CABOMETYX8 (kabozantinib) tablete life : 60 mg 40 mg 20 mg CABOMETYX® pomembno izboljša PFS, OS in ORR v drugi liniji zdravljenja napredovalega karcinoma ledvičnih celic1 ORR: objektivna stopnja odziva; OS: celokupno preživetje; PFS: preživetje brez napredovanja bolezni Referenci: 1. Choueiri TK, Escudier B, ;P6wles T, et al. Cabozantinib versus evero|3jmus in advanced renal cell c phase 3 triaf^The Lancet Onco^gy. 2016;17(7):917-27. 2. Povzetek glavnih značilnosti zdravila Cabometyx. Skrajšan povzetek glavnih značilnosti zdravila CABOMETYX 20 mg filmsko obložene tablete CABOMETYX 40 mg filmsko obložene tablete CABOMETYX 60 mg filmsko obložene tablete (kabozantinib) TERAPEVTSKE INDIKACIJE Zdravljenje napredovalega karcinoma ledvičnih celic (KLC) pri predhodno nezdravljenih odraslih bolnikih s srednje ugodnim ali slabim prognostičnim obetom ter pri odraslih bolnikih po predhodnem zdravljenju, usmerjenem v vaskularni endotelijski rastni faktor (VEGF). V monoterapiji zdravljenje hepatocelularnega karcinoma (HCK) pri odraslih bolnikih, ki so se predhodno že zdravili s sorafenibom. ODMERJANJE IN NAČIN UPORABE Pri bolnikih s KLC in HKC je priporočeni odmerek 60 mg enkrat na dan Zdravljenje je treba nadaljevati tako dolgo, dokler bolnik več nima kliničnih koristi od terapije ali do pojava nesprejemljive toksičnosti. Pri sumu na neželene reakcije na zdravilo bo morda treba zdravljenje začasno prekiniti in/ali zmanjšati odmerek. Če je treba odmerek zmanjšati se priporoča zmanjšanje na 40 mg na dan in nato na 20 mg na dan. Prekinitev odmerka se priporoča pri obravnavi toksičnosti 3. ali višje stopnje po CTCAE (common terminology criteria for adverse events) ali nevzdržni toksičnosti 2. stopnje. Zmanjšanje odmerka se priporoča za dogodke, ki bi lahko čez čas postali resni ali nevzdržni. V primeru pojava neželenih učinkov 1. in 2. stopnje, ki jih bolnik prenaša in jih je možno enostavno obravnavati prilagoditev odmerjanja običajno ni potrebna. Treba je uvesti podporno oskrbo. V primeru pojava neželenih učinkov 2. stopnje, ki jih bolnik ne prenaša in jih ni mogoče obravnavati z zmanjšanjem odmerka ali podporno oskrbo, je treba zdravljenje prekiniti, dokler neželeni učinki ne izzvenijo do < 1. stopnje, uvesti podporno oskrbo in razmisliti o ponovni uvedbi zdravljenja z zmanjšanim odmerkom. V primeru pojava neželenih učinkov 3. stopnje je treba zdravljenje prekiniti, dokler neželeni učinki ne izzvenijo do < 1. stopnje, uvesti podporne oskrbo in ponovno uvesti zdravljenje z zmanjšanim odmerkom. V primeru pojava neželenih učinkov 4. stopnje je treba zdravljenje prekiniti, uvesti ustrezno zdravniško oskrbo, in če neželeni učinki izzvenijo do < 1. stopnje, ponovno uvesti zdravljenje z zmanjšanim odmerkom. Če neželeni učinki ne izzvenijo, je treba trajno prenehati z uporabo zdravila. Pri bolnikih z blago ali zmerno ledvično okvaro je treba kabozantinib uporabljati previdno Uporaba se ne priporoča pri bolnikih s hudo ledvično okvaro. Pri bolnikih z blago okvaro jeter odmerka ni treba prilagajati. Pri bolnikih z zmerno okvaro jeter (Child Pugh B) priporočil za odmerjanje ni možno podati. Pri teh bolnikih je priporočljivo skrbno spremljanje celokupne varnosti. Pri bolnikih s hudo okvaro jeter (Child Pugh C) uporaba kabozantiniba ni priporočljiva. Način uporabe: Tablete je treba pogoltniti cele in jih ni dovoljeno drobiti. Bolnikom je treba naročiti, naj vsaj 2 uri pred uporabo zdravila in 1 uro po tem ničesar ne jedo. KONTRAINDIKACIJE Preobčutljivost na učinkovino ali katero koli pomožno snov. POSEBNA OPOZORILA IN PREVIDNOSTNI UKREPI Večina dogodkov se pojavi zgodaj v teku zdravljenja, zato mora zdravnik bolnika v prvih 8 tednih zdravljenja skrbno spremljati, da oceni, ali je treba odmerek prilagoditi. Dogodki, ki se običajno pojavijo zgodaj, vključujejo hipokalciemijo, hipokaliemijo, trombocitopenijo, hipertenzijo, sindrom palmarno-plantarne eritrodisestezije (PPES), proteinurijo in gastrointestinalne dogodke (bolečine v trebuhu, vnetje sluznice, zaprtje, driska, bruhanje). Pred uvedbo zdravljenja s kabozantinibom je priporočljivo izvesti preiskave delovanja jeter (ALT, AST in bilirubin) vrednosti skrbno spremljati med zdravljenjem in po potrebi prilagoditi odmerek. Bolnike je treba spremljati glede znakov in simptomov jetrne encefalopatije. Bolnike, ki imajo vnetno bolezen črevesja (npr. Crohnovo bolezen, ulcerozni kolitis, peritonitis, divertikulitis ali apendicitis), ki imajo tumorsko infiltracijo prebavil ali so imeli pred posegom na prebavilih zaplete (zlasti v povezavi z zapoznelim ali nepopolnim celjenjem), je treba pred uvedbo zdravljenja skrbno oceniti, nato pa natančno spremljati za pojav simptomov perforacij ir fistul, vključno z abscesi in sepso. Trajna ali ponavljajoča se driska med zdravljenjem je lahko dejavnik tveganja za nastanek analne fistule. Uporabo kabozantiniba je treba pri bolnikih, pri 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. I F» SE IM Innovation for patient care katerih se pojavi gastrointestinalna perforacija ali fistula, ki je ni možno ustrezne obravnavati, prekiniti. Driska, navzea/bruhanje, zmanjšanje apetita in vnetje ustne sluznice/ bolečina v ustni votlini so nekateri od najpogosteje poročanih neželenih učinkov na prebavila. Nemudoma je treba uvesti ustrezne medicinske ukrepe, vključno s podpornim zdravljenjem z antiemetiki, antidiaroiki ali antacidi, da se prepreči dehidracija, neravnovesje elektrolitov in izguba telesne mase. Če pomembni neželeni učinki na prebavila vztrajajo ali se ponavljajo, je treba presoditi o prekinitvi odmerjanja, zmanjšanju odmerka ali trajni ukinitvi zdravljenja s kabozantinibom. Kabozantinib je treba uporabljati previdno pri bolnikih, pri katerih obstaja tveganje za pojav venske trombembolije, vključno s pljučno embolijo, in arterijske trombembolije ali imajo te dogodke v anamnezi. Z uporabo je treba prenehati pri bolnikih, pri katerih se razvije akutni miokardni infarkt ali drugi klinično pomembni znaki zapletov trombembolije. Kabozantiniba se ne sme dajati bolnikom, ki hudo krvavijo, ali pri katerih obstaja tveganje za hudo krvavitev. Uporaba zaviralcev poti VEGF pri bolnikih s hipertenzijo ali brez nje lahko spodbudi nastanek anevrizem in/ali disekcij arterij Pred uvedbo kabozantiniba je treba to tveganje skrbno preučiti pri bolnikih z dejavniki tveganja, kot sta hipertenzija ali anamneza anevrizme. Med zdravljenjem s kabozantinibom je treba spremljati vrednosti trombocitov in odmerek prilagoditi glede na resnost trombocitopenije. Zdravljenje s kabozantinibom je treba ustaviti vsaj 28 dni prec načrtovanim kirurškim posegom, vključno z zobozdravstvenim, če je mogoče. Kabozantinib je treba ukiniti pri bolnikih z zapleti s celjenjem rane, zaradi katerih je potrebna zdravniška pomoč. Pred uvedbo kabozantiniba je treba dobro obvladati krvni tlak. Med zdravljenjem je treba vse bolnike spremljati za pojav hipertenzije in jih po potrebi zdraviti s standardnimi antihipertenzivi. V primeru trdovratne hipertenzije, kljub uporabi antihipertenzivov, je treba odmerek kabozantiniba zmanjšati. Z uporabo je treba prenehati, če je hipertenzija resna ali trdovratna kljub zdravljenju z antihipertenzivi in zmanjšanemu odmerku kabozantiniba. V primeru hipertenzijske krize je treba zdravljenje prekiniti. Pri resni PPES je treba razmisliti o prekinitvi zdravljenja. Nadaljevanje zdravljenja naj se začne z nižjim odmerkom, ko se PPES umiri do 1. stopnje. V času zdravljenja je treba redno spremljati beljakovine v urinu. Pri bolnikih, pri katerih se razvije nefrotični sindrom, je treba z uporabo kabozantiniba prenehati. Pri uporabi kabozantiniba so opazili sindrom reverzibilne posteriorne levkoencefalopatije (RPLS), znan tudi kot sindrom posteriorne reverzibilne encefalopatije (PRES). Na ta sindrom je treba pomisliti pri vseh bolnikih s številnimi prisotnimi simptomi vključno z epileptičnimi napadi, glavobolom, motnjami vida, zmedenostjo ali spremenjenim mentalnim delovanjem. Pri bolnikih z RPLS je treba zdravljenje prekiniti. Kabozantinib je treba uporabljati previdno pri bolnikih s podaljšanjem intervala QT v anamnezi, pri bolnikih ki jemljejo antiaritmike, in pri bolnikih z relevantno obstoječo boleznijo srca, bradikardijo ali elektrolitskimi motnjami. Uporaba kabozantiniba je bila povezana z večjo pojavnostjo elektrolitskih nepravilnosti (vključno s hipokaliemijo, hiperkaliemijo, hipomagneziemijo hipokalciemijo in hiponatriemijo), zato je priporočljivo spremljati biokemijske parametre ir po potrebi uvesti ustrezno nadomestno zdravljenje v skladu s standardno klinično prakso Bolniki z redko dedno intoleranco za galaktozo, laponsko obliko zmanjšane aktivnosti laktaze ali malabsorpcijo glukoze/galaktoze ne smejo jemati tega zdravila. Plodnost, nosečnost in doienie: Ženskam v rodni dobi je treba svetovati, da v času zdravljenja s kabozantinibom ne smejo zanositi. Zanositev morajo preprečiti tudi ženske partnerice moških bolnikov, ki uporabljajo kabozantinib. Med zdravljenjem in še vsaj 4 mesece po končanju terapije morajo tako bolniki in bolnice kot tudi njihovi partnerji uporabljati zanesljiv način kontracepcije. Kabozantiniba se ne sme uporabljati med nosečnostjo, razen če zdravljenje ni nujno potrebno zaradi kliničnega stanja ženske. Matere med zdravljenjem s kabozantinibom in še 4 mesece po končanju terapije ne smejo dojiti. Zdravljenje s kabozantinibom lahko predstavlja tveganje za plodnost pri moških in ženskah INTERAKCIJE Kabozantinib je substrat za CYP3A4. Pri sočasni uporabi močnih zaviralcev CYP3A4 (npr. ritonavirja, itrakonazola, eritromicina, klaritromicina, soka grenivke) je SAMO ZA STROKOVNO JAVNOST CAB1219-05, december 2019 potrebna previdnost. Kronični sočasni uporabi močnih induktorjev CYP3A4 (npr. fenitoina karbamazepina, rifampicina, fenobarbitala ali pripravkov zeliščnega izvora iz šentjanževke) se je treba izogibati. Razmisliti je treba o sočasni uporabi alternativnih zdravil, ki CYP3A4 ne inducirajo in ne zavirajo ali pa inducirajo in zavirajo le neznatno. Pri sočasni uporabi zaviralcev MRP2 (npr. ciklosporina, efavirenza, emtricitabina) je potrebna previdnost, saj lahko povzročijo povečanje koncentracij kabozantiniba v plazmi. Učinka kabozantiniba na farmakokinetiko kontraceptivnih steroidov niso preučili, vendar pa se priporoča dodatna kontracepcijska metoda (pregradna metoda). Zaradi visoke stopnje vezave kabozantiniba na plazemske beljakovine je možna interakcija z varfarinom v obliki izpodrivanja s plazemskih beljakovin, zato je treba spremljati vrednosti INR. Kabozantinib morda lahke poveča koncentracije sočasno uporabljenih substratov P-gp v plazmi. Osebe je treba opozoriti na uporabo substratov P-gp (npr. feksofenadina, aliskirena, ambrisentana, dabigatran eteksilata, digoksina, kolhicina, maraviroka, posakonazola, ranolazina saksagliptina, sitagliptina, talinolola, tolvaptana) sočasno s kabozantinibom NEŽELENI UČINKI Za popolno informacijo o neželenih učinkih, prosimo, preberite celoten povzetek glavnih značilnosti zdravila Cabometyx. Najpogostejši resni neželeni učinki zdravila v populaciji bolnikov s KLC so bili driska, hipertenzija, dehidracija hiponatriemija, navzea, zmanjšanje apetita, embolija, utrujenost, hipomagneziemija in PPES. Najpogostejši neželeni učinki katere koli stopnje (ki so se pojavili pri vsaj 25 % bolnikov) v populaciji bolnikov s KLC so bili driska, hipertenzija, utrujenost, zvišanje vrednosti AST, zvišanje vrednosti ALT navzea, zmanjšanje apetita, PPES, paragevzija, zmanjšanje števila trombocitov, stomatitis, anemija, bruhanje, zmanjšanje telesne mase dispepsija in konstipacija. Najpogostejši resni neželeni učinki zdravila v populaciji bolnikov s HCK so bili jetrna encefalopatija, PPES, astenija in driska. Najpogostejši neželeni učinki katere koli stopnje (ki so se pojavili pri vsaj 25 % bolnikov) v populaciji bolnikov s HCK so bili driska, PPES, utrujenost, zmanjšanje apetita, hipertenzija in navzea. Zelo pogosti (> 1/10): anemija, hipotiroidizem, zmanjšan apetit, hipomagneziemija, hipokaliemija, paragevzija glavobol, omotica, hipertenzija, krvavitev, disfonija, dispneja, kašelj, driska, navzea bruhanje, stomatitis, konstipacija, bolečine v trebuhu, dispepsija, bolečina v zgornjem predelu trebuha, PPES, izpuščaj, bolečine v okončinah, utrujenost, vnetje sluznice, astenija periferni edem, zmanjšanje telesne mase, zvišanje vrednosti ALT v serumu, zvišanje vrednosti AST. Pogosti (> 1/100, < 1/10): absces, trombocitopenija, nevtropenija, dehidracija hipoalbuminemija, hipofosfatem ija, hiponatriemija, hipokalciemija, hiperkaliem ija hiperbilirubinemija, hiperglikemija, hipoglikemija, periferna senzorična nevropatija, tinitus venska tromboza, arterijska tromboza, pljučna embolija, gastrointestinalna perforacija fistula, gastroezofagealna refluksna bolezen, hemoroidi, bolečina v ustni votlini, suha usta jetrna encefalopatija, pruritus, alopecija, suha koža, akneiformni dermatitis, sprememba barve las oz. dlak, mišični krči, artralgija, proteinurija, zvišanje vrednosti ALP v krvi, GGT kreatinina v krvi, amilaze, lipaze, holesterola v krvi, zmanjšanje števila belih krvnih celic Občasni (> ¡/¡.000, < ¡/¡00): limfopenija, konvulzije, pankreatitis, glosodinija, holestatični hepatitis, osteonekroza čeljusti, zvišanje vrednosti trigliceridov v krvi, zapleti z ranami Neznana pogostnost (ni mogoče oceniti iz razpoložljivih podatkov): možganska kap miokardni infarkt, anevrizme in disekcije arterij. Vrsta ovojnine in vsebina: Plastenka vsebuje 30 filmsko obloženih tablet. Režim izdaje: Rp/Spec Imetnik dovoljenja za promet z zdravilom: Ipsen Pharma, 65 quai Georges Gorse, 92100 Boulogne-Billancourt, Francija Pred predpisovanjem, prosimo, preberite celoten povzetek glavnih značilnosti zdravila! CAB-301019 Oh PharmaSwiss Choose More Life PharmaSwiss d.o.o., Brodišče 32, 1236 Trzin telefon: +386 1 236 47 00, faks: +386 1 236 47 05 ADIOLOGY AND NCOLOGY June 2020 Vol. 54 No. 2 Pages 135-252 ISSN 1318-2099 UDC 616-006 CODEN: RONCEM Publisher Association of Radiology and Oncology Aims and Scope Radiology and Oncology is a multidisciplinary journal devoted to the publishing original and high quality scientific papers and review articles, pertinent to diagnostic and interventional radiology, computerized tomography, magnetic resonance, ultrasound, nuclear medicine, radiotherapy, clinical and experimental oncology, radiobiology, medical physics and radiation protection. Therefore, the scope of the journal is to cover beside radiology the diagnostic and therapeutic aspects in oncology, which distinguishes it from other journals in the field. Editor-in-Chief Gregor Sersa, Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia (Subject Area: Experimental Oncology) Executive Editor Viljem Kovac, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia (Subject Areas: Clinical Oncology, Radiotherapy) Deputy Editors Andrej Cor, University of Primorska, Faculty of Health Science, Izola, Slovenia (Subject Areas: Clinical Oncology, Experimental Oncology) Božidar Casar, Institute of Oncology Ljubljana, Department for Dosimetry and Quality of Radiological Procedures, Ljubljana (Subject Area: Medical Physics) Maja Čemažar, Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia (Subject Area: Experimental Oncology) Igor Kocijančič, University Medical Center Ljubljana, Institute of Radiology, Ljubljana, Slovenia (Subject Areas: Radiology, Nuclear Medicine) Karmen Stanič, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia (Subject Areas: Radiotherapy; Clinical Oncology) Primož Strojan, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia (Subject Areas: Radiotherapy, Clinical Oncology) Editorial Board Subject Areas: Radiology and Nuclear Medicine Sotirios Bisdas, University College London, Department of Neuroradiology, London, UK Boris Brkljacic, University Hospital "Dubrava", Department of Diagnostic and Interventional Radiology, Zagreb, Croatia Maria Gódény, National Institute of Oncology, Budapest, Hungary Gordana Ivanac, University Hospital Dubrava, Department of Diagnostic and Interventional Radiology, Zagreb, Croatia Luka Lezaic, University Medical Centre Ljubljana, Department for Nuclear Medicine, Ljubljana, Slovenia Katarina Surlan Popovic, University Medical Center Ljubljana, Clinical Institute of Radiology, Ljubljana, Slovenia Jernej Vidmar, University Medical Center Ljubljana, Clinical Institute of Radiology, Ljubljana, Slovenia Subject Areas: Clinical Oncology and Radiotherapy Serena Bonin, University of Trieste, Department of Medical Sciences, Cattinara Hospital, Surgical Pathology Blg, Molecular Biology Lab, Trieste, Italy Luca Campana, Veneto Institute of Oncology (IOV-IRCCS), Padova, Italy Christian Dittrich, Kaiser Franz Josef - Spital, Vienna, Austria Blaž Grošelj, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana Luka Milas, UT M. D. Anderson Cancer Center, Houston, USA Miha Oražem, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana Gaber Plavc, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana Csaba Polgar, National Institute of Oncology, Budapest, Hungary Dirk Rades, University of Lubeck, Department of Radiation Oncology, Lubeck, Germany Luis Souhami, McGill University, Montreal, Canada Borut Štabuc, University Medical Center Ljubljana, Division of Internal Medicine, Department of Gastroenterology, Ljubljana, Slovenia 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 Subject Area: Experimental Oncology Metka Filipic, National Institute of Biology, Department of Genetic Toxicology and Cancer Biology, Ljubljana, Slovenia Janko Kos, University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia Tamara Lah Turnsek, National Institute of Biology, Ljubljana, Slovenia Damijan Miklavcic, University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia Justin Teissie, CNRS, IPBS, Toulouse, France Gillian M. Tozer, University of Sheffield, Academic Unit of Surgical Oncology, Royal Hallamshire Hospital, Sheffield, UK Subject Area: Medical Physics Robert Jeraj, University of Wisconsin, Carbone Cancer Center, Madison, Wisconsin, USA Mirjana Josipovic, Rigshospitalet, Department of Oncology, Section of Radiotherapy, Copenhagen, Denmark Hakan Nystrom, Skandionkliniken, Uppsala, Sweden Ervin B. Podgorsak, McGill University, Medical Physics Unit, Montreal, Canada Matthew Podgorsak, Roswell Park Cancer Institute, Departments of Biophysics and Radiation Medicine, Buffalo, NY ,USA Advisory Committee Tullio Giraldi, University of Trieste, Faculty of Medicine and Psyhology, Department of Life Sciences, Trieste, Italy Vassil Hadjidekov, Medical University, Department of Diagnostic Imaging, Sofia, Bulgaria Marko Hocevar, Institute of Oncology Ljubljana, Department of Surgical Oncology, Ljubljana, Slovenia Miklos Kasler, National Institute of Oncology, Budapest, Hungary Maja Osmak, Ruder Boškovic Institute, Department of Molecular Biology, Zagreb, Croatia Tomaž Benulič, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia Radiol Oncol 2020; 54(2): 3-143. doi: 10.2478/raon-2020-0025 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 Klemen čič Zvezdana Vukmirovič 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: Baidu Scholar Case Chemical Abstracts Service (CAS) - CAplus Chemical Abstracts Service (CAS) - SciFinder CNKI Scholar (China National Knowledge Infrastructure) CNPIEC - cnpLINKer Dimensions DOAJ (Directory of Open Access Journals) EBSCO (relevant databases) EBSCO Discovery Service Embase Genamics JournalSeek Google Scholar Japan Science and Technology Agency (JST) J-Gate Journal Citation Reports/Science Edition JournalGuide JournalTOCs KESLI-NDSL (Korean National Discovery for Science Leaders) Medline Meta Agency. Microsoft Academic Naviga (Softweco) Primo Central (ExLibris) ProQuest (relevant databases) Publons PubMed PubMed Central PubsHub QOAM (Quality Open Access Market) ReadCube Reaxys SCImago (SJR) SCOPUS Sherpa/RoMEO Summon (Serials Solutions/ProQuest) TDNet Ulrich's Periodicals Directory/ulrichsweb WanFang Data Web of Science - Current Contents/Clinical Medicine Web of Science - Science Citation Index Expanded WorldCat (OCLC) This journal is printed on acid- free paper On the web: ISSN 1581-3207 https://content.sciendo.com/raon http://www.radioloncol.com Radiol Oncol 2020; 54(2): 4-143. doi: 10.2478/raon-2020-0025 contents contents review 135 Current and innovative approaches in the treatment of non-muscle invasive bladder cancer: the role of transurethral resection of bladder tumor and organoids Milena Taskovska, Mateja Erdani-Kreft, Tomaž Smrkolj 144 Mechanical recanalization for acute bilateral cerebral artery occlusion -literature overview with a case Miran Jeromel, Zoran V. Milosevic, Janja Pretnar Oblak radiology 149 Major and ancillary features according to LI-RADS in the assessment of combined hepatocellular cholangiocarcinoma Vincenza Granata, Roberta Fusco, Sergio Venanzio Setola, Fabio Sandomenico, Maria Luisa Barretta, Andrea Belli, Raffaele Palaia, Fabiana Tatangelo, Roberta Grassi, Francesco Izzo, Antonella Petrillo 159 Relation of the chondromalatia patellae to proximal tibial anatomical parameters, assessed with MRI Mohammadreza Tabary, Azadehsadat Esfahani, Mehdi Nouraie, Mohammad Reza Babaei, Ali Reza Khoshdel, Farnaz Araghi, Mostafa Shahrezaee experimental oncology 168 Pulsed low dose-rate irradiation response in isogenic HNSCC cell lines with different radiosensitivity Vesna Todorovic, Ajda Prevc, Martina Niksic Zakelj, Monika Savarin, Simon Bucek, Blaz Groselj, Primoz Strojan, Maja Cemazar, Gregor Sersa clinical oncology 180 The prevalence of occult ovarian cancer in the series of 155 consequently operated high risk asymptomatic patients - Slovenian population based study Andreja Gornjec, Sebastijan Merlo, Srdjan Novakovic, Vida Stegel, Barbara Gazič, Andraž Perhavec, Ana Blatnik, Mateja Krajc 187 Dietary iodine intake, therapy with radioiodine, and anaplastic thyroid carcinoma Nikola Besic and Barbara Gazic Radiol Oncol 2020; 54(2): C. contents 194 Significance of nuclear factor - kappa beta activation on prostate needle biopsy samples in the evaluation of Gleason score 6 prostatic carcinoma indolence Marko Zupancic, Boris Pospihalj, Snezana Cerovic, Barbara Gazic, Primoz Drev, Marko Hocevar, Andraz Perhavec 201 Evaluation of the training program for p16/Ki-67 dual immunocytochemical staining interpretation for laboratory staff without experience in cervical cytology and immunocytochemistry Veronika Kloboves Prevodnik, Ziva Pohar Marinsek, Janja Zalar, Hermina Rozina, Nika Kotnik, Tine Jerman, Jerneja Varl, Urska Ivanus 209 Care of patients with non-small-cell lung cancer stage III - the Central European real-world experience Milada Zemanova, Robert Pirker, Lubos Petruzelka, Zuzana Zbozinkova, Dragana Jovanovic, Mirjana Rajer, Krisztina Bogos, Gunta Purkalne, Vesna Ceriman, Subhash Chaudhary, Igor Richter, Jin Kufa, Lenka Jakubikova, Marius Zemaitis, Marketa Cernovska, Leona Koubkova, Zdenka Vilasova, Karin Dieckmann, Attila Farkas, Jelena Spasic, Katerina Fröhlich, Andreas Tiefenbacher, Virag Hollosi, Juraj Kultan, Iveta Kolarova, Jiri Votruba 221 Diagnostic accuracy of (1^3)-ß-D-glucan to predict Pneumocystis jirovecii pneumonia in non-HIV-infected patients Petra Rogina, Miha Skvarc 227 Stereotactic body radiation therapy (SBRT) for the treatment of primary lung cancer in recipients of lung transplant Assaf Moore, Mordechai R. Kramer, Dror Rosengarten, Osnat Shtraichman, Alona Zer, Elizabeth Dudnik, Yasmin Korzets, and Aaron M. Allen 233 Sorafenib for the treatment of hepatocellular carcinoma: a single-centre real-world study Jurij Hanzel, Tajda Kosir Bozic, Borut Stabuc, Rado Jansa 237 Sarcopenia and myosteatosis at presentation adversely affect survival after esophagectomy for esophageal cancer Matevz Srpcic, Taja Jordan, Karteek Popuri, Mihael Sok radiophysics 247 The influence of shielding reinforcement in a vault with limited dimensions on the neutron dose equivalent in vicinity of medical electron linear accelerator Ana Ivkovic, Dario Faj, Mladen Kasabasic, Marina Poje Sovilj, Ivana Krpan, Marina Grabar Branilovic, Hrvoje Brkic I Slovenian abstracts Radiol Oncol 2020; 54(2): C. review Current and innovative approaches in the treatment of non-muscle invasive bladder cancer: the role of transurethral resection of bladder tumor and organoids Milena Taskovska12, Mateja Erdani Kreft3, Tomaz Smrkolj12 1 Department of Urology, University Medical Centre Ljubljana, Ljubljana, Slovenia 2 Department of Surgery, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Institute for Cell Biology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2020; 54(2): 135-143. Received 19 January 2020 Accepted 30 January 2020 Correspondence to: for organoids - Prof. Mateja Erdani-Kreft, Ph.D., Institute for Cell Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana, Slovenia. E-mail: mateja.erdani@mf.uni-lj.si Correspondence to: for monopolar/bipolar TURB - Assoc. Prof. Tomaž Smrkolj, M.D., Ph.D., Department of Urology, University Medical Centre Ljubljana, Zaloška 7, 1000 Ljubljana, Slovenia or Department of Surgery, Faculty of Medicine, University of Ljubljana, Zaloška 7, SI-1000 Ljubljana, Slovenia. Email: tomaz.smrkolj@kclj.si Disclosure: No potential conflicts of interest were disclosed. Background. Bladder cancer is the 7th most common cancer in men. About 75% of all bladder cancer are non-muscle invasive (NMIBC). The golden standard for definite diagnosis and first-line treatment of NMIBC is transurethral resection of bladder tumour (TURB). Historically, the monopolar current was used first, today bipolar current is preferred by most urologists. Following TURB, depending on the tumour grade, additional intravesical chemo- or/and immunotherapy is indicated, in order to prevent recurrence and need for surgical resection. Development of new technologies, molecular and cell biology, enabled scientists to develop organoids - systems of human cells that are cultivated in the laboratory and have characteristics of the tissue from which they were harvested. In the field of urologic cancers, the organoids are used mainly for studying the course of different diseases, however, in the field of bladder cancer the data are scarce. Conclusions. Different currents - monopolar and bipolar, have different effect on urothelium, that is important for oncological results and pathohistological interpretation. Specimens of bladder cancer can be used for preparation of organoids that are further used for studying carcinogenesis. Bladder organoids are step towards personalised medicine, especially for testing effectiveness of chemo-/immunotherapeutics. Key words: bladder cancer; transurethral resection of bladder tumour; monopolar/bipolar current; organoids, mitomycin C; BCG Introduction Bladder cancer is 7th most common cancer in men. In the European Union (EU) age-standardised incidence rate is 19.1 for men and 4.0 in women.1 In Europe, bladder cancer incidence is 27.1 and mortality 8.9.2 The highest incidence rate in the EU is reported in Belgium and lowest in Finland. On the global level, incidence and mortality rates vary due to different methodologies and diagnos- tic practices.12 Approximately three-quarters of all bladder cancer are non-muscle invasive (NMIBC) - the disease is confined to the mucosa (stage Ta, CIS) and submucosa (stage T1) (Table 1, Figure 1), in patients younger than 40 years this proportion is even higher.13-5 According to the data from Cancer registry of the Republic of Slovenia, bladder cancer is the 8th most common cancer in men and the 13th most common when both genders are considered. Age standardised incidence rate is Radiol Oncol 2020; 54(2): 135-143. doi: 10.2478/raon-2020-0025 136 Taskovska M et al. / Current and innovative treatment of non-muscle invasive bladder cancer 11.74 in men and 2.97 in women. Age-standardised mortality rate is 5.46 in men and 1.52 in women.6 Although many European countries experience incidence rise, the projected growth rate of bladder cancer incidence rates by 2030 in Slovenia is extremely high, i.e. 92% for men and 256% for women. Standard treatment of NMIBC is transurethral resection of bladder tumour (TURB). Depending on the histopathological tumour characteristics, additional treatment with intravesical chemothera-peutics or immunotherapeutics is indicated. The aim of intravesical therapy is to decrease the rate of recurrence and need for surgical intervention.1 Organoids are 3D models which consist of cells derived from specific tissue or organ and are grown in the laboratory with the aim to study different cell biological mechanisms, homeosta-sis, development of disease and effect of different medications. Organoids have characteristics of the cells from which are derived, although differences could appear because of the effect of microenviron-ment.7 There are some new data about organoids used to study oncogenesis and different treatments in urological cancers (prostate, kidney, bladder).8 Aim of this review is to present different modalities of TURB for treatment of NMIBC, and the role of bladder/urothelium organoids for studying oncogenesis, therapeutic modalities and personalised medicine in NMIBC. Transurethral resection of bladder tumour (TURB) TURB is a golden standard in diagnosis, treatment, and staging of NMIBC as well as in diagnosis of muscle invasive bladder cancer (MIBC).1,9 The first published report involving the application of electric current for endoscopic resection of papillary bladder tumours through cystoscope originates in 1910s. The initial case was performed using water as medium. Since then, TURB has become standard in evaluation and treatment of patients with bladder cancer.9 Guidelines of three urologic associations (European Association of Urology, EAU; American Urology Association, AUA; and Canadian Urology Association, CUA) emphasize the importance of TURB for diagnosis, staging and treatment of NMIBC (1, 9, 10, 11).1,9,10,11 TABLE 1. 2017 TNM classification of urinary bladder cancer 5 T - primary tumour TX Primary tumour cannot be assessed T0 No evidence of primary tumour Ta Non-invasive papillary carcinoma Tis Carcinoma in situ: 'flat tumour' T1 Tumour invades subepithelial connective tissue T2 Tumour invades muscle T2a Tumour invades superficial muscle (inner half] T2b Tumour invades deep muscle (outer half] T3 Tumour invades perivesical tissue T3a Microscopically T3b Macroscopically (extravesical mass] T4 Tumour invades any of the following: prostate stroma, seminal vesicles, uterus, vagina, pelvic wall, abdominal wall T4a Tumour invades prostate stroma, seminal vesicles, uterus or vagina T4b Tumour invades pelvic wall or abdominal wall N - regional lymph nodes NX Regional lymph nodes cannot be assessed N0 No regional lymph node metastasis N1 Metastasis in a single lymph node in the true pelvis (hypogastric, obturator, external iliac, or presacral] N2 Metastasis in multiple regional lymph nodes in the true pelvis (hypogastric, obturator, external iliac, or presacral] N3 Metastasis in common iliac lymph node(s) M - distant metastasis M0 No distant metastasis M1a Non-regional lymph nodes M1b Other distant metastases Radiol Oncol 2020; 54(2): 135-143. 9 Taskovska M et al. / Current and innovative treatment of non-muscle invasive bladder cancer TURB is a relatively safe procedure with few complications, in some countries is performed in an outpatient setting. The most common complications are haematuria and urinary tract infection. One of the most important aims of TURB is to resect the whole tumour (depending on the size and invasion in muscle layer) and resect muscle layer in order to obtain the correct pathological stage.11011 Since its introduction in the 1910s, technology has developed and so has equipment and technique of TURB.9 In 1900 Joseph Rivere discovered that a spark arcing from an electrode coagulates skin and he used it for treatment of skin lesions. In the following decades, this technique was used to treat skin lesions, lesions in the oral cavity, bladder, coagulation of vascular tumours and haemorrhoids. In 1920s Clark was one of the first who observed the tissues exposed to current under a microscope and found that they shrink from dehydration. Bovie constructed the first diathermy unit that was used for cutting, coagulation, and dissection and was first used on October 1st 1926 in Boston. Since then, this instrument is used in everyday surgical practice (12).12 The clinical effect of electrocautery is a consequence of heat. When oscillating current is applied to the tissue, the rapid movement of electrons through the cytoplasm causes an increase of intracellular temperature. The effect on the tissue depends upon the amount of thermal energy delivered and the time rate of delivery. Temperature below 45°C causes reversible thermal damages, when increased it causes denaturation and loss of protein structure, above 90°C liquid evaporates, resulting in vaporization if heated rapidly or desiccation if heated slowly. Temperatures over 200°C cause car-bonization.12 Electric energy could be monopolar or bipolar. Monopolar energy delivery requires the current to pass from the generator to the active electrode through the patient and out of the body through a dispersive electrode pad which is connected to the generator in order to complete the circuit. On the other hand, bipolar delivery does not require a dispersive return electrode because both active and return electrodes are integrated into energy delivery forceps with target tissue between them (Figure 2).12 Monopolar electrocautery for TURB In urology, TURB was introduced in the 1910s by Beer using monopolar current for fulguration (Figure 2).1314 Monopolar electrocautery requires NMIBC MIBC FIGURE 1. Classification of bladder cancer. MIBC = Muscle invasive bladder cancer; NMIBC = Non muscle invasive bladder cancer FIGURE 2. Monopolar vs. bipolar transurethral resection of bladder tumor. high energy and voltage to allow current transmission from the loop to the tissue. In monopolar electrocautery glycine is used as a medium that is associated with limitations, such as short resection time due to the risk of development of TURB syndrome. The heat generated while cutting the tissue causes damage to the surrounding cells. Because of the thermal artefacts, pathologic assessment of the specimen is sometimes difficult.13 There are studies which compared monopolar and bipolar elec- Radiol Oncol 2020; 54(2): 135-143. 138 Taskovska M et al. / Current and innovative treatment of non-muscle invasive bladder cancer TABLE 2. Comparison of monopolar and bipolar current for TURB1-9-14 Variable Monopolar Bipolar Dispersive electrode pad yes no Energy high low Voltage high low Working medium glycine saline Temperature at thermal effect (°C) 400 40-70 ,. , extended Time of resection limited . , , . ,. ., (not strictly limited) TUR syndrome common rare Obturator jerk common rare Quality of haemostasis and coagulum poor good tissue desiccation and collateral tissue damage. Radiofrequency energy of bipolar electrocautery systems converts the conductive medium into a plasma field with highly ionised particles that disrupt the organic bonds between tissues and allows thermal effect to occur at lower temperatures (between 40 and 70°C). Pathologists sometimes classify thermal damage into three categories: (1) cautery artefact less than 1/3 of the specimen, (2) cautery artefact of 1/3 to 2/3 of the specimen and (3) over 2/3 of the specimen.13 Although different elec-trocautery systems are used for more than 30 years we have no data on the effect of different electro-cautery systems on cellular level. trocautery for TURB and concluded that there is very little or no difference in thickness of thermal artefacts.14 One study has shown mean depth of thermal artefacts of 0.237 mm when using bipolar electrocautery and 0.26 mm when using monopolar electrocautery.9 Monopolar electrocautery is not inferior to bipolar electrocautery regarding intra and postoperative bleeding, and perforation of bladder wall (Table 2).14 Bipolar electrocautery for TURB Bipolar electrocautery has been introduced about 30 years ago and a few years after its introduction has gained popularity over monopolar electro-cautery.12 Bipolar electrocautery was first used for transurethral resection of the prostate (TURP) for benign prostate enlargement and obstruction. It was quickly adopted for TURB because of advantages: (1) isotonic medium could be used (such as saline), (2) electric circuit is completed using only resection loop and sheath of the device itself, so the patient is not included in the circuit, (3) risk for TUR syndrome is low, (4) time for resection is extended, which is of essential importance in case of large bladder tumours, (5) incidence of obturator jerk is lower, (6) there are fewer bladder perforations. It is also related to fewer postoperative complications such as clot retention and contracture of the bladder neck (Table 2).9 One of the main differences between monopolar electrocautery and bipolar electrocautery systems is the coagulation mechanism. In bipolar electro-cautery system voltage is low and energy dissipates as heat in the tissue leading to the formation of coagulum and haemostasis.13 In monopolar electrocautery, electrical injury directed into the tissues and electrical resistance creates temperature as high as 400°C which causes Adjuvant treatment of NMIBC Intravesical chemotherapy TURB is used as definitive therapy in TaT1 tumours. The adjuvant treatment of NMIBC is indicated when dealing with high grade NMIBC. Immediate single intravesical instillation of chemotherapy is used to destroy circulating tumour cells after TURB and has an ablative effect on residual tumour cells at the resection site and on small overlooked tumours. Immediate single intravesical instillation should be performed within the first 24 hours after TURB to maximise its effect. Meta-analyses were performed which have shown that after immediate single intravesical instillation the recurrence rate is lower. Recent reviews and meta-analysis have shown that immediate single intravesical instillation also reduces 5-year recurrence rate.115 The most commonly used chemotherapeutics for immediate single intravesical instillation are mitomycin C (MMC), epirubicin and pirarubicin.1 In Slovenia, we use MMC for immediate single intravesical instillation. In literature, there is data that further repeat instillations of chemotherapy also have an impact on the recurrence rate. The length and frequency of chemotherapy instillation are still controversial. According to data available, the length of this treatment should not exceed one year.116 There is evidence that intravesical chemotherapy combined with microwave-induced hyperthermia in high-risk patients has enhanced efficacy - it has improved recurrence-free survival at 24 months. There are also undergoing trials regarding use of different methods of hyperthermic intravesical chemotherapy and electromotive drug administration but data about their efficacy is still lacking.117 In Slovenia, we are not using combinations with microwave, hyperthermia or electromotive drug Radiol Oncol 2020; 54(2): 135-143. 139 Taskovska M et al. / Current and innovative treatment of non-muscle invasive bladder cancer administration, since the use of these combinations is not confirmed by randomised controlled trials. Intravesical bacillus Calmette-Guerin (BCG) immunotherapy There is evidence in the literature that BCG after TURB is superior to TURB alone or in combination with chemotherapy for preventing recurrence of NMIBC. BCG maintenance therapy reduces the recurrence rate for 32% in comparison to MMC, but increases risk for 28% in patients who did not receive maintenance therapy with BCG.118 The main disadvantage of BCG intravesical immunotherapy is in its side effects. According to data in the literature, serious side effects are encountered in less than 5% of treated patients. Side effects are a consequence of systemic absorption of BCG.119 Caution is needed in immunocompromised patients, although some studies have not shown that immunocompromised patients are more prone to experience side effects.1 For BCG instillation is used 6-week schedule introduced by Morales.120 Many studies were conducted but none has shown advantages or disadvantages of this schedule compared to others.121 Meta-analysis has shown that at least one year of maintenance BCG is required to obtain superiority of BCG over MMC for prevention of recurrence or progression. Regarding BCG dose, studies have shown that one-third dose is required to be effective for intermediate-risk tumors, full dose is needed for high-risk tumors.122 According to the EAU guidelines on NMIBC, BCG intravesical immunotherapy is recommended to patients with intermediate and high-risk tumors, three year maintenance therapy is more effective in patients with high-risk tumors to prevent recurrence.1 There are cases when BCG intravesical chemotherapy fails: (1) muscle invasive bladder cancer (MIBC) detected during follow-up, (2) BCG-refractory tumor - (a) if high-grade NMIBC is detected at three months, (b) CIS is present at three and six months and (c) high grade tumor is detected after BCG therapy. Therefore, radical cystec-tomy is indicated.1 Personalized treatment -precision medicine in the treatment of bladder cancer The efficacy of cancer management is challenging and depends upon genomic, molecular and immu- nologic characteristics of cancer. New discoveries in these fields are making cancer treatment more targeted and efficient. Concerning bladder cancer, mutations in genes of DNA repair pathway (e.g. ERCC2, FANCC, ATM, RB1 and etc.) can predict response to neoadjuvant platinum-based systemic chemotherapies. Therapies that influence the immune system such as immune checkpoint inhibitors (PDA, PDL1, CTLA4) are approved for use in treatment of bladder cancer and represent renaissance in medical oncological treatment of this disease.23 Cancer treatment based on the response of bladder cancer organoid could contribute to more personalized approach in the treatment of this disease. Role of organoids in studying bladder cancer Organoids In order to understand the role of cancer-specific genetic alterations in tumorigenesis, maintenance of tumor and sensibility age-standardized response to different therapeutics, development of in vitro and in vivo model systems that accurately reflect genetic diversity and lineage specificity of cancer was required.78 For this purpose, cell lines are used. Their disadvantages is that they are mainly long term 2D cultures, and there is a lack of clinical data regarding the organ of origin. To overcome these disadvantages in vivo models are used.8 The advantage of in vivo models is that they are able to recapitulate histological and therapeutic response but there are species-specific differences and inaccurate recapitulation of in vivo human tumour biology that are main disadvantage.78 In combination with organoids derived from normal cells, tumour cell organoids can be used to study transformation from normal to malignant, when exposed to different carcinogens.8 Sato et al. in 2009 discovered that single leucine-rich repeat containing G-protein coupled receptor 5-positive intestinal stem cell is able to generate a continuously expanding, self-organizing, physiological epithelial structure that was similar to normal gut tissue. It was named organoid cul-ture.24 Liu et al. demonstrated that combination of ROCK inhibitor and feeder fibroblast culture conditions enables the infinite growth of multiple primary human epithelial cell types. Based on this, organoids from normal and tumour cells might be able to proliferate indefinitely in vitro, with no need to transduct exogenous viral or cellular genes Radiol Oncol 2020; 54(2): 135-143. 140 Taskovska M et al. / Current and innovative treatment of non-muscle invasive bladder cancer normal bladder organoid FIGURE 3. Shematic presentation of bladder organoid preparation. Sample of bladder wall (healty and /tumorous) is taken with transurethral resection of bladder tumour. The cells are cultivated under special conditions in laboratory to form organoids. Organoids are used for studying the characteristics of normal and tumorous bladder wall, pathogenesis, response to different treatment approaches. This is step toward personalized treatment of bladder cancer. (Figure 3).82526 Organoids are nowadays used for research of different cancers.78 In the field of urology there are studies/reports on kidney, prostate and bladder organoids.8,27,28 3D organoids could be derived from cell lines, primary tissues, induced pluripotent stem cells, embryonic stem cells and embryonic whole organs such as organ explants that consist of many tissue types. Organoid is composed of multiple cell types and contains multicellular organ structures, which mimic the tissue of origin and functions in a similar manner. Organoids can be generated in different manners.78 Cell lines In 3D culture conditions, some immortalized cell lines are able to form polarized 3D structures.8 Smith et al. cultured human bladder cells that were used to investigate how terminally differentiated human urothelial cells interact with uropathogenic E.coli. They cultured 5637 cells under microgravity conditions within a rotating wall vessel bioreactor. Under these conditions, cells remain in suspension and form organoids that reflect characteristics of in vivo tissue-specific determination. Human bladder cells in this model had developed into a model that expresses specific markers and structures, characteristic of differentiated human urothelium.829 Lee et al. have shown that patient-derived bladder tumour organoids have most characteristics of parental tumour, although in a culture they may change their marker phenotype. Organoids could be used for studying the biology of the tumour and the effect of different therapies on tumour of individual patient.7 In the future, these models could be used for personalized treatment of the patient according to tumour characteristics.7 Radiol Oncol 2020; 54(2): 135-143. 141 Taskovska M et al. / Current and innovative treatment of non-muscle invasive bladder cancer Primary adult stem cells Primary adult stem cells could also be used for organoids.8 Matulay et al. collected bladder tumour specimens during cystoscopy, enzymatically digested them into single cells and cell clusters and then cultured them in organoid-prompting, embedded-cell culture conditions. They used these bladder organoids to analyse genetic mutations in bladder cancer.830 Pluripotent stem cells Pluripotent stem cells have the ability to form all cellular components of an organ (epithelial, stromal and endothelial cells). Organoid could be generated from embryonic or induced pluripotent stem cells (iPSC). Using organoid 3D system, scientists are able to induce pluripotent stem cells to develop into organoids of the desired organ. Until 2014 there were two protocols to induce human embryonic stem cells or human iPSC into urothe-lium.8 Osborn et al. have developed in vitro culture system that was matrix-free and cell-contact-free, and was able to induce human embryonic stem cells or human iPSC to differentiate into definitive endoderm and then urothelium using directed differentiation in urothelium specific medium.831 Kang et al. have developed a protocol that used a chemically defined culture system to induce human iPSC to differentiate into endoderm and bladder urothelial cells.832 Application of organoids in diagnosis and management of bladder cancer Organoids could be used to predict the response to treatment and guide the medicine regimen. For colorectal cancer, 3D organoid based drug sensitivity screen was developed. It identifies molecular signatures associated with altered drug responses. A similar strategy could be used with bladder cancer organoids.3334 Resistance to chemotherapy is promoted by cancer stem cells. Models with origin from cancer stem cells may be helpful for identifying effective prognostic biomarkers and for individual treatment.8 In case of prostate cancer, there are already organoids being used to test different drugs, even further, there are models developed to screen for prostate cancer instead of cored biopsy, so-called liquid biopsy.83036 Matulay et al. have established urothelial cancer organoids from patient-derived tissue samples. Using DNA sequencing analysis, they have shown that organoid lines have similar mutational profiles to those of tumour sample and can provide a platform for personalized drug-response assays in urothelial cancers.830 There are few studies aim of which was to identify bladder cancer stem cells, but the results are inconclusive.8 In one study, researchers have described Sonic hedgehog (Shh) expressing and cytokeratin-5 (Ck5) expressing basal urothelial cells from ShhCreER/WT; R26mTmG/WT mice in organoid culture. Those were multipotent stem cells capable of self-renewal and regeneration into all cell types within urothelium in response to chemical injury or bacterial infection. Individual Shh-expressing cells formed cyst-like organoids after 5-7 weeks of 3D culture. CK5 was expressed in the outer layer. They also formed a luminal space in which CK5 and Shh were not expressed. Individual cells of these organoids were capable of self-renewal and differentiation, however, the origin and formation of bladder cancer stem cells remain unknown.837 Mullenders et al. collected samples of tumours of MIBC and NMIBC patients who underwent radical cystectomy and TURB, and normal macroscop-ically looking urothelium and established a sample of 50 human bladder organoids. Besides histologi-cal and functional investigations, they also used organoids for testing the efficacy of different intra-vesical chemotherapeutics. They applied different concentrations of anticancer drugs for time period of 5 days and found different responses in different organoids.38 Neal et al. purposed an in vitro model of different cancers for studying immunotherapy. One of them was also bladder cancer and responsiveness to immunotherapy with checkpoint inhibitors.34 Limitations of organoids Organoids have a potentially important role in urological research, clinical decision-making, and treatment of urological cancers. Limitations of or-ganoids are that the spatial orientation of tissues is random, also in many cases, the cellular components that are present in in vivo systems such as stromal, vascular endothelial and immune cells are missing.8 Conclusions Bladder cancer presents public health problem because incidence and mortality are constant despite Radiol Oncol 2020; 54(2): 135-143. 142 Taskovska M et al. / Current and innovative treatment of non-muscle invasive bladder cancer the development of technology, molecular and cell biology, pharmacology and improvement of surgical technique. TURB is the golden standard for diagnosis, staging, and treatment of NMIBC. Both monopolar and bipolar current are equally effective, bipolar current having fewer complications. Development of molecular and cell biology leads to the construction of organoids which are a step towards personalized medicine. We expect that they will enable us to treat our patients based on the data acquired from organoids, regarding on-cogenesis, responsiveness to different therapeutic modalities and possibilities for reconstruction. References 1. European Association of Urology. Guidelines. Non-invasive muscle bladder cancer. [cited 2019 Dec 15]. Available at: https://uroweb.org/guideline/ non-muscle-invasive-bladder-cancer/ 2. European Commission. ECIS - European Cancer Information System. Incidence and mortality of bladder cancer in Europe. [cited 2018 Sep 15]. Available at: https://ecis.jrc.ec.europa.eu/explorer. php?$1-All$2-All$4-1,2$3-38$6-0,14$5-2008,2008$7-7,8$0-0$CEstByCountry$X0_8-3$CEstRelative$X1_8-3$X1_9-AE28 3. 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Electrosurgery: history, principles, and current and future uses. J Am Coll Surg 2006; 202: 520-30. doi: 10.1016/j. jamcollsurg.2005.11.017 13. Osman Y, Harraz AM. A review comparing experience and results with bipolar versus monopolar resection for treatment of bladder tumors. Curr Urol Rep 2016; 17: 21. doi: 10.1007/s11934-016-0579-1 14. Mashni J, Godoy G, Haarer C, Dalbagni G, Reuter VE, Al-Ahmadie H, et al. Prospective evaluation of plasma kinetic bipolar resection of bladder cancer: comparison to monopolar resection and pathologic findings. Int Urol Nephrol 2014; 46: 1699-705. doi: 10.1007/s11255-014-0719-9 15. Sylvester RJ, Oosterlinck W, Holmang S, Sydes MR, Birtle A, Gudjonsson S, et al. Systematic review and individual patient data meta-analysis of randomized trials comparing a single immediate instillation of chemotherapy after transurethral resection with transurethral resection alone in patients with stage pTa-pT1 urothelial carcinoma of the bladder: which patients benefit from the instillation? Eur Urol 2016; 69: 231-44. doi: 10.1016/j. eururo.2015.05.050 16. Sylvester RJ, Oosterlinck W, Witjes JA. The schedule and duration of intravesical chemotherapy in patients with non-muscle-invasive bladder cancer: a systematic review of the published results of randomized clinical trials. Eur Uro! 2008; 53: 709-19. doi: 10.1016/j.eururo.2008.01.015 17. Arends TJ, Nativ O, Maffezzini M, de Cobelli O, Canepa G, Verweij F, et al. Results of a randomised controlled trial comparing intravesical chemohyperthermia with mitomycin C versus bacillus Calmette-Guerin for adjuvant treatment of patients with intermediate- and high-risk non-muscle-invasive bladder cancer. Eur Uro! 2016; 69: 1046-52. doi: 10.1016/j. eururo.2016.01.006 18. Malmstrom PU, Sylvester RJ, Crawford DE, Friedrich M, Krege S, Rintala E, et al. An individual patient data meta-analysis of the long-term outcome of randomised studies comparing intravesical mitomycin C versus bacillus Calmette-Guerin for non-muscle-invasive bladder cancer. Eur Uro! 2009; 56: 247-56. doi: 10.1016/j.eururo.2009.04.038 19. van der Meijden AP, Sylvester RJ, Oosterlinck W, Hoeltl W, Bono AV; EORTC Genito-Urinary Tract Cancer Group. Maintenance Bacillus Calmette-Guerin for Ta T1 bladder tumors is not associated with increased toxicity: results from a European Organisation for Research and Treatment of Cancer Genito-Urinary Group Phase III Trial. Eur Uro! 2003; 44: 429-34. doi: 10.1016/s0302-2838(03)00357-9 20. Morales A, Eidinger D, Bruce AW. Intracavitary bacillus Calmette-Guerin in the treatment of superficial bladder tumors. J Uro! 1976; 116: 180-3. doi: 10.1016/s0022-5347(17)58737-6 21. Sylvester, RJ, van der Meijden AP, Lamm DL. Intravesical bacillus Calmette-Guerin reduces the risk of progression in patients with superficial bladder cancer: a meta-analysis of the published results of randomized clinical trials. J Uro! 2002; 168: 1964-70. doi: 10.1097/01.ju.0000034450.80198.1c 22. Ojea A, Nogueira JL, Solsona E, Flores N, Gomez JM, Molina JR, et al. A mul-ticentre, randomised prospective trial comparing three intravesical adjuvant therapies for intermediate-risk superficial bladder cancer: low-dose bacillus Calmette-Guerin (27 mg) versus very low-dose bacillus Calmette-Guerin (13.5 mg) versus mitomycin C. Eur Uro! 2007; 52: 1398-406. doi: 10.1016/j. eururo.2007.04.062 23. Flsenstein KM, Theodorescu D. Precision medicine for urothelial bladder cancer: update on tumour genomics and immunotherapy. Nat Rev Urol 2018; 15: 92-111. doi: 10.1038/nrurol.2017.179 24. Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 2009; 459: 262-5. doi: 10.1038/nature07935 25. Liu X, Ory V, Chapman S, Yuan H, Albanese C, Kallakury B, et al. ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells. Am J Patho! 2012; 180: 599-607. doi: 10.1016/j.ajpath.2011.10.036 26. Chapman S, Liu X, Meyers C, Schlegel R, McBride AA. Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor. J C!in Invest 2010; 120: 2619-26. doi: 10.1172/JCI42297 27. Santos CP, Lapi E, Martinez de Villarreal J, Alvaro-Espinosa L, Fernandez-Barral A, Barbachano A, et al. Urothelial organoids originating from Cd49fhigh mouse stem cells display Notch-dependent differentiation capacity. Nat Commun 2019; 10: 4407. doi: 10.1038/s41467-019-12307-1. 28. Vasytin I, Zerihun L, Ivan C, Atala A. Bladder organoids and spheroids: potential tools for normal and diseased tissue modelling. Anticancer Res 2019; 39: 1105-18. doi: 10.21873/anticanres.13219 29. Smith YC, Grande KK, Rasmussen SB, O'Brien AD. Novel three-dimensional organoid model for evaluation of the interaction of uropathogenic Escherichia coli with terminally differentiated human urothelial cells. Infect Immun 2006; 74: 750-7. doi: 10.1128/IAI.74.1.750-757.2006 30. Matulay JT, Barlow LJ, Silva MV, Chua CW, Benson MC, McKiernan JM, et al. Genetic mutations in patient-derived bladder tumor organoids mimic parental tumor samples [abstract PD38-07]. J Uro! 2016; 195(Suppl): e926. doi: 10.1016/j.juro.2016.02.1487 Radiol Oncol 2020; 54(2): 135-143. 143 Taskovska M et al. / Current and innovative treatment of non-muscle invasive bladder cancer 31. Osborn SL, Thangappan R, Luria A, Lee JH, Nolta J, Kurzrock EA, et al. Induction of human embryonic and induced pluripotent stem cells into urothelium. Stem Cells Transl Med 2014; 3: 610-9. doi: 10.5966/ sctm.2013-0131 32. Kang M, Kim HH, Han YM. Generation of bladder urothelium from human pluripotent stem cells under chemically defined serum- and feeder-free system. Int J Mol Sci 2014; 15: 7139-57. doi: 10.3390/ijms15057139 33. Banerjee S, Southgate J. Bladder organoids a step towards personalised cancer therapy? Transl Androl Urol 2019; 8: S300-2. doi: 10.21037/ tau.2019.06.10 34. Neal JT, Li X, Zhu J, Giangarra V, Grzeskowiak CL, Ju J, et al. Organoid modeling of the tumor immune microenvironment. Cell 2018; 175: 1972-88. doi: 10.1016/j.cell.2018.11.021 35. Scher HI, Heller G, Molina A, Attard G, Danila DC, Jia X, et al. Circulating tumor cell biomarker panel as an individual-level surrogate for survival in metastatic castration-resistant prostate cancer. J Clin Oncol 2015; 33: 134855. doi: 10.1200/JCO.2014.55.3487 36. Lozar T, Gersak K, Cemazar M, Grasic Kuhar C, Jesenko T. The biology and clinical potential of circulating tumor cells. Radiol Oncol 2019; 53: 131-47. doi: 10.2478/raon-2019-0024 37. Shin K, Lee J, Guo N, Kim J, Lim A, Qu L, et al. Hedgehog/Wnt feedback supports regenerative proliferation of epithelial stem cells in bladder. Nature 2011; 472: 110-4. doi: 10.1038/nature09851 38. Mullenders J, de Jongh E, Brousali A, Roosen M, Blom JPA, Begthel, et al. Mouse and human urothelial cancer organoids: a tool for bladder cancer research. Proc Natl Acad Sci USA 2019; 116: 4567-74. doi: 10.1073/ pnas.1803595116. Radiol Oncol 2020; 54(2): 135-143. 144 review Mechanical recanalization for acute bilateral cerebral artery occlusion - literature overview with a case Miran Jeromel12, Zoran V. Milosevic1, Janja Pretnar Oblak3 1 Department of Diagnostic and Interventional Neuroradiology, University Medical Centre Ljubljana, Ljubljana, Slovenia 2 Department of Diagnostic and Interventional Radiology, General Hospital Slovenj Gradec, Slovenj Gradec, Slovenia 3 Department for Vascular Neurology and Intensive Neurological Therapy, University Medical Centre Ljubljana, Ljubljana, Slovenia Radiol Oncol 2020; 54(2): 144-148. Received 19 January 2020 Accepted 5 March 2020 Correspondence to: Miran Jeromel, M.D., Ph.D., Department of Diagnostic and Interventional Radiology, General Hospital Slovenj Gradec, Gosposvetska cesta 1, SI-2380 Slovenj Gradec, Slovenia. E-mail: miran.jeromel@gmail.com Disclosure: No potential conflicts of interest were disclosed. Background. Acute bilateral internal carotid artery (ICA) and/or middle cerebral artery (MCA) occlusion is extremely rare and associated with poor clinical outcomes. There are only a few reports in the literature about mechanical thrombectomy being performed for acute bilateral occlusions. The treatment strategies and prognoses (clinical outcomes) are therefore unclear. Methods. A systematic review of the literature was performed through several electronic databases with the following search terms: acute bilateral stroke, mechanical recanalization and thrombectomy. Results. In the literature, we identified five reports of six patients with bilateral ICA and/or MCA occlusion treated with mechanical recanalization. Additionally, we report our experience with a subsequent contralateral large brain artery occlusion during intravenous thrombolytic therapy, where the outcome after mechanical thrombectomy was not dependent on the time from stroke onset but rather on the capacity of collateral circulation exclusively. Conclusions. Acute bilateral cerebral (ICA and/or MCA) occlusion leads to sudden severe neurological deficits (comas) with unpredicted prognoses, even when mechanical recanalization is available. As the collateral capacity seems to be more important than the absolute time to flow restoration in determining the outcomes, simultaneous thrombectomy by itself probably does not lead to improved functional outcomes. Key words: acute bilateral stroke; mechanical recanalization; thrombectomy Introduction Acute embolic bilateral internal carotid artery (ICA) and/or middle cerebral artery (MCA) occlusion leads to sudden comas with poor prognoses.1-9 The reported incidence of this condition in stroke patients treated with intravenous or intra-arterial therapy is 0.34%.8 There are few reports on the en-dovascular treatment (mechanical recanalization - thrombectomy) of this rare condition. The treat- ment strategies and prognoses (clinical outcomes) are therefore unclear. The aim of the present article was to discuss our experience with the treatment of this severe condition through a few published reports. To the best of our knowledge, we report the first case of subsequent contralateral large brain artery occlusion during intravenous thrombolytic therapy, where the outcome after mechanical thrombectomy was not dependent on the time from stroke onset but Radiol Oncol 2020; 54(2): 144-148. doi: 10.2478/raon-2020-0017 Jeromel M et.al. / Acute bilateral cerebral artery occlusion 145 rather on the capacity of collateral circulation exclusively. Methods A systematic review of the literature was performed through several electronic databases: PubMed (US National Library of Medicine, http ://www.ncbi.nlm.nih.gov/pubmed), Google Scholar (https://scholar.google.com/), Scopus (Elsevier, http://www.scopus.com/), DeGruyer (https://www.degruyer.com) and Cochrane Library (http://www.cocranelibrary.com). The following search terms were used: acute bilateral stroke, mechanical recanalization and thrombectomy. Results Altogether, five reports of six patients with bilateral ICA and/or MCA occlusion that met the inclusion criteria were identified (Table 1). Four patients were females (age range 64-78 years), one was male (72 years), and one was a middle-aged patient of an undetermined sex. All patients were treated with mechanical recanalization (thrombectomy) using different endovascular techniques (stent retriever, aspiration or a combination of both). Flow in the occluded artery was completely or partially restored in all cases. The clinical outcome ranged from complete recovery (without neurological deficits) to a coma (a fatal outcome). We present a case of a 77-year-old female patient with a medical history of hypertension, diabetes FIGURE 1. Initial imaging workup upon arrival at the general hospital. CT angiography (CTA) shows right Ml occlusion (arrow). mellitus type 2, hyperlipidaemia and atrial fibrillation who was admitted to the general hospital with an acute onset of left-sided hemiplegia and dysarthria (National Institutes of Health Stroke Scale [NIHSS]: 4, Modified Rankin Scale [mRS]: 3). The patient was last seen without deficits 80 minutes prior to admission. The initial computed tomography (CT) scan revealed no ischaemic brain damage, and CT angiography showed right M1 MCA occlusion (Figure 1). Intravenous thrombolysis (IVT) was administered after 173 minutes and discontinued due to the sudden loss of conscience, the deviation of the head toward the left side and tonic-clonic spasms of the left extremities. The control CT scan performed under general anaesthesia TABLE 1. Comparing 6 reported cases of mechanical thrombectomy in acute bilateral ICA and/or MCA occlusions Author, (Year), Sex/age (years) Site of occlusion Mechanical Clinical outcome reference Clinical presentation ICA MCA thrombectomy (technique) Dietrich et al. (2014)5 left hemiparesis, progressing to coma M/72 - + (M1) aspiration+stent- retriever minor deficit Pop et al. (2014)6 impaired consciousness F/78 + + (M2) stent-retriever no deficit Pop et al. (2014)6 right sided weakness F/66 + + (M1) stent-retriever severe deficit Braksick et al. (2018)7 coma F/76 - + (M1) - (no data) coma Larrew et al. (2019) 8 coma - (no data) / middle age + + - aspiration fatal Storey et al. 2019)9 hemiparesis / hemiplegia F/64 + + (M1,M2) aspiration+stent-retriever minor deficit F = female; ICA = internal carotid artery; M = male; MCA = middle cerebral artery Radiol Oncol 2020; 54(2): 144-148. 