Cilja na 2 procesa nastanka CINV* v 1 odmerku Zagotavlja učinkovito 5-dnevno preprečevanje CINV1-5 En odmerek Dvojno delovanje 5-dnevno preprečevanje SKRAJŠAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA ^Fza to zdravilo se izvaja dodatno spremljanje varnosti. Tako bodo hitreje na voljo nove informacije o njegovi varnosti. Zdravstvene delavce naprošamo, da poročajo o katerem koli domnevnem neželenem učinku zdravila. Akynzeo 300 mg/0,5 mg trde kapsule (netupitant/palonosetron) TERAPEVTSKE INDIKACIJE Pri odraslih za preprečevanje akutne in zakasnjene navzee in bruhanja, povezanih z zelo emetogeno kemoterapijo na osnovi cisplatina za zdravljenje raka ter z zmerno emetogeno kemoterapijo za zdravljenje raka. ODMERJANJE IN NAČIN UPORABE Eno 300 mg/0,5 mg kapsulo je treba dati približno eno uro pred začetkom vsakega cikla kemoterapije. Trdo kapsulo je treba pogoltniti celo. Kapsulo je mogoče vzeti s hrano ali brez nje. Priporočeni peroralni odmerek deksametazona je treba ob sočasni uporabi z Akynzeom zmanjšati za približno 50 %. Prilagoditev odmerka pri starejših bolnikih ni potrebna. Pri uporabi tega zdravila pri bolnikih, starejših od 75 let, je potrebna previdnost zaradi dolgega razpolovnega časa zdravilnih učinkovin in omejenih izkušenj s to populacijo. Varnost in učinkovitost Akynzea pri pediatrični populaciji nista bili dokazani. Prilagoditev odmerka pri bolnikih z blago do hudo okvaro ledvic predvidoma ni potrebna. Potrebno se je izogibati uporabi Akynzea pri bolnikih s končnim stadijem bolezni ledvic, ki potrebujejo hemodializo. Pri bolnikih z blago ali zmerno okvaro jeter (stopnje 5-8 po lestvici Child-Pugh) prilagoditev odmerka ni potrebna. Pri bolnikih s hudo okvaro jeter (stopnja po lestvici Child-Pugh) je treba Akynzeo uporabljati previdno. KONTRAINDIKACIJE Preobčutljivost na zdravilni učinkovini ali katero koli pomožno snov, nosečnost. POSEBNA OPOZORILA IN PREVIDNOSTNI UKREPI Ker lahko palonosetron podaljša čas prehoda skozi debelo črevo, je treba bolnike z anamnezo zaprtja ali znaki subakutne zapore črevesa po dajanju zdravila spremljati. Pri uporabi antagonistov 5-HT3 samih ali v kombinaciji z drugimi serotonergičnimi zdravili (vključno s selektivnimi zaviralci ponovnega privzema serotonina (SSRI) in zaviralci ponovnega privzema serotonina in noradrenalina (SNRI)) so poročali o serotoninskem sindromu. Priporočamo ustrezno opazovanje bolnikov glede simptomov, podobnih kot pri serotoninskem sindromu. Ker Akynzeo vsebuje antagonist receptorjev 5-HT3, je potrebna previdnost pri sočasni uporabi z zdravili, ki podaljšujejo interval QT, ali pri bolnikih, ki so razvili podaljšan interval QT, oziroma je verjetno, da ga bodo. Tega zdravila ne smemo uporabljati za preprečevanje navzee in bruhanja v dneh po kemoterapiji, razen v povezavi z dajanjem naslednjega cikla kemoterapije. Ne smemo ga uporabljati za zdravljenje navzee in bruhanja po kemoterapiji. Pri bolnikih s hudo okvaro jeter je potrebna previdnost, saj je za te bolnike na voljo malo podatkov. To zdravilo je treba uporabljati previdno pri bolnikih, ki sočasno peroralno prejemajo zdravilne učinkovine, ki se primarno presnavljajo prek CYP3A4 in imajo ozko terapevtsko območje. Netupitant je zmeren zaviralec CYP3A4 in lahko poveča izpostavljenost kemoterapevtskim zdravilom, ki so substrati za CYP3A4, npr. docetakselu. Zaradi tega je treba bolnike spremljati glede povečane toksičnosti kemoterapevtskih zdravil, ki so substrati za CYP3A4, vključno z irinotekanom. Poleg tega lahko netupitant vpliva tudi na učinkovitost kemoterapevtskih zdravil, pri katerih je potrebna aktivacija prek presnove s CYP3A4. Akynzeo vsebuje sorbitol in saharozo. Bolniki z redko dedno intoleranco za fruktozo, malabsorpcijo glukoze/galaktoze ali pomanjkanjem saharoza-izomaltaze ne smejo jemati tega zdravila. Poleg tega lahko vsebuje tudi sledi lecitina, pridobljenega iz soje. Zaradi tega je treba bolnike z znano preobčutljivostjo na arašide ali sojo skrbno spremljati glede znakov alergijske reakcije. Ženske v rodni dobi ne smejo biti noseče ali zanositi med zdravljenjem z Akynzeom. Pred začetkom zdravljenja je treba opraviti test nosečnosti pri vseh ženskah, ki še niso imele menopavze. Ženske v rodni dobi morajo uporabljati učinkovito kontracepcijo med zdravljenjem in še do en mesec po njem. Akynzeo je kontraindiciran med nosečnostjo. Med zdravljenjem z Akynzeom in še 1 mesec po zadnjem odmerku je treba prenehati z dojenjem. INTERAKCIJE Ob sočasni uporabi Akynzea z drugim zaviralcem CYP3A4 lahko pride do zvišanja plazemskih koncentracij netupitanta. Pri sočasni uporabi Akynzea in zdravil, ki spodbujajo delovanje CYP3A4, lahko pride do znižanja plazemskih koncentracij netupitanta, kar lahko privede do zmanjšane učinkovitosti. Akynzeo lahko zviša plazemske koncentracije sočasno uporabljenih zdravil, ki se presnavljajo prek CYP3A4. Ob sočasnem dajanju deksametazona z Akynzeom je treba peroralni odmerek deksametazona zmanjšati za približno 50 %. Ob sočasnem dajanju z Akynzeom se je izpostavljenost docetakselu in etopozidu povečala za 37 % oziroma 21 %. Pri ciklofosfamidu po sočasnem dajanju netupitanta niso opazili konsistentnih učinkov. Pri eritromicinu, midazolamu ali drugih benzodiazepinih, ki se presnavljajo prek CYP3A4 (alprazolam, triazolam), je treba ob sočasnem dajanju Akynzea upoštevati možne učinke njihovih zvišanih plazemskih koncentracij. Pri sočasnem dajanju Akynzea z močnimi zaviralci CYP3A4 (npr. ketokonazol) je potrebna previdnost, sočasnemu dajanju z močnimi spodbujevalci CYP3A4 (npr. rifampicin) pa se je treba izogibati. Priporočamo previdnost pri uporabi netupitanta v kombinaciji s peroralnim substratom encima UGT2B7 (npr. zidovudin, valprojska kislina, morfin), ker in vitro podatki kažejo, da netupitant zavira UGT2B7. Priporočamo previdnost pri kombiniranju netupitanta z digoksinom ali drugimi substrati P-gp, kot sta dabigatran ali kolhicin, ker podatki in vitro kažejo, daje netupitant zaviralec P-gp. NEŽELENI UČINKI Pogosti (¿1/100 do <1/10): glavobol, zaprtje, utrujenost. Občasni&1/1.000 do <1/100): nevtropenija, levkocitoza, zmanjšan apetit, nespečnost, omotica, vrtoglavica, atrioventrikularni blok prve stopnje, kardiom¡opatija, motnja prevajanja, hipertenzija, kolcanje, bolečina v trebuhu, driska, dispepsija, napenjanje, navzea, alopecija, urtikarija, astenija, zvišane jetrne transaminaze, zvišana alkalna fosfataza v krvi, zvišan kreatinin v krvi, podaljšanje QT na elektrokardiogramu. Redki (¿1/10.000 do <1/1.000): cistitis, levkopenija, limfocitoza, hipokaliemija, akutna psihoza, sprememba razpoloženja, motnja spanja, hipestezija, konjuktivitis, zamegljen vid, aritmija, atrioventrikularni blok druge stopnje, kračni blok, popuščanje mitralne zaklopke, miokardna ishemija, ventrikularne ekstrasistole, hipotenzija, disfagija, obložen jezik, bolečina v hrbtu, občutek vročine, nekardialna bolečina v prsnem košu, nenormalen okus zdravila, zvišan bilirubin v krvi, zvišana kreatin fosfokinaza MB v krvi, depresija segmenta ST na elektrokardiogramu, nenormalen segment ST-T na elektrokardiogramu, zvišan troponin. Vrsta ovojnine in vsebina: Škatla z eno kapsulo v pretisnem omotu iz aluminija. Režim izdaje: Rp Imetnik dovoljenja za promet: Helsinn Birex Pharmaceuticals Ltd, Damastovvn, Mulhuddart, Dublin 15, Irska AKY-062016 Pred predpisovanjem in uporabo zdravila prosimo preberite celoten povzetek glavnih značilnosti zdravila! Samo za strokovno javnost! AKY0816-01, avgust 2016 HELSINN Building quality cancer care together Hh PharmaSwiss Choose More Life Odgovoren za trženje v Sloveniji: PharmaSwiss d.o.o., Brodišče 32,1236 Trzin telefon: +3861236 47 00, faks: +386 1283 38 10 ADIQLQGY AND NCOLOGY June 2016 Vol. 50 No. 3 Pages 247-346 ISSN 1318-2099 UDC 616-006 CODEN: RONCEM 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 Sersa, Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia Executive Editor Viljem Kovac, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia Editorial Board Sotirios Bisdas, National Hospital for Neurology and Neurosurgery, University College London Hospitals, London, UK Karl H. Bohuslavizki, Facharzt für Nuklearmedizin, Hamburg, Germany Serena Bonin, University of Trieste, Department of Medical Sciences, Trieste, Italy Boris Brkljacic, 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 Filipic, National Institute of Biology, Department of Genetic Toxicology and Cancer Biology, Ljubljana, Slovenia Maria Gódény, 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 Turnsek, National Institute of Biology, Ljubljana, Slovenia Damijan Miklavcic, University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia Luka Milas, UT M. D. Anderson Cancer Center, Houston , USA Damir Miletic, Clinical Hospital Centre Rijeka, Department of Radiology, Rijeka, Croatia Hakan Nystrom, Skandionkliniken, Uppsala, Sweden Maja Osmak, Ruder Boskovic Institute, Department of Molecular Biology, Zagreb, Croatia Dusan Pavcnik, 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. Podgorsak, 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 Miklos Kasler, 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 Stabuc, University Medical Centre Ljubljana, Department of Gastroenterology, Ljubljana, Slovenia Katarina Surlan Popovic, University Medical Center Ljubljana, Clinical Institute of Radiology, Ljubljana, Slovenia Justin Teissie, 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 2G16; 50(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 Vukmirovic Design Monika Fink-Serša, Samo Rovan, Ivana Ljubanovič 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 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 Agency. 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) 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 Radiol Oncol 2G16; 50(3): A. contents contents review 247 18F-fluorodeoxyglucose and 18F-flumazenil positron emission tomography in patients with refractory epilepsy Marina Hodolic, Raffi Topakian, Robert Pichler 254 Uncertainties in target volume delineation in radiotherapy - are they relevant and what can we do about them? Barbara Segedin, Primoz Petric radiology 263 Non-contrast computed tomography in the diagnosis of cerebral venous sinus thrombosis Andrej Vogrin, Hana Besic, Nikola Besic, Maja Marolt Music 269 Recurrence rate in regional lymph nodes in 737 patients with follicular or HUrthle cell neoplasms Jernej Avsenik, Janja Pretnar Oblak, Katarina Surlan Popovic experimental oncology 274 Electrochemotherapy with bleomycin is effective in BRAF mutated melanoma cells and interacts with BRAF inhibitors Tanja Dolinsek, Lara Prosen, Maja Cemazar, Tjasa Potocnik, Gregor Sersa 280 Discovery of 'click' 1,2,3-triazolium salts as potential anticancer drugs Ivana Steiner, Nikolina Stojanovic, Aljosa Bolje, Anamaria Brozovic, Denis Polancec, Andreja Ambriovic-Ristov, Marijana Radic Stojkovic, Ivo Piantanida, Domagoj Eljuga, Janez Kosmrlj, Maja Osmak clinical oncology 289 Functional polymorphisms in antioxidant genes in Hurthle cell thyroid neoplasm - an association of GPX1 polymorphism and recurrent Hurthle cell thyroid carcinoma Blaz Krhin, Katja Goricar, Barbara Gazic, Vita Dolzan, Nikola Besic 297 Association between polymorphisms in segregation genes BUB1B and TTK and gastric cancer risk Petra Hudler, Nina Kocevar Britovsek, Snjezana Frkovic Grazio, Radovan Komel Radiol Oncol 2016; 50(3): C. contents 308 Metastatic sebaceous cell carcinoma, review of the literature and use of electrochemotherapy as possible new treatment modality Simone Ribero, Eugenio Sportoletti Baduel, Matteo Brizio, Franco Picciotto, Emi Dika, Maria Teresa Fierro, Giuseppe Macripo, Pietro Quaglino 313 The proliferation marker Ki67, but not neuroendocrine expression, is an independent factor in the prediction of prognosis of primary prostate cancer patients Mariarosa Pascale, Cinzia Aversa, Renzo Barbazza, Barbara Marongiu, Salvatore Siracusano, Flavio Stoffel, Sando Sulfaro, Enrico Roggero, Serena Bonin, Giorgio Stanta 321 Pancreaticoduodenectomy for ductal adenocarcinoma of the pancreatic head with venous resection Vojko Flis, Stojan Potrc, Nina Kobilica, Arpad Ivanecz 329 Interdisciplinary consensus statement on indication and application of a hydrogel spacer for prostate radiotherapy based on experience in more than 250 patients Arndt-Christian Müller, Johannes Mischinger, Theodor Klotz, Bernd Gagel, Gregor Habl, Gencay Hatiboglu, Michael Pinkawa 337 Excellent outcomes after radiotherapy alone for malignant spinal cord compression from myeloma Dirk Rades, Antonio J. Conde-Moreno, Jon Cacicedo, Barbara Segedin, Volker Rudat, Steven E. Schild 341 The role of neoadjuvant chemotherapy in patients with advanced (stage IIIC) epithelial ovarian cancer Erik Skof i Slovenian abstracts Radiol Oncol 2016; 50(3): C. 247 review article 18F-fluorodeoxyglucose and 18F-flumazenil positron emission tomography in patients with refractory epilepsy Marina Hodolic12, Raffi Topakian3, Robert Pichler4 1 Nuclear Medicine Research Department, lason, Graz, Austria 2 Department of Nuclear Medicine, Palacky University Olomouc, Czech Republic 3 Department of Neurology, Klinikum Wels-Grieskirchen, Wels, Austria 4 Institute of Nuclear Medicine, Kepler Universitätsklinikum, Neuromed Campus, Linz, Austria Radiol Oncol 2016; 50(3): 247-253. Received 23 February 2016 Accepted 29 April 2016 Correspondence to: Marina Hodolic, M.D., Ph.D., Nuclear Medicine Research Department, lason, Graz-Seiersberg, Feldkirchner Straße 4, Austria. Phone: +43 (0) 664 830 94 93; Fax: +43 316 284 300 14; E-mail: marina.hodolic@gmail.com Disclosure: No potential conflicts of interest were disclosed. Background. Epilepsy is a neurological disorder characterized by epileptic seizures as a result of excessive neuronal activity in the brain. Approximately 65 million people worldwide suffer from epilepsy; 20-40% of them are refractory to medication therapy. Early detection of disease is crucial in the management of patients with epilepsy. Correct localization of the ictal onset zone is associated with a better surgical outcome. The modern non-invasive techniques used for structural-functional localization of the seizure focus includes electroencephalography (EEG) monitoring, magnetic resonance imaging (MRI), single photon emission tomography/computed tomography (SPECT/CT) and positron emission tomography/computed tomography (PET/CT). PET/CT can predict surgical outcome in patients with refractory epilepsy. The aim of the article is to review the current role of routinely used tracer 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG) as well as non routinely used 18F-Flumazenil (18F-FMZ) tracers PET/CT in patients with refractory epilepsy. Conclusions. Functional information delivered by PET and the morphologic information delivered by CT or MRI are essential in presurgical evaluation of epilepsy. Nowadays 18F-FDG PET/CT is a routinely performed imaging modality in localization of the ictal onset zone in patients with refractory epilepsy who are unresponsive to medication therapy. Unfortunately, 18F-FDG is not an ideal PET tracer regarding the management of patients with epilepsy: areas of glucose hypometabolism do not correlate precisely with the proven degree of change within hippocampal sclerosis, as observed by histopathology or MRI. Benzodiazepine-receptor imaging is a promising alternative in nuclear medicine imaging of epileptogenic focus. The use of 11C-FMZ in clinical practice has been limited by its short half-life and necessitating an on-site cyclotron for production. Therefore, 18F-FMZ might be established as one of the tracers of choice for patients with refractory epilepsy because of better sensitivity and anatomical resolution. Key words: epilepsy; nuclear medicine; PET/CT; 18F-FDG; 18F-Flumazenil Introduction Epilepsy is a neurological disorder characterized by epileptic seizures as a result of excessive neuronal activity in the brain (the word "epilepsy" is derived from the Greek word meaning to be attacked or seized). For diagnosis of epilepsy at least two unprovoked seizures are required. Metabolic, genetic or structural conditions can be recognized as causes of epilepsy, but in 60% of patients, the cause remains unknown.1 Approximately 65 million people worldwide suffer from epilepsy2, 20-40% of them are refractory to medication therapy.3 Depending on the epilepsy syndrome, dietary changes, neurostimulation or surgery may be considered as treatment options in patients whose seizures do not Radiol Oncol 2016; 50(3): 247-253. doi:10.1515/raon-2016-0032 248 Hodolic M et al. / 18F-FDG and 18F-Flumazenil PET in patients with refractory epilepsy respond to medication therapy. With the advancement of surgical techniques and devices, surgical treatment has become the treatment of choice in patients who are unresponsive to medication therapy. In selected patients with focal structural lesions such as cortical dysplasia, mesial temporal sclerosis, vascular malformations and in some paediatric epilepsy syndromes, surgery may substantially reduce the frequency of epileptic seizures and improve the patient's quality of life. Several studies over the last decades support the statement that surgical treatment significantly improved long-term outcomes of seizure control.4-8 Early detection of disease is crucial in the management of patients with epilepsy. Correct localization of the ictal onset zone is associated with a better surgical outcome. Epilepsy surgery may lead to seizure freedom. When "surgical cure" is impossible, epilepsy surgery may help achieve palliative goals such as minimizing the frequency and severity of seizures. Following successful epilepsy surgery, quality of life, cognition and behaviour may improve substantially.9 However, careful patient selection and weighing of risks and benefits are of paramount importance, as surgery may not only fail in terms of improving seizure control, but come with serious adverse events such as intrac-ranial bleeding. Despite international guidelines recommending early and systematic assessment of patient's eligibility, epilepsy surgery is still being underused and referral of patients with drug refractory epilepsy is often delayed with deleterious consequences on outcome and quality of life.10 The scope of this paper on PET imaging in refractory epilepsy patients cannot cover all aspects of patient management including the range of indications, specific issues in children and adults, surgical techniques or predictors of outcome. For this, the interested reader is referred to a recent review of Ryvlin et al.11 Different modalities in diagnosis of epilepsy Non-invasive focus localisation of seizures precedes invasive intracranial electrodes procedures. The modern non-invasive techniques used for structural-functional localization of the seizure focus includes electroencephalography (EEG) monitoring, magnetic resonance imaging (MRI), single photon emission tomography/computed tomography (SPECT/CT) and positron emission tomography/computed tomography (PET/CT). EEG has low sensitivity in the diagnosis of seizure disorders (25-56%). MRI is a sensitive and specific imaging modality for identifying hippocampal sclerosis as well as other lesions responsible for epilepsy. However 1-1.5 T MRI still fails to reveal approximately 20% of abnormalities in patients with medically refractory epilepsy12; MRI may miss mild changes and subtle lesions. In patients with intractable extratemporal epilepsy, the most common underlying pathology is microscopic cortical dysplasia which sometimes cannot be detected by MRI and hence may pass unnoticed by 18F-FDG PET/CT as well. A recent study of 194 adult patients with medically refractory focal epilepsy showed that 18F-FDG PET/CT is helpful for decision making in 53% of presurgical patients with normal or discordant MRI.13 Advanced MRI technologies (MR spectroscopy, MR volumetry, MR perfusion) may provide additional and more precise information. In recent years brain perfusion SPECT imaging has been widely used for detection of epileptic focus. In their meta-analysis, Devous et al. reported a 44% (interictal), 75% (postictal) and 97% (ictal) sensitivity of SPECT in patients with temporal lobe epilepsy.14 Extratemporal lobe epilepsy showed SPECT sensitivity in 66% (ictal) and in 40% (inter-ictal) of patients.1516 PET procedures in patients with epilepsy In recent years, multiple studies17-22 have demonstrated that 18F-FDG PET/CT can predict surgical outcome in patients with refractory epilepsy. Actually 18F-FDG PET/CT has proven to be the most sensitive imaging technique for presurgical localization of epileptogenic foci in patients with medically refractory partial epilepsy who have non-contributory MRI and EEG. In comparison to SPECT, PET technology provides much better resolution and allows quantitative measurement.23-26 There is a variety of PET tracers used for imaging of epileptic focus: tracers that measure glucose metabolism, serotonine receptors and transport, oxygen metabolism, cerebral blood flow and other receptor binding.27 PET scan findings at the area of a seizure focus are different according to the PET tracer used (Table 1). Multiple PET radiotracers for neurotransmitter and neuromodulator systems still form part of preclinical trials. In this review article we focus on the routinely used tracer 18F-FDG as well as on 18F-labelled Radiol Oncol 2016; 50(3): 247-253. 249 Hodolic M et al. / 18F-FDG and 18F-Flumazenil PET in patients with refractory epilepsy flumazenil tracers which are less available but becoming popular. 18F-FDG PET/CT in patients with epilepsy The first application of 18F-FDG PET/CT in patients with epilepsy dates back to the early 1980s.28'29 As glucose is the main energy source for the brain, a radioactive glucose analogue has been the most widely used tracer for PET imaging in patients with refractory epilepsy. There is a good match of glucose metabolism and neuronal activity. Glucose transporters (predominantly GLUT1) transfer 18F-FDG from the blood into cells. Once in the cell, FDG is phosphorylated by hexokinase and forms FDG-6-phosphate. Further metabolism of FDG-6-phosphate is stopped and FDG-6-phosphate is essentially trapped in the cell. In 2009 the European Association of Nuclear Medicine established an imaging protocol for 18F-FDG PET/CT imaging of the brain.30 According to this protocol patients should fast at least 4 hours before scanning. Psychotropic pharmaceuticals may influence reagional metabolism of the glucose. Serum glucose levels must be checked before 18F-FDG PET/CT examination. If the value is greater than 160 mg/dl, the patient must be rescheduled. Diabetic patients should undergo scanning in euglycemic state. At least 30 minutes before examination as well as 30 minutes after tracer injection patients must rest in a quiet dimly-lighted room. They should be instructed not to talk, read or to be otherwise active. 18F-FDG dose for adults patients is 300-600 MBq in 2-D mode and 125-250 MBq in 3-D mode. For children, dose is calculated by body wight (EANM dosage card). Continuous EEG recording is required 2 hours before tracer injection (to exclude that tracer is not injected postic-tal state) and at least until 20 minutes after tracer injection. Static PET scan acquisition starts 30 up to 60 minutes after tracer injection and lasts for 15-30 minutes. There are small variations in the protocols depending on quality of imaging technology. In PET scans interpretation, combination of visual inspection and semiquantitive analysis is crucial. Semiquantitative analysis helps to detect abnormalities which are not present on visual inspection. Physiological18F-FDG distribution is as follows: high in cerebral and cerebellar cortices and subcortical grey matter and mild in the white matter. In children and with normal aging there is decrease in TABLE 1. Some PET tracers used for imaging of epileptic focus and PET scan findings PET scan Findings on PET scan 18F-FDG interictal Decreased metabolism 18F-FDG ictal Increased and decreased metabolism (complex pattern) 18F-FDG postictal Increased and decreased metabolism (complex pattern) Serotonin receptor (e.g. 18F-Mefway) Reduced binding Dopamine receptor (e.g. 18F-Fallypride) Reduced binding 18F-Flumazenil (GABA receptor) Decreased binding 15O-H20 interictal Reduced perfusion 15O-H20 ictal Increased perfusion r I » rto Wf FIGURE 1. Focal epilepsy in 17 year old male patient. Interictal 18F-FDG PET (A): physiological distribution of 18F-FDG in the brain. Ictal 18F-FDG PET (B): hypermetabolism frontolateral in the right hemisphere (arrow). cerebral metabolic rates (particularly in lateral, medial frontal cortex and anterior cingulate cortex).31,32 Regions involving epileptic foci may present increased, reduced, or, absent metabolic activity. 18F-FDG PET/CT scans in patients with epilepsy are usually obtained in the interictal phase. Ictal 18F-FDG PET/CT appears to be highly sensitive (Figure 1), but is difficult to obtain because of unpredictability and very rapid onset of major seizures. In addition, medical personnel and the radiotracer must be available at the patient's bedside at the time of seizure onset. Postictal 18F-FDG PET/ CT scans can be very complex, and it represents a mishmash of increased and/or decreased metabolism, depending on the time of 18F-FDG injection after the onset of seizure. Radiol Oncol 2016; 50(3): 247-253. 250 Hodolic M et al. / 18F-FDG and 18F-Flumazenil PET in patients with refractory epilepsy Interictal 18F-FDG PET/CT in patients with epilepsy The goal of implementing 18F-FDG PET/CT in the management of patients with drug refractory epilepsy was to obtain functional imaging of interictal brain glucose metabolism. Multiple studies have shown that interictal 18F-FDG PET/CT is more sensitive than interictal perfusion SPECT33-34,15, because in the interictal period, reduction of regional glucose cerebral metabolism is more pronounced than reduction of cerebral perfusion.35-37 Neuronal loss due to chronic seizure activity, reduction in density of synapses, inhibitory processes in the interictal period and diaschisis also influence glucose hypometabolism, the characteristic sign of epilepsy on interictal 18F-FDG PET/ CT. Interictal 18F-FDG PET/CT cannot precisely define the surgical margins, because some areas of hypometabolism extend beyond epileptic zones. Drzezga et al. showed that automated analysis of 18F-FDG PET/CT scans in patients with epilepsy is more sensitive than visual analysis in patients with temporal lobe epilepsy and extra temporal lobe epilepsy.36 The combination of MRI and interictal 18F-FDG PET/CT will probably evolve as the future modality of choice to get the best results for presur-gical evaluation. Ictal 18F-FDG PET/CT in patients with epilepsy Ictal 18F-FDG PET in the presurgical workup of refractory epilepsy is rarely performed; it is performed either in status epilepticus or in a status of induced epileptic seizures. As status epilepticus is defined as seizure longer than 30 minutes or more than one seizure within thirty minutes without the person returning to normal, 18F-FDG PET is difficult to be obtained in those moments. Despite technical difficulties ictal 18F-FDG PET has the advantage of a high spatial resolution. Ictal 18F-FDG PET/CT shows hypermetabolism, although an ictal scan reveals a complex combination of hyper and hypometabolism and for that reason it is important to continuously monitor the patient with scalp EEG. A study from 2013 showed that in patients with incompatible EEG, MRI and clinical features, ictal 18F-FDG PET/CT helped to localize the origin of status epilepticus.38 Definition of an ictal onset zone is usually made visually with the support of semiquantitative analysis. A difference above 15% between the affected and the contralat- eral side suggests significant physiological asymmetry. Unfortunately, 18F-FDG is not an ideal PET tracer regarding the management of patients with epilepsy. Areas of glucose hypometabolism do not correlate with the proven degree of change within hippocampal sclerosis, as observed by histopathol-ogy or MRI; the area of hypometabolism may be larger than the pathological seizure focus.39 So, the presurgical definition of epileptogenic foci cannot be based on 18F-FDG PET/CT imaging alone, because 18F-FDG changes may appear larger than the real ictal onset zone. The reason for this anatomical-functional discrepancy has not been explained yet. However, 18F-FDG PET/CT can reveal and confirm surgical targets and therefore represents a valuable tool in the management of patients with intractable epilepsy. 18F- Flumazenil tracers for PET/CT of patients with epilepsy Gamma-aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the central nervous system.40-43 Its main role consists of reducing neuronal excitability and regulating muscle tone. There are two types of GABA receptors: GABAA and GABAB; benzodiazepine receptors are modulatory sites on GABAA receptors. Benzodiazepine distribution in the human brain includes the occipital cortex, temporal cortex, cerebellum, thalamus, and the pons. The majority of GABAA receptors are benzodiazepine-sensitive, but there are also GABAA receptors which are insensitive to classical benzodiazepines. Benzodiazepines enhance the action of GABA on its receptors, thus having anticonvulsant, sedative, hypnotic, anxiolytic and muscle-relaxant effects. Flumazenil blocks the benzodiazepine sites on GABAA receptors and thus antagonizes the action benzodiazepines have on the central nervous system. Flumazenil was introduced in 1987 and over time it was used as an antidote for the treatment of benzodiazepine overdoses. In epileptogenic regions a reduced level of benzodiazepine receptors is found44; also, irreversible ischemic cortical damage after stroke45, Alzheimer disease4647 chronic alcoholism48 and schizophrenia49 may influence benzodiazepine complex density. In the 1980s, Flumazenil was proposed as a promising new marker for imaging of benzodi-azepine receptors by PET. The substance was ini- Radiol Oncol 2016; 50(3): 247-253. 251 Hodolic M et al. / 18F-FDG and 18F-Flumazenil PET in patients with refractory epilepsy tially labelled with Carbon-11 (UC-FMZ)50 and used in some studies: the majority of patients with seizures refractory to medical treatment had hippocampal sclerosis characterized by neuronal loss and gliosis, which was detected by this positron-labelled GABAa receptor antagonist.51 Six years later a SPECT agent, 123I-iomazenil, was synthetized.5253 Both compounds showed high benzodiazepine binding potential in the brain. However, use of 11C-FMZ in clinical practice has been limited by its short half-life, necessitating an on-site cyclotron for production, and multiple syntheses if several subjects have to be examined. The first paper on a fluorinated analogue of flumazenil was published in 1992.54 18F-labelled PET radiotracers are more suited for clinical use. Also, the shorter positron range of 18F provides better image resolution, which may enhance the detection of small pathological complexes such as epileptic foci. Following this, several 18F-labelled FMZ tracers, including 5-(2'-[18F]fluoroethyl) flumazenil (18F-FEFMZ), 3-(2'-[18F]fluorofluma-zenil (18F-FFMZ), 5-(2'-[18F]fluoroethyl)fluma-zenil (18F-FEF) and [18F]flumazenil (18F-FMZ) have been developed.55-60 Compared to 11C-FMZ, 18F-FEFMZ has lower receptor affinity, and higher nonspecific binding due to faster metabolism and faster kinetics. 18F-FFMZ also has faster kinetics than 11C-FMZ. Compared to 11C-FMZ, 18F-FEF has lower affinity to benzodiazepine receptors, rapid kinetics in the brain and faster metabolism.55-60 18F-FMZ, with an identical structure to 11C-FMZ, has been shown to have similar pharmacokinet-ics and peripheral metabolism and PET imaging characteristics as 11C-FMZ. 18F-FMZ has been established as the tracer of choice for patients with refractory epilepsy in some neurological centres in Europe. PET/CT imaging using flumazenil tracers identify a more restricted region of abnormality in the epileptogen-ic zone than 18F-FDG PET/CT.61 In comparison to 18F-FDG PET/CT scans benzodiazepine-receptor scans appears "sharper" (Figure 2, 3). Imaging protocol for PET/CT with 18F-labelled-flumazenil radiotracers is almost similar to protocol for 18F-FDG PET/CT, except no serum glucose level measurement is required. All flumazenil tracers bind non-selectively to all benzodiazepine receptor subtypes, so pre-treatment with unla-belled flumazenil can result in reduced tracer uptake on benzodiazepine receptors. As a young modality, benzodiazepine-receptor PET imaging is still strengthening its place in neurology. FIGURE 2. 18F-FDG PET (B) scan vs. 18F-Flumazenil PET (C) in a 19 year old female patient with bilateral hippocampal sclerosis as shown by MRI (A). Both PET modalities present low temporomesial uptake being larger on the left side, but benzodiazepine-receptor imaging appears sharper and presents a focal defect. / J ' âi\ c /jr ' 1 # jbL> , V&l f t r, A ikU l&ii K 7% (A © @ FIGURE 3. Focal epilepsy in 56 year old male patient. (A) MRI: astrogliosis of the right hippocampus (later proven by histology); (B) 18F-FDG PET: minal temporomesial hypometabolism of both sides; (C) 18F-Flumazenil PET: focal defect of tracer uptake at the right hippocampus. Conclusions Functional information delivered by PET and the morphologic information delivered by CT or MR are essential in presurgical evaluation of epilepsy. Nowadays 18F-FDG PET/CT is a routinely performed imaging modality in localization of the ic-tal onset zone in patients with refractory epilepsy who are unresponsive to medication therapy. Unfortunately, 18F-FDG is not an ideal PET tracer regarding the management of patients with epilepsy: areas of glucose hypometabolism do not correlate precisely with the proven degree of change within hippocampal sclerosis, as observed by histopathol-ogy or MRI. Benzodiazepine-receptor imaging is a promising alternative in nuclear medicine imaging of epileptogenic focus. The use of 11C-FMZ in clinical practice has been limited by its short half-life and necessitating an on-site cyclotron for production. 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Barbara Segedin1, Primoz Petric2 1 Department of Radiation Oncology, Institute of Oncology Ljubljana, Slovenia 2 Department of Radation Oncology, National Centre for Cancer Care and Research, Doha, Qatar Radiol Oncol 2016; 50(3): 254-262. Received 29 December 2015 Accepted 1 February 2016 Correspondence to: Primož Petrič, M.D., M.Sc., Department of Radiation Oncology, National Centre for Cancer Care and Research, P.O.box 3050, Doha, Qatar. Phone: +974 44397720. E-mail: pp.visoko@gmail.com Disclosure: No potential conflicts of interest were disclosed. Background. Modern radiotherapy techniques enable delivery of high doses to the target volume without escalating dose to organs at risk, offering the possibility of better local control while preserving good quality of life. Uncertainties in target volume delineation have been demonstrated for most tumour sites, and various studies indicate that inconsistencies in target volume delineation may be larger than errors in all other steps of the treatment planning and delivery process. The aim of this paper is to summarize the degree of delineation uncertainties for different tumour sites reported in the literature and review the effect of strategies to minimize them. Conclusions. Our review confirmed that interobserver variability in target volume contouring represents the largest uncertainty in the process for most tumour sites, potentially resulting in a systematic error in dose delivery, which could influence local control in individual patients. For most tumour sites the optimal combination of imaging modalities for target delineation still needs to be determined. Strict use of delineation guidelines and protocols is advisable both in every day clinical practice and in clinical studies to diminish interobserver variability. Continuing medical education of radiation oncologists cannot be overemphasized, intensive formal training on interpretation of sectional imaging should be included in the program for radiation oncology residents. Key words: target volume; interobserver variability; delineation uncertainties; imaging; training Introduction Modern radiotherapy techniques such as intensity modulated radiotherapy (IMRT), volumetric modulated arch therapy (VMAT) and image guided adaptive brachytherapy (IGABT) enable delivery of high doses to the target volume without escalating dose to organs at risk (OAR), offering the possibility of better local control while preserving good quality of life.12 Highly conformal radiation techniques and sharp dose falloff make the accuracy and precision of every step in treatment planning and delivery extremely important. Uncertainties in the process of radiotherapy include patient set-up error, inter- and intra-fraction organ movement, patient movement and uncer- tainties in target volume delineation. Image guided radiation therapy (IGRT) addresses the uncertainties arising from patient set-up, patient and organ movement and improves target localisation during treatment. However, reduction of margins introduced with the use of IGRT is limited by the ability to adequately define the target. Accurate target volume delineation is a precondition for the use of IMRT, VMAT, IGABT and other high precision radiotherapy techniques, since all subsequent steps in treatment planning and delivery are based on target volume contours. Inadequate definition of the target introduces a systematic geographic miss that could potentially lead to reduction of the dose delivered to the tumour, lower local control and/ or increased morbidity for an individual patient.3-6 Radiol Oncol 2016; 50(3): 254-262. doi:10.1515/raon-2016-0023 Segedin B et al. / Uncertainties in target volume delineation in radiotherapy 255 In addition, such uncertainties can undermine meaningful comparison of treatments within and between institutions and interpretation of clinical studies. Uncertainties in target volume delineation have been demonstrated for most tumour sites, and various studies indicate that inconsistencies in target volume delineation may be larger than errors in all other steps of the treatment planning and delivery process.7-22 The aim of this paper is to summarize the degree of delineation uncertainties for different tumour sites reported in the literature and review the effect of strategies to minimize them. Magnitude of uncertainties Direct comparison of published data is difficult, since a variety of methods is used to quantify interobserver variability. Most papers report parameters describing the distribution of delineated volumes including mean, range, standard deviation (SD), the ratio of the largest and the smallest delineated volume (Vmax/Vmin), coefficient of variation (COV) etc. Also commonly used are different concordance measures such as conformity, concordance or similarity index (CI, SI - ratio between common and encompassing volume), Dice-Jaccard coefficient (DJC), percent overlap, ratio of encompassing and common volume (1/CI), geographical miss index and mean discordance index or statistical measures of agreement i.e. kappa (k) - statistics.23 Less commonly, methods for local interobserver variation assessment are used i.e. local standard deviation (SD), inter-delineation distance or radial line measurement variation, all expressed in mm.24-27 A wide range of interobserver variability is observed for various tumour sites, the largest variation being reported for target volume delineation in oesophageal, head and neck and lung cancer, Hodgkin's lymphoma and sarcoma, where the V /V ratios are 6, 18.3, > 7, 15 and > 8, respec- max min ' ' ' ' r tively.3,18,28'30 Gastrointestinal tumours In rectal cancer, reported conformity indices are from 0.29 to 0.98 for clinical target volume (CTV) and from 0.26 to 0.81 for primary tumour gross target volume (GTV), depending on the use of consensus guidelines and chosen imaging modality.1931-33 The ratio of V to V . is 1.93 - 2.65 for GTV and max min 1.75 - 4.71 for CTV depending on imaging modal- ity used for treatment planning.19 Interobserver variability in CTV and planning target volume (PTV) delineation for gastric cancer was assessed as a part of the CRITICS trial. Despite delineation atlas provided for participants Vmax/Vmin ratio was 3.4 for CTV and 2.6 for PTV and the authors speculated the reason was unfamiliarity with target volumes in the upper abdomen.6 For oesophageal cancer, median Jaccard conformity index for GTV was 0.69 in a study by Gwynne et al.34, with the highest observer agreement in the middle section of the GTV, which is a marked improvement compared to results reported by Tai et al.18 at the start of 3D planning era, when Vmax/Vmin ratio was up to 6. Cervix cancer Considerable interobserver variability was described by Weiss et al.10 in CTV for cervix carcinoma, with the ratio of common to encompassing volume from 0.11 to 0.57 and Vmax/Vmin ratio 1.3 - 4.9. The max min main reason for large variability was wide variation in caudal and cranial CTV borders, resulting from varying inclusion of specific nodal regions (para-aortic, iliac and inguinal) by the observers. In a study of cervix cancer IGABT, CI was 0.6 - 0.8 for high risk CTV (HR CTV) and 0.6 - 0.7 for GTV and intermediate risk CTV (IR CTV), demonstrating a relatively good interobserver agreement considering that CI is sensitive to volume size and volumes in brachytherapy tend to be much smaller than in EBRT. Mean inter-delineation distance was 4.2 mm, 3.8 mm and 5.2 mm for GTV, HR CTV and IR CTV, respectively.2535 Head and neck tumours Interobserver variability in CTV delineation in oro-pharyngeal cancer (tonsillar tumour) is one of the largest described in the literature. With the primary GTV already provided, Vmax/Vmin ratio for CTV reported by Hong et al.30 was 18.3. Recommended PTV expansion from the contoured CTV also varied considerably in different institutions (mean 4.11 mm, range 0 - 15 mm). Smaller but still significant variability was reported by Thiagarajan et al.9 for oropharyngeal primary tumour GTV with CI 0.54 -0.62, depending on imaging modality. Agreement on nodal GTV was higher with CI > 0.75 for all imaging modalities. For nasopharyngeal carcinoma local SD was 3.3 - 4.4 mm for CTV (visible tumour + potential microscopic extension) and 4.9 - 5.9 mm for elective CTV (CTV + 1 cm margin and the entire nasopharynx), depending on imaging modality.24 Radiol Oncol 2016; 50(3): 254-262. 256 Segedin B et al. / Uncertainties in target volume delineation in radiotherapy 256 FIGURE 1. MR images showing interobserver variability between an unexperienced RO and the reference contour in IGABT of 4 cervix cancer patients (from a workshop for RO residents at the Institute of Oncology Ljubljana). Lung tumours In lung cancer the range of reported interobserver variability is quite large with Vmax/Vmin ratio from 1.8 to 2.3 for primary GTV alone and from 5.2 to > 7 for primary and nodal GTV. Reported conformity indices range from 0.04 to 0.70 for the same target volumes, depending on imaging modality, with some authors describing cases where there was no common volume for all observers.31415'27'36 Like in cervix cancer the reason for large variability is inclusion of different nodal regions in the target volume. In a study by Van De Steene et al.3 the observers included only 63% of involved nodal regions in the target volume (generating 37% false negative nodes), on the other hand 22% of included nodal regions were considered false positive after a review. The authors suggested lack of knowledge being one of the main reasons for interobserver variability, beside problems of methodology (interpretation of GTV definition, drawing precision etc.) and difficulty in discriminating the tumour from surrounding pathological (i.e. atelectasis, peritu-moral reaction) and normal structures (i.e. medias-tinal vessels). Other tumour sites Interobserver variability for target delineation in brain tumours is similar to the one described for prostate with V /V ratio from 1.3 to 2.8 and CI r max min from 0.14 to 0.47 depending on imaging modal-ity.16,37,38 Despite being one of the smallest reported variations, it is still larger than the patient set-up error and/or organ motion. In prostate interobserver variability for CTV delineation seems to be smaller than in other tumour sites with V /V ratio from 1.2 to 1.6, which is max min probably due to a better circumscribed CTV.2126 The largest variation is described at the apex and the base of the prostate.39 40 Valicenti et al.21 found that interobserver variability is 4 times larger for seminal vesicles delineation compared to prostate delineation. For breast cancer the largest interobserver variability is reported for lumpectomy cavity with CI from 0.19 to 0.56, followed by CTV with CI from 0.38 to 0.87 and PTV with CI from 0.45 to 0.92.11'13,41,42 In partial breast brachytherapy CI for lumpectomy cavity ranges from 0.48 to 0.52 and for PTV from 0.55 to 0.59, with V /V ratio for all volumes max min 2.2. - 2.8.43 Lower CI for lumpectomy cavity compared to other target volumes could be attributed to the fact that lumpectomy cavity is the smallest target volume in postoperative breast carcinoma and CI is sensitive to volume size. How described interobserver variability affected delivered dose to the target and/or OAR is only reported in a few papers. Steenbakkers et al.44 observed a reduction of mean dose to the rectal wall by 5.1 Gy and to the penile bulb by 11.6 Gy when reducing interobserver variability by using MRI for delineation in EBRT for prostate cancer. Allowing the same dose to OAR as in CT based delineation the dose prescribed to the target volume (prostate) could be escalated from 78 to 85 Gy. With improved target volume delineation due to the use of CT/MRI fusion in nasopharyngeal carcinoma, the mean PTV D95 improved from 60 to 69.3 Gy, while D5 to the brainstem and spinal cord was reduced by 19%, dose to the parotid glands and cochlea was reduced below their dose constraint.45 In lung cancer the probability of delivering at least 95% of prescribed dose to at least 95% of the target volume was reduced from 96% to 88% when using a plan designed to cover another observer's GTV. Mean interobserver range of irradiated normal tissue volume was 12%, with a maximum variability Radiol Oncol 2016; 50(3): 254-262. Segedin B et al. / Uncertainties in target volume delineation in radiotherapy 257 of 66%.3 In cervix IGABT, a mean relative SD of 8-10% in D90 for GTV and HR CTV was observed in a single fraction analysis. For bladder and rectum mean relative SD for D2cc was 5 - 8%, whereas for sigmoid it was 11%. When taking into account the whole treatment course, interobserver variability generated an uncertainty of +/-5 Gy (a|3 = 10) for HRCTV and +/-2-3 Gy (a|3 = 3) for OAR.46 Strategies to improve target volume delineation Several strategies to reduce uncertainties in target volume delineation have been proposed by different authors 7A25 and there have been a few attempts to implement those strategies to improve quality assurance in clinical trials in radiation oncolo-gy.2647-49 Three major areas that could contribute to improving the accuracy of target delineation have been identified: optimisation of imaging, implementation of standardized protocols and delineation guidelines and specialized training. Optimisation of imaging High quality imaging with reproducible protocols is a pre-requisite for accurate target volume delineation. In the last decades, radiotherapy planning was mostly CT based, recently, new imaging techniques i.e. MRI, PET-CT, functional MRI are increasingly being used to improve visibility of the target. Potential advantages of functional imaging modalities are reduction of interobserver variability, indentification of tumour extensions missed by CT and/or MRI and possibly identification of GTV subvolumes requiring higher radiation dose. Even in the absence of modern imaging modalities for treatment planning, simple measures such as the use of intravenous and/or intracavitary contrast, fiducial markers and reproducible imaging protocols can markedly increase the quality of imaging. When contouring, the use of zoom levels, simultaneous viewing in multiple planes (use of sagittal and coronal plane) and use of adequate level and window settings on the planning CT reduce interobserver variability.50 In a series of 42 patients with rectal cancer, the use of PET-CT significantly reduced the size of GTV compared to CT alone, better interobserver agreement was observed (mean CI 0.79 vs. 0.82 vs. 0.93 for CT, PET-CT and PET-CT with auto-contours, respectively). Additionally, in almost one third of patients GTV based on PET-CT extended FIGURE 2. Interobserver variability in delineation of the prostate. MR and CT images in different planes of the same patient are shown. Ability to discriminate prostate apex, base and lateral borders is superior on MRI. cor = coronal; sag = sagittal; tra = transverse40 outside CT based GTV. The addition of MRI to CT did not result in significant improvement of CI.31 Patel et al.33 also compared CT and PET-CT for delineation of primary and nodal GTV (GTVp and GTVn) in rectal cancer. Similarity index for GTVp was modestly better, but statistically significant on PET CT e.g. 0.81 vs. 0.77, and notably better for GTVn e.g. 0.70 vs. 0.22. Several studies show a good correlation between PET-CT and pathology based tumour length in oesophageal cancer5152, but to our knowledge, there are no studies comparing interobserver variability on different imaging modalities. The benefit of PEC-CT based delineation was also demonstrated for GTV in lung cancer patients, where registration of PET-CT images with the planning CT improved median interobserver percentage of concordance form 61% to 70% compared to CT alone.36 In RTOG 0515 trial the lung cancer GTV volumes contoured on PET CT were significantly smaller when compared to CT derived volumes and nodal GTV was altered in over 50% of patients on PET-CT.53 When compared to pathological findings both CT and PET-CT based contours overestimated tumour size for 46.6% and 32.5%, respectively. Both GTV volumes and maximal tumour diameters were larger on CT.54 There are several publications evaluating the effect of addition of PET-CT and/or MRI on interobserver variability in delineation of the GTV or CTV for head and neck tumours.92455-57 In a study by Daisne et al.55 GTV was contoured on CT, MRI and Radiol Oncol 2016; 50(3): 254-262. 258 Segedin B et al. / Uncertainties in target volume delineation in radiotherapy 258 PET-CT in 29 patients with head and neck tumours. Mean GTV volume was not significantly different on CT and MRI, mean GTVs on PET-CT were significantly smaller. For nine patients where surgical specimen after total laryngectomy was available, no imaging modality adequately depicted the extension of the tumour. The average GTVs for anatomic imaging were over 100% larger and for functional imaging almost 50% larger than the surgical specimen. For laryngeal and hypopharyngeal tumours mean GTV volume was 21.4 cm3 for both CT and MRI, 16.4 cm3 for PET-CT and 12.6 cm3 in surgical specimen. PET-CT was the most accurate modality in patients where comparison with the surgical specimen was available. In a similar comparison Thiagajaran et al.9 compared contouring GTVs in oropharyngeal carcinoma on CT + PET vs. CT + MR vs. CT + PET + MR to a reference contour and found no significant difference in the size of the GTV when contouring using any combination of two imaging modalities. Interobserver agreement between GTVctpet and GTVctmr was low, with CI = 0.62. When compared to the reference contour CICTPETMR was low (0.62), but still significantly higher than CI for either CT + PET or CT + MR (0.54 and 0.55, respectively), which implicates that none of the imaging modalities should be used alone. For nodal GTV CI was > 0.75 for all tested imaging modalities compared to the reference contour, the added benefit to contrast enhanced CT alone was small. Anderson et al.58 also compared CT, PET-CT and MRI for definition of GTV in head and neck tumours. Interobserver variability was present for all imaging modalities, with CT being least consistent. PET-CT derived target volumes were the smallest in size, interobserver agreement was the highest with CI = 0.46, compared to CI = 0.36 and 0.35 for MRI and CT, respectively. In nasopharyngeal carcinoma the use of CT and co-registered MRI decreased local SD from 4.4, to 3.3 mm and from 5.9 to 4.9 mm for CTV and elective CTV, respectively, and resulted in a higher agreement between observers.24 Two published studies observed no significant difference between observers across imaging modalities when comparing CT to PET-CT and CT to MRI for GTV delineation in head and neck tumours.5657 Giezen et al.4142 compared CT and MRI for delineation of CTV and lumpectomy cavity (LC) after breast-conserving surgery and found that both imaging modalities provided similar visibility of LC, CI was lower for MRI than for CT, but the difference was not significant. These results have to be interpreted with caution, as the participating radiologists had no experience in LC contouring and the radiation oncologists were not familiar with breast MRI, which gives the results limited value. In postoperative brain gliomas radiotherapy the use of registered CT and postoperative MR images reduced interobserver variability compared to contouring on CT with the aid of preoperative MRI (CI 0.47 vs. 0.14, respectively). However, in delineation of inoperable supratentorial brain tumours the addition of MRI did not reduce interobserver variability with Vmax remaining up to 2.7 times larger than Vmin.38 For prostate cancer all studies demonstrate up to almost 75% larger volumes on CT compared to MRI, but while some found better interobserver agreement on MRI others found less interobserver variability on CT, demonstrating that current delineation guidelines might not be applicable to MRI planning.405960 Implementation of delineation protocols and guidelines Delineation guidelines have been published on a national or international level for several tumour sites both in EBRT and BT.61-67 Different reports show that the use of site specific anatomical atlases, consensus delineation guidelines and standardized contouring protocols diminish variability between observers in various tumour sites.3268-71 In rectal carcinoma, the implementation of site specific consensus atlas significantly reduced interobserver variability in a pilot study68, which was later confirmed in a larger study, in which Nijkamp et al.32 demonstrated that the use of a digital delineation atlas twice or more during target volume contouring significantly improves CI. The addition of delineation guidelines significantly reduced interobserver variation in caudal CTV border (from 1.8 to 1.2 cm) and the size of average CTV volume by 25% (620 vs. 460 cc). In lung cancer, re-contouring of the GTV with the use of a protocol, aimed at minimizing variation, reduced the degree of interobserver variation from 20% to 13%. In the second contouring session the differences between observers were not statistically sig-nificant.72 Comparison of contouring seroma cavity in partial breast radiotherapy with and without guidelines showed that radiation oncologists (ROs) contouring without guidelines contoured significantly larger CTVs and PTVs in more than 50% of patients.69 When all participating ROs were provided with guidelines, the differences in sizes of the target volumes were no longer significant. In breast brachytherapy conformity indices increased significantly with the use of guidelines Radiol Oncol 2016; 50(3): 254-262. Segedin B et al. / Uncertainties in target volume delineation in radiotherapy 259 TABLE 1. Interobserver variation for various tumour sites Tumoursite Target volume No of pts No of obs Imaging modality Results Author (publication date) Rectum GTV, CTV 2 10 CT, PETCT CI(GTV) = 0.26-0.33 CI(CTV) = 0.29-0.35 Krengli et al 2010 GTV 52 5 CT, PETCT, MRI CI = 0.79-0.93 Bujisen et al 2012 CTV 8 10 CT CI = 0.63-0.66 Nijkamp et al 2012 GTV 6 4 CT, PETCT SI(GTV-P) = 0.77-0.81 SI(GTV-N) = 0.22-0.70 Patel et al 2007 Stomach CTV, PTV 1 10 CT Vmax/Vmin(CTV) = 3.4 Vmax/Vmin(PTV) = 2.6 Jansen et al 2010 Oesophagus GTV 1 50 CT JCI = 0.69 Gwynne et al 2012 GTV, CTV, PTV 1 48 CT Vmax/Vmin(PTV) = 5.25-6.03 Tai et al 1998 Cervix EBRT CTV 3 7 CT CI = 0.11-0.57 Vmax/Vmin = 3.6-4.9 Weiss et al 2003 IGABT GTV, HRCTV, IRCTV 6 10 MRI CI(GTV) = 0.6-0.8 CI(HR&IRCTV) = 0.6-0.7 Petric et al 2012, 2013 Head and GTV,CTV,PTV 1 20 CT Vmax/Vmin(CTV) = 18.3 Hong et al 2012 neck GTV 41 3 CT, PETCT, MRI CI(GTV-P) = 0.54-0.62 CI(GTV-N)>0.75 Thiagajaran et al 2012 CTV, CTVE 10 10 CT, MRI localSD(CTV) = 3.3-4.4mm localSD(CTVe) = 4.9-5.9mm Rasch et al 2012 Lung GTV 12 8 CT, CBCT CI = 0.27-0.39 CIgen = 0.58-0.70 Altorjai et al 2012 GTV 8 5 CT Vmax/Vmin>7 Van De Steene et al 2002 GTV 10 17 CT Vmax/Vmin = 5.2 CI = 0.04-0.48 Giraud et al 2002 GTV 22 11 CT, PETCT meanCI = 0.17(CT),0.29(PETCT) localSD = 1cm(CT),0.4cm(PETCT) Steenbakers et al 2006 GTV 19 2 CT, PETCT medianCI(CT) = 0.61, medianCI(PETCT) = 0.70 Fox et al 2005 Brain CTV 7 5 CT + MRI CI = 0.14-0.47 Cattaneo et al 2005 GTV 5 9 CT, MRI Vmax/Vmin(CT) = 1.7-2.8 Vmax/Vmin(MR) = 1.5-2.7 Weltens et al 2001 Prostate Prostate, seminal vesicles (SV) 10 7 CT Vmax/Vmin(P) = 1.18-1.63 Vmax/Vmin(SV) = 2.02-6.43 Valicenti et al 1999 Prostate 3 2 CT Vmax/Vmin = 1.39-1.65 Seddon et al 2000 Prostate 5 5 CT, MRI MeanCI(MR)CI = 0.83 MeanCI(CT) = 0.69 Segedin et al 2011 Breast Lumpectomy cavity (LC), CTV Lumpectomy cavitiy Lumpectomy cavity, CTV, PTV Lumpectomy cavity, PTV Lumpectomy cavity, CTV 15 30 3 5 13 CT, MRI CT CT CT CT CI(LC) = 0.32(MR),0.52(CT) CI(CTV) = 0.77(CT),0.79(MR) MeanCI = 0.36 CI(LC) = 0.19-0.77 CI(CTV) = 0.38-0.80 CI(PTV) = 0.45-0.81 CI(LC) = 0.48-0.52 CI(PTV) = 0.55-0.59 Vmax/Vmin = 2.2-2.8 MeanCI(LC) = 0.56 MeanCI(CTV) = 0.87 Giezen et al 2011,2012 Boersma et al 2012 VanMourik et al 2010 Major et al 2015 Struikmans et al 2005 CI = conformity/concordance index; CTV = clinical target volume; GTV = gross target volume; local SD = local standard deviation; max = maximum; min = minimum; obs = observers; pts = patients; PTV = planning target volume; SI = similarity index; V = volume; 8 5 both for lumpectomy cavity contours and PTV. The increase was 14% and 11% for the cavity and 28% and 17% for PTV on preimplant and postimplant CT images, respectively.43 Even for site-specialized ROs, a reduction in interobserver variability was noticed in CTV delineation for postprotatectomy radiotherapy when adhering to the RADICALS trial delineation protocol.71 Mean V /V for all cases was reduced from 3.7 at first max min delineation to 2.0 at the second delineation. Radiol Oncol 2016; 50(3): 254-262. 260 Segedin B et al. / Uncertainties in target volume delineation in radiotherapy 260 Training A survey of radiotherapy planning and delivery undertaken in the UK in 2007 showed a lack of formal education in target volume and OAR delineation in different staff groups.73 Only 4% of NHS radiotherapy departments offered structured training on image interpretation, while 6% offered informal sessions with radiologists. 