sequence could not be included as a minimum criterion); (2) T2-w images, fluid attenuated inversion recovery (FLAIR), and (3) T1-w images pre and post contrast in at least two different section planes. Enlargement of the pineal gland was defined as described previously by Sumida et al.6 Single and multisystem LCH as well as CNS risk lesions were defined according to the guidelines of the LCH study protocols.4 The study has been ap­proved by the local Ethics committee. Statistical analysis In addition to the neuroradiologist who refer­ences cerebral MRI studies of children with LCH in Germany (LP), MRIs were assessed by a second senior neuroradiologist. The inter-rater agreement was evaluated by the percent agreement (uncor­ rected) and the Cohen’s Kappa . index (corrected for chance effects). Substantial or almost per­ fect agreement was defined for . values between 0.61–1.0.7 For the analysis, the software R Statistics 2.15.1 (http://www.R-project.org/) in combination with functions from the packages “irr” and “caret” were used.8 The results of both the reference neuro­radiologist (LP) and the inter-rater agreement are reported. Results Patients´ characteristics Clinical data and imaging studies were available in a total of 94 children with LCH. Cerebral MRI was performed in these children for a variety of reasons such as the presence of neurological abnormalities (e.g., diabetes insipidus (DI)), for further evaluation in patients with involvement of the skull/craniofa­cial bones, or according to the physician´s discre­tion (e.g. the involvement of the mandibula or the cervical vertebrae). Only brain imaging studies of 31 patients (22 of whom were boys) met the inclu­sion criteria of the study. Eleven children suffered from unifocal and 5 other from multifocal bone disease. Localized LCH of the skull was seen in 11 patients. Nine children were affected by multisys­tem LCH. Eleven children suffered from DI, and 2 other children had endocrine disorders other than DI. With the exception of 2 children presenting with absence epilepsy and muscular hypotonia, respectively, no neurological abnormalities were detected in any patient (Table 1). The median age of the patients at the time of cranial MRI was 7 years (range 0.5–17 years), and the median time between the diagnosis of LCH and cranial MRI was 70 days (range 0–10 years). 11 pa­tients had not received treatment for LCH at the time of MRI. MR imaging and correlation with clinical data The most common MRI changes were osseous (55%), followed by solid enlargement of the pin­eal gland (45%), a thickened enhancing stalk (32%) and signal changes of the dentate nucleus (29%). Occasionally, hyperintensity in the hippocampus, parenchymal/meningeal enhancement and white matter hyperintensity were observed. TabLe 1. Patients´ characteristics and cerebral magnetic resonance imaging (MRI) finding in 31 children and adolescents with Langerhans cell histiocytosis (LCH) 1 M 17 <10 d y y y Y 2 M 7 <10 d y y y 3 M 11 <10 d y y 4 M 15 10 yrs y y y Y y Y 5 F 3 170 d y y Y 6 M 1 <10 d y 7 M 3 <10 d y 8 M 8 7 yrs y y y y Y 9 F 9 18 mths y y Absence seizures y Y 10 M 11 8 mths y Somatomegaly y Y 11 M 15 3 mths y y Y 12 F 3 3 yrs y y y y Y Y 13 F 14 2 mths y y Y y 14 M 3 2 mths y y y Muscular hypotonia y y Y 15 F 1 10 d y y Y 16 M 3 2 yrs y y Y 17 M 15 3 yrs y 18 M 5 <10 d y Y 19 M 1 < 10d y y y Y 20 F 10 5 yrs y y y Y 21 M 9 <10 d y y 22 F 0,5 < 10d y y y 23 M 3 2 yrs y y Y Y 24 F 10 2 mths y y Y 25 M 17 3 yrs y y y y 26 M 11 <10 d y y 27 M 6 22 d y y 28 M 5 3 yrs y y Hypopituitarism y Y y Y 29 M 14 <10 d y y y 30 M 11 <10 d y y y 31 F 6 3 mths y y y y y represents “present”, blanks represent “not present”. 1 The pattern of LCH single- and multi-system LCH as well as central nervous system (CNS) risk lesions were defined according to the guidelines of the LCH study protocols [Ref. 5]; 2 Therapy given prior to or at the time of cerebral imaging consisted of regimens according to LCH or to modified LCH protocols; d = day; DI = diabetes insipidus; f = female; m = male; mth = month; yr = year Tumorous/granulomatous lesions Hypothalamic-pituitary axis involvement, hypothalamus Enhancement and thickening of the pituitary stalk > 3 mm were seen in 10 patients (32%) (Table 1). Seven of the 12 available pre contrast MR sagit­tal images demonstrated loss of bright spot. Nine patients with abnormalities of the pituitary stalk suffered from DI, whereas 1 patient had no clini­cal manifestation of DI. Seven patients had DI and CNS risk lesions, of whom five patients had addi­tional multisystem LCH. Pineal abnormalities Solid enlargement of the pineal gland with en­hancement was observed in 14 patients (45%). Enlargement was defined when the height of the pineal gland was more than 3.5 mm in patients younger than two years and as more than 4.5 mm in patients older than 2 years, respectively.6 In 6 patients, abnormalities were seen in both the pitui­tary stalk and pinealis, whereas in 12 patients, only one of the structures was affected (pituitary stalk [4], pineal enlarged enhancement [8]). Extra-axial space involvement 4 patients (13%) presented with dural enhance­ment. All these patients had osseous lesions com­bined with epidural and subdural involvement (Figure 1). None of the patients presented with iso­lated dura-base masses or hypointensity on T2w at the choroid plexus. 245 FIGURe 1. T1-w MR-images in an 11-year-old boy with LCH (patient ID #26). (a) Coronal enhanced T1-MR image reveals an osseous enhancing mass (arrow) combined with epidural und subdural involvement along the left side of the superior sagittal sinus. (b) Enhanced T1-MPRage-Image with reconstruction shows the mass (arrow) closely related to the superior sagittal sinus. Note the infiltration of the dural venous plexus, which is located within the inner portion of the dura. The dural plexus enhances in particular parasagittally on the left, where it connects to the sagittal sinus. There was no thrombus within the sagittal sinus. Enhancement due to intracerebral granulomatous lesions 5 patients (16%) presented with parenchymal en­hancement. Pontine enhancement was seen in 2 cases (Figures 2 and 3), and supratentorial enhance­ment in 4 patients. One patient presented with si­multaneous infra- and supratentorial enhancement. Lytic lesions of skull Osseous lesions in the skull, skull base and crani­ofacial bones were seen in 17 patients (55%); of FIGURe 2. Cerebral MRI in a 15-year-old-boy with LCH (patient ID #4). (a-C) FLAIR images show high-signal lesions (arrows) in the deep white matter, in pons and hippocampus. After contrast note the parenchymal (partial nodular pattern, curved arrow) and perivascular enhancement. The classical finding, enhancement and thickening of the pituitary stalk, was also present (straight arrow). 246 those, 4 had dural infiltration, 4 had extension of the disease within the intraconal space with en­hancement, and 3 had bone destruction with in­volvement of the mastoid. Non-tumorous, non-granulomatous lesions Dentate nucleus The evaluation of the dentate nucleus as a classical location of deep grey matter affected by LCH was limited by artefacts resulting in a relatively low inter-rater agreement (. < 0.61). None of the 9 chil­dren with dentate nucleus abnormalities showed neurological symptoms such as tremor, dysarthria, or ataxia. Whereas in 6 of the children, LCH had been diagnosed more than 1 year prior to the ab­normal MRI finding; the affection of the dentate nucleus was seen in 1 untreated patient at the time of diagnosis of LCH. White matter changes A hyperintense signal on T2w and FLAIR images in the supratentorial white matter were observed in 2 patients (Figure 2) and were likely in 6 pa­tients (representing a total of 25% of patients). The lesions were symmetrical and showed a vascular pattern. These white matter changes were associ­ated with pons involvement in 2 patients, of which one showed enhancement (Figure 2). Basal ganglia and hypothalamus Changes in basal ganglia or in the hypothalamus were not observed in any patient. atrophy No signs of cerebellar, midbrain, or supratentorial atrophy were noted for any patient Inter-rater agreement The diversity of CNS lesions may lead to difficul­ties in diagnosis, which may have a significant impact on treatment and outcome. The inter-rater agreement in this study was 69–100%. Substantial inter-rater agreement (. > 0.61) was found for the following variables: enlarged pituitary stalk/ mass, bone changes, pineal enlarged enhance­ment, white matter and hippocampal hyperin­tensity on T2w, as well as for parenchymal and meningeal enhancement. Partial volume effects and artefacts limited the MRI evaluation and the inter-rater reliability in the area of the dentate nu­cleus (. = 0.31). Other variables, such as the hyper­intense signal on T2w and FLAIR in the lentiform nucleus, hypothalamus or cerebellum had low oc­currence rates and are therefore unreliable agree­ment indices. Discussion Since LCH is a rare disease, research on radiologi­cal CNS abnormalities is limited. In addition, the variable quality of diagnostics makes comparing treatment and outcome difficult. Our aim was to describe CNS manifestations in children with LCH based on images sent to a reference center and meeting minimum requirements. The second and more important goal was to evaluate the reliability of the MRI findings based on the inter-rater agree­ment of two senior neuroradiologists. 247 Only a total of 33% of the cerebral MRIs sent for reference met the minimum requirements of the study. Unfortunately, we had to omit pre contrast sagittal T1-w as a minimum criterion, since most imaging studies did not include this sequence. The lack of thin T1 sagittal pre contrast images can be explained by the fact that the German public health system only pays for a maximum of 4 MR sequenc­es. Hypothalamic-pituitary axis Diabetes insipidus, as the most common endo­crinopathy in LCH, is caused by inadequate anti­diuretic hormone (ADH) secretion. Corroborating our data, DI occurs in approximately 25% of all patients with LCH or in approximately 50% of pa­tients with multisystem disease, mainly in those with skull and orbital involvement.9-12 The typical MRI finding in DI is the lack of high signal inten­sity of the posterior pituitary on T1w images before contrast (“loss of bright spot”), which correlates with the loss of ADH-containing granules, and is often associated with enhancement and thickening of the pituitary stalk >3 mm (Figure 4).13 In our se­ries, loss of bright spot was seen in 7 patients, but it is important to note that this result may be biased as usually only children with DI or anterior pitui­tary hormone deficiency have a typical targeted MRI examination of the pituitary region which includes T1w without gadolinium. Enhancement and thickening of the pituitary stalk were seen in 10 of 31 (32%) of our patients with high inter-rater agreement. Abnormalities of the pituitary stalk were present in 9 of the patients with DI, but no­tably, also in one patient without DI. Interestingly, over the three year follow-up period, this patient, who received prolonged chemotherapy for prima­ry and relapsed LCH, did not develop DI. Future research is needed to address which children with LCH and associated abnormalities of the pituitary stalk will develop DI at a later time-point,14 and, more importantly, whether treatment in these chil­dren could prevent this specific complication. Pineal abnormalities Enhancement of the solid enlarged pineal gland was seen with a high inter-rater agreement in 14 patients (45%), which is considerably higher than reported previously (3% and 15%).15,16 This differ­ence could be due to several reasons: Firstly, differ­ent MRI protocols make it difficult to evaluate pin­eal enhancement, and the lack of high-resolution thin-section imaging may result in false-negative findings. Secondly, the normal pineal gland tissue enhances with gadolinium on MRI because of the lack of a blood-brain barrier. This means that con­trast enhancement within the pineal gland, espe­cially in teenagers, does not necessarily mean ab­normality, e.g. infiltration. There is a greater preva­lence of ring-like pineal glands in children than in adults, and it was postulated that these glands may form pineal cysts in the future, which would ac­count for the higher percentage enhancing mass in children and teenagers, and of cysts in adulthood and the increased incidence on autopsy reports.17 It is also important to note that the results of the pineal gland should be interpreted carefully, since there is a large variation of the size of the gland in all age groups.6 In contrast to a previous study, we did not find a correlation between abnormalities in the pituitary stalk and the pineal gland.15 Whereas in 6 patients of our series, both structures showed an abnormal­ity, 12 patients presented with an irregularity in one structure only. It was speculated whether co­existing changes, which have also been observed in other diseases, might be caused by the functional interactions of both structures.18 However, as not­ed before, the intrinsic enhancement of the pineal gland makes the evaluation of this structure diffi­cult. Lytic lesions of skull Craniofacial involvement is the most common presentation of CNS-LCH. In line with other re­ports, the frequency of these abnormalities in the present study was 55%.19,20 Intracranial non-tumorous lesions including neurodegenerative changes Intra-axial neurodegenerative parenchymal chang­es are among the most frequent patterns of CNS­LCH.16 Neurodegenerative grey-matter changes mainly involve the dentate nucleus and basal ganglia, with a bilaterally symmetrical, hyperin­tense signal in T1w and T2w as key radiological features.4 Alterations in the signal-intensity may reflect the loss of neurons, demyelination, gliosis, and inflammation.4,21 It was speculated that chronic or recurrent granulomatous lesions in the craniofa­cial bones result in an intracranial process which includes chemokine-mediated tissue damage or an autoimmune response to brain tissue induced by antigen-presentation through Langerhans cells.4 If this is the case, both the frequency and severity of neurodegenerative lesions might increase with older age of patients with LCH. This hypothesis could explain the lower frequency of grey-matter 248 changes in the dentate nucleus in our study (29%) compared to a previous analysis (40%).19 The old­est patient in the present study was 17 years old, whereas Prayer et al included patients up to 47 years of age.19 In addition, the short follow-up is a limiting factor of our study. On the other hand, it is important to note that the inter-rater agree­ment assessing the dentate nucleus was moder­ate due to potential volume effects and artefacts. Interestingly, one 14-year-old patient demonstrat­ed radiological signs of neurodegeneration with­out having received treatment for LCH. In contrast to a previous study, we did not see a correlation be­tween radiological signs of neurodegeneration and pituitary involvement of LCH.22 We are currently designing a study which closely monitors children with LCH and radiological signs of neurodegener­ation over a long period of time in order to identify risk factors for patients who will ultimately devel­op clinical symptoms of neurodegeneration. Inter-rater variability To date, no study has evaluated the inter-rater variability of CNS abnormalities of children with LCH. This, however, is important, since a valid evaluation is the prerequisite for comparing clini­cal trials. Although our study demonstrated a high inter-rater agreement for most of the MRI-findings in which minimal criteria were met, the data sug­gest that cerebral imaging should be centrally ref­erenced as is the case for many pediatric tumors. The inter-rater agreement may even be significant­ly lower when MRI is performed outside a tertiary referral hospital or by a general radiologist. In ad­dition, it would be interesting to evaluate the inter-rater variability between different reference cent­ers, but this is beyond the scope of this study. Limitations of this study As in other studies in children with LCH, cerebral MRI was not performed in all patients, but only in those who had already developed or were at high risk for CNS complications, or according to the treating physician´s discretion. There was there­fore an inherent selection bias as not all LCH pa­tients had an MRI. Although we included only patients with a min­imum number of MR-image sequences, the study included imaging from various institutions with heterogeneous protocols making it difficult to as­sess all the CNS features of LCH. In addition, T1 pre-gadolinium imaging was not performed in most patients and it was therefore often not pos­sible to assess the posterior pituitary bright spot. Conclusions The majority of the CNS-images sent for reference did not follow previous guidelines5, and only one-third of the MRI could be included in this analysis. In order to improve the comparison of clinical trials in the future, all cerebral MRI should meet stand­ardized protocols, which include the assessment of the posterior pituitary bright spot, and should be centrally reviewed. The following protocol is rec­ ommended: axial T2w, Fluid Attenuated Inversion Recovery (FLAIR) and T1 w of the entire brain; axi­al and coronal T1W post contrast of the entire brain (at least one with fat saturation to evaluate lesions of skull). In addition, the hypothalamo-pituitary region should be evaluated with . 3 mm slice thick­ness with and without contrast enhancement. In conclusion, (1) CNS manifestations are fre­quent, but result in variable findings in children with LCH, (2) the assessment of the posterior pi­tuitary was not possible in most of the referred im­ages, and (3), although the inter-rater agreement between neuroradiologists was high, the MRI di­agnosis based on referred images was not suitable to rule out all LCH CNS manifestations. References 1. Delprat C, Arico M. Blood spotlight on Langerhans cell histiocytosis. Blood 2014; 124: 867-72. 2. Badalian-Very G, Vergilio JA, Degar BA, Rodriguez-Galindo C, Rollins BJ. Recent advances in the understanding of Langerhans cell histiocytosis. Br J Haematol 2012; 156: 163-72. 3. Vaiselbuh SR, Bryceson YT, Allen CE, Whitlock JA, Abla O. Updates on histio­cytic disorders. Pediatr Blood Cancer 2014; 61: 1329-35. 4. Grois N, Fahrner B, Arceci RJ, Henter JI, McClain K, Lassmann H, et al. Central nervous system disease in Langerhans cell histiocytosis. J Pediatr 2010; 156: 873-81, 881.e1. 5. Haupt R, Minkov M, Astigarraga I, Schafer E, Nanduri V, Jubran R, et al. Langerhans cell histiocytosis (LCH): guidelines for diagnosis, clinical work­up, and treatment for patients till the age of 18 years. Pediatr Blood Cancer 2013; 60: 175-84. 6. Sumida M, Barkovich AJ, Newton TH. Development of the pineal gland: measurement with MR. Am J Neuroradiol 1996; 17: 233-6. 7. Landis J, Koch G. An application of hierarchical kappa-type statistics in the assessment of majority agreement among multiple observers. Biometrics 1977; 33: 363-74. 8. Kuhn M. Building predictive models in R using the caret package. J Statistical Software 2008; 28: 1-26. 9. Nanduri VR, Bareille P, Pritchard J, Stanhope R. Growth and endocrine disorders in multisystem Langerhans’ cell histiocytosis. Clin Endocrinol (Oxf) 2000; 53: 509-15. 10. Grois N, Potschger U, Prosch H, Minkov M, Arico M, Braier J, et al. Risk factors for diabetes insipidus in langerhans cell histiocytosis. Pediatr Blood Cancer 2006; 46: 228-33. 11. Donadieu J, Rolon MA, Thomas C, Brugieres L, Plantaz D, Emile JF, et al. Endocrine involvement in pediatric-onset Langerhans’ cell histiocytosis: a population-based study. J Pediatr 2004; 144: 344-50. 249 12. Haupt R, Nanduri V, Calevo MG, Bernstrand C, Braier JL, Broadbent V, et al. Permanent consequences in Langerhans cell histiocytosis patients: a pilot study from the Histiocyte Society-Late Effects Study Group. Pediatr Blood Cancer 2004; 42: 438-44. 13. Tien R, Kucharczyk J, Kucharczyk W. MR imaging of the brain in patients with diabetes insipidus. Am J Neuroradiol 1991; 12: 533-42. 14. Fahrner B, Prosch H, Minkov M, Krischmann M, Gadner H, Prayer D, et al. Long-term outcome of hypothalamic pituitary tumors in Langerhans cell histiocytosis. Pediatr Blood Cancer 2012; 58: 606-10. 15. Grois N, Prosch H, Waldhauser F, Minkov M, Strasser G, Steiner M, et al. Pineal gland abnormalities in Langerhans cell histiocytosis. Pediatr Blood Cancer 2004; 43: 261-6. 16. Chaudhary V, Bano S, Aggarwal R, Narula MK, Anand R, Solanki RS, et al. Neuroimaging of Langerhans cell histiocytosis: a radiological review. Jpn J Radiol 2013; 31: 786-96. 17. Sener RN. The pineal gland: a comparative MR imaging study in children and adults with respect to normal anatomical variations and pineal cysts. Pediatr Radiol 1995; 25: 245-8. 18. Warmuth-Metz M, Gnekow AK, Muller H, Solymosi L. Differential diagnosis of suprasellar tumors in children. Klin Padiatr 2004; 216: 323-30. 19. Prayer D, Grois N, Prosch H, Gadner H, Barkovich AJ. MR imaging presenta­tion of intracranial disease associated with Langerhans cell histiocytosis. Am J Neuroradiol 2004; 25: 880-91. 20. D’Ambrosio N, Soohoo S, Warshall C, Johnson A, Karimi S. Craniofacial and intracranial manifestations of langerhans cell histiocytosis: report of findings in 100 patients. Am J Roentgenol 2008; 191: 589-97. 21. Prosch H, Grois N, Wnorowski M, Steiner M, Prayer D. Long-term MR imaging course of neurodegenerative Langerhans cell histiocytosis. Am J Neuroradiol 2007; 28: 1022-8. 22. Wnorowski M, Prosch H, Prayer D, Janssen G, Gadner H, Grois N. Pattern and course of neurodegeneration in Langerhans cell histiocytosis. J Pediatr 2008; 153: 127-32. 250 research article Correlation of diffusion MRI with the Ki-67 index in non-small cell lung cancer Adem Karaman1, Irmak Durur-Subasi1, Fatih Alper1, Omer Araz2, Mahmut Subasi3, Elif Demirci4, Mevlut Albayrak4, Gökhan Polat1, Metin Akgun2, Nevzat Karabulut5 1 Department of Radiology, Ataturk University, Medical Faculty, Erzurum, Turkey 2 Department of Pulmonary Diseases, Ataturk University, Medical Faculty, Erzurum, Turkey 3 Department of Thoracic Surgery, Erzurum Regional Training and Research Hospital, Erzurum, Turkey 4 Department of Pathology, Ataturk University, Medical Faculty, Erzurum, Turkey 5 Department of Radiology, Pamukkale University, Medical Faculty, Denizli, Turkey Radiol Oncol 2015; 49(3): 250-255. Received 11 March 2015 Accepted 9 July 2015 Correspondence to: Assist. Prof. Irmak Durur-Subasi, M.D., Department of Radiology, Ataturk University, Medical Faculty, Erzurum, Turkey. Phone: +90 533 460 386; Fax: +90 442 236 1301; E-mail: irmakdurur@yahoo.com Disclosure: No potential conflicts of interest were disclosed. Background. The primary objective of the study was to evaluate the association between the minimum apparent diffusion coefficient (ADCmin) and Ki-67, an index for cellular proliferation, in non-small cell lung cancers. Also, we aimed to assess whether ADCmin values differ between tumour subtypes and tissue sampling method. Methods. The patients who had diffusion weighted magnetic resonance imaging (DW-MRI) were enrolled retrospec­tively. The correlation between ADCmin and the Ki-67 index was evaluated. Results. Ninety three patients, with a mean age 65 ± 11 years, with histopathologically proven adenocarcinoma and squamous cell carcinoma of the lungs and had technically successful DW-MRI were included in the study. The numbers of tumour subtypes were 47 for adenocarcinoma and 46 for squamous cell carcinoma. There was a good negative correlation between ADCmin values and the Ki-67 proliferation index (r = -0.837, p < 0.001). The mean ADCmin value was higher and the mean Ki-67 index was lower in adenocarcinomas compared to squamous cell carcinoma (p < 0.0001). There was no statistical difference between tissue sampling methods. Conclusions. Because ADCmin shows a good but negative correlation with Ki-67 index, it provides an opportunity to evaluate tumours and their aggressiveness and may be helpful in the differentiation of subtypes non-invasively. Key words: diffusion weighted-magnetic resonance imaging; apparent diffusion coefficient; Ki-67 index; adenocar­cinoma; squamous cell carcinoma Introduction Diffusion weighted magnetic resonance imaging (DW-MRI) is a promising MRI technique used in the evaluation of lung tumours. It has been in­creasingly used for the detection, differential di­agnosis and evaluation of tumour characteristics, including grading and prediction of the therapeu­tic response.1-7 DW-MRI is a functional imaging technique that reveals physiological information by quantifying the diffusion of water molecules in tissues. The extent of this diffusion is measured using the apparent diffusion coefficient (ADC). Malignant tissues tend to have a lower ADC and demonstrate higher signal intensity on a DW-MRI image due to their increased cellularity and larger nuclei with abundant macromolecular proteins.8,9 The Ki-67 protein (also known as MKI67) is a cellular proliferation marker. During interphase, the Ki-67 antigen can only be detected within the cell nucleus; however, in mitosis, most of the Ki-67 is relocated to the surface of the chromosomes. Ki­67 protein is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent in 251 resting cells (G0). The Ki-67 proliferation index, one of the biological markers used in histopathological evaluation, is an important criterion in the differ­entiation of benign and malignant tumours.10-12 It is also correlated with the clinical course of cancer and has been shown to have prognostic value for treatment response, tumour recurrence and surviv­al in brain, breast, bladder and prostate tumours, meningioma and nephroblastoma.13-19 The Ki-67 index has also been used routinely in the evalua­tion of lung tumours and has been shown to be an important prognostic factor for lung cancer.3,6,20-27 Although a few studies have evaluated the associa­tion of ADC with Ki-67 index in lung tumours3,6, no study has previously investigated differences in the ADC/Ki 67 correlation in different tumour subtypes. In this study, our primary objective was to evaluate whether there is an association between the minimum ADC (ADCmin), determined on DW­MRI, and Ki-67, which is a cellular proliferative index. Our secondary aim was to assess whether ADCmin values differ between the adenocarcino­mas and squamous cell carcinomas of the lungs and also differ according to the pathologic sam­pling method used, surgical excision specimen and biopsied material. Methods Study population Between January 2012 and December 2013, records for 104 consecutive patients with histopathologi­cally proven primary adenocarcinoma and squa­mous cell carcinoma of the lungs, and who had technically successful images on DW-MRI were retrieved from the hospital’s pathology database. The patients who were previously treated (n = 5) and\or had an interval of more than 15 days be­tween DW-MRI and biopsy (n = 6) were excluded from the study. All measurements, including cal­culation of Ki-67 index and ADCmin values, were done in the same lesion for each patient. The proto­col of the retrospective study was approved by the institutional ethics committee and the requirement for informed consent was waived. Imaging technique, DW-MRI It was performed with a 3 tesla scanner (MAGNETOM Skyra, Siemens Healthcare, Erlangen, Germany). Conventional MRI included an axial T1-weighted sequence (repetition time, 104 ms; echo time, 4.92 ms; 1 excitation) and an axial T2-weighted sequence (repetition time, 1400 ms; echo time, 101 ms; 1 excitation). Breath-free DW-MRI was performed in the axial plane using a single-shot, spin-echo echo-planar imaging se­quence with the following parameters: repetition time, 6500 ms; echo time, 61 ms; real spatial resolu­tion in the phase-encoding direction, 3.7 mm; flip angle, 900; diffusion gradient encoding in three or­thogonal directions; b value b = 50, b = 400 and b = 800 s/mm2; field of view, 380 mm x 380 mm x 310 mm; matrix size, 113 x 192; slice thickness, 6 mm; section gap, 0 mm; 2 signals acquired. Image analysis We analysed the lesions using DW-MRI images in association with T1- and T2-weighted images in or­der to identify accurately. The ADC of the tumour was then calculated to quantitatively analyse the degree of diffusion, using the following formula: ADC = -ln(S/S0) / (b-b0), where S0 and S are the signal intensities, obtained at three different diffu­sion gradients (b = 50, b = 400 and b = 800 s/mm2). The ADC maps were reconstructed at a worksta­tion. While establishing the size and region for the ROI, positioning in the larger area was considered in order to minimize the effect of region on hemo­dynamic inhomogeneity of tumour by avoiding necrotic, cystic or calcific areas by referring to T2 and T1-weighted images.28,29 The ADCmin values within the ROI were then used in statistical analy­ses (Figure 1). In analyses workstation (Syngo Via Console, software version 2.0, Siemens AG Medical Solutions, Erlangen, Germany) was used. Calculation of Ki-67 index Archived paraffin blocks belonging to the patients were transferred to polylysine glass slides with 4-micron thick sections. Immunochemistry was performed using a Lecia Bond-max automated immunostainer (Leica Microsystems, Newcastle, UK), as described manufacturers protocol. For Ki­67 staining, Ki-67 antibody (NCL-L-Ki67-MM1, monoclonal, 1:60, Novocostra, Newcastle, UK) was used. The sections prepared for examination were evaluated by two pathologists who were blinded to each-other. Firstly, ten areas having highest ex­pression of Ki-67 were determined at low magnifi­cation. Then, these areas were further analysed at a single high power field (400 x magnification). Ki­67 expression was defined as the percent of Ki-67­positive tumour cells divided by the total number 252 of tumour cell within one high power field.26,30 In the last step, Ki-67 index was calculated as the av­erage percentage of those fields. Statistical Analysis Analyses were performed using IBM SPSS 20.0 for Mac software. The correlation between ADCmin and the Ki-67 index was evaluated using Spearman’s correlation coefficient. Mann-Whitney U tests were used to assess the difference between the ADCmin and the Ki-67 index for the different tumour sub­types. A p value of less than 0.05 was considered statistically significant. Results Ninety three patients, with a mean age 65 ± 11 years ranged between 43 and 84, with histopatho­logically proven primary adenocarcinoma (n = 47) and squamous cell carcinoma (n = 46) of the lungs and had technically successful DW-MRI were in­cluded in the study. Histopathological diagnoses were obtained through transthoracic or transbron­chial biopsy in 65 subjects and 28 patients under­went surgery. The mean ADCmin value for all the lesions was 0.77 ± 0.15 x 10-3 mm2/sec (range, 0.50–1.00 x 10-3 mm2/sec). The mean ADCmin value for adenocarci­nomas was 0.83 ± 0.12 x 10-3 mm2/sec and that of squamous cell carcinomas was 0.70 ± 0.16 x 10-3 mm2/sec; there was a significant difference be­tween these values (p < 0.0001). The mean Ki-67 was 43.5 ± 22.2 for all the tumours (range, 5–96), with a mean of 30.8 ± 14.1 for adenocarcinomas and 55.9 ± 21.8 for squamous cell carcinoma; the differ­ence between tumour subtypes was significant (p < 0.0001). There was a negative correlation between ADCmin values and the Ki-67 proliferation index (p < 0.001, r = -0.837) (Figure 2). The ADCmin values were lower in the cases with higher Ki-67 grades. The mean ADCmin values and Ki-67 index for ade­nocarcinomas and squamous cell carcinomas of the lung are shown in Figure 3. There was no statistical difference of Ki-67 and ADCmin values between bi­opsied material and surgical specimen. The mean Ki-67 was 45.3 ± 22.8 vs 39.3 ± 19.8 and the mean value was 0.76 ± 0.16x10-3 vs 0.78 ± 0.14 x 10-3 ADCmin for biopsied material and surgical specimen, re­spectively. In the comparative evaluation of corre­lation between ADCmin and the Ki-67 proliferation index that measured either in surgical specimen or biopsied material, the Ki-67 index of surgical speci­mens was slightly better correlated with ADCmin values without statistical significance (r = -0.870 vs. -0.617) compared to biopsied material. 253 Discussion Our results showed that there is a negative cor­relation between the ADCmin and the Ki-67 index of lung cancers, which reflects aggressiveness of a tumour. ADCmin values for adenocarcinomas were higher than those for squamous cell carcino­mas. This finding indicates that ADCmin may have a role in discriminating adenocarcinomas from squamous cell carcinomas, as well as being used for evaluating the aggressiveness of the tumour. Also, a low ADCmin value can potentially be used as a non-invasive surrogate biomarker for the Ki-67 index for the evaluation of lung tumour character­istics, regardless of subtype. Lung cancer is the leading cause of cancer-re­lated deaths.31 Until now, the Ki-67 proliferation index, reflecting aggressiveness of a tumour has been used to determine the prognosis. Malignant tumours are characterized by increased Ki-67 pro­liferation index due to their cellularity, larger nu­clei with more abundant macromolecular proteins, a larger nuclear/cytoplasmic ratio and less extracel­lular space relative to normal tissue. As these char­acteristics also restrict the diffusion of water mole­cules, ADCmin decreases in malignant tumours.8,9,32 Because ADCmin is found to have stronger cor­relation with Ki-67 index compared to ADCmean, we used ADCmin in our study.15 Apparent diffusion co­efficient can be used in the non-invasive assessment of suspicious masses, for example, to differentiate metastatic lymph nodes from those that are benign when they cannot be differentiated by size criteria.5 ADC values also correlate with tumour grades.4,17,18 Recent studies have shown that ADC may be more useful than FDG-PET in the differentiation of ma­lignant tumours from benign lesions3,6 and the new approaches using PET\MRI may provide more promising results in the future.33 Among primary lung cancers, ADC values are usually low in cases with small cell carcinomas, but the values for ad­enocarcinomas and squamous cell carcinomas are usually similar.3,4 However Matoba et al. stated that ADCs of well-differentiated adenocarcinoma appear to be higher than those of other histologic lung carcinoma types.23 Our findings demonstrate that adenocarcinomas showed higher ADC values than squamous cell carcinomas, and had weaker staining diffusivity and intensity of Ki-67. A high Ki-67 and low ADCmin value indicates that a tumour has a high proliferation rate. Ki-67 values obtained using an invasive method reflect only the level in the sampled tissue; this is a particu­lar problem when using biopsy. Since lung carci­nomas are not always homogenous, the biopsy site can influence the results. This could be reflected in the fact that in our study the correlation between and Ki-67 proliferation index was stronger ADCmin for surgical than for biopsy samples. Unlike these invasive sampling methods, ADCmin values ob­tained by DW-MRI in a non-invasive manner can be calculated from anywhere in the tumour, provid­ing an entire and reproducible assessment of the tu­mour. Furthermore, since the region with the low­est ADCmin value is likely to be the most aggressive portion.17,34 DWI could also help in the selection of an appropriate biopsy site within the tumour. 