146 Jeromel M et.al. / Acute bilateral cerebral artery occlusion 146 FIGURE 2. (A) Control images taken in the general hospital after clinical deterioration during intravenous thrombolysis and before the transfer to the tertiary institution. There were still no signs of ischaemic brain damage in the right cerebral hemisphere but there were subtle signs of stroke in the left middle cerebral artery (MCA) territory (white line delineates loss of cortical grey matter - white matter differentiation in the frontoparietal lobe with sulcal effacement). (B) Control images taken in the general hospital after clinical deterioration during intravenous thrombolysis and before the transfer. CT angiography (CTA) showed persistent right Ml occlusion (black arrow) but also left carotid "T" occlusion (white arrow). (C) Control images taken in the general hospital after clinical deterioration during intravenous thrombolysis and before the transfer. CT perfusion imaging (CTP) showed a penumbra in the right MCA territory (black circle) and irreversible brain damage in the left MCA territory (white circle). showed no haemorrhagic complications. Since it was assumed that the patient had a symptomatic epileptic seizure, IVT was continued. The sedated patient was immediately transported to a tertiary institution, where a multimodal CT protocol (native CT scan, CT angiography [CTA] and CT perfusion imaging [CTP]) was performed, and the results revealed no signs of ischaemic brain damage in the symptomatic right cerebral hemisphere despite the presence of Ml occlusion and subtle (newly appeared) signs of irreversible brain damage in the contralateral MCA territory due to left ICA ("T") occlusion. It was obvious that an additional embolic occlusion occurred during the IVT treatment. Although the time of occlusion was long, the CTP imaging results showed a penumbra in the right MCA territory and not in the contralateral left MCA territory, where irreversible brain damage occurred within 3 hours (Figure 2). Conventional (digital subtraction) angiography confirmed right MCA occlusion with good collateral flow provided by the right anterior cerebral artery (AC A). However, the situation on the left side had changed. Namely, a complete recanaliza-tion of the carotid "T" occlusion was seen angio-graphically as a consequence of thrombolysis and distal migration of the thrombembolus (now presenting with proximal M2 segment occlusion of the major MCA branch). However, no collateral flow was observed despite complete left ACA patency (Figure 3). Endovascular mechanical recanaliza-tion with aspiration was successfully performed on the right side (Figure 4A). The same procedure was not performed on the left side because there was irreversible brain damage in the whole occluded arterial territory and a high risk of haemorrhagic complications. The postprocedural thrombolysis in cerebral infarction (TICI) scores were 3 (right MCA) and 2b (left MCA). The control CT and magnetic resonance imaging (MRI) scans performed on the next and subsequent days revealed no ischaemic damage on the right side and acute ischaemic stroke in the corresponding left MCA territory (Figure 4B). The patient was discharged with an improved clinical condition. However, severe neurological deficits (global dys-phasia, dysarthria, spastic hemiplegia of the right limbs) were a consequence of permanent brain damage on the left side (NIHSS 5, mRS 5). Discussion To the best of our knowledge, there are only six reports about mechanical thrombectomy being performed for acute bilateral ICA and/or MCA occlusions.5-9 A 2014 report by Dietrich et al. describes two Ml MCA thrombectomy procedures being performed in sequence. Aspiration was attempted without success, so multiple passes with Radiol Oncol 2020; 54(2): 144-148. Jeromel M et.al. / Acute bilateral cerebral artery occlusion 147 stent retrievers were performed for successful re-canalization.5 Another report from the same year (2014) by Pop et al. demonstrates two cases for which ICA-MCA (M1, M2) bilateral thrombectomy procedures with stent retrievers were successful.6 A recent report by Larrew et al. (2019) describes a novel successful method for bilateral ICA occlusion, simultaneous recanalization, which utilizes two interventionalists and technicians simultaneously for aspiration thrombectomy.8 In the latest report by Storey et al. (2019), sequential M1-M2 MCA mechanical thrombectomy was successfully performed using a combination of the stent retriever and aspiration techniques.9 A case of stroke similar to that described in our study was reported by Braksick et al. (2018), where M1 MCA occlusion also occurred during the thrombolytic treatment of contralateral M1 MCA occlusion. Despite attempts at clot retrieval, flow was not completely restored, and the patient remained comatose.7 As so few related cases have been reported, it is unclear whether the outcomes can be improved by the optimization of the assessment and endovascu-lar approach.10 A comparison of our case with the abovementioned cases shows that the initial clinical presentations are similar, as they are cases of rapid deterioration with a loss of conscience. A prompt, adequate workup and treatment (a successful combination of standard care and mechanical re-canalization) have led to clinical improvements and minimize morbidity and mortality.9 However, the clinical outcomes reported in the literature are still diverse.5-9 It is unclear whether simultaneous thrombectomy, as described by Larrew et al.8, can improve the outcome by means of faster recanali-zation. The answer can possibly be found in our case study. Namely, an important conclusion from our case study is that the collateral capacity was more important than the time from stroke onset to successful flow restoration. It is well known that good collateral circulation in acute stroke patients is associated with better clinical and functional out-comes.11 The results of the DAWN trial show that thrombectomy plus standard care compared to standard care alone, even 6 to 24 hours after acute ischaemic stroke, yields better functional improvements in people with mismatch between clinical deficit and infarction.12 It is becoming obvious that the presence of the collateral flow, which defines the minimal blood flow in the penumbra, is equally important as the time in stroke patients being assessed for IVT.13 The clinical outcome in our patient with bilateral stroke during the same thrombolytic time window therefore confirms the importance FIGURE 3. (A) Digital subtraction angiography (DSA) at the beginning of mechanical recanalization. Right internal carotid contrast injection confirming right Ml occlusion. (B) DSA at the beginning of mechanical recanalization. Left side contrast injection showing complete spontaneous recanalization of the carotid "T" occlusion with thrombembolar distal migration (occlusion of the proximal M2 segment of the major MCA branch) (arrow). of collaterals. Namely, the region within the brain with good collateral circulation showed a complete recovery after mechanical recanalization at 7 hours after stroke onset, while the other (contralateral) region without collateral circulation exhibited a poor outcome no more than 3 hours after stroke onset. Our case shows that even in the same individual, the presence of collateral circulation varies across regions. Namely, the affected brain territory, even with comparable anatomic vascular architecture (patent ACA with possible collateral inflow) as the contralateral territory, exhibited shorter time from onset of occlusion to recanalization (with distal thrombus migration) but did not exhibit collaterals, leading to an immediate and irreversible inju- l'ï Ar <3 •"i i V f ® ' % ® FIGURE 4. (A) Digital subtraction angiography (DSA) after mechanical recanalization. Right Ml mechanical recanalization (aspiration device) led to complete flow restoration. (B) MR diffusion weighted imaging (DWI) scan taken 6 days after mechanical recanalization: complete salvage of the affected right middle cerebral artery (MCA) brain parenchyma (recanalization at 7 hours after stroke onset). In contrast, subsequent persistent left M2 occlusion without collateral flow resulted in significant stroke within 3 hours after stroke onset. Radiol Oncol 2020; 54(2): 144-148. 148 Jeromel M et.al. / Acute bilateral cerebral artery occlusion 148 ry. To the best of our knowledge, we presented the first case of subsequent contralateral large cerebral artery (MCA-ICA/MCA) occlusion during IVT, where the outcome after standard care and mechanical thrombectomy was not dependent on the time from stroke onset but rather on the capacity of collateral circulation exclusively. Simultaneous thrombectomy, as described by Larrew et al.8, offers an efficient and feasible means to reduce the time to recanalization. However, our case study confirms the fact that the outcome is still very much dependent on the capacity of collateral circulation. The main drawback of simultaneous thrombectomy (compared to subsequent thrombec-tomy) is that it requires two neurointerventionalists and technicians (nurses). The procedure is technically challenging, and the team must be methodical and organized and communicate effectively to allow effective, efficient, simultaneous and safe progression on both sides.10 It is likely that simultaneous thrombectomy would not have any impact on the outcome in our case study of a patient with poor collaterals. Interestingly, the TICI scores for the patient in our study were the same as those reported for patients treated with simultaneous thrombecto-my in previous study (TICI 3 for one side and TICI 2b for contralateral side). The presence of collateral capacity, the location of the residual occlusion (MCA vs ACA-MCA), and a concomitant disease (better cardiac function) were key determinants of better outcomes in the patient in our study. Understanding the importance of collaterals and preprocedural imaging with techniques that enable collateral flow assessment is becoming extremely important. Multiphase CT angiography (CTA), which enables the evaluation of collateral circulation within a single contrast injection, is a simple example.14 It is already accepted that for good outcomes, the onset-to-reperfusion time window should be adjusted according to the collateral status.15 In the future, perhaps a pretreatment imaging assessment of the thrombus itself will make stroke treatment planning easier.16 However, it seems that even in the era of mechanical recanali-zation, bilateral stroke is a severe condition with unpredicted outcomes. Conclusions Acute bilateral cerebral (ICA and/or MCA) occlusion leads to sudden severe neurological deficits (comas) with unpredicted prognoses, even in the era of mechanical recanalization. As a the collateral capacity seems to be more important than the absolute time to flow restoration in determining the outcomes, simultaneous thrombectomy by itself probably does not lead to improved functional outcomes. Because there are only a few reported cases, additional experience is needed to fully understand the outcomes of sequential and simultaneous thrombectomy. References 1. Kwon SU, Lee SH, Kim JS. Sudden coma from acute bilateral internal carotid artery territory infarction. Neurology 2002; 58: 1846-9. doi: 10.1212/ wnl.58.12.1846 2. Hu WT, Wijdicks EF. Sudden coma due to acute bilateral M1 occlusion. Mayo Clin Proc 2007; 10: 1155. doi: 10.4065/82.10.1155 3. Zubkov AY, Klassen BT, Burnett MS, Rabinstein AA. Bilateral internal carotid artery occlusions resulting in near total acute brain infraction. Neurocrit Care 2007; 7: 247-9. doi: 10.1007/s12028-007-0076-y 4. Nawashiro H, Wada K, Kita H. Decerebrate posture following bilateral middle cerebral artery occlusion. Intern Med 2011; 50: 2063. doi: 10.2169/ internalmedicine.50.5843 5. Dietrich U, Graf T, Schabitzb WR. Sudden coma from acute bilateral M1 occlusion: successful treatment with mechanical thrombectomy. 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Kim BM, Beak JH, Heo JH, Nam HS, Kim YD, Yoo J, et al. Collateral status affects the onset-to-reperfusion time window for good outcome. J Neurol Neurosurg Psychiatry 2018; 89: 903-9. doi: 10.1136/jnnp-2017-317627 16. Vidmar J, Bajd F, Milosevic ZV, Kocijancic IJ, Jeromel M, Sersa I. Retrieved cerebral thrombi studied by T2 and ADC mapping: preliminary results. Radiol Oncol 2019; 53: 427-33. doi: 10.2478/raon-2019-0056 Radiol Oncol 2020; 54(2): 144-148. 149 research article Major and ancillary features according to LI-RADS in the assessment of combined hepatocellular-cholangiocarcinoma Vincenza Granata1, Roberta Fusco1, Sergio Venanzio Setola1, Fabio Sandomenico1, Maria Luisa Barretta1, Andrea Belli2, Raffaele Palaia2, Fabiana Tatangelo3, Roberta Grassi4, Francesco Izzo2, Antonella Petrillo1 1 Radiology Division, "Istituto Nazionale Tumori IRCCS Fondazione Pascale - IRCCS di Napoli", Naples, Italy 2 Hepatobiliary Surgical Oncology Division, "Istituto Nazionale Tumori IRCCS Fondazione Pascale - IRCCS di Napoli", Naples, Italy 3 Pathology Diagnostic Division, "Istituto Nazionale Tumori IRCCS Fondazione Pascale - IRCCS di Napoli", Naples, Italy 4 Division of Radiology, University of Campania Luigi Vanvtelli, Naples, Italy Radiol Oncol 2020; 54(2): 149-158. Received 6 February 2020 Accepted 22 April 2020 Correspondence to: Roberta Fusco, M.D., Department of Radiology, Istituto Nazionale Tumori Fondazione G. Pascale, Naples, I-80131, Italy. E-mail: r.fusco@istitutotumori.na.it Disclosure: No potential conflicts of interest were disclosed. Background. The aim of the study was to investigate the performance of the Liver Imaging Reporting and Data System (LI-RADS) v2018 for combined hepatocellular-cholangiocarcinoma (cHCC-CCA) identifying the features that allow an accurate characterization. Patients and methods. Sixty-two patients (median age, 63 years; range, 38-80 years), with pre-surgical biopsy diagnosis of hepatocellular carcinoma (HCC) that underwent hepatic resection, comprised our retrospective study. All patients were subject to multidetector computed tomography (MDCT); 23 patients underwent to magnetic resonance (MR) study. The radiologist reported the presence of the HCC by using LIRADS v2018 assessing major and ancillary features. Results. Final histological diagnosis was HCC for 51 patients and cHCC-CCA for 11 patients. The median nodule size was 46.0 mm (range 10-190 mm). For cHCC-CCA the median size was 33.5 mm (range 20-80 mm), for true HCC the median size was 47.5 mm (range 10-190 mm). According to LIRADS categories: 54 (87.1%) nodules as defined as LR-5, 1 (1.6%) as LR-3, and 7 (11.3%) as LR-M. Thirty-nine nodules (63%) showed hyper-enhancement in arterial phase; among them 4 were cHCC-CCA (36.4% of cHCC-CCA) and 35 (68.6%) true HCC. Forty-three nodules (69.3%) showed washout appearance; 6 cHCC-CCAs (54.5% of cHCC-CCA) and 37 true HCC (72.5%) had this feature. Only two cHCC-CCA patients (18.2% of cHCC-CCA) showed capsule appearance. Five cHCC-CCA (71.4% of cHCC-CCA) showed hyperintensity on T2-W sequences while two (28.6%) showed inhomogeneous signal in T2-W. All cHCC-CCA showed restricted diffusion. Seven cHCC-CCA patients showed a progressive contrast enhancement and satellite nodules. Conclusions. The presence of satellite nodules, hyperintense signal on T2-W, restricted diffusion, the absence of capsule appearance in nodule that shows peripheral and progressive contrast enhancement are suggestive features of cHCC-CCA. Key words: hepatocellular carcinoma; combined hepatocellular-cholangiocarcinoma; multidetector computed tomography; magnetic resonance imaging. Introduction liver tumour consisting of mixed elements of hepa- tocellular carcinoma (HCC) and cholangiocarci-Combined hepatocellular-cholangiocarcinoma noma (CCA) or cancer cells with hepatic progeni-(cHCC-CCA) is considered a rare entity of primary tor/stem cell traits.12 The incidence of cHCC-CCA Radiol Oncol 2020; 54(2): 144-148. doi: 10.2478/raon-2020-0017 150 Granata V et al. / LI-RADS in the assessment of combined hepatocellular-cholangiocarcinoma ranging from 1.0%-4.7% of all primary hepatic tumours.34 Described risk factors are male gender, cirrhosis, hepatitis infection (hepatitis B virus [HBV] and hepatitis C virus [HCV]), family history of liver cancer, heavy alcohol consumption and diabetes mellitus.5-10 Therefore, that cHCC-CCA is associated with overlapping clinical features of both HCC and CCA.3 An exact pre-surgical diagnosis is very complicated and, it is due to its heterogeneous imaging characteristics with overlapping features of HCC and CCA. The predominant histologic elements within the tumour determine the predominant radiographic features.3 Therefore, in this scenario the hallmark radiological findings of HCC show an overlapping with those of CCA.3 Since cHCC-CCAs are predominant in patients at high risk of HCC, and the possibility that this tumour can mimic HCC in imaging appearance, this is problematic considering the current dependence on the non-invasive diagnosis of HCC.11-17 Specifically, given that surgical resection is the current standard of care for cHCC-CCAs and controversies surround the appropriateness of other therapies, such us ablative therapies, imaging misdiag-nosis of cHCC-CCA can lead to non standard treatments for cHCC-CCA.18 The current imaging-based criteria to characterize a HCC lesion have several limitations, including the lack of established consensus regarding the exact definitions of imaging features, binary categorization (either definite or not definite HCC), and failure to address non-HCC malignancies and vascular invasion.13 Therefore, The American College of Radiology sustained the spread of Liver Imaging Reporting and Data System (LI-RADS) to homogenizing the interpreting, reporting and data collection of HCC imaging. LI-RADS is a scheme for interpreting and reporting of imaging features on multidetector computed tomography (MDCT) and magnetic resonance (MR) studies in patients at risk for HCC. In the current (v2018) LI- RADS19, the diagnosis of HCC is based on the presence of major imaging features. These are features used to categorize LI-RADS-category 3 (LR-3), LI-RADS- category 4 (LR-4), and LI-RADS-category 5 (LR-5) and include arterial-phase hyperenhancement, tumour diameter, washout appearance, capsule appearance, and thresh-old growth. Ancillary features favoring HCC diagnosis include the hepatobiliary phase hypointensity (after administration of liver-specific MR contrast agent), transitional phase hypointensity, mild to moderate T2 hyperintensity, restricted diffusion, distinctive rim, corona enhancement, mosaic architecture, nodule-in-nodule architecture, intra-lesional fat, lesional iron or fat sparing, blood products, and diameter increase less than the threshold growth. The presence of ancillary features favoring malignancy may be used to up- grade by one category, but not beyond LR-4 (e.g. from LR-3 to LR-4). Absence of ancillary features must not be used to downgrade an LR category.19 The purpose of this study is to investigate the performance of the LI-RADS v2018 for cHCC-CCA identifying which features allow an exact characterization respected to HCC. Patients and methods Study population The institutional review board approved this retrospective study, and the requirement for patient informed consent was waived. We searched the surgical database at our institution from January 2013 to September 2018 and selected 74 patients with pre surgical biopsy and radiological diagnosis of HCC, who underwent hepatic resection. The inclusion criteria for the study population were as follows: (a) patients who had pathologically-proven HCC; (b) patients who had undergone MR imaging and liver MDCT with less than a 1-month interval between imaging modalities; (c) patients who had less than a 1-month interval between imaging and pathologic diagnosis; and (d) availability of diagnostic quality pictures of the cut sections of the resected specimens in patients who underwent surgical resection for matching of imaging and pathology findings. The exclusion criteria were as follows: (a) conflict between the imaging-based diagnosis and the pathologically confirmed diagnosis, (b) no available MR or MDCT images. In total, 73 patients with HCC confirmed at pathology fulfilled the inclusion criteria during the study period. Among them, 11 patients were excluded for the following reasons: (a) 4 patients no had available MR or MDCT images and (b) 7 patients because the final diagnosis were not HCC. Finally, 62 patients (14 women, 48 men; median age, 63 years; range, 38-80 years), with pre-surgi-cal biopsy and radiological diagnosis of HCC comprised our study population. Characteristics of the 62 patients are summarized in Table 1. Lesion confirmation: reference standard All original pathological samples were reviewed by one experienced hepatic pathologist (F.T.). Lesions were confirmed histopathologically as he- Radiol Oncol 2020; 54(2): 149-158. 23 Granata V et al. / LI-RADS in the assessment of combined hepatocellular-cholangiocarcinoma patic tumours comprising unequivocal elements of both HCC and CC according to the tumour classification of the World Health Organization. The CC component was defined as glandular differentiation with mucin production, while the HCC component was defined as trabecular, solid sheet, or pseudoacinar arrangements with interspersed sinusoids. All pathological samples displayed an intimate intermingling of trabecular hepatocellu-lar and true glandular elements (type C). The bidirectional differentiation was further supported by immunohistochemical stain. For each specimen, biliary differentiation was confirmed with mucin positivity or with immunohistochemical stains characteristic of bile duct differentiation (cy-tokeratin 7, cytokeratin 19), whereas hepatocellular differentiation was confirmed with immunohisto-chemical stains characteristic of hepatocyte differentiation (Hepatocyte-Paraffin-1). MDCT and MR examinations All patients underwent to MDCT and 23 to MR. TABLE 1. Characteristics of the 62 selected patients Description Gender Age Number of hepatic nodules Single nodule Multiple nodules Nodule size [mm] Risk factor for HCC Chronic hepatitis B; HBV-related liver cirrhosis Chronic hepatitis C; HCV-related liver cirrhosis Alcoholic liver cirrhosis Child-Pugh Classification A B Numbers (%)/ range Men 48 [77.4%] Women 14 [22.6%] 63 y; range. 38-80 y 62 [100%] / median size 46.0 mm; range 10-190 mm 37 [59.7%] 23 [37.1] 2 [3.2%] 62 [100%] HBV = hepatitis B virus; HCC = hepatocellular carcinoma; hepatitis C virus MDCT protocol MDCT was performed with a 64-detector row scanner (Optima 660, GE Healthcare, United States). MDCT scanning parameters were 120 kVp, 100470 mAs (NI 16.36), 2.5 mm slice thickness and table speed 0.984/1 mm/rotation. Scans were carried out including a region encompassing the liver from diaphragm to iliac crests. Liver protocol examinations were composed of quadruple phases, including the unenhanced, arterial, portal venous, and equilibrium phases. CT images were obtained after injection of 120 mL of a nonionic contrast medium (iomeprol, Iomeron 400, Bracco, Milan, Italy) at a rate of 3.0-4.0 mL/sec by using an automatic power injector (Empower CTA, E-Z-EM Inc., New York, United States). Image acquisition in the arterial phase was initiated 19 seconds after attenuation in the descending aorta reached 100 HU, as measured with the bolus tracking method; in the portal venous phase, images were acquired 33 seconds after the arterial phase; in the equilibrium phase, images were acquired 180 seconds after administration of contrast media. MR imaging protocol MR imaging was performed by using a 1.5 T scanner (Magnetom Symphony, with Total Imaging Matrix Package, Siemens, Erlangen, Germany) with an 8-element body coil and a phased array coil. Our routine liver MR imaging protocol consisted of a breath-hold fat-saturated and not fat-saturated T2-weighted turbo spin-echo sequence, an in- and opposed-phase T1-weighted gradient-echo sequence, dynamic imaging with a fat-saturated T1-weighted gradient-echo sequence, and diffusion-weighted imaging. Diffusion weighted imaging (DWI) was obtained with planar echo-pulse sequence (b values 0, 50, 100, 200, 400, 600, and 800 s/mm2). A non-specific agent the Gd-BT-DO3A (Gadovist, Bayer Schering Pharma, Germany) was employed. All patients received 0.1 ml/kg of Gd-BT-DO3A by means of a power injector (Spectris Solaris® EP MR, MEDRAD Inc., Indianola, IA, USA), at an infusion rate of 2 ml/s followed by a 30-mL saline flush. Arterial phase images were acquired 7 seconds after contrast material arrival at the thoracic aorta by using an MR fluoroscopic monitoring system. Thereafter, portal venous phase and equilibrium phase were obtained 60 seconds and 3 minutes after contrast material administration, respectively. Detailed information regarding the MR imaging parameters are summarized in Table 2. Image analysis For each patient, MDCT and MR images were independently and blindly evaluated in random or- Radiol Oncol 2020; 54(2): 149-158. 152 Granata V et al. / LI-RADS in the assessment of combined hepatocellular-cholangiocarcinoma der within and between three radiologists (V.G., S.V.S., A.P.; 10, 15, and 20 years of experience in abdominal imaging). A consensus evaluation was performed when there was disagreement between the readers. The readers were blinded to previous radiological examination, pathologic results and history of previous treatment but were aware that the patients had cirrhosis and thus were at higher risk for HCC. To reduce recall bias, all three readers maintained an interval of more than 2 weeks between interpretation sessions of MR and MDCT images. Each radiologist was asked to identify the presence of lesion, that was considered to be detectable if the nodule had attenuation or signal intensity that differed from that of the surrounding liver parenchyma. Thereafter, they reported the presence of the HCC by using LIRADS v2018 assessing major and ancillary features19; also the radiologists reported any radiological accessory findings if detected. Readers assessed and recorded the following parameters: greatest nodule diameter, attenuation at unenhanced CT, signal intensity on T1- and T2-weighted images, vascular hyperenhancement pattern during arterial phase (wash-in), wash-out appearance during portal phase, vascular enhancement during equilibrium or late phases.13 Region of interests (ROIs) have been manually drawn by an expert radiologist on T1-w and T2-w images and on DW images at the highest b value (including hyperintense voxels at b value 800 s/ mm2) considering the same slices position. The contours of lesions were validated by another expert radiologist of 25 years of experience. The signal intensity of the lesions in T1-w and T2-w images was categorized subjectively as isointense, hypointense, and hyperintense compared to surrounding liver parenchyma. We assessed the signal on DWI sequences and measured the apparent diffusion coefficient (ADC) of each lesion. The diffusion weighted signal decay was analyzed using the mono-exponential model, according to the equation, the apparent diffusion coefficient ADC = (ln [S0/ Sb])/b, where Sb is the signal intensity with diffusion weighting b and S0 is the non-diffusion-weighted signal intensity. This analysis was ROI-based using median value of single voxel signals for each b value. Median diffusion parameters of ROI were used as representative values for each lesion. No motion correction algorithm was used but ROIs were drawn taking care to exclude areas in which movement artifacts or blurring caused voxel misalignments. We analysed the enhancement pattern during arterial, portal, equilibrium or late phase and described it as homogeneous, heterogeneous, or progressive. We described the capsule appearance, defined as a peripheral rim of smooth hyperenhancement in the portal or delayed phase, as complete or partial. In addition, we recorded the number and segmental location of the nodule for all detected lesions and the presences of satellite nodules. Statistical analyses Each continuous variable was expressed in terms of median value ± range while each variable categorical was summarized by frequencies and percentages. Fisher's exact test was performed to assess statistically significant difference between percentage values. Mann Whitney non parametric test were used to compare a continuous variable between 2 groups. A p value < 0.05 was considered statistically significant. All statistical analysis was performed with SPSS for Windows (Version 23.0; SPSS Inc, Chicago, Ill). Results We assessed 62 patients that underwent surgical treatment with preoperative diagnosis of HCC. According to the surgical procedure, 10 patients underwent to lobectomy, 3 meso-hepatectomy, 23 bi-segmentectomy and 27 segmentectomy (8 for VII, 1 for I, 2 for II, 2 for III, 6 for IV, 6 for V and 2 for VI hepatic segment). Pathological features After pathological evaluation the final diagnosis was HCC for 51 patients and cHCC-CCA for 11 patients (17.7%). Twelve patients were classified as G3 (19.4%) and 50 G2 (80.6%) according to the grading system of Edmondson-Steiner.21 Among cHCC-CCA 8 patients were classified as G2 (72.7%) and 3 as G3 (27.3%). In 8 out of 11 (72.7%) cHCC-CCA microvascular infiltration was reported. In two cHCC-CCA patients were reported nodal metastases. Imaging features All lesions were detected and analyzed by readers. The consensus in the assessment of the nodules Radiol Oncol 2020; 54(2): 149-158. 153 Granata V et al. / LI-RADS in the assessment of combined hepatocellular-cholangiocarcinoma FIGURE 1. Man 56 y with combined hepatocellular-cholangiocarcinoma (cHCC-CCA) on VI hepatic segment. MRI study. The nodule is iso-hyperintense (arrow) in T2-W sequence (A), with inhomogeneous hypervascular appearance (arrow) during arterial phase of contrast study (B), without wash-out or capsule appearance (arrow) during portal phase of contrast study (C). The nodule shows restricted (arrow) diffusion (D, E and F) in diffusion weighted imaging (DWI) sequences. was 100%. The median nodule size was 46.0 mm (range 10-190 mm). For cHCC-CCA the median size was 33.5 mm (range 20-80 mm), for true HCC the median size was 47.5 mm (range 10-190 mm). The consensus between pre-surgical radiological report and second evaluation was 100% for each LIRADS categories: LR-5 for 54 (87.1%) nodules, LR-3 for 1 (1.6%) lesion, and LR-M for 7 (11.3%) nodules. Thirty-nine nodules (63%) showed hyperenhancement in arterial phase; among them 4 were cHCC-CCA (36.4% of cHCC-CCA) and 35 (68.6%) true HCC. Twenty nodules (32.2%) showed an inhomo-geneous hyperenhancement in arterial phase; 7 of them were cHCC-CCA (63.6% of cHCC-CCA) (Figure 1). We found this feature in 13 (25.5%) true HCC. Forty-three nodules (69.3%) showed wash-out appearance; 6 cHCC-CCAs (54.5% of cHCC-CCA) and 37 true HCC (72.5%) had this feature. We found inhomogeneous wash-out in 13 (20.9%) nodules; 4 nodules with inhomogeneous wash-out were cHCC-CCA (35.4% of cHCC-CCA). A one cHCC-CCA (9.1% of cHCC-CCA) patient did not show this feature. Nine true HCC (17.6%) showed inhomogeneous wash-out. Thirty-three (53.2%) nodules showed capsule appearance, 28 (45.2%) did not show this feature and in one patient we found a peripheral halo sign. Only two cHCC-CCA patients (18.2% of cHCC-CCA) showed capsule appearance while 9 cHCC-CCAs (81.8% of cHCC-CCA) did not have this feature (Figure 2). Thirty-one (60.8%) true HCC showed capsule appearance and 19 (37.2%) true HCC did not show this feature. Only 23 patients underwent MR study, among them 7 out of cHCC-CCA. We found T2 hyperin-tensity of signal in 20 nodules (86.9%), two lesions (8.6%) were isointense and one (4.3%) hypointense. Five cHCC-CCA (714 % of cHCC-CCA) showed hyperintensity on T2-W sequences while two (28.6%) showed inhomogeneous signal in T2-W (Figure 3). Fifteen true HCC (93.7%) had hyperintense signal on T2-W and one true HCC (6.2%) inhomoge-neous signal on T2-W. We found restricted diffusion in 23 (100%) nodules with median ADC of 975.6 x 10-3mm2/s. All cHCC-CCA showed restricted diffusion with median ADC of 880.7 x 10-3 mm2/s. All true HCC showed restricted diffusion with median ADC of 1210.0 x 10-3 mm2/s. Radiol Oncol 2020; 54(2): 149-158. 154 Granata V et al. / LI-RADS in the assessment of combined hepatocellular-cholangiocarcinoma FIGURE 2. Woman 68 y with combined hepatocellular-cholangiocarcinoma (cHCC-CCA) on VI hepatic segment. Multidetector computed tomography (MDCT) study. The nodule shows hypervascular appearance (arrow) during arterial phase of contrast study (A), with wash-out appearance (arrow) and without capsule appearance (arrow) during portal and late phase of contrast study (B and C). Progressive contrast enhancement: nine (14.5%) patients showed a progressive contrast enhancement; among them 7 were cHCC-CCA (63.6% of cHCC-CCA) (Figure 4) and 2 (3.9% of HCC) true HCC. In ten patients (16.1%) we found satellite nodules (neighboring micrometastases), among them 7 were cHCC-CCA (63.6% of cHCC-CCA) (Figure 5) and 3 (5.9% of HCC) were true HCC. FIGURE 3. Woman 58 y with combined hepatocellular-cholangiocarcinoma (cHCC-CCA) on IV-V-VIII hepatic segment. MRI study. Pre surgical radiological diagnosis was cholangiocarcinoma (CCA). The lesion shows inhomogeneous hyperintense signal (arrow) in T2-W sequence (A) with central more hyperintense area. In T1-W in-out phase sequence (B and C) the lesion is inhomogeneous hypointense (arrow). During contrast study (D: arterial phase, E: portal phase; F: late phase) the lesion shows progressive contrast enhancement (arrow). In diffusion weighted imaging (DWI) (G, H and I) it shows restricted diffusion (arrow). Radiol Oncol 2020; 54(2): 149-158. 155 Granata V et al. / LI-RADS in the assessment of combined hepatocellular-cholangiocarcinoma FIGURE 4. Man 71 y with combined hepatocellular-cholangiocarcinoma (cHCC-CCA) on VI hepatic segment. MRI study. The nodule shows hyperintense signal (arrow) in T2-W sequence (A) and target like pattern of enhancement (arrow) during arterial (B) and portal (C) phase of contrast study. Restricted diffusion (arrow) in diffusion weighted imaging (DWI) (D, E and F) sequence. FIGURE 5. Man 69 y with combined hepatocellular-cholangiocarcinoma (cHCC-CCA) on VI hepatic segment. MRI study. The nodule shows inhomogeneous hyperintense signal (arrow) in T2-W sequence (A) and progressive pattern of enhancement (arrow] during arterial (B) and portal (C) phase of contrast study. In (D, E and F) arrow shows a nodule satellite. Restricted diffusion (arrow) in diffusion weighted imaging (DWI) (G, H and I) sequence. Radiol Oncol 2020; 54(2): 149-158. 28 Granata V et al. / LI-RADS in the assessment of combined hepatocellular-cholangiocarcinoma TABLE 2. Imaging features in study population True HCC (n 51) cHCC-CCA (n 11) P value* Arterial hyperenhancement Yes No Inhomogeneous Wash-out appearance Yes No Inhomogeneous Capsule appearance Yes No Inhomogeneous MR features T2-W hyperintensity Yes Inhomogeneous Diffusion restricted Yes No Median ADC Progressive contrast enhancement Satellite nodules 35 [68.6%] 3 [5.8%] 13 [25.5%] 37 [72.5%] 5 [17.6%] 9 [17.6%] 31 [60.8%] 19 [37.2%] 1 [1.9%] alo-sign 16 15 [93.7%] 1 [6.2%] 15 [100%] 1210 x 10-3mm2/s 2 [3.9%] 3 [5.9%] 4 [36. 4%] 0 [0%] 7 [63.6%] 6 [54.5%] 1 [9.1%] 4 [35.4%] 2 [18.2%] 9 [81.8%] 0.04 0.38 0.03 5 [71.4%] 0.14 2 [28.6%] 7 [100%] ).7 x 10-3mm2/s 0.03 7 [63.6%] <<0.001 7 [63.6%] <<0.001 * Fisher's exact test ADC = apparent diffusion coefficient; hepatocellular-cholangiocarcinoma; HCC = hepatocellular carcinoma; cHCC-CCA = combined MR = magnetic resonance Statistical analysis The hyperenhancement in arterial phase (p value = 0.04), the absence of the pseudocapsule (p value = 0.03), progressive contrast enhancement (p value < 0.001) and satellite nodules (neighboring microme-tastases, p value < 0.001) showed percentages statistically different respect to the presence of combined HCC and cholangiocarcinoma at Fisher's exact test (see Table 2). A statistically significant difference (p value = 0.03 at Mann Whitney test) was detected between ADC median value of the two groups pure HCC and cHCC-CCA group. Discussion In this study, we assessed 62 patients with liver single nodule that underwent surgical treatment with preoperative biopsy diagnosis of HCC and pre-surgical radiological diagnosis of HCC in 54 lesion and LR-M in 7 lesions. All 7 lesions classified as LR-M were cHCC-CCA; 4 cHCC-CCA patients were wrongly identified as HCC patients. In this subgroup the median size lesion was 21 mm, while the subgroup LR-M showed a median size of 54 mm. In this scenario we think that radiological features are also related to median lesion diameter. When we assessed the major features, we found that arterial hyperenhancement, portal wash-out and capsule appearance were more frequent for true HCC group (68.6%, 72.5%, and 60.8%, respectively) than cHCC-CCA group (36.4%, 54.5%, and 18.2%, respectively). Our results are different from what is reported by Jeon et al.18 In fact, these researches reported that a substantial proportion of cHCC-CCAs was categorized as LR-5 or LR-4 showing higher frequencies of major HCC features: arterial hyperenhancement was present in 96.2% of cHCC-CCA, washout appearance in 80.8% of cHCC-CCA and enhancing capsule in 34.6% of cHCC-CCA.18 Arterial phase hyperenhancement is considered a crucial precondition to define HCC19, and it is considered the most important feature for imaging diagnosis. This feature reflects the neoangiogenesis, which is associated with the stepwise process of carcinogenesis and becomes the dominant blood supply in overt HCC lesions.13 However, it is non-specific condition and may be detected in benign pathologies. Also in our previous study we demonstrated that arterial phase hyperenhancement is a prerequisite but not sufficient for LR-5 classification.12 The post-contrast feature, "rim arterial phase hyperenhancement" is a subtype of LR-M in which arterial phase enhancement is most pronounced in observation periphery and it is defined as a''target appearance''. ''Target appearance'' or ''targetoid mass'' includes other features as peripheral "Washout", in which apparent washout is most pronounced in observation periphery, and delayed central enhancement, in which we find a central area of progressive post-arterial phase enhancement. In our study we found that cHCC-CCAs showed in 63.6% of lesions inhomogeneous arterial contrast enhancement, with a peripheral rim during arterial phase of contrast study and a progressive contrast enhancement (63.6% of lesions). Our findings 7 Radiol Oncol 2020; 54(2): 149-158. 157 Granata V et al. / LI-RADS in the assessment of combined hepatocellular-cholangiocarcinoma are comparable with the results of Lee et al.21 that found that in cHCC-CCA group the more frequent radiological findings were suggestive of LR-M.21 In fact. Also Potretzke et al. found that 93.4% of cHCC-CCA showed at least one ancillary feature favouring non-HCC malignancy.22 Conversely, Sammon et al.23 reported that arterial enhancement was seen in 90.9% (n. 30) of the cHCC-CCA group, although the most common enhancement patterns in the cHCC-CCA group were peripheral persistent and heterogeneous hyperenhancement with washout. In our study, the wash-out appearance was seen in 54.5% (n. 6) of patients, so we think it is not a feature that allow identifying that patients as a cHCC-CCA. Conversely, the absence of capsule appearance is more frequent (81.8%) in cHCC-CCA group than in true HCC group (37.2%). Therefore, according to our results, the absence of capsule appearance in nodule that shows peripheral and progressive contrast enhancement should guide the radiologist in differential diagnosis, since these features are more specific for cHCC-CCA. Also Fowler et al. confirmed that peripheral arterial enhancement was the most common pattern observed in their study population. The target like or reverse target like pattern of enhancement, was a common feature of cHCC-CCA; this pattern was rarely seen with HCC in their population.24 In our study, only 23 patients were subject to pre-surgical MR study, among them only 7 cHCC-CCA patients. According to our protocol study for lesion characterization, we employed a non-hepatospecific contrast medium, so no one of our patients was subject to EOB-MR. In our previous study we showed that when we analyzed the degree of arterial phase hyperenhancement, we found that the degree was higher with Gd-BT-DO3A than GD-EOB-DTPA, with significant statistically difference. In addition, the image quality degradation was lower with Gd-BT- DO3A than with GD-EOB-DTPA. There was significant statistically difference between the quality on arterial phase with Gd-BT-DO3A and the quality on arterial phase with GD-EOB-DTPA.12 Regarding the signal observed on T2-W sequences we found that 15/16 (93.7%) of true HCC showed hyperintense signal on T2-W and 5/7 (71.4%) of cHCC-CCA had this feature. T2-W hyperintensity was a highly specific marker of nodule malignancy, although poorly sensitive.13 Kim et al.25 evaluated the most predictive finding among hyperintensity on T2-W, DWI, washout, capsular enhancement, and hypointensity on gadoxetic acid-enhanced hepatobiliary phase images in the detailed char- acterization of arterial phase enhancing nodules 1 cm in diameter and smaller. They showed that for hypervascular lesions 1 cm in diameter or smaller, T2-weighted images have the highest sensitivity among tests with an odds ratio statistically separable from 1 for differentiating HCC from benign hypervascular lesions 1 cm or smaller.25 Our results confirmed that hypeintensity on T2-W is suggestive of malignancy, in fact 20/23 nodules (86.9%) showed this feature, however it is not allow characterizing cHCC-CCA as a separate lesion to HCC. In our study, all cHCC-CCA patients showed restricted diffusion with a median ADC value of 880.7 x 10-3mm2/s so as all true HCC showed restricted diffusion with median ADC of 1210.0 x 10-3 mm2/s. DWI has been applied to liver imaging as an excellent tool for detection and characterization of focal liver lesions, increasing clinical confidence and decreasing false positives.11 DWI could be used as a helpful diagnostic tool for HCC in patients with chronic liver disease, since DWI can accurately detect HCC in patients with chronic liver disease regardless of the lesion size.1112 A potential reason for the better accuracy of DWI is that this does not rely on morphologic features only. Malignant tissues tend to be hypercellular with an accumulation of macromolecular proteins leaving a small extracellular space resulting in a decrease of the ADC value. The major limits of DWI are the different parameters used in DWI sequences that may affect the results of ADC calculation.1112 In our study, ADC value could allow to distinguish cHCC-CCA as a separate group from true HCC patients. An interesting radiological finding that we found was the presence in cHCC-CCA group of satellite nodules, expression of micrometastases, as confirmed by pathologist. In fact, we found this finding in 63.6% (n. 7) cHCC-CCA patients and only in 5.9% (n. 3) true HCC. At the best of our knowledge, no previous study reported this result. We think that it is linked to more aggressiveness of this type of lesion, data confirmed also by the presence of nodal metastases in two cHCC-CCA patients. There are several limitations to our study. First, the sample size was small because of the rarity of cHCC-CCA and it is a single-center experience. Second that this is a retrospective study and the readers were aware, the cohort comprised of cHCC-CC and HCC. Third, the study is defective of radiology-pathology correlation. Our future prospective is to assess radiology-pathology correlation to better show how the relative composition of these mixed tumours translates into their imaging appearance. Radiol Oncol 2020; 54(2): 149-158. 158 Granata V et al. / LI-RADS in the assessment of combined hepatocellular-cholangiocarcinoma Conclusions A proper diagnosis of cHCC-CCA is mandatory given that surgical resection is the current standard of care for cHCC-CCAs and controversies surround the appropriateness of other therapies. An exact pre-surgical diagnosis is very complicated due to its heterogeneous imaging characteristics with overlapping features of HCC and CCA. However according to our results in patients at risk for HCC, when the lesion shows satellite nodules, hyperintense signal on T2-W, restricted diffusion and especially the absence of capsule appearance in nodule that shows peripheral and progressive contrast enhancement, the radiologist should consider the diagnosis of cHCC-CCA. Acknowledgements The authors are grateful to Alessandra Trocino, librarian at the National Cancer Institute of Naples, Italy. Moreover, for the collaboration, authors are grateful to Assunta Zazzaro and Dr Ivano Rossi, TSRM at Radiology Division, "Istituto Nazionale Tumori IRCCS Fondazione Pascale - IRCCS di Napoli", Naples, I-80131, Italy. References 1. Garancini M, Goffredo P, Pagni F, Romano F, Roman S, Sosa JA, et al. Combined hepatocellular-cholangiocarcinoma: a population-level analysis of an uncommon primary liver tumor. Liver Transpl 2014; 20: 952-9. doi: 10.1002/lt.23897 2. Joo I, Kim H, Lee JM. Cancer stem cells in primary liver cancers: pathological concepts and imaging findings. Korean J Radiol 2015; 16: 50-68. doi: 10.3348/kjr.2015.16.1.50 3. Gera S, Ettel M, Acosta-Gonzalez G, Xu R. Clinical features, histology, and his-togenesis of combined hepatocellular-cholangiocarcinoma. World J Hepatol 2017; 9: 300-9. doi: 10.4254/ wjh. v9. i6.300 4. Akiba J, Nakashima O, Hattori S, Tanikawa K, Takenaka M, Nakayama M, et al. Clinicopathologic analysis of combined hepatocellular-cholangiocarcino-ma according to the latest WHO classification. Am J Surg Pathol 2013; 37: 496-505. doi: 10.1097/PAS.0b013e31827332b0 5. Portolani N, Baiocchi GL, Coniglio A, Piardi T, Grazioli L, Benetti A, et al. Intrahepatic cholangiocarcinoma and combined hepatocellular-cholangio-carcinoma: a Western experience. Ann Surg Oncol 2008; 15: 1880-90. doi: 10.1245/s10434-008-9933-y 6. Yeh MM. Pathology of combined hepatocellular-cholangiocarcinoma. J Gastroenterol Hepatol 2010; 25: 1485-92. doi: 10.1111/j.1440-1746. 2010.06430.x 7. Yoon YI, Hwang S, Lee YJ, Kim KH, Ahn CS, Moon DB, et al. Postresection outcomes of combined hepatocellular carcinoma-cholangiocarcinoma, hepato-cellular carcinoma and intrahepatic cholangiocarcinoma. J Gastrointest Surg 2016; 20: 411-20. doi: 10.1007/s11605-015-3045-3 8. Allen RA, Lisa JR. Combined liver cell and bile duct carcinoma. Am J Pathol 1949; 25: 647-55. PMID: 18152860 9. Okuda K. Natural history of hepatocellular carcinoma including fibrolamellar and hepato-cholangiocarcinoma variants. J Gastroenterol Hepatol 2002; 17: 401-5. doi: 10.1046/j.1440-1746.2002.02734.x 10. Zhou YM, Zhang XF, Wu LP, Sui CJ, Yang JM. Risk factors for combined hepatocellular-cholangiocarcinoma: a hospital-based case-control study. World J Gastroenterol 2014; 20: 12615-20. doi: 10.3748/wjg.v20.i35.12615 11. Granata V, Fusco R, Filice S, Catalano O, Piccirillo M, Palaia R, et al. The current role and future prospectives of functional parameters by diffusion weighted imaging in the assessment of histologic grade of HCC. Infect Agent Cancer 2018; 13: 23. doi: 10.1186/s13027-018-0194-5 12. Granata V, Fusco R, Avallone A, Filice F, Tatangelo F, Piccirillo M, et al. Critical analysis of the major and ancillary imaging features of LI-RADS on 127 proven HCCs evaluated with functional and morphological MRI: lights and shadows. Oncotarget 2017; 8: 51224-37. doi: 10.18632/oncotarget.17227 13. Granata V, Fusco R, Avallone A, Catalano O, Filice F, Leongito M, et al. Major and ancillary magnetic resonance features of LI-RADS to assess HCC: an overview and update. Infect Agent Cancer 2017; 12: 23. doi: 10.1186/ s13027-017-0132-y 14. Granata V, Fusco R, Catalano O, Guarino B, Granata F, Tatangelo F, et al. Intravoxel incoherent motion (IVIM) in diffusion-weighted imaging (DWI) for hepatocellular carcinoma: correlation with histologic grade. Oncotarget 2016; 7: 79357-64. doi: 10.18632/oncotarget.12689 15. Granata V, de Lutio di Castelguidone E, Fusco R, Catalano O, Piccirillo M, Palaia R, et al. Irreversible electroporation of hepatocellular carcinoma: preliminary report on the diagnostic accuracy of magnetic resonance, computer tomography, and contrast-enhanced ultrasound in evaluation of the ablated area. Radiol Med 2016; 121: 122-31. doi: 10.1007/s11547-015-0582-5. 16. Zhang XY, Luo Y, Wen TF, Jiang L, Li C, Zhong XF, et al. Contrast-enhanced ultrasound: improving the preoperative staging of hepatocellular carcinoma and guiding individual treatment. World J Gastroenterol 2014; 20: 1262836. doi: 10.3748/wjg.v20.i35.12628 17. Ayuso C, Rimola J, García-Criado A. Imaging of HCC. Abdom Imaging 2012; 37: 215-30. doi: 10.1007/s00261-011-9794-x 18. Jeon SK, Joo I, Lee DH, Lee SM, Kang HJ, Lee KB, et al. Combined hepato-cellular cholangiocarcinoma: LI-RADS v2017 categorisation for differential diagnosis and prognostication on gadoxetic acid-enhanced MR imaging. Eur Radiol 2019; 29: 373-82. doi: 10.1007/s00330-018-5605-x 19. The American College of Radiology. Liver Reporting & Data System (LI-RADS). [cited 2020 Jan 15]. Available at: https://www.acr.org/Clinical-Resources/Reporting-and-Data-Systems/LI-RADS 20. Edmondson HA, Steiner PE. Primary carcinoma of the liver: a study of 100 cases among 48,900 necropsies. Cancer 1954; 7: 462-503. doi: 10.1002/1097-0142(195405)7:3<462::aid-cncr2820070308>3.0.co;2-e 21. Lee HS, Kim MJ, An C. How to utilize LR-M features of the LI-RADS to improve the diagnosis of combined hepatocellular-cholangiocarcinoma on gadoxetate-enhanced MRI? Eur Radiol 2019; 29: 2408-16. doi: 10.1007/ s00330-018-5893-1 22. PotretzkeTA, Tan BR, Doyle MB, Brunt EM, Heiken JP, Fowler KJ. Imaging features of biphenotypic primary liver carcinoma (hepatocholangiocarcinoma) and the potential to mimic hepato- cellular carcinoma: LI-RADS analysis of CT and MRI features in 61 cases. AJR Am J Roentgenol 2016; 207: 25-31. doi: 10.2214/AJR.15.14997 23. Sammon J, Fischer S, Menezes R, Hosseini-Nik H, Lewis S, Taouli B, et al. MRI features of combined hepatocellular-cholangiocarcinoma versus mass forming intrahepatic cholangiocarcinoma. Cancer Imaging 2018; 18: 8. doi: 10.1186/s40644-018-0142-z 24. Fowler KJ, Sheybani A, Parker RA 3rd, Doherty S, M Brunt E, Chapman WC, et al. Combined hepatocellular and cholangiocarcinoma (biphenotypic) tumors: imaging features and diagnostic accuracy of contrast-enhanced CT and MRI. AJR Am J Roentgenol 2013; 201: 332-9. doi: 10.2214/AJR.12.9488 25. Kim JE, Kim SH, Lee SJ, Rhim H. 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AJR Am J Roentgenol 2011; 196: W758-65. doi: 10.2214/AJR.10.4394 Radiol Oncol 2020; 54(2): 149-158. research article Relation of the chondromalatia patellae to proximal tibial anatomical parameters, assessed with MRI Mohammadreza Tabary1, Azadehsadat Esfahani2, Mehdi Nouraie3, Mohammad Reza Babaei4, Ali Reza Khoshdel5, Farnaz Araghi6, Mostafa Shahrezaee17 1 Department of Science and Research Branch, AJA University of Medical Sciences, Tehran, Iran 2 School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 3 Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA 4 Department of Interventional Radiology, Firouzgar Hospital, Iran University of Medical Sciences, Tehran, Iran 5 Modern Epidemiology Research Center, AJA University of Medical Sciences, Tehran, Iran 6 School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 7 Department of Orthopedics, AJA University of Medical Sciences, Tehran, Iran Radiol Oncol 2020; 54(2): 159-167. Received 13 February 2020 Accepted 18 March 2020 Correspondence to: Mostafa Shahrezaee, M.D., Professor of Orthopedics, Department of Science and Research Branch, AJA University of Medical Sciences, Etemadzadeh St., Tehran, Iran. E-mail: moshahrezayee@yahoo.com Disclosure: No potential conflicts of interest were disclosed. Background. Magnetic resonance imaging (MRI) is a non-invasive highly sensitive tool for diagnosing chondromalacia patellae in the early stages. Many studies have evaluated patellar and trochlear morphology with different radiologic indices. We aimed to assess the discriminative power of tibial, patellar, and femoral indices in MRI for chondromalacia patellae. Patients and methods. 100 cases of chondromalacia, as well as 100 age-matched controls among the patients who underwent knee MRI between February 2017 and March 2019, were included. The standard protocol of knee MRI was applied and the diagnosis of chondromalacia was made on MRI findings. Chondromalacia subjects were also classified as grade 1 to 4 according to the Modified Outerbridge's MRI grading system. We measured 25 MRI parameters in the knee and adjacent structures to determine the relation between chondromalacia patellae and anatomical MRI parameters. Results. Tibial slope, trochlear depth, lateral trochlear inclination, and lateral patellar tilt angle had significant correlation with chondromalacia. Any increase in lateral trochlear inclination and lateral patellar tilt angle could increase the probability of the disease (Odds ratio [OR] 1.15, 1.13; 95% CI: 1.03-1.30; 1.02-1.26, respectively), while any increase in medial tibial slope and trochlear depth could decrease the probability of chondromalacia (OR 0.85, 0.06; 95% CI: 0.73-0.98, 0.02-0.17, respectively). We also designed a model for the severity of disease by using the patellar height index (relative odds ratio: 75.9). Conclusions. The result of this study showed the novelty role of tibial anatomy in developing chondromalacia and its mechanism. We also concluded that patellar height might be an important factor in defining disease severity. Key words: magnetic resonance imaging; chondromalacia patellae; anatomical indices Introduction Chondromalacia patellae is a common reason for patellofemoral pain syndrome. It is defined as the disruption of patellar cartilage due to repeated stress to the articular surface.1 These patients may experience frequent recurrence and chronic pain, which limits daily life activities.2 Radiographs Radiol Oncol 2020; 54(2): 159-143. doi: 10.2478/raon-2020-0025 160 Tabary M et al. / Chondromalacia patellae and tibial anatomy were traditionally used to diagnose chondromalacia; however, this modality was unable to visualize patellar alignment and congruency angles3, and the complete anatomy was not visualized using X-ray. Magnetic resonance imaging (MRI), a non-invasive tool to detect chondromalacia patellae with a high soft tissue contrast, can detect chondromalacia in the lower stages with a sensitivity of 66%. Moreover, this sensitivity rises to 85-100% for the higher stages.4 Many key findings can be detected in the early stage of chondral loss including signal irregularities, fissures, and chondral thinning; thus, leading to earlier diagnosis.5 Multiple mechanisms can cause chondromala-cia, including vascular insufficiency, trauma, and structural abnormalities.6 In line with the mechanism of structural abnormalities, many studies have evaluated patellar and femoral trochlear morphology with different radiologic indices. Femoral intertrochlear notch angle, femoral trochlear depth, femoral sulcus angle, femoral trochlear angle, patellar tilt angle, and patellar height indices were among these factors. Many patellar height indices, measured as patellar tendon to patellar length ratio, seemed to correlate with cartilage defects.7 Furthermore, the correlation between tibial structural abnormalities and patellar cartilage stress, but not cartilage defects, was evaluated pre-viously.8 On the other hand, some studies focused on the correlation between soft tissue structure and chondromalacia, particularly subcutaneous fat and adjacent muscles.8, 9 Radiologic findings of the knee joint differ between low-grade (grade 1, 2) and high-grade (grade 3,4) chondromalacia. For instance, the sul-cus angle and trochlear depth were shown to be significantly different between mild and severe cartilage defects.10 The role of tibial anatomy is not well-known in developing chondromalacia. Some studies evaluated the role of tibial anatomy in dynamic models, but not in human subjects.11 To our knowledge, there is no published diagnostic model of radiological findings for chon-dromalacia that includes all important findings including tibial anatomical parameters. We aimed to develop a model and assess the discriminative power of tibial, patellar, and femoral indices for chondromalacia and emphasize the role of tibial anatomy for the first time. We also hypothesize that patellar height is a substantial factor, affecting the development of the disease. Patients and methods Patients and population We reviewed all the radiology records of the patients who underwent knee MRI in our institute between February 2017 and March 2019. We reviewed almost 2000 MRI images of the patients who referred to our institution. After reviewing all the images, we included 200 cases of chondromalacia patellae without any other accompanied diseases in MRI images. The diagnosis of chondromalacia was made on MRI findings including irregularity of the cartilage and the loss of cartilage thickness in at least two consecutive slices. The exclusion criteria were as follows: age more than 65 or less than 18, history of trauma to the knee and adjacent structures in the last 6 months, history of knee surgery, osteoarthritis grade 3 and 4, presence of a fracture or space-occupying lesion in MRI, and patellar subluxation. Patients were contacted and referred to our clinic for clinical evaluation. Patients with other clinical signs not related to chondromalacia were also excluded. After applying exclusion criteria and considering clinical signs, we included 100 cases of chondromalacia patellae. Chondromalacia cases were also classified as grade 1 to 4 according to the Modified Outerbridge's MRI grading sys-tem.12 In case of the presence of more than a single grade lesion, the higher grade was selected. After reviewing MRI images, we selected 200 controls without any significant changes in bony, ligamentous, chondral, tendinous, and muscular structures around the knee. Selection bias was minimized by using case-matched controls regarding age, gender, and BMI. The patellar cartilage was evaluated intact in the control group. After clinical evaluation, we excluded 100 controls with positive clinical finding in the knee. We finally included 100 healthy matched controls in the final analysis. A radiologist experienced in musculoskeletal imaging (A.E.) performed all the measurements. The same radiologist repeated the MRI measurement after 4 weeks in a random sample of the whole population to test the intra-rater reliability. The reader was blinded to participant's names, sex, age. The ethical approval for this study was obtained from local ethics committee and written consent was obtained from the patients. MRI evaluation A 1.5 Tesla Achieva (Philips, Best, Netherlands) MRI device with an extremity superficial coil was used to perform the scans. All the measurements Radiol Oncol 2020; 54(2): 159-167. 161 Tabary M et al. / Chondromalacia patellae and tibial anatomy 2 Ns^i 0 -if---- - / I if Y Pv ) & • * FIGURE 1. (A, B, C) Axial PD fat sat MR images illustrating lateral trochlear inclination, trochlear depth, and lateral patellofemoral angle. (Lateral trochlear inclination, z: the angle formed between the lateral trochlear facet and a parallel line to the line connecting the posterior-most cortical surfaces of the femoral condyles; Trochlear depth, y: the distance between the deepest point of trochlear sulcus and the line connecting the anterior points of the medial and lateral condyles; Lateral patellar tilt angle, x: the angle between the line parallel to the patellar lateral facet and the line connecting the most posterior parts of femoral condyles) and processing were performed in the Picture Archiving Communication System (Marco PACS, Version 10). The standard protocol of knee MRI was applied (neutral knee position) with a slice thickness of 3 mm. T1 and T2 (sagittal), Proton density (PD) fat saturation (axial, coronal, and sagittal) sequences were recorded. We measured 25 MRI parameters in the knee and adjacent structures. The definition and the procedure of all measurements were described elsewhere and are summarized in Figure 1,2,3 (Figure 1 & Figure 2 illustrates the significant MRI measurements in the final model). Intercondylar notch angle (INA), medial tibial slope (MTS), lateral tibial slope (LTS), anterior tibial slope (ATS), coronal tibial slope (CTS), intercon-dylar depth (ID), condylar width (CW), intercon-dylar width (IW), medial condylar width (MCW), lateral condylar width (LCW), notch width index (NWI), and patellar tendon tibial shaft (PTTS) angle were evaluated in previous studies in cruciate ligament injuries13-15, but not in chondromalacia subjects. Subchondral and cartilaginous Wiberg-angle (SWA and CWA) were evaluated in trochlear dysplasia16, but not in chondromalacia patients. Tilting deformity was evaluated in healthy subject by using patellar-patellar tendon (P-PT) angle17; however, it was not evaluated in chondromalacia cases. Lateral patellar tilt angle (LPTA), sulcus angle (SA), trochlear depth (TD), lateral trochlear in- FIGURE 2. (A, B) Sagittal T1-Weighted MR images illustrating medial tibial slope and Insall-Salvati index. (Medial tibial slope, z: the angle formed between the line running along the tibial slope of the medial tibial condyle and the perpendicular line to the tibial axis; Insall-Salvati index: the ratio of x to y; x, the length of patellar tendon and y, the greatest diagonal length of patella) clination (LTI), medial trochlear inclination (MTI), patellar facet angle (PFA) and trochlear angle (TA) were studied in chondromalacia cases; however, the controls were not matched to the cases regarding gender and BMI.18 Also, they were analyzed in a single variable model using student t-test method and not in a multi-variable model. Tibial tuberosity-trochlear groove (TTTG) distance was described in patellofemoral pain syndrome19, but not assessed specifically in chondromalacia cases. Patellar height index was also described in previous studies20; however, we evaluated the predictive power of Insall-Salvati index (ISI) for the severity (grading) of the disease. Statistical analysis MRI measurements were presented in the case and control group by median (interquartile range). Odds ratio (95% CI) for each measurement was calculated including the clustering of observations within subjects. We applied multiple alternative variable selection approaches, including backward and forward stepwise approach to develop the most parsimonious predictive model with the highest calibration. Final model was selected based on Akaike Information Criterion. Then we applied bootstrapping (with 50 replications) to assess the internal validity of the model. The final Odds ratio (95%CI) was reported from bootstrapping results. In the final model, we calculated the Area Under Curve using bootstrapping internal validation. Same analysis approach was replicated to compare two group of cases with low disease severity (grade 1, 2) vs. high severity (grade 3,4). Intra-class correlation coefficient was used to assess intra-rater reli- Radiol Oncol 2020; 54(2): 159-167. 