90% of participating ROs stated they wanted formal training in interpretation of cross sectional imaging and almost 85% were interested in online training modules. More than half of junior ROs considered their training in cross sectional imaging to be inadequate Some publications evaluated the effect of clinical experience on interobserver variability, the results, however, were ambiguous.15'3774 While Hurkmans et al.74 reported that more experienced ROs delineate smaller volumes than unexperienced in breast carcinomas, Giraud et al.15 found experienced ROs to delineate larger volumes than their younger colleagues in lung carcinoma. In brain tumours, Leunens et al. found no significant difference between experienced and unexperienced ROs.37 Only a few publications have addressed the subject of training, some in the course of pre-accrual quality assurance delineation exercises (dummy run).263447' 49,75,76 In dummy run for a randomised multicentre PET-plan clinical trial in lung cancer, they found considerable differences despite providing detailed contouring guidelines. After a teaching session at a study group meeting, they observed an improvement in overall interobserver agreement, demonstrated by reduction of target volumes and an increase in kappa (k) indices for GTV and two CTVs (0.63 vs. 0.71, 0.60 vs. 0.65 and 0.59 vs. 0.63, respectively).48 Similarly, Khoo et al. reported reduced encompassing to intersecting volume ratio (VR) at re-contouring the prostate after education sessions focusing on MRI prostate anatomy with CT correlation. Mean VR was reduced by 15% for CT (from 2.74 to 2.33) and 40% for MRI (from 2.38 to 1.41).49 Dewas et al.75, however, found no significant difference for delineation of the target volumes in lung cancer before and after training. The residents k- indices were lower compared to senior ROs both before and after the training, V20 for lung was higher in the residents group. The authors speculated there was no improvement because initial delineations by the residents were good. However, they offered no hands-on training for the residents and most reports showing improvement included hands-on training in their educational sessions. During training, special at- tention needs to be payed to predilection areas for larger interobserver variability, identified in available literature.25'26'30'3940 Conclusions The main goal of improving accuracy in radiotherapy treatment planning and delivery is better local control with less morbidity, resulting in better quality of life. Our review shows that interobserver variability in target volume contouring represents the largest uncertainty in the process for most tumour sites, potentially resulting in geographic miss in dose delivery, which could hamper local control for individual patients. Studies on use of multi-modality imaging and image co-registration show promising results, however, for most tumour sites the optimal combination of imaging modalities still needs to be determined. Strict introduction and use of imaging and delineation protocols and guidelines reduces interobserver variability, therefore it is advisable in every day practice and mandatory in the frame of clinical studies. Especially in multi-centric studies, efforts to unify target volume delineation in different institutions in a dummy run should be maximized as interobserver variability influences reliability of dose reporting, comparison of treatment outcomes and interpretation of study results, hence diminishing the value of a study. To assure adherence to study protocols and delineation guidelines, a central reviewing board for contour correction is useful. Continuing medical education of ROs cannot be overemphasized, intensive formal training on interpretation of sectional imaging should be included in the program for radiation oncology residents. 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Consensus guidelines and contouring atlas for pelvic node delineation in prostate and pelvic node intensity modulated radiation therapy. Int J Radiat Onco! 2015; 92: 874-83. 64. Grégoire V, Ang K, Budach W, Grau C, Hamoir M, Langendijk JA, et al. Delineation of the neck node levels for head and neck tumors: A 2013 update. DAHANCA, EORTC, HKNPCSG, NCIC CTG, NCRI, RTOG, TROG consensus guidelines. Radiother Onco! 2014; 110: 172-81. 65. Nielsen MH, Berg M, Pedersen AN, Andersen K, Glavicic V, Jakobsen EH, et al. Delineation of target volumes and organs at risk in adjuvant radiotherapy of early breast cancer: National guidelines and contouring atlas by the Danish Breast Cancer Cooperative Group. Acta Onco! (Madr) 2013; 52: 703-10. 66. Lim K, Small W, Portelance L, Creutzberg C, Jürgenliemk-Schulz IM, Mundt A, et al. Consensus guidelines for delineation of clinical target volume for intensity-modulated pelvic radiotherapy for the definitive treatment of cervix cancer. Int J Radiat Onco! 2011; 79: 348-55. 67. Wu AJ, Bosch WR, Chang DT, Hong TS, Jabbour SK, Kleinberg LR et al. Expert consensus contouring guidelines for intensity modulated radiation therapy in esophageal and gastroesophageal junction cancer. Int J Radiat Onco! 2015; 92: 911-20. 68. Fuller CD, Nijkamp J, Duppen J, Rasch CR, Thomas CR Jr, Wang SJ, et al. Prospective randomized double-blind pilot study of site-specific consensus atlas implementation for rectal cancer target volume delineation in the cooperative group setting. Int J Radiat Onco! Bio! Phys 2011; 79: 481-9. 69. Wong EK, Truong PT, Kader HA, Nichol AM, Salter L, Petersen R, et al. Consistency in seroma contouring for partial breast radiotherapy: impact of guidelines. Int J Radiat Onco! Bio! Phys 2006; 66: 372-6. 70. Senan S, van Sornsen de Koste J, Samson M, Tankink H, Jansen P, Nowak PJ, et al. Evaluation of a target contouring protocol for 3D conformal radiotherapy in non-small cell lung cancer. Radiother Onco! 1999; 53: 247-55. 71. Mitchell DM, Perry L, Smith S, Elliott T, Wylie JP, Cowan RA, et al. Assessing the effect of a contouring protocol on postprostatectomy radiotherapy clinical target volumes and interphysician variation. Int J Radiat Onco! 2009; 75: 990-3. 72. Bowden P, Fisher R, Mac Manus M, Wirth A, Duchesne G, Millward M, et al. Measurement of lung tumor volumes using three-dimensional computer planning software. Int J Radiat Onco! Bio! Phys 2002; 53: 566-73. 73. Jefferies S, Taylor A, Reznek R. Results of a national survey of radiotherapy planning and delivery in the UK in 2007. C!in Onco! 2009; 21: 204-17. 74. Hurkmans CW, Borger JH, Pieters BR, Russell NS, Jansen EPM, Mijnheer BJ. Variability in target volume delineation on CT scans of the breast. Int J Radiat Onco! Bio!Phys 2001; 50: 1366-72. 75. Dewas S, Bibault J-E, Blanchard P, Vautravers-Dewas C, Pointreau Y, Denis F, et al. Delineation in thoracic oncology: a prospective study of the effect of training on contour variability and dosimetric consequences. Radiat Onco! 2011; 6: 118. 76. Szumacher E, Harnett N, Warner S, Kelly V, Danjoux C, Barker R, et al. Effectiveness of educational intervention on the congruence of prostate and rectal contouring as compared with a gold standard in three-dimensional radiotherapy for prostate. Int J Radiat Onco! Bio! Phys 2010; 76: 379-85. Radiol Oncol 2016; 50(3): 254-262. 263 research article Non-contrast computed tomography in the diagnosis of cerebral venous sinus thrombosis Jernej Avsenik1, Janja Pretnar Oblak2, Katarina Surlan Popovic1 1 Clinical Institute of Radiology, University Medical Centre, Ljubljana, Slovenia 2 Department of Neurology, University Medical Centre, Ljubljana, Slovenia Radiol Oncol 2016; 50(3): 263-268. Received 9 February 2015 Accepted 6 March 2016 Correspondence to: Jernej Avsenik, M.D., Clinical Institute of Radiology, University Medical Centre, Zaloska cesta 7, SI-1000 Ljubljana, Slovenia. Phone: +386 1 522 8530; E-mail: jernej.avsenik@gmail.com Disclosure: No potential conflicts of interest were disclosed. Background. The aim of the study was to investigate the sensitivity and specificity of non-contrast computed tomography (NCCT) in the diagnosis of cerebral venous sinus thrombosis (CVST). Methods. Screening our neurological department database, we identified 53 patients who were admitted to neurological emergency department with clinical signs of CVST. Two independent observers assessed the NCCT scans for the presence of CVST. CT venography and/or MR venography were used as a reference standard. Interobserver agreement between the two readers was assessed using Kappa statistic. Attenuation inside the cerebral venous sinuses was measured and compared between the patient and the control group. Results. CVST was confirmed in 13 patients. Sensitivity and specificity of NCCT for overall presence of CVST were 100% and 83%, respectively, with Kappa value of 0.72 (a good agreement between observers). The attenuation values between CVST patients and control group were significantly different (73.4 ± 14.12 HU vs. 58.1 ± 7.58 HU; p = 0.000). The ROC analysis showed an area under the curve (AUC) of 0.916 (95% CI, 0.827 - 1.00) and an optimal cutoff value of 64 HU, leading to a sensitivity of 85% and specificity of 87%. Conclusions. NCCT as a first-line investigation has a high value for diagnosis of CVST in the emergency setting. The additional measurement of the sinus attenuation may improve the diagnostic value of the examination. Key words: cerebral venous sinus thrombosis; computed tomography; stroke Introduction Due to the diversity of underlying factors and the absence of a uniform treatment approach, diagnosis and management of patients with cerebral venous sinus thrombosis (CVST) remain a challenging task.1 CVST represents 0.5% to 1% of all strokes and affects approximately 5 patients per million every year, but has a higher frequency among younger patients.1-3 Typical acquired risk factors include recent surgery, trauma, pregnancy, postpartum state, antiphospholipid syndrome, cancer and use of oral contraceptives. Cases of inherited thrombophilia include Antithrombin III, Protein C and Protein S deficiency, factor V Leiden positivity, prothrombin gene mutation and hyper-homocisteinemia.1-4 Infection of parameningeal spaces (ears, paranasal sinuses, oral cavity, face and neck) is common cause of CVST in pediatric population2'56, but rare in adults.6 The symptoms of CVST are not specific. The most common complaint is headache which occurs in up to 90% of patients.7 Additionally, abnormal vision, any of the symptoms of stroke and seizures have been described.7 In the past D-dimer levels appeared to be of value as an initial screening test. A study in 2004 evaluated the sensitivity of D-dimer to be 97.1% and specificity 99.1%.8 However, later studies showed that up to 10% of patients with CVST have a normal D-dimer.9 As it is fast, affordable and widely available, non-contrast computed tomography (NCCT) is the most frequently performed imaging study for evaluation of patients with new headache, focal neuro- Radiol Oncol 2016; 50(3): 254-262. doi:10.1515/raon-2016-0023 264 Avsenik J et al. / Non-contrast CT in cerebral venous sinus thrombosis Subjects Screening our neurological department database, we retrospectively identified 53 patients (37 women, 16 men; mean age, 42 years; age range, 17—82 years) who were admitted to neurological emergency department from July 2008 to May 2013 with clinical signs of CVST. The following inclusion criteria were defined: NCCT had to be performed on admission and at least one of the reference imaging modalities, i.e. CT venography and/or MR venography had to be performed in 24 hours from admission. CT image acquisition All CT images were acquired on Somatom Sensation 40 Open system (Siemens, Erlangen, Germany). The following scanning parameters were used: 120 kV, 220 mAs, section thickness of 3 mm below the tentorium and 120 kV, 260 mAs and section thickness of 4.8 mm above the tentorium. FIGURE 1. 19-year-old female with thrombosis (arrow) of the left transverse sinus (LTS) (A) and superior sagittal sinus (C), confirmed by CT venography in sagittal (B) and axial (D) reconstruction. Average attenuation inside the LTS was 83.6 HU. logical abnormalities, seizure, or change in mental status. A typical imaging finding in patients with CVST is direct visualization of a hyperattenuat-ing thrombus in the occluded sinus (dense sinus sign).23 Occasionally, NCCT may only show indirect signs of thrombosis, including diffuse brain edema and parenchymal hemorrhage.10 Sensitivity of NCCT in the diagnosis of CVST was previously considered rather poor.210-13 However, using modern multidetector row CT scanners, recent studies report higher sensitivity and specificity values.14 In addition, Buyck et al. suggest measurement of the venous sinus attenuation to increase the diagnostic yield of the examination.15 Therefore, the goal of the present study was to evaluate the diagnostic accuracy of NCCT in the diagnosis of CVST in the emergency setting. Patients and methods The study was performed in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board Committee. Image interpretation NCCTs were assessed for the presence of direct and indirect signs of CVST by two experienced neuro-radiologists, using a standard picture archiving and communication system (PACS) workstation. Observers were blinded to the clinical data and patient identification. Readings were randomized, and standardized evaluation forms were used to ensure the systematic evaluation of the following structures: superior sagittal sinus (SSS), straight sinus (SS), inferior sagittal sinus (ISS), right and left transverse sinuses (RTS, LTS), and right and left sigmoid sinuses, (RSS, LSS). Additionally, right and left internal cerebral vein (RICV, LICV), vein of Galen (VG), right and left basal vein of Rosenthal (BVR), right and left thalamostriate vein (TSV) and cortical veins were assessed. Attenuation inside the thrombosed venous sinus was measured. If no venous structure was classified as thrombosed, mean attenuation of up to three venous sinuses that could be reliably differentiated from surrounding brain parenchyma was documented. Presence of parenchymal hemorrhage or edema was also noted. Finally, observers had to decide regarding the overall presence or absence of CVST and rate their diagnostic confidence on a scale from 1 (absolutely certain) to 5 (uncertain). After having evaluated all NCCTs, readers reviewed all available imaging data, including follow-ups of any respective patient to obtain a ref- Radiol Oncol 2016; 50(3): 263-268. 265 Avsenik J et al. / Non-contrast CT in cerebral venous sinus thrombosis erence standard. In this manner, they determined the overall presence of CVST, the involvement of individual venous structures and the presence of parenchymal hemorrhage or edema. Statistical analysis Statistical analysis was performed using IBM SPSS Statistics for Macintosh, Version 20.0. (IBM Corp., Armonk, NY, USA) software package. Based on collected data, we calculated the specificity and sensitivity of NCCT for diagnosis of CVST. Agreement between the two observers was assessed using Kappa statistic.16 Patients in whom the diagnosis of CVST was excluded were used as a control group for the second part of the study. Average venous sinus attenuation value of individual subject from the CVST patient group and the control group was used for statistical evaluation. Comparison between the two groups was made by unpaired t-test. Receiver operating characteristic curves (ROC) were used to define optimal cutoff value for which sensitivity and specificity was calculated. A p value < 0.05 was considered significant. Results CVST was confirmed in 13 patients (9 women, 4 men; mean age, 36.5 years; age range, 19 - 82 years). Patients presented with headache (12 patients, 93%), papiloedema (7 of 9 evaluated patients, 78%), objective neurological deficit (8 patients, 62%), vomiting (8 patients, 62%), somnolence or stupor (6 patients, 46%), confusion (5 patients, 38%), disturbance of vision (3 patients, 23%) and epileptic seizures (2 patients, 15%). Risk factors of patients with CVST included oral contraception (6 of 9 female patients), pregnancy, puerperium or recent abortion (4 of 9 female patients). Laboratory values were retrospectively available for 12 of 13 patients. Thrombophilia was found in 6 of 12 evaluated patients (50%). Average D-dimer value was 2644 mmol/L, but it was normal in 2 of 12 patients (17%). CRP levels were elevated in 9 of 12 patients (75%). Transverse sinus was the most commonly thrombosed structure, followed by sigmoid sinus (Table 1). Typical NCCT findings of CVST are depicted in Figure 1. All 13 patients with CVST were accurately diagnosed by both observers. Therefore, the calculated sensitivity was 100% in both readings, with specificity of 80 and 87.5%, respectively. Kappa value regarding the presence TABLE 1. Location of thrombus and parenchymal changes in 13 patients with cerebral venous sinus thrombosis (CVST) Patient No. Location of thrombus Parenchymal changes 1 RTS, RSS None 2 LTS Hemorrhage, edema 3 SSS, RTS, RSS None 4 RTS None 5 SSS, RTS, RSS None 6 SSS, LTS, LSS, right ICV, left ICV, VG None 7 RTS, RSS None 8 RTS, RSS None 9 LTS, LSS None 10 SSS, RTS, RSS, cortical veins None 11 SSS, LTS, LSS Hemorrhage, edema 12 RTS Hemorrhage, edema 13 SSS, cortical veins Hemorrhage, edema ICV = internal cerebral vein; LSS = left sigmoid sinus; LTS = left transverse sinus; RSS = right sigmoid sinus; RTS = right transverse sinus; SSS = superior sagittal sinus; VG = vein of Galen or absence of thrombosis was 0.72, which is considered to represent a good agreement between observers. Sensitivity and specificity of NCCT for overall presence of thrombosis were 100% and 83%. Average diagnostic confidence level regarding the presence or absence of CVST was 2.1 ± 1.2 (very certain). A case with false positive finding is depicted in Figure 2. Attenuation values were available for all 13 CVST patients and 23 controls and significant difference was found between the two groups (Figure 3). The ROC analysis of the attenuation showed an area under the curve (AUC) of 0.916 (95% CI 0.827, 1.00) and an optimal cutoff value of 64 HU, leading to a sensitivity of 85% and specificity of 87%. Discussion CVST is a distinct cerebrovascular disorder that, unlike arterial stroke, most often affects young adults and children and is associated with significant morbidity and mortality, especially when the diagnosis is not made in time.17 In order to initiate appropriate therapy as soon as possible, early diagnosis is essential. The clinical presentation of CVST is highly variable. In our series, headache was the most frequently reported symptom. Similar to recent study by Linn et al.14, there was no single symptom or sign, present in all patients with CVST. Radiol Oncol 2016; 50(3): 263-268. 266 Avsenik J et al. / Non-contrast CT in cerebral venous sinus thrombosis FIGURE 2. Left transverse sinus (LTS) of a 26-year-old male appeared hyperattenuated on non-contrast computed tomography (arrow) (A) and was interpreted as thrombosed by both readers. However, CT venography in axial (B) and sagittal (C) reconstruction showed patency of the LTS. Average attenuation inside the LTS was 60 HU. Normal D-dimer levels have previously been considered to have a high negative predictive value in patients with suspected CVST.7 Recently, normal D-dimer levels were reported in patients with isolated thrombosis of deep cerebral venous system and were explained by the relatively small thrombus volume.14 However, we found normal D-dimer levels in 2 patients with CVST, suggesting that D-dimer is of limited value in excluding the diagnosis. The American Heart Association (AHA)/ American Stroke Association (ASA) 2011 Scientific Statement on diagnosis and management of cerebral venous thrombosis recommends imaging of the cerebral venous system in patients with suspected cerebral venous thrombosis.1 Recently, MRI in combination with MR venography has largely replaced digital subtraction angiography as a gold standard for imaging of CVST17 and is currently considered the most sensitive examination tech-nique.1819 In situations when MRI is not readily available, CT venography has been shown to be equivalent to MRI in establishing the diagnosis.20 Nevertheless, in most institutions NCCT remains the first-line imaging method in the emergency evaluation of patients with unspecific neurological symptoms14, because of its cost-effectiveness and availability.15 Using a blinded reader approach, we examined the value of NCCT in diagnosis of CVST, based on presence or absence of classic hyperattenuating signs. These are observed on NCCT scans when an acute thrombus has formed in a blood vessel. The increase in attenuation is caused by clot retraction, eliminating water and thereby raising the concentrations of red blood cells and hemoglobin. This mechanism results in increased attenuation of the thrombus to 60 - 90 HU.15 In CVST, hyperattenuat-ing sign (dense sinus sign) can serve as a unique finding indicating an acute stage, at a time when treatment is most likely to be effective and to have a significant effect on clinical outcome.21 In the past, sensitivity and specificity of NCCT was considered rather low11-13, and according to the literature, direct signs of CVST were present in only one third of patients.11 A study from 1987 reported a sensitivity of approximately 25%.22 However, using modern multidetector row CT scanner, recently reported sensitivity of the direct signs for CVST was 64.6%, which was higher than reported in older stud-ies13,22, but still insufficient in excluding the diag-nosis.14 Authors of this work emphasized the value of NCCT in diagnosis of deep venous thrombosis, reporting the 100% sensitivity and 99.4% specificity in this subgroup of patients. In our series however, there was only one patient who, in addition to venous sinus thrombosis, also had deep venous system thrombosis (Table 1). Our study revealed higher sensitivity (100%) of NCCT for overall presence of thrombosis than previously reported (25% - 64%).13 14 22 Specificity, on the other hand, was lower than expected (83.8%). The observers, although blinded to clinical and imaging data, knew the purpose of the study, which may partially explain these results. Also, if the venous structure was recognized as thrombosed, it was considered a positive finding even in cases, where diagnostic confidence was low (4 or 5 on our scale). Interestingly enough, if we interpreted these cases as negative, the sensitivity and specificity would be 88.5% and 95%, which is closer to previously published data. Apparent increase in attenuation of venous sinuses can be misleading and is not always visually Radiol Oncol 2016; 50(3): 263-268. 267 Avsenik J et al. / Non-contrast CT in cerebral venous sinus thrombosis FIGURE 3. The attenuation values between cerebral venous sinus thrombosis (CVST) patients and control group were significantly different (73.4 ± 14.12 HU vs. 58.1 ± 7.58 HU; p = 0.000). HU, Hounsfield units. recognized, therefore recently additional measurement of the sinus attenuation was proposed to increase the sensitivity of the examination in the diagnosis of CVST.15 In our series, mean attenuation of 73.4 HU was found in thrombosed venous sinuses. This degree of increased attenuation was previously found in clotted sinuses of patients with CVST1523, as well as in acute thrombosis elsewhere in the body.23 Significant difference in the average sinus attenuation was found between the CVST patient group and the control group. However, an overlap between the two groups and the presence of outliers in the control group (Figure 3) may limit the reliability of this method in some patients. Our results are similar to the findings of Buyck et al., who suggested a threshold of 62 HU to differentiate the patients with CVST from those without.15 Based on our data, the optimal threshold was 64 HU. The drawback of this method is the possibility of false negative finding in patients with anemia who have low attenuation of blood due to low hemoglobin.15 Similarly, the most common cause of false positive readings is the high attenuation of blood in patients with high hemoglobin or hematocrit level.24 These limitations may partially be avoided by calculating the Hounsfield unit-to-hematocrit ratio. However, the improvement in accuracy with this method has been shown to be minor.15 Additionally, such calculation may often be impractical in emergency setting and was therefore not included in our study. We acknowledge the following limitations of our work. Relatively small size of the studied population was a drawback. However, CVST is a relatively uncommon disease and the size of our group is comparable to previously published studies. Furthermore, the impact of relatively low number of patients was minimized using the blind and multiple observer approach. DSA was not performed and so the consensus reading of CT venog-raphy or MR venography was used as a reference standard. Nonetheless, the diagnostic value of these methods for the diagnosis of CVST has been shown to be very high.2526 In conclusion, prompt therapy of CVST has a profound impact on clinical outcome, therefore early diagnosis is important. Our study has shown high sensitivity of NCCT for diagnosis of CVST and thus confirmed the role of NCCT as a investigation of choice in the emergency setting. The additional measurement of the sinus attenuation may improve the diagnostic value of the examination and help decide on the need for confirmatory study. References 1. Saposnik G, Barinagarrementeria F, Brown RD, Jr., Bushnell CD, Cucchiara B, Cushman M, et al. Diagnosis and management of cerebral venous thrombosis: a statement for healthcare professionals from the American Heart Association/American Stroke Association. Stroke 2011; 42: 1158-92. 2. Bousser MG, Ferro JM. Cerebral venous thrombosis: an update. Lancet Neurol 2007; 6:162-70. 3. Ferro JM, Canhao P, Stam J, Bousser MG, Barinagarrementeria F. Prognosis of cerebral vein and dural sinus thrombosis: results of the International Study on Cerebral Vein and Dural Sinus Thrombosis (ISCVT). Stroke 2004; 35: 664-70. 4. de Freitas GR, Bogousslavsky J. Risk factors of cerebral vein and sinus thrombosis. Front Neurol Neurosci 2008; 23: 23-54. 5. Boncoraglio G, Carriero MR, Chiapparini L, Ciceri E, Ciusani E, Erbetta A et al. Hyperhomocysteinemia and other thrombophilic risk factors in 26 patients with cerebral venous thrombosis. Eur J Neurol 2004; 11: 405-9. 6. Damak M, Crassard I, Wolff V, Bousser MG. Isolated lateral sinus thrombosis: a series of 62 patients. Stroke 2009; 40: 476-81. 7. Stam J. Thrombosis of the cerebral veins and sinuses. N Engl J Med 2005; 352: 1791-8. 8. Kosinski CM, Mull M, Schwarz M, Koch B, Biniek R, Schlafer J, et al. Do normal D-dimer levels reliably exclude cerebral sinus thrombosis? Stroke 2004; 35: 2820-5. 9. Crassard I, Soria C, Tzourio C, Woimant F, Drouet L, Ducros A, et al. A negative D-dimer assay does not rule out cerebral venous thrombosis: a series of seventy-three patients. Stroke 2005; 36: 1716-9. 10. Leach JL, Fortuna RB, Jones BV, Gaskill-Shipley MF. Imaging of cerebral venous thrombosis: current techniques, spectrum of findings, and diagnostic pitfalls. Radiographics 2006; 26 (Suppl 1): S19-41; discussion S2-3. 11. Piazza G. Cerebral venous thrombosis. Circulation 2012 125: 1704-9. 12. Renowden S. Cerebral venous sinus thrombosis. Eur Radiol 2004;14: 21526. 13. Teasdale E. Cerebral venous thrombosis: making the most of imaging. J R Soc Med 2000; 93: 234-7. 14. Linn J, Pfefferkorn T, Ivanicova K, Muller-Schunk S, Hartz S, Wiesmann M, et al. Noncontrast CT in deep cerebral venous thrombosis and sinus thrombosis: comparison of its diagnostic value for both entities. Am J Neuroradiol 2009; 30: 728-35. Radiol Oncol 2016; 50(3): 263-268. 268 Avsenik J et al. / Non-contrast CT in cerebral venous sinus thrombosis 15. Buyck PJ, De Keyzer F, Vanneste D, Wilms G, Thijs V, Demaerel P. CT density measurement and H:H ratio are useful in diagnosing acute cerebral venous sinus thrombosis. Am J Neuroradiol 2013; 34: 1568-72. 16. Viera AJ, Garrett JM. Understanding interobserver agreement: the kappa statistic. Fam Med 2005; 37: 360-3. 17. Gaikwad AB, Mudalgi BA, Patankar KB, Patil JK, Ghongade DV. Diagnostic role of 64-slice multidetector row CT scan and CT venogram in cases of cerebral venous thrombosis. Emerg Radiol 2008; 15: 325-33. 18. Lafitte F, Boukobza M, Guichard JP, Hoeffel C, Reizine D, Ille O, et al. MRI and MRA for diagnosis and follow-up of cerebral venous thrombosis (CVT). Clin Radiol 1997; 52: 672-9. 19. Dormont D, Anxionnat R, Evrard S, Louaille C, Chiras J, Marsault C. MRI in cerebral venous thrombosis. J Neuroradiol 1994; 21: 81-99. 20. Ozsvath RR, Casey SO, Lustrin ES, Alberico RA, Hassankhani A, Patel M. Cerebral venography: comparison of CT and MR projection venography. Am J Roentgenol 1997; 169: 1699-707. 21. Goldstein M, Quen L, Jacks L, Jhaveri K. Acute abdominal venous thrombo-ses--the hyperdense CT sign. J Comput Assist Tomogr 36: 8-13. 22. Virapongse C, Cazenave C, Quisling R, Sarwar M, Hunter S. The empty delta sign: frequency and significance in 76 cases of dural sinus thrombosis. Radiology 1987; 162: 779-85. 23. Black DF, Rad AE, Gray LA, Campeau NG, Kallmes DF. Cerebral venous sinus density on noncontrast CT correlates with hematocrit. Am J Neuroradiol 2011; 32: 1354-7. 24. Healy JF, Nichols C. Polycythemia mimicking venous sinus thrombosis. Am J Neuroradiol 2002; 23: 1402-3. 25. Greiner FG, Takhtani D. Neuroradiology case of the day. Superior sagittal sinus thrombosis and infarcts. Radiographics 1999; 19: 1098-101. 26. Wetzel SG, Kirsch E, Stock KW, Kolbe M, Kaim A, Radue EW. Cerebral veins: comparative study of CT venography with intraarterial digital subtraction angiography. Am J Neuroradiol 1999; 20: 249-55. Radiol Oncol 2016; 50(3): 263-268. 269 research article Recurrence rate in regional lymph nodes in 737 patients with follicular or Hürthle cell neoplasms Andrej Vogrin1, Hana Besic2, Nikola Besic2, Maja Marolt Music1 1 Department of Radiology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Department of Surgical Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia Radiol Oncol 2016; 50(3): 269-273. Received 5 November 2015 Accepted 7 March 2016 Correspondence to: Maja Music Marolt, M.D., Ph.D., Institute of Oncology Ljubljana, Zaloska 2, 1000 Ljubljana, Slovenia. Phone: +386 1 5879 696; Fax: +386 1 5879 400; E-mail: mmusic@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. Preoperative ultrasound (US) evaluation of central and lateral neck compartments is recommended for all patients undergoing a thyroidectomy for malignant or suspicious for malignancy cytologic or molecular findings. Our aim was to find out how frequent was recurrence in regional lymph nodes in patients with follicular or Hurthle cell neoplasm and usefulness of preoperative neck US investigation in patients with neoplasm. Patients and methods. Altogether 737 patients were surgically treated because of follicular or Hurthle cell neoplasms from 1995 to 2014 at our cancer comprehensive center, among them 207 patients (163 females, 44 males; mean age 52 years) had thyroid carcinoma. Results. Carcinoma was diagnosed in follicular and Hurthle cell neoplasm in 143/428 and 64/309 of cases, respectively. A recurrence in regional lymph nodes occurred in 12/207 patients (6%) during a median follow-up of 55 months. Among patients with carcinoma a recurrence in regional lymph nodes was diagnosed in follicular and Hurthle cell neoplasms in 2% and 14%, respectively (p = 0.002). Recurrence in regional lymph nodes was diagnosed in 3/428 of all patients with follicular neoplasm and 9/309 of all patients with Hurthle cell neoplasm. Conclusions. Recurrence in lymph nodes was diagnosed in 0.7% of patients with a preoperative diagnosis of fol-licular neoplasm and 3% of patients with a Hurthle cell neoplasm. A recurrence in regional lymph nodes is rare in patients with carcinoma and preoperative diagnosis of follicular neoplasm. Preoperative neck ultrasound examination in patients with a follicular neoplasm is probably not useful, but in patients with Hurtle cell neoplasm it may be useful. Key words: thyroid neoplasms; ultrasonography; recurrence; diagnosis; pathology Introduction High-resolution ultrasound (US) examination with a 10-13 MHz linear probe detected thyroid nodules in 68% of randomly selected individuals and 18% of nodes were larger than 10 mm.1 Diagnostic fine needle aspiration biopsy (FNAB) and cytology of a thyroid nodule is recommended for nodules > 1cm with a high suspicion US pattern.23 The Bethesda system for reporting thyroid cytopathol-ogy is used to report thyroid nodule FNAB cytology.45 The Bethesda IV category comprises follicular neoplasm or suspicious for a follicular neoplasm and also encompasses the diagnosis of Hurthle cell neoplasm/suspicious for Hurthle cell neoplasm. Confirmation of malignancy in these types of primary thyroid tumors is possible only with histological examination of the tumor.45 Demonstration of transcapsular and/or vascular invasion confirms malignancy.45 Risk of malignancy in Bethesda IV tumors was 26% among 2751 patients.6 At our institute the risk of malignancy was 33% and 25% in follicular and Hurthle cell neoplasms, respectively.78 According to the National Cancer Comprehensive Network guidelines for patients with thyroid cancer a preoperative US examination of central and Radiol Oncol 2016; 50(3): 254-262. doi:10.1515/raon-2016-0023 270 Vogrin A et al. /Preoperative neck ultrasound in follicular or Hurthle cell neoplasms lateral neck compartments is recommended for all patients undergoing thyroidectomy for malignant or suspicious for malignancy cytological findings.5 Our aim was to find out the frequency of recurrence in regional lymph nodes in patients with carcinoma who had preoperative diagnosis of follicular or Hurthle cell neoplasm and to estimate usefulness of preoperative US examination of the neck region in patients with follicular or Hurthle cell neoplasm. Patients and methods Altogether 737 patients were surgically treated because of follicular (N = 428) or Hurthle cell (N = 309) neoplasms as shown by cytology from 1995 to 2014 at our cancer comprehensive center. Altogether 207 patients (163 females, 44 males; mean age 52 years, range 12-84 years) had thyroid carcinoma in a dominant nodule as shown by a definitive his-topathology. According to the TNM classification system using the UICC criteria from 2009 pT1, pT2, pT3 and pT4 tumor was diagnosed in 69 patients, 68, 64 and 6 patients, respectively.9 Metastases in regional lymph nodes and distant metastases were diagnosed in 2 and 10 patients, respectively. A majority of patients were treated by total or near-total thyroidectomy (87%) and radioiodine (RAI) ablation of the thyroid remnant (91%) followed by a suppressive therapy with L-thyroxine. None of our patients had central or lateral neck dissection during thyroidectomy. All the patients with carcinoma were followed-up at our institute at least once a year (median follow-up period 55 months, range 6-180 months). The follow-up consisted of a medical history, physical examination and determination of serum thy-roglobulin (Tg) concentration and Tg antibodies. The criteria for disease-free survival were: Tg levels of less than 1 ng/mL, negative whole-body RAI scans, and exclusion of cervical lymph node metastases detected by US as defined by 2009 American Thyroid Association guidelines.2 Neck US was always performed within first 6 months after surgery because of thyroid cancer. Neck US was performed at least once per year in case of positive Tg antibodies. Imaging (X-ray, US, CT, MRI, bone scintigraphy, PET-CT and/or RAI scintigraphy) was performed whenever Tg concentration was elevated or clinical symptoms of possible recurrence were present in order to determine the site and extent of the suspected recurrence. A retrospective chart review of all patients with neoplasms was carried out and data about preop- erative US neck examination and recurrence in regional lymph nodes was collected for this study. Predictive factors for the presence of carcinoma in follicular (N = 388) and Hurthle cell (N = 279) neoplasms in our patients have already been published.78 Twelve of them had thyroid lesions incidentally detected by 18F-FDG PET-CT.10 A follicular or Hurthle cell neoplasm was diagnosed by FNAB and cytology. FNABs were performed by an endocrinologist, radiologist, and/ or cytopathologist using a 21-23-gauge needle attached to a 10-mL syringe. All cytological slides were examined by cytopathologists and histologi-cal slides by pathologists experienced in thyroid pathomorphology. Routine cytological and final pathology reports from our Institute were used in this study. Preoperative US examination of central and lateral neck compartments were performed in 27 patients with carcinoma who had a preoperative diagnosis of follicular or Hurthle cell neoplasm. Benign cervical LN were sonographically seen as oval shaped, well defined structures with different amount of hilar fat, hilar type of vascularization and transversal diameter in upper regions limited to 9 mm. US criteria for malignant lymph node were: marked hypoechogenicity, rounded shape, absent hilum and irregular, blurred, angular or invasive margins, and presence of microcalcifications. Doppler criteria for malignant lymph node were: peripheral flow, multiple vascular pedicles, chaotic vascular pattern and high impedance values. Probability of malignancy is higher with increasing number of malignant features. The study was reviewed and approved by the Institutional Review Board and Medical Ethics Committee and was performed in accordance with the medical ethics standards laid down in an appropriate version of the 1964 Declaration of Helsinki. All our patients were asked during the first admission to our institute or a follow-up visit to give consent to use their charts and biopsy material for scientific purposes. Since the Institutional Review Board of the Institute of Oncology Ljubljana approved this specific study, our patients were not asked to give written consent on this specific study. Statistical analysis The chi-square test was used to compare the observed and expected frequencies of recurrence in regional and lymph nodes in patients with follicu-lar and Hurthle cell neoplasms. A p-value < 0.05 Radiol Oncol 2016; 50(3): 269-273. 31 Vogrin A et al. /Preoperative neck ultrasound in follicular or Hurthle cell neoplasms was considered as statistically significant. SPSS 16.0 for Windows was used for statistical analysis. Results Carcinoma was diagnosed in follicular and Hurthle cell neoplasms in 143/428 (33%) and 64/309 (21%) of cases, respectively. A follicular variant of papillary thyroid carcinoma, Hurthle cell carcinoma, follicular carcinoma, classical type of papillary carcinoma and other types of papillary carcinoma were diagnosed in 90, 50, 39, 21 and 7 cases, respectively (Table 1). Mean size of carcinoma was 3.46 cm (range 0.4-11 cm). Microcarcinoma in a dominant nodule was diagnosed in only 8.2% of patients. Twelve patients had focal thyroid lesions incidentally detected by 18F-FDG PET-CT. Five of them had carcinoma (2 follicular carcinoma, 2 papillary carcinoma and 1 medullary carcinoma), while seven patients had benign tumor (5 adenoma, 2 multinodular goiter). Median tumor size in malignant and benign tumor was 30 mm and 15 mm, respectively. Median maximal standardized uptake value in malignant and benign tumor was 11 and 6.4, respectively. Recurrence in regional lymph nodes Recurrence in lymph nodes was diagnosed in 3/428 (0.7%) of patients with a preoperative diagnosis of follicular neoplasm and 9/309 (3%) of patients with a Hurthle cell neoplasm. Among patients with carcinoma, a recurrence in lymph nodes was detected in 2% and 14% of those who had preoperative diagnosis of follicular and Hurthle cell neoplasms, respectively (p = 0.002). Recurrence rate in regional lymph nodes according to subtype of carcinoma, tumor size and pT tumor stage is shown in Table 2. Neither primary tumor diameter or pT tumor stage was correlated with a recurrence in regional lymph nodes. Primary tumor diameter was 4 cm or more in 4/12 patients with a recurrence in regional lymph nodes. A recurrence in regional lymph nodes was diagnosed in only 2/12 patients earlier than three years after a thyroidectomy and radioiodine ablation of the thyroid remnant. These two patients had a Hurthle cell neoplasm and concentration of Tg remained elevated after initial treatment. One of them had a very aggressive Hurthle cell carcinoma which had a regional recurrence in lymph nodes 6 months after a thyroidectomy and radioiodine ablation and bone metastases 11 months thereafter. TABLE 1. Distribution of follicular and Hurthle cell neoplasms and carcinoma subtype Type of neoplasm Carcinoma type Follicular neoplasm Hürthle cell neoplasm Total Follicular 31 8 39 Hurthle cell 12 38 50 Papillary - classical variant 16 5 21 Papillary - follicular variant 81 9 90 Papillary - other variant* 3 4 7 Total 143 64 207 * Oncocytic variant in 4 cases, carcinoma in 1 case trabecular variant in 2 cases, poorly differentiated thyroid TABLE 2. Recurrence rate in regional lymph nodes and subtype of carcinoma, pT, pN, M and tumor size in 207 patients with malignant follicular or Hurthle cell neoplasm Carcinoma type Total Recurrence in regional lymph nodes p-value No Yes Follicular Hurthle cell Papillary - classical variant Papillary - follicular variant Papillary - other variant pT1 pT2 pT3 pT4 pN0 pN1 M0 Ml Tumor diameter 0.4-4 cm Tumor diameter 4.01-11 cm Total 39 50 21 90 7 69 68 64 6 205 2 197 10 145 62 207 37 42 20 89 7 69 62 59 5 194 1 137 58 195 4 12 0.008 0.067 0.113 0.014 0.75 TABLE 3. Preoperative neck US and recurrence in regional lymph nodes with follicular and Hurthle cell neoplasms (Fisher's exact test p = 0.296) in patients Type of neoplasm Recurrence in regional lymph nodes Total No Yes Follicular 19 0 19 Hüthle cell 71 8 Total 26 1 27 From our cohort of patients, these two cases are the only ones in whom a preoperative US examination would have shown the presence of metastases in regional lymph nodes and therefore the treatment would have been changed. After initial treatment altogether 8/12 patients had suppressed thyroglobulin (Tg) < 1 ng/mL, TSH-stimulated Tg < 10 ng/ml, or stable or decreasing levels of Tg antibodies and an absence of structural Radiol Oncol 2016; 50(3): 269-273. 272 Vogrin A et al. /Preoperative neck ultrasound in follicular or Hurthle cell neoplasms disease. Two of the remaining four patients had distant metastases soon after a regional relapse. Preoperative neck US examination Preoperative US examination of the neck region was performed in 27 patients with thyroid carcinoma (Table 3): in 12 , 6, 5, 1 and 3 cases with a follicular variant of papillary carcinoma, Hurthle cell carcinoma, follicular carcinoma, classical type of papillary carcinoma and other types of papillary carcinoma, respectively. In patients who underwent preoperative US examination of the neck region no pathological lymph nodes were detected. However, a recurrence in regional lymph nodes was diagnosed in one of 26 patients six years after thyroid surgery. This patient had a follicular variant of papillary carcinoma. In only 2 of 12 patients with a recurrence in regional lymph nodes it was diagnosed earlier than three years after thyroidectomy. After modified radical neck dissection in one of them, there is no evidence of disease. However, the other one had a very aggressive Hurthle cell carcinoma in whom a pre-operative US would probably have shown the presence of metastases in regional lymph nodes which could have changed the treatment and possibly his outcome. However, it is very doubtful if, in the remaining 10 patients with a recurrence, metastases in regional lymph could have been detected with a preoperative US examination. Among patients with a recurrence in regional lymph nodes after a thy-roidectomy and radioiodine ablation of the thyroid remnant, altogether 8/12 patients had suppressed thyroglobulin (Tg) < 1 ng/mL, TSH-stimulated Tg < 10 ng/ml, or stable or decreasing Tg antibody levels or the presence of structural disease. Therefore we believe that there are only two patients from our cohort of patients in whom a preoperative US examination would have shown the presence of metastatic lymph nodes which would probably have changed the treatment and his outcome. Discussion Preoperative US examination of central and lateral neck compartments is recommended for all patients undergoing a thyroidectomy because of malignant or suspicious for malignancy cytology. Namely, cervical lymph node metastases were found in as many as 50% of patients with carci- noma.11-13 Because preoperative US examination of the neck region detects metastases in cervical lymph nodes in 23-33% of patients the surgical approach is changed.1415 In the literature, there is very limited data on the frequency of involvement of cervical lymph nodes in patients with follicular or Hürthle cell neoplasms. Paunovic et al. reported the presence of regional lymph node metastases in 2% of patients with a Hürthle cell neoplasm.16 Our study was observational and not randomized, thus it is not possible to draw conclusions about the impact of preoperative US examination on the surgical approach or patients' outcomes. However, it has shown in a large dataset of patients that recurrence in regional lymph nodes was detected in only 0.7% of patients with preoperative diagnosis of a follicular neoplasm and 3% of patients with a Hürthle cell neoplasm. Based on these findings our opinion is that preoperative US examination of the neck region is not useful in patients with a follicular neoplasm. On the other hand, in patients with a Hürthle cell neoplasm preoperative US examination of the neck region may be useful. A recurrence in regional lymph nodes occurred in 6% of our patients with a carcinoma during a median follow-up of 55 months. The outcomes of our patients are in accordance with reports from the literature. During a median follow-up period of 7 years after a total thyroidectomy and radioiodine ablation of the thyroid remnant a recurrence rate was reported to occur in low risk patients in 3%, in intermediate risk patients in 21%, and in high risk patients in 68%.17 In papillary thyroid carcinoma (PTC), a recurrence was diagnosed in papillary mi-crocarcinoma in 1-3% of patients.1819 The ten-year regional recurrence rate of T1, T2, and T3 patients with PTC was 1.9, 4.6, and 8.1%, respectively.20 In our patients with PTC a recurrence in regional lymph nodes was diagnosed in only 2 of 118 (1.7%) patients. But it should be stressed that the median follow-up in our study was only 55 months. It is well known that in thyroid carcinoma recurrences occur years after initial treatment.20-22 Our study confirmed the well-known fact that the recurrence rate in regional lymph nodes is common in Hürthle cell carcinoma.23-26 It was diagnosed in as much as 16% of patients with Hürthle cell carcinoma. The recurrence rate in our patients is comparable to reports in the literature. Khafif et al., Stojadinovic et al. and Mills et al. reported that locoregional recurrence occurred in 10.5%, 16% and 34% of cases, respectively.23-25 Radiol Oncol 2016; 50(3): 269-273. 273 Vogrin A et al. /Preoperative neck ultrasound in follicular or Hurthle cell neoplasms Conclusions Preoperative neck ultrasound examination in patients with a follicular neoplasm is probably not useful, but in patients with Hurtle cell neoplasm it may be useful. Recurrence in lymph nodes was diagnosed in 0.7% of patients with a preoperative diagnosis of follicular neoplasm and 3% of patients with a Hürthle cell neoplasm. A recurrence in regional lymph nodes is rare in patients with carcinoma and preoperative diagnosis of follicular neoplasm. Acknowledgement This paper is a part of Research Study No. P3-0289 supported by the Ministry of Higher Education, Science, and Technology of the Republic of Slovenia. References 1. Guth S, Theune U, Aberle J, Galach A, Bamberger CM. Very high prevalence of thyroid nodules detected by high frequency (13 MHz) ultrasound examination. Eur J Clin Invest 2009; 39: 699-706. 2. American Thyroid Association (ATA) Guidelines Taskforce on Thyroid Nodules and Differentiated Thyroid Cancer, Cooper DS, Doherty GM, Haugen BR, et al. Revised American Thyroid Association management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid 2009; 19: 1167-214. 3. I to Y, Amino N, Yokozawa T, Ota H, Ohshita M, Murata N, et al. Ultrasonographic evaluation of thyroid nodules in 900 patients: comparison among ultrasonographic, cytological, and histological findings. Thyroid 2007; 17: 1269-76. 4. Baloch ZW, LiVolsi VA, Asa SL, Rosai J, Merino MJ, Randolph G, et al. Diagnostic terminology and morphologic criteria for cytologic diagnosis of thyroid lesions: a synopsis of the National Cancer Institute Thyroid Fine-Needle Aspiration State of the Science Conference. Diagn Cytopathol 2008; 36: 425-37. 5. National Comprehensive Cancer Network (2015) NCCN Clinical practice guidelines in oncology (NCCN Guidelines) Thyroid carcinoma. Version 2.2015. National Comprehensive Cancer Network, Washington. Available at http://www.nccn.org/professionals/physician_gls/f_guidelines. asp#thyroid. Accessed on 21 August 2015 6. Bongiovanni M, Spitale A, Faquin WC, Mazzucchelli L, Baloch ZW. The Bethesda System for Reporting Thyroid Cytopathology: a meta-analysis. Acta Cytol 2012; 56: 333-9. 7. Petric R, Perhavec A, Gazic B, Besic N. Preoperative serum thyroglobulin concentration is an independent predictive factor of malignancy in follicular neoplasms of the thyroid gland. J Surg Oncol 2012; 105: 351-6. 8. Strazisar B, Petric R, Sesek M, Zgajnar J, Hocevar M, Besic N. Predictive factors of carcinoma in 279 patients with Hürthle cell neoplasm of the thyroid gland. J Surg Onco! 2010; 101: 582-6. 9. 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. 10. Jamsek J, Zagar I, Gaberscek S, Grmek M. Thyroid lesions incidentally detected by (18)F-FDG PET-CT - a two centre retrospective study. Radiol Oncol 2015; 49: 121-7. 11. Nam-Goong IS, Kim HY, Gong G, Lee HK, Hong SJ, Kim WB, Shong YK. Ultrasonography-guided fine-needle aspiration of thyroid incidentaloma: correlation with pathological findings. Clin Endocrinol (Oxf) 2004; 60: 21-8. 12. Scheumann GF, Gimm O, Wegener G, Hundeshagen H, Dralle H. Prognostic significance and surgical management of locoregional lymph node metastases in papillary thyroid cancer. World J Surg 1994; 18: 559-67. 13. Ito Y, Miyauchi A. Lateral lymph node dissection guided by preoperative and intraoperative findings in differentiated thyroid carcinoma. World J Surg 2008; 32: 729-39. 14. Stulak JM, Grant CS, Farley DR, Thompson GB, van Heerden JA, Hay ID, et al. Value of preoperative ultrasonography in the surgical management of initial and reoperative papillary thyroid cancer. Arch Surg 2006; 141: 489-94. 15. O'Connell K, Yen TW, Quiroz F, Evans DB, Wang TS. The utility of routine pre-operative cervical ultrasonography in patients undergoing thyroidectomy for differentiated thyroid cancer. Surgery 2013; 154: 697-701. 16. Paunovic I, Krgovic K, Tatic S, Diklic A, Zivaljevic V, Kalezic N, Havelka M. Surgery for thyroid Hürthle cell tumours - a single institution experience. Eur J Surg Oncol 2006; 32: 458-61. 17. Tuttle RM, Tala H, Shah J, Leboeuf R, Ghossein R, Gonen M, et al. Estimating risk of recurrence in differentiated thyroid cancer after total thyroidec-tomy and radioactive iodine remnant ablation: using response to therapy variables to modify the initial risk estimates predicted by the new American Thyroid Association staging system. Thyroid 2010; 20: 1341-9. 18. Roti E, degli Uberti EC, Bondanelli M, Braverman LE. Thyroid papillary microcarcinoma: a descriptive and meta-analysis study. Eur J Endocrinol; 159: 659-73. 19. Besic N, Pilko G, Petric R, Hocevar M, Zgajnar J. (2008) Papillary thyroid microcarcinoma: prognostic factors and treatment. J Surg Oncol 2008; 97: 221-5. 20. Ito Y, Kudo T, Kihara M, Takamura Y, Kobayashi K, Miya A, Miyauchi A. Prognosis of low-risk papillary thyroid carcinoma patients: its relationship with the size of primary tumors. Endocr J 2012; 59: 119-25. 21. Mazzaferri EL. An overview of the management of papillary and follicular thyroid carcinoma. Thyroid 1999; 9: 421-7. 22. Hay ID, Thompson GB, Grant CS, Bergstralh EJ, Dvorak CE, Gorman CA, et al. Papillary thyroid carcinoma managed at the Mayo Clinic during six decades (1940-1999): temporal trends in initial therapy and long-term outcome in 2444 consecutively treated patients. World J Surg 2002; 26: 879-85. 23. Khafif A, Khafif RA, Attie JN. Hurthle cell carcinoma: a malignancy of low-grade potential. Head Neck 1999; 21: 506-11. 24. Stojadinovic A, Hoos A, Ghossein RA, Urist MJ, Leung DH, Spiro RH, et al. Hürthle cell carcinoma: a 60-year experience. Ann Surg Oncol 2002; 9: 197-203. 25. Mills SC, Haq M, Smellie WJ, Harmer C. Hurthle cell carcinoma of the thyroid: retrospective review of 62 patients treated at the Royal Marsden Hospital between 1946 and 2003. Eur J Surg Oncol 2009; 35: 230-4. 26. Petric R, Gazic B, Besic N. Prognostic factors for disease-specific survival in 108 patients with Hürthle cell thyroid carcinoma: a single-institution experience. BMC Cancer 2014; 14: 777. Radiol Oncol 2016; 50(3): 269-273. 274 research article Electrochemotherapy with bleomycin is effective in BRAF mutated melanoma cells and interacts with BRAF inhibitors Tanja Dolinsek1, Lara Prosen1, Maja Cemazar12, Tjasa Potocnik1, Gregor Sersa1 1 Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Faculty of Health Sciences, University of Primorska, Izola, Slovenia Radiol Oncol 2016; 50(3): 274-279. Received 23 May 2016 Accepted 14 June 2016 Correspondence to: Prof. Gregor Sersa, Ph.D., Institute of Oncology Ljubljana, Department of Experimental Oncology, Zaloska 2, SI-1000 Ljubljana, Slovenia. Phone: +386 1 5879 434; Fax: +386 1 5979 434; E-mail: gsersa@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. The aim of the study was to explore the effectiveness of electrochemotherapy (ECT) during the treatment of melanoma patients with BRAF inhibitors. Its effectiveness was tested on BRAF mutated and non-mutated melanoma cells in vitro and in combination with BRAF inhibitors. Materials and methods. ECT with bleomycin was performed on two human melanoma cell lines, with (SK-MEL-28) or without (CHL-1) BRAF V600E mutation. Cell survival was determined using clonogenic assay to determine the effectiveness of ECT in melanoma cells of different mutation status. Furthermore, the effectiveness of ECT in concomitant treatment with BRAF inhibitor vemurafenib was also determined in BRAF mutated cells SK-MEL-28 with clonogenic assay. Results. The survival of BRAF V600E mutated melanoma cells was even lower than non-mutated cells, indicating that ECT is effective regardless of the mutational status of melanoma cells. Furthermore, the synergistic interaction between vemurafenib and ECT with bleomycin was demonstrated in the BRAF V600E mutated melanoma cells. Conclusions. The effectiveness of ECT in BRAF mutated melanoma cells as well as potentiation of its effectiveness during the treatment with vemurafenib in vitro implies on clinical applicability of ECT in melanoma patients with BRAF mutation and/or during the treatment with BRAF inhibitors. Key words: electrochemotherapy; BRAF inhibitors; vemurafenib; melanoma Introduction Recently, there have been great advances in the treatment of metastatic melanoma, including targeted therapy with BRAF and MEK inhibitors, and immunomodulation either with anti-CTLA-4 or anti-PD-1 or anti-PD-L1 antibodies.1-5 BRAF inhibitors are based on the fact that 50% of melanoma tumors harbor BRAF V600 mutations, which cause increased activation of MAP kinase signaling pathway that results in melanoma cell proliferation.6 Inhibitors of BRAF V600 mutated melanoma, such as vemurafenib and dabrafenib, increase progression-free survival and overall sur-viva!178, but unfortunately the resistance mecha- nisms usually appear to re-establish the signaling pathway and the disease progresses in the matter of months after the start of treatment.9 For the progressive disease after treatment with BRAF inhibitors, other locoregional treatments are usually needed to control tumor relapses. Electrochemotherapy (ECT) provides this approach. It is a combined local treatment in which locally-applied high voltage electric pulses are used to facilitate the uptake of non-permeant chemotherapeutic drugs.10 It is used mainly for the treatment of cutaneous and subcutaneous metastases of different tumor histology, with a complete and objective response rate of 59.4% and 84.1%, respectively.11 Specifically for melanoma, the com- Radiol Oncol 2016; 50(3): 254-262. doi:10.1515/raon-2016-0023 Dolinsek T et al. / Electrochemotherapy in BRAF mutated cells 275 plete and objective response rates of 56.8% and 80.6% were obtained.11 The unexplored question is whether ECT is effective in BRAF mutated melanoma cells, and whether ECT is effective as concomitant treatment to therapy with BRAF inhibitors. In the first reported case regarding treatment of a patient undergoing dabrafenib treatment, ECT proved to be effective on progressing tumor nodules.12 Based on this report, studies on effectiveness of ECT with bleo-mycin in BRAF V600 mutated, compared to non-mutated melanoma cells are warranted. Besides this aim, we also investigated whether the concomitant vemurafenib treatment of cells can affect the effectiveness of ECT in melanoma cells in vitro. Materials and methods Cell lines and culturing Human melanoma cell line SK-MEL-28 (American Type Culture Collection (ATCC), Manassas, VA, USA) with BRAF V600E mutation was cultivated in an Advanced MEM medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 5% fetal bovine serum (FBS, Gibco), 10 mM L-glutamine (GlutaMAX, Gibco), 100 U/ml penicillin (Grünenthal, Aachen, Germany) and 50 |jg/ml gentamicin (Krka, Novo mesto, Slovenia) in a 5% CO2 humidified incubator at 37°C. Human melanoma cell line CHL-1 (ATCC) without BRAF V600 mutations was cultured in an Advanced RPMI 1640 medium (Gibco), supplemented with 5% FBS, 10 mM L-glutamine, 100 U/ ml penicillin and 50 ^g/ml gentamicin in a 5% CO2 humidified incubator at 37°C. Electrochemotherapy (ECT) Melanoma cells were grown as a monolayer until they reached at least 80% confluence. The medium was removed and the cells were washed with phosphate-buffered saline (PBS, Merck Millipore, Darmstadt, Germany). After that, cells were detached from the surface with 0.25% trypsin/EDTA in Hank's buffer (Gibco). After detachment, trypsin was inactivated with an equal amount of cell culture medium with FBS and cells were collected and cen-trifuged. For electroporation, 88 |jl of cell suspension (25 x 106 cells/ml) was prepared in electropo-ration buffer (125 mM sucrose, 10 mM K2HPO4, 2.5 mM KH2PO4, 2 mM MgCl2 x 6H20) at 4°C. Ce4l suspension was mixed with 22 |jl of different stock concentrations of bleomycin (Bleomycin medac, Medac, Wedel, Germany) to reach a final concentration of 1.4 x 10-12 M, 1.4 x 10-11 M, 1.4 x 10-10 M, 1.4 x 10-9 M, 1.4 x 10-8 M, 1.4 x 10-7 M, 1.4 x 10-6 M. Out of 110 |jl of mixture, 50 |jl served as a control for bleomycin treatment and other 50 |jl was pipetted between two stainless steel parallel plate electrodes (2 mm apart) and 8 square-wave electric pulses (amplitude over distance ratio of 1300 V/ cm, duration of 100 |js and frequency of 1 Hz) were applied. Electric pulses were generated with the electric pulse generator GT-01 (Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia). The cells were incubated 5 min after electroporation at room temperature, and then cell culture medium was added. Afterwards, clonogen-ic assay was performed. Experimental groups were denoted: BLM (different concentrations of bleomycin); BLM + EP (ECT with bleomycin of different concentrations). Each group was normalized to the control group of same treatment regimen with 0 M bleomycin (Ctrl, EP). Clonogenic assay After the treatment, cells were plated in 6 cm petri dishes with 4 ml of culture medium for clonogenic assay. The number of plated cells was in a range of 300-4000 cells, based on predicted cytotoxicity of the treatment. Colonies were formed after 12 and 8 days for SK-MEL-28 and CHL-1, respectively. After the colonies were formed, they were fixed and stained with crystal violet solution (Sigma-Aldrich, St. Louis, MO, USA) and counted. The colonies containing less than 50 cells were disregarded. Plating efficiency was calculated for each experimental group as the ratio between counted colonies and the number of plated cells. Plating efficiency of each treatment group was normalized to the untreated cells group, representing cell survival. IC90 value was determined (drug concentration required to reduce cell survival for 90%). Vemurafenib sensitivity Cell lines were tested for their sensitivity to vemu-rafenib treatment in order to confirm the selectivity of vemurafenib on BRAF V600E mutated cells and to determine the optimal concentration for the combination treatment. Cells were detached from cell culture dishes and prepared for clonogenic assay. Vemurafenib (MedChem Express, Monmouth Junction, NJ, USA) was obtained in 10 mM DMSO solution and was added to 6 cm culture dishes with 4 ml of culture medium to reach a final concentra- Radiol Oncol 2016; 50(3): 274-279. 