254 An association between the ADC value and the Ki-67 index has been shown for various kinds of tu­mours2,14-18,34-38, including lung cancer.3,6 Wang et al., in their study on DWI in pancreatic endocrine tu­mours, reported a correlation coefficient of -0.702, while Onishi et al. reported a correlation coefficient of -0.825 for mucinous breast carcinoma.15 Previous studies reporting ADC values of lung carcinoma have been conducted under various magnet strengths, and reported ADC values are lower in magnets with a stronger field. Matoba et al. reported mean ADC values of 1.63 × 10-3 mm2/ sec ± 0.5 (mean ± SD) for squamous cell carcinomas, 2.12 × 10-3 mm2/sec ± 0.6 for adenocarcinomas, 1.30 × 10-3 mm2/sec ± 0.4 for large-cell carcinomas, and 2.09 × 10-3 mm2/sec ± 0.3 for small-cell carcinomas, using a 1.5 T scanner. Usuda et al.6 found that ma­lignant nodules had a mean ADC of 1.27 ± 0.35 ×10­3 mm2/sec on a 1.5T system. Using a 3.0 T scanner, Zhang et al. reported that malignant pulmonary nodules had a mean ADC of 0.87.±.0.16 × 10-3 mm2/ sec. Similarly, we found a mean ADCmin of 0.77 ± 0.12 x 10-3 mm2/sec in our study conducted on a 3.0 T scanner. These values are lower than those were reported by the studies conducted using 1.5 T sys­tems.6,23 However, Kivrak et al. noted that ADC val­ues vary for different MRI systems with the same magnetic field strength (1.5 T).39 On the other hand, some authors reported that ADC values might not change for different organ systems under different magnetic fields.40 However, they only used healthy volunteers and neither pathologic conditions nor image quality was not assessed. Further work is still needed to investigate the effect of magnetic field strength on the ADC of different organ sys­tems. One of the strongest side of our study was that we used 3 tesla MRI, which has increased signal to noise ratio, spatial resolution, temporal resolution, etc. Thus, decreased imaging time increased pa­tients’ cooperation and we had better qualified im­ages. Our study had a few limitations. Our study population was relatively small and, although our results are robust, prospective studies with larger series are warranted to confirm our results. Additionally, to be able to generalize our results to all subtypes of lung cancer, such as small cell car­cinomas and the other subtypes of non-small cell lung cancer, which we had very limited number of such cases during the study period, need to be in­cluded in future studies. Because we had no data about survival of the cases, we could not conclude any association between ADCmin or Ki-67 and sur­vival. However, use of ADCmin may provide new insight to the evaluation of lung cancer including benign-malignant discrimination, the possibility of evaluation all lesions and lymph nodes non­invasively, even in the cases that tissue sampling is difficult, as well as predicting the prognosis of tumour by using it as a surrogate marker of Ki-67 index. In conclusion, our results suggested that ADCmin values were inversely correlated with Ki-67 index in non-small cell lung cancer and may be used as a surrogate marker of Ki-67 index in the evaluation of tumour aggressiveness with the advantage of its non-invasiveness and without requirement of tis­sue sampling of all the lesions. References 1. Yabuuchi H, Hatakenaka M, Takayama K, Matsuo Y, Sunami S, Kamitani T, et al. Non-small cell lung cancer: detection of early response to chemotherapy by using contrast-enhanced dynamic and diffusion-weighted MR imaging. Radiology 2011; 26: 598-604. 2. Wang Y, Chen ZE, Yaghmai V, Nikolaidis P, McCarthy RJ, Merrick L, et al. Diffusion-weighted MR imaging in pancreatic endocrine tumors corre­lated with histopathologic characteristics. J Magn Reson Imaging 2011; 33: 1071-9. 3. Zhang J, Cui LB, Tang X, Ren XL, Shi JR, Yang HN, et al. DW MRI at 3.0 T ver­sus FDG PET/CT for detection of malignant pulmonary tumors. Int J Cancer 2014; 134: 606-11. 4. Li F, Yu T, Li W, Zhang C, Cao Y, Su D, et al. Correlation of apparent diffusion coefficient with histologic type and grade of lung cancer. Zhongguo Fei Ai Za Zhi 2012; 15: 646-51. 5. Xu L, Tian J, Liu Y, Li C. Accuracy of diffusion-weighted (DW) MRI with background signal suppression (MR-DWIBS) in diagnosis of mediastinal lymph node metastasis of nonsmall-cell lung cancer (NSCLC). J Magn Reson Imaging 2014; 40: 200-5. 6. Usuda K, Sagawa M, Motono N, Ueno M, Tanaka M, Machida Y, et al. Diagnostic performance of diffusion weighted imaging of malignant and benign pulmonary nodules and masses: comparison with positron emission tomography. Asian Pac J Cancer Prev 2014; 15: 4629-35. 7. Türkbey B, Aras Ö, Karabulut N, Turgut AT, Akpinar E, Alibek S, et al. Diffusion-weighted MRI for detecting and monitoring cancer: a review of current applications in body imaging. Diagn Interv Radiol 2012; 18: 46-59. 8. Koh DM, Collins DJ. Diffusion-weighted MRI in the body: applications and challenges in oncology. AJR Am J Roentgenol 2007; 188: 1622-35. 9. Padhani AR, Liu G, Koh DM, Chenevert TL, Thoeny HC, Takahara T, et al. Diffusion-weighted magnetic resonance imaging as a cancer biomarker: consensus and recommendations. Neoplasia 2009; 11: 102-25. 10. Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol 2000; 182: 311-22. 11. Raikhlin NT, Bukaeva IA, Smirnova EA, Gurevich LE, Delektorskaia VV, Polotskii BE, et al. Significance of the expression of nucleolar argyrophilic proteins and antigen Ki-67 in the evaluation of cell proliferative activity and in the prediction of minimal (T1) lung cancer. Arkh Patol 2008; 70: 15-18. 12. Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 1984; 133: 1710-15. 13. Zhu L, Ren G, Li K, Liang ZH, Tang WJ, Ji YM, et al. Pineal parenchymal tumours: minimum apparent diffusion coefficient in prediction of tumour grading. J Int Med Res 2011; 39: 1456-63. 14. Choi SY, Chang YW, Park HJ, Kim HJ, Hong SS, Seo DY. Correlation of the apparent diffusion coefficiency values on diffusion-weighted imaging with prognostic factors for breast cancer. Br J Radiol 2012; 85(1016): e474-9. 255 15. Onishi N, Kanao S, Kataoka M, Iima M, Sakaguchi R, Kawai M, et al. Apparent diffusion coefficient as a potential surrogate marker for Ki-67 index in muci­nous breast carcinoma, J Magn Reson Imaging 2015; 41: 610-5. 16. Mesko S, Kupelian P, Demanes DJ, Huang J, Wang PC, Kamrava M. Quantifying the ki-67 heterogeneity profile in prostate cancer. Prostate Cancer 2013: 2013: 717080. 17. Kobayashi S, Koga F, Kajino K, Yoshita S, Ishii C, Tanaka H, et al. Apparent diffusion coefficient value reflects invasive and proliferative potential of bladder cancer. J Magn Reson Imaging 2014; 39: 172-8. 18. Tang Y, Dundamadappa SK, Thangasamy S, Flood T, Moser R, Smith T, et al. Correlation of apparent diffusion coefficient with Ki-67 proliferation index in grading meningioma. AJR Am J Roentgenol 2014; 202: 1303-8. 19. Martin B, Paesmans M, Mascaux C, Berghmans T, Lothaire P, Meert AP, et al. Ki-67 expression and patients survival in lung cancer: systematic review of the literature with meta-analysis. Br J Cancer 2004; 91: 2018-25. 20. Usuda K, Zhao XT, Sagawa M, Aikawa H, Ueno M, Tanaka M, et al. Diffusion-weighted imaging (DWI) signal intensity and distribution represent the amount of cancer cells and their distribution in primary lung cancer. Clin Imaging 2013; 37: 265-72. 21. Ohno Y, Koyama H, Yoshikawa T, Matsumoto K, Aoyama N, Onishi Y, et al. Diffusion-weighted MRI versus 18F-FDG PET/CT: performance as predictors of tumor treatment response and patient survival in patients with nonsmall cell lung cancer receiving chemoradiotherapy. AJR Am J Roentgenol 2012; 198: 75-82. 22. Tanaka R, Horikoshi H, Nakazato Y, Seki E, Minato K, Iijima M, et al. Magnetic resonance imaging in peripheral lung adenocarcinoma: correlation with histopathologic features. J Thorac Imaging 2009; 24: 4-9. 23. Matoba M, Tonami H, Kondou T, Yokota H, Higashi K, Toga H, et al. Lung carcinoma: diffusion-weighted MR imaging—preliminary evaluation with apparent diffusion coefficient. Radiology 2007; 243: 570-7. 24. Martin B, Paesmans M, Mascaux C, Berghmans T, Lothaire P, Meert AP, et al. Ki-67 expression and patients survival in lung cancer: systematic review of the literature with meta-analysis. Br J Cancer 2004; 91: 2018-25. 25. Warth A, Cortis J, Soltermann A, Meister M, Budczies J, Stenzinger A, et al. Tumour cell proliferation (Ki-67) in non-small cell lung cancer: a critical reap­praisal of its prognostic role. Br J Cancer 2014; 111: 1222-9. 26. Tabata K, Tanaka T, Hayashi T, Hori T, Nunomura S, Yonezawa S, et al. Ki-67 is a strong prognostic marker of non-small cell lung cancer when tissue heterogeneity is considered. BMC Clin Pathol 2014; 14: 23-30. 27. Ahn HK, Jung M, Ha SY, Lee JI, Park I, Kim YS, et al. Clinical significance of Ki-67 and p53 expression in curatively resected non-small cell lung cancer. Tumour Biol 2014; 35: 5735-40. 28. Alper F, Kurt AT, Aydin Y, Ozgokce M, Akgun M. The role of dynamic magnetic resonance imaging in the evaluation of pulmonary nodules and masses. Med Princ Pract 2013; 22: 80-6. 29. Karaman A, Kahraman M, Bozdogan E, Alper F, Akgün M. Diffusion magnetic resonance imaging of thorax. Tuberk Toraks 2014; 62: 215-30. 30. Araz O, Demirci E, Ucar EY, Calik M, Karaman A, Durur-Subasi I, et al. Roles of Ki-67, p53, transforming growth factor-ß and lysyl oxidase in the metastasis of lung cancer. Respirology 2014; 19: 1034-9. 31. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin 2013; 63: 11-30. 32. Zhang Z, Zhou Y, Qian H, Shao G, Lu X, Chen Q, et al. Stemness and induc­ing differentiation of small cell lung cancer NCI-H446 cells. Cell Death Dis 2013; 16: e633. 33. Schaarschmidt BM, Buchbender C, Nensa F, Grueneien J, Gomez B, Köhler J, et al. Correlation of the apparent diffusion coefficient (ADC) with the standardized uptake value (SUV) in lymph node metastases of non-small cell lung cancer (NSCLC) patients using hybrid 18F-FDG PET/MRI. PLoS One 2015; 10(1): e0116277. 34. Yoshida S, Kobayashi S, Koga F, Ishioka J, Ishii C, Tanaka H, et al. Apparent diffusion coefficient as a prognostic biomarker of upper urinary tract cancer: a preliminary report. Eur Radiol 2013; 23: 2206-14. 35. Yoshida S, Koga F, Kobayashi S, Ishii C, Tanaka H, Tanaka H, et al. Role of diffusion weighted magnetic resonance imaging in predicting sensitivity to chemoradiotherapy in muscle-invasive bladder cancer. Int J Radiat Oncol Biol Phys 2012; 83: e21-e7. 36. Wieduwilt MJ, Valles F, Issa S, Behler CM, Hwang J, McDermott M, et al. Immunochemotherapy with intensive consolidation for primary CNS lym­phoma: a pilot study and prognostic assessment by diffusion-weighted MRI. Clin Cancer Res 2012; 18: 1146-55. 37. Srinivasan A, Chenevert TL, Dwamena BA, Eisbruch A, Watcharotone K, Myles JD, et al. Utility of pretreatment mean apparent diffusion coefficient and apparent diffusion coefficient histograms in prediction of outcome to chemoradiation in head and neck squamous cell carcinoma. J Comput Assist Tomogr 2012; 36: 131-7. 38. Pope WB, Lai A, Mehta R, Qiao J, Young JR, Xue X, et al. Apparent diffusion coefficient histogram analysis stratifies progression-free survival in newly diagnosed bevacizumab-treated glioblastoma. AJNR Am J Neuroradiol 2011; 32: 882-9. 39. Kivrak AS, Paksoy Y, Erol C, Koplay M, Özbek S, Kara F. Comparison of ap­parent diffusion coefficient values among different MRI platforms: a multi­center phantom study. Diagn Interv Radiol 2013; 19: 433-7. 40. Rosenkrantz AB, Oei M, Babb JS, Niver BE, Taouli B. Diffusion-weighted imaging of the abdomen at 3.0 Tesla: image quality and apparent diffusion coefficient reproducibility compared with 1.5 Tesla. J Magn Reson Imaging 2011; 33: 128-35. 256 research article The influence of cytokine gene polymorphisms on the risk of developing gastric cancer in patients with Helicobacter pylori infection David Stubljar1, Samo Jeverica1, Tomislav Jukic2, Miha Skvarc1, Tadeja Pintar3, Bojan Tepes4, Rajko Kavalar5, Borut Stabuc6, Borut Peterlin7, Alojz Ihan1 1 Institute of Microbiology and Immunology, Ljubljana, Slovenia 2 Medical faculty of Osijek, Osijek, Croatia 3 Department of Abdominal Surgery, University Clinical Centre Ljubljana, Ljubljana, Slovenia 4 Abacus Medico Diagnostic Centre Rogaska, Rogaska Slatina, Slovenia 5 Department of Pathology, University medical Centre Maribor, Maribor, Slovenia 6 Department of Gastroenterology, University Medical Centre Ljubljana, Ljubljana, Slovenia 7 Clinical Institute of Medical Genetics, University Clinical Centre Ljubljana, Ljubljana, Slovenia Radiol Oncol 2015; 49(3): 256-264. Received 24 June 2014 Accepted 27 August 2014 Correspondence to: David Štubljar, University of Ljubljana, Faculty of Medicine, Institute of Microbiology and Immunology, Zaloška 4, Ljubljana, Slovenia. E-mail: d.stubljar@gmail.com Disclosure: No potential conflicts of interest were disclosed. Background. Helicobacter pylori infection is the main cause of gastric cancer. The disease progression is influenced by the host inflammatory responses, and cytokine single nucleotide polymorphisms (SNPs) may have a role in the course of the disease. The aim of our study was to investigate proinflammatory cytokine polymorphisms, previously associated with the development of gastric cancer, in a Slovenian population. Patients and methods. In total 318 patients and controls were selected for the study and divided into three groups: (i) patients with gastric cancer (n = 58), (ii) patients with chronic gastritis (n = 60) and (iii) healthy control group (n = 200). H. pylori infection in patient groups was determined by serology, histology and culture. Four proinflammatory gene polymorphisms were determined (IL-1ß, IL-1ra, TNF-., TLR-4) in all subjects. Results. We found a statistically significant difference between males and females for the groups (p = 0.025). Odds ratio (OR) for gastric cancer risk for females was 0.557 (95% confidence interval [CI]: 0.233.1.329) and for chronic gastritis 2.073 (95% CI: 1.005.4.277). IL-1B-511*T/T homozygous allele for cancer group had OR = 2.349 (95% CI: 0.583.9.462), heterozygous IL-1B-511*T had OR = 1.470 (95% CI: 0.583.3.709) and heterozygotes in TNF-A-308 genotype for chronic gastritis had OR = 1.402 (95% CI: 0.626.3.139). Other alleles had OR less than 1. Conclusions. We could not prove association between gastric cancer and chronic gastritis due to H. pylori in any cytokine SNPs studied in Slovenian population. Other SNPs might be responsible besides infection with H. pylori for the progression from atrophy to neoplastic transformation. Key words: Helicobacter pylori; gene polymorphisms; gastric cancer; chronic gastritis Introduction Gastric cancer is the fourth most commonly diag­nosed cancer and the second most common cause of cancer-related death worldwide.1 The incidence of gastric cancer in Slovenia is among the high­est in Europe with the crude incidence rate in male population of 28.6/100 000.2-4 Gastric cancer is multifactorial disease. Environmental and host genetic factors influence the development of gas­tric cancer. The most important is Helicobacter py­lori (H. pylori) infection. It is belived that roughly 65.80% of gastric cancers are associated with H. pylori infection. However, only a minority (1.2%) of infected individuals will develop gastric cancer during their lifetime.5,6 Gastric carcinogenesis is a 257 multistep process that starts with chronic active gastritis and continues through the development of gastric atrophy, and metaplasia, to reach gas­tric cancer stage at the end of that process, lasting tipically between 30.50 years.7-10 In addition to H. pylori infection, several host genetic factors are im­portant for the development of gastric cancer, es­pecially several single nucleotide polymorphisms (SNPs) and/or point mutations in genes that affect gastric acid secretion and innate immune response to infection.11-14 Polymorphisms in cytokine genes may influence the level of the cytokine production by the host, and consequently influence the dis­ease outcome.15 The immune response to H. pylori is important for the development of gastric cancer due to the recognition of pathogenic elements and induced synthesis and secretion of inflammatory cytokines, resulting inflammation, what can lead to severe gastric immunopathology and cancer.16 Interleukin 1b (IL-1b) is the main cytokine se­creted in response to H. pylori infection. It has a strong pro-inflamatory activity and inhibits gastric acid secretion. IL-1b is 100-times more potent in­hibitor of acid secretion than proton pump inhibi­tors.17 Inhibition of acid secretion may lead to the spread of bacteria from the antrum to the corpus, and consequently the development of corpus pre­dominant gastritis which further leads to the devel­opment of gastric cancer.18,19 Three polimorphisms were described in the IL-1B gene at positions -31, -511 and +3954 from the transcription start site.18,20 IL-1B-31*C and IL-1B-511*T alleles are associated with decreased acidity in the stomach (hypochlo­hydria) in response to the infection with H. pylori.18 IL-1b receptor antagonist (IL-1ra) polymorphisms have also been associated with the level of IL-1b secretion. Genotype IL-1RN*2 is associated with higher secretion of IL-1b, most probably through the reduction of its receptor antagonist IL-1ra.20,21 Tumor necrosis factor-a (TNF-.) is a central mediator of the immune response. Several poly­morphisms are known in the promoter region of TNF-A gene of which -308*G>A was associated with increased production of TNF-. in response to the infection, and increased gastric cancer risk.22-24 El-Omar et al.25 and Machado et al.26 found that sub­ject with this polymorphism have almost two-fold increased risk of gastric cancer. Recently, a functional polymorphism at the po­sition +896, in exon 4 of the Toll-like receptor-4 (TLR­4) gene, has been described. This A>G transition results in an alteration of the extracellular domain of TLR-4, that causes hyporesponsiveness to LPS, reduced epithelial TLR-4 density and exaggerated inflammatory cytokine response.27 A recent study has reported an association of TLR-4 gene poly­morphisms with gastroduodenal diseases such as gastric atrophy and hypochlorhydria.28,29 TLR-4 substitution was associated with noncardia gastric cancer.30,31 The aim of our study was to determine the prev­alence of the selected pro-inflammatory cytokine polymorphisms in the Slovenian population of pa­tients with gastric cancer and chronic gastritis, and compare its prevalence with the prevalence in the normal healthy population, to see if high incidence of gastric cancer in Slovenian population could be, at least partially, attributed to the higher preva­lence of those proinflammatory polymorphisms in the genes for IL-1ß, IL-1ra, TNF-. and TLR-4. Patients and methods Patients In total 318 patients and controls were included in the study devided into three groups: (i) consecutive patients with gastric cancer (n = 58), (ii) consecutive patients with chronic gastritis due to H. pylori (n = 60) and (iii) healthy control group (n = 200). Study was conducted as a case-control study, where the cancer patients represented one group and the gas­tritis patients represented the other group. Subjects for the healthy control group were randomly se­lected from the pool of representative blood sam­ples of Slovenian healthy adults, to be matched for age and sex. All subjects were informed about the inclusion in the study and agreed to it in writing form. National medical ethics committee reviewed and cleared the protocol of the study. Histopathology, serology and culture Patients in the gastric cancer group had the his­tological type of cancer determined using the Lauren’s classification that differentiates among intestinal, diffuse and mixed or indetermined type adenocarcinoma. In the group of patients with chronic gastritis two biopsies were obtained from corpus and antrum, and the histological diagnosis was determined in accordance with the Huston modification of Sydney classification for gastri­ tis.7,32 Serological confirmation of H. pylori infection in the gastric cancer group was confirmed by the quantitative IgG ELISA test GAP®-IgG (Biomerica, USA) from human serum. Test was performed in accordance to instructions by the manufacturer.33 TABLE 1. Primers and probes sequences used in the KASP assays FAM IL-1ß -511 C/T 5’-GGGTGCTGTTCTCTGCCTCG-3’ VIC 5’-GCCCCAGCCAAGAAAGGTCAATTTT-3’ 5’-GGGTGCTGTTCTCTGCCTCA-3’ FAM TNF-. -308 G/A 5’-GGAGGCTGAACCCCGTCCT-3’ VIC 5’-GAGGCAATAGGTTTTGAGGGGCAT-3’ 5’-GAGGCTGAACCCCGTCCC-’3 FAM TLR-4 +896 A/G 5’-GCATACTTAGACTACTACCTCGATGA-3’ VIC 5’-CACTCACCAGGGAAAATGAAGAAACATT-3’ 5’-CATACTTAGACTACTACCTCGATGG-3’ H. pylori culture was performed in the gastritis group from two biopsy samples of antrum and cor­pus, respectively. Biopsy samples were transport­ed to the laboratory in Portagerm pylori transport medium (Biomerieux, France). In the laboratory, samples were homogenized in 1 mL of phosphate buffer (PBS) and 0.5 mL of the homogenate was in­oculated onto two selective agar plates: Pylori agar (Biomerieux, France) and Brucella agar supple­mented with human blood and antibiotic mixture (BBL, USA). Culture media were incubated at 37 °C for 72 hours in microaerophilic conditions. The identification of typical colonies was confirmed us­ing Gram stain and the proof of enzymes: urease, catalase and oxidase. Genotyping Genomic DNA was extracted from the whole blood samples with EDTA using automated system for DNA isolation Magna Pure Compact Nucleic Acid Isolation Kit I (Roche Applied Science, Germany) on fully automated platform MagNa Pure Compact System in accordance to the instruc­tions by the manufacturer.34 Complete nucleotide sequences of individual genes for inflammatory cytokine IL-1ß (rs16944), TNF-. (rs1800629) and TLR-4 (rs4986790) were looked into online databas­es National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov) and ENSEMBLE (www.ensembl.org). The sequences were examined with the help of the software package Vector NTI Advance 11 (Invitrogen, Carlsbad, CA, USA).21,25,26 Polymorphisms genotyping was performed using the KASP technology (KBioscience competitive Allele-Specific PCR) using primers and reagents Kasp On Demand (KOD) (KBioscience, UK). 120 bp long reference sequences were sent to the man­ufacturer, upon which the appropriate primers and probes were designed (Table 1). The amplification of genomic DNA and the detection of polymorphisms were performed us­ing the real-time polymerase chain reaction (PCR) apparatus LightCycler 480II (Roche Diagnostics GmbH, Germany). A touchdown protocol provid­ed by the manufacturer was used: 94 °C for 15 min; 10 cycles of 94 °C for 10 s, 61 °C for 60 s (the anniling temperature dropped 0.6 °C per cycle to reach the annealing temperature of 55 °C) then; 26 cycles of 94 °C for 10 s, 55 °C for 60 s. IL-1RN gene contains a variable number of 86 base pair long tandem re­peats (VNTR).19 Genomic DNA was amplified and PCR products were separated by the 1.5% agarose gel electrophoresis. Primers to detect IL-1RN*2/2 (TIB Molbiol, Germany) were used. We have used forward primer: 5’-CCCCTCAGCAACACTCC-3’, reverse primer: 5’-GGTCAGAAGGGCAGAGA-3’. Cycling conditions for the PCR were 95 °C for 15 min; 30 cycles of 94 °C for 30 s and 61 °C for 30 s; 72 °C for 60 s and 15 min at 72 °C. PCR reaction with the final volume of 25 µl was used, containing 12.5 µl of twice the reaction mixture of HotStartTaq Plus, 0.75 µl of each primer with a concentration of 10 µM, 8.5 µl of ddH2O and 2.5 µl of sample DNA. There are 5 versions of alleles. Allele 1, 2, 3, 4 and 5 carries 4, 2, 5, 3 and 6 repeats, respectively.20,35 Due to easier statistical analysis the allele polymor­phisms were divided into short and long, the short allele being allele 2 and the long allele being those with 3 repeats or more (alleles 1, 3, 4, and 5).26 Statistical analysis The SPSS Statistics 21 (IBM, USA) software pack­age was used for the statistical analysis. The Hardy-Weinberg equilibrium (HWE) of alleles in each individual locus was assessed. The degrees of freedom for HWE were calculated as the number of genotypes subtracted with the number of al­leles. If the value of the c2 was less than 3.84, the 259 TABLE 2. Demographic profiles of subjects No. of subjects 32 (55%) 26 (45%) 51 (85%) 9 (15%) 200 Age (years) 52 ± 10 52 ± 10 52 ± 10 58 ± 13 49 ± 5 Gender Male 22 (38%) 18 (31%) 20 (33%) 2 (3%) 100 (50%) Female 10 (17%) 8 (14%) 31 (52%) 7 (12%) 100 (50%) TABLE 3. Pearson’s .2 analysis for association between frequencies of cytokine polymorphisms and patients with intestinal type gastric adenocarcinoma, atrophic chronic gastritis and healthy controls IL-1B -511 8.214 0.084 C/C 10 31.3% 30 58.8% 42 38.9% C/T 17 53.1% 18 35.3% 53 49.1% T/T 5 15.6% 3 5.9% 13 12.0% IL-1RN 4.377 0.357 L/L 17 53.1% 33 64.7% 63 58.3% L/2 13 40.6% 15 29.4% 32 29.6% 2/2 2 6.3% 2 3.9% 13 12.0% TNF-A -308 4.796 0.309 G/G 27 84.4% 36 70.6% 83 76.9% G/A 5 15.6% 15 29.4% 22 20.4% A/A 0 0.0% 0 0.0% 3 2.8% TLR-4 +896 3.355 0.500 A/A 30 93.8% 46 90.2% 90 83.3% A/G 2 6.3% 5 9.8% 17 15.7% G/G 0 0.0% 0 0.0% 1 0.9% Gender 7.355 0.025 M 22 68.8% 20 39.2% 60 55.6% F 10 31.3% 31 60.8% 48 44.4% F = female; M = male frequencies of the population were in HWE. For all genotypes, the homozygote of the common al­lele was used as the reference. The IL-1B, IL-1RN, TNF-A and TLR-4 genotype frequencies for each polymorphism were compared by 2-sided Pearson c2 test, to evaluate the genotype distributions of categorical variables between each group of cases and controls, and to see if there was any association between the tested variables. The odds ratios (ORs) and the 95% confidence interval (95% CI) were as­sessed using logistic regression analysis with the reference category being healthy controls. ORs for different groups were adjusted for sex only. Statistical differences were considered to be signifi­cant at a P value < 0.05. Results Patients with diagnosed chronic gastritis due to H. pylori and gastric cancer were investigated com­pared to healthy controls. The average age of in­dividuals and gender ratio were comparable in all groups (Table 2). We included 198 subjects and 260 TABLE 4. Genotype polymorphisms odds ratios (ORs) and 95% confidence intervals (CIs) for gastric cancer and atrophic gastritis subjects IL-1B -511 C/C 10 (31.3) reference 30 (58.8) reference C/T 17 (53.1) 1.470 0.583-3.709 0.414 18 (35.3) 0.489 0.228-1.050 0.067 T/T 5 (15.6) 2.349 0.583-9.462 0.230 3 (5.9) 0.416 0.099-1.757 0.233 IL-1RN L/L 17 (53.1) reference 33 (64.7) reference L/2 13 (40.6) 1.064 0.436-2.597 0.891 15 (29.4) 1.052 0.473-2.341 0.900 2/2 2 (6.3) 0.394 0.072-2.162 0.394 2 (3.9) 0.400 0.081-1.988 0.263 TNF-A -308 G/G 27 (84.4) reference 36 (70.6) reference G/A 5 (15.6) 0.704 0.236-2.099 0.528 15 (29.4)% 1.402 0.626-3.139 0.411 A/A 0 (0.0) 0 0 0 0 (0.0) 0 0 0 TLR-4 +896 A/A 30 (93.8) reference 46 (90.2) reference A/G 2 (6.3) 0.326 0.066-1.603 0.168 5 (9.8) 0.499 0.149-1.668 0.259 G/G 0 (0.0) 0 0 0 0 (0.0) 0 0 0 Gender M 22 (68.8) reference 20 (39.2) reference F 10 (31.3) 0.557 0.233-1.329 0.187 31 (60.8) 2.073 1.005-4.277 0.048 Reference category for groups was set to control group. Referent allele was common homozygote; F = female; M = male controls in the study meeting the necessary initial criteria: 108 healthy control subjects with no under­lying conditions, 32 patients with intestinal type of gastric adenocarcinoma and 58 patients with chron­ic gastritis and positive H. pylori infection were in­cluded and processed for statistical analysis. The genotype frequencies distribution among cytokine polymorphisms are presented in Table 3. Comparison of genotype frequencies between in­testinal adenocarcinoma group and atrophic gas­tritis group and healthy controls showed no sig­nificant difference (p > 0.05). P-value of 0.084 for IL-1ß showed closest statistical difference between the diagnosis severe progression and influence of genetic polymorphisms. However, there was a sta­tistically significant difference between males and females compared between the groups (p = 0.025) (Table 3). The sex-adjusted OR of gastric cancer among H. pylori positive subjects was 0.557 (95% CI: 0.233.1.329; p = 0.187) and of chronic gastritis 2.073 (95% CI: 1.005.4.277; p = 0.048). Males were taken as reference. In the gastric carcinoma patients, IL-1B-511*T/T homozygous allele represented 15.6% (5/32) of the case subjects, which was proportionally higher than in control group (12.0%; 13/108), however sta­tistically with an OR of 2.349 (95% CI: 0.583.9.462) was not confirmed. Carriers of heterozygous IL­1B-511*T allele in cancer group (53.1%, 17/32) also showed no difference against control group (49.1%, 53/108) despite the OR = 1.470 (95% CI: 0.583.3.709). For atrophic gastritis group there was no statistical­ly difference compared to control group (Table 4). Carriers of the proinflammatory IL-1B-511*T allele (both IL-1B-511T homozygotes and IL-1B-511 het­erozygotes) had also no increased risk for gastric cancer (OR = 1.570; 95% CI: 0.644.3.825) or chronic gastritis (OR = 0.480; 95% CI: 0.232.0.996). The as­sociated OR value was even smaller than for ho­mozygotes alone with low frequency of homozy­ gous controls (Table 4). According to Pearson’s .2 frequency distribution of IL-1B-511*T carriers were statistically significant in combination for specific diagnose (p = 0.021; F = 7.760) (data not shown). The observed associations between IL-1RN VNTR genotype carriers (IL-1RN*L/2) and the risk of gastric carcinoma or atrophic gastritis had mean­ingless OR = 1.064 (95% CI: 0.436.2.597), OR = 1.052 261 TABLE 5. Frequencies of genotype carriers, odds ratios (ORs) and 95% confidence intervals (CIs) for gastric cancer and atrophic gastritis subjects IL-1B -511 C/C 10 (31.3) reference 30 (58.8) reference T carrier 22 (68.7) 1.570 0.644-3.825 0.321 21 (41.2) 0.480 0.232-0.996 0.049 IL-1RN L/L 17 (53.1) reference 33 (64.7) reference 2 carrier 15 (46.9) 0.947 0.408-2.200 0.900 17 (33.3) 0.905 0.429-1.912 0.794 TNF-A -308 G/G 27 (84.4) reference 36 (70.6) reference A carrier 5 (15.6) 0.590 0.201-1.730 0.336 15 (29.4) 1.217 0.556-2.667 0.623 TLR-4 +896 A/A 30 (93.8) reference 46 (90.2) reference G carrier 2 (6.3) 0.318 0.068-1.487 0.145 5 (9.8) 0.435 0.135-1.407 0.165 Gender M 22 (68.8) reference 20 (39.2) reference F 10 (31.3) 0.561 0.237-1.329 0.189 31 (60.8) 2.068 1.015-4.213 0.045 Reference category for groups was set to control group. Referent allele was common homozygote; F = female; M = male (95% CI: 0.473.2.341), respectively. Furthermore short allele had no statistical association with de­veloping the disease. In a logistic regression model that included the other genetic markers (TNF-A and TLR-4), there were no statistical significant differences adjusted to control group and common alleles. Heterozygotes in TNF-A-308 genotype had also no statistically significant excess for the chronic gastri­tis (OR = 1.402; 95% CI: 0.626.3.139) (Table 3). TNF­A-308*A carriers (both TNF-A-308*A homozygotes and TNF-A-308 heterozygotes) had even less prob­ability with an OR of 1.217 (95% CI: 0.556.2.667) for gastritis (Table 5). Pearson correlation model for all IL-1B-511, IL-1RN VNTR, TNF-A-308 and TLR-4+896 geno­types was performed and showed no statistical significance between them (p > 0.01). However correlation between IL-1B and IL-1RN was found (Pearson’s R = 0.300; p < 0.001). Furthermore, there was no evidence of the association between the 55 (28.9%) carriers of IL-1B-511*T and IL-1RN*2 al­leles (OR = 1.489; 95% CI: 0.660-3.361) for the risk of gastric cancer (data not shown). There was also no association for chronic gastritis. Moreover, com­bined T and 2 allele carriers had even lesser risk as­sociated with developing gastric cancer than each allele separately. Discussion This is the first study on Slovenian population that checked variants or polymorphisms in genes re­sponsible for cytokine secretion that may contrib­ute to the different outcomes of infection and the development of gastric lesions. Our results showed that there was a statistical difference between gen­ders on the outcome of infection with H. pylori (p = 0.025). Males were more predominant to develop gastric cancer than females (female OR = 0.557). Meanwhile females had 2-fold greater probability to develop chronic gastritis (OR = 2.073; 95% CI: 1.005.4.277). Our results were consistent with re­ported results in studies stated by Chandanos and Lagergren4, and Dixon et al.32 All investigated poly­morphism unfortunately showed no associations with disease prediction. IL-1B polymorphisms were not statistically as­sociated with the prediction of each diagnose as ac­cording, however p-value to determine association between polymorphism and outcome of infection (diagnose severity: gastritis or cancer) was 0.084. Frequency distribution in our population showed that IL-1B-511*C homozygote allele was most fre­quent in chronic gastritis group (58.8%). According to our knowledge such results were not found in any other study. Genotype frequencies for cancer 262 group were coincided with control group. Studies in Caucasian and Asian populations have shown that polymorphisms in the genes IL-1B and IL-1RN were in conjunction with an increased risk for hy­pochlohydria and gastric carcinoma.13 According to our findings, individuals carrying the IL-1B­511*T/T allele compared to control group showed an increased OR for gastric cancer. Heterozygotes for IL-1B gene (IL-1B-511*T carriers) and both ho­mozygotes and heterozygotes for T allele, also showed increased OR for developing gastric cancer. Although the OR values were evaluated it would be exaggerated to affirm that these polymorphisms could indicate on the risk for developing intestinal adenocarcinoma, because the power of our statisti­cal anysis was really poor with p-values less then 0.05 and wider 95% CI. However allele combina­tion (T/T and C/T) showed statistically significant association with diagnose prediction (p = 0.021). Percent of IL-1B-511*T carriers in cancer group has reached almost 69% of tested individuals. El-Omar et al.24 have identified the inflammatory profile of genetic polymorphisms in the genes for IL-1ß (IL-1B -511*T) and IL-1ra (IL-1RN*2/2) to in­crease the risk of developing gastric cancer.17 In our population there was 40.6% of short allele carriers diagnosed with cancer but no statistical difference to predict the disease was observed (Table 4). The correlated association between IL-1B and IL-1RN proinflammatory genotypes (IL-1B-511*T carri­ers and IL-1RN*2 homozygotes) and risk for gas­tric cancer was also determined (p < 0.001 and Pearson’s R = 0.300). These results indicated that IL-1RN*2/2 gene is recessive in combination with T carriers in IL-1B.36 The genotype frequencies for individuals with gastric cancer or even chronic gastritis were even smaller than in control group. Results should be taken caustiously because in our population only 2% of cancer patients or patients with gastritis and 12% of controls had IL-1RN*2/2. The present study has showed that TLR-4 pol­ymorphism is not associated with the develop­ment of the premalignant gastric abnormalities of hypochlorhydria and atrophy, or with increased risk of gastric adenocarcinoma. No association was seen with cancer although this polymorphism has been associated with risk of other inflamma­tory conditions. The polymorphism was associated with hyporesponsiveness to bacterial LPS.37 The association of the TLR-4+896A>G polymorphism identifies subjects who have an increased risk of se­vere inflammation and subsequently, development of hypochlorhydria and gastric atrophy, which are regarded as the most important precancerous ab­normalities.27 However, our results were compa­rable to those by Garza-Gonzales28 that the TLR-4 polymorphism did not play a role in the develop­ment of gastric premalignancies. H. pylori infection also enhances the mucosal production of TNF-.. TNF-. is not as potent inhib­itor of gastric acid secretion as IL-1ß.38 Although El-Omar et al.20 and Machado et al.26 found an al­most two-fold increase in risk for gastric cancer, several studies have not found an association be­tween TNF-A-308*A and gastric cancer risk.39-41 The TNF-A-308*A allele has been found in association with an increased risk of cagA positive infections and gastric cancer by Zambon et al.23 and Yea et al.42 also found no significant association between the TNF-A-308 polymorphism and the severity of gastric disease (carcinoma, gastritis, gastric ulcers, duodenal ulcers). However our results have not confirmed that and were coincided with results of Tseng et al.43, who investigated polymorphisms in Jamaican children. Meanwhile the G allele has been found to be associated with peptic ulcer, which commonly accompanies gastritis32, and concomitant H. pylori infection, compared to those without ulcerations.44 Mucosal expression levels of TNF-. was lower in H. pylori-infected individuals with duodenal ulcers. Heterozygous G carriers in our population were slightly drawn near with de­velopment of chronic gastritis (OR = 1.402; 95% CI: 0,626.3,139), but again the p-value was 0.411 and the association was not confirmed. The reduced number of samples available for statistical analysis may have harmed our results. We have found no indications that the infection with H. pylori in a given inflammatory genotype of could result in an inflammatory response, and then gastritis or cancer. We have also showed that the presence of IL-1B-511 genotype for the inflam­matory cytokine was inclined to the difference between intestinal type of gastric cancer, chronic gastritis and healthy controls. However statisti­cally it was not associated entirely and could not be used to identify people at increased risk. On the other hand, cytokine gene polymorphisms repre­sent just one component of complex interactions among host, pathogen, and environmental fac­tors involved in gastric carcinogenesis, what was definitly confirmed with statistical difference be­tween genders. Only combination of H. pylori and host-associated risk factors do not always allow evaluation of gastric carcinoma risk. The progres­sion from atrophy to neoplastic transformation depends on other factors, including diet and dif­ferent pathogenesis of H. pylori strains.5,7 Ando et 263 al.45 have found that patients who develop duode­nal ulcer disease are protected from gastric cancer. Both conditions are associated with H. pylori, but duodenal ulcers are associated with an antrum predominant gastritis, low prevalence of gastric atrophy, and very high acid secretion. On the con­trary, gastric cancer patients develop corpus pre­dominant gastritis, multifocal atrophic gastritis, and hypochlorhydria. Proinflammatory genotypes of the IL-1B gene, through its induction of gastric atrophy and gastric acid inhibition, increase the risk of gastric atrophy. The number of cases in our study was small. In the study, in cancer group, we only included pa­tients with intestinal type of gastric cancer, how­ever in gastritis group we included all gastritis types, not only those with accompanied atrophy. Individuals with extensive corpus gastritis devel­op hypochlorhydria and gastric atrophy, which are presumptive precursors of gastric cancer.20 Another drawback is that we have not determined bacterial strain (vac A, cag A) as it was done by Figueiredo et al.46 and Zambon et al.23 Anyway, now we have learnt that the assessment of patients with H. pylori infection and its strain is very impor­tant and concluded that eradication of bacteria has essential meaning. We recommend that not only screening for H. pylori also the strain determina­tion should have some diagnostic value, especially in the patients who already developed gastritis. Furthermore, for such patients assessment of dis­ease progression (atrophic or metaplastic gastri­tis) could be followed by polymorphism deter­mination. The statistical power of our pilot study was very poor and we could not evaluate it to the whole Slovenian population, but for further poly­morphism investigations it is necessary to include more patients with different disease progression. Our study design was considered good, because our study population was not heterogenic. Untill now we cannot predict the disease based only on single polymorphism. Conclusions Altogether, our findings indicated that host geno­type as well as H. pylori infection could be impor­tant for greater risk for developing gastric cancer. However, those parameters alone could not pre­dict the incidence of the disease. 