162 Tabary M et al. / Chondromalacia patellae and tibial anatomy ability. All analyses were performed in Stata 15.0 (StataCorp, College Station, TX). Results Patients' characteristics Firstly we evaluated the records of 2000 knee MRIs. Overall, 400 patients were eligible to enter the study, while after applying inclusion and exclusion criteria and clinical examination, 100 cases of chondromalacia were eligible for the final analysis. We evaluated MRI findings of 200 participants, including 100 cases (50 male) with chondromalacia aged between 21 to 64, and 100 healthy controls (51 FIGURE 3. Measurements of nonsignificant MRI parameters in the final model. (A) Axial MRI plane of the knee showing Intercondylar notch angle (INA) and Intercondylar depth (ID): The posterior bicondylar line is drawn. ID is the distance between top of the notch to the bi-epicondylar line. Intercondylar notch angle is defined as the angle formed by the 2 lines going from the top of the notch to the most inferior aspect of the notch at the medial and lateral condyles; (B) Axial MRI plane of the knee showing Intercondylar Width (IW), Medial condyle width (MCW), and Lateral condyle width (LCW): IW is the distance between the medial and lateral femoral condyle walls at the anterior third of the intercondylar depth (the middle line), the other two distances in this axial cut represent LCW, and MCW; (C) Sagittal MRI plane of the knee showing anterior tibial slope (ATS): At first the tibial axis is drawn by joining to midpoints between the anterior and posterior cortex of the tibial diaphysis with at least 5cm distance from each other. The anterior tibial slope is then defined as the angle between a perpendicular line (inferior line) to the longitudinal axis and a line passing through the anterior cruciate ligament tibial footprint (superior line); (D) Sagittal MRI plane of the knee showing coronal tibial slope (CTS): The coronal tibial slope is defined as the angle between a line joining the highest points on the medial and lateral aspects of the tibial plateau (inferior horizontal line] and a perpendicular line (superior horizontal white line) to the longitudinal axis as mentioned above; (E) Sagittal MRI plane of the knee showing lateral tibial slope (LTS): the angle formed between the line running along the tibial slope of the medial tibial condyle and the perpendicular line to the tibial axis; (F) Sagittal MRI plane of the knee showing patellar tibial tendon shaft angle (PTTS angle): PTTS angle is measured as the angle between Proximal tibial anatomical axis described above and patellar tendon axis in the mid-sagittal section; (G) Coronal MRI plane of the knee showing notch width index (NWI): first Bicondylar width parallel to the joint line at the level of popliteal groove is measured (1). Then notch width is measured at the level of popliteal groove, using the line joining the innermost margins of the femoral condyles at the borders of the intercondylar notch (2). The NWI is ratio of 2/1; (H) Axial MRI plane of the knee showing sulcus angle (SA): The angle formed between the medial and lateral trochlear facets; (I) Axial MRI plane of the knee showing patellar facet angle (PFA): the angle between two line connecting the central ridge to the middle of patellar facets; (J) Axial MRI plane of the knee showing patellofemoral index (PFI): the ratio of medial to lateral interspaces; (K, L) Axial MRI plane of the knee showing tibial tuberosity trochlear groove distance (TTTG): posterior line was drawn at posterior border of femoral condyles. A vertical line is drawn at trochlear groove and the other parallel line is drawn along the tibial tuberosity that transferred to this level. Distance between the two lines is measured as TT-TG; (M) Sagittal MRI plane of the knee showing Patella-patellar tendon angle (P-PT angle): The P-PT angle was defined as the angle between the upper patellar pole and the lower patellar pole, and the tibial tuberosity; (N) Sagittal MRI plane of the knee showing Patellotrochlear index (PTI): it is defined as the length of patellar cartilage overlapping the trochlear cartilage divided by length of patellar cartilage; (O) Axial MRI plane of the knee showing medial trochlear inclination (MTI): the angle formed between the medial trochlear facet and a parallel line to the line connecting the posterior-most cortical surfaces of the femoral condyles. Radiol Oncol 2020; 54(2): 159-167. 163 Tabary M et al. / Chondromalacia patellae and tibial anatomy TABLE 1. MRI measurements of the control patients versus chondromalacia cases Median (IQR) MRI Measurements Odds ratio 95% CIt P-value Control Case INA (degree) 52.0 [48.0-57.0] 53.0 [48.0-56.0] 1.00 0.96-1.05 0.863 IW (mm) 21.5 [20.1-23.4] 20.3 [19.0-21.7] 0.77 0.47-0.88 < 0.001 ID (mm) 28.2 [26.7-29.8] 25.9 [26.7-29.8] 0.76 0.68-0.85 < 0.001 MCW (mm) 26.5 [24.9-28.5] 24.8 [23.2-26.7] 0.79 0.70-0.88 < 0.001 LCW (mm) 27.2 [25.5-28.8] 24.4 [23.3-26.1] 0.77 0.49-0.86 < 0.001 ATS (degree) 8.0 [5.5-11.5] 8.0 [6.0-10.5] 1.01 0.94-1.09 0.727 CTS (degree) 3.5 [2.0-5.0] 4.0 [2.5-5.2] 1.13 0.96-1.32 0.144 MTS (degree) 7.5 [5.2-9.5] 7.0 [4.5-9.5] 0.95 0.87-1.04 0.252 LTS (degree) 5.5 [3.5-7.5] 5.5 [3.5-7.5] 0.96 0.88-1.05 0.448 PTTS angle (degree) 27.0 [23.0-30.0] 27.0 [24.0-30.0] 1.01 0.95-1.08 0.46 NWI 0.29 [0.27-0.31] 0.29 [0.27-0.31] 0.18 0.00-6399.00 0.749 TD (mm) 5.30 [4.82-5.87] 4.04 [3.51-4.60] 0.14 0.08-0.24 < 0.001 SA (degree) 140.0 [134.0-145.0] 142.0 [137.0-150.0] 1.04 1.01-1.08 0.009 LTI (degree) 20.0 [18.0-23.0] 20.0 [17.5-24.0] 0.99 0.93-1.05 0.683 MTI (degree) 18.0 [15.0-21.0] 16.0 [13.5-20] 0.93 0.89-0.99 0.020 LPTA (degree) 13.0 [10.0-16.0] 14.0 [10.5-17.0] 1.03 0.97-1.08 0.294 PFA (degree) 138.0 [133.0-142.0] 136.0 [132.5-141.0] 0.97 0.93-1.01 0.160 PFI 1.14 [1.01-1.35] 1.32 [1.07-1.59] 3.02 1.37-4.45 0.004 TTTG (mm) 12.5 [10.2-15.2] 12.3 [10.5-16.1] 1.01 0.95-1.09 0.603 SCF (mm) 20.5 [17.3-25.3] 26.7 [21.7-34.8] 1.14 1.09-1.19 < 0.001 CWI 0.52 [0.50-0.54] 0.52 [0.50-0.54] 0.76 0.00-3481.00 0.949 SWI 0.57 [0.54-0.59] 0.56 [0.54-0.58] 0.01 0.00-34.80 0.270 P-PT angle (degree) 140.5 [137.0-144.0] 140.0 [137.5-143.5] 0.99 0.93-1.05 0.802 ISI 0.98 [0.89-1.07] 1.00 [0.89-1.14] 4.62 0.60-35.20 0.139 PTI 0.31 [0.26-0.36] 0.30 [0.25-0.35] 0.04 0.00-2.21 0.115 t IQR represents 25th-75th interquartile range, 95% CI represents 95% confidence interval for odds ratio ATS = Anterior tibial slope; CTS = Coronal tibial slope; CWI = Cartilaginous Wiberg index; ID = Intercondylar depth; INA = Intercondylar notch angle; ISI = Insall-Salvati index; IW = Intercondylar width; LCW = Lateral condyle width; LPTA = Lateral patellar tilt angle; LTI = Lateral trochlear inclination; LTS = Lateral tibial slope; MCW = Medial condyle width; MTI = Medial trochlear inclination; MTS = Medial tibial slope; NWI = Notch width index; P-PT angle = Patella-patellar tendon angle; PTTS angle = Patellar tibial tendon shaft angle; PFA = Patellar facet angle; PFI = Patellofemoral index; PTI = Patellotrochlear index; SA = Sulcus angle; SCF = Subcutaneous fat pad; SWI = Subchondral Wiberg index; TD = Trochlear depth; TTTG = Tibial tuberosity trochlear groove distance male) aged between 20 and 57. The mean age of the chondromalacia subjects and cases were 32.7 ± 9.3 and 30.2 ± 7.2, respectively (P-value = 0.21) Radiographic measurements We measured 25 radiographic parameters. Table 1 summarizes the radiographic features in patients and controls. Intercondylar width, intercondy-lar depth, medial condyle width, lateral condyle width, trochlear depth, and medial trochlear inclination pad were significantly lower in chondroma-lacia cases (P-value = < 0.001, < 0.001, 0.020), while sulcus angle, patellofemoral index, and subcutaneous fat were significantly higher in cases compared to controls (P-value = 0.009, 0.006, < 0.001). Predictive model for chondromalacia We developed a predictive model for chondro-malacia. This model included medial tibial slope, trochlear depth, lateral trochlear inclination, and lateral patellar tilt angle and age. This model predicted the disease with an excellent AUC (area under curve) of 0.92 (95% CI: 0.85-0.94). Any increase in lateral trochlear inclination and lateral patellar Radiol Oncol 2020; 54(2): 159-167. 164 Tabary M et al. / Chondromalacia patellae and tibial anatomy TABLE 2. Predictive model for chondromalacia including MRI measurements MRI ,. „.„,_. _ . Intra-rater Contribution of any increase in this . Odds ratio 95% CI P-value .. , .... . . , ' , . measurements reliability parameter to chondromalacia LTI 1.15 1.03-1.30 0.014 0.992 Increases disease probability LPTA 1.13 1.02-1.26 0.018 0.996 Increases disease probability MTS 0.85 0.73-0.98 0.026 0.997 Decreases disease probability TD 0.06 0.02-0.17 0.000 0.995 Decreases disease probability Age 1.10 1.02-1.20 0.015 - Increases disease probability LTI = Lateral trochlear inclination; LPTA = Lateral patellar tilt angle; MTS = Medial tibial slope; TD = Trochlear depth The Area under curve (AUC) for this model is estimated as 0.92 (bootstrap bias-corrected 95% CI: 0.85-0.94). Any increase in lateral trochlear inclination and lateral patellar tilt angle could increase the probability of the disease (positive correlation), while any increase in medial tibial slope and trochlear depth could decrease the probability of the disease (negative correlation) TABLE 3. Predictive model for chondromalacia severity including MRI measurements MRI measurements Odds ratio 95%CI P-value Intra-rater reliability Contribution of any increase in this parameter to chondromalacia severity ISI 75.89 2.17-2652.69 0.017 0.997 Increases disease grade Age 1.14 1.07-1.21 0.000 - Increase disease grade ISI = Insall-Salvati index The Area under curve (AUC) for this model is estimated as 0.82 (bootstrap bias-corrected 95% CI: 0.64-0.86). Grade 1 and 2 are considered as non-severe, while grade 3 and 4 are assumed as severe. Increase in patellar height and age will increase disease grade (positive correlation with disease severity) tilt angle could increase the probability of the disease (positive correlation with chondromalacia) (Odds ratio 1.15, 1.13; 95% CI: 1.03-1.30; 1.02-1.26, respectively). However, any increase in medial tibial slope and trochlear depth could decrease the probability of chondromalacia (negative correlation with chon-dromalacia) (Odds ratio 0.85, 0.06; 95% CI: 0.730.98, 0.02-0.17, respectively), (Table 2). Increasing age could also increase the probability of the disease (positive correlation with chondromalacia) (Odds ratio 1.10; 95%CI: 1.02-1.20). INA, IW, ID, MCW, LCW, ATS, CTS, LTS, PTTS angle, NWI, SA, MTI, PFA, PFI, TTTG, SCF, CWI, SWI, P-PT angle, PTI, and ISI were not significant (no correlation with chondromalacia) in the multivariable analysis regarding the presence of chon-dromalacia (P-value = 0.20, 0.24, 0.15, 0.19, 0.56, 0.30, 0.09, 0.39, 0.43, 0.32, 0.21, 0.55, 0.07, 0.15, 0.11, 0.11, 0.19, 0.66, 0.09, 0.27, 0.29; respectively). Predictive model for the severity of the disease Patients were classified as grade 1 lesion (38 patients), grade 2 lesion (28 patients), grade 3 lesion (14 patients), and grade 4 lesion (20 patients). We combined grade 1 and 2 vs. grade 3 and 4 to develop a predictive model for severity of disease in patients with chondromalacia. We designed a two-variable model for predicting the severity of the disease using age and Insall-Salvati index concurrently. Insall-Salvati index and age were two factors that could predict disease severity (Odds ratio = 75.89, 1.14; 95% CI: 2.17-2652.69, 1.07-1.21, respectively) with an AUC of 0.82 (95% CI: 0.64-0.86) (Table 3). Any increase in Insall-Salvati index (i.e. any increase in patellar height) and age could increase the severity of the disease (resulted in higher grades of chondroma-lacia). It means that if patellar height increases, patients will experience more severe disease (higher grades). Discussion Prediction models are becoming more popular in medicine. Previous studies focused their attention on the correlation between trochlear morphology and chondromalacia patellae, in a single variable analysis model. We developed a model to predict chondromalacia. We included medial tibial slope, lateral trochlear inclination, lateral patellar tilt an- Radiol Oncol 2020; 54(2): 159-167. 165 Tabary M et al. / Chondromalacia patellae and tibial anatomy gle, trochlear depth, and age in the final predictive model for chondromalacia. We found that any increase in lateral trochlear inclination and lateral patellar tilt angle could increase the probability of the disease, while any increase in medial tibial slope and trochlear depth could decrease the probability of the disease. We also found that if patellar height increases, patients would experience more severe disease (higher grades). Lateral patellar tilt angle (LPTA) was suggested as one of the predictive factors in the final model. LPTA was shown to be increased in chondromalacia subjects. Abnormal position of the patella in the femoral trochlear groove may play a role in the progression of chondromalacia patellae.2122 Studies have shown that lateral patellar displacement is associated with cartilage loss.23 This can be justified by the fact that lateral tilting happens as a result of lateral compressive forces which acts on the lateral facet and increases shear forces in the central ridge area. These forces will increase chondro-cyte activity, until the functional requests exceed chondrocyte potential and results in chondrocyte degeneration.24 Moreover, previous studies have found that LPTA correlates with the sulcus angle in different knee flexion angles.25 Sulcus angle has been reported to be greater in lateral maltrackers compared to non-lateral maltrackers26, and it also correlates with cartilage lesions.27 A wider sulcus is responsible for the increased pressure on patel-lofemoral articular surface and will predispose the patient to cartilage loss.27 This correlation between the sulcus angle and LPTA may justify our result. However, LPTA has been reported to correlate significantly with chondral lesions in the lateral compartment of the patellofemoral joint (odds ratio < 1) and non-significantly with chondral lesions in the medial compartment of the patellofemoral joint (odds ratio > 1) in previous studies.27 In this study, we did not define the side of cartilage defect. As chondromalacia impacts the medial side more frequently28, we may speculate that this relationship in our study should be judged according to the side of involvement. In addition, LTI was positively correlated with chondromalacia according to our results. Kuroda et al. showed that elevation of lateral trochlea facet (increased LTI) could increase average patellofem-oral contact pressure (40% increased with 10 mm lateral trochlear elevation). This may also induce the degradation of the cartilage.29 The most important finding of the present study is that the tibial slope may play an important role in the progression of chondromalacia. We found that with the medial tibial slope increasing, the risk of chondromalacia decreases. Studies suggest that an increase in the tibial slope leads to a decrease in patellofemoral contact force.30 Posterior positioning of femoral components and increase in quadriceps lever arm are responsible for this associa-tion.31-33 We deduce that the tibial slope correlates with chondromalacia patellae; however, the effect of tibial slope on medial and lateral patellar facets may be different. Furthermore, correcting the tibial slope with a greater angle has been shown to reduce anterior tibial strains, which are imposed on the patella.34 Moreover, studies in patients with osteoarthritis suggested that increasing the tibial slope in unicompartmental knee arthroplasty of medial patellar facet will reduce the tension on the medial side.11 This may also show the clinical importance of tibial slope on decreasing patellofemo-ral stress. Our study suggests that trochlear depth would be sufficient for measuring the association of troch-lear dysplasia with chondromalacia. Sulcus angle and trochlear depth are the two important factors associated with trochlear dysplasia in previous studies35, and the congruency of the patella and femoral trochlea has been underscored in the development of cartilage lesions.10 Patellofemoral tracking is also considered an important factor in patellofemoral pain syndrome.36 Trochlear depth has been reported to be an important determinant of patellofemoral stability37, thus, it may be an important factor in the process of tracking. Tuna et al. reported sulcus angle and trochlear depth to be significantly different in patients with and without the disease.18 They reported greater mean of sulcus angle and lower mean of trochlear depth in chon-dromalacia subjects. Our findings are consistent with the latter study. We found that with the troch-lear depth decreasing, the risk of chondromalacia increases to a great extent. This may be explained by the fact that when the trochlear surface becomes shallower, the surface area for articulation at the patellofemoral joint increases. Although it may provide a better distribution of joint load37, the increased friction may result in cartilage defects. Further studies are needed to evaluate this proposed kinematics in active knee movements. This finding may underscore the clinical importance of trochlear depth. Takahashi et al. also showed better adaptation of the implants featuring deep trochlea to the native patella (low-contact stress implant and NexGen implant).38 We also proposed a predictive model of disease severity. In our study, ISI and age could predict Radiol Oncol 2020; 54(2): 159-167. 166 Tabary M et al. / Chondromalacia patellae and tibial anatomy higher disease severity. To our knowledge, this is the first predictive model for the severity of chon-dromalacia with an excellent AUC. A model was proposed in the previous study by Lu et al. for chondromalacia but not severity by using Insall-Salvati index (ISI). This model was based on the patella alta and baja and could diagnose chondro-malacia with an AUC of 0.596.7 In both predictive models proposed by our findings, age was remained as an important factor. A study in the United States population showed that the contribution of chondromalacia to the patel-lofemoral pain syndrome increases by age up to 59 years.39 We also excluded patients older than 65 years and the results may not be applicable to older age groups. There are some limitations to our study. This was a single center study, we used MRI to detect chondral lesion and early lesions might be considered as normal. MRI evaluations were performed during rest. Muscle contractions may alter these findings as described by previous studies.42 We did not evaluate the exact site of the cartilage lesions. Moreover, medial and lateral facet chondromalacia might have different natures. Previous studies reported the predominance of chondromalacia in the medial patellar facet.22 We did not define the side of chondromalacia in our subjects, so more pieces of evidence are needed to define the exact correlation of tibial slope with both medial- and lateral-facet chondromalacia. The advantage of this study in knowledge is that when more diverse anatomical factors are analyzed regarding the presence of chondromala-cia, tibial anatomical factors play a role in the progression of the disease. This study may clarify the mechanism of chondromalacia and emphasize the role of tibial slopes in the mechanism of cartilage loss. Previous studies focused on femoral trochlear dysplasia, and did not include tibial anatomical parameters in their analysis. The result of this study meight also affect the treatment of chondromala-cia, as new treatment plans for healing cartilage and bone defects are being introduced and focus on altering tibial anatomy including tubercle oste-otomy.4041 The result of this study shew the role of tibial anatomy in developing chondromalacia. In addition, the clinical importance of this study was the fact that tibial slope should be precisely set in knee surgeries; however, more studies are needed to determine the best cut-off for tibial slope. References 1. Endo Y, Stein BE, Potter HG. Radiologic assessment of patel-lofemoral pain in the athlete. Sports Health 2011; 3: 195-210. doi: 10.1177/1941738110397875 2. Crossley KM, Stefanik JJ, Selfe J, Collins NJ, Davis IS, Powers CM, et al. Patellofemoral pain consensus statement from the 4th International Patellofemoral Pain Research Retreat, Manchester. 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J Sport Rehabil 2010; 19: 1-11. doi: 10.1123/jsr.19.1.1 Radiol Oncol 2020; 54(2): 159-167. 168 research article Pulsed low dose-rate irradiation response in isogenic HNSCC cell lines with different radiosensitivity Vesna Todorovic1, Ajda Prevc1, Martina Niksic Zakelj1, Monika Savarin1, Simon Bucek2, Blaz Groselj3, Primoz Strojan34, Maja Cemazar15, Gregor Sersa16 1 Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia 2 Institute of Oncology Ljubljana, Department of Cytopathology, Ljubljana, Slovenia 3 Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia 4 University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia 5 University of Primorska, Faculty of Health Sciences, Izola, Slovenia 6 University of Ljubljana, Faculty of Health Sciences, Ljubljana, Slovenia Radiol Oncol 2020; 54(2): 168-179. Received 10 February 2020 Accepted 1 March 2020 Correspondence to: Prof. Gregor Sersa, Ph.D., Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. E-mail: gsersa@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. Management of locoregionally recurrent head and neck squamous cell carcinomas (HNSCC) is challenging due to potential radioresistance. Pulsed low-dose rate (PLDR) irradiation exploits phenomena of increased radiosensitivity, low-dose hyperradiosensitivity (LDHRS), and inverse dose-rate effect. The purpose of this study was to evaluate LDHRS and the effect of PLDR irradiation in isogenic HNSCC cells with different radiosensitivity. Materials and methods. Cell survival after different irradiation regimens in isogenic parental FaDu and radioresistant FaDu-RR cells was determined by clonogenic assay; post irradiation cell cycle distribution was studied by flow cytometry; the expression of DNA damage signalling genes was assesed by reverse transcription-quantitative PCR. Results. Radioresistant Fadu-RR cells displayed LDHRS and were more sensitive to PLDR irradiation than parental FaDu cells. In both cell lines, cell cycle was arrested in G2/M phase 5 hours after irradiation. It was restored 24 hours after irradiation in parental, but not in the radioresistant cells, which were arrested in G,-phase. DNA damage signalling genes were under-expressed in radioresistant compared to parental cells. Irradiation increased DNA damage signalling gene expression in radioresistant cells, while in parental cells only few genes were under-expressed. Conclusions. We demonstrated LDHRS in isogenic radioresistant cells, but not in the parental cells. Survival of LDHRS-positive radioresistant cells after PLDR was significantly reduced. This reduction in cell survival is associated with variations in DNA damage signalling gene expression observed in response to PLDR most likely through different regulation of cell cycle checkpoints. Key words: DNA damage; isogenic cell lines; low dose irradiation; pulsed low dose-rate irradiation; radiosensitivity Introduction Low dose hyperradiosensitivity (LDHRS) is a phenomenon of increased radiosensitivity to single doses below 0.5 Gy.1 LDHRS has been demonstrated in various normal and tumour cell lines, tumour spheroids and human tumours.2-7 LDHRS was not observed in the intrinsically radiosensitive cell lines, whereas radioresistant cell lines demonstrated the most marked LDHRS.38 LDHRS precedes the occurrence of increased radioresistance (IRR) to cell killing by radiation over the dose range of 0.5 - 1 Gy.1 Transition from LDHRS to IRR is cell type-dependent and has been typically observed in the dose range of 0.2 Gy to 0.6 Gy.12910 Radiol Oncol 2G2G; 54(2): 168-179. doi: 1G.2478/raon-2G2G-GG15 Todorovic V et al. / Pulsed low dose-rate irradiation of isogenic cells with different radiosensitivity 169 0.3 Gy 7x0.3 Gy 2.1 Gy time interval irradiation FIGURE 1. Schematic diagram of irradiation schedule. Another phenomenon of increased radiosensitivity, especially in some LDHRS-positive tumor cells, is the inverse dose-rate effect. In contrary to normal tissue sparing due to repair of sublethal DNA damage during low dose-rate irradiation, increased radiosensitivity of tumour cells was observed when the dose-rate was decreased.11 The inverse dose-rate effect can be observed at dose-rates below 1 Gy/h in cells showing LDHRS.1112 The LDHRS and the inverse dose-rate effect were exploited in pulsed low-dose rate (PLDR) radiotherapy as a treatment strategy combining multiple low doses (hyperfractionation) in a pulsed delivery to reduce the effective dose-rate.13 Its effectiveness was evaluated first in the radioresist-ant gliomas.14 The delivery of low dose fractions in a pulsed fashion significantly reduced surviving fraction of glioma cell lines in vitro13, greatly inhibited tumour growth of orthotopic xenografts, preserved vascular density, caused less neuronal cell death in vivo15,16, and allowed retreatment of recurrent glioma tumors.14 A similar low-dose fractionated regime significantly increased tumour growth delay in metastatic melanoma, leiomyosarcoma, breast cancer, and non-Hodgkin lymphoma.6 In the last decade, PLDR irradiation has been used clinically for re-irradiation of recurrent tumours in the previously irradiated areas.1417 Both glioblastoma and head and neck squamous cell carcinoma (HNSCC) are known for tumour recurrences within the previously irradiated area.18-20 Based on the promising glioblastoma results using PLDR radiotherapy, this approach could be beneficial also to improve HNSCC management, namely to decrease regrowth of recurrent tumours and to reduce normal tissue toxic-ity. Management of HNSCC remains challenging due to complex anatomy of the region, the need for preserving function of the involved organs, lo-coregional recurrence of radioresistant tumours, and normal tissue toxicity.19 In HNSCC cell lines with different radiosensitivity, so far no apparent 0.4 0.6 Dose (Gy) FIGURE 2. Surviving fraction of parental FaDu and radioresistant FaDu-RR cells after exposure to low doses of ionizing radiation. Symbols are mean ± standard error of the mean from four independent experiments. * - significantly different from FaDu cells. FIGURE 3. Surviving fraction of parental FaDu and radioresistant FaDu-RR after exposure to different PLDR irradiation regimes. (A) Surviving fraction of parental FaDu cells after 7x0.3 Gy PLDR and (B) after 10x0.2 Gy PLDR irradiation. (C) Surviving fraction of radioresistant FaDu-RR cells after 7x0.3 Gy PLDR and (D) after 10x0.2 Gy PLDR irradiation. Bars present mean ± SEM from four independent experiments. ** = significantly different from 0 Gy and low dose IR (0.3 Gy or 0.2 Gy); * = significant difference between the groups; n. s. = non-significant difference. Radiol Oncol 2020; 54(2): 168-179. 170 Todorovic V et al. / Pulsed low dose-rate irradiation of isogenic cells with different radiosensitivity 170 iE 30 "E 3Ï ® i A 0 Gy . 0.3 Oy 5.1 Oy 7*0.3 Gy il fi i % 0: % G JU Coli cyclo phaso (D I OGy I 0 3 Gy I 2.1 Gy i 7x0.3 Gy il X s Coll cyclo pno 60 GjiM be attributed to the activation of different cellular mechanisms. The purpose of this study was first: to evaluate the LDHRS status of two isogenic HNSCC cell lines with different radiosensitivity, followed by the evaluation of cell survival after PLDR irradiation in the isogenic cell lines. Second, with the aim to explore the underlying mechanisms of radiosen-sitivity of radioresistant cells to PLDR irradiation, we determined cell cycle progression and DNA damage signalling gene expression in response to low dose, conventional and PLDR irradiation. FaDu-RR 5 h Fa D Li-RR 24 h I OGy I 0.3 Gy I 2.1 Gy I 7x0.3 Gy il %S Cell cycle phase % 5 % G .'M Cell cycle phasp FIGURE 4. Cell cycle distribution in parental FaDu and radioresistant FaDu-RR cells after exposure to different PLDR irradiation regimes. (A) Cell cycle distribution in FaDu cells 5 h and (B) 24 h after irradiation. (C) Cell cycle distribution in FaDu-RR cells 5 h and (D) 24 h after irradiation. Bars present mean ± SEM from four independent experiments. * = significantly different from 0 Gy; ** = significantly different from 0 Gy and 0.3 Gy; # = significantly different from 0 Gy, 0.3 Gy and 2.1 Gy; n. s. = nonsignificant difference. difference was observed between conventional and low dose irradiation, however, LDHRS status of these HNSCC cell lines was unknown.21 Therefore, proper selection of LDHRS-positive cell lines and tumours is crucial to evaluate the effect of PLDR radiotherapy and/or ultrahyperfraction-ated irradiation in HNSCC. The exact mechanisms causing the LDHRS are not clear yet. Most prominently LDHRS appears in G2-phase cells, where the threshold amount of DNA damage needs to occur to overcome LDHRS and induce IRR.2223 DNA damage signalling network is involved in cell cycle checkpoint activation and plays an important role in cellular radio-sensitivity.2425 Isogenic cell lines with different LDHRS status are an attractive model to study the mechanisms involved in the LDHRS response. Due to the same genetic background, observed difference in the response to PLDR irradiation can Materials and methods Cell lines Human pharyngeal HNSCC cell line FaDu (ATCC, HTB-43) and 2.6-fold more radioresistant FaDu-RR cells, were established in our laboratory from the parental FaDu cells after repeated exposure to ionizing radiation as previously described.26 Both cell lines were grown in Advanced Dulbecco's Modified Eagle Medium (DMEM, Gibco, Thermo Fisher, MA, USA) supplemented with 5% fetal bovine serum (FBS, Gibco, Thermo Fisher), 10 mM L-glutamine (GlutaMAX, Gibco), penicillin (100 U/mL) (Grünenthal, Germany) and gentamicin (50 mg/mL) (Krka, Slovenia). Cells were routinely sub-cultured twice a week and incubated in a humidified atmosphere at 37°C and 5% CO2. Low dose irradiation Irradiation was delivered using a Gulmay MP1-CP225 X-ray unit (Gulmay Medical Ltd, UK) with a filter consisting of Cu thickness of 0.55 mm and Al thickness of 1.8 mm at 200 kV and 1.0 mA to achieve the low dose rate 0.185 Gy/min. The low dose rate was used to allow precise delivery of single low dose (0.1 Gy was delivered in 0.5 min). To determine the radiosensitivity of parental FaDu and radioresistant FaDu-RR cells, cells were exposed to single doses of 0.1 - 1 Gy in steps of 0.1 Gy and plated for clonogenic assay as described below. Pulsed low dose-rate irradiation Cells were exposed to three different irradiation schedules (Figure 1). The control, non-irradiated cells, were handled as irradiated samples but were not exposed to any irradiation. The irradiated cells were exposed to either a single dose of 0.3 Gy, a series of seven 0.3 Gy pulses (7x0.3 Gy) or a single dose of 2.1 Gy. A series of 0.3 Gy pulses was sep- Radiol Oncol 2020; 54(2): 168-179. Todorovic V et al. / Pulsed low dose-rate irradiation of isogenic cells with different radiosensitivity 43 ules, they were plated for clonogenic assay as described below. Clonogenic assay For all irradiation doses, 350 cells/dish were plated onto 60-mm tissue culture dish and irradiated with a specific single dose or specific irradiation schedule using Gulmay MP1-CP225 X-ray unit, as described above. After 10 days, the resulting colonies were stained with crystal violet and counted. Surviving fraction was calculated as a ratio of the plating efficiencies for irradiated and control non-irradiated cells. The experiments were repeated 3 to 4 times in triplicates. Cell cycle Cell cycle distribution of parental FaDu and radioresistant FaDu-RR cells after irradiation was determined by flow cytometry as previously described.26 Briefly, the samples were prepared following the standard procedure using fluoro-chrome DAPI (4',6-diamidino-2-phenylindole-dihydrochloride). The samples were acquired using a flow cytometer Partec PAS II (Partec GmbH, Germany) and at least 30,000 cells per sample were collected during sample acquisition. Results were analyzed with MultiCycle AV DNA analysis software (Phoenics Flow Systems, Inc., CA, USA) and percent of cells in G1, S and G2/M phases of the cell cycle were calculated. The experiment was repeated 4 times. DNA damage signalling gene expression An array of 84 pathway-specific and 5 reference genes (Human DNA Damage Signalling Pathway RT2 ProfilerTM PCR Array, PAHS-029Z, Qiagen, Germany) was used to study the DNA damage response in parental FaDu and radioresistant FaDu-RR cells after the low dose and PLDR irradiation. Genomic DNA control, reverse transcription control, and positive PCR controls were included in the array. Samples for gene expression analysis were prepared as previously described.26 Briefly, 5 hours after different irradiation protocols, total RNA was isolated from the cells using RNeasy Plus Mini Kit (Qiagen), and RNA concentration and sample purity (A260/280) were determined spec-trophotometrically. For cDNA synthesis, 2 |jg total RNA was used using the RT2 First Strand Kit (Qiagen). Reverse transcription-quantitative PCR was carried out on QuantStudio 3 Real-time PCR FaDu A.BL1 -APEX1-ATM-ATR-ATRIP-ATRX-BARD1 -BAX-BBC3-BLM BRCA1-BRIP1 -CDC25A-CDC25C- COK7 CDKN1A-CHEK1-CHEK2-CIB1-CRY-M CSNK2A2-1 DDS1-□ □82* ODIT3-ERCC1-ERCC2- EX01 FANCA-FANCD2-FANCG-FEN1-GADD4SA GAOD45G H2AFX-HUS1-L1G1-MAPK12-MBD4-MCPH1-MDC1 -MLH1-MLH3-MPG MRE11A-MSH2-MSH3-NBN-HTHL1-OGG1-PARP1 -PCNA-PMS1 PMS2 PNKP-PPMID -PPP1R1SA-PRKDC-RA01 RAD 17 RAD18-RAD21 RAOSO-RAD51-RAD51B RAD9A- REV1-RNF168-RNFfi RPA1-SIRT1-SMC1A-SUM01-TOPBP1 TPS3 TP53BP1 TP73 UNG-XPA-XPC~ XRCC1 -XRCC2-XRCC3-XRCC6- 1 ® " 2.7 2.4 2.1 1.8 1.5 1.2 09 0 6 0.3 FaDu-RR ABM-APEX!- ATM- ATR- ATRIP- ATRX- BARD1 - BAX- BBC3- 8LM- BRCA1 - BRIPt- CDC25A- CDC25C- CDK7- CDKN1A- CHEK1 - CHEK2- CIB1- CRY1- CSNK2A2- DDB1 - DDB2- DDIT3- ERCCt- ERCC2- EX01- FANCA- FANCD2- FANCG- FEN1- GADD45A- G ADD45G - H2AFX - KUS1- UC1- MAPK12- MCPH1- MOC1- MLHl- MLH3- MPG- HRE11A- MSH2- MSH3- NBN- NTHL1 - OGGt - PARP1 - PCNA- PMSt - PMS2- PNKP- PPM1D- PPP1R15A- PRKDC- RADt- RAD17- RAD18- RAD21 - RAD 50 - RAD 51 - RAD51B- RAD9A- RBBPfl- REVl - RNF166- RNF8- RPA1- S4RT1- SMC1A- SUMOt- TOPBP1- TP53- TP53BP1- TP73- UNG- XPA- XPC- XRCC1 - XRCC2- XRCC3- XRCC6- UJ f 2,7 2.4 2,1 1.8 1.5 1,2 0.9 0,6 |.3 Gy 2.1 Gy 7x0.3 Gy 0,3 Gy 2.1 Gy 7x0.3 Gy — 0.3 FIGURE 5. Heat maps of DNA damage signalling gene expression in parental FaDu (A) and radioresistant FaDu-RR cells (B) in 0.3 Gy, 2.1 Gy and 7x0.3 Gy irradiated cells relative to the gene expression in control non-irradiated cells. The magnitude of the fold change in gene expression of each gene from three independent experiments is represented by the colour. Green indicates under-expressed genes, and red indicates over-expressed genes. arated by 4.5 min intervals to create an apparent dose rate of 0.055 Gy/min. Additionally, a series of 0.2 Gy pulses was separated by 3 min intervals, to create an apparent dose rate of 0.053 Gy/min, and was compared to the effect of single 2 Gy dose. A 4.5-minute and 3 min interval between the doses for each of the above PLDR irradiation protocols, was chosen to create a similar apparent dose-rate as proposed by Tome et al.13 To determine radio-sensitivity of the cells to these irradiation sched- Radiol Oncol 2020; 54(2): 168-179. Todorovic V et al. / Pulsed low dose-rate irradiation of isogenic cells with different radiosensitivity 44 FaDu FaDu-RR log fold change 0.3 Gy/control} log2(fold change 0.3 Gy,'control) cran B6C3 » »J .........MWfl— unchanged over-expressed under-expressed unchanged over-expressed under-expressed log7(fold change 2.1 Gy/control) log3(fold change 2,1 Gy/control) unchanged over-expressed under-expressed a 2 "S I ■ unchanged • over-expressed • —BBC 3 • under-expressed CRY1—• • —PMS1 ■- GAOD4SA tog^ifala change 7x0.3 GyfcontroM logjIFold change 7x0.3 Gy.'control) FIGURE 6. DNA damage signalling gene expression in parental FaDu and radioresistant FaDu-RR cells in response to different irradiation protocols. (A) Gene expression of FaDu cells in response to 0.3 Gy, 2.1 Gy, and 7x0.3 Gy irradiation relative to the control non-irradiated FaDu cells. (B) Gene expression in FaDu-RR cells in response to 0.3 Gy, 2.1 Gy, and 7x0.3 Gy irradiation relative to the control non-irradiated FaDu-RR cells. Volcano plots show the fold change in gene expression and statistical significance (p value). The horizontal line shows the statistical significance threshold (p value < 0.05). Two vertical dashed lines show the threshold of over-expressed (right) and under-expressed genes (left), while the solid vertical line shows no change in gene expression. Symbols represent the mean gene expression of each tested gene in irradiated cells relative to control non-irradiated cells from three independent experiments. System (Applied Biosystems, USA) using RT2 qP-CR Sybr Green ROX Mastermix (Qiagen) and cycling conditions as described previously.26 GeneGlobe Data Analysis Center (Qiagen) was used to analyze the results. Data were normalized to the gene expression of the reference gene with the most stable expression (HPRT1). Fold change in gene expression was calculated using the AACT method.27 We used 1.5 fold-change in gene expression as a threshold and p values less than 0.05 to identify significantly different gene expression. Statistics GraphPad Prism 8.1.2 (GraphPad Software, Inc., CA, USA) was used for graphs and statistical analysis. Normal distribution of data was tested using the Shapiro-Wilk test. For normally distributed data, data are shown as the mean ± standard error of the mean (SEM). Differences between parental and radioresistant cells were identified by unpaired two-tailed t-test. One Way ANOVA with Tukey test for posthoc multiple comparisons were used to identify the difference between groups. Differences were considered significant for p values less than 0.05. For statistical analysis of DNA damage signalling gene expression data, Student's t-test (two-tail distribution and equal variances between the two samples) was used on the replicate 2AACT values for each gene in each irradiation protocol compared to the control non-irradiated cells from 3 independent experiments. Results Low dose irradiation We observed similar radiosensitivity to single low doses of ionizing radiation in parental FaDu and radioresistant FaDu-RR cells, except at 0.3 Gy and 0.4 Gy doses (Figure 2). Surviving fraction of radioresistant FaDu-RR at 0.3 Gy was significantly lower (p=0.006) than the surviving fraction of parental FaDu cells, exposed to the same irradiation dose. From 0.3 Gy up to 0.5 Gy, an increase in the surviving fraction of radioresistant FaDu-RR cells was observed compared to parental FaDu cells, the difference was significant at 0.4 Gy (p=0.048), but not at 0.5 Gy (p=0.160). Pulsed low-dose rate irradiation Based on our experimental results, the radioresist-ant FaDu-RR cells showed the highest radiosen-sitivity at 0.3 Gy, therefore we used this dose to deliver PLDR irradiation. In both parental FaDu Radiol Oncol 2020; 54(2): 168-179. Todorovic V et al. / Pulsed low dose-rate irradiation of isogenic cells with different radiosensitivity 173 and radioresistant FaDu-RR, the surviving fraction of cells irradiated with either a single dose of 2.1 Gy or a PLDR dose of 7x0.3 Gy was significantly reduced in comparison to control non-irradiated cells or cells irradiated with a single dose of 0.3 Gy (p<0.0001). However, no difference in surviving fraction was observed between parental FaDu cells irradiated with a single dose of 2.1 Gy or PLDR irradiation of 7x0.3 Gy (p=0.607) (Figure 3A). On the contrary, surviving fraction of radioresistant FaDu-RR cells irradiated with PLDR dose of 7x0.3 Gy was significantly reduced (p=0.028) in comparison to cells irradiated with a single dose of 2.1 Gy (Figure 3C). Similarly, a significant reduction of surviving fraction after irradiation with 0.3 Gy was observed in radioresistant FaDu-RR cells (p=0.020), but not in parental FaDu cells (p=0.178) compared with the control non-irradiated cells. Modifying PLDR irradiation to 10x0.2 Gy abolished the difference in cell survival between PLDR and singledose irradiation in radioresistant FaDu-RR cells (p=0.951) (Figure 3B and 3D). Cell cycle Differences in cell cycle distribution in parental FaDu and radioresistant FaDu-RR were evaluated at 5- and 24-hour time point after different irradiation protocols (Figure 4). Asynchronous populations of non-irradiated FaDu and FaDu-RR cells did not differ in the cell cycle distribution. In response to different irradiation schemes, perturbations of cell cycle were observed in both FaDu and FaDu-RR cells. Namely, 5 hours after irradiation with a single dose of 2.1 Gy and a PLDR dose of 7x0.3 Gy, the percent of G1-phase FaDu cells was significantly reduced (p=0.021 and p=0.027, respectively), while the percent of G2/M-phase FaDu cells was significantly increased (p=0.023 and p=0.035, respectively) in comparison to control, non-irradiated FaDu cells. Contrary to FaDu cells, the percent of G1-phase radioresistant FaDu-RR cells was significantly reduced only after PLDR irradiation (p=0.047), while the percent of G2/M-phase cells was increased after both, a single dose of 2.1 Gy and a PLDR dose of 7x0.3 Gy (p=0.037 and p=0.014, respectively). No difference was observed in S-phase in FaDu nor FaDu-RR cells in all treatment groups. Cell cycle phase distribution was restored 24 hours after different irradiation protocols in FaDu cells, but not in FaDu-RR cells where an increase in G1-phase and a decrease in S-phase cells was observed after 2.1 Gy irradiation regimen (p=0.007 and p=0.0001). A similar increase in G1- FaDu FaDu-RR FIGURE 7. Venn diagrams of DNA damage signalling gene expression in parental FaDu and radioresistant FaDu-RR cells showing overlapping and differential gene expression. Only genes significantly over-expressed or under-expressed relative to control non-irradiated cells are shown. Genes in bold red are over-expressed, genes in bold green are under-expressed. phase and decrease in S-phase cells was observed also in PLDR-irradiated FaDu-RR cells, however not as prominent as after a single dose of 2.1 Gy. No difference in G2/M-phase of FaDu-RR cells was observed in any group. DNA damage signalling gene expression Different DNA damage signalling gene expression pattern was observed in response to different irradiation protocols relative to the control non-irradiated cells. In parental FaDu cells, more DNA damage signalling genes were under-expressed (Figure 5A), while in the radioresistant FaDu-RR cells, DNA damage signalling genes were predominantly over-expressed in response to irradiation (Figure 5B). In parental FaDu cells, significant under-expression of 2, 4, and 3 genes was observed in response to 0.3 Gy, 2.1 Gy, and 7x0.3 Gy irradiation, respectively (Figure 6A). In radioresistant FaDu-RR cells, significant under-expression of 2 genes and over-expression of 11, 8, and 2 genes was observed in response to 0.3 Gy, 2.1 Gy, and 7x0.3 Gy irradiation, respectively (Figure 6B). Specifically, BBC3 and CRY1 genes were under-expressed in both parental FaDu and radioresistant FaDu-RR cells in response to all irradiation schedules (Figure 7). PMS1 was over-expressed in radi-oresistant FaDu-RR cells in response to all three irradiation schedules, while ATR, BLM, CDC25A, H2AFX, MCPH1, and XRCC2 were over-expressed in 0.3 Gy and 2.1 Gy irradiated FaDu-RR cells. Radiol Oncol 2020; 54(2): 168-179. 174 Todorovic V et al. / Pulsed low dose-rate irradiation of isogenic cells with different radiosensitivity 174 FIGURE 8. Heat maps of DNA damage signalling gene expression in radioresistant FaDu-RR cells relative to the gene expression in parental FaDu cells. The magnitude of the fold change in gene expression of each gene from three independent experiments is represented by colour. Green indicates under-expressed genes, and red indicates over-expressed genes. GADD45A was over-expressed in 2.1 Gy and 7x0.3 Gy irradiated FaDu-RR cells, while ATM, MSH3, NBN, and RAD18 were over-expressed in 0.3 Gy irradiated FaDu-RR cells only. Direct comparison of the DNA damage gene expression in radioresistant FaDu-RR relative to parental FaDu cells identified differences in gene expression profile in non-irradiated cells and 7x0.3 Gy irradiated cells, but not 0.3 Gy and 2.1 Gy irradiated cells (Figure 8). Specifically, 71% of the tested DNA damage signalling genes in the control non-irradiated FaDu-RR cells were under-expressed, of which 7 genes (BLM, ERCC2, H2AFX, HUS1, RNF168, TOPBP1, XRCC3) were significantly under-expressed (Figure 9A). No significant difference in gene expression was observed in 0.3 Gy (Figure 9B) and 2.1 Gy (Figure 9C) irradiated FaDu-RR cells relative to FaDu cells. In 7x0.3 Gy irradiated FaDu-RR cells, 6 genes (ERCC1, EXO1, MBD4, PCNA, PPM1D, TOPBP1) were under-expressed and 1 gene (XPA) was over-expressed relative to parental FaDu cells irradiated with the same irradiation scheme (Figure 9D). TOPBP1 was the only gene under-expressed in both non-irradiated and PLDR-irradiated radioresistant FaDu cells relative to parental FaDu cells (Figure 10). Discussion Understanding molecular mechanisms of cellular response to low dose irradiation is important in order to evaluate risks and benefits of such exposure.28 In radioresistant tumors this could provide the basis for a more tailored and effective radiotherapy. Re-irradiation of recurrent tumours in the previously irradiated areas is a feasible approach that improves survival, but is limited due to normal tissue toxicity.19 However, altered fractiona-tion regimen could improve the therapeutic outcome of re-irradiated tumours and reduce normal tissue toxicities.14152930 In this study, we confirmed the presence of LDHRS in the experimentally established radiore-sistant FaDu-RR cells in vitro, but not in its parental FaDu cells. Furthermore, radioresistant FaDu-RR cells were more sensitive to PLDR irradiation than parental FaDu cells, likely due to the observed perturbations of the cell cycle and changes in the expression of DNA damage signalling genes observed in these cells. The use of PLDR irradiation in local recurrent HNSCC has been recently tested in a clinical trial in order to evaluate safety and treatment efficacy.31-33 PLDR irradiation was initially proposed for the treatment of recurrent radioresistant gliomas.1316 It exploits two phenomena, LDHRS, and the inverse dose-rate effect. First, the low dose fractions used in this approach fall within the LDHRS region, generally observed in the more radioresistant tu- Radiol Oncol 2020; 54(2): 168-179. Todorovic V et al. / Pulsed low dose-rate irradiation of isogenic cells with different radiosensitivity 47 mour cells.38 Second, the short intervals between low dose pulses create an apparently reduced dose-rate, which contributes to the normal tissue sparing and results in increased radiosensitivity of tumour cells.11 PLDR irradiation can be delivered over multiple days to increase the total irradiation dose, and improve the antitumor effects compared to conventional fractionation.4 LDHRS has to be confirmed prior to PLDR irradiation. Tailoring the PLDR parameters, such as the low dose and time intervals between the low doses, can further increase radiosensitivity.34 Low dose pulses should be applied within the LDHRS range of specific cell type, however, this might not be straightforward. The transition dose from LDHRS to IRR is cell type-specific and has been observed in the range of 0.2 Gy to 0.6 Gy for different tumour cells.12910 In the clinics, this transition dose might differ between tumours as well as within the tumour due to tumour cell heterogeneity; identification of specific LDHRS markers is needed to select patients, which could benefit from PLDR irradiation. Deciphering mechanisms contributing to the LDHRS could provide a better starting point to determine the efficient low irradiation doses used for PLDR clinically. In our study, dose reduction from 0.3 Gy to 0.2 Gy abolished the difference in cell survival between PLDR and single-dose irradiation in parental FaDu and radioresistant FaDu-RR cells. To describe the survival curve of LDHRS-positive cell lines, the linear-quadratic model fails in the low dose region and has to be adjusted to account for the increased radiosensitivity and IRR bellow 1 Gy. To take account for these specific processes, the induced repair model was proposed by Joiner et al.35 In addition to the induced repair model, alternative models have been proposed53637, such as the variable induced repair, which is more complex, but does not account for the dose rate effect.5 As a proof of LDHRS, different approaches can be considered. Namely, the condition aS > aR, confidence limits of aS and aR not overlapping, and DC value significantly greater than zero can be used to deduce the presence of LDHRS.45 Due to the variability in the measurements made by conventional clonogenic assay, which is typical at such high survival levels35, the experimental data fit the induced repair model in a variable extent. Because LDHRS is prevalent in radioresistant tumour cell lines38, we first evaluated the LDHRS status in the isogenic FaDu and radioresistant FaDu-RR cells, which was confirmed in the latter but not in the parental cell line. The observed transition from LDHRS to IRR in the 0.3 to 0.4 Gy dose range is similar to obser- FIGURE 9. DNA damage signalling gene expression in radioresistant FaDu-RR cells relative to parental FaDu. (A) Gene expression in control non-irradiated cells. (B) Gene expression in 0.3 Gy irradiated cells. (C) Gene expression in 2.1 Gy irradiated cells. (D) Gene expression in 7x0.3 Gy irradiated cells. Volcano plots show the fold change in gene expression in radioresistant FaDu-RR relative to parental FaDu cells and statistical significance (p value). The horizontal line shows the statistical significance threshold (p value < 0.05). Two vertical dashed lines show the threshold of over-expressed (right) and under-expressed genes (left), while the solid vertical line shows no change in gene expression. Symbols represent the mean gene expression of each tested gene in radioresistant FaDu-RR cells relative to parental FaDu cells from three independent experiments. vations in other reports.12910 In this preliminary experiment, we focused on the low dose response and did not evaluate cell survival in response to doses above 1 Gy due to technical limitations of our X-ray unit. Fitting these experimental data to the induced repair model is not balanced due to the lack of high dose response, and the parameters describing LDHRS (aS, aR, and DC) cannot be estimated with confidence intervals, which is a drawback of this study. In addition, the model-derived DC and the experimental dose with the lowest survival are not always the same. Mathematically, DC is defined as the dose required for 63% induction of radioresistance,35 therefore variations are expected Radiol Oncol 2020; 54(2): 168-179. 176 Todorovic V et al. / Pulsed low dose-rate irradiation of isogenic cells with different radiosensitivity 176 FIGURE 10. Venn diagrams of DNA damage signalling gene expression in radioresistant FaDu-RR cells showing overlapping and different gene expression after different irradiation protocols relative to parental FaDu cells. Only genes significantly over-expressed or under-expressed relative to parental FaDu cells are shown. Gene in bold red is over-expressed, genes in bold green are under-expressed. between model-derived and experimentally observed transition points from increased radiosensitivity to increased radioresistance.4 LDHRS can be efficient, if the proposed pulsed irradiation scheme delivers pulses of smaller dose than the transition dose from LDHRS to IRR.13 In our study, the PLDR irradiation scheme therefore consisted of a series of 0.3 Gy pulses. While surviving fraction of parental FaDu cells did not differ between single-dose and PLDR irradiation, a significantly lower survival was observed in PLDR-irradiated radioresistant FaDu-RR cells in comparison to single-dose irradiation. Similarly, several in vivo studies showed PLDR irradiation tumour volume reduction, resulting in a longer tumour growth delay in comparison to continuous irradiation.14,15 Ample scientific evidence supports an important role of cell cycle checkpoints and DNA damage signalling networks in the mechanisms of LDHRS.2 Cellular repair processes are induced above a certain threshold dose as described by the induced repair model.9 Below this threshold dose, cells can show increased radiosensitivity, while above this dose cell survival is increased due to induced signalling and repair. In the IRR range, DNA doublestrand break (DSB) repair is reportedly more efficient than in the LDHRS dose range.38 Evaluation of LDHRS in isogenic cell lines has not been studied extensively and therefore the isogenic cell lines with different LDHRS statuses are an attractive model to study the mechanisms of LDHRS in more detail. Novel insights into the unknown mechanisms of LDHRS could thus be gained. DNA repair is tightly coordinated with the cell cycle checkpoints.9 In our study, low dose irradiation did not affect cell cycle in isogenic cells, while irradiation with a higher single dose and PLDR irradiation resulted in cell cycle perturbations. Following G2/M arrest 5 hours after single and PLDR irradiation in both FaDu and FaDu-RR cells, the cell cycle was restored 24 hours after irradiation in FaDu, but not in FaDu-RR cells. This indicates a differential regulation of the cell cycle in radioresistant FaDu-RR cells in comparison to parental cells. Differences in cell cycle checkpoints in LDHRS-positive and LDHRS-negative cells have been observed previously. Most notably, in LDHRS-positive cells G2/M checkpoint was activated at irradiation doses higher than transition dose.39 Because LDHRS is associated with the G2-phase enriched populations40, it is likely that the observed LDHRS is due to inactive G2/M checkpoint in response to irradiation below the threshold dose.39 This data indicate on important role of DNA damage signalling mechanisms in LDHRS. Activation of G2/M checkpoint in cells with damaged DNA prevents entry into mitosis and provides an opportunity for DNA repair during the cell cycle delay. Increased radiosensitivity, observed in the LDHRS-positive cells, could be associated with inactive DNA damage-induced cell cycle checkpoints. Functional DNA damage signalling and repair mechanisms constitute DNA damage recognition, recruitment of specific signalling and repair proteins to the damage site and effective repair. LDHRS is not associated with reduced recognition of DSB breaks as seen by the same extent of phosphorylated H2AX.10,41 Persistent gammaH2AX foci after low dose irradiation despite the functional DNA repair mechanisms support different DSB repair kinetics.39,41 The unchanged level of phospho-rylated ATM in response to low dose irradiation indicates an inactive ATM signalling cascade.38 In the present study we focused on the expression of DNA damage signalling and repair genes in Radiol Oncol 2020; 54(2): 168-179. Todorovic V et al. / Pulsed low dose-rate irradiation of isogenic cells with different radiosensitivity 177 isogenic cell lines with different LDHRS status. The gene panel included DNA repair, apoptosis and cell cycle-associated genes. In LDHRS-negative parental FaDu cells, under-expression of DNA damage signalling genes was observed, while over-expression of DNA damage signalling genes was observed in LDHRS-positive radioresistant cells in response to irradiation. Specifically, DNA damage sensor genes (ATM, ATR, and H2AFX), cell cycle checkpoint regulator genes (CDC25A, BLM, GADD45A, MCPH1) and genes involved in homologous recombination (BLM, XRCC2) were over-expressed in response to 0.3 Gy and 2.1 Gy irradiation. On the other hand, after PLDR irradiation the expression of DNA damage sensor genes and homologous recombination genes was not increased, indicating inactive DNA repair mechanisms and decreased cell survival after PLDR. The reduction in cell survival can be associated also with aberrant regulation of cell cycle checkpoints. The observed G1 cell cycle arrest after 2.1 Gy and PLDR irradiation is likely mediated by over-expression of GADD45A in radioresistant FaDu-RR cells.42 Inactivation of GADD45A was also associated with chemosensitization and radiosensitization.43 44 Exact mechanisms of PLDR irradiation contributing to reduced cell survival of radioresistant cells are not clear yet. The role of GADD45A in the observed G1 cell cycle arrest should be further confirmed by RNA interference. Differential DNA damage signalling gene expression analysis demonstrated an early radiation-induced expression of various genes involved in the recognition of DNA damage, DNA repair and cell cycle regulation in radioresistant cells. However, after PLDR irradiation only 2 genes were over-expressed indicating inactive DNA damage response. To support the results of this preliminary study, the response to PLDR irradiation should be evaluated in other ra-dioresistant and LDHRS-positive tumour cell lines. Furthermore, since PLDR irradiation is a promising approach for re-irradiation of previously irradiated tissues, in vivo analysis of the effects of PLDR irradiation would greatly contribute to the promotion of PLDR irradiation scheme in the clinical setting. However, in vivo studies using human HNSCC tumours are limited by the use of immu-nocompromised animals to enable engraftment of human xenografts. In addition, the antitumor effects of PLDR irradiation might differ from the effects of PLDR irradiation observed in the clinical settings, because immunostimulatory effects of low dose irradiation would be limited in immuno-compromised animals.45 Also, the role of tumour microenvironment should be taken into account, as cell-cell and cell-microenvironment interactions importantly contribute to the radiosensitivity of cells.46 Modifications of irradiation schemes to improve the therapeutic index in the clinical management is an emerging approach for the treatment of HPV-positive oropharyngeal tumors.47-50 Considering the prevalence of LDHRS in radioresistant cells and tumours, PLDR irradiation could be more effective in radioresistant tumours than conventional radiotherapy. Modifications of irradiation schemes to reduce the effective dose rate and increase daily treatment time, such as PLDR irradiation, allow safer retreatment of previously irradiated areas, including recurrent radioresistant tumours of different origin.61617 In this respect, by using PLDR irradiation, a normal tissue damage could be minimised, and tumour control elevated.51 Benefits of PLDR irradiation, such as less normal tissue damage, were confirmed in in vivo studies of human orthotopic xenografts in nude mice.1415 A limit of the PLDR irradiation is the prolonged radiation delivery of one fraction composed of several pulses, which would lead to a larger burden of medical facilities. Although enhanced cyto-toxic effects were observed with shorter intervals of several minutes between low dose fractions, it is possible to introduce variations in time intervals between consecutive doses, dose per fraction and dose rate.34 Reduced cell survival can be observed also when low doses are separated by intervals of several hours, and additional benefit can be observed when combining this approach with chem-otherapy.52-55 In this study, we demonstrated LDHRS in iso-genic radioresistant cells, but not in the parental cells. Cell survival of LDHRS-positive radioresist-ant cells after PLDR was significantly reduced in comparison to parental cells. This reduction in cell survival of LDHRS-positive radioresistant cells was associated with variations in DNA damage signalling gene expression observed in response to PLDR. Variations in the DNA damage signalling response could be further exploited for the development of combined treatment approaches to radi-osensitizing recurrent and radioresistant HNSCC to improve the therapeutic index. Acknowledgements Authors would like to acknowledge Ilija Vojvodic for his help with setting up X-ray unit for low Radiol Oncol 2020; 54(2): 168-179. 178 Todorovic V et al. / Pulsed low dose-rate irradiation of isogenic cells with different radiosensitivity dose irradiations. This work was supported by the Slovenian Research Agency (program no. P3-0003 and P3-0307). References 1. Joiner MC, Marples B, Lambin P, Short SC, Turesson I. Low-dose hypersensitivity: Current status and possible mechanisms. Int J Radiat Oncol Biol Phys 2001; 49: 379-89. doi: 10.1016/S0360-3016(00)01471-1 2. Dai X, Tao D, Wu H, Cheng J. Low dose hyper-radiosensitivity in human lung cancer cell line A549 and its possible mechanisms. J Huazhong Univ Sci Technolog Med Sci 2009; 29: 101-6. doi: 10.1007/s11596-009-0122-4 3. Martin LM, Marples B, Lynch TH, Hollywood D, Marignol L. Exposure to low dose ionising radiation: Molecular and clinical consequences. Cancer Lett 2014; 349: 98-106. doi: 10.1016/j.canlet.2013.12.015 4. Schoenherr D, Krueger SA, Martin L, Marignol L, Wilson GD, Marples B. Determining if low dose hyper-radiosensitivity (HRS) can be exploited to provide a therapeutic advantage: A cell line study in four glioblastoma multiforme (GBM) cell lines. Int J Radiat Biol 2013; 89: 1009-16. doi: 10.3109/09553002.2013.825061 5. Guirado D, Aranda M, Ortiz M, Mesa J a, Zamora LI, Amaya E, et al. Low-dose radiation hyper-radiosensitivity in multicellular tumour spheroids. Br J Radiol 2012; 85: 1398-406. doi: 10.1259/bjr/33201506 6. Harney J, Short SC, Shah N, Joiner M, Saunders MI. Low dose hyperradiosensitivity in metastatic tumors. 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Cell Cycle 2004; 3: 477-83. doi: 10.4161/cc.3.4.786 Radiol Oncol 2020; 54(2): 168-179. 