276 Dolinsek T et al. / Electrochemotherapy in BRAF mutated cells 276 ® FIGURE 1. Survival of melanoma cells after vemurafenib (VMF) treatment. Survival of (A) BRAF V600E mutated melanoma cells SK-MEL-28 and (B) non-mutated melanoma cells CHL-1. tion of 0.5 jaM, 2.5 jaM, 5 jaM and 10 jaM. The final amount of DMSO in the cell culture medium represented 20000x — 1000x dilution and was not toxic to the cells. Afterwards, cells were cultured in a 5% CO2 humidified incubator at 37°C until the formation of colonies, which were counted and analyzed as described. Each group was normalized to the control group with 0 jM vemurafenib. Combination of ECT and vemurafenib ECT was performed as described. After that, cells were plated in 6 cm petri dishes for clonogenic assay and vemurafenib was added in each dish to reach a final concentration of 0.5 jM. Afterwards, cells were cultured in a 5% CO2 humidified in- cubator at 37°C until the formation of colonies, which were counted and analyzed as described. Experimental groups were denoted: BLM (different concentrations of bleomycin); BLM + EP (ECT with bleomycin of different concentrations); BLM + VMF (bleomycin of different concentrations and addition of 0.5 jM vemurafenib), BLM + EP + VMF (ECT with bleomycin of different concentrations and addition of 0.5 jM vemurafenib). Each group was normalized to the control group of same treatment regimen with 0 M bleomycin (Ctrl, EP, Ctrl + VMF, EP + VMF). Statistical analysis The mode of interaction between the treatments with independent mode of action was calculated at the level of IC90 by the method developed by Spector.13 For the analysis and graphical representation, SigmaPlot Software (version 12.0, Systat Software, London, UK) was used. Results Sensitivity of BRAF V600E mutated melanoma cells to vemurafenib Vemurafenib treatment was tested for selective cytotoxicity to BRAF V600E mutated cells. Selectivity of BRAF inhibitor vemurafenib was confirmed on SK-MEL-28 BRAF V600E mutated melanoma cells. Cell survival was reduced with increasing vemurafenib concentration with IC90 5 jM (Figure 1A). In contrast, vemurafenib treatment did not reduce the survival of BRAF V600E non-mutated cell line CHL-1. The cell survival was higher than 94%, also with the highest concentration of vemurafenib used (Figure 1B). These results also provided bases for the combined vemurafenib and ECT treatment of cells, the concentration of 0.5 jM was selected, which reduced cell survival to 50%. BRAF V600E mutated melanoma cells are more sensitive to ECT than non-mutated melanoma cells The effectiveness of ECT was tested on BRAF V600E mutated melanoma cells (SK-MEL-28) and non-mutated melanoma cells (CHL-1). ECT effectively reduced survival of both cell lines with IC90 3.8 x 10-10 M and 7.7 x 10-10 M for SK-MEL-28 and CHL-1, respectively. ECT was even more effective on BRAF V600E mutated SK-MEL-28 cells that required 2 times lower concentration of BLM Radiol Oncol 2016; 50(3): 274-279. Dolinsek T et al. / Electrochemotherapy in BRAF mutated cells 277 at IC90,compared to non-mutated cells CHL-1 (Figure 2), confirming that ECT is an effective method for treatment of also BRAF V600E mutated melanoma cells. Concomitant vemurafenib treatment increased ECT effectiveness Concomitant treatment with ECT and vemurafenib was simulated in vitro on cells in the way, that ECT treated cells were seeded into dishes containing 0.5 |xM vemurafenib. The vemurafenib treatment decreased survival of SK-MEL-28 cells for 50%. If the effect of the vemurafenib was eliminated (normalized to control groups with added vemurafenib for groups BLM + VMF and BLM + EP + VMF), as shown in Figure 3, then an increased effectiveness of ECT was observed on BRAF mutated SK-MEL-28 cells. A 4.5 times lower concentration of BLM was needed at the IC90 for cells treated with vemurafenib (BLM + EP + VMF; IC90 value 8.5 x 10-11 M) compared to cells without vemurafenib treatment (BLM + EP; IC90 value 3.8 x 10-10 M). The potentiation was more than additive (Figure 3), in fact, according to the method developed by Spector et al. was synergistic.13 Discussion This study demonstrates the effectiveness of ECT with bleomycin on BRAF V600E mutated melanoma cells. The effectiveness was higher to that on BRAF non-mutated cells. Furthermore, an interaction of ECT and vemurafenib treatment was observed in BRAF mutated melanoma cells, indicating on more than additive or synergistic effectiveness. BRAF inhibitors provide a clear benefit to patients with disseminated disease. The effect is mediated by inhibition of cell proliferation through the inhibition of the MAPK pathway.14 Patients often present with multiple metastases, that may not all respond to the treatment either due to their bigger size or due to the development of the resistance to BRAF inhibitors. In such cases additional therapy is needed, that has proven effectiveness also in BRAF mutated melanoma cells. Our study demonstrates that ECT with BLM is as effective, or even more effective on BRAF mutated, compared to non-mutated melanoma cells, although the exact biological mechanism still needs to be explored. The data support the recent observation on melanoma patient undergoing dabrafenib treatment FIGURE 2. Survival of BRAF V600E mutated SK-MEL-28 melanoma cells and non-mutated CHL-1 cells after ECT with bleomycin. BLM (bleomycin); BLM + EP (ECT with bleomycin). Dotted line represents the IC90 value. where some nodules were treated with ECT. ECT proved effective even on tumor nodules, which were in progression during the dabrafenib treat-ment12, which indicates that it could be effective also on dabrafenib resistant tumor clones. The other aspect that was clarified is that ECT can be successfully implemented also during the treatment with BRAF inhibitors. The in vitro results demonstrated that vemurafenib and ECT treatment has synergistic effectiveness. This is of clinical importance, since the in vitro data indicate that there is no need to wait for the discontinuation of FIGURE 3. Survival of BRAF V600E mutated SK-MEL-28 melanoma cells after concomitant treatment with ECT and vemurafenib. BLM (bleomycin); BLM + VMF (bleomycin and 0.5 pM vemurafenib); BLM + EP (ECT with bleomycin); BLM + EP + VMF (ECT with bleomycin and 0.5 pM vemurafenib). Dotted line represents the IC90 value. Radiol Oncol 2016; 50(3): 274-279. 278 Dolinsek T et al. / Electrochemotherapy in BRAF mutated cells 278 treatment with BRAF inhibitors and can be given concomitantly. Based on the observation of the Valpione et al, the tolerability of the combined treatment is of great importance, as was observed in the described case.12 The possibility of the enhanced effectiveness further supports the approach to use ECT concomitantly during the treatment with BRAF inhibitors; however, further clinical studies with larger number of patients are needed to fully support the fact that the combined treatment does not cause additional undesired side effects. If the interaction of the treatments will be observed also in vivo, this may lead also to reduction of BLM dosage in ECT. The interaction of BRAF inhibitors with radiotherapy has also been demonstrated, both on tumor and normal tissue. In vitro study demonstrated radiosensitization of BRAF mutated melanoma cells with BRAF inhibitor PLX-4032 by clonogenic and invasion assay and was associated with enhancement of G1 cell cycle arrest.15 Furthermore, the combination of BRAF inhibitor and irradiation was proven to be effective also in high-grade gliomas, harboring BRAF V600E mutation. Radiosensitization was observed by PLX-4720 BRAF inhibitor in vitro, whereas in BRAF non-mutated glioma cells the radiosensitizing effect was not observed.16 The clinical studies, however, demonstrated the radiosensitization of normal tissue as well. There is still not consensus whether the normal tissue damage is acceptable17, or that they are so severe that this requires profound investigations in the future.18 Eventually ECT will most probably find its place in combination with systemic treatments, with targeted therapies as well as with recently emerging immune checkpoint inhibitors. ECT was recently evaluated also combined with the immunothera-peutic approaches, immune checkpoint inhibitors. The first clinical study of Mozillo et al. reported on safety of the combined ipilimumab and ECT with good therapeutic responsiveness.19 The second report also demonstrated effectiveness and safety of a sequential treatment with ECT plus ipilimumab, which induced a durable complete response of multiple cutaneous metastases with vitiligo-like lesions indicating on involvement of immune re-sponse.20 Obviously ECT is progressing into concomitant treatment with melanoma targeted therapies and immunotherapies, therefore its safety and effectiveness needs to be established. Specifically, due to induction of the immunogenic cell death induced in tumors21, ECT of tumors may serve as in situ vaccination that could be boosted by con- comitant immunotherapeutic approach.22 The consequences may be both, the potentiated local response and also increased side effects. In conclusion, the effectiveness of ECT in BRAF mutated cells implies on clinical applicability of ECT in BRAF mutated melanoma tumors. Furthermore, its effectiveness also during the treatment with BRAF inhibitors was demonstrated, with synergistic effectiveness. These results are encouraging, but need to be extended to more cell lines, and in vivo studies on experimental tumors, evaluating both the tumor and normal tissue response. The study should also be extended to patient-derived melanoma cell lines and also on clones which develop resistance to the therapy with BRAF inhibitors to verify if ECT maintains its effectiveness on the resistant clones. Such studies will then predict the tumor response, and possible side effects. Acknowledgements The authors acknowledge the financial support from the state budget by the Slovenian Research Agency (program no. P3-0003). The research was conducted in the scope of LEA EBAM (French-Slovenian European Associated Laboratory: Pulsed Electric Fields Applications in Biology and Medicine). The research is a result of the networking efforts of the COST Action TD1104. We wish to thank Mira Lavric (Institute of Oncology Ljubljana, Ljubljana, Slovenia) for the help with cell culturing. References 1. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 2011; 364: 2507-16. 2. Hauschild A, Grob JJ, Demidov LV, Jouary T, Gutzmer R, Millward M, et al. Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial. Lancet 2012; 380: 358-65. 3. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 2010; 363(8): p. 711-23. 4. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 2012; 366: 2443-54. 5. Ugurel S, Rohmel J, Ascierto PA, Flaherty KT, Grob JJ, Hauschild A, et al. Survival of patients with advanced metastatic melanoma: the impact of novel therapies. Eur J Cancer 2016; 53: 125-34. 6. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al. Mutations of the BRAF gene in human cancer. Nature 2002; 417: 949-54. 7. Puzanov I, Amaravadi RK, McArthur GA, Flaherty KT, Chapman PB, Sosman JA, et al. Long-term outcome in BRAF(V600E) melanoma patients treated with vemurafenib: patterns of disease progression and clinical management of limited progression. Eur J Cancer 2015; 51: 1435-43. Radiol Oncol 2016; 50(3): 274-279. Dolinsek T et al. / Electrochemotherapy in BRAF mutated cells 279 8. Scholtens A, Geukes Foppen MH, Blank CU, van Thienen JV, van Tinteren H, Haanen JB. Vemurafenib for BRAF V600 mutated advanced melanoma: results of treatment beyond progression. Eur J Cancer 2015; 51: 642-52. 9. Trunzer K, Pavlick AC, Schuchter L, Gonzalez R, McArthur GA, Hutson TE, et al. Pharmacodynamic effects and mechanisms of resistance to vemurafenib in patients with metastatic melanoma. J Clin Oncol 2013; 31: 1767-74. 10. Sersa G, Miklavcic D, Cemazar M, Rudolf Z, Pucihar G, and Snoj M. Electrochemotherapy in treatment of tumours. Eur J Surg Oncol 2008; 34: 232-40. 11. Mali B, Jarm T, Snoj M, Sersa G, Miklavcic D. Antitumor effectiveness of electrochemotherapy: a systematic review and meta-analysis. Eur J Surg Oncol 2013; 39: 4-16. 12. Valpione S, Campana LG, Pigozzo J, Chiarion-Sileni V. Consolidation electro-chemotherapy with bleomycin in metastatic melanoma during treatment with dabrafenib. Radiol Oncol 2015; 49: 71-4. 13. Spector SA, Tyndall M, Kelley E. Effects of acyclovir combined with other antiviral agents on human cytomegalovirus. Am J Med 1982; 73: 36-9. 14. Hertzman Johansson C, Egyhazi Brage S. BRAF inhibitors in cancer therapy. Pharmacol Ther 2014; 142: 176-82. 15. Sambade MJ, Peters EC, Thomas NE, Kaufmann WK, Kimple RJ, Shields JM. Melanoma cells show a heterogeneous range of sensitivity to ionizing radiation and are radiosensitized by inhibition of B-RAF with PLX-4032. Radiother Oncol 2011; 98: 394-9. 16. Dasgupta T, Olow AK, Yang X, Hashizume R, Nicolaides TP, Tom M, et al. Survival advantage combining a BRAF inhibitor and radiation in BRAF V600E-mutant glioma. J Neurooncol 2016; 126: 385-93. 17. Hecht M, Zimmer L, Loquai C, Weishaupt C, Gutzmer R, Schuster B, et al. Radiosensitization by BRAF inhibitor therapy-mechanism and frequency of toxicity in melanoma patients. Ann Oncol 2015; 26: 1238-44. 18. Zahnreich S, Mayer A, Loquai C, Grabbe S, Schmidberger H. Radiotherapy with BRAF inhibitor therapy for melanoma: progress and possibilities. Future Oncol 2016; 12: 95-106. 19. Mozzillo N, Simeone E, Benedetto L, Curvietto M, Giannarelli D, Gentilcore G, et al. Assessing a novel immuno-oncology-based combination therapy: Ipilimumab plus electrochemotherapy. Oncoimmunol 2015; 4: e1008842. 20. Brizio M, Fava P, Astrua C, Cavaliere G, Savoia P. Complete regression of melanoma skin metastases after electrochemotherapy plus ipilimumab treatment: an unusual clinical presentation. Eur J Dermatol 2015; 25: 271-2. 21. Calvet CY, Famin D, Andre FM, Mir LM. Electrochemotherapy with bleomy-cin induces hallmarks of immunogenic cell death in murine colon cancer cells. Oncoimmunol 2014; 3: e28131. 22. Sersa G, Teissie J, Cemazar M, Signori E, Kamensek U, Marshall G, et al. Electrochemotherapy of tumors as in situ vaccination boosted by immunogene electrotransfer. Cancer Immunol Immunother 2015; 64: p. 1315-27. Radiol Oncol 2016; 50(3): 274-279. 280 research article Discovery of 'click' 1,2,3-triazolium salts as potential anticancer drugs Ivana Steiner1, Nikolina Stojanovic1, Aljosa Bolje2, Anamaria Brozovic1, Denis Polancec3, Andreja Ambriovic-Ristov1, Marijana Radic Stojkovic4, Ivo Piantanida4, Domagoj Eljuga5, Janez Kosmrlj2, Maja Osmak1 1 Division of Molecular Biology, Ruder Boškovič Institute, Zagreb, Croatia 2 Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia 3 Department for Translational Medicine, Children's Hospital Srebrnjak, Zagreb, Croatia 4 Division of Organic Chemistry and Biochemistry, Ruder Boškovič Institute, Zagreb, Croatia 5 Department for Oncoplastic and Reconstructive Surgery, University Hospital for Tumors, University Clinical Hospital Centre Sisters of Mercy, Zagreb, Croatia Radiol Oncol 2016; 50(3): 280-288. Received 23 November 2015 Accepted 17 March 2016 Correspondence to: Maja Osmak, Ph.D., Division of Molecular Biology, Ruder Boškovic Institute, Bijenička cesta 54, 10000 Zagreb, Croatia. Phone: + 385 1 456 0939; Fax: + 385 1 456 1 177; E-mail: maja.osmak@irb.hr and Janez Košmrlj, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia. Phone: +386 1 479 8558; E-mail: janez.kosmrlj@fkkt.uni-lj.si Ivana Steiner and Nikolina Stojanovic: equal contributing authors Disclosure: No potential conflicts of interest were disclosed. Background. In order to increase the effectiveness of cancer treatment, new compounds with potential anticancer activities are synthesized and screened. Here we present the screening of a new class of compounds, 1-(2-picolyl)-, 4-(2-picolyl)-, 1-(2-pyridyl)-, and 4-(2-pyridyl)-3-methyl-1,2,3-triazolium salts and 'parent' 1,2,3-triazole precursors. Methods. Cytotoxic activity of new compounds was determined by spectrophotometry MTT assay on several tumour and one normal cell line. Effect of the selected compound to bind double stranded DNA (ds DNA) was examined by testing its influence on thermal stability of calf thymus DNA while its influence on cell cycle was determined by flow cytometric analysis. Generation of reactive oxygen species (ROS) was determined by addition of specific substrate 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA). Results. Parent triazoles were largely inactive, while some of the triazolium salts were highly cytotoxic for HeLa cells. Triazolium salts exhibited high cell-type dependent cytotoxicity against different tumour cells. Selected compound (4-(4-methoxyphenyl)-3-methyl-1-(2-picolyl)-1H-1,2,3-triazolium hexafluorophosphate(V) (2b) was significantly more cytotoxic against tumour cells than to normal cells, with very high therapeutic index 7.69 for large cell lung carcinoma H460 cells. Additionally, this compound was similarly cytotoxic against parent laryngeal carcinoma HEp-2 cells and their drug resistant 7T subline, suggesting the potential of this compound in treatment of drug resistant cancers. Compound 2b arrested cells in the G1 phase of the cell cycle. It did not bind ds DNA, but induced ROS in treated cells, which further triggered cell death. Conclusions. Our results suggest that the 'click' triazolium salts are worthy of further investigation as anti-cancer agents. Key words: triazoles; triazolium salts; anticancer activity; cell cycle; ROS Introduction Cancer is one of the major causes of death in developed countries.1 Despite the fact that several decades of investigations and massive funding have been devoted to the cancer research, the de- crease in cancer mortality is relatively modest.2 Based on numerous different genes implicated in sustained proliferative signalling, evading growth suppression, resisting cell death, enabling replica-tive immortality, inducing angiogenesis, and activating invasion and metastasis, cancers exhibit Radiol Oncol 2016; 50(3): 254-262. doi:10.1515/raon-2016-0023 41 Steiner I et al. / 1,2,3-triazolium salts as potential anticancer drugs FIGURE 1. The structures of triazoles 1 and triazolium salts 2. considerable genetic complexity and genome instability, which generates the genetic diversity.23 Consequently, amongst all diseases, cancer is one of the most difficult to treat and cure, because it is not a single disease but rather consists of numerous different types and subtypes. Targeted therapy with novel drugs directed at specific molecular pathways opens promising new avenues to improve the efficacy of therapy. However, in spite of the initial enthusiasm, the clinical application of such target oriented anticancer drugs did not fulfil the expectations. Although the targeted therapy has been successful, it is still limited to some very specific types of tumours.3 Hence, in order to increase the effectiveness of cancer treatment, new compounds with potential anticancer activities have to be synthesized and screened. Modern synthetic chemistry is powered by facile synthetic protocols that allow for a rapid generation of compound scaffolds. Catalyzed azide-alkyne cycloaddition reaction producing 1,2,3-tri-azoles ('click' triazoles) is one of such example.45 Moreover, this chemistry paves the way to a variety of derivatives including 1,3,4-trisubstituted 1,2,3-triazolium salts ('click' triazolium salts).6 1,2,3-Triazole has drug-like properties7 and its de- rivatives are recognized for their broad range of biological activities, including antiviral, antibacterial, antifungal, anti-inflammatory and analgesic, anti-convulsant, antiparasitic, antidiabetic, antiobesitic, antihistaminic, antineuropathic, antihypertensive, and anticancer activities8, presenting a promising group of potential anticancer drugs. Despite the fact that there are numerous examples on cytotoxic activities of compounds with a triazole subunit9-15, to our knowledge, only one such report exists for the 1,2,3-triazolium salts. Namely, Shrestha and Chang reported an interesting anticancer activity of the compounds having triazolium ring fused to 1,4-naphthoquinone.16 No cytotoxic activity of the 1,3,4-trisubstituted 1,2,3-triazolium salts has been reported to date. We recently developed a strategy for the 'click' triazolium salts synthesis, and prepared a library of twelve isomeric and homologous pyridine tethered 1,2,3-triazoles (1), which were then subjected to the selective N-methylation into the 3-methyl-1,4-disubstituted 1,2,3-triazolium salts 2a - l.1718 The library constituents differed in the 1,4-substitution pattern consisting of phenyl, 4-methoxyphenyl and 4-(trifluoromethyl)phenyl functionalized 1-(2-pi-colyl) (2a - c), 4-(2-picolyl) (2d - f), 1-(2-pyridyl) (2g - i) and 4-(2-pyridyl) (2j - l) isomers (Figure 1). The unique properties of this class of compounds, including the charge and hydrogen bonding ability, prompted us to evaluate their anticancer activity and to gain more insight into the mode of action that underlies their antiproliferative activity. Materials and methods Triazoles and triazolium salts Triazoles 1 and triazolium salts 2 were prepared as described previously.1718 Compounds 1b, 1f, 2b, 2c, 2f - i and 2l were dissolved in ethanol while compounds 1a, 2a, 2d, 2e, 2j and 2k were dissolved in DMSO. The solutions were stored at -20°C, and diluted to the appropriate concentrations with growth medium just before use. Cell culture Human cervical carcinoma HeLa and laryngeal carcinoma HEp-2 cells were obtained from cell culture bank (GIBCO BRL, Invitrogen, Grand Island, NY, USA). HEp-2 cell line was recently recognized and categorized by ATCC as human laryngeal carcinoma cell line cross-contaminated with HeLa cells. The development of HEp-2 subline resist- Radiol Oncol 2016; 50(3): 280-288. 282 Steiner I et al. / 1,2,3-triazolium salts as potential anticancer drugs ant to carboplatin (7T) has been published previously.19 These cells are cross-resistant to anticancer drug cisplatin, transplatin, mitomycin C and the natural compound curcumin as well.19-21 Large cell lung carcinoma H460 cells and colorectal carcinoma HCT-116 were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). Normal human skin fibroblasts were isolated from the upper arm of a 7-years-old female donor at the Neurochemical Laboratory, Department of Chemistry and Biochemistry, School of Medicine, University of Zagreb. They were used for the cytotoxicity assay at 32 and 36 population doublings. All cell lines were grown as a monolayer culture in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) in a humidified atmosphere of 5% CO2 at 37°C and were sub-cultured every 3 - 4 days. Cytotoxicity assay 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay Cytotoxic activity of triazoles and triazolium salts was determined by MTT assay22, modified as described. Cells were seeded into 96-well tissue culture plates (3 x 103 cells/0.18 mL medium/well). The next day different concentrations of triazoles or triazolium salts were added (0.02 mL) to each well and each concentration was tested in quadruplicate. Following 72 h incubation at 37°C, the medium was aspirated, and 20 |jg of the MTT dye (Sigma-Aldrich) /0.04 mL medium/well was added. Three hours later, formazan crystals were dissolved in DMSO (0.17 mL/well), the plates were mechanically agitated for 5 min and the optical density at 545 nm was determined on a microtiter plate reader (Awareness Technology Inc, Palm City, FL, USA). To examine the effect of reactive oxygen species (ROS) scavengers on survival of 2b treated cells, the same procedure was used as described above, except that two hours prior to addition of 2b, 5 mM of N-acetyl-cysteine (NAC, Sigma-Aldrich), or 1mM tempol (Santa Cruz Biotechnology, Dallas, TX, USA) was added in wells. Experiment was repeated at least three times. Colony-forming assay For determination of colony formation 1000 cells were seeded in 6 cm dish. The next day the cells were pretreated either with 5 mM NAC or with 1 mM (HEp-2 cells) or 0.125 mM (H460 cells) tem- pol. Two hours later different concentrations of 2b were added in dishes with or without ROS scavengers. The effect of antioxidants on colony formation alone was determined as well. After ten days of continuous treatment, the colonies were washed with PBS, fixed with methanol, stained with Giemsa-crystal violet and counted. Untreated samples were used for determination of plating efficiency. Each concentration was tested in triplicate. Experiment was repeated at least three times. Cell cycle analysis Human large cell lung carcinoma H460 cells were seeded into 6-well tissue culture plates (105 cells/2 mL medium/well) and treated with 29.7 and 110 |jM of 2b on the following day for 24, 48 and 72 hours. Thereafter, both adherent and floating cells were collected, washed with PBS and fixed overnight in 70% ethanol at -20°C. Fixed cells were treated with RNase A (0.1 mg/mL, Sigma-Aldrich) for 1 h at room temperature and afterward stained with pro-pidium iodide (50 |jg/mL, Sigma-Aldrich) for 30 min in the dark. The DNA content was analysed by flow cytometry (FACS Calibur, Becton Dickinson, Mountain View, CA, USA). Data were analysed with ModFitLTTM program (Verity Software House Inc., Topsham, ME, USA). Experiment was repeated more than three times. Determination of DNA binding The calf thymus (ct)-DNA was purchased from Sigma-Aldrich, dissolved in Na-cacodylate buffer, I = 0.05 mol/dm3, pH = 7, additionally sonicated and solution filtered through a 0.45 mm filter. Polynucleotide concentration was determined spectroscopically as the concentration of phosphates by e260 nm = 6600 dm3/mol1 cm-1. Thermal denaturation curves for ct-DNA and its complexes with studied compounds were determined in Na-cacodylate buffer, I = 0.05 mol/dm3, pH = 7 by following the absorption change at 260 nm as a function of temperature, as previously described.2324 The absorbance of the compound was subtracted from each curve and the absorbance scale was normalized. Measured Tm values are the midpoints of the transition curves determined from the maximum of the first derivative and checked graphically by the tangent method. The DTm values were calculated subtracting Tm of the free nucleic acid from Tm of the complex. Every ATm value reported here was an average of at least two measurements. The error in DT is ± 0.5°C. m Radiol Oncol 2016; 50(3): 280-288. 283 Steiner I et al. / 1,2,3-triazolium salts as potential anticancer drugs TABLE 1. IC50 values of triazolium salts and some parent triazoles against cervical carcinoma HeLa cells Cmpd. IC50 (|iM)a 1a > 100 1b > 100 1f > 100 2a 91.6 ± 3.9 2b 57.0 ± 12.9 2c > 100 2d > 100 2e 55.4 + 9.4 2f —b 2g —c 2h 88.9 + 7.5 2i 54.4 + 14.7 2j 54.9 + 3.5 2k > 100 2l —c a IC50 is the concentration of the triazoles and triazolium salts inducing 50% cell growth inhibition after 72 h incubation. The results are shown as mean values of at least three experiments (± SD). b Triazolium salt precipitated promptly after the addition to the growth medium and thus the cytotoxicity could not be measured accurately. c The range of concentrations 10 - 1000 [jM reduced survival from about 60 to 40%, and therefore the exact IC50 was difficult to determine. Induction of reactive oxygen species (ROS) Generation of ROS was determined by addition of 5-(and-6)-chloromethyl-2',7'-dichlorodihydro-fluorescein diacetate, acetyl ester (CM-H2DCFDA) (Invitrogen). Briefly, logarithmically growing H460 cells were incubated with 10 mM CM-H2DCFDA for 1 hour according to manufacturer's instructions. Afterward, cells were incubated with or without different concentrations of 2b during indicated time periods. After trypsinization and cen-trifugation, the cells were fixed in cold 80% methanol. Shortly before measurement, they were centri-fuged and resuspended in PBS. The fluorescence of the product, developed by removal of the acetate groups from CM-H2DCFDA by intracellular esterases and oxidation, was measured by flow cytom-etry on BC Navios instrument (Beckman Coulter, Inc., Miami, FL, USA). To further examine whether toxicity of 2b is coupled with formation of ROS, two ROS scavengers were used: NAC, a drug that has been known for years to directly reduce the level of ROS25 or the new ROS scavenger tempol.26 Their effect was determined by MTT assay or colony-forming assay as described in Cytotoxicity assay. Statistical analysis All data were analysed by unpaired Student's t-test, and expressed as the mean ± standard error of the mean. Data were considered significant when P values were lower than 0.05, and in the figures these are designated as * = P < 0.05 or ** = P < 0.01. Experiments were repeated at least three times. Results and discussion Cancer is the second leading cause of death in developed countries.1 Primary or acquired drug resistance and heavy side-effects strongly limit the effectiveness of classical chemotherapy. The success of advanced, target-oriented cancer therapy is at present limited only to the special types of can-cers.3 This provides a great impetus for investigation of new compounds with potential anticancer activities. 1,2,3-Triazoles are very important class of heterocycles, which have been well-recognized for their broad range of biological activities, including anticancer activity.8-16 This and the fact that no cytotoxic activity of the unfused 1,3,4-trisubstituted 1,2,3-triazolium salts has yet been reported encouraged us to examine the cytotoxic activity of compounds 2a - l along with some selected parent 1,4-disubstituted triazoles 1a, 1b and 1f. Antiproliferative effects of triazoles and triazolium salts The effect of tested triazoles and triazolium salts was first evaluated in HeLa cells, the cell model system that we previously found suitable for screening of new compounds.2728 The results are collected in Table 1. It appears that in the picolyl series of the triazolium cations (2a - f) the aryl substituent modulated the cytotoxicity against the tumour cells, with the electron donating 4-methoxyphenyl group being greater as compared to the electron neutral phenyl and the electron withdrawing 4-(trifluoromethyl)-phenyl groups. In this series of the compounds the pyridine ring was separated from the triazole core by a methylene bridge and thus less likely influences the electron density at the latter. The situation dras- Radiol Oncol 2016; 50(3): 280-288. 284 Steiner I et al. / 1,2,3-triazolium salts as potential anticancer drugs tically changes in the pyridyl series (2g - l) were the pyridine ring was attached directly to the triazole, now playing an important role in the cytotoxicity. In 2g - l the triazole core was functionalized with pyridine in two different ways, either through the triazole N1 nitrogen atom as in 2g - i, or through the triazole C4 carbon atom (2j - l). Interestingly, in the former (2g - i), the electron deficient 4-(trifluo-romethyl)phenyl group (2i) increased the cytotox-icity whereas in the latter series of compounds the member with the electron neutral phenyl group (2j) was identified as the most active. Apparently, no clear structure-activity correlation, based purely on the electronic considerations of the triazolium cations, could be drawn at this point. Noteworthy, different chemical reactivity patterns of the four isomeric triazolium cations have been previously observed in some chemical transformations, with the structure-reactivity relation still remaining to be fully understood.29 It is reasonable to expect that in a far more complex system such as the living cell, the structure-activity relation is likely to be a result of combined stereo-electronic effects and could be addressed after considerably larger library of derivatives have been assayed. TABLE 2. Cytotoxic activity of 2b against different cell lines Time (h) 24 48 72 110 IU-Vt FIGURE 2. Effects of 2b on the cell cycle of H460 cells. Logarithmically growing H460 cells were treated with 29.7 and 110 |jM of 2b for indicated period of time. Afterwards they were harvested for cell cycle analysis measured by FACS as described in Materials and methods. Representative data of three experiments are shown. Cell line IC50 (MM)' T.I.c HeLa HEp-2 7T H460 HCT-116 Fibroblasts 57.0 ± 12.9 87.0 ± 28.5 111.7 ± 7.6 29.7 + 4.5 51.9 ± 7.2 228.5 ± 5.6 4.01 2.63 2.05 7.69 4.40 HeLa = cervical carcinoma cells; HEp-2 = laryngeal carcinoma cells; 7T = carboplatin and cisplatin-resistant HEp-2 subline; H460 = large cell lung carcinoma cells; HCT-116 = colorectal carcinoma cells; Fibroblasts = normal primary fibroblasts. The results are shown as mean values of three experiments (±SD). b IC50 is the concentration of 2b that induces 50% cell growth inhibition after 72 h incubation. c T.I. refers to therapeutic index, calculated from the ratio of cytotoxicity (IC50) on normal fibroblasts and cytotoxicity (IC50) on tumour cells. In order to compare the results with those for the triazolium salts 2, three parent triazoles 1a, b, f were also selected for the biological screening. None of them exhibited cytotoxic activity against HeLa cells with IC50 below 100 |jM. To shed more light on the mechanisms responsible for the cytotoxic effect of above examined compounds, we decided to proceed further with compound 4-(4-methoxyphenyl)-3-methyl-1-(2-picolyl)-1H -1,2,3-triazolium hexafluorophosphate(V) (2b) as a representative compound. First, we explored its antiproliferative activity on several tumour cell lines from different origin, as well as on the normal human fibroblasts. The results are shown in Table 2. Compound 2b strongly inhibited the growth of all examined tumour cell lines and this effect was cell-type specific. Human large cell lung carcinoma H460 cells were the most sensitive toward 2b (and were selected for further studies), while HEp-2 cells were most resistant. Difference in sensitivity between most sensitive H460 and most resistant 7T cells (for IC50 value) was almost 4 times. Drug resistance is the major cause of failure in successful treatment of cancer patients.3031 It is based on the variety of complex mechanisms.30-35 The compounds that might be efficient against drug-resistant cells could be of great help in improvement of cancer treatment. Therefore we also included carboplatin, cisplatin and mitomycin C resistant 7T subline of HEp-2 cells in our study. As shown in Table 2, both, parental HEp-2 and drug-resistant 7T cells are similarly sensitive to 2b compound, suggesting a potential future application of Radiol Oncol 2016; 50(3): 280-288. 285 Steiner I et al. / 1,2,3-triazolium salts as potential anticancer drugs TABLE 3. Effect of compound 2b on the cell cycle of H460 cells Conc. (MM) 24 h 48 h 72 h G1 S G2/M subGl G1 S G2/M subGl G1 S G2/M subGl 0 54 31 15 2 59 26 15 2 73 13 14 2 29.7 61 24 15 2 61 26 13 5 65 17 18 6 110 78 9 13 3 78 9 13 15 79 5 16 35 H460 cells were treated for the indicated time period with 2b, stained with propidium iodide and analysed by flow cytometry. Cell cycle distribution was assessed as described in the Materials and methods section. 0.24 0 22 0.08 Ct-DNA+2b Ct-DNA V 65 70 95 100 75 80 85 9( Temperature (° C) FIGURE 3. DNA as possible target of compound 2b. Thermal > M, 0.05 denaturation curves of ct-DNA (c(ct-DNA) = 2 x 10-=0.3) at pH 7.0 (sodium cacodylate buffer, I = [compound]/[ct-DNA] M) upon addition of compound 2b. Error in DTm values: ±0.5°C. 2b compound in clinical treatment of cisplatin and carboplatin resistant tumours. For comparison, 7T cells were 3.3 fold more resistant to cisplatin as shown previously.19 One of the most requested characteristics of potential anticancer drug is its higher efficacy against tumour than the normal cells. In this study all examined tumour cell lines were more sensitive to compound 2b than normal cell line, with the therapeutic index higher than 2 (Table 2). Specifically, for H460 cells it was particularly high, 7.69, fulfilling the request of this essential characteristic for a potential anticancer compound. Effect of compound 2b on the cell cycle of H460 cells To gain more insight into the mode of action that underlies the antiproliferative activity of 2b, we investigated its effect on the cell cycle in H460 cells. The flow cytometric analysis is presented in Figure 2, and Table 3. They show that compound 2b arrested the cells in the G1 phase of the cell cycle in dose-dependent manner, even after 24 hours of treatment. At later time points a dose- and time-dependent increase was detected in a fraction of cells with reduced DNA content (subGl), which represents the apoptotic cells subG1 fraction. These results suggest that 2b induces apoptosis in treated cells. DNA as possible target of compound 2 b According to their structure, triazolium salts resemble the structures of DNA minor groove binders like those studied by Chenoweth and Dervan36, that show precise recognition of the DNA sequence by thermodynamically controlled "H-bond based reading" of predesigned heterocycle-polyamide molecules. Some non-condensed heterocyclic molecules also proved to be DNA intercalators.837 Therefore we studied the interactions of 2b with double strand (ds) DNA. Thus, in thermal dena-turation experiment compound 2b was mixed with ct-DNA in a ratio r[compound]/[ct-DNA] = 0 3, at which any DNA binding mode should give a measurable change in DNA melting point transition. However, in the thermal denaturation experiment no measurable change in DNA melting point transition was observed, and no influence on the thermal stability of ct-DNA (Figure 3), indicating that at biologically relevant conditions (pH 7, c(compound) = 6 x 10-6 M) stable non-covalent complex with ds DNA was not formed, suggesting that DNA was not the target of 2b compound. Formation of ROS by compound 2b Although 2b does not bind to DNA, alternative mechanisms of 2b action and cytotoxicity were examined. Literature data indicate that diverse compounds can induce cell damage due to formation of ROS.3839 ROS may irreversibly oxidize DNA, nucleic acids, proteins, and lipids, thereby representing the primary source of damage in biological systems that may eventually lead to cell death.40,41 Accordingly, we directly measured the induction of ROS formation following the treatment with 2b. For this, we stained the cells with 10 mM CM- Radiol Oncol 2016; 50(3): 280-288. 286 Steiner I et al. / 1,2,3-triazolium salts as potential anticancer drugs m 185 ^M 2b 110 |jM 2b / \ 05 MM 2b / \ 40 mM 2b / \ 0 |jM 2b / V FIGURE 4. Formation of ROS by 2b in H460 cells. Logarithmically growing H460 cells were stained for 1 hour with 10 mM CM-H2DCFDA and then either treated with 110 |jM 2b during indicated time points (A) or treated with indicated concentrations of 2b for 180 min (3 hours) (B). Afterward ROS formation was determined by flow cytometry as described in Materials and methods section. Dose-dependent formation of ROS was additionally presented by cell count and fluorescence intensity of CM-H2DCFDA. M1 line is positioned to designate MFI value of the non-treated sample (white histogram) compared to signals obtained upon cell treatment with indicated concentrations of 2b. FIGURE 5. The effects of ROS scavenger on survival of H460 cells treated with 2b determined by MTT assay. H460 cells were seeded and next day pretreated for 2 hours with 5 mM of NAC or 1 mM tempol. Afterwards different concentrations of 2b were added. The cell survival was determined 72 hours later by MTT assay as described in Materials and methods section. Each point represents the mean ±SD of at least three independent experiments. All data are expressed as the average percentage of survival values relative to an untreated control ± SD or samples treated with antioxidants alone. The significance in differences is indicated (*, P < 0.05; **, P < 0.01). H2DCFDA for one hour and then treated them either with 110 |jM 2b during different time points (Figure 4A) or with different concentrations of 2b during 3 hours (Figure 4B). For better illustration of ROS formation upon 2b treatment during 3 hours we presented additionally results as CM-H2DCFDA fluorescence intensity compared to cell number (count). As shown in Figure 4, 2b induced ROS in time- and dose-dependent manner. Dose-dependent skewing of signals toward higher fluorescence intensity with increased concentration of compound is noticeable (Figure 4B). In order to confirm the formation of ROS and validity of detection we incubated the cells in each experiment with 0.01% H2O2 for 30 min (data not shown). To approve this result and additionally examine the possible role of 2b-induced formation of ROS in cell toxicity, we pretreated for 2 hours H460 cells with two different ROS scavengers: NAC and tem-pol. As shown in Figure 5, the pretreatment of cells with either NAC or tempol increased survival of 2b treated cells compared to the cells treated only with 2b, indicating that 2b induced ROS and that the cytotoxicity of 2b can be reduced by addition of ROS scavenger. To confirm the data that we obtained by MTT assay (Figure 5), we have done additional experiments using colony-forming assay and chronical exposure to 2b with ROS scavenger. The preteratment of HEp-2 cells (a less responsive cell line to 2b compound) with either NAC or tem-pol increased the survival of 2b treated cells compared to the survival of cells treated only with 2b (Figure 6A). Similar results were obtained with the most sensitive cell line to 2b compound, i. e. H460 cells treated with NAC. Again, the pretreatment with this ROS scavenger increases the survival of cells as compared to the survival of cells treated only with 2b. However H460 cells were highly sensitive to tempol. The highest non-toxic concentration of tempol for chronic treatment of H460 cells was 0.125 mM, which is 8 times lower concentration than could be applied for HEp-2 cells (see Figure 6B). It is possible that this concentration of tempol was too low to scavenge ROS induced by 2b. Perhaps, higher concentration could protect the cells from toxic effect of 2b, but higher concentration used during 10 days incubation was too toxic for H460 cells. H460 and HEp-2 cells differ in their p53 status: H460 has wild type p53, while HEp-2 cells have mutated p53. While p53 has important role in cell response to oxidative stress and apop-tosis42, we can assume that wild type p53 can be involved in increased H460 sensitivity to chronic treatment with tempol. Radiol Oncol 2016; 50(3): 280-288. 287 Steiner I et al. / 1,2,3-triazolium salts as potential anticancer drugs Thus, experiments in which the cells were pre-treated with ROS scavengers suggest that 2b indeed induced ROS in treated cells and that ROS induction was involved in cytotoxicity, but that induction of ROS is not the only mechanism of action of the selected compound 2b. Conclusions In conclusion, pyridine tethered 'click' triazolium salts have been tested for their anticancer activity for the first time. As revealed on human cervical carcinoma HeLa cells, selected compound (4-(4-methoxyphenyl)-3-methyl-1-(2-picolyl)-1H-1,2,3-triazolium hexafluorophosphate(V) (2b), exhibits high cytotoxicity. Its antiproliferative activity was cell type dependent, being mostly cytotoxic against large cell lung carcinoma H460 cells. It is of utmost importance that 2b was significantly more cytotoxic against tumour cells than normal cells, having very high therapeutic index, such as 7.69 for H460 cells. Additionally, this compound was similarly cytotoxic against parental laryngeal carcinoma HEp-2 cells and their drug resistant 7T subline which is, having in mind the importance of inhibitory effect of drug resistance on the success of cancer treatment, a very valuable result. Compound 2b arrested tumour cells in the G1 phase of the cell cycle and induced programmed cell death. This compound does not form a complex with ds DNA, but rather induced ROS in treated cells which further triggers cell death. In short, our results suggest that the 'click' triazolium salts are simple to make compounds that are worth of further investigation as anticancer agents. Work is in progress to design and examine an extended library of their analogues. 100 -| 80 - I (Ö 60 - > tn 40 - flj O 20 - 0 - nh ■ 2b ] 2b + 5 mM NAC i2b +0.125 mM tempol 18 24 Concentration (jjM) 30 ® ® FIGURE 6. The effect of ROS scavenger on survival of HEp-2 (A) and H460 (B) cells treated with 2b determined by colony-forming assay. HEp-2 and H460 cells were seeded and next day pretreated for 2 hours with 5 mM of NAC or 1 mM (HEp-2 cells) or 0.125 mM (H460 cells) tempol. Afterwards different concentrations of 2b were added. Ten days later the colonies were counted. Non-treated cells and cells treated with antioxidants alone were used as controls. Each point represents the mean ±SD of at least three independent experiments. All data are expressed as the average percentage of survival values relative to an untreated control ± SD or samples treated with antioxidants alone. The significance in differences is indicated (*, P < 0.05; **, P < 0.01). References Acknowledgments This study was supported by the Ministry of Science, Education and Sport of the Republic of Croatia (Projects 098-0982913-2748, 098-09829132850 and 098-0982914-2918). The Ministry of Education, Science and Sport, Republic of Slovenia, the Slovenian Research Agency is gratefully acknowledged for the financial support (Project P1-0230; Young Researcher Grant to A.B.). Financial support from Joint Projects BI-HR/12-13-028 and BI-HR/14-15-007 is also acknowledged. We thank Ms. Snjezana Juler and Ana Tupek for technical assistance. 1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin 2012; 62: 10-29. 2. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; 144: 646-74. 3. Duffy MJ. 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Radiol Oncol 2016; 50(3): 280-288. 289 research article Functional polymorphisms in antioxidant genes in Hurthle cell thyroid neoplasm -an association of GPX1 polymorphism and recurrent Hurthle cell thyroid carcinoma Blaz Krhin1, Katja Goricar2, Barbara Gazic1, Vita Dolzan2, Nikola Besic1 1 Institute of Oncology Ljubljana, Slovenia 2 Pharmacogenetics Laboratory, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Slovenia Radiol Oncol 2016; 50(3): 289-296. Received 4 January 2016 Accepted 2 May 2016 Correspondence to: Blaž Krhin, Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. Phone: +386 1 587 9838; Fax: +386 1 587 9810; E-mail: bkrhin@onko-i.si; Disclosure: The authors declare no conflict of interest. Background. Hurthle cells of the thyroid gland are very rich in mitochondria and oxidative enzymes. As a high level oxidative metabolism may lead to higher level of oxidative stress and can be associated with an increased risk for cancer, we investigated whether common functional polymorphisms in antioxidant genes (SOD2, CAT, GPX, GSTPI, GSTM! and GSTTI) are associated with the development or clinical course of Hurthle cell thyroid carcinoma (HCTC). Methods. A retrospective study was performed in 139 patients treated by thyroid surgery for a Hurthle cell neoplasm. HCTC, Hurthle cell thyroid adenoma (HCTA) or Hurthle cell thyroid nodule (HCTN) were diagnosed by pathomorphology. DNA was extracted from cores of histologically confirmed normal tissue obtained from formalin-fixed paraffin-embedded specimens and genotyped for investigated polymorphisms. Logistic regression was used to compare genotype distributions between patient groups. Results. HCTC, HCTA and HCTN were diagnosed in 53, 47 and 21 patients, respectively. Metastatic disease and recurrence of HCTC were diagnosed in 20 and 16 HCTC patients, respectively. Genotypes and allele frequencies of investigated polymorphisms did not deviate from Hardy-Weinberg equilibrium in patients with HCTC, HCTA and HCTN. Under the dominant genetic model we observed no differences in the genotype frequency distribution of the investigated polymorphisms when the HCTA and HCTN group was compared to the HCTC group for diagnosis of HCTC or for the presence of metastatic disease. However, GPX! polymorphism was associated with the occurrence of recurrent disease (p = 0.040). Conclusions. GPX! polymorphism may influence the risk for recurrent disease in HCTC. Key words: Hurthle cell thyroid carcinoma; Hurthle cell neoplasm; thyroid; oxidative stress; antioxidant genes Introduction Hurthle cell thyroid carcinoma (HCTC) is a rare type of differentiated thyroid cancer (DTC). Traditionally, HCTC was regarded as a subtype of follicular thyroid cancer, while new evidence indicates that HCTCs may have a distinct molecular profile compared to other DTCs.1 Clinically, and compared to other DTCs, HCTCs are considered more aggressive, with worse prog- nosis, requiring more stringent follow-up. HCTCs are also more likely to metastasize to neck soft tissue and distant sites, are more iodine resistant and have higher tumour-related mortality.1-4 A definitive way to differentiate a HCTC from a benign Hurthle cell thyroid adenoma (HCTA) is based on vascular and/or transcapsular invasion.5-9 For HCTA, a lobectomy is a sufficient surgical procedure. However, if a HCTC is diagnosed on histo-logic sections after a lobectomy, then a complete Radiol Oncol 2016; 50(3): 254-262. doi:10.1515/raon-2016-0023 290 Krhin B et al. / Antioxidant genes polymorphisms in Hurthle cell thyroid neoplasm thyroidectomy is performed as a second surgical procedure. Therefore, when a follicular neoplasm is detected with a cytological analysis of material obtained by fine-needle aspiration biopsy, the use of predictive clinical410 or genetic markers11 has been proposed, before deciding on the extent of the thyroid surgical procedure. A Hurthle (oncocytic) cell has abundant granular eosinophilic cytoplasm, which has such an appearance because of the accumulation of a large number of mitochondria. A full-blown Hurthle cell has 4000 to 5000 mitochondria, while a human cell rich in mitochondria (oocyte) has about 1500 mitochondria only.712 Enzyme histochemistry studies have shown that Hurthle cells contain high concentrations of oxidant enzymes.13 The respiratory redox chain in the mitochondria is considered the major source of reactive oxygen species (ROS) and other free radicals in the cell.14 ROS and other free radicals can oxidize target cellular proteins, membrane lipids, nucleic acids and damage their cellular structure and function. Effective protective mechanisms, comprising anti-oxidative molecules and compartmentalization of potentially toxic molecules, have been developed to maintain a balance between generation and detoxification of reactive oxygen species (ROS) under physiological conditions. In case of excessive ROS oxidative stress occurs.1516 To prevent this, complex defence mechanisms including many enzymes, proteins and antioxidants are involved. Antioxidant enzymes such as manganese superoxide dismutase (Mn-SOD), glutathione peroxidase (GPX) and catalase (CAT) directly eliminate ROS, while glutathione-S-transferases (GSTs) detoxify cytotoxic secondary metabolites. Numerous functional polymorphisms in the genes coding for an-tioxidant enzymes have been described that may also modify their ROS detoxification capacity.17 Oxidative stress and ROS have been associated with several cancers and also many complex diseases like cardiovascular disease, diabetes melli-tus and neurodegenerative disorders.1518 Several studies also found a connection between oxidative stress and thyroid diseases including neoplasia and thyroid cancer.1619-25 However all these studies have been done on papillary thyroid carcinoma and/or follicular thyroid carcinoma. As Hurthle cells are very rich in mitochondria and oxidative enzymes, it is possible that antioxidant enzymes may have an important role in defence against oxi-dative stress. To our knowledge, there are no data in the literature about oxidative stress and HCTC or HCTA. Furthermore, there are no data about the association between HCTC/HCTA and polymorphisms of genes coding for antioxidant enzymes. Patients and methods Patients A retrospective study included Slovenian patients treated by thyroid surgery for a Hurthle cell neoplasm at the Institute of Oncology Ljubljana. The medical records of all the patients were reviewed and a total of 167 patients with cytological features for a Hurthle cell neoplasm were selected for molecular analysis. As 28 patients had no sufficient formalin-fixed and paraffin-embedded (FFPE) material for DNA extraction, they were excluded from the study. Eventually, 139 patients were included. All the patients had a Hurthle cell neoplasm diagnosed by fine-needle aspiration cytology and the majority of fine-needle aspiration biopsies were ultrasound guided.410 The cytological criteria for Hurthle cell neoplasms were hypercellularity, with a predominance of Hurthle cells (at least 75%), few or no lymphocytes, and scant or no colloid.26 Cytological slides were examined by cytopatholo-gist, experienced in thyroid pathomorphology. Final diagnosis of HCTC/HCTA/other was obtained by definitive histology of thyroid tissue obtained by surgical procedure. The histological features for HCTC were based on vascular invasion and/or transcapsular invasion.26 Histology slides were examined by a pathologist, experienced in thyroid pathomorphology. All patients with HCTC diagnosis were regularly monitored for possible recurrent or meta-static disease. The median follow-up time was 105 (1-337) months.4 The study was reviewed and approved by the Slovenian Ethics Committee for Research and Medicine (No: KME 32/12/11) and was carried out according to the Declaration of Helsinki. The study was also approved by the Institute of Oncology Ljubljana Protocol Review Board. Methods Hematoxylin and eosin (H&E) stained slides from FFPE samples were examined by a pathologist, experienced in thyroid pathomorphology, to confirm the diagnosis and to select areas representative of normal tissue. Two to three cores (1 mm in diameter) of histologically confirmed normal tissue were obtained from each specimen for DNA extraction using a QiaAmp Mini kit (Qiagen, Hilden, Radiol Oncol 2016; 50(3): 289-296. 291 Krhin B et al. / Antioxidant genes polymorphisms in Hurthle cell thyroid neoplasm Germany) according to the manufacturer's instructions. Genotyping of SNPs in SOD2 rs4880 (c.47C>T; p.Val16Ala), CAT rs1001179 (c.-262C>T; c.-262G>A), GPX1 rs1050450 (c.599C>T; p.Pro200Leu), GSTP1 rs1695 (c.341C>T; p.Ile105Val) and GSTP1 rs1138272 (c.313A>G; p.Ala114Val) was carried out using a fluorescence-based competitive allele-specific (KASPar) assay (Kbiosciences, Herts, UK) according to the manufacturer's instructions. Amplifications were performed in a PCR system 9700 AB (Applied Biosystems, California, USA) as recommended by the manufacturer (Kbiosciences). Fluorescence was measured on a 7500 Real Time PCR System AB and allele discrimination data analyzed with 7500 System SDS Software (both Applied Biosystems). GSTM1 and GSTT1 gene deletions were detected using a multiplex PCR simultaneously amplifying GSTM1, GSTT1 and BGLO genes as described previously.27 With this approach, we could identify homozygous GSTM1 or GSTT1 gene deletion, but we were not able to distinguish between carriers of one or two copies of each gene. Genotyping was repeated in 20% of samples to check for genotyp-ing accuracy. Statistical analysis Median and interquartile ranges were used to describe central tendency and variability of continuous variables, while frequencies were used to describe the distribution of categorical variables. A standard chi-square test was used to assess the deviation from Hardy-Weinberg equilibrium (HWE). Logistic regression was used to compare genotype distributions between patient groups and to calculate odds rations (ORs) and 95% confidence intervals (CIs). All statistical analyses were carried out using IBM SPPS Statistics version 19.0 (IBM Corporation, Armonk, NY, USA). A dominant genetic model was used in all statistical analyses and the level of statistical significance was set at 0.05. Haplotype analysis was performed using Thesias software28 as previously described.29 Results In total 139 patients with cytological features for Hurthle cell neoplasm were included in the study. The female to male sex ratio was 3.8:1. Median (range) age was 54 (42-66) years. Median diameter TABLE 1. Clinical and demographic characteristics of patients with Hurthle cell neoplasms HCTA + HCTN HCTC Number [N] (%) 68 (56.2) 53 (43.8) Median age [years] (range ) 49.5 (38.5-57.8) 62 (45.5-70.5) Gender F/M [N] (%) 58/10 (85.3/14.7) 37/16 (69.8/30.2) Median tumor diameter [mm] (range ) 26.0 (16.0-34.8) 40.0 (25.5-65.0) Metastasis (%) / 20 (37.7) Recurrence (%) / 16 (30.2) Concomitant disease N (%) 16 (23.5) 20 (37.7) Hashimoto thyroiditis 11 (16.2) 12 (22.6) Diabetes mellitus 1 (1.5) 7 (13.2) Graves' disease 2 (2.9) 3 (5.7) Non-thyroid Malignancy 2 (2.9) 2 (3.8) F= female; HCTA = Hurthle cell thyroid adenoma; HCTC = Hurthle cell thyroid carcinoma; HCTN = Hurthle cell thyroid nodule; M = male of the tumour was 28 (20-45) mm. The final diagnosis was established by definitive histology of the thyroid tissue obtained by a surgical procedure. Patients were diagnosed as follows: 53 (38.1%) had HCTC, 47 (33.8%) HCTA, 21 (15.1%) Hurthle cell thyroid nodule (HCTN), 11 (7.9%) multi nodular goiter, 4 (2.9%) follicular thyroid adenoma, while 2 (1.4%) patients had lymphocyitic thyroiditis. In 46 (33%) patients, concomitant disease was recorded: 31 (22.3%) had Hashimoto thyroiditis, 12 (8.6%) diabetes mellitus, 7 (5.0%) Graves' disease, and 4 (2.9%) patients had other malignant disease not present in the thyroid tissue. Only patients with a final diagnosis of HCTC, HCTA or HCTN were selected for molecular analysis. The group of patients with HCTA or HCTN was compared to the group of patients with HCTC. Altogether 20 of 53 (37.7%) patients with HCTC had metastatic disease. Recurrent disease was observed in 16 (30.0%) patients with HCTC. The clinical and demographic characteristics of those patients are summarized in Table 1. The patients from the HCTC group had a different gender (F/M) ratio (p = 0.043), were older (p = 0.004) and had a larger tumour diameter (p < 0.001) in comparison to the patients from the HCTA or HCTN group (Table 2). In the HCTC group, independent risk factors for both metastatic disease and recurrent disease were the patient's age and tumour diameter as shown by logistic regression analysis (Table 2). Genotype frequencies of the investigated polymorphisms in patients with HCTC, HCTA and Radiol Oncol 2016; 50(3): 289-296. 