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Assessment of the toll-like receptor Asp299Gly, Thr399Ile and interleukin-8 -25 I polymoprhisms in the risk for the development of distal gastric cancer. BMC Cancer 2007; 7: 70. 29. Hishida A, Matsuo K, Goto Y, Mitsuda Y, Hiraki A, Naito M, et al. Toll-like receptor 4 +3725 G/C polymorphism, Helicobacter pylori seropositivity, and the risk ofgastric atrophy and gastric cancer in Japanese. Helicobater 2009; 14: 47-53. 30. Achyut BR, Ghoshal UC, Moorchung N, Mittal B. Association of toll-like receptor-4 (Asp299Gly and Thr399Ileu) gene polymorphisms with gastritis and precancerous lesions. Hum Immunol 2007; 68: 901-7. 31. El-Omar EM, Ng MT, Hold GL. Polymorphisms in Toll-like receptor genes and risk of cancer. Oncogene 2008; 27: 244-52. 32. Dixon MF, Genta R, Yardley JH, Correa P. Classification and grading of gas­tritis: The updated Sydney system. Am J Surg Pathol 1996; 20: 1161-81. 33. Szeto ML, Lee CK, Yee YK, Li K., Lee WK, Lee CC, et al. Evaluation of five com­mercial serological tests for the detectionof Helicobacter pylori infection in Chinese. Aliment Pharmacol Ther 2001; 15: 703-6. 34. Kirchgesser M, Adem C, Baumgartner A, Girgnhuber H, Malmberg W, Schmitt I, et al. Automated isolation of DNA from tissue samples in 35-50 minutes: Fast and easy purification combining the MagNA Lyser and the MagNA Pure Compact System. Biochemica 2006; 1: 9-10. 35. Highet AR, Gibson CS, Goldwater PN. Variant interleukin 1 receptor antago­nist gene alleles in sudden infant death syndrome. Arch Dis Child 2010; 95: 1009-12. 36. Machado JC, Pharoah P, Sousa S, Carvalho R, Oliveira C, Figueiredo C, et al. Interleukin 1B and interleukin 1RN polymorphisms are associated with increased risk of rastric carcinoma. Gastroenterology 2001; 121: 823-9. 37. Kutikhin AG. Impact of Toll-like receptor 4 polymorphisms on risk of cancer. Hum Immunol 2011; 72: 193-206. 38. Queiroz DM, Guerra JB, Rocha GA, Rocha AM, Santos A, De Oliveira AG, et al. 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J Natl Cancer Instit 2002; 94: 1680-7. 265 case report Inflammatory myofibroblastic tumor of the pancreatic head – a case report of a 6 months old child and review of the literature Ales Tomazic1, Diana Gvardijancic1, Joze Maucec1, Matjaz Homan2 1 Department of Abdominal Surgery, University Medical Center Ljubljana, Ljubljana, Slovenia 2 Department of Gastroenterology, Hepatology and Nutrition, University Children’s Hospital, Ljubljana, University Medical Center Ljubljana, Ljubljana, Slovenia Radiol Oncol 2015; 49(3): 265-270. Received 14 March 2013 Accepted 22 March 2014 Correspondence to: Assist. Prof. Aleš Tomažič, M.D., Ph.D., Department of Abdominal Surgery, University Medical Center Ljubljana, Zaloška cesta 7, SI-1000 Ljubljana, Slovenia. E-mail: ales.tomazic@kclj.si Disclosure: The authors have no conflict of interest to disclose. Tomazic A and Homan M contributed equally in preparation of the mansuscript Background. Inflammatory myofibroblastic tumors are rare in the pediatric population. Most common localizations were reported in the lungs. A localization in the pancreas needs differentiation from other tumors and chronic pancre­atitis. Treatment is surgical resection, although there are reports of treatment with oral steroids and radiation therapy. Case report. A 6-month-old child was treated due to a tumor in the head of the pancreas. On admission he was jaundiced with pruritus. US and MRI confirmed pancreatic tumor. Preoperative biopsy wasn’t conclusive regarding the nature of the tumor. Duodenopancreatectomy was performed. Postoperative course was uneventful. Histologic examination confirmed the diagnosis of inflammatory myofibroblastic tumor. On follow up, he remained with no evi­dence of recurrence. Conclusions. A literature review revealed 10 cases of pancreatic inflammatory myofibroblastic tumors in the pediat­ric age group. Our patient is the youngest reported. Despite major resection, there were no complications. However, management of this child might be possible with steroids, but conservative treatment might be insufficient, especially in aggressive forms of tumors. Key words: child; inflammatory myofibroblastic tumor; duodenopancreatectomy Introduction Inflammatory myofibroblastic tumors (IMT’s) are rare solid lesions that occur primarily in visceral and soft tissue. Most frequently they occur in the first two decades of life. The most common locali­zations of IMTs have been reported in lung, mesen­tery and omentum.1 These lesions have also been termed inflammatory pseudotumors, fibroxan­thomas, fibrous histiocytomas, postinflammatory tumors and plasma cell granulomas. There are few hypotheses of the etiological factors responsible for development of the IMT. IMT can develop as a con­sequence of an inflammatory reaction to an under­lying low grade malignancy. Human herpes viruses 3 and 8, Eikenella corrodens and Epstain Barr virus have been also proposed as possible infectious trig­gers of the IMT.2,3,4 It is speculated that the disease is provoked by deregulation of cytokine produc­tion caused by infection. Clinically and radiologically, an IMT can be con­fused with malignancy. A localization of IMT in the pancreas is very rare and needs differentiation from other tumors and chronic pancreatitis. The macroscopic appearance of IMT is usually well-circumscribed or multinodular, white, firm mass. Histological, IMT is composed of spindle-shaped myofibroblasts or fibroblasts accompanied by a mixed inflammatory infiltrate of eosinophils, plasma cells, and lymphocytes.1,5 Treatment is 266 usually in the form of surgical resection, although there are recent reports of treatment with oral ster­oids.6 Some authors report also palliative treatment with radiation therapy.7 We present a case of 6 months old male, who was referred to our department due to an IMT of the pancreatic head, which caused jaundice and pruritus. To the best of our knowledge, this is the youngest child with this type of tumor published so far in the literature. Case report A 6-months old boy was transferred to our hospital with a 4-days history of jaundice and pruritus. On examination he was jaundiced with no organomeg­aly. There was a rash on the trunk and extremities. Liver function tests revealed a direct bilirubin level of 109 µmol/L (normal below 17), alkaline phos­phatas level of 11.62 µkat/L (normal up to 1.74) and .GT level of 3.23 µkat/L (normal up to 0.63). An ultrasound scan (US) of his abdomen identi­fied a 40 mm large mass in the head of the pan­creas. The common bile duct was dilated, the gall­bladder was extremely enlarged, but there was no dilatation of intrahepatic bile ducts and pancreatic duct (Figure 1). The results of US-guided fine nee­dle aspiration biopsy wasn’t conclusive regarding the nature of the tumor. MRI confirmed well cir­cumscribed tumor mass, with a diameter of 37 mm. The tumor originated from the head of the pancre­as and uncinate process. 3D reconstruction showed no infiltration in the surrounding tissue, including major vessels (Figure 2). However, there was evi­dence, that the tumor impressed on the caval vein 267 and pushed the superior mesenteric artery and vein ventrally and laterally. Whipple’s procedure was performed due to biliary obstruction and possible malignancy (Figure 3). Histological examination revealed an infiltrative growth in the pancreatic head, mainly surrounding and destroying pancreatic acini (Figure 4), but also encroaching on papila of Vater and duodenal wall. The lesion was composed of bland spindle cells forming a storiform (Figure 5) and vague fascicu­lar growth pattern, admixed with areas display­ing more epithelioid morphology (Figure 6) and variably prominent inflammatory cell infiltrate (Figure 7), composed of lymphocytes, plasma cells and eosinophilic granulocytes (Figure 8). By immunohistochemistry, the lesional cells were smooth muscle actin positive proliferation of bland spindle cells forming a storiform and vague fascicular growth pattern, admixed with areas dis­playing more epithelioid morphology and variably prominent inflammatory cell infiltrate, composed of lymphocytes, plasma cells and eosinophilic granulocytes, while stainings for cytokeratins, S100, desmin, H-caldesmon and ALK were nega­tive, confirming myofibroblastic differentiation of the lesional cells. Although the histological and immunohistochemical features were suggestive of inflammatory myofibroblastic tumour, an unusual form of chronic pancreatitis could not be reliably excluded. The postoperative course was uneventful. The boy was discharged on the 14th postoperative day. Over the next 3.5 years of follow up, he remains well and with no clinical or radiological evidence of recurrence. Discussion and review of literature Pancreatic tumors are rare in childhood, account­ing for only 0.2% of childhood malignancies.8 Inflammatory myofibroblastic tumors are usually benign solid lesions of unclear etiology, commonly found in the lungs. The term inflammatory myofi­broblastic tumor, commonly referred to as inflam­matory pseudotumor in the previous literature, was initially proposed in 1990 in the study of in­flammatory lesions of the pulmonary system.9 The majority of the cases that were reported in the liter­ature as »inflammatory pseudotumor« of the pan­creas, would probably now be classified as autoim­mune pancreatitis and in rare cases they represent 268 true »inflammatory myofibroblastic tumors«.10,11 Another nosological problem with inflammatory myofibroblastic tumor is differentiation from in­flammatory fibrosarcoma, which was first reported as an invasive tumor with greater atypia of constit­uent fibroblasts or myofibroblasts than seen in in­flammatory myofibroblastic tumor.12 According to Coffin, inflammatory myofibroblastic tumors are characterized with local invasion, vascular inva­sion and multifocal onset.13 Invasion of retroperi­toneal connective tissue, duodenal wall and Vater’s papila was also seen in our case. This indicated, that the lesion was neoplastic. However in inflam­matory fibrosarcoma, more aggressive behaviour is seen, including higher incidence of recurrence and death.12 Inflammatory myofibroblastic tumor and inflammatory fibrosarcoma have been speculated to be two lesions occupying the same spectrum, with reported cases of inflammatory myofibroblas­tic tumors probably including some low-grade ex­amples of inflammatory fibrosarcoma.14 Histologically, inflammatory myofibroblastic tumors are characterized by irregular proliferation of myofibroblasts intermixed with inflammatory cells, mainly lymphocytes and plasmacytes. They are subcategorized into fibrohystiocytic type, plas­ma cell granuloma type, largely sclerosed or fibro­sed type, hypocellular fibrous type and myxoid/ vascular type.15 Discovery of cytogenetic aberra­tions in inflammatory myofibroblastic tumors and the recognition of ALK gene rearrangements so­lidified the concept of inflammatory myofibroblas­tic tumor as a neoplastic lesion. It most frequently occurs in the lung or the mesenterium of children or young adults and rarely metastasizes (<5%).16 The liver is also relatively frequently involved17, but other sites such as the stomach18, spleen19, blad­der20, kidney21, maxillary sinuses22, heart23, para-pharyngeal space24, retrorectal space25 and periph­eral nerve26 have also been recorded. Only 28 cases of pancreatic inflammatory myofi­broblastic tumors have been reported so far, 60% being located in the pancreatic head.7 Inflammatory fibroblastic tumor equally affects males and fe­males. The age distribution resembled that of in pulmonary system ranging 2.5 to 70 years.27 A lit­erature review revealed 10 documented cases of pancreatic inflammatory myofibroblastic tumor in the pediatric age group (Table 1). Compared to this data, our patient is the young­est child with inflammatory myofibroblastic tumor of the pancreas reported in the literature. The main features at presentation were pruritus, jaundice, abdominal mass, lethargy, vomiting, fever and ane­mia.6 Curative resection is treatment of choice for inflammatory myofibroblastic tumors. Whipple’s procedure or distal pancreatectomy is performed, according to the site of the tumor. The prognosis of inflammatory myofibroblastic tumors is generally good, with rare incidence of malignant transfor­mation.28 However, a significant recurrence rate of 25% was reported.29 It was suggested that the pres­ence of atypia, ganglion-like cells and p53 expres­sion may suggest more aggressive behaviour.30,31 These lesions may be indistinguishable from in­flammatory fibrosarcoma due to a high degree of clinical and morphological overlap.28 269 TABle 1. Reported cases of pediatric pancreatic inflammatory myofibroblastic tumors in the literature 10 F body Abdominal mass Distal pancreatectomy Abrebanel et al. 1984 2.5 F body Anemia, fever, abdominal mass Distal pancreatectomy Scott et al. 1988 5 F head vomiting Whipple Stringer et al. 1992 8 F head Jaundice, anemia, weight loss Whipple Uzoaru et al. 1993 8 F body Abdominl mass Distal pancreatectomy Shankar eta al. 1998 11 F head Jaundice, pruritus, anorexia, pain Whipple McLain et al. 2000 4 F head Malaise, lethargy Whipple Slavotinek et al. 2000 11 M body Lethargy, anemia, abdominal mass Distal pancreatectomy Morris-Stiff et al. 1998 13 F head Jaundice, vomiting, weight loss Whipple Dagash eta l. 2009 10 M head Jaundice, pain Steroids Dagash eta l. 2009 Besides surgical resection, alternative therapeu­tic regimens are still lacking. While systemic im­munosuppresive treatment with steroids, chemo­therapy and radiation therapy have been reported for unresected or recurrent cases of extrapancreatic inflammatory myofibroblastic tumors.28,32,33 In the literature there are only two reported cases of pancreatic inflammatory myofibroblastic tumors that were not treated with resection.6,7 The first reported case was a child treated with high dose steroids. The mass gradually resolved and the patient remains disease free 6 years after treat­ment.6 The second case was an adult treated with palliative radiation and corticoid therapy because of unresectable mass in the head of the pancreas.7 In this case, long term results were not published. Chronic pancreatitis could not be completely excluded according to histological examination. Anyway, specific causative factor for chronic pan­creatitis was not identified. In addition, pediatric patients present with chronic pancreatitis much later (average age 6 ± 4 years) than it developed in our patient (6 months).34Therefore, chronic pancre­atitis was very unlikely the reason for pancreatic head mass in our patient. Conclusions We report a case of pancreatic inflammatory my­ofibroblastic tumors in a six month old male child treated with surgical resection. This is the first case report of an infant with IMT. In addition, the tumor is rarely described in the pancreas. Despite major surgery no complications evolved in long term fol­low up. references 1. Coffin CM, Humphrey PA, Dehner LP. Extrapulmonary inflammatory my­ofibroblastic tumor: a clinical and pathological survey. Semin Diagn Pathol 1998, 15: 85-101. 2. Slavotinek JP, Bourne AJ, Sage MR, Freeman JK. Inflammatory pseudotu­mour of the pancreas in a child. Pediatr Radiol 2000; 30: 801-3. 3. Gomez-Roman JJ, Sanchez-Velasco P, Ocejo-Vinyals G, Hernandez-Nieto E, Leyva-Cobian F, Val-Bernal JF. Human herpesvirus-8 genes are expressed in pulmonary inflammatory myofibroblastic tumor (inflammatory pseudotu­mor). Am J Surg Pathol 2001; 25: 624-9. 4. Lewis JT, Gaffney RL, Casey MB, Farrell MA, Morice WG, Macon WR. Inflammatory pseudotumor of the spleen associated with a clonal Epstein-Barr virus genome. Case report and review of the literature. Am J Clin Pathol 2003; 120: 56-61. 5. Gomez-Roman JJ, Ocejo-Vinyals G, Sanchez-Velasco P, Nieto EH, Leyva-Cobian F, Val-Bernal JF. Presence of human herpesvirus-8 DNA sequences and overexpression of human IL-6 and cyclin D1 in inflammatory myofibro­blastic tumor. Lab Invest 2000; 80: 1121-6. 6. Dagash H, Koh C, Cohen M, Sprigg A, Walker J. Inflammatory myofibroblastic tumor of the pancreas: a case report of 2 pediatric cases-steroids or sur­gery? J Ped Surg 2009; 44: 1839-41. 7. Schutte K, Kandulski A, Kuester D, Meyer F, Wieners G, Schulz HU, et al. Inflammatory myofibroblastic tumor of the pancreatic head: An unusual cause of recurrent acute pancreatitis- case presentation of a palliative ap­proach after failed resection and review of the literature. Case Rep Gastroenterol 2010; 4: 443-51. 8. Mammen A, Kalisadan V, Beasley SW. Rare pancreatic tumors in children (other than nesidioblastosis). Aust N Z J Surg 1997; 67: 720-1. 9. Petinato G, Manivel JC, De Rosa N, Dehner LP. Inflammatory myofibroblastic tumor (plasma cell granuloma). Clinicopathologic study of 20 cases with immunohistocemical and structural observations. Am J Clin Pathol 1990; 94: 538-46. 10. Volkan Adsay N, Bastruk O, Klimstra DS, Kloppel G. Pancreatic pseudotu­mors: non-neoplastic solid lesions of the pancreas that clinically mimic pancreas cancer. Sem Diagn Pathol 2004; 21: 260-7. 11. Petter LM, Martin JK, Menke DM. Localized lymphoplasmacellular pan­creatitis forming a pancreatic inflammatory pseudotumor. Mayo Clin Proc 1998; 73: 447-50. 12. Meis JM, Enzinger FM. Inflammatory fibrosarcoma of the mesentery and retroperitoneum; A tumor closely simulating inflammatory pseudotumor. Am J Surg Pathol 1991; 15: 1146-56. 13. Coffin CM, Waterson J, Priest JR, Dehner LP. Extrapulmonary inflammatory myofibroblastic tumor (inflammatory pseudotumor); A clinicopathologic and immunohistochemical study of 84 cases. Am J Surg Pathol 1995; 19: 859-72. 270 14. Nakamura Y, Inui K, Yoshino J, Tokoro T, Sabater L, Takeda S, et al. Inflammatory myofibroblastic tumor (inflammatory fibrosarcoma) of the pancreas: A case report. Hepato-Gastroenetrology 2005; 52: 625-8. 15. Travis WD, Colby TV, Koss MN, Rosado-de-Christenson ML, Muller NL, King TE. Miscellaneous disease of uncertain etiology. In: Atlas of Nontumor Pathology. Non-neoplastic Disorders of the Lower Respiratory Tract. 1st ed. King DW, ed. American Registry of Pathology and Armed Forced Institute of Pathology, Washington DC, 2002. p. 857-900. 16. Coffin CM, Hornick JL,Fletcher CDM. Inflammatory myofibroblastic tumor: comparison of clinicopathologic, histologic, and immunohistochemical fea­tures including ALK expression in atypical and aggressive cases. Am J Surg Pathol 2007; 31: 509-20. 17. Krech RH, Erhardt-Domagalsky M, Neumann H. Inflammatory pseudotumor of the liver. Morphologic and cytophotometry studies and differential diag­nosis. Pathologe 1995; 16: 415-20. 18. Taratuta E, Krinsky G, Genega E, Roche K, Geneisier N. Pediatric inflamma­tory pseudotumor of the stomach: contrast-enhanced CT and MR imaging findings. Am J Radiol 1996; 167: 919-20. 19. Glazer M, Sagar V. SPECT imaging of the spleen in inflammatory pseudo-tumor. Correlation with ultrasound, CT, and MRI. Clin Nucl Med 1993; 18: 527-9. 20. Foschini MP, Scarpellini F, Rinaldi P, Mancini AF, Accinelli G, Eusebi V. Inflammatory pseudotumor of the urinary bladder . Study of 4 cases and review of the literature. Pathologica 1995; 87: 653-8. 21. Vujanic GM, Berry PJ, Frank JD. Inflammatory pseudotumor of the kidney with extensive metaplastic bone. Pediatr Pathol 1992; 12: 557-61. 22. Som PM, Brandwein MS, Maldijan C, Reino AJ, Lawson W. Inflammatory pseudotumor of the maxillary sinus: CT and MR findings in six cases. Am J Radiol 1994; 163: 689-92. 23. Jenkins PC, Dickinson AE, Flanagan MF. Cardiac inflammatory pseudotumor: rapid appearance in an infant with congenital heart disease. Pediatr Cardiol 1996; 17: 399-401. 24. Hytiroglou P, Brandwein MS, Strauchen JA, Mirante JP, Urken ML, Biller HF. Inflammatory pseudotumor of the parapharyngeal space: Case report and review of the literature. Head Neck 1992; 14: 230-4. 25. Georgia JD, Lawrence DP, DeNobile JW. Case report. Inflammatory pseu­dotumor in the retrorectal space: CT and MR appearance. J Comput Assist Tom 1996; 20: 410-2. 26. Weiland TL, Scheithauer BW, Rock MG, Sargent JM. Inflammatory pseudo-tumor of nerve. Am J Surg Pathol 1996; 20: 1212-8. 27. Hassan KS, Cohen HI, Hassan FK, Hassan SK. Unusual case of inflammatory myofibroblastic tumor associated with spontaneous splenic rupture. World J Emerg Surg 2010; 5: 28. 28. DiFiore LW, Goldblum JR. Inflammatory myofibroblastic tumor of the small intestine. J Am Coll Surg 2002; 194: 502-6. 29. Wreesmann V, van Eijck CH, Naus DC, van Velthuysen ML, Jeekel J, Mooi WJ. Inflammatory pseudotumor (inflammatory myofibroblastic tumor) of the pancreas: a case report of six cases associated with obliterative phlebitis. Histopathology 2001; 38: 105-10. 30. Biselli R, Ferlini C, Fattorossi A, Boldrini R, Bosman C. Inflammatory my­ofibroblastic tumor (inflammatory pseudotumor): DNA flow cytometric analysis of nine pediatric cases. Cancer 1996; 77: 778-84. 31. Hussong JW, Brown M, Perkins SL, Dehner LP, Coffin CM. Comparison of DNA ploidy, histologic and clinical outcome in inflammatory myofibroblastic tumors. Mod Pathol 1999; 12: 279-86. 32. Tang TT, Segura AD, Oechler HW, Harb JM, Adair SE, Gregg DC, et al. Inflammatory myofibrohistiocytic proliferation simulating sarcoma in chil­dren. Cancer 1990; 65: 1626-34. 33. Doski JJ, Priebe CJ, Driessnack M, Smith T, Kane P, Romero J. Corticosteroids in the management of unresected plasma cell granuloma (inflammatory pseudotumor) of the lung. J Pediatr Surg 1991; 26: 1064-6. 34. Clifton MS, Pelayo JC, Cortes RA, Grethel EJ, Wagner AJ, Lee H, et al. Surgical treatment of childhood recurrent pancreatitis. J Pediatr Surg 2007; 42: 1203-7. 271 research article Neoadjuvant chemotherapy in 13 patients with locally advanced poorly differentiated thyroid carcinoma based on Turin proposal - a single institution experience Nikola Besic1, Marta Dremelj2, Andreja Schwartzbartl-Pevec3, Barbara Gazic4 1 Department of Surgery, 2Department of Radiotherapy, 3Department of Nuclear Medicine, 4Department of Pathology, Institute of Oncology Ljubljana, Ljubljana, Slovenia Radiol Oncol 2015; 49(3): 271-278. Received 3 May 2014 Accepted 29 October 2014 Correspondence to: Prof. Nikola Bešić, M.D., Ph.D., Department of Surgical Oncology, Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. Phone: +386 1 5879 953; Fax: +386 1 5879 400; E-mail: nbesic@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. There is a paradigm that chemotherapy is ineffective in thyroid carcinoma. The aim of our study was to find out whether neoadjuvant chemotherapy before thyroid surgery had an effect on the size of primary tumour in patients with poorly differentiated thyroid carcinoma (PDTC) based on Turin proposal. Patients and methods. Altogether, 13 patients (8 women, 5 men; median age 61 years) with PDTC based on Turin proposal were treated with neoadjuvant chemotherapy between 1986 and 2005. Tumour diameter was from 4.5 to 18 cm (median 9 cm). Regional and distant metastases were detected in 6 and 9 patients, respectively. Eight patients had pT4 tumour. Results. Altogether, 29 (range 1–5) cycles of chemotherapy were given. Tumour diameter decreased in all the pa­tients and by more than 30% in 5 patients (= 38%). Two of these five patients had also preoperative external beam irra­diation (EBRT). Total thyroidectomy, lobectomy and neck dissection were performed in 10, 3 and 5 cases, respectively. R0 and R1 resection was done in 5 and 8 cases, respectively. Eight patients had postoperative EBRT of the neck and upper mediastinum. The 5-year and 10-year cause-specific survival rates of patients were 66% and 20%, respectively. Conclusions. After neoadjuvant chemotherapy a partial tumour regression was observed in 38% of patients with PDTC based on Turin proposal. Key words: poorly differentiated thyroid carcinoma; neoadjuvant; chemotherapy; survival; pathology Introduction A clinicopathologic entity of poorly differenti­ated thyroid carcinoma (PDTC) was proposed by Sakamoto et al. 30 years ago.1 They found that the differences in the survival rates among well differ­entiated, poorly differentiated and anaplastic car­cinomas were significant and of value in determin­ing management and survival of thyroid cancer patients.1 The World Health Organization (WHO) classification of tumours of endocrine organs in 2004 introduced PDTC as a separate entity and de­fined it as a follicular-cell neoplasm showing limit­ed evidence of structural follicular cell differentia­tion, and morphologically and behaviourally at an intermediate position between differentiated (fol­licular and papillary carcinomas) and undifferen­tiated (anaplastic) carcinoma.2 At an international consensus meeting, uniform diagnostic criteria for PDTC (Turin Proposal criteria) were defined in the presence of solid/trabecular/insular growth pat­tern, absence of conventional nuclear features of papillary carcinoma, and in the presence of at least one of the following features: convoluted nuclei, 272 mitotic activity .3/10 high-power fields, or tumour necrosis.3 PDTC is a rare disease that carries a poor prog­nosis.4 The incidence of PDTC as defined by the Turin Proposal criteria in Japan, USA and Northern Italy was 0.3%, 1.8% and 6.7%, respectively.5,6 In the literature, there are only limited data on the treatment of patients with PDTC.7-11 Recently, Ibrahimpasic et al. reported the results of treatment of 27 patients with PDTC presenting with gross ex­trathyroidal extension during the period 1986-2009 at the Memorial Sloan-Kettering Cancer Center.9 The majority of their patients (60%) who presented with or subsequently developed distant metastases eventually died of distant disease, therefore they concluded that systemic therapies to target distant metastatic disease are required to achieve improve­ments in the outcome.9 The aim of the present study was to find out whether neoadjuvant chemo­therapy before thyroid surgery had an effect on the size of primary tumour in patients with locally ad­vanced and/or initially metastatic PDTC based on Turin proposal. Patients and methods During the period 1979-2005, 45 patients (33 wom­en, 12 men; mean age 61.6 years) were treated with neoadjuvant chemotherapy for thyroid carcinoma at our tertiary comprehensive cancer center.12,13 Among them, 13 patients (8 women, 5 men; medi­an age 61 years) had poorly differentiated thyroid carcinoma. They were initially treated during the period 1986-2005. The histological slides of all 13 patients with PDTC, who were the subject of this study, were reviewed by a pathologist experienced in thyroid pathology. All tumours fulfilled the Turin proposal criteria.3 In all patients in the primary chemotherapy group, the tumour was considered inoperable be­cause of infiltration into the surrounding tissues. Altogether, ten patients were treated with neoadju­vant chemotherapy, while two patients were treat­ed with preoperative chemotherapy and preopera­tive external beam radiotherapy (EBRT). Surgery was performed whenever the tumour was reduced after chemotherapy and/or EBRT and the surgeon deemed it resectable. The median interval between the beginning of chemotherapy and the surgical procedure was 30 days (range 8.85 days). Data on the patients’ gender, age, history and extent of the disease, morphological characteristics, therapy, locoregional control, and survival were collected. The tumour size, presence of regional or distant metastases and residual tumour after sur­gery were assessed by TNM clinical classification according to the Union for International Cancer Control (UICC) criteria from 2009.14 Clinical char­acteristics of patients with PDTC based on Turin proposal and therapy are presented in Table 1. The aim of the study was to evaluate the effect of chemotherapy in patients with PDTC. Because of rarity of PDTC we collected data of patients who were treated at our Institute and were included in the following consecutive clinical trials: Rational diagnostic and therapy of patients with thyroid tumours (J3-7842), Importance of cytomorphol­ogy, measurements of DNA, Ki 67 and apoptosis for planning and evaluation of effect of chemother­apy in thyroid carcinoma (J3-3026), Genetic and radio-nuclear methods in diagnostics and therapy of thyroid carcinoma (J3-3460), all supported by the Ministry of Science of Slovenia. The Medical Ethics Committee of the Republic Slovenia and the Protocol Review Board and Ethics Committee of the Institute of Oncology Ljubljana reviewed and approved all three studies, which were performed in accordance with the ethical standards laid down in an appropriate version of the 1964 Declaration of Helsinki. These studies were conducted with the understanding and consent of the involved human subjects. Our chart review for the present publi­cation was approved by the Institutional Review Board. Surgery Surgery was considered the most effective treat­ment of PDTC and has therefore remained its mainstay. At our Institute, treatment of each pa­tient with advanced thyroid carcinoma depends on the decision of the multidisciplinary team consist­ing of a surgical oncologist, a specialist in nuclear medicine, a medical oncologist and a radiothera­pist. Our patients were treated with surgery, radi­oiodine (RAI), EBRT, chemotherapy, or a combina­tion of these modalities as dictated by the circum­stances.12 Before RAI ablation of thyroid remnant and RAI therapy all patients were on low-iodine diet for two weeks in order to achieve moderate io­dine deficiency.15 Chemotherapy and radiotherapy The treatment was started with a non-toxic sched­ule (i.e. 2 mg vinblastine over 12- or 24-h infusions in 1000 mL of 0.9% saline) as already reported 273 TABLE 1. Clinical characteristics and therapy of patients with poorly differentiated thyroid carcinoma (PDTC) based on Turin proposal 1 F 62 Hurthle cell T3N1M1 SD no TT+mRND R1 yes 0 no 131 DM 2 F 80 Papillary T3N1M1 PR no TT+mRND R1 yes 2 yes 24 DM 3 F 78 Papillary T3N1M1 SD no TT+mRND R1 yes 0 yes 26 DM+LR 4 F 45 Follicular T4N0M0 SD no TT R1 yes 1 yes 119 DM 5 F 47 Hurthle cell T4N0M1 SD no TT R1 yes 5 no 52 DM 6 M 69 Follicular T4N1M0 PR yes TT R1 yes 0 yes 101 Other causes 7 M 65 Papillary T4N1M1 SD no lobectomy+mRND R1 yes 2 yes 49 Other causes 8 M 61 Follicular T4N0M0 PR no lobectomy R0 yes 3 yes 104 DM 9 M 56 Papillary T4N1M1 SD no lobectomy+mRND R1 yes 6 yes 92 DM 10 F 57 Papillary T4N0M0 PR yes TT R0 yes 2 yes 103 DM 11 F 63 Follicular T3N0M1 SD no TT R0 yes 3 no 183 DM 12 F 37 Follicular T3N0M1 SD no TT R0 yes 1 no 54 DM 13 M 59 Follicular T4N0M1 PR no TT R0 yes 7 no 118 DM DM = distant metastases; F = female; LR = locoregional disease; M = male; mRND = modified radical neck dissection; PR = partial response; RAI = radioiodine; SD = stable disease; TT = total thyroidectomy in our previous publications.8,12,13,16 Vinblastine shows a cytostatic effect in cell lines models, which is reflected in rapid decrease of relative cell vi­ability during prolonged exposure.17 At all tested concentrations, the relative cell viability was re­duced by 20% or more already after 48 h expo­sure.17 However, vinblastine may cause cardiac ar­rhythmia, therefore we did not use vinblastine in a patient (number 7) with ischemic heart disease. Instead, in this patient, 20 mg of adriamycin in a 2-hour infusion was used once a week. In such doses, adriamycin does not cause nausea, vomit­ing, alopecia, hematopoietic side effects or conges­tive heart failure according to our extensive expe­rience with adriamycin in patients with anaplastic thyroid carcinoma. The tumour increased in one of our patients (number 8) despite treatment with vinblastine, therefore a combination of vinblastine and cisplatin of 90 mg over a 24-h infusion with EBRT was used. After treatment with this combina­tion, the tumour size decreased by more than half. Hematologic (anaemia, leukopenia, neutrope­nia, and thrombocytopenia) and non-hematologic (nephrotoxicity defined by serum creatinine lev­el, alopecia, and nausea/vomiting) toxic effects were evaluated according to the National Cancer Institute - Common Toxicity Criteria, version 4.0. The local effect of chemotherapy used to be as­sessed by clinical findings only. The size of the primary tumour was measured clinically each day during the first week after chemotherapy and once a week thereafter during the visits to the outpatient clinic and before the next cycle of chemotherapy. Resectability of a tumour was clinically evaluated by a surgeon once a week. The extent of the disease and the potential effectiveness of chemotherapy were evaluated before the first chemotherapy and before the surgical procedure by clinical exami­nation, X-ray, CT scan, ultrasonography and/or serum thyroglobulin (Tg) concentration measure­ments, as dictated by the circumstances. The over­all effect of chemotherapy on the primary tumour size was defined according to Response Evaluation Criteria in Solid Tumours (RECIST) criteria18: (1) progressive disease (PD): at least a 20% increase in the sum of longest diameter of target lesions, tak­ing as reference the smallest sum of longest diame­ter recorded before the treatment started; (2) stable disease (SD): neither sufficient shrinkage to qual­ify for partial regression nor sufficient increase to qualify for progressive disease; (3) partial response (PR): at least a 30% decrease in the sum of the long­est diameter of target lesions; and as (4) complete response if the tumour disappeared. 