180 research article The prevalence of occult ovarian cancer in the series of 155 consequently operated high risk asymptomatic patients - Slovenian population based study Andreja Gornjec1, Sebastijan Merlo1, Srdjan Novakovic2, Vida Stegel2, Barbara Gazic3, Andraz Perhavec4, Ana Blatnik5, Mateja Krajc5 1 Department for Gynaecological Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Molecular Diagnostics Department, Institute of Oncology Ljubljana, Ljubljana, Slovenia 3 Pathology Department, Institute of Oncology Ljubljana, Ljubljana, Slovenia 4 Department for Surgical Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 5 Cancer Genetics Clinic, Institute of Oncology Ljubljana, Ljubljana, Slovenia Radiol Oncol 2020; 54(2): 180-186. Received 11 December 2019 Accepted 22 March 2020 Correspondence to: Assist. Prof. Mateja Krajc, M.D., Ph.D., Cancer Genetics Clinic, Institute of Oncology Ljubljana, Zaloška cesta 2, SI-1000 Ljubljana, Slovenia. E-mail: mkrajc@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. We assessed the prevalence, localization, type and outcome of occult cancer at risk-reducing sal-pingo-oophorectomy or salpingectomy (RRSO) in asymptomatic carriers of pathogenic or likely pathogenic BRCA1/2 variants and high-risk BRCA1/2 negative women. Patients and methods. A retrospective analysis of all consecutive gynaecologic preventive surgeries from January 2009 to December 2015 was performed. Participants underwent genetic counselling and BRCA1/2 testing before the procedure. Data on clinical parameters, adjuvant treatment and follow-up were collected and analysed. Results. One hundred and fifty-five RRSO were performed in 110 BRCA1, 35 BRCA2 carriers of pathogenic or likely pathogenic variants and 10 high-risk BRCA1/2 negative women, at the mean age of 48.3 years. Nine occult cancers (9/155, 5.8%) were identified; eight in BRCA1 positive women and one in high-risk BRCA1/2 negative woman. We identified four non-invasive serous intraepithelial tubal carcinomas (3 in BRCA1 carriers and 1 in a high-risk BRCA1/2 negative woman) and five invasive tubo-ovarian high grade serous cancers (all detected in BRCA1 carriers). Only one out of nine patients (11.1%) with occult cancer had a slightly elevated CA-125 value preoperatively. Conclusions. A 5.8% prevalence of occult invasive and noninvasive tubo-ovarian serous cancer after RRSO was found in high risk asymptomatic and screen negative women. We conclude that RRSO should be performed in BRCA1/2 carriers and in high-risk BRCA1/2 negative women. Age of preventive gynaecologic surgery should be carefully planned, taking into account the completion of childbearing age and type of mutation. The results favour the tubal hypothesis of tubal origin of high grade serous ovarian and peritoneal cancer. Cytology result of peritoneal cavity washing was important for the decision making process in determining treatment. Cytology examination should be performed in all cases of RRSO. CA-125 assay did not prove to be an effective screening tool for early cancer detection in our patients. Key words: risk-reducing salpingo-oophorectomy (RRSO); occult serous cancer; serous tubal intraepithelial cancer (STIC); BRCA1/2 pathogenic or likely pathogenic variant Radiol Oncol 2020; 54(2): 144-148. doi: 10.2478/raon-2020-0017 Gornjec A et al./ Occult ovarian cancer in operated high risk asymptomatic patients 181 Introduction Worldwide, ovarian cancer is the seventh most common cancer and the eighth cause of death from cancer in women.1 According to the Slovenian cancer registry, the median age at the time of diagnosis for ovarian cancer patients is 62 years.2 Epithelial ovarian cancer (EOC), being the commonest, is thought to be hereditary in at least 10% of cases, mostly due to BRCA1 or BRCA2 germline pathogenic or likely pathogenic variants. Ovarian cancer risk in BRCA carriers ranges from 36%-53% in BRCA1 and 11%-25% in BRCA2 carriers by the age of 80 years, compared to 1-2% in the general population.3 In addition, it presents at a younger age than in patients without a genetic predisposition. Genetic counselling, testing and appropriate screening and preventive strategies can highly reduce the risk.3-6 In more than 70%, EOC is diagnosed in advanced stages (III/IV). This is mainly due to vague and non-specific symptoms, and because of an ineffective ovarian cancer screening. BRCA carriers are therefore recommended to undergo salpingo-oophorectomy or salpingectomy as a risk-reducing strategy for ovarian cancer.378 A new paradigm of high grade pelvic serous cancer carcinogenesis puts fallopian tubes as an anatomic origin of the primary lesion. Salpingectomy with delayed oo-phorectomy might therefore be offered to BRCA carriers younger than 40 (after given informed consent in a research setting) who have completed their reproduction.910 So far, no precancerous lesions, like intraepithe-lial carcinoma, have ever been found in the ovaries. Precancerous lesions were found only in fallopian tubes. The first report of precancerous lesions in fallopian tubes after risk-reducing salpingo-oopho-rectomy (RRSO) was as a non-invasive serous tubal intraepithelial carcinoma (STIC).1112 Occult ovarian and tubal cancers where also found in specimens of BRCA carriers after RRSO. Occult cancers have been reported to occur between 2% and 17% and STICs between 3% and 12%, respectively.13-22 The occult cancer detection is probably influenced by the age at RRSO, gynaecological screening prior to RRSO, extent of the surgical removal of specimens and the accuracy of tubal pathohistological assessment. It is known that the protocol for sectioning and extensively examining the fimbriated end of the Fallopian tube (SEE-FIM) enables a more exact histopathological examination of the distal tube with its fimbrial part.23 Ovarian cancer risk reduction after RRSO is reported to be between 80%-96%. The risk of primary peritoneal cancer after RRSO is between 1%-4%.24' 25 Recurrence rate of ovarian cancer after the diagnosis of an occult invasive carcinoma at RRSO is relatively high (16%-47%), despite predominantly early stage, and small volume disease.17 25 On the other hand, the STICs rarely recur as carcinoma (5.8%-9%), and therefore chemotherapy may not be needed.24 26 Recommendations about the optimal treatment of STICs lesions remain unclear. Opinions for treatment of STIC lean toward staging procedures and observations, if no other lesions are found. Our study aimed to assess the prevalence, localization, type and outcome of occult cancer at RRSO in asymptomatic carriers of pathogenic or likely pathogenic BRCA1/2 variants and high-risk BRCA1/2 negative women. Patients and methods All consecutively operated women (asymptomatic carriers of pathogenic or likely pathogenic BRCA1/2 variants and high-risk BRCA1/2 negative) who underwent RRSO or salpingectomy from January 2009 to December 2015 at the Institute of Oncology Ljubljana, Slovenia, were included in our study. From 1999 genetic counselling and testing is offered at the Institute for women with positive family history of ovarian and breast cancer. Women with confirmed BRCA pathogenic or likely pathogenic variants and BRCA negative women with high ovarian cancer risk (at least two first or second-degree relatives with ovarian cancer) are assessed in a multidisciplinary setting by an onco-genetic team. In accordance with the guidelines high risk women are advised to perform risk reducing procedures (RRSO).7827 Until 2014, RRSO was offered only after women turned 40 years of age. From 2014 tubec-tomy with delayed oophorectomy is offered in a research setting to women younger than 40 years who have completed their reproduction. Clinical data were retrospectively collected from women with occult ovarian/tube/peritoneal cancer or non-invasive high-grade serous intraepithelial tube carcinoma diagnosed at the time of RRSO. Collected data included age at RRSO, mutation status, type of mutation, preoperative cancer antigen 125 (CA-125) level, histopathology result, staging, treatment (surgery and adjuvant chemotherapy), Radiol Oncol 2020; 54(2): 180-186. 182 Gornjec A et al./ Occult ovarian cancer in operated high risk asymptomatic patients 182 recurrence rate, prior history or development of breast cancer after RRSO, disease status and vital status. Asymptomatic women with negative ovarian cancer screening test (normal CA-125 and gynaecological ultrasound) 6 months prior to RRSO were included. Patients with ovarian or tubal cancer diagnosis prior to RRSO and those whose RRSO was a part of breast cancer treatment were excluded from the analysis. All RRSOs had the same surgical and pathological protocol. During surgery, before the salpingo-oophorectomy took place, peritoneal washing was performed and the material was sent for cytologi-cal examination. If there was no free fluid in the cavum Douglasi, we eluted the pelvis with 10 mL of physiologic solution and the material was sent for cytological examination. At our institution SEE-FIM protocol is used for RRSO specimens.23 When occult cancer and STIC are diagnosed, staging procedure is offered. After staging procedure is performed, cancers are managed according to the national guidelines.7 In women with STIC, when no other lesion is found at staging procedure, only observation with regular follow up is offered. When malignant cells are found at cytological examination of peritoneal cavity washing with no other lesion at staging procedure, chemotherapy with car-boplatin and paclitaxel is offered. The start of follow-up after RRSO was defined as the date of RRSO, where no other treatment was required. After cancer treatment with chemotherapy, the start of follow-up was defined as the date of the last given chemotherapy. The end of follow-up was defined as the last outpatient visit at our institution. The study was approved by the Ethical Committee of the Institute of Oncology Ljubljana (Number ERID-EK/15). Statistics Statistical analysis was performed using SPSS 22.0 for Windows. Descriptive statistics was used to describe the basic features of the data in the study. Differences between the groups were investigated with Student-t test. P-values <0.05 were considered to be statistically significant. Results In the period of our study (January 2009 - December 2015), 155 women underwent RRSO due to high ovarian cancer risk. Characteristic of patients and RRSO procedures In our cohort, there were 110/155 (71.0%) BRCA1 mutation carriers, 35/155 (22.6%) BRCA2 mutation carriers and 10/155 (6.5%) high-risk BRCA negative patients. A variant of uncertain significance (VUS) was detected in 4/10 high risk BRCA negative women. Mean age at RRSO among our patients was 48.3 (29-72); 47.6 (29-72) for BRCA1 carriers and 49.1 (38-66) for BRCA2 carriers, the difference did not differ significantly (p = 0.4). In high-risk BRCA negative women mean age at RRSO was 52.2 (3664) years. Median age at RRSO was also assessed for further comparison with other studies and accounted 47 years. Before RRSO was performed, 110/155 (71.0%) women had already been diagnosed with breast cancer, 4/155 (2.6%) women had the first breast cancer diagnosis before and the second breast cancer diagnosis after RRSO and 1/155 (0.6%) woman was diagnosed with breast cancer after RRSO. Among women where RRSO was performed, the mean age at breast cancer diagnosis was 54.0 (33-64) among women with STIC and 49.3 (38-61) among women with invasive cancer. The mean age of breast cancer diagnosis in BRCA1 carriers was 42.0 (27-62) in BRCA2 carriers 43.7 (26-57), which was not significantly different. In BRCA negative women it was 42.7 (26-64) years. Of all the RRSO procedures (N = 155), there were 141 (91.0%) bilateral laparoscopic salpingo-oophorectomies, 5 (3.2%) unilateral laparoscopic salpingo-oophorectomies and 7 (4.5%) laparoscopic salpingectomies. One patient (0.6%) had a bilateral and one had (0.6%) a unilateral laparoscopic salpingo-oophorectomy. Pathological findings at RRSO Non-invasive or invasive serous high-grade cancer was diagnosed in 9 out of 155 (5.8%) operated women (Table 1). There were five (3.2%) occult ovarian cancers and 4 (2.6%) STICs. All cancers were detected among BRCA1 positive women as described in Table 1. Among STICs there were three BRCA1 carriers and one without a known mutation, but from the high risk group. Among patients with occult invasive cancers, two were 39 years of age, all the other were older, mean age being 50.4 years. Mean age of patients with STICs was 57.8 years. The difference was not Radiol Oncol 2020; 54(2): 180-186. Gornjec A et al./ Occult ovarian cancer in operated high risk asymptomatic patients 183 TABLE 1. Clinical characteristics of occult findings after RRSO Patient Number Age at RRSO (years) Occult finding BRCA gene involved Type of pathogenic variant FIGO STAGE Cytology Treatment Vital status 1 53 STIC BRCA1 deletion exons 4-9 STIC NEG Surgery NED 2 69 STIC BRCA1 c.3018_3021delTTCA STIC NEG Surgery NED 3 64 STIC negative/high risk STIC NEG Surgery NED 4 45 STIC BRCA1 deletion exons 4-9 (Staging procedure NEG) I C PC Surgery+ACT NED 5 56 HGSC BRCA1 c.181T>G III B PC Surgery+ACT DOOD 6 39 HGSC BRCA1 c.5266dup.C III B PC Surgery+ACT OT 7 57 HGSC BRCA1 c.5266dup.C I C PC Surgery+ACT NED 8 39 HGSC BRCA1 c.1687C>T III A PC Surgery+ACT NED 9 61 HGSC BRCA1 deletion exons 4-9 III A PC Surgery+ACT OT ACT = adjuvant chemotherapy; DOOD = died of other disease; FIGO = International Federation of Gynecology and Obstetrics; HGSC = high grade serous cancer; NED = no evidence of disease; NEG = negative; OT = on treatment; PC = peritoneal carcinomatosis; RRSO = risk reducing oophorectomy; STIC = serous intraepithelial tubal cancer statistically significant. There were no occult cancers diagnosed among BRCA2 carriers. Stages of cancer Three of four women with STIC underwent surgical staging procedure (Table 1). In three cases with STIC, staging procedure did not find any additional neoplastic cells and observation with no adjuvant treatment was recommended. In one case, the malignant cells were detected in the peritoneal cavity by cytological examination only. Stage was assessed to be I C. This patient received adjuvant chemotherapy with paclitaxel and carboplatin. Among patients with occult cancers, one was assessed as stage I C, two as stage III A and two as stage III B. All received the adjuvant treatment with six courses of paclitaxel and carboplatin (Table 1). Localisation of findings Two STICs were found at the fimbrial part of fallopian tube and in the other two STICs, fallopian tube only was reported as a localisation. In invasive cancer patients, cancer cells were found in (i) two cases at the fimbrial part of the Fallopian tube and in one ovary, (ii) in one case the disease was present in both ovaries and in both Fallopian tubes, (iii) in one case cancer cells were present on the surface of both ovaries with normal Fallopian tubes and, (iv) in one case cancer cells were present on the surface of one ovary and in the Fallopian tube. Pathohistological findings included one patient with focally atypical epithelium of the fallopian tube with the addition of transitional cell metaplasia, one patient with adenomatoid hyperplasia with bilateral proliferation of Sertoli cells in both hiluses of ovaries that represented embryonal remnants and one patient with transitional cell metaplasia. CA-125 and cytology results All except one patient with STIC had a negative cytology result; on the other hand, all occult cancers had positive cytological findings. Before the RRSO CA-125 measurement was performed in 83.9% of women. It was negative in all occult cancers and STICs except in one occult cancer, where it was slightly elevated, being 48 kU/L (normal value being < 35 kU/L). When considering CA-125 specificity in premenopausal years and normal vaginal ultrasound, patient was considered screen negative. Follow-up time of women with occult invasive and non-invasive cancer In our study the follow-up period was 23 to 73 months. Until December 2018 one woman with occult cancer died of gastric cancer which was diagnosed after adjuvant treatment for ovarian cancer. Two out of nine (22.2%) are being treated for their third recurrence of disease, the rest (6/9, 66.7%) are alive with no signs of disease. Mean follow-up of patients with occult invasive and non-invasive ovarian cancer was 29 months (15-51). After RRSO, no woman developed peritoneal cancer. Radiol Oncol 2020; 54(2): 180-186. 184 Gornjec A et al./ Occult ovarian cancer in operated high risk asymptomatic patients 184 Discussion We are presenting a population based study which aimed to address the prevalence, localization, type and outcome of occult cancer at RRSO in asymptomatic carriers of pathogenic or likely pathogenic BRCA1/2 variants and high-risk BRCA1/2 negative women. Our main outcome was the detection of pathologic serous changes in tubes and ovaries in 5.8% of all operated women. At RRSO we found occult serous cancer in 5.5% of BRCA carriers, with 3.4% (5/145) having highgrade cancers and 2.1% (3/145) STICs; all were found in BRCA1 positive women. The prevalence of occult cancers in BRCA1 positive women in our study was 7.3% (8/110). According to the available literature, the prevalence of occult cancer found after RRSO varies from 2% to 17%.13-2228 In our study, the prevalence of occult cancer in BRCA1/2 positive women was 5.5%, which is similar to what Conner et al. have found.29 We detected fewer occult cancers than Powell et al. who reported a rate of 7.9% and more than Reitsma et al. (2.2%) and Finch et al. (4.2%).141624 It would be expected that studies that reported lower prevalence of occult cancers also had a lower median age at RRSO. Reitsma et al. reported the median age at RRSO to be 44 years of age, which is less than in our study where the median age at RRSO was 47.14 Since only asymptomatic women and screen negative women were included, the age at RRSO seems to be the most important factor which determines the higher prevalence of pathologic findings. When considering BRCA1/2 carriers, all pathological changes in our study were detected among the BRCA1 positive patients. In contrast, there were no pathological changes in 35 BRCA2 positive women. The speculative reason for this might be lower ovarian cancer penetrance and the later age of onset in BRCA2 carriers in comparison with BRCA1 carriers and therefore critical number of cases for one case to be found was probably not achieved until the end of this evaluation. Among high-risk BRCA negative women, the prevalence of occult serous disease was 10% (1/10). Limited data is available for the comparison of prevalence of occult cancers after RRSO in BRCA negative women. Only Reitsma's study from Netherlands found one (1/57) case of STIC and one (1/57) case of atypical hyperplasia in RRSO specimens of BRCA-negative women with VUS.14 Our finding suggests the benefit and the importance of preventive surgeries in these women as well, though the number of our patients was very small. An interesting and counterintuitive finding is the comparison of mean age among invasive and noninvasive cancers found at RRSO. Surprisingly, women diagnosed with STIC were older than women diagnosed with cancer. Mean age was 57.8 (45-69) and 50.4 (39-61) years, respectively. The difference was not statistically significant, most probably due to relatively small sample size. Furthermore, there was also insignificant trend of noninvasive cancer patients having breast cancer at an older age than those with invasive cancer. Similar findings were mentioned also in study from Powell et al.16 The localization of occult serous pelvic disease was coherent with the literature. Occult cancers were found in the tubal epithelium in 60% (3/5) and only in 40% (2/5) in the ovaries. In two cases the ovary was infiltrated only on the surface epithelium, in all other cases the cortex and stroma were also infiltrated. Intraepithelial serous lesions were found only in the tubal epithelium. In 50% (2/4) of cases, STICs were found at the fimbrial part of tubes. In other two cases exact localization was not defined and is being revised.17 International Federation of Gynaecology and Obstetrics (FIGO) stage distribution among occult cancers diagnosed after RRSO was undoubtedly different and much more favourable than among population that presents with symptoms. There were 55.6% (4/9) of cancers staged I, II or in situ. There were no cancers staged higher than III B, two were III B and two were III A. The long term outcome is therefore expectedly better among patients diagnosed with ovarian/fallopian/peritoneal cancer after RRSO. Cytological examination of peritoneal cavity washing was found to be very important. When all pathologic findings are negative, positive cytology finding is the only one that may determine further treatment. Women with positive cytology with all other specimens being negative are advised systemic chemotherapy based on cytology findings.26 Screening for ovarian cancer in the general population with CA-125 (and possibly transvaginal ultrasound) is generally not recommended due to its low sensitivity and specificity. It is, however, seen as a reasonable temporary alternative for women at high risk, who wish to delay RRSO.30 In our study, only one patient with an occult cancer had serum CA-125 levels above the cut-off value. CA-125 assay did not prove to be an effective screening tool for early cancer detection in our patients. Genotype-phenotype correlations in BRCA carriers are not well defined and it is therefore difficult Radiol Oncol 2020; 54(2): 180-186. Gornjec A et al./ Occult ovarian cancer in operated high risk asymptomatic patients 185 to estimate the exact risk of ovarian cancer associated with specific pathogenic variants.31 The mutational spectrum in patients with occult carcinoma in our study is in line with what is otherwise known about Slovenian BRCA carriers. Three of the detected variants are very common in Slovenian BRCA1 carriers, i.e. c.181T>G p.(Cys61Ser), c.1687C>T p.(Gln563*) and c.5266dupC p.(Gln1756Profs*74).32 In contrast, the deletion of exons 4-9 is only seen in 3.4% of our BRCA positive families but appears to be associated with a particularly high ovarian cancer risk. In this study, three out of eight (37.5%) BRCA positive patients with occult cancer carried this variant, which further supports the hypothesis that deletion 4-9 is highly penetrant with regards to ovarian cancer. The main limitation of our study is already mentioned relatively small sample size that limits statistical evaluation. On the other hand, we were able to obtain an accurate clinical data for the sample studied. All studied women were tested and operated in our centre, where genetic testing and preventive follow up is performed on a national level. Conclusions In conclusion, a 5.8% prevalence of occult invasive and noninvasive serous cancer after RRSO was found in high risk asymptomatic and screen negative BRCA1/2 carriers. Most of occult invasive and noninvasive serous cancers were detected in BRCA1 positive patients, yet the RRSO should also be considered as a preventive method for BRCA negative high risk women. Age at preventive gy-naecologic surgery should be carefully considered, taking into account the completion of childbearing age, and ideally, performed soon after 35, at least in BRCA1 patients. Cytology examination of peritoneal cavity washing should be performed in all cases of RRSO. 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Long term follow up of BRCA1 and BRCA2 mutation carriers with unsuspected neoplasia identified at risk reducing salpingo-oophorectomy. Gynecol Oncol 2013; 129: 364-71. doi.: org/10.1016/j.ygyno.2013.01.029 17. Sherman ME, Piedmonte M, Mai PL, loffe OB, Ronnett BM, Van Le L, et al. Pathologic findings at risk-reducing salpingo-oophorectomy: Primary results from Gynecologic Oncology Group trial GOG-0199. J Clin Oncol 2014; 32: 3275-83. doi: 10.1200/JCO.2013.54.1987 18. Thompson C, McCormick C, Kamran W, O'Riain C, Norris L, Gallagher D, et al. Risk reduction surgery (RRS) for tubo-ovarian cancer in an Irish gynaecological practice: an analysis of indications and outcomes. Ir J Med Sci 2018; 187: 789-94. doi: 10.1007/s11845-017-1717-6 19. Ricciardi E, Tomao F, Aletti G, Bazzurini L, Bocciolone L, Boveri S, et al. Risk-reducing salpingo-oophorectomy in women at higher risk of ovarian and breast cancer: a single institution prospective series. 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Int J Surg Case Rep 2018; 52: 107-10. doi: 10.1016/j.ijscr.2018.10.014 23. Clarke BA, Crum CP, Nucci, MR, Oliva MR. Protocol for the examination of specimens from patients with carcinoma of the fallopian tube. Based on AJCC/UICC TNM, 7th edition, and FIGO 2006 Annual Report. [cites 2019 Nov 15]. Available from: http://webapps.cap.org/apps/docs/committees/ cancer/cancer_protocols/2013/FallopianTube_13protocol_3101.pdf 24. Finch APM, Lubinski J, M0ller P, Singer CF, Karlan B, Senter L, et al. Impact of oophorectomy on cancer incidence and mortality in women with a BRCA1 or BRCA2 mutation. J Clin Oncol 2014; 32: 1547-53. doi: 10.1200/ JCO.2013.53.2820 25. Domchek SM, Friebel TM, Singer CF, Evans DG, Henry T, Isaacs C, et al. Europe PMC funders group association of risk-reducing surgery in BRCA1 or BRCA2 mutation carriers with cancer risk and mortality. JAMA 2010; 304: 967-75. doi: 10.1001/jama.2010.1237 26. Patrono MG, Corzo C, Iniesta M, Ramirez PT. Management of preinvasive lesions. Clin Obstet Gynecol 2017; 60: 771-9. doi: 10.1097/ GRF.0000000000000316 27. Genetic/Familial High-Risk Assessment: Breast and Ovarian, version 3.2019, NCCN Guidelines, January 18, 2019. [cites 2019 Nov 15]. Available from: https://www.nccn.org/professionals/physician_gls/default.aspx#detection 28. Lavie O, Moskoviz MG, Auslender R, Gemer O, Bitterman A, Younes G, et al. Clinical and pathological characteristics of incidental diagnostic early occult malignancy after risk-reducing salpingo-oophorectomy in BRCA mutation carriers. Int J Gynecol Cancer 2016; 26: 233-9. doi: 10.1097/ IGC.0000000000000624 29. Conner JR, Meserve E, Pizer E, Garber J, Roh M, Urban N, et al. Outcome of unexpected adnexal neoplasia discovered during risk reduction salpingo-oo-phorectomy in women with germ-line BRCA1 or BRCA2 mutations. Gynecol Oncol 2014; 132: 280-6. doi: 10.1016/j.ygyno.2013.12.009 30. Drescher CW, Anderson GL. The yet unrealized promise of ovarian cancer screening. JAMA Oncol 2018; 4: 456-7. doi: 10.1001/jamaoncol.2018.0028 31. Rebbeck TR, Mitra N, Wan F, Sinilnikova OM, Healey S, McGuffog L, et al. Association of type and location of BRCA1 and BRCA2 mutations with risk of breast and ovarian cancer. JAMA 2015; 313: 1347-61. doi: 10.1001/ jama.2014.5985 32. Stegel V, Krajc M, Zgajnar J, Teugels E, De Grève J, Hocevar M, et al. The occurrence of germline BRCA1 and BRCA2 sequence alterations in Slovenian population. BMC Med Genet 2011; 12: 9. doi: 10.1186/1471-2350-12-9 Radiol Oncol 2020; 54(2): 180-186. 187 research article Dietary iodine intake, therapy with radioiodine, and anaplastic thyroid carcinoma Nikola Besic1,Barbara Gazic2 1 Department of Surgical Oncology, Institute of Oncology Ljubljana, Slovenia 2 Department of Pathology, Institute of Oncology Ljubljana, Slovenia Radiol Oncol 2020; 54(2): 187-193. Received 16 November 2019 Accepted 30 March 2020 Correspondence to: Prof. Nikola Bešic, M.D., Ph.D., Department of Surgical Oncology, Institute of Oncology Ljubljana, Zaloška 2, 1000 Ljubljana, Slovenia. E-mail: nbesic@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. Anaplastic thyroid cancer (ATC) is one of the most aggressive tumors. The aim of the study was to determine the correlation between a higher dietary intake of iodine, frequency of ATC and the characteristics of ATC, and to find out how often patients with ATC had a history of radioiodine (RAI) therapy. Patients and methods. This retrospective study included 220 patients (152 females, 68 males; mean age 68 years) with ATC who were treated in our country from 1972 to 2017. The salt was iodinated with 10 mg of potassium iodide/ kg before 1999, and with 25 mg of potassium iodide/kg thereafter. The patients were assorted into 15-year periods: 1972-1986, 1987-2001, and 2002-2017. Results. The incidence of ATC decreased after a higher iodination of salt (p = 0.04). Patients are nowadays older (p = 0.013) and have less frequent lymph node metastases (p = 0.012). The frequency of distant metastases did not change over time. The median survival of patients in the first, second, and third periods was 3, 4, and 3 months, respectively (p < 0.05). The history of RAI therapy was present in 7.7% of patients. Conclusions. The number of patients with a history of RAI therapy did not change statistically over time. The incidence of ATC in Slovenia decreased probably because of higher salt iodination. Key words: anaplastic thyroid carcinoma; iodination of salt; treatment, survival Introduction Anaplastic thyroid cancer (ATC) is one of the most aggressive tumors known in humans.1 It is a locally widely invasive disease which progresses despite treatment and finally results in metastatic disease in the majority of patients.2 Fortunately, ATC is a rare disease and the estimated annual incidence is about two per million of the population.34 In the USA and Japan, ATC represents less than 2% of thyroid carcinomas.56 On the other hand, the incidence of ATC has been stable in the last decades.17 In the literature, there are only limited data about the history of radioiodine (RAI) therapy in patients with ATC. The aim of the study was to find out how often patients had a history of RAI therapy. In one of our recent studies we compared the incidence of ATC during the periods when the intake of potassium iodide in salt was 10 and 25 mg/kg in the Republic of Slovenia.8 It was observed that the incidence of ATC decreased with a higher iodination of salt in Slovenia.8 Another aim of the study was to determine the correlation between a higher dietary intake of iodine, frequency of ATC and the characteristics of ATC. Patients and methods The data on the patients with ATC treated at the Institute of Oncology in Ljubljana in the years 1972-2017 were collected retrospectively. During this period, there were 220 patients (152 females, 68 males; median age 69 years; mean age 68 years) with ATC. The Cancer Registry of Republic of Slovenia is one of the oldest population-based Radiol Oncol 2020; 54(2): 144-148. doi: 10.2478/raon-2020-0017 188 Besic N and Gazic B / Dietary iodine intake and anaplastic carcinoma cancer registries in Europe.9 It was founded in 1950 at the Institute of Oncology in Ljubljana as a special service for collecting and processing data on cancer incidence and cancer patients' survival. Notification of cancer has been compulsory in Slovenia since the foundation of the Registry and prescribed by law.9 The main sources of data are notifications of cancer, gathered from all hospitals and diagnostic centers in Slovenia.9 Furthermore, all the patients with thyroid cancer are treated at the Institute of Oncology in Ljubljana, so our data represent a population based study. For each patient, the data on sex, age, history of treatment with radioiodine, clinical and tumor characteristics, and duration of survival were collected. Distant metastases were diagnosed by clinical examination and additional diagnostic procedures, including lung and/or bone X-ray, radionu-clide investigations, ultrasonography, computed tomography, and/or nuclear magnetic resonance imaging. Data about the treatment of our patients have already been reported.1011 The Protocol Review Board and Ethics Committee of the Institute of Oncology on 12th December 2018 (ERID-KSOPKR/43, OIRIKE 00448) reviewed and approved the study, which was conducted in accordance with the ethical standards prescribed in the Declaration of Helsinki. For retrospective studies, informed consent is not necessary according to the national regulations. The need for consent was waived by the Institutional Review Board and Ethics Committee of the Institute of Oncology Ljubljana. All cases were reviewed by pathologists and cy-topathologists at our comprehensive cancer center, experienced in thyroid pathology. Histological specimens were retrieved by surgical removal of the thyroid tumor, surgical biopsy, or autopsy, whereas cytological samples were obtained by fine-needle aspiration biopsy of the primary tumor or its metastases. The diagnosis of ATC was confirmed by both histology and cytology in 75 patients, by cytology alone in 97 patients, and by histology alone in 48 patients. In Slovenia, salt was iodinated with 10 mg of potassium iodide/kg and 25 mg of potassium iodide/ kg during the periods 1972-1998 and 1999-2017, respectively.1213 All salt that was on the market in Slovenia during the first and the second 15-year periods had the required content of iodine. Slovenia has been considered to be an area with an adequate iodine supply since 1999.1213 Since 2004, when Slovenia became a member of the European Union, salt with a lower content of iodine and Himalayan salt with no iodine added at all have also been available in health food stores. Furthermore, in the last decade, the use of prefabricated or frozen food became more popular in Slovenia. Fortunately, almost all households in Slovenia use salt with 25 mg of potassium iodide/kg, which is evident from epi-demiological studies in schoolchildren.1214 For the purposes of the present study, patients were assorted into one of three 15-year periods according to the year of diagnosis of ATC: 1972-1986, 1987-2001, and 2002-2017. The frequency of ATC during these three periods and the characteristics of the patients during these periods were compared. The characteristics of the patients and tumors and the history of RAI therapy according to 15-year periods were statistically analyzed using contingency tables and analysis of variance. SPSS 16.0 for Windows (SPSS, Chicago, IL) was also used in Kaplan-Meier univariate analysis for the assessment of patients' survival. Results The incidence of ATC in Slovenia has decreased. ATC was diagnosed in the periods 1972-1987, 1988-2003, and 2004-2017 in 95, 87, and 38 patients, respectively (p = 0.04). The mean incidence of ATC in the periods 1972-1986, 1987-2001, and 20022017 was 6.3 (range 2-12), 5.8 (range 3-10), and 2.5 (range 1-10) patients per year, respectively. The characteristics of patients and their outcomes according to 15-year periods are presented in Table 1. Patients with ATC are older now than they used to be (p = 0.013). During the periods 1972-1986, 1987-2001, and 2002-2017, the patients' mean age was 66 (SD ±11.8) years, 69 (SD ±8.4) years, and 72 (SD ±11.8) years. The mean tumor diameter in the three time periods was 9.1 cm, 9.9 cm, and 8.5 cm respectively. The difference was not statistically significant (p = 0.26). However, a tumor diameter larger than 10 cm was more frequent in patients during the 1972-2001 periods than thereafter (p < 0.05). Lymph node metastases were less commonly diagnosed in the last period (p = 0.012). However, the frequency of distant metastases did not change over time (p = 0.65). The median survival of patients during the first, second, and third 15-year periods was 3 months, 4 months, and 3 months, respectively (p < 0.05). Cumulative yearly doses of 131I applied in medicine from 1994 to 2017 in Slovenia are presented in Figure 1. Altogether, 17/220 (7.7%) of patients had a Radiol Oncol 2020; 54(2): 187-193. Besic N and Gazic B / Dietary iodine intake and anaplastic carcinoma 189 TABLE 1. Clinical characteristics of patients and 15-year periods Clinical characteristic Subgroup All patients N = 220 Number Patients 1972-1984 N=95 Patients 1987-2001 N = 87 Patients 2002-2017 N = 38 p-value Number Number Number Gender Male Female 68 152 27 68 29 58 12 26 0.77 Age 70 years or less 124 59 49 16 0.11 71 years or more 96 36 38 22 History of radioiodine therapy No Yes 203 17 89 6 83 4 31 7 0.023 Previous thyroid enlargement No or no data 110 46 49 26 0.036 (> 1 year) Yes 110 49 38 12 Good 92 47 33 12 General condition Moderate 58 25 20 13 0.13 Poor 70 23 34 13 Tumor growth > 3 months 173 70 72 31 0.29 < 3 months 47 25 15 7 Local tumor extension Extrathyroid Intrathyroid 199 21 87 8 78 9 34 4 0.88 < 5 cm 35 20 12 3 Tumor size 5-10 cm 105 40 39 26 0.043 > 10 cm 80 35 36 9 < 50 ml 16 7 5 4 Tumor volume (width2 x length)/2 50-99 ml 100-149 ml 150-199 ml 37 22 18 19 6 9 14 12 7 4 4 2 0.68 200 ml or more 127 54 49 24 Lymph nodes No 127 45 54 28 0.012 metastases Yes 93 50 33 10 Distant metastases No Yes 113 107 46 49 48 39 19 19 0.65 Presentation of anaplastic Incidental 8 4 4 0 0.50 carcinoma Evident 212 91 83 38 IVA 17 5 9 3 TNM stage IVB 96 41 39 16 0.73 IVC 107 49 39 19 Without surgery 130 48 52 30 Biopsy 23 13 10 0 Thyroid surgery Subtotal thyroidectomy 13 10 3 0 Extracapsular lobectomy with isthmusectomy 17 9 7 1 0.029 Total or near-total thyroidectomy 37 15 15 7 Biopsy or no surgery 153 61 62 30 Residual tumor after surgery R0 R1 32 18 18 9 10 6 4 3 0.55 R2 17 7 9 1 Lymph node dissection No 211 90 83 38 0.36 Yes 9 5 4 0 External beam irradiation No Yes 37 183 22 73 11 76 4 36 0.087 Without or < 20 Gy 54 33 15 6 External beam irradiation > 20 Gy and 45 Gy 74 41 26 7 0.001 > 45 Gy 92 21 46 25 Chemotherapy No Yes 81 139 38 57 24 63 19 19 0.04 Death because of anaplastic No (alive, other causes, lost from follow-up) 13 3 7 13 0.32 carcinoma Yes 207 92 80 25 Radiol Oncol 2020; 54(2): 1 87-193. 190 Besic N and Gazic B / Dietary iodine intake and anaplastic carcinoma TABLE 2. Clinical characteristics of the patients and the history of radioiodine therapy Clinical characteristic Subgroup All patients N = 220 Without history of RAI therapy N = 203 With history of RAI therapy N = 17 p-value Gender Male Female 68 152 66 137 2 15 0.10 Age 70 years or less 124 114 10 1.00 71 years or more 96 89 7 1972-1986 95 89 6 Year of diagnosis 1987-2001 87 83 4 0.023 2002-2017 38 31 7 Previous thyroid No or no data 110 108 2 0.001 enlargement (> 1 year) Yes 110 95 15 Good 92 85 7 General condition Moderate 58 57 1 0.063 Poor 70 61 9 Tumor growth > 3 months 173 157 16 0.13 < 3 months 47 16 1 Local tumor extension Extrathyroid Intrathyroid 199 21 183 20 16 1 1.00 < 5 cm 35 34 1 Tumor size 5-10 cm 105 96 9 0.50 > 10 cm 80 73 7 < 50 ml 16 16 0 Tumor volume (width2 x length)/2 50-99 ml 100-149 ml 150-199 ml 37 22 18 36 20 18 1 2 0 0.19 200 ml or more 127 113 14 Lymph nodes No 127 117 10 1.00 metastases Yes 93 86 7 Distant metastases No Yes 113 107 105 98 8 9 0.83 Presentation of Incidental 8 8 0 0.51 anaplastic carcinoma Evident 212 195 17 IVA 17 16 1 TNM stage IVB 96 89 7 0.91 IVC 107 98 9 Without surgery 130 117 13 Biopsy 23 22 1 Subtotal thyroidectomy 13 12 1 Thyroid surgery Extracapsular lobectomy with isthmusectomy 17 17 0 0.56 Total or near-total thyroidectomy 37 35 2 Biopsy or no surgery 153 139 14 Residual tumor after surgery R0 R1 32 18 32 17 0 1 0.32 R2 17 15 2 Lymph node dissection No 211 194 17 1.00 Yes 9 9 0 External beam irradiation No Yes 37 183 30 173 7 10 0.005 Without or < 20 Gy 54 47 7 Dose of radiotherapy > 20 Gy and < 45 Gy 74 70 4 0.244 > 45 Gy 92 86 6 Chemotherapy No Yes 81 139 75 128 6 11 1.00 Death because of ATC No (alive, other causes, lost from follow up) 13 12 1 1.00 Yes 207 191 16 ATC = anaplastic thyroid cancer; RAI = radioiodine Radiol Oncol 2020; 54(2): 187-193. 191 Besic N and Gazic B / Dietary iodine intake and anaplastic carcinoma history of radioiodine therapy from 4 months to 40 years before the diagnosis of ATC. The number of patients with ATC who had a history of RAI therapy did not change statistically over time, while the incidence of patients with ATC decreased over time (p = 0.023). Data about patients with regard to the history of RAI therapy are presented in Table 2. Previous enlargement of thyroid gland was more common in patients with a history of RAI therapy in comparison to those who received no RAI (p < 0.001). There was no difference in survival of patients with and without a history of RAI therapy (p = 0.49). Discussion Salt was iodinated in Slovenia with 10 mg of potassium iodide/kg and 25 mg of potassium iodide/ kg during the periods 1972-1998 and 1999-2017, respectively.1213 As expected and reported in other countries15, ten years after the beginning of higher salt iodination in Slovenia, the incidence of diffuse goiter in adolescents and adults decreased.12-1416 Furthermore, from 1999 to 2009, the incidence of thyroid autonomy in Slovenia decreased from 32.7/100.000 to a 27 % lower value.12 During the same time period, the baseline incidence of Graves' disease (27.8/100.000) did not change significantly.12 On the other hand, the incidence of Hashimoto's thyroiditis (73.2/100.000 in 1999) gradually increased to levels more than twice as high as be-fore.12 During the same time period, the incidence of thyroid carcinoma increased from 5.1/100.000 to 7.25/100.000,17 but the incidence of anaplastic carcinoma decreased after higher iodination of salt in Slovenia.9 A similar observation about the incidence of ATC after higher salt iodination was reported in other endemic goiter regions, namely the Tyrol region in Austria and Argentina.1819 In contrast to differentiated thyroid cancer, which often has a subtle clinical presentation and may be difficult to detect, ATC is correctly diagnosed in nearly all cases in countries with adequate health care because of rapid tumor growth and the clinical presentation.920 Risk factors for ATC are: a history of goiter or a prior co-existing differentiated thyroid cancer21-23, insufficient iodine in the diet9182425, low level of education21, type B blood group21, and presence of TERT mutation in coexisting thyroid papillary carcinoma26. We think that the drop in ATC incidence in Slovenia was mainly caused by higher salt iodination. A lower rate of goiter in Slovenia, which was also due to higher io- 1311 (GBq) 1800 16W> 14CO 120C 1000 - — — — — — — — — - — — — - — — — — - FIGURE 1. Cumulative yearly doses of 131I applied in medicine from 1994 to 2017 in Slovenia. dination of salt, is another risk factor that contributed to a lower incidence of ATC in our country.1213 Other risk factors for ATC, namely the educational level of the population, the socioeconomic status of the general population, or the rate of persons with type B blood group, did not change over time in Slovenia. RAI treatment for benign thyroid disease is considered to be a safe procedure.27 28 A meta-analysis showed no increase in the overall cancer risk after RAI treatment for hyperthyroidism. However, there was a trend towards increased risk of thyroid, stomach, and kidney cancer.2728 In 1990, Venkatesh et al.29 reported in a series of 121 cases with ATC that seven (6%) patients had received prior RAI treatment. Even 7.7% of patients from our present study also had a history of RAI therapy. A history of RAI therapy in patients with ATC is more frequent than the proportion of persons treated with RAI therapy in the Slovenian population. The use of 131I increased in Slovenia in the 1990s and was at 1630.9 GBq in 1999. Thereafter, the use of 131I has slowly been decreasing as seen in Figure 1. In 2011, a total of 531 diagnostic procedures with 131I or 123I for thyroid imaging with an accumulative effective dose of 2.29 manSv were done in Slovenia.30 131I and 123I were used in 48% and 52% of diagnostic procedures, which contributed to 69% and 31% of the collective effective dose for diagnostic procedures, respectively.30 On the other hand, the therapy with 131I was done in 512 patients with a benign disease and 151 patients with a carcinoma in 2014. The main difference between RAI therapy in benign thyroid disease and thyroid carcinoma is the dose Radiol Oncol 2020; 54(2): 187-193. 192 Besic N and Gazic B / Dietary iodine intake and anaplastic carcinoma of RAI. In benign disease and thyroid carcinoma, 10-15 mCi (370-555 MBq)28 and 50-200 mCi (1.87.4 GBq)31 of RAI is used, respectively. In 1982, Kapp et al.32 reported that in two patients with a differentiated carcinoma, ATC occurred after irradiation. In one of them, the transformation to ATC occurred five years after 39.6 Gy of external beam irradiation, while in the other patient, ATC was diagnosed one year after 149 mCi of RAI. A component of differentiated thyroid carcinoma is usually identified in the primary ATC on histology examination.22 But, the transformation of differentiated thyroid cancer to ATC may be found also in metastatic lymph nodes.33 It was detected in two of five patients during initial surgery of primary tumor and regional lymph node dissection, while in three of five cases, the interval between treatment of differentiated carcinoma and occurrence of ATC was 46, 74, and 266 months after initial surgery. None of them had a history of RAI therapy, while one patient received external beam irradiation because of recurrent papillary carcinoma before the occurrence of ATC.33 In our patients, RAI therapy was applied from 4 months to 40 years before the diagnosis of ATC. For some patients with a very short interval between RAI therapy and the diagnosis of ATC, we could suspect that they already had a differentiated carcinoma and possibly a very small ATC which was not detected. Namely, in patients with Graves' disease without clinically evident nodes, a thyroid ultrasound investigation is not recommended according to the ATA Guidelines for diagnosis and management of hyperthyroidism and other causes of thyrotoxicosis.28 Furthermore, the use of thyroid scintigraphy to preselect only the cold nodules for cytology is advocated by some authors.34 However, there are at least eight case reports in the literature about patients with a follicular variant of papillary thyroid carcinoma as autonomous functioning thyroid nodule.35 On the other hand, ATC was also reported in a long-standing multinodular goiter or a Hürthle cell tumor following RAI therapy.36-38 This study has some limitations. Because it is a retrospective analysis over a very long time period, we do not have reliable data about the reasons for RAI therapy in our patients. Furthermore, diagnostic radiological methods have changed dramatically over last decades. Modern precise imaging investigations have an impact on the detection of very small regional and/or distant metastases. The detection of small distant metastases possibly influences therapeutic decisions, which might explain why a lesser proportion of patients were treated with radical surgery during the last 15-year period in comparison to prior periods. In the literature, data on how the patients' and tumors' characteristics in ATC have changed over time are very limited.39 We observed that, nowadays, patients are older and have less frequent regional metastases in comparison to previous periods. However, the mean tumor diameter has not changed significantly over time, and ATC was inoperable because of infiltration to the surrounding structures at the time of diagnosis in the majority of our patients. Unfortunately, the frequency of distant metastases has not changed over time. Thus, in Slovenia, the survival of patients with ATC remains short. By contrast, in South Korea where an opportunistic screening is performed in the general population for thyroid carcinoma, the proportion of cases with a differentiated thyroid carcinoma and only anaplastic foci has increased over time, while that of evident ATC has decreased from 1985 to 2013.39 As a consequence, the survival rate is significantly higher than it used to be. Obviously, in patients with a very early ATC, lymphatic invasion was the most significant postoperative prognosticator, so the choice of ATC treatment has to be modified based on resectability and the lymphatic invasion of cancer.39 Conclusions A history of RAI therapy was present in 7.7% of patients with ATC, and the number of patients with a history of RAI therapy did not change over time. The incidence of ATC in Slovenia probably decreased because of higher salt iodination. Patients are nowadays older and have less frequent lymph node metastases than in the past. The frequency of distant metastases did not change over time. The median survival of patients remains short. Acknowledgements This research was funded by the Ministry of Education, Science and Sport of the Republic of Slovenia, grant number P3-0289. References 1. Nikiforov YE, Seethala RR. Anaplastic (undifferentiated) carcinoma. In: Nikiforov YE, Biddinger PW, Thompson LDR, editors. 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Thyroid 2016; 26: 404-13. doi: 10.1089/ thy.2015.0316 Radiol Oncol 2020; 54(2): 187-193. 194 research article Significance of nuclear factor - kappa beta activation on prostate needle biopsy samples in the evaluation of Gleason score 6 prostatic carcinoma indolence Marko Zupancic1, Boris Pospihalj2, Snezana Cerovic3, Barbara Gazic4, Primoz Drev4, Marko Hocevar5, Andraz Perhavec5 1 Department of Urology, General Hospital Slovenj Gradec, Slovenj Gradec, Slovenia 2 Department of Pathology, General Hospital Slovenj Gradec, Slovenj Gradec, Slovenia 3 Institute of Pathology and Forensic Medicine, Military Medical Academy Belgrade, Belgrade, Serbia 4 Department of Pathology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 5 Department of Surgical Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia Radiol Oncol 2020; 54(2): 194-200. Received 3 January 2020 Accepted 24 March 2020 Correspondence to: Marko Zupančič, M.D., M.Sc., Department of Urology, General Hospital Slovenj Gradec, Gosposvetska cesta 1, SI-2380 Slovenj Gradec, Slovenia. E-mail: marko.zupancic@sb-sg.si Disclosure: No potential conflicts of interest were disclosed. Background. The goal of our study was to find out whether the immunohistochemical expression of nuclear factor-kappa beta (NF-kB) p65 in biopsy samples with Gleason score 3 + 3 = 6 (GS 6) can be a negative predictive factor for Prostate cancer (PCa) indolence. Patients and methods. Study was conducted on a retrospective cohort of 123 PCa patients with initial total PSA < 10 ng/ml, number of needle biopsy specimens > 8, GS 6 on biopsy and T1/T2 estimated clinical stage who underwent laparoscopic radical prostatectomy and whose archived formalin-fixed and paraffin-embedded (FFPE) prostate needle biopsy specimens were used for additional immunohistochemistry staining for detection of NF-kB p65. Both cytoplasmic and nuclear NF-kB p65 expression in biopsy cores with PCa were correlated with postoperative pathological stage, positive surgical margins, GS and biochemical progression of disease. Results. After follow-up of 66 months, biochemical progression (PSA > 0.2 ng/ml) occurred in 6 (5.1%) patients, 3 (50%) with GS 6 and 3 (50%) with GS 7 after radical prostatectomy. Both cytoplasmic and nuclear NF-kB p65 expressions were not significantly associated with pathological stage, positive surgical margin and postoperative GS. Patients with positive cytoplasmic NF-kB reaction had significantly more frequent biochemical progression than those with negative cytoplasmic NF-kB reaction with PSA 0.2 ng/ml as cutoff point (p = 0.015) and a trend towards more biochemical progression with PSA > 0.05 ng/ml as cutoff point (p = 0.068). Conclusions. Cytoplasmic expression of NF-kB is associated with more biochemical progression and might be an independent prognostic factor for recurrence-free survival (RFS), but further studies including larger patient cohorts are needed to confirm these initial results. Key words: nuclear factor-kappa beta; prostatic cancer; Gleason 6; needle biopsy sample Introduction Prostate cancer (PCa) is the most common cancer in men in developed European countries, particularly those with a high proportion of elderly population, with incidence rate up to 189 per 100.000.1 In last two decades the disease has emerged as the most frequent cancer amongst men following rapid increases in the detection of a substantial number of early-stage PCa, particularly due to the increased Radiol Oncol 2020; 54(2): 194-200. doi: 10.2478/raon-2020-0019 Zupancic M et al. / Nuclera factor-kappa beta activation in Gleason 6 prostatic cancer 195 number of PSA testing. Recognizing that the expected men's life is obviously increasing, PCa also means increasing financial burden for individual countries.2 Majority of men newly diagnosed with PCa will be candidates for primary curative therapy, either with radical prostatectomy or radiation, but many PCa, however, are low-grade, even indolent and the number of newly diagnosed PCa far outnumbers the number of lethal cases. Indolent PCa may exist for a long period without causing any symptoms or death, so the prediction of low risk and indolent PCa is needed to avoid overtreat-ment by unnecessary invasive therapies, and select men for active surveillance (AS).3-5 The nuclear factor-kappa beta (NF-kB) family of transcription factors plays a crucial role in inflammation as well as in the development and progression of cancer. Extensive evidence indicates that the NF-kB pathway is implicated in controlling the expression of genes involved in cell survival, proliferation, angiogenesis, and invasion.6 Many studies indicate that activation of NF-kB signaling in PCa cells correlates with PCa progression, including chemoresistance, advanced stage, biochemical progression, and metastatic spread.7-11 NF-kB is critical for human health, and aberrant NF-kB activation contributes to development of various autoimmune, inflammatory and malignant disorders including rheumatoid arthritis, atherosclerosis, inflammatory bowel diseases, multiple sclerosis and malignant tumors.12 Despite the growing evidence for a role of NF-kB in prostate tumorigenesis and resistance to therapy, the mechanisms underlying the activation of NF-kB in PCa remain only partially understood. The main goal of our study was to find out whether the immunohistochemical expression of NF-kB p65 in biopsy samples with Gleason score 3 + 3 = 6 (GS 6) is inversely correlated with prostatic carcinoma indolence. Patients and methods Patients Our study was based on a retrospective cohort of 178 consecutive PCa patients whose archived formalin-fixed and paraffin-embedded (FFPE) prostate needle biopsy specimens were used for additional immunohistochemistry staining. All consecutive patients underwent the extraperito-neal laparoscopic radical prostatectomy (ELRP) or "nerve-sparing" extraperitoneal laparoscopic radical prostatectomy (N-S ELRP) without lymph node dissection between 2006 and 2012 as a first treatment of PCa. All patients were follo wed-up for at least five years after surgery at Department of Urology in General Hospital Slovenj Gradec. Hospital patient's files were used for clinical data. The inclusion criteria were total PSA < 10 ng/ml, number of biopsy specimen > 8, histopathological result of prostate cancer GS 6 on biopsy and T1/T2 estimated clinical stage, based on clinical examination only. The exclusion criterion was the presence of chronical diseases in which the activation of NF-kB is common. After the screening review of the clinical data and histopathological revision of prostate needle biopsy specimens, done by two unrelated pathologists with extensive experience in PCa, 15 patients were excluded from the study, as they did not meet the criterion of GS. During the additional microtome cutting of archived FFPE prostate needle biopsy specimens for immunohistochemis-try due to the lack of the tissue, another 40 patients were excluded. The final analysis in this study was performed on 123 patients. Based on a PSA levels, two biochemical progression were defined, at PSA cutoff point > 0.05 ng/ml and > 0.2 ng/ml, 6 months or more after radical prostatectomy. Recurrence-free survival (RFS) was defined as the period between the surgery and biochemical progression (i.e. first increase of PSA above one or both PSA cutoff points). 5 patients were excluded due to the initiation of hormonal treatment immediately after surgery, so regarding the biochemical progression 118 patients were analyzed For control group archived FFPE prostate needle biopsy specimens from 60 patients with PCa GS 7, 30 with 3 + 4 and 30 with 4 + 3, were used. Study was approved by the Slovene National Medical Ethics Committee No 109/14. Tissue preparation and immuno-histochemical staining IHC staining for detection of NF-k|3 p65 was performed on 2-4 |om FFPE tissue sections, dried at 560C for 2 hours, using fully automated IHC system Ventana Benchmark XT (manufacturer Ventana ROCHE inc.). Epitope was retrieved on board employing heat-mediated epitope retrieval using high pH Cell Conditioning Solution 1 (cat No 950-124, manufacturer Ventana ROCHE inc.) for 88 minutes at 1000C. Epitope was detected using commercially available mouse monoclonal antibody NF-k|3 p65 (clone F-6; cat No sc-8008; manufacturer Santa Cruz Biotechnology inc.) directed against amino acids 1-286 of NF-k|3 p65 of human origin. Primary Radiol Oncol 2020; 54(2): 194-200. 196 Zupancic M et al. / Nuclera factor-kappa beta activation in Gleason 6 prostatic cancer 196 TABLE 1. Patient characteristics General, N = 123 Mean Age, year, (range) 63.6 (50-75) Mean init. PSA, ng/ml, (range) 5.32 (1,32-9,51) Mean Prostate V, ml, (range) 38.3 (14-97) Mean Biopsy cores, n, (range) 9.6 (8-10) Total Biopsy cores, n 1.180 Clinical stage T1, n, (%) 113 (91.9) T2, n, (%) 10 (8.1) Biopsy GS 3+3=6, n, (%) 123 (100) Surgery ELRP, n, (%) 87 (70.7) N-S ELRP, n, (%) 36 (29.3) Pathological result after RP N = 123 N of total P % of total P pT classification T2 96 78.0 T3a 23 18.7 T3b 4 3.3 Surgical margins Positive 13 10.6 Negative 110 89.4 Gleason score 3+3=6 79 64.2 3+4=7 37 30.1 4+3=7 7 5.7 Biochemical progression after RP N = 118 N (%) GS 6 GS 7 BP N( %) N( %) PSA 0,05-0.19 ng/ml 14 (11.8) 8(57.1) 6(42.9) PSA > 0,2 ng/ml 6 (5.1) 3(50) 3(50) BP = biochemical progression; ELRP = laparoscopic radical prostatectomy; GS = Gleason score; N-S ELRP = "nerve-sparing" extraperitoneal laparoscopic radical prostatectomy; PSA = Prostate-specific antigen; RP = radical prostatectomy; V = volume antibody was diluted 1:200 using DAKO REAL™ antibody diluent (cat No S2022; manufacturer DAKO Agilent technologies inc.) and incubated on board for 60 minutes at 370C. Primary antibody was visualized using 3-step multimer detection system OptiView DAB IHC Detection Kit (cat No 760-700; manufacturer Ventana ROCHE inc.) according to manufacturer's instructions. The staining was analysed by pathologist with extensive experience in PCa who was not familiar with patient's clinical data. For nuclear staining, positive result was reported when at least 5% nuclei of cancer cells showed unequivocal brown coloration.13 To consider reaction as positive, nuclear brown coloration should exceed the effect of cyto-plasmic overlapping. The intensity of cytoplasmic staining was assessed as negative, weak, moderate and strong, and for statistical analysis grouped as negative (negative, weak) and positive (moderate, strong).14 Statistical analyses Clinical, laboratory and pathological characteristics were summarized using frequency and percentage for categorical variables, and mean and range for continuous variables. RFS was calculated from the time of primary tumour excision, and was censored at the last contact date if there were no events. Association of NF-kB expression status with pathological findings was tested using Chi-square test. Survival curves were calculated by Kaplan-Meier's method and tested for statistical significance using log-rank test. Multivariate Cox regression model was used to test whether NF-kB expression status is an independent predictor of RFS; other covariates included in the model are known prognostic factors in prostate cancer: final Gleason score, surgical margin status and pathologic stage. The differences were considered statistically significant if the p values were less than 0.05. Software package SPSS 22.0 for Windows was used. Results Table 1 shows patients characteristics and biochemical progression in patients after radical prostatectomy. Postoperative pathological stage 3 was noticed in 27 (22%) and positive surgical margins were detected in 13 patients (10.6%). biochemical progression (PSA > 0.05 ng/ml) occurred in 20 (16.9%) patients (11 with GS 6 after radical prostatectomy and 9 with GS 7). Among 118 patients, clinically significant postoperative PSA > 0.2 ng/ml was detected in six patients (5.1%), with GS 6 in 3 and GS 7 in 3. Positive cytoplasmic NF-kB staining was detected in 173 (56.9%) and positive nuclear Radiol Oncol 2020; 54(2): 194-200. Zupancic M et al. / Nuclera factor-kappa beta activation in Gleason 6 prostatic cancer 69 TABLE 2. Association of nuclear factor-kappa beta (NF-kB) p65 expression status in cytoplasm with pathological findings N = 123 N NF-kB p65 expression negative positive P pT status pT2 96 48 48 pT3 27 12 15 0.667* Surg. m. Negative 110 53 7 Positive 13 7 6 0.774** GS 3 + 3 = 6 79 39 40 3 + 4 = 7 37 17 20 4 + 3 = 7 7 5 2 0.465*** N NF-kB p65 expression P negative positive 3 + 3 = 6 123 60 63 3 + 4 = 7 30 3 27 <0.001* 4 + 3 = 7 30 0 30 <0.001** TABLE 4. Multivariate analysis of cytoplasmic nuclear factor-kappa beta (NF-kB) p65 expression and other clinicopathologic variables associated with recurrence-free survival (RFS] Hazard ratio (95% CI) P Cytoplasmic NF-kB p65 expression (negative vs. positive] Postoperative Gleason score (6 vs. 7] Surgical margin (negative vs. positive] Pathologic stage (T2 vs. T3) 2.367 (0.908-6.170) 0.078 1.105 (0.406-3.008) 0.845 4.845 (1.646-14.260) 0.004 1.041 (0.339-3.194) 0.944 * pT2 versus pT3, **negative versus positive surgical margin, ***3 + 3 = 6 versus 3 + 4 = 7 versus 4 + 3 = 7 GS = Gleason score; Surg. M. = surgical margin TABLE 3. Cytoplasmic nuclera factor-kappa beta (NF-kB) p65 expression status in biopsy group postoperative Gleason score (GS) 6 and control biopsy group postoperative GS 7 * 3 + 3 = 6 versus 3 + 4 = 7, **3 + 3 = 6 versus 4 + 3 = 7 NF-kB staining in 57 (18.7%) of the 304 analyzed biopsy cores with GS 6. Cytoplasmic NF-kB staining Cytoplasmic NF-kB p65 expression was not correlated with pathological stage, positive surgical margin and postoperative GS (Table 2). Cytoplasmic NF-kB p65 expression was significantly more common in biochemical progression with PSA cut off point > 0.2 ng/ml (P = 0.015) and there was a trend towards biochemical progression with PSA cut off point > 0.05 ng/ml (P = 0.068) (Figure 1). Cytoplasmic NF-kB p65 expression was positive in 57/60 control group patients with GS 7 (Table 3). In multivariate analysis only positive surgical margin was significantly associated with worse RFS with a PSA cut off point > 0.05 ng/ml, while postoperative Gleason score 7 and pathologic stage of the disease were not significantly associated with RFS (Table 4). Positive cytoplasmic NF-kB p65 expression negatively affects RFS with borderline statistical significance (p = 0.078). When PSA cut off point was set to > 0.2 ng/ml, none of the prognostic factors was significantly associated with RFS in multivariate analysis. Nuclear NF-kB staining Nuclear NF-kB p65 expression was not associated with pathological stage, positive surgical margins and postoperative GS (Table 5), neither with biochemical progression (Figure 2) and did not differ from control group patients with GS 7 (Table 6). FIGURE 1. Recurrence-free survival (RFS) in patients with positive and negative nuclear factor-kappa beta (NF-kB) p65 expression status in cytoplasm (N = 118). Radiol Oncol 2020; 54(2): 194-200. Zupancic M et al. / Nuclera factor-kappa beta activation in Gleason 6 prostatic cancer 70 TABLE 5. Association of nuclear factor-kappa beta (NF-kB) p65 expression status in nucleus with pathological findings TABLE 6. Nuclear factor-kappa beta (NF-kB) p65 expression status in biopsy group GS 6 and control biopsy group GS 7 N = 123 N NF-kB p65 expression P N NF-kB p65 expression P negative positive negative positive pT status 3+3-6 123 103 20 pT2 96 81 15 3+4-7 30 24 6 0.596* pT3 27 22 5 0.769* 4+3-7 30 17 13 0.003** Surg. m. Negative 110 92 18 * 3+3-6 versus 3+4-7, **3+3-6 versus 4+3-7 Positive GS 3 + 3 = 6 3 + 4 - 7 13 79 37 11 66 31 2 13 6 1 1** TABLE 7. Multivariate analysis of nuclear factor-kappa beta (NF-kB) p65 expression and other clinicopathologic variables associated with recurrence-free survival (RFS; recurrence defined as PSA > 0.05] 4 + 3 = 7 7 6 0.989*** Hazard ratio (95% CI) P * pT2 versus pT3, **negative versus positive surgical margin, *** 3 + 3 - 6 versus 3 + 4 - 7 versus 4 + 3 - 7 GS - Gleason score; Surg. M. - surgical margin In multivariate analysis only positive surgical margin was significantly associated with worse RFS with a PSA cut off point > 0.05 ng/ml, while positive nuclear NF-kB p65 expression, postoperative Gleason score 7 and pathologic stage of the disease were not significantly associated with RFS (Table 7). When PSA cut off point was set to > 0.2 ng/ml, none of the prognostic factors was significantly associated with RFS in multivariate analysis. Figure 3 shows difference in positive cytoplasmic and nuclear staining in patient with GS 6 and GS 7. Discussion Increase in incidence of PCa since 1990s mostly starts with PSA testing, either in the form of all types of screening or on the basis of a suspicious NF-kB p65 expression (negative vs. positive] Postoperative Gleason score (6 vs. 7] Surgical margin (negative vs. positive] Pathologic stage (T2 vs. T3) 0.254 (0.034-1.915) 0.184 1.078 (0.400-2.907) 0.882 4.838 (1.674-13.983) 0.004 1.232 (0.409-3.705) 0.711 digital rectal examination. Nevertheless, the most important part of diagnostic procedure is accurate histopathologic diagnosis, particularly in low-risk PCa where AS could be an option.15 Despite the fact that in our study biopsy samples were evaluated by two experienced uropathologists, we recorded the postoperative upgrade of GS 6 to GS 7 in 44 patients (3 + 4 in 37 and 4 + 3 in 7), so biopsy under-grading was present in 35.8%. This is in concordance with literature reports where GS from needle biopsies underestimates the GS of the radical prostatectomy specimen in 28% to 57%.16 Postoperative pathological stage 3 was noticed in 27 (22%), positive surgical margins were detected in 13 (10.6%) FIGURE 2. Recurrence-free survival (RFS) in patients with positive and negative nuclear factor-kappa beta (NF-kB] status in nucleus (N = 118). P = 0,991 p65 expression Radiol Oncol 2020; 54(2): 194-200. Zupancic M et al. / Nuclera factor-kappa beta activation in Gleason 6 prostatic cancer 199 and clinically significant biochemical progression (PSA > 0.2 ng/ml) in 6 (5.1%) patients. Molecular biomarkers offer the possibility to further stratify patients with similar clinicopatho-logical parameters. Domingo-Domenech et al.9 and Ross et al.10 reported that tumors with nuclear NF-kB expression and no additional risk factors (i.e. low GS and low preoperative PSA) had the lowest rate of biochemical recurrence in the nuclear NF-kB positive group. In addition to nuclear staining, we included also the NF-kB cytoplasmic staining and contrary to reported results found that only the cytoplasmic NF-kB variable was associated with worse RFS. This association was statistically significant when PSA cut-off point for recurrence was set to > 0.2 ng/ml (p = 0.013) and borderline significant when PSA cut-off point was set to > 0.05 (p = 0.057). In multivariate analysis positive cytoplasmic NF-kB p65 expression remained negatively associated with RFS (PSA cut-off point > 0.05) with borderline statistical significance (p = 0.078). The p65 subunit of NF-kB was expressed in the cytoplasm of 173 (56.9%) biopsy cores with GS 6. Only 57 (18.7%) biopsy samples also showed a nuclear staining of NF-kB p65, suggesting a constitutive activation of NF-kB in these tissues. Our results showed a positive correlation between NF-kB cytoplasmic staining and biochemical progression. The main differences with other studies relate to tissue sample type and biochemical progression definition. Unlike Domingo-Domenech et al.9 and Ross et al.10 the studies that used tissue samples from radical prostatectomy, our analysis is preoperative and is based on diagnostic biopsies and their predictive significance. Also, we used more stringent parameters for the determination of biochemical progression, which in previous works was only determined as a value of 0.4 ng/ml in two consecutive measurements. In the present study, NF-kB expression and its subcellular localization were highly variable among different specimens. In another study, nuclear NF-kB was found in 40% of PCa.8-13 As in the current study, nuclear NF-kB did not significantly correlate with GS. The functional relevance of this immunoreactivity on NF-kB activation is not known. This limitation is based on the fact that p65/NF-KB nuclear translocation is necessary but not sufficient for NF-kB induced transcriptional activity, since both recruitment of NF-kB to target genes and NF-KB-induced tran-scriptional events after recruitment are needed for this to occur. Furthermore, the minimum percentage of tumor cells with nuclear p65 staining required to potentially result in detectable NF-kB- ¿A ¿z't»- -r + V. frnùm^ ' J ii » *.