292 Krhin B et al. / Antioxidant genes polymorphisms in Hurthle cell thyroid neoplasm TABLE 2. Association of clinical and demographic characteristics with Hurthle cell thyroid neoplasms, metastatic disease and recurrent disease HCTA+HCTN versus HCTC Metastatic disease Recurrent disease OR (95% CI) Pa OR (95% CI) Pa OR (95% CI) pa Gender 2.51 (1.03-6.12) 0.043 2.08 (0.63-6.90) 0.230 2.42 (0.70-8.37) 0.163 Age 1.04 (1.01-1.06) 0.004 1.07 (1.02-1.12) 0.005 1.05 (1.01-1.10) 0.026 Tumor diameter 1.05 (1.02-1.07) < 0.001 1.09 (1.04-1.14) < 0.001 1.04 (1.01-1.07) 0.005 Concomitant disease 1.97 (0.90-4.34) 0.092 0.83 (0.26-2.63) 0.749 0.83 (0.26-2.63) 0.523 CI = confidence interval; HCTA = Hurthle cell thyroid adenoma; HCTC = Hurthle cell thyroid carcinoma; HCTN = Hurthle cell thyroid nodule; OR = odds ratio; a = p less than 0.05 was considered statistically significant HCTN are shown in Table 3. The observed genotype frequencies did not deviate from Hardy-Weinberg equilibrium in the whole cohort of patients (p > 0.050, Table 3). The association of SOD2, CAT, GPX1 and GST polymorphisms with diagnosis of Hurthle cell neoplasm and with the presence of metastatic or recurrent disease are presented in Table 4. These associations were also adjusted for clinical parameters. Since gender, age and tumour diameter were correlated in a multivariable model, only tumour diameter was used for adjustment. Under the dominant genetic model, no significant differences in the genotype frequency distribution of the investigated polymorphisms were observed when the HCTA and HCTN group was compared to the HCTC group (all p > 0.050). These polymorphisms were also not associated with metastatic disease (all p > 0.050). However, GPX1 polymorphism was associated with the presence of recurrent disease (p = 0.040). The association of GPX1 polymorphism and recurrent disease was even greater when adjusted for tumour diameter (p = 0.036). TABLE 3. Genotype frequencies in patients with Hurthle cell neoplasms Gene Polymorphism Genotype All patients (%) P 1 HWE HCTA+HCTN (%) HCTC (%) CC 26 (21.7) 0.903 12 (17.9) 14 (26.4) SOD2 rs4880; c.47C>T; p.Val16Ala CT 59 (49.2) 34 (50.7) 25 (47.2) TT 35 (29.2) 21 (31.3) 14 (26.4) CC 70 (58.3) 0.907 35 (52.2) 35 (66.0) CAT rs1001179; c.-262C>T; c.-262G>A CT 43 (35.8) 30 (44.8) 13 (24.5) TT 7 (5.8) 2 (3) 5 (9.4) CC 63 (52.1) 0.424 35 (51.5) 28 (52.8) GPX1 rs1050450; c.599C>T; p.Pro200Leu CT 51 (42.1) 32 (47.1) 19 (35.8) TT 7 (5.8) 1 (1.5) 6 (11.3) CC 54 (44.6) 0.653 28 (41.2) 26 (49.1) GSTP1 rs1695; c.341C>T; p.Ile105Val CT 52 (43.0) 32 (47.1) 20 (37.7) TT 15 (12.4) 8 (11.8) 7 (13.2) AA 103 (85.1) 0.159 58 (85.3) 45 (84.9) GSTP1 rs1138272; c.313A>G; p.Ala114Val AG 16 (13.2) 8 (11.8) 8 (15.1) GG 2 (1.7) 2 (2.9) 0 (0) GSTM1 Gene deletion Wild type 55 (50.9) /a 33 (50.8) 22 (51.2) Gene deletion 53 (49.1) 32 (49.2) 21 (48.8) GSTT1 Gene deletion Wild type 93 (86.1) /a 54 (83.1) 39 (90.7) Gene deletion 15 (13.9) 11 (16.9) 4 (9.3) HCTA = Hurthle cell thyroid adenoma; HCTC = Hurthle cell thyroid carcinoma; HCTN = Hurthle cell thyroid nodule; HWE = Hardy-Weinberg equilibrium a HWE could not be evaluated for GSTM1 and GSTT1 as we were not able to distinguish between carriers of one or two copies of each gene. Radiol Oncol 2016; 50(3): 289-296. 293 Krhin B et al. / Antioxidant genes polymorphisms in Hurthle cell thyroid neoplasm TABLE 4. Association of SOD2, CAT, GPX1 and GST polymorphisms with diagnosis of Hurthle cell neoplasm, presence of metastatic disease and occurrence of recurrent disease Gene Genotype Diagnosis (HCTA+HCTN vs. HCTC) Metastatic disease Recurrent disease OR a OR-adjb ..„ (95% CI) P (95% CI) p-adj OR a OR-adjb d.b (95% CI) P (95% CI) p-adj OR a OR-adjb d.b (95% CI) P (95% CI) p-adj SOD2 rs4880 CC CT+TT 0.61 (0.25-1.46) 0.264 0.65 (0.25-1.67) 0.373 1.12 (0.32-4.00) 0.856 0.72 (0.12-4.09) 0.706 1.11 (0.29-4.26) 0.878 0.82 (0.18-3.62) 0.788 CAT rs1001179 CC CT+TT 0.56 (0.27-1.18) 0.129 0.81 (0.36-1.81) 0.600 0.34 (0.09-1.24) 0.102 0.57 (0.11-2.91) 0.499 1.25 (0.37-4.25) 0.721 2.95 (0.66-13.1) 0.155 GPXI rs1050450 CC CT+TT 0.95 (0.46-1.94) 0.882 1.02 (0.46-2.24) 0.962 0.63 (0.20-1.93) 0.417 0.72 (0.15-3.52) 0.682 0.25 (0.07-0.94) 0.040 0.19 (0.04-0.89) 0.036 GSTP1 rs1695 CC CT+TT 0.73 (0.35-1.50) 0.388 0.82 (0.37-1.82) 0.628 1.30 (0.43-3.96) 0.646 2.40 (0.47-12.13) 0.291 0.46 (0.14-1.52) 0.202 0.49 (0.13-1.89) 0.300 GSTP1 rs1138272 AA AG+GG 1.03 (0.38-2.83) 0.952 1.15 (0.39-3.45) 0.800 0.99 (0.21-4.67) 0.988 1.24 (0.17-9.19) 0.836 0.29 (0.03-2.54) 0.261 0.24 (0.02-2.64) 0.244 GSTMI Wild type Gene deletion 0.98 (0.46-2.13) 0.968 0.91 (0.39-2.12) 0.819 1.59 (0.47-5.39) 0.456 1.40 (0.23-8.57) 0.716 1.32 (0.38-4.64) 0.666 1.24 (0.28-5.41) 0.774 GSTTI Wild type Gene deletion 0.50 (0.15-1.70) 0.269 0.44 (0.11-1.82) 0.257 1.44 (0.18-11.29) 0.730 0.83 (0.02-39.34) 0.923 2.00 (0.25-15.85) 0.512 1.42 (0.1-20.98) 0.798 CI = confidence interval; HCTA = Hurthle cell thyroid adenoma; HCTC = Hurthle cell thyroid carcinoma; HCTN = Hurthle cell thyroid nodule; OR = odds ratio; a = p less than 0.05 was considered statistically significant; b = adjusted for tumor diameter TABLE 5. Association of GSTP1 haplotypes and diagnosis of Hurthle cell neoplasm, presence of metastatic disease and occurrence of recurrent disease Haplotype Estimated frequency Diagnosis (HCTA+HCTN vs. HCTC) Metastatic disease Recurrent disease OR (95% CI) pa OR (95% CI) pa OR (95% CI) pa AC 0.68 Reference Reference Reference GC 0.25 0.88 (0.49-1.60) 0.686 1.04 (0.38-2.86) 0.935 0.45 (0.13-1.64) 0.230 GT 0.07 0.83 (0.33-2.13) 0.704 0.99 (0.21-4.72) 0.988 028 (0.03-2.89) 0.288 CI = confidence interval.; HCTC = Hurthle cell thyroid carcinoma; HCTA = Hurthle cell thyroid adenoma; HCTN = Hurthle cell thyroid nodule; OR = odds ratio a - p less than 0.05 was considered statistically significant Haplotype analysis was performed to assess the combined effect of SNPs within the GSTP1 gene. As shown in Table 5, no associations were observed between GSTP1 haplotypes and diagnosis of HCTA/HCTN versus HCTC, the presence of metastatic disease or the occurrence of recurrent disease. Discussion In the present study, we investigated whether common functional polymorphisms in antioxidant genes could be used as molecular markers for the development of HCTC or its clinical course in patients with Hurthle cell neoplasms. In patients with cytological features for Hurthle cell neoplasm, different final diagnoses are made by definitive histology of thyroid tissue obtained by a surgical procedure. In our study group, 87% of patients with cytological features for Hurthle cell thyroid neoplasm had HCTC, HCTA or HCTN and were eligible for our study. Patient groups with benign HCTA and HCTN were combined and compared to a group with HCTC. The malignancy rate in our HCTC group was 44% and within the incidence rate of malignancy reported in the literature, where it ranged from 13%30 up to 70%.31 A significant difference in age was observed between the HCTA+HCTN group and the HCTC group, with patients in the HCTC group being nearly 12 years older and having a significantly larger median size of initial tumour (26 versus 40 mm). These findings are consistent with previous reports.103233 We also found a small gender difference, with a significantly larger F/M ratio in the HCTA+HCTN group as compared to the HCTC group. The two groups did not differ regarding the presence of concomitant disease. Metastases were diagnosed in 38% of patients with HCTC. Furthermore, 30% of patients developed a recurrent disease. These two groups of patients had a significantly larger initial Radiol Oncol 2016; 50(3): 289-296. 294 Krhin B et al. / Antioxidant genes polymorphisms in Hurthle cell thyroid neoplasm tumour diameter (69 versus 30 mm and 62 versus 30 mm, respectively) or were significantly older (67 versus 53 years and 65 versus 54 years, respectively) at initial diagnosis than the HCTC patients that did not have metastatic or recurrent disease. However, it has to be noted that our HCTC group with metastatic or recurrent disease was relatively small compared to non-metastatic or non-recurrent HCTC group. To establish whether common functional polymorphisms in genes coding for antioxidant genes could be used as molecular markers for the development of HCTC or its clinical course, we investigated associations between SOD2, CAT, GSTP1, GSTM1, GSTT1 and GPX1 genotypes and the clinical characteristics of patients with definite diagnosis of HCTC, HCTA or HCTN. CAT -262C>T genotype frequencies observed in our patient group were in accordance with those previously published for a healthy population.34-36 In our study CAT -262C>T polymorphism was not associated with HCTC, or metastatic or recurrent disease. To our knowledge CAT -262C>T has not been studied in HCTC, but higher CAT activity has been associated with papillary thyroid carcinoma and follicular carcinoma.2437 It has been demonstrated that CAT -262C>T polymorphism influences the binding of transcriptional factors and is associated with a decrease in enzyme expression353839, but also with higher CAT activity.3440 Also SOD2 Val16Ala genotype frequencies in our patients with Hurthle cell neoplasms were similar to frequencies previously reported in Caucasian patients.41-44 In our study SOD2 Val16Ala polymorphism was not associated with the occurrence of HCTC, or with metastatic or recurrent disease. To our knowledge this polymorphism has not been studied in HCTC yet. SOD2 Val16Ala polymorphism leads to less efficient transport of SOD2 into mitochondrial matrix in vitro45, but association studies of SOD2 in thyroid cancers gave inconclusive results. Two groups showed an increased SOD2/SOD level or activity in follicular and papillary thyroid cancer, while one group found no change of SOD activity in papillary thyroid can-cer.37 4647 On the other hand a reduced level of SOD2 was found in poorly differentiated thyroid cancers.48 Frequencies of GSTP1, GSTM1 and GSTT1 polymorphisms in our patients were similar to previously reported studies.49 However, in the HCTC group we noticed a lower percentage of GSTT1 gene deletion, compared to the HCTN/HCTA group. GSTP1 genotypes and haplotypes as well as GSTT1 and GSTM1 deletions were not associated with the occurrence of HCTC and neither with metastatic nor recurrent disease. Our findings are in agreement with a previous study that also found no association between GSTM1 and GSTT1 polymorphisms and HCTC.50 Both GSTP1 Ile105Val and GSTP1 Ala114Val decrease enzymatic activity5152, while GSTM1 and GSTT1 deletion polymorphisms result in the complete loss of enzymatic activity in homozygous carriers.53 Some previous studies have shown possible associations of GSTP1, GSTM1 or GSTT1 polymorphisms, or a combination of GSTT1 and GSTM1 null allele with papillary and/or follicular thyroid cancer54-58, while others found no association between these polymorphisms and primary or secondary thyroid can-cers.59-62 Frequencies of GPX1 Pro198Leu genotypes in our study group were also similar to the ones previously reported.63 We did not find any association of GPX1 Pro198Leu polymorphism with the occurrence of HCTC or with metastatic disease, even though several groups have found decreased activity or decreased expression of GPX1 in thyroid carcinomas24,47,64,65, while one group reported increased levels of GPX1 in papillary thyroid carci-noma.37 Several groups also reported that Leu variant could lead to lower GPX1 activity in patients with lung cancer, breast cancer, prostate cancer, bladder cancer and some other cancers.66-68 We observed an interesting association between GPX1 Pro198Leu polymorphism and lower probability for recurrent disease. Our findings are consistent with a previous report on the association of GPX1 198Leu variant with lower risk of recurrence in cancer patients.69 A possible explanation may be that some HCTC therapies (radioiodine ablation and radiotherapy) are large ROS generators with antineoplastic effects and may also influence the patient's prognosis after these treatments. As GPX1 198Leu variant is associated with reduced removal of ROS and their secondary products produced by some HCTC therapies, patients with variant allele may have a better prognosis and longer recurrence-free survival time. To sum up, in our study we did not find any association between common functional polymorphism antioxidant genes (SOD2, CAT, GPX1, GSTP1, GSTM1, and GSTT1) and the development of HCTC. A possible explanation could be that these polymorphisms may influence an initial and shared phase of HCTC and HCTA/HCTN development. Common functional polymorphisms in SOD2, CAT, GSTP1, GSTM1 or GSTT1 were also Radiol Oncol 2016; 50(3): 289-296. 295 Krhin B et al. / Antioxidant genes polymorphisms in Hurthle cell thyroid neoplasm not associated with metastatic or recurrent disease development, while GPX1 Pro198Leu polymorphism may modulate the risk of HCTC recurrence. However, the group of patients with recurrent disease was relatively small, so it is possible that the results may result from sampling error. Ideally, our findings relating both to statistically significant associations and not significant associations, should be confirmed in an independent sample cohort. Because of the rarity of these tumours, it was impossible to perform a validation study in a single institution. Further research in a larger group is needed before we can conclude that GPX1 Pro198Leu polymorphism could be used as an additional molecular marker in clinical practice to support decisions about follow-up procedures in patients with HCTC. Conclusions In conclusion, GPX1 Pro198Leu polymorphism may influence the risk for recurrent disease in HCTC, however, these results must be validated in an independent sample cohort. Acknowledgments This work was financially supported by the Slovenian Research Agency (ARRS Grant No. P3-0289 and P1-0170). References 1. Chindris AM, Casler JD, Bernet VJ, Rivera M, Thomas C, Kachergus JM, et al. Clinical and molecular features of Hürthle cell carcinoma of the thyroid. J Clin Endocrino! Metab 2015; 100: 55-62. 2. Cannon J. The significance of hurthle cells in thyroid disease. Oncologist 2011; 16: 1380-7. 3. Barnabei A, Ferretti E, Baldelli R, Procaccini A, Spriano G, Appetecchia M. Hurthle cell tumours of the thyroid. 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Cooper DS, Doherty GM, Haugen BR, Kloos RT, Lee SL, Mandel SJ, et al. American Thyroid Association (ATA) Guidelines Taskforce on Thyroid Nodules and Differentiated Thyroid Cancer; Revised American Thyroid Association management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid 2009; 19: 1167-214. 27. Jazbec J, Aplenc R, Dolzan V, Debeljak M, Jereb B. GST polymorphisms and occurrence of second neoplasms after treatment of childhood leukemia. Leukemia 2003; 17: 2540-2. 28. Tregouet DA, Garelle V. A new JAVA interface implementation of THESIAS: testing haplotype effects in association studies. Bioinformatics 2007; 23: 1038-9. 29. Erčulj N, Kovač V, Hmeljak J, Franko A, Dodič-Fikfak M, Dolžan V. The influence of gemcitabine pathway polymorphisms on treatment outcome in patients with malignant mesothelioma. Pharmacogenet Genomics 2012; 22: 58-68. 30. Carcangiu ML, Bianchi S, Savino D, Voynick IM, Rosai J. Follicular Hurthle cell tumors of the thyroid gland. Cancer 1991; 68: 1944-53. Radiol Oncol 2016; 50(3): 289-296. 296 Krhin B et al. / Antioxidant genes polymorphisms in Hurthle cell thyroid neoplasm 31. Elliott DD, Pitman MB, Bloom L, Faquin WC. Fine-needle aspiration biopsy of Hurthle cell lesions of the thyroid gland: A cytomorphologic study of 139 cases with statistical analysis. Cancer 2006; 108: 102-9. 32. Lopez-Penabad L, Chiu AC, Hoff AO, Schultz P, Gaztambide S, Ordonez NG, et al. Prognostic factors in patients with Hürthle cell neoplasms of the thyroid. Cancer 2003; 97: 1186-94. 33. Zhang YW, Greenblatt DY, Repplinger D, Bargren A, Adler JT, Sippel RS, et al. Older age and larger tumor size predict malignancy in hürthle cell neoplasms of the thyroid. Ann Surg Oncol 2008; 15: 2842-6. 34. Forsberg L, Lyrenäs L, de Faire U, Morgenstern R. A common functional C-T substitution polymorphism in the promoter region of the human catalase gene influences transcription factor binding, reporter gene transcription and is correlated to blood catalase levels. Free Radic Biol Med 2001; 30: 500-5. 35. Bastaki M, Huen K, Manzanillo P, Chande N, Chen C, Balmes JR, et al. Genotype-activity relationship for Mn-superoxide dismutase, glutathione peroxidase 1 and catalase in humans. Pharmacogenet Genomics 2006; 16: 279-86. 36. Gavalas NG, Akhtar S, Gawkrodger DJ, Watson PF, Weetman AP, Kemp EH. Analysis of allelic variants in the catalase gene in patients with the skin depigmenting disorder vitiligo. Biochem Biophys Res Commun 2006; 345: 1586-91. 37. Sadani GR, Nadkarni GD. Role of tissue antioxidant defence in thyroid cancers. Cancer Lett 1996; 109: 231-5. 38. Ahn J, Gammon MD, Santella RM, Gaudet MM, Britton JA, Teitelbaum SL, et al. 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J Pathol 1993; 169: 341-5. 47. Akinci M, Kosova F, Cetin B, Sepici A, Altan N, Aslan S, et al. Oxidant/antioxidant balance in patients with thyroid cancer. Acta Cir Bras 2008; 23: 551-4. 48. Russo D, Bisca A, Celano M, Talamo F, Arturi F, Scipioni A, et al. Proteomic analysis of human thyroid cell lines reveals reduced nuclear localization of Mn-SOD in poorly differentiated thyroid cancer cells. J Endocrinol Invest 2005; 28: 137-44. 49. Li J, Long J, Hu Y, Tan A, Guo X, Zhang S. Glutathione S-transferase M1, T1, and P1 polymorphisms and thyroid cancer risk: a meta-analysis. Cancer Epidemiol 2012; 36: e333-40. 50. Stankov K, Landi S, Gioia-Patricola L, Bonora E, Volante M, Papotti M, et al. GSTT1 and Ml polymorphisms in Hürthle thyroid cancer patients. Cancer Lett 2006; 240: 76-82. 51. Ali-Osman F, Akande O, Antoun G, Mao JX, Buolamwini J. 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Glutathione S-transferase polymorphisms and risk of differentiated thyroid carcinomas: a case-control analysis. Arch Otolaryngol Head Neck Surg 2006; 132: 756-61. 56. Granja F, Morari J, Morari EC, Correa LA, Assump^ao LV, Ward LS. GST profiling may be useful in the screening for thyroid nodule malignancy. Cancer Lett 2004; 209: 129-37. 57. Lemos MC, Coutinho E, Gomes L, Carrilho F, Rodrigues F, Regateiro FJ, et al. Combined GSTM1 and GSTT1 null genotypes are associated with a lower risk of papillary thyroid cancer. J Endocrinol Invest 2008; 31: 542-5. 58. Gaspar J, Rodrigues S, Gil OM, Manita I, Ferreira TC, Limbert E, et al. Combined effects of glutathione S-transferase polymorphisms and thyroid cancer risk. Cancer Genet Cytogenet 2004; 151: 60-7. 59. Gon^alves AJ, Carvalho LH, Serdeira K, Nakai MY, Malavasi TR. Comparative analysis of the prevalence of the glutathione S-transferase (GST) system in malignant and benign thyroid tumor cells. Sao Paulo Med J 2007; 125: 289-91. 60. 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Radiol Oncol 2016; 50(3): 289-296. 297 research article Association between polymorphisms in segregation genes BUB1B and TTK and gastric cancer risk Petra Hudler1, Nina Kocevar Britovsek1, Snjezana Frkovic Grazio2, Radovan Komel1 1 University of Ljubljana, Faculty of Medicine, Institute of Biochemistry, Ljubljana, Slovenia 2 University Clinical Hospital Ljubljana, Department of Obstetrics and Gynecology, Department of Gynecological Pathology and Cytology, Ljubljana, Slovenia Radiol Oncol 2016; 50(3): 297-307. Received 13 March 2015 Accepted 9 August 2015 Correspondence to: Petra Hudler, Ph.D., Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana, Slovenia. Phone: +386 1 543 7664; Fax: +386 1 543 7641; E-mail: petra.hudler@mf.uni-lj.si Disclosure: No potential conflicts of interest were disclosed. Background. Malignant transformation of normal gastric cells is a complex and multistep process, resulting in development of heterogeneous tumours. Susceptible genetic background, accumulation of genetic changes, and environmental factors play an important role in gastric carcinogenesis. Single nucleotide polymorphisms (SNPs) in mitotic segregation genes could be responsible for inducing the slow process of accumulation of genetic changes, leading to genome instability. Patients and methods. We performed a case-control study of polymorphisms in mitotic kinases TTK rs151658 and BUBIB rs1031963 and rs1801376 to assess their effects on gastric cancer risk. We examined the TTK abundance in gastric cancer tissues using immunoblot analysis. Results. C/G genotype of rs151658 was more frequent in patients with diffuse type of gastric cancer and G/G genotype was more common in intestinal types of gastric cancers (p = 0.049). Polymorphic genotype A/A of rs1801376 was associated with higher risk for developing diffuse type of gastric cancer in female population (p = 0.007), whereas A/A frequencies were increased in male patients with subserosa tumour cell infiltration (p = 0.009). T/T genotype of rs1031963 was associated with well differentiated tumours (p = 0.035). TT+CT genotypes of rs1031963 and GG+AG genotypes of rs1801376 were significantly associated with gastric cancer risk (dominant model; OR = 2,929, 95% CI: 1.281-6.700; p = 0.017 and dominant model; OR = 0,364, 95% CI: 0.192-0.691; p = 0.003 respectively). Conclusions. Our results suggest that polymorphisms in mitotic kinases TTK and BUBIB may contribute to gastric tu-morigenesis and risk of tumour development. Further investigations on large populations and populations of different ethnicity are needed to determine their clinical utility. Key words: cancer susceptibility; chromosomal instability; chromosome segregation; mitotic checkpoint; serine/ threonine kinase; genetic association Introduction Gastric cancer is one of the major contributors to cancer-related deaths worldwide with estimated 989 600 new cases and 738 000 deaths in 2008.12 It is believed that complex interplay of genetic and environmental factors triggers the accumulation of numerous genetic and epigenetic alterations in cells, resulting in deregulation of normal cell func- tions and disruption of stomach linen homeosta-sis.3-6 Individual genetic factors probably contribute to aberrant processes in the genesis of malignant phenotype. Among them, single nucleotide polymorphisms (SNPs) and other genetic variants play an important role as the main genetic elements in the aetiology of several complex diseases, including gastric cancer.7-11 In gastric carcinogen-esis this is further supported by the fact that only Radiol Oncol 2016; 50(3): 254-262. doi:10.1515/raon-2016-0023 298 Hudler P et al. / Segregation genes and gastric cancer risk a small proportion of individuals exposed to the known environmental risk factors develop adenocarcinoma.510 Therefore, there is continuing interest for determining simple genetic tests for identifying individuals at high risk for the development of gastric tumours and for identifying patients with high risk for recurrence in order to ensure improved and early diagnosis as well as better survival of patients. A majority of gastric cancer patients show chromosomal instability (CIN) resulting in aneu-ploidy.41213 It has been suggested that tumour cells acquire aberrant chromosome numbers and other chromosomal defects as a result of deregulation of mechanisms responsible for maintaining the chromosomal number stability, such as spindle assembly checkpoint and chromosome segregation.1415 However, mutations in mitotic genes are rare, due to the fact that severe defects of these genes would trigger cell death by cell-surveillance early in the development.14-17 Studies revealed that subtle changes in mitotic segregation genes, controlling chromatids separation or regulating the progress of mitosis, could be prime candidates for inducing the slow process of accumulation of genetic changes, leading to CIN.1518-20 The novel hypothesis is further supported by the fact that this process is slow, and explains the late onset of sporadic epithelial cancers21,22, as well as heterogeneous mutation load observed in different sections of tumours from individual patients. The multidomain protein kinase BUB1B (BUB1-related kinase, known as MAD3 in yeast) plays a central role in the process of spindle assembly checkpoint (SAC), which prevents defects in the segregation of sister chromatids by delaying their separation until all chromatids have achieved correct attachments to the mitotic spindle.23,24 BUB1B is part of the mitotic checkpoint complex (MCC), which together with BUB3, MAD2 and CDC20 inhibits the anaphase-promoting complex/cyclo-some (APC/C), delaying the onset of anaphase and ensuring proper chromosome segregation.25 The protein BUB1B has also been localized to the kinetochores and is important for stabilizing the kinetochore-microtubule interactions and chromosome alignment.26 A dual specificity protein kinase TTK (alias MPS1) is crucial for the spindle assembly checkpoint, for chromosome biorienta-tion on the mitotic spindle and for ensuring accurate chromosome segregation.2728 Inhibitor and chemical genetics studies showed that TTK activity facilitates the conformational activation of MAD2 from open to closed form (C-MAD2) capable of CDC20 binding and inhibition, thus delaying the onset of anaphase.29 TTK is probably implicated in the recruitment of the MAD1-C-MAD2 complex to kinetochores and during mitosis its activity is continuously required to recruit O-Mad2 to the Mad1-C-Mad2 core.30 Furthermore, TTK is required for CENP-E recruitment, whose activity is essential for metaphase chromosome alignment.30 In the present study we examined polymorphisms rs151658 (C>G) in TTK gene, rs1031963 (C>T) and rs1801376 (A>G) in BUB1B gene in the population of Slovenian patients with an advanced gastric cancer and their impact on gastric cancer risk. We also examined the associations of these genetic variants with clinico-histopathological features of patients. Patients and methods Research subjects The study population (n = 284) consisted of 108 Slovenian patients with gastric cancer and 176 control subjects who at the time of peripheral blood extraction did not have cancer. Tumour and corresponding non-tumour tissues at least 7 cm away from the edge of the adenocarcinoma were collected from patients who were admitted to the Clinical Department for Abdominal Surgery at the University Medical Centre Ljubljana and Department for Pathology at the Institute of Oncology Ljubljana during the years 2000-2008. Samples were macrodissected by pathologist, frozen in liquid nitrogen and stored at -70°C. Comprehensive medical data were obtained from registries and pathologist's evaluation. The following clinico-histopathological parameters were recorded: tumour differentiation (grade), location, blood and lymphatic vessel invasion (vascular invasion, perineural invasion), occurrence of tumour cells in the lymphatic vessels (lymphatic invasion), depth of invasion (pT), lymph node involvement (pN), and presence of distant metastases (pM). The gastric cancer cases were classified into diffuse type (n = 46) and intestinal (n = 58) according to Lauren classification. The mean age ± standard deviation (SD) of patients was 66.12 ± 12.02 (range, 33-87 years), and the percentage of men was 63.0%. Cases lost to follow-up (n = 6) and those, who died within 30 days after surgery (n = 2), were excluded from survival analyses. The control population was randomly selected during the years 1999-2007 and shared the ethnic and geographic background of the gastric cancer patients. The research was ap- Radiol Oncol 2016; 50(3): 297-307. 299 Hudler P et al. / Segregation genes and gastric cancer risk proved by the National Medical Ethics Committee of the Republic of Slovenia and confidentiality of personal medical data as well as other data relating to individual identification has been assured in accordance with the Helsinki Declaration. Genotyping Genomic DNA from gastric tumour and non-tumour tissues was extracted using a Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA) and QuickGene™ DNA Tissue Kit S (Fujifilm Corporation, Tokyo, Japan) on QuickGene-810 DNA isolation system (Fujifilm) according to manufacturer's protocol. Genomic DNA from control population was extracted from peripheral blood samples using Wizard® Genomic DNA Purification Kit (Promega) following the manufacturer's protocol. The DNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc.). Genotyping for polymorphism rs151658 (C>G) in TTK gene, and polymorphisms rs1031963 (C>T) and rs1801376 (A>G) in BUB1B gene was performed using TaqMan-based allele-specific polymerase chain reaction assays on the ABI Prism 7000 Sequencing Detection System apparatus (Applied Biosystems, Foster City, CA, USA) according to the procedure recommended by Applied Biosystems. The 10 |jL reaction volume contained 100 ng of DNA. Assay IDs were: C_3181603_10, C_1237153_10, and C_3052718_1. In order to confirm the veracity of the results, the polymorphisms were re-genotyped by direct sequencing on a randomly selected smaller batch of samples. Immunoblot analysis A total of 21 paired gastric adenocarcinoma (GA) and adjacent control tissue samples were ground with a mortar and pestle in liquid nitrogen and lysed with 7 mol/L urea, 2 mol/L thiourea, 40 g/L CHAPS, with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). For every 10 mg tissue, 50 |jl lysis buffer was added. After sonication on ice (3 x 10 s), the samples were incubated for 1 h on ice with occasional vortexing, and then cen-trifuged at 20,000xg for 1 h at 4°C. The superna-tants were collected and the protein concentrations were determined using the commercial Bradford reagent (Thermo Fisher Scientific, Waltham, MA, USA) with BSA used as the standard. Immunoblot analysis was performed on 42 samples. A total of 30 |jg protein per sample was loaded onto 10% gels, separated using SDS-PAGE, and transferred onto PDVF membranes (Millipore, Billerica, MA, USA), which were then blocked in 50 g/L skimmed milk 1 h. The primary antibody was used in the following dilution: anti-Mps1 (anti-TTK) antibody, 1 |jg/ml (ab11108, Abcam, Cambridge, UK). Horseradish peroxidase-conjugated secondary antibody was used in the following dilution: goat anti-mouse antibody, 1:5000 (115-035-062, Jackson ImmunoResearch, Newmarket, Suffolk, UK). The proteins were revealed by chemiluminescence using LAS-4000 CCD camera (Fujifilm, Tokyo, Japan). The blots were then quantified with Multi Gauge software (Fujifilm) and the intensities were normalized to Ponceau-S-stained membranes, to allow for loading and transfer variations. Statistical and bioinformatic analyses Statistical evaluation of the genotyping data was carried out using the c2 or Fischer's exact tests to compare the groups regarding genotype frequencies. Hardy-Weinberg (HW) equilibrium was calculated with an online program (http://www. genes.org.uk/software/hardy-weinberg.shtml).31 Survival was assessed by the Kaplan-Meier method and differences between groups were evaluated using the log-rank test. Multivariate survival analyses were further performed using the Cox proportional-hazards regression model. In the Cox multivariate analyses, forced entry procedure was used to determine the predictor variables. Only the variables that resulted in p-values < 0.05 in the Kaplan-Meier test were entered into the Cox proportional hazard model for the determination of independent prognostic factors for gastric cancer. The postoperative period was measured from the date of surgery to the date of the last follow-up or death. Statistical software used for calculations was IBM® SPSS® Statistics Version 20. For all statistical tests, a probability level (p-value) of less than 0.05 was considered significant. To assess the statistical significance of altered protein abundance in the immunoblotting (as the tumour vs. non-tumour paired samples), non-parametric Wilcoxon signed-rank test was used. The tests were double-sided and the values with p < 0.05 with a confidence level of 95% were considered to be statistically significant. To assess the correlation of the altered protein abundance from the immu-noblotting with the histopathological parameters, repeated measures ANOVA was used. The values with p < 0.05 were considered to be statistically significant. Bonferroni post-tests were used to deter- Radiol Oncol 2016; 50(3): 297-307. 300 Hudler P et al. / Segregation genes and gastric cancer risk TABLE 1. Clinicopathological characteristics of patients with gastric cancer Parameter Number of patients (%) Age (years ± standard deviation) (n = 108) 66.12 ± 12.02 Gender (n = 105) Male 68 (63.0) Age (years ± standard deviation) (n = 66) 65.07 ± 12.03 Female 40 (37.0) Age (years ± standard deviation) (n = 39) 67.90 ± 11.94 Lauren's classification (n = 104) Intestinal 58 (55.8) Diffuse 46 (44.2) Location (n = 101) Upper 40 (39.6) Lower 34 (33.7) Mixed 27 (26.7) Grade/differentiation (n = 105) Well 9 (8.6) Moderate 24 (22.9) Poor 72 (68.6) Vascular invasion (n = 80) Present 27 (33.8) Not present 53 (66.3) Perineural invasion (n = 95) Present 44 (46.3) Not present 51 (53.7) Lymphatic invasion (60) Present 53 (88.3) Not present 7 (11.7) pN (n = 105) 0 24 (22.9) 1-2 15 (14.3) 3-6 20 (19.0) > 7 46 (43.8) pT (n = 105) Muscularis propria 6 (3.7) Subserosa 50 (42.6) Serosa 49 (36.1) pN = number of positive regional lymph nodes; pT = tumour invasion TABLE 2. Multivariate survival analysis of clinic-pathological variables in gastric cancer patients Variable B* SE (B) OR (95% CI) P pN 0.670 0.127 1.954 (1.525-2.504) 0.000 Lauren's classification 0.591 0.246 1.807 (1.116-2.925) 0.016 'Predicted change in the hazard for a unit increase in the predictor. CI = confidence interval; OR = odds ratio; pN = number of positive regional lymph nodes; SE = standard error tein binding motifs. We used web-based software PROMO, which is part of the ALGGENE web-serv-er.32,33 The search for putative binding sites was performed using the following parameters: human species, all motifs, and all factors. The data for comparisons of genotype frequencies in European populations of examined SNPs in this study was extracted from the 1000 Genomes Project data platform using a specific version of the Ensembl browser (http://browser.1000genomes.org).34 Results Patients' survival is associated with certain clinico-pathological features The clinical information and demographic characteristics of selected patients with gastric cancer in this study are summarized in Table 1. At the end of a period of up to 11 years of follow-up, a total of 69 patients out of 100 have died. The overall 5-year survival was 33.5%. No statistically significant association between tested genetic variations and survival was observed (p > 0.05). Univariate survival analysis showed that only Lauren's classification and lymph node involvement (pN) were significant prognostic factors. Diffuse type predicted shorter 5-year survival (logrank test, c2 = 5.516, p = 0.019) with overall mean estimate of survival for patients with intestinal type 64.67 months ± 7.74 (SE) (CI = 49.50-79.84) and 39.73 months ± 6.83 (SE) (CI = 26.75-53.11) for patients with diffuse type of gastric cancer. Regarding the parameter pN, patients with 7 or more positive lymph nodes had shorter survival time of 21.93 months ± 4.30 (SE) with CI = 13.5030-36 (log-rank test, c2 = 34.169, p = 0.000). Multivariate analysis was performed for the same set of patients with complete clinical data sets. Cox regression model included both significant variables, pN and tumour classification. The enter method showed significant improvement (p < 0.05) if both parameters were entered into the model (Table 2). mine where the differences were significant. All of the analyses were performed using Microsoft Office Excel 2007 (Microsoft Corporation, Washington, USA) and GraphPad Prism 5 (GraphPad Software, Inc., California, USA). In order to functionally evaluate intronic polymorphisms we identified their effect in the context of polymorphic biological sequences on pro- SNPs in TTK and BUB1B are associated with type, grade, and location of gastric cancer Associations between clinicopathological parameters and genotypes of SNPs are presented in Table 3. Statistical analysis revealed a weakly significant association for rs151658 genotypes C/G and risk of developing diffuse type of gastric cancer, and genotype G/G and risk of developing intestinal type of Radiol Oncol 2016; 50(3): 297-307. 301 Hudler P et al. / Segregation genes and gastric cancer risk cancer (p = 0.049). Similar results were obtained for both male and female populations of patients (p = 0.047 and p = 0.024, respectively). Genotype A/A of rs1801376 polymorphism was significantly associated with higher risk of developing the diffuse type of gastric cancer in total and female populations of patients (p = 0.007). Interestingly, A/G genotype was under-represented in populations with diffuse type of gastric cancer. Genotype A/A of this polymorphism was also associated with the invasion of tumour cells into subserosa layer of stomach in male population (p = 0.009). A/A genotype was also associated with tumour location, namely, A/A frequencies were increased in patients with tumours disseminated across the whole stomach (p = 0.035). Genotype T/T of rs1031963 was associated with well differentiated tumours in total population (p = 0.035); however, when we stratified it into female and male populations, we observed a significant association of this genotype with moderately differentiated tumours in the female population (p = 0.004). Clinico-pathological features lymph node involvement (pN), depth of invasion (pT), vascular invasion, perineural invasion and lymphatic invasion did not show significant associations with investigated polymorphisms. SNPs in BUB1B are associated with gastric cancer risk The analyses of genotype frequencies in selected SNPs between cases and controls are shown in Tables 4 and 5. The frequencies of all genotypes in cases and control groups were in Hardy-Weinberg equilibrium. The tested polymorphisms did not show significant differences between gastric cancer patients and control group. In contrast, when we stratified the population for gender, we found significant association between BUB1B rs1801376 genotypes and higher risk for developing gastric tumours (p = 0.029). Similarly, dominant model combining genotypes A/G and G/G showed comparable results (p = 0.010; p [Yates correction] = 0.017). Furthermore, tests for association showed analogous results and confirmed significantly higher frequency of G al-lele in female population of patients with gastric cancer (0.41 vs. 0.28 in control group). We also observed allele frequency difference in male patient population for BUB1B rs1031963. The dominant model, combining genotypes TT+CT versus CC, showed that patients with C/C homozygous allele had significantly higher risk for developing gastric cancer. TABLE 3. Comparison of clinic-pathological features and genotypes TTK rs151658, BUBIB rs1031963, and BUBIB rs1801376 in patients with gastric cancer Parameter Subject Variant/Genotype P TTK rs151658 GG CG CC Lauren's classification Intestinal Diffuse Total 17 5 21 25 20 16 0.049 C2=6.033 Intestinal Diffuse Male 9 5 10 20 14 8 0.047 C2=6.113 Intestinal Diffuse Female 8 0 11 5 6 8 0.024 F=7.499 BUB1B rs1801376 AA AG GG Intestinal Diffuse Total 18 28 33 13 7 5 0.007 C2=9.951 Intestinal Male 15 14 4 0.472 Diffuse 20 9 4 F=1.836 Intestinal Diffuse Female 3 8 19 4 3 1 0.007 F=9.688 BUB1B rs1031963 CC CT TT Tumour Well Total 1 4 4 0.035 F=9.642 differentiation Moderate Poor 7 23 9 39 7 7 Well Male 0 2 3 0.139 F=6.439 Moderate Poor 6 19 7 20 2 6 Well Moderate Poor Female 1 1 19 2 2 20 1 5 6 0.004 F=12.549 BUB1B rs1801376 AA AG GG PT Muscularis propria Subserosa Serosa Total 1 27 18 4 18 25 1 5 6 0.232 F=5.250 Muscularis propria Subserosa Male 0 22 3 6 1 2 0.009 F=11.832 Serosa 13 14 5 Muscularis propria Subserosa Female 1 5 1 12 0 3 0.816 F=1.967 Serosa 5 11 1 BUB1B rs1801376 AA AG GG Tumour location Upper Lower Whole Total 14 13 18 23 15 6 3 6 3 0.035 F=10.104 pT = tumour invasion Radiol Oncol 2016; 50(3): 297-307. 302 Hudler P et al. / Segregation genes and gastric cancer risk p =0.03 ® ® 0.0015-1 0.0010- 0.0005- 0.0000 0.0015-1 0.0010- 0.0005- □ N □ N FIGURE 1. Immunoblotting of TTK. (A) Densitometry quantification analysis for the relative band densities from the protein abundance immunoblotting for the indicated protein in the non-tumour (N) and tumour (T) gastric tissues. The p value given (Wilcoxon signed-rank test) indicates the significance of the difference between the non-tumour (N) and tumour (T) gastric tissue samples. (B, C) Densitometry quantification analysis for the relative band densities from the protein abundance immunoblotting for TTK in the non-tumour (N) and tumour (T) gastric tissues samples according to lymph node involvement (pN) and location of the tumours. Distribution and genotype frequencies of SNPs in TKK and BUB1B between European and Slovenian populations Comparisons of the SNPs' genotype frequencies between our test groups and European populations are presented in Figure 3. Genotype frequen- cies of rs151658, rs1031963 and rs1801376 in our groups of populations showed significant differences from European population (Table 6). The frequency of rs151658 C/C genotype was higher than expected in the Slovenian population of patients compared to total European population (p = 0.015). Similarly, we observed more rs1031963 C/C genotypes in the male population of Slovenian patients with gastric cancer (p = 0.042) compared with total European population and European population stratified for males. The rs1801376 A/G genotype was higher and A/A genotype was under-represented in female population of patients with gastric cancer compared to the total European population (p = 0.034) and female European population (p = 0.014). TTK abundance is altered in tumour tissues of gastric cancer patients Immunobloting data on individual samples (Figure 1A) demonstrated statistical significance for the increased abundance of TTK (p = 0.03) in the tumour tissues. No statistically significant correlation of TTK abundance with clinical his-topathological parameters or rs151658 genotypes was observed. However, some trends were observed (Figures 1B and C) for lymph node involvement (pN) and antral tumour location: TTK abundance was higher in normal tissues compared to tumour tissues when no regional nodes were invaded with tumour cells (pN = 0) and when the tumours were located at the bottom of the stomach (antrum). Prediction of binding motifs showed that polymorphic sites in TTK and BUB1B bind different transcription factors To determine if different intronic polymorphisms could affect binding of transcription factors, we performed in silico analysis of conserved human motifs using polymorphic sequences as templates (Figure 2). We identified distinct recognition sites for different proteins for both TTK rs151658 and BUB1B rs1031963. TFII-I, c-Myb, NFI/CTF, and HNF-4 alpha binding motifs were recognized if rs151658 polymorphic site contained C allele. In contrast, if G allele was present, GR, TFII-I, NCFI/CTF, and HNF-4 alpha were identified. Comparison of rs1031963 alleles showed that if allele T was present, several binding motifs were predicted, whereas in the case of allele C, there were no recognizable binding patterns. Radiol Oncol 2016; 50(3): 297-307. 303 Hudler P et al. / Segregation genes and gastric cancer risk Discussion In this study, we investigated the effects of selected polymorphisms in mitotic kinases TTK and BUB1B and risk of developing gastric cancer in Slovenian population. We also determined the associations between tested polymorphisms and clinic-pathological features of patients. The results provide evidence that TTK rs151658, BUB1B rs1031963, and rs1801376 could potentially serve as prognostic biomarkers for determining tumour differentiation and invasion. Furthermore, rs1801376 G allele could be used as one of determinants for gastric cancer screening in female population and CC genotype in rs1031963 could be used for selection of male population at higher risk for developing gastric cancer. TTK gene harbours more than 600 different one-nucleotide polymorphisms (data obtained from GeneCards, http://www.genecards.org/). We investigated intronic polymorphism, because this gene has many alternative transcripts, and intronic one-nucleotide variants could have an effect on splicing and/or ubiquitination.35,36 SNP BUBl B rsl031963 ... ATGTGGAGGACTTTGATGTAAAG... TCF-4E C/EBPp LEF-1 TCF-4 TTK rsl51658 ...TCAAAGGCCAAACTCCTGTGATG.. TFII-I C-Myb BUBl B rsl031963 ...ATGTGGAGGACTTCGATGTAAAG.. FIGURE 2. Predicted binding sites for transcription factors for polymorphic alleles rs1031963 and rs151658. TTK rsl51 ¿58 ...TCAAAGGCCAAAGTCCTGTGATG.. NFI/CTF rs151658 lies between exons 12 and 13. In its vicinity there are binding sites for TFII-I, c-Myb, NFI/ CTF, and HNF-4 alpha transcription factors, if C allele is present and binding sites for GR, TFII-I, NFI/CTF, and HNF-4 alpha, if G allele is present TABLE 4. Distribution of genotype frequencies of TTK rs151658, BUBIB rs1031963, and BUBIB rs1801376 between gastric cancer patients and control subjects Variants Genotype Cases (n) Controls (n) P HWE (cases) HWE (controls) GG 24 55 TTK rs151 658 CG C/C CC 48 36 32 76 44 48 C2=3,628 0.163 X2=1.08 0.299 C2=2.87 0.090 BUBIB rs1031963 CT TT AA 54 18 47 89 39 89 C2=1.059 0.589 C2=0.345 0.557 C2=0.035 0.852 BUBIB rs1801376 AG GG AA 49 12 36 69 13 49 C2=2.291 0.318 C2=0.021 0.885 C2=0.005 0.941 Male population AG GG AA 24 8 11 39 7 40 C2=1.186 0.553 X2=1.530 0.216 C2=0.040 0.841 Female population AG GG 25 4 30 6 F=6.955 0.029 C2=3.352 0.067 C2=0.013 0.909 F = Fisher statistics; HWE = Hardy-Weinberg Equilibrium; c2 = chi-square statistics; Radiol Oncol 2016; 50(3): 297-307. 304 Hudler P et al. / Segregation genes and gastric cancer risk TABLE 5. Odds ratios for TTK rs151658, BUB1B rs1031963, and BUB1B rs1801376 between the cases and controls and their effect on gastric cancer risk Genotype Cases (n)/Control model group (n) OR (95% CI)* P Py BUB1B rs1031963 Dominant 72/120 0.900 C2=0.149 C2=0.062 TT+CT vs. CC vs. 32/48 (0.527-1.536) 0.699 0.803 Recessive 18/36 0.797 C2=0.507 C2=0.309 TT vs. CT+CC vs. 86/137 (0.426-1.491) 0.476 0.578 Heterozygous 54/89 0.910 C2=0.108 C2=0.035 CT vs. CC vs. 32/48 (0.520-1.594) 0.742 0.853 Male population Dominant 41/101 0.364 C2=9.848 C2=8.834 TT+CT vs. CC vs. 26/26 (0.192-0.691) 0.002 0.003 Recessive 11/26 0.763 C2=0.467 C2=0.241 TT vs. CT+CC vs. 56/101 (0.351-1.659) 0.494 0.623 Heterozygous 30/75 0.400 C2=6.959 C2=6.057 CT vs. CC vs. 26/26 (0.201-0.797) 0.008 0.014 Female population Dominant 31/57 1.994 C2=1.862 C2=1.281 TT+CT vs. CC vs. 6/22 (0.731-5.437) 0.172 0.258 Recessive 7/13 1.185 C2=0.107 C2=0.004 TT vs. CT+CC vs. 30/66 (0.429-3.269) 0.743 0.949 Heterozygous 24/44 2.000 C2=1.775 C2=1.188 CT vs. CC vs. 6/22 (0.714-5.606) 0.183 0.276 BUB1B rs1801376 Dominant 61/82 1.409 C2=1.927 C2=1.601 GG+AG vs. AA vs. 47/89 (0.868-2.287) 0.165 0.206 Recessive 12/13 1.519 C2=0.999 C2=0.615 GG vs. AA+AG vs. 96/158 (0.666-3.465) 0.318 0.433 Heterozygous 49/69 1.345 C2=1.304 C2=1.025 AG vs. AA vs. 47/89 (0.808-2.237) 0.253 0.311 Male population Dominant 32/46 0.947 C2=0.029 C2=0.0002 GG+AG vs. AA vs. 36/49 (0.508-1.766) 0.864 0.990 Recessive 8/7 1.676 C2=0.917 C2=0.466 GG vs. AA+AG vs. 60/88 (0.577-4.868) 0.338 0.495 Heterozygous 24/39 0.838 C2=0.272 C2=0.124 AG vs. AA vs. 36/49 (0.430-1.630) 0.602 0.725 Female population Dominant 29/36 2.929 C2=6.719 C2=5.737 GG+AG vs. AA vs. 11/40 (1.281-6.700) 0.010 0.017 Recessive 4/6 1.296 C2=0.147 c2=0.001 GG vs. AA+AG vs. 36/70 (0.344-4.889) 0.701 0.971 Heterozygous 25/30 3.030 C2=6.732 C2=5.709 AG vs. AA vs. 11/40 (1.292-7.108) 0.009 0.017 * p value with Yates correction C2 = chi-square statistics; OR = odds ratio; CI = confidence interval (PROMO, ALGGEN server) (Figure 2).3233 This indicated that different polymorphic alleles bind different proteins, which could in turn affect splicing or gene expression. Studies, performed on breast cancer patients, confirmed the significant association of this polymorphism with cancer risk15; however, we did not find any studies regarding the effect of rs151658 on gastric cancer risk. In our study, we identified the association of G/G geno- type with intestinal type of gastric cancer, while C/G genotype was significantly increased in cases with diffuse type of gastric cancer. Interestingly, comparison of genotype distribution for rs151658 between Slovenian patients with gastric cancer and European population showed that C/C genotype was over-represented in patients with gastric cancer. The significance of this finding is not clear and further analyses are needed on larger cohorts of patients in order to determine its usefulness in clinical setting. To assess if the above mentioned genotypes perhaps had an effect on TTK protein levels, immuno-blotting was performed. While the results regarding the effect of genotypes on protein abundance remain inconclusive, it should be noted that polymorphisms usually exert low-penetrance effects, which could more profoundly affect the pathogenesis of gastric cancer in early stages; however, when the disease progresses, the mutation load and aberrant expression of other genes mask their effects. We did, however, confirm higher abundance of TTK in tumour tissues, which is in accordance with several other studies and points out the deregulation of cell cycle homeostasis, higher proliferative trend of tumour cells and weakened spindle assembly checkpoint leading to increased genome instability and aneuploidy.3738 Furthermore, this study showed a trend of increased TTK abundance associated with the spread of cancer cells to regional lymph nodes indicating a possible link between TTK levels and metastatic potential of malignant gastric cells. Homozygous mutations of critical spindle-assembly BUB1B are extremely rare and associated with the diseases such as mosaic variegated ane-uploidy syndrome 1 (biallelic mutations) and premature chromatid separation trait, which are both characterized by aneuploidy and chromosomal in-stability.39 BUB1B overexpression has been found in gastric cancers, although the results are often conflicting. In one study, the overexpression of BUB1B was associated with tumour proliferation40, however, Enjoji et al. observed that patients with higher expression of BUB1B had improved relapse-free survival.41 Furthermore, Ando et al. found that high expression of BUB1B correlated with invasion, lymph node metastasis, liver metastasis, and poor prognosis.14 Bohers et al. confirmed that the function of BUB1B is dosage-dependent by gradual reduction of BUB1B expression by shRNA in cell lines.42 In their experiment, residual levels of BUB1B protein below 50% of the normal level indicated premature chromatid separation and ane- Radiol Oncol 2016; 50(3): 297-307. 305 Hudler P et al. / Segregation genes and gastric cancer risk TABLE 6. Comparison of TTK rs151658, BUBIB rs1031963, and BUBIB rs1801376 genotypes between the European population and examined groups of Slovenian population Population N Genotype counts P TTK rs151658 C2 = 8.391; P = 0.015 a EUR 503 97 (C|C) / 246 (C|G) / 160 (G|G) c2 = 11.143; P = 0.004 b NS c SI (total)a 283 80 (C|C) / 124 (C|G) / 79 (G|G) SI (cases)b 108 36 (C|C) / 48 (C|G) / 24 (G|G) SI (controls)0 175 44 (C|C) / 76 (C|G) / 55 (G|G) BUB1B rs1031963 EUR 503 125 (C|C) / 259 (C|T) / 119 (T|T) NS d NS e C2 = 5.715; P = 0.057 f EUR - male 240 56 (C|C) / 124 (C|T) / 60 (T|T) _ c _ d C2 = 6.348; P = 0.042 f SI (total)d SI (cases - female)e SI (cases - male)' BUB1B rs1801376 EUR 280 36 65 503 80 (C|C) / 143 (C|T) / 57 (T|T) 6 (C|C) / 23 (C|T) / 7 (T|T) 25 (C|C) / 29 (C|T) / 11 (T|T) 240 (A|A) / 217 (A|G) / 46 (G|G) NS g F = 6.569; P = 0.034 h NS 1 EUR - female 263 135 (A|A) / 109 (A|G) / 19 (G|G) F = 8.277; P = 0.014 h g SI (total)g 279 136 (A|A) / 118 (A|G) / 25 (G|G) SI (cases _ female)h 40 11 (A|A) / 25 (A|G) / 4 (G|G) SI (cases _ male)' 68 36 (A|A) / 24 (A|G) / 8 (G|G) EUR = European population; F = Fisher statistics; SI (cases) = gastric cancer patients; SI (controls) = control population; SI (total) = combined populations of patients and controls; C2 = chi-square statistics; superscript letters indicate comparisons between European population and Slovenian populations BUBIB rsl031963 BUBIB rsl801376 TTK rsl516S8 cc CT TT AA AG GG GG CG CC EUR 0.249 0.515 0.237 EUR 0.477 0.431 0.091 EUR 0.318 0.489 0.193 CEU 0.242 0.556 0.202 CEU 0.566 0.364 0.071 CEU 0.253 0.485 0.263 FIN 0.232 0.545 0.222 FIN 0.455 0.455 0.091 FIN 0.253 0.485 0.263 GBR 0.286 0.473 0.242 GBR 0.505 0.363 0.132 GBR 0.418 0.484 0.099 IBS 0.271 0.467 0.262 IBS 0.4Î7 0.421 0.112 IBS 0.299 0.523 0.178 TSI 0.215 0.533 0.252 TSI 0.402 0.542 0.056 TSI 0.374 0.467 0.159 SI (total) 0.286 0.510 0.204 SI (total) 0.487 0.423 0.090 SI (total) 0.279 0.438 0.283 SI (cases) 0.308 0.519 0.173 SI (cases) 0.435 0.454 0.111 SI (cases) 0.222 0.444 0.333 SI (controls) 0.273 0.506 0.221 SI (controls) 0.520 0.404 0.076 SI (controls) 0.314 0.434 0.251 FIGURE 3. Distribution of genotype frequencies of polymorphisms rs151658, rs1031963, and rs1801376 between European populations and Slovenian population. CEU = Utah Residents (CEPH) with Northern and Western European Ancestry; EUR = European population; FIN = Finnish in Finland; GBR = British in England and Scotland; IBS = Iberian Population in Spain; SI (cases) = gastric cancer patients; SI (controls) = control population; SI (total) = combined populations of patients with gastric cancer and healthy controls; TSI = Tuscany in Italy Radiol Oncol 2016; 50(3): 297-307. 306 Hudler P et al. / Segregation genes and gastric cancer risk uploidy. These conflicting effects of BUB1B could be mediated by different polymorphisms, present in the nucleotide sequence of the gene. BUB1B rs1031963 polymorphism is in 5'-promoter region, which harbours binding sites for C/EBPbeta, GR, HNF-4alpha, LEF-1, SRY, TCF-4E, and TCF4 if T allele is present (PROMO, ALGGEN server).32-33 Interestingly, if C allele is present, the DNA sequence harbours no transcription factor motifs. In our study the T/T genotype was associated with well differentiated tumours in total population, whereas in female population, when analysed separately, it was associated with moderately differentiated tumours. Well differentiated adeno-carcinomas tend to have a better prognosis than infiltrative poorly differentiated adenocarcinomas. Furthermore, T/T+C/T genotypes were nominally associated with reduced risk of gastric cancer in male population, whereas C/C genotype was more common in male patient population. Comparisons with European population showed similar results. BUB1B rs1801376 A/A genotype was significantly higher in female patients with diffuse gastric cancer. A/A genotype was also increased in samples, which were characterized by invasion of tumour cells into subserosa in male population, and was associated with tumours, growing throughout whole stomach tissue. The consequence of this functional polymorphism is amino acid substitution Q349R in conserved region KEN, which is the binding site for CDC20.43 CDC20 is co-activator of anaphase promoting complex APC/C.24 Impaired function of KEN region in BUB1B could thus affect the regulation of anaphase delay, which ensures genome stability by providing time for correct spindle assembly, chromosome alignment and segregation. In addition, A/G genotype showed significant association with gastric cancer risk in female population of gastric cancer patients compared to Slovenian control group and European population. 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Hum Genet 2008; 124: 473-8. 43. D'Arcy S, Davies OR, Blundell TL, Bolanos-Garcia VM. Defining the molecular basis of BubR1 kinetochore interactions and APC/C-CDC20 inhibition. J Biol Chem 2010; 285: 14764-76. Radiol Oncol 2016; 50(3): 297-307. 