274 TABLE 2. Presence of distant metastases in 13 patients with poorly differentiated thyroid carcinoma (PDTC) based on Turin proposal treated with neoadjuvant chemotherapy Mean age (years) Mean tumour size (cm) Gender Age (years) Tumour diameter (cm) pT tumour stage N stage M stage Thyroid surgical procedure Residual tumour after surgery Neck dissection Radioiodine ablation after surgery Therapy with radioiodine Effect of chemotherapy Recurrence Outcome Disease-free interval in months: mean (range) Cause-specific survival in months: mean (range) Female Male 44 or less 45 or more 0 . 4 More than 4 pT3 pT4 N0 N1 or N2 M0 M1 Total or near-total thyroidectomy Lobectomy R0 R1 No Yes No Yes No Yes < 50% or no effect 50.99% No Yes - distant Disease present permanently Death of disease Death of other causes According to our study protocol, if primary tumour progressed after chemotherapy, the pa­tient was treated with a combination of EBRT and chemotherapy. Two patients received preoperative EBRT with a median dose of 35 Gy (range 30.40 Gy) over three to four weeks. In one patient, intu­bation was necessary one week after the initiation of external irradiation with a daily dose of 2.5 Gy. Altogether, eight patients had preoperative and/or postoperative EBRT of the neck and superior me­diastinum with a total tumour dose of 30.6.59.4 Gy (median 50 Gy). The radiation field included the whole neck up to the level of the mastoid process, bilateral supraclavicular and infraclavicular re­gions, and the superior mediastinum using a 60Co unit and two opposed fields. 59 10 2 2 0 4 0 4 0 4 3 1 4 0 3 1 2 2 4 0 0 4 1 3 1 3 1 3 0 3 1 106 (101.119) 106 (101.119) 62 9.7 6 3 1 8 0 8 5 4 4 5 0 9 7 2 3 6 4 5 0 9 2 7 7 2 0 0 9 8 1 - 81 (24.183) 0.64 0.64 1.00 1.00 1.00 0.105 0.56 - 1.00 1.00 0.105 1.00 1.00 0.22 1.00 -0.75 Follow-up and survival For all patients, follow-up examinations were per­formed at our Institute at least once a year. They consisted of obtaining a detailed medical history, a physical exam, and determination of the serum Tg concentration. Whenever the Tg concentration was elevated, imaging (X-ray, ultrasound, RAI scin­tigraphy, computed tomography, magnetic reso­nance imaging, bone scintigraphy and/or positron emission tomography - computed tomography (PET-CT) scan) was performed to determine the lo­cation and extent of residual disease or suspected recurrence. Disease-specific survival was defined as the pe­riod from the first day of treatment with preopera­ 275 tive chemotherapy to the death or last follow-up of the patient. Overall survival was defined as the pe­riod from the first day of primary treatment preop­erative chemotherapy to death of any cause or the last follow-up. Disease-free interval was defined as the period from the beginning of chemotherapy to recurrence or the last follow-up. The disease-free interval excludes those patients with distant metas­tases at initial presentation. Statistical analysis Characteristics of patients and treatments were compared by Fisher’s exact or chi-square test, where appropriate. The age of the patients and size of their tumours were compared using the Mann-Whitney rank-sum test. Survival and disease-free intervals were compared using a log-rank test. Survival curves were calculated according to the Kaplan-Meier method. For statistical analysis, SPSS 16.0 for Windows was used. Results Patients Tumour diameter was from 4.5 to 18 cm (median 9 cm). Regional and distant metastases were de­tected in 6 and 9 patients, respectively. Six patients had lung metastases and three patients had bone metastases. Eight (61%) patients had pT4 tumour (Table 2). Actual chemotherapy and toxicity Chemotherapy consisted of vinblastine, vinblastine with adriamycin or vinblastine with cisplatin in 11, 1 and 1 cases, respectively. The interval between the first chemotherapy and surgical procedure was 1.12 weeks (median 4 weeks). Altogether, 29 (range 1.5) cycles of chemotherapy were given. The following toxic effects of cisplatin were ob­served in our patient number 8: leukopenia grade 1, nausea/vomiting grade 1-2, nephrotoxicity grade 1 and alopecia grade 1. None of other patients had any toxic side effects of chemotherapy because doses of chemotherapy used were very low. Response to treatment, survival, additional treatment and follow up Survival of patients with PDTC according to the effect of chemotherapy is presented in Figure 1. Tumour size decreased in all of patients, but PR was observed in 5 patients (38%). Two of these five patients had also preoperative EBRT. Total thyroidectomy, lobectomy and neck dis­section were performed in 10, 3 and 5 cases, re­spectively. R0 and R1 resection was done in 5 and 8 cases, respectively. Radioiodine (RAI) therapy was used in patients with initially distant metastatic disease and distant dissemination during follow-up in 7 out of 9 and 3 out of 3 patients, respectively. They received 1.7 (median 2.5) therapies with RAI in a dose of 3.7.7.4 GBq. Eight patients received postoperative EBRT of the neck and upper mediastinum. Distant metastases were diagnosed in three pa­tients during follow-up of 7.189 months (median 118 months). Ten patients died of distant metastases, one of distant metastases with small locoregional recur­rence, and two patients died of other causes. The 5-year and 10-year cause-specific survival rates of patients were 66% and 20%, respectively. Survival of patients with PDTC based on Turin proposal and presence of metastases are shown in Figure 2. Discussion Fortunately, aggressive locally advanced differen­tiated, poorly differentiated and anaplastic thyroid carcinomas are rare. However, because of this rar­ity, it is very unlikely that randomized trials will be conducted in patients with these rare tumours. One way to test the effectiveness of the therapy is to use a specific drug in a neoadjuvant setting. In 276 two of our previous studies, we found out that ne­oadjuvant chemotherapy reduced the size of pri­mary tumour by more than half in 44% of patients with differentiated thyroid carcinoma.12,13 The aim of the present study was to report the effectiveness of neoadjuvant chemotherapy in re­ducing the size of primary tumour in patients with locally advanced and/or initially metastatic PDTC based on Turin proposal. We found that in 38% of patients with PDTC based on Turin proposal, neoadjuvant chemotherapy decreased the size of primary tumour and PR was achieved. Based on this data we believe that chemotherapy may be the treatment of choice in locoregionally advanced and metastatic PDTC. Our data are not the only ones that oppose the paradigm that chemotherapy is ineffective in well, moderately or poorly differ­entiated thyroid carcinoma. Santini et al. reported a 37% response rate after a combination of carbo­platin, epirubicin and thyroid-stimulating hor­mone (TSH) stimulation in fourteen patients with PDTC and RAI-resistant diffuse lung metastases.19 Carboplatin (300 mg/m2) and epirubicin (75 mg/ m2) were given at 4- to 6-week intervals for a total of six courses. To raise serum TSH, either endog­enous TSH stimulation was obtained by reducing the daily dose of L-thyroxin therapy or exogenous TSH stimulation was induced by recombinant human TSH. Lung computed tomography scans before and after therapy showed that one patient had a complete remission, while five patients had a partial remission.19 We believe that extensive surgery is not enough to obtain long-lasting locoregional control of dis­ease in advanced PDTC. Namely, Ibrahimpasic et al. reported that 19 patients had only micro­scopic residual disease and 8 (42%) of them had locoregional recurrence.9 They also reported that 63% of patients had only RAI therapy, 11% un­derwent RAI therapy and EBRT, while 11% had only EBRT.9 On the other hand, our 8 patients with microscopic residual disease after thyroid surgery received a more extensive additional therapy: all of them had initial chemotherapy, postopera­tive RAI ablation of thyroid remnant and EBRT. Additionally, 5 patients (62.5%) also received RAI therapy. A more extensive additional therapy in our patients might be the reason that locoregional recurrence occurred in only 25% of cases. Locoregional recurrence of thyroid carcino­ma may lead to an uncontrollable disease and frequently often correlates with poor outcome. EBRT is used to prevent locoregional recurrence. According to the American Thyroid Association guidelines20, EBRT treatment of the primary tu­mour should be considered in patients aged over 45 years with grossly visible extrathyroidal exten­sion at the time of surgery and a high likelihood of microscopic residual disease, or in patients with gross residual tumour in whom further surgery or RAI would likely be ineffective. In a recent review article, Sanders et al. concluded that EBRT should probably be considered also in patients with PDTC who have pT3 carcinoma, extracapsular extension of lymph node disease or extensive lymph node involvement.7 One reason that supports the initial multimodal approach is the fact that PDTC is often composed of a poorly differentiated as well as moderately differentiated component. It is well known that poorly differentiated cells are sensitive to chemo­therapy. Neoadjuvant chemotherapy proved to be effective in all our patients, while in 38% PR of primary tumour was observed. RAI was used whenever possible to treat the differentiated com­ponent of PDTC. Our study has several limitations. It is not ran­domized and there is no control group of patients (without chemotherapy). Furthermore, number of patients is too small to draw any conclusions whether the prognosis of R0 tumours is superior to that of R1 tumours and whether R1 tumours can be controlled by EBRT. However, we believe that in order to prevent locoregional recurrence and/ or dissemination, the initial treatment should be based on prognostic and predictive factors also in patients with thyroid carcinoma. This principle is widely applied to many solid malignancies, i.e. breast cancer, colorectal cancer, head and neck cancer and many others. For example, after surgi­cal procedure, a patient with breast cancer will al­so be treated with EBRT, chemotherapy, hormonal therapy and targeted therapy based on prognostic and predictive factors.21 However, American Thyroid Association and European Thyroid Association guidelines for treatment of differentiated thyroid carcinoma do not recommend initial multimodal approach in more aggressive types of differentiated thyroid carcinoma.21,22 It is well known that PDTC based on Turin proposal and anaplastic thyroid carci­noma are aggressive tumours that cause locore­gional recurrence and dissemination4, therefore, we believe that an adequate initial multimodal treatment is justified. Naturally, in locoregionally advanced and/or metastatic PDTC, multimodal treatment should be used whenever possible. With such an approach, excellent locoregional 277 control of disease was achieved in our patients. None of our patients had uncontrollable locore­gional PDTC. In locally advanced patients with PDTC based on Turin proposal from the Memorial Sloan-Kettering Cancer Center9, 5-year disease-specific survival was only 49%, while it was 66% in our patients, although initially distant metastases were more common in our series (37% vs. 61%). Possibly, longer survival of our patients was a result of our multimodal treatment approach. All our patients had initial chemotherapy, and 92% of them re­ceived adjuvant therapy: 31% RAI only, 15% EBRT only, while as many as 46% received both RAI therapy and EBRT. On the other hand, only 77% of patients reported by Ibrahimpasic et al. underwent adjuvant therapy: 55% RAI only, 11% EBRT only, while only 11% underwent both RAI therapy and EBRT.9 Jung et al. reported treatment results in 49 pa­tients with PDTC not based only on Turin propos­al.23 RAI therapy was used in 38 patients. Patients with RAI therapy had significantly longer survival in comparison to patients without RAI therapy (5-year survival: 73% vs. 60%).22 However, in a multivariate analysis, RAI therapy was not an in­dependent factor for survival.22 Similarly, Lai et al. reported that RAI therapy was not an independ­ent prognostic factor for survival in a retrospective review consisting of 82 patients with insular carci­noma, possibly because patients with worse-prog­nosis tumours were selected for a more extensive adjuvant treatment, obscuring any potential bene­fit.10 RAI scanning was performed at the Memorial Sloan-Kettering Cancer Center in eight patients with PDTC based on Turin proposal with distant metastases at presentation, and seven (87.5%) pa­tients had RAI-avid metastases.9 Similarly, at our institute, 83% of cases with distant disease had RAI-avid metastases, so these patients underwent RAI therapy. Like many others studies7,9,10,23 ours also shows that distant disease is the main cause of death in patients with locally advanced and metastatic PDTC. Initially or after disease progression, PDTC was a systemic disease in 92% of our patients. In order to treat systemic PDTC based on Turin pro­posal effectively, not only RAI but also other sys­temic treatment modalities are needed. Of course, there is a place for targeted therapy in PDTC, but at present time, there are only limited data about its use in poorly differentiated thyroid carcinoma.24 Sorafenib was reported to be effective treatment in radioiodine-refractory PDTC.25 Conclusions After neoadjuvant chemotherapy and preopera­tive EBRT a partial response of primary tumour was observed in 38% of patients with PDTC based on Turin proposal. Surgical procedure is the main­stay of treatment in PDTC, but postoperative RAI therapy, EBRT, or both, seem to be associated with prolonged survival. Acknowledgement This paper is a part of the Research studies No. P3-0289 supported by the Ministry of Education, Science and Sport of Republic of Slovenia. References 1. Sakamoto A, Kasai N, Sugano H. Poorly differentiated carcinoma of the thy­roid. A clinicopathologic entity for a high-risk group of papillary and follicular carcinomas. Cancer 1983; 52: 1849-55. 2. Sobrinho-Simoes M, Albores-Saavedra J, Tallini G. Poorly differentiated carcinoma. In: DeLellis RA, Lloyd RV, Heitz U, Eng C, editors, Pathology and genetics. Tumours of endocrine organs. Lyon: World Health Organization, IARC Press, France; 2004. p. 73-6. 3. Volante M, Collini P, Nikiforov YE, Sakamoto A, Kakudo K, Katoh R, et al. Poorly differentiated thyroid carcinoma: the Turin proposal for the use of uniform diagnostic criteria and an algorithmic diagnostic approach. Am J Surg Pathol 2007; 31: 1256-64. 4. Patel KN, Shaha AR. Poorly differentiated and anaplastic thyroid cancer. Cancer Control 2006; 13: 119-28. 5. Asioli S, Erickson LA, Righi A, Jin L, Volante M, Jenkins S. Poorly differentiated carcinoma of the thyroid: validation of the Turin proposal and analysis of IMP3 expression. Mod Pathol 2010; 23: 1269-78. 6. Ito Y, Hirokawa M, Fukushima M, Inoue H, Yabuta T, Uruno T, et al. Prevalence and prognostic significance of poor differentiation and tall cell variant in papillary carcinoma in Japan. World J Surg 2008; 32: 1535-43. 7. Sanders EM Jr, LiVolsi VA, Brierley J, Shin J, Randolph GW. An evidence-based review of poorly differentiated thyroid cancer. World J Surg 2007; 31: 934-45. 8. Auersperg M, Us-Krasovec M, Petric G, Pogacnik A, Besic N. Results of com­bined modality treatment in poorly-differentiated and anaplastic thyroid carcinoma. Wien Klin Wochenschr 1990; 102: 267-70. 9. Ibrahimpasic T, Ghossein R, Carlson DL, Chernichenko N, Nixon I, Palmer FL, et al. Poorly differentiated thyroid carcinoma presenting with gross ex­trathyroidal extension: 1986-2009 Memorial Sloan-Kettering Cancer Center experience. Thyroid 2013; 23: 997-1002. 10. Lai HW, Lee CH, Chen JY, Tseng LM, Yang AH. Insular thyroid carcinoma: col­lective analysis of clinicohistologic prognostic factors and treatment effect with radioiodine or radiation therapy. J Am Coll Surg 2006; 203: 715-22. 11. Justin EP, Seabold JE, Robinson RA, Walker WP, Gurll NJ, Hawes DR. Insular carcinoma: a distinct thyroid carcinoma with associated iodine-131 localiza­tion. J Nucl Med 1991; 32: 1358-63. 12. Besic N, Auersperg M, Dremelj M, Vidergar-Kralj B, Gazic B. Neoadjuvant chemotherapy in 16 patients with locally advanced papillary thyroid carci­noma. Thyroid 2013; 23: 178-84. 13. Besic N, Auersperg M, Gazic B, Dremelj M, Zagar I. Neoadjuvant chemo­therapy in 29 patients with locally advanced follicular or Hürthle cell thyroid carcinoma: a phase 2 study. Thyroid 2012; 22: 131-7. 278 14. Sobin LH, Gospodarowitz MK, Witekind C. Thyroid gland (ICD-O C73). In: Sobin LH, Gospodarowitz MK, Witekind C, editors. TNM classification of malignant tumours. 7th edition. New York: Wiley Blackwell; 2009. p. 58-62. 15. Dobrenic M, Huic D, Zuvic M, Grosev D, Petrovic R, Samardzic T. Usefulness of low iodine diet in managing patients with differentiated thyroid cancer ­initial results. Radiol Oncol 2011; 45: 189-95. 16. Auersperg M, Us-Krasovec M, Pogacnik A, Hocevar M, Novak B, Besic N, et al. Induction chemotherapy in primarily inoperable differentiated thyroid carcinomas. Radiol Oncol 1993; 27: 187-91. 17. Zager V, Cemazar M, Hreljac I, Lah TT, Sersa G, Filipic M. Development of human cell biosensor system for genotoxicity detection based on DNA damage-induced gene expression. Radiol Oncol 2010; 44: 42-51. 18. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009; 45: 228-47. 19. Santini F, Bottici V, Elisei R, Montanelli L, Mazzeo S, Basolo F. Cytotoxic effects of carboplatinum and epirubicin in the setting of an elevated se­rum thyrotropin for advanced poorly-differentiated thyroid cancer. J Clin Endocrinol Metab 2002; 87: 4160-5. 20. Cooper DS, Doherty GM, Haugen BR, Kloos RT, Lee SL, Mandel SJ, et al. Revised American Thyroid Association Management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid 2009; 19: 1167-214. 21. Marinko T, Dolenc J, Bilban-Jakopin C. Cardiotoxicity of concomitant ra­diotherapy and trastuzumab for early breast cancer. Radiol Oncol 2014; 48: 105-12. 22. Pacini F, Schlumberger M, Dralle H, Elisei R, Smit JW, Wiersinga W, et al. The European Thyroid Cancer Taskforce: European consensus for the manage­ment of patients with differentiated thyroid carcinoma of the follicular epithelium. Eur J Endocrinol 2006; 154: 787-803. 23. Jung TS, Kim TY, Kim KW, Oh YL, Park do J, Cho BY, et al. Clinical features and prognostic factors for survival in patients with poorly differentiated thyroid carcinoma and comparison to the patients with the aggressive variants of papillary thyroid carcinoma. Endocr J 2007; 54: 265-74. 24. Kim KB, Cabanillas ME, Lazar AJ, Williams MD, Sanders DL, Ilagan JL, et al. Clinical responses to vemurafenib in patients with metastatic papillary thy­roid cancer harboring BRAF(V600E) mutation. Thyroid 2013; 23: 1277-83. 25. Liu M, Shen Y, Ruan M, Li M, Chen L. Notable decrease of malignant pleural effusion after treatment with sorafenib in radioiodine-refractory follicular thyroid carcinoma. Thyroid 2014; 24: 1179-83. 279 research article Fibulin-3 as a biomarker of response to treatment in malignant mesothelioma Viljem Kovac1, Metoda Dodic-Fikfak2, Niko Arneric2, Vita Dolzan3, Alenka Franko2 1 Institute of Oncology Ljubljana, Zaloška cesta 2, Ljubljana, Slovenia 2 Clinical Institute of Occupational Medicine, University Medical Center Ljubljana, Ljubljana, Slovenia 3 Pharmacogenetics Laboratory, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2015; 49(3): 279-285. Received 16 March 2015 Accepted 30 March 2015 Correspondence to: Assist. Prof. Alenka Franko, M.D., Ph.D., Clinical Institute of Occupational Medicine, University Medical Center Ljubljana, Poljanski nasip 58, Ljubljana, Slovenia. Phone: +386 1 522 2119; Fax: +386 1 522 2478; Email: alenka.franko@siol.net Disclosure: No potential conflicts of interest were disclosed. Background. Fibulin-3 is a new potential biomarker for malignant mesothelioma (MM). This study evaluated the potential applicability of fibulin-3 plasma levels as a biomarker of response to treatment and its prognostic value for progressive disease within 18 months. The potential applicability of fibulin-3 in comparison with or in addition to soluble mesothelin-related peptides (SMRP) was also assessed. Patients and methods. The study included 78 MM patients treated at the Institute of Oncology Ljubljana between 2007 and 2011. Fibulin-3 levels in plasma samples obtained before treatment and in various responses to treatment were measured with the enzyme-linked immunosorbent assay. Results. In patients evaluated before the treatment, fibulin-3 levels were not influenced by histopathological sub­types, tumour stages or the presence of metastatic disease. Significantly higher fibulin-3 levels were found in progres­sive disease as compared to the levels before treatment (Mann-Whitney [U] test = 472.50, p = 0.003), in complete response to treatment (U = 42.00, p = 0.010), and in stable disease (U = 542.00, p = 0.001). Patients with fibulin-3 levels exceeding 34.25 ng/ml before treatment had more than four times higher probability for developing progressive disease within 18 months (odds ratio [OR] = 4.35, 95% confidence interval [CI] 1.56–12.13). Additionally, patients with fibulin-3 levels above 34.25 ng/ml after treatment with complete response or stable disease had increased odds for progressive disease within 18 months (OR = 6.94, 95% CI 0.99–48.55 and OR = 4.39, 95% CI 1.63–11.81, respectively). Conclusions. Our findings suggest that in addition to SMRP fibulin-3 could also be helpful in detecting the progres­sion of MM. Key words: fibulin-3; biomarker; malignant mesothelioma; response to treatment. Introduction Malignant mesothelioma (MM) is an aggressive malignant disease that has been associated with oc­cupational and environmental exposure to asbes­tos.1-8 Most commonly it arises from serosal cells of the pleura and less frequently from peritoneum or other serosal surfaces such as pericardium and tunica vaginalis.6,9 Malignant mesothelioma remains a fatal disease that is hard to treat with favourable outcome.9,10 Hence, potential new biomarkers for earlier diag­nosis and following the response to treatment have been intensively investigated. One of the most ex­tensively studied blood-based biomarkers is solu­ble mesothelin-related peptides (SMRP); however, the poor sensitivity limits its added value to early diagnosis.10,11 Nevertheless, the results of our pre­vious study suggest that SMRP may be a useful tu­mour marker for detecting the progression of MM and evaluating tumour response to treatment.12 Fibulin-3, also known as epidermal growth fac­tor containing fibulin-like extracellular matrix pro­tein 1 (EFEMP1), is suggested to be a new potential 280 biomarker for MM.13 Fibulin- 3 belongs to a family of extracellular matrix glycoproteins14 that have re­cently been shown to act as tumour suppressors or activators in different cancers.15-17 It has restricted expression in the body and is predominately local­ized in the extracellular matrix of elastic tissue.18 The levels of fibulin-3 expression have been found to be decreased in many cancer types due to pro­moter hypermethylation and have been correlated with poor survival of patients with lung cancer19,20, breast cancer21, and hepatocellular carcinoma.22 On the other hand, an increase in fibulin-3 was observed in malignant gliomas23, cervical carcino­mas24, and pancreatic cancer.25 Fibulin-3 was first studied as a biomarker of MM by Pass et al. who reported that plasma fibu­lin-3 levels can distinguish a healthy person with exposure to asbestos from patients with MM.13 They found that in conjunction with fibulin-3 lev­els in pleural effusions, plasma fibulin-3 levels can further differentiate MM effusion from other ma­lignant and benign effusions.13 Recent studies iden­tified soluble mesothelin as a superior diagnostic biomarker for MM compared to fibulin-3, whereas fibulin-3 provided superior prognostic informa­tion compared to mesothelin.26 According to our knowledge and available lit­erature, fibulin-3 has not been studied so far as a biomarker for evaluating tumour response to treat­ment. This study aimed to determine fibulin-3 levels in plasma of patients with MM before treat­ment and in various responses to treatment (com­plete response, partial response, stable disease, and progressive disease), to evaluate its potential applicability as a biomarker of tumour response to treatment, and to assess if plasma level of fibulin-3 could predict the probability of progressive disease after the response to treatment in the period of 18 months. We also assessed the potential applicabil­ity of fibulin-3 as a biomarker of tumour response to treatment in comparison with or in addition to SMRP. Patients and methods Patients A panel study was performed. Patients eligible for inclusion in the study had histologically proven MM and each subject acted as her/his own con­trol in an ongoing longitudinal study.12 Briefly, the study included 78 patients with MM treated at the Institute of Oncology Ljubljana in the period be­tween March 2007 and June 2011. Eligibility criteria included biopsy-proven MM. In all patients, thoracoscopy or laparoscopy/lapa­rotomy was performed. The immunohistochemis­try methods were used (Cytokeratin 5/6 [CK5/6], Epithelial Membrane Antigen [EMA], Calretinin, Vimentin, Wilms tumour gene–1 [WT1], CD15, Ber-EP4, B72.3, MOC-31, actin, desmin, S-100, Carcinoembryonic Antigen [CEA], thyroid tran­scriptor factor1 [TTF-1]). The patients had no history of another cancer during the past 5 years or breast cancer ever; the Eastern Cooperative Oncology Group (ECOG) performance status (PS) was 0–2. Tumour extension was classified according to TNM classification, based on the results from chest and upper abdominal CT scan and thoracoscopy.27 For comparison with subsequent scanning, the thickness of the tumour on three CT levels was re­corded considering the modified RECIST criteria.28 Sporadically a NMR was done to evaluate the op­erability of some patients29 and a PET-CT was also done in some patients to evaluate the extent of dis­ease and response to treatment like in patients with lung cancer.30 The patients were treated with 4 to 9 cycles of chemotherapy comprising cisplatin and low dose gemcitabine in prolonged infusion, or cisplatin and pemetrexed.31-34 In one patient with pleural MM, extrapleural pleuropneumonectomy was carried out before chemotherapy and in four patients with pleural MM, it was carried out after chemothera­py. Peritonectomy was performed in two patients with peritoneal MM before chemotherapy and in three patients after chemotherapy. Four patients received best supportive care only. Twenty-nine patients with pleural MM were treated with sec­ond-line chemotherapy and two of them received palliative radiotherapy.12 For all the patients, data on smoking were ob­tained using a standardized questionnaire. The du­ration of smoking and the number of pack-years of smoking were calculated for each subject.35,36 To determine occupational and/or environmental as­bestos exposure, a semi-quantitative method was used as previously described.12 Methods Blood specimen collection was carried out in pa­tients before treatment (before the 1st cycle of chem­otherapy or surgery) and/or after treatment (after the third and/or the sixth cycle of chemotherapy or surgical procedure) and/or at the progress of the disease. In total, 135 blood samples from 78 pa­ 281 TABLE 1. Fibulin-3 levels (ng/ml) before treatment at different histopathological subtypes, at different tumour stages and according to the presence of metastatic disease in patients with malignant mesothelioma Subtype Epitheloid (N = 25) Biphasic (N = 6) Sarcomatoid (N = 2) 41.52 41.04 27.36 24.26 16.57 1.60 36.42 35.24 27.36 1.65–92.32 22.72–65.22 26.23–28.49 23.00–58.14 28.41–59.41 26.23–27.36 69.00a 2.00b 17.50c 0.789 0.286 0.519 Tumour staged Ie (N = 1) II (N = 8) III (N = 13) IV (N = 7) 28.88 46.39 40.97 8.32 28.09 21.71 28.67 47.41 30.31 15.69–40.78 1.65–92.32 22.72–84.33 21.98–35.32 22.03–66.43 28.49–54.93 32.00f 42.00g 19.00h 0.156 0.817 0.320 Metastatic disease Present (N = 7) Not present (N = 22) 40.97 39.89 21.70 23.38 30.31 35.25 22.72–84.33 1.65–92.32 28.49–54.93 22.21–54.12 73.00i 0.854 N = number of plasma samples; a Mann-Whitney (U) test calculated for epitheloid subtype vs. biphasic subtype; b Mann-Whitney (U) test calculated for biphasic vs. sarcomatoid subtype; c Mann-Whitney (U) test calculated for epitheloid subtype vs. sarcomatoid subtype; d Pleural malignant mesothelioma only; e Stage I was found only in one patient with fibulin-3 level 43.44 ng/ml; f Mann-Whitney (U) test calculated for stage II vs. III; g Mann-Whitney (U) test calculated for stage III vs. IV; h Mann-Whitney (U) test calculated for stage II vs. IV; i Mann-Whitney (U) test calculated for metastatic disease present vs. not present tients were collected in different periods of disease and treatment. Plasma was prepared immediately after blood sampling and stored in aliquots frozen at -30 oC until the fibulin-3 assay was performed. Fibulin-3 levels in plasma were measured with the use of enzyme-linked immunosorbent assay (Uscn Life Science Inc., Wuhan, China). The median value of fibulin-3 in complete response or after the surgery was chosen as the cut-off level. For all the patients, the information on SMRP levels was available from our previous study12 for the same time-points before and/or after treatment. A level of 1.50 nmol/L was considered as a cut-off value for positive SMRP. Using receiver operat­ing characteristic (ROC) curve analysis, we deter­mined the fibulin-3 cut-off values for prediction of disease progression. We compared serum lev­els in progressive disease with levels in complete response, partial response or stable disease and calculated the area under the curve (AUC), sensi­tivity and specificity. As our aim was to determine the usefulness of serum fibulin-3 for screening for progressive disease, cut-off value with at least 80% sensitivity was selected to limit the potential for false negative results. On the other hand, lower specificity would not be as problematic, as patients would have a more detailed check-up after initial screening. Statistics and ethical consideration Standard descriptive statistics were used to de­scribe each variable. Mann-Whitney test (U) test was performed to determine the differences in fibulin-3 levels before treatment and in various responses to treatment. The correlations between fibulin-3 and SMRP levels were calculated using Pearson’s correlation coefficient. Logistic regres­sion analysis was used to assess the odds for differ­ent responses to treatment. Prior to inclusion, all patients were fully in­formed about the study and signed informed consent to participate. The study was approved by the Slovenian Ethics Committee for Research in Medicine and was carried out according to the Helsinki Declaration. Results Patients The study included 78 patients with MM, 57 (73%) male and 21 (27%) female. The overall median (min–max range) age was 66 (23–84) years. Among them, 35 (44.9%) were ever smokers and 43 (55.1%) of them never smoked. The median duration of smoking was 18 (1–69) years, the median number of smoked cigarettes per day was 20 (1–29) and 282 TABLE 2. Fibulin-3 levels (ng/ml) before treatment and in different responses to treatment in 78 patients with MM All phases (N = 135) 44.57 21.31 40.78 0.00–105.00 29.18–56.27 Before treatment (N = 33) 40.57 22.26 35.09 1.65–92.32 24.23–56.21 877.00a 0.598 Complete response orafter surgery (N = 5) 32.43 9.98 34.25 18.16–45.50 23.55–40.40 Partial response (N = 13) 45.13 26.48 41.18 0.00–105.00 27.90–56.42 Stable disease (N = 39) 40.00 16.11 37.10 6.52–73.44 29.40–47.56 Progressive disease (N = 45) 53.56 21.67 47.19 16.26–105.00 37.78–67.93 813.00b 0.001 a N = number of plasma samples; Mann-Whitney (U) test calculated for fibulin-3 before treatment vs. stable disease + partial response + complete response or after surgery; b Mann-Whitney (U) test calculated for fibulin-3 in progressive disease vs. stable disease + partial response + complete response or after surgery TABLE 3. The odds for developing different responses to treatment for fibulin-3 levels > 34.25 ng/ and SMRP levels >1.50 nmol/L in univariate and multivariate analysis Fibulin-3 SMRP Fibulin-3 SMRP OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI) Before treatment vs. complete response 0.63 (0.09–4.26) 0.14 (0.01–1.43) 0.74 (0.1–5.48) 0.15 (0.02–1.48) Before treatment vs. partial response 1.51 (0.41–5.58) 0.67 (0.18–2.45) 1.56 (0.42–5.84) 0.64 (0.17–2.39) Before treatment vs. stable disease 0.99 (0.39–2.51) 0.74 (0.29–1.91) 1.04 (0.41–2.67) 0.73 (0.28–1.93) Before treatment vs. progressive disease 4.35 (1.56–12.13) 5.86 (1.68–22.40) 3.74 (1.28–10.93) 4.94 (1.35–18.08) Complete response vs.progressive disease 6.94 (0.99–48.55) 41.00 (3.65–461.03) 7.77 (0.58–104.98) 43.99 (3.07–629.57) Partial response vs. progressive disease 2.89 (0.75–11.19) 8.79 (1.97–39.28) 2.90 (0.65–13.00) 8.81 (1.89–41.11) Stable disease vs. progressive disease 4.39 (1.63–11.81) 7.92 (2.37–26.46) 3.52 (1.22–10.14) 6.58 (1.90–22.83) CI = confidence interval; OR = odds ratio; SMRP = soluble mesothelin-related peptides the median pack-years of smoking amounted to 15 (1–45). Asbestos exposure was confirmed in 67 (85.9%) of the patients with MM. The assessed exposure was low in 24 (30.8%) patients, median in 21 (26.9%) patients, and high in 22 (28.2%) patients, while in 11 patients (14.1%) asbestos exposure could not be proven with certainty. In the exposed group, the median duration of exposure was 90.50 (0.1–528) months. Regarding the location of the disease, 70 (89.7%) patients had pleural and 8 (10.3%) peritoneal MM. Epitheloid MM was found in 64 (82.0%), biphasic in 7 (9.0%), and sarcomatoid in 7 (9.0%) patients. Five (6.4%) patients were diagnosed with stage I, 17 (21.8%) with stage II, 28 (35.9%) with stage III and 20 (25.6%) with stage IV, while 8 (10.3%) patients had MM of peritoneum and therefore, the stage could not been determined. The median survival of all patients was 20.1 (2.8–86.1) months. Among 33 patients evaluated before treatment, no significant differences in fibulin-3 levels were observed between histopathological subtypes or between tumour stages. Fibulin-3 levels before treatment were not significantly different between patients with and without evidence of metastatic disease (U = 73.00, p = 0.854) (Table 1). 