*-v. -PU' ' |vH&kM © i* S '''>4 ? i . ■ ViTw ® . FIGURE 3. Immunohistochemistry of nuclear factor-kappa beta (NF-kB) p65. (A) Positive cytoplasmic staining (GS 6). (B) Positive nuclear staining. (C) Positive cytoplasmic staining (GS 7). (D) Positive nuclear staining. induced transcriptional activity remains uncharac-terized. An important limitation of our study is a significant reduction in the size of diagnostic biopsies during microscopic reevaluation and diagnosis of PCa with GS 6.1314 In our group of 20 patients who developed the biochemical progression with PSA cutoff point > 0.05 ng/ml cytoplasmic NF-kB staining was detected in 14 (70%) and nuclear NF-kB staining in only 1 (5%), while among those 6 patients who had the biochemical progression with PSA cutoff point > 0.2 ng/ml cytoplasmic NF-kB staining was noticed in all 6 (100%) and nuclear NF-kB staining in 1 (16.7%). Among several available selection criteria for AS worldwide, at our institution the EAU AS guidelines were used.17 According to them all patients in our cohort had initial PSA below 10 ng/ ml, biopsy GS 6, estimated clinical stage T1c-T2 and 82 of them (66.7%) had < 2 positive cores on biopsy. From clinical point of view the biochemical progression of PCa after radical prostatectomy is defined with PSA > 0.2 ng/ml.18 In our group of 6 patients with biochemical progression at PSA cutoff point > 0.2 ng/ml, positive cytoplasmic NF-kB staining was present in all 6 and all AS criteria were met in 5 (83.3%) patients. There was no positive nuclear NF-kB staining in any of these 5 patients. As a control group we used 60 patients with biopsy GS 7 and positive cytoplasmic NF-kB staining was present in 57 (95%) of them. Since patients with bi- Radiol Oncol 2020; 54(2): 194-200. 200 Zupancic M et al. / Nuclera factor-kappa beta activation in Gleason 6 prostatic cancer 200 opsy GS 7 are not candidates for AS according to EAU AS guidelines there is no need for additional prognostic factor (i.e. positive cytoplasmic NF-kB staining). However, in patients with biopsy GS 6, an additional prognostic factor is needed in order to stratify these patients to a group where only AS is enough. According to our results, positive cytoplasmic NF-kB staining could be a negative predictive factor for the GS 6 PCa indolence and these patients are candidates for primary curative therapy. There are several limitations of our study. Most importantly, all patients underwent surgical treatment, so the significance of NF-kB activation on prostate needle biopsy samples for disease progression was found only indirectly, based on biochemical progression and is most probably underestimated. Another important limitation is a small number of patients and single institution results. Conclusions Cytoplasmic expression of NF-kB is associated with worse RFS, while it is not significantly associated with standard prognostic factors and remains an independent prognostic factor for RFS in multi-variate analysis with borderline statistical significance. However, further studies, including larger patient cohorts are needed to confirm these initial results. References 1. Ferlay J, Colombet M, Soerjomataram I, Dyba T, Randi G, Bettio M, et al. Cancer incidence and mortality patterns in Europe: Estimates for 40 countries and 25 major cancers in 2018. Eur J Cancer 2018; 103: 356-87. doi: 10.1016/j.ejca.2018.07.005. 2. Luengo-Fernandez R, Leal J, Gray A, Sullivan R. Economic burden of cancer across the European Union: a population-based cost analysis. Lancet Oncol 2013; 14: 1165-74. doi: 10.1016/S1470-2045(13)70442-X. 3. Cooperberg MR, Broering JM, Litwin MS, Lubeck DP, Mehta SS, Henning JM, et al. The contemporary management of prostate cancer in the United States: lessons from the cancer of the prostate strategic urologic research endeavor (CapSURE), a national disease registry. J Urol 2004; 171: 1393-401. doi: 10.1097/01.ju.0000107247.81471.06 4. Albertsen PC, Hanley JA, Fine J. 20-year outcomes following conservative management of clinically localized prostate cancer. JAMA 2005; 293: 2095101. doi: 10.1001/jama.293.17.2095 5. Bangma CH, Roobol MJ. Defining and predicting indolent and low risk prostate cancer. Crit Rev Oncol Hematol 2012; 83: 235-41. doi: 10.1016/j. critrevonc.2011.10.003 6. Karin M, Lin A. NF-kB at the crossroads of life and death. Nat. Immunol 2002; 3: 221-7. doi: 10.1038/ni0302-221 7. May MJ, Ghosh S. Rel/NF-kappa B and I kappa B proteins: an overview. Semin Cancer Biol 1997; 8: 63-73. doi: 10.1006/scbi.1997.0057 8. Lessard L, Karakiewicz PI, Bellon-Gagnon P, Alam-Fahmy M, Ismail HA, Mes-Masson AM, et al. Nuclear localization of nuclear factor-kappaB p65 in primary prostate tumors is highly predictive of pelvic lymph node metastases. Clin Cancer Res 2006; 12: 5741-5. doi: 10.1158/1078-0432.CCR-06-0330 9. Domingo-Domenech J, Mellado B, Ferrer B, Truan D, Codony-Servat J, Sauleda S, et al. Activation of nuclear factor-kappaB in human prostate carcinogenesis and association to biochemical relapse. Br J Cancer 2005; 93: 1285-94. doi: 10.1038/sj.bjc.6602851 10. Ross JS, Kallakury BV, Sheehan CE, Fisher HA, Kaufman RP Jr, Kaur P, et al. Expression of nuclear factor-kappa B and I kappa B alpha proteins in prostatic adenocarcinomas: correlation of nuclear factor-kappa B immuno-reactivity with disease recurrence. Clin Cancer Res 2004; 10: 2466-72. doi: 10.1158/1078-0432.ccr-0543-3 11. Setlur SR, Royce TE, Sboner A, Mosquera JM, Demichelis F, Hofer MD, et al. Integrative microarray analysis of pathways dysregulated in metastatic prostate cancer. Cancer Res 2007; 67: 10296-303. doi: 10.1158/1078-0432. ccr-0543-3 12. Park MH, Hong JT. Roles of NF-kB in cancer and inflammatory diseases and their therapeutic approaches. Cells 2016; 5: E15. doi: 10.3390/cells5020015. 13. Lessard L, Begin LR, Gleave ME, Mes-Masson AM, Saad F. Nuclear localisation of nuclear factor-kappaB transcription factors in prostate cancer: an immunohistochemical study. Br J Cancer 2005; 93: 1019-23. doi: 10.1038/ sj.bjc.6602796 14. Gannon PO, Lessard L, Stevens LM, Forest V, Bégin LR, Minner S, et al. Large-scale independent validation of the nuclear factor-kappa B p65 prognostic biomarker in prostate cancer. Eur J Cancer 2013; 49: 2441-8. doi: 10.1016/j. ejca.2013.02.026 15. Ganz PA, Barry JM, Burke W, Col NF, Corso PS, Dodson E, et al. NIH State-of-the-Science Conference Statement: role of active surveillance in the management of men with localized prostate cancer. NIH Consens State Sci Statements 2011; 28: 1-27. PMID: 23392076 16. Lucia MS, Bostwick DG, Somerville MC, Fowler IL, Rittmaster RS. Comparison of classic and international society of urological pathology 2005 modified Gleason grading using needle biopsies from the reduction by dutasteride of prostate cancer events (REDUCE) trial. Arch Pathol Lab Med 2013; 137: 1740-6. doi: 10.5858/arpa.2012-0447-OA 17. Komisarenko M, Martin LJ, Finelli A. Active surveillance review: contemporary selection criteria, follow-up, compliance and outcomes. Transl Androl Urol 2018; 7: 243-55. doi: 10.21037/tau.2018.03.02 18. McCormick BZ, Mahmoud AM, Williams SB, Davis JW. Biochemical recurrence after radical prostatectomy: Current status of its use as a treatment endpoint and early management strategies. Indian J Urol 2019; 35: 6-17. doi: 10.4103/iju.IJU_355_18 Radiol Oncol 2020; 54(2): 194-200. 201 research article Evaluation of the training program for p16/ Ki-67 dual immunocytochemical staining interpretation for laboratory staff without experience in cervical cytology and immunocytochemistry Veronika Kloboves Prevodnik12, Ziva Pohar Marinsek1, Janja Zalar1, Hermina Rozina1, Nika Kotnik3, Tine Jerman4, Jerneja Varl25, Urska Ivanus42 1 Department of Cytopathology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Department of Experimental Allergology and Immunodermatology Oldenburg, Carl von Ossietzky Universität, Oldenburg, Germany 4 Epidemiology and Cancer Registry, Institute of Oncology Ljubljana, Ljubljana, Slovenia 5 Department of Experimental Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia Radiol Oncol 2020; 54(2): 201-208. Received 22 January 2020 Accepted 4 March 2020 Correspondence to: Assoc. Prof. Veronika Kloboves Prevodnik, M.D. Ph.D., Department of Cytopathology, Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. E-mail: vkloboves@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. p16/Ki-67 dual immunocytochemical staining (DS) is considered easy to interpret if evaluators are properly trained, however, there is no consensus on what constitutes proper training. In the present study we evaluated a protocol for teaching DS evaluation on students inexperienced in cervical cytology. Methods. Initial training on 40 DS conventional smears was provided by a senior cytotechnologist experienced in such evaluation. Afterwards, two students evaluated 118 cases. Additional training consisted mainly of discussing discrepant cases from the first evaluation and was followed by evaluation of new 383 cases. Agreement and accuracy of students' results were compared among the participants and to the results of the reference after both evaluations. We also noted time needed for evaluation of one slide as well as intra-observer variability of the teacher's results. Results. At the end of the study, agreement between students and reference was higher compared to those after initial training (overall percent agreement [OPA] 81.4% for each student, kappa 0.512 and 0.527 vs. OPA 78.3% and 87.2%, kappa 0.556 and 0.713, respectively). However, accuracy results differed between the two students. After initial training sensitivity was 4.3% points and 2.9% points higher, respectively compared to the reference, while specificity was 30.6% points and 24.4% points lower, respectively, compared to the reference. At the end of the study, the sensitivity reached by one student was the same as that of the reference, while it was 2.6% points lower for the other student. There was a statistically significant difference in specificity between one student and the reference and also between students (16.7 and 15.1% points). Towards the end of the study, one student needed 5.2 min for evaluating one slide while the other needed 8.2 min. The intra-observer variability of the senior cytotechnologist was in the range of "very good" in both arms of the study. Conclusions. In teaching DS evaluation, the students' progress has to be monitored using several criteria like agreement, accuracy and time needed for evaluating one slide. The monitoring process has to continue for a while after students reach satisfactory results in order to assure a continuous good performance. Monitoring of teacher's performance is also advisable. Key words: training protocol; p16/Ki-67 dual immunocytochemical staining; agreement; accuracy; inter-observer reproducibility Radiol Oncol 2020; 54(2): 194-200. doi: 10.2478/raon-2020-0019 202 Kloboves Prevodnik V et al. / Evaluation of the training program for p16/Ki-67 dual staining interpretation Introduction p16/Ki-67 dual immunocytochemical staining (DS) is considered easy to interpret if evaluators are properly trained. However, there is no consensus on what constitutes proper training. Authors have used different training approaches in studies investigating inter-observer reproducibility and accuracy of DS.1-6 Most training protocols described in these studies consisted of initial and additional training. The initial training was provided by the manufacturer, however, it was not exactly the same in all cases except that it was completed by a proficiency test. The information on the initial training is sparse. Three of the above mentioned studies do not describe the initial training.1'24 In two studies, participants were shown 155 or 406 microscope-projected images, while in the third study participants examined a teaching set of slides.3 The number of cases in the teaching set is not mentioned. In all three studies the training was completed in one or two-half day sessions.356 Four authors described additional training which consisted of evaluating from 80 to 469 slides as well as reviewing and discussing cases with discrepant results.1-36 Agreement in DS interpretation among evaluators improved after additional training in all three studies (kappa range: 0.43-0.73 compared to 0.50-0.87).1-36 In the study of Wentzensen et al.2 agreement was evaluated only at the end of the study. In our recently published study which assessed reproducibility of the DS test we described a training protocol which was designed to introduce DS in three Slovenian cytopathological laboratories participating in the national organized cervical cancer screening program.6 At the time we designed the protocol we found only one similar study by Waldstrom et al.1 The results of our study demonstrated that initial training by the manufacturer was not enough for achieving accurate results of DS interpretation. Furthermore, the manufacturer provides training when an institution is ready to implement the test. Later on, when we have the need to teach additional personnel, we have to have our own training protocol which will assure the students receive the necessary expertise. On the basis of the results of our previous study, we proposed a training protocol for staff inexperienced in DS reading.6 In the present study, we aimed to test the proposed training protocol for DS interpretation on two students inexperienced in DS reading and to discern how improvement in DS evaluation influences the time needed for DS reading of one slide. An additional end point of the study was also monitoring the performance of the senior cytotechnologist involved in teaching DS interpretation to new personnel. Material and methods Study design and setting We used DS slides on conventional cervical smears taken from 501 women who underwent colposcopy at Celje General Hospital or at University Medical Centre Maribor between April 2014 and December 2015. Samples from 118 women were the same ones we have used in our previous study.6 These women were invited to colposcopy per screening program guidelines, either due to high-grade (HG) cytology, a human Papillomavirus (HPV)-positive triage test after low-grade pathological changes or due to a positive HPV test during follow-up after treatment of high grade cervical intraepithelial ne-oplasia (CIN). An additional set of samples came from 383 women, of which 87 were referred to colposcopy from the screening program and 296 were non-responders. All non-responders were invited to colposcopy after they have taken their own cervical sample. 250 were HPV positive and 46 were HPV negative.7 Sample acquisition, procedures following abnormal colposcopy, reasons for excluding patients from the study and classification of histopathology results were the same as already described in our previous study.6 In both sets of women the same gynecologists participated in col-poscopy examinations and the same criteria were used for performing biopsy for histological examinations. After the initial colposcopy, all women were followed via the Cervical Cancer Screening Registry ZORA that registers all cervical cytology, HPV test results and cervical histology results of all Slovenian women. Two students, a biologist (S1) and a medical doctor (S2) as well as a senior cytotechnologist (SC) participated in the study. SC was employed at the Department of cytopathology, Institute of Oncology Ljubljana, where the study was conducted. She was trained in DS evaluation during our previous study. The two students had no previous knowledge of cervical cytology or of DS evaluation. Four cytopathologist from the Institute of Oncology Ljubljana reviewed all DS slides. Their consensus results were considered as reference. Fixation of slides, immunocytochemical staining and the rules for slide interpretation have already been described in our previous paper.6 Radiol Oncol 2020; 54(2): 201-208. 203 Kloboves Prevodnik V et al. / Evaluation of the training program for p16/Ki-67 dual staining interpretation The reading of DS slides was divided into primary reading of 118 slides after initial training and the secondary reading of new 383 slides after additional training. In each reading, students spotted the DS cells and passed slides on to the SC who reviewed all of them. The SC reviewed both sets of slides separately after each student and therefore, each case had four readings. Results of both students and of the SC were evaluated after initial and after secondary reading and compared to the reference results. For both students we also noted the time needed to interpret each of the 501 slides. S1 evaluated 501 slides during the course of four months while S2 evaluated all the slides in five months because none of the students were evaluating slides continuously eight hours per day. The p16/Ki-67 DS study was nested within the randomized trial of HPV self-sampling among non-attenders of ZORA. It was conducted in compliance with the Helsinki Declaration, and was approved by the institutional review board and the National Medical Ethics Committee at the Slovenian Ministry of Health (consents Nos. 155/03/13 and 136/04/14). All women signed informed consent to participate in the study. This research was financed by the Slovenian Research Agency and the Slovenian Ministry of Health (trial No. L3-5512). Training design The initial training program for DS interpretation started by lectures and by demonstration of morphology of normal and atypical cervical cytology and of DS interpretation. Afterwards, the students examined 40 teaching slides and discussed difficult cases with SC at a multi-head microscope. Training was completed in one week. Additional training took place after we evaluated the results of the primary reading. It was also provided by SC and lasted two days. Additional training included a troubleshooting slide review of discordant cases of the primary reading as well as a theoretical repetition of the criteria for DS evaluation. Study outcomes and statistical analysis The primary outcomes were: (1) agreement in DS interpretation between all three evaluators (S1, S2, SC), between each evaluator and the reference as well as intra-observer agreement for SC; (2) accuracy prior to and after the additional training. For evaluating agreement and accuracy we used the same statistical methods as already described in FIGURE 1. Study flow chart with histopathology follow-up results. our previous article.6 The secondary outcome was measuring screening time per slide of both students. We assessed the mean screening time with standard deviation (SD) for primary and secondary evaluation and also for the last 100 slides. The screening time trends for positive and negative p16/Ki-67 DS results were characterized in terms of an average percent change per slide estimated by the log-linear joinpoint regression. Sensitivity, specificity, positive (PPV) and negative (NPV) predictive values were calculated for one year histo-pathological follow-up for both evaluation sets. In addition, sensitivity and specificity were calculated at each of the 501 ratings, taking into account only 100 most recent ratings, and presented on a line plot. We conducted all our analyses with R v3.5.18, using 2-tailed tests and the significance level a = 0.050. Joinpoint Regression program9 was used for the assessment of the screening time trends. Results Study population The average age of the 501 women in the study was 44.3 years (SD 11.9). The average age of women whose smears were subject to primary DS evaluation was 36.5 years (SD 11.1) while the average age of women in secondary DS evaluation was 46.7 years (SD 11.2). The study flow chart with histo-pathology results is shown in Figure 1. Women whose smears were included into the primary DS evaluation had higher prevalence of cervical in-traepithelial neoplasia grade 2 or worse (CIN2+) compared to women whose samples were material for the secondary evaluation (58.5 % vs. 20.1%) (Figure 1). Radiol Oncol 2020; 54(2): 201-208. 204 Kloboves Prevodnik V et al. / Evaluation of the training program for p16/Ki-67 dual staining interpretation TABLE 1. p16/Ki-67 study results and CIN2+ outcome for students, senior cytotechnologist and reference Categories Reviewer of p16/Ki67 dual staining result Initial training (N = 118) Additional training (N = 383) p16/Ki67 dual staining CIN2+ outcomes result N, (%) N (PV, %)* p16/Ki67 dual staining CIN2+ outcomes result (N, %) N (PV, %)* positive suspicious S1 negative unsatisfactory 91 (77.1) 65 (71.4) 7 (5.9) 2 (28.6) 20 (16.9) 2 (10.0) 0 (0.0) 0 (0.0) 171 (44.6) 65 (38.0) 7 (1.8) 2 (28.6) 205 (53.5) 10 (4.9) 0 (0.0) 0 (0.0) S2 positive 92 (78.0) 66 (71.7) 129 (33.7) 64 (49.6) suspicious 2 (1.7) 0 (0.0) 1 (0.3) 1 (100.0) negative 24 (20.3) 3 (12.5) 253 (66.1) 12 (4.7) unsatisfactory 0 (0) 0 (0.0) 0 (0.0) 0 (0.0) SC (S1) positive suspicious negative unsatisfactory 83 (70.3) 2 (1.7) 32 (27.1) 1 (0.8) 65 (78.3) 2 (100.0) 2 (6.3) 0 (0.0) 143 (37.9) 5 (1.3) 235 (61.4) 0 (0.0) 65 (45.5) 0 (0.0) 12 (5.1) 0 (0.0) SC (S2) positive 83 (70.3) 65 (78.3) 145 (37.9) 64 (44.1) suspicious 2 (1.7) 2 (100.0) 4 (1.0) 0 (0.0) negative 32 (27.1) 2 (6.3) 234 (61.1) 13 (5.6) unsatisfactory 1 (0.8) 0 (0.0) 0 (0.0) 0 (0.0) Reference positive suspicious negative unsatisfactory 78 (66.1) 2 (1.7) 38 (32.2) 0 (0.0) 64 (82.1) 0 (0) 5 (13.2) 0 (0.0) 114 (29.8) 13 (3.4) 255 (66.6) 1 (0.3) 64 (56.1) 3 (23.1) 10 (3.9) 0 (0.0) N = number of cases; Reference = results of four cytopathologists at the Deptment of Cytopathology, Institute of Oncology Ljubljana; S1 = student 1; S2 = student 2; SC (S1) = senior cytotechnologist results obtained during revision of student 1 results; SC (S2) = senior cytotechnologist results obtained during revision of student 2 results; * PV = predictive value (number of CIN2+detected within specific category of p16/Ki-67 dual staining result divided by the number of test results in specific category) TABLE 2. Individual comparison of p16/Ki-67 agreement and performance between students, senior technologist and reference Training Reviewers OPA McNemar's test p k (cohen) (95% CI) S1/Reference 81.4% 0.000 0.512 (CI: 0.329-0,696) S2/Reference 81.4% 0.006 0.527 (CI: 0.349-0,705) Initial training (N = 118) SC (S1)/ Reference SC (S2)/ Reference 94.1% 94.1% 0.131 0.131 0.859 (CI: 0.758-0.960) 0.859 (CI: 0.758-0.960) SC (S1)/SC (S2) 100.0% / 1.000 (CI: /) S1/Reference 78.3% 0.000 0.556 (Cl: 0.472-0.641) S2/Reference 87.2% 0.775 0.713 (Cl: 0.638-0.788) Addtional (N = 383) SC (S1)/ Reference 86.2% 0.006 0.700 (Cl: 0.625-0,775) SC (S2)/ Reference 86.4% 0.004 0.707 (Cl: 0.632-0.781) SC (S1)/SC (S2) 98.7% 1.000 0.973 (CI: 0.949-0.996) N = number of cases; OPA = overall percent agreement; S1 = student 1; S2 = student 2; SC (S1) = senior cytotechnologist results obtained during revision of student 1 results; SC (S2) = senior cytotechnologist results obtained during revision of student 2 results; SC1/SC2 = intra-observer variability; Reference = results of four cytopathologists at the Deptment of Cytopathology, Institute of Oncology Ljubljana; * Scale for interpretation of k values = below 0.20 (poor), 0.21-0.40 (fair), 0.41-0.60 (moderate), 0.61-0.80 (good), >0.81 (very good)10 p16/Ki-67 dual staining results and agreement In primary and secondary evaluation both students (S1 and S2) had more positive p16/Ki-67 DS results compared to the reference (Table 1). However, in secondary evaluation, S2 had less positive results (33.7%) compared to the outcome in primary evaluation. Her percentage of positive results was even closer to the reference results (29.8%) than that of SC (37.9%). All evaluators used the suspicious category sparsely, only 0.3-5.9% of results fell into this category. In primary evaluation, the agreement of DS results between reference and each of the students was moderate, while the agreement between reference and SC was very good. However, S2 reached good agreement in secondary evaluation which even slightly surpassed the agreement result between SC and the reference (Table 2). The agreement between S1 and the reference remained mod- Radiol Oncol 2020; 54(2): 201-208. 205 Kloboves Prevodnik V et al. / Evaluation of the training program for p16/Ki-67 dual staining interpretation erate. Intra-observer variability between the two evaluations performed by the SC after each student was very good in primary, as well as in secondary evaluation (Table 2). Accuracy of p16/Ki-67 results Reference results showed higher sensitivity and positive predictive value for CIN2+ in primary compared to secondary evaluation (92.8% vs. 87.0% and 80.0% vs. 52.8%, respectively) (Supplementary Table 1, Figure 2). Specificity and negative predictive value for CIN2+ were lower in primary compared to secondary evaluation (67.3% vs. 80.4% and 86.8% vs. 96.1%, respectively). In primary evaluation, the results of both students had slightly higher sensitivity and negative predictive values for CIN2+ but much lower specificity and positive predictive values compared to reference results (Supplementary Table 1, Figure 2, Figure 3). While students preformed similarly in terms of sensitivity and specificity after initial training, after additional training the results of S2 were closer to the results of the reference compared to the results of S1. In secondary evaluation, S2 even reached higher sensitivity and specificity than SC (84.4% vs. 83.1% and 78.8% 1 vs. 72.2%, respectively). S1 had the highest sensitivity in both evaluations (97.1% and 87.0%) combined with the lowest specificity (36.7% and 63.7%, respectively) among all evaluators. The specificity of S1 was significantly lower than the reference's in both primary and secondary 1 evaluation. Her specificity came close to the specificity of the reference after evaluating approximately 250 slides. However, after this point her performance started to decline (Figure 3). Screening time 51 evaluated 501 slides in 96 hours and 40 minutes while 52 needed 95 hours and 56 minutes for evaluating all the slides. In primary evaluation S1 needed less screening time 100- 80 60 40 • ■ A X • S ■ < 40 Initial training Additional training 60 Specificity (%) 80 100 51 52 O A SC(S1) SC (S2) X Reference FIGURE 2. Sensitivity and specificity of p16/Ki-67 dual immunocytochemical staining (DS) for detecting CIN2+ for both students and the teacher (senior cytotechnologist). S1 = student 1; S2 = student 2; SC (S1) = senior cytotechnologist - slide review after S1; SC (S2) = senior cytotechnologist - slide review after S2 1 ' '-'V'" ■ . rLJJ^— FIGURE 3. Sensitivity and specificity for the results of both students and the reference according to the number of evaluated slides. S1 = student 1; S2 = student 2 0 0 100 200 400 500 Student Sensitivity Radiol Oncol 2020; 54(2): 201-208. 206 Kloboves Prevodnik V et al. / Evaluation of the training program for p16/Ki-67 dual staining interpretation 0 100 200 300 400 500 Consecutive slide rating FIGURE 4. Joinpoint regression analysis of students' screening times. S1 = student 1; S2 = student 2 per slide (18.8 min; SD 10.3) compared to S2 (24.2 min; SD 13.0; p < 0.001). In secondary evaluation, the screening times decreased for both students, however, S1 now needed more time (9.3 min; SD 3.9) compared to S2 (7.6 min; SD 3.5; p < 0.001). At the end of the training, during the evaluation of the last 100 slides, the average time of two students was 6.7 minutes per slide. However, the difference between them was statistically significant (S1: 8.2 min vs. S2: 5.2 min; SD 2.8 and 0.4, respectively; p < 0.001). Joinpoint regression analysis of screening times of S1 showed the joinpoint at 163rd slide for negative slides. There was a 0.7% decrease per each slide (p < 0.05) in the first segment and 0.0% decrease in the second segment. The joinpoint for positive slides was at 109th slide with 1.0% decrease per slide (p < 0.05) in the first segment and 0.1% decrease (p < 0.05) in the second segment (Figure 4). For S2, the joinpoint for negative slides was at 207th slide with 0.6% decrease per slide (p < 0.05) in the first segment and 0.1% decrease (p < 0.05) in the second segment. The joinpoint for positive slides in case of S2 was at 248th slide with 0.6% decrease (p < 0.05) in the first segment and 0.1% decrease (p < 0.05) in the second segment. Discussion The results of our study confirmed that the training protocol we have used was adequate for teaching the interpretation of p16/Ki-67 DS. At the end of the training one student was competent for independent DS interpretation as evidenced by comparable accuracy results and by good agreement between her results and those of the reference. Towards the end of the training this student needed 5.2 min for evaluating one slide. The training protocol is suitable also for monitoring the performance of the teacher. We do not have an explanation as to why the results of the other student showed a statistically significant difference in agreement and accuracy when compared to the results of the reference. Since environmental factors were the same for both students, it seems that internal factors played an important role in performance difference. It is well known that interpreting slides is partly a subjective method. Furthermore, in our previous study we demonstrated a number of reasons that contributed significantly to the difficulty of DS inter-pretation.6 Weak p16 staining, less preserved cell morphology and strong background staining were important drawbacks of decision making. Similar observations were also made by McMenamin et al.4 and Benevolo et al.5 An additional important information from our previous study was also the fact that 45% of cases which were marked as suspicious for p16/Ki-67 positivity had only one such cell.6 Therefore, this type of training is not suitable for every person without prior knowledge of DS evaluation. However, by monitoring student's results, we can assess if additional experience to reach the necessary expertise is needed. Most articles which describe agreement and accuracy of DS interpretation among observers briefly mention the training program they have used. Only the training programs in three studies had some similarities with our own.123 However, the initial training provided by the manufacturer is not described in detail in any of them. The secondary training can be compared to ours because it consisted of reviewing a certain number of slides (80, 150 and 469) and of discussing discrepant cases of the first viewing. In the second reading, Allia et al. 3 mention evaluation of 350 new slides, while Wentzesen et al.2 used 480 new slides, however, not all reviewers red all of them. In the study of Waldstrom et al.1 they randomly selected 185 slides from set of 469 slides from the first reading. Only the study of Allia et al.3 included three evaluators inexperienced in cervical cytology (two medical students and one biologist) in addition to the four experienced ones. Unfortunately, we can compare our results to those of Allia et al.3 only to a limited extent due to Radiol Oncol 2020; 54(2): 201-208. 207 Kloboves Prevodnik V et al. / Evaluation of the training program for p16/Ki-67 dual staining interpretation differences in methodology. Allia et al. compared agreement between results of four evaluators with experience in DS evaluation and between results of three evaluators without experience. Agreement improved after additional training in each group. In our study, the population of 118 women from the first arm of the study had a higher percentage of histologically confirmed CIN2+ compared to the population of 383 women in the second arm (58.5% vs. 20.1%). This difference in the prevalence of the disease has to be taken into account when comparing the results of primary and secondary evaluation, especially Kappa values and predictive values of a test. In the Allia's et al. paper, the study population contained 14.7% of women with histological diagnoses of CIN2+.3 Since the populations of women in the two studies were not totally alike, we can compare only the end results indirectly. At the end of Allia's et al. study, the specificity of DS for CIN2+ was 66.7% for the experienced evalua-tors, while the students reached a specificity of 60.5%, a difference of 5.2% points. On the basis of these data Allia et al. concluded that DS evaluation "can be performed even by staff not trained in the morphological interpretation of cytology" after a short training phase.3 At the end of our study the difference in specificity between the reference and the successful student was 1.6% points and 16.7% points for the unsuccessful student. Therefore, we agree with Allia et al. that it is possible to perform DS evaluation with personnel not experienced in cervical cytology, however, not after a short training period. If we translate 97 hours needed for reviewing 501 slides into 12 days with an eight-hour working day, the whole training period in our study would last 19 days. The evaluation of slides after initial training, as well as partly after additional training, has to be considered part of the learning process. This is clearly demonstrated in the graph of continual monitoring of the students' accuracy results. For the successful student the specificity continued rising during the evaluation of the first 118 slides. It continued to rise also for a period after the secondary training until approximately the time when the student evaluated roughly 350 slides altogether. At this point the accuracy results of the student were very similar to the results of the reference (same sensitivity, 87.0%) and slightly lower specificity (84.4 vs. 85.7%). The rest of the time used in the evaluation of the last 150 slides was necessary for monitoring the student's performance. The necessity of such action has proven to be correct in the case of the other student. Her accuracy results came close to those of the reference after she evaluated approximately 250 slides, however, her performance started to decline afterwards. For monitoring the students' progress in DS evaluation it is advisable to use more than one criterion of successfulness. Using only agreement between the students and the reference will be reliable only when positive and negative predictive values of the reference results are high. However, these values depend partly on the percentage of CIN2+ cases within the population of women from which samples for DS evaluation originate. Therefore, comparing accuracy results between students and reference is beneficial because it will demonstrate more exactly where a particular student has difficulties in DS interpretation. For example, high sensitivity and low specificity point to the fact that a student is signing too many cases as positive. An additional measure of a student's successfulness is also the time needed to evaluate one slide. Since both students were inexperienced in DS evaluation it is reasonable that the time needed to evaluate one slide was progressively decreasing. The decrease was sharper towards the beginning and less pronounced latter on. In addition to the agreement and the accuracy results, time needed for evaluating one slide also showed the difference between the two students. The ultimately unsuccessful student reached faster the point after which her time per slide started to decrease very slowly, compared to the successful student. However, during the evaluation of the last 100 slides, the successful student needed significantly less average time per slide compared to the other student (5.2 min vs. 8.2 min). Only McMenamin et al. made a quick mention of the time needed per slide evaluation.4 They reported that their experienced DS evaluators needed less than 1 minute for evaluating a clearly positive slide and 3-4 min for more challenging ones. Their evaluation time per slide is shorter than in our study not only because our students were unexperienced but mainly because we used conventional smears while in the study of Mc Menamin et al. ThinPrep specimens were used.4 In addition to teaching DS interpretation to students, our training program was designed also to monitor the performance of the cytotechnolo-gist involved in the teaching process. We believe that teacher monitoring is an important element of the training program which helps to assure the students will receive the best training. The intra-observer agreement was within the range of "very good" in both arms of our DS evaluation (OPA 100.0% and 98.7%, kappa 1.000 and 0.937, respec- Radiol Oncol 2020; 54(2): 201-208. 208 Kloboves Prevodnik V et al. / Evaluation of the training program for p16/Ki-67 dual staining interpretation tively). This result is even slightly better than the results obtained by McMenamin et al.4 where agreement for three cytotechnologists was 82.8% (0.65), 83.8% (0.67) and 94.9%, (0.91), respectively. Conclusions In conclusion we would like to say that teaching p16/Ki-67 interpretation should be a closely monitored process in which students' results have to be compared to the reference results with known accuracy for CIN2+. The students' progress has to be monitored using several criteria like agreement, accuracy and time needed for evaluating one slide. The monitoring process has to continue for a while after students reach satisfactory results in order to assure a continuous good performance. Monitoring of teacher's performance is also advisable. References 1. Waldstr0m M, Christensen RK, 0rnskov D. Evaluation of p16 INK4a/Ki-67 dual stain in comparison with an mRNA human papillomavirus test on liquid-based cytology samples with low-grade squamous intraepithelial lesion. Cancer Cytopathol 2013; 121: 136-45. doi: 10.1002/cncy.21233 2. Wentzensen N, Fetterman B, Tougawa D, Shiffman M, Castle PE, Wood SN, et al. Interobserver reproducibility and accuracy of p16/Ki67 dual-stain cytology in cervical cancer screening. Cancer Cytopathol 2014; 122: 914-20. doi: 10.1002/cncy.21473 3. Allia E, Ronco G, Coccia A, Luparia P, Macri L, Fiorito C, et al. Interpretation of p16INK4a/Ki-67 dual immunostaining for the triage of human papilloma-virus-positive women by experts and nonexperts in cervical cytology. Cancer Cytopathol 2015; 123: 212-8. doi: 10.1002/cncy.21511 4. McMenamin M, McKenna M, McDowell A, Dawson C, McKenna R. Intra-and inter-observer reproducibility of CINtecR PLUS in ThinPrep cytology preparations. Cytopathology 2017; 28: 284-90. doi: 10.1111/cyt.12426 5. Benevolo M, Allia E, Gustinucci D, Rollo F, Bulletti S, Cesarini E, et al. Interobserver reproducibility of cytologic p16INK4a /Ki-67 dual immu-nostaining in human papillomavirus-positive women. Cancer Cytopathol 2017; 125: 212-20. doi: 10.1002/cncy.21800 6. Kloboves Prevodnik V, Jerman T, Nolde N, Repse Fokter A, Jezersek J, Pohar Marinsek Z, et al. Interobserver variability and accuracy of p16/Ki-67 dual immunocytochemical staining on conventional cervical smears. Diagn Pathol 2019; 14: 1-9. doi: 10.1186/s13000-019-0821-5 7. Ivanus U, Jerman T, Fokter AR, Takac I, Prevodnik VK, Marcec M, et al. Randomised trial of HPV self-sampling among non-attenders in the Slovenian cervical screening programme ZORA: comparing three different screening approaches. Radiol Oncol 2018; 52: 399-412. doi: 10.2478/ raon-2018-0036 8. R Core Team. The R project for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2019. [cited 2019 Dec 15]. Avalable at http://www.R-project.org/ 9. National Cancer Institute. Joinpoint Trend Analysis Software. Joinpoint Regression Program, Version 4.6.0.0 - April 2018; Statistical Methodology and Applications Branch, Surveillance Research Program. 10. Altman DG. Practical statistics for medical research. Boca Raton: Chapman and Hall/CRC; 1991. Radiol Oncol 2020; 54(2): 201-208. 209 research article Care of patients with non-small-cell lung cancer stage III - the Central European real-world experience Milada Zemanova1, Robert Pirker2, Lubos Petruzelka1, Zuzana Zbozinkova3, Dragana Jovanovic4, Mirjana Rajer5, Krisztina Bogos6, Gunta Purkalne7, Vesna Ceriman4, Subhash Chaudhary8, Igor Richter9, Jiri Kufa10, Lenka Jakubikova11, Marius Zemaitis12, Marketa Cernovska13, Leona Koubkova14, Zdenka Vilasova15, Karin Dieckmann16, Attila Farkas17, Jelena Spasic18, Katerina Fröhlich3, Andreas Tiefenbacher2, Virag Hollosi6, Juraj Kultan10, Iveta Kolarova15, Jiri Votruba1 1 1st Faculty of Medicine of Charles University in Prague, Czech Republic 2 Department of Medicine I, Medical University of Vienna, Vienna, Austria 3 Institute of Biostatistic and Analyses, Faculty of Medicine, Masaryk University, Brno, Czech Republic 4 Clinic for Pulmonology, Clinical Centre of Serbia, Belgrade, Serbia 5 Institute of Oncology, Ljubljana, Slovenia 6 National Koranyi Institute of TB and Pulmonology, Budapest, Hungary 7 Pauls Stradins Clinical University Hospital, Riga, Latvia 8 Comprehensive Oncology Center, Novy Jicin, Czech Republic 9 Comprehensive Oncology Center, Liberec, Czech Republic 10 Clinic for Pneumology & Tuberculosis, Faculty of Medicine, Palacky University, Olomouc, Czech Republic 11 Clinic for Pneumology & Tuberculosis, Faculty of Medicine, Masaryk University, Brno, Czech Republic 12 Hospital of LUHS Kauno Klinikos, Kauno, Lithuania 13 Thomayer Hospital, Prague, Czech Republic 14 Department of Pulmonology, University Hospital Motol, Prague, Czech Republic 15 Comprehensive Oncology Center and Multiscan, Pardubice, Czech Republic 16 Department of Radiotherapy, Medical University of Vienna, Vienna, Austria 17 Department of Thoracic Surgery, Semmelweis University, Budapest, Hungary 18 Institute for Oncology and Radiology of Serbia, Belgrade, Serbia Radiol Oncol 2020; 54(2): 209-220. Received 9 January 2020 Accepted 14 April 2020 Correspondence to: Milada Zemanova, Oncology VFN, U Nemocnice 2, 128 08, Prague, Czech Republic. E-mail: milada.zemanova@vfn.cz Disclosure: No potential conflicts of interest were disclosed. Background. Management of non-small-cell lung cancer (NSCLC) is affected by regional specificities. The present study aimed at determining diagnostic and therapeutic procedures including outcome of patients with NSCLC stage III in the real-world setting in Central European countries to define areas for improvements. Patients and methods. This multicentre, prospective and non-interventional study collected data of patients with NSCLC stage III in a web-based registry and analysed them centrally. Results. Between March 2014 and March 2017, patients (n=583) with the following characteristics were entered: 32% females, 7% never-smokers; ECOG performance status (PS) 0, 1, 2 and 3 in 25%, 58%, 12% and 5%, respectively; 21% prior weight loss; 53% squamous carcinoma, 38% adenocarcinoma; 10% EGFR mutations. Staging procedures included chest X-ray (97% of patients), chest CT (96%), PET-CT (27%), brain imaging (20%), bronchoscopy (89%), endobronchial ultrasound (EBUS) (13%) and CT-guided biopsy (9%). Stages IIIA/IIIB were diagnosed in 55%/45% of patients, respectively. N2/N3 nodes were diagnosed in 60%/23% and pathologically confirmed in 29% of patients. Most patients (56%) were treated by combined modalities. Surgery plus chemotherapy was administered to 20%, definitive chemoradiotherapy to 34%, chemotherapy only to 26%, radiotherapy only to 12% and best supportive care (BSC) to 5% of patients. Median survival and progression-free survival times were 16.8 (15.3;18.5) and 11.2 (10.2;12.2) months, respectively. Stage IIIA, female gender, no weight loss, pathological mediastinal lymph node verification, surgery and combined modality therapy were associated with longer survival. Conclusions. The real-world study demonstrated a broad heterogeneity in the management of stage III NSCLC in Central European countries and suggested to increase the rates of PET-CT imaging, brain imaging and invasive mediastinal staging. Key words: diagnostic procedures; multimodality treatment; non-small-cell lung cancer; stage III Radiol Oncol 2020; 54(2): 194-200. doi: 10.2478/raon-2020-0019 210 Zemanova M et al. / NSCLC stage III - the Central European real-world experience Introduction Management of locally advanced (stage III) non-small cell lung carcinoma (NSCLC) includes a broad spectrum of diagnostic procedures and therapeutic modalities. Diagnosis is primarily based on computer tomography (CT) scan of chest plus upper abdomen and brain, bronchoscopy and CT-guided biopsies. For more precise exclusion of distant metastases, the evaluation with PET-CT is useful. Detailed locoregional staging is done by means of PET-CT1, endobronchial or endoscopic ultrasound (EBUS/EUS), mediastinoscopy, thora-coscopy, mediastinotomy or other biopsies of suspected nodal lesions in order to distinguish early stages (stages I and II) from stage IIIA/IIIB.2 Treatment of NSCLC stage III requires multidis-ciplinary co-operation in order to deliver appropriate local and systemic therapies for the various subgroups. Occurrence of distant metastases with and without local progression is frequent, thereby leading to 5-year survival rates often less than 20%.3 According to the 7th edition of the TNM classifica-tion3 stage III was subdivided into stage IIIA and IIIB. The 5-year survival rates were 36% and 19%, respectively.4 According to the 8th edition, stage III is divided into IIIA, IIIB and IIIC. The 5-year survival rates are 36%, 26% and 13%, respectively.4 In operable stage IIIA, induction or adjuvant chemotherapy improve overall survival5,6 and are established as standard treatments. Important prognostic factors associated with prolonged survival are pathological down-staging of mediastinal lymph nodes and/or primary tumors, and complete tumour re-section.7-10 Some trials assessing induction therapy followed by surgery have also included patients with stage IIIB disease and suggested that patients with operable stage IIIB NSCLC have outcomes similar to those with stage IIIAN2 disease.11 For patients with inoperable stage IIIA or stage IIIB and good performance status (PS), definitive chemora-diotherapy is the treatment of choice.2 Concurrent chemoradiotherapy is associated with longer survival at increased toxicity compared to the sequential approach.12 Radiotherapy dose escalation has no clear benefit.13 Sequential chemoradiotherapy or radiotherapy alone remain options for selected patients.2 Consolidation therapy with durvalumab after concurrent chemoradiotherapy has recently been shown to improve survival of patients14 and has been established as standard treatment. There is evidence that management of NSCLC stage III varies between countries, geographical regions, cancer centres and even treating physicians. Reasons for these variations include differences in regional standards, access to diagnostic procedures as well as therapeutic modalities, and resources. The aim of the present study was to determine diagnostic and therapeutic procedures as well as clinical outcome including survival of patients with NSCLC stage III in the real-world setting in Central European countries and to define areas for future improvements in routine management of these patients. Patients and methods Patients The present study was prospective, observational, non-interventional, multicentric, multinational and registry based. The study had been approved by ethics committees of participating centres and was performed in accordance with the Declaration of Helsinki. Study entry criteria were patients of any age, histological and/or cytological diagnosis of NSCLC stage III according to 7th edition of the TNM classification3 and signed written informed consent. The study allowed enrolling patients who have been treated between March 2014 and March 2017. Follow-up of patients continued until February 2018. Data collection Registration of all data was fully anonymous and performed in an electronic case report form (CRF [eCRF]) by qualified personnel. Patient identification was in the responsibility of each investigator. The following data were collected: age, gender, race, smoking status, PS, weight loss, date of NSCLC diagnosis, histology and mutational status of tumors, staging procedures, method of medias-tinal lymph-node examination, TNM and tumor stage. Data collected on therapeutic procedures were primary therapy, surgical procedures (date, extent, completeness of resection, information on repeated resections), thoracic radiotherapy (date, dose, fractions, technique, energy), prophylactic cranial irradiation, and chemotherapy (dates of start and end, number of cycles, cytotoxic drugs). Combined modality therapy was assessed according to investigator statement and/or dates of overlapping therapies. Type of best treatment response, date of recurrence or progression, and dates of death were collected. Radiol Oncol 2020; 54(2): 209-220. Zemanova M et al. / NSCLC stage III - the Central European real-world experience 211 TABLE 1. Patient characteristics Characteristic IIIA (N = 321) IIIB (N = 262) IIIA + IIIB (N = 583) P * N (%) N (%) N (%) Age < 65 years > 65 years Mean ± SD (years) Median (years) 155 (48.3) 166 (51.7) 64.8 ± 10.5 65.4 117 (44.7) 145 (55.3) 65.1 ± 10.1 66.0 272 (46.7) 311 (53.3) 64.9 ± 10.3 65.6 0.429 Gender Female 92 (28.7) 96 (37.0) 188 (32.2) Male 229 (71.3) 166 (63.6) 395 (67.8) 0.050 Smoking status Current smoker 161 (50.2) 147 (56.3) 308 (52.8) Former smoker 127 (39.6) 92 (35.2) 219 (37.6) Never smoker 26 (8.1) 16 (6.1) 42 (7.2) Unknown 7 (2.2) 7 (2.7) 14 (2.4) 0.446 Weight loss >10% within prior three months No 243 (75.7) 163 (62.5) 406 (69.6) Yes 50 (15.6) 70 (26.8) 120 (20.6) Unknown 28 (8.7) 29 (11.0) 57 (9.8) <0.001 WHO performance status 0 86 (26.8) 60 (23.0) 146 (25.0) 1 194 (60.4) 144 (54.9) 338 (58.0) 2 31 (9.7) 41 (15.7) 72 (12.3) 3 10 (3.1) 17 (6.5) 27 (4.6) 0.025 Histology Squamous cell carcinoma 176 (54.8) 133 (51.0) 309 (53.0) Adenocarcinoma 115 (35.8) 105 (40.0) 220 (37.7) NSCLC NOS 18 (5.6) 18 (6.9) 36 (6.2) Other 12 (3.7) 6 (2.3) 18 (3.1) 0.477 * = Chi-square test for IIIA vs. IIIB; NOS = not otherwise specified; NSCLC = non-small-cell lung cancer; SD = standard deviation Statistical analyses Descriptive statistics and frequency tables were used to characterize the sample data set. Statistical significances of differences for categorical and continuous parameters were assessed by means of Fisher's exact test and Mann-Whitney test, respectively. Overall survival (OS) was defined as the time from treatment initiation until death of any cause. Progression-free survival (PFS) was defined as the time from treatment initiation until first documented progression and/or death of any cause. Patients without an event were censored at the time of last follow-up visit. Patients lost to follow-up were counted as interval-censored observations. The assumption was made that patients were lost to follow-up due to their treatment failure and that, therefore, no information is available about them. Interval set (interval between visits) was 6 months.15 PFS and OS were estimated by KaplanMeier analysis and 95% confidence intervals (95% CI) were provided for all point estimates. Statistical significance of differences in survival between the subgroups was assessed by means of the log-rank test. The multivariable Cox proportional hazard model was used to evaluate the effects of potential prognostic factors on survival measures. Hazard ratios (HR) were complemented with 95% confidence intervals and supported with significance levels. Overall response rate (ORR) was defined as the sum of complete response rate (CR) and partial response rate (PR) and disease control rate as ORR plus stable disease rate (SD). All statistical tests were performed at the significance level of P = 0.05. Results Patients A total of 617 patients were enrolled but 34 patients were excluded due to various violations of the study protocol. Thus the study population comprises 583 patients from 16 centres of seven Central European countries: eight centres from Czech Republic (269 patients), two centres from Serbia (109 patients), two centres from Hungary (48 patients) and one centre each from Slovenia (53 patients), Latvia (43 patients), Lithuania (38 patients) and Austria (23 patients). The date for final analysis was February 19, 2018. Minimum follow-up since initial diagnosis was 11 months. Patient characteristics are shown in Table 1: 53.3% aged > Radiol Oncol 2020; 54(2): 209-220. 212 Zemanova M et al. / NSCLC stage III - the Central European real-world experience TABLE 2. Diagnostic procedures Procedure Patients (N = 583) N (%) range of % in centres Chest CT scan 567 (97) 67 - - 100 Chest X-ray 559 (96) 79 - - 100 Bronchoscopy 521 (89) 65 - - 100 Upper abdominal CT scan 389 (67) 0 - 100 Upper abdominal US 160 (27) 2 87 PET-CT or PET scan 163 (28) 0 78 Brain CT or MRI 117 (20) 0 91 Bone scan 88 (15) 0 79 EBUS or EUS 80 (14) 0 71 CT-guided biopsy 54 (9) 0 31 VATS 19 (3) 0 35 Mediastinoscopy 13 (2) 0 13 Others 47 (8) 0 30 EBUS = endobronchial ultrasound; EUS = endoscopic ultrasound; VATS = video-assisted thoracoscopic surgery TABLE 3. TNM subgroups (Union for International Cancer Control [UICC] 7] TNM Total patient population Patients undergoing surgery N (%) N % of total population % in stage subgroup IIIA+IIIB 583 (100) 135 32.2 NA IIIA 321 (55.1) 119 20.4 37.1 T4N0 T3N1 T4N1 T1N2 T2N2 T3N2 33 (5.7) 39 (6.7) 26 (4.5) 25 (4.3) 98 (16.8) 100 (17.2) 10 16 7 14 45 27 1.7 2.7 1.2 2.4 7.7 4.6 30.3 41.0 26.9 56.0 45.9 27.0 IIIB 242 (44.9) 16 2.8 6.1 T4N2 T1N3 T2N3 T3N3 T4N3 129 (22.2) 17 (2.9) 32 (5.5) 29 (5.0) 55 (9.4) 15 0 1 0 0 2.6 0 0.2 0 0 11.6 0 3.1 0 0 T stage TTTT -N GJ ho —' 42 (7.2) 130 (22.3) 168 (28.8) 243 (41.7) 14 46 43 32 2.4 7.9 7.4 5.5 33.3 35.4 25.6 13.1 N stage -Z.-Z.~Z.~Z. 33 (5.7) 65 (11.1) 352 (60.4) 133 (22.8) 10 23 101 1 1.7 3.9 17.3 0.2 30.3 35.4 28.7 0.8 NA = not applicable 65 years, 32.2% females, 7.2% never-smokers, 20.6% weight loss (> 10% within prior three months), 83% PS WHO 0-1. Stages IIIA and IIIB were diagnosed in 321 (55.1%) and 262 (44.9%) patients, respectively. Females, patients with weight loss and patients with poor PS were slightly more frequent among patients with stage IIIB. Squamous cell carcinomas were diagnosed in 309 (53.0%) patients, adenocarcinomas in 220 (37.7%) patients, NSCLC not otherwise specified in 36 (6.2%) patients, and other types (e.g. adeno-squamous, large cell) in 18 (3.1%) patients. Results of molecular analyses were documented for 150 (25.7%) patients (data not shown). EGFR mutations were detected in 14/142 (9.9%) patients, KRAS mutations in 11/31 (35.5%) patients and ALK aberrations in 2/88 (2.3%) patients (data not shown). Staging procedures Diagnostic and staging procedures are summarized in Table 2. Widely used procedures were chest x-ray (96%), chest CT scan (97%), bronchoscopy (89%) and upper abdominal CT scan (67%). PET-CT or PET were performed in 28% of patients. Brain imaging by means of CT or MRI was done in 20% of patients. EBUS or EUS were performed in 14% of patients, CT-guided biopsy in 9%, videoassisted thoracoscopic surgery (VATS) in 3%, and mediastinoscopy in 2%. Other procedures included diagnostic thoracotomy in 5%, radical thoracic surgery with gain of histology in 1%, ultrasonography (other than abdominal) (1%) and extracranial MRI examinations. The frequencies of the procedures showed great variations between centres with particularly great variations for PET or PET-CT (0-78%), brain imaging (0-91%) and EBUS/EUS (0-71%) (Table 2). Stage IIIA and stage IIIB were diagnosed in 55.1% and 44.9% of patients, respectively (Table 1). Subgroups based on T and N descriptors are shown in Table 3. Stages T1-T3 were found in 340 patients (58.3%) mostly having N2 or N3 nodal stage in 301 (51.6%) patients. T4N0, T4N1 and T4N2 tumours were found in 33 (5.7%), 26 (4.5%) and 129 (22.2%) patients, respectively. T3N3 and T4N3 tumours were diagnosed in 29 (5%) and 55 (9.4%) patients, respectively. T3-4N3 tumours (stage IIIC according to 8th TNM classification) were diagnosed in 84 (14.4%) patients. N2 or N3 lymph nodes were found in 485 (83.2%) patients. Histopatho logical involvement of mediastinal lymph nodes was confirmed in 172 (29.5%) patients by surgery (96 patients), transbronchial biopsy (58 patients), medias- Radiol Oncol 2020; 54(2): 209-220. 213 Zemanova M et al. / NSCLC stage III - the Central European real-world experience tinoscopy (11 patients) or other types of biopsy (10 patients) (data not shown). Treatment Therapeutic modalities are shown in overview for the total study population in Figure 1, for patients with stage IIIA and those with IIIB in detail in Table 4. Time from diagnosis to treatment initiation ranged from 0 to 369 (median 23) days. Combined therapies of any type were delivered to 326 (55.9%) patients. With regard to a single treatment modality only, surgery was delivered to 1.5% thoracic radiotherapy to 11.8%, chemotherapy to 25.7% and best supportive care (BSC) to 5.0% of patients of the total study population. Prophylactic cranial irradiation was not delivered to any patient. Surgery was performed in 135 (23.2%) patients (119 IIIA, 16 IIIB) and resulted in radical tumour resection in 121 patients and explorative surgery or non-radical resections in 14 patients (Table 4 and Figure 1). The types of surgery included lobectomy (77 patients), bi-lobectomies (6 patients), pneumo-nectomy (32 patients) and atypical radical resec- TABLE 4. Therapeutic modalities and combinations Therapeutic modalities IIIA (N=321) IIIB (N=242) N (%) IIIA+IIIB (N=583) Any combination 207 (64.5) 119 (45.4) 324 (55.9) Surgery 119 (37.1) 14 (4.1) 135 (23.2) Surgery alone 9 [2.8] 0 9 [1.5] Surgery plus radiotherapy 7 [2.2] 1 [0.4] 8 [1.4] Surgery plus chemotherapy 70 [21.8] 7 [2.7] 77 [13.2] Preoperative chemotherapy 22 [6.9] 4 [1.5] 26 [4.4] Perioperative chemotherapy 3 [0.9] 0 3 [0.5] Adjuvant chemotherapy 45 [14.0] 3 [1.1] 48 [8.2] Surgery plus RT plus CT (trimodality) 33 [10.3] 8 [3.1] 41 [7.0] Sequential preoperative RT plus CT 2 [0.6] 0 2 [0.3] Concurrent preoperative RT plus CT 3 [0.9] 1 [0.4] 4 [0.7] Sequential postoperative RT plus CT 23 [7.2] 3 [1.1] 26 [4.5] Concurrent postoperative RT plus CT 5 [1.6] 4 [1.5] 9 [1.5] Thoracic RT (including other modalities] 174 (54.2) 144 (55.0) 318 (54.4) Radiotherapy alone 37 [11.5] 32 [12.2] 69 [11.8] Chemoradiotherapy 97 (30.2) 103 (39.3) 200 (34.3) Sequential 74 [23.1] 72 [27.5] 146 [25.0] Concurrent 23 [7.2] 31 [11.8] 54 [9.3] Chemotherapy (including other modalities) 254 (79.7) 212 (80.9) 448 (80.3) Chemotherapy alone 58 [18.1] 92 [35.1] 150 [25.7] Best supportive care alone 9 (2.8) 20 (7.4) 29 (5.0) Type of surgery IIIA (N = 119) IIIB (N = 14) IIIA+IIIB (N = 135) Lobectomy 74 [62.3] 3 [18.7] 77 [57.0] Bi-lobectomy 6 [5.0] 0 6 [4.5] Pneumonectomy 28 [23.5] 4 [25.0] 32 [23.7] Atypical radical resection 5 [4.2] 1 [6.2] 6 [4.5] Non-radical surgery* 6 [5.0] 8 [50.0] 14 [10.4] * = Non-radical surgery (n = 14) as biopsy (n = 4, all IIIB), exploration (n = 7, IIIA = 5, IIIB = 2) or palliative resection (n = 3, IIIA = 1, IIIB = 2); CT = chemotherapy; RT = radiotherapy Radiol Oncol 2020; 54(2): 209-220. BSC = best supportive care; CT = chemotherapy; pre = preoperative; post = postoperative; peri = perioperative; RT = radiotherapy FIGURE 1. Treatment modalities and combinations. tions (6 patients) (Table 4). Surgery was delivered as single modality (nine patients), in combination with chemotherapy (77 patients) or radiotherapy (eight patients) or as trimodality therapy (41 patients). Adjuvant chemotherapy was about twice as frequent as pre-operative chemotherapy among patients undergoing surgery. 214 Zemanova M et al. / NSCLC stage III - the Central European real-world experience FIGURE 2. Overall survival and progression-free survival. Thoracic radiotherapy was delivered to 318 (54.6%) patients with similar proportions among stage IIIA and stage IIIB (Table 4). Thoracic radiotherapy was delivered as single modality (69 patients), as definitive chemoradiotherapy (200 patients) and as trimodality therapy (41 patients). Among patients undergoing definitive chemo-radiotherapy, the sequential administration was much more frequent than the concurrent one (146 versus 54 patients). Among patients undergoing tri- modality therapy, concurrent chemoradiotherapy was delivered in 13 patients (Table 4). Chemotherapy as the most frequent treatment modality was delivered to 468 (80.3%) patients with similar percentages among IIIA and IIIB (Table 4). Chemotherapy was the only treatment modality in 150 patients. Carboplatin was used in 43.6% and cisplatin in 37.4% of patients (data not shown). Platins were combined with vinorel-bine (33%), gemcitabine (22%), etoposide (11%) or paclitaxel (11%). Pemetrexed was used in 7.7% of patients in stage IIIB and 2.5% in stage IIIA. Other drugs including docetaxel, vinblastine and bevaci-zumab were each used in less than 2% of patients. No relevant differences in chemotherapy protocols between stage IIIA and IIIB patients were seen (data not shown). Treatment outcome At a median follow-up time of 30 months, 154 (26.4%) patients were alive, 295 (50.6%) patients were dead and 134 (23%) patients were lost to follow-up. Among 334 patients evaluable for overall best response, complete remission, partial remis- Good = PS 0/1 and no weight loss; poor = PS 2/3 and/or weight loss >10% within prior three months FIGURE 3. Overall survival and progression-free survival by performance status and weight loss in all patients, stage IIIA and stage IIIB. Radiol Oncol 2020; 54(2): 209-220. 