308 research article Metastatic sebaceous cell carcinoma, review of the literature and use of electrochemotherapy as possible new treatment modality Simone Ribero12, Eugenio Sportoletti Baduel12, Matteo Brizio1, Franco Picciotto2, Emi Dika3, Maria Teresa Fierro1, Giuseppe Macripo2, Pietro Quaglino1 1 University of Turin, Department of Medical Sciences, Section of Dermatology, Turin, Italy 2 Citta della Salute e della Scienza di Torino University Hospital, Department of Oncology, Section of Dermatologic Surgery, Turin, Italy 3 Unit of Dermatology, Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy Radiol Oncol 2016; 50(3): 308-312. Received 24 April 2016 Accepted 14 May 2016 Correspondence to: Simone Ribero, M.D., Ph.D., Department of Medical Sciences, Section of Dermatology, University of Turin, Turin, Italy. E-mail: simone.ribero@unito.it Disclosure: No potential conflicts of interest were disclosed. Background. Metastatic extraorbital sebaceous carcinoma is a rare event that could involve the head and neck. The treatment of choice for the initial stage of the disease is surgery and/or radiotherapy. The treatment of recurrent or advanced disease is still controversial. Material and methods. Extensive literature search was done, and the treatment options are discussed. Results. Results. The literature search found several treatment modalities in use for the treatment of metastatic extraorbital sebaceous carcinoma. Electrochemotherapy was not included in the reported treatments. We used this technique for a man of 85 years old with a recurrent and locally metastatic extraorbital sebaceous carcinoma of the scalp.During the period of 8 months, two sessions of electrochemotherapy were employed, which resulted in an objective response of the tumour and good quality of life. Conclusions. Electrochemotherapy has shown to be a interesting tools for treatment of metastatic extraorbital sebaceous carcinoma when other radical options are not available or convenient. Key words: electrochemotherapy; head and neck tumour; extraorbital tumour; sebaceous carcinoma Introduction The sebaceous carcinoma (SC) is a rare and potentially aggressive adnexial neoplasm of sebaceous gland. Its prevalence varies from 0.05% to 0.7% of all the skin cancers.1 Approximately, the 25% of sebaceous carcinomas occur in extra-orbital sites, in 15% of these cases the torso and in 10% the extremities are affected.2 Extraorbital SC is considered as a less aggressive neoplasm when compared with its periorbital counterpart (a reduced tendency for regional metastasis, 1.4% for extraorbital vs. 4.4% for periorbital).3 Despite this, highly aggressive extraorbital SC has been reported in the literature.4 The typical clinical presentation is indistinguishable from other more common dermatologic conditions. Usually, clinical features are a painless pink or yellow firm papule, gradually enlarging and ranging from skin-coloured to red papules, plaques, or nodules. This aspecific presentation delays the time of diagnosis. Histologically an unencapsulated, lobular, dermally based collections of sebaceous and undifferentiated cells that may contain lipid granules in the cytoplasm with a characteristic "frothy" appearance.5 SC requires vigilant follow-up after treatment due to its potentially aggressive nature. Local recurrence rate is 4%-28% for both periorbital and extraorbital lesions.6 Radiol Oncol 2016; 50(3): 308-312. doi:10.1515/raon-2016-0039 Ribero S et al. / Electrochemotherapy for sebaceous cell carcinoma 309 Pathogenesis of SC is poorly understood, however associations with Muir-Torre syndrome7 is described as well as irradiation8, immunosuppression9, familial retinoblastoma.10 This neoplasm can occur on any sebaceous glands rich skin, particularly face, scalp, and neck. For unknown reasons SC is more prevalent in periorbital skin (39%), especially eyelid, and in extraorbital skin of the head and neck (41%). Some genitalis and extremities cases are reported.11 The management of skin cancer of the head and neck region is still challenging. Surgery is commonly considered the treatment of choice and significantly improves the locoregional control, if associated with adjuvant radiotherapy.12 In the head and neck area, where it is difficult to obtain safety margins wide enough, radiotherapy can be the first treatment option. In particular some areas have been included in the last American Joint Committee on Cancer (AJCC) classification as more at risk of involved margins after surgery in squamous cell carcinoma.13 Methods A systematic literature review for case reports of extra-ocular, cutaneous, multifocal metastasizing SC in the time between 1960 and 2015 was conducted. We therefore searched the literature for all cases mentioning a skin directed therapy and response results for metastatic sebaceous carcinoma utilizing PubMed's MEDLINE database. TABLE 1. Clinical cases of extra-ocular metastatic sebaceous carcinoma treated with skin directed therapies Authors Sex Age Localization / metastases Treatment Prognosis Mellette et al. (1981) 14 M 63 Nose / cutaneous and paratiroid metastases Curettage and surgical excision Parotidectomy Radiotherapy nod Moreno et al. (2001) 16 M 45 Left axilla / lymph node metastases Surgery 6 months Moura et al. (2002) 17 M 71 Forehead / cutaneous, nodal and skeletal metastases Surgery Local radiation Chemo and cryosurgery 4 years without recurrence Khan et al. (2003) 18 F 49 Vulva / subcutaneous and nodal metastases Surgery Radiotherapy Palliative chemotherapy nod Murphy et al. (2004) 19 M 71 Right nostril / left nostril metastases Surgery 18 months without recurrence Swick et al. (2009) 20 M 83 Right flank / subcutaneous, nodal, pulmonary and hepatic metastases Palliative radiotherapy nod Bhat et al. (2011) 21 M 32 Left feet / cutaneous metastases Surgery Chemotherapy nod Bolm et al. (2015) 15 M 87 Left ear / nodal, pulmonary and cutaneous metastases Surgery 18 months without recurrence Our case M 85 Scalp / cutaneous and subcutaneous metastases Electrochemotherapy 2 months Radiol Oncol 2016; 50(3): 308-312. FIGURE 1. Metastatic sebaceous carcinoma, before electrochemotherapy. Results Local and distant cutaneous/subcutaneous metastases were described in the course of disease in 8 cases (Table 1).14-21 Heterogeneity regarding managing of skin cutaneous metastases in head and neck is due to the variability in size, site and clinical comorbidities. Although a few reported cases have shown that skin directed therapy regimens help in palliative treatment, prospective studies have not been performed and their role has not yet been settled due to the rarity of this disease. 310 Ribero S et al. / Electrochemotherapy for sebaceous cell carcinoma 310 FIGURE 2. Response to treatment after two electrochemotherapy sessions (2 months). Electrochemotherapy for sebaceous carcinoma Herein we report a case of a 85-years old Caucasian male came to our attention for the first time presenting 9 papulo-nodular lesions on the scalp ranging from 1 to 3 cm in diameter (Figure 1 A-B). Moreover, some of them bled frequently. The clinical scenario was suggestive for locally metastatic cutaneous carcinoma but the eruptive emergence of skin lesions necessitated a better diagnostic determination. One year before, he had a prior surgical excision of a nodular lesion of the scalp carried out in another hospital treated with skin grafting and histologically diagnosed as cutaneous carcinoma. Thus, we performed three skin biopsies on three different lesions with histopathological diagnosis of SC. The total body PET-CT scan confirmed the lack of visceral metastatic spreading. A month after the first visit we observed the appearance of new lesions on the scalp and those previously described were increased in size. After collecting the informed consent for the off label procedure, we performed electrochemotherapy with a 30% reduced dose of the 15000 IU/m2 intravenous bleomycin due to the patient impaired renal function using the Cliniporator TM device (IGEA Ltd, Carpi, Italy). The treatment of tumour area of 20 cm2 was performed with hexagonal electrodes in 80 runs of electric pulse applications. Neither post-treatment complications, nor cranial nerves injuries nor post-procedural pain were observed. Globally 16 lesions were treated with a complete bleeding control. After one month the lesions treated were flat (Figure 1). Despite this other 4 new lesions appeared in the area on the previous skin graft. A second section of ECT was performed with the same parameters. Two months after the second electrochemo-therapy session the patients has not showed any relapse and was disease free (Figure 2). Discussion Herein we described the first case of Electro-chemotherapy in the treatment of metastatic extraorbital SC. SC typically consists of roundish nests of tumour-cells with central necrosis, in proximity to normal sebaceous glands. The tumour cells of not well-differentiated SC may display high- grade features with a high frequency of mi-totic figures. Angiolymphatic invasion is a consistent finding in SC, like an intraepidermal pagetoid tumour-spread. The role of Immunohistochemistry in the diagnosis of SC is fundamental as it consistently expresses Epithelial Membrane Antigen (EMA) (which is absent in squamous cell carcinoma and basal cell carcinoma) and, in the centres of the tumour-nests, CD15. Surrounding sebaceous glands are used as a positive internal control for CD15. CD 10 is usually absent in SC. Ansai et al.22 showed that positivity for adipophilin is most useful in the diagnosis of sebaceous neoplasms. Because of the rarity of this tumour, no standard therapy exists specially in its metastatic onset. In the last years, electrochemotherapy has been proposed as a novel therapeutic option for the control of recurrent cutaneous, subcutaneous or mucosal neoplastic lesions of different histologies.23-24 There is an increasing body of published clinical data on electrochemotherapy.25 This procedure achieves rates of objective response between the 56% and the 100%, depending on the tumour size and his-tology.26-27 Electrochemotherapy combines the administration of a poorly permeant cytotoxic agent, such as bleomycin with the local application of electric pulses that induce reversible electropora-tion, thus improving drug diffusion into cells.28 Electrochemotherapy has demonstrated a high rate of efficacy and favorable toxicity profile in a European multicenter study on skin metastases from different tumour histotypes.29 In this study, the objective response (OR) rate on treated tumour Radiol Oncol 2016; 50(3): 308-312. Ribero S et al. / Electrochemotherapy for sebaceous cell carcinoma 311 nodules was 89.0% with complete regression in 73.3% of cases. A recently published meta-analysis including 47 prospective studies comparing five skin-directed therapies (electrochemotherapy, radiation, photodynamic therapy, intralesional therapy, and topical therapy), electrochemother-apy demonstrated an OR rate of 75.4% (complete response [CR] rate 47.5%) with a low toxicity profile (grade 3 in less than 6% of patients).30 The main advantages of electrochemotherapy include: • high success rate in local tumour control after a single session; • no damage to healthy peripheral tissue (using low doses of chemotherapeutic agents electro-chemotherapy is very specific for dividing tumour cells, sparing the surrounding normal tissue); • no protein denaturation, so that tumour antigens are not destroyed and may elicit an immune response; • excellent safety profile (in clinical use, no serious adverse events were reported in association with electrochemotherapy; • advantageous cost/benefit ratio: the technology and the drugs used, in particular bleomycin, do not require large investments; • improvement of patient's quality of life. Reported studies showed clinical activity, positive impact on patients subjective clinical perception and low toxicity profile of electrochemo-therapy and encourages us to propose to patients this technique with an palliative intent, after the failure of conventional treatment options, such as surgery, radiotherapy, and systemic therapies.31 Especially in Kaposi Sarcoma, where skin lesions often cause pain and disfigurement and may lead to functional disability, electrochemotherapy has become the standard of care as first line treatment strategy.32 To the best of our knowledge no data on skin metastases from extracutaneous SC treated with electrochemotherapy are documented in the literature. Extraorbital SC has a high risk of local recurrence and it usually appears in elderly. Herein, we reported a CR at 2 months time from two sessions of electrochemotherapy for SC skin metastases. The choice of using electrochemotherapy in our patient was determined by the locally advanced pathology that would not permit a surgical resection and by the patient's refusal of radiotherapy. This case demonstrates that electrochemotherapy can be considered as an effective palliative treatment op- tion for patients with recurrent or advanced-stage tumour, not suitable for conventional treatments. References 1. Warren S, Warvi WN. Tumors of sebaceous glands. Am J Pathol 1943; 19: 441-59. 2. Nelson BR, Hamlet KR, Gillard M, Railan D, Johnson TM. Sebaceous carcinoma. J Am Acad Dermatol 1995; 33: 1-15. 3. Tryggvason G, Bayon R, Pagedar NA. Epidemiology of sebaceous carcinoma of the head and neck: implications for lymph node management. Head Neck 2012; 34: 1765-8. 4. Samarasinghe V, Marsden J, Roberts C. Sebaceous carcinoma of the scalp presenting with nodal metastasis. J Plast Reconstr Aesthet Surg 2010; 63: 2193-4. 5. Buitrago W, Joseph AK. Sebaceous carcinoma: the great masquerader: emerging concepts in diagnosis and treatment. Dermatol Ther 2008; 21: 459-66. 6. Erovic BM, Goldstein DP, Kim D, Al Habeeb, Waldron J, Ghazarian D, A, et al. Sebaceous gland carcinoma of the head and neck: the Princess Margaret Hospital experience. Head Neck 2013; 35: 316-20. 7. Ansai S, Takeichi H, Arase S, Kawana S, Kimura T. Sebaceous carcinoma: an immuno-histochemical reappraisal. Am J Dermatopathol 2011; 33: 579-87. 8. Rumelt S, Hogan NR, Rubin PA, Jakobiec FA. Four-eyelid sebaceous cell carcinoma fol-lowing irradiation. Arch Ophthalmol 1998; 116: 1670-2. 9. Landis MN, Davis CL, Bellus GA, Wolverton SE. Immunosuppression and sebaceous tu-mors: a confirmed diagnosis of Muir-Torre syndrome unmasked by immunosuppressive therapy. J Am Acad Dermatol 2011; 65: 1054-8. 10. Shields JA, Demirci H, Marr BP, Eagle RC Jr, Shields CL. Sebaceous carcinoma of the eyelids: personal experience with 60 cases. Ophthalmology 2004; 111: 2151-7. 11. Dasgupta T, Wilson LD, Yu JB. A retrospective review of 1349 cases of sebaceous carcinoma. Cancer 2009; 115: 158-65. 12. Wang LS, Handorf EA, Wu H, Liu JC, Perlis CS, Galloway TJ. Surgery and adjuvant radiation for high-risk skin adnexal carcinoma of the head and neck. Am J Clin Oncol 2015; [Epub ahead of print]; PMID: 25599317 13. Ribero S, Osella-Abate S, Di Capua C, Dika E, Balagna E, Senetta R, et al. Squamocellu-lar carcinoma of the skin: clinicopathological features predicting the involvement of the surgical margins and review of the literature. Dermatology 2016; [Epub ahead of print]; PMID: 27028227 14. Mellette JR, Amonette RA, Gardner JH, Chesney TM. Carcinoma of sebaceous glands on the head and neck. A report of four cases. J Dermatol Surg Onco! 1981; 7: 404-7. 15. Bolm I, Babaryka G, Moergel M, Al-Nawas B, Kämmerer PW. Multifocal me-tastasizing extra-ocular facial sebaceous carcinoma as diagnostic challenge: case report and systematic review. J Maxillofac Oral Surg 2015; 14(Suppl 1): 331-7. 16. Moreno C, Jacyk WK, Judd MJ, Requena L. Highly aggressive extraocular sebaceous carcinoma. Am J Dermatopathol 2001; 23: 450-5. 17. Moura C, Pecegueiro MM, Sachse MF, Amaro J, Fonseca I, Fernandes A, et al. Report of a case of Muir-Torre syndrome. J Eur Acad Dermatol Venereol 2002; 16: 638-40. 18. Khan Z, Misra G, Fiander AN, Dallimore NS. Sebaceous carcinoma of the vulva. BJOG 2003; 110: 227-8. 19. Murphy J, Bleach NR, Thyveetil M. Sebaceous carcinoma of the nose: multi-focal presentation? J Laryngol Otol 2004; 118: 374-6. 20. Swick JM, Lang PG Jr. Sebaceous gland carcinoma of the right flank. South Med J 2009; 102: 312-4. 21. Bhat IP, Madhukara J, Elizabeth J, Kini U, Anuradha A. Multifocal extra-ocular sebaceous carcinoma. Indian J Dermatol Venereol Leprol 2011; 77: 403. 22. Ansai S, Takeichi H, Arase S, Kawana S, Kimura T. Sebaceous carcinoma: an immuno-histochemical reappraisal. Am J Dermatopathol 2011; 33: 579-87. Radiol Oncol 2016; 50(3): 308-312. 312 Ribero S et al. / Electrochemotherapy for sebaceous cell carcinoma 312 23. Macripo G, Caliendo V, Grassi M, Lista P, Ribero S, Giacone E, et al. Squamous cell car-cinoma of the umbilicus: management of an unusual localization. Tumori 2011; 97: 236-8. 24. Rotunno R, Marenco F, Ribero S, Calvieri S, Amerio P, Curatolo P, et al. Electrochemotherapy in non-melanoma head and neck skin cancers: a three centers experience and literature review. G Ital Dermatol Venereol 2015; [Epub ahead of print]; PMID: 26376042 25. Campana LG, Clover AJ, Valpione S, Quaglino P, Gehl J, Kunte C, et al. Recommendations for improving the quality of reporting clinical electro-chemotherapy studies based on qualitative systematic review. Radiol Oncol 2016; 50: 1-13. 26. Gargiulo M, Moio M, Monda G, Parascandolo S, Cubicotti G. Electrochemotherapy: actual considerations and clinical experience in head and neck cancers. Ann Surg 2010; 251: 773. 27. Mali B, Miklavcic D, Campana LG, Cemazar M, Sersa G, Snoj M, et al. Tumor size and effectiveness of electrochemotherapy. Radiol Oncol 2013; 47: 3241. 28. Mir LM, Orlowski S. Mechanisms of electrochemotherapy. Adv Drug Deliv Rev 1999; 35: 107-18. 29. Marty M, Sersa G, Garbay JR, Gehl J, Collins CG, Snoj M, et al. Electrochemotherapy -an easy, highly effective and safe treatment of cutaneous and subcutaneous metastases: results of ESOPE (European Standard Operating Procedures of Electrochemotherapy) study. Eur J Cancer 2006; 4(Suppl 11): 3-13. 30. Spratt DE, Gordon Spratt EA, Wu S, DeRosa A, Lee NY, Lacouture ME, et al. Efficacy of skin-directed therapy for cutaneous metastases from advanced cancer: a meta-analysis. J Clin Ocol 2014; 32: 3144-55. 31. Solari N, Spagnolo F, Ponte E, Quaglia A, Lillini R, Battista M, et al. Electrochemotherapy for the management of cutaneous and subcutaneous metastasis: a series of 39 patients treated with palliative intent. J Surg Oncol 2014; 109: 270-4. 32. Di Monta G, Caraco C, Benedetto L, La Padula S, Marone U, Tornesello ML, et al. Electrochemotherapy as "new standard of ca-re" treatment for cutaneous Kaposi's sarcoma. Eur J Surg Oncol 2014; 40: 61-6. Radiol Oncol 2016; 50(3): 308-312. 313 research article The proliferation marker Ki67, but not neuroendocrine expression, is an independent factor in the prediction of prognosis of primary prostate cancer patients Mariarosa Pascale1, Cinzia Aversa2, Renzo Barbazza2, Barbara Marongiu1, Salvatore Siracusano2, Flavio Stoffel3, Sando Sulfaro4, Enrico Roggero1, Serena Bonin2, Giorgio Stanta2 1 Oncology Institute of Southern Switzerland (IOSI), Bellinzona, Switzerland 2 Department of Medical Sciences, University of Trieste, Cattinara Hospital, Trieste, Italy 3 Department of Urology, Ospedale San Giovanni, Bellinzona, Switzerland 4 Department of Laboratory Medicine, S.C. Pathology, Santa Maria degli Angeli Hospital, Pordenone, Italy. Radiol Oncol 2016; 50(3): 313-320. Received 8 February 2016 Accepted 28 April 2016 Correspondence to: Serena Bonin, M.D., Department of Medical Sciences, Cattinara University Hospital, Strada di Fiume 447, 34149 Trieste, Italy. Phone: +39 40 3996266; E-mail: sbonin@units.it Disclosure: No potential conflicts of interest were disclosed. Background. Neuroendocrine markers, which could indicate for aggressive variants of prostate cancer and Ki67 (a well-known marker in oncology for defining tumor proliferation), have already been associated with clinical outcome in prostate cancer. The aim of this study was to investigate the prognostic value of those markers in primary prostate cancer patients. Patients and methods. NSE (neuron specific enolase), ChrA (chromogranin A), Syp (Synaptophysin) and Ki67 staining were performed by immunohistochemistry. Then, the prognostic impact of their expression on overall survival was investigated in 166 primary prostate cancer patients by univariate and multivariate analyses. Results. NSE, ChrA, Syp and Ki67 were positive in 50, 45, 54 and 146 out of 166 patients, respectively. In Kaplan-Meier analysis only diffuse NSE staining (negative vs diffuse, p = 0.004) and Ki67 (< 10% vs > 10%, p < 0.0001) were significantly associated with overall survival. Ki67 expression, but not NSE, resulted as an independent prognostic factor for overall survival in multivariate analysis. Conclusions. A prognostic model incorporating Ki67 expression with clinical-pathological covariates could provide additional prognostic information. Ki67 may thus improve prediction of prostate cancer outcome based on standard clinical-pathological parameters improving prognosis and management of prostate cancer patients. Key words: primary prostate cancer; prognosis; Ki67; NSE Introduction Conventional clinical parameters alone are inadequate for differentiating indolent and aggressive prostate cancer. Therefore, molecular biomarkers are needed to better define prognosis of prostate cancer patients. Neuroendocrine markers could be used to detect particularly aggressive variants of prostate cancer. Typical markers used to identify neuroendocrine differentiation (NED) in tumor tissue are neuron specific enolase (NSE), chromogranin A (CgA) and synaptophysin (Syp).1-3 Neuroendocrine differentiation, measured by one or more of those markers, has been associated with disease progression4 or poor survival in prostate cancer5, but up to now its prognostic value has not been clarified because of controversial results6;78. However, Epstein et al.9 Radiol Oncol 2016; 50(3): 308-312. doi:10.1515/raon-2016-0039 314 Pascale M et al. / Prognostic markers in primary prostate cancer patients have recently suggested using neuroendocrine markers to better characterize and classify NED in prostate cancer. They also outlined Ki67 ranges in those tumors, which usually have a high proliferative index.49-13 Ki67 is a well-known marker in oncology for defining tumor proliferation. Expression of Ki67 detection by immunohistochemistry14 is used as a prognostic marker for cell proliferation in many tumors, especially in breast carcinoma15 and cervical cancer.16 In prostate cancer it has also been associated with clinical outcome, irrespective of treatment.417-27 The aims of this study were to 1) investigate the immunohistochemical expression of neuroendocrine and Ki67 markers in primary prostate cancer patients in order to identify tumors characterized by biological aggressiveness and poor prognosis, 2) evaluate neuroendocrine expression with respect to Ki67 staining. Patients and methods Patients Detailed histopathological and clinical data were retrospectively collected for 166 patients, who were diagnosed with primary prostate cancer in a single institution of the North-eastern Italy from January 1992 to December 1994, therefore associated to a long follow-up period. Inclusion criteria for this study were: a) diagnosis of prostate cancer and b) availability of formalin-fixed and paraffin-embedded tissues for immunohistochemical staining and molecular analyses. Only TURP (N = 122, 73.9%) and prostatectomy (N = 43, 26.1%) specimens were used (missing information for one patient). Fine needle biopsies were excluded because of the low amount of tissue. Patients did not receive any treatment before diagnosis. The use of formalin-fixed and paraffin-embedded prostate cancer tissues and FIGURE 1. Determination of Ki67, NSE, CgA, SYP in primary prostate cancer patients. Number of concurrent biomarkers evaluated. The number of patients is reported above each column. their related clinical information were approved by the Ethical Committee of the University of Trieste (Report 23; 5.10.2009) before the beginning of the study. Tissue microarray and immunohistochemical staining Representative multiple areas of the primary tumours were selected by two pathologists (G.S. and R.B.) for TMA construction. Tissue cores were chosen at the border of the primary tumour. Tissue cylinders of 1.0 mm in diameter were taken from the selected regions of the donor's paraffin block and were placed into a recipient paraffin block using a tissue-arrayer (Galileo TMA CK3500; Integrated Systems Engineering, Milano, Italy), as previously described.28 Multiple cores were taken for cases as representative of heterogeneous histological areas. Neuroendocrine differentiation (NED) was evaluated using NSE, ChrA, Syp as neuroendocrine markers. Immunostainings for Ki67 (clone MIB-1; DakoDenmark A/S, Glostrup, Denmark), 1:200 dilution; NSE (clone E27; NeoMarker; ThermoScientific, Waltham, MA, USA) 1:2500; ChrA (clone LK2H10; NeoMarker; ThermoScientific, Waltham, MA, USA), 1:500; Syp (clone SY 38; Thermo-Fisher; ThermoScientific, Waltham, MA, USA) 1:75 were performed in a Lab Vision Autostainer 480S (Thermo Scientific, Waltham, MA, USA) with the UltraVision LP Large Volume Detection System HRP Polymer (Lab Vision Corporation, Thermo Scientific) according to manufacturer's recommended protocol. For evaluation of the immu-nostaining, positively stained cells were counted across 3 high-power fields. Staining intensity was not taken into consideration. Due to technical issues related to the detachment of tissue cores it was not possible to analyze the four biomarkers in all samples (Figure 1). The percentage of the positively stained cells was reported for each specimen. Ki67 expression was dichotomized for assessing its prognostic value using a cut-off of 10%.7,29 Statistical analysis The Kaplan-Meier method was used to generate overall survival (OS) curves, which was defined as the time between the date of diagnosis and the date of death or the last follow-up (FU) observation. Patients were censored if they were still alive or they were lost to FU. The log-rank test was used to evaluate differences between groups. Association Radiol Oncol 2016; 50(3): 313-320. 315 Pascale M et al. / Prognostic markers in primary prostate cancer patients TABLE 1. Neuroendocrine marker staining in our cohort of primary prostate cancer patients Staining Neuroendocrine markers NSE CgA Syp Negative Diffuse Focal Spotty Total missing 28 (31.5) 50 (56.1) 3 (3.4) 8 (9) 89 77 49 (40.5) 45 (37.2) 19 (15.7) 8 (6.6) 121 45 44 (34.6) 54 (42.5) 17 (13.4) 12 (9.5) 127 39 % Ki67-positive cells Number of samples N % 0 14 9.6 < 5 70 47.9 > 5 and < 10 24 16.5 > 10 and < 20 18 12.3 > 20 and < 30 13 8.9 > 30 7 4.8 Total 146 100% Data reported as N (%) TABLE 2. Distribution of Ki67 positively stained cells in our cohort of primary prostate cancer patients >S jnC S10 >20jnds}0 % positive KI67 cells FIGURE 2. Distribution of Ki67 staining score. Positive Ki67 cells in TURP and prostatectomy specimens from a cohort of 1 66 primary prostate cancer patients. TABLE 3. Association between Ki67 expression and clinicopathological variables (N = 146) of OS with each prognostic factor was evaluate in univariate and multivariate analyses by using the Cox proportional hazards model. All variables associated with univariate value of p < 0.05 were included in the multivariate model using a stepwise method. The proportional hazards assumptions were checked before applying the Cox regression model. The goodness of fit was assessed using a likelihood-ratio test. The discrimination ability was quantified by calculating the concordance index that ranges from 0.5 (no discrimination) to 1 (perfect discrimination). Possible correlations of the expression of Ki67 and neuroendocrine markers with the other prognostic variables were assessed by x2 test or Wilcoxon rank sum. All statistical tests were performed using STATA software (StataCorp. 2011. Stata Statistical Software: Release 12. College Station, TX: StataCorp LP) and p values < 0.05 were considered as statistically significant. Variable Ki67 expression < 10% > 10% Tot P-value Nuclear grading 1 22 1 23 2 72 23 95 0.001* 3 14 14 28 Gleason Score < 7 56 8 64 0.001* > 7 51 30 81 missing 1 Age at diagnosis (median, years) < 71 53 19 72 0.922 > 71 55 19 74 Age at diagnosis (continuous) 108 38 146 0.973 NSE expression Negative 21 4 25 0.073 Positivea 28 16 44 missing 77 CgA expression Negative 37 10 47 0.217 Positivea 24 12 36 missing 40 Syp expression Negative 31 8 39 0.326 Positivea 33 14 47 missing 60 Type of intervention TURP 78 33 111 0.081 Prostatectomy 29 5 34 missing 1 Total 108 38 146 a Only diffuse expression was considered as positive staining; *Significant value Results Neuroendocrine marker staining NSE expression was assessable in 89 prostate cancer cases. Of those, 28 cases (31.5%) were completely negative, whereas 61 (68.5%) revealed a cytoplasmic positivity, referring to diffuse, focal and spotty staining (Table 1). Detachment of tissue cores did not allow analyzing NSE in some cases. ChrA was negative in 49 out of 121 cores (40.5%) and it was positive on the cytoplasmic level in 72 cores (59.5%). Positive cases had weakly to highly diffused, or focal or spotty reactivity (Table 1). Syp was analyzed in 127 cases of which 44 (34.6%) were negative and 83 (65.4%) showed a cytoplasmic pos-itivity, from spotty to focal to diffused (Table 1). Only diffused expression was considered as posi- Radiol Oncol 2016; 50(3): 313-320. 316 Pascale M et al. / Prognostic markers in primary prostate cancer patients TABLE 4. Univariate analysis of prognostic factors in patients with primary prostate cancer based on Cox proportional hazards regression model Variable UNIVARIATE HR (95% CI) P-value Nuclear grading 2 vs 1 3 vs 1 1.45 (0.90-2.34) 3.49 (1.94-6.27) 0.125 < 0.001* Gleason score > 7 vs < 7 2.97 (2.07-4.26) < 0.001* Ki67 expression > 10% vs < 10% 2.75 (1.85-4.09) < 0.001* Age at diagnosis (continuous) 1.04 (1.02-1.07) < 0.001* NSE expression Positive0 vs negative 1.99 (1.19-3.34) 0.009* CgA expression Positivea vs negative 0.96 (0.62-1.47) 0.840 Syp expression Positivea vs negative 1.18 (0.77-1.81) 0.447 Type of intervention Prostatectomy vs TURP 0.45 (0.30-0.67) < 0.001* CI = confidence interval; HR = hazard ratio a Only diffuse expression was considered as positive staining; *Significant value TABLE 5. Multivariate analysis of prognostic factors in patients with primary prostate cancer based on Cox proportional hazards regression model stratified by type of intervention (N = 144) MULTIVARIATE Variable Adjusted HR (95%CI) Adjusted P-value Nuclear grading 2 vs 1 3 vs 1 1.27 (0.75-2.13) 1.93 (1.01-3.68) 0.373 0.045* Gleason score > 7 vs < 7 2.41 (1.56-3.74) < 0.001* Ki67 expression > 10% vs < 10% 2.14 (1.41-3.25) < 0.001* Age at diagnosis (continuous) 1.04 (1.02-1.07) 0.001* CI = confidence interval; HR = hazard ratio; *Significant value tive for each marker. Focal or spotty stainings were evaluated as negative. No significant associations were found between neuroendocrine markers and clinicopathological variables. A slight association was revealed between NSE expression and Gleason score (p = 0.04). Moreover, a positive relationship was found be- tween CgA and SYP expression (p = 0.01). The type of intervention was significantly associated with CgA (p = 0.009) and NSE (p = 0.001) expression. Ki67 staining Ki67 staining analysis was measured in 146 out of 166 prostatic cases. The median Ki67 staining score was 5% with an IQR of 9 whereas the mean value was 10.3% with a standard deviation of 14.2% (range of 0 - 90%). The percentage of the positively stained cells was recorded for each sample (Table 2, Figures 2,3). Ki67 was scored and stratified into two groups (low < 10%; high > 10%) as already reported.2324 No statistically significant difference was observed for Ki67 staining between TURP and prostatectomy specimens (p = 0.08). Ki67 expression was significantly associated with Gleason score and nuclear grading, but not with age at diagnosis or with neuroendocrine markers (Table 3). Univariate analysis In univariate analysis, diffuse expression of NSE, Ki67 expression > 10%, Gleason score > 7 and nuclear grading > 2 predicted for shorter OS (Table 4). Considering neuroendocrine markers, only NSE staining was significantly associated with OS. Patients with diffuse NSE expression had a reduced survival time (median OS 2 years; 95% CI, 2-4) compared to patients with negative expression (median OS 7; 95% CI, 5-10; p = 0.004), showing nearly double-fold increased risk of death (Table 4, Figure 4). Additionally, NED measured as positive at least at one of the three markers did not result significant. Kaplan-Meier survival curves showed a significant difference (p < 0.001) between low and high levels of Ki67 staining with median survival time of 6 years (95% CI, 5-9) and 2 years (95% CI, 1-2) for patients with < 10% and > 10% of positive cells, respectively (Figure 5A). Moreover, splitting group four categories according to Ki67 staining were obtained: negative, 1-10%, 11-20%, > 20%. Negative patients showed a median overall survival (10 years, 95% CI: 4-9) which was four years longer than in patients with Ki67 staining of 1-10% (6 years, 95% CI: 5-8; p < 0.001) (Figure 5B). An improved discrimination was also reached in the category > 10%. Patients with Ki67 staining > 20% were associated with a 2.5-fold increased risk of death, compared with patients showing Ki67 expression of 10-20% (the overall 2-year survival Radiol Oncol 2016; 50(3): 313-320. 77 Pascale M et al. / Prognostic markers in primary prostate cancer patients rate of 50%-95% CI: 25-70 vs 20%, 95% CI: 6-39, p < 0.001). These data further support the potential role of Ki67 immunostaining in selecting patients according to proliferation rate, and thus to tumor aggressiveness. However, we decided to assess the prognostic value of Ki67 for overall survival of prostate cancer patients by using the previous binary variable because of harmonization with already published studies.7,29 Age at diagnosis and type of intervention significantly impacted on OS (p < 0.001). Thus, considering the relationship existing with other variables, they were included in multivariate analysis to take into account their possible confounding effect. Multivariate analysis Multivariate analysis was done by stratifying according to type of intervention, because of no proportional risks between TURP and prostatectomy groups (Test of proportional-hazards assumption, C2 = 8.86, df = 1, p = 0.003). Ki67 expression, but not NSE, resulted as an independent prognostic factor for OS. In the final multivariable model the risk of death was higher for older patients (p = 0.001) with a nuclear grading of 3 (p = 0.04), a Gleason score > 7 (p < 0.001) and Ki67 expression > 10% (p < 0.001) (Table 5). Unfortunately, our dataset did not include information on PSA before surgical intervention for all patients, because they were diagnosed many years ago before the routinely application of PSA screening. Comparison of the multivariate model incorporating Ki67 expression with a base model including conventional variables only (nuclear grading, Gleason score, age at diagnosis stratified by type of intervention) showed an improved fit which suggested an enhanced prognostic ability over the models containing clinicopathological variables only (x2 = 11.33, df = 1, p = 0.0006). These data indicated that in a multivariate analysis Ki67 is a relevant and independent prognostic factor for OS of primary prostate cancer patients undergoing TURP or radical prostatectomy. The concordance index (0.72) revealed a good accuracy of the model in predicting OS. Discussion This study shows that Ki67 expression, but not NED, is an independent prognostic factor for OS in primary prostate cancer patients who underwent TURP or prostatectomy. fW V - rw ■ * ÏÈÛ ' w ffl, - Vv (i y >■■ ¿if- C ® ■■ r*'C/&S. v ■i* .f "if 4«* p * - f" " b rr t A* ', : W^t; FIGURE 3. Representative immunohistochemical staining for Ki67. (A) prostate adenocarcinoma with Ki67 > 10% 20 x and 40 x (B) magnification; (C) prostate adenocarcinoma with Ki67 < 10% 20 x and 40 x (D) magnification; (E) prostate adenocarcinoma negative for Ki67 20 x and 40 x (F) magnification. FIGURE 4. Survival curves by NSE staining in patients with primary prostate cancer. p-value from log-rank test is reported. Numbers of at risk (still alive) patients are indicated below the x-axis. Radiol Oncol 2016; 50(3): 313-320. 318 Pascale M et al. / Prognostic markers in primary prostate cancer patients LO = Is- 20% p< 0.001 No. at risk negative 14 1-10% 94 11-20% 18 > 20% 20 11 59 7 1 10 Time (years) 31 2 0 15 5 19 1 0 20 FIGURE 5. Survival curves by Ki67 expression in patients with primary prostate cancer. (A) Ki67 staining dichotomized in <10% and > 10%; (B) Ki67 staining divided into negative, 1-10%, 11%-20%, > 20%. p-value from log-rank test is reported. Numbers of at risk (still alive) patients are indicated below the x-axis. Our data demonstrate that NED, as measured by NSE, CgA and SYP immunohistochemistry, is present at the time of diagnosis in a large proportion of our cohort (over 50%), but without influence on OS. NSE staining seems to influence OS, but it was not confirmed as an independent prognostic factor in the multivariate analysis. These results are consistent with current published literature where strong evidence of NSE as potential prognostic factor is lacking (reviewed in 68), especially at early stages.30 NED in PCa increases with higher histological grades31 and disease progression, especially in response to androgen deprivation therapy.32-34 It seems that androgen deprivation therapy may promote the transformation of prostate adeno-carcinoma into a neuroendocrine cancer, defined as t-NEPC (transformed neuroendocrine prostate cancer), as a mechanism of resistance.35 Ki67 expression has already been proposed as a candidate marker for gastroenteropancreatic36 and lung neuroendocrine cancers.37 An increased proliferation using Ki67 expression has been shown in prostate tumors with high NED compared to tumors with low or without NED.41011 In this study we did not find any association between NED and Ki67 expression, although a trend for a positive relationship between NSE staining and Ki67 expression (p = 0.08) has been observed. Limitations of this study are: small sample size, missing data for neuroendocrine IHC, statistically significant results obtained by using the less specific marker for NED.9 Another limitation is the analysis based on mixed sample population of prostatectomy and TURP which was partially solved using a stratified Cox model to investigate the risk difference in two groups. Of relevance, our cohort represents a long-term time series which allows investigating OS in cancers with a long life expectancy. The prognostic significance of concurrent presence of NED features in prostate adenocarcinoma is currently very controversial.6 Although NED may have an adverse effect on prognosis of newly diagnosed prostate cancer, other mechanisms probably influence the prognosis by favoring the selection of the neuroendocrine pattern transformation under a specific stimulus, such as the pressure by androgen deprivation therapy.38 The mechanisms that are currently involved are not known. Interestingly, we found that cell proliferation measured by Ki67 staining scored as a dichoto-mous variable with a 10% cut-off is an independent prognostic factor for OS in our cohort. Comparing prognostic models with and without Ki67 demonstrated that Ki67 expression could yield additional prognostic information to that provided by conventional clinicopathological parameters improving prognosis of prostate cancer patients. Furthermore, our data show that Ki67 expression correlates with Gleason score and nuclear grading, highlighting its association with prostate cancer aggressiveness, in agreement with others.4,23253940 Several studies have shown that Ki67 is useful to predict prostate cancer prognosis either on TURP19,23 or needle biop- sy 21,23,25,40 specimens. Furthermore, it has been pro- posed as a candidate prognostic marker both for overall and specific survival endpoints17-22 as well as disease progression.4,17,18,20,22-27 Its prognostic rel- Radiol Oncol 2016; 50(3): 313-320. 319 Pascale M et al. / Prognostic markers in primary prostate cancer patients evance does not seem to be influenced by therapy, as it has been reported to predict prognosis in patients treated by prostatectomy, alone4'24'2527 or with adjuvant therapy26, or radiotherapy, alone172223 or with androgen deprivation therapy1718,20,22, or conservatively managed.1921 Consistent with our results, Ki67 emerges as a powerful marker of biological aggressiveness that could provide supplemental prognostic information, concerning the cellular proliferation rate, in addition to that provided by currently used markers, which are related to tumor pattern and extension only. Therefore, Ki67 may improve prediction of prostate cancer outcome based on standard clinical parameters, and may help stratify and select patients for more aggressive treatments. The utility of Ki67 was also demonstrated in selecting candidates with clinically insignificant cancer suitable for active surveillance among patients with PSA < 4 ng/ml at diagnosis.40 The validity of Ki67 expression as an indicator of prognosis for prostate cancer patients in different treatment cohorts, including both radical4,17,18,20,22-27 and conservative thera- pies1921, and in many investigative materials, such as biopsy21,23,25,40, TURP1923 or prostatectomy424-27 specimens, further supports its implementation in clinical practice, as recently sustained also in a me-ta-analysis.41 However, larger prospective studies are needed to validate its use in routine pathology. Despite unresolved issues on cut-offs, we suggest the analysis of Ki67 in routine diagnostic practice as an additional factor for improving prognosis and management of prostate cancer patients. Acknowledgments This work was partially supported by grants from ABREOC 2011. References 1. Schmechel D, Marangos PJ, Brightman M. Neurone-specific enolase is a molecular marker for peripheral and central neuroendocrine cells. Nature 1978; 276: 834-6. 2. Kimura N, Funakoshi A, Aunis D, Tateishi K, Miura W, Nagura H. Immunohistochemical localization of chromostatin and pancreastatin, chromogranin A-derived bioactive peptides, in Normal and Neoplastic Neuroendocrine Tissues. Endocr Pathol 1995; 6: 35-43. 3. Wiedenmann B, Franke WW, Kuhn C, Moll R, Gould VE. Synaptophysin: a marker protein for neuroendocrine cells and neoplasms. Proc Natl Acad Sci USA 1986; 83: 3500-4. 4. May M, Siegsmund M, Hammermann F, Loy V, Gunia S. Prognostic significance of proliferation activity and neuroendocrine differentiation to predict treatment failure after radical prostatectomy. Scand J Urol Nephrol 2007; 41: 375-81. 5. Krauss DJ, Amin M, Stone B, Ye H, Hayek S, Cotant M, et al. Chromogranin A staining as a prognostic variable in newly diagnosed Gleason score. Prostate 2014; 74: 520-7. 6. Berruti A, Vignani F, Russo L, Bertaglia V, Tullio M, Tucci M, et al. Prognostic role of neuroendocrine differentiation in prostate cancer, putting together the pieces of the puzzle. Open Access J Urol 2010; 2: 109-24. 7. Cindolo L, Cantile M, Franco R, Chiodini P, Schiavo G, Forte I, et al. Parallel determination of NeuroDl, chromogranin-A, KI67 and androgen receptor expression in surgically treated prostate cancers. Int Braz J Urol 2011; 37: 57-66. 8. Jeetle SS, Fisher G, Yang ZH, Stankiewicz E, Moller H, Cooper CS, et al. Neuroendocrine differentiation does not have independent prognostic value in conservatively treated prostate cancer. Virchows Arch 2012; 461: 103-7. 9. Epstein JI, Amin MB, Beltran H, Lotan TL, Mosquera J-M, Reuter VE, et al. Proposed morphologic classification of prostate cancer with neuroendocrine differentiation. Am J Surg Pathol 2014; 38: 756-67. 10. Helpap B, Kollermann J. Undifferentiated carcinoma of the prostate with small cell features: immunohistochemical subtyping and reflections on histogenesis. Virchows Arch 1999; 434: 385-91. 11. Grobholz R, Griebe M, Sauer CG, Michel MS, Trojan L, Bleyl U. Influence of neuroendocrine tumor cells on proliferation in prostatic carcinoma. Hum Pathol 2005; 36: 562-70. 12. Gunia S, Albrecht K, Koch S, Herrmann T, Ecke T, Loy V, et al. Ki67 staining index and neuroendocrine differentiation aggravate adverse prognostic parameters in prostate cancer and are characterized by negligible interobserver variability. World J Urol 2008; 26: 243-50. 13. Beltran H, Tomlins S, Aparicio A, Arora V, Rickman D, Ayala G, et al. Aggressive variants of castration-resistant prostate cancer. Clin Cancer Res 2014; 20: 2846-50. 14. Gerdes J, Schwab U, Lemke H, Stein H. Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer 1983; 31: 13-20. 15. Dowsett M, Nielsen TO, A'Hern R, Bartlett J, Coombes RC, Cuzick J, et al. Assessment of Ki67 in Breast Cancer: Recommendations from the International Ki67 in Breast Cancer Working Group. J Natl Cancer Inst 2011; 103: 1656-64. 16. Ikenberg H, Bergeron C, Schmidt D, Griesser H, Alameda F, Angeloni C, et al. Screening for cervical cancer precursors with p16/Ki-67 dual-stained cytology: results of the PALMS study. J Natl Cancer Inst 2013; 105: 1550-7. 17. Li R, Heydon K, Hammond ME, Grignon DJ, Roach M 3rd, Wolkov HB, et al. Ki-67 staining index predicts distant metastasis and survival in locally advanced prostate cancer treated with radiotherapy: an analysis of patients in radiation therapy oncology group protocol 86-10. Clin Cancer Res 2004; 10: 4118-24. 18. Pollack A, DeSilvio M, Khor LY, Li R, Al-Saleem TI, Hammond ME, et al. Ki-67 staining is a strong predictor of distant metastasis and mortality for men with prostate cancer treated with radiotherapy plus androgen deprivation: Radiation Therapy Oncology Group Trial 92-02. J Clin Oncol 2004; 22: 2133-40. 19. Berney DM, Gopalan A, Kudahetti S, Fisher G, Ambroisine L, Foster CS, et al. Ki-67 and outcome in clinically localised prostate cancer: analysis of conservatively treated prostate cancer patients from the Trans-Atlantic Prostate Group study. Br J Cancer 2009; 100: 888-93. 20. Khor LY, Bae K, Paulus R, Al-Saleem T, Hammond ME, Grignon DJ, et al. MDM2 and Ki-67 predict for distant metastasis and mortality in men treated with radiotherapy and androgen deprivation for prostate cancer: RTOG 9202. J Clin Oncol 2009; 27: 3177-84. 21. Fisher G, Yang ZH, Kudahetti S, M0ller H, Scardino P, Cuzick J, et al. Prognostic value of Ki-67 for prostate cancer death in a conservatively managed cohort. Br J Cancer 2013; 108: 271-7. 22. Verhoven B, Yan Y, Ritter M, Khor LY, Hammond E, Jones C, et al. Ki-67 is an independent predictor of metastasis and cause-specific mortality for prostate cancer patients treated on Radiation Therapy Oncology Group (RTOG) 94-08. Int J Radiat Onco! Bio! Phys 2013; 86: 317-23. 23. Cowen D, Troncoso P, Khoo VS, Zagars GK, von Eschenbach AC, Meistrich ML, et al. Ki-67 staining is an independent correlate of biochemical failure in prostate cancer treated with radiotherapy. Clin Cancer Res 2002; 8: 1148-54. Radiol Oncol 2016; 50(3): 313-320. 320 Pascale M et al. / Prognostic markers in primary prostate cancer patients 24. Laitinen S, Martikainen PM, Tolonen T, Isola J, Tammela TL, Visakorpi T. EZH2, Ki-67 and MCM7 are prognostic markers in prostatectomy treated patients. Int J Cancer 2008; 122: 595-602. 25. Zellweger T, Günther S, Zlobec I, Savic S, Sauter G, Moch H, et al. Tumour growth fraction measured by immunohistochemical staining of Ki67 is an independent prognostic factor in preoperative prostate biopsies with small-volume or low-grade prostate cancer. Int J Cancer 2009; 124: 2116-23. 26. Antonarakis ES, Keizman D, Zhang Z, Gurel B, Lotan TL, Hicks JL, et al. An immunohistochemical signature comprising PTEN, MYC, and Ki67 predicts progression in prostate cancer patients receiving adjuvant docetaxel after prostatectomy. Cancer 2012; 118: 6063-71. 27. Rubio J, Ramos D, Lopez-Guerrero JA, Iborra I, Collado A, Solsona E, et al. Immunohistochemical expression of Ki-67 antigen, cox-2 and Bax/Bcl-2 in prostate cancer; prognostic value in biopsies and radical prostatectomy specimens. Eur Urol 2005; 48: 745-51. 28. Pascale M, Pracella D, Barbazza R, Marongiu B, Roggero E, Bonin S, et al. Is human papillomavirus associated with prostate cancer survival? Dis Markers 2013; 35: 607-13. 29. Zellweger T, Ninck C, Mirlacher M, Annefeld M, Glass AG, Gasser TC, et al. Tissue microarray analysis reveals prognostic significance of syndecan-1 expression in prostate cancer. Prostate 2003; 55: 20-9. 30. Surcel CI, van Oort IM, Sooriakumaran P, Briganti A, De Visschere PJL, Futterer JJ, et al. Prognostic effect of neuroendocrine differentiation in prostate cancer: A critical review. Urol Oncol 201; 33: 265.e1-7. 31. Hirano D, Jike T, Okada Y, Minei S, Sugimoto S, Yamaguchi K, et al. Immunohistochemical and ultrastructural features of neuroendocrine differentiated carcinomas of the prostate: an immunoelectron microscopic study. Ultrastruct Pathol 2005; 29: 367-75. 32. Hirano D, Okada Y, Minei S, Takimoto Y, Nemoto N. Neuroendocrine differentiation in hormone refractory prostate cancer following androgen deprivation therapy. Eur Urol 2004; 45: 586-92. discussion 592. 33. Beltran H, Tagawa ST, Park K, MacDonald T, Milowsky MI, Mosquera JM, et al. Challenges in recognizing treatment-related neuroendocrine prostate cancer. J Clin Oncol 2012; 30: e386-9. 34. Komiya A, Yasuda K, Watanabe A, Fujiuchi Y, Tsuzuki T, Fuse H. The prognostic significance of loss of the androgen receptor and neuroendocrine differentiation in prostate biopsy specimens among castration-resistant prostate cancer patients. Mol Clin Oncol 2013; 1: 257-62. 35. Aggarwal R, Zhang T, Small EJ, Armstrong AJ. Neuroendocrine prostate cancer: subtypes, biology, and clinical outcomes. J Natl Compr Canc Netw 2014; 12: 719-26. 36. Ezziddin S, Attassi M, Yong-Hing CJ, Ahmadzadehfar H, Willinek W, Grünwald F, et al. Predictors of long-term outcome in patients with well-differentiated gastroenteropancreatic neuroendocrine tumors after peptide receptor radionuclide therapy with 177Lu-Octreotate. J Nucl Med 2014; 55: 183-90. 37. Pelosi G, Rindi G, Travis WD, Papotti M. Ki-67 antigen in lung neuroendocrine tumors: unraveling a role in clinical practice. J Thorac Oncol 2014; 9: 273-84. 38. Mosquera JM, Beltran H, Park K, MacDonald TY, Robinson BD, Tagawa ST, et al. Concurrent AURKA and MYCN gene amplifications are harbingers of lethal treatment-related neuroendocrine prostate cancer. Neoplasia 2013; 15: 1-10. 39. Khoo VS, Pollack A, Cowen D, Joon DL, Patel N, Terry NH, et al. Relationship of Ki-67 labeling index to DNA-ploidy, S-phase fraction, and outcome in prostate cancer treated with radiotherapy. Prostate 1999; 41: 166-72. 40. Nagao K, Yamamoto Y, Hara T, Komatsu H, Inoue R, Matsuda K, et al. Ki67 and BUBR1 may discriminate clinically insignificant prostate cancer in the PSA range < 4 ng/ml. Jpn J Clin Oncol 2011; 41: 555-64. 41. Zhao L, Yu N, Guo T, Hou Y, Zeng Z, Yang X, et al. Tissue biomarkers for prognosis of prostate cancer: a systematic review and meta-analysis. Cancer Epidemiol Biomarkers Prev 2014; 23: 1047-54. Radiol Oncol 2016; 50(3): 313-320. 321 research article Pancreaticoduodenectomy for ductal adenocarcinoma of the pancreatic head with venous resection Vojko Flis1, Stojan Potrc2, Nina Kobilica1, Arpad Ivanecz2 1 Department of Vascular Surgery, Surgical clinics, University Clinical Centre Maribor, Slovenia 2 Department of Abdominal Surgery, Surgical clinics, University Clinical Centre Maribor, Slovenia Radiol Oncol 2016; 50(3): 321-328. Received 17 December 2014 Accepted 26 March 2015 Correspondence to: Vojko Flis, M.D., Ph.D., Department of Vascular Surgery, Surgical Clinics, University Clinical Centre Maribor, Slovenia. E-mail: vojko.flis@ukc-mb.si Disclosure: No potential conflicts of interest were disclosed. Background. Recent reports have shown that patients with vascular tumour invasion who undergo concurrent vascular resection can achieve long-term survival rates equivalent to those without vascular involvement requiring pancreaticoduodenectomy alone. There is no consensus about which patients benefit from the portal-superior mes-enteric vein resection and there is no consensus about the best surgical technique of vessel reconstruction (resection with or without graft reconstruction). As published series are small the aim of this study was to evaluate our experience in pancreatectomies with en bloc vascular resection and reconstruction of vessels. Methods. Review of database at University Clinical Centre Maribor identified 133 patients (average age 65.4 ± 8.6 years, 69 female patients) who underwent pancreatoduodenectomy between January 2006 and August 2014. Clinical data, operative results, pathological findings and postoperative outcomes were collected prospectively and analyzed. Current literature and our experience in pancreatectomies with en bloc vascular resection and reconstruction of portal vein are reviewed. Results. Twenty-two patients out of 133 (16.5%) had portal vein-superior mesenteric vein resection and portal vein reconstruction (PVR) during pancreaticoduodenectomy. In fourteen patients portal vein was reconstructed without the use of synthetic vascular graft. In these series two types of venous reconstruction were performed. When tumour involvement was limited to the superior mesenteric vein (SPV) or portal vein (PV) such that the splenic vein could be preserved, and vessels could be approximated without tension a primary end-to-end anastomosis was performed. When tumour involved the SMV-splenic vein confluence, splenic vein ligation was necessary. In the remaining eight procedures interposition graft was needed. Dacron grafts with 10 mm diameter were used. There was no infection after dacron grafting. One patient had portal vein thrombosis after surgery: it was thrombosis after primary reconstruction. There were no thromboses in patients with synthetic graft interposition. There were no significant differences in postoperative morbidity, mortality or grades of complication between groups of patients with or without a PVR. Median survival time in months was in a group with vein resection 16.13 months and in a group without vein resection 15.17 months. Five year survival in the group without vein resection was 19.5%. Comparison of survival curves showed equal hazard rates with log-rank p = 0.090. Conclusions. Survival of patients with pancreatic cancer who undergo an R0 resection with reconstruction was comparable to those who have a standard pancreaticoduodenectomy with no added mortality or morbidity. Synthetic graft appeared to be an effective and safe option as an interposition graft for portomesenteric venous reconstruction after pancreaticoduodenectomy. Key words: pancreaticoduodenectomy; pancreatic cancer; vein resection Radiol Oncol 2016; 50(3): 308-312. doi:10.1515/raon-2016-0039 322 Flis V et al. / Pancreaticoduodenectomy with vein resection Introduction In cases of pancreatic cancer pancreaticoduodenectomy with complete resection offers the only chance for cure. Historically, involvement of regional vascular structures by pancreatic carcinoma has been considered a contraindication for reconstruction.1 At the time of diagnosis more than three quarters of patients have locally advanced disease or distant metastasis that preclude radical surgery and 5-year survival after "curative" surgery ranges from 10 to 20% even in recent large series.2 Advances in surgical techniques, perioperative care and the institution of tertiary specialized centres have been the key for a substantial improvement in mortality and morbidity rate. Venous resection (VR) is performed to achieve negative resection margins because the tumour involves the vessel or inflammatory adhesions preclude a safe separation of the vein. Another theoretical benefit of VR is to achieve clearance of surrounding perivascular and perineural tissue. Venous resections (VR) include excision of portal vein (PV), superior mesenteric vein (SMV) or the superior mesenteric-portal vein confluence (SMPV).2 Although the utility of aggressive vascular resection in pancreatic adenocarcinoma continues to be debated34 several institutional series have demonstrated the feasibility of margin negative resection with acceptable morbidity rates comparable to those after isolated pancreaticoduodenectomy. Recent reports also have shown that patients with vascular tumour invasion who undergo concurrent vascular resection can achieve long-term survival rates equivalent to those without vascular involvement requiring PD alone.5-9 Reconstruction of the PV or SMV is a challenge for the vascular surgeon because of the lack of size-matched autogenous conduit. In addition, concerns about graft infection have restricted the use of prosthetic grafts during the intra-abdom-inal surgery.9 Numerous techniques of VR have been described, ranging from partial excision of the lateral wall to major segmental resections.10-12 The resultant defects can be repaired with either a primary anastomosis or a graft. A variety of different native vessels and synthetic grafts have been described to bridge the defect. Each method, however, has limitations and the optimal conduit and surgical methods remain a controversy.13-15 As published series are small the aim of this study was to evaluate our experience in pancreatectomies for ductal adenocarcinoma with en bloc vascular resection and reconstruction of vessels. Methods Approval of the Research Council of Surgical Clinics was obtained to perform the audit of patients with pancreatic adenocarcinoma undergoing surgery between January 2006 and August 2014. Clinical data, operative results, pathological findings and postoperative outcomes were collected prospectively and analyzed. Preoperative evaluation All patients underwent contrast-enhanced CT as a routine preoperative work-up. Magnetic resonance imaging, endoscopic ultrasound scan, and laparos-copy were performed on an individual basis based on the multidisciplinary team discussion. The final operative decision lay with the surgeon at lapa-ratomy. Only patients deemed respectable preop-eratively were included. The criteria for en bloc resection where there was no evidence of metastatic disease were the following: tumour not involving the root of the small bowel mesentery; tumour not involving the superior mesenteric artery, celiac axis, or hepatic artery; and intention of obtaining R0 resection margin status. Patients with portal vein occlusion were not included. The general condition of the patients was determined by American Society of Anesthesiologists (ASA) score.16 For study purposes regarding pre-operative level of bilirubin, Carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 199) subgroups were formed. Perioperative data The operative approach was a median laparotomy until the 2007 later a bilateral subcostal laparotomy was preferred. Ultrasound examination of the pancreas was always used for evaluation of vascular involvement and for possible liver metastases. Pancreatic head resections were done in a conventional manner.17 In the last two years the artery first approach (posterior approach) was used whenever the infiltration and resection of superior mesenteric vein were planned.18 Jejunum was exclusively used for the anastomosis to the pancreas (duct to mucosa type, two layers) and for bile reconstruction (on layer) successively. A separate Roux en Y loop for pancreatojejuno anastomosis was done only in 2 patients. All gastro/pyloro-jeju-no anastomoses were placed above the colon. In all patients with pylorus-preserving pancreaticoduo-denectomy (PPPD)/Whipple resection a small en- Radiol Oncol 2016; 50(3): 321-328. 