283 The results of descriptive statistics for fibulin-3 levels before treatment and/or in different respons­es to treatment for all 78 malignant mesothelioma patients are presented in Table 2. No significant difference was observed between the fibulin-3 lev­els before treatment as compared to the levels in patients in complete response to treatment (U = 71.00, p = 0.641), partial response to treatment (U = 186.00, p = 0.496), or stable disease (U = 597.00, p = 0.603). On the other hand, significantly higher fibulin-3 levels were found in progressive disease as compared to the levels before treatment (U = 472.50, p = 0.006). Fibulin-3 levels were also signifi­cantly higher in progressive disease as compared to the levels in complete response to treatment (U = 42.00, p = 0.020) or stable disease (U = 542.00, p = 0.002), while no significant difference was ob­served between progressive disease and partial re­sponse to treatment (U = 229.00, p = 0.241). No correlation (r = 0.364, p < 0.001) was detected between fibulin-3 levels and SMRP levels as de­termined at the same time-points in our previous study.12 In ROC curve analysis comparing progressive disease with complete response, partial response or stable disease, AUC for fibulin-3 was 68.3% (95% CI = 57.9–78.7, p = 0.002). Cut-off value of 34.25 ng/ ml had sensitivity of 82.2%, thus passing the sensi­tivity threshold of 80%. On the other hand, speci­ficity for this cut-off value was 47.7% (Figure 1). For mesothelin levels, AUC was 84.2% (95% CI = 76.8–91.7, p < 0.001, Figure 1). Previously deter­mined cut-off value of 1.5 nmol/L had high sensi­tivity of 91.1% and specificity of 49.1%, thus also limiting the chance of false negative results. For further logistic regression analysis, fibu­lin-3 levels were categorized into two categories based on ROC curve analysis: . 34.25 ng/ml and > 34.25 ng/ml. Patients with fibulin-3 levels before treatment exceeding 34.25 ng/ml had more than four times higher probability for developing pro­gressive disease during the period of 18 months (OR = 4.35, 95% CI 1.56–12.13, p = 0.005). However, fibulin-3 levels before treatment were not associat­ed with complete response to treatment (OR = 0.63, 95% CI 0.09–4.26, p = 0.633), partial response to treatment (OR = 1.51, 95% CI 0.41–5.58, p = 0.540), or stable disease (OR = 0.99, 95% CI 0.39–2.51, p = 0.984). Nevertheless, patients with fibulin-3 levels higher than 34.25 ng/ml after the treatment with complete response to treatment or with stable dis­ease showed increased odds for developing pro­gressive disease during the period of 18 months (OR = 6.94, 95% CI 0.99–48.55, p = 0.051 and OR = 4.39, 95% CI 1.63–11.81, p = 0.003 respectively) (Table 3). The analysis also showed that patients with pre­treatment SMRP levels >1.50 nmol/L had almost six times higher odds for progressive disease during the period of 18 months (OR = 5.86, 95% CI 1.68– 22.40, p = 0.005). Additionally, patients with SMRP levels >1.50 nmol/L after the treatment and with complete response to treatment, partial response to treatment, and stable disease were at higher risk for developing progressive disease compared with those with SMRP . 1.50 nmol/L during the period of 18 months (OR = 41.00, 95% CI 3.65–461.03, p = 0.003, OR = 8.79, 95% CI 1.97–39.28, p = 0.004, and OR = 7.92, 95% CI 2.37–26.46, p = 0.001 respective­ly) (Table 3). To evaluate the combined effect of fibulin-3 and SMRP levels in evaluating tumour response to treat­ment, we constructed multivariate logistic regres­sion models that included two categories of fibu­lin-3 (> 34.25 ng/ml vs. . 34.25 ng/ml) and two cat­egories of SMRP (>1.50 nmol/L vs. . 1.50 nmol/L). The odds for developing different responses to treatment (complete response, partial response, stable disease, progressive disease) did not change considerably compared to the results of univarate logistic regression analysis when pretreatment fibulin-3 and SMRP were both above the respective cut-off levels (34.25 ng/ml and 1.50 nmol/L respec­tively). Similarly, the probability for developing progressive disease did not change significantly 284 compared with the results of univarate logistic re­gression analysis when fibulin-3 and SMRP were both above the respective cut-off levels in different responses to treatment (Table 3). Discussion As expected the occupational and/or environmen­tal exposure to asbestos was confirmed in almost 86% of patients with MM. This is in agreement with the results of the studies published so far that have proposed asbestos as a major cause for devel­oping this aggressive disease.1-8 Fibulin-3 has recently been suggested as a new tumour biomarker for MM.13,26 Pass et al. presented that plasma fibulin-3 levels can distinguish an as­bestos exposed healthy person from patients with MM.13 Creaney et al. recognized soluble mesothelin as a superior diagnostic biomarker for MM com­pared with fibulin-3, while fibulin-3 was indicated to provide superior prognostic information com­pared with mesothelin.26 However, to our knowl­edge and available literature, fibulin-3 has not been studied yet as a biomarker for evaluating tumour response to treatment. The results of the current study show signifi­cantly higher fibulin-3 levels in progressive disease as compared with the levels before treatment, in complete response to treatment, and in stable dis­ease, which indicates that fibulin-3 could be help­ful in identifying the progression of MM. On the other hand, no significant difference was observed between the fibulin-3 levels before treatment as compared with the levels in complete response to treatment, partial response to treatment, and sta­ble disease. The results of our previous study in­vestigating SMRP as a tumour biomarker for MM, showed significantly higher SMRP levels before treatment than the levels in complete response, partial response, and a borderline significant dif­ference between levels before treatment and stable disease.12 These findings suggest SMRP not only as a superior diagnostic biomarker for MM compared with fibulin-3 as presented in the study of Creaney et al.26, but also as a superior biomarker for evaluat­ing tumour response to treatment. An important finding of the current study shows that the probability for the development of progressive disease during the period of 18 months was more than four times higher when fibulin-3 levels before treatment exceeded 34.25 ng/ml, and almost five times higher when SMRP level before treatment was higher than 1.50 nmol/L. The analy­sis also showed increased odds for developing pro­gressive disease during the period of 18 months when fibulin-3 levels after the treatment and with complete response to treatment or stable disease were higher than 34.25 ng/ml. The same holds true of SMRP levels above 1.50 nmol/L in complete re­sponse, partial response, and stable disease. These results suggest that in addition to SMRP, the fibu­lin-3 levels before treatment, in complete response to treatment, and in stable disease could help pre­dict the risk of developing progressive disease in MM. When including fibulin-3 levels above 34.25 ng/ml and SMRP levels above 1.50 nmol/L in multivariate logistic regression models, the odds for both fibulin-3 and SMRP did not change signifi­cantly, suggesting independent effects. However, we have to indicate that the confidence intervals were wide because the number of involved sub­jects was low. In conclusion, the findings of our current study show that in addition to SMRP12, fibulin-3 could also be helpful in detecting the progression of MM. Contrary to SMRP12, fibulin-3 has not been proven as a useful biomarker for evaluating tu­mour response to treatment. The results of the pre­sent study also indicate that fibulin-3 levels before treatment, in complete response to treatment, and in stable disease could be beneficial in predicting the risk of developing progressive disease in pa­tients with MM. To increase the power of the study and to validate these results, a larger sample size is needed. Acknowledgements The authors thank Barbara Možina, M.Sc., head of the Biochemistry Laboratory, Institute of Oncology Ljubljana, Slovenia, for her help with blood sam­ples collection and handling, Savica Soldat, B.Sc., for her help with enzyme-linked immunosorbent assays and Katja Goričar, Ph.D., for her support with the statistical analysis. This work was financially supported by The Slovenian Research Agency (ARRS Grants Nos. L3­3648, P1-0170 and P3-0003). References 1. Berry M. Mesothelioma incidence and community asbestos exposure. Environ Res 1997; 75: 34-40. 2. Iwatsubo Y, Pairon JC, Boutin C, Menard O, Massin N, Caillaud D, et al. Pleural mesothelioma: dose-response relation at low levels of asbestos exposure in a French population-based case-control study. Am J Epidemiol 1998; 148: 133-42. 285 3. Howel D, Gibbs A, Arblaster L, Swinburne L, Schweiger M, Renvoize E, et al. Mineral fibre analysis and routes of exposure to asbestos in the develop­ment of mesothelioma in an English region. Occup Environ Med 1999; 56: 51-8. 4. Agudo A, González CA, Bleda MJ, Ramirez J, Hernandez S, Lopez F, et al. Occupation and risk of malignant pleural mesothelioma: a case-control study in Spain. Am J Ind Med 2000; 37: 159-68. 5. Magnani C, Dalmasso P, Biggeri A, Ivaldi C, Mirabelli D, Terracini B. Increased risk of malignant mesothelioma of the pleura after residential or domestic exposure to asbestos: a case-control study in Casale Monferrato, Italy. Environ Health Perspect 2001; 109: 915-9. 6. Zellos L, Christiani DC. Epidemiology, biologic behaviour, and natural history of mesothelioma. Thorac Surg Clin 2004; 14: 469-77. 7. Pan XL, Day HW, Wang W, Beckett LA, Schenker MB. Residential proximity to naturally occurring asbestos and mesothelioma risk in California. Am J Respir Crit Care Med 2005; 172: 1019-25. 8. Maule MM, Magnani C, Dalmasso P. Modeling mesothelioma risk associ­ated with environmental asbestos exposure. Environ Health Perspect 2007; 115: 1066-71. 9. Robinson BW, Lake RA. Advances in malignant mesothelioma. N Engl J Med 2005; 353: 1591-603. 10. Kovac V, Zwitter M, Zagar T. Improved survival after introduction of chemo­therapy for malignant pleural mesothelioma in Slovenia: population-based survey of 444 patients. Radiol Oncol 2012; 46: 136-44. 11. Hollevoet K, Reitsma JB, Creaney J, Grigoriu BD, Robinson BW, Scherpereel A, et al. Serum mesothelin for diagnosing malignant pleural mesothelioma: an individual patient data meta-analysis. J Clin Oncol 2012; 30: 1541-9. 12. Franko A, Dolzan V, Kovac V, Arneric N, Dodic-Fikfak M. Soluble mesothelin­related peptides levels in patients with malignant mesothelioma. Dis Markers 2012; 32: 123-31. 13. Pass HI, Levin SM, Harbut MR, Melamed J, Chiriboga L, Donington J, et al. Fibulin-3 as a blood and effusion biomarker for pleural mesothelioma. N Engl J Med 2012; 367: 1417-27. 14. Timpl R, Sasaki T, Kostka G, Chu ML. Fibulins: a versatile family of extracel­lular matrix proteins. Nat Rev Mol Cell Biol 2003; 4: 479-89. 15. Argraves WS, Greene LM, Cooley MA, Gallagher WM. Fibulins: physiological and disease perspectives. EMBO Rep 2003; 4: 1127-31. 16. Zhang Y, Marmorstein LY. Focus on molecules: fibulin-3 (EFEMP1). Exp Eye Res 2010; 90: 374-5. 17. Kim IG, Kim SY, Choi SI, Lee JH, Kim KC, Cho EW. Fibulin-3-mediated inhibi­tion of epithelial-to-mesenchymal transition and self-renewal of ALDH+ lung cancer stem cells through IGF1R signaling. Oncogene 2014; 33: 3908-17. 18. Kobayashi N, Kostka G, Garbe JH, Keene DR, Bachinger HP, Hanisch FG, et al. A comparative analysis of the fibulin protein family. Biochemical charac­terization, binding interactions, and tissue localization. J Biol Chem 2007; 282: 11805-16. 19. Yue W, Dacic S, Sun Q, Landreneau R, Guo M, Zhou W, et al. Frequent inactivation of RAMP2, EFEMP1 and Dutt1 in lung cancer by promoter hypermethylation. Clin Cancer Res 2007; 13: 4336-44. 20. Xu S, Yang Y, Sun YB, Wang HY, Sun CB, Zhang X. Role of fibulin-3 in lung cancer: in vivo and in vitro analyses. Oncol Rep 2014; 31: 79-86. 21. Sadr-Nabavi A, Ramser J, Volkmann J, Naehrig J, Wiesmann F, Betz B, et al. Decreased expression of angiogenesis antagonist EFEMP1 in sporadic breast cancer is caused by aberrant promoter methylation and points to an impact of EFEMP1 as molecular biomarker. Int J Cancer 2009; 124: 1727-35. 22. Nomoto S, Kanda M, Okamura Y, Nishikawa Y, Qiyong L, Fujii T, et al. Epidermal growth factor-containing fibulin-like extracellular matrix protein 1, EFEMP1, a novel tumor-suppressor gene detected in hepatocellular carcinoma using double combination array analysis. Ann Surg Oncol 2010; 17: 923-32. 23. Hu B, Thirtamara-Rajamani KK, Sim H, Viapiano MS. Fibulin-3 is uniquely upregulated in malignant gliomas and promotes tumor cell motility and invasion. Mol Cancer Res 2009; 7: 1756-70. 24. En-lin S, Sheng-guo C, Hua-qiao W. The expression of EFEMP1 in cervical carcinoma and its relationship with prognosis. Gynecol Oncol 2010; 117: 417-22. 25. Seeliger H, Camaj P, Ischenko I, Kleespies A, De Toni EN, Thieme SE, et al. EFEMP1 expression promotes in vivo tumor growth in human pancreatic adenocarcinoma. Mol Cancer Res 2009; 7: 189-98. 26. Creaney J, Dick IM, Meniawy TM, Leong SL, Leon JS, Demelker Y, et al. Comparison of fibulin-3 and mesothelin as markers in malignant mesothe­lioma. Thorax 2014; 69: 895-902. 27. UICC International Union Against Cancer. Pleurak mesothelioma. In: Sobin LH, Gospodarowicz MK, Wittekind C, editors. TNM classification of malig­nant tumours, 7th edition. Chichester: Wiley-Blackwell; 2009. p. 147-50. 28. Byrne MJ, Nowak AK. Modified RECIST criteria for assessment of response in malignant pleural mesothelioma. Ann Oncol 2004; 15: 257-60. 29. Podobnik J, Kocijancic I, Kovac V, Sersa I. 3T MRI in evaluation of asbestos-related thoracic diseases - preliminary results. Radiol Oncol 2010; 44: 92-6. 30. Zwitter M, Stanic K, Rajer M, Kern I, Vrankar M, Edelbaher N, et al. Intercalated chemotherapy and erlotinib for advanced NSCLC: high propor­tion of complete remissions and prolonged progression-free survival among patients with EGFR activating mutations. Radiol Oncol 2014; 48: 361-8. 31. Vogelzang NJ, Rusthoven JJ, Symanowski J, Denham C, Kaukel E, Ruffie P, et al. Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma. J Clin Oncol 2003; 21: 2636-44. 32. Goricar K, Kovac V, Dolzan V. Polymorphisms in folate pathway and pem­etrexed treatment outcome in patients with malignant pleural mesothe­lioma. Radiol Oncol 2014; 48: 163-72. 33. Lee CW, Murray N, Anderson H, Rao SC, Bishop W. Outcomes with first-line platinum-based combination chemotherapy for malignant pleural mesothelioma: A review of practice in British Columbia. Lung Cancer 2009; 64: 308-13. 34. Kovac V, Zwitter M, Rajer M, Marin A, Debeljak A, Smrdel U, et al. 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Ann Occup Hyg 2007; 51: 261-8. 286 research article Impact of tumour volume on prediction of progression-free survival in sinonasal cancer Florian Hennersdorf1, Paul-Stefan Mauz2, Patrick Adam3, Stefan Welz4, Anne Sievert2, Ulrike Ernemann1, Sotirios Bisdas1 1 Department of Diagnostic and Interventional Neuroradiology, University Hospital Tübingen, Germany 2 Department of Otorhinolaryngology, University Hospital Tübingen, Germany 3 Institute of Pathology, University Hospital Tübingen, Germany 4 Department of Radiation Oncology, University Hospital Tübingen, Germany Radiol Oncol 2015; 49(3): 286-290. Received 16 February 2015 Accepted 13 June 2015 Correspondence to: Florian Hennersdorf, M.D., Department of Diagnostic and Interventional Neuroradiology, Universitätsklinikum Tübingen, Hoppe-Seyler-Straße 3, 72076 Tübingen, Germany. E-mail: florian.hennersdorf@med.uni-tuebingen.de Disclosure: No potential conflicts of interest were disclosed. Background. The present study aimed to analyse potential prognostic factors, with emphasis on tumour volume, in determining progression free survival (PFS) for malignancies of the nasal cavity and the paranasal sinuses. Patients and methods. Retrospective analysis of 106 patients with primary sinonasal malignancies treated and followed-up between March 2006 and October 2012. Possible predictive parameters for PFS were entered into univariate and multivariate Cox regression analysis. Kaplan-Meier curve analysis included age, sex, baseline tumour volume (based on MR imaging), histology type, TNM stage and prognostic groups according to the American Joint Committee on Cancer (AJCC) classification. Receiver operating characteristic (ROC) curve analysis concerning the predictive value of tumour volume for recurrence was also conducted. Results. The main histological subgroup consisted of epithelial tumours (77%). The majority of the patients (68%) showed advanced tumour burden (AJCC stage III-IV). Lymph node involvement was present in 18 cases. The mean tumour volume was 26.6 ± 21.2 cm3. The median PFS for all patients was 24.9 months (range: 2.5–84.5 months). The ROC curve analysis for the tumour volume showed 58.1% sensitivity and 75.4% specificity for predicting recurrence. Tumour volume, AJCC staging, T- and N- stage were significant predictors in the univariate analysis. Positive lymph node status and tumour volume remained significant and independent predictors in the multivariate analysis. Conclusions. Radiological tumour volume proofed to be a statistically reliable predictor of PFS. In the multivariate analysis, T-, N- and overall AJCC staging did not show significant prognostic value. Key words: tumour volume; sinonasal carcinoma; prognostic value Introduction With an annual incidence rate of 0.5–1.0 per 100.000, malignancies of the nasal cavity and paranasal si­nuses are rare entities constituting only 3% of head and neck carcinomas and 0.5% of all malignant tu­mours.1 Sinonasal neoplasms show a wide variety of histological subtypes comprising carcinomas, melanomas, lymphomas, sarcomas and esthe­sioneuroblastomas.1 Unspecific related symptoms and asymptomatic tumour growth within the large air-filled spaces of the viscerocranium result in late diagnosis and poor prognosis.1,2 5-year survival rates reported in the literature range from 10–75% and depend significantly on tumour histology.3 Despite different subgroups, sinonasal tumours are commonly uniformly staged according to the TNM classification as published by the American Joint Committee on Cancer (AJCC).4 Retrospective studies have identified patient age, sex and tumour stage as predictive factors for progression-free and overall survival.5-11 Specifically, poor outcome was 287 observed when cervical lymph node involvement was present.7 However, preliminary evidence has shown no reliable prognostic value of the widely used staging systems.4,10 In the present work, we sought to validate and extend previous evidence regarding the prognos­tic factors in sinonasal malignancies by examining the prognostic value of epidemiological (age, sex) and clinical (staging systems) criteria in conjunc­tion with baseline imaging parameters like tumour volume based on MR imaging. Patients and methods Patients We conducted a retrospective analysis of all pa­tients who were imaged, diagnosed, and treated with sinonasal tumours between March 2006 and October 2012 at the Head and Neck Cancer centre at University Hospital Tübingen. The study was conducted according to the Helsinki Declaration. Each patient’s informed consent was obtained and Institutional Review Board approval was granted. It also approved the use of images and medical records. The inclusion criteria were: a) primary malignancy of the sinonasal tract with histological verification; b) patients undergone either primary surgery, primary radiotherapy or combined ad­juvant radiotherapy with or without concomitant chemotherapy; c) baseline MRI with gadolinium-enhanced T1-weighted sequences for tumour volu­metry, performed not longer than 1 week before surgical resection; d) adequate clinical follow-up on a 3- or 6-month time interval. Informed consent was obtained from all patients for the MR exams. Imaging studies and tumour volumetry All MR imaging examinations were performed by using the same 1.5 T MR scanners (Avanto and Aera, Siemens Medical Systems, Erlangen, Germany), with a 12-channel-array head coil. Along with a number of conventional T2- and T1-sequences before and after contrast agent, 3D isotropic T1-weighted image datasets (TR/TE 1300/2.6 ms, voxel size 1 mm3) were acquired after intravenous administration of gadobutrol. Apart from the head and neck MRI studies, patients re­ceived whole-body CT imaging with iodinated contrast agent in order to exclude distant disease. Radiological tumour volumetry in the contrast-enhanced 3D T1-weighted images was performed offline by two radiologists in consensus using a dedicated workstation and commercially avail­able software (Advantage Windows, GE Medical Systems, Milwaukee, WI). Therapy Standard treatment for epithelial tumours con­sisted of radical surgery and depending on tumour stage of subsequent radiotherapy with doses of 50– 67 Gy. Chemotherapy was not part of the standard therapeutic regimen and was only administered on adjuvant setting or for palliation to the patients with advanced tumour stages (stage IV). In these cases protocols containing cisplatin or carboplatin were used. Only patients with sinonasal lympho­ma received chemotherapy according to the rituxi­mab, cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP) protocol as the standard therapeutic regimen. Statistical analysis Progression-free survival (PFS) was defined as the number of months between the tumour resection and the diagnosis of locoregional tumour progress in follow-up surveillance and was analysed using the Kaplan-Meier method with log-rank (Mantel-Cox) test. Univariate and multivariate Cox regres­sion analysis with forward entry (Wald test) was conducted and the metrics were primarily treated as continuous or categorical variables without pre­determined cut-off values. The model, adjusted for age and sex, included baseline tumour volumetry, tumour histology and histological grading, T-stage and N-stage as “stand-alone” parameters, TNM and stage grouping according to the AJCC clas­sification. Receiver operating characteristic (ROC) curve analysis for the prediction of locoregional recurrence was conducted to determine the cut-off value of tumour volume that yielded optimal sensi­tivity and specificity. Overall survival was not used as an outcome owing to the small number of pa­tients being observed for five years or longer and to variations in treatment after patients experienced a disease progression, which would confound the direct evaluation of the stated hypothesis. Data normality was examined by Kolmogorov- Smirnov test and Q-Q plots. All statistical computations were conducted with commercially available soft­ware (MedCalc Statistical Software version 12.7.2, MedCalc Software bvba, Ostend, Belgium) and results were declared statistically significant at the 2-sided 5% comparison-wise significance level (P < 0.05). 288 Results One-hundred and six patients (45 females, 61 males) were identified (mean age: 64.8 years, range: 31–77 years). The main histological subgroup consisted of carcinomas comprising squamous cell carcino­mas (SCCA) (42 cases), adenocarcinomas (22 cases), adenoid cystic carcinomas (3 cases), anaplastic car­cinomas (2 cases), neuroendocrine carcinomas (5 cases) as well as other rare subtypes (8 cases). The remaining tumour entities included melanomas (10 cases), sarcomas (4 cases) and one esthesioneuro­blastoma. Patients diagnosed with lymphoma or plasmocytoma (6 and 3 respectively) were exclud­ed from the analysis due to the completely differ­ent therapeutic approach. Comprising 55% of pa­tients, the nasal cavity was the more common site of origin compared to 38% of tumours originating in the paranasal sinuses. In 7 patients the tumour could not be assigned to being nasal or paranasal in origin due to advanced tumour stage. The major­ity of the patients (68%) showed advanced tumour burden (stage III-IV). The distribution of T stages was as follows: 16% T1, 22% T2, 11% T3 and 41% T4. Most tumours were graded G2 (55%) and G3 (28%). Cervical lymph node involvement was pre­sent in 18 cases. The mean (± standard error, SE) radiological volume of primary tumours was 26.6 ± 21.2 cm3. Tumour cells at the surface of the resec­tion margin (R1) occurred in 13 cases, consisting of 8 epithelial tumours and 5 other than epithelial. The median PFS for all patients was 24.9 months (range: 2.5–84.5 months). Six patients with advanced dis­ease in the primary radiological staging received only palliative care and had short overall survival and thus, were excluded from further analysis in order to avoid statistical bias. Therefore a total of 91 patients were included into the statistical analy­sis. To further exclude bias due to different tumour subtypes we conducted subgroup analyses includ­ing only SCCA (42 patients) and adenocarcinomas (22 patients). In subgroup analysis patients with R1 resections were also excluded. Kaplan-Meier analysis demonstrated a sig­nificant (P = 0.003) prolongation of the PFS in pa­tients with T1-T2 tumours (mean PFS 68.6 months, standard error [SE] 5.7 months, 95% confidence interval [CI] 57.5–79.8) compared to those with T3-T4 tumours (mean PFS of 44.9 months, SE 5.6 months, 95% CI 34–55.9). Similarly, AJCC stage I-II patients had mean PFS of 68.9 months (SE 5.6, 95% CI 57.9–79.9) vs. 43.3 months (SE 5,6, 95% CI 32,3–54) for patients with AJCC stage III-IV tu­mours (P = 0.002). Tumour volume < 25.4 cm3 was associated with a mean PFS of 63 months (SE 5.1 months, 95% CI 53–73.1), whereas patients with larger tumour volumes had significantly lower PFS of 38.7 months (SE 6.4 months, 95% CI 26.1–51.3) (P = 0.004). Figure 1 shows Kaplan- Meier curves for T-stages (A), AJCC stage groups (B), different tumour volumes (C) and for N- stages (D) in all studied patients. In addition, Kaplan- Meier curves for different tumour volumes for SCCA subgroup (E, P = 0.0001) and adenocarcinoma subgroup (F, P = 0.057) are shown. ROC curves for the sum of covariates are pre­sented in Figure 2: for all studied patients (A) and separately for those with epithelial tumours (SCCA and adenocarcinoma). The ROC curve analysis for the tumour volume revealed 25.4 cm3 as the trade-off value with optimal sensitiv­ity (58.1%) and specificity (75.4%) rates for pre­dicting locoregional recurrence. Furthermore, TABLE 1. Univariate analysis of prognostic factors for progression­free-survival. The statistically significant predictors are indicated in bold italics Age (in years) < 67 . 67 0.32 Sex Male 0.06 Female Histology Epithelial Non-epithelial 0.49 T stage T . 2 T > 2 0.02 0.23 (0.11–0.68) N stage N = 0 N = 1 0.002 4.56 (1.75–11.94) AJCC AJCC = 1 AJCC > 1 0.004 0.27 (0.11–0.65) Volume (in cm3) < 25.4 . 25.4 0.0072 2.66 (1.31–5.41) AJCC = American Joint Committee on Cancer; CI = confidence interval multiple ROC curve analyses demonstrated that the largest area under the curve (AUC) was ob­served for tumour volume (0.687, SE 0.0857, 95% CI 0.519–0.855) followed by AJCC stage (0.607, SE 0.0824, 95% CI 0.445–0.768). The significant prognostic factors were entered into a multivariate model (overall model fit: P = 0.0008) where T-stage and AJCC stage were not significant covariates (P . 0.09). On contrary, posi­tive lymph node status at diagnosis proved to be a significant predictor for tumour recurrence (P = 0.04, odds ratio 2.6, 95% CI 1.06–13.6). Also, tumour volume was a significant predictor for tumour pro­gression (P = 0.03, odds ratio 1.05, 95% CI 0.15–6.7). The subgroup analyses revealed similar results to those for all patients. Notably, when the SCCA­adenocarcinoma subgroup of patients with com­plete tumour resection (R0) where included in a univariate analysis, tumour volume was highly sig­nificant for predicting PFS with a P-value of 0.0003. In the multivariate analysis for these patients, tu­mour volume remained the strongest prognostic parameter (P = 0.01, overall model fit < 0.0001). Discussion Prognosis of malignant neoplasms of the nasal cav­ity and paranasal sinuses is moderate to poor de­ 289 pending on factors such as histology, tumour stage and patient’s age.2,12-14 The crucial point in manag­ing sinonasal tumours is local tumour control.15 Despite improvement in therapy only marginal improvement of survival has been achieved over the past decades.3,15 The standard therapeutic regi­men includes surgery followed by radiotherapy. The potential benefit of chemotherapy adminis­tered neoadjuvantly and/or concomitantly in treat­ing epithelial neoplasms has been shown to be only marginal and is therefore controversial.5 Consistent with the literature epithelial neo­plasms were the most common entity constituting more than two thirds of all tumours with SCCA being the most frequent histology in our popu­lation. Adenoid cystic carcinoma which is com­monly found to be the second most frequent entity after SCCA was markedly underrepresented with only 3% of cases. Surprisingly, we had only one case of esthesioneuroblastoma but as much as 5 patients with neuroendocrine carcinoma. Though we did not perform revision of histologies, this aberration might be explained by observation of Cohen et al. Who reviewed 12 patients previously diagnosed with olfactory neuroblastoma. In this study only 2 cases were confirmed as neuroblasto­ma whereas 10 patients had in fact other tumours such as neuroendocrine carcinoma or others.16 Consistently with this theory, we found 5 cases of neuroendocrine carcinoma. In agreement with the literature, our population showed a male: female ratio of 3:2.15 The lag time between initial symptoms onset and surgery of tumours in nasal cavity or para-nasal sinuses is crucial for surgeon to obtain clear margins. Usually, many tumours undergo surgery in advanced stage, which precludes margin-free 290 tumour eradication.5 On the other hand, surgery is the most effective treatment modality.3 Therefore, to plan optimal oncologic treatment it is impor­tant to know factors with impact on the patient’s prognosis. Many potential factors for an unfa­vorable outcome such as stage of disease, histol­ogy, intracranial extension and recently molecular markers such as EGFR have been studied in the literature.15,17 Our results indicate that besides the known predictive factors, including T-, N-, M- and overall AJCC-stage15, tumour volume is an impor­tant predictive factor that should be encountered in the staging system in future. Compared to N- and M-stage status, which are rarely positive in sinona­sal tumours except in some rare histologic types, tumour volume seems to be a robust predictive biomarker. Khademi et al. found only therapy response and stage of disease as independent predictors on mul­tivariate analysis.15 In our dataset, neither T- nor overall AJCC- staging system proved to be signifi­cant on multivariate analysis which is in accord­ance to previously published data.4,10 However, radiologic tumour volume and N-stage showed the highest significance in predicting PFS, though N-stage outperformed tumour volume. The mains limitation of our study is that multi­ple histological subgroups were analysed together. Due to small patient numbers subgroup analysis was only possible for SCCA and adenocarcinoma where the results from the overall analysis were confirmed (see Figure 1). The outstanding signifi­cance of radiological tumour volume is somehow surprising taking into account that T-staging sys­tem incorporates detailed information of local tumour extension such as orbital or skull base in­volvement whereas radiologic volumetry reflects only tumour size. As mentioned above, the prog­nosis is mostly influenced by ability to assure lo­cal tumour control with radical surgical resection being often limited due to the close proximity of midfacial anatomic structures and the skull base. Therefore, it appears reasonable that tumour size plays an essential role in patient’s outcome. Based on presented results, we recommend us­ing imaging-based tumour volumetry as an essen­tial factor when planning therapeutic strategy and aggressiveness of oncologic therapy. Although our data are mainly based on SCCA and adenocarci­noma histologies, we consider our results to be a reasonable platform to integrate primary tumour volume into therapeutic considerations when deal­ing with other, non-epithelial histological entities. References 1. Dulguerov P, Jacobsen MS, Allal AS, Lehmann W, Calcaterra T. Nasal and paranasal sinus carcinoma: are we making progress? A series of 220 pa­tients and a systematic review. Cancer 2001; 92: 3012-29. 2. 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Cancer of the nasal cavity: survival and factors influencing prognosis. Arch Otolaryngol Head Neck Surg 2002; 128: 1079-83. 8. Jethanamest D, Vila PM, Sikora AG, Morris LG Predictors of survival in mu­cosal melanoma of the head and neck. Ann Surg Oncol 2011; 18: 2748-56. 9. Alvarez I, Suárez C, Rodrigo JP, Nunez F, Caminero MJ. Prognostic factors in paranasal sinus cancer. Am J Otolaryngol 1995; 16: 109-14. 10. Suarez C, Llorente JL, Fernandez De Leon R, Maseda E, Lopez A. Prognostic factors in sinonasal tumors involving the anterior skull base. Head Neck 2004; 26: 136-44. 11. Lee CH, Hur DG, Roh HJ, Rha KS, Jin HR, Rhee CS, et al. Survival rates of sinonasal squamous cell carcinoma with the new AJCC staging system. Arch Otolaryngol Head Neck Surg 2007; 133: 131-4. 