215 Zemanova M et al. / NSCLC stage III - the Central European real-world experience TABLE 5. Overall survival according to stage and treatment modalities Characteristic N Median 1 -year 2-year 3-year Months (95% CI) % (95% CI) IIIA Total* 321 20.0 (17.1; 21.4] Good 223 21.4 (20.1; 25.8] Poor 75 13.3 (11.4; 17.7] 70.7 (65.9; 75.9] 39.4 (34.3; 45.2] 27.0 (21.8; 33.3] 76.3 (70.9; 82.1] 45.6 (39.4; 52.8] 33.8 (27.7; 41.3] 57.3 (47.2; 69.7 23.2 (15.2; 35.2] 9.1 (3.2; 26.0] IIIB Total* 262 14.4 (13.0; 15.9] 58.2 (52.3 ; 64.5] 27.5 (22.5 ■; 33.7] 13.5 (9.0; 20.2] Good 146 17.8 (14.8; 19.6] 67.0 (59.8 ; 75.1] 33.1 (26.1 ; 42.0] 19.6 (13.2; 29.1] Poor 97 10.4 (8.8; 19.6] 44.1 (35.2 ; 55.2] 17.3 (11.1 ; 27.0] 8.1 (3.0; 21.6] T3N3 and T4N3** 84 11.6 (9.8; 15.9] 48.5 (38.8 ; 60.5] 22.6 (15.2 :; 33.8] 9.4 (3.9; 22.7] IIIA+IIIB Total* 583 16.8 (15.3; 18.5] Good 369 20.1 (18.0; 21.5] Poor 172 11.8 (10.2; 14.2] 65.1 (61.3; 69.1] 34.1 (30.4; 38.2] 21.0 (17.3; 25.4] 72.6 (68.2; 77.3] 40.7 (35.9; 46.2] 28.3 (23.6; 34.0] 50.0 (42.9; 57.9] 19.9 (14.6; 27.0] 8.0 (3.6; 17.8] Surgery All surgeries 135 29.0 (27.1; NA] 82.2 (76.0; 88.9] 60.6 (52.8; 69.6] 43.9 (35.2; 54.8] Alone 9 13.9 (3.2; NA] 55.6 (31.0; 99.7] 22.2 (6.6; 75.4] 22.2 (6.6; 75.4] Surgery plus CT or RT 85 27.8 (23.6; NA] 80.0 (71.9; 89.0] 57.2 (47.5; 68.9] 37.0 (26.7; 51.3] Surgery plus adjuvant CT 77 28.3 (24.7; NA] 81.8 (73.6; 90.9] 60.6 (50.6; 72.7] 38.9 (28.0; 54.0] Trimodality therapy 41 Not reached 92.7 (85.0; 100] 76.9 (64.7; 91.5] 64.8 (49.9; 84.2] Non-surgical therapy All non-surgical CT alone RT alone CRT all CRT sequential CRT concurrent 448 150 69 200 146 54 14.6 (13.7; 15.9] 12.7 (11.3; 13.9] 12.7 (9.6; 18.4] 19.6 (17.5; 21.6] 20.5 (17.2; 22.8] 17.9 (14.6; 22.2] 59.9 (55.5; 64.7] 56.5 (49.1; 65.0] 52.2 (41.6; 65.4] 72.9 (67.0; 79.3] 75.2 (68.5; 82.5] 66.7 (55.2; 80.5] 26.0 (22.1; 30.5] 19.1 (13.7; 26.8] 18.8 (11.6; 30.7] 36.9 (30.7; 44.5] 38.8 (31.5; 47.9] 32.0 (21.6; 47.6] 13.8 (10.2; 18.8] 6.2 (2.7; 14.4] 11.8 (5.9; 23.5] 22.6 (16.1; 31.6] 23.5 (16.2; 34.0] 22.5 (12.7; 39.9] HR (95% CI) p-value IIIA vs. IIIB 0.70 (0.58; 0.85] < 0.001 IIIA Good vs. IIIA Poor 0.54 (0.40; 0.73] < 0.001 IIIB Good vs. IIIB Poor 0.57 (0.43; 0.76] < 0.001 IIIA vs. IIIB Good 0.86 (0.68; 1.09] 0.212 Tri-modality vs. Surgery plus adjuvant CT 0.47 (0.25; 0.89] 0.021 CRT sequential vs. concurrent 0.83 (0.57; 1.20] 0.326 Good = performance status 0-1 and no weight loss; Poor = performance status 2-3 and/or weight loss > 10%; CRT = chemoradiotherapy; CT = chemotherapy; RT = radiotherapy * = Total is not sum of Good and Poor, as some patients had no data about weight loss; ** = Stage IIIC in Union for International Cancer Control [UICC] 8 sion, stable disease and progressive disease were seen in 27.5%, 35.9%, 24.9% and 11.7% patients, respectively. Information on progression during follow-up was available for 338 patients, while no information was available for the other patients due to death (171 patients) or loss to follow-up (74 patients). Eighty-two (24.3%) patients were without progression and 256 (75.7%) patients had progression, either local (40.2%), distant (26.6%) or both (8.9%). Progression after end of treatment was more frequent among patients with stage IIIB than those with stage IIIA (91.2% versus 81.6%). Survival analyses OS and PFS are shown in Table 5, Table 6 and Figure 2. Median OS was 16.8 months and the 3-year OS rate was 21%. Median PFS was 11.2 months and the 3-year PFS rate was 11.5%. OS and PFS were longer among patients with IIIA than among those with IIIB and the corresponding hazard ratios were 0.70 (95% CI 0.58-0.85; p < 0.001) and 0.71 (95% CI 0.59-0.85; p < 0.001), respectively (Table 5; Table 6). Among patients with stage IIIA, median OS was 20 months and the 3-year OS rate was 27%. The corresponding values for patients with stage IIIB were 14.4 months and 13.5%, respectively. Among patients with T3-4/N3 tumours who are classified as stage IIIC based on the 8th edition of the TNM classification, survival outcome (median OS 11.6 months; 3-year OS rate 9.4%) was worse than the outcome of patients with stage IIIB based on the 7th TNM classification. OS and PFS were longer among patients with PS 0-1 and no weight loss (good prognostic group) than among those with PS 2-3 and/or weight loss > 10% (poor prognostic group) (Table 5; Table 6; Figure 3). These differences were observed in the Radiol Oncol 2020; 54(2): 209-220. 216 Zemanova M et al. / NSCLC stage III - the Central European real-world experience TABLE 6. Progression free survival according to stage and risk groups Characteristic N Progression-free survival Median 1 -year 2-year 3-year Months (95% CI) % (95% CI) IIIA Total* 321 12.5 (11.3; 13.6) 52.9 (47.7; 58.7) 24.1 (19.8; 29.3) 16.8 (13.0; 21.7) Good 223 12.9 (12; 15.2) 55.8 (49.6; 62.7) 27.6 (22.3; 34.3) 19.6 (14.8; 26.0) Poor 75 11.4 (7.9; 13.2) 45.3 (35.4; 58.1) 14.2 (8.1; 25.0) 8.3 (3.8; 18.1) IIIB Total* 262 9.9 (9.2; 10.8) 38.5 (33.1; 44.9) 12.8 (9.3; 17.7) 4.3 (1.8; 9.9) Good 146 10.7 (9.6; 12.6) 43.8 (36.5; 52.6) 13.6 (9.0; 20.8) 8.6 (4.7; 15.5) Poor 97 8.6 (7.8; 9.9) 26.8 (19.3; 37.2) 9.9 (5.4; 18.2) 0 T3N3 and T4N3** 84 9.4 (7.9; 11.3) 34.5 (25.7; 46.3) 10.7 (5.8; 20.0) 2.5 (0.4; 15.1) IIIA+IIIB Total* 583 11.2 (10.2; 12.2) 46.5 (42.6; 50.7) 19.0 (16.0; 22.6) 11.5 (8.9; 14.9) Good 369 12.2 (11.0; 13.3) 51.0 (46.2; 56.4) 22.1 (18.2; 26.9) 15.4 (11.9; 19.9) Poor 172 9.2 (8.3; 10.5) 34.9 (28.4; 42.8) 11.7 (7.7; 17.7) 3.7 (1.3; 10.8) HR (95% CI) p-value IIIA vs IIIB 0.71 (0.59; 0.85) < 0.001 IIIA Good vs IIIA Poor 0.69 (0.52; 0.92) 0.011 IIIB Good vs IIIB Poor 0.69 (0.53; 0.90) 0.006 IIIA vs IIIB Good 0.80 (0.65; 0.99) 0.037 Good = performance status 0-1 and no weight loss; Poor = performance status 2-3 and/or weight loss > 10%; * = Total is not sum of Good and Poor, as some patients had no data about weight loss; ** = Stage IIIC in Union for International Cancer Control [UICC] 8 Time {months) ^me {months) PS = performance status FIGURE 4. Overall survival by variables. Time [months) Time {months) Time [months) Time [months) Radiol Oncol 2020; 54(2): 209-220. 217 Zemanova M et al. / NSCLC stage III - the Central European real-world experience TABLE 7. Overall survival according to diagnostic variables Survival Variable N Median Univariate analysis P-value Multivariate analysis P-value months (95% CI) HR (95% Wald CI) HR (95% Wald CI) IIIA Stage IIIB 321 262 20.0 (17.1; 21.4) 14.4 (13.0; 15.9) 0.70 (0.58; 0.85) IIIA vs. IIIB: < 0.001 0.77 (0.62;0.95) 0.017 Gender Female Male 188 395 20.4 (16.8; 23.8) 15.4 (14.3; 17.6) 0.76 (0.62; 0.94) Female vs. male: 0.010 0.78 (0.64;0.96) 0.044 Smoking Current smoker 308 15.3 (14.2; 17.7) Never-smokers vs: status Former smoker 219 18.9 (15.9; 20.6) Current: 0.54 (0.34; 0.84) 0.007 Current: 0.71 (0.42;0.22) 0.218 Never smoker 42 27.1 (17.0; NA) Former: 0.61 (0.39; 0.97) 0.036 Former: 0.75 (0.44;1.30) 0.308 Weight loss > 10% No Yes 406 120 20.0 (17.9; 21.1) 12.7 (10.8; 14.8) 0.54 (0.43; 0.68) No vs. yes: < 0.001 0.68 (0.53;0.88) 0.003 0 146 22.4 (17.7; 26.1) 0 vs. > 2: 0.37 (0.28; 0.50) < 0.001 0.62 (0.43;0.88) 0.007 PS 1 338 15.4 (13.9; 17.2) 1 vs. > 2: 0.52 (0.41; 0.66) < 0.001 0.69 (0.52;0.91) 0.009 >2 99 11.6 (8.8; 14.7) 0 vs. 1: 0.72 (0.57; 0.92) 0.007 0.90 (0.68;1.17) 0.422 Squamous 309 16.3 (15.0; 19.0) Adeno vs.: Histology Adenoca 220 18.1 (14.7; 20.6) Squam: 0.96 (0.79; 1.18) 0.726 Squam: 0.93(0.68;1.26) 0.630 NOS/others 54 15 (10.1; 20.1) NOS/oth: 0.79 (0.57; 1.11) 0.18 N0S/oth:0.96(0.63;1.48) 0.857 EGFR Positive 14 27.1 (20.3; NA) Positive vs: mutation Negative 128 15.8 (13.9; 19.7) Negative: 0.51 (0.24; 1.10) 0.087 Negative:0.57(0.25;1.28) 0.176 positivity Not tested 441 16.5 (15.0; 18.9) Not tested: 0.52 (0.25; 1.10) 0.087 Not tested:0.83(0.36;1.89) 0.651 Nodes No 411 14.4 (13.3;15.9) Yes vs. no: confirmed Yes 172 24.7 (21.0; 28.8) 0.56 (0.45; 0.70) < 0.001 0.67 (0.52;0.87) 0.002 PET-CT or Yes 163 19.6 (17.8; 22.8) Yes vs. no: PET scan No 420 15.4 (14.1; 17.6) 0.73 (0.58-0.91) 0.005 0.84 (0.66;1.07) 0.160 NOS = not otherwise specified; oth = others; nodes confirmed = mediastinal lymph-nodes histologically confirmed; PS = performance status total study population, in IIIA patients and in IIIB patients. Among the good prognostic group of IIIB patients, the survival outcome was similar to the survival outcome of IIIA patients. Additional outcome data are summarized in Tables 5, 6 and 7 and Figures 3 and 4. Univariate analysis demonstrated longer OS for females, nev-er-smokers, good PS, stage IIIA, mediastinal node verification, and for those with PET/CT or PET staging exam. In contrast, OS was not different between patients with adenocarcinomas and those with squamous cell carcinomas. Including these variables into the multivariate survival proportional hazard model, the differences in OS remained significant for females versus males, no weight loss versus weight loss, PS 0-1 versus 2-3, stage IIIA versus IIIB, and pathological verification of medi-astinal nodes versus no verification. EGFR mutation status was not significant in the multivariate analysis but trend for better survival was found in the univariate model (Table 7). OS was also assessed in various treatment subgroups. Significant survival benefits were seen in patients undergoing surgery compared to patients without surgery (HR = 0.43; 95% CI 0.33-0.56; p-value < 0.001) and in patients receiving combined treatment modalities compared to patients with a single treatment modality (Table 5, hazard ratios not shown). The best OS outcome was shown for patients receiving tri-modality therapy (Table 5). A significant OS difference between concurrent and sequential chemora-diotherapy could not be demonstrated. Time from diagnosis to initiation of treatment start had no impact on outcome (analysis by quartiles, p = 0.585). Discussion Our observational study demonstrated a large heterogeneity in both diagnostic procedures and treatment modalities among patients with locally advanced NSCLC in the real-world setting in Central European countries. The results were based on 583 patients from 16 cancer centres of seven countries. The patient characteristics of our study were slightly different from those of recent phase 3 tri-als14,16, and these differences might be explained, at least partly, by higher smoking rates in Central European countries compared to other countries and the real-world nature of our study. In our study, two thirds were male, about half of the patients were older than 65 years and 7% were never-smokers. These rates are lower in terms of females and never-smokers than in the PROCLAIM trial16, but similar to those of the PACIFIC trial.14 The ma- Radiol Oncol 2020; 54(2): 209-220. 218 Zemanova M et al. / NSCLC stage III - the Central European real-world experience jority of our patients (53%) had squamous cell carcinomas and this frequency is slightly higher than the one (47%) of the PACIFIC trial.14 The low rate (6%) of NSCLC not otherwise specified confirms that accurate pathological examination is well established in Central European countries. Most patients (83%) had good (WHO 0-1) PS and 70% had no weight loss prior to diagnosis. Tumour stages IIIA and IIIB were diagnosed in 55% and 45% of patients, respectively, and these percentages are similar to those of the PACIFIC trial.14 Although not mandatory for patients with stage III NSCLC, molecular analyses were performed in selected patients. The EGFR mutation rate of 10% is consistent with the previously reported rate of 13.8% among patients with advanced NSCLC.17 The percentage of patients with ALK translocations (2%) was in the lower range of stage IV patients1819, but similar to another study.20 PD-L1 testing was not yet standard for patients with NSCLC stage at the time of our study. Our real-world data can also be compared with those from other recent real-world studies.20-23 All studies were similar in terms of median age (around 65 years), good PS (75-86%), never-smok-ers (4-9%) and percentage of adenocarcinomas (around 40%). In comparison to our study, the proportion of females were slightly higher in the American study (44.8%) and the Canadian study (46%).20,22 The percentages of stage IIIA patients were similar between our and the American study but higher (77%) in the Canadian study.2022 A major goal of our study was to determine the diagnostic and staging procedures (Table 2). Computer tomography of the chest, supplemented by CT or ultrasonography of the upper abdomen, was performed in most patients. PET-CT (or PET) and brain imaging were performed in only 28% and 20% of patients, respectively. These percentages, however, greatly varied between centres and reached almost 100% in some centres (Table 2). In the Canadian real-world study, 58% of patients received a PET scan as part of staging and 74% had baseline brain imaging.22 Bronchoscopy was performed in 89% of patients, thereby being the most frequent intervention for obtaining tumour material for histopathological diagnosis. Only 13.5% of patients had histopathological mediastinal nodal staging prior to therapy. The American and Canadian real-world studies did not provide data on histopathological assessment of mediastinal lymph nodes prior to treatment.2022 In a retrospective analysis of 106 patients who had been treated with definitive radiotherapy (plus chemotherapy in most patients), 48.1% of patients had pathological confirmation of nodal disease.24 Treatment of our patients involved surgery, radiotherapy and chemotherapy (Table 4). Surgery with and without any other treatment was performed in 23% of all patients, 37.1% and 6.1% of patients with stage IIIA and IIIB, respectively. This percentage is similar to the Canadian study in which 21% of patients underwent surgery, either alone or as part of combined modality therapy. This similarity is somewhat surprising based on the fact that the percentage of our stage IIIB patients was higher than the Canadian one (45% versus 33%). Lobectomy and pneumonectomy were performed in 57% and 24% of patients, respectively. The rates of pneumonectomies were similar between our and the Canadian study (23% versus 18%). Like in the Canadian study22, most of our surgical patients (87%) received chemotherapy, more frequently as adjuvant than induction chemotherapy. Chemoradiotherapy was delivered to 34.3% of our patients (Table 4). The concurrent approach was chosen only in 27% of these patients. This low frequency compared to other studies21,22 may be explained by patients unfit for the concurrent approach, limitations in the infrastructure of several centres including long waiting lists, and a stricter definition of concurrent versus sequential chemora-diotherapy than in other studies. Overall, 80.3% of patients received systemic chemotherapy, either alone or combined with other treatment modalities. Radiotherapy alone and BSC alone were given to 11.8% and 5% of patients, respectively (Table 4). These percentages are in agreement with the percentage of patients with poor (WHO >2) performance status. Although the proportion of patients with BSC was small and most likely caused by patient selection, it was similar to another study in which radiotherapy and BSC alone were delivered to 11% and 21% of patients, respectively.24 Our study confirmed tumour stage (IIIA versus IIIB), PS, prior weight loss and gender as prognostic factors. OS of patients with adenocarcino-mas did not differ from the one of patients with squamous cell NSCLC. Patients undergoing surgery had better survival than those without surgery (Table 5). The combination of surgery with chemotherapy and/or radiotherapy was also associated with improved survival outcome. The lack of a survival benefit for our patients undergoing concurrent chemoradiotherapy compared to those undergoing sequential chemoradiotherapy in our real-world study (Table 5) might be explained by Radiol Oncol 2020; 54(2): 209-220. 219 Zemanova M et al. / NSCLC stage III - the Central European real-world experience the multicentre nature of our study, low number of patients, and the heterogeneity of stage III NSCLC. The survival outcomes were similar between our study and the American real-world study.20 Median OS times were 20.0 and 22.3 months for stage IIIA patients, respectively, and 14.4 and 14.7 months for stage IIIB patients, respectively. The longer median OS of 27.3 months in the Canadian study can, at least partly, be explained by the higher percentage of stage IIIA patients. The best OS in our study with 64% of patients being alive at three years was seen among patients undergoing trimodal-ity therapy and was consistent with the Canadian study.22 It remains unclear, however, whether this survival benefit is due to treatment or just selection of patients with good prognostic features. In a randomized trial, the addition of surgery to chemora-diotherapy failed to improve overall survival.25 Our observational study has several limitations. Firstly, the findings were based on major academic cancer centres and, therefore, might not be representative for smaller centres. Secondly, there was no even distribution of patients between centres and an abundance of Czech patients was seen. Finally, other patient selection biases including patients lost to follow-up might have also played some role. Our real-world findings also define areas for future improvements in the management of patients with locally advanced NSCLC in Central Europe. Firstly, the rates of staging by means of PET-CT must be increased, mainly through improving access to these procedures. Secondly, brain imaging should be implemented prior to treatment in all patients who are planned for aggressive treatments such as surgery or chemoradiotherapy. Thirdly, invasive staging of mediastinal lymph nodes prior to treatment should be performed more frequently. In order to achieve these goals, awareness among doctors has to be raised, opportunities for education as well as training of doctors must be increased, and the infrastructure of some centres has yet to be improved. Conclusions The present real-world study confirmed known prognostic factors and the broad heterogeneity in diagnostic and therapeutic strategies among patients with stage III NSCLC in Central European countries. Rates of PET-CT staging, invasive staging of mediastinal nodes, and brain imaging prior to combined treatments should be increased in the future. Acknowledgements We would like to thank the heads of the all participating centres for permission to use data of patients from their respective regional networks and for the support of this project. We are also indebted to all physicians who provided data for the registry. This work was supported by the Czech Lung Cancer Cooperative Group (Kooperativni skupina pro lečbu plicni rakoviny, spolek). References 1. Nappi A, Gallicchio R, Simeon V, Nardelli A, Pelagalli A, Zupa A, et al. [F-18] FDG-PET/CT parameters as predictors of outcome in inoperable NSCLC patients. Radiol Oncol 2015; 49: 320-6. doi: 10.1515/raon-2015-0043 2. Eberhardt WE, De Ruysscher D, Weder W, Le Péchoux C, De Leyn P, Hoffmann H, et al. Panel members. 2nd ESMO consensus conference in lung cancer: locally advanced stage III non-small-cell lung cancer. Ann Oncol 2015; 26: 1573-88. doi: 10.1093/annonc/mdv187 3. Goldstraw P, Crowley J, Chansky K, Giroux DJ, Groome PA, Rami-Porta R, et al. The IASLC Lung Cancer Staging Project: proposals for the revision of the TNM stage groupings in the forthcoming (seventh) edition of the TNM classification of malignant tumours. J Thorac Oncol 2007; 2: 706-14. doi: 10.1097/JTO.0b013e31812f3c1a 4. Goldstraw P, Chansky K, Crowley J, Rami-Porta R, Asamura H, Eberhardt WE, et al. International Association for the Study of Lung Cancer Staging and Prognostic Factors Committee, Advisory Boards, and Participating Institutions. The IASLC Lung Cancer Staging Project: proposals for the revision of the TNM stage groupings in the forthcoming (eighth) edition of the TNM classification for lung cancer. J Thorac Oncol 2016; 11: 39-51. doi: 10.1016/j.jtho.2015.09.009. 5. Pignon JP, Tribodet H, Scagliotti GV, Douillard JY, Shepherd FA, Stephens RJ, et al. Lung adjuvant cisplatin evaluation: a pooled analysis by the LACE collaborative group. J Clin Oncol 2008; 26: 3552-9. doi: 10.1200/ JCO.2007.13.9030 6. NSCLC Meta-analysis Collaborative Group. Preoperative chemotherapy for non-small-cell lung cancer: a systematic review and meta-analysis of individual participant data. Lancet 2014; 383: 1561-71. doi: 10.1016/S0140-6736(13)62159-5 7. Betticher DC, Hsu Schmitz SF, Totsch M, Hansen E, Joss C, von Briel C, et al. Prognostic factors affecting long-term outcomes in patients with resected stage IIIA pN2 non-small-cell lung cancer: 5-year follow-up of a phase II study. Br J Cancer 2006; 94: 1099-106. 8. Sher DJ, Fidler MJ, Liptay MJ, Koshy M. Comparative effectiveness of neo-adjuvant chemoradiotherapy versus chemotherapy alone followed by surgery for patients with stage IIIA non-small cell lung cancer. Lung Cancer 2015; 88: 267-74. doi: 10.1016/j.lungcan.2015.03.015 9. Thomas M, Rube C, Hoffknecht P, Macha HN, Freitag L, Linder A, et al. Effect of preoperative chemoradiation in addition to preoperative chemotherapy: a randomised trial in stage III non-small-cell lung cancer. Lancet Oncol 2008; 9: 636-48. doi: 10.1016/S1470-2045(08)70156-6 10. Garrido P, Gonzalez-Larriba JL, Insa A, Provencio M, Torres A, Isla D, et al. Long-term survival associated with complete resection after induction chemotherapy in stage IIIA (N2) and IIIB (T4N0-1) non-small-cell lung cancer patients: the Spanish Lung Cancer Group Trial 9901. J Clin Oncol 2007; 25: 4736-42. Radiol Oncol 2020; 54(2): 209-220. 220 Zemanova M et al. / NSCLC stage III - the Central European real-world experience 11. Barlési F, Doddoli C, Torre JP, Giudicelli R, Fuentes P, Thomas P, et al. Comparative prognostic features of stage IIIAN2 and 111B non-small-cell lung cancer patients treated with surgery after induction therapy. Eur J Cardiothorac Surg 2005; 28: 629-34. doi: 10.1016/j.ejcts.2005.06.018 12. Auperin A, Le Pechoux C, Rolland E, Curran WJ, Furuse K, Fournel P, et al. Meta-analysis of concomitant versus sequential radiochemotherapy in locally advanced non-small-cell lung cancer. J Clin Oncol 2010; 28: 2181-90. doi: 10.1200/JC0.2009.26.2543 13. Ma L, Men Y, Feng L, Kang J, Sun X, Yuan M, et al. A current review of dose-escalated radiotherapy in locally advanced non-small cell lung cancer. Radiol Oncol 2019; 53: 6-14. doi: 10.2478/raon-2019-0006 14. Antonia SJ, Villegas A, Daniel D, Vicente D, Murakami S, Hui R, et al. PACIFIC Investigators. Durvalumab after chemoradiotherapy in stage III non-small-cell lung cancer. N Engl J Med 2017; 377: 1919-29. doi: 10.1056/ NEJMoa1709937 15. Zhang Z, Sun J. Interval censoring. Stat Methods Med Res 2010; 19: 53-70. doi: 10.1177/0962280209105023 16. Senan S, Brade A, Wang LH, Vansteenkiste J, Dakhil S, Biesma B, et al. PROCLAIM: Randomized phase III trial of pemetrexed-cisplatin or etopo-side-cisplatin plus thoracic radiation therapy followed by consolidation chemotherapy in locally advanced nonsquamous non-small-cell lung cancer. J Clin Oncol 2016; 34: 953-62. doi: 10.1200/JC0.2015.64.8824 17. Ramlau R, Cufer T, Berzinec P, Dziadziuszko R, Olszewski W, Popper H, et al. INSIGHT study team. Epidermal growth factor receptor mutation-positive non-small-cell lung cancer in the real-world setting in Central Europe: The INSIGHT Study. 10: 1370-4. J Thorac Oncol 2015; 10: 1370-4. doi: 10.1097/ JT0.0000000000000621 18. Barlesi F, Mazieres J, Merlio JP, Debieuvre D, Mosser J, Lena H, et al. Biomarkers France contributors. Routine molecular profiling of patients with advanced non-small-cell lung cancer: results of a 1-year nationwide programme of the French Cooperative Thoracic Intergroup (IFCT). Lancet 2016; 387: 1415-26. doi: 10.1016/S0140-6736(16)00004-0 19. Hofman P. ALK in Non-Small Cell Lung Cancer (NSCLC): pathobiology, epidemiology, detection from tumor tissue and algorithm diagnosis in a daily practice. Cancers 2017; 9: pii: E107. doi: 10.3390/cancers9080107 20. Ryan KJ, Skinner KE, Fernandes AW, Punekar RS, Pavilack M, Walker MS, et al. Real-world outcomes in patients with unresected stage III non-small cell lung cancer. Med Oncol 2019; 36: 24. doi: 10.1007/s12032-019-1249-1 21. Ryan KJ, Skinner KE, Fernandes AW, Punekar RS, Pavilack M, Walker MS, et al. Real-world treatment patterns among patients with unresected stage III non-small-cell lung cancer. Future Oncol 2019; 15: 2943-53. doi: 10.2217/ fon-2018-0939 22. Moore S, Leung B, Wu J, Ho C. Real-world treatment of stage III NSCLC: the role of trimodality treatment in the era of immunotherapy. J Thorac Oncol 2019; 14: 1430-9. doi: 10.1016/j.jtho.2019.04.005 23. Fernandes AT, Mitra N, Xanthopoulos E, Evans T, Stevenson J, Langer C, et al. The impact of extent and location of mediastinal lymph node involvement on survival in stage III non-small cell lung cancer patients treated with definitive radiotherapy. Int J Radiat Oncol Biol Phys 2012; 83: 340-7. doi: 10.1016/j.ijrobp.2011.05.070 24. Van der Meer FS, Schramel FM, Van Vulpen M, El Sharouni SY. Feasibility of concomitant chemoradiotherapy in daily practice for patients with NSCLC stage III. Anticancer Res 2016; 36: 4673-6. doi: 10.21873/anticanres.11019 25. Albain KS, Swann RS, Rusch VW, Turrisi AT 3rd, Shepherd FA, Smith C, et al. Radiotherapy plus chemotherapy with or without surgical resection for stage III non-small-cell lung cancer: a phase III randomised controlled trial. Lancet 2009; 374: 379-86. doi: 10.1016/S0140-6736(09)60737-6 Radiol Oncol 2020; 54(2): 209-220. 221 research article Diagnostic accuracy of (1^3)-ß-D-glucan to predict Pneumocystis jirovecii pneumonia in non-HIV-infected patients Petra Rogina1, Miha Skvarc2 1 Infectious Disease Department, General Hospital Novo mesto, Novo mesto, Slovenia 2 Infection Control and Microbiology Unit, General Hospital Jesenice, Jesenice, Slovenia Radiol Oncol 2020; 54(2): 221-226. Received 31 December 2019 Accepted 14 April 2020 Correspondence to: Assist. Prof. Miha Skvarč, M.D., Ph.D., General Hospital Jesenice, Cesta maršala Tita 112, Jesenice, Slovenia. E-mail: mihaskvarc@hotmail.com Disclosure: No potential conflicts of interest were disclosed. Background. Pneumocysfisjirovecii pneumonia (PCP) is a common and potentially fatal opportunistic infection in immunocompromised non-HIV individuals. There are problems with clinical and diagnostic protocols for PCP that lack sensitivity and specificity. We designed a retrospective study to compared several methods that were used in diagnostics of PCP. Patients and methods. One hundred and eight immunocompromised individuals with typical clinical picture for PCP and suspicious radiological findings were included in the study. Serum samples were taken to measure the values of (1^3)-^-D-glucan (Fungitell, Associates of Cape Cod, USA). Lower respiratory tract samples were obtained to perform direct immunofluorescence (DIF, MERIFLUOR® Pneumocystis, Meridian, USA) stain and real-time PCR (qPCR). Results. Fifty-four (50%) of the 108 patients in our study had (1^3)-£-D-glucan > 500 pg/ml. Patients that had (1^3)-^-D-glucan concentrations < 400 pg/ml in serum, had mean threshold cycles (Ct) 35.43 ± 3.32 versus those that had (1^3)-£-D-glucan concentrations >400 pg/mL and mean Ct of 28.97 ± 5.27 (P < 0.001). If we detected P.jirovecii with DIF and qPCR than PCP was proven. If the concentration of (1 ^3)-^-D-glucan was higher than 400 pg/ml and Ct of qPCR was below 28.97 ± 5.27 than we have been able be certain that P. jirovecii caused pneumonia (odds ratio [OR] 2.31, 95% confidence interval [CI] 1.62-3.27, P < 0.001). Conclusions. Measurement of (1^3)-^-D-glucan or qPCR alone could not be used to diagnose PCP. Diagnostic cut-off value for (1^3)-^-D-glucan > 400pg/ml and qPCR below 30 Ct, allow us to conclude that patient has PCP. If the values of (1^3)-^-D-glucan are < 400 pg/ml and qPCR is above 35 Ct than colonization with P. jirovecii is more possible than PCP. Key words: Pneumocysfisjirovecii pneumonia; (1 ^3)-^-D-glucan; DIF; real-time PCR; colonization with P.jirovecii; non-HIV-infected patients Introduction Pneumocystis jirovecii is a cause of Pneumocystis pneumonia (PCP) in immunocompromised patients. PCP most often occurs in human immunodeficiency virus (HIV) infected patients associated with high pathogen burdens but also in non-HIV immunocompromised patients.1-3 The diagnosis is mainly based on clinical and radiographic examinations, which are in majority of cases inconclusive, so microscopic or molecular detection of P. jirovecii in lower respiratory tract samples are necessary.23 The mechanism by which Pneumocystis organisms induce lung inflammation remains incompletely understood, but studies imply that Pneumocystis spp. cell wall constituent (1^3)-|3-D-glucans are released and contribute to the development of extensive lung inflammatory reaction.4-10 Various studies have reported high serum (1^3)-|3-D-glucan levels in patients with PCP.7-15 The (1^3)-|3-D-glucan has Radiol Oncol 2020; 54(2): 194-200. doi: 10.2478/raon-2020-0019 222 Rogina P and Skvarc M / Pneumocystis jirovecii pneumonia high sensitivity for PCP but, it is a not species-specific marker for P. jirovecii.2'11'13'15 Detection of P. jiroveci DNA in respiratory tract specimens without signs and symptoms of PCP is defined as colonization. Molecular diagnostic techniques like quantitative PCR (qPCR) are more sensitive than staining methods like direct immu-nofluorescence method (DIF). However, qPCR can give sometimes false signal that patient has PCP' although patient is just colonized with fungi.16-21 Studies have reported prevalence of colonization in susceptible adults from 10.0% to 43.8%.17-23 Realtime qPCR assays have been shown as most accurate for discrimination of true PCP from colonization with P. jirovecii. Flori et al. compared the performance of standard staining' standard PCR' and qPCR using 173 BAL specimens from 150 patients. Real-time qPCR gave the best results.24 A systematic review, that evaluated PCR kits to confirm PCP' revealed that positive PCR results do not always confirm PCP. Low amounts of DNA can be expected in patients with P. jirovecii colonization of and no clinical signs of PCP.25 (1^3)-|3-D-glucan can be helpful to distinguish PCP from P. jirovecii colonization. However, not as standalone test. (1^3)-|3-D-glucan is positive in colonization of gastrointestinal tract with candida. It is positive when patients are colonized with P. jirovecii.13,16 Combining best tests is a advisable in immunocompromised cancer patients to be able to treat patients with the right therapy. We designed a retrospective multicentric study that included patients from 3 tertiary care hospitals and 2 secondary care hospitals. The objective of our study was to distinguish true PCP from colonization with P. jirovecii with diagnostic algorithm that includes measurement of (1^3)-|3-D-glucan and real-time PCR (qPCR). Patients and methods Patients We included 108 consecutive non-HIV patients on immunosuppression therapy from 2006 to end of 2014 with clinical diagnosis of possible PCP. Possible PCP diagnosis was given to patients at admission to hospital or during diagnostic process that had typical suggestive symptoms and signs and radiological findings for PCP (fever, dry cough, dyspnoea on exertion and bilateral interstitial pneumonia on chest X-ray, and/or ground glass look on thorax CT scan). Patients receiving antifun-gal therapy or therapy that works against P. jirovecii were excluded from the study. Patients with excessive colonization with Candida spp. on mucosal barriers were also excluded from the study. Methods Microbiological diagnosis was based on the detection of P. jirovecii with DIF (MERIFLUOR® Pneumocystis, Meridian, USA) and on in-house developed qPCR from lower respiratory tract sample (induced sputum, aspiration of trachea or bron-choalveolar lavage - BAL). DNA was extracted from lower respiratory tract samples using QIAamp DNA mini kit (Qiagen, Germany). In-house qPCR developed after article of Alanio et al.16 was performed using a reaction mix Platinum® Quantitative PCR SuperMix-UDG with ROX (Invitrogen, USA) and PCR primers to detect Pneumocystis PjFI and PjRI with hybridization probe PjSL (TIB MolBiol, Germany). The qPCR assay developed for this study amplifies a 121-bp fragment of the P. jirovecii mito-chondrial large-subunit rRNA gene. The primers PjF1 (5'-CTGTTTCCCTTTCGAC TATCTACCTT-3') and PjR1 (5'-CACTGAATATCTCGAGG GAGTATGAA-3') and the TaqMan-MGB probe PjSL (5'-TCGCACATAGTCTGATTAT-3') were designed by using Primer Express software (Applied Biosystems, Foster City, CA, USA). DNA sample volume was 5 |xL. PCR reaction with a final volume of 25 |xL was carried out with following conditions: 50°C for 2 min, 95°C for 2 min, followed by 45 cycles consisting of 15 sec at 95°C, 60 sec at 60°C. The results are presented as threshold cycles (Ct). We used Quality Control for Molecular Diagnostics (QCMD, UK) samples from Pneumocystis jirovecii pneumonia (PCP) DNA scheme to evaluate our qPCR (sensitivity 97%, specificity 100%). Serum samples were collected to measure the values of (1^3)-|3-D-glucan (Fungitell, Associates of Cape Cod, USA). In all patients 2 sets of blood cultures were collected. Lower respiratory tract samples were collected to detect potential bacterial cause of deteriorating health. Institutional review board approved of General Hospital Jesenice approved the study under condition that patients' data were anonymized. The results of the study did not influence the course of the treatment. Statistical analysis Microbiologically proven PCP was defined as positive DIF and positive qPCR. Colonization was suspected when DIF was negative and if any amount Radiol Oncol 2020; 54(2): 221-226. 223 Rogina P and Skvarc M / Pneumocystis jirovecii pneumonia of DNA with qPCR was found. (1^3)-|3-D-glucan in patients with possible colonization with P. jirovecii had to be positive (above 80 pg/ml). Mean values for (1^3)-|3-D-glucan and Ct values between different patients' groups and (1^3)-|3-D-glucan ranges were compared using an independent T-test. Receiver operating curve (ROC) analysis with area under the curve (AUC) and confidence interval (CI) were performed to calculate diagnostic accuracy. Univariate logistic regression was performed to explore the association of laboratory parameters with the disease. The statistical significance level was set at 0.05 (two-tailed). All analyses were conducted with SPSS version 23.0 statistical software. Results One hundred and eight immunosuppressed patients (mean age 53 ± 15 years) with possible PCP were included into final analysis. Majority of patients were in some way immunocompromised. They received immunosuppressive therapy because of transplanted solid organ (53 patients, 49%) with mean (1^3)-|3-D-glucan value of 330.01 ± 191 pg/mL. The rest of patients had autoimmune diseases or malignant disease (Table 1). Patient's data were divided into 6 groups according to (1^3)-|3-D-glucan values. In the first group were patients with (1^3)-|3-D-glucan value < 100 pg/mL, followed by group of patients with (1^3)-|3-D-glucan value from 100 pg/mL till 200 pg/mL and so on till the last group with (1^3)-|3-D-glucan values > 500 pg/mL. The largest group of patients (50% of all patients) were in the range of (1^3)-|3-D-glucan > 500 pg/mL. Twenty-four patients with possible PCP had (1^3)-|3-D-glucan < 100 pg/mL, with mean value of 81.14 ± 3.41 pg/mL (Table 2). Results for patients in different (1^3)-|3-D-glucan ranges and mean qPCR Ct values are presented in Table 3. Patients in the last 2 ranges ((1^3)-|3-D-glucan 400-500pg/m and > 500pg/ml) had significantly more P. jirovecii DNA detected in lower respiratory tract ((1^3)-|3-D-glucan 400-500 pg/mL, mean Ct 29.41 ± 6.25; (1^3)-|3-D-glucan > 500 pg/ml, mean Ct 28.90 ± 5.18, P < 0.001) in comparison to other groups of patients. Statistically significant difference in Ct values was noticed when (1^3)-|3-D-glucan concentrations exceeded 400pg/ml ((1^3)-|3-D-glucan < 400 pg/mL, mean Ct 35.43 ± 3.32 versus (1^3)-|3-D-glucan > 400 pg/ mL, mean Ct 28.97 ± 5.27, P < 0.001). Moreover, analysis showed that majority of patients (57%) TABLE 1. Average (1 ^3)-|-D-glucan values for each group of immunocompromised patients Diagnosis N = 108 patients (1^3)-ß-D-glucan (pg/mL) ± SD Malignant disease 20 (18%) 240.34 ± 162.45 Transplantation 53 (49%) 330.01 ± 191.26 Autoimmune disease 35 (33%) 427.73 ± 152.23 TABLE 2. Average (1 ^3)-|-D-glucan values for each (1 ^3)-|-D-glucan range (1^3)-ß-D-glucan group N = 108 patients (1^3)-ß-D-glucan (pg/mL) ± SD < 100 pg/mL 24 81.14 ± 3.41 100-200 pg/mL 11 124.4 ± 22.37 200-300 pg/mL 4 238,75 ± 16,55 300-400 pg/mL 7 336.75 ± 20.18 400-500 pg/mL 8 454.13 ± 31.3 > 500 pg/mL* 54 > 500 pg/mL * Mean could not be calculated since samples dilutions to measure real (1 ^3)-|-D-glucan concentrations were not performed. TABLE 3. Average Ct values for each (1 ^3)-|-D-glucan range (1^3)-ß-D-glucan group N = 108 patients Mean Ct ± SD Group 1 < 100 pg/mL 24 35.43 ± 3.51 Group 2 100-200 pg/mL 11 34.82 ± 3.65 Group 3 200-300 pg/mL 4 37.33 ± 2.36 Group 4 300-400 pg/mL 7 35.27 ± 2.75 Group 5 400-500 pg/mL 8 29.41 ± 6.25* Group 6 > 500 pg/mL 54 28.90 ± 5.18* Ct = threshold cycle; SD = standard deviation * Statistically significant difference seen when Ct from group 1 to 4 are compared to group 5 and 6 combined, P < 0.001. Odds ratio (OR) is the greatest to predict PCP if (1 ^3)-|3-D-glucan > 400 pg/mL, OR 2.31 (95%CI 1.62-3.27), p < 0.001). had (1^3)-|3-D-glucan > 400 pg/ml and had the greatest chance to have PCP ((1^3)-|3-D-glucan > 400 pg/mL: OR 2.31 (95% CI 1.62-3.27), p < 0.001). We compared (1^3)-|3-D-glucan values and qPCR Ct of 42 patients with microbiologically confirmed PCP (DIF positive, qPCR positive) to those 66 patients that had possible PCP at initial presentation to predict true PCP (Table 4). Forty-two patients had statistically significant higher concentrations of (1^3)-|3-D-glucan and P. jirovecii DNA (P < 0.001). Subgroup of 26 patients, that had DIF positive and qPCR Ct < 30, had slightly higher Radiol Oncol 2020; 54(2): 221-226. 224 Rogina P and Skvarc M / Pneumocystis jirovecii pneumonia TABLE 4. (1 ^3)-|-D-glucan values for patients with microbiologically confirmed PCP and possible PCP PCP 108 patients (1^3)-p-D-glucan (pg/mL) ± SD qPCR Ct ± SD Possible PCP Microbiologically confirmed PCP (DIF and qPCR positive) Subgroup definite PCP (DIF positive and qPCR Ct < 30 and response to treatment) P value Ct = threshold cycle; DIF = direct immunofluorescence; PCP = Pneumocystis pneumonia; qPCR = real time PCR; SD = standard deviation * Area under the curve (AUC) to predict PCP (positive DIF and qPCR) with measurement of (1^3)-|3-D-glucan: AUC = 0.817, 95% CI: 0.736-0.898, sensitivity = 83.3%, specificity = 64.6%, cut off 496.45 pg/mL, p < 0.001 66 (61%) 42 (39%) 26 (24%) 256.06 ± 183.48 447.5 ± 95.5 494.81 ± 17.73* 35.01 ± 3.8 27.2 ± 4.6 < 0.001 < 0.001 (1^3)-|3-D-glucan mean values than the group of microbiologically confirmed PCP. The subgroup cut-off for (1^3)-|3-D-glucan was set to 496.45 pg/ mL, and determined PCP with greatest AUC and statistically significance (AUC = 0.817, 95% CI: 0.736-0.898, sensitivity = 83.3%, specificity = 64.6%, p < 0.001). Discussion Our experience with (1^3)-|3-D-glucan and qPCR indicated that cut-off of > 400 pg/mL for (1^3)-|3-D-glucan could be used to predict micro-biologically confirmed PCP with greatest statistical significance. Results of the analysis indicated that colonization is possible when levels of (1^3)-|3-D-glucan are moderate, in our case < 400 pg/mL, and low concentrations of P. jirovecii DNA with qPCR are detected, in our case > 35 cycles. Fifty percent of the patients in our study had (1^3)-|3-D-glucan > 500 pg/mL and mean qPCR Ct 28.9 ± 5.18. The second largest group of patients (24 patients) with possible PCP were those with (1^3)-|3-D-glucan < 100 pg/mL, and mean qPCR Ct 35.43 ± 3.51. Patients with microbiologically confirmed PCP had mean (1^3)-|3-D-glucan of 467.5 ± 95.5 and mean qPCr Ct 27.2 ± 4.6. Even more, true PCP where patients with it, had response to therapy, can be predicted when cut-off for (1^3)-|3-D-glucan is set to 496.45 pg/ml. At this cut-off, AUC to predict PCP was 0.817. Similar results were produced in recent studies.2627 In an older study cut-off value of 340 copies/ mL was used to discriminate probable PCP from colonization. At this qPCR cut-off, (1^3)-|3-D-glucan levels were significantly higher in patients with both definite PCP and probable PCP than in colonized patients.26 It was recently shown that serum (1^3)-|3-D-glucan levels of 143 pg/mL can be used to distinguish PCP from colonisation with P. jirovecii.28 To diagnose and treat PCP you have to have a lot of experience.29 Our results were not concordant with latter studies because we measured much higher (1^3)-|3-D-glucan levels in the patients with true PCP. The clinicians in majority of included institutions seldomly get the chance to treat PCP and are not experienced enough. When PCR was introduced, it remarkably increased sensitivity to diagnose PCP, resulting in an increase in the number of documented PCP cases.30-34 On the other hand multiple evidences indicate that P. jirovecii can colonize the mucosal epithelium of both healthy individuals and those with compromised immunity.34-36 Results of qPCR in the area where differentiation between PCP and colonization is demanding, if we look at them separately from (1^3)-|3-D-glucan. It was suggested that every (1^3)-|3-D-glucan values should be interpreted with qPCR and vice versa in patients with possible PCP.162629 Montesinos et al. also showed with their in-house PCR test cut-off value Ct = 34 to discriminate definite PCP from unlikely PCP with 65% sensitivity and 85% specificity.37 Other studies have suggested applying two qPCR cut-off values to increase sensitivity and specificity of qPCR. There is a grey zone of unclear clinical significance between these two cut-off values where the differential diagnosis of PCP versus colonization cannot be determined.162438-40 In recent literature two cut-offs were proposed to predict definite PCP. The two cut-offs for qPCR provide 100% sensitivity and 100% specificity to diagnosis definite PCP. The higher cut-off value represents the value below which the diagnosis of PCP is unlikely. The authors suggested that the range between these two cut-off values represents an indeterminate zone.163941-42 Radiol Oncol 2020; 54(2): 221-226. 225 Rogina P and Skvarc M / Pneumocystis jirovecii pneumonia According to our study, P. jirovecii DNA copy number above the cut-off value Ct >35 would support a diagnosis of colonization, and a copy number below the cut-off value would support a diagnosis of PCP. However, good sensitivity of qPCR can be misleading, since qPCR detects very low fungal load that can lead to over-diagnosis of PCP due to misinterpretation of findings.33 42 Our cut-off was determined with the help of (1^3)-|3-D-glucan, which is a good indicator for PCP if the values are high and we detect a lot of P. jirovecii DNA in lower respiratory tract. Our study had some limitations. We could not gather the information that treatment with antimicrobials helped patients which would confirm that patients really had PCP for all included individuals. The influence of different samples from lower respiratory tract on qPCR was not assessed. Although our qPCR displayed high accuracy for discriminating colonization from PCP, the cut-off values used in our study should be standardized to copies/ml and compared to other qPCRs. The cutoff values for (1^3)-|3-D-glucan was not always measured the same day as qPCR was done which could influence on the concentration of (1^3)-|3-D-glucan. For future studies, patients that have their clinical picture and response to therapy consistent with possible PCP, and are randomized according to the analysed samples, should be included to resolve the discrepancy seen in our study. Conclusions The protocol we presented here could better support clinicians in their decisions whether patient in front of them has PCP. Our diagnostic cut-off values allow identification of true PCP versus colonization with P. jirovecii with better diagnostic accuracy. However, according to our results qPCR cannot be used alone to confirm PCP. PCP has to be confirmed with qPCR and with (1^3)-|3-D-glucan. Acknowledgment We thank Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana for their routine diagnostic support when needed. References 1. Tamburrini E, Mencarini P, Visconti E, Zolfo M, De Luca A, Siracusano A, et al. Detection of Pneumocystis carinii DNA in blood by PCR is not of value for diagnosis of P. carinii pneumonia. J Clin Microbiol 1996; 34: 1586-8. PMID: 8735128 2. Maschmeyer G, Helweg-Larsen J, Pagano L, Robin C, Cordonnier C, Schellongowski P, et al. ECIL guidelines for treatment of Pneumocystis jirovecii pneumonia in non-HIV-infected haematology patients. J Antimicrob Chemother 2016; 71: 2405-13. doi: 10.1093/jac/dkw158 3. Maertens J, Cesaro S, Maschmeyer G, Einsele H, Donnelly JP, Alanio A, et al. ECIL guidelines for preventing Pneumocystis jirovecii pneumonia in patients with haematological malignancies and stem cell transplant recipients. J Antimicrob Chemother 2016; 71: 2397-404. doi: 10.1093/jac/dkw157. 4. Wang J, Gigliotti F, Maggirwar S, Johnston C, Finkelstein JN, Wright TW. Pneumocystis carinii activates the NF-kappaB signaling pathway in alveolar epithelial cells. Infect Immun 2005; 73: 2766-77. doi: 10.1128/IAI.73.5.2766-2777.2005 5. Vassallo R, Standing JE, Limper AH. Isolated Pneumocystis carinii cell wall glucan provokes lower respiratory tract inflammatory responses. J Immunol 2000; 164: 3755-63. doi: 10.4049/jimmunol.164.7.3755 6. Tasaka S, Hasegawa N, Kobayashi S, Yamada W, Nishimura T, Takeuchi T, et al. Serum indicators for the diagnosis of Pneumocystis pneumonia. Chest 2007; 131: 1173-80. doi: 10.1378/chest.06-1467 7. Cuetara MS, Alhambra A, Chaves F, Moragues MD, Ponton J, del Palacio A. 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Blood (1^3)-ß-D-glucan as a diagnostic test for HIV-related Pneumocystis jirovecii pneumonia. Clin Infect Dis 2011; 53: 197-202. doi: 10.1093/cid/ cir335 16. Alanio A, Desoubeaux G, Sarfati C, Hamane S, Bergeron A, Azoulay E, et al. Real-time PCR assay-based strategy for differentiation between active Pneumocystis jirovecii pneumonia and colonization in immunocompro-mised patients. Clin Microbiol Infect 2011; 17: 1531-7. doi: 10.1111/j.1469-0691.2010.03400.x 17. Morris A, Wei K, Afshar K, Huang L. Epidemiology and clinical significance of pneumocystis colonization. J Infect Dis 2008; 197: 10-7. doi: 10.1086/523814 Radiol Oncol 2020; 54(2): 221-226. 226 Rogina P and Skvarc M / Pneumocystis jirovecii pneumonia 18. Can H, Caner A, Dôçkaya M, Degirmenci A, Karaçali S, Polat C, et al. Detection of Pneumocystis in the nasal swabs of immune-suppressed rats by use of PCR and microbiology. Med Sci Monit Basic Res 2013; 19: 62-7. doi: 10.12659/MSMBR.883777. 19. 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Totet A, Meliani L, Lacube P, Pautard JC, Raccurt C, Roux P, et al. Immunocompetent infants as a human reservoir for Pneumocystis jirovecii: rapid screening by non-invasive sampling and real-time PCR at the mito-chondrial large subunit rRNA gene. J Eukaryot Microbiol 2003; 50(Suppl): 668-9. doi: 10.1111/j.1550-7408.2003.tb00678.x 24. Flori P, Bellete B, Durand F, Raberin H, Cazorla C, Hafid J, et al. Comparison between real-time PCR, conventional PCR and different staining techniques for diagnosing Pneumocystis jiroveci pneumonia from bronchoal-veolar lavage specimens. J Med Microbiol 2004; 53: 603-7. doi: 10.1099/ jmm.0.45528-0 25. Fan LC, Lu HW, Cheng KB, Li HP, Xu JF. Evaluation of PCR in bronchoalveolar lavage fluid for diagnosis of Pneumocystis jiroveciii pneumonia: a bivari-ate meta-analysis and systematic review. PloS one 2013; 8: e73099. doi: 10.1371/journal.pone.0073099 26. Matsumura Y, Ito Y, Iinuma Y, Yasuma K, Yamamoto M, Matsushima A, et al. 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Azoulay E, Bergeron A, Chevret S, Bele N, Schlemmer B, Menotti J. Polymerase chain reaction for diagnosing pneumocystis pneumonia in non-HIV immunocompromised patients with pulmonary infiltrates. Chest 2009; 135: 655-61. doi: 10.1378/chest.08-1309 31. Jarboui MA, Sellami A, Sellami H, Cheikhrouhou F, Makni F, Ben Arab N, et al. Molecular diagnosis of Pneumocystis jirovecii pneumonia in immu-nocompromised patients. Mycoses 2010; 53: 329-33. doi: 10.1111/j.1439-0507.2009.01715.x 32. Carmona EM, Limper AH. Update on the diagnosis and treatment of Pneumocystis pneumonia. Ther Adv Respir Dis 2011; 5: 41-59. doi: 10.1177/1753465810380102 33. Nevez G, Raccurt C, Jounieaux V, Dei-Cas E, Mazars E. Pneumocystosis versus pulmonary Pneumocystis carinii colonization in HIV-negative and HIV-positive patients. AIDS 1999; 13: 535-6. doi: 10.1097/00002030199903110-00020 34. Huang L, Crothers K, Morris A, Groner G, Fox M, Turner JR, et al. Pneumocystis colonization in HIV-infected patients. 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A prospective, blinded study of quantitative touch-down polymerase chain reaction using oralwash samples for diagnosis of Pneumocystis pneumonia in HIV-infected patients. j Infect Dis 2004; 189: 1679-83. doi: 10.1086/383322 42. Huggett JF, Taylor MS, Kocjan G, Evans HE, Morris-Jones S, Gant V, et al. Development and evaluation of a real-time PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveolar lavage fluid of HIV-infected patients. Thorax 2008; 63: 154-9. doi: 10.1136/thx.2007.081687 Radiol Oncol 2020; 54(2): 221-226. 227 research article Stereotactic body radiation therapy (SBRT) for the treatment of primary lung cancer in recipients of lung transplant Assaf Moore1'3, Mordechai R. Kramer23, Dror Rosengarten23, Osnat Shtraichman23, Alona Zer13, Elizabeth Dudnik13, Yasmin Korzets1, Aaron M. Allen13 1 Institute of Oncology, Davidoff Cancer Center, Rabin Medical Center, Petach Tiqva, Israel 2 The Institute of Pulmonary Medicine, Rabin Medical Center, Petach Tiqva, Israel 3 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Radiol Oncol 2020; 54(2): 227-232. Received 21 December 2019 Accepted 29 February 2020 Correspondence to: Aaron M. Allen, Radiotherapy Department, Davidoff Center, 39 Jabotinski St., Petach Tikvah, Israel. E-mail: ahron.alon@gmail.com Disclosure: No potential conflicts of interest were disclosed. Background. Lung transplantation is a life-saving treatment for patients with end stage lung disease. There may be a higher incidence of lung cancer in lung transplant recipients, and these cancers tend to be diagnosed at a more advanced stage. There is very little data on the safety and efficacy of stereotactic body radiation therapy (SBRT) for lesions in the native lung in lung-transplant recipients. Patients and methods. A retrospective chart review of all patients who have undergone lung transplantation and were treated with SBRT for lung cancer in the native lung in the Davidoff Cancer Center was performed. Results. Four patients who were treated with SBRT to a total of 5 lesions were included. Two patients were treated without histological confirmation of malignancy. All cases were discussed in a multidisciplinar/ tumor board before being referred for radiotherapy. Standard SBRT dosing was used. Responses were assessed by imaging. Three lesions exhibited a complete response and two lesions had a partial response. The patients who had partial responses developed distant metastases and died shortly. No patient developed measurable toxicity. Conclusions. SBRT is effective and safe for the management of lung cancer in lung-transplant patients. Standard dose and fractionation can be used. Key words: Stereotactic Body Radiation Therapy (SBRT); lung transplantation; lung cancer; radiotherapy Introduction Lung transplant is a life-saving last resort treatment for patients with end stage lung disease. The most common indications are chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF).1 This risk for lung cancer thus arises mainly from the native lung.1 There is a higher incidence of lung cancer in lung transplant recipients compared with the general population.2-6 This finding might be related to smoking, immunosuppression and/or underlying lung disease.45 Due to the improved life expectancy of transplanted patients and these predisposing factors, the incidence of lung cancer in lung transplant recipients is expected to increase.1 These cancers are often diagnosed in a more advanced stage and with a worse prognosis.34 For patients diagnosed with a potentially curable disease, curative treatment options include surgery and radiotherapy. We hereby present our experience with SBRT for the definitive treatment of four patients who underwent single lung transplantation and were diagnosed with lung cancer in the native lung. Radiol Oncol 2020; 54(2): 194-200. doi: 10.2478/raon-2020-0019 228 Moore A et al. / Lung SBRT in lung transplant recipients Patients and methods Patients The study includes all consecutive patients located in the registry of the Institute of Pulmonary Medicine who underwent lung transplantation and were treated with SBRT for lung cancer in the native lung at the Davidoff Cancer Center (DCC) at Rabin Medical Center (RMC) between June 2011 and June 2015. Data collection and outcomes This retrospective study was approved by the medical center's institutional Helsinki review board. No informed consent was required. Data were collected from medical records and included demographics, medical comorbidities, location and extent of disease, imaging findings, radiation treatment details, imaging and clinical follow-up, performance status, response to treatment, survival, and cause of death. Treatment planning Patients were immobilized for simulation using a customized vacuum cushion for CT simulation. Patients were simulated using a multiphase 4-di-mentional CT simulation to monitor breathing-related tumor motion. Images were reconstructed on the Advantage Workstation (GE Healthcare, Chicago, IL). An expansion in the cranio-caudal and axial dimensions for the internal target volumes (ITVs) was created based on tumor motion and location in 10 phases of breathing. The planning treatment volume (PTV) was defined as a 3-mm margin around the ITV. The PTV was reduced in case of proximity to vital normal tissue. Patients were treated with IMRT using dynamic sliding window multileaf collimator (MLC) or volumetric modulated arc therapy VMAT). Image guidance with cone beam CT preceded each fraction. Specification of the dose-volume histogram Spinal Cord Max dose < 18 Gy Esophagus Dose to 1cc < 27 Gy Heart Dose to 1cc < 30 Gy Gy = Gray; V20 = proportion of the lung receiving 20Gy (DVH) constraints is available in Table 1. Dose calculations were performed using the Eclipse™ treatment planning system (Varian, Palo Alto, CA), AAA algorithm version 8. Treatment was prescribed to the 95% isodose line with PTV tolerance of ± 5%. Quality assurance verification plans were performed with the ArcCHECK™ dosimeter (Sun Nuclear Corporation, Melbourne, FL). Before each treatment, image guided radiotherapy was used based on cone beam CT (CBCT) to position the patients. Post RT evaluation The treated tumors were assessed by CT or PET-CT eight weeks from completion of SBRT. Further imaging studies were scheduled at the treating physician's discretion. Results Four consecutive cases of lung transplant recipients who underwent SBRT for a lung lesion are included. All cases were discussed in a multidis-ciplinary team including pulmonologists, thoracic surgeons, radiation oncologists, medical oncologists and radiologists before being referred for radiotherapy. All patients were asymptomatic at diagnosis. Radiotherapy treatment parameters are summarized in Table 2. Case 1 A 72-year-old man with a history of heavy smoking, COPD, hypertension, diabetes mellitus type 2, chronic renal failure, peripheral vascular disease and fatty liver disease. The patient underwent left lung transplant in 2006 due to severe emphysema, and was treated with tacrolimus and azathioprine to prevent rejection. In November 2011, an 8 mm nodule was detected in the right lower lobe. The lesion increased in size to 26 mm in May 2015. A biopsy yielded moderately differentiated squamous cell carcinoma. A positron emission tomography-computed tomography (PET-CT) demonstrated high fluorodeoxyglucose (FDG) uptake in the nodule with no evidence of disease outside the lung. The patient was treated in June 2015 with a dose of 54 Gray (Gy) in three 18 Gy fractions. Treatment was well tolerated with no adverse events or measurable toxicity. Follow-up imaging demonstrated a complete response (CR). In February 2017, a new nodule appeared in the right lower lobe that in- TABLE 1. Dose-volume histogram constraints for organs at risk Organ Constraints Total Lung Dose (both lungs) V20 < 10% Radiol Oncol 2020; 54(2): 227-232. 229 Moore A et al. / Lung SBRT in lung transplant recipients TABLE 2. SBRT treatment parameters Case 1 1st course Case 1 2nd course Case 2 Case 3 Case 4 Prescribed dose (Gy), number of fractions 54 Gy, 3 54 Gy, 3 60 Gy, 5 54 Gy, 3 60 Gy, 5 MLD - both lungs (Gy) 3.3 3.6 4.4 2.5 6 V5 - both lungs, (%) 15.6 21.5 18.9 10.2 27.1 V20 - both lungs, (%) 4.9 3.2 5.7 2.9 8 MLD - transplanted lung (Gy) 4.5 0.8 2.2 0.6 1.8 V5 - transplanted lung, (%) 20.4 1.4 9.6 0.1 6.1 V20 - transplanted lung, (%) 7 0 2.3 0 0 Gy = Gray; MLD = mean lung dose, V5 = the % of a structure's volume that receives 5 Gy; V20 = the % of a structure's volume that receives 20 Gy creased in size on follow-up imaging. A PET-CT in April 2017 demonstrated high FDG uptake in the nodule with no evidence of disease outside the lung. The patient was treated in July 2017 to a dose of 54 Gy in three 18 Gy fractions. The post SBRT PET-CT demonstrated shrinkage of the treated nodule, however, clear metastases in the liver and bone. A liver biopsy yielded adenocarcinoma of lung origin. The patient succumbed to metastatic disease in January 2018. Case 2 A 76-year-old woman with a history of heavy smoking, COPD, diabetes mellitus type 2, chronic renal failure and atrial fibrillation. The patient underwent left lung transplant in 2002 due to severe emphysema, and was treated with tacrolimus and mycophenolic acid to prevent rejection. In June 2010, a central 0.5 mm nodule was detected in the right lower lobe. The lesion increased in size up to 13 mm in October 2011, and demonstrated high FDG uptake with no evidence of disease outside the lung. The patient was discussed in a multidisci-plinary tumor board and it was decided to treat the lesion with SBRT without histological confirmation. The patient was treated in February 2012 to a dose of 60 Gy in five 12 Gy fractions. Treatment was well tolerated with no adverse events or measurable toxicity. Follow-up imaging demonstrated a complete response (Figure 1). The patient passed away in May 2018 secondary to pneumonia and sepsis. Case 3 A 72-year-old man with a history of hypertension, diabetes mellitus type 2, chronic renal failure and ischemic heart disease. Underwent right lung transplant in 2002 due to IPF, and was treated with tacrolimus and mycophenolic acid to prevent rejection. In 2013 a nodule was detected in the left lower lobe and increased in size to 15 mm in October 2013. There was no FDG uptake in the nodule. The patient was discussed in a multidisciplinary tumor board and it was decided to treat the lesion FIGURE 1. (A) Pretreatment CT demonstrating a central nodule in the right lower lobe (B) Radiation field arrangement and dose color wash for SBRT (C) CT 2 months after treatment completion demonstrating a complete disappearance of the target nodule. Radiol Oncol 2020; 54(2): 227-232. 230 Moore A et al. / Lung SBRT in lung transplant recipients with SBRT without histological confirmation. The patient was treated in February 2012 to a dose of 54 Gy in three 18 Gy fractions. Treatment was well tolerated with no adverse events or measurable toxicity. The post SBRT, CT scans yielded a disappearance of the treated nodule (Figure 2). The patient passed away in October 2015 secondary to bacteremia and sepsis. Case 4 A 65-year-old man with a history of hypertension and diabetes mellitus type 2. Underwent left lung transplant in 2009 due to severe IPF, and was treated with tacrolimus and mycophenolic acid to prevent rejection. In 2011, a mass was detected in the anterior right mediastinum with involvement of the pleura and right upper lobe. A PET-CT demonstrated high FDG uptake in the mass that increased in size to 3.8 cm with no evidence of disease outside the lung. A biopsy yielded moderately differentiated small cell lung cancer (SCLC). The patient was discussed in a multidisciplinary tumor board, and determined to be a poor surgical candidate, was referred for SBRT and then planned for "adjuvant" chemotherapy. The patient was treated in June 2011 to a dose of 60 Gy in five 12 Gy fractions. Treatment was well tolerated with no adverse events or measurable toxicity. The post SBRT, CT scans yielded a partial response (PR) of the treated nodule (Figure 3), however, appearance of metastases in the liver and nodes above and below the diaphragm. The patient succumbed to a rapid progression of metastatic disease in August 2011. FIGURE 3. (A) Pretreatment CT demonstrating a mass in the anterior right mediastinum with involvement of the pleura and right upper lobe (B) Radiation field arrangement and dose color wash for SBRT (C) CT 2 months after treatment completion demonstrating regression of the target nodule. Radiol Oncol 2020; 54(2): 227-232. 