323 Flis V et al. / Pancreaticoduodenectomy with vein resection teroentero anastomosis was added to connect the afferent end efferent loop of gastro/pyloro-jejuno anastomosis. In total and left pancreatectomy by rule the spleen and the splenic vessels were resected "en block", however in three 3 total pan-cratectomies with additional intraductal papillary mucinous neoplasia of the left pancreas the spleen was preserved. The pancreatic stump was almost exclusively closed with sutures. At the end of operation abdominal drains were always placed. In 3 cases spleen could be preserved, however in others ligation of splenic artery at the origin and splenic vein at the confluence was done. For prevention of pancreatic fistula in cases with the soft texture of the pancreas somatostatin 0.6 mg daily for 5 to 8 days was administrated.19 Vascular resections were carried out as primary closure of the vein, end to end anastomosis, or a segmental resection and reconstruction with interposition graft. Dacron grafts with 10 mm diameter were used. Postoperative follow up After surgery, the patients were followed up to detect complications, local recurrence, distant metastasis and survival rate. The surgical complications were noted and classified.20 Laboratory tests and control of the tumour markers CEA and CA 19-9 as well as ultrasound and/or CT scans were obtained at three to four month intervals within 2 years after the operation and then later at six month intervals. The samples of fluid on drains were regularly examined for amylase on the day 4 and anytime in the course if the volume on drains was more than 50ml to rule out the possible pancreatic fistula (PF).21 Adjuvant chemotherapy was given according to final patohistological stage (pTNM) and was gem-citabine based on the majority of cases. Hospital stay was defined as time from operation to final dismissal from the hospital. 30 and 60 day mortality was defined as any postoperative death within 30 or 60 days after the operation. All resected specimens were sent to standardized pathohistological work up to the Department of pathology in Maribor. Statistical analysis Perioperative and clinicopathological parameters were evaluated and further compared between the two groups of patients. Categorical data were compared using x2 test or Fischer's exact test. TABLE 1. Selected clinical characteristics and preoperative data in patients undergoing pancreaticoduodenectomy with or without vein resection (VR). There is no statistically important differences between both groups Variable Without VR (n = 111) With VR (n = 22) P-value test Demographics Age (average years) Sex (male:female) ASA score 1 2 3 4 Bilirubin level Below 100mmol/l Above 100mmol/l CEA (ng/l) Increased (> 5ng/l) CA 19-9 (IU/l) Increased (> 30IU/l) 65.6 ± 7.7 63.95 ± 9.5 P = 0.45; t test 53:58 9:13 P = 0.64; Chi square 34 61 16 0 77 34 30 78 7 12 2 0 P = 0.83; Chi square 4 P = 0.306; Chi square 5 P = 0.795; Chi square 16 P = 1.0; Chi square ASA = American Society of Anesthesiologists; CA 19-9 = carbohydrate antigen 19-9 ; CEA = carcinoembryonic antigen Comparison of two different means was done by t-test. Survival curves were computed according to the Kaplan-Meier method. SPSS version 20 software (SPSS, IBM Corp, Armonk, NY, USA) was used to collect data and perform statistical analyses. Results Review of database at University Clinical Centre Maribor identified 133 patients (average age 65.4 ± 8.6 years, 69 female patients) who underwent pan-creaticoduodenectomy for ductal adenocarcinoma of the pancreatic head between January 2006 and August 2014. In the first group there were one 111 patients (83.5%), (53 male, 58 female patients, average 65.6 ± 7.7 years) with a standard pancreaticoduodenectomy without portal vein resection (PD - VR). In the second group there were 22 patients out of 133 (16.5%) (10 male, 12 female patients, average 63.95 ± 9.5 years) who had portal vein - superior mes-enteric vein resection and portal vein reconstruction (PD + VR) during pancreaticoduodenectomy. There was no statistically important difference in preoperative patient characteristics between the PD - VR and PD + VR groups (Table 1). The dif- Radiol Oncol 2016; 50(3): 321-328. 324 Flis V et al. / Pancreaticoduodenectomy with vein resection TABLE 2. TNM stage in 111 patients who underwent pancreaticoduodenectomy without vein resection in comparison with vein resection (VR) group. There is no statistically important difference in NO and N1 stage between both groups (P = 0.432, Fischer's exact test, no statistical significance). TNM stage Without VR (n = 111) With VR (n = 22) TO 0 0 T1 1O O T2 26 0 T3 75 22 T4 O 0 NO 32 4 N1 79 18 TABLE 3. List of surgical complications developed after pancreatoduodenectomy. Comparison between group without vein resection (PD [pacreaticoduodenectomy] - VR [vein resection]) and group with vein resection (PD + VR) Type of surgical complication Without VR (n = 111) With VR (n = 22) Pancreatic fistula 5 (4.5%) 1 (4.5%) Bile leak 3 (2.7%) 1 (4.5%) Intraperitoneal bleeding 6 (5.4%) 0 Abdominal abscess 5 (4.5%) 0 Gastric emptying syndrome 1 (0.9%) 1 (4.5%) Rupture of the laparatomy 4 (3.6%) 0 Necrosing pancreatitis 1 (0.9%) 0 Ileus of Roux-Y 1 (0.9%) 0 Critical ischemia of the colon 1 (0.9%) 0 Poral vein thrombosis 0 1 (4.5%) 60 day mortality 5 (4.5%) 1 (4.5%) ference between groups in TNM staging is shown in Table 2. All patients in PD + VR group are in stage T3, however, there was no difference in N and M classification (P = 0.432, Fischer's exact test, no statistical significance) (Table 2). Surgical complications are listed in Table 3. The occurrence of surgical complications in the second group was to low for valid statistical comparison. There was no statistically important difference in histology (extension) of resection margins between groups (Table 4), (Chi-square 2.79, p = 0.247). There was one early death in vein reconstruction group (4.5%) and 5 deaths in another group (4.5%). There was no statistically significant difference between both TABLE 4. Resection margins of extirpated tumors. There is no statistically important difference bewteen both groups (with or without venous resection [VR]) (Chi-square 2.79, p = 0.247) Resection Without VR (n = 111) With VR (n = 22) R0 102 18 R0,1 5 3 R1 4 1 TABLE 5. Cause of intrahospital deaths between both groups. (P = 1.0, Fischer's exact test, no statistical significance in death rate) Cause of death Without VR With VR Massive pulmonary embolia Cerebrovascular insult Myocardial infarction Bronchopneumonia VR = vein resection groups (P = 1.0, Fischer's exact test, no statistical significance in death rate). Causes of intrahospital deaths are listed in Table 5. Adjuvant chemotherapy didn't impact the long term survival. In fourteen patients portal vein was reconstructed without the use of synthetic vascular graft. In these series two types of venous reconstruction were performed. When tumour involvement was limited to the superior mesenteric vein (SPV) or portal vein (PV) such that the splenic vein could be preserved, and vessels could be approximated without tension a primary end-to-end anastomosis was performed. When tumour involved the SMV - splenic vein confluence, splenic vein li-gation was necessary (Figure 1). In the remaining eight procedures interposition graft was needed. Dacron grafts with 10 mm diameter were used. There was no infection after dacron grafting. One patient had portal vein thrombosis after surgery: it was thrombosis after primary reconstruction. There were no thromboses in patients with synthetic graft interposition. Survival analysis Median survival time in months was in a group with vein resection (PD + VR) 16.1 months and in a group without vein resection (PD - VR) 15.2 months. Five year survival in the group without Radiol Oncol 2016; 50(3): 321-328. 325 Flis V et al. / Pancreaticoduodenectomy with vein resection vein resection was 19.5%. Comparison of survival curves showed equal hazard rates with log-rank p = 0.090 (z = 1.659 at 5% C; C = 1.96) (Figure 2 and Figure 3). Discussion Pancreatic cancer is the 4th most common cause of cancer death in the Western world.22 The mortality rate closely approximates the incidence, but surgical resection is generally accepted as having a beneficial effect on survival.12 However, due to the presence of metastatic disease or invasion of local structures, most patients are not operative candidates at presentation. Historically, involvement of regional vasculature by pancreatic carcinoma has been considered a contraindication to resection.1 Advances in surgical technique, intensive care and neoadjuvant chemotherapy have increased the rate of resectability, particularly for patients whose pancreatic cancer involves the portal vein (PV) and superior mesenteric vein (SMV). Vascular resection has become routine for locally advanced pancreatic tumours. Venous resections are supported only when an R0 resection is achieved. Many recent studies have shown that venous resection does not alter overall mortality and is therefore not a contraindication to extended tumour resection.1,15 However the resection and reconstruction of the PV is a technically challenging procedure and the number of patients undergoing this type of operation in any given series is small.1415 Currently, venous resection has been reported in up to 20% of pancreaticoduodenectomies at high-volume pancreatic surgery centers.1222 It has been suggested that pancreatic head resection (PHR) with venous resection (VR) might be associated with a higher complication rate when compared with pancreatic head resection alone.14 In our study as in some other studies the morbidity of PHR combined with VR was similar to PHR alone.22 In the meta-analysis by Zhou et al., of 19 studies that reported on mortality, no difference was observed between PHR with VR and PHR alone.23 Arterial resection is more rarely performed; they can include the celiac trunk, superior mesenteric and hepatic arteries, but usually arterial involvement is regarded as a contraindication to surgery as it carries a higher postoperative mortality, lack of survival benefit and are more likely associated to R1 resections.12 Some studies show a greater proportion of R1 resections in pancreatectomies with vein resection FIGURE 1. In presented series basically two types of venous reconstruction were performed. When tumour involvement was limited to the superior mesenteric vein (SMV) or portal vein (PV) such that the splenic vein (SplV) could be preserved, and vessels could be approximated without tension a primary end-to-end anastomosis was performed (V1). In the remaining cases interposition graft (IG) was needed (V2). FIGURE 2. Kaplan-Meier survival plot for patients with vein reconstruction (pacreaticoduodenectomy [PD]+ vein resection [VR]). Median survival time in months was in this group 16.1 months. than in pancreatectomies alone.22 However, the greater proportion of R1 resections in PHR with VR group might be connected with differences in histopahtological reporting. The nature of tissue sampling of the circumferential resection margins differs between institutions. As a result, R1 rates vary considerably in the literature ranging from 37% to 75%.22'24 Additionally, some studies have shown that R1 resections have had no adverse effect on survival.1222 In contrast, the ESPAC-1 trial suggested that resection margin status was a nega- Radiol Oncol 2016; 50(3): 321-328. 326 Flis V et al. / Pancreaticoduodenectomy with vein resection A - B - PD-VR (A) and PD+VR (B) survival curve i o lao l~40 "eo Tso 1100 Months FIGURE 3. Comparison of Kaplan-Meier survival plots for both groups. Median survival time in months was in group with vein resection (pacreaticoduodenectomy [PD]+ vein resection [VR]) 16.1 months (line B) and in group without vein resection (PD - VR) 15.2 months (line A). Five year survival in group without vein resection (line B) was 19.5%. Comparison of survival curves showed equal hazard rates with log-rank p = 0.090 (z = 1.659 at 5% C; C = 1.96). tive predictor of survival.25 In our study R1 status had no adverse effect on survival. However, with such discrepancies in the literature with regard to the resection margin status it could be postulated that until histopathologic reporting is more standardized universally its role as a prognostic indicator remains equivocal.22 The opponents of the PHR with VR also argue that these tumours are larger with worse prognosis because of vessel-wall invasion and higher potential of developing liver me-tastases.26 Several studies have shown that true his-tologic venous invasion has no impact on survival rates. Yekebas et al. found no statistically significant impact of tumour size, resection margin status and histologic vascular wall invasion on life expectancy.27 Tseng and colleagues found no difference in median survival between patients with who did and who did not have histopahtologic evidence of vein invasion.12 In our study life expectancy of PHR combined with VR was similar to PHR alone. Few studies have analyzed the durability of the venous reconstruction or reported on the morbidity associated with graft thrombosis.1112 In these series two types of venous reconstruction were performed. When tumour involvement was limited to the superior mesenteric vein (SPV) or portal vein (PV) such that the splenic vein could be preserved, and vessels could be approximated without tension a primary end-to-end anastomosis was performed. When tumour involved the SMV-splenic vein confluence, splenic vein ligation was necessary. In the remaining eight procedures interposition graft was needed. Dacron grafts with 10 mm diameter were used. There was no infection after dacron grafting. Of the 6 thromboses observed all were in the acute setting (less than 30 days), however, none of these six patients died secondary to acute thrombosis. The literature documenting portal vein graft thrombosis rates is sparse.1 122829 DiPerna et al. observed patency rates of 93% and 90% at 12 and 24 months, respectively. However, in this series, there were only eight portal vein resections with recon-struction.29 Tseng et al. noted occlusion in 6.9% of portal vein grafts, but specific timing and morbidity were not discussed.12 The thrombosis rate in this series was lower than in those previously reported (4.5%). Recommendations for anticoagulation following major venous reconstruction for malignancy have varied.130 No difference was observed in thrombosis rates when comparing patients receiving therapy and those who did not.1 Currently, our approach to patients with SMV-PV involvement is similar to other published series.1 Primary end-to-end anastomosis is performed in those patients requiring segmental resection if it can be accomplished without tension. In those patients who cannot be reconstructed with primary end-to-end anastomosis, an interposition graft is used, with the synthetic dacron graft being our first preference due to its acceptable results in portal decompression surgery.31 Generally, the use of a synthetic graft such as dacron or polytetrafluoroethylene (PTFE) is discouraged because of fear from infection or anastomosis disruption from pancreatic juices, and just a few small reports exist.915 When portomesenteric vein resection is necessary during PD, primary anastomosis of the portomesenteric veins is always the first choice for reconstruction. However portal vein thrombosis was observed frequently after primary vein anastomosis, for several reasons. The most important is probably the anastomotic tension that may go unrecognized when intestines are returned to their original position after pancreaticoduo-denectomy.15 Some centres use vein interposition graft harvested from the jugular or renal location. However, additional resection of vein is connected with potentially higher morbidity. Additionally the need for vein resection is often not known until the last stage of resection. Because PV clamping time should be kept to a minimum, the suitability and ready availability of synthetic grafts make Radiol Oncol 2016; 50(3): 321-328. 327 Flis V et al. / Pancreaticoduodenectomy with vein resection them a desirable conduit for PVR. Synthetic graft provides the necessary length to bridge any gap between the mesenteric vessels and the PV, thus avoiding tension.15 The potential risk of infection has restricted the use of synthetic grafts in PVR. Another disadvantage in this scenario is the potential risk of anastomosis disruption following a pancreatic leak. There were no graft infections or anastomotic leaks in this series. It is interesting to note that ligation of the splenic vein, not only in presenting series wasn't presented with long term complications.12 Similar survival times after surgical resection in both groups raises once again the question about which factors independently influence the long term outcome in patients with pancreatic cancer.32 Survival after surgical resection is related to several factors: most important seem to be the extent of local invasion of the primary tumour, lymph node involvement, vascular invasion, perineural invasion, cellular differentiation, and uninvolved surgical margins. El Ghazzawy et al. reviewed experience in the US Department of Veterans affairs hospitals from 1987-1991. In the group that underwent surgical resection, perineural invasion, microlymphatic invasion, vascular invasion, or tumour differentiation did not independently influence survival when tumours were controlled for stage.33 Exactly which factors are truly independent remains controversial.2232 Conclusions Survival of patients with pancreatic cancer who undergo a resection with reconstruction was comparable to those who have a standard pancreati-coduodenectomy with no added mortality or morbidity. Synthetic graft appeared to be an effective and safe option as an interposition graft for por-tomesenteric venous reconstruction after pancrea-ticoduodenectomy. References 1. Smoot LR, Christein JD, Farnell MB. Durability of portal venous reconstruction following resection during pancreaticodudenectomy. J Gastrointest Surg 2006; 10: 1371-5. 2. Marangoni G, O'Sullivan A, Faraj W, Heaton W, Rela M. Pancreatectomy with synchronous vascular resection-An argument in favor. Surgeon 2012; 10: 102-6. 3. Katz MH, Pisters PW, Evans DB, Sun CC, Lee JE, Fleming JB, et al. Borderline resectable pancreatic cancer: the importance of this emerging stage of disease. J Am Co!!Surg 2008; 206: 833-46. 4. Christians K, Evans DB. Pancreaticoduodenectomy and vascular resection: persistent controversy and current recommendations. Ann Surg Oncol 2009; 16: 789-91. 5. Muller SA, Hartel M, Mehrabi A, Welsch T, Martin DJ, Hinz U, et al. Vascular resection in pancreatic cancer surgery: survival determinants. J Gastrointest Surg 2009; 13: 784-92. 6. Martin RC 2nd, Scoggins CR, Egnatashvili V, Staley CA, McMasters KM, Kooby DA. Arterial and venous resection for pancreatic adenocarcinoma: operative and long-term outcomes. Arch Surg 2009; 144: 154-9. 7. Al-Haddad M, Martin JK, Nguyen J, Pungpapong S, Raimondo M, Woodward T, et al. Vascular resection and reconstruction for pancreatic malignancy: a single center survival study. J Gastrointest Surg 2007; 11: 1168-74. 8. Howard TJ, Villanustre N, Moore SA, DeWitt J, LeBlanc J, Maglinte D, et al Efficacy of venous reconstruction in patients with adenocarcinoma of the pancreatic head. J Gastrointest Surg 2003; 7: 1089-95. 9. Chu CK, Farnell MB, Nguyen JH, Stauffer JA, Kooby DA, Sclabas GM, et al. Prosthetic Graft Reconstruction after Portal Vein Resection in Pancreaticoduodenectomy: A Multicenter Analysis. J Am Coll Surg 2010; 211: 316-24. 10. Lai CSE. Vascular resection and reconstruction at pancreatico-duodenecto-my: technical issues. Hepatobiliary Pancreat Dis Int 2012; 11: 234-42. 11. Wang F, Arianayagam R, Gill A, Puttaswamy V, Neale M, Gananadha S, et al. Grafts for mesenterico-portal vein resections can be avoided during pancreatoduodenectomy. J Am Coll Surg 2012; 215: 569-79. 12. Tseng JF, Raut CP, Lee JE, Pisters PW, Vauthey JN, Abdalla EK, et al. Pancreaticoduodenectomy with vascular resection: margin status and survival duration. J Gastrointest Surg 2004; 8: 935-50. 13. Kim SH, Min SK, Park D, Min SI, Jang JY, Kim SW, et al. Reconstruction of portal vein and superior mesenteric vein after extensive resection for pancreatic cancer. J Korean Surg Soc 2013; 84: 346-52. 14. Siriwardana HPP, Siriwardena AK. Systematic review of outcome of synchronous portal-superiormesenteric vein resection during pancreatectomy for cancer. Brit J Surg 2006; 93: 662-73. 15. Stauffer JA, Dougherty MK, Kim GP, Nguyen JH. Interposition graft with polytetrafluoroethylene for mesenteric and portal vein reconstruction after pancreaticoduodenectomy. Brit J Surg 2009; 96: 247-52. 16. Dripps RD. New classification of physical status. Anesthesiology 1963; 24: 111. 17. Christians KK, Tsai S, Tolat PP, Evans DB. Critical steps for pancreaticoduo-denectomy in the setting of pancreatic adenocarcinoma. J Surg Oncol 2013; 107: 33-8. 18. Rose JB, Rocha F, Alseidi A, Helton S. Posterior 'superior mesenteric artery first' approach for resection of locally advanced pancreatic cancer. Ann Surg Oncol 2014; 21: 1927-8. 19. Malleo G, Pulvirenti A, Marchegiani G, Butturini G, Salvia R, Bassi C. Diagnosis and management of postoperative pancreatic fistula. Langenbecks Arch Surg 2014; 399: 801-10. 20. Clavien PA, Barkun J, de Oliveira ML, Vauthey JN, Dindo D, Schulick RD, et al. The Clavien-Dindo classification of surgical complications: five-year experience. Ann Surg 2009; 250: 187-96. 21. Kim WS, Choi DW, Choi SH, Heo JS, Kim MJ, Song SC, et al. Clinical validation of the ISGPF classification and the risk factors of pancreatic fistula formation following duct-to-mucosa pancreaticojejunostomy by one surgeon at a single center. J Gastrointest Surg 2011; 15: 2187-92. 22. Ravikumar R, Sabin C, Hilal MA, Bramhall S, White S, Wigmore S, et al. Portal vein resection in borderline respectable pancreatic cancer: a United Kingdom multicenter study. J Am Coll Surg 2014; 218: 401-11. 23. Zhou Y, Zhang Z, Liu Y, Li B, Xu D. Pancreatectomy combined with superior mesenteric vein - portal vein resection for pancreatic cancer: a meta-analy-sis. World J Surg 2012; 36: 884-91. 24. Esposito I, Kleef J, Bergmann F, Reiser C, Herpel E, Friess H, et al. Most pancreatic cancer resections are R1 resections. Ann Surg Oncol 2008; 15: 1651-60. Radiol Oncol 2016; 50(3): 321-328. 328 Flis V et al. / Pancreaticoduodenectomy with vein resection 25. Neoptolemos JP, Stocken DD, Dunn JA, Alomd J, Beger HG, Pederzoli P, et al. Influence of resection margins on survival for patients with pancreatic cancer treated by adjuvant chemoradiation and/or chemotherapy in the ESPAC-1 randomized controlled trial. Ann Surg 2001; 234: 758-68. 26. Ouaissi M, Hubert C, Verhelst R, Astarci P, Sempoux C, Jouret-Mourin A, et al. Vascular reconstruction during pancreaticoduodenectomy for ductal ad-enocarcinoma of the pancreas improves resectability but does not achieve cure. World J Surg 2010; 34: 2648-61. 27. Yekebas EF, Bogoevski D, Caraldegirmen G, Kunze C, Marx A, Vashist YK, et al. En bloc vascular resection for locally advanced pancreatic malignancies infiltrating major blood vessels: perioperative outcome and long-term survival in 136 patients. Ann Surg 2008; 247: 300-9. 28. Evans DB, Lee JE, Tamm EP, Pisters PW. Pancreaticoduodenectomy (Whipple operation) and total pancreatectomy for cancer. In: Fischer JE, editor. Mastery of surgery. Philadelphia: Lippincott Williams and Wilkins; 2007. p. 1299-317. 29. DiPerna CA, Bowdish ME, Weaver FA, Bremner RM, Jabbour N, Skinner D, et al. Concomitant vascular procedures for malignancies with vascular invasion. Arch Surg 2002; 137: 901-7. 30. Quinones-Baldrich WJ, Alktaifi A, Eilber E, Eilber F. Inferior vena cava resection and reconstruction for retroperitoneal tumor excision. J Vasc Surg 2012; 55: 1386-93. 31. Graziano JL, Sullivan HJ. Portal decompression: clinical experience with the "H" graft. Ann Surg 1973; 178: 209-14. 32. Lazaryan A, Kalmadi S, Almhanna K, Pelley R, Kim R. Predictors of clinical outcomes of resected ampullary adenocarcinoma: a single-institution experience. Eur J Surg Oncol 2011; 37: 791-7. 33. el-Ghazzawy AG, Wade TP, Virgo KS, Johnson FE. Recent experience with cancer of the ampulla of Vater in a national hospital group. Am Surg 1995; 61: 607-11. Radiol Oncol 2016; 50(3): 321-328. 329 research article Interdisciplinary consensus statement on indication and application of a hydrogel spacer for prostate radiotherapy based on experience in more than 250 patients Arndt-Christian Müller1, Johannes Mischinger2, Theodor Klotz3, Bernd Gagel4, Gregor Habl5, Gencay Hatiboglu6, Michael Pinkawa7 1 Department of Radiation Oncology, Eberhard Karls University, Tübingen, Germany 2 Department of Urology, Eberhard Karls University, Tübingen, Germany 3 Department of Urology, Hospital Weiden, Weiden, Germany 4 Department of Radiation Oncology, Hospital Weiden, Weiden, Germany 5 Department of Radiation Oncology, Technical University Munich, Munich, Germany 6 Department of Urology, University of Heidelberg, Heidelberg, Germany 7 Department of Radiation Oncology, RWTH Aachen University, Aachen, Germany Radiol Oncol 2016; 50(3): 329-336. Received 28 February 2016 Accepted 17 April 2016 Correspondence to: Arndt-Christian Müller, M.D., Department of Radiation Oncology, Eberhard-Karls-University, 72076 Tübingen, Germany. Phone: +49 7071 298 2165; Fax: +49 7071 295 026; E-mail: arndt-christian.mueller@med.uni-tuebingen.de Disclosure: No potential conflicts of interest were disclosed. Background. The aim of the study was to reach a consensus on indication and application of a hydrogel spacer based on multicentre experience and give new users important information to shorten the learning curve for this innovative technique. Methods. The interdisciplinary meeting was attended by radiation oncologists and urologists, each with experience of 23 - 138 hydrogel injections (SpaceOAR®) in prostate cancer patients before dose-escalated radiotherapy. User experience was discussed and questions were defined to comprise practical information relevant for successful hydrogel injection and treatment. Answers to the defined key questions were generated. Hydrogel-associated side effects were collected to estimate the percentage, treatment and prognosis of potential risks. Results. The main indication for hydrogel application was dose-escalated radiotherapy for histologically confirmed low or intermediate risk prostate cancer. It was not recommended in locally advanced prostate cancer. The injection or implantation was performed under transrectal ultrasound guidance via the transperineal approach after prior hydrodissection. The rate of injection-related G2-toxicity was 2% (n = 5) in a total of 258 hydrogel applications. The most frequent complication (n = 4) was rectal wall penetration, diagnosed at different intervals after hydrogel injection and treated conservatively. Conclusions. A consensus was reached on the application of a hydrogel spacer. Current experience demonstrated feasibility, which could promote initiation of this method in more centres to reduce radiation-related gastrointestinal toxicity of dose-escalated IGRT. However, a very low rate of a potential serious adverse event could not be excluded. Therefore, the application should carefully be discussed with the patient and be balanced against potential benefits. Key words: prostate cancer; hydrogel spacer; dose-escalated radiotherapy; proctitis; toxicity Radiol Oncol 2016; 50(3): 308-312. doi:10.1515/raon-2016-0039 330 Müller AC et al. / Interdisciplinary consensus on application of a hydrogel spacer Background Dose escalated intensity-modulated radiation treatment (IMRT with radiation doses > 76 Gy) is a highly effective, curative treatment option for localized prostate cancer. Biochemical control is directly related to radiation dose with a dose effect per each additional Gy.1 For example, escalation from 70 to 80 Gy is connected with a 15% increase in PSA control. This dose effect is described for all risk groups. However, an increased radiation dose is also associated with rising levels of grade > 2 acute and chronic toxicity.1 Lower gastrointestinal toxicity rates can result from smaller posterior safety margins or even no safety margins2, potentially compromising local tumour control. A novel method to reduce rectal toxicity during dose-escalated IMRT is the insertion of a hydrogel spacer between the Denonvilliers' fascia and anterior rectal wall to separate these structures.3 The created space generates a distance of 10 - 15 mm between both organs.4-6 Recent studies unequivocally demonstrated a significant reduction in high-dose areas on the anterior rectal wall.4'5'7'8 As expected, better rectal sparing from higher radiation doses was associated with only mild toxicity from the dose-escalated treatment.4'9'10 The application technique3, dosimetric stud-ies4'8 and some early toxicity data4'10, as mentioned above, were all published within the last two years. However' despite rising numbers of hydrogel injections, reports on practical aspects or pitfalls of hy-drogel application as well as frequency and management of side effects of the administration were not or were only provided for single cases.11 Therefore, the first consensus meeting was held in July 2013 to discuss this practical issue and to generate answers for users on the indication' application and management of side effects of a hydrogel spacer for dose-escalated radiotherapy. Thereafter' toxicity data of the injection technique was collected from > 250 patients of four centres to better balance the benefit and potential risks of this new method. The aim of this consensus report is to offer new users of this technique easy access to relevant information on practical application and patient management to shorten the learning curve7 and to carefully balance potential benefits against potential risks of this technique. Patients and methods The interdisciplinary meeting was attended by radiation oncologists and urologists' each with experi- ence of 23 - 138 hydrogel injections (SpaceOAR®) in prostate cancer patients before dose-escalated IMRT. In the first part of the meeting, user experiences were discussed and questions were defined to comprise practical information relevant for successful gel injection and treatment. In the second part, answers to the defined key questions were developed. Prospective data from the multi-institutional clinical trial10, prospective mono-institutional data (German Clinical Trials Register DRKS00003273)4 and data collected retrospectively from patient files were considered in this interdisciplinary process to evaluate hydrogel application in current practice. The prospective studies were approved by each institution's ethics committee. With regard to the participating centres approvals were given by the University of Aachen10, the University of Heidelberg10 and the University of Tübingen.4 All of these patients (n = 62) gave their written informed consent to participate in these studies.410 After discussing the intended analysis of retrospectively collected data (n = 196) the institutional review board (Ethics Committee of the University of Tübingen) had no objections (266/2015B02). Patients gave informed consent to standardized data documentation and evaluation of treatment related toxicity. After the meeting, participants were asked to state the incidence of side effects to better balance risks and beneficial effects. Finally, the statement was revised and consented. Recommendations derived from prospective studies were indicated as level of evidence (LOE) 2a (evidence obtained from at least one well-designed controlled study without randomisation). Consensus statements based on expert opinions were indicated as LOE 4. The SpaceOAR® System (resulting in 10 mL hydrogel) is FDA cleared and CE Mark approved, and commercially available in the US and most countries of Western Europe. Results The following key questions were developed with regard to practical aspects of hydrogel application and patient management: 1. Indication: what criteria are required to recommend the injection of a hydrogel spacer in an individual patient? 2. Injection technique: how should the injection be optimally applied? 3. Potential toxicity: which side effects could theoretically occur? Radiol Oncol 2016; 50(3): 329-336. 331 Müller AC et al. / Interdisciplinary consensus on application of a hydrogel spacer 9 ' f 7 p hydnodissection 1 * . ■ - ^Ov > - W p / SV fjMw- hydrogel ® ! 1 ï Î . 4. Prophylaxis: are prophylactic procedures reasonable? 5. Actual toxicity: what is the current grade 2 or higher toxicity rate of hydrogel injection measured according to CTC v 4.0.12 6. Treatment of side effects: how should side effects be treated? 7. Absolute exclusion criteria: what are absolute exclusion criteria for the injection? 8. Relative exclusion criteria: what are relative exclusion criteria for the injection? 9. Special aspects of radiation treatment planning: Which aspects of radiation treatment planning should be considered? The following key answers were developed: Indication A hydrogel spacer can be considered for dose-escalated radiotherapy (radiation doses > 76 Gy in conventional 1.8 - 2.0 Gy fractions) for histologi-cally confirmed low or intermediate risk prostate cancer (LOE 2a). A hydrogel spacer can be considered for dose-escalated radiotherapy (radiation doses > 76 Gy in conventional 1.8 - 2.0 Gy fractions) for histologi-cally confirmed prostate cancer with any localized disease (LOE 4). The risk of a microscopic T3 stage with risk of adhesions potentially impairing the hydrodissection should be considered. Following hydrogel injection, other forms of dose-escalated radiotherapy as hypofraction-ated radiotherapy, particle beam radiotherapy or brachytherapy were also carried out.13-17 Injection technique Hydrogel injection can be performed under local (possibly additional sedation), spinal or general anaesthesia. Additional procedures that are planned at the same time (i.e. brachytherapy, marker implantation etc.) determine the selected anaesthesia and should be performed in advance or a few days later since hydrogel injection might worsen visibility by air contamination. For preparation of the patient see also 3. Prophylaxis. Generally, the patient is placed in the lithotomy position. The injection is performed transperineally under transrectal ultrasound (TRUS) guidance using a linear side-fire TRUS probe and a stand-off balloon to optimize visibility. A stepper unit stabilizes the probe, so that both hands are free for the procedure.318 The transperineal route is well known for procedures such as prostate biopsies, fiducial placement or prostate brachytherapy.19 FIGURE 1. Hydrogel injection. Sagittal transrectal ultrasound images showing (A) the needle placed at the Denonvillier's fascia at the start of hydrodissection, after complete hydrodissection (B), at the start (C) and after successful hydrogel injection (D). Air contamination after hydrogel injection worsens visibility (D). P = prostate; SV = seminal vesicles All centres involved in this consensus used the hydrodissection technique before spacer injection to separate Denonvilliers' fascia and the anterior rectal wall. This fluid-mediated tissue separation technique is also used in other settings like cataract surgery and carpal tunnel syndrome treatment.2021 In short, an 18 gauge needle is inserted 1 - 2 cm above the patient's anus through his perineum. The needle is advanced either parallel to the probe or slightly angled towards the prostate apex. The correct needle position is below the prostatic apex in midaxial and midsagital position of prostate (so called midgland position). Lowering the probe before hydrodissection might facilitate the procedure. Hydrodissection is performed with 10 - 20 ml of saline or lidocaine (as local anaesthesia) diluted in saline. A slow injection of the fluid is necessary to ensure later a symmetric distribution of the spacer. Only in case of a successful hydrodissection, the hydrogel can be applied. Radiol Oncol 2016; 50(3): 329-336. 332 Müller AC et al. / Interdisciplinary consensus on application of a hydrogel spacer Hydrogel is formed during the simultaneous injection i.e. mixing of the precursor (polyethylene glycol powder) and accelerator solutions (diluent). The solutions are mixed as they pass through a Y-connector prior to passing through the injection needle. Both solutions polymerise to a soft PEG-based gel within 10 seconds. An injection of 10 mL hydrogel results in a separation of about 9 - 10 mm between the prostate and rectal wall (Figure 1).6 The injection procedure can be completed within a few minutes. Potential toxicity Depending on the type of and experience with anaesthesia, patients might experience pain and discomfort during needle insertion and hydrogel injection. After spacer injection, patients may feel discomfort and rectal tenesmus. Data on pain frequency and pain intensity after injection was not routinely collected. Therefore, only retrospective data on pain management indicating the use of am-pyrone sulfonate analgesics (metamizole) for the day of the procedure and sometimes afterwards was available. During spacer injection, there might be a risk of the needle and hydrogel penetrating the rectal wall, urethra, bladder or prostate. Bleeding, necrosis or ulceration of the bladder or rectal wall may follow. Lower urinary tract symptoms or even urinary retention could result from pressure on the prostate or the bladder from the spacing gel. Local inflammation or infection is possible, as with every invasive procedure. Air or hydrogel might be potentially injected into vessels. Prophylaxis Anticoagulants should be discontinued. Antibiotic prophylaxis is applied in some centres with fluoro-quinolones or cephalosporines. However, no infections have been diagnosed up to now, even in centres with > 100 hydrogel injections without antibiotics. A rectal enema might be used to optimize TRUS conditions during the procedure. Constipation and hard stools need to be avoided during treatment to decrease pressure on the rectal wall and a low residue diet and/or laxatives may be indicated. Actual toxicity Experience from all centres were participating in this consensus statement included 258 cases of hy-drogel application before external beam radiotherapy for localized prostate cancer. All patients were treated with photons. Hydrogel associated complications, defined as grade 2 or higher toxicity, were experienced by 5 patients (2%). Hydrogel was injected intraprostati-cally in one single case. In 4 cases, rectum penetration was diagnosed at different intervals following injection. An injection into the rectal wall was observed in a single patient shortly after injection and radiotherapy was therefore started several weeks later. Two rectum penetrations were diagnosed during an external beam photon treatment after reports of passing mucous discharge. The patients were treated conservatively and radiotherapy was interrupted in one case. One patient reported increased bowel urgency 3 - 4 weeks after the end of radiotherapy before the diagnosis of a rectum penetration on proctoscopy. All patients with the mentioned complications were followed-up with proctoscopies and/or pelvic MRI (magnetic resonance imaging). Rectal wall defects healed in all patients completely after several weeks. Observation and treatment of side effects Post-injection care comprises usually the first day with examination of potential urological side effects (bleeding, obstruction, pain) including the removal of a urinary catheter (if present). Side effects must be treated symptomatically. Urinary catheterization is needed in cases of urinary obstruction. Hydrogel (PEG) is not toxic or allergenic and all known injections into the prostate, bladder or rectal wall resolved without further sequelae.6 Patience is required as the hydrogel remains stable for three months and subsequently liquefies within 6 months. This was documented in 98% of patients (n = 43/44) in the multi-center study.10 Antibiotic treatment is indicated in cases of penetration, perforation or ulceration of the rectal wall and depending on the extent, patients could be kept on parenteral nutrition or a low residue diet. Radiotherapy should not be started during an infection or after inadvertent injection into the bladder or rectal wall before the healing process of a defect is complete. Absolute exclusion criteria (complication risk exceeds potential benefits) locally advanced prostate cancer (space cannot be effectively created, tumour cell dissemination cannot be excluded) active bleeding disorder or clinically significant coagulopathy Radiol Oncol 2016; 50(3): 329-336. 333 Müller AC et al. / Interdisciplinary consensus on application of a hydrogel spacer Relative exclusion criteria anticoagulants (discontinuation usually possible) active inflammatory or infectious disease in the perineum or injection area (prostatitis, anorectal inflammatory disease with increased risk of ulceration, fistula or bleeding such as ulcerative colitis or Crohn's disease) previous treatment of prostate with high risk of adhesions (high-intensity focused ultrasound, cryotherapy, radiotherapy). Presently, very limited experience exists in hy-drogel application after previous radiotherapy or high-intensity focused ultrasound.1622 Hydrogel injection was performed without problems; however adhesions can make an injection difficult or impossible. 9. Special aspects of radiation treatment planning Radiation treatment planning CT should start approximately five days after hydrogel injection to allow for decreasing of post-procedural swelling (and not to overestimate prostate volume).22 An post-injection MRI (T2-sequence sufficient without contrast media) fused to the planning CT could help to better identify the spacer (because the hydrogel is sometimes not distinguishable from the rectal wall due to same density in CT). An additional advantage of an MRI is the capability to evaluate the properness of injection. Circumferential CTV-PTV-margins depend on the verification strategy (with IGRT usually 7-10 mm, posterior if necessary less). Monitoring of the spacer volume is not necessary during radiation treatment. Stability over 3 months after injection was shown for the gel in the multicenter study.10 Discussion The most relevant practical aspects of hydrogel injection after 258 applications were summarized in this consensus statement. A detailed description of indications, prophylaxis and management of side effects should provide new users with a fast and comprehensive introduction to the successful application of this new method. After a short learning period, the procedure can be performed to a high standard, ensuring low toxicity. Most data used are derived from well-defined controlled but not randomized studies or prospective investigations, leading to Level IIA evidence for indication and application of the hydrogel spacer. In the multi-institutional phase II trial (52 patients recruited, 49 patients after successful spacer injection), patients were informed of higher probability of grade 2+ toxicity, as no experience existed. With a carefully estimated probability of 6 - 20%, it included an injection into the rectal wall, bladder wall and urethra, ulceration and necrosis of the rectal wall, bleeding and urinary retention. Three patients who were initially treated within this study experienced procedure-related events after hydro-gel injection including focal rectal necrosis due to inadvertent injection of hydrogel into the rectal wall, bladder piercing during injection with hy-drogel leak into the bladder, urinary retention and a device-related proctitis.6 All of these events occurred during the initial experience (learning curve in the first patient cohort) and resolved completely. Adaptations of the injection procedure (side-fire TRUS probe, stepper, stand-off balloon) were conducted which facilitated handling of the needle and hydrogel insertion. A learning curve has been reported for the application and treatment with a hydrogel, again stressing several technical aspects to achieve homogenous hydrogel distribution. This report summarizes important issues that need to be considered to achieve satisfactory spacer distribution. Radiotherapy planning should not include the usual objectives for the dose to the rectal wall. A dose of 70Gy can be allowed for 20% of the rectal wall volume according to RTOG (Radiation Therapy Oncology Group) recommendations.23 With a prescription dose of 76-78Gy, mean rectum volumes within the 70Gy isodose can range by about 1% with good spacer placement and adequate treatment planning.7 However, the findings of this multi-institutional evaluation of spacer-related toxicity (no G3+ event) were based on conventional fractionated dose-escalated IGRT and cannot be simply adopted to other radiation treatment schedules (hypofraction-ation) or treatment with other ionizing radiation sources. In a study with hypofractionation using particle beam therapy (without CT-image guidance) two cases (2/92; 2%) of G3-toxicity (colos-tomy) occurred, a relation to the hydrogel spacer injection cannot be excluded.24 Therefore, it is extremely important that patients are closely followed up at their centre after hydro-gel injection. As the hydrogel is not tissue-toxic or allergenic, conservative management in case of inaccurate injection should be initiated as described above. Patience is required in case of inadvertent injection to the rectum or bladder wall, or in case Radiol Oncol 2016; 50(3): 329-336. 334 Müller AC et al. / Interdisciplinary consensus on application of a hydrogel spacer of rectal wall penetration or ulceration. All cases in this analysis where this occurred healed without long-term sequelae. A currently published randomized trial demonstrated well toleration of spacer application (10% mild transient procedural perineal discomfort) in 149 patients suggesting safety of this method with conventional fractionated dose-escalated IGRT, too.25 For optimized injection results, one expert in each centre was trained by another expert. The procedure was performed by only one or two experts at each centre, guaranteeing a high degree of experience. Last but not least, correct patient selection is essential. The optimal patient for this new method is at low risk of adhesions (inflammation, tumour spread due to locally advanced disease) and has a low risk of bleeding. The risk of tumour displacement by hydrodissection is very small, since prostatectomy series with limited pT3 stages reported in less than one fifth of patients an invasion and in no case a progression through the full thickness of the Denonvilliers' fascia.26 After successful injection, the benefit for the patients was measured by acute toxicity scores and by radiation planning parameters (dose-volume histograms). In brief, the theoretical benefit of an additional space between prostate and rectum translated into improved radiation treatment plans with approximately 10% reduction in relevant high-dose areas (dose level from 40-70Gy).8 These improved radiation treatment plans with lower rectal doses converted into reduced acute toxicity rates. Grade 2 proctitis resulting from radiotherapy was a rare event compared to standard conformal or intensity-modulated radiotherapy, for example 12.5% acute toxicity in the multicenter phase II trial10 in comparison to occasionally 50% or more in studies without a spacer.2728 The prevention of acute proctitis with this procedure is a benefit for the patient. Further benefits for the patients are conceivable. Consequential late side effects derive from persisting acute toxicity29 and reduced acute toxicity will usually be associated with a lower risk of late toxicity. However, the evaluation of this potential long-term benefit needs longer follow-up. Another beneficial effect of improved rectum protection is the facilitation of dose escalation to the prostate. Since increased radiation doses improve outcome in the range of approximately 1.5% better biochemical control per Gy after a mean follow-up of five years1, these dosimetric changes are relevant for improved tumour control with a lower risk of toxicity. This spacer consensus focuses on the use of Polyethylene-glycol (PEG) hydrogel spacers in dose-escalated radiotherapy of prostate cancer. However, at least four different bio-resorbable spacer materials (PEG-hydrogel, balloon of copolymer of polylactic acid or similar poly (a-hydroxy acids), hyaluronic acid and collagen) are currently evaluated. PEG hydrogel spacers and bioresorbable balloons have demonstrated an excellent bio-compatibility profile in humans compared to other spacers made of hyaluronic acid or collagen.30 Direct comparison of PEG hydrogel spacer and bio-resorbable balloon demonstrated the following. PEG spacers were less invasive (smaller needle diameter with 1.3 vs. 2 - 3mm). The balloon spacer was superior in reducing rectum dose (-28%), but exhibited an average volume loss of > 50% during the full course of treatment (37-40 fractions), while the volume of gel spacers remained fairly constant.31 Displacement of radiosensitive organs by spacers is not limited to primary prostate cancer alone. Further applications being investigated include treatment of recurrent prostate cancers32, gynecological malignancies33 and esophageal gel-shifting facilitating treatment of mediastinal nodes.34 The principle to displace radiosensitive organs from high dose areas is also used in case of adhesions of small intestinum and radiation targets. For such special situations are invasive surgical techniques available like laparoscopic mesh placement.35 We conclude that hydrogel injection can be considered for dose-escalated radiotherapy. Well trained physicians, correct patient selection and knowledge of the management of potential side effects are essential for optimal application. The benefit for the patient is improved protection of the rectal wall, which is associated with low radiation related proctitis rates. This allows dose-escalation associated with improved tumour control. However, a very low rate of a potential serious adverse event cannot be excluded and should carefully be discussed with the patient and be balanced against potential benefits. The evaluation of this potential long-term benefit needs longer follow-up. Disclosures The consensus meeting was supported by CSDiagnostics, Neuss, Germany. Augmenix, Inc., Waltham, Massachusetts, United States is the sponsor of the multicentre-study and sponsored with Radiol Oncol 2016; 50(3): 329-336. 335 Müller AC et al. / Interdisciplinary consensus on application of a hydrogel spacer CSDiagnostics, Neuss, Germany additional hydrogels for patients treated outside the multicentre study. TK reports grants from CSDiagnostics, Neuss, Germany for another meeting. Acknowledgment Dr. Elizabeth Krämer performed the copyedit-ing. We acknowledge support by Deutsche Forschungsgemeinschaft (DFG) and Open Access Publishing Fund of University of Tübingen. References 1. Viani GA, Stefano EJ, Afonso SL. Higher-than-conventional radiation doses in localized prostate cancer treatment: a meta-analysis of randomized, controlled trials. Int J Radiat Oncol Biol Phys 2009; 74: 1405-18. 2. Dearnaley D, Syndikus I, Sumo G, Bidmead M, Bloomfield D, Clark C, et al. Conventional versus hypofractionated high-dose intensity-modulated radiotherapy for prostate cancer: preliminary safety results from the CHHiP randomised controlled trial. Lancet Oncol 2012; 13: 43-54. 3. Hatiboglu G, Pinkawa M, Vallee JP, Hadaschik B, Hohenfellner M. Application technique: placement of a prostate-rectum spacer in men undergoing prostate radiation therapy. BJU Int 2012; 110: E647-52. 4. Eckert F, Alloussi S, Paulsen F, Bamberg M, Zips D, Spillner P, et al. Prospective evaluation of a hydrogel spacer for rectal separation in dose-escalated intensity-modulated radiotherapy for clinically localized prostate cancer. BMC Cancer 2013; 13: 27. 5. Pinkawa M, Piroth MD, Holy R, Escobar-Corral N, Caffaro M, Djukic V, et al. Spacer stability and prostate position variability during radiotherapy for prostate cancer applying a hydrogel to protect the rectal wall. Radiother Oncol 2013; 106: 220-4. 6. Uhl M, van Triest B, Eble MJ, Weber DC, Herfarth K, De Weese TL. Low rectal toxicity after dose escalated IMRT treatment of prostate cancer using an absorbable hydrogel for increasing and maintaining space between the rectum and prostate: results of a multi-institutional phase II trial. Radiother Oncol 2013; 106: 215-19. 7. Pinkawa M, Klotz J, Djukic V, Schubert C, Escobar-Corral N, Caffaro M, et al. Learning curve in the application of a hydrogel spacer to protect the rectal wall during radiotherapy of localized prostate cancer. Urology 2013; 82: 963-8. 8. Song DY, Herfarth KK, Uhl M, Eble MJ, Pinkawa M, van Triest B, et al. A multi-institutional clinical trial of rectal dose reduction via injected polyethylene-glycol hydrogel during intensity modulated radiation therapy for prostate cancer: analysis of dosimetric outcomes. Int J Radiat Oncol Biol Phys 2013; 87: 81-7. 9. Pinkawa M, Piroth MD, Holy R, Escobar-Corral N, Caffaro M, Djukic V, et al. Quality of life after intensity-modulated radiotherapy for prostate cancer with a hydrogel spacer. Matched-pair analysis. Strahlenther Onkol 2012; 188: 917-25. 10. Uhl M, Herfarth K, Eble MJ, Pinkawa M, van Triest B, Kalisvaart R, et al. Absorbable hydrogel spacer use in men undergoing prostate cancer radiotherapy: 12 month toxicity and proctoscopy results of a prospective multicenter phase II trial. Radiat Oncol 2014; 9: 96. 11. Klotz T, Mathers MJ, Lazar Y, Gagel B. Use of hydrogel as spacer in Denovier's space: optimization of IMRT radiotherapy of localized prostate cancer. Urologe ^ 2013; 52: 1690-97. 12. National Cancer Institute. Common terminology criteria for adverse events v.4.0 (CTCAE). 2011. [citated 14 Feb 2016]. Available from: http://ctep.can-cer.gov/protocolDevelopment/electronic_applications/ctc.htm. 13. Beydoun N, Bucci JA, Chin YS, Malouf D, Enari E, Painter SD. First report of transperineal polyethylene glycol hydrogel spacer use to curtail rectal radiation dose after permanent iodine-125 prostate brachytherapy. Brachytherapy 2013; 12: 368-74. 14. Christodouleas JP, Tang S, Susil RC, McNutt TR, Song DY, Bekelman J, et al. The effect of anterior proton beams in the setting of a prostate-rectum spacer. Med Dosim 2013; 38: 315-9. 15. Habl G, Hatiboglu G, Edler L, Uhl M, Krause S, Roethke M, et al. Ion Prostate Irradiation (IPI) - a pilot study to establish the safety and feasibility of primary hypofractionated irradiation of the prostate with protons and carbon ions in a raster scan technique. BMC Cancer 2014; 14: 202. 16. Nguyen PL, Devlin PM, Beard CJ, Orio PF, 3rd, O'Leary MP, Wolfsberger LD, et al. High-dose-rate brachytherapy for prostate cancer in a previously radiated patient with polyethylene glycol hydrogel spacing to reduce rectal dose: case report and review of the literature. Brachytherapy 2013; 12: 77-83. 17. Rucinski A, Bauer J, Campbell P, Brons S, Unholtz D, Habl G, et al. Preclinical investigations towards the first spacer gel application in prostate cancer treatment during particle therapy at HIT. Radiat Oncol 2013; 8: 134. 18. Pinkawa M. Spacer application for prostate cancer radiation therapy. Future Oncol 2014; 10: 851-64. 19. Ng M, Brown E, Williams A, Chao M, Lawrentschuk N, Chee R. Fiducial markers and spacers in prostate radiotherapy: current applications. BJU Int 2014; 113 (Suppl 2): 13-20. 20. Malone D, Clark T, Wei N. Ultrasound-guided percutaneous injection, hydrodissection, and fenestration for carpal tunnel syndrome: description of a new technique. J Appl Res 2010; 10: 116-23. 21. Vasavada AR, Singh R, Apple DJ, Trivedi RH, Pandey SK, Werner L. Effect of hydrodissection on intraoperative performance: randomized study. J Cataract Refract Surg 2002; 28: 1623-8. 22. Pinkawa M, Bornemann C, Escobar-Corral N, Piroth MD, Holy R, Eble MJ. Treatment planning after hydrogel injection during radiotherapy of prostate cancer. Strahlenther Onkol 2013; 189: 796-800. 23. Lawton CA, Michalski J, El-Naqa I, Buyyounouski MK, Lee WR, Menard C, et al. RTOG GU Radiation oncology specialists reach consensus on pelvic lymph node volumes for high-risk prostate cancer. Int J Radiat Oncol Biol Phys 2009; 74: 383-7. 24. Habl G, Uhl M, Hatiboglu G, Ellerbrock M, Debus J, Herfarth K. Hypofractionated radiotherapy of the prostate using carbon ions or protons - acute toxicities of the IPI-study. [German], [Abstract]. 20th Annual Congress of the German Society for Radiation Oncology. Dusseldorf, Germany; Jul 3-6, 2014. Strahlenther Onkol 2014; 190(Suppl 1): 136. 25. Mariados N, Sylvester J, Shah D, Karsh L, Hudes R, Beyer D, et al. Hydrogel Spacer Prospective Multicenter Randomized Controlled Pivotal Trial: Dosimetric and Clinical Effects of Perirectal Spacer Application in Men Undergoing Prostate Image Guided Intensity Modulated Radiation Therapy. Int J Radiat Oncol Biol Phys 2015; 92: 971-7. 26. Villers A, McNeal JE, Freiha FS, Boccon-Gibod L, Stamey TA. Invasion of Denonvilliers' fascia in radical prostatectomy specimens. J Urol 1993; 149: 793-8. 27. Vora SA, Wong WW, Schild SE, Ezzell GA, Halyard MY. Analysis of biochemical control and prognostic factors in patients treated with either low-dose three-dimensional conformal radiation therapy or high-dose intensity-modulated radiotherapy for localized prostate cancer. Int J Radiat Oncol Biol Phys 2007; 68: 1053-8. 28. Zietman AL, Bae K, Slater JD, Shipley WU, Efstathiou JA, Coen JJ, et al. Randomized trial comparing conventional-dose with high-dose conformal radiation therapy in early-stage adenocarcinoma of the prostate: long-term results from proton radiation oncology group/american college of radiology 95-09. J Clin Oncol 2010; 28: 1106-11. 29. Pinkawa M, Holy R, Piroth MD, Fischedick K, Schaar S, Szekely-Orban D, et al. Consequential late effects after radiotherapy for prostate cancer - a prospective longitudinal quality of life study. Radiat Oncol 2010; 5: 27. 30. Mok G, Benz E, Vallee JP, Miralbell R, Zilli T. Optimization of radiation therapy techniques for prostate cancer with prostate-rectum spacers: a systematic review. Int J Radiat Oncol Biol Phys 2014; 90: 278-88. Radiol Oncol 2016; 50(3): 329-336. 336 Müller AC et al. / Interdisciplinary consensus on application of a hydrogel spacer 31. Wolf F, Gaisberger C, Ziegler I, Krenn E, Scherer P, Hruby S, et al. Comparison of two different rectal spacers in prostate cancer external beam radiotherapy in terms of rectal sparing and volume consistency. Radiother Oncol 2015; 116: 221-5. 32. Arcangeli S, Agolli L, Donato V. Retreatment for prostate cancer with stereotactic body radiation therapy (SBRT): Feasible or foolhardy? Rep Pract Oncol Radiother 2015; 20: 425-9. 33. Viswanathan AN, Damato AL, Nguyen PL Novel use of a hydrogel spacer permits reirradiation in otherwise incurable recurrent gynecologic cancers. J Clin Oncol 2013; 31: e446-7. 34. Kishi K, lida T, Ojima T, Sonomura T, Shirai S, Nakai M, et al. Esophageal gel-shifting technique facilitating eradicative boost or reirradiation to upper mediastinal targets of recurrent nerve lymph node without damaging esophagus. J Radiat Res 2013; 54: 748-54. 35. Bachmann R, Heinzelmann F, Muller AC, Ladurner R, Schneider CC, Konigsrainer A, et al. Laparoscopic pelvic mesh placement with closure of pelvic floor entrance to prevent small intestine radiation trauma - A retrospective cohort analysis. Int J Surg 2015; 23: 62-7. Radiol Oncol 2016; 50(3): 329-336. 