12. Dirix P, Nuyts S, Geussens Y, Jorissen M, Vander Poorten V, Fossion E, et al. Malignancies of the nasal cavity and paranasal sinuses: long-term outcome with conventional or three-dimensional conformal radiotherapy. Int J Radiat Oncol Biol Phys 2007; 69: 1042-50. 13. Snyers A, Janssens GO, Twickler MB, Hermus AR, Takes RP, Kappelle AC, et al. Malignant tumors of the nasal cavity and paranasal sinuses: long-term out­come and morbidity with emphasis on hypothalamic-pituitary deficiency. Int J Radiat Oncol Biol Phys 2008; 73: 1343-51. 14. Guntinas-Lichius O, Kreppel MP, Stuetzer H, Semrau R, Eckel HE, Mueller RP. Single modality and multimodality treatment of nasal and paranasal sinuses cancer: a single institution experience of 229 patients. Eur J Surg Oncol 2007; 33: 222-8. 15. Khademi B, Moradi A, Hoseini S, Mohammadianpanah M. Malignant neo­plasms of the sinonasal tract: report of 71 patients and literature review and analysis. Oral Maxillofac Surg 2009; 13: 191-9. 16. Cohen ZR, Marmor E, Fuller GN, DeMonte F. Misdiagnosis of olfactory neu­roblastoma. Neurosurg Focus 2002; 12(5): e3. 17. Takahashi Y, Bell D, Agarwal G, Roberts D, Xie TX, El-Naggar A, et al. Comprehensive assessment of prognostic markers for sinonasal squamous cell carcinoma. Head Neck 2013; 36: 1094-102. 291 research article Comparison of hybrid volumetric modulated arc therapy (VMAT) technique and double arc VMAT technique in the treatment of prostate cancer Christopher Amaloo1, Daryl P. Nazareth2, Lalith K. Kumaraswamy3 1 Department of Radiation Medicine, Roswell Park Cancer Institute, Buffalo, NY, USA 2 Department of Radiation Medicine, Roswell Park Cancer Institute and Department of Biophysics and Physiology, University at Buffalo, Buffalo, USA 3 Department of Radiation Medicine and Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, USA Radiol Oncol 2015; 49(3): 291-298. Received 22 October 2014 Accepted 26 February 2015 Correspondence to: Assist. Prof. Lalith K. Kumaraswamy, M.Sc., DABR, Department of Radiation Medicine, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA; or Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA. Email: lalith.kumaraswamy@roswellpark.org Disclosure: No potential conflicts of interest were disclosed. Background. Volumetric modulated arc therapy (VMAT) has quickly become accepted as standard of care for the treatment of prostate cancer based on studies showing it is able to provide faster delivery with adequate target coverage and reduced monitor units while maintaining organ at risk (OAR) sparing. This study aims to demonstrate the potential to increase dose conformality with increased planner control and OAR sparing using a hybrid treatment technique compared to VMAT. Methods. Eleven patients having been previously treated for prostate cancer with VMAT techniques were replanned with a hybrid technique on Varian Treatment Planning System. Multiple static IMRT fields (2 to 3) were planned initially based on critical OAR to reduce dose but provide some planning treatment volume (PTV) coverage. This was used as a base dose plan to provide 30-35% coverage for a single arc VMAT plan. Results. The clinical VMAT plan was used as a control for the purposes of comparison. Average of all OAR sparing between the hybrid technique and VMAT showed the hybrid plan delivering less dose in almost all cases except for V80 of the bladder and maximum dose to right femoral head. PTV coverage was superior with the VMAT technique. Monitor unit differences varied, with the hybrid plan able to deliver fewer units 37% of the time, similar results 18% of the time, and higher units 45% of the time. On average, the hybrid plan delivered 10% more monitor units. Conclusions. The hybrid plan can be delivered in a single gantry rotation combining aspects of VMAT with regions of dynamic intensity modulated radiation therapy (IMRT) within the treatment arc. Key words: VMAT; hybrid; prostate; planning Introduction Prostate cancer is one of the most prevalent malig­nant diseases that occur among men with a new case diagnosed every 2.2 minutes, affecting 1 in 6 men in their lifetime.1,2 Traditionally, radiotherapy has been a vital part of the treatment of prostate cancer with three-dimensional conformal radio­therapy (3D-CRT) as the historical standard. Data from dose escalation studies suggests an associa­tion between increased dose and an improvement in prostate cancer control3 with an increased effi­cacy in prostate-specific antigen (PSA) control at the cost of increased toxicity.4,5 By utilizing tech­niques such as intensity modulated radiation ther­apy (IMRT) or volumetric modulated arc therapy 292 (VMAT) with image guided radiotherapy (IGRT), the amount of normal tissue treated can be reduced and thus limit this increased toxicity.6,7 Due to these improved outcomes, IMRT and VMAT techniques are becoming the new standard for curative ex­ternal beam radiation therapy.7-9 Given the wide­spread and prolific nature of the disease, it has proven to be a vital site for the validation of new treatment modalities. Previous studies have shown that VMAT offers reduced monitor units (MU) and delivery time in comparison to IMRT10-15, at the cost of low dose spillage and potentially reduced con­formation of dose for all treatment sites. RapidArc® is a form of VMAT that delivers intensity modulated radiation arcs by simultane­ously changing gantry speed, multileaf collimator (MLC) position, and dose rate.11 While this tech­nique offers increased dose conformality and spar­ing of organs at risk (OAR) compared to 3D-CRT16, one drawback of such a technique is the spread of low dose to the surrounding normal tissue.11-17 In the treatment of prostate cancer, the spread of such a large low dose region can lead to issues with the intestinal tract, causing such secondary issues as di­arrhea, intestinal strictures, and incontinence.9,18 In order to reduce the low dose volume, and achieve better control to the respective OARs, a hybrid technique was developed similar in nature to Chan et al. with the use of dynamic IMRT in place of 3D conformal fields.19 Our hybrid technique features a pair of non-opposing dynamic IMRT fields where the beam axis covers the planning treatment vol­ume (PTV) while minimizing the overlap with the OARs. It contributed approximately 1/3 of the total TABLE 1. Patient characteristics 1 T1c 4+3=7/10 6.0 70 2 T1c 3+3=6/10 4.4 67 3 T1c 3+4=7/10 9.9 61 4 T2a 4+5=9/10 24.4 81 5 T2c 3+3=6/10 10.8 67 6 T2a 5+4=9/10 9.5 74 7 T1c 3+4=7/10 6.1 70 8 T1c 4+3=7/10 8.8 60 9 T1c/T2a 4+3=7/10 4.8 69 10 T1c 4+3=7/10 6.8 63 11 T2b/T3a 4+5=9/10 14.2 63 PSA = prostate-specific antigen; Pt = patient dose to the targets, with the remaining 2/3 of the dose coming from a single overlaying VMAT arc. The aim of this study is to retrospectively com­pare the dosimetric parameters of this hybrid treat­ment technique combining the use of dynamic IMRT fields supplementing a single modulated arc pass to the standard VMAT plan containing two full arcs frequently utilized clinically at our insti­tute for the treatment of prostate cancer. Methods Eleven patients, previously treated at our institute for prostate cancer, were chosen for this study. A variety of treatment plans were chosen to include a combination of patient size, target size, and com­promised critical OARs that require special con­sideration. Patient age ranged from 60 to 81 with a mean age of 68. Patients had PSA scores ranging from of 4.4 to 24.4, with Gleason scores from 6 to 9 (Table 1). All patients were treated clinically with a VMAT technique, utilizing two arcs to achieve a confor­mal dose to the target structure. These patients were then retrospectively re-planned with a hybrid technique on Varian Eclipse Treatment Planning Software (TPS) (Varian Medical Systems, Palo Alto, CA) Version 10.0. The hybrid technique consisted of dual dynamic IMRT fields with geometry de­signed to limit OAR dose but provide some PTV coverage. Dose was calculated and subsequently used as a base dose plan, providing initial coverage for a single overlay volume modulated arc. Patients were simulated with a GE Lightspeed computed tomography simulator in the supine position on a flat tabletop. A custom formed vac­loc bag was utilized to ensure consistent setup and stabilization. The bladder at time of simulation was filled to a degree that was maintainable and reproducible for daily treatment. CT slices were ac­quired at a thickness of 2.5 mm covering a region from above the iliac crests superiorly to below the perineum inferiorly. A physician contoured the gross tumor volume (GTV) to include all known disease, as defined by the planning CT, encompassing the entirety of the prostate gland. A urethrogram was used in plan­ning to aid in delineation of the inferior border of the prostate to include a volume 5 mm superior to the tip of the dye. The clinical treatment volume (CTV) is the GTV and areas of microscopic disease extension including the proximal 1 cm of the semi­nal vesicles. The PTV included a 1 cm radial expan­ 293 sion of the CTV in all directions, except a posterior margin of 6 mm, to allow for treatment set up vari­ation as well as internal motion of organs. The con­fidence in the reduced size of the posterior margin is due, in part, to IGRT techniques of weekly CBCT and daily kV orthogonal matching to imbedded ra­diopaque fiducial prostate seed markers. OARs contoured on the treatment planning CT include the left and right femoral heads to the level of the ischial tuberosity, the bladder, and the rectum from the superior rectosigmoid flexure to the infe­rior level of the ischial tuberosities. Additionally, an external body structure was also contoured as a normal tissue for the purposes of dose volume his­togram (DVH) analysis. All structures contoured exist as solid organs in their entirety. The prescription dose was 1.8 Gy per fraction for 44 fractions (79.2 Gy total dose) to cover 95% of the PTV, with the maximum dose in the PTV no more than 107% of the prescription dose. VMAT planning All treatment planning was performed on Varian Eclipse TPS 10.0. The original VMAT plans were copied and dose was calculated based on the previ­ously generated arc parameters with the following criteria: The treatment isocenter was placed at the center of the PTV. Two full arcs were planned using the Eclipse Arc Geometry Tool, with the initial arc rotating clockwise from 181° to 179°, a collimator rotation of 30° and the second arc rotating counter clockwise from 179° back to 181° with a collimator rotation of 320°. Additional structures created for planning pur­poses only included a radial expansion of 1 mm on the PTV to enhance dose coverage and fall off. Regions of overlap between treatment volumes and OARs were contoured to control dose effectively within these areas. Additionally, to better control dose to the rectum, two additional structures were contoured as illustrated in Figure 1. Rectum_Out was a radial expansion of 5 mm on the rectum mi­nus the overlap contour, subsequently cropped out of the overlap area with an additional 1 cm mar­gin. Rectum_Mid was a radial expansion of 5 mm on the rectum minus the overlap contour, subse­quently cropped out of both the overlap area with a 3 mm margin and Rectum_Out. Finally, support structures were added to account for the treatment couch in the path of the arcs. Within the VMAT optimizer, using calculation model algorithm PRO_10028, upper and lower dose constraints were set on all tumor and treat-ment volumes and expansions thereof. Coverage of targets thus defined received topmost priority, with OARs receiving lesser priority in the order of rectum, bladder, and finally, bilateral femoral head and necks. The Normal Tissue Objective was utilized, with a priority value matching that of the target coverage, with automatic tissue sparing se­lected. Dose was calculated for an intermediate optimi­zation, and final dose calculated after the VMAT optimizer was run a second time to completion. A normalization point was selected such that 100% of the prescribed dose would be delivered to 95% the PTV. Hybrid planning The hybrid planning technique was comprised of two dynamic IMRT fields with a single overlying volume modulated arc delivered to the same iso­center as in the VMAT plans. Beam arrangement of the dynamic fields was chosen such that the major­ity of the PTV on the central axis received coverage while minimizing direct OAR exposure within the fields. Directly opposed fields were avoided, and in general, left and right anterior oblique fields gave the best geometry. The two dynamic IMRT fields provided ap­proximately 1/3 of the total dose, with the remain­ing dose supplied by the overlying arc. The IMRT fields served as a base dose for the VMAT opti­mizer, with the same structures and constraints as utilized for the VMAT planning process. Again, dose was calculated for an intermediate optimization, and final dose calculated with the 294 VMAT optimizer run to completion on a second pass. A normalization point was chosen to achieve the same coverage as in the VMAT plan. Isodose lines were analyzed and, if possible, the IMRT fluences were adjusted manually to increase target coverage or reduce hot spots. Analysis For each case, the two competing treatment plans were compared on the basis of several criteria. For target coverage, PTV min (D2), max (D98), and mean, as recommended by ICRU 8320 for dose re­porting, as a percentage of prescribed dose were cross referenced against a conformality index (CI) as defined below. For OAR analysis, the data was examined based on the specific organ tolerances as per tables in RTOG 0815. For the rectum, the DVH points of D15, D25, D35, and D50 as well as the V80, V75, V70 and V65 were examined. For the bladder, the DVH points of interest were again D15, D25, D35, and D50 as well as V75, V70, V65, and V60. For the bilateral femora both the max and mean values were compared. To gauge low dose to the body, the body V5 and V10 was used as a point of comparison, as well as a calculation for integral dose (ID) as defined below. Finally, a monitor unit comparison was made between the hybrid and VMAT plan as an indicator of modulation. [1] Where VRx is the volume in cc receiving the pre­scription dose, and VPTV is the PTV volume in cc. [2] Where V, p, and are respectively the volume, density of the organ, and mean organ dose. Results Dose color wash at 95% of the prescription dose is shown for the hybrid treatment and for the double arc VMAT comparison in Figure 2. The breakdown of target coverage with CI, PTV minimum, maxi­mum, and mean along with MU delivered and ID are shown in Table 2. The OAR study parameters for the two techniques are tabulated in Table 3 with cor­responding differences. The average DVHs for the PTV and bladder and rectum are shown in Figure 3. Target coverage The hybrid plan had better conformality compared to the double arc VMAT plan with a relative im­provement of 5.5% in CI. All of the plans were con- 295 TABLE 2. Planning treatment volume (PTV) coverage, monitor units, and integral dose for delivery of plans 1 Hybrid 0.98 92.8% 108.5% 103.0% 686 308.1 VMAT 1.08 95.5% 108.1% 101.4% 867 303.7 2 Hybrid 0.99 91.3% 107.1% 102.3% 817 186.5 VMAT 1.15 97.5% 107.6% 102.0% 697 198.8 3 Hybrid 1.00 94.4% 107.6% 102.3% 616 255.8 VMAT 1.05 96.2% 106.2% 101.3% 592 255.3 4 Hybrid 1.05 95.0% 107.7% 102.5% 743 200.3 VMAT 1.08 97.5% 109.4% 101.5% 574 196.3 5 Hybrid 1.07 94.9% 106.6% 102.4% 521 232.0 VMAT 1.12 96.9% 106.9% 101.3% 796 233.1 6 Hybrid 1.15 96.8% 111.0% 101.6% 600 216.3 VMAT 1.32 91.6% 109.8% 102.0% 602 208.6 7 Hybrid 0.97 94.2% 107.1% 102.8% 542 201.3 VMAT 0.97 94.7% 106.1% 102.3% 487 201.8 8 Hybrid 1.03 95.2% 110.0% 102.6% 729 133.5 VMAT 1.03 94.8% 107.4% 101.7% 598 132.2 9 Hybrid 1.09 95.3% 109.7% 102.3% 786 254.7 VMAT 1.17 97.4% 106.2% 101.6% 622 263.5 10 Hybrid 1.13 96.2% 109.3% 101.8% 603 178.5 VMAT 1.03 92.6% 107.3% 102.2% 610 175.6 11 Hybrid 1.00 96.0% 106.6% 102.1% 521 165.6 VMAT 1.14 98.1% 106.0% 101.7% 587 175.5 AVE Hybrid 1.04 94.74% 108.29% 102.34% 651.27 212.05 VMAT 1.10 95.71% 107.36% 101.73% 639.27 213.13 AVE = average; CI = confidence interval; MU = monitor units; Pt = patient; VMAT = volumetric modulated arc therapy sidered acceptable with 95% of the PTV volume re­ceiving 100% of the prescribed dose, but the VMAT plan achieved better dose homogeneity with an increase in 1% to PTV minimum and a reduction of 1% in PTV maximum (Table 2). Organs at risk Critical structure dose constraints followed RTOG protocol 0815 such that volumes of 15%, 25%, 35%, and 50% shall receive doses less than 80, 75, 70 and 65 Gy for the bladder and 75, 70, 65, 60 Gy for the rectum, respectively. The clinical VMAT plan served as control for the purposes of comparison. On av­erage, the hybrid technique provided greater OAR sparing for both the rectum and bladder at all vol­umes and doses of interest (Table 3). For the volume constraints, the p-value of each difference is pro­vided. A low p-value indicates that the VMAT and hybrid values are statistically significantly different. Monitor units, integral dose, low dose spillage Monitor unit differences varied, with the hybrid plan able to deliver fewer MU 45% of the time. Integral dose was slightly lower with the hybrid plan. For low doses of radiation to the whole body, the hybrid plan fared slightly better than the dou­ble arc VMAT plans, with a reduction in V5 of 0.8%, and a reduction in V10 of 1.9% on average (Table 2, 3). Discussion Previous studies comparing VMAT to IMRT for prostate treatment have highlighted the fact that VMAT delivery is more efficient than that of IMRT.16, 21-33 However, all of these studies have 296 TABLE 3. Organ-at-Risk constraints for bladder, rectum, femora and body Bladder Rectum Left F. Head Right F. Head D50 D35 D25 D15 D50 D35 D25 D15 Max Mean Max Mean VMAT (Gy) 30.66 42.35 52.12 69.11 30.75 42.98 54.31 69.50 38.49 17.31 37.13 17.18 HYBRID (Gy) 29.10 40.56 50.62 66.72 30.66 41.69 52.83 67.34 44.86 15.41 46.02 18.25 DIFF (Gy) 1.56 1.79 1.50 2.40 0.09 1.28 1.48 2.16 -6.37 1.90 -8.89 -1.07 Bladder Rectum Body V65 V70 V75 V80 V50 V60 V65 V70 V75 V5 V10 VMAT (%) 16.92 14.60 12.28 6.73 28.08 21.15 17.97 14.92 11.57 25.34 20.40 HYBRID (%) 15.88 13.40 10.96 6.43 26.24 19.47 16.10 12.93 9.69 24.51 18.51 DIFF (P-Val) 1.04 (0.36) 1.20 (0.32) 1.32 (0.27) 0.30 (0.39) 1.84 (0.31) 1.68 (0.25) 1.87 (0.17) 1.99 (0.09) 1.88 (0.06) 0.84 1.89 DIFF = difference; f. = femur; VMAT = volumetric modulated arc therapy mixed dosimetric results. Some studies have shown better sparing of OARs with IMRT23, while some have shown equivalent sparing of OARs.26,33 For example, the study by Ishii et al.33 showed that PTV coverage was similar between RapidArc, 7 field IMRT, and 9 field IMRT. For the rectum Dmean, V65Gy and V45Gy and bladder V45Gy, their results in­dicated that 9 field IMRT plans had significantly lower values than the RapidArc and 7 field IMRT. Our aim was to develop a technique that would further reduce the delivery time and maintain the same level of dosimetric outcome as the conven­tional RapidArc delivery. The hybrid technique enables the planner to more easily control dose to critical OARs. The use of dynamic IMRT allows for additional input in­to the TPS and serves as a portal for the planner to more directly control dose distributions. The combination of directly changing fluencies for the IMRT portion of delivery, as well as a choice of beam geometry allows the planner to better control where dose falls. Greater OAR sparing was seen in the hybrid technique for both bladder and rectum points across the range of interest. The hybrid technique provides greater confor­mality compared to the standard VMAT. As IGRT localization improves, this allows for reduced OAR volumes exposed to escalated doses, potentially reducing toxicity to normal tissues. This not only creates the opportunity for reduced patient com­plications, but also room for dose escalation lead­ing to potential increased local control. One way of measuring the dose-modulation potential of a plan delivery modality is to consider the number of con­trol points it allows. The control points are created by the TPS software and contained in a treatment plan’s DICOM file. Each control point specifies the state of the linac at a given instant of treatment (i.e., jaw settings, MLC positions, MUs, gantry angle and rotation speed, etc.). An IMRT plan and full-arc VMAT plan contain 320 and 178 control points, respectively. Therefore, a hybrid plan contains 2x320+178 = 818 control points, while double-arc VMAT plan contains 178x2 = 356 control points. Thus, the hybrid plan provides better dose modu­lation and control of dose fall off around the PTV. With Varian Eclipse treatment planning, the hy­brid plan was created utilizing separate optimiz­ers from the constituent VMAT and IMRT por­tions. With this method of planning, the IMRT base dose plan is unaware of the subsequent VMAT arc and therefore cannot effectively yield homogene­ous coverage to the PTV. This inhomogeneous base dose presents an additional restriction on the VMAT optimizer to achieve constraints as seen by the decrease in PTV homogeneity of 2% with the hybrid technique. A typical prostate VMAT field has a beam on time of 1.2 minutes (72 seconds) during treatment. Since most of our prostate VMAT plans require two arcs to achieve a homogeneous dose distribu­tion to the target, the combined beam on time is about 2.4 minutes (144 seconds). The hybrid plan, on the other hand, can be delivered in one gantry sweep consisting of two dynamic IMRT fields with a single overlying VMAT field. A typical dynam­ic IMRT field from this technique has a beam on time of 0.3 minute. Therefore, the hybrid technique would have a typical total beam on time of 1.2 min + 0.3 min + 0.3 min = 1.8 min, or 108 seconds, which is approximately 0.5 min lower than the typical prostate VMAT delivery in our clinic. Thus, this reduction in time would reduce the chance of in-tra-fraction motion and increasing patient comfort. Delivery of the treatment in one gantry rotation can also reduce machine wear and potential down­time for machine maintenance. Total MUs for both techniques are similar, but with a lower ID with the hybrid technique, as well as reduced volume receiving 5 Gy and 10 Gy over­all. This is critical due to the radiosensitivity of the OAR’s, particularly the rectum and bladder. Conclusions Dosimetrically the hybrid and double arc VMAT plans are similar, with the hybrid plan achieving better constraints on the OARs without significant­ly higher MUs. Clinically, the hybrid plan offered slightly poor­er homogeneity compared with the VMAT plans, yielding a greater shoulder on the PTV coverage. This issue is due to the nature of the two disparate optimizers attempting to achieve overall dose ho­mogeneity. Changing the nature of the optimiza­tion to a single overall algorithm would correct the difficulty in achieving completely uniform cover­age. The hybrid treatment delivered in a single gan­try rotation with short pauses for small IMRT cor­rections could potentially reduce treatment time and increase target localization compared to mul­tiple arc VMAT, while providing superior sparing of critical OARs. 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RapidArc radiotherapy planning for prostate cancer: single-arc and double-arc techniques vs. intensity-modulated radiotherapy. Med Dosim 2012; 37: 87-91. 32. Fontenot JD, King ML, Johnson SA, Wood CG, Price MJ, Lo KK. Single-arc volumetric-modulated arc therapy can provide dose distributions equiva­lent to fixed-beam intensity-modulated radiation therapy for prostatic irradiation with seminal vesicle and/or lymph node involvement. Br J Radiol 2012; 85: 231-6. 33. Ishii K, Ogino R, Okada W, Nakahara R, Kawamorita R, Nakajima T. A dosi­metric comparison of RapidArc and IMRT with hypofractionated simultane­ous integrated boost to the prostate for treatment of prostate cancer. Br J Radiol 2013; 86: 20130199. 299 research article Rounded leaf end effect of multileaf collimator on penumbra width and radiation field offset: an analytical and numerical study Dong Zhou, Hui Zhang, Peiqing Ye Department of Mechanical Engineering, Tsinghua University, Beijing, China Radiol Oncol 2015; 49(3): 299-306. Received 1 February 2015 Accepted 22 March 2015 Correspondence to: Prof. Peiqing Ye, Ph.D., Department of Mechanical Engineering, Tsinghua University, Beijing, China. Phone: +86 010 6277 3269; E-mail: yepq@tsinghua.edu.cn Disclosure: No potential conflicts of interest were disclosed. Background. Penumbra characteristics play a significant role in dose delivery accuracy for radiation therapy. For treatment planning, penumbra width and radiation field offset strongly influence target dose conformity and organ at risk sparing. Methods. In this study, we present an analytical and numerical approach for evaluation of the rounded leaf end effect on penumbra characteristics. Based on the rule of half-value layer, algorithms for leaf position calculation and radiation field offset correction were developed, which were advantageous particularly in dealing with large radius leaf end. Computer simulation was performed based on the Monte Carlo codes of EGSnrc/BEAMnrc, with groups of leaf end radii and source sizes. Data processing technique of curve fitting was employed for deriving penumbra width and radiation field offset. Results. Results showed that penumbra width increased with source size. Penumbra width curves for large radius leaf end were U-shaped. This observation was probably related to the fact that radiation beams penetrated through the proximal and distal leaf sides. In contrast, source size had negligible impact on radiation field offset. Radiation field offsets were found to be constant both for analytical method and numerical simulation. However, the overall resulting values of radiation field offset obtained by analytical method were slightly smaller compared with Monte Carlo simulation. Conclusions. The method we proposed could provide insight into the investigation of rounded leaf end effects on penumbra characteristics. Penumbra width and radiation field offset calibration should be carefully performed to commission multileaf collimator for intensity modulated radiotherapy. Key words: multileaf collimator; rounded leaf end effect; penumbra width; radiation field offset; Monte Carlo simulation Introduction Multileaf collimator system was introduced as a re­placement of shielding block for beam shaping and beam intensity modulation, which has become an essential component for modern radiation therapy and a standard of care for radiation oncology fa­cilities.1 Penumbra characteristics of multileaf colli­mator are closely related to healthy tissues involve­ment, which is of interest to medical physicists, do­simetrists and radiation oncologists.2 Single-focused multileaf collimator is character­ized by linear leaf motion perpendicular to colli­mator rotation axis, which has been widely used by virtue of its compact space and simplified structures. The rounded leaf end design of single-focused multileaf collimator for following beam di­vergence has a strong impact on penumbra charac­teristics.3 In order to avoid tumour underdose and normal tissue overdose, the rounded leaf end effect of single-focused multileaf collimator on penum­bra characteristics should be carefully modelled 300 FIGURE 1. Components of treatment head are comprised of source, diaphragm, multileaf collimator, and scoring plane. Leaf positions on scoring plane are classified into nominal leaf position N, geometric leaf position G and physical leaf position P. SAD = source to axis distance; SCD = source to collimator distance; SDD = source to diaphragm distance in treatment planning system, otherwise it would result in dose error particularly when sharp dose gradient is intended for stereotactic body radio­therapy. For the purpose of precision radiation therapy, intensive research efforts have been made on the dosimetric measurement and Monte Carlo simula­tion of multileaf collimator systems.4,5 Studies have revealed that dosimetric characteristics of multileaf collimator are influenced by the factors, including but not limited to geometry of treatment modality, radiation source properties, leaf end shape and leaf position with respect to central axis.6 It was found that dosimetric penumbra of multileaf collimator is the combined effect of geometric penumbra, trans­mission penumbra and phantom scatter.7 Quality assurance has been implemented to determine pe­numbra width and the offset between light field edge and radiation field edge during commission­ing of multileaf collimator.8,9 Results have shown that penumbra width and radiation offset are leaf position dependent and largely attributed to leaf end shape. It is reported that the projected leaf position on scoring plane, light field edge and ra­diation field edge follow a nonlinear relationship. Calibrations of leaf position offset and radiation field offset were performed to minimize the error between planned doses and delivered doses.10,11 Rule of half-value layer12 has been proposed for calculation of radiation field offset based on geo­metrical approach.13 However, previous studies were confined to single source energy distribu­tion, normally simplified as Gaussian shaped, and limited leaf ends in the shape of circular arc were investigated. There is a lack of consistency in the quantitative study into rounded leaf end effect on penumbra characteristics of multileaf collimator in literature. Besides, there is no literature available, to our knowledge, reporting on algorithms of leaf position calculation and radiation field offset cor­rection for large radius leaf end. Consequently, the aim of this study was to ex­plore the rounded leaf end effect and efforts were made to reveal the source energy distribution and leaf end shape related penumbra characteristics. An analytical method for radiation field offset cor­rection was developed and numerical simulation with various leaf end radii and source sizes was conducted based on Monte Carlo codes. Materials and methods In this section, leaf positions are classified and ge­ometry based algorithms for radiation field offset correction are developed. With treatment head modelling, Monte Carlo simulation is introduced to investigate the rounded leaf effect on penumbra characteristics. Data processing techniques for de­riving penumbra width and radiation field offset are proposed. Algorithms for leaf position calculation and radiation field offset correction Leaf positions on scoring plane are divided into projected leaf end position (nominal leaf position), light field edge (geometric leaf position), and radi­ation field edge (physical leaf position).14 Nominal leaf position is usually calibrated so that it corre­sponds to the light field edge or the radiation field edge. In this study, nominal leaf position is desig­nated to coincide with the projected leaf position without calibration. As depicted in Figure 1, mechanical leaf posi­tion is referred to as the leaf tip location relative to collimator rotation axis, which is shown as point E. Nominal leaf position, geometric leaf position and physical leaf position on the scoring plane are represented by point N, point G, and point P, re­spectively. Leaf position offset (LPO) is defined as the distance between geometric leaf position and nominal leaf position. Radiation field offset (RFO) is defined as the distance between physical leaf position and geometric leaf position. The term of physical-nominal offset (PNO) is proposed, which is defined as the distance between physical leaf po­sition and nominal leaf position. Place the origin of coordinate in coincidence with isocenter O. Therefore, the Z-coordinates are zero for point N, G and P. Point N is obtained by projecting of mechanical leaf position E onto the scoring plane. Point G is obtained by deriving the tangent line of circular arc leaf end from source S. Point P is obtained by rule of half-value layer. 301 Equations for the LPO, RFO and PNO derivation are presented, [1] Algorithms for calculation the X-coordinates of leaf positions are illustrated as follows. Consider a specific nominal leaf position, which is designated as point N in Figure 1, Point E is obtained by back-projecting leaf end point N onto the collimator middle plane, that is [2] where SCD is used to stand for source to collima­tor distance, while SAD stands for source to axis distance. The circular arc center C is shifted to the positive side of point E with a length of radius R, that is, [3] Denote the distance between source S and arc cen­tre C as D, which is used as a reference, [4] Firstly, point G is obtained by deriving the tangent line of circular arc leaf end from source S. The rela­tionship between point G and point T is, [5] Thus, the prerequisite for point G derivation is to obtain point T. The X-coordinate of point T should satisfy the condition of . Point T can be ob­tained by the following equations, [6] In case that the tangent point falls out of circu­lar arc or S falls within circle, denote the tangent point as the intersection point of circular arc with proximal or distal leaf side. The intersection points are depicted as point U and point V, respectively. Algorithm is illustrated as follows, for leaf height of lh, [7] [8] Secondly, point P is obtained by the half-value layer rule. Draw a secant line from source S to the point P, the secant point A and B fall on the left side of C, that is, , . Find the equation of se­cant line satisfying the condition that path length AB equals half-value layer L, [9] where path length is calculated according to the inverse exponential power law and m denotes the attenuation coefficient of tungsten leaf. A, B and S are on the secant line. It is written in the following form, [10] Suppose the A and B are both on the circular arc, that is, [11] Combine [9], [10] and [11] into a system of nonlin­ear equations, solve it with iterative methods. In case that the resulting A or B is not on circular arc, conditional statements are performed as follows. If the resulting or , it means point A should be on the proximal leaf side. Substitute equation [12] for [11] in the system of equations and solve it. [12] Else, if , substitute equation [13] for [11] in the system of equations and solve it. [13] Else, return with the solutions of equations. Monte Carlo simulation Numerical simulation is performed based on Monte Carlo codes EGSnrc/BEAMnrc.15,16 Multileaf col­limator component module of VARMLC is adopt­ed. Leaf ends in the shape of circular arc are used, with radius values in a range of 4 cm to 25 cm. The geometry of Monte Carlo simulation is depicted as Figure 1. Multileaf collimator is comprised of 40 pairs of leaves. Symmetric field size of 10x10 is adopted, which remains constant for different leaf positions. It means that the central 10 pairs of leaves shift for field shaping, while the peripheral leaves stay still in close state. For central leaf pair, the gap between leading leaf and trailing leaf corresponds to the square field edge of 10 cm, measured along leaf travel direction on scoring plane. Diaphragms are extracted to the maximum position in order to TABLE 1. Parameters for Monte Carlo simulation Parameter Value Unit Source to axis distance 100 cm Source to collimator distance 46 cm Source to diaphragm distance 33.9 cm Diaphragm height 7.8 cm Leaf height 8 cm Leaf pairs 40 ­Leaf density 19.3 g/cm3 Attenuation coefficient m 0.96 cm-1 Maximum field size 40 cm Photon source average energy 1.5 MeV Source divergence angle 15.8° ­Recording histories 109 ­ FIGURE 2. Data processing for leaf end radius of 10 cm with nominal leaf position at 10 cm and source size of 2 mm full width at half maximum. MC = Monte Carlo AB D FIGURE 3A-D. (A). 