231 Moore A et al. / Lung SBRT in lung transplant recipients Discussion Lung cancer in lung transplant recipients is a unique clinical scenario, in which patients suffer from at least two major life-threatening conditions. Treatment options for these patients may be limited due to the underlying condition, other comorbidi-ties and their immunosuppressed state. Treating these patients requires a multi-disciplinary effort. Characterizing this group of patients is beyond the scope of this report. We present a series of 4 consecutive patients who were treated with SBRT to a total of 5 lesions in the native lungs. One patient had proven squa-mous cell carcinoma and then adenocarcinoma, one had small cell carcinoma, and two were treated without histological confirmation of malignancy. Surgery remains the standard-of-care treatment for medically-operable early stage non-small cell lung cancer. Radiotherapy for the definitive treatment of early lung cancer was traditionally indicated for the medically-inoperable or those refusing surgery.7 Lung resection is challenging in patients with a major pulmonary disease, and especially when these patients have undergone lung transplantation and are immunosuppressed. Specific concerns include Impaired wound healing and anastomotic complications.8 Data on the safety of these procedures is scarce. In one series that included both malignant and benign/infectious causes for pulmonary resection of the native or allograft, of the 11 patients included, 3 patients died of post-procedural infectious complications, 2 died of acute respiratory distress syndrome and organ failure and 1 died as a result of bronchiolitis obliterans organizing pneumonia.8 Radiotherapy is an integral modality in the treatment of early lung cancer. SBRT has been studied extensively in medically inoperable patients, and may in fact achieve better control than standard ra-diotherapy.7910 SBRT has also been proven effective for medically operable patients, even compared with surgery.1112 While it is unknown whether radiotherapy and SBRT specifically can be safely and effectively used in lung transplant recipients, toxicity in clinical trials has been low with no treatment-related mortality.7910 Data on the interaction between immunosuppression and advanced onco-logic treatments is scarce. This applies to systemic therapy such as tyrosine kinase inhibitors, but even more concerning - with immunotherapeutic agents facing a risk for organ rejection.13 The immune system's role in mediating response to radiotherapy is being studied, however the exact implica- tions of immunosuppression during radiotherapy are poorly understood.14 In this series, all lesions achieved excellent response by imaging - 3 lesions exhibited a complete response and two lesions partial responses. The patients who had partial responses developed distant metastases and died shortly, thus; the maximal response may not have been achieved during the short follow-up. None of the lesions who exhibited a complete response recurred locally. Based on this small series, even in immunosuppressed patients, SBRT to lung lesions is effective at achieving local control. There have been several reports of SBRT in the treatment of patients with lung cancer who had previously undergone solid organ transplant. In a series of 15 patients (9 of which underwent lung transplant), no patients experienced grade 3 or 4 toxicity, however, one patient with a history of single lung transplant died of radiation pneumonitis 7 months after SBRT to a lesion within the transplanted lung. The cumulative incidence of local failure was 7% and 13% at 1 and 2 year and the incidence of distant failure was 40% at 1 year.15 In another series, two patients were treated with SBRT-range dosing sequentially with chemotherapy. One achieved a durable complete response, while another progressed shortly.4 One case ineligible for surgical management of stage IB adenocarcinoma arising from a donor lung post-double lung transplantation, was safely and effectively treated to a dose of 60 Gy in 8 fractions.16 The doses and organs at risk constraints we have used did not differ from our institutional policy towards non-transplanted patients and seemed to be effective and safe. However, whether an optimal dose and fractionation exists or whether tighter constraints are required, has not been determined and no clear recommendation can be made. While there were no cases of grade 2 or higher pneumonitis in our patients, organ transplanted patients, and specifically lung transplanted patients could prove a diagnostic challenge for this type of toxicity, as the differential diagnosis for clinical deterioration is broad and might not be distinguishable by imaging. The main limitations of this study relate to its retrospective design and lack of a control arm, which are generally associated with methodological biases and difficulties in results interpretation. The most concerning bias in our study is clearly associated with patient selection, as patients who have undergone transplantation usually have other comorbidities and their cancers may have a more aggressive course. Radiol Oncol 2020; 54(2): 227-232. 232 Moore A et al. / Lung SBRT in lung transplant recipients It is clear that SBRT is a safe and efficient modality for the treatment of recipients of lung transplant and could offer long-term control and potentially a definitive solution. There are no randomized comparative reports comparing it with invasive procedures. Physicians must consider the potential early and late toxicities of thoracic radiation, including pneumonitis, esophagitis and vascular toxicity. These may be avoided by minimizing volumes and strictly maintaining dose constraints. SBRT could serve to achieve these objectives. Conclusions SBRT for the management of lung cancer in the native lung in lung-transplanted patients is effective and safe and can offer long-term control. Standard dose and fractionation can be used. It should be considered in clinical situations where surgical procedures are not feasible. These results should encourage clinicians to investigate its role further, potentially in place of invasive procedures. 10. Ball D, Mai GT, Vinod S, Babington S4, Ruben J5, Kron T, et al. Stereotactic ablative radiotherapy versus standard radiotherapy in stage 1 non-small-cell lung cancer (TROG 09.02 CHISEL): a phase 3, open-label, randomised controlled trial. Lancet Oncol 2019; 20: 494-503. doi: 10.1016/S1470-2045(18)30896-9 11. Chang JY, Senan S, Paul MA, Mehran RJ, Louie AV, Balter P, et al. Stereotactic ablative radiotherapy versus lobectomy for operable stage I non-small-cell lung cancer: a pooled analysis of two randomised trials. Lancet Oncol 2015; 16: 630-7. doi: 10.1016/S1470-2045(15)70168-3 12. Zheng X, Schipper M, Kidwell K, Lin J, Reddy R, Ren Y, et al. Survival outcome after stereotactic body radiation therapy and surgery for stage I non-small cell lung cancer: a meta-analysis. Int J Radiat Oncol Biol Phys 2014; 90: 60311. doi: 10.1016/j.ijrobp.2014.05.055 13. Ros J, Matos I, Martin-Liberal J. Immunotherapy in organ-transplanted cancer patients: efficacy and risk of organ rejection. Ann Oncol 2019; 30: 1173-7. doi: 10.1093/annonc/mdz129 14. Walle T, Martinez Monge R, Cerwenka A, Ajona D, Melero I, Lecanda F. Radiation effects on antitumor immune responses: current perspectives and challenges. Ther Adv Med Oncol 2018; 10: 1758834017742575. doi: 10.1177/1758834017742575 15. Fleming CW, Stephans KL, Broughman JR, Rybicki L, Budev M, Ahmad U, et al. Outcomes after SBRT for inoperable early stage lung cancers arising in organ transplant patients. Int J Radiat Oncol Biol Phys 2019; 105: E486. doi: 10.1016/j.ijrobp.2019.06.1382 16. Chen H, Tikkanen J, Boldt RG, Louie A V. Stereotactic ablative radiotherapy for early-stage lung cancer following double lung transplantation. Radiat Oncol 2018; 13: 142. doi: 10.1186/s13014-018-1089-8 References 1. Mathew J, Kratzke RA. Lung cancer and lung transplantation: a review. J Thorac Oncol 2009; 4: 753-60. doi: 10.1097/JTO.0b013e31819afdd9 2. Choi YH, Leung AN, Miro S, Poirier C, Hunt S, Theodore J. Primary bronchogenic carcinoma after heart or lung transplantation: radiologic and clinical findings. J Thorac Imaging 2000; 15: 36-40. doi: 10.1097/00005382200001000-00008 3. Raviv Y, Shitrit D, Amital A, Fox B, Rosengarten D, Fruchter O, et al. Lung cancer in lung transplant recipients: experience of a tertiary hospital and literature review. Lung Cancer 2011; 74: 280-3. doi: 10.1016/j.lung-can.2011.02.012 4. Du L, Pennell NA, Elson P, Hashemi-Sadraei N. Lung cancer treatment outcomes in recipients of lung transplant. Transl Lung Cancer Res 2015; 4: 784-91. doi: 10.3978/j.issn.2218-6751.2015.12.08 5. Collins J, Kazerooni EA, Lacomis J, McAdams HP, Leung AN, Shiau M, et al. Bronchogenic carcinoma after lung transplantation: frequency, clinical characteristics, and imaging findings. Radiology 2002; 224: 131-8. doi: 10.1148/ radiol.2241011189 6. Dickson RP, Davis RD, Rea JB, Palmer SM. High frequency of bronchogenic carcinoma after single-lung transplantation. J Heart Lung Transplant 2006; 25: 1297-301. doi: 10.1016/j.healun.2006.09.009 7. Timmerman RD, Hu C, Michalski J, Straube W, Galvin J, Johnstone D, et al. Long-term results of RTOG 0236: a phase II trial of stereotactic body radiation therapy (SBRT) in the treatment of patients with medically inoperable stage I non-small cell lung cancer. Int J Radiat Oncol Biol Phys 2014; 90: S30. doi: 10.1016/j.ijrobp.2014.05.135 8. Fitton TP, Bethea BT, Borja MC, Yuh DD, Yang SC, Orens JB, et al. Pulmonary resection following lung transplantation. Ann Thorac Surg 2003; 76: 1680-6. doi: 10.1016/s0003-4975(03)00975-5 9. Nyman J, Hallqvist A, Lund J-A, Brustugun OT, Bergman B, Bergstrom P, et al. SPACE - A randomized study of SBRT vs conventional fractionated radiotherapy in medically inoperable stage I NSCLC. Radiother Oncol 2016; 121: 1-8. doi: 10.1016/j.radonc.2016.08.015 Radiol Oncol 2020; 54(2): 227-232. 233 research article Sorafenib for the treatment of hepatocellular carcinoma: a single-centre real-world study Jurij Hanzel1, Tajda Kosir Bozic1, Borut Stabuc12, Rado Jansa12 1 Department of Gastroenterology, University Medical Centre Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2020; 54(2): 233-236. Received 18 November 2019 Accepted 1 December 2019 Correspondence to: Rado Janša, Ph.D., M.D., Department of Gastroenterology, University Medical Centre Ljubljana, Japljeva ulica 2, SI-1000 Ljubljana, Slovenia. E-mail: rado.jansa@kclj.si Disclosure: No potential conflicts of interest were disclosed. Background. Sorafenib is an oral multi-kinase inhibitor used for the treatment of hepatocellular carcinoma. Its efficacy in randomised controlled trials was demonstrated in patients with well-preserved liver function and good functional status. In the real-world setting, treatment is often offered to patients outside these criteria. We therefore performed a single-centre real-world cohort study on the efficacy of sorafenib in patients with hepatocellular carcinoma. Patients and methods. We identified all patients with hepatocellular carcinoma initiating treatment with sorafenib between January 2015 and January 2018. The primary endpoint was overall survival (OS) since starting sorafenib. Clinical and demographic variables associated with survival were studied. Results. The median OS was 13.4 months (95% CI 8.2-18.6). Multivariable Cox's regression identified worse ECOG performance status (HR 2.21; 95% CI 1.56-3.16; P < 0.0001), Child-Pugh class C (HR 52.4; 95% CI 3.20-859; P = 0.005) and absence of prior locoregional treatment (HR 2.30; 95% CI 1.37-3.86; P = 0.002) to be associated with increased mortality. Conclusions. Careful selection of patients for treatment with sorafenib is of paramount importance to optimize outcomes. Key words: survival; multivariable analysis; real-world cohort study Introduction Sorafenib is an oral multi-kinase inhibitor, which inhibits tumours angiogenesis through inhibition of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) signalling pathways. It has demonstrated a significant prolongation in overall survival of patients with advanced-stage hepatocellular carcinoma (HCC) up to 2.8 months.12 The two landmark trials included mainly patients with compensated liver cirrhosis of viral aetiology and an excellent baseline functional status. Consequently, sorafenib is formally indicated only in patients with well-preserved liver function (Child-Pugh A) and advanced tumours (Barcelona Clinic Liver Cancer [BCLC] C) or intermediate stage tumours (BCLC B) progressing after locoregional therapy.3 Nevertheless, sorafenib is often used outside these criteria in the real-world setting, mainly due to the absence of alternative treatment options. As these patient subgroups were not studied in regis-trational trials, only large observational non-randomized cohort studies can help inform practice.4-6 We therefore a retrospective real-world cohort study of patients treated with sorafenib for advanced HCC, investigating its efficacy and variables associated with OS. Patients and methods Patients and study design We performed a retrospective cohort study of all patients with HCC initiating treatment with sorafenib between January 2015 and January 2018, Radiol Oncol 2020; 54(2): 194-200. doi: 10.2478/raon-2020-0019 234 Hanzel J et al. / Sorafenib in HCC - single-centre real-world experience who were followed until October 2018 at a single tertiary centre. Data collection was approved by the institutional ethics committee, while treatment did not differ from the standard of care and thus did not require additional approval. We included all consecutive patients aged at least 18 years with a histologically or radiologically confirmed diagnosis of HCC, who were treated with sorafenib. The decision for initiation of the drug was made based on the consensus of the Liver Multidisciplinary Team. Patient records were retrospectively reviewed for demographic and clinical information. The date of death was extracted from the national health insurance database. The primary outcome was OS from initiation of sorafenib. We explored the relationship of clinical characteristics with OS. Statistical analysis Continuous variables are given as medians with interquartile ranges (IQR). Univariable association analyses with survival were performed using the Kaplan-Meier method with log-rank testing. Multivariable analysis was performed using a Cox-proportional hazards model with stepwise backward selection where variables were removed if they did not achieve statistical significance at P < 0.05. All analyses were performed on an intention-to-treat basis. Analyses were performed using SPSS, Version 25 (IBM, Chicago, USA). Results Patient characteristics We included 115 patients, who were predominantly male with Child-Pugh class A alcoholic cirrhosis with good performance status (Table 1). Survival outcomes A total of 83 patients (72%) died during the study period. The median OS since initiation of sorafenib was 13.4 months (95% CI 8.2-18.6). In univariable analysis, reduced OS was associated with worse ECOG performance status (P < 0.0001), higher Child-Pugh class (P < 0.0001), higher baseline AFP (P = 0.003) and absence of prior locoregional treatment (P < 0.0001) (Table 2). The associations of liver disease aetiology (P = 0.192), BCLC stage (P = 0.539), gender (P = 0.944) and age at treatment initiation (P = 0.201) with OS were not statistically significant. TABLE 1. Patient characteristics at initiation of sorafenib (n = 115) Variable Male gender, n (%) 96 (84) Age, years, median (IQR) 67 (60-72) ECOG performance status 0 31 (27) 1 47 (40.9) 2 36 (31.3) 3 1 (0.9) Aetiology of underlying liver disease, n (%) Alcoholic liver disease 56 (49) Hepatitis B 11 (9.6) Hepatitis C 7 (6.1) Non-alcoholic steatohepatitis 18 (15.7) Cryptogenic 10 (8.7) Wilson's disease 1 (0.9) Primary biliary cholangitis 1 (0.9) HCC in non-cirrhotic liver 11 (9.6) Child-Pugh class, n (%) A 77 (66.9) B 37 (32.2) C 1 (0.9) BCLC stage, n (%) A 3 (2.6) B 42 (36.5) C 70 (60.9) Prior treatment, n (%) 45 (39.1) Resection 10 (8.7) RFA 2 (1.8) Transplant 3 (2.6) TACE 29 (25.2) Radioembolization 5 (4.3) AFP, kU/L, median (IQR) 6-1518) AFP = alpha-fetoprotein; BCLC = Barcelona Clinic Liver Cancer; ECOG = Eastern Cooperative Oncology Group; HCC = hepatocellular carcinoma; IQR = interquartile range; RFA = radiofrequency ablation; TACE = trans-arterial chemoembolization Multivariable analysis demonstrated significant associations between mortality and ECOG performance status (HR 2.21; 95% CI 1.56-3.16; P < 0.0001), Child-Pugh class C (HR 52.4; 95% CI 3.20859; P = 0.005) and absence of prior locoregional treatment (HR 2.30; 95% CI 1.37-3.86; P = 0.002), but not baseline AFP (HR 1.00; 95% CI 0.8-1.2; P = 0.278) (Table 2, Figure 1). Discussion HCC is among the leading causes of cancer-related deaths. It primarily develops from cirrhosis, and many patients are infected with hepatitis C virus (HCV) or hepatitis B virus (HBV). Treatment with the multikinase inhibitor sorafenib is a systemic therapy option for patients with advanced HCC since 2008. Radiol Oncol 2020; 54(2): 233-236. 235 Hanzel J et al. / Sorafenib in HCC - single-centre real-world experience FIGURE 1. Kaplan-Meier plots of overall survival (OS) after initiating sorafenib stratified by (A) ECOG performance status, (B) Child-Pugh class, and (C) prior locoregional treatment. Systemic therapy has helped prolong survival after disease progression. Clinical management of patients should target improvement of patient OS. Sorafenib therapy is recommended in guidelines as the first-line option in patients who cannot benefit from resection, transplantation, ablation or TACE, and still have preserved liver function and significantly prolonged OS and TTP. Sorafenib monotherapy remains the standard of care in unresectable HCC. Sorafenib has demonstrated survival benefit in patients with unre-sectable HCC in two (2) randomized, placebo-con- TABLE 2. Factors associated with overall survival (OS) after initiation of sorafenib Univariable analysis Multivariable analysis Median survival in . , „ . Hazard ratio (95% Cox's regression P ,, ,,._<- Log rank P value v , months (95% CI) a CI) value ECOG performance status 0 1 2 3 25.1 (12.8-37.4) 17.0 (7.0-26.9) 5.5 (3.7-7.3) 7.3 (/) <0.0001 2.21 (1.56-3.16) <0.0001 Child-Pugh class A B C 16.9 (12.8-21.0) 6.7 (4.7-8.7) 1.0 (/) <0.0001 1.00 1.34 (0.80-2.26) 52.4 (3.20-859) 0.271 0.005 Baseline AFP < 200 > 200 17.0 (9.3-24.6) 6.7 (5.6-7.8) 0.003 1.00 (0.8-1.2) 0.278 Prior locoregional treatment Yes No 24.0 (20.1-27.9) 7.3 (5.0-9.5) <0.0001 1.00 2.30 (1.37-3.86) 0.002 Liver disease aetiology Alcoholic Other 8.6 (3.80-13.3) 16.7 (13.5-19.9) 0.192 BCLC stage A B C 22.4 (7.0-37.7) 14.5 (5.1-23.9) 13.4 (7.1-19.7) 0.539 Gender Female Male 8.8 (7.0-10.7) 13.8 (10.1-17.6) 0.944 Age < 70 years > 70 years 15.0 (11.2-18.8) 8.4 (6.0-10.8) 0.201 AFP = alpha-fetoprotein; BCLC = Barcelona Clinic Liver Cancer; ECOG = Eastern Cooperative Oncology Group; IQR = interquartile range Radiol Oncol 2020; 54(2): 233-236. 236 Hanzel J et al. / Sorafenib in HCC - single-centre real-world experience trolled, double-blind, phase III trials: SHARP and AP. The use of sorafenib significantly increased OS: 10.7 months vs. 7.9 months (SHARP study) and radiologic progression was significantly lower in the sorafenib group of patients.8 The use of Sorafenib also significantly increased OS in Asian-Pacific study. However, the results compared with SHARP study were worst especially because of different demographic characteristics of patients, more extrahepatic spread, greater number of hepatic tumor lesions and poorer ECOG performance status. In GIDEON, real life analysis of the sorafenib group of patients median OS was 8.6 month vs. 10.4 in SHARP study. Clinical outcomes of advanced HCC patients treated with sorafenib in real-life practice are better compared to the other studies conducted in the Asia-Pacific region in terms of survival and tolerability. Extrahepatic spread and combination with other therapies are of predictive value for OS of advanced HCC. Further studies are required to maximize the effect of sorafenib in combination with other modalities.78 In our retrospective study, we collected and analyzed the clinical outcomes of advanced HCC patients who underwent treatment with sorafenib in real-life clinical setting. We found that HCC patients with Child-Pugh A exhibited a significantly higher median survival. In the present study, factors that are predictive of OS in HCC patient treated with sorafenib include gender, extrahepatic spread, and combined other therapies.78 In the Slovenian study, HCC patients treated with sorafenib had median OS of 13.4 months, which is longer than that reported in SHARP (10.5 months) and GIDEON (Global Investigation of Therapeutic Decisions in HCC and of its treatment with sorafenib) (10.8 months). Multivariable analysis of the Slovenian group of patients demonstrated significant associations between mortality and ECOG performance status, Child-Pugh class C and absence of prior locore-gional treatment, but not baseline AFP. There are several limitations in this retrospective designed analysis. Being a retrospective study, it is difficult to ascertain the actual cause of death in our cohort. The population size examined in our study is relatively small, which may limit the statistical power. Small population size may have influences on subgroup analysis. Other limitations include the reduced initial dose of sorafenib based on clinical decision made by individual physicians and adjustment of dosages during treatment due to intolerance. However, our results are comparable with results of other worldwide studies. In conclusions, careful selection of patients for sorafenib treatment is important. Treatment of HCC patients should be performed in experienced centers, where the decision of treatment of each patients should be made after previous presentation of patients at multidisciplinary board of experts. References 1 Llovet JM, Hilgard P, de Oliveira AC, Hilgard P, Gane E, Blanc JF, et al. Sorafenib in Advanced Hepatocellular Carcinoma. N Engl J Med 2008; 13: 378-90. doi: 10.1056/NEJMoa0708857 2 Cheng AL, Kang YK, Chen Z, Tsao CJ, Qin S, Kim JS, et al. Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial. Lancet Oncol 2009; 10: 25-34. doi: 10.1016/S1470-2045(08)70285-7 3 Vogel A, Cervantes A, Chau I, Daniele B, Llovet JM, Meyer T, et al. Hepatocellular carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2018; 29(Suppl 4): iv238-55. doi: 10.1093/annonc/mdy308 4 Marrero JA, Kudo M, Venook AP, Ye SL, Bronowicki JP, Chen XP, et al. Observational registry of sorafenib use in clinical practice across Child-Pugh subgroups: The GIDEON study. J Hepatol 2016; 65: 1140-7. doi: 10.1016/j. jhep.2016.07.020 5 Lee S, Kim BK, Kim SU, Park SY, Kim JK, Lee HW, et al. Clinical outcomes and prognostic factors of patients with advanced hepatocellular carcinoma treated with sorafenib as first-line therapy: a Korean multicenter study: Sorafenib as a first-line therapy. J Gastroenterol Hepatol 2014; 29: 1463-9. doi: 10.1111/jgh.12542 6 Doyle A, Marsh P, Gill R, Rodov M, Mohsen W, Varma P, et al. Sorafenib in the treatment of hepatocellular carcinoma: a multi-centre real-world study. Scand J Gastroenterol 2016; 51: 979-85. doi: 10.3109/00365521.2016.1166518 7 Llovet LM, Bruix J. Management of HCC. Hepathology 2008; 48: 1312-27. doi: 10.1002/hep.22506. 8 Forner A, Llovet LM, Bruix J. Hepatocellular carcinoma. Lancet 2012; 379: 1245-55. doi: 10.1016/S0140-6736(11)61347-0 Radiol Oncol 2020; 54(2): 233-236. 237 research article Sarcopenia and myosteatosis at presentation adversely affect survival after esophagectomy for esophageal cancer Matevz Srpcic12, Taja Jordan3, Karteek Popuri4, Mihael Sok12 1 Department of thoracic surgery, Surgical clinic, University Medical Centre Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Slovenia 3 Institute of radiology, University Medical Centre Ljubljana, Slovenia 4 Simon Fraser University, Burnaby, Canada Radiol Oncol 2020; 54(2): 237-246. Received 7 January 2020 Accepted 3 March 2020 Correspondence to: Matevž Srpčič, M.D., Department of Thoracic Surgery, Surgical Clinic, University Medical Centre Ljubljana, Zaloška 7, SI-1000 Ljubljana, Slovenia. Phone: +386 1 522 3813; fax +386 1 522 2485; E-mail: matevz.srpcic@kclj.si Disclosure: MSr, TJ and MSo declare that they have no competing interests. KP is a co-founder of and actively directs Voronoi Health Analytics Incorporated, a Canadian corporation that sells commercial licenses for the ABACS (Automated Body Composition Analyzer using Computed tomography image Segmentation) software. Background. Esophageal cancer remains a disease with poor survival and many complications. Measuring muscle mass and quality can identify patients with diminished muscle mass (sarcopenia) and muscle fat infiltration (myosteatosis). We studied the impact of sarcopenia and myosteatosis in resectable esophageal cancer on overall survival and complications. Patients and methods. 139 patients received a radical esophagectomy. Skeletal muscle area (SMA) and muscle attenuation (MA) in CT images at L3 level were recorded and groups with and without sarcopenia and myosteatosis were compared for overall survival (OS), perioperative mortality, conduit complications, pleuropulmonary complications, respiratory failure requiring mechanical ventilation and other significant complications. Results. Prevalence of sarcopenia and myosteatosis at presentation was 16.5% and 51.8%, respectively. Both were associated with decreased OS. Median survival was 18.3 months (CI 5.4-31.1) vs. 31.0 months (CI 7.4-54.6) for sarco-penia/no sarcopenia (log rank p = 0.042) and 19.0 months (CI 13.3-24.7) vs. 57.1 months (CI 15.2-99.0) for myosteatosis (log rank p = 0.044), respectively. A relationship between sarcopenia and myosteatosis and other negative outcomes after esophagectomy could not be established. Conclusions. Sarcopenia and myosteatosis before esophagectomy are associated with decreased overall survival but not with more frequent perioperative complications. Identification of patients at risk can guide therapeutic decisions and interventions aimed at replenishing muscle reserves. Key words: sarcopenia; myosteatosis; esophagectomy; survival; esophageal cancer; muscle depletion Introduction Constant gradual improvements of operative techniques and perioperative care have reduced the dangers of esophagectomy, the cornerstone of radical treatment of resectable esophageal cancer, but it remains a major procedure burdened with high morbidity and mortality.1 Overall 5-year survival in resectable esophageal cancer has improved in recent years by about 2-3 fold.2 This improvement was attributed to centralization of surgical treatment and introduction of neoadjuvant chemoradi-otherapy.3 Advances were also made in perioperative care and better understanding and prevention of the detrimental effects of muscle depletion so typical of esophageal malignancies.4 Further improvement in outcomes can be achieved by tailoring the treatment to patients' ability to withstand the trauma of surgery and to return to a functional life after treatment. Adequate Radiol Oncol 2020; 54(2): 194-200. doi: 10.2478/raon-2020-0019 238 Srpcic M et al. / Sarcopenia and myosteatosis impair survival after esophagectomy fitness for treatment has traditionally been assessed from various performance scores, risk scores as well as more basic patients' characteristics like age and body mass index.5 Body mass index (BMI) at presentation has proven to be an inaccurate predictor of outcomes since it does not correspond to body composition well.67 Better methods to assess the most important parameter of body composition, the skeletal muscle content, have been introduced. They include functional tests like muscle strength measurements and measurements of muscle mass with dual energy x-ray imaging (DEXA), bioim-pedance analysis or cross-sectional imaging (CT or MRI).8 Cross-sectional imaging (or planimetry) has the advantage of being readily available in cancer patients for staging purposes. This has encouraged many studies to examine the relationship between overall muscle mass, its quality and their effect on outcomes. A reliable relationship between planimetrically determined muscle mass and quality and its function, determined by other methods available, has been established. Muscle area at the level of 3rd lumbal vertebra, normalized for height (skeletal muscle index (SMI)) is highly correlated with total body skeletal muscle mass.9 Estimating survival chances for a patient presenting with resectable esophageal cancer is important in planning appropriate treatment strategies and interventions aimed at improving survival and quality of life. Pronounced weight loss is a hallmark of malignant disease, especially pronounced in digestive tract tumors, among them in esophageal and pancreatic cancers in particular.10 In their seminal work, the team from University of Alberta have shown that skeletal muscle depletion (sarcopenia and low muscle attenuation) is the real negative predictor of survival regardless of overall body weight in cancer patients.6 Sarcopenia is defined by the European Working Group on Sarcopenia in Older people as the presence of low muscle mass (under the 5th percen-tile) and low muscle function (strength or per-formance)11 typically presenting in advanced age but also in cancer and other diseases. It is a well established predictor of poor survival and treatment outcomes in cancer patients.6 Myosteatosis is defined as abnormal fat infiltration in skeletal muscle. It is negatively associated with muscle strength and quality and is brought on by aging12, diabetes13, obesity14 and malignant disease.61516 Radiodensity of human muscle on CT scan (or muscle attenuation, MA) correlates well with its triglyceride con-tent.14 Measuring the attenuation values of muscle tissue corresponds well to the extent of myosteato- sis, which is a sign of muscle wasting and again a predictor of poor outcome.17 By assessing muscle mass and quality before treatment an individualized risk assessment for overall survival and complications during treatment can be improved, patients at risk identified and appropriate interventions (mainly directed towards maintaining and gaining muscle mass) undertaken.18 Our aim was to study the impact of muscle depletion (sarcopenia and myosteatosis) on outcomes (overall survival [OS], perioperative mortality and rate of complications) in resectable esophageal cancer. Patients and methods Study population All patients who received an esophagectomy with curative intent for esophageal or esophago-gastric junction cancer at Clinical Department of Thoracic Surgery at University Medical Centre Ljubljana were eligible for inclusion in the study. Patients received either upfront surgery or neoadjuvant chemoradiotherapy followed by esophagectomy according to national guidelines. All patients received individualized nutritional support and counselling according to ESPEN best practice guidelines19 and in all patients a catheter feeding jejunostomy was placed during esophagectomy. Clinical parameters were recorded prospectively in a database since 2003. Out of the 162 patients operated on consecutively between 2008 and 2018 CT images suitable for analysis of muscle mass and quality were available for 139 patients which were included in the study. Requirements for adequate images were the inclusion of L3 level and availability of non-contrast images for attenuation analysis. Only images recorded at presentation before the initiation of any treatment were considered. Our study design was approved and the need for obtaining informed consent from participants waived by the Slovenian National medical ethics committee (approval number 0120-301/2016-2). Definitions We grouped complications into following groups. Conduit complications included clinically silent fis-tulae seen on esophagograms and/or CT scans, clinically important leaks that required interventions and frank gastric necroses. Respiratory complications included respiratory failure requiring mechanical ventilation and pneumonia, defined as the Radiol Oncol 2020; 54(2): 237-246. 239 Srpcic M et al. / Sarcopenia and myosteatosis impair survival after esophagectomy presence of new infiltrates on chest radiography and a positive culture result from bronchoalveolar lavage or sputum requiring antibiotics. Respiratory failure requiring mechanical ventilation was recorded separately as well. Other complications were defined as other serious complications (Dindo Clavien 2 or greater)20 requiring intervention (i.e. early reoperation, cardioversion, endoscopic intervention) or directoscopi-cally proven laryngeal nerve paralysis.21 OS was defined as the time interval between esophagectomy and death of any cause. Patients alive on 1.10.2018 as reported by Cancer registry of Slovenia were censored at that date. BMI was calculated as patient weight [kg]/ height [m]2, recorded at admission one day before surgery. CT body composition analysis (planimetry) Pre-operative abdominal CT or whole body PET-CT scans were obtained. In each patient a single slice at the level of the 3rd lumbar vertebra (L3) was selected for automatic segmentation. CT scans were analyzed using the "Automated Body Composition Analyzer using Computed tomography image Segmentation" (ABACS) software2223, which uses a priori information about the skeletal muscle shape in the L3 region and predefined Hounsfield units (HU) values to recognize different tissues. HU values used to assess the total cross-sectional area for muscular tissue (SMA - skeletal muscle area) were -29 to +150 HU. Muscle attenuation (MA) was assessed by averaging HU of skeletal muscle. Additionally, SMI was calculated using the following formula: (SMA [cm2])/(patient height [m2]). All abdominal CT and PET-CT scans were analyzed by one blinded independent radiologist. The following planimetry data were reported: number of days between CT and esophagectomy, SMA (skeletal muscle area) reported in cm2, SMI (skeletal muscle index) is SMA corrected for height (i.e. divided by height squared) and expressed in cm2/m2. MA (muscle attenuation) was reported in Hounsfield units. Previously defined muscle index cut-off values for sarcopenia in a healthy non-elderly Caucasian population were used to define limits for SMI in men at less than 43.1 cm2/m2and less than 32.7 cm2/ m2 in women. Cutoff values for myosteatosis from the same study were used with myosteatosis defined as MA of less than 30.9 HU in men and 24.8 HU in women.24 Outcomes and statistical analysis Standard descriptive statistics of demographic and clinical characteristics for patients with and without sarcopenia and myosteatosis were summarized. Differences in demographic and clinical characteristics between groups (sarcopenia/no sar-copenia and myosteatosis/no myosteatosis) were evaluated with Pearson's Chi-square tests for categorical and t-tests for parametric variables. Primary outcome studied was overall survival. It was reported in each group with the KaplanMeier curve and the survival of groups with/without sarcopenia and with/without myosteatosis was compared using the log rank Mantel Cox test. Secondary outcomes of interest were the incidences of complications in groups with/without sar-copenia and with/without myosteatosis. They were compared with Pearson's Chi-square test. P value of < 0.05 was considered significant. All statistical analyses were performed using Statistical Package for the Social Sciences (SPSS, version 22.0, Armonk NY). Results Patient characterictics One hundred and thirty-nine patients underwent esophagectomy with primary reconstruction with curative intent. Overall demographic, clinical and complication characterictics are summarized in Table 1. Mean BMI was 26.3 ± 4.8 with only 7 (5.0%) having a BMI less than 18.5. As many as 46 (33.1%) patients reported having lost 10% or more of their normal body weight prior to esophagectomy. Average time between CT and esophagectomy was 76.9 ± 52.3 days with a much shorter time in those receiving primary resection compared to those with neoadjuvant treatment. Sarcopenia was present in 23 (16.5%) patients and myosteatosis in 72 (51.8%). Surgery and pathology Eighty-seven (62.6%) patients received an open esophagectomy and 52 (37.4%) had a hybrid or completely minimally invasive procedure. Type of procedure data, radicality rates, numbers of lymph nodes harvested and histology and staging data are given in Table 1. Complications and survival 9 patients died after esophagectomy during the initial hospitalization (in hospital mortality of 6.5%). Radiol Oncol 2020; 54(2): 237-246. 240 Srpcic M et al. / Sarcopenia and myosteatosis impair survival after esophagectomy TABLE 1. Demographic, preoperative, procedure and outcome data in all patients (N = 139) Demographic and preoperative data Age at Surgery (mean ± SD) [years] 63.9 ± 9.5 min-max 30-83 Gender (N, % female) 22 (15.8%] BMI (mean ± SD] [kg/ m2] 26.3 ± 4.8 Weight loss > 10% (N, %] 46 (33.1%) Neoadjuvant therapy (N, %] 74 (53.2 %] Planimetry data Days between CT and esophagectomy all (mean ± SD] min-max median Neoadjuvant (mean ± SD] min-max median No neoadjuvant (mean ± SD] min-max median SMA [cm2] (mean ± SD] male female SMI [cm2/m2] (mean ± SD] male female Muscle attenuation [HU] (mean ± SD] male female sarcopenia (N, %] myosteatosis (N, %] 76.9 ± 52.3 6-192 84 115.2 ± 36.0 14-192 125 33.5 ± 28.5 6-141 23 157.6 ± 28.0 103.9 ± 16.3 52.1 ± 9.5 39.8 ± 6.8 31.2 ± 8.3 27.8 ± 8.7 23 [16.5%] 72 [51.8%] Almost half or 65 patients (46.8%) experienced a complication of Dindo-Clavien grade 2 severity or greater20 after the procedure. Rates of other complications and survival rates are shown in Table 1. Survival is shown as a Kaplan-Meier curve in Figure 1. Median follow up was 18.1 months (range 0-115). 72 patients (51.8%) died during the observation period and 67 (48.1%) were censored. Sarcopenia and myosteatosis subgroups Demographic and clinical data was compared between patients with and without sarcopenia and with and without myosteatosis (Table 2). Patients with myosteatosis were significantly older than patients without it whereas in patients with or with- Procedure data Surgical approach (N, %) open 87 [62.6%] MIE 52 [37.4%] Type of esophagectomy (N, %) Ivor-Lewis 109 [78.4%] McKeown 26 [18.7%] Transhiatal 4 [2.9%] Radicality (N, %) R0 130 [93.5%] R1 5 [3.6%] R2 4 [2.9%] Lymph nodes (mean ± SD) (N, %) 23.4 ± 12.3 min-max 0-76 median 21 Cancer type (N, %) Adenocarcinoma 74 [53.2%] Squamous cell carcinoma 64 [46.0%] GIST 1 [0.7%] Pathological Stage [AJCC 2017] [N, %] 51 [36.7%] II 27 [19.4%] III 36 [25.9%] IVA 23 [16.5%] IVB 2 [1.4%] Complications (N, %) In hospital mortality 9 [6.5%] Any complication 65 [46.8%] Conduit complications 21 [15.1%] Pleuropulmonary complications 37 [26.6%] Respiratory failure 26 [18.7%] Any other complications 42 [30.2%] Median survival [months] 24.8 (95% CI 8.1-45,7) 1 year survival 73.7% 3 year survival 45.1% 5 year survival 40.3% AJCC = American joint committee on cancer; BMI = body mass index; CI = confidence interval; CT = computed tomography; GIST = gastrointestinal stromal tumor; HU = Hounsfield units; MIE = minimally invasive esophagectomy; SD = standard deviation; SMA = skeletal muscle area; SMI = skeletal muscle index out sarcopenia age difference didn't reach statistical significance. BMI was significantly lower in sar-copenic patients but significantly higher in patients with myosteatosis. Radiol Oncol 2020; 54(2): 237-246. 241 Srpcic M et al. / Sarcopenia and myosteatosis impair survival after esophagectomy 1 1 V —r_1no sarc operas —n sarcopenia —f—i» sarcopewa-censwid —f— soícopenia 10% (n (%)] 11 (47.8%] 35 (30.2%] 0.100 25 (34.7%] 21 (31.3%] 0.672 Neoadjuvant Therapy (n (%]] 14 (60.9%] 60 (51.7%] 0.422 34 (47.2%] 40 (59.7%] 0.141 Cancer Type (n (%]] 0.864 0.500 Adenocarcinoma 13 (56.6%] 61 (52.6%] 37 (51.4%] 37 (55.2%] Squamous cell carcinoma 10 (43.4%] 54 (46.6%] 35 (48.6%] 29 (43.3%] GIST 1 (0.8%] 1 (1.5%] Pathological Stage (AJCC 2017] (n (%]] 0.650 0.546 I 8 (34.8%] 43 (37.1%] 26 (36.1%] 25 (37.3%] II 6 (26.1%] 21 (18.1%] 11 (15.3%] 16 (23.9%] III 4 (17.4%] 32 (27.6%] 21 (29.2%] 15 (22.4%] IVA 4 (17.4%] 19 (16.4%] 12 (16.7%] 11 (16.4%] IVB 1 (4.3%] 1 (0.8%] 2 (2.8%] 0 Lymph nodes (mean ± SD] 28.8 ± 10.5 23.9 ± 12.6 0.266 24.4 ± 11.1 22.4 ± 13.5 0.337 Surgical approach 0.258 0.167 open 12 (52.2%] 75 (64.7%] 49 (68.1%] 38 (56.7%] MIE 11 (47.8%] 41 (35.3%] 23 (31.9%] 29 (43.3%] AJCC = American joint committee on cancer; BMI = body mass index; CT = computed tomography; GIST-gastrointestinal stromal tumor; HU = Hounsfield units; MIE = minimally invasive esophagectomy; SD = standard deviation; SMA = skeletal muscle area; SMI = skeletal muscle index proposed using the 5th percentile for cutoff values for SMI and MA in non-elderly (age 20-60) to avoid age related muscle loss. These values (SMI 43.1 cm2/m2 for men and 32.7 cm2/m2 for women) are markedly lower then ones used in most previous studies. Consequently, the prevalence of sarcopenia in our study (16.5%) is also lower than 26-75% reported in other studies in resectable esophageal cancer. Mean SMA and SMI was 157.6 ± 28.0 cm2 and 52.1 ± 9.5 cm2/m2 in males and 103.9 ± 16.3 cm2 and 39.8 ± 6.8 cm2/m2 in females (both significantly different between sexes with p < 0.001) which correlates well with studies in similar populations. We believe that choosing the right population with which patients are compared is crucial in determining the real prevalence of sarcopenia (e.g., the study by Nishigori et al. in Japanese esophageal cancer patients33 used the cutoff points obtained in Canadian obese patients34 and reported sarcopenia in 75% of patients). Defining myosteatosis is even more difficult, since the term is not used much yet and reports are scarcer. We chose cutoffs according to the same principle, i.e. at the 5th percentile of a healthy population. We did not find a statistically significant difference in muscle attenuation between males and females (31.2 ± 8.3 HU vs. 27.8 ± 8.7 HU, p = 0.082), but with small numbers in our groups and the availability of sex-specific cutoff values for attenuation we opted for those. Myosteatosis was present in 51.8% of our patients and there was no significant relationship between sarcopenia and myosteatosis (OR 1.256 (CI 0.510-3.093, p = 0.620)). This is in contrast with the study by Stretch et al. where the proportions of patients with sarcopenia and myosteatosis were inverse (40.7% vs. 25.2%) but they similarly reported no correlation between muscle mass and muscle radiodensity. A possible reason for this are the higher cutoffs they used for sarcopenia (40th percentile of their patients or 47.7 cm2/m2 and 36.5 cm2/m2).28 On univariate analysis sarcopenia and myostea-tosis were associated with lower overall survival in our study group (Kaplan Meier log rank p = 0.042 Radiol Oncol 2020; 54(2): 237-246. 243 Srpcic M et al. / Sarcopenia and myosteatosis impair survival after esophagectomy TABLE 3. Complication and survival data compared between sarcopenia/no sarcopenia and myosteatosis/no myosteatosis groups Sarcopenia No Sarcopenia Odds Ratio p (N = 23 (14.5%)) (N = 114 (83.5%)) (OR. 95% CI) Complications (n (%)) In hospital mortality 1 [4.3%] Any complication 11 [47.8%] Conduit complications 4 [17.4%] Pleuropulmonary complications 8 [34.8%] Respiratory failure 5 [21.7%] Any other complications 4 [17.4%] Median survival [months] 18.3 [CI 5.4-31 1 year survival 50.8% 3 year survival 32.9% 5 year survival 32.9% 8 [6.9%] 0.614 [0.073-5.158] 0.650 54 [46.6%] 1.052 [0.430-2.578] 0.911 17 [14.7%] 1.226 [0.371-4.049] 0.738 29 [25.0%] 1.600 [0.615-4.160] 0.332 21 [18.1%] 1.230 [0.410-3.689] 0.711 38 [32.8%] 0.432 [0.137-1.359] 0.143 31.0 [CI 7.4-54.6] 0.042 78.5% 47.7% 42.2% myosteatosis no myosteatosis odds ratio p (N = 72 (51.8%)) (N = 47 (48.2%)) (OR. 95% CI) Complications (n (%)) In hospital mortality 7 [9.7%] 2 [3.0%] 3.500 [0.701-17.486] 0.107 Any complication 32 [44.4%] 33 [49.3%] 0.824 [0.423-1.607] 0.570 Conduit complications 5 [6.9%] 16 [23.9%] 0.238 [0.082-0.692] 0.005 Pleuropulmonary complications 17 [23.6%] 20 [30.0%] 0.726 [0.341-1.545] 0.406 Respiratory failure 14 [19.4%] 12 [17.9%] 1.066 [0.453-2.510] 0.884 Any other complications 24 [33.3%] 18 [26.9%] 1.361 [0.656-2.822] 0.407 Median survival [months] 19.0 [CI 13.3-24.7] 57.1 [CI 15.2-99.0] 0.044 1 year survival 64.2% 84.0% 3 year survival 36.9% 53.7% 5 year survival 33.9% 46.9% CI = confidence interval; OR = odds ratio and p = 0.044, respectively). For sarcopenia this is in accordance with previously published data and for myosteatosis this is one of the first published reports. Dijksterhuis et al. have published a report on body composition, survival and toxicity in advanced esophagogastric cancer patients receiving palliative chemotherapy where they used BMI-specific cutoff values to define myosteatosis (< 41 HU in non obese (BMI < 25) and < 33 HU in overweight patients). Prevalence of myosteatosis in their group was 50% and they found a lower risk of grade III and IV toxicity in patients with higher muscular density but no association between sar-copenia or myosteatosis and survival was found.35 Tamandl et al. published a study with 200 patients receiving an esophagectomy. They stratified patients in low- and high-muscle attenuation groups with a cutoff of 40HU in a population similar to ours. Average MA was 36 HU (31-41) and patients with MA < 40 HU had significantly poorer overall survival.36 The percentage of patients with MA over and under 40 HU is not given, so we cannot compare the prevalence to our results but this definition of reduced muscle attenuation uses a cutoff considerably higher than ours. On the other hand, a study by Gabiatti et al. in patients with locally advanced esophageal cancer receiving definitive chemoradiotherapy demonstrated favorable progression free survival and overall survival in a subgroup of patients with my-osteatosis but without systemic inflammation.37 Sarcopenia has been studied extensively as a predictive factor in esophageal cancer. A recently published meta-analysis by Boshier et al. reviewed Radiol Oncol 2020; 54(2): 237-246. 244 Srpcic M et al. / Sarcopenia and myosteatosis impair survival after esophagectomy 29 studies with 3193 patients (38% sarcopenic) in which various methods were used to diagnose sarcopenia.38 Sarcopenic patients had more pulmonary complications and lower overall survival. A similar meta-analysis by Deng et al. reviewed 11 cohort studies including 1520 patients (52.3% sarcopenic). Patients with sarcopenia had lower 3-year and 5-year survival after resection.39 Complications and perioperative mortality were compared in our study between sarcopenia/no sarcopenia and myosteatosis/no myosteatosis groups and no statistically significant negative effect of muscle depletion was found. This is in concordance with most other studies who failed to show a connection even in studies who showed differences in long term survival.2940 Insufficient statistical power in most studies including ours to detect a potential difference in complication rates is no doubt a strong factor. For conduit complications however, the incidence in our cohort was significantly lower in the myosteatosis group (5/72 (6.9%) vs. 16/67 (23.9%) in patients without myosteatosis, (OR 0.238 (0.082-0.692), p = 0.005). It is difficult to explain the reason for this observation. A higher BMI in patients with myosteatosis could indicate a better nutritional status at presentation. Despite the lower incidence of this dangerous complication perioperative mortality in patients with myostea-tosis was not different than in patients without it. General clinical data in our cohort does not differ significantly from similar published series in re-sectable esophageal cancer. Patients with myostea-tosis were significantly older than patients without it (67.1 ± 7.7 vs. 60.5 ± 10.0 (p < 0.001)) whereas in patients with or without sarcopenia age difference didn't reach statistical significance (67.1 ± 7.8 vs. 63.3 ± 9.7 (p = 0.076)). BMI was significantly lower in sarcopenic patients (23.8 ± 5.9 vs. 26.7 ± 4.4 (p = 0.006)) but significantly higher in patients with myosteatosis (27.3 ± 4.9 vs. 25.2 ± 4.4 (p = 0.006)). 13 patients (9.4%) had both sarcopenia and myosteatosis, their BMI was 25.5 ± 6.1 (range 18.1-37.1). 33.1% of our patients lost 10% or more of their body weight but this did not confer a greater risk of having sarcopenia (OR 2.12 (CI 0.855-5.266), p = 0.100) or myosteatosis (OR 1.165 (CI 0.574-2.366), p = 0.672). As suggested elsewhere28 sarcopenia and myosteatosis are probably two separate entities with different causes and effects reflecting different disturbances in metabolic processes. Underlying causes of sarcopenia and myostea-tosis are most likely overlapping to some extent. Possible mechanisms, through which they nega- tively affect survival, are various. Diminished food intake due to dysphagia and loss of appetite as well as a chronic inflammation state in esophageal cancer lead to sarcopenia. This in turn causes diminished mobility and rehabilitation after surgery41, respiratory complications33, inferior wound healing42 and diminished tolerance of chemo and radiotherapy.35 Skeletal muscle has been described as an endocrine organ43 and it is the derangement of this function that is also a possible cause of inferior survival. Carefully designed studies are needed to corroborate this hypothesis. The inclusion of myosteatosis assessment is in our opinion a strength of our study. We see that myosteatosis is more prevalent than sarcopenia and is a more sensitive marker of muscle degradation which precedes muscle mass and overall body mass loss. It is nevertheless at least as detrimental to prognosis as sarcopenia. Our study also uses recently published cut-off values that in our opinion assess the incidence of sarcopenia better than previous studies. However, this hinders the comparability of our results with others. It is not without weaknesses either. All CT images were recorded at staging with approximately half the patients going straight to resection and the other half receiving neoadjuvant treatment first. No repeat CT images were taken after neoadjuvant treatment if there were no clinical signs of progression according to our group's guidelines. The distribution of intervals from CT to esophagectomy is therefore bimodal and the planimetric data reflects patients' muscle reserves at beginning of any treatment and not necessarily at esophagectomy. This is a shortcoming when assessing the impact on perioperative mortality and complications since muscle mass loss is a well known process during neoadjuvant therapy.44-47 The large variation in times between CT and esophagectomy should in our opinion however not be regarded as a weakness when assessing the impact on overall survival of radical esophageal cancer treatment. Our study also lacks statistical power to detect a potential difference in mortality and complications, an issue that has fraught all previous studies as well. With growing numbers of cases in which CT images are available for analysis and with potential pooling of data these statistical issues can be overcome in the future. Lastly, due to the univariate nature of our analysis no causal effect between survival and muscle depletion markers can be established, but the association shown can serve as an incentive for further research. Radiol Oncol 2020; 54(2): 237-246. 245 Srpcic M et al. / Sarcopenia and myosteatosis impair survival after esophagectomy Conclusions In a prospective cohort study from a dedicated database on esophagectomies we studied the association of sarcopenia and myosteatosis with outcomes after curative esophagectomies with or without neoadjuvant chemoradiotherapy. Prevalence of sarcopenia and myosteatosis at presentation was 16.5% and 51.8%, respectively. Both sarcopenia and myosteatosis were associated with decreased overall survival. For sarcopenia this is in accordance with previously published data and for my-osteatosis this is one of the first published reports. Identifying novel predictors of outcomes can be beneficial for tailoring treatment options in patients with esophageal cancer as well as for planning intervention strategies targeted at improving functional body reserves. Authors' contributions MSr and MSo designed the study. 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Gabiatti CTB, Martins MCL, Miyazaki DL, Silva LP, Lascala F, Macedo LT, et al. Myosteatosis in a systemic inflammation-dependent manner predicts favorable survival outcomes in locally advanced esophageal cancer. Cancer Med 2019; 8: 6967-76. doi: 10.1002/cam4.2593 38. Boshier PR, Heneghan R, Markar SR, Baracos VE, Low DE. Assessment of body composition and sarcopenia in patients with esophageal cancer: a systematic review and meta-analysis. Dis Esophagus 2018; 31. doi: 10.1093/ dote/doy047 39. Deng H-Y, Zha P, Peng L, Hou L, Huang K-L, Li X-Y. Preoperative sarcopenia is a predictor of poor prognosis of esophageal cancer after esophagectomy: a comprehensive systematic review and meta-analysis. Dis Esophagus 2019; 32. doi: 10.1093/dote/doy115 40. Tsukioka T, Nishiyama N, Izumi N, Mizuguchi S, Komatsu H, Okada S, et al. Sarcopenia is a novel poor prognostic factor in male patients with pathological Stage I non-small cell lung cancer. Jpn J Clin Oncol 2017; 47: 363-8. doi: 10.1093/jjco/hyx009 41. JR, Bathe OF, Fassbender K, Winget M, Baracos VE. Sarcopenia is associated with postoperative infection and delayed recovery from colorectal cancer resection surgery. Br J Cancer 2012; 107: 931-6. doi: 10.1038/bjc.2012.350 42. Achim V, Bash J, Mowery A, Guimaraes AR, Li R, Schindler J, et al. Prognostic indication of sarcopenia for wound complication after total laryngectomy. JAMA Otolaryngol Head Neck Surg 2017; 143: 1159-65. doi: 10.1001/ jamaoto.2017.0547 43. Pedersen BK, Febbraio MA. Muscles, exercise and obesity: skeletal muscle as a secretory organ. Nat Rev Endocrinol 2012; 8: 457-65. doi: 10.1038/ nrendo.2012.49 44. Guinan EM, Doyle SL, Bennett AE, O'Neill L, Gannon J, Elliott JA, et al. Sarcopenia during neoadjuvant therapy for oesophageal cancer: characterising the impact on muscle strength and physical performance. Support Care Cancer 2018; 26: 1569-76. doi: 10.1007/s00520-017-3993-0 45. Reisinger KW, Bosmans JWAM, Uittenbogaart M, Alsoumali A, Poeze M, Sosef MN, et al. Loss of skeletal muscle mass during neoadjuvant chemora-diotherapy predicts postoperative mortality in esophageal cancer surgery. Ann Surg Oncol 2015; 22: 4445-52. doi: 10.1245/s10434-015-4558-4 46. Yip C, Goh V, Davies A, Gossage J, Mitchell-Hay R, Hynes O, et al. Assessment of sarcopenia and changes in body composition after neoadjuvant chemotherapy and associations with clinical outcomes in oesophageal cancer. Eur Radiol 2014; 24: 998-1005. doi: 10.1007/s00330-014-3110-4 47. Awad S, Tan BH, Cui H, Bhalla A, Fearon KCH, Parsons SL, et al. Marked changes in body composition following neoadjuvant chemotherapy for oe-sophagogastric cancer. Clin Nutr Edinb Scotl 2012; 31: 74-7. doi: 10.1016/j. clnu.2011.08.008 Radiol Oncol 2020; 54(2): 237-246. 247 research article The influence of shielding reinforcement in a vault with limited dimensions on the neutron dose equivalent in vicinity of medical electron linear accelerator Ana Ivkovic1'2, Dario Faj13, Mladen Kasabasic12, Marina Poje Sovilj4, Ivana Krpan13, Marina Grabar Branilovic5, Hrvoje Brkic13 1 Faculty of Medicine, J. J. Strossmayer University of Osijek, Osijek, Croatia 2 University Hospital Osijek, Osijek, Croatia 3 Faculty of Dental Medicine and Health, J. J. Strossmayer University of Osijek, Osijek, Croatia 4 Department of Physics, J. J. Strossmayer University of Osijek, Osijek, Croatia 5 Department of Organic Chemistry and Biochemistry, Rudjer Boskovic Institute, Zagreb, Croatia Radiol Oncol 2020; 54(2): 247-252. Received 2 February 2020 Accepted 11 March 2020 Correspondence to: Hrvoje Brkic, Faculty of Medicine in Osijek, J. Huttlera 4, 31000 Osijek, Croatia. E-mail: hbrkic@mefos.hr Disclosure: No potential conflicts of interest were disclosed. Background. High energy electron linear accelerators (LINACs) producing photon beams with energies higher than 10 MeV are widely used in radiation therapy. In these beams, fast neutrons are generated, which results in undesired contamination of the therapeutic beam. In this study, measurements and Monte Carlo (MC) simulations were used to obtain neutron spectra and dose equivalents in vicinity of linear accelerator. Materials and methods. LINAC Siemens Oncor Expression in Osijek University Hospital is placed in vault that was previously used for 60Co machine. Then, the shielding of the vault was enhanced using lead and steel plates. Measurements of neutron dose equivalent around LINAC and the vault were done using CR-39 solid state nuclear track detectors. To compensate energy dependence of detectors, neutron energy spectra was calculated in measuring positions using MC simulations. Results. The vault is a source of photoneutrons, but a vast majority of neutrons originates from accelerator head. Neutron spectra obtained from MC simulations show significant changes between the measuring positions. Annual neutron dose equivalent per year was estimated to be less than 324 |jSv in the measuring points outside of the vault. Conclusions. Since detectors used in this paper are very dependent on neutron energy, it is extremely important to know the neutron spectra in measuring points. Though, patient dosimetry should include neutrons, estimated annual neutron doses outside the vault were far below exposure limit of ionizing radiation for workers. Key words: vault reconstruction; 60Co decommission; Monte Carlo simulations; CR-39; neutron dose equivalent Introduction High energy electron linear accelerators (LINACs) producing photon beams with energies higher than 10 MeV are widely used in radiation therapy (RT). In these beams, fast neutrons are generated, which results in undesired contamination of the therapeutic beam.1-6 Neutron contamination in high-energy RT implies an increase of secondary radiation-induced cancer risk.7 It is also well known that an essential requirement for successful radiation therapy is that the discrepancies between dose distributions calculated at the treatment planning stage and those delivered to the patient are minimized.8 All modern RT modalities aim to be highly conformal, which is achieved by using Radiol Oncol 2020; 54(2): 194-200. doi: 10.2478/raon-2020-0019 248 Ivkovic A et al. / Neutron dose equivalent after vault reconstruction many small fields and requires longer beam-on times. The additional dose from photoneutrons is proportional to the beam-on time9, causing higher photoneutron doses then expected in modern RT techniques, so it is important to determine the full radiation field correctly in order to evaluate the exposure of patients and medical personnel. The head of the LINAC is the primary source of photoneutrons in vicinity of linear accelerators since it consists mainly of high-Z materials such as tungsten and steel.110 Also, the vault in which the machine is placed can be the source of photoneu-trons.11 In Croatia, three linear accelerators were placed in vaults that were previously used for 60Co machines.5 Because of the difference in photon energies between the devices, shielding of the vault had to be enhanced. Also, 60Co and LINAC machines and their auxiliary systems differ in size. 60Co vaults are smaller than the vaults designed for high energy photon LINACs and during the reconstruction of 60Co vaults for LINACs, the main limitation was the space. The vault walls had to be enhanced for photon shielding, but there was no space for adding more concrete to the walls. Therefore, lead and steel panels were added into the walls instead. LINAC Siemens Oncor Expression 18 MV placed in Osijek University Hospital is one of the LINAC's placed in the reconstructed vaults. Measurements of the neutron flux and dose equivalent that were done in Osijek University Hospital are already published.2 Measurements were done during all stages of installation, namely before and after lead shielding was added to the walls. It was done using ® lead panel D accelerator's head paraffin leap panel C door concrete wall © FIGURE 1. Top view (down) of the vault where Siemens Oncor Expression accelerator at Osijek University Hospital is placed with marked positions of measurements. (A), (B) and (D) are positions outside the vault, (C) and E are inside the vault. Position E is in isocenter. solid state nuclear track detector (SSNTD) LR115.5 Back then, the neutron spectra at the positions of measurements were unknown and the results had a large uncertainty due to the energy dependence of detectors. In this paper the measurements were repeated with SSNTD CR-39, but energy dependence was taken into consideration using Monte Carlo (MC) simulations. Neutron dose equivalents for patients and staff were assessed and compared to the results presented before.2 Additionally, we investigated the origin of the photoneutrons to show if the steel panels added to the vault walls could be significant sources of photoneutrons. The MC simulations were done for different orientations of the gantry and different points around the accelerator and vault in order to show whether these panels increase total radiation dose to patients and staff. Materials and methods The model of dual photon beam (6MV and 18 MV) medical LINAC Siemens ONCOR Expression at Osijek University Hospital was built using MCNP611® beta code.12 Due to negligible cross sections for neutron productions in a low energy photon beam (6 MV), only the high energy photon beam (18 MV) was modeled. Data used for accelerator's geometry building and materials of which accelerator's head consists were provided by manufacturer. Model of the accelerator head was built as it was described in our previous publications.11013 For the purpose of this study the accelerator vault was also constructed using MCNP Code (Figure 1). The model of the vault was built using macrobod-ies, such as boxes and cylinders. The materials for the model were taken from Compendium of Material Composition Data for radiation transport modeling.14 The majority of vault walls is made of concrete and bricks. As seen in the Figure 1, lead panels were placed in the vault wall (D) and the wall that defines the maze (C). The door at the maze entrance is made of lead and filled with paraffin. All the data used in construction of the vault were obtained from the technical service of Osijek University Hospital. Each simulation had at least 5108 initial events (electrons incident on target). The criteria for acceptance of the simulations were that R value (relative error) falls below 0.1 and that all 10 statistical checks are met.12 The energy cut-off for both electrons and photons was 1 keV, and for neutrons it remained 0 MeV. The continuous energy neutron cross sections Radiol Oncol 2020; 54(2): 247-252. 249 Ivkovic A et al. / Neutron dose equivalent after vault reconstruction library ENDF/B-VII (Evaluated Nuclear data file B-VII) was used for neutron transport.15 In Figure 1, measuring positions (A, B, C and D) used previously are presented.5 In this research, the measurements were done using SSNTD CR-39 in the same measuring positions as before, with additional measuring position E in isocenter. Therefore, F4 tallies for detecting neutrons in positions A, B, C, D and E were modeled. Tallies in positions A, B, C and D were modeled as boxes with dimensions 20x20x20 cm3 while the tally at the isocenter was modeled as a box with dimensions 1x1x1 cm3. The efficiency of the calculations is very low because only a few percent of all the electrons impinging on target produce photons in the beam, and only a few of those photons with high energies produce photoneutrons. To improve efficiency of MC simulations, i.e. particle sampling in the detector region, DXTRAN spheres were setup around all neutron detectors. Neutron spectra were collected in energy bins ranging from 110-9 to 18 MeV in logarithmic scale that corresponds to energy bins for the NCRP flux to dose conversion factors.16 Each detector had all the model cells flagged, in order to determine the place of origin of neutrons. 18 MV photon beam with field size 10x10 cm2 was modeled. Simulations were run with accelerator head pointing down (gantry angle 0°) and in the direction of point D (gantry angle 270°). As seen in the Figure 1, the measurements were performed in five positions: at the operator's console (A), on both sides of the vault door approximately 150 cm above the floor (B -outside the vault, C - inside the vault), on the outside wall of the accelerator vault at the central axis of the beam (D) (5) and isocenter (E). Position E is important for radiation protection of a patient while positions A, B, C and D are important for the staff working in the area around the vault. In our previous measurements with SSNTD LR 115 and boron converter, the dose was calculated without known neutron spectra for the aforementioned positions.5 In this research, spectra for measuring positions are obtained and taken into account when calculating neutron dose. The neutron detector consisted of CR-39 solid state nuclear track detector with dimensions 2x3 cm2 and boron foil BN-1 (ALGADE Laboratoire DOSIRAD, France) that is a 10B converter for reaction (n,a). Detectors that contain a 10B converter are very sensitive to low energy neutrons and less sensitive to fast neutrons. After the irradiation, CR-39 detectors were separated from boron foils and etched in 30% KOH aqueous solution at 70°C for 6 hours. After etching, detectors were rinsed in distilled water and dried. The detectors were scanned with built-in microscope camera (Zeiss Axiovert 200) using 10x16 magnification. 