337 research article Excellent outcomes after radiotherapy alone for malignant spinal cord compression from myeloma Dirk Rades1, Antonio J. Conde-Moreno2, Jon Cacicedo3, Barbara Segedin4, Volker Rudat5, Steven E. Schild6 1 Department of Radiation Oncology, University of Lübeck, Lübeck, Germany 2 Department of Radiation Oncology, Consorcio Hospital Provincial de Castellón, Castellón, Spain 3 Department of Radiation Oncology, Cruces University Hospital, Barakaldo, Vizcaya, Spain 4 Department of Radiotherapy, Institute of Oncology Ljubljana, Ljubljana, Slovenia 5 Department of Radiation Oncology, Saad Specialist Hospital Al Khobar, Saudi Arabia 6 Department of Radiation Oncology, Mayo Clinic, Scottsdale, AZ, USA Radiol Oncol 2016; 50(3): 337-340. Received 13 December 2015 Accepted 25 January 2016 Correspondence to: Prof. Dirk Rades, M.D., Department of Radiation Oncology, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. Phone: +49 451 5006 661; Fax: +49 451 5003 324; E-mail: rades.dirk@gmx.net Disclosure: No potential conflicts of interest were disclosed. Background. Uncertainty exists whether patients with spinal cord compression (SCC) from a highly radiosensitive tumor require decompressive spinal surgery in addition to radiotherapy (RT). This study addressed the question by evaluating patients receiving RT alone for SCC from myeloma. Patients and methods. Data of 238 patients were retrospectively analyzed for response to RT and local control of SCC. In addition, the effect of RT on motor function (improvement, no further progression, deterioration) was evaluated. Overall response was defined as improvement or no further progression of motor dysfunction. Prior to RT, patients were presented to a neurosurgeon for evaluation whether upfront decompressive surgery was indicated (e.g. vertebral fracture or unstable spine). Results. In the entire cohort, the overall response rate was 97% (53% improvement plus 44% no further progression). Following RT, 88% of the patients were able to walk. Of the 69 non-ambulatory patients 44 patients (64%) regained the ability to walk. Local control rates at 1, 2 and 3 years were 93%, 82% and 82%, respectively. A trend towards better local control was observed for patients who were ambulatory before starting RT (p = 0.08) and those with a more favorable performance status (p = 0.07). Conclusions. RT alone provided excellent response rates, functional outcomes and local control in patients with SCC from myeloma. These results should be confirmed in a prospective randomized trial. Key words: myeloma; spinal cord compression; radiotherapy alone; overall response; local control Introduction Myeloma patients account for about ten percent of patients presenting with malignant spinal cord compression (SCC).1 Radiotherapy (RT) alone is the most frequently used treatment for these patients worldwide. Ten years ago, a small randomized trial of 101 patients was published that compared RT alone to decompressive surgery plus stabilization followed by RT in highly selected patients.2 In that trial the combined approach resulted in significantly better functional outcome (ambulation) and survival than irradiation alone. Therefore, upfront neurosurgery has become significantly more popular in several countries. Although patients with highly radiosensitive tumors such as myeloma, Radiol Oncol 2016; 50(3): 308-312. doi:10.1515/raon-2016-0039 338 Rades D et al. / Spinal cord compression from myeloma 1.0 0,8 ï 0,6 £ S 0.4 0.2 0,0 h ambulatory not ambulatory p=0.08 100 0 20 40 90 80 Time to recurrence (months) FIGURE 1. Kaplan-Meier curves of patients who were ambulatory prior to RT and of those patients who were not ambulatory. lymphoma and germ cell tumors were excluded from the randomized trial of 101 patients, many neurosurgeons extrapolated from these findings and perform decompressive surgery in myeloma patients.2 The question remains whether RT alone is sufficient or needs to be supplemented by upfront decompressive surgery in malignant SCC from a highly radiosensitive tumor. This study aims to contribute to this open question by investigating overall response and local control of SCC in patients treated with RT alone for SCC from myeloma. Patients and methods Data of 238 patients presenting with motor deficits of the lower extremities in consequence of SCC from vertebral body myeloma were retrospectively analyzed. Prior to the start of RT, the patients were presented to a neurosurgeon for evaluation whether upfront decompressive surgery was indi- 1.0 <18 . aa iT 02 oo h ECOG 1 -2 FCOCÎ 3-d p=0.07 so 100 0 20 « 60 Titneic recurrence (months} FIGURE 2. Kaplan-Meier curves of patients with an ECOG performance score of 1-2 and those patients with an ECOG performance score of 3-4. cated, e.g. in case of vertebral body fracture, unstable spine or sphincter dysfunction. Patient who did not require surgery were included in this study. RT was performed with 6-18 MV photon beams from a linear accelerator (mostly after 3D-treatment planning), and target volumes included the vertebrae affected by SCC plus on additional vertebra on either side. The study has been approved by the local ethics committee. For this retrospective study, specific written informed consent was not required. The primary endpoint local control was defined as freedom from a symptomatic in-field recurrence of SCC in the irradiated parts of the vertebral column. In addition, the effect of radiation treatment on motor function (improvement, no further progression, deterioration) was measured. Improvement and deterioration of motor function were defined as a change of one point on a five-point scale (0 = normal strength; 1 = ambulation without aid; 2 = ambulation with aid; 3 = no ambulation; 4 = complete paraplegia).3 Patients with complete paraplegia who did not improve after RT were rated as deteriorated. Motor function at about one month (three to six weeks) following RT was compared to motor function at baseline (i.e. before the start of RT Overall response to RT was defined as either improvement or no further progression of motor dysfunction. RT was administered without upfront neurosur-gery and performed either as short-course RT (1 x 8Gy, 5 x 4Gy) or longer-course RT (10 x 3Gy, 15 x 2.5Gy, 20 x 2Gy). The RT regimen plus ten other factors were analyzed for local control of SCC. The other factors included age at the time of RT (< 64 years vs. > 65 years, median age: 64 years), gender, myeloma subtype (IgG subtype vs. other subtypes), time from first diagnosis of myeloma to SCC (< 15 vs. > 15 months), presence of extra-osseous lesions before RT (no vs. yes), further osseous lesions before RT (no vs. yes), gender, time developing motor deficits before RT (faster: < 14 vs. slower: > 14 days), gait function before the start of RT (ambulatory vs. not ambulatory), number of vertebrae involved by SCC (1-2 vs. > 3) and performance status (Eastern Cooperative Oncology Group (ECOG) performance score 1-2 vs. 3-4). The univariate analyses of local control of SCC were done with the KaplanMeier method4, and the Kaplan-Meier curves were compared with the log-rank test. Those factors being significant or showing a trend (p < 0.09) for local control were additionally analyzed in a multi-variate manner with the Cox proportional hazards model. Radiol Oncol 2016; 50(3): 337-340. 339 Rades D et al. / Spinal cord compression from myeloma TABLE 1. Impact of the eleven factors on local control of SCC (univariate analysis) At 1 year (%) At 2 years (%) At 3 years (%) p-value Age < 64 years (n = 125) > 65 years (n = 113) 94 92 86 75 86 75 0.81 Gender Female (n = 88) Male (n = 150) 90 94 78 85 78 85 0.47 Myeloma subtype IgG subtype (n = 153) Other subtypes (n = 85) 96 88 84 78 84 78 0.14 Time from myeloma diagnosis to SCC < 15 months (n = 128) > 15 months (n = 110) 95 91 81 85 81 85 0.83 Extra-osseous lesions No (n = 218) Yes (n = 20) 94 67 82 n.a. 82 n.a. 0.19 Further osseous lesions No (n = 91) Yes (n = 147) 93 93 86 76 86 76 0.73 Time developing motor deficits Faster (< 14 days) (n = 112) Slower (> 14 days = (n = 126) 93 93 81 83 81 83 0.79 Gait function before the start of RT Ambulatory (n = 169) Not ambulatory (n = 69) 95 85 85 57 85 57 0.08 Number of vertebrae involved by SCC 1-2 (n = 112) > 3 (n = 126) 95 92 87 77 87 77 0.17 ECOG performance score 1-2 (n = 150) 3-4 (n = 88) 96 83 86 62 86 62 0.07 Radiotherapy regimen Short-course RT (n = 84) Longer-course RT (n = 154) 94 93 69 90 69 90 0.29 Entire cohort (n = 238) 93 82 82 n.a. = not available Results Two-hundred-and-thirty-seven patients were available for evaluation of response to RT. The overall response rate at one month was 97% (230 of 237 patients); 53% of patients (n = 126) showed improvement and 44% (n = 104) no further progression. Following RT, 88% of the patients (209 of 237) were able to walk. Of 69 non-ambulatory patients 44 patients (64%) regained the ability to walk after RT. In the entire cohort of 238 patients, the local control rates at 1, 2 and 3 years following RT of SCC were 93%, 82% and 82%, respectively. In the uni-variate analysis, no factor was significantly associated with local control of SCC. A trend towards better local control was observed for patients who were ambulatory before RT was started (p = 0.08, Figure 1) and for patients with a more favorable performance status (p = 0.07, Figure 2). These two factors were additionally evaluated in the multi-variate analysis, where both pre-RT gait function (risk ratio: 2.34; 95%-confidence interval: 0.80-6.10; p = 0.11) and performance status (risk ratio: 2.36; 95%-confidence interval: 0.85- 6.09; p = 0.09) did not reach significance (significance = defined as p < 0.05). Discussion Malignant SCC represents a serious complication for patients with a malignant disease.15 A rapid start of treatment is required. Until 2005, radiotherapy alone has been considered the unquestioned standard treatment for SCC. In 2005, a randomized trial of 101 selected patients with SCC from different primaries, who had a good performance status and a relatively good survival prognosis, suggested that the results of RT alone can be improved by upfront decompressive surgery.2 In that trial, 84% (42 of 50) of patients were able to walk after surgery plus RT compared to 57% (29 of 51) of patients Radiol Oncol 2016; 50(3): 337-340. 340 Rades D et al. / Spinal cord compression from myeloma after RT alone (p = 0.001). The results were supported by a meta-analysis including 24 surgical series (n = 999) and four radiotherapy series (n = 543), mostly uncontrolled cohort studies.6 These data have led to a fundamental change of practice. In several countries, neurosurgery proceeding RT has become very popular. In some centers, the majority patients with SCC receive the combined treatment rather than RT alone. This new trend includes also patients with highly radiosensitive tumors such as myeloma, although these patients were excluded from the previously mentioned randomized trial.2 The question is whether these patients really need surgery in addition to RT? One should bear in mind that spinal surgery is associated with significant risks and complications such as severe wound infections. A second surgery, extensive bleeding, postoperative pneumonia, and major thromboembolic events occurred in more than 10% of patients.278 In addition, iatrogenic neurologic complications were reported for 9% of patients receiving surgery of the lumbar spine.9 In the current study, functional outcomes were excellent with a post-RT overall ambulation rate of 88% and a rate of regaining ambulatory status of 64%. Furthermore, local control of SCC achieved with RT alone was long lasting. At 3 years following RT, local control was still 82%. These excellent local control rates were achieved irrespectively of patient characteristics. None of the eleven investigated characteristics was significantly associated with local control of SCC. However, pre-RT gait function and performance status showed a trend. Three-year local control rates were 57% in initially non-ambulatory patients and 62% in patients with an ECOG performance score of 3-4, respectively. The question whether these patients would benefit from the addition of upfront decompressive surgery to RT can be properly answered only in a prospective trial. Clear indications for neurosurgery also for very radiosensitive tumors include vertebral fractures, unstable spine, sphincter dysfunction, and impairment of the spinal cord by bony fragments. According to a recent retrospective study focusing on surgery for vertebral involvement of myeloma, the probability of receiving surgery was about 40%.10 No differences in disability and quality of life were observed between patients receiving RT alone and those receiving RT plus up-front surgery. For highly selected patients, stereo-tactic body radiation surgery (SBRT) may also be an option.1112 However, it has been recommended to use SBRT for malignant SCC only within clinical trials.12 In summary, in patients with malignant SCC from myeloma, RT alone provides excellent response rates, functional outcomes such as post-RT ambulation, and local control of SCC. These results should be confirmed in a prospective randomized trial. References 1. Rades D, Abrahm JL. The role of radiotherapy for metastatic epidural spinal cord compression. Nat Rev Clin Oncol 2010; 7: 590-8. 2. Patchell R, Tibbs PA, Regine WF, Payne R, Saris S, Kryscio RJ, et al. Direct de-compressive surgical resection in the treatment of spinal cord compression caused by metastatic cancer: a randomised trial. Lancet 2005; 366: 643-8. 3. Tomita T, Galicich JH, Sundaresan N. Radiation therapy for spinal epidural metastases with complete block. Acta Radiol Oncol 1983; 22: 135-43. 4. Kaplan EL, Meier P. Non parametric estimation from incomplete observations. J Am Stat Assoc 1958; 53: 457-81. 5. Rades D, Huttenlocher S, Bajrovic A, Karstens JH, Bartscht T. A new instrument for estimating the survival of patients with metastatic epidural spinal cord compression from esophageal cancer. Radiol Oncol 2015; 49: 86-90. 6. Klimo P Jr, Thompson CJ, Kestle JR, Schmidt MH. A meta-analysis of surgery versus conventional radiotherapy for the treatment of metastatic spinal epidural disease. Neuro Oncol 2005; 7: 64-76. 7. Rades D, Huttenlocher S, Bajrovic A, Karstens JH, Adamietz IA, Kazic N, et al. Surgery followed by radiotherapy versus radiotherapy alone for metastatic spinal cord compression from unfavorable tumors. Int J Radiat Oncol Biol Phys 2011; 81: e861-8. 8. Rades D, Huttenlocher S, Dunst J, Bajrovic A, Karstens JH, Rudat V, et al. Matched pair analysis comparing surgery followed by radiotherapy and radiotherapy alone for metastatic spinal cord compression. J Clin Oncol 2010; 28: 3597-604. 9. Ghobrial GM, Williams KA Jr, Arnold P, Fehlings M, Harrop JS. Iatrogenic neurologic deficit after lumbar spine surgery: A review. Clin Neurol Neurosurg 2015; 139: 76-80. 10. Donnarumma P, Tarantino R, Rullo M, Grisaro A, Petrucci MT, Santoro A, Delfini R. Surgery for vertebral involvement in multiple myeloma. J Neurosurg Sci 2015; in press. 11. Lee SH, Lee KC, Choi J, Ahn SH, Lee SH, Sung KH, et al. Clinical applicability of biologically effective dose calculation for spinal cord in fractionated spine stereotactic body radiation therapy. Radiol Oncol 2015; 49: 185-91. 12. Lutz S, Berk L, Chang E, Chow E, Hahn C, Hoskin P, et al. Palliative Radiotherapy for Bone Metastases: An ASTRO Evidence-Based Guideline. Int J Radiat Oncol Biol Phys 2011; 79: 965-76. Radiol Oncol 2016; 50(3): 337-340. 341 research article The role of neoadjuvant chemotherapy in patients with advanced (stage IIIC) epithelial ovarian cancer Erik Škof1, Sebastjan Merlo2, Gasper Pilko2, Borut Kobal3 1 Department of Medical Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Department of Surgery, Institute of Oncology Ljubljana, Ljubljana, Slovenia 3 Division of Gynaecology and Obstetrics, University Medical Centre Ljubljana, Ljubljana, Slovenia Radiol Oncol 2016; 50(3): 341-346. Received 29 May 2015 Accepted 5 September 2015 Correspondence to: Borut Kobal, M.D., Division of Gynaecology and Obstetrics, University Medical Centre Ljubljana, Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. E-mail: borut.kobal@kclj.si Disclosure: The authors declare no conflict of interest. Background. Primary treatment of patients with advanced epithelial ovarian cancer consists of chemotherapy either before (neoadjuvant chemotherapy, NACT) or after primary surgery (adjuvant chemotherapy). The goal of primary treatment is no residual disease after surgery (R0 resection) what is associated with an improvement in survival of patients. There is, however, no evidence of survival benefits in patients with R0 resections after prior NACT. Methods. We retrospectively reviewed the records of patients who were treated with diagnosis of epithelial ovarian cancer at Institute of Oncology Ljubljana in the years 2005-2007. The differences in the rates of R0 resections, progression free survival (PFS), overall survival (OS) and in five-year and eight-year survival rates between patients treated with NACT and patients who had primary surgery were compared. Results. Overall 160 patients had stage IIIC epithelial ovarian cancer. Eighty patients had NACT and eighty patients had primary surgery. Patients in NACT group had higher rates of R0 resection (42% vs. 20%; p = 0.011) than patients after primary surgery. PFS was 14.1 months in NACT group and 17.7 months after primary surgery (p = 0.213). OS was 24.8 months in NACT group and 31.6 months after primary surgery (p = 0.012). In patients with R0 resections five-year and eight-year survival rates were 20.6% and 17.6% in NACT group compared to 62.5% and 62.5% after primary surgery (p < 0.0001), respectively. Conclusions. Despite higher rates of R0 resections achieved by NACT, survival of patients treated with NACT was inferior to survival of patients who underwent primary surgery. NACT should only be offered to patients with advanced epithelial cancer who are not candidates for primary surgery. Key words: advanced ovarian cancer; neoadjuvant chemotherapy; primary surgery Introduction The standard treatment of patients with advanced epithelial ovarian cancer is a combination of primary surgery followed by chemotherapy. In the recent years it became clear that the goal of surgery is to achieve no macroscopic residual disease, since the survival of patients with no residual disease is superior to survival of patients with visible residual disease.1 To achieve this goal, several aggressive surgical techniques have been proposed. Often multivisceral resections are performed (diaphragm resection, splenectomy, colon resection, extensive peritonectomy, etc.)2, which increase morbidity of patients. Not all patients are candidates for primary surgery, either due to extend of their disease (unlikely to achieve no residual disease) or due to poor general condition (too ill to undergo an extensive operation). In this situation patients are treated with initial (neoadjuvant) chemotherapy, typically a combination of a platinum-based drug and a tax- Radiol Oncol 2016; 50(3): 308-312. doi:10.1515/raon-2016-0039 342 Skof E et al. / Advanced ovarian cancer and neoadjuvant chemotherapy ane.3 Patients, who are treated with neoadjuvant chemotherapy, are more likely to undergo surgery with no residual disease than patients with primary surgery.4 Therefore many authors believe that neoadjuvant chemotherapy is justified in order to have best chance to achieve a status of no residual disease.45 However, treatment with neoadjuvant chemotherapy has not yet been shown to provide better survival than treatment with primary surgery.46 It has been shown that survival of patients with stage III or IV who have no residual disease after primary surgery can be up to 50% at 10 years.7 It is not known, if patients who achieve a status of no residual disease with neoadjuvant chemotherapy, Primary surgery? 0-no, 1-yes _ni Months FIGURE 1. Progression free survival of patients after primary surgery and neoadjuvant chemotherapy (NACT). Primary surgery? 0-no, 1-yes _ni -f- 0-eertsored -f- 1 -censored "i-1---1---1---r---r 0.C0 20,00 40,00 60,00 30.00 100,00 120,00 Months FIGURE 2. Overall survival of patients after primary surgery and neoadjuvant chemotherapy (NACT). have the same or equally good prognosis. There are data from one randomized trial which showed similar survival of patients treated with neoadju-vant chemotherapy to those treated with primary surgery, but, this study was criticized due to poor survival rates in both groups.4 We compared the differences in the rates of no residual disease (R0 resection) after surgery and the differences in five-year and eight-year survival rates in patients treated with neoadjuvant chemotherapy or primary surgery in correlation to the extent of residual disease post-surgery. Methods Patients We retrospectively reviewed the records of patients who were treated with diagnosis of epithelial ovarian cancer at Institute of Oncology Ljubljana in the period from 1st of January 2005 until 31st of December 2007. During this period we identified 346 patients who were treated for epithelial ovarian cancer. Of these 160 patients had stage FIGO IIIC disease - they were eligible for analysis. Of the 160 eligible patients, 80 patients had neoadjuvant chemotherapy (NACT) and 80 patients had primary surgery. Patients had primary surgery in seven different hospitals performed by many gynecologic surgeons. In all patients, hysterectomy, bilateral oophorectomy, infracolic omentectomy with limited peritonectomy was performed. In only few of the patients multivisceral resections (diaphragm resection, splenectomy, colon resection, extensive peritonectomy) were performed - therefore no comparison between different surgical techniques outcomes was carried out. In the NACT group 40% of patients received platinum and taxane therapy (paclitaxel 175mg in 3h i.v. infusion and carboplatin AUC 6 i.v. infusion) and 55% of patients received carboplatin (AUC 6) monotherapy before surgery. Post-surgery all patients received 3 additional cycles of chemotherapy - the same regimen as in neoadjuvant setting. Median number of cycles in NACT group was 7 (range 1-13). Of the 80 patients, who had primary surgery, 82% of them received platinum and taxane therapy (paclitaxel 175mg in 3h i.v. infusion and carboplatin AUC 6 i.v. infusion), 16% of patients received carboplatin (AUC 6) monotherapy. Median number of cycles of chemotherapy was 6 (range 1-9). All patients were treated with chemotherapy at Radiol Oncol 2016; 50(3): 341-346. 343 Skof E et al. / Advanced ovarian cancer and neoadjuvant chemotherapy Institute of Oncology Ljubljana by medical oncologist specialized for gynecologic oncology. Reasons given for NACT included extent of disease (50%), co-morbidities (10%), and poor performance (40%). For patients who had NACT, stage was established by combination of clinical examination, imaging (US, CT of abdomen), cytology and biopsy. For patients who were treated with primary surgery, stage was established by intraoperative examination and review pf pathology reports. The extent of residual disease was based on the diameter of the single largest lesion. Patients without macroscopic evident residual lesions had R0 resection, patients with less than 10 mm residual lesions had R1 resection, patients with residual lesions of 10 mm or more had R2 resection. Our retrospective study was approved by the Institutional Review Board and Ethics Committee. TABLE 1. The characteristics of patients and surgical outcomes Primary surgery N = 80 NACT N = 80 p Age (years) 60.2 64.8 0.006 High grade serous 43 (54%) 53 (66%) 0.307 Performance 0 or 1 >1 66 (83%) 14 (17%) 32 (40%) 48 (60%) < 0.0001 R0 resection 16 (20%) 34 (42%) 0.011 R1 resection 18 (23%) 18 (22%) 1.0 R2 resection 46 (57%) 11 (14%) < 0.0001 Inoperable - 17 (21%) - PFS (months) 17.7 14.1 0.213 OS (months) 31.6 24.8 0.012 NACT = neoadjuvant chemotherapy; OS = overall survival; PFS = progression-free survival; R0 = no macroscopic residual disease; R1= <1 cm residual disease; R2= >1 cm residual disease Analysis We compared patients who had NACT with those who had primary surgery for a range of clinical variables. The extent of residual disease post-surgery was measured. We observed the differences in the rates of no residual disease after surgery, differences in progression free survival (PFS), overall survival (OS) and in five-year and eight-year survival rates in patients after various treatments. Patients were followed from the date of diagnosis until death from ovarian cancer, death from another cause or lost from follow-up. The primary endpoints were OS, five-year and eight-year survival rates. The secondary end points were R0 resection rates and PFS. Survival curves were calculated by Kaplan-Meier's method. Univariate and multivariate analyses using log-rank test and Cox's regression model were used for the assessment of the factors associated with OS and for comparison of factors between patients who had NACT with those who had primary surgery. Quantitative variables were compared using Student or the Wilcoxon test. Categorical variables were compared using Chi-square test. The differences were considered statistically significant if the p values were less than 0.05. Software package SPSS 15.0 for Windows was used. Results We analyzed 160 patients with stage IIIC epithelial ovarian cancer who were treated at Institute of Oncology Ljubljana in the period from 1st of TABLE 2. Five-year and eight-year survival of patients after different surgical outcomes Five-year survival Eight year survival p Surgery -NACT - - R0 resection R0 resection 62.5% 20.6% 62.5% 17.1% P < 0.0001 Surgery -NACT - ■ R1 resection R1 resection 38.9% 16.7% 27.8% 11.1% P < 0.0001 Surgery -NACT - R2 resection R2 resection 15.5% 0% 0% 0% P < 0.0001 Inoperable disease 0% 0% - NACT = neoadjuvant chemotherapy; R0 = no macroscopic residual disease; R1= <1 cm residual disease; R2= >1 cm residual disease January 2005 until 31st of December 2007. Median follow-up of patients was 8.4 years (range 6.7-10 years). Of 160 patients, 80 patients had primary surgery and 80 patients had NACT. The characteristics of patients and surgical outcomes of patients who had primary surgery or NACT are shown in Table 1. Patients treated with primary surgery were younger (60.2 vs. 64.8 years; p < 0.001). Patients treated with primary surgery also had a better performance status according to WHO classification (p < 0.001). In patients treated with NACT higher rates of R0 resection were observed (42% vs. 20%; p < 0.001) and lower rates of R2 resection rates (14% vs. 57%; p < 0.001) compared to patients who had primary surgery. After NACT in 21% of patients disease remained to be inoperable. Median PFS of patients was 14.1 months after NACT and 17.7 months after primary surgery (Figure 1). The difference was not statistically sig- Radiol Oncol 2016; 50(3): 341-346. 344 Skof E et al. / Advanced ovarian cancer and neoadjuvant chemotherapy TABLE 3. Factors correlated with survival on univariate analysis Age (years) < 40 0.008 > 60 Histology High grade serous 0.066 Other histology Performance status 0 or 1 > 1 < 0.0001 Extent of residual disease R0 < 0.0001 Kl R2 Therapy Primary surgery 0.012 NACT NACT = neoadjuvant chemotherapy TABLE 4. Factors correlated with survival on multivariate analysis Age (years) < 40 0.775 > 60 Histology High grade serous 0.370 Other histology Performance status 0 or 1 > 1 0.003 Extent of residual disease R| < 0.0001 R2 Therapy Primary surgery 0.038 NACT NACT = neoadjuvant chemotherapy nificant (p = 0.213). Median OS of patients was 24.8 months after NACT and 31.6 months after primary surgery (Figure 2). The difference was statistically significant (p = 0.012). Five-year and eight-year survival rates of patients after different surgical outcomes are shown in Table 2. Patients treated with primary surgery had superior five-year and eight-year survival rates in all types of surgical outcomes compared to patients treated with NACT. Five-year and eight year survival rates were 62.5% and 62.5% vs. 20.6% and 17.1% after R0 resection (p < 0.001), 38 .9% and 27.8% vs. 16.7% and 11.1% after R1 resection (p < 0.001) and 15.5% and 0% vs. 0% and 0% after R2 resection (p < 0.0001), respectively. Survival curves of patients after different surgical outcomes are shown in Figure 3. Univariate analysis showed that factors associated with survival were: performance status according to WHO (p < 0.0001), extent of residual disease (p < 0.0001), primary surgery (p = 0.012) and age (p = 0.008) (Table 3). We conducted multivariate survival analysis using Cox's regression model. The following prognostic factors in the multivariable model: patient age at diagnosis (< 60 vs. > 60 years); extent of residual disease (R0, R1, R2); chemotherapy (NACT vs. primary surgery); performance status according to WHO (0 or 1 vs. > 1) and histopathological subtype (high grade serous vs. other) were entered. Patients treated with primary surgery had better survival also on multivariate survival analysis. Besides that, other independent predictors of survival were extent of residual disease and performance status (Table 4). Discussion Our results show that treatment with NACT doubles the chance to have no visible residual disease (R0 resection) post-surgery compared to primary surgery in patients with stage IIIC epithelial ovarian cancer (42% vs. 20%). This is in concordance with already published studies.4,5 Despite higher rates of R0 resections achieved by NACT, survival of patients treated with NACT was inferior to survival of patients who underwent primary surgery by almost 7 months (24.8 months vs. 31.6 months). Even in patients who had R0 resection post-surgery, there was a huge difference in probability to be five-year or eight-year survivor in favor of primary surgery. Patients with R0 resection at primary surgery had three-fold higher rates of five-year and eight-year survival rates than patients with R0 resection after NACT (62.5% vs. 20.6% and 62.5% vs. 17.1%, respectively). Our results are in concordance with many authors who reported similar results with inferior overall survival of patients after NACT despite higher rates of optimal debulking surgery.5-8 There have been published results of a randomized trial which showed similar survival of patients treated with NACT to those treated with primary surgery, but, this study was criticized due to poor survival rates in both groups.4 The limitation of our study was, that this was not a randomized trial, therefore at least to some extent the difference in the outcomes for the different groups might be the result of an imbalance in the baseline characteristics. Patients who received p p Radiol Oncol 2016; 50(3): 341-346. 345 Skof E et al. / Advanced ovarian cancer and neoadjuvant chemotherapy NACT were more likely to have more extensive disease at diagnosis than patients who had primary surgery, and therefore it was expected to do relatively worse. Patients who received NACT were almost five years older (64.8 years vs. 60.2 years), had more co-morbidities and had worse performance than patients who had primary surgery. Half of the patients who received NACT did not receive combination of taxane and carboplatin chemotherapy due to poor performance or co-morbidities, whereas vast majority of patients received combination of taxane and carboplatin after primary surgery. Multivariate analysis showed that independent predictors of survival were primary surgery, performance status and extent of residual disease. Therefore, a profound differences in survival among patients after R0 resection (and also R1 resection) between primary surgery and NACT can be at least to some extend explained by factors, other than disease itself. Authors believe that it is improper to compare survival of patients who had R0 resection at primary surgery to patients who had R0 resection after prior NACT if the patients are not balanced regarding performance status, age, comorbidities, etc. It is like comparing apples with oranges. It is not just the extent of the disease at diagnosis that is important for prognosis. The biology of the disease may also play an important role. At the moment there are no biologic markers that would help us to choose the best treatment strategy for patients with advanced epithelial ovarian cancer. It is known that the most common histologic type of epithelial ovarian cancer, high grade serous adenocarcinoma, is sensitive to chemotherapy, while other histologic types such as clear cell and mucinous ovarian cancer are resistant to chemotherapy. In the last years much effort has been done in discovering of predictive and prognostic molecular markers with molecular profiling of epithelial ovarian cancer. Recently published data have shown that at least three molecular subtypes of high-grade serous ovarian cancer exist, which may have different predictive and prognostic values in systemic treatment of patients with advanced epithelial ovarian cancer.9 At the moment there is still no consensus regarding the use of NACT. The lack of consensus on who are candidates for NACT was reflected at the fourth Gynecologic Cancer InterGroup (GCIG) consensus conference.10 While the majority of attendees felt that NACT was a standard option for all patients with advanced epithelial ovarian cancer, others felt that NACT should be offered to a more clearly defined subgroup of women in whom p<0.0001 O 0,4> o 0,0- _n R0 _n R1 _n R2 J! NACT R0 _n NACT R1 _n NACT R2 _f"l Inoperable -(- 0-censored -(- 1-censored 2-censored -(- 3-censored 4-censored I 0,00 20,00 40,00 60,00 80,00 100,00 120,00 Months FIGURE 3. Survival curves of patients after different surgical outcomes. NACT = neoadjuvant chemotherapy; R0 = no macroscopic residual disease; R1= <1 cm residual disease; R2= >1 cm residual disease upfront surgery is contraindicated. The decision on whether to treat with NACT is based on the clinical status of the patient and whether or not disease is resectable at the time of presentation. Therefore, all patients require clinical staging. There are widely accepted criteria for unresectability.11 It is often difficult to preoperatively assess whether patients with advanced epithelial ovarian cancer can be optimally cytoreduced at the time of primary sur-gery.12 Therefore, many authors perform a staged surgical assessment for these patients and perform a diagnostic laparoscopy to further evaluate for resectability. If the surgeon conducting the assessment feels disease is resectable, primary surgery should be performed. If complete resectability is unlikely, NACT can be administered. Despite better overall survival of our patients who had primary surgery, two-thirds of patients in this group had R2 resection (> 1 cm residual disease) which is considered as a sub-optimal resection. These patients had five-year survival rates of only 15.5% with none being alive at eight years post-surgery. In NACT group there were only 14 % of patients with R2 resection. We believe that the main reason for high rate of R2 resections at primary surgery was that primary surgery was performed in different hospitals mainly by surgeons not skilled with principles of oncology surgery, whereas all patients who had NACT underwent surgery provided by surgeon experienced in oncology. There are convincing data showing that surgical expertise plays a major role in outcome of patients with advanced epithelial ovarian cancer. In 1,0— 0,8 r 0,6 — 0,2 — Radiol Oncol 2016; 50(3): 341-346. 346 Skof E et al. / Advanced ovarian cancer and neoadjuvant chemotherapy multidisciplinary cancer centers with advanced expertise in gynecologic oncology, optimal debulking rates in excess of 70% have been reported even for patients with bulky stage IIIC disease.7 Therefore, it is strongly recommended that a gynecologic oncologist must be involved in surgical decision making and treatment in these circumstances. We believe there is enough evidence that in operable disease primary surgery with aim of no residual disease should be performed, since this offers best possible survival of patients with stage IIIC epithelial ovarian cancer.1 Of course patients must be fit enough for surgery, in which often multivisceral resections are needed with intention to achieve no residual disease post-surgery, which increase morbidity of patients.2 The NACT should only be offered to patients who are not candidates for primary surgery for whatever reason (advanced disease, poor performance, comorbidities, etc.). The aim of NACT should be to convert inoperable advanced disease to operable disease with goal to achieve optimal debulking (R0 resection). We believe that in patients with advanced epithelial ovarian cancer the aim of treatment should not only be the survival benefit but also the improvement in quality of life. Latter can be achieved by direct effect of chemotherapy on downsizing tumor burden, which offers higher chance for improvement in performance by lessening of the disease symptoms, offers higher chance to achieve optimal debulking at surgery, with less postoperative complications.4513 Since this was a retrospective study, a comparison of the quality of life between patients treated with NACT or primary surgery was not performed. One of the remarks towards NACT was that patients who started their treatment with NACT never had the chance for complete or optimal cy-toreductive surgery. Since 48% of patients who received NACT in our study population were not candidates for radical or ultraradical surgical procedures as a result of poor performance status or comorbidities, they should probably be excluded from comparison with patients who were capable to undergo primary surgery. Perhaps the survival of patients who had NACT, but were otherwise fit for primary surgery, should be compared to survival of patients who had suboptimal primary surgery (R2 resection), thus comparing different treatments for similar burden of the disease. If we compare these two groups of patients we can see that five-years survival rates in NACT group with R0 or R1 post-surgery (NACT-R0 and NACT-R1) were somewhat higher compared to patients who had R2 resection at primary surgery (20.6% after NACT-R0 surgery, 16.7% after NACT-R1 surgery and 15.5% after R2 primary surgery), the difference was not statistically significant. To conclude, our results show that treatment with NACT doubles the chance to have no visible residual disease (R0 resection) post-surgery compared to primary surgery in patients with advanced (stage IIIC) epithelial ovarian cancer. Despite higher rates of R0 resections achieved by NACT, survival of patients treated with NACT was inferior to survival of patients who underwent primary surgery by almost 7 months. Therefore we strongly believe that NACT should only be offered to patients with advanced epithelial cancer who are not candidates for primary cytoreductive surgery, or when the disease is unresectable after staged surgical assessment performed by oncology surgeon. References 1. Aletti GD, Dowdy SC, Gostout BS, Jones MB, Stanhope CR, Wilson TO, et al. Aggressive surgical effort and improved survival in advanced-stage ovarian cancer. Obstet Gynecol 2006; 107: 77-85. 2. Eisenkop SM, Spirtos NM. Procedures required to accomplish complete cytoreduction of ovarian cancer: is there a correlation with "biological aggressiveness" and survival? Gynecol Oncol 2001; 82: 435-41. 3. Ozols RF, Bundy BN, Greer BE, Fowler JM, Clarke-Pearson D, Burger RA, et al. Phase III trial of carboplatin and paclitaxel compared with cisplatin and paclitaxel in patients with optimally resected stage III ovarian cancer: a Gynecologic Oncology Group study. J Clin Oncol 2003; 21: 3194-200. 4. Vergote I, Trope CG, Amant F, Kristensen GB, Ehlen T, Johnson N, et al. Neoadjuvant chemotherapy or primary surgery in stage IIIC or IV ovarian cancer. N Engl J Med 2010; 363: 943-53. 5. Vergote I, Leunen K, Amant F. Primary surgery or neoadjuvant chemotherapy in ovarian cancer: what is the value of comparing apples with oranges? Gynecol Oncol 2012; 124: 1-2. 6. Bristow RE, Chi DS. Platinum-based neoadjuvant chemotherapy and interval surgical cytoreduction for advanced ovarian cancer: a meta-analysis. Gynecol Oncol 2006; 103: 1070-6. 7. Chi DS, Musa F, Dao F, Zivanovic O, Sonoda Y, Leitao MM, et al. An analysis of patients with bulky advanced stage ovarian, tubal, and peritoneal carcinoma treated with primary debulking surgery (PDS) during an identical time period as the randomized EORTC-NCIC trial of PDS vs neoadjuvant chemotherapy (NACT). Gynecol Oncol 2012; 124: 10-4. 8. Rosen B, Laframboise S, Ferguson S, Dodge J, Bernardini M, Murphy J, et al. The impacts of neoadjuvant chemotherapy and of debulking surgery on survival from advanced ovarian cancer. Gynecol Oncol 2014; 134: 462-7. 9. GourleyC, McCavigan A, Perren T, Paul J, OgilvieMichie C, Churchman M, et al. Molecular subgroup of high-grade serous ovarian cancer (HGSOC) as a predictor of outcome following bevacizumab. [Abstract]. J Clin Oncol 2014; 32(5 Suppl): abstract No. 5502. 10. Vergote I, du Bois A, Amant F, Heitz F, Leunen K, Harter P. Neoadjuvant chemotherapy in advanced ovarian cancer: On what do we agree and disagree? Gynecol Oncol 2013; 128: 6-11. 11. Stuart GC, Kitchener H, Bacon M, duBois A, Friedlander M, Ledermann J, et al. 2010 Gynecologic Cancer InterGroup (GCIG) consensus statement on clinical trials in ovarian cancer: report from the Fourth Ovarian Cancer Consensus Conference. Int J Gynecol Cancer 2011; 21: 750-5. 12. Salani R, Bristow RE. Surgical management of epithelial ovarian cancer. Clin Obstet Gynecol 2012; 55: 75-95. 13. Thrall MM, Gray HJ, Symons RG, Weiss NS, Flum DR, Goff BA. Neoadjuvant chemotherapy in the Medicare cohort with advanced ovarian cancer. Gynecol Oncol 2011; 123: 461-6. Radiol Oncol 2016; 50(3): 341-346. Slovenian abstracts III Radiol Oncol 2016; 50(3): 274-279. doi:10.1515/raon-2016-0032 Pozitronska emisijska tomografija z 18F-FDG in 18F-flumazenilom pri bolnikih z neodzivno epilepsijo Hodolič M, Topakian R, Pichler R Izhodišča. Epilepsija je nevrološka motnja, za katero so značilni epileptični napadi, ki so posledica prekomerne nevronske aktivnosti v možganih. Približno 65 milijonov ljudi po svetu trpi zaradi epilepsije; 20-40 % se jih na terapijo z zdravli ne odziva. Zgodnje odkrivanje bolezni je ključnega pomena pri zdravljenju bolnikov z epilepsijo, saj pravilna lokalizacija mesta epilepto-genega žarišča izboljša obravnavo teh bolnikov. Sodobne neinvazivne tehnike, ki jih uporabljajmo za strukturno in funkcionalno lokalizacijo žarišča, so elektroencefalografija (EEG), slikanje z magnetno resonanco (MRI), nuklearnomedicinska tomografija v kombinaciji z računalniško tomografijo (SPECT/CT) in pozitronska emisijska tomografija s CT ali MRI (PET/CT oz. PET/MRI). V zadnjih letih številne raziskave opisujejo, da lahko s pomočjo PET/CT napovemo izhod kirurškega zdravljenja bolnikov z neodzivno epilepsijo. Namen članka je sistematično preučiti vlogo dveh PET/CT radiofarmakov: 18F-fluorodeoksiglukoze (18F-FDG), ki jo pri bolnikih z neodzivno epilepsijo uporabljamo rutinsko, in 18F-flumazenila (18F-FMZ), ki ga uporabljamo le v kliničnih študijah. Zaključki. Informacije o delovanju, ki jih dobimo s pomočjo PET in informacije o morfologiji, ki jih dobimo s CT ali MRI, so bistvenega pomena za predkirurško oceno bolnika z epilepsijo. 18F-FDG PET/CT je danes rutinska metoda slikanja za določitev mesta epileptogenega žarišča pri bolnikih z neodzivno epilepsijo. Na žalost 18F-FDG PET/CT ni idealna metoda: področja z zmanjšanim metabolizmom glukoze se ne ujemajo natančno s histopatološko ali MRI dokazano stopnjo sprememb skleroze hipokampusa. Nova obetavna nuklearnomedicinska metoda je prikaz epileptogenega žarišča z gostoto benzodiazepinskih receptorjev. Zaradi boljše občutljivosti in anatomske ločljivosti bi bil lahko 18F-FMZ pomemben radiofarmak pri bolnikih z neodzivno epilepsijo. Radiol Oncol 2016; 50(3): 254-262. doi:10.1515/raon-2016-0023 Razlike pri vrisovanju tarčnih volumnov v radioterapiji. Kako pomembne so in kaj lahko storimo? Šegedin B, Petrič P Izhodišča. Moderne obsevalne tehnike omogočajo obsevanje tarčnega volumna z visoko dozo ob upoštevanju doznih omejitev za rizične organe, kar omogoča boljšo lokalno kontrolo ob ohranjevanju kakovosti življenja. Neujemanje med vriso-valci pri vrisovanju tarčnih volumnov je opisano za različne tumorske lokalizacije. Nekatere raziskave kažejo, da so razlike pri vrisovanju večje kot napake v vseh ostalih korakih načrtovanja in izvajanja obsevanja. Namen članka je povzeti nivo razlik pri vrisovanju tarčnih volumnov opisanem v literaturi in oceniti učinkovitost strategij za njihovo zmanjševanje. Zaključki. Pregled je potrdil pomembne razlike pri vrisovanju tarčnih volumnov za večino tumorskih lokalizacij, kar bi lahko vplivalo na lokalno kontrolo pri posameznih bolnikih. Kljub obetavnim rezultatom raziskav glede uporabe različnih anatomskih in funkcionalnih slikovnih metod pri vrisovanju tarčnih volumnov, bodo potrebne dodatne raziskave za opredelitev optimalne kombinacije le-teh. Dosledna uporaba priporočil za vrisovanje zmanjša neujemanje med vrisovalci. Njihova uporaba je priporočljiva tako v vsakdanji klinični praksi kot v sklopu kliničnih raziskav, saj je interpretacija rezultatov raziskav ob obstoječi stopnji razlik med vrisovalci lahko vprašljiva. Pomanjkanje znanja pri interpretaciji različnih slikovnih metod je pogost vzrok za neujemanje med vrisovalci, kar kaže, da sedanji obseg izobraževanja v sklopu specializacije radioterapije in onkologije ter v rednem kliničnem delu ni zadosten. Radiol Oncol 2016; 50(3): I-VIII. II Slovenian abstracts Radiol Oncol 2016; 50(3): 263-268. doi:10.1515/raon-2016-0026 Vloga nativne računalniške tomografije v diagnostiki tromboze možganskih venskih sinusov Avsenik J, Pretnar Oblak J, Šurlan Popovič K Izhodišča. Namen raziskave je bil preučiti senzitivnost in specifičnost nativne računalniške tomografije glave (CT) v diagnostiki tromboze možganskih venskih sinusov. Metode. Pregledali smo klinične podatke in radiološke preiskave 53 bolnikov, ki so bili obravnavani v urgentni nevrološki ambulanti zaradi suma na trombozo možganskih venskih sinusov. Dva neodvisna ocenjevalca sta pregledala nativne CT preiskave bolnikov in ocenila prisotnost znakov tromboze možganskih venskih sinusov. Za referenčno preiskavo smo upoštevali CT venografijo ali magnetnoresonančno venografijo. Strinjanje med ocenjevalcema smo ocenili s pomočjo Kappa statistike. Dodatno smo izmerili atenuacijske vrednosti znotraj venskih sinusov ter jih primerjali med skupino bolnikov s potrjeno trombozo možganskih venskih sinusov in kontrolno skupino. Rezultati. Trombozo možganskih venskih sinusov smo potrdili pri 13 bolnikih. Senzitivnost in specifičnost nativne CT preiskave sta bili 100 % in 83 %. Vrednost Kappa je bila 0,72 (dobro strinjanje med ocenjevalcema). Atenuacijske vrednosti znotraj možganskih venskih sinusov so bile pri bolnikih s trombozo značilno višje (73,4 ± 14,12 HU) kot v kontrolni skupini (58,1 ± 7,58 HU; p = 0,000). S pomočjo analize ROC smo določili pražno vrednost 64 HU, pri kateri sta bili senzitivnost in specifičnost metode 85 % in 87 %. Zaključki. Nativni CT glave je učinkovita prva preiskava pri urgentni obravnavi bolnikov s sumom na trombozo možganskih venskih sinusov. Merjenje atenuacijskih vrednosti znotraj sinusov lahko pripomore k večji diagnostični vrednosti preiskave. Radiol Oncol 2016; 50(3): 269-273. doi:10.1515/raon-2016-0025 Ponovitev bolezni v področnih bezgavkah pri 737 bolnikih s folikularno neoplazmo ali neoplazmo HUrthlejevih celic Vogrin A, Bešic H, Bešic N, Marolt Mušič M Izhodišča. Predoperativno ultrazvočno (UZ) preiskavo osrednjega in stranskega področja vratu priporočamo pri vseh bolnikih pred tiroidektomijo, kjer smo že dokazali malignom ali postavili citološki oziroma molekularni sum na malignost. Cilj raziskave je bil ugotoviti, kako pogosto se je rak ponovil v področnih bezgavkah pri bolnikih s folikularno neoplazmo ali neoplazmo Hurthlejevih celic in oceniti uporabnost predoperativne UZ preiskave pri bolnikih z neoplazmo. Bolniki in metode. Zaradi folikularne neoplazme ali neoplazme Hurthlejevih celic smo od leta 1995 do leta 2014 na Onkološkem inštitutu operirali 737 bolnikov. Med njimi je imelo raka ščitnice 207 bolnikov (163 žensk, 44 moških, povprečna starost 52 let). Rezultati. Med bolniki s folikularno neoplazmo smo rak diagnosticirali v 143/428 primerih, med bolniki z neoplazmo Hurthlejevih celic pa v 64/309 primerih. Ponovitev bolezni v področne bezgavke smo ugotovili pri 12/207 bolnikih (6 %) po srednji vrednosti spremljanja 55 mesecev. Med bolniki, ki so imeli raka, se je le-ta ponovil v regionalnih bezgavkah pri 2% bolnikov s folikularno neoplazmo in pri 14% bolnikov z neoplazmo Hurthlevih celic (p = 0,002). Po zdravljenju smo ponovno ugotovili rak v področnih bezgavkah v 3/428 (0,7 %) primerih bolnikov s folikularno neoplazmo in v 9/309 (3%) primerih bolnikov z neoplazmo Hurthlejevih celic. Zaključki. Ponovitev bolezni v bezgavkah smo diagnosticirali pri 0,7 % bolnikov s predoperativno diagnozo folikularne neo-plazme in pri 3 % bolnikih z neoplazmo Hurthlejevih celic. Ponovitev v področnih bezgavkah je redka tudi pri bolnikih z rakom, ki so imeli predoperativno diagnozo folikularna neoplazma. Predoperativna preiskava vratnih bezgavk pri bolnikih s folikularno neoplazmo verjetno ni uporabna, pri bolnikih z neoplazmo Hurthlejevih celic pa bi lahko bila koristna. Radiol Oncol 2016; 50(3): I-VIII. Slovenian abstracts III Radiol Oncol 2016; 50(3): 274-279. doi:10.1515/raon-2016-0042 Elektrokemoterapija z bleomicinom je učinkovita na BRAF mutiranih melanomskih celicah in ima potencirano delovanje z inhibitorji BRAF Dolinšek T, Prosen L, Čemažar M, Potočnik T, Serša G Izhodišča. Namen raziskave je bil ugotoviti učinkovitost elektrokemoterapije (ECT) med zdravljenjem bolnikov z melano-mom z inhibitorji BRAF. Testirali smo učinkovitost ECT na BRAF mutiranih in nemutiranih melanomskih celicah in vitro in v kombinaciji z inhibitorjem BRAF. Materiali in metode. ECT z bleomicinom smo izvedli na dveh humanih melanomskih celičnih linijah, eni z BRAF V600E mutacijo (SK-MEL-28) in eni brez BRAF mutacije (CHL-1). Učinkovitost ECT in ECT v kombinaciji z inhibitorjem BRAF vemurafenibom smo ovrednotili s preživetjem celic, ki smo ga določevali s testom klonogenosti. Rezultati. Preživetje melanomskih celic z BRAF V600E mutacijo je bilo po ECT manjše kot pri celicah brez mutacije, kar kaže na to, da je ECT učinkovita ne glede na mutacijski status melanomskih celic. Dokazali smo tudi sinergistično delovanje ECT z bleomicinom in vemurafeniba v BRAF V600E mutiranih melanomskih celicah. Zaključki. Učinkovitost ECT v BRAF mutiranih melanomskih celicah in njena potencirana učinkovitost v kombinaciji z ve-murafenibom in vitro kaže na klinično uporabnost ECT pri bolnikih z melanomom z mutacijo BRAF in/ali med zdravljenjem z inhibitorji BRAF. Radiol Oncol 2016; 50(3): 280-288. doi:10.1515/raon-2016-0027 Odkritje 'klik' 1,2,3-triazolijevih soli kot potencialnih antitumorskih učinkovin Steiner I, Stojanovič N, Bolje A, Brozovic A, Polančec D, Ambriovič-Ristov A, Radič Stojkovič M, Piantanida I, Eljuga D, Košmrlj J, Osmak M Izhodišča. Da bi povečali učinkovitost zdravljenja raka, smo pripravili in testirali nove potencialno antitumorsko aktivne spojine. Poročamo o testiranju novega tipa spojin, 1-(2-pikolil)-, 4-(2-pikolil)-, 1-(2-piridil)- in 4-(2-piridil)-3-metil-1,2,3-triazolijevih soli ter njihovih 1,2,3-triazolskih prekurzorjev. Metode. Citotoksičnost novih spojin smo določali s spektrometričnim testom 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolijev bro-mid (MTT) na več tumorskih in eni normalni celični liniji. Učinek vezave izbranih spojin na dvojno vijačnico DNA (ds DNA) smo ugotavljali s testiranjem vpliva na termično stabilnost DNA telečjega timusa, medtem ko smo vpliv na celični ciklus določevali s pretočno citometrično analizo. Tvorbo reaktivnih kisikovih spojin (ROS) smo določevali z dodatkom specifičnega substrata, 5-(in-6)-klorometil-2',7'-diklorodihidrofluorescein diacetata, acetil estra (CM-H2DCFDA). Rezultati. Osnovni triazoli na splošno niso aktivni, medtem ko so triazolijeve soli zelo citotoksične na celice HeLa. Triazolijeve soli so pokazale veliko specifično citotoksičnost na različne tumorske celice. Ena od spojin, 3-metil-4-(4-metoksifenil)-1-(2--pikolil)-1H-1,2,3-triazolijev heksafluorofosfat(V) (2b), je bila bistveno bolj citotoksična na tumorske kot pa na normalne celice, z visokim terapevtskih indeksom 7,69 za celice pljučnega raka H460. Ta spojina je bila podobno aktivna na osnovne celice raka grla HEp-2 in na njihovo na zdravila odporno podlinijo 7T, kar kaže na njen potencial v zdravljenju na zdravila odpornih rakov. Spojina 2b je ustavila celice v G1 fazi celičnega cikla. Ni vezala ds DNA, temveč je inducirala ROS v obdelovanih celicah, kar je sprožilo celično smrt. Zaključki. Naši rezultati nakazujejo, da je smiselno nadaljevati raziskave 'klik' triazolijevih soli kot antitumorskih učinkovin. Radiol Oncol 2016; 50(3): I-VIII. II Slovenian abstracts Radiol Oncol 2016; 50(3): 289-296. doi:10.1515/raon-2016-0031 Funkcionalni polimorfizmi antioksidativnih genov pri neoplazmi Huerthlejevih celic ščitnice - povezava med polimorfizmom gena GPX1 in ponovitvijo raka Huerthlejevih celic ščitnice Krhin B, Goričar K, Gazic B, Dolžan V, Bešic N Izhodišča. Za Huerthlejeve celice ščitnice je značilno veliko število mitohondrijev in oksidativnih encimov. Ker povečan oksi-dativni metabolizem lahko vodi v povečan oksidativni stres oziroma ga lahko povezujemo z večjo verjetnostjo razvoja raka, smo v naši raziskavi preverjali, ali obstaja povezava med funkcionalnimi polimorfizmi antioksidativnih genov (SOD2, CAT, GPX, GSTP1, GSTM1 in GSTT1) in nastankom ali kliničnim potekom raka Huerthlejevih celic ščitnice (HCTC). Bolniki in metode. Retrospektivno raziskavo smo izvedli pri 139 bolnikih, pri katerih smo zaradi suma na neoplazmo Huerthlejevih celic ščitnice opravili operacijo ščitnice. Diagnozo HCTC, adenoma Huerthlejevih celic ščitnice (HCTA) ali gomolja Huerthlejevih celic ščitnice (HCTN) smo postavili s histopatomorfološko analizo. DNA smo izolirali iz stebričkov histološko potrjenega zdravega dela ščitnice, pridobljenega iz arhiviranih parafinskih blokov tumorjev, fiksiranih v formalinu. S postopki genotipizacije smo določali prisotnost polimorfizmov v antioksidativnih genih. Z logistično regresijo pa smo primerjali porazdelitve posameznih genotipov med različnimi skupinami bolnikov. Rezultati. HCTC smo ugotovili pri 53, HCTA pri 47 in HCTN pri 21 bolnikih. Pri 20 bolnikih s HCTC smo ugotovili prisotnost zasevkov, pri 16 pa ponovitev bolezni. Pri skupinah bolnikov s HCTC, HCTA in HCTN frekvence genotipov in alelov preučevanih polimorfizmov niso odstopale od Hardy-Weinbergovega ravnotežja. Dominantni genetski model ni pokazal povezave med porazdelitvijo frekvenc genotipov preučevanih polimorfizmov in prisotnostjo HCTC v primerjavi s HCTA in HCTN, prav tako ni bilo povezave s prisotnostjo zasevkov pri HCTC. Ugotovili pa smo povezavo med polimorfizmom GPX1 in ponovitvijo HCTC (p = 0,040). Zaključki. Polimorfizem GPX1 lahko vpliva na možnost ponovitve HCTC. Radiol Oncol 2016; 50(3): 297-307. doi:10.1515/raon-2015-0047 Vpliv polimorfizmov v segregacijskih genih BUB1B in TTK na dovzetnost za razvoj želodčnega raka Hudler P, Kočevar Britovšek N, Frkovic Grazio S, Komel R Izhodišča. Maligna preobrazba normalnih želodčnih celic je zapleten večstopenjski proces, ki vodi v nastanek heteroge-nih tumorjev. Na razvoj želodčnega raka vplivajo poleg dejavnikov okolja tudi genetsko ozadje in genetske spremembe. Polimorfizmi enega baznega para (angl. Single nucleotide polymorphisms, SNP) v mitotskih segregacijskih genih bi lahko bili odgovorni za počasno kopičenje genetskih sprememb, ki vodijo v genomsko nestabilnost. Bolniki in metode. V raziskavi primerov s kontrolami smo opredelili vpliv polimorfizmov rs151658 v mitotski kinazi TTK in rs1031963 ter rs1801376 v kinazi BUB1B na razvoj želodčnega raka. Z metodo imunskega odtisa smo določili količino TTK v rakavih tkivih bolnikov. Rezultati. Odkrili smo, da genotipa C/G in G/G polimorfizma rs151658 značilno vplivata na dovzetnost za razvoj difuzne oziroma intestinalne oblike želodčnega raka (p = 0,049). Genotip A/A polimorfizma rs1801376 je bil značilno povezan z višjim tveganjem za razvoj želodčnega raka pri bolnicah (0,007), medtem ko se je pri moških z želodčnim rakom pogosteje pojavljal le pri preiskovancih, pri katerih so tumorske celice preraščale v subserozo (0,009). Pri nosilcih genotipa T/T polimorfizma rs1031963 so se pogosteje razvili dobro diferencirani tumorji (0,035). V dominantnem modelu sta bila genotipa TT+CT polimorfizma rs1031963 (razmerje obetov [OR] = 2,929, 95 % interval zaupanja [CI]: 1,281-6,700; p = 0,017) in genotipa GG+AG polimorfizma rs1801376 (OR = 0,364, 95 % CI: 0,192-0,691; p = 0,003) značilno povezana z višjim tveganjem za razvoj bolezni. Zaključki. Rezultati raziskave kažejo, da polimorfizmi v mitotskih kinazah TTK in BUB1B v naši skupini preiskovancev statistično značilno prispevajo k povišanemu tveganju za razvoj želodčnega raka in mogoče vplivajo na potek razvoja tumorjev. Za opredelitev njihove klinične uporabnosti so potrebne nadaljnje raziskave v večjih skupinah bolnikov z želodčnim rakom različnih ras. Radiol Oncol 2016; 50(3): I-VIII. Slovenian abstracts III Radiol Oncol 2016; 50(3): 274-279. doi:10.1515/raon-2016-0039 Metastatski sebacijski rak. Pregled literature in elektrokemoterapija kot nova možna oblika zdravljenja Ribero S, Sportoletti Baduel E, Brizio M, Pcciotto F, Dika E, Fierro MT, Macripo G, Quaglino P Izhodišča. Metastatski sebacijski rak je redka bolezen glave in vratu. V začetni stopnji razvoja je terapija izbora kirurgija in/ ali radioterapija. Zdravljenje ponovljene ali napredovale bolezni pa je še vedno različno. Metode. Naredili smo izčrpno poizvedbo objavljene literature, ki je obravnavala terapevtske možnosti te redke bolezni. Rezultati. V literature je opisanih več oblik zdravljenja metastatskega sebacijskega raka. Elektrokemoterapija do sedaj še ni bila opisna kot možen način zdravljenja. Prikažemo 85 let starega bolnika s ponovljeno, lokalno metastatsko boleznijo na temenu, ki smo ga zdravili z elektrokemoterapijo. To smo aplicirali dvakrat v obdobju 8. mesecev. Dosegli smo delni odgovor tumorja in dobro kakovost življenja bolnika. Zaključki. Pregled literature nazorno nakazuje potrebo po novih načinih zdravljenja metastatskega sebacijskega raka. Na osnovi naše prve in pozitivne izkušnje predlagamo nadaljnje raziskave, ki bi uporabile elektrokemoterapijo za zdravljenje te redke entitete tumorja in bi jo nato uporabile kot terapijo izbora v kliničnih situacijah, kjer je potrebna lokalna kontrola tumorjev ali pa radikalni posegi niso možni ali zaželeni. Radiol Oncol 2016; 50(3): 313-320. doi:10.1515/raon-2016-0033 Proliferacijski faktor Ki67, vendar ne neuroendokrina ekspresija, je neodvisni napovedni dejavnik za primarni rak prostate Pascale M, Aversa C, Barbazza R, Marongiu B, Siracusano S, Stoffel F, Sulfaro S, Roggero E, Bonin S, Stanta G Izhodišča. Neuroendokrine označevalce in proliferacijski faktor Ki67 so že povezovali s potekom bolezni primarnega raka prostate. Namen raziskave je bil raziskati napovedno vrednost teh označevalcev pri bolnikih s primarnim rakom prostate. Bolniki in metode. Neuron specifično enolazo (NSE), kromagranin A (ChrA), sinaptofizin (Syp) in Ki67 smo določevali imunohistokemično. S pomočjo univariantne in multivariantne analize smo ovrednotili njihovo izražanje in povezanost s celokupnim preživetjem pri 166 bolnikih s primarnim rakom prostate. Rezultati. NSE, ChrA, Syp in Ki67 so bili pozitivni pri 50, 45, 54 in 146 od skupno 166 bolnikov. S Kaplan-Meier analizo smo dokazali, da sta samo difuzno barvanje NSE (negativni proti difuzno barvani vzorci p = 0,004) in proliferacijski označevalec Ki67 (< 10 % vs. > 10 %, p < 0,0001) povezana s celokupnim preživetjem bolnikov. V multivariatni analizi se je izražanje Ki67 pokazalo kot neodvisni napovedni dejavnik celokupnega preživetja ne pa izražanje NSE. Zaključki. Napovedni model, ki vključuje izražanje Ki67 ob upoštevanju klinično patoloških parametrov, lahko predstavlja dodatno napovedno informacijo oz. lahko izboljša napoved izhoda bolezni raka prostate ter obravnavo bolnikov z rakom prostate. Radiol Oncol 2016; 50(3): I-VIII. II Slovenian abstracts RadiolOncol 2016; 50(3): 321-328. doi:10.1515/raon-2015-0017 Cefalna pankreatektomija z resekcijo ven pri duktalnem raku trebušne slinavke Flis V, Potrč S, Kobilica N, Ivanecz A Izhodišča. Nekatere raziskave kažejo, da imajo bolniki z rakom trebušne slinavke, pri katerih se rakava rašča širi v portalni venski sistem, po kirurški odstranitvi tumorja podobno preživetje kot bolniki, kjer ni razraščanja v vene. Mnenja o tem so deljena, katerim bolnikom z vraščanjem tumorja v venski sistem bi kirurška odstranitev tumorja koristila. Prav tako ni soglasja o tem, kateri kirurški postopek poprave odstranjenih ven je ustreznejši (rekonstrukcija z žilno protezo ali brez nje). Ker so so dosedanje raziskave vsebovale majhno število bolnikov, je bil namen pričujoče raziskave predstavitev izkušenj posamične ustanove pri cefalni duodenopankreatektomiji s hkratno resekcijo ven. Bolniki in metode. Pregled računalniške podatkovne zbirke Univerzitetnega kliničnega centra v Mariboru za obdobje od januarja 2006 do avgusta 2014 je pokazal, da smo v omenjenem obdobju zaradi raka trebušne slinavke s cefalno duodeno-pankreatektomijo operirali 133 bolnikov (poprečna starost 65,4 ± 8,6 let; 69 žensk). Razčlenili smo njihove demografske, klinične in biokemijske podatke, histološke izvide in pooperativni izhod. Primerjali smo podatke bolnikov, kjer smo hkrati z duodenopan-kreatektomijo opravili tudi resekcijo ven, z bolniki, kjer resekcije ven nismo naredili. Rezultati. Izmed 133 bolnikov je bilo 22 (16,5 %) takih, kjer smo hkrati s cefalno duodenopankreatektomijo opravili tudi resekcijo ven in njihovo rekonstrukcijo. Tako smo opredelili skupino 111 bolnikov (poprečna starost 65,6 ± 7, let; 58 žensk), kjer ven nismo resecirali in skupino 22 bolnikov (63,95 ± 9.5 let; 13 žensk), kjer smo resekcijo ven naredili. V slednji skupini smo pri 14 bolnikih portalno veno popravili brez uporabe umetnega žilnega vsadka, pri osmih pa smo vstavili umetni žilni vsadek iz dakrona. Pri bolnikih, pri katerih uporaba vsadka ni bila potrebna, smo vedno uporabili anastomozo konec s koncem, če smo jo lahko varno naredili brez tenzije na anastomozni črti. Kadar smo operirali področje na stičišču zgornje mezenterične in vranične vene, smo vranično veno podvezali. Pri osmih bolnikih smo uporabili umetni žilni vsadek iz dakrona s premerom 10 mm. Pri njih nismo videli zapletov na anastomozi. V skupini z direktnim šivom vene smo ugotovili eno trombozo vene. Neposredna primerjava obeh skupin (vsi bolniki z resekcijo ven v primerjavi z bolniki brez resekcije) je pokazala, da ni bilo statistično pomembnih razlik med skupinama v pooperativni obolevnosti in umrljivosti. Srednji čas preživetja je bil v skupini z resekcijo 16,13 mesecev in v skupini brez resekcije 15,17 mesecev. V skupini brez resekcije je bilo celokupno petletno preživetje 19,5 %. Primerjava krivulj preživetja med skupinama ni pokazala pomembnih statističnih razlik (test log-rank p = 0,090). Zaključki. Preživetje bolnikov z rakom trebušne slinavke, kjer je bila poleg cefalne pankreatektomije opravljena tudi resek-cija ven, je bilo primerljivo s skupino, kjer resekcija ven ni bila potrebna. Dodaten poseg na venah ni vplival na pooperativno obolevnost in umrljivost. Uporaba umetnega žilnega vsadka iz dakrona je bila varna alternativa neposredni venski popravi. Radiol Oncol 2016; 50(3): I-VIII. Slovenian abstracts III Radiol Oncol 2016; 50(3): 274-279. doi:10.1515/raon-2016-0036 Interdisciplinarna soglasna izjava o indikaciji in uporabi hidrogelnega vmesnika pri radioterapiji prostate. Izkušnje pri več kot 250 bolnikih Müller AC, Mischinger J, Klotz T, Gagel B, Habl G, Hatiboglu G, Pinkawa M Izhodišča. Namen raziskave je bil doseči soglasje o indikaciji in uporabi hidrogelnega vmesnika na podlagi multicentričnih izkušenj ter posredovati uporabnikom pomembno informacijo, s katero bi skrajšali učenje te inovativne tehnike. Metode. Interdisciplinarnega srečanja so se udeležili radioterapevti in urologi, ki so opravili 23-138 aplikacij hidrogela (SpaceOAR®) pri bolnikih z rakom prostate pred radioterapijo, kjer smo zviševali dozo. Želeli smo pridobiti praktične informacije, ki so pomembne za zaporedno injiciranje hidrogela in zdravljenje, zato smo obravnavali izkušnje uporabnikov in odgovorili na zastavljena ključna vprašanja. Pregledani smo stranske učinke, povezane s hidrogelom in ocenili delež, zdravljenje in napoved možnih tveganj. Rezultati. Najpomembnejša indikacija za aplikacijo hidrogela je bila radioterapija z naraščajočo dozo pri histološko potrjenem raku prostate z nizkim ali vmesnim tveganjem. Priporočili smo jo pri lokalno napredovalem raku prostate. Injiciranje ali implantacijo smo opravljali pod nadzorom transrektalnega ultrazvoka in s transperinealnim pristopom po predhodni hid-rodisekciji. Pri skupno 258 aplikacijah hidrogela je bil delež toksičnosti stopnje 2, ki je bila povezana z injiciranjem 2 % (n = 5). Najpogostejši zaplet (n = 4) je bilo predrtje stene rektuma, ki smo ga ugotovili ob različnih časovnih intervalih po injiciranju hidrogela in smo ga zdravili konzervativno. Zaključki. Dosegli smo soglasje o aplikaciji hidrogelnega vmesnika. Dosedanje izkušnje kažejo, da je metoda izvedljiva, kar lahko spodbudi njeno uvedbo v več centrih. Na ta način bi lahko znižali z obsevanjem povezano gastrointestinalno toksičnost slikovno vodene radioterapije z naraščajočo dozo. Še vedno pa je možen zelo nizek delež resnih neželenih dogodkov. Zato bi morali skrbno proučiti aplikacijo in bolnika ter upoštevati možne prednosti takšnega zdravljenja. Radiol Oncol 2016; 50(3): 337-340. doi:10.1515/raon-2016-0029 Odlični rezultati zdravljenja kompresije hrbtenjače pri mielomu z radioterapijo Rades D, Conde-Moreno AJ, Cacicedo J, Šegedin B, Rudat V, Schild SE Izhodišča. Ni znano, ali bolniki s kompresijo hrbtenjače in radiosezibilnimi tumorji poleg radioterapije potrebujejo tudi kirurško dekompresijo hrtenjače. Zato smo v raziskavi analizirali potek bolezni pri bolnikih, ki so zboleli zaradi mielomoma in pri katerih smo ugotovili kompresijo hrbtenjače ter smo jih zdravili samo z obsevanjem. Bolniki in metode. Retrospektivno smo analizirali podatke 238 bolnikov glede odgovora na RT in lokalno kontrolo kompresije hrbtenjače. Ocenili smo učinek radioterapije na motorično funkcijo (izboljšanje, brez nadaljnega slabšanja, poslabšanje). Odgovor smo opredelili kot izoboljšanje ali odsotnost nadaljnega slabšanja motorične okvare. Pred radioterapijo smo bolnike predstavili nevrokirurgu, da je ocenil, ali je indicirana takojšnja operacija z dekompresijo (zaradi zloma vretenca, nestabilne hrbtenice). Rezultati. V celotni kohorti je bil odgovor na radioterapijo 97 % (izboljšanje 53 %, brez nadaljnega slabšanja 44 %). Po radioterapiji je lahko hodilo 88 % bolnikov. Izmed 69 hospitaliziranih bolnikov je shodilo 44 bolnikov (64 %). Lokalna kontrola po 1, 2 in 3 letih je bila 93 %, 82 % in 82 %. Trend izboljšanja lokalne kontrole smo opazili pri bolnikih, ki smo jih obravnavali pred začetkom radioterapije oambulantno (p = 0.08), in pri tistih z boljšim stanjem zmogljivosti (p = 0.07). Zaključki. Pri bolnikih z mielomom in kompresijo hrbtenjače zagotavlja radioterapija odličen odgovor na zdravljenje, funkcionalne rezltate in lokalno kontrolo. Rezultate bo potrebno potrditi s prospektivno randomizirano raziskavo. Radiol Oncol 2016; 50(3): I-VIII. VIII Slovenian abstracts Radiol Oncol 2016; 50(3): 341-346. doi:10.1515/raon-2016-0034 Vloga neoadjuvantne kemoterapije pri bolnicah z napredovalim epitelijskim rakom jajčnika (stadij IIIC) Škof E, Merlo S, Pilko G, Kobal B Izhodišča. Primarno zdravljenje bolnic z napredovalim epitelijskim rakom jajčnika vključuje kemoterapijo pred (neoadju-vantna kemoterapija) ali po zamejitveni operaciji (adjuvantna kemoterapija). Cilj primarnega zdravljenja epitelijskega raka jajčnika je odstranitev vsega rakastega tkiva brez makroskopskega ostanka. Popolna resekcija (R0) po primarnem kirurškem zdravljenju predstavlja pomemben dejavnik v izboljšanju preživetja bolnic, medtem ko za enkrat še ni dokazov o enakem vplivu R0 resekcije, če primarno zdravljenje začnemo z neoadjuvantno kemoterapijo. Bolnice in metode. V letih od 2005 do 2007 smo na Onkološkem inštitutu pregledali popise bolnic z diagnosticiranim epitelijskim rakom jajčnika. Med bolnicami, ki so zdravljenje začele z neoadjuvantno kemoterapijo in tistimi, ki so bile primarno operirane, smo primerjali stopnjo dosežene kirurške resekcije, čas do ponovitve bolezni, ter petletno in osemletno preživetje. Rezultati. V raziskavo smo zajeli 160 bolnic s stadijem IIIC epitelijskega raka jajčnika. 80 bolnic smo pričeli zdraviti z neoadjuvantno kemoterapijo, pri preostalih 80-ih bolnicah pa je bilo primarno zdravljenje kirurško. Bolnice v skupini z neoadjuvantno kemoterapijo so imele višjo stopnjo resekcije R0 (42 % vs. 20 %; p = 0,011) kot bolnice s primarnim kirurškim zdravljenjem. Čas do ponovitve bolezni je bil v skupini z neoadjuvantno kemoterapijo 14,1 mesecev, po primarnem kirurškem zdravljenju pa 17,7 (p = 0,213). Celokupno preživetje je bilo 24,8 meseca v skupini z neoadjuvantno kemoterapijo in 31,6 meseca po primarni kirurgiji (p = 0,012). Pri bolnicah z doseženo resekcijo R0 sta bila petletno in osemletno preživetje v skupini z neoadjuvantno kemoterapijo 20,6 % in 17,6 % , v skupini s primarno kirurgijo pa 62,5 % in 62,5 % (p < 0,0001). Zaključki. Kljub večjemu deležu resekcije R0, ki smo jo dosegli v skupini, ki je začela zdravljenje z neoadjuvantno kemoterapijo, je bilo preživetje teh bolnic nižje od preživetja bolnic s primarnim kirurškim zdravljenjem. Neoadjuvantna kemoterapija tako ostaja prednostna za bolnice z napredovalim epitelijskim rakom jajčnika, ki niso primerne za primarno radikalno kirurško zdravljenje. Radiol Oncol 2016; 50(3): I-VIII. FUNDACIJA DR. J. CHOLEWA Fundacija "Docent dr. J. Cholevva" je neprofitno, neinstitucionalno in nestrankarsko združenje posameznikov, ustanov in organizacij, ki želijo materialno spodbujati in poglabljati raziskovalno in izobraževalno dejavnost v onkologiji. Fundacija »docent dr. Josip Cholevva« v sodelovanju z Medicinsko fakulteto Ljubljana, Medicinsko fakulteto Maribor, univerzitetnim kliničnim centrom ljubljana, univerzitetnim kliničnim centrom Maribor in Onkološkim Inštitutom Ljubljana PRIREJA STROKOVNI SIMPOZIJ Z NASLOVOM: DIAGNOSTIKA IN ZDRAVLJENJE ZGODNJEGA RAKA Simpozij bo potekal v Ljubljani, dne 7. oktobra 2016 V MODRI DVORANI DOMUS MEDICA, DUNAJSKA CESTA 1 62. Dunajska 106 1000 Ljubljana TRR: 02033-001 787943 1 FUNDACIJA DR. J. CHOLEWA Activity of "Dr. J. Cholewa" Foundation for Cancer Research and Education - a report for the third quarter of 2016 Dr. Josip Cholewa Foundation for cancer research and education continues with its planned activities in the third quarter of 2016. Its primary focus remains the provision of grants and scholarships and other forms of financial assistance for basic, clinical and public health research in the field of oncology. In parallel, it also makes efforts to provide financial and other support for the organisation of congresses, symposia and other forms of meetings to spread the knowledge about prevention and treatment of cancer, and finally about rehabilitation for cancer patients. In Foundation's strategy the spread of knowledge should not be restricted only to the professionals that treat cancer patients, but also to the patients themselves and to the general public. The Foundation continues to provide support for »Radiology and Oncology«, a quarterly scientific magazine with a long tradition and with a respectable impact factor that publishes research and review articles about all aspects of cancer. The magazine is edited and published in Slovenia. The Foundation will continue with its activities in the future, especially since the problems associated with cancer affect more and more people in Slovenia and elsewhere. Ever more successful treatment results in longer survival in many patients with previously incurable cancer conditions, thus adding many new dimensions in life of cancer survivors and their families. Andrej Plesničar, M.D., M.Sc. Viljem Kovač M.D., Ph.D. Borut Štabuc, M.D., Ph.D. Tomaž Benulič, M.D. XGEVA® VAŠE BOLNIKE BOLJE ZAŠČITI PRED KOSTNIMI ZAPLETI1*0 XGEVA (denosumab) NATANČEN. MOČAN. DOKAZAN. Podatki integrirane analize preskušanj III. faze, ki so vključevala bolnike z rakom dojke, rakom prostate, ostalimi solidnimi tumorji ali diseminiranim plazmocitomom, ki so imeli zasevke v kosteh.1 ' XGEVA® značilno podaljša čas do pojava prvega zapleta kostnih zasevkov (ZKZ) za 8,21 mesecev in zmanjša tveganje za pojav prvega ZKZ za 17% (HR; 0,83 [95% CI; 0,76-0,90]; p < 0,001) v primerjavi z zoledronsko kislino. •Opredelitev kostnih zapletov: Preprečevanje zapletov kostnih zasevkov (ZKZ) in z njimi povezane bolečine pri odraslih s kostnimi zasevki solidnih tumorjev. XGEVA® 120 mg raztopina za injiciranje (denosumab) - SKRAJŠAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA Samo za strokovno javnost. Pred predpisovanjem si preberite celoten Povzetek glavnih značilnosti zdravila. VZa to zdravilo se izvaja dodatno spremljanje varnosti. Poročati je potrebno o vseh domnevnih neželenih učinkih zdravila. SESTAVA ZDRAVILA: Ena viala vsebuje 120 mg denosumaba v 1,7 ml raztopine (70 mg/ml]. Pomožne snovi z znanim učinkom: 1,7 ml raztopine vsebuje 78 mg sorbitola (E420).TERAPEVTSKE INDIKACIJE: Preprečevanje skeletnih dogodkov (patoloških zlomov, obsevanja kosti, kompresije hrbtenjače ali operacije kosti) pri odraslih s kostnimi metastazami solidnih tumorjev. Zdravljenje odraslih bolnikov in skeletno dozorelih mladostnikov z gigantocelularnim kostnim tumorjem, ki ni operabilen, ali pri katerem bi kirurška odstranitev verjetno povzročila hudo obolevnost. ODMERJANJE IN NAČIN UPORABE: Zdravilo XGEVA® mora aplicirati zdravstveni delavec. Vsi bolniki morajo prejemati dodatek vsaj 500 mg kalcija in 400 i.e. vitamina D dnevno, razen če ima bolnik hiperkalc iemijo. Bolniki morajo dobiti navodilo za uporabo in opozorilno kartico za bolnika. Preprečevanje skeletnih dogodkov pri od&slih s kostnimi metastazami solidnih tumorjev: Priporočeni odmerek je 120 mg enkrat na 4 tedne v enkratni subkutani injekciji v stegno, trebuh ali nadlaket. Gigantocelularni kostni tumor: Priporočeni odmerek zdravila XGEVA® je 120 mg enkrat na 4 tedne v enkratni subkutani injekciji v stegno, trebuh ali nadlaket ter dodaten odmerek 120 mg 8. in 15. dan zdravljenja v prvem mesecu terapije. Bolnike z gigantocelularnim kostnim tumorjem je treba v rednih presledkih ocenjevati, da bi ugotovili, ali jim zdravljenje še koristi. Pri bolnikih, ki imajo bolezen z zdravilom XGEVA® obvladano, niso ocenili učinka prekinitve ali prenehanja zdravljenja, toda omejeni podatki pri teh bolnikih ne kažejo povratnega učinka po prenehanju zdravljenja. Bolniki z okvaro ledvic: Prilagoditev odmerka ni potrebna. Bolniki z okvaro jeter: Varnost in učinkovitost denosumaba nista raziskani. Starejši bolniki (stari » 65 leti: Prilagoditev odmerka ni potrebna. Pediatrični bolniki: Varnost in učinkovitost zdravila XGEVA® pri pediatričnih bolnikih (starih < 18 let) še nista bili dokazani z izjemo skeletno dozorelih mladostnikov z gigantocelularnim kostnim tumorjem. Zdravilo XGEVA® ni priporočljivo za pediatrične bolnike (stare < 18 let) z izjemo skeletno dozorelih mladostnikov z gigantocelularnim kostnim tumorjem. Odmerjanje za zdravljenje skeletno dozorelih mladostnikov z gigantocelularnim kostnim tumorjem, ki ni operabilen, ali pri katerem bi kirurška odstranitev verjetno povzročila hudo obolevnost, je enako kot za odrasle. Za subkutano uporabo. KONTRAINDIKACIJE: Preobčutljivost na zdravilno učinkovino ali katero koli pomožno snov. Huda, nezdravljena hipokalciemija. Nezaceljene lezije po zobnih ali ustnih kirurških posegih. POSEBNA OPOZORILA IN PREVIDNOSTNI UKREPI: Vsi bolniki morajo prejemati dodatek kalcija in vitamina D, razen če ima bolnik hiperkalciemijo. Obstoječo hipokalciemijo je treba odpraviti še pred začetkom zdravljenja z zdravilom XGEVA®. Hipokalciemija se lahko pojavi kadarkoli med zdravljenjem z zdravilom XGEVA®. Kontrolo koncentracije kalcija je treba izvesti (i) pred prvim odmerkom zdravila XGEVA®, (ii) v dveh tednih po prvem odmerku, (m) če se pojavijo simptomi, sumljivi za hipokalciemijo. O dodatnih kontrolah koncentracije kalcija med zdravljenjem je treba razmisliti pri bolnikih, ki imajo dejavnike tveganja za hipokalciemijo, ali če so takšne kontrole sicer umestne glede na bolnikovo klinično stanje. Bolnikom je treba naročiti, naj zdravnika obvestijo o simptomih, ki kažejo na hipokalciemijo. Če se med prejemanjem zdravila XGEVA® pojavi hipokalciemija, je lahko potrebno dodatno dodajanje kalcija in dodatne kontrole. Bolniki s hudo okvaro ledvic (očistek kreatinina < 30 ml/min) ali bolniki na dializi imajo večje tveganje za pojav hipokalciemije. Tveganje za pojav hipokalciemije in spremljajočega zvišanja paratiroidnega hormona se povečuje s povečano stopnjo okvare ledvic. Pri takšnih bolnikih so redne kontrole koncentracije kalcija posebej pomembne. Pri bolnikih, ki imajo nezaceljene lezije mehkih tkiv v ustih, je treba začetek zdravljenja/nov ciklus zdravljenja odložiti. Pred zdravljenjem z zdravilom XGEVA® je priporočljivo opraviti zobozdravstveni pregled in preventivno zobozdravstveno oskrbo ter individualno oceno koristi in tveganja. Pri ocenjevanju bolnikovega tveganja za pojav osteonekroze čeljustnice je treba upoštevati naslednje dejavnike tveganja: moč zdravila, ki zavira resorpcijo kosti (tveganje je večje z zelo močnimi spojinami), pot uporabe (tveganje je večje v primeru parenteralne uporabe) in kumulativni odmerek zdravila, uporabljenega za zdravljenje resorpcije kosti, rak, sočasne bolezni (npr. anemijo, koagulopatije, okužbo), kajenje, sočasna zdravljenja: kortikosteroide, kemoterapijo, zaviralce angiogeneze, radioterapijo glave in vratu, slabo ustno higieno, periodontalno bolezen, slabo prilegajoče se zobne proteze, že obstoječo zobno bolezen, irvazivne zobozdravstvene posege, npr. ekstrakcije zob. ^em bolnikom je treba naročiti, da morajo med zdravljenjem z zdravilom XGEVA® vzdrževati dobro ustno higieno, redno opravljati zobozdravniške preglede in nemudoma obvestiti zdravnika, če se pojavi kakršen koli simptom v ustih, na primer majanje zob, bolečina, oteklina, rana, ki se ne celi, ali izcedek. Med zdravljenjem je izvajanje invazvnih zobozdravniških posegov dovoljeno le po skrbnem razmisleku in se jim je treba izogniti v bližini termina za odmerjanje zdravila XGEVA®. Načrt vodenja bolnikov, ki se jim pojavi osteonekroza čeljustnice, je treba oblikovati na podlagi tesnega sodelovanja med lečečim zdravnikom in zobozdravnikom ali ustnim kirurgom, ki ima izkušnje z osteonekrozo čeljustnice. Razmisliti je treba o začasnem prenehanju zdravljenja z zdravilom XGEVA®, dokler se to stanje ne razreši in se sovpleteni dejavniki tveganja ublažijo, če je mogoče. Atipični zlomi stegnenice se lahko pojavijo že ob majhni poškodbi ali celo brez poškodbe, in sicer v subtrohanternem in diafiznem predelu stegnenice. Za te dogodke so značilni specifični radiografski izvidi. O njih so poročali tudi pri bolnikih z določenimi sočasnimi bolezenskimi stanji (npr. s pomanjkanjem vitamina D, revmatoidnim artritisom, hipofosfatazijo) in med uporabo določenih zdravil (npr. bisfosfonatov, glukokortikoidov, zaviralcev protonske črpalke). Ti dogodki so se pojavili tudi brez antiresorpcijskega zdravljenja. Podobni zlomi, opisani v zvezi z bisfosfonati, so pogosto obojestranski, zato je treba pri bolnikih, ki se zdravijo z denosumabom in so imeli zlom srednjega dela stegnenice, opraviti tudi pregled druge stegnenice. Pri bolnikih, pri katerih obstaja sum na atipičen zlom stegnenice, je treba razmisliti o prenehanju uporabe zdravila XGEVA® ob vrednotenju bolnika glede na individualno oceno koristi in tveganja. Bolnikom je treba naročiti, da morajo med zdravljenjem z zdravilom XGEVA® zdravniku poročati o novi ali nenavadni bolečini v stegnu, kolku ali dimljah. Bolnike s takšnimi simptomi je treba preiskati glede nepopolnega zloma stegnenice. Zdravilo XGEVA® ni priporočljivo pri bolnikih, ki se jim skelet še razvija. Bolniki, zdravljeni z zdravilom XGEVA®, sočasno ne smejo prejemati drugih zdravil, ki vsebujejo denosumab (za indikacije pri osteoporozi), in bisfosfonatov. Malignost pri gigantocelularnem kostnem tumorju ali napredovanje do metastatske bolezni je redek dogodek in je znano tveganje pri bolnikih z gigantocelularnim kostnim tumorjem. Bolnike je treba kontrolirati glede radioloških znakov malignosti, nove radiolucentnosti ali osteolize. Razpoložljivi klinični podatki ne kažejo povečanega tveganja za malignost pri bolnikih z gigantocelularnim kostnim tumorjem, zdravljenih z zdravilom XGEVA®. Zdravilo XGEVA®vsebuje sorbitol. Bolniki z redko prirojeno motnjo intolerance za fruktozo ne smejo uporabljati zdravila XGEVA®. Zdravilo vsebuje manj kot 1 mmol natrija (23 mg) na 120 mg, kar pomeni, da je praktično »brez natrija. INTERAKCIJE: Študij medsebojnega delovanja niso izvedli. Sočasna kemoterapija in/ali hormonsko zdravljenje ali predhodna intravenska izpostavljenost bisfosfonatom niso klinično pomembno spremenili najmanjše koncentracije denosumaba v serumu in farmakodinamike denosumaba (N-telopeptid v urinu, prilagojen na kreatinin, uNTx/Cr). POVZETEK NEŽELENIH UČINKOV: Zelo pogosti (» 1/10): dispneja, driska, mišično-skeletna bolečina. Pogosti (» 1/100 do < 1/10): hipokalciemija, hipofosfatemija, ekstrakcija zoba, hiperhidroza, osteonekroza čeljustnice. Redki (» 1/10.000 do < 1/1.000): preobčutljivost na zdravilo, anafilaktična reakcija, atipični zlom stegnenice. FARMACEVTSKI PODATKI: Shranjujte v hladilniku (2 °C - 8 °C). Ne zamrzujte. NAČIN IN REŽIM PREDPISOVANJA TER IZDAJE ZDRAVILA: Predpisovanje in izdaja zdravila je le na recept s posebnim režimom - ZZ. IMETNIK DOVOLJENJA ZA PROMET: Amgen Europe B.V., Minervum 7061, NL-4817 ZK Breda, Nizozemska. Dodatna pojasnila lahko dobite v lokalni pisarni: Amgen zdravila d.o.o., Šmartinska 140, SI-1000 Ljubljana. DATUM ZADNJE REVIZIJE BESEDILA: April 2016. DATUM PRIPRAVE INFORMACIJE: Julij 2016. Podrobne informacije o zdravilu so objavljene na spletni strani Evropske agencije za zdravila http://www.ema.europa.eu. LITERATURA: 1. Lipton A, et al. Eur J Cancer. 2012; 48: 3082-3092. rRIAD" Iclusig® (ponatinib) Ključ do učinkovitega zdravljenja bolnikov s KML in Ph + ALL Zdravilo Iclusig® je peroralni zaviralec tirozin-kinaze (TKI) za doziranje enkrat dnevno z učinkovitim delovanjem pri odraslih bolnikih s KML in Ph+ ALL1 ICLUSIG (ponatinib) tablete Za bolnike s kronično mieloidno levkemijo (KML) v kronični, pospešeni ali blastni fazi, ki: • so odporni na dasatinib ali nilotinib ali • ne prenašajo dasatiniba ali nilotiniba in pri katerih nadaljnje zdravljenje z imatinibom ni klinično ustrezno ali • imajo mutacijo T315I Za bolnike z akutno limfoblastno levkemijo s prisotnim kromosomom Philadelphia (Ph+ ALL), ki: • so odporni na dasatinib ali • ne prenašajo dasatiniba in pri katerih nadaljnje zdravljenje z imatinibom ni klinično ustrezno ali • imajo mutacijo T315I SKRAJŠAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA Iclusig 15 mg, 30 mg in 45 mg filmsko obložene tablete Pred predpisovanjem natančno preberite celoten Povzetek glavnih značilnosti zdravila. Samo za strokovno javnost. ▼ Za to zdravilo se izvaja dodatno spremljanje varnosti. Tako bodo hitreje na voljo nove informacije o njegovi varnosti. Zdravstvene delavce naprošamo, da poročajo o katerem koli domnevnem neželenem učinku zdravila. Sestava: Ena filmsko obložena tableta vsebuje 15mg, 30mg ali 45 mg ponatiniba (v obliki ponatinibijevega klorida). Indikacije: Zdravilo Iclusig je indicirano pri odraslih bolnikih s kronično mieloidno levkemijo (KML) v kronični fazi, pospešeni fazi ali blastni fazi, ki so odporni na dasatinib ali nilotinib; ki ne prenašajo dasatiniba ali nilotiniba in pri katerih nadaljnje zdravljenje z imatinibom ni klinično ustrezno; ali ki imajo mutacijo T315I ter pri odraslih bolnikih z akutno limfoblastno levkemijo s prisotnim kromosomom Philadelphia (Ph+ ALL), ki so odporni na dasatinib; ki ne prenašajo dasatiniba in pri katerih nadaljnje zdravljenje z imatinibom ni klinično ustrezno; ali ki imajo mutacijo T315I. Odmerjanje in način uporabe: Terapijo mora uvesti zdravnik z izkušnjami v diagnosticiranju in zdravljenju bolnikov z levkemijo. Med zdravljenjem se lahko bolniku nudi hematološka podpora, če je to klinično indicirano. Pred začetkom zdravljenja s ponatinibom je treba oceniti kardiovaskularni status bolnika, vključno z anamnezo in telesnim pregledom, in aktivno obravnavati kardiovaskularne dejavnike tveganja. Kardiovaskularni status je treba še naprej spremljati in med zdravljenjem s ponatinibom optimizirati zdravljenje z zdravili in podporno zdravljenje stanj, ki prispevajo h kardiovaskularnim tveganjem. Odmerjanje: Priporočeni začetni odmerek ponatiniba je 45 mg enkrat na dan. Potrebno je razmisliti o ukinitvi ponatiniba, če v 3 mesecih ni celovitega hematološkega odgovora. Z zdravljenjem je treba prenehati, če se pojavijo znaki napredovanja bolezni ali v primeru hudih neželenih učinkov. Prilagoditev odmerjanja: tveganje za žilni okluzivni dogodek je verjetno povezano z odmerkom. Zdravljenje z zdravilom Iclusig je treba pri sumu, da se je pri bolniku razvil arterijski ali venski okluzivni dogodek, takoj prekiniti. Ko se dogodek razreši, je treba pri odločitvi o ponovni uvedbi zdravljenja upoštevati oceno koristi in tveganj. Pri obravnavi hematoloških in nehematoloških toksičnosti je treba razmisliti o prilagoditvi ali prekinitvi odmerjanja. V primeru hudih neželenih učinkov je treba z zdravljenjem prekiniti. Prilagajanje odmerka je priporočljivo v primeru nevtropenije ali trombocitopenije, ki nista povezani z levkemijo, pri pankreatitisu in zvišani ravni lipaze/amilaze. Način uporabe: tablete je treba pogoltniti cele, ne sme se jih drobiti ali raztapljati, lahko pa se jih jemlje s hrano ali brez nje. Kontraindikacije: Preobčutljivost na ponatinib ali katero koli pomožno snov. Posebna opozorila in previdnostni ukrepi: Mieoupresita - Zdravilo Iclusig je povezano s hudo trombocitopenijo, nevtropenijo in anemijo. Prve 3 mesece je treba vsaka 2 tedna opraviti pregled celotne krvne slike, nato pa mesečno ali kot je klinično indicirano.Žilna okiuzija - Pojavili so se arterijska in venska tromboza in okluzija, vključno s smrtnim miokardnim infarktom, možgansko kapjo, retinalna žilna okluzija, v nekaterih primerih povezana s trajno okvaro vida ali slepoto, stenozo velikih arterijskih žil v možganih, hudo periferno žilno boleznijo in potrebo po nujnem postopku revaskularizacije. Zdravila Iclusig se ne sme uporabljati pri bolnikih z miokardnim infarktom, predhodno revaskularizacijo ali možgansko kapjo v anamnezi, razen če so možne koristi zdravljenja večje od možnih tveganj. Med zdravljenjem s ponatinibom je treba spremljati znake trombembolije in žilne okluzije in zdravljenje je treba takoj prekiniti, če se pojavi žilna okluzija. V primeru, da se pojavi poslabšanje vida ali zamegljen vid, je treba opraviti oftalmološki pregled (vključno s fundoskopijo). Hipertenzija - Pri zdravljenju z zdravilom Iclusig, se je pojavila z zdravljenjem povezana hipertenzija (vključno s hipertenzivno krizo), ki lahko prispeva k tveganju arterijskih trombotičnih dogodkov. Zato je treba ob vsakem obisku zdravnika spremljati krvni tlak. Zdravljenje z zdravilom Iclusig je treba prekiniti, če hipertenzija ni pod zdravniškim nadzorom. Kongestivno srčno popuščaj - Pojavilo se je smrtno in resno srčno popuščanje ter dogodki, povezani s predhodnimi vaskularni-mi okluzivnimi dogodki. Bolnike je treba spremljati in jih zdraviti, kot je klinično ustrezno, vključno s prekinitvijo zdravljenja z zdravilom Iclusig. Pri bolnikih, pri katerih se razvije resno srčno popuščanje, je treba razmisliti o ukinitvi ponatiniba. Pankreatitis in serumska Ijpaza - Pogostnost pojava pankreatitisa je večja prva 2 meseca uporabe. Prva 2 meseca vsaka 2 tedna preverjajte serumsko lipazo, nato pa periodično. Morda bo treba odmerek prekiniti ali zmanjšati. Če zvišanje ravni lipaz spremljajo abdominalni simptomi, je treba z uporabo zdravila Iclusig prenehati in preveriti, ali ima bolnik pankreatitis. Pri bolnikih s pankreatitisom ali zlorabo alkohola v anamnezi se priporoča previdnost. Bolnike s hudo ali zelo hudo hipertriglic-eridemijo je treba ustrezno obravnavati. Laktoza - Zdravilo Iclusig vsebuje laktozo monohidrat. Bolniki z redkimi dednimi težavami neprenašanja galaktoze, laponsko obliko zmanjšane aktivnosti laktaze ali slabo absorpcijo glukoze-galaktoze ne smejo jemati tega zdravila. Podaljšanje intervala QT -Klinično pomembnih učinkov na interval QT ni mogoče izključiti. Hepatotok-sjičnol - Lahko se zvišajo ravni ALT, AST, bilirubina in alkalne fosfataze. Opazili so jetrno odpoved (vključno s smrtnim izidom). Teste delovanja jeter je treba opraviti pred uvedbo zdravljenja in nato periodično, kot je klinično indicirano. Krvavitev - Pojavili so se smrtni ter resni hemoragični dogodki. Pri resni ali hudi krvavitvi je treba zdravljenje z zdravilom Iclusig prekiniti. Okvara jeter - Pri bolnikih s hudo okvaro jeter se priporoča previdnost. Okvara ledvic - Pri bolnikih z ocenjenim očistkom kreatinina < 50 ml/min ali ledvično boleznijo v zadnjem stadiju se priporoča previdnost. Starejši bolniki - Verjetnost neželenih učinkov je večja. pediatrična populacija - Varnost in učinkovitost zdravila Iclusig pri bolnikih, starih do 18 let, še nista bili dokazani. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Sočasni uporabi zdravila Iclusig z močnimi induktorji CYP3A4 se je treba izogniti; pri sočasni uporabi močnih zaviralcev CYP3A je potrebna previdnost, razmisliti pa je treba tudi o uporabi zdravila Iclusig z začetnim odmerkom 30 mg; potrebna je previdnost pri sočasno uporabljenih substratih P-glikoproteina (P-gp) ali beljakovine rezistence za raka dojke (BCRP). Pri sočasni uporabi ponatiniba z zdravili proti strjevanju krvi pri bolnikih, pri katerih obstaja tveganje za krvavitev, je potrebna previdnost. Plodnost, nosečnost in dojenje: Ženskam v rodni dobi je treba svetovati, da naj v času zdravljenja z zdravilom Iclusig ne zanosijo, moškim pa, da naj v času zdravljenja ne zaplodijo otroka. Med zdravljenjem je treba uporabljati alternativno ali dodatno metodo kontracepcije. Ni zadostnih podatkov o uporabi zdravila Iclusig pri nosečnicah. Študije na živalih so pokazale vpliv na sposobnost razmnoževanja. Če se zdravilo uporablja med nosečnostjo, je treba bolnico obvestiti o možnem tveganju za plod. Z dojenjem je treba med zdravljenjem z zdravilom Iclusig prenehati. Vpliv na sposobnost vožnje in upravljanja s stroji: Pri vožnji ali upravljanju strojev je potrebna previdnost. Neželeni učinki: Zelo pogosti (> 1/10): okužba zgornjih dihal, nespečnost, anemija, zmanjšanje števila trombocitov, zmanjšanje števila nevtrofilcev, zmanjšan apetit, glavobol, omotica, hipertenzija, dispneja, kašelj, bolečine v trebuhu, driska, bruhanje, zaprtje, navzea, zvišanje ravni lipaz, zvišanje ravni alanin aminotransferaze, zvišanje ravni aspartat-ami-notransferaze, izpuščaj, suha koža, bolečine v kosteh, artralgija, mialgija, bolečine v okončinah, bolečine v hrbtu, mišični krči, utrujenost, astenija, periferni edem, pireksija, bolečine. Pogosti (> 1/100 do < 1/10): pljučnica, sepsa, folikulitis, pancitopenija, febrilna nevtropenija, zmanjšanje števila levkocitov, dehidracija, zastajanje tekočine, hipokalciemija, hiperglikemija, hiperurikemija, hipofosfatemija, hipertrigliceridemija, hipokaliemija, zmanjšanje telesne mase, cerebrovaskularni dogodek, cerebralni infarkt, periferna nevropatija, letargija, migrena, hiperestezija, hipoestezija, parestezija, prehodni ishemični napad, zamegljen vid, suhe oči, periorbitalni edem, edem veke, srčno popuščanje, miokardni infarkt, kongestivno srčno popuščanje, bolezen koronarnih arterij, angina pektoris, perikardni izliv, atrijska fibrilacija, zmanjšanje iztisnega deleža, periferna arterijska okluzivna bolezen, periferna ishemija, stenoza periferne arterije, intermitentna klavdikacija, globoka venska tromboza, vročinski oblivi, zariplost, pljučna embolija, plevralni izliv, epistaksa, disfonija, pljučna hipertenzija, pankreatitis, zvišanje amilaz v krvi, gastroezofagealna refluksna bolezen, stomatitis, dispepsija, trebušna distenzija, nelagodje v trebuhu, suha usta, zvišanje ravni bilirubina v krvi, zvišanje ravni alkalne fosfataze v krvi, zvišanje ravni gama-glutamiltransferaze, pruritični izpuščaj, eksfoliativni izpuščaj, eritem, alopecija, pruritis, eksfoliacija kože, nočno potenje, hiperhidroza, petehija, ekhimoza, boleča koža, eksfoliativni dermatitis, mišično-skeletne bolečine, bolečine v vratu, mišično-skeletne bolečine v prsnem košu, erektilna disfunkcija, mrzlica, gripi podobna bolezen, nekardiogena bolečina v prsnem košu, tipljiv vozlič, obrazni edem. Občasni (> 1/1000 do < 1/100): sindrom tumorske lize, cerebralna arterijska stenoza, tromboza mrežnične vene, okluzija mrežnične vene, okluzija mrežnične arterije, okvara vida, ^ miokardna ishemija, akutni koronarni sindrom, kardialno nelagodje, «— ishemična kardiomiopatija, spazem koronarnih arterij, disfunkcija levega jm prekata, atrijska undulacija, slaba periferna cirkulacija, vranični infarkt, ¡L venska embolija, venska tromboza, hipertenzivna kriza, krvavitev v želodcu, .3, hepatotoksičnost, odpoved jeter, zlatenica. Režim izdaje zdravila: ^ Predpisovanje in izdaja zdravila je le na recept. Imetnik dovoljenja za ® promet z zdravilom: ARIAD Pharma Ltd., Riverbridge House, Guildford JS Road, Leatherhead, Surrey KT22 9AD, Velika Britanija. Zadnja revizija Oi besedila: marec 2016. Informacija pripravljena: april 2016. ® Podrobnejše informacije o zdravilu Iclusig so na voljo pri predstavniku imetnika dovoljenja za promet z zdravilom: Angelini Pharma d.o.o., Koprska 3 ulica 108A, 1000 Ljubljana, Tel.: +386 1 544 65 79, E-pošta: info@angelini.si ^ ANGELINI Predstavnik: Angelini Pharma d.o.o. Koprska ulica 108 A, Ljubljana ' / PRVA REGISTRIRANA TERAPIJA V 2. LINIJI ZA ZDRAVLJENJE ADENOKARCINOMA ŽELODCA ALI GASTRO-EZOFAGEALNEGA PREHODA1 IMW USPOSO CYRAMZA" (ramucirumab) UKREPAJTE ZDAJ MBE, ZKUSNJE Skrajšan povzetek glavnih značilnosti zdravila ▼ Za to zdravilo se izvaja dodatno spremljanje varnosti. Tako bodo hitreje na voljo nove informacije o njegovi varnosti. Zdravstvene delavce naprošamo, da poročajo o katerem koli domnevnem neželenem učinku zdravila. Cyramza 10 mg/ml koncentrat za raztopino za infundiranje En mililiter koncentrata za raztopino za infundiranje vsebuje 10 mg ramucirumaba. Ena 10-mililitrska viala vsebuje 100 mg ramucirumaba. Terapevtske indikacije Zdravilo Cyramza je v kombinaciji s paklitakselom indicirano za zdravljenje odraslih bolnikov z napredovalim rakom želodca ali adenokarcinomom gastro-ezofagealnega prehoda z napredovalo boleznijo po predhodni kemoterapiji, ki je vključevala platino in fluoropirimidin. Monoterapija z zdravilom Cyramza je indicirana za zdravljenje odraslih bolnikov z napredovalim rakom želodca ali adenokarcinomom gastro-ezofagealnega prehoda z napredovalo boleznijo po predhodni kemoterapiji s platino ali fluoropi-rimidinom, za katere zdravljenje v kombinaciji s paklitakselom ni primerno. Zdravilo Cyramza je v kombinaciji s shemo FOLFIRI indicirano za zdravljenje odraslih bolnikov z metastatskim kolorektalnim rakom (mCRC), z napredovanjem bolezni ob ali po predhodnem zdravljenju z bevacizumabom, oksaliplatinom in fluoropirimidinom. Zdravilo Cyramza je v kombinaciji z docetakselom indicirano za zdravljenje odraslih bolnikov z lokalno napredovalim ali metastatskim nedrobnoceličnim pljučnim rakom, z napredovanjem bolezni po kemoterapiji na osnovi platine. Odmerjanje in način uporabe Zdravljenje z ramucirumabom morajo uvesti in nadzirati zdravniki z izkušnjami v onkologiji. Odmerjanje Rak želodca in adenokarcinom gastro-ezofagealnega prehoda Priporočeni odmerek ramucirumaba je 8 mg/kg 1. in 15. dan 28-dnevnega cikla, pred infuzijo paklitaksela. Priporočeni odmerek paklitaksela je 80 mg/m2 in se daje z intravenskim infundiranjem, ki traja približno 60 minut, 1., 8. in 15. dan 28-dnevnega cikla. Pred vsakim infundiranjem paklitaksela je treba pri bolnikih pregledati celotno krvno sliko in izvide kemičnih preiskav krvi, da se oceni delovanje jeter. Priporočeni odmerek ramucirumaba kot monoterapije je 8 mg/kg vsaka 2 tedna. Kolorektalni rak Priporočeni odmerek ramucirumaba je 8 mg/kg vsaka 2 tedna, dan z intravensko infuzijo pred dajanjem sheme FOLFIRI. Pred kemoterapijo je treba bolnikom odvzeti kri za popolno krvno sliko. Nedrobnocelični pljučni rak (NSCLC) Priporočeni odmerek ramucirumaba je 10 mg/kg na 1. dan 21-dnevnega cikla, pred infuzijo docetaksela. Priporočeni odmerek docetaksela je 75 mg/m2, dan z intravensko infuzijo v približno 60 minutah na 1. dan 21-dnevnega cikla. Premedikacija Pred infundiranjem ramucirumaba je priporočljiva premedikacija z antagonistom histaminskih receptorjev H1. Način uporabe Po redčenju se zdravilo Cyramza daje kot intravenska infuzija v približno 60 minutah. Zdravila ne dajajte v obliki intravenskega bolusa ali hitre intravenske injekcije. Da boste dosegli zahtevano trajanje infundiranja približno 60 minut, največja hitrost infundiranja ne sme preseči 25 mg/minuto, saj morate sicer podaljšati trajanje infundiranja. Bolnika je med infundiranjem treba spremljati glede znakov reakcij, povezanih z infuzijo, zagotoviti pa je treba tudi razpoložljivost ustrezne opreme za oživljanje. Kontraindikacije Pri bolnikih z NSCLC je ramucirumab kontraindiciran, kjer gre za kavitacijo tumorja ali prepletenost tumorja z glavnimi žilami. Posebna opozorila in previdnostni ukrepi Trajno prekinite zdravljenje z ramucirumabom pri bolnikih, pri katerih se pojavijo resni arterijski trombembolični dogodki, gastrointestinalne perforacije, krvavitev stopnje 3 ali 4, če zdravstveno pomembne hipertenzije ni mogoče nadzirati z antihipertenzivnim zdravljenjem ali če se pojavi fistula, raven beljakovin v urinu > 3 g/24 ur ali v primeru nefrotskega sindroma. Pri bolnikih z neuravnajio hipertenzijo zdravljenja z ramucirumabom ne smete uvesti, dokler oziroma v kolikor obstoječa hipertenzija ni uravnana. Pri bolnikih s ploščatocelično histologijo obstaja večje tveganje za razvoj resnih pljučnih krvavitev. Če se pri bolniku med zdravljenjem razvijejo zapleti v zvezi s celjenjem rane, prekinite zdravljenje z ramucirumabom, dokler rana ni povsem zaceljena. V primeru pojava stomatitisa je treba takoj uvesti simptomatsko zdravljenje. Pri bolnikih, ki so prejemali ramucirumab in docetaksel za zdravljenje napredovalega NSCLC z napredovanjem bolezni po kemoterapiji na osnovi platine, so opazili trend manjše učinkovitosti z naraščajočo starostjo. Plodnost, nosečnost in dojenje Ženskam v rodni dobi je treba svetovati, naj se izognejo zanositvi med zdravljenjem z zdravilom Cyramza in jih je treba seznaniti z možnim tveganjem za nosečnost in plod. Ni znano, ali se ramucirumab izloča v materino mleko. Neželeni učinki Zelo pogosti(> 1/10) nevtropenija, levkopenija, trombocitopenija, hipoalbuminemija, hipertenzija, epistaksa, gastrointestinalne krvavitve, stomatitis, driska, proteinurija, utrujenost/astenija, periferni edem, bolečina v trebuhu. Pogosti(> 1/100do< 1/10)hipokaliemija, hiponatriemija, glavobol. Rok uporabnosti 3 leta Posebna navodila za shranjevanje Shranjujte v hladilniku (2 °C-8 °C). Ne zamrzujte. Vialo shranjujte v zunanji ovojnini, da zagotovite zaščito pred svetlobo. Pakiranje 2 viali z 10 ml IMETNIK DOVOLJENJA ZA PROMET Z ZDRAVILOM Eli Lilly Nederland B.V., Papendorpseweg 83, 3528 BJ Utrecht, Nizozemska DATUM ZADNJE REVIZIJE BESEDILA 25.01.201 6 Režim izdaje: Predpisovanje in izdaja zdravila je le na recept, zdravilo pa se uporablja samo v bolnišnicah. Pomembno obvestilo: Pričujoče gradivo je namenjeno samo za strokovno javnost. Zdravilo Cyramza se izdaja le na recept. Pred predpisovanjem zdravila Cyramza vas vljudno prosimo, da preberete celotni Povzetek glavnih značilnosti zdravila Cyramza. Podrobnejše informacije o zdravilu Cyramza in o zadnji reviziji besedila Povzetka glavnih značilnosti zdravila so na voljo na sedežu podjetja Eli Lilly (naslov podjetja in kontaktni podatki spodaj) in na spletni strani European Medicines Agency (EMA): www.ema.europa.eu. in na spletni strani European Commission http://ec.europa.eu/health/documents/community-register/html/alfregister.htm. Eli Lilly farmacevtska družba, d.o.o., Dunajska cesta 1 67, 1 000 Ljubljana, telefon: (01) 5800 01 0, faks: (01) 5691 705 Referenca: 1. Cyramza, Povzetek glavnih značilnosti zdravila, zadnja odobrena verzija. EERAM0001 0a, 12.02.2016. IRBITUX CETUXIMAB See the difference Individualizirano zdravljenje za bolnike z metastatskim kolorektalnim rakom Merck Serono Onkologija | Ključ je v kombinaciji Erbitux 5 mg/ml raztopina za infundiranje Skrajšan povzetek glavnih značilnosti zdravila Sestava: En ml raztopine za infundiranje vsebuje 5 mg cetuksimaba in pomožne snovi. Cetuksimab je himerno monoklonsko IgGi protitelo. Terapevtske indikacije: Zdravilo Erbitux je indicirano za zdravljenje bolnikov z metastatskim kolorektalnim rakom z ekspresijo receptorjev EGFR in nemutiranim tipom RAS v kombinaciji s kemoterapijo na osnovi irinotekana, kot primarno zdravljenje v kombinaciji s FOLFOX in kot samostojno zdravilo pri bolnikih, pri katerih zdravljenje z oksaliplatinom in zdravljenje na osnovi irinotekana ni bilo uspešno in pri bolnikih, ki ne prenašajo irinotekana. Zdravilo Erbitux je indicirano za zdravljenje bolnikov z rakom skvamoznih celic glave in vratu v kombinaciji z radioterapijo za lokalno napredovalo bolezen in v kombinaciji s kemoterapijo na osnovi platine za ponavljajočo se in/ali metastatsko bolezen. Odmerjanje in način uporabe: Zdravilo Erbitux pri vseh indikacijah infundirajte enkrat na teden. Pred prvo infuzijo mora bolnik prejeti premedikacijo z antihistaminikom in kortikosteroidom najmanj 1 uro pred uporabo cetuksimaba. Začetni odmerek je 400 mg cetuksimaba na m2 telesne površine. Vsi naslednji tedenski odmerki so vsak po 250 mg/m2. Kontraindikacije: Zdravilo Erbitux je kontraindicirano pri bolnikih z znano hudo preobčutljivostno reakcijo (3. ali 4. stopnje) na cetuksimab. Kombinacija zdravila Erbitux s kemoterapijo, ki vsebuje oksaliplatin, je kontraindicirana pri bolnikih z metastatskim kolorektalnim rakom z mutiranim tipom RAS ali kadar status RAS ni znan. Posebna opozorila in previdnostni ukrepi: Pojav hude reakcije, povezane z infundiranjem, zahteva takojšnjo in stalno ukinitev terapije s cetuksimabom. Če pri bolniku nastopi blaga ali zmerna reakcija, povezana z infundiranjem, lahko zmanjšate hitrost infundiranja. Priporočljivo je, da ostane hitrost infundiranja na nižji vrednosti tudi pri vseh naslednjih infuzijah. Če se pri bolniku pojavi kožna reakcija, ki je ne more prenašati, ali huda kožna reakcija (> 3. stopnje po kriterijih CTCAE), morate prekiniti terapijo s cetuksimabom. Z zdravljenjem smete nadaljevati le, če se je reakcija izboljšala do 2. stopnje. Če ugotovite intersticijsko bolezen pljuč, morate zdravljenje s cetuksimabom prekiniti, in bolnika ustrezno zdraviti. Zaradi možnosti pojava znižanja nivoja elektrolitov v serumu se pred in periodično med zdravljenjem s cetuksimabom priporoča določanje koncentracije elektrolitov v serumu. Pri bolnikih, ki prejemajo cetuksimab v kombinaciji s kemoterapijo na osnovi platine, obstaja večje iMerckSerono tveganje za pojav hude nevtropenije. Takšne bolnike je potrebno skrbno nadzorovati. Pri predpisovanju cetuksimaba je treba upoštevati kardiovaskularno stanje in indeks zmogljivosti bolnika in sočasno dajanje kardiotoksičnih učinkovin kot so fluoropirimidini. Če je diagnoza ulcerativnega keratitisa potrjena, je treba zdravljenje s cetuksimabom prekiniti ali ukiniti. Cetuksimab je treba uporabljati previdno pri bolnikih z anamnezo keratitisa, ulcerativnega keratitisa ali zelo suhih oči. Cetuksimaba ne uporabljajte za zdravljenje bolnikov s kolorektalnim rakom, če imajo tumorje z mutacijo RAS ali pri katerih je tumorski status RAS neznan. Interakcije: Pri kombinaciji s fluoropirimidini se je v primerjavi z uporabo fluoropirimidinov, kot monoterapije, povečala pogostnost srčne ishemije, vključno z miokardnim infarktom in kongestivno srčno odpovedjo ter pogostnost sindroma dlani in stopal. V kombinaciji s kemoterapijo na osnovi platine se lahko poveča pogostnost hude levkopenije ali hude nevtropenije. V kombinaciji s kapecitabinom in oksaliplatinom (XELOX) se lahko poveča pogostnost hude driske. Neželeni učinki: Zelo pogosti (> 1/10): hipomagneziemija, povečanje ravni jetrnih encimov, kožne reakcije, blage ali zmerne reakcije povezane z infundiranjem, mukozitis, v nekaterih primerih resen. Pogosti (> 1/100 do < 1/10): dehidracija, hipokalciemija, anoreksija, glavobol, konjunktivitis, driska, navzeja, bruhanje, hude reakcije povezane z infundiranjem, utrujenost. Posebna navodila za shranjevanje: Shranjujte v hladilniku (2 °C - 8 °C). Pakiranje: 1 viala z 20 ml ali 100 ml raztopine. Način in režim izdaje: Izdaja zdravila je le na recept-H. Imetnik dovoljenja za promet: Merck KGaA, 64271 Darmstadt, Nemčija. Datum zadnje revizije besedila: november 2014. Pred predpisovanjem zdravila natančno preberite celoten Povzetek glavnih značilnosti zdravila. Samo za strokovno javnost. Podrobnejše informacije so na voljo pri predstavniku imetnika dovoljenja za promet z zdravilom: Merck d.o.o., Ameriška ulica 8, 1000 Ljubljana, tel.: 01 560 3810, faks: 01 560 3830, el. pošta: info@merck.si www.merckserono.net www.Erbitux-international.com I Merck Serono is a division of Merck .MERCK I I KER TEGA NE MORETE NAREDITI... JE TU ZYDELIG®V Prvo zdravilo v svojem razredu, peroralni zaviralec PI3K512 Referenci: 1. Herman SEM, Gordon AL, Wagner AJ in sod. Phosphatidylinositol 3-kinase-6 inhibitor CAL-101 shows promising preclinical activity in chronic lymphocytic leukemia by antagonizing intrinsic and extrinsic cellular survival signals. Blood. 2010;116:2078-2088. 2. Povzetek glavnih značilnosti zdravila Zydelig, junij 2016T ZYDELIG je indiciran v kombinaciji z rituksimabom za zdravljenje odraslih bolnikov s kronično limfocitno levkemijo (KLL):2 • ki so se pred tem vsaj enkrat zdravili, ali • za nadaljevanje zdravljenja pri bolnikih z delecijo 17p ali mutacijo TP53, pri katerih kemoimunoterapija ni primerna in so se že začeli zdraviti z zdravilom Zydelig kot zdravilom prve izbire. ZYDELIG je indiciran kot monoterapija za zdravljenje odraslih bolnikov s folikularnim limfomom (FL), neodzivnim na dve predhodni obliki zdravljenja.2 SkrqjSani povzetek glavnih značilnosti zdravila Zydelig m dHavca ruprofamo, di poročajo o IhE ZDRAVILA: Zydollg 100 mg filmsko obložene tablete ali Zydellg 1E0 mg filmsko obložene tablet. KAKOVOSTNA IN KOLIČINSKA SESTAVA: Ena filmske obložena tableta vsebuje ali 100 mg ali ISO mg Idelallziba. Za celoten seznam pomožnih snovi glejte SrnPC za zdravilo Zydellg 100 ali 150 mg filmsko obložene tablete. TERAPEVTSKE IM9IKACUE: Zdravilo Zydellg Je Indldrano v kombinaciji z rituksimabom za zdravljenje odraslih bolnikov s krortino limfocitno levkemijo (KLL): H so se pred tem vsaj enkrat zdravili, aH za zdravljenja'' ODMERJAM* IN NAČIN UPORABEfaravljerje zzdravitom zyOal!g mt^rad^ zdrSnlt"? izkuinlarnl pri uporaN^rairti prati rajaT^n^a^f Prtooroaln! odmerek zdravila Zydellg Je 150 mg peroralno dvakratna dan. Z zdravljenjem Je treba nadaJJevatl do napredovanja bolezni ali nesprejemljive toksičnosti. Ce bolnik bpusti odmerek baoSm^S -Zdravljenje ustaviT« n pojavi pov^ireiCH^^ferezrl!™ 4? stoo° ia° amlnotransfaraza [AST] > 5 * zgornja meja normalne vrednosti [ULN, uppar Hnlit o t normalJ>. Ko se vrednosti vrnejo na 1. ali nižjo stopnjo (ALT/AST t 3 X IILN), se lahko zdravljenje nadaljuje z odmerkom 100 mg dvakrat na dan. če se ta dogodek ne pojavi ponovno, se lahko odmerek po presoji lečečega zdravnika ponovno poveča na 150 mg nadaljuje z jemanjem 100 mg dvakrat na dan. Drista/toAfe* Zdravljenja z zdravitem Zydelig je toba začasno ^vi'i driskaAolit!« id 4, stcpnja^OH^rcdg^tolitil vrne ne 1. ali nižjo stopnjo, se lahko zdravljenje nadaljuje z odmerkom 100 mg dvekret na «en. Ce se driska/koliti* ne ponovi, se lehko odmerek po presoji lečečeaa zdravnike ponovno poveča na 150 mg dvakrat na dan Pnavmonltls - Zdravljenja z zdravilom Zydollg ja treba pri sumu na pnevmonlds začasno ustaviti. Ko je pnevmonltls minil In & jo ponovno zdravljene ustrezno, se lahko razrasli o nadaljevanju zdravljenja z odmerkom 100 mg dvakrat na dan. izpufi^-Zdravljenje z zdravilom Zydella Je treba začasno ustaviti, iesepojavllzpuKaJ3.all4.stopnJe.Kose IzpuKaj vrne na 1. ali nižjo stopnjo, se lahko zdravljenje nadaljuje z odmerkom 100 mgdvakrat na dan. Ce se IzpuitaJ ne ponovi, se lahko odmerek po presoji lečečega zdravnika ponovno poveča na lbO mg dvakrat na dan. Nmtmoenija: Zdravljenja z zdravilom Zydolig jg trn Da začasno ustaviti pri bolnikih z absolutnim Številom nevtrofilcev(ANOpOd500ne mm=.ANCje treba spremljati najmanj en k re t tedensko, dokler nTANC s SOOna mm', neto se zdravljenje lahko nadaljuj« »100 • • - • .........- - .......staram boMki - Pri starejših bolnikih (starost i 65 let) o<..... .......... Zato w pri dajanju zdravila Zydelig tej populaciji priporoča previdnost in intenzivnejše spremljanja neželenih učinkov. Podiatritna oopuhoja - Varnost in ufinkovitost zdravila Zydeiig pri otrocih, starih do 18 let Se nista bili dokazani. Podatkov nI ne voljo. N»an unorahe: zdravilo Zydeiifl le za peroralno uporabo- Bolnikom je treba neroStI, da naj pogoltnejo celo tableto. Filmsko obložene tablete se ne smo žvečiti ali zdrobiti. Filmsko obložena tableta se lahko jemlje s hrano ali brez nje. KONTRAINDIKACIJE: Preobčutljivost okuOaniMnL sta pitačntiTla^p^ profllakso za PJP. Pri bolnikih je treba ves čas zdravljena spremljati respli-etorne znake In simptome. Bolnike je treba opozoriti, da morejo nemudoma poroiatl o novih .......h. Troba je Izv^atl redno klinične In laboratorijske preglede za okužbo s CMV. Pri znakih okužbo ali vlramlje je treba zdravljene z zdravilom Zydollg ........ >rl bolnikih z ANC pod 1.000 na mm1 pa najmanj enkrat tedensko (glejte poglavje a - V kliničnih Kudijah z idelalizibom so opazili povečanja ravni ALT in AST 3, in 4. stopnje O 5 x ULN), Ti laboratorijski izsledki, ki so jih običajno opazili ponovnega pojava. Pri vseh bolnikih Jo troba prve 3 mesece zdravljenja vsaka2tednaspramljati vra*wsti ALT, AST In skupnega bilirubina, na............. " ' " iti ALT In/all AST 2. ali vlije stopnje Jb tn..... ...................." "" ...... in toksični epidermalni nekrolizi (TEN) s smrtnimi izidi, koso idelalizib dajali sočasno z drugimi zdravili, po nemudoma prekiniti in bolnika ustrezno zdraviti, Induktorii P/P3A - izpostavljenost idalalizibu se lahko zmenila pri sočasni uporabi z induktorji CVP3A. kot so rifarreicin, fanitoin, Sentjanžovka (Hypertcum parforatorri) ali karbamazepln. Ker lahko zmanjšanje koncentracij Idelallziba v plazmi zmanjša ufinkovitost, se Je treba sočasnemu dajanju zdravila Zydellg z zmernimi ali močnimi Induktorji CVP3A Izogniti. Substrati CYP3A - Primarni presnovek Idelallziba. GS 583117, Je močan zaviralec CVP3A4. Zato lahko klelallzlb medsebojno deluje z z*avllL ki Jih CYP3A presnavlja. kar lahko povzroči povečane koncentracije drugih zdravil v serumu. Pri sočasni uporabi Idelallziba z dmglml zdravili Je treba upoitovati povzetek glavnih značilnosti zdravila za druga zdravila glede priporočil o sočasni uporabi z zavirala CVP3A4. Sočasnemu zdravljenju z idelalizibom in substrati CYP3A. slmvastatin, slldenafíl^mldazotam, trt^olam), se je treba Izogniti In uporabiti alternativna zdravllMd so' manj občutljiva na zSrtranJe'cVP3A4. če"jo mogoče. Ji 65 let): Med starejšimi in mlajšimi bolniki niso opazili pomembnih razlik v varnosti in učinkovitosti. Okvara jeter: Pri bolnikih z jetrno okvaro razreda A in B po Child-Pughu prilagoditev odmerka ni potrebna; pri bolnikih z okvaro razreda C sunitinib ni bil preizkušen, zato njegova uporaba ni priporočljiva. Okvara ledvic: Prilagajanje začetnega odmerka ni potrebno, nadaljnje prilagajanje odmerka naj temelji na varnosti in prenašanju pri posameznem bolniku. Način uporabe: Zdravilo Sutent se uporablja peroralno, bolnik ga lahko vzame s hrano ali brez nje. Če pozabi vzeti odmerek, ne sme dobiti dodatnega, temveč naj vzame običajni predpisani odmerek naslednji dan. Kontraindikacije: Preobčutljivost na zdravilno učinkovino ali katerokoli pomožno snov. Posebna opozorila in previdnostni ukrepi: Bolezni kože in tkiv: obarvanje kože, gangrenozna pioderma (običajno izgine po prekinitvi zdravljenja), hude kožne reakcije (multiformni eritem (EM), Stevens-Johnsonov sindrom (SJS) in toksična epidermalna nekroliza (TEN)). Če so prisotni znaki EM, SJS ali TEN, je treba zdravljenje prekiniti. Krvavitve v prebavilih, dihalih, sečilih, možganih; najpogosteje epistaksa; krvavitve tumorja, včasih s smrtnim izidom. Pri bolnikih, ki se sočasno zdravijo z antikoagulanti, se lahko redno spremlja celotna krvna slika (trombociti), koagulacijski faktorji (PT / INR) in opravi telesni pregled. Bolezni prebavil: poleg diareje, navzee/bruhanja, bolečine v trebuhu, dispepsije, stomatitisa/bolečine v ustih in ezofagitisa tudi hudi zapleti (včasih s smrtnim izidom), vključno z gastrointestinalno perforacijo. Hipertenzija: pri bolnikih s hudo hipertenzijo, ki je ni mogoče urediti z zdravili, je priporočljivo začasno prenehanje zdravljenja. Hematološke bolezni: zmanjšanje števila nevtrofilcev, trombocitov, anemija. Bolezni srca in ožilja: srčno-žilni dogodki, vključno s srčnim popuščanjem, kardiomiopatijo, miokardno ishemijo in miokardnim infarktom, v nekaterih primerih s smrtnim izidom; sunitinib povečuje tveganje za pojav kardiomiopatije; previdna uporaba pri bolnikih s tveganjem za te dogodke, ali ki so te dogodke imeli v preteklosti. Podaljšanje intervala QT: previdna uporaba pri bolnikih z znano anamnezo podaljšanja intervala QT, tistih, ki jemljejo antiaritmike ali zdravila, ki lahko podaljšajo interval QT, in tistih z relevantno, že obstoječo srčno boleznijo, bradikardijo ali elektrolitskimi motnjami. Venski in arterijski trombembolični dogodki; arterijski včasih s smrtnim izidom. Trombotična mikroangiopatija (TMA): TMA, vključno s trombotično trombocitopenično purpuro in hemolitično-uremičnim sindromom, v nekaterih primerih z odpovedjo ledvic ali smrtnim izidom. Dogodki na dihalih: dispneja, plevralni izliv, pljučna embolija ali pljučni edem; redki primeri s smrtnim izidom. Moteno delovanje ščitnice: bolnike je treba med zdravljenjem rutinsko spremljati glede delovanja ščitnice vsake 3 mesece. Pankreatitis, tudi resni primeri s smrtnim izidom. Hepatotoksičnost, nekateri primeri s smrtnim izidom. Holecistitis, vključno z akalkuloznim in emfizemskim holecistitisom. Delovanje ledvic: primeri zmanjšanega delovanja ledvic, odpovedi ledvic in/ali akutne odpovedi ledvic, v nekaterih primerih s smrtnim izidom. Fistula: če nastane fistula, je treba zdravljenje s sunitinibom prekiniti. Oteženo celjenje ran: pri bolnikih, pri katerih naj bi bil opravljen večji kirurški poseg, je priporočljiva začasna prekinitev zdravljenja s sunitinibom. Osteonekroza čeljustnic: pri sočasnem ali zaporednem dajanju zdravila Sutent in intravenskih bisfosfonatov je potrebna previdnost; invazivni zobozdravstveni posegi predstavljajo dodatni dejavnik tveganja. Preobčutljivost/angioedem. Motnje okušanja. Konvulzije: obstajajo poročila, nekatera s smrtnim izidom, o preiskovancih s konvulzijami in radiološkimi znaki sindroma reverzibilne posteriorne levkoencefalopatije. Sindrom lize tumorja, v nekaterih primerih s smrtnim izidom. Okužbe: hude okužbe z ali brez nevtropenije (okužbe dihal, sečil, kože in sepsa), vključno z nekaterimi s smrtnim izidom; redki primeri nekrotizitajočega fasciitisa, vključno s prizadetostjo presredka, ki so bili včasih smrtni. Hipoglikemija: če se pojavi simptomatska hipoglikemija, je treba zdravljenje s sunitinibom začasno prekiniti. Pri sladkornih bolnikih je treba redno preverjati raven glukoze v krvi in, če je treba, prilagoditi odmerek antidiabetika. Medsebojno delovanje z drugimi zdravili: (Študije so izvedli le pri odraslih.) Zdravila, ki lahko zvečajo koncentracijo sunitiniba v plazmi (ketokonazol, ritonavir, itrakonazol, eritromicin, klaritromicin ali sok grenivke). Zdravila, ki lahko zmanjšajo koncentracijo sunitiniba v plazmi (deksametazon, fenitoin, karbamazepin, rifampin, fenobarbital, Hypericum perforatum oz. šentjanževka). Plodnost, nosečnost in dojenje: Zdravila Sutent ne smemo uporabljati med nosečnostjo in tudi ne pri ženskah, ki ne uporabljajo ustrezne kontracepcije, razen če možna korist odtehta možno tveganje za plod. Ženske v rodni dobi naj med zdravljenjem z zdravilom Sutent ne zanosijo. Ženske, ki jemljejo zdravilo Sutent, ne smejo dojiti. Neklinični izsledki kažejo, da lahko zdravljenje s sunitinibom poslabša plodnost samcev in samic. Vpliv na sposobnost vožnje in upravljanja s stroji: Sutent lahko povzroči omotico. Neželeni učinki: Najbolj resni neželeni učinki (nekateri s smrtnim izidom) so: odpoved ledvic, srčno popuščanje, pljučna embolija, gastrointestinalna perforacija in krvavitve (npr. v dihalih, prebavilih, tumorju, sečilih in možganih). Najpogostejši neželeni učinki (ki so se pojavili v registracijskih preskušanjih) so: zmanjšan tek, motnje okušanja, hipertenzija, utrujenost, prebavne motnje (npr. diareja, navzea, stomatitis, dispepsija in bruhanje), sprememba barve kože in sindrom palmarno-plantarne eritrodisestezije. Med najbolj pogostimi neželenimi učinki so tudi hematološke motnje (nevtropenija, trombocitopenija, anemija in levkopenija). Ostali zelo pogosti (> 1/10) neželeni učinki so: hipotiroidizem, nespečnost, omotica, glavobol, dispneja, epistaksa, kašelj, bolečina v trebuhu, zaprtje, izpuščaj, spremembe barve las, suha koža, bolečine v udih, artralgija, bolečine v hrbtu, vnetje sluznice, edem, pireksija. Način in režim izdaje: Predpisovanje in izdaja zdravila je le na recept, zdravilo pa se uporablja samo v bolnišnicah. Izjemoma se lahko uporablja pri nadaljevanju zdravljenja na domu ob odpustu iz bolnišnice in nadaljnjem zdravljenju. Imetnik dovoljenja za promet: Pfizer Limited, Ramsgate Road, Sandwich, Kent, CT13 9NJ, Velika Britanija. Datum zadnje revizije besedila: 25.02.2016 Pred predpisovanjem se seznanite s celotnim povzetkom glavnih značilnosti zdravila. Pfizer Luxembourg SARL, GRAND DUCHY OF LUXEMBOURG 51, Avenue IF. Kennedy, L-1855 PFIZER, Podružnica Ljubljana, Letališka cesta 3c, 1000 Ljubljana, Slovenija