3D graph of penumbra width with source size of 0.5 to 3 mm full width at half maximum. (B). Penumbra width for leaf end of radius 4 cm. (C). Penumbra width for leaf end of radius 15 cm. (D). Penumbra width for leaf end of radius 25 cm. avoid interference with radiation beams. Source sizes with a range of 0.5 to 3 mm full width at half maximum (FWHM) are adopted. Parameters for configuration are listed in Table 1. Data processing Data processing software BEAMDP is utilized for deriving energy fluence versus position from the acquired phase space files grouped by source size and leaf end radius. Curve fitting is performed to obtain penumbra characteristics, including pe­numbra width and radiation field offset. Figure 2 shows a sample result for data processing. Normalize the Monte Carlo data by scaling be­tween 0 and 1. The penumbra width is referred to as the distance between relative intensity of 0.2 and 0.8, and the radiation field edge is referred to as the position with relative intensity of 0.5. Gaussian function is employed for curve fitting, with the fol­lowing equation: [14] Three-peak Gaussian curve fitting is used for the sample data, with coefficients listed in Table 2. Result shows that the goodness of fit is 0.0154 measured by Root Mean Squared Error. Penumbra width is 1.98 mm and radiation field edge is at 9.932 cm, with PNO offset of 0.68 mm. Results Penumbra width The results of penumbra width are illustrated as Figure 3A. Note that penumbra width varies ac­cording to field location, leaf end radius and source size. The minimum and maximum penumbra width are obtained with source sizes of 0.5 mm and 3 mm FWHM, respectively. Figures 3B, 3C and 3D show penumbra width for leaf end radius of 4, 15, 25 cm, respectively. Observe that penumbra width is a function of distance from central axis. The overall trend is that penumbra width with positive distance from central axis is smaller than its nega­tive counterpart. Figure 4 shows the penumbra width with for source size of 1 mm FWHM. Curve E with leaf end radius of 15 cm demonstrates the minimum sum of penumbra width, which is the optimum in terms of penumbra characteristics. Observe that for large radius leaf end and nominal leaf position far from central axis, penumbra width curve are U-shaped, as shown by curve F and curve G. 303 Radiation field offset Results of physical-nominal offset are depicted in Figure 5A. It is noted that source size has little im­pact on physical-nominal offset and the surfaces of PNO with source size of 0.5, 1, 2, 3 mm FWHM coincide with each other. Physical-nominal offset maximum of -10.1 mm is obtained at radius of 25 cm with nominal leaf position at -20 cm. Figures 5B, 5C and 5D show PNO for source size of 0.5 to 3 mm with leaf end radius of 4 cm, 15 cm and 25 cm, respectively. Observe that the maximum dis­crepancy of PNO curves occurs at central axis for radius of 4 cm, which is 0.4 mm. Figure 6 shows the physical-nominal offset of Monte Carlo simulation and calculation using ana­lytical method for source size of 1 mm FWHM. Note that PNO curve for small radius tends to be flat, and leaf end with large radius follows a quasi-quadratic function between PNO and leaf projected position. Although overall trends of calculation curves are in good agreements with Monte Carlo data, the results of Monte Carlo data are slightly larger compared with analytical values, with maximum discrepancy of 2 mm found for curve pair G at the point with a distance of -20 cm from central axis. As illustrated in Figure 7, the analytical results agree well with Monte Carlo simulation for leaf end of radius 15 cm with source size of 1 mm. The curves of calculation PNO and RFO are shifted up­wards with a constant gap of 0.16 mm, compared with simulation PNO and RFO. Note that the RFO curve is almost parallel with axis, which implies that a constant RFO value could be assigned and physical-nominal offset could be quickly deduced from leaf position offset. The RFO values derived from analytical method and numerical simulation are 0.10 and 0.26 mm, respectively. Figure 8 shows the radiation offset results using Monte Carlo simulation and analytical method. It is noted that the RFO values of Monte Carlo data are slightly larger than the calculation data. As for leaf end with radius of 25 cm, the curve of radiation field offset is U-shaped with maximum RFO of 2 mm. Discussion The results demonstrate that algorithms for leaf position calculation and radiation field offset cor­rection serve well the purpose of investigating rounded leaf end effect on penumbra characteris­tics. Compared with previous works10-14, the algo­rithms we proposed are advantageous particularly TABLE 2. Three-peak Gaussian curve fitting coefficients Coefficient Value Coefficient Value Coefficient Value a1 0.406 a2 0.397 a3 0.996 b1 9.813 b2 9.586 b3 9.054 c1 0.169 c2 0.269 c3 0.668 FIGURE 4. Penumbra width for source size of 1mm full width at half maximum. Curve A to G denote radius of 4, 6, 8, 10, 15, 20, 25 cm. AB C D FIGURE 5A-D. (A). 3D graph of physical-nominal offset with source size of 0.5, 1, 2, 3 mm full width at half maximum. (B). physical-nominal offset (PNO) for radius of 4 cm. (C). PNO for radius of 15 cm. (D). PNO for radius of 25 cm. Source size ranges from 0.5 to 3 mm. in dealing with large radius leaf end. It is shown that penumbra width of multileaf collimator is a function of radiation source size, geometry of treat­ment head, leaf end shape and projected leaf posi­tion on scoring plane. Optimal radius of leaf end shape could be found by examining the penumbra width curves of Monte Carlo simulation. The re­sults also reveal that radiation source size has a FIGURE 6. Physical-nominal offset for 1mm full width at half maximum source. Curve pair A to G are referred to as circular arc leaf ends with radius of 4, 6, 8, 10, 15, 20, 25 cm, for Monte Carlo (MC) simulation (solid line) and analytical method (dashed line). FIGURE 7. Comparison of leaf position offset (LPO), physical-nominal offset (PNO) and radiation field offset (RFO) for leaf end of radius 15 cm with source size of 1 mm. Calc. = calculation; Sim. = simulation FIGURE 8. Comparison of radiation field offset (RFO) for Monte Carlo simulation and analytical method with source size of 1 mm full width at half maximum. MC = Monte Carlo negligible impact on radiation field offset, while for penumbra width, source size counts. In our study, virtual source model (VSM) is applied, which has been intensively reported by previous studies.16 The VSM technique for dose calculation is computational efficient and able to simulate the same dose profile without explic­itly taking into consideration the realistic treat­ment head geometry. Virtual source in our study is Gaussian shaped. However, for realistic treat­ment head, source energy distribution could more complex than single Gaussian source. In order to accommodate realistic system properties, firstly, virtual source modelling should be conducted to identifying focal source and extra-focal source en­ergy distribution. Secondly, Monte Carlo simula­tion would be performed according to results of VSM. The geometry of treatment head for numeri­cal simulation is simplified so that the impact of treatment head components, such as primary colli­mator and flattening filter, on extra-focal radiation, is minimized. However, it is quick to implement by modifying the component properties in Monte Carlo simulation codes. Monoenergetic photon source is designated as Gaussian shaped with full width at half maximum in a range of 0.5 mm to 3 mm, which is in accord­ance with the dosimetric results.17 Average source energy of 1.5 MeV is adopted corresponding to 6MeV medical linear accelerator. However, energy spectrum and angular distribution of radiation beams would be implemented in the future works, which is not a trivial task. For leaf end with large radius, the U-shaped curves appear both for penumbra width and radia­tion field offset, which are not preferred for clini­cal application. This observation is probably re­lated with the fact that radiation beams penetrated through the proximal and distal leaf sides. Although the fluence energy distribution has been intensively studied, the round leaf end effect on penumbra characteristics in phantom or in vivo has not been explored, which could be realized by dose calculation algorithms in future works. Scatter effect on dose profile could be calculated using kernel-based convolution and superposition algorithms. It is noted that the results of RFO correction algorithm are generally in good agreement with numerical simulation. However, the values ob­tained using analytical method are slightly small­er compared with the corresponding Monte Carlo results, which means that analytical method may underestimates radiation field offset. In order to better predict the radiation field offset, analytical RFO can be placed to match numerical RFO by moving in the direction of from irradiation area to shielded area. This observation implies that ana­lytical method should be applied with care. The error between analytical and numerical methods is probably related the empirical rule of half-value layer or “geometric optics” formulae. A simple physical explanation for the underestimation is given as follows. Denote P as the physical leaf position obtained by “geometric optics” formulae, which means the path length AB equals half value layer. Denote as the radiation field edge, or physical leaf P50 position, obtained by Monte Carlo simulation. Consequently, the question being proposed could be rephrased as “why is ?”. Firstly, as illustrated in Figure 9, draw a line CJ from circle arc centre C that is perpendicular with path length AB, with intersection point of K. Denote the length of JK as H. It is obvious that H is monotonically decreasing for R > 0. For clinical ap­plication, leaf end radius is commonly larger than half of leaf width. The maximum of H is written, 305 Normally, source energy distribution for treat­ment modality is with FWHM ranging from 1 to 3 mm. Note that the H is small compared with source size. Secondly, suppose that source energy distribution is approximately symmetric about central axis. Divide source energy into three parts, the left part S1, the middle part S2 and the right part S3 with re­spect to the central axis. Denote that total source energy as 1, it is written that, [16] [17] The attenuation weight for the beams from source part S1, S2 and S3 to the point P are defined as w1, w2 and w3, respectively. The rule of half value layer tells that w2 = 0.5. On account that H is small, beams irradiate from the left part of source are sup­posed to reach P without attenuation, that is w1 = 1, while for the right part, beams penetrate through leaf entity to reach P with path length larger than half value layer, that is, 0 < w3 < 0.5. Consequently, the radiation intensity EP of point P is written as follows, (18) Therefore, it is implied that the physical edge of radiation field P50 should be on the right side of P, that is, . This is a simple physical expla­nation why “geometric optics” formulae system­atically underestimate the physical-nominal offset. Since the segmentation of source energy is coarse, further study is suggested with Ray Tracing al­gorithm, which is implemented by computation of the weighed beam integral based on the law of exponential attenuation. It is suggested that modi­fications for analytical RFO correction should be performed in order to fit in well with treatment modalities. Path length larger than the half-value layer would be beneficial. The rounded leaf end design of multileaf col­limators leads to partial transmission of radiation beams, which have a significant impact on dose de­livery accuracy of IMRT, SBRT and VMAT. Based on Monte Carlo simulation for SBRT multileaf col­limator, Asnaashari et al.5 have revealed that dosi­metric penumbra is influenced by source energy, beam collimators and field size. This observation is in good agreement with our study. It is suggested that dosimetric characteristics of multileaf collima­tor should be calibrated and comprehensive rou­ [15] FIGURE 9. Geometry of treatment head for simple physical explanation of why the rule of half value layer systematically underestimates the physical-nominal offset. SAD = source to axis distance; SCD = source to collimator distance; SDD = source to diaphragm distance tine quality assurance should be performed before they are implemented for IMRT applications.3 Further study is needed both for theoretical inves­tigation and dosimetric measurement of rounded leaf end effect. In our study, penumbra width and radiation field offset of single leaf are intensively studied. In contrast, Szpala et al.11 investigated the value of dosimetric leaf gap (DLG) for leaf pairs in treat­ment planning. It was demonstrate that the DLG depends on the size of mulileaf collimator slit. Such effect is probably caused by scatter variation from the opposite leaf with different slit widths. Furthermore, they proposed a method by expand­ing the DLG parameter from a single value to a function of distance from the nominal leaf position and displacement of the opposite leaf. However, efforts should made to improve dose calculation accuracy in VMAT treatment planning, not merely by adjusting single parameter, such as leaf trans­mission or DLG. Better modeling rounded leaf end effect is of significance for future works. Conclusions In summary, the algorithms we proposed for leaf position calculation and radiation field offset cor­rection are effective for leaf end with large radi­us. Results of Monte Carlo simulation show that source size influences penumbra width, while for radiation field offset, the source size impact is negligible. Penumbra width performance could be improved by carefully choosing the radius of circular arc leaf end. In this study, the leaf posi­tions, including mechanical leaf position, nominal leaf position, geometric leaf position and physical 306 leaf position are classified and rigorously deduced. Correction of leaf position offset, radiation field off­set and physical-nominal offset are realized based on analytical method. In general, results of analyti­cal method agree well with numerical simulation. However, a slight gap exists between analytical radiation field offset and numerical radiation field offset, which implies that modification should be introduced when applying the empirical rule of half-value layer. For better treatment planning, the rounded leaf end effect on penumbra characteris­tics should be taken with care in order to achieve dose delivery accuracy. Acknowledgments This work was partially supported by the Beijing Municipal Science and Technology Commission of China, grant Z141100000514015, State Key Laboratory of Tribology of China, grant SKLT12A03 and Tsinghua University Initiative Scientific Research Program, grant 2011Z01013. References 1. Jeraj M, Robar V. Multileaf collimator in radiotherapy. Radiol Oncol 2004; 3: 235-40. 2. Clark BG, Teke T, Otto K. Penumbra evaluation of the varian millennium and BrainLAB M3 multileaf collimators. Int J Radiat Oncol Biol Phys 2006; 66(4 Suppl): S71-5. 3. Pasquino M, Borca VC, Catuzzo P, Ozzello F, Tofani S. Transmission, penum­bra and leaf positional accuracy in commissioning and quality assurance program of a multileaf collimator for step-and-shoot IMRT treatments. Tumori 2006; 92: 511-16. 4. Mohan R, Jayesh K, Joshi RC, Al-idrisi M, Narayanamurthy P, Majumdar SK. Dosimetric evaluation of 120-leaf multileaf collimator in a Varian linear ac­celerator with 6-MV and 18-MV photon beams. J Med Phys 2008; 33: 114-8. 5. Asnaashari K, Chow JCL, Heydarian M. Dosimetric comparison between two MLC systems commonly used for stereotactic radiosurgery and radiothera­py: A Monte Carlo and experimental study. Phys Medica 2013; 29: 350-6. 6. Topolnjak R, van der Heide UA. An analytical approach for optimizing the leaf design of a multi-leaf collimator in a linear accelerator. Phys Med Biol 2008; 53: 3007-21. 7. Sun J, Zhu Y. Study of dosimetric penumbra due to multileaf collimation on a medical linear accelerator. Int J Radiat Oncol Biol Phys 1995; 32: 1409-17. 8. Bailey D, Kumaraswamy L, Podgorsak M. A fully electronic intensity-modu­lated radiation therapy quality assurance (IMRT QA) process implemented in a network comprised of independent treatment planning, record and verify, and delivery systems. Radiol Oncol 2010; 44: 124-30. 9. Sumida I, Yamaguchi H, Kizaki H, Koizumi M, Ogata T, Takahashi Y, et al. Quality assurance of MLC leaf position accuracy and relative dose effect at the MLC abutment region using an electronic portal imaging device. J Radiat Res 2012; 53: 798-806. 10. Graves MN, Thompson AV, Martel MK, McShan DL, Fraass BA. Calibration and quality assurance for rounded leaf-end MLC systems. Med Phys 2001; 28: 2227-33. 11. Szpala S, Cao F, Kohli K. On using the dosimetric leaf gap to model the rounded leaf ends in VMAT/RapidArc plans. J Appl Clin Med Phys 2014; 15: 4484. 12. Boyer AL, Li S. Geometric analysis of light-field position of a multileaf col­limator with curved ends. Med Phys 1997; 24: 757-62. 13. Wu JM, Lee TF, Yeh SA, Hsiao KY, Chen HH, Chao PJ, et al. A Light-Field-Based Method to Adjust On-Axis Rounded Leaf End MLC Position to Predict Off-Axis MLC Penumbra Region Dosimetric Performance in a Radiation Therapy Planning System. Biomed Res Int 2013; 2013: 461801. 14. Vial P, Oliver L, Greer PB, Baldock C. An experimental investigation into the radiation field offset of a dynamic multileaf collimator. Phys Med Biol 2006; 51: 5517-38. 15. Rogers DWO, Walters B, Kawrakow I. BEAMnrc Users manual NRCC report PIRS-0509(A)revL. Ottawa: National Research Council of Canada; 2013. 16. Rucci A, Carletti C, Cravero W, Strbac B. Use of IAEA’s phase-space files for the implementation of a clinical accelerator virtual source model. Phys Medica 2014; 30: 242-8. 17. Sterpin E, Chen Y, Lu W, Mackie TR, Olivera GH, Vynckier S. On the relation­ships between electron spot size, focal spot size, and virtual source position in Monte Carlo simulations. Med Phys 2011; 38: 1579-86. 307 research article A comparison of the quality assurance of four dosimetric tools for intensity modulated radiation therapy Jaeman Son1,2, Taesung Baek1,4, Boram Lee1,5, Dongho Shin2, Sung Yong Park3, Jeonghoon Park2, Young Kyung Lim2, Se Byeong Lee2, Jooyoung Kim2, Myonggeun Yoon1 1 Department of Bio-Convergence Engineering, Korea University, Seoul, Korea 2 Proton Therapy Center, National Cancer Center, Goyang, Korea 3 McLaren Proton Therapy Center, Karmanos Cancer Institute, Flint, MI, USA 4 Department of Radiation Oncology, Ilsan Hospital, Goyang, Korea 5 Department of Radiation Oncology, Sun Hospital, Daejeon, Korea Radiol Oncol 2015; 49(3): 307-313. Received 10 November 2014 Accepted 18 January 2015 Correspondence to: Myonggeun Yoon, Department of Bio-convergence Engineering, Korea University, Anam Ro 145, Seongbuk-gu, Seoul 136-701, Korea. Phone: +82 2 3290 5651; Fax: +82 2 940 2829; E-mail: radioyoon@korea.ac.kr Disclosure: The authors declare no conflict of interest. Background. This study was designed to compare the quality assurance (QA) results of four dosimetric tools used for intensity modulated radiation therapy (IMRT) and to suggest universal criteria for the passing rate in QA, irrespective of the dosimetric tool used. Materials and methods. Thirty fields of IMRT plans from five patients were selected, followed by irradiation onto radiochromic film, a diode array (Mapcheck), an ion chamber array (MatriXX) and an electronic portal imaging de­vice (EPID) for patient-specific QA. The measured doses from the four dosimetric tools were compared with the dose calculated by the treatment planning system. The passing rates of the four dosimetric tools were calculated using the gamma index method, using as criteria a dose difference of 3% and a distance-to-agreement of 3 mm. Results. The QA results based on Mapcheck, MatriXX and EPID showed good agreement, with average passing rates of 99.61%, 99.04% and 99.29%, respectively. However, the average passing rate based on film measurement was significantly lower, 95.88%. The average uncertainty (1 standard deviation) of passing rates for 6 intensity modulated fields was around 0.31 for film measurement, larger than those of the other three dosimetric tools. Conclusions. QA results and consistencies depend on the choice of dosimetric tool. Universal passing rates should depend on the normalization or inter-comparisons of dosimetric tools if more than one dosimetric tool is used for pa­tient specific QA. Key words: intensity modulated radiation therapy; quality assurance; dosimetric tool; gamma index Introduction Radiation therapy is one of the most widely used cancer treatment methods. Among the methods used are 3D conformal radiation therapy (3D CRT), intensity modulated radiotherapy (IMRT), and particle beam therapy. IMRT uses a multi-leaf collimator (MLC) to vary the intensity of the beam delivered to the tumour.1 Since IMRT requires fine control of the MLC during irradiation, cau­tion must be exercised in delivering radiation.2-4 Treatment quality assurance (QA) is therefore nec­essary to determine the difference between calcu­lated and actual dose distributions.5,6 QA for conventional IMRT treatment is widely performed using an ion chamber and film, with the ion chamber used to measure absolute dose at each location and film used for 2D relative dose com­parisons.7-9 Although film has the great advantage of high resolution, it has several disadvantages, 308 including the need to change film for every beam test and its dependence on beam energy, process­ing conditions, and external light. Recently de­veloped radiation therapy methods have greater accuracy, with new dosimetry tools developed to overcome the disadvantages of film measure­ments. Among these tools are a diode detector-based 2D diode array dosimeter10 (MapCheck2; Sun Nuclear Corporation, Melbourne, Florida), a 2D ion chamber array dosimeter11 (I’mRT MatriXX; IBA) and portal dosimetry using an electronic por­tal imaging device12 (EPID). The gamma evaluation method is generally used to verify the actual dose distribution that will be delivered to the patient during IMRT.13 This method is based on a compari­son of the calculated 2D dose map from treatment planning system (TPS) with the measured 2D dose map from each dosimetric tool. Although there is no general consensus, QA results are considered acceptable when the passing rate is greater than 95% using as criteria a tolerance of dose difference (DD) of 3% and a tolerance for distance to agree­ment (DTA) of 3 mm.14 Table 1 shows an example of QA results using film, Mapcheck, MatriXX and portal dosimetry based on gamma evaluation for 10 randomly se­lected treatment plans undergoing IMRT at four different institutions in Korea. Each of 4 hospitals sorted out 10 IMRT QA results using one of four dosimetric tools and analysed the passing rates depending on the QA tools. Therefore, this data shows the general passing rates of IMRT QA de­pending on the dosimetric tools. The mean ± stand­ard deviation (SD) passing rates (.% . 1) for film, Mapcheck, MatriXX and EPID were 96.80 ± 0.94%, 98.90 ± 0.55%, 99.40 ± 0.48% and 97.10 ± 1.12%, re­spectively. Thus, passing rates are dependent on the dosimetric tool used, with mean passing rates being lowest for film and highest for MatriXX. This example suggests that passing rates of 95% for film measurement and portal dosimetry are not equiva­lent.15,16 Many institutions, however, have not set an acceptance level for each tool but have univer­sally set 95% as the acceptance level for all tools. The current emphasis on treatment QA for patients suggests the need for specific guidelines for each specific dosimetric tool. Although guidelines have been proposed17, they were only for acceptable doses and allowable errors for each body part, and did not include passing rates for different dosimet­ric tools. In this study, four different dosimetric tools were used for quality assurance method of IMRT plans from five patients and their patient spe- TABLE 1. Average passing rates for film, diode array (Mapcheck), ion chamber array (MatriXX) and electronic portal imaging device (EPID) for intensity modulated radiation therapy (IMRT) quality assurance (QA) in four different institutions in Korea Mean 96.80 98.90 99.40 97.10 Standard 0.94 0.55 0.48 1.12 deviation cific QA results were compared. The correlations among these dosimetric tools were used to deter­mine reasonable tolerance levels for each. Materials and methods The IMRT plans for five patients undergoing radia­tion therapy, modelled using the Eclipse treatment planning system Ver. 8.9. (Varian Medical Systems, Salt Lake City, UT, USA) with anisotropic analyti­cal algorithm (AAA), were used for this study.18 Figure 1 shows the experimental setup of IMRT QA with film, Mapcheck, MatriXX and EPID. The gamma index method was used to compare the TPS dose with measured doses, using dose dif­ference (DD) criteria of 3% and distance-to-agree­ment criteria (DTA) of 3 mm. The reference dose map was the calculated dose maps from TPS in our analysis. The gamma index value was calculated at all 2-dimensional points, with the percentage of points with a gamma index value . 1 and meeting the DD and DTA criteria being the passing rate. The passing rates of the four dosimetric tools were compared. Film QA The traditional method of QA for IMRT is 2-di­mensional testing using film. We used commer­cial Gafchromic EBT2 film (International Specialty Products, New Jersey, USA)19 and an Epson Expression 10000-XL flatbed film scanner (Epson, California, USA), with a resolution of 0.38 x 0.38 mm2. Film was calibrated using an ion chamber, with more calibration points used for low dose areas to enhance accuracy. Doses were measured at a source to axis distance (SAD) of 100 cm, with film located at a depth of 10 cm of solid slab phan­tom and a gantry angle of 0.. Radiological Imaging Technology IMRT software (Ver. 5.2, Colorado Springs, CO, USA) was used to verify dose delivery after the beam measurement. 309 MapCheck QA MapCheck2 (Sun Nuclear; Melbourne, FL, USA) is a relatively new dosimetric tool, consisting of di­ode detectors in a 2D array and a field size of 32 cm x 26 cm. The matrix is composed of 1,527 diodes, spaced 7.07 mm apart, with each having an active detector area of 0.64 mm2 and the entire matrix hav­ing active detector size of 24.4 x 24.4 cm2.20 Similar to film QA, dose was measured at 8 cm depth of Mapcheck dedicated phantom, Mapphan (Sun Nuclear, Melbourne, FL, USA) at a gantry angle of 0°. Mapcheck dedicated software (MapCHECK2, Ver 5.01.05, Sun Nuclear, Melbourne, FL, USA), was used to verify the dose delivery after the beam measurement. MatriXX QA The MatriXX (IBA Dosimetry GmbH, Schwarzen­bruck, Germany) is similar to Mapcheck but has ionization chambers rather than diode detectors in a 2D array.21,22 Although MatriXX has fewer ion chambers than Mapcheck has diode detectors, the ion chambers yield more stable data than the di­odes.23 The 1,024 ionization chambers of MatriXX are aligned in a parallel pattern in a 32 x 32 grid, with the diameter, height, volume and detector spacing of each ion chamber being 4.5 mm, 7.62 mm, 0.08 cc and 7.62 mm, respectively. The gantry angle was 0° and the beam was investigated using a 5 cm solid water phantom on top of the MatriXX. OmniPro-I’mRT (Ver 1.7.0021, IBA Dosimetry, Germany), a MatriXX-dedicated software pro­gram, was used to verify dose delivery after beam irradiation. EPID QA (portal dosimetry) We used an aSi-based EPID (aS500, Varian Medical Systems, Palo Alto, CA) attached to a Varian Clinac iX Linear accelerator (Varian Medical Systems, Palo Alto, CA, USA).12 This EPID has a resolution of 0.784 x 0.784 mm2 with an array of 512 x 384 pixels, thus having higher resolution than Mapcheck or MatriXX, and a field size of 40 x 30 cm2. The source to axis distance (SAD) was set at 100 cm and the beam was investigated at a gantry angle of 0°. Measurement with EPID was measured with no phantom and EPID dedicated software (Eclipse, Ver 8.9, Varian Medical System, USA) was used to verify dose delivery after the beam measurement. 310 TABLE 2. Mean passing rates based on the gamma index method for the treatment fields of each patient using film, diode array (Mapcheck), ion chamber array (MatriXX), and electronic portal imaging device (EPID) Film 97.42 95.42 95.83 94.80 95.92 95.88 Mapcheck 100.00 99.45 100.00 98.90 99.70 99.61 MatriXX 99.10 99.26 98.84 98.82 99.20 99.04 EPID 99.42 99.12 99.20 99.52 99.20 99.29 Results Doses calculated using TPS were compared with doses measured by the four dosimetric tools based on gamma evaluation (3%/3mm, threshold 15%). Figure 2 shows examples of gamma evaluation results using film, Mapcheck, MatriXX and EPID for IMRT QA. Although Table 1 shows the general passing rates of IMRT QA depending on the do­simetric tools, the result can be dependent on the patient selected in each institution. To clarify the dependency of QA result on dosimetric tools, one should carry out IMRT QA of same patient using different dosimetric tools. Table 2 shows the mean passing rates, based on the gamma index method, for the treatment fields of each patient using film, FIGURE 2. 2D images of the passing rate based on gamma evaluation for various dosimetric tools. (A) Film, (B) diode array (Mapcheck), (C) ion chamber array (MatriXX), (D) Portal dosimetry. Mapcheck, MatriXX, and EPID. The values meas­ured with the four dosimetry tools showed good agreement with the calculated values for all five patients. The mean ± standard deviation (SD) pass­ ing rates (.% . 1) for film, Mapcheck, MatriXX and EPID for 30 IMRT fields of five patients were 95.88 ± 0.87%, 99.61 ± 0.41%, 99.04 ± 0.18% and 99.29 ± 0.15%, respectively. Although all four dosimetry tools met the acceptable passing rate of 95%, these tools showed some differences in measuring the same beam, with the gamma index being much lower for film than for the three other tools. To show fluctuations for each dosimetric tool, we assessed the passing rates of three consecu­tive measurement results using film, Mapcheck, MatriXX and EPID based on gamma index values for 6 fields of patient 1 from Table 2. This means that QA was carried out 3 times, repetitively. As shown in Table 3, the gamma index results were similar, regardless of the number of measure­ments, for Mapcheck, MatriXX and EPID. Film, however, showed higher a standard deviation (i.e., fluctuation) for three consecutive measurements than for the other dosimetirc tools. For example, the fluctuation of film measurement for field 1 was 0.59 which is much higher than 0.00, 0.00, 0.05 for Mapcheck, MatriXX and EPID, respectively. Discussion This comparison of gamma indices for EBT film, Mapcheck, MatriXX, and EPID showed differenc­es in dose distribution when using these various dosimetric tools to carry out the quality assurance for the same patients undergoing IMRT. Even with using the same dosimetry tool, the results differed slightly for each measurement of the same field. The passing rates based on film measurement were much lower than those using the three other dosimetric tools (Table 2). To check the uncertainty of film for the exact same beam, QA was carried out only one time but 3 films were inserted in the 311 TABLE 3. Passing rates of three consecutive measurement results using film, diode array (Mapcheck), ion chamber array (MatriXX) and electronic portal imaging device (EPID) based on gamma index values for 6 fields of patient 1. The data shown for patient 1 in Table 2 is the average of first measurement set of data in Table 3 1st 98.95 98.97 91.20 97.86 98.88 98.66 2nd 97.90 98.93 91.18 98.74 98.28 98.71 Film 3rd 97.58 98.42 91.15 99.24 98.65 98.26 Mean (SD) 98.14 (0.59) 98.77 (0.25) 91.18 (0.02) 98.61 (0.57) 98.60 (0.25) 98.54 (0.20) 1st 100.00 100.00 100.00 100.00 100.00 100.00 2nd 100.00 100.00 100.00 100.00 100.00 100.00 Mapcheck 3rd 100.00 100.00 100.00 100.00 100.00 100.00 Mean (SD) 100.00 (0.00) 100.00 (0.00) 100.00 (0.00) 100.00 (0.00) 100.00 (0.00) 100.00 (0.00) 1st 99.29 98.41 99.57 99.30 99.10 98.97 2nd 99.30 99.25 99.57 99.28 99.06 99.01 MatriXX 3rd 99.29 99.24 99.58 99.29 99.00 98.99 Mean (SD) 99.29 (0.00) 98.97 (0.39) 99.57 (0.00) 99.29 (0.01) 99.05 (0.04) 98.99 (0.02) 1st 99.80 99.40 99.60 98.90 98.80 100.00 2nd 99.90 99.60 99.70 99.20 99.10 100.00 EPID 3rd 99.80 99.60 99.45 99.05 98.90 99.90 Mean (SD) 99.83 (0.05) 99.53 (0.09) 99.58 (0.10) 99.05 (0.12) 98.93 (0.12) 99.97 (0.05) SD = standard deviation phantom. To irradiate the exactly same beam, one should remove the unstable low dose part of the beam caused by scattering, leakage, etc. To do that, we did carry out QA not with individual field but with composite fields, which may remove the low dose part in the analysis. Assessment of the passing rates based on gamma evaluation showed uncertainties ranging from 0.11 to 0.39 (Table 4). Despite these uncertainties, however, the results were reasonably stable, suggesting that a single measurement would be sufficient for QA. TABLE 4. Passing rates based on gamma evaluation using 3 films in the same location for intensity modulated radiation therapy (IMRT) quality assurance (QA) of each patient 98.36 95.29 98.51 98.08 99.41 Film 97.47 95.47 98.55 98.19 99.14 97.63 95.65 98.87 98.36 99.27 Mean 97.82 95.47 98.64 98.21 99.27 SD 0.39 0.15 0.16 0.12 0.11 In general, uncertainties tend to be higher for low dose film measurements.20 We therefore in­vestigated a beam with a universally-tripled-beam intensity (i.e., tripled monitor unit) on each field to confirm the decrease of gamma index with low dose. Table 5 shows the passing rate using film measurements for 3-fold increased beam intensity for 6 fields of patient 1, 2, 4. Compared with the results shown in Table 3, the mean passing rate in­creased, indicating that, in general, high and sta­ble gamma index results with film can be obtained at higher beam intensity (i.e., increased monitor units), comparable to the results from the three other dosimetric tools. Table 5 also shows that standard deviation is decreased with increased beam intensity. Therefore, QA using film should be performed at a sufficiently high intensity beam to reduce the uncertainties from low doses. Although raising the threshold level will lead to clearing of low dose, it may not appropriate since it can cause only the high dose area to be set as region of in­terest. If one wants to use normal treatment MU in QA, careful calibration of film should be used to decrease the standard deviation of film for low dose area. While the QA results based on Mapcheck, MatriXX and EPID showed good agreement among themselves by showing average passing rates of 99.61%, 99.04% and 99.29%, respectively, the aver­ 312 TABLE 5. Passing rates of film measurements based on gamma evaluation for 3-fold increased beam intensity (i.e., monitor unit) for 6 fields of patient 1 Patient1 99.23 99.45 99.20 99.09 99.04 99.21 99.20 0.13 Patient2 98.63 98.04 98.03 97.91 96.80 97.15 97.76 0.61 Patient4 98.77 98.67 99.23 99.40 99.93 98.30 99.05 0.53 age passing rate based on film measurement was significantly lower, 95.88%. The QA result seems to depend on the unique characteristics of each dosimetric tool such as depth of measurement, its resolution, etc. These experimental results suggest that the criteria of the passing rate based on film measurement should be decreased about 3% com­pared to the criteria for other dosimetric tools. If one wants to use universal criteria for the passing rate for all four dosimetric tools, one should in­crease the beam intensity about three times if the film is used as a measurement tool. Further study is needed to suggest the more reliable universal passing rate with large number of case studies. Conclusions We have used film, Mapcheck, MatriXX and EPID to evaluate their dosimetric properties for IMRT QA. While the measured dose maps with Mapcheck, MatriXX and EPID agreed well with the calculated dose maps of TPS, the passing rate was noticeably lower for film measurements. It seems that different passing rates in QA results may par­tially stem from the different resolution (or meas­ured dose matrix) of four dosimetric tools. Use of film for IMRT QA should be implemented using beams of sufficiently high intensity to be compat­ible with the results of Mapcheck, MatriXX, and EPID. Our results suggest that setting an accept­ance level based on the correlation of various dosi­metric tools is a more correct method than simply setting the same acceptance level for all of these tools. Acknowledgment This work was supported by the Nuclear Safety Research Program through the Korea Radiation Safety Foundation (KORSAFe) and the Nuclear Safety and Security Commission(NSSC), Republic of Korea (Grant No. 1305033) and by the National Nuclear R&D Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (NRF-2013M2A2A7067089), and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF­2013R1A1A2007630). References 1. Lee N, Terezakis S. Intensity-modulated radiation therapy. J Surg Oncol 2008; 97: 691-6. 2. Langer M, Brown R, Urie M, Leong J, Stracher M, Shapiro J. Large scale optimization of beam weights under dose-volume restrictions. Int J Radiat Oncol Biol Phys 1990; 18: 887-93. 3. Morrill SM, Lane RG, Wong JA, Rosen II. Dose-volume considerations with linear programming optimization. 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Characterization and use of EBT radiochromic film for IMRT dose verification. Med Phys 2006; 33: 4064-72. 20. Letourneau D, Gulam M, Yan D, Oldham M, Wong JW. Evaluation of a 2D diode array for IMRT quality assurance. Radiother Oncol 2004; 70: 199-206. 21. Saminathan S, Manickam R, Chandraraj V, Supe SS. Dosimetric study of 2D ion chamber array matrix for the modern radiotherapy treatment verifica­tion. J Appl Clin Med Phys 2010; 11: 3076. 22. Herzen J, Todorovic M, Cremers F, Platz V, Albers D, Bartels A, et al. Dosimetric evaluation of a 2D pixel ionization chamber for implementation in clinical routine. Phys Med Biol 2007; 52: 1197-208. 23. Li JG, Yan G, Liu C. Comparison of two commercial detector arrays for IMRT quality assurance. J Appl Clin Med Phys 2009; 10: 2942. 314 Erratum Anoop Haridass, Jillian Maclean, Santanu Chakraborty, John Sinclair, Janos Szanto, Daniela Iancu, Shawn Malone. Dynamic CT angiography for cyberknife radiosurgery planning of intracranial arteriovenous malformations: a technical/ feasibility report. Radiology and Oncology. Volume 49, Issue 2, Pages 192– 199, ISSN (Online) 1581-3207. doi: 10.1515/raon-2015-0006 The corresponding author should be Santanu Chakraborty instead of Jillian Mclean. The corresponding author should be: Dr. Santanu Chakraborty, Department of Radiology, University of Ottawa, Department of Medical Imaging, The Ottawa Hospital, 1053 Carling Avenue, Ottawa, ON K1Y 4E9, Canada. Phone: (613) 737 8571; Fax: (613) 761 4476; E-mail: schakraborty@toh.on.ca Radiol Oncol 2015; 49(3): 209-216. doi:10.1515/raon-2015-0022 Z obsevanjem pospešena diferenciacija matičnih celic Mieloch AA, Suchorska WM Izhodišča. Učinkovita diferenciacija matičnih celic je zelo pomembna v regenerativni medicini. V mnogih raziskavah so preučevali usmerjeno diferenciacijo matičnih celic in izdelali učinkovito zaporedje postopkov. Kljub temu je potrebno, da te postopke izboljšujemo. V članku razlagamo, kako z obsevanjem pospešujemo diferenciacijo matičnih celic in kakšni molekularni procesi pri tem potekajo. Opisujemo z ionizirajočim sevanjem inducirane odgovore različnih vrst matičnih celic. Opredeljujemo vlogo proteina p53 pri diferenciaciji embrionalnih in somatskih matičnih celicah. Na koncu postavimo hipote­zo o vlogi mitohondrijev pri razvoju matičnih celic in pri odgovoru na ionizirajoče sevanje. Zaključki. Kljub temu, da poznamo nevarnost poškodb in nevarnost genomske nestabilnosti zaradi ionizirajočega sevanja, pa lahko ionizirajoče sevanje ob primerni dozi postane učinkovito orodje za spodbujanje usmerjene diferenciacije nekaterih vrst matičnih celic. Radiol Oncol 2015; 49(3): 217-226. doi:10.1515/raon-2015-0035 Gama-enolaza. Dobro poznan tumorski označevalec, z manj poznano vlogo pri raku Vižin T, Kos J Izhodišča. Gama-enolaza, poznana tudi kot nevronska specifična enolaza (NSE), je encim glikolitične poti, ki se izraža predvsem v nevronih in celicah nevroendokrinega sistema. Kot tumorski označevalec jo uporabljamo ob diagnozi raka in napovedi izida bolezni pri bolnikih z rakom, mehanizmi preko katerih je vključena v napredovanje malignega procesa pa ostajajo nepojasnjeni. Gama-enolaza je kot citoplazemski encim vključena v povečano aerobno glikolizo, ki je glavni vir ener­gije rakavih celic in podpira njihovo proliferacijo. Različne lokalizacije gama-enolaze v patofizioloških stanjih pa nakazujejo na dodatne funkcije. Zaključki. Ugotovili so, da C-končni del molekule, ki ni povezan z glikolitično aktivnostjo, spodbuja preživetje nevronskih celic z uravnavanjem signalnih poti, ki so odvisne od receptorja nevronskega rastnega faktorja, kar vodi tudi v obsežno preo­blikovanje aktinskega citoskeleta. Ta dodatna funkcija gama-enolaze je lahko pomembna tudi pri rakavih celicah bodisi za zaščito celic pred stresnimi pogoji in terapijo raka bodisi za spodbujanje migracije in invazije tumorskih celic. Gama-enolaza ima zato najverjetneje večfunkcijsko vlogo pri napredovanju raka, saj podpira povečane presnovne zahteve tumorskih celic, jih ščiti v stresnih pogojih ter spodbuja njihovo invazijo in migracijo. Radiol Oncol 2015; 49(3): 227-233. doi:10.1515/raon-2015-0014 Vpliv rekonstrukcijskega algoritma in vključitve informacije časa preleta na kakovost slik PET/CT Suljič A, Tomše P, Jensterle L, Škrk D Izhodišča. Namen raziskave je bil ugotoviti vpliv različnih rekonstrukcijskih algoritmov z in brez vključitve informacije časa preleta (TOF, time of flight) na kakovost slik pozitronske emisijske tomografije z računalniško tomografijo (PET/CT). Materiali in metode. Meritve smo opravili s trilinijskim fantomom, ki je imel kapilare notranjega premera ~ 1 mm, in s stan­dardnim fantomom Jaszczak. Zajete podatke smo rekonstruirali z analitičnim algoritmom filtrirane povratne projekcije (FBP), iterativnim algoritmom ukazane pričakovane maksimizacije (OSEM, Ordered Subsets Expectation Maximization) (4 iteracije, 24 podmnožic), iterativnim algoritmom True-X, ki vključuje specifično funkcijo razširitve točke (PSF, point spread function), in izhodiščnim iterativnim algoritmom OSEM (2 iteraciji, 8 podmnožic). Rezultati. Meritve prostorske ločljivosti izražene v polni širini pri polovičnem maksimumu (FWHM, full width at half maximum) so dosegle vrednosti 5,2 mm pri FBP; 4,5 pri OSEM in 2,9 pri True-X; pri FBP+TOF je bila vrednost prostorske ločljivosti 5,1 mm, pri OSEM+TOF 4,5 mm in pri True-X+TOF 2,9 mm. Ocena parametrov rekonstruiranih slik so pokazale, da informacija TOF predvsem izboljša hladni kontrast, medtem ko je vpliv izboljšanja pri vročem kontrastu manjši. Največje izboljšanje se pokaže pri variabil­nosti ozadja (zniževanju šuma). Zaključki. Rezultati raziskave kažejo, da vključitev informacije TOF v rekonstrukcijski algoritem ne izboljša prostorske ločljivosti. Informacija TOF ima največji vpliv pri zniževanju variabilnosti ozadja oz. nivoja šuma na sliki. Primerjava med tradicionalnimi in modernimi rekonstrukcijskimi metodami je pokazala, da analitična FBP prikaže primerljive ali celo boljše rezultate pri meritvah hladnega kontrasta in relativne napake števnosti (relative count error). Iterativne metode so prikazale najvišje ravni kontrastne ločljivosti, ko smo informaciji TOF in PSF upoštevali hkrati. Radiol Oncol 2015; 49(3): 234-241. doi:10.1515/raon-2015-0031 Natančno načrtovanje zdravljenja omogoča varno ablacijo jetrnih tumorjev v bližini večjih žil s perkutano ireverzibilno elektroporacijo Kos B, Voigt P, Miklavčič D, Moche M Izhodišča. Ireverzibilna elektroporacija je metoda, ki jo lahko uporabljamo za ablacijo tkiva na osnovi elektroporacije. Kadar celice izpostavimo dovolj močnemu električnemu polju, poškodujemo celično membrano in celice odmrejo po me­hanizmu apoptotične ali nekrotične celične smrti. Čeprav med terapijo z ireverzibilno elektroporacijo prihaja do segrevanja tkiva, metodo uvrščamo med netermične metode ablacije tkiva in jo največ uporabljamo na mestih, kjer so termične metode kontraindicirane. Materiali in metode. Proizvajalec trenutno edinega generatorja električnih pulzov za ireverzibilno elektroporacijo pripo­roča razmerje napetost-razdalja med elektrodami 1500 do 1700 V/cm za zdravljenje tumorjev v jetrih. Vendar pa lahko bližina žil vpliva na porazdelitev električnega polja in posledično na izid terapije. Predstavljamo metodo za načrtovanje zdravljenja ireverzibilne elektroporacije, ki upošteva vpliv žil na električno polje in ilustriramo metodo na primeru 48 letne bolnice z meta­stazo v neposredni bližini zadnje preostale hepatične vene po desni hemihepatektomiji. Rezultati. Rezultati numerične metode za načrtovanje zdravljenja kažejo, da je bila z električnimi pulzi ustvarjena 19,9 cm3 velika lezija in da je bila celotna tumorska masa pokrita z električnim poljem vsaj 900 V/cm. Volumen lezije v modelu se dobro ujema z volumnom lezije vidne na računalniškotomografskih slikah s kontrastom zajetih neposredno po terapiji z ireverzibilno elektroporacijo. V bližini elektrod prihaja do pomembnega dviga temperature, vendar ostane hepatična vena pretočna tudi po terapiji; po šestih mesecih sledenja ni dokazov o ponovitvi bolezni. Zaključki. Načrtovanje zdravljenja z natančnimi računalniškimi modeli prepoznavamo kot pomembno za uspešno zdra­vljenje z ireverzibilno elektroporacijo. Pomemben rezultat raziskave je, da v okolici elektrod prihaja do bistvenega segrevanja. Zato naj se klinični uporabniki izogibajo vstavljanju elektrod manj kot 4 mm stran od rizičnih struktur, kadar sledijo navodilom proizvajalca. Radiol Oncol 2015; 49(3): 242-249. doi:10.1515/raon-2015-0024 Slikovna diagnostika histiocitoze Langerhansovih celic pri otrocih. Analiza referenčnega centra Porto L, Schöning S, Hattingen E, Sörensen J, Jurcoane A, Lehrnbecher T Izhodišča. Namen raziskave je bil (1) opisati histiocitozo Langerhansovih celic pri otrocih v centralnem živčnem sistemu na osnovi minimalnega števila opravljenih sekvenc MRI, zbranih v referenčnem centru ter (2) na osnovi izvidov MRI oceniti medsebojno usklajenost ocenjevalcev. Bolniki in metode. Retrospektivno smo analizirali posnetke MRI otrok s histiocitozo Langerhansovih celic, ki so izpolnjevali kriterije minimalnega števila opravljenih sekvenc MRI. Spremembe centralnega živčnega sistema sta ločeno opisala dva izkušena nevroradiologa, na podlagi česar smo presodili medsebojno usklajenost ocenjevalcev. Rezultati. Od 94 opravljenih slikanj MRI jih je le 31 izpolnjevalo kriterije minimalnega števila opravljenih sekvenc MRI za vklju­čitev v raziskavo. Ti kriteriji so bili: T2 poudarjeno slikanje, metoda MR za izločanje signalnih tekočin (angl. FLAIR), T1 poudarjeno slikanje pred/po kontrastu v vsaj dveh različnih ravninah ter tanke po-kontrastne T1 poudarjene rezine v sagitalni ravnini skozi sello. Najpogosteje opažene spremembe so bile spremembe kostnega mozga, nato solidno povečanje češarike, zadebeljen in obarvan infundibulum ter spremenjen signal dentatnega jedra. Medsebojna usklajenost ocenjevalcev večine sprememb centralnega živčnega sistema je bila razmeroma visoka (. > 0,61). Uporabo minimalnih kriterijev MRI pogosto ni omogočila ocene posteriorne hipofize. Zaključki. Raznolikost radioloških protokolov v različnih institucijah otežuje diagnostiko sprememb v centralnem živčnem sistemu pri otrocih s histiocitozo Langerhansovih celic. Medsebojna usklajenost med nevroradiologi je bila razmeroma vi­soka, kljub temu pa na osnovi minimalno izpolnjenih kriterijev MRI ni bilo mogoče izključiti vseh pojavnih oblik histiocitoze Langerhansovih celic. Ustrezen predpisani postopek MRI naj bi zato vključeval T1 poudarjeno predkontrastno slikanje v sagi­talni ravnini po priporočilih za diagnosticiranje histiocitoze Langerhansovih celic. Potrebna je tudi centralna preverba slikovnih preiskav, ki bi omogočila lažjo primerjavo kliničnih raziskav. Radiol Oncol 2015; 49(3): 250-255. doi:10.1515/raon-2015-0032 Soodvisnost difuzijskega magnetnoresonančnega slikanja in indeksa Ki-67 pri nedrobnoceličnem raku pljuč Karaman A, Durur-Subasi I, Alper F, Araz O, Subasi M, Demirci E, Albayrak M, Polat G, Akgun M, Karabulut N Izhodišča. Primarni cilj raziskave je bil oceniti povezavo med minimalno difuzijsko konstanto (angl. minimum apparent dif­fusion coefficient, ADCmin) in indeksom celične proliferacije Ki-67 pri nedrobnoceličnem pljučnem raku. Zanimalo nas je tudi, ali se vrednost ADCmin razlikuje pri različnih histoloških tipih in različnih tkivnih vzorčenjih. Metode. V raziskavo smo retrospektivno vključili bolnike, pri katerih smo predhodno naredili difuzijsko poudarjeno magnetno resonančno slikanje (angl. diffusion weighted magnetic resonance imaging, DW-MRI). Ocenili smo soodvisnost med ADCmin in indeksom Ki-67. Rezultati. Analizirali smo 93 bolnikov s povprečno starostjo 65 ± 11 let. Pri 47 smo patohistološko dokazali žlezni in pri 46 skva­moznocelični rak pljuč. Pri vseh smo naredili preiskavo DW-MRI. Ugotovili smo izrazito negativno soodvisnost med vrednostmi ADCmin in proliferativnim indeksom Ki-67 (r = -0,837; p < 0,001). Povprečna vrednost ADCmin je bila višja in povprečna vrednost indeksa Ki-67 nižja pri žleznem raku, če smo ga primerjali s skvamoznočeličnim (p < 0,0001). Nismo našli statistično značilne razlike pri različnih tkivnih vzorčenjih. Zaključki. ADCmin je pokazal znatno negativno soodvisnost z indeksom Ki-67, zato bi lahko bil način za ocenjevanje agre­sivnosti tumorja in bi na neinvazivni način prispeval k lažji opredelitvi vrste tumorja. Radiol Oncol 2015; 49(3): 256-264. doi:10.2478/raon-2014-0041: Vpliv polimorfizmov citokinskih genov na razvoj želodčnega raka pri bolnikih z okužbo Helicobacter pylori Štubljar D, Jeverica S, Jukić T, Skvarč M, Pintar T, Tepeš B, Kavalar R, Štabuc B, Ihan A Izhodišča. Baktetrija Helicobacter pylori je glavni vzrok za nastanek želodčnega raka. Na napredovanje bolezni predvsem vplivajo vnetni odzivi gostitelja, pri katerih imajo ključno vlogo polimorfizmi citokinskih genov. Namen raziskave je bil v slovenski populaciji bolnikov preučiti polimorfizme vnetnih citokinov, ki so povezani z razvojem želodčnega raka. Bolniki in metode. V klinično raziskavo smo vključili 318 bolnikov in kontrolnih primerov. Razdelili smo jih v tri skupine: (i) bol­niki z rakom želodca (n = 58), (ii) bolniki s kroničnim gastritisom (n = 60) in (iii) zdrava kontrolna skupina (n = 200). Helicobacter pylori smo v prvih dveh skupinah bolnikov določili s serološkimi in histološkimi preiskavami ter dokazovanjem bakterije na kulturi. Genske polimorfizme štirih provnetnim citokinov (IL-1ß, IL-1 ra, TNF-. in TLR-4) smo določili pri vseh osebah. Rezultati. Ugotovili smo statistično pomembne razlike med moškimi in ženskami (p = 0,025). Razmerje obetov (RO) za tve­ganje za nastanek želodčnega raka pri ženskah je bilo 0,557 (95% interval zaupanja [CI]: 0,233.1,329) in kroničnega gastritisa 2,073 (95% CI: 1,005.4,277). IL-1B-511*T/T homozigotni alel za skupino raka je imel RO = 2,349 (95% CI: 0,583.9,462), heterozigo­ten IL-1B-511*T pa RO = 1,470 (95% CI: 0,583.3,709). Heterozigoti v TNF-A-308 genotipu za kronični gastritis so imeli RO = 1,402 (95% CI: 0,626.3,139). Med drugimi aleli je bilo razmerje obetov manjše kot 1. Zaključki. Čeprav smo odkrili statistično pomambno razliko med moškimi in ženskami, pa v slovenski populaciji nismo uspeli dokazati povezave med želodčnim rakom in kroničnim gastritisom zaradi okužbe s Helicobacter pylori ter polimorfizmi cito­kinskih genov. Zaključili smo, da so drugi genski polimorfizmi - poleg okužbe s Helicobacter pylori - lahko odgovorni za razvoj atrofije črevesne sluznice v neoplastično transformacijo Radiol Oncol 2015; 49(3): 265-270. doi:10.2478/raon-2014-0017 Inflamatorni miofibroblastni tumor v glavi trebušne slinavke. Prikaz primera pri 6-mesečnem otroku in pregled literature Tomažič A, Gvardijančič D, Maučec J, Homan M Izhodišča. Inflamatorni miofibroblastni tumorji so pri otrocih redki. Najpogosteje se pojavijo v pljučih. Če se pojavijo v glavi trebušne slinavke, jih je diferencialno diagnostično potrebno ločiti od drugih vrst tumorjev in kroničnega pankreatitisa. Običajna metoda zdravljenja je kirurška resekcija, v zadnjem času so opisana tudi zdravljenja s steroidi in obsevanjem. Prikaz primera. Šest mesecev starega dečka smo zdravili zaradi tumorja v glavi trebušne slinavke. V klinični sliki sta bila ob sprejemu izražena pruritus in ikterus. Ultrazvok in magnetnoresonančna preiskava sta odkrili tumor v glavi trebušne slinavke. Preoperativna biopsija ni jasno opredelila izvora tumorja. Dečku smo naredili duodenopankreatektomijo. Pooperativni potek je minil brez zapletov. Patohistološki pregled resektata je potrdil diagnozo inflamatornega miofibroblastnega tumorja. Na kontrolnih pregledih ni bilo znakov za ponovitev bolezni. Zaključki. Pri pregledu literature smo zasledili 10 primerov inflamatornih miofibroblastnih tumorjev v trebušni slinavki pri otro­cih, vendar še nikoli pri dojenčku. Kljub večji resekciji pooperativnih zapletov ni bilo. Zdravljenje bi bilo morda mogoče tudi s steroidi, vendar bi bil takšen pristop vprašljiv, posebej pri agresivnih oblikah tumorjev. Radiol Oncol 2015; 49(3): 271-278. doi:10.1515/raon-2015-0001 Neoadjuvantna kemoterapija pri 13 bolnikih z lokalno napredovalim slabo diferenciranim rakom ščitnice opredeljenim s torinskimi izhodišči. Izkušnje posamične bolnišnice Besić N, Dremelj M, Schwartzbartl-Pevec A, Gazić B Izhodišča. Uveljavljeno je prepričanje, da kemoterapija ni učinkovita pri zdravljenju raka ščitnice. Namen raziskave je bil ugotoviti, ali neoadjuvantna kemoterapija pred operacijo ščitnice vpliva na velikost primarnega tumorja pri bolnikih s slabo diferenciranim rakom ščitnice (SDRŠ) opredeljenim s torinskimi izhodišči. Bolniki in metode. Skupno 13 bolnikov (8 žensk, 5 moških; srednja starost 61 let) s slabo diferenciranim rakom ščitnice opredeljenim s torinskimi izhodišči smo v obdobju od leta 1986 do 2005 zdravili z neoadjuvantno kemoterapijo. Premer tumorja je bil 4,5.18 cm (srednji 9 cm). Šest bolnikov je imelo zasevke v področnih bezgavkah, 9 bolnikov pa oddaljene zasevke. Osem bolnikov je imelo tumor pT4. Rezultati. Bolniki so prejeli 29 (razpon 1.5) krogov kemoterapije. Premer tumorja se je zmanjšal pri vseh bolnikih, za več kot 30 % pa pri 5 bolnikih (38%). Dva od teh petih bolnikov smo obsevali z zunanjim žarkovnim snopom. Totalno tiroidektomijo smo naredili pri 10 bolnikih, lobektomijo pri treh in modificirano disekcijo vratnih bezgavk pri petih bolnikih. Histopatološki pregled je pokazal, da je bila v 5 primerih resekcija R0, v 8 primerih pa R1. Osmim bolnikom smo z zunanjim žarkovnim snopom poope­rativno obsevali vrat in zgornji mediastinum. Petletno specifično preživetje bolnikov glede raka je bilo 66 %, 10-letno pa 20 %. Zaključki. Po neadjuvantni kemoterapiji se je tumor zmanjšal za več kot 30% pri 38% bolnikih s slabo diferenciranim rakom ščitnice opredeljenim s torinskimi izhodišči. Radiol Oncol 2015; 49(3): 279-285. doi:10.1515/raon-2015-0019 Fibulin-3 kot tumorski označevalec odgovora na zdravljenje pri bolnikih z malignim mezoteliomom Kovač V, Dodič-Fikfak M, Arnerić N, Dolžan V, Franko A Izhodišča. Fibulin-3 je novi možni tumorski označevalec pri bolnikih z malignim mezoteliomom. V raziskavi smo želeli ovredno­titi morebitno uporabo plazemskega nivoja fibulina-3 pri ocenjevanju odgovora na zdravljenje in njegovo napovedno vre­dnost za napredovanje bolezni do 18 mesecev po diagnozi malignega mezotelioma. Primernost fibulina-3 smo tudi primerjali s topnimi z mezotelinom povezanimi peptidi (SMRP) in ocenili njihovo skupno napovedno vrednost. Bolniki in metode. V raziskavo smo vključili 78 bolnikov z malignim mezoteliomom, ki smo jih zdravili na Onkološkem inšti­tutu Ljubljana v letih 2007 do 2011. Nivo plazemskega fibulina-3 smo določevali pred zdravljenjem in pri različnih odgovorih na zdravljenje. Uporabljali smo encimski imunski test. Rezultati. Pred zdravljenjem se nivo plazemskega fibulina-3 ni razlikoval glede na histološki podtip, tumorski stadij in priso­tnost oddaljenih zasevkov. Statistično značilno višji nivo pa smo ugotovili pri napredovanju bolezni v primerjavi z nivojem pred zdravljenjem (test Mann-Whitney [U] = 472,50, p = 0,003) ter v primerjavi z nivojem ob popolnem odgovoru na zdravljenje (U = 42,00, p = 0,010) in pri stagnaciji bolezni (U = 542,00, p = 0,001). Bolniki, pri katerih je nivo fibulina-3 pred zdravljenjem prese­gel 34,25 ng/ml, so imeli več kot štirikrat večjo verjetnost, da bo bolezen napredovala v času 18 mesecev (razmerje obetov [OR] = 4,35; 95 % interval zaupanja [CI] 1,56–12,13). Prav tako so bolniki z višjim nivojem fibulina-3 po zdravljenju in s popolnim odgovorom na zdravljenje ali stagnacijo bolezni imeli večjo verjetnost, da bo bolezen napredovala v času 18 mesecev (OR = 6,94; 95 % CI 0,99–48,55 in OR = 4,39; 95 % CI 1,63–11,81). Zaključki. Raziskava je pokazala, da bi fibulin-3 ob SMRP lahko bil dodatni tumorski označevalec za ugotavljanje napredo­vanja bolezni pri bolnikih z malignim mezoteliomom. Radiol Oncol 2015; 49(3): 286-290.. doi:10.1515/raon-2015-0028 Vpliv tumorskega volumna na napoved preživetja brez napredovanja bolezni pri sinonazalnem raku Hennersdorf F, Mauz PS, Adam P, Welz S, Sievert A, Ernemann U, Bisdas S Izhodišča. Namen raziskave je bil analizirati napovedne dejavnike, s poudarkom na tumorskem volumnu, ki vplivajo na preživetje brez napredovanja bolezni pri raku nosne votline in obnosnih votlin. Bolniki in metode. Retrospektivno smo analizirali 106 bolnikov s primarnim sinonazalnim rakom, ki smo jih zdravili in spre­mljali med marcem 2006 in oktobrom 2012. Možne napovedne dejavnike za preživetje brez ponovitve bolezni smo vključili v univariatno in multivariatno Coxovo regresijsko analizo. S Kaplan-Meierjevo metodo smo izračunali, kakšen vpliv na preživetje so imeli starost, spol, izhodiščni tumorski volumen (ki smo ga odčitali iz magnetnoresonančnih slik), histološki tip, stadij TNM in napovedne skupine po razvrstitvi Ameriškega odbora za raka (American Joint Committee on Cancer, AJCC). Analizirali smo tudi krivulje za občutljivost in specifičnost (receiver operating characteristic, ROC), s katerimi smo opredelili napovedno vrednost tumorskega volumna. Rezultati. Najštevilčnejša histološka podskupina so bili epitelijski tumorji (77 %). Večina bolnikov (68 %) je imela napredovale tumorje (stadij AJCC II-IV). 18 bolnikov je imelo prizadete bezgavke. Povprečen tumorski volumen je bil 26,6 ± 21,2 cm3. Srednji čas preživetja brez ponovitve bolezni je bil 24,9 mesecev (razpon: 2,5–84,5 mesecev). Analiza krivulj ROC za tumorski volumen je pri ponovitvi bolezni pokazala občutljivost 58,1 % in specifičnost 75.4 %. V univariatni analizi so bili značilni napovedni dejavni­ki tumorski volumen, stadij AJCC, stadij T in stadij N. V multivariatni analizi sta bila značilna in neodvisna napovedna dejavnika pozitiven bezgavčni status in tumorski volumen. Zaključki. Radiološki tumorski volumen se je pokazal statistično zanesljiv napovedni dejavnik preživetja brez ponovitve bo­lezni. Stadij T, stadij N in celokupni stadij AJCC v multivariatni analizi niso zadržali napovedne vrednosti. Radiol Oncol 2015; 49(3): 291-298. doi:10.1515/raon-2015-0018 Primerjava hibridne volumetrične modulirane ločne radioterapevtske tehnike (VMAT) in dvoločne tehnike VMAT za zdravlejnje raka prostate Amaloo C, Nazareth D, Kumaraswamy LK Izhodišča. Volumetrično modulirano ločno terapijo (angl. VMAT) so hitro sprejeli kot standarden način zdravljenja raka prostate. Raziskave so pokazale, da VMAT lahko zagotovi hitrejše obsevanje ob ustrezni pokritosti tarče in znižanem številu monitorskih enot ter ohranjeni zaščiti kritičnih organov. Namen raziskave je bil predstaviti zmožnost hibridne radioterapevtske tehnike, da zagotovi (v primerjavi z VMAT) večjo konformnost ob hkratni boljši kontroli načrtovanja in zaščiti kritičnih organov. Metode. Pri 11 bolnikih, ki smo jih predhodno zdravili zaradi raka prostate s tehniko VMAT, smo ponovno načrtovali obsevanje s hibridno tehniko in s pomočjo Varianovega načrtovalnega sistema (Varian Treatment Planning System). Multipla statična polja (2 do 3) intenzitetno modulirane radioterapije (angl. IMRT) smo načrtovali glede na kritične organe. Na ta način smo zmanjšali obsevalno dozo vendar načrtovali dovolj veliko dozo na obsevalne tarčne volumne (planirane tarčne volumne, PTV). Tako smo dosegli bazično načrtovano dozo, ki je predstavljala 30-35 % doze za posamični ločni plan obsevanja tehnike VMAT. Rezultati. Klinični načrt obsevanja VMAT smo uporabili za kontrolo oz. primerjavo. Primerjava povprečnih doz pri hibridni tehniki in VMAT, ki so jih prejeli rizični organi, je pokazala, da s hibridnim načrtom obsevanja dosežemo nižjo dozo pri vseh primerih, razen pri V80 za mehur in za desno glavo stegnenice. Obsevalna doza na PTV je bila primernejša pri tehniki VMAT. Zaključki. Hibridni načrt je lahko izsevan v času ene same rotacije gantrija, pri čemer so prvine VMAT znotraj terapevtskega loka kombinirane z vložki dinamične IMRT. Radiol Oncol 2015; 49(3): 299-306. doi:10.1515/raon-2015-0023 Učinek zaokroženega konca lističev pri večlistnem kolimatorju na širino polsence in na premik meje obsevalnega polja. Analitična in numerična analiza Zhou D, Zhang H, Ye P Izhodišča. V radioterapiji imajo značilnosti polsence pomembno vlogo pri natančni aplikaciji doze. Ob načrtovanju zdra­vljenja širina polsence in premik meje obsevalnega polja znatno vplivata na konformnost dozne porazdelitve v področjih tarče in kritičnih organov. Metode. V raziskavi analitično in numerično ocenjujemo učinek zaokroženih koncev lističev na značilnosti polsence. Izhajajoč iz pravila razpolovne debeline smo razvili algoritme za izračun lege lističev in korekcije meje obsevalnega polja, kar je še posebej pomembno v primeru lističev z velikim polmerom. Naredili smo računalniško simulacijo, ki je temeljila na programih Monte Carlo EGSnrc/BEAMnrc za različne polmere koncev lističev in različne velikosti virov sevanja. Za izračun širine polsence in premika meje obsevalnega polja smo uporabili tehniko obdelave podatkov s prileganjem krivulji. Rezultati. Rezultati so pokazali, da se širina polsence povečuje z velikostjo vira. Krivulje širine polsence za lističe velikih pol­merov so imele obliko črke U. To je bilo verjetno povezano z dejstvom, da žarki prehajajo na proksimalni in distalni strani lističa. Nasprotno pa je imela velikost vira zanemarljiv vpliv na premik meje obsevalnega polja. Premiki meje obsevalnega polja so bili v primeru analitične in numerične simulacije nespremenjeni. Kljub temu so bile celokupne končne vrednosti premikov meje obsevalnega polja, ki smo jih pridobili z analitično metodo, nekoliko manjše kot vrednosti pridobljene s simulacijo Monte Carlo. Zaključki. Predlagana metoda bi lahko omogočila vpogled v raziskovanje učinkov zaokroženih koncev lističev na značilno­sti polsence. Ob pripravi večlistnega kolimatorja za potrebe intenziteto modulirajoče radioterapije morata biti širina polsence in kalibracija premikov meje obsevalnih polj opravljena natančno. Radiol Oncol 2015; 49(3): 307-313. doi:10.1515/raon-2015-0021 Primerjava zagotavljanja kakovosti štirih dozimetričnih orodij pri intenzitetno moduliranem obsevanju Son J, Baek T, Lee B, Shin D, Park SY, Park J, Lim YK, Lee SB, Kim J, Yoon M Izhodišča. Raziskavo smo zasnovali, da bi primerjali rezultate štirih dozimetričnih orodij zagotavljanja kakovosti, ki jih upora­bljamo pri intenzitetno moduliranem obsevanju (IMRT) in da bi predlagali univerzalne kriterije za zagotavljanje kakovosti, ne glede na dozimetrična orodja, ki jih uporabljamo. Materiali in metode. Izbrali smo trideset polj načrtov IMRT pri petih bolnikih in nadaljevali s postopki za zagotavljanje kakovosti. Uporabili smo (1) obsevanje na radiokromatskem filmu, (2) diodno matriko (Mapcheck), (3) matriko ionske celice (MatriXX) in (4) napravo za elektronsko portalno slikanje (EPID). Izmerjene doze iz štirih dozimetričnih orodij smo primerjali z do­zo, ki smo jo izračunali s sistemom načrtovanja zdravljenja. Stopnje menjavanja (passing rates) štirih dozimetričnih orodij smo izračunali z metodo indeksa gama, ob uporabi razlike v odmerku 3 % in dogovorne razdalje do 3 mm kot merila. Rezultati. Rezultati zagotavljanja kakovosti, ki so temeljili na Mapcheck, MatriXX in EPID so pokazali dobro ujemanje s povprečnimi stopnjami menjavanja 99,61 %, 99,04 % in 99,29 %. Povprečna stopnja menjavanja, ki temeljila na merjenju ra­diokromatskega filma, pa je bila precej nižja, 95,88 %. Povprečna negotovost (1 standardni odklon) za minljive stopnje pri 6 intenzitetno modulirajočih področjih je bila okrog 0,31 pri merjenju s filmom in večja kot pri drugih treh dozimetričnih orodjih. Zaključki. Rezultati zagotavljanja kakovosti in doslednosti so odvisni od izbire dozimetričnega orodja. Univerzalna stopnja menjavanja bi morala biti odvisna od normalizacije ali medsebojnih primerjav dozimetričnih orodij, če uporabljamo več kot eno dozimetrično orodje za zagotavljanje kakovosti pri posameznem bolniku. 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. 9. 2015 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 third quarter of 2015 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, case reports, short reports 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. Andrej Plesničar, M.D., M.Sc. Borut Štabuc, M.D., Ph.D. Tomaž Benulič, M.D. Viljem Kovač, M.D., Ph.D. 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. 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. The Editorial Board requires that the paper has not been published or submitted for publication elsewhere; the authors are responsible for all statements in their papers. Accepted articles become the property of the journal and, therefore cannot be published elsewhere without the written permission of the editors. Submission of the manuscript The manuscript written in English should be submitted to the journal via online submission system Editorial Manager avail­able for this journal at: www.radioloncol.com. In case of problems, please contact Sašo Trupej at saso.trupej@computing.si or the Editor of this journal at gsersa@onko-i.si All articles are subjected to the editorial review and when the articles are appropriated they are reviewed by independent ref­erees. In the cover letter, which must accompany the article, the authors are requested to suggest 3-4 researchers, competent to review their manuscript. However, please note that this will be treated only as a suggestion; the final selection of reviewers is exclusively the Editor’s decision. The authors’ names are revealed to the referees, but not vice versa. Manuscripts which do not comply with the technical requirements stated herein will be returned to the authors for the cor­rection before peer-review. The editorial board reserves the right to ask authors to make appropriate changes of the contents as well as grammatical and stylistic corrections when necessary. Page charges will be charged for manuscripts exceeding the recommended length, as well as additional editorial work and requests for printed reprints. Articles are published printed and on-line as the open access (www.degruyter.com/view/j/raon). All articles are subject to 700 EUR + VAT publication fee. Exceptionally, waiver of payment may be negotiated with editorial office, upon lack of funds. 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It should discuss the results of the study in the light of previously published work. instructions Charts, Illustrations, Images and Tables Charts, Illustrations, Images and Tables must be numbered and referred to in the text, with the appropriate location indi­cated. Charts, Illustrations and Images, provided electronically, should be of appropriate quality for good reproduction. Illustrations and charts must be vector image, created in CMYK color space, preferred font “Century Gothic”, and saved as .AI, .EPS or .PDF format. Color charts, illustrations and Images are encouraged, and are published without additional charge. Image size must be 2.000 pixels on the longer side and saved as .JPG (maximum quality) format. In Images, mask the identities of the patients. Tables should be typed double-spaced, with a descriptive title and, if appropriate, units of numeri­cal measurements included in the column heading. The files with the figures and tables can be uploaded as separate files. References References must be numbered in the order in which they appear in the text and their corresponding numbers quoted in the text. Authors are responsible for the accuracy of their references. References to the Abstracts and Letters to the Editor must be identified as such. Citation of papers in preparation or submitted for publication, unpublished observations, and personal communications should not be included in the reference list. If essential, such material may be incorporated in the appropri­ate place in the text. References follow the style of Index Medicus. All authors should be listed when their number does not exceed six; when there are seven or more authors, the first six listed are followed by “et al.”. The following are some examples of references from articles, books and book chapters: Dent RAG, Cole P. In vitro maturation of monocytes in squamous carcinoma of the lung. Br J Cancer 1981; 43: 486-95. Chapman S, Nakielny R. A guide to radiological procedures. London: Bailliere Tindall; 1986. Evans R, Alexander P. Mechanisms of extracellular killing of nucleated mammalian cells by macrophages. In: Nelson DS, editor. Immunobiology of macrophage. New York: Academic Press; 1976. p. 45-74. Authorization for the use of human subjects or experimental animals When reporting experiments on human subjects, authors should state whether the procedures followed the Helsinki Declaration. Patients have the right to privacy; therefore the identifying information (patient’s names, hospital unit num­bers) should not be published unless it is essential. In such cases the patient’s informed consent for publication is needed, and should appear as an appropriate statement in the article. Institutional approval and Clinical Trial registration number is required. 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