100 images were taken on average for each film. The number of images depended on the number of tracks registered on the film. In order to get a less than 10% error, the measured area had to be large enough, so that the number of counted tracks is over 100 (N>100).17 Tracks were counted using ImageJ/Fiji 1.46 software. First, the tracks were counted manually, i.e. track by track. Manual track counting was used as a reference. Tracks were counted so that each track in the image was marked. Marked tracks were added to a tally sheet. Manual counting is time consuming and impractical, especially if there are many images to process. Therefore, counting was automated. Images were converted to grayscale and a grayscale threshold was applied along with other criteria (track size, shape). Tracks in processed images were counted automatically by the software. Track density is a quantity obtained by division of counted tracks and total area of film used for counting. As in previous measurements CR-39 detectors were placed in positions A, B, C, D and E approximately 150 cm above the floor as in.5 Measurements for gantry angles 0° and 270° with collimator opening 10cm x 10cm were done for all 5 measuring points. Since track densities in measuring positions A, B and D are comparable to background measurements, we investigated the total neutron fluence in these positions and how many neutrons reaching these positions are going to produce alpha particles that will eventually form track in SSNTD CR-39. Total neutron fluencies, number of alpha particles and the spectra for positions A, B, C, D and E, for gantry angles 0° and 270°, are obtained from MCNP simulations. The spectra for 0° and 270° are convoluted with cross section of neutron on boron for reaction 10B (n, a) 7Li. Cross section data is taken from ENDF/B-VII (Evaluated Nuclear Data File B-VII).15 Therefore, the probability of alpha particles production per 1 atom of boron is obtained. The number of tracks on CR-39 is proportional to the number of alpha particles originating from boron foil. The quantity used in experimental part is track density in units track/mm2 (mm2 is approximately the size of the field of view that was used for detector scanning on microscope). In order to compare experimental data with simulation, the number of alpha particles per mm2 of boron has to be calculated and normalized to 2500 MU. Using the data obtained from the manufacturer, the num- Radiol Oncol 2020; 54(2): 247-252. 250 Ivkovic A et al. / Neutron dose equivalent after vault reconstruction 1 J [ , —ÎÎT _J A »a] a [oi Il . f. FIGURE 2. Neutron spectra in measuring positions (A), (B), (C), (D) and (E) for photon field size 10 x 10 cm2 and gantry angles 0° and 270°. (E - 1) is normalized neutron spectrum in measuring position E for simulations with and without vault. ber of alpha particles produced in (n,a) reaction on 1 mm2 of boron foil was calculated. Statistical analysis Relative error of MC simulations is below 10% which is in agreement with statistical checks performed by code itself.12 All measurements using SSNTD were repeated at least 6 times and relative error did not exceed 8.7%. Relative error in experimental measurements was estimated the maximum deviation from mean. Results Calculated normalized neutron fluence spectra for 10x10 cm2 field and gantry angles 0° and 270° in TABLE 1. Total MC calculated neutron fluence in measuring positions A, B, C, D and E for gantry angles 0° and 270°. Neutron fluence is in number of neutrons per cm2 per electron impinging on target. Results are normalized to source particle Measuring position Gantry angle A B C D E 0' 5.6 • 10-13 5.3 • 10-14 3.6 • 10-10 8.4 • 10-24 1.5 • 10-8 270' 6.6 • 10-13 3.4 • 10-14 4.2 • 10-10 1.6 • 10-22 1.5 • 10-8 measuring points A,B,C, D and E are presented in Figure 2 as well as neutron spectrum in isocenter (measuring position E) calculated in simulations with and without vault (Figure 2, E-1). Total MC calculated neutron fluence per electron impinging on target in measuring positions A, B, C, D and E for gantry angles 0° and 270° is presented in Table 1. Normalized neutron fluence obtained from MC calculations according to the place of origin is presented in Figure 3. Data is given for all measuring positions and gantry angles 0° and 270°. Track density caused by background irradiation was 0.47 tracks per mm2 with its standard deviation 0.09 tracks per mm2. Track density caused by background radiation is subtracted from measured track density. In measuring positions A, B and D measured track density is slightly larger than background. Mean value of detector sensitivities for all measuring position is 0.0002. Neutron dose equivalents for all measuring positions for gantry angles 0° and 270° together with already published data2 are presented in Table 2. Discussion Neutron spectra obtained from MC simulations show significant changes between the measuring positions (Figure 2). There are also changes in spectra (Figure 2) when gantry angle changes from 0° to 270°. Since detectors used in this paper are very dependent on neutron energy, it is extremely important to know the neutron energy in measuring points. Simulations with and without LINAC vault have shown that surrounding structures also influence the neutron fluence and energy spectrum (Figure 2, E-1). Therefore, it is important to simulate not only the accelerator, but all surrounding structures as well. Furthermore, neutron spectrum with vault has a significant neutron component in lower energy part of spectrum, around 0.1 eV. Simulations that include the vault have 38% larger neutron fluence than simulations without vault. The reason for that is because the vault serves as a box that does not allow neutrons to escape so they are bouncing back and forth in the vault. Therefore, more neutrons are detected on the tally in isocenter (E). When simulation doesn't include the vault, neutrons are dispersed and it is easy for them to escape. Furthermore, the vault itself is a source of photoneutrons with small energies, but only 1% of all neutrons detected in isocenter originate from Radiol Oncol 2020; 54(2): 247-252. 251 Ivkovic A et al. / Neutron dose equivalent after vault reconstruction the vault walls (Figure 3, position E). Therefore, the small energy peak mainly comes from the wall attenuation of neutrons originating from accelerator head. It means that a part of the neutron fluence will move from high to low energy part of the spectrum which can be observed in Figure 2, E-1. According to Table 1, the change of the gantry angle did not have significant influence on total neutron fluence in measuring positions A, B, C and E, but in position D total neutron fluence is 20 times larger for gantry angle 270° than for angle 0°. This was an expected result since the photon beam is directly pointed to the position D when gantry is rotated to 270°. Position D is on the outer side of the wall which contains a lead panel used for high energy photon shielding purposes. At the same time, the lead panel has become a source of photoneutrons (Figure 3). According to MC simulations in measuring positions C and E there is a great number of alpha particles that can produce track in detector and consequently in these positions, track densities are significantly larger than track density caused by background radiation. However, in positions A, B and D (especially D) number of alpha particles is 1 or several orders of magnitude smaller. Mean value of detector sensitivities for all measuring position is 0.0002, i.e. every 5000th neutron reaching detector will make track in the detector. According to Table 2 our previous measure-ments5 overestimated neutron dose equivalent, especially for measuring position D. The underlying reason for overestimation was the unknown neutron spectra in measuring positions. Measuring position E (isocenter) was added to this research, since the neutron dose to patients was one of the interests of this study. Neutron dose equivalent in isocenter is 3.3 mSv per Gy photon dose in isocenter. If we take a radiotherapy treatment of prostate for example, with prescribed dose of 74 Gy and all rectangular fields 10x10 cm2 4 field box technique, 250 MU daily, then the equivalent neutron dose is 0.3 Sv. The neutron dose is comparable to iL Ij 1=1 1 II / ¿5* f S Y f j / v/s / / y ✓ / / í> Amu 1 /// y a/ © /y ïï s / s & f =»=4 y-y/ ' / s /  FIGURE 3. Normalized neutron fluence obtained from MC calculations according to the place of origin for all measuring positions and gantry angles 0° and 270°. the published results.18 Measurements with neutron dosimeters that consist of SSNTD CR-39 and boron foil BN-1 showed that neutron dose rate in isocenter is large enough that these detectors can be used in in vivo patient dosimetry. Neutron dose to the patient can be calculated from track density and calibration coefficients listed in Table 3. Outside the vault, in positions A, B and D, it is difficult to measure the neutron dose because the track density on detectors is of the same order of magnitude as track density caused by the background radiation. Therefore, application of neutron dosimeters described in this study in personal dosimetry is questionable. According to the MC simulations, mean neutron dose per year in is 324 |jSv, 83 |jSv and 2.710-5 |jSv in positions A, B and D respectively. Calculated neutron doses are far below exposure limit of ionizing radiation for workers which is 20 mSv per year.19 Therefore, neutron personal dosimetry is not necessary. In our previ- TABLE 2. Neutron dose equivalents in pSv per Gy photon dose in isocenter for all measuring positions and gantry angles 0° and 270° Gantry 0° Gantry 270° A B C D E A B C D E (|jSv/Gy) (|iSv/Gy) (|iSv/Gy) (|iSv/Gy) (|iSv/Gy) (||Sv/Gy) (||Sv/Gy) (||Sv/Gy) (||Sv/Gy) (||Sv/Gy) Poje LR 115 0.04 0.08 10 0.04 0.1 0.17 20 0.13 et al Active detector 0.052 0.1 14.3 0.052 Our CR-39 0.008 0.0006 8.2 1.3-10-12 3297 0.01 0.004 10.3 4.21 -10-11 3333 results (0.0006) (0.00004) (0.6) (7-10-14) (264) (0.008) (0.0002) (0.5) (2-10-12) (202) Data from Table 2 are obtained by using calibration coefficients for neutron detector CR-39. In Table 3 calibration coefficients for measuring positions C and E are presented. Radiol Oncol 2020; 54(2): 247-252. 252 Ivkovic A et al. / Neutron dose equivalent after vault reconstruction TABLE 3. Calibration coefficients for neutron detector CR-39 for measuring positions C and E for gantry angles 0° and 270° Position G 0° G 270° |iSv/(track/mm2) |iSv/(track/mm2) C 6,52 11,20 E 151,61 194,97 ously published data5, estimated dose for workers was 2 mSv per year which is higher than the doses estimated in this study. Conclusions Problem of placing high energy linear accelerators in small vaults that are not originally built for linear accelerators is still very present in Southern European countries. Since the space is limited, photon shielding problems are often solved by inserting lead or iron plates in vault walls. High Z elements are new sources of photoneutrons which complicate assessment of neutron spectra and dose in vicinity of linear accelerators. In this study, MC simulations of linear accelerator Siemens Oncor Expression in Osijek University Hospital were done and neutron spectra and dose equivalents in vicinity of linear accelerator were obtained. It is important to include the vault in MC model when assessing neutron dose to patients, otherwise the dose can be overestimated. Simulations showed that lead panels inserted as photon shielding in vault walls are the source of photoneutrons and they contribute to patient and staff dose. However, neutron dose to staff working in vicinity of accelerator vaults is small and there is no need for personal neutron dosimetry. In experimental part of this study, SSNTD CR-39 was used to measure neutron doses in positions inside and outside the vault. Neutron dose rate outside the vault was of the same order of magnitude as the background radiation and their use in personal dosimetry is questionable. 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Slovenian abstracts III Radiol Oncol 2020; 54(2): 159-167. doi: 10.2478/raon-2020-0025 Sodobni in inovativni pristopi k zdravljenju • V* V • • I V I • miSicno neinvazivnega raka sečnega mehurja. Vloga transuretralne resekcije tumorja mehurja in organoidi Taskovska M, Kreft ME, Smrkolj T Izhodišča. Rak sečnega mehurja je sedmi najpogostejši rak pri moških. Približno 75 % vseh rak sečnega mehurja je mišično neinvazivnih. Zlati standard za dokončno diagnostiko in prva linija zdravljenja mišično neinvazivnega raka sečnega mehurja je transuretralna resekcija tumorja mehurja (TURM). V preteklosti so za TURM uporabljali monopolarni resektoskop, danes večina urologov uporablja bipolarnega. Po TURM je glede na gradus tumorja indicirano dodatno zdravljenje z intravezikalno aplikacijo kemo- in/ali imuno-terapevtika. Cilj intravezikalne aplikacije kemo- in/ali imunoterapevtika je preprečiti ponovitev tumorja in potrebo po ponovnem kirurškem zdravljenju. Razvoj tehnologije, molekularne in celične biologije, omogoča raziskovalcem gojenje organoidov - sistemov humanih celic, ki jih gojijo v laboratoriju in imajo lastnosti tkiv, iz katerih izhajajo. Čeprav se organoidi že uporabljajo za preučevanje različnih bolezni na področju urološke onkologije, pa je podatkov za uporabo organoidov raka sečnega mehurja v literaturi zelo malo. Zaključki. Monopolarni in bipolarni tok resektoskopa imata različen vpliv na urotelij, posledično na onkološki učinek ter patohistološko interpretacijo preparata. Vzorci tumorja sečnega mehurja so uporabni za pripravo organoidov in preučevanje onkogeneze. Organoidi sečnega mehurja so osnova za perso-nalizirano medicino in so uporabni za testiranje učinkovitosti kemo-/imunoterapije pri bolnikih z rakom sečnega mehurja. Radiol Oncol 2020; 54(2): 144-148. doi: 10.2478/raon-2020-0017 Mahansko odstranjevanje strdka pri nenadni obojestranski zapori možganske arterije Jeromel M, Miloševič ZV, Pretnar Oblak J Izhodišča. Nenadna zapora obeh notranjih karotidnih in/ali srednjih možganskih arterij je zelo redka in povezana s slabim kliničnim izhodom. V literaturi je le nekaj tovrstnih primerov bolnikov, ki so bili zdravljeni z znotrajžilnim mehanskim odstranjevanjem strdka. Strategija znotrajžilnega pristopa in klinični izhod zato nista jasna. Metode. Sistematični pregled literature smo izvedli preko več elektronskih baz podatkov, pri čemer smo uporabljali naslednje iskalne zahteve: nenadna obojestranska kap, mehanska ponovna vzpostavitev pretoka, odstranitev strdka. Rezultati. V literaturi smo našli pet poročil o šestih bolnikih z obojestransko nenadno zaporo notranje karotidne in/ali srednje možganske arterije, ki so jih zdravili z mehansko odstranitvijo strdka. Dodajamo tudi primer bolnika, ki je po našem vedenju prvi primer obojestranske zapore velike možganske arterije, nastale med intravenskim trombolitičnim zdravljenjem, kjer je bil klinični izhod po mehanski odstranitvi strdka popolnoma odvisen od kolateralnih možganskih pretokov in ne od časa, ki je minil od začetka nastanka zapore. Zaključki. Nenadna zapora obeh notranjih karotidnih in/ali srednjih možganskih arterij vodi v hudo nevrološko stanje (komo) z nepredvidljivim izhodom bolezni tudi v času, ko imamo na voljo metode za mehansko odstranitev strdka. Videti je, da je prisotnost kolateralnih pretokov bolj pomembna kot pa čas do ponovne vzpostavitve pretoka, zato sočasna odstranitev strdka sama po sebi verjetno ne izboljša kliničnega izhoda bolnika. Radiol Oncol 2020; 54(2): I-XII. XII Slovenian abstracts Radiol Oncol 2020; 54(2): 149-158. doi: 10.2478/raon-2020-0029 Glavne in dodatne značilnosti kriterijev LI-RADS za oceno kombiniranega hepatocelularnega-holangiokarcinoma Granata V, Fusco R, Setola SV, Sandomenico F, Barretta ML, Belli A, Palaia R, Tatangelo F, Grassi R, Izzo F, Petrillo A Izhodišča. Namen raziskave je bil določiti, katere značilnosti kriterijev LI-RADS verzije 2018 (v2018) omogočajo natančno karakterizacijo kombiniranega hepatocelularnega-holangiokarcinoma (cHCC-CCA). Bolnikii in metode. V retrospektivno raziskavo smo zajeli 62 bolnikov (srednja starost 63 let; razpon 38-80 let) z operabilnimi HCC-ji, ki so bili pred operacijo potrjeni z biopsijo. Pri vseh bolnikih smo naredili računalniško tomografijo (CT), pri 23 bolnikih tudi magnetnoresonančna preiskavo (MR). HCC smo v radioloških izvidih opisali s pomočjo glavnih in dodatnih značilnosti glede na kriterije LI-RADS v2018. Rezultati. Dokončna histološka diagnoza je pokazala, da je imelo 51 bolnikov HCC in 11 bolnikov cHCC-CAA. Srednja velikost vseh lezij je bila 46,0 mm (razpon 10-190 mm), srednja velikost cHCC-CCA je bila 33,5 mm (razpon 20-80 mm) in srednja velikost HCC 47,5 mm (razpon 10-190 mm). Glede na kategorije LI-RADS je bilo 54 (87,1 %) lezij opredeljenih kot LR-5, 1 (1,6 %) lezija kot LR-3 in 7 (11,3 %) kot LR-M. 39 (63 %) lezij je bilo hipervaskularnih v arterijski fazi preiskave; med njimi so tako značilnost kazali 4 cHCC-CCA (36,4 % izmed vseh cHCC-CCC) in 35 pravih HCC (68,6 % izmed vseh HCC). 43 (69,3 %) lezij je izplavljalo kontrastno sredstvo; izplavljanje je bilo vidno pri 6 cHCC-CCAs (54,5 % izmed vseh cHCC--CCA) in 37 pravih HCC (72,5 % izmed vseh HCC). Pri samo dveh bolnikih s cHCC-CCA (18,2 % izmed vseh cHCC-CCA) je bila vidna kapsula. Pet cHCC-CCA (71,4 % izmed vseh cHCC-CCA) je bilo hiperin-tenzivnih na T2 poudarjenih sekvencah (T2-w), dve leziji (28,6 %) sta bili heterogenega signala na T2-w. Pri vseh cHCC-CCA je bila vidna restrikcija difuzije. Pri sedmih bolnikih s cHCC-CCA je bilo vidno progresivno obarvanje in satelitni noduli. Zaključki. Značilnosti, ki nakazujejo možnost cHCC-CCA so prisotnost satelitnih nodulov, hiperintenziven signal na T2-w, restrikcija difuzije, odsotnost kapsule in progresivno obarvanje po kontrastnem sredstvu. Radiol Oncol 2020; 54(2): I-XII. Slovenian abstracts III Radiol Oncol 2020; 54(2): 159-167. doi: 10.2478/raon-2020-0021 Povezava hondromalacije pogačice z anatomskimi parametri proksimalne tibije, ocenjenimi z magnetno resonanco Tabary M, Esfahani A, Nouraie M, Babaei MR, Khoshdel AR, Araghi F, Shahrezaee M Izhodišča. Magnetna resonance (MR) je neinvazivna visoko občutljiva preiskava za oceno zgodnje hondromalacije pogačice. Morfologijo pogačice in sosednje femuralne trohlee je ocenjevalo več razsikav. Namen naše študije je ocena povezave hondromalacijo pogačice s tibialnimi, patelarnimi in femoralnimi anatomskimi indeksi s pomočjo MR. Bolniki in metode. Med letoma 2017 in 2019 smo v raziskavo vključili 100 preiskav MR kolena pri bolnikih s hondromalacijo pogačice in 100 preiskav MR pri bolnikih brez hondromalacije ob ustrezni starosti. Preiskave MR smo opravili po standardnem protokolu. Hondromalacijo pogačice smo ocenili po modificirani Outerbridgeovi klasifikaciji (stopnje 1-4). V kolenskem sklepu ter okolnih strukturah smo izmerili 25 parametrov in jih primerjali s stopnjo hondromalacije pogačice. Rezultati. Hondromalacija pogačice je bila omembno povezana z nagibom tibialnega platoja, globino trohlee femurja, nagibom lateralne fasete trohlee femurja ter lateralnim nagibom pogačice. Povečan nagib lateralne fasete trohlee femurja (Razmerje obetov [OR] 1,15, 1,03-1,30 pri 95 % intervalu zaupanja), povečan lateralni nagib pogačice (OR 1,13, 1,02-1,26 pri 95 % intervalu zaupanja), povečan medialni nagib tibialnega platoja (OR 0,85, 0,73 in 0,98 pri 95 % intervalu zaupanja) ter povečana globina trohlee (0,06, 0,02 in 0,17 pri 95 % intervalu zaupanja) so bili povezani z zvišano možnostjo za hondromalacijo pogačice. Model za oceno stopnje hondromalacije v odvisnosti od višine pogačice je pokazal OR 75,9. Zaključki. Rezultati raziskave so pokazali novo povezavo anatomskih parametrov proksimalne tibije s hondromalacijo pogačice. Pokazali so tudi na povezavo višine pogačice s stopnjo hondromalacije. Radiol Oncol 2020; 54(2): I-XII. XII Slovenian abstracts Radiol Oncol 2020; 54(2): 168-179. doi: 10.2478/raon-2020-0015 Odziv na pulzno obsevanje z nizko hitrostjo doze v • • ■ ■ • v • i 1***1 * i v v , v | • v «i 1*1 izogemh celicnih linijah pri ploscatocelicnih rakih glave in vratu z različno radiosenzitivnostjo Todorovič V, Prevc A, Nikšič Žakelj M, Savarin M, Buček S, Grošelj B, Strojan P, Čemažar M, Serša G Izhodišča. Zdravljenje lokoregionalnih ponavljajočih ploščatoceličnih rakov glave in vratu predstavlja izziv zaradi razvoja radiorezistence. Pulzno obsevanje z nizko hitrostjo doze temelji na pojavu povečane radiosenzitivnosti na nizke doze sevanja ter učinku hitrosti doze sevanja. Namen te raziskave je bil ovrednotiti pojav povečane radiosenzitivnosti na nizke doze sevanja in učinek pulznega obsevanja z nizko hitrostjo doze na modelu izogenih celic ploščatoceličnih rakov glave in vratu z različno radiosenzitivnostjo. Materiali in metode. Preživetje izogenih starševskih celic FaDu in radiorezistentnih celic FaDu-RR po obsevanju z različnimi protokoli smo določili s testom klonogenosti. S pretočno citometrijo smo preučili učinek obsevanja na razporeditev celic v celičnem ciklu. Z obratno transkripcijo in kvantitativno verižno reakcijo s polimerazo smo določili izražanje genov, ki so vključeni v signaliziranje poškodb DNA. Rezultati. V radiorezistentnih celicah FaDu-RR smo pokazali pojav povečane radiosenzitivnosti na nizke doze sevanja ter povečano občutljivost na pulzno obsevanje z nizko hitrostjo doze v primerjavi s starševskimi celicami FaDu. Pri obeh celičnih linijah smo zaustavili celični cikel v fazi G2/M pet ur po obsevanju. Normalen celični cikel se je ponovno vzpostavil 24 ur po obsevanju v starševskih, ne pa v radiorezistentnih celicah, ki so zastale v fazi G,. Izražanje genov, ki so vključeni v signaliziranje poškodb DNA, je v radiorezistentnih celicah zmanjšano v primerjavi s starševskimi celicami. Obsevanje je povečalo izražanje teh genov v radiorezistentnih celicah, medtem ko se je v starševskih celicah zmanjšalo izražanje le nekaj genov. Zaključki. Pokazali smo prisotnost pojava povečane radiosenzitivnosti na nizke doze sevanja v izogenih radiorezistentnih celicah, ne pa v starševskih celicah. Preživetje celic, kjer smo ugotovili povečano radi-osenzitivnost na nizke doze sevanja, a so bile sicer radiorezistentne, se je po pulznem obsevanju z nizko hitrostjo doze značilno zmanjšalo. Manjše preživetje celic po obsevanju z nizko hitrostjo doze je povezano s spremembami v izražanju genov, ki so vključeni v signaliziranje poškodb DNA, najverjetneje preko spremenjene regulacije kontrolnih točk celičnega cikla. Radiol Oncol 2020; 54(2): I-XII. Slovenian abstracts III Radiol Oncol 2020; 54(2): 159-167. doi: 10.2478/raon-2020-0020 Prevalenca okultnega raka jajčnikov pri 155 preventivnih operacijah visoko ogroženih asimptomatskih bolnicah. Slovenska populacijska raziskava Gornjec A, Merlo S, Novakovič S, Stegel V, Gazic B, Perhavec A, Blatnik A, Krajc M Izhodišča. Določali smo prevalenco, lokalizacijo, patohistološke značilnosti in napoved izhoda bolezni pri bolnicah z okultnim rakom jajčnikov. Jajčnike in/ali jajcevode smo preventivno odstraniili pri asimptomatskih nosilkah BRCA patogenih in verjetno patogenih različic ter visoko ogroženih BRCA negativnih ženskah. Bolnice in metode. Retrospektivno smo analizirali vse ginekološke preventivne operacije od januarja 2009 do decembra 2015. Bolnice smo spremljali do januarja 2019. Vse vključene bolnice so imele pred posegom opravljeno genetsko svetovanje in BRCA testiranje. Zbrali in analizirali smo podatke o kliničnih lastnostih bolnic, dodatnem zdravljenju in spremljanju po končanem zdravljenju. Rezultati. Odstranili smo 155 jajčnikov in jajcevodov pri 110 nosilkah patogenih in verjetno patogenih različic na BRCA!, 35 na BRCA2 in 10 visoko ogroženih BRCA negativnih ženskah. Povprečna starost bolnic je bila 48,3 let. Odkrili smo devet okultnih rakov jajčnikov in jajcevodov (5,8 % vseh operiranih); osem pri nosilkah BRCA! in en pri visoko rizični BRCA negativni bolnici. Od devetih so bili štirje serozni tubarni in-traepitelni karcinomi (trije pri BRCA! nosilkah in enega pri visoko rizični BRCA negativni bolnici), pet je bilo invazivnih tubo-ovarijskih seroznih karcinomov (vsi pri BRCA! nosilkah). Le ena izmed devetih bolnic (11,1 %) z okultnim rakom je imela predoperativno blago povišano vrednost tumorskega označevalca CA-125. Zaključki. Prevalenca okultnih seroznih karcinomov po preventivni ginekološki operaciji pri visoko rizič-nih asimptomatskih bolnicah je bila 5,8 %. Starost ob preventivnih ginekoloških operacijah mora biti skrbno načrtovana po smernicah za določen tip mutacije, oziraje se na končano rodno obdobje. Pri nosilkah BRCA patogenih in verjetno patogenih različic in visoko rizičnih BRCA negativnih bolnicah bi morala biti operacija opravljena po 35. letu starosti in pri BRCA! nosilkah pred 40. letom starosti. Ugotavljamo, da je smiselno ponuditi preventivno ginekološko operacijo tudi visoko rizičnim BRCA negativnim bolnicam. Rezultati analize potrjujejo tubarno hipotezo nastanka seroznega raka jajčnikov/jajcevodov. Citološka preiskava izpirka peritonealne votline je pomembno vplivala na odločitev o vrsti dodatnega zdravljenja in bi naj bila narejena ob vsaki preventivni ginekološki operaciji. Merjenje vrednosti tumorskega označevalca CA-125 se pri naših preiskovankah ni izkazalo za učinkovito presejanje za zgodnje odkrivanje raka jajčnikov. Radiol Oncol 2020; 54(2): I-XII. XII Slovenian abstracts Radiol Oncol 2020; 54(2): 187-193. doi: 10.2478/raon-2020-0023 Prehranski vnos joda, zdravljenje z radiojodom in anaplastični rak ščitnice Bešic N, Gazic B Izhodišča. Anaplastični rak ščitnice je eden najbolj agresivnih tumorjev. Namen raziskave je bil ugotoviti, ali obstaja povezava med večjim prehranskim vnosom joda, pogostostjo anaplastičnega raka ščitnice in značilnostmi tega raka ter ugotoviti, kako pogosto smo bolnike z anaplastičnim rakom ščitnice v preteklosti zdravili z radiojodom. Bolniki in metode. V retrospektivno raziskavo smo vključili 220 bolnikov (152 žensk, 68 moških; povprečna starost 68 let) z anaplastičnim rakom ščitnice, ki so se na Onkološkem inštitutu Ljubljana zdravili od leta 1972 do 2017. Sol je bila jodirana z 10 mg kalijevega jodida / kg pred letom 1999 in s 25 mg kalijevega jodida / kg po letu 1999. Bolnike smo razvrstili v 15-letna obdobja: 1972-1986, 1987-2001 in 2002-2017. Rezultati. Incidenca anaplastičnega raka ščitnice se je zmanjšala po večjem jodiranju soli (p = 0,04). Bolniki so sedaj starejši (p = 0,013) in imajo redkeje zasevke v bezgavkah (p = 0,012) kot v preteklosti. Pogostost oddaljenih zasevkov se sčasoma ni spreminjala. Srednje preživetje bolnikov v prvem, drugem in tretjem obdobju je bilo tri, štiri in tri mesece (p < 0,05). Predhodno smo z radiojodom zdravili 7,7 % bolnikov. Zaključki. Število bolnikov z anamnezo zdravljenja z radiojodom se sčasoma ni spreminjalo. Incidenca anaplastičnega raka ščitnice v Sloveniji se je verjetno zmanjšala zaradi uporabe bolj jodirane soli kot v preteklosti. Radiol Oncol 2020; 54(2): I-XII. Slovenian abstracts III Radiol Oncol 2020; 54(2): 159-167. doi: 10.2478/raon-2020-0019 Pomen aktivacije nuklearnega faktorja kappa beta na vzorcih igelne biopsije pri ugotavljanju indolentnosti raka prostate, ki smo ga po Gleasonu ocenili 6 Zupančič M, Pospihalj B, Cerovič S, Gazič B, Drev P, Hočevar M, Perhavec A Izhodišča. Namen raziskave je bil ugotoviti, ali je imunohistokemično izražanje nuklearnega faktorja kappa beta (NF-kB) na vzorcih igelnih biopsije negativni napovedni dejavnik za indolentnost raka prostate z oceno po Gleasonu 3 + 3 = 6 (GS 6). Bolniki in metode. Raziskavo smo zasnovali na retrospektivni analizi podatkov pri 123 bolnikih z rakom prostate, ki so imeli začetno celokupno vrednost prostatičnega specifičnega antigena (PSA) < 10 ng/ml, število vzorcev igelne biopsije prostate > 8, GS 6 pri biopsiji in ocenjen klinični stadij T1/T2. Vsem smo naredili laparoskopsko radikalno prostatektomijo in njihova arhivirana, s formalinom fiksirana tkiva v parafinskih blokih uporabili za dodatno imunohistokemično barvanje na NF-kB p65. Citoplazemsko in jedrno izražanje NF-kB p65 v vzorcih igelne biopsije prostate s prisotnim rakom prostate smo ločeno primerjali s poopera-tivnim patološkim stadijem, pozitivnimi kirurškimi robovi, GS in biokemičnim napredovanjem bolezni. Rezultati. Po 66 mesecih spremljanja je do biokemičnega napredovanja bolezni (PSA > 0,2 ng/ml) prišlo pri 6 (5,1 %) bolnikih, pri 3 (50 %) s pooperativnim GS 6 in pri 3 (50 %) s pooperativnim GS 7. Tako citoplazemsko kot tudi jedrno izražanje NF-kB p65 nista bili statistično povezani s patološkim stadijem, pozitivnim kirurškim robom in pooperativnim GS. Pri bolniki s pozitivno citoplazemsko NF-kB reakcijo smo ugotovili biokemična napredovanja bolezni z mejno vrednostjo PSA 0,2 ng/ml značilno več (p = 0,015) kot pri bolnikih z negativno reakcijo, medtem ko je bil pri mejni vrednosti PSA 0,05 ng/ml nakazan le trend večjega števila biokemičnih napredovanj bolezni (p = 0,068). Zaključki. Citoplazemsko izražanje NF-kB je povezano z večjim številom biokemičnih napredovanj bolezni in bi lahko bilo neodvisen napovedni dejavnik za preživetje brez ponovitve bolezni. Za potrditev teh začetnih rezultatov pa bo potrebna analiza na večjem številu bolnikov. Radiol Oncol 2020; 54(2): I-XII. VIII Slovenian abstracts Radiol Oncol 2020; 54(2): 201-208. doi: 10.2478/raon-2020-0018 Testiranje programa usposabljanja laboratorijskega osebja brez znanja cervikalne citologije in imunocitokemije za ocenjevanje dvojnega imunocitokemičnega barvanja p16/Ki-67 Kloboves Prevodnik V, Pohar Marinšek Ž, Zalar J, Rozina H, Kotnik N, Jerman T, Varl J, Ivanuš U Izhodišča. Ustrezno usposobljeni laboratorijski delavci naj ne bi imeli težav pri ocenjevanju p16/Ki-67 dvojnega imunocitokemičnega barvanja (DB), vendar trenutno še ni soglasja, kakšno usposabljanje je najbolj primerno. V raziskavi smo na dveh študentih brez znanja cervikalne citologije testirali program učenja ocenjevanja DB. Metode. Začetno usposabljanje, ki ga je vodil starejši presejalec, je potekalo na 40 konvencionalnih brisih materničnega vratu pobarvanih z DB. Po začetnem usposabljanju sta dva študenta brez znanja cervikalne citologije in imunocitokemije ocenila 118 preparatov z DB. Sledilo je dodatno usposabljanje, ki je zajemalo predvsem pregled preparatov DB z neskladnimi ocenami iz prvega ocenjevanja na večglavem diskusijskem mikroskopu. Nato sta študenta ocenila dodatnih 383 preparatov. Ujemanje in zanesljivost ocen DB za odkrivanje CIN2+ obeh študentov smo po obeh ocenjevanjih primerjali med seboj in z referenčnimi rezultati. Pozorni smo bili tudi na čas potreben za oceno enega preparata in na variabilnost rezultatov učitelja. Rezultati. Ujemanje rezultatov med študentoma in referenco je bilo po zaključku raziskave večje v primerjavi s tistim po začetnem usposabljanju. Po začetnem usposabljanju je bil odstotek ujemanja za vsakega študenta 81,4 % ter kapa 0,512 in 0,527, po dodatnem usposabljanju pa je bil odstotek ujemanja 78.3 % in 87,2 % ter kapa 0,556 in 0,713. Zanesljivost ocen testa za odkrivanje CIN2+ se je na koncu raziskave med študentoma razlikovala. Po začetnem usposabljanju je bila občutljivost ocen obeh študentov za 4,3 % in 2,9 % točk višja v primerjavi z referenco, nasprotno pa je bila specifičnost nižja za 30,6 % in 24.4 % točk. Ob koncu raziskave je bila občutljivost ocen testa enega študenta enaka kot pri referenci, medtem ko je bila pri drugem študentu za 2,6 % točk nižja. Med enim študentom in referenco ter med obema študentoma je prišlo do statistično pomembne razlike v specifičnosti (16,7 in 15,1 % točk). Proti koncu raziskave je en študent za oceno enega preparata potreboval 5,2 min, drugi pa 8,2 min. V obeh rokah raziskave je bila notranja variabilnost ocen starejšega presejalca v območju „zelo dobro". Zaključki. Med usposabljanjem za ocenjevanje DB je napredek študentov potrebno spremljati z več različnimi merili, kot so ujemanje in zanesljivost rezultatov za odkrivanje CIN2+ ter čas, potreben za ocenjevanje enega preparata. Spremljanje napredka je potrebno izvajati še nekaj časa po tem, ko študenti že dosežejo zadovoljive rezultate. Le tako lahko zagotovimo nadaljnje dobro delo. Priporočljivo je tudi spremljanje rezultatov učitelja. Radiol Oncol 2020; 54(2): I-XII. Slovenian abstracts III Radiol Oncol 2020; 54(2): 159-167. doi: 10.2478/raon-2020-0026 Obravnava bolnikov z nedrobnoceličnim pljučnim rakom stadija III. Srednjeevropska izkušnja v klinični praksi Zemanova M, Pirker R, Petruželka L, Zbožinkova Z, Jovanovic D, Rajer M, Bogos K, Purkalne G, Ceriman V, Chaudhary S, Richter I, Kufa I, Jakubikova L, Zemaitis M, Černovska M, Koubkova L, Vilasova Z, Dieckmann K, Farkas A, Spasic J, Fröhlich K, Tiefenbacher A, Hollosi V, Kultan J, Kolarova I, Votruba J Izhodišča. Na obravnavo nedrobnoceličnega raka pljuč vplivajo regionalne posebnosti. Cilj raziskave je bil določiti diagnostične in terapevtske postopke, vključno z izidi zdravljenja bolnikov z nedrobnoceličnim rakom pljuč stadija III v vsakodnevni klinični praksi v srednjeevropskih državah, z namenom opredelite področja, kjer bi lahko dosegli izboljšave. Bolniki in metode. V multicentrični, prospektivni in neintervencijski raziskavi smo v spletnem registru zbrali podatke bolnikov z nedrobnoceličnim rakom pljuč stadija III in jih centralno analizirali. Rezultati. Med marcem 2014 in marcem 2017 smo vključili 583 bolnikov z nedrobnoceličnim rakom pljuč stadija III z naslednjimi značilnostmi: 32 % je bilo žensk; 7 % bolnikov ni nikoli kadilo; splošno stanje zmogljivosti po ECOG 0, 1, 2 in 3 je bilo 25 %, 58 %, 12 % in 5 %; 21 % bolnikov je predhodno izgubljalo težo; 53 % bolnikov je imelo ploščatocelični rak, 38 % žlezni rak; 10 % bolnikov je imelo mutacijo EGFR. Stadij smo določili s pomočjo rentgenskega slikanje prsnega koša (97 % bolnikov), CT-jem prsnega koša (96 %), PET-CT-jem (27 %), slikovno preiskavo možgan (20 %), bronhoskopijo (89 %), endobronhialnim ultrazvokom (13 %) in CT-vodeno biopsijo (9 %). Stadij IIIA / IIIB smo diagnosticirali pri 55 % oz. 45 % bolnikih, N2 / N3 pa pri 60 % / 23 % in patološko potrdili pri 29 % bolnikih. Večino bolnikov (56%) smo zdravili kombinirano. Operativno in s kemoterapij smo zdravili 20 % bolnikov, s kemoradioterapijo 34 %, samo s kemoterapijo 26 %, samo z radioterapijo 12 % in samo z najboljšo podporno terapijo 5 % bolnikov. Srednji čas preživetja je bil 16,8 (15,3-18,5) mesecev, čas preživetja brez napredovanja bolezni pa 11,2 (10,2-12,2) mesecev. Stadij IIIA, ženski spol, odsotnost hujšanja, preverjanje patoloških mediastinalnih bezgavk, operacija in kombinirana terapija so bili povezani z daljšim preživetjem. Zaključki. Raziskava vsakodnevne klinične prakse je pokazala široko heterogenost pri obravnavi bolnikov z nedrobnoceličnim rakom pljuč stadija III v srednjeevropskih državah. Pomembno bi bilo povečati število preiskav s pomočjo PET-CT-ja, slikovne preiskave možgan in invazivno mediastinalno diagnostiko. Radiol Oncol 2020; 54(2): I-XII. XII Slovenian abstracts Radiol Oncol 2020; 54(2): 221-226. doi: 10.2478/raon-2020-0028 Natančnost diagnosticiranja pljučnice povzročene s Pneumocystis jirovecii s testom (1^3)-ß-D-glucan pri bolnikih, ki niso okuženi s HIV Rogina P, Skvarč M Izhodišča. Pljučnica Pneumocystis jirovecii (P. jirovecii) je potencialno usodna oportunistična okužba pri imunsko ogroženih posameznikih, ki niso okuženi s HIV. Običajni diagnostični in klinični protokoli so premalo občutljivi in specifični, da bi zaznali to pljučnico. Naredili smo retrospektivno raziskavo in proučili več metod, ki smo jih uporabili v diagnostiki pljučnice povzročene s Pneumocystis jirovecii (PCP). Bolniki in metode. V raziskavo smo vključili 108 bolnikov z oslabljenim imunskim sistemom, s tipično klinično sliko pljučnice P. jirovecii in s sumljivimi radiološkimi izvidi. Vzorce seruma smo odvzeli za merjenje vrednosti (1^3)-ß-D-glukana (Fungitell, Associates of Cape Cod, ZDA). Vzorce iz spodnjih dihal smo pridobili za dokazovanje genoma P. jirovecii s pomočjo verižne polimerazne reakcije (qPCR). Rezultati. V raziskavi je 54 bolnikov (50 %) od 108 imelo (1^3)-ß-D-glukan > 500 pg/ml. Bolniki, ki so imeli v serumu koncentracije (1^3)-ß-D-glukana < 400 pg/ml, so imeli nižje koncentracije genoma P. jirovecii v dihalih. Število cilklov pomnoževanja genoma (ang. Cycle threshold [Ct]) je bilo 35,43 ± 3,32 v primerjavi s tistimi, ki so imeli (1 ^3)-ß-D- koncentracije glucan > 400 pg/ml in povprečen Ct 28,97 ± 5,27 (P < 0,001), in s tem večjo verjetnost, da imajo PCP. Če je koncentracija (1^3)-ß-D-glukana bila višja od 400 pg/ ml in je bila Ct vrednost qPCR-ja pod 28,97 ± 5,27, smo lahko bili z veliko verjetnostjo prepričani, da je P. jirovecii povzročila pljučnico (razmerje obetov [OR] 2,31, 95 % interval zaupanja [CI] 1,62-3,27, P < 0,001). Zaključki. Izključno merjenje (1^3)-ß-D-glukana ali samo rezultat qPCR-ja nista zadostna, da bi potrdili ali izključili pljučnico P. jirovecii. Vrednosti (1^3)-ß-D-glukana > 400 pg/ml in qPCR pod 30 Ct nam omogočata, da z veliko gotovostjo sodimo, da ima bolnik PCP. Če je vrednosti (1 ^3)-ß-D-glukana < 400 pg/ ml in je qPCR nad 35 Ct, je bolj verjetna kolonizacija dihal s P. jirovecii kot pa PCP. Radiol Oncol 2020; 54(2): 227-232. doi: 10.2478/raon-2020-0022 Stereotaktično obsevanje (SBRT) za zdravljenje primarnega pljučnega raka pri prejemnikih presajenih pljuč Moore A, Kramer MR, Rosengarten D, Shtraichman O, Zer A, Dudnik E, Korzets Y, Allen AM Izhodišča. Presaditev pljuč je reševalno zdravljenje za bolnike s pljučno boleznijo v terminalni fazi. Incidenca pljučnega raka pri prejemnikih presaditve pljuč bi lahko bila večja. Ti raki se po navadi odkrijejo v napredovalem stadiju. Podatkov o varnosti in učinkovitosti stereotaktičnega obsevanja (SBRT) za lezije preostalih lastnih pljuč pri prejemnikih presajenih pljuč je zelo malo. Bolniki in metode. Naredili smo retrospektivni pregled vseh bolnikov v Centru Davidoff Cancer, ki so bili predhodno zdravljeni s transplantacijo pljuč in so razvili rak v lastnih pljučih ter smo jih zdravili s tehniko SBRT. Rezultati. V analizo smo vključili štiri bolnike, ki smo jih zdravili s SBRT na skupno 5 lezij. Dva bolnika nista imela histološke potrditve malignosti. Vse primere smo pred napotitvijo na obsevanje obravnavali na multidisciplinarnem konziliju. Uporabili smo standardno zdravljenje SBRT. Odgovor smo ocenili s slikovno diagnostiko. Tri lezije so pokazale popoln odziv, dve leziji pa delen. Bolniki, ki so imeli delen odziv, so razvili oddaljene metastaze in so kmalu umrli. Pri nobenem bolniku nismo zabeležili toksičnosti. Zaključki. Tehnika SBRT j e učinkovita in varna za zdravljenje pljučnega raka pri bolnikih s presajenimi pljuči. Uporabljamo lahko standardno dozo in frakcionacijo. Radiol Oncol 2020; 54(2): I-XII. Slovenian abstracts III Radiol Oncol 2020; 54(2): 159-167. doi: 10.2478/raon-2020-0027 Uporaba sorafeniba pri zdravljenju hepatoceličnega raka. Retrospektivna monocentrična analiza Hanžel J, Košir Božič T, Štabuc B, Janša R Izhodišča. Sorafenib je peroralni zaviralec multikinaze, ki ga uporabljamo pri zdravljenje hepatoceličnega raka. Njegovo učinkovitost smo preizkušali v naključnih kontroliranih preizkusih pri bolnikih z dobro ohranjenim delovanjem jeter in dobrim funkcionalnim statusom. V praksi je bolnikom pogosto na voljo zdravljenje tudi zunaj teh meril. Zato smo izvedli kohortno raziskavo o učinkovitosti sorafeniba pri bolnikih s hepatocelularnim rakom. Bolniki in metode. V raziskavo smo vključili vse bolnike s hepatocelularnim rakom, ki so pričeli zdravljenje s sorafenibom med januarjem 2015 in januarjem 2018 na Kliničnem oddelku za gastroenterologijo UKC Ljubljana. Primarni cilj raziskave je bil ugotoviti celokupno preživetje od začetka uporabe sorafeniba. Preučevali smo klinične in demografske spremenljivke, povezane s preživetjem. Rezultati. Srednje celokupno preživetje je bilo 13,4 meseca (95 % interval zaupanja [CI] 8,2-18,6). Pri multivariatni Cox regresiji so bili neugodni napovedni dejavniki slabo stanje telesne zmogljivosti (ECOG PS) (razmerje ogroženosti [HR] 2,21; 95 % CI 1,56-3,16; P < 0,0001), razred Child-Pugh C (HR 52,4; 95% CI 3,20-859; P = 0,005) in odsotnost predhodnega locoregionalnega zdravljenja (HR 2,30; 95% IZ 1,37-3,86; P = 0,002) ter so bili povezani s povečano smrtnostjo. Zaključki. Za optimizacijo zdravljenja oziroma boljše preživetje je potrebna skrbna izbira bolnikov za zdravljenje s sorafenibom. Radiol Oncol 2020; 54(2): 237-246. doi: 10.2478/raon-2020-0016 Sarkopenija in miosteatoza ob začetku zdravljenja negativno vplivata na preživetje po ezofagektomiji zaradi raka požiralnika Srpčič M, Jordan T, Popuri K, Sok M Izhodišča. Rak požiralnika ostaja bolezen s slabim preživetjem in številnimi zapleti. Merjenje mišične mase in in njene kakovosti lahko prepozna bolnike z zmanjšano mišično maso (sarkopenijo) in infiltracijo mišic z maščevjem (miosteatozo). Preučevali smo vpliv sarkopenije in miosteatoze pri bolnikih z resektabilnim rakom požiralnika na celokupno preživetje in zaplete. Bolniki in metode. Pri 139 bolnikih smo napravili radikalno ezofagektomijo. Izmerili smo površino skeletne mišičnine in mišično atenuacijo na posnetkih CT v višini vretenca L3 in primerjali celokupno preživetje, perioperativno smrtnost, zaplete na presadku, plevropulmonalne zaplete, dihalno odpoved in druge značilne zaplete med skupinami z in brez sarkopenije in miosteatoze. Rezultati. Prevalenci sarkopenije in miosteatoze ob začetku zdravljenja sta bili 16,5 % in 51,8 %. Obe sta bili povezani z zmanjšanim celokupnim preživetjem. Mediano preživetje je bilo 18,3 mesecev (interval zaupanja [CI] 5,4-31,1) proti 31,0 mesecev (CI 7,4-54,6) za sarkopenijo/brez sarkopenije (log rank p = 0,042) in 19,0 mesecev (CI 13,3-24,7) proti 57,1 mesecev (CI 15,2-99,0) za miosteatozo (log rank p=0,044). Povezave med sarkopenijo in miosteatozo ter drugimi negativnimi izidi po ezofagektomiji nismo uspeli odkriti. Zaključki. Sarkopenija in miosteatoza ob diagnozi pred ezofagektomijo sta povezani s slabšim celokupnim preživetjem, ne pa z bolj pogostimi perioperativnimi zapleti. Prepoznava bolnikov s povečanim tveganjem lahko pomaga pri odločitvah o zdravljenju in ukrepih, ki naj povrnejo mišične zaloge. Radiol Oncol 2020; 54(2): I-XII. XII Slovenian abstracts Radiol Oncol 2020; 54(2): 247-252. doi: 10.2478/raon-2020-0024 Vpliv povečanja ščitenja v majhnem obsevalnem prostoru na ekvivalentno nevtronsko dozo v bližini linearnega pospeševalnika Ivkovič A, Faj D, Kasabašič M, Poje Sovilj M, Krpan I, Grabar Branilovič M, Brkič H Izhodišča. Visoko energijske linearne pospeševalnike (LINAC), ki proizvajajo fotonske žarke z energijami višjimi od 10 MeV, pogosto uporabljamo v radioterapiji. Pri teh energijah nastajajo hitri nevtroni, ki neželeno kontaminirajo terapevtske žarke. V raziskavi smo želeli določiti nevtronski spekter in dozne ekvivalente v okolici linearnega pospeševalnika z dvema metodama, z meritvami in s simulacijo Monte Carlo (MC). Materiali in metode. Linearni pospeševalnik Siemens Oncor Expression univerzitetne bolnišnice v Osijeku smo postavili v obsevalni prostor, v katerem je bil predhodno obsevalni aparat z virom 60Co. Zaščito v prostoru smo povečali z vgradnjo svinčenih in jeklenih plošč. Meritve nevtronske doze smo izvedli z detektorji CR-39. Energijsko odvisnost detektorjev smo kompenzirali z izračuni energijskega spektra nevtronov z uporabo simulacij Monte Carlo. Rezultati. Večina nevtronov ima izvor v glavi linearnega pospeševalnika. Simulacije Monte Carlo so pokazale pomembne razlike v nevtronskem spektru v odvisnosti od mesta meritev. Letna ekvivalentna nevtronska doza na mestih izven obsevalne sobe je bila ocenjena na manj kot 324 |jSv. Zaključki. Odziv detektorjev, ki smo jih uporabili v raziskavi je bil močno odvisen od energije nevtronov, zato je zelo pomembno, da poznamo nevtronski spekter na mestih, kjer smo izvajali meritve. Čeprav bi nevtronsko dozo načeloma morali upoštevati pri klinični dozimetriji, smo v razsikavi pokazali, da je ocenjena letna nevtronska doza precej nižja od mejnih doz ionizirajočega sevanja, ki so predpisane za izpostavljene delavce. Radiol Oncol 2020; 54(2): I-XII. Fundacija DR J,CHOI>m Fundacija "Docent dr. J. ChoLewa" je neprofitno, neinstitucionamo in nestrankarsko združenje posameznikov, ustanov in organizacij, ki želijo materiaLno spodbujati in pogLabLjati raziskovaLno dejavnost v onkologui. DunAjska 106 1000 Ljubljana IBAN: SI56 0203 3001 7879 431 fundacija dh.j.cholkwa Activity of "Dr. J. Cholewa" Foundation for Cancer Research and Education - a report for the second quarter of 2020 Doc. Dr. Josip Cholewa Foundation for cancer research and education continues with its planned activities in the second quarter of 2020. 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 respectable impact factor that publishes research and review articles about all aspects of cancer. The magazine is edited and published in Ljubljana, Slovenia. »Radiology and Oncology« is an open access journal available to everyone free of charge. Its long tradition represents a guarantee for the continuity of international exchange of ideas and research results in the field of oncology for all in Slovenia that are interested and involved in helping people affected by many different aspects of cancer. 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 treatment that is successful reflects in results with longer survival in many patients with previously incurable cancer conditions. Thus adding many new dimensions in life of cancer survivors and their families. Viljem Kovač, M.D., Ph.D. Borut Štabuc, M.D., Ph.D. Tomaž Benulič, M.D. Andrej Plesničar, M.D., M.Sc. TANTUM VERDE' Za lajšanje bolečine in oteklin v ustni votlini in žrelu, ki so posledica radiomukozitisa Bistvene informacije iz Povzetka glavnih značilnosti zdravila Tantum Verde 1,5 mg/ml oralno pršilo, raztopina Tantum Verde 3 mg/ml oralno pršilo, raztopina Sestava 1,5 mg/ml: 1 ml raztopine vsebuje 1,5 mg benzidaminijevega klorida, kar ustreza 1,34 mg benzidamina. V enem razpršku je 0,17 ml raztopine. En razpršek vsebuje 0,255 mg benzidaminijevega klorida, kar ustreza 0,2278 mg benzidamina. Sestava 3 mg/ml: 1 ml raztopine vsebuje 3 mg benzidaminijevega klorida, kar ustreza 2,68 mg benzidamina. V enem razpršku je 0,17 ml raztopine. En razpršek vsebuje 0,51 mg benzidaminijevega klorida, kar ustreza 0,4556 mg benzidamina. Terapevtske indikacije: Samozdravljenje: Lajšanje bolečine in oteklin pri vnetju v ustni votlini in žrelu, ki so lahko posledica okužb in stanj po operaciji. Po nasvetu in navodilu zdravnika: Lajšanje bolečine in oteklin v ustni votlini in žrelu, ki so posledica radiomukozitisa. Odmerjanje in način uporabe: Odmerjanje 1.5 ma/ml: Odrasli: 4 do 8 razprškov 2- do 6-krat na dan (vsake 1,5 do 3 ure). Pediatrična populacija: Mladostniki, stari od 12 do 18 let: 4-8 razprškov 2- do 6-krat na dan. Otroci od 6 do 12 let: 4 razprški 2- do 6-krat na dan. Otroci, mlajši od 6 let: 1 razpršek na 4 kg telesne mase; do največ 4 razprške 2- do 6-krat na dan. Odmerjanje 3 ma/ml: Uporaba 2- do 6-krat na dan (vsake 1,5 do 3 ure). Odrasli: 2 do 4 razprški 2- do 6-krat na dan. Pediatrična populacija: Mladostniki, stari od 12 do 18 let: 2 do 4 razprški 2- do 6-krat na dan. Otroci od6do12 let: 2 razprška 2- do 6-krat na dan. Otroci, mlajši od 6 let: 1 razpršek na 8 kg telesne mase; do največ 2 razprška 2- do 6-krat na dan. Starejši bolniki, bolniki z jetrno okvaro in bolniki z ledvično okvaro: Uporabo oralnega pršila z benzidaminijevim kloridom se svetuje pod nadzorom zdravnika. Način uporabe: Za orofaringealno uporabo. Zdravilo se razprši v usta in žrelo. Kontraindikacije: Preobčutljivost na učinkovino ali katero koli pomožno snov. Posebna opozorila in previdnostni ukrepi: Če se simptomi v treh dneh ne izboljšajo, se mora bolnik posvetovati z zdravnikom ali zobozdravnikom, kot je primerno. Benzidamin ni priporočljiv za bolnike s preobčutljivostjo nasalicilno kislino ali druga nesteroidna protivnetna zdravila. Pri bolnikih, ki imajo ali so imeli bronhialno astmo, lahko pride do bronhospazma, zato je potrebna previdnost. To zdravilo vsebuje majhne količine etanola (alkohola), in sicer manj kot 100 mg na odmerek. To zdravilo vsebuje metilpar-ahidroksibenzoat (E218). Lahko povzroči alergijske reakcije (lahko zapoznele). Zdravilo z jakostjo 3 mg/ml vsebuje makrogolglicerol hidroksistearat 40. Lahko povzroči želodčne težave in drisko. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Študij medsebojnega delovanja niso izvedli. Nosečnost in dojenje: O uporabi benzidamina pri nosečnicah in doječih ženskah ni zadostnih podatkov. Uporaba zdravila med nosečnostjo in dojenjem ni priporočljiva. Vpliv na sposobnost vožnje in upravljanja strojev: Zdravilo v priporočenem odmerku nima vpliva na sposobnost vožnje in upravljanja strojev. Neželeni učinki: Neznana pogostnost (ni mogoče oceniti iz razpoložljivih podatkov): anafilaktične reakcije, preobčutljivostne reakcije, odrevenelost, laringospazem, suha usta, navzea in bruhanje, angioedem, fotosenzitivnost, pekoč občutek v ustih. Neposredno po uporabi se lahko pojavi občutek odrevenelosti v ustih in v žrelu. Ta učinek se pojavi zaradi načina delovanja zdravila in po kratkem času izgine. Način in režim izdaje zdravila: BRp-Izdaja zdravila je brez recepta v lekarnah in specializiranih prodajalnah. Imetnik dovoljenja za promet: Aziende Chimiche Riunite Angelini Francesco - A.C.R.A.F. S.p.A., Viale Amelia 70, 00181 Rim, Italija Datum zadnje revizije besedila: 14. 10. 2019 Pred svetovanjem ali izdajo preberite celoten Povzetek glavnih značilnosti zdravila. CM 5 čo o O! A i m ANGELINI £ Samo za strokovno javnost. Datum priprave informacije: november 2019 Odgovoren za trženje: Bonifar d.o.o. EDINI zaviralec CDK4 & 6, ki se jemlje NEPREKINJENO VSAK DAN, 2x NA DAN1'2 3 abemaciklib dvakrat na dan 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. Glejte poglavje 4.8, kako poročati o neželenih učinkih. IME ZDRAVILA: Verzenios 50 mg/100 mg/150 mg filmsko obložene tablete KAKOVOSTNA IN KOLIČINSKA SESTAVA: Ena filmsko obložena tableta vsebuje 50 mg/100 mg/150 mg abemacikliba. Ena filmsko obložena tableta vsebuje 14 mg/28 mg/42 mg laktoze (v obliki monohidrata). Terapevtske indikacije: Zdravilo Verzeniosje indicirano za zdravljenje žensk z lokalno napredovalim ali metastatskim, na hormonske receptorje (HR - Hormone Receptor) pozitivnim in na receptorje humanega epidermalnega rastnega faktorja 2 (HER2 - Human Epidermal Growth Factor Receptor 2) negativnim rakom dojk v kombinaciji z zaviralcem aromataze ali s fulvestrantom kot začetnim endokrinim zdravljenjem ali pri ženskah, ki so prejele predhodno endokrino zdravjenje. Pri ženskah v pred- in perimenopavzi je treba endokrino zdravjenje kombinirati z agonistom gonadoliberina (LHRH - Luteinizing Hormone-Releasing Hormone). Odmerjanje in način uporabe: Zdravjenje z zdravilom Verzenios mora uvesti in nadzorovati zdravnik, ki ima izkušnje z uporabo zdravil za zdravjenje rakavih bolezni. Zdravilo Verzenios v kombinaciji z endokrinim zdravljenjem: Priporočeni odmerek abemacikliba je 150 mg dvakrat na dan, kadar se uporabja v kombinaciji z endokrinim zdravljenjem. Zdravilo Verzenios je treba jemati, dokler ima bolnica od zdravjenja klinično korist ali do pojava nesprejemljive toksičnosti. Če bolnica bruha ali izpusti odmerek zdravila Verzenios, ji je treba naročiti, da naj naslednji odmerek vzame ob predvidenem času; dodatnega odmerka ne sme vzeti. Obvladovanje nekaterih neželenih učinkov lahko zahteva prekinitev in/ali zmanjšanje odmerka. Zdravjenje z abemaciklibom prekinite v primeru povišanja vrednosti AST in/ali ALT >3 x ZMN SKUPAJ s celokupnim bilirubinom > 2,0 x ZMNI v odsotnosti holestaze ter pri bolnicah z intersticjsko pjučno boleznjo (ILD)/pnevmonitis stopnje 3 ali 4. Sočasni uporabi močnih zaviralcev CYP3A4 se je treba izogibati. Če se uporabi močnih zaviralcev CYP3A4 ni mogoče izogniti, je treba odmerek abemacikliba znižati na 100 mg dvakrat na dan. Pri bolnicah, pri katerih je bil odmerek znižan na 100 mg abemacikliba dvakrat na dan in pri katerih se sočasnemu dajanju močnega zaviralca CYP3A4 ni mogoče izogniti, je treba odmerek abemacikliba dodatno znižati na 50 mg dvakrat na dan. Pri bolnicah, pri katerih je bil odmerek znižan na 50 mg abemacikliba dvakrat na dan in pri katerih se sočasnemu dajanju močnega zaviralca CYP3A4 ni mogoče izogniti, je mogoče z odmerkom abemaciklibanadajevati ob natančnem spremjanju znakov toksičnosti. Alternativno je mogoče odmerek abemacikliba znižati na 50 mg enkrat na dan ali prekiniti dajanje abemacikliba. Če je uporaba zaviralca CYP3A4 prekinjena, je treba odmerek abemacikliba povečati na odmerek, kakršen je bil pred uvedbo zaviralca CYP3A4 (po 3-5 razpolovnih časih zaviralca CYP3A4). Prilagajanje odmerka glede na starost in pri bolnicah z blago ali zmerno ledvično okvaro ter z blago (Child Pugh A) ali zmerno (Child Pugh B) jetrno okvaro ni potrebno. Pri dajanju abemacikliba bolnicam s hudo ledvično okvaro sta potrebna previdnost in skrbno spremjanje glede znakov toksičnosti. Način uporabe: Zdravilo Verzenios je namenjeno za peroralno uporabo. Odmerek se lahko vzame s hrano ali brez nje. Zdravilo se ne sme jemati z grenivko ali grenivkinim sokom. Bolnice naj odmerke vzamejo vsak dan ob približno istem času. Tableto je treba zaužiti celo (bolnice je pred zaužitjem ne smejo gristi, drobiti ali deliti). Kontraindikacije: Preobčutljivost na učinkovino ali katero koli pomožno snov. Posebna opozorila in previdnostni ukrepi: Pri bolnicah, ki so prejemale abemaciklib, so poročali o nevtropenji, o večji pogostnosti okužb kot pri bolnicah, zdravjenih s placebom in endokrinim zdravljenjem, o povečanih vrednostih ALT in AST. Pri bolnicah, pri katerih se pojavi nevtropenja stopnje 3 ali 4, je priporočljivo prilagoditi odmerek. Bolnice je treba spremjati za znake in simptome globoke venske tromboze in pjučne embolje ter jih zdraviti, kot je medicinsko utemejeno. Glede na povečanje vrednosti ALT ali AST je mogoče potrebna prilagoditev odmerka. Driska je najpogostejši neželeni učinek. Bolnice je treba ob prvem znaku tekočega blata začeti zdraviti z antidiaroiki, kot je loperamid, povečati vnos peroralnih tekočin in obvestiti zdravnika. Sočasni uporabi induktorjev CYP3A4 se je treba izogibati zaradi tveganja za zmanjšano učinkovitost abemacikliba. Bolnice z redkimi dednimi motnjami, kot so intoleranca za galaktozo, popolno pomanjkanje laktaze ali malapsorpcja glukoze/galaktoze, tega zdravila ne smejo jemati. Bolnice spremjajte glede pljučnih simptomov, ki kažejo na ILD/pnevmonitis, in jih ustrezno zdravite. Glede na stopnjo ILD/pnevmonitisa je morda potrebno prilagajanje odmerka abemacikliba. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Abemaciklib se primarno presnavja s CYP3A4. Sočasna uporaba abemacikliba in zaviralcev CYP3A4 lahko poveča plazemsko koncentracijo abemacikliba. Uporabi močnih zaviralcev CYP3A4 sočasno z abemaciklibom seje treba izogibati. Čeje močne zaviralce CYP3A4 treba dajati sočasno, je treba odmerek abemacikliba zmanjšati, nato pa bolnico skrbno spremjati glede toksičnosti. Pri bolnicah, zdravjenih z zmernimi ali šibkimi zaviralci CYP3A4, ni potrebno prilagajanje odmerka, vendar jih je treba skrbno spremjati za znake toksičnosti. Sočasni uporabi močnih induktorjev CYP3A4 (vkjučno, vendar ne omejeno na: karbamazepin, fenitoin, rifampicin in šentjanževko) se je treba izogibati zaradi tveganja za zmanjšano učinkovitost abemacikliba. Abemaciklib in njegovi glavni aktivni presnovki zavirajo prenašalce v ledvicah, in sicer kationski organski prenašalec 2 (OCT2) ter prenašalca MATE1. In vivo lahko pride do medsebojnega delovanja abemacikliba in klinično pomembnih substratov teh prenašalcev, kot je dofelitid ali kreatinin. Trenutno ni znano, ali lahko abemaciklib zmanjša učinkovitost sistemskih hormonskih kontraceptivov, zato se ženskam, ki uporabjajo sistemske hormonske kontraceptive, svetuje, da hkrati uporabjajo tudi mehansko metodo. Neželeni učinki: Najpogostejši neželeni učinki so driska, okužbe, nevtropenja, anemja, utrujenost, navzea, bruhanje in zmanjšanje apetita. Zelo pogosti: okužbe, nevtropenja, levkopenja, anemija, trombocitopenja, driska, bruhanje, navzea, zmanjšanje apetita, disgevzja, omotica, alopecja, pruritus, izpuščaj, utrujenost, pireksja, povečana vrednost alanin-aminotransferaze, povečana vrednost aspartat-a-minotransferaze Pogosti: limfopenja, povečano solzenje, venska trombembolja, intersticjska pjučna bolezen (ILD)/pnevmonitis, suha koža, mišična šibkost Občasni:febrilna nevtropenja Rok uporabnosti: 3 leta Posebna navodila za shranjevanje: Za shranjevanje zdravila niso potrebna posebna navodila. Imetnik dovoljenja za promet z zdravilom: Eli Lilly Nederland B.V., Papendorpseweg 83, 3528BJ, Utrecht, Nizozemska. Datum prve odobritve dovojenja za promet: 27. september 2018 Datum zadnje revizije besedila: 16.1.2020 Režim izdaje: Rp/Spec - Predpisovanje in izdaja zdravila je le na recept zdravnika specialista ustreznega področja medicine ali od njega pooblaščenega zdravnika. Reference: 1. Povzetek glavnih značilnosti zdravila Verzenios. Datum zadnje revizije besedila: 16.1.2020. 2. Povzetek glavnih značilnosti zdravila Ibrance. Dostop preverjen 10.4.2020. 3. Povzetek glavnih značilnosti zdravila Kisqali. Dostop preverjen 10.4.2020. Pomembno: Predpisovanje in izdaja zdravila je le na recept zdravnika specialista ustreznega področja medicine ali od njega pooblaščenega zdravnika. Pred predpisovanjem zdravila Verzenios si preberite zadnji veljavni Povzetek glavnih značilnosti zdravil. Podrobne informacije o zdravilu so objavljene na spletni strani Evropske agencije za zdravila http://www.ema.europa.eu Eli Lilly farmacevtska družba, d.o.o., Dunajska cesta 167, 1000 Ljubljana, telefon 01 / 580 00 10, faks 01 / 569 17 05 PP-AL-SI-0059, 20.04.2020, Samo za strokovno javnost. Pomaga spreminjati pričakovanja o preživetju (pembrolizumab, MSD) • pri metastatskem NSCLC *1,2 • in napredovalem melanomu3 Reference: 1. Gandhi L, Rodriguez-Abreu D, Gadgeel S, et. al.; for the KEYNO-TE-189 investigators. Pembrolizumab plus chemotherapy in metastatic non-small-cell lung cancer. N Engl J Med. 2018;378(22):2078-2092. 2. Keytruda EU SmPC 3. Hamid O, Robert C, Daud A, et. al. 5-year survival outcomes for patients with advanced melanoma treated with pembrolizumab in KEYNOTE-001. Annals of Oncology 2019; 30: 582-588. SKRAJŠAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA Pred predpisovanjem, prosimo, preberite celoten Povzetek glavnih značilnosti zdravila! Ime zdravila: KEYTRUDA 25 mg/ml koncentrat za raztopino za infundiranje vsebuje pembrolizumab. Terapevtske indikacije: Zdravilo KEYTRUDA je kot samostojno zdravljenje indicirano za zdravljenje: napredovalega (neoperabilnega ali metastatskega) melanoma pri odraslih; za adjuvantno zdravljenje odraslih z mela-nomom v stadiju III, ki se je razširil na bezgavke, po popolni kirurški odstranitvi; metastatskega nedrobnoceličnega pljučnega raka (NSCLC) v prvi liniji zdravljenja pri odraslih, ki imajo tumorje z > 50 % izraženostjo PD-L1 (TPS) in brez pozitivnih tumorskih mutacij EGFR ali ALK; lokalno napredovalega ali metastatskega NSCLC pri odraslih, ki imajo tumorje z > 1 % izraženostjo PD-L1 (TPS) in so bili predhodno zdravljeni z vsaj eno shemo kemoterapije, bolniki s pozitivnimi tumorskimi mutacijami EGFR ali ALK so pred prejemom zdravila KEYTRUDA morali prejeti tudi tarčno zdravljenje; odraslih bolnikov s ponovljenim ali neodzivnim klasičnim Hodgkinovim limfomom (cHL), pri katerih avtologna presaditev matičnih celic (ASCT) in zdravljenje z brentuksimabom vedotinom (BV) nista bila uspešna, in odraslih bolnikov, ki za presaditev niso primerni, zdravljenje z BV pa pri njih ni bilo uspešno; lokalno napredovalega ali metastatskega urotelijskega raka pri odraslih, predhodno zdravljenih s kemoterapijo, ki je vključevala platino; lokalno napredovalega ali metastatskega urotelijskega raka pri odraslih, ki niso primerni za zdravljenje s kemoterapijo, ki vsebuje cisplatin in imajo tumorje z izraženostjo PD-L1 > 10, ocenjeno s kombinirano pozitivno oceno (CPS); ponovljenega ali metastatskega ploščatoceličnega raka glave in vratu (HNSCC) pri odraslih, ki imajo tumorje z > 50 % izraženostjo PD-L1 (TPS), in pri katerih je bolezen napredovala med zdravljenjem ali po zdravljenju s kemoterapijo, ki je vključevala platino. Zdravilo KEYTRUDA je kot samostojno zdravljenje ali v kombinaciji s kemoterapijo s platino in 5-fluorouracilom (5-Fu) indicirano za prvo linijo zdravljenja metastatskega ali neoperabilnega ponovljenega ploščatoceličnega raka glave in vratu pri odraslih, ki imajo tumorje z izraženostjo PD-L1 s CPS > 1. Zdravilo KEYTRUDA je v kombinaciji s pemetreksedom in kemoterapijo na osnovi platine indicirano za prvo linijo zdravljenja metastatskega neploščatoceličnega NSCLC pri odraslih, pri katerih tumorji nimajo pozitivnih mutacij EGFR ali ALK; v kombinaciji s karbopla-tinom in bodisi paklitakselom bodisi nab-paklitakselom je indicirano za prvo linijo zdravljenja metastatskega ploščatoceličnega NSCLC pri odraslih; v kombinaciji z aksitinibom je indicirano za prvo linijo zdravljenja napredovalega raka ledvičnih celic (RCC) pri odraslih. Odmerjanje in način uporabe: Testiranje PD-L1 pri bolnikih z NSCLC, urotelijskim rakom ali HNSCC: Za samostojno zdravljenje z zdravilom KEYTRUDA je priporočljivo opraviti testiranje izraženosti PD-L1 tumorja z validira-no preiskavo, da izberemo bolnike z NSCLC ali predhodno nezdravljenim urotelijskim rakom. Bolnike s HNSCC je treba za samostojno zdravljenje z zdravilom KEYTRUDA ali v kombinaciji s kemoterapijo s platino in 5-fluorouracilom (5-FU) izbrati na podlagi izraženosti PD-L1, potrjene z validirano preiskavo. Odmerjanje: Priporočeni odmerek zdravila KEYTRUDA za samostojno zdravljenje je bodisi 200 mg na 3 tedne ali 400 mg na 6 tednov, apliciran z intravensko infuzijo v 30 minutah. Priporočeni odmerek za kombinirano zdravljenje je 200 mg na 3 tedne, apliciran z intravensko infuzijo v 30 minutah. Za uporabo v kombinaciji glejte povzetke glavnih značilnosti sočasno uporabljenih zdravil. Če se uporablja kot del kombiniranega zdravljenja skupaj z intravensko kemoterapijo, je treba zdravilo KEYTRUDA aplicirati prvo. Bolnike je treba zdraviti do napredovanja bolezni ali nesprejemljivih toksičnih učinkov. Pri adjuvantnem zdravljenju melanoma je treba zdravilo uporabljati do ponovitve bolezni, pojava nesprejemljivih toksičnih učinkov oziroma mora zdravljenje trajati do enega leta. Če je aksitinib uporabljen v kombinaciji s pembrolizumabom, se lahko razmisli o povečanju odmerka aksiti-niba nad začetnih 5 mg v presledkih šest tednov ali več. Pri bolnikih starih > 65 let, bolnikih z blago do zmerno okvaro ledvic, bolnikih z blago okvaro jeter prilagoditev odmerka ni potrebna. Odložitev odmerka ali ukinitev zdravljenja: Zmanjšanje odmerka zdravila KEYTRUDA ni priporočljivo. Za obvladovanje neželenih učinkov je treba uporabo zdravila KEYTRUDA zadržati ali ukiniti, prosimo, glejte celoten Povzetek glavnih značilnosti zdravila. Kontraindikacije: Preobčutljivost na učinkovino ali katero koli pomožno snov. Povzetek posebnih opozoril, previdnostnih ukrepov, interakcij in neželenih učinkov: Imunsko pogojeni neželeni učinki (pnevmonitis, kolitis, hepatitis, nefritis, endokrinopatije, neželeni učinki na kožo in drugi): Pri bolnikih, ki so prejemali pembrolizumab, so se pojavili imunsko po- gojeni neželeni učinki, vključno s hudimi in smrtnimi primeri. Večina imunsko pogojenih neželenih učinkov, ki so se pojavili med zdravljenjem s pembrolizumabom, je bila reverzibilnih in so jih obvladali s prekinitvami uporabe pembroli-zumaba, uporabo kortikosteroidov in/ali podporno oskrbo. Pojavijo se lahko tudi po zadnjem odmerku pembrolizumaba in hkrati prizadanejo več organskih sistemov. V primeru suma na imunsko pogojene neželene učinke je treba poskrbeti za ustrezno oceno za potrditev etiologije oziroma izključitev drugih vzrokov. Glede na izrazitost neželenega učinka je treba zadržati uporabo pembrolizumaba in uporabiti kortikosteroide - za natančna navodila, prosimo, glejte Povzetek glavnih značilnosti zdravila Keytruda. Zdravljenje s pembrolizumabom lahko poveča tveganje za zavrnitev pri prejemnikih presadkov čvrstih organov. Pri bolnikih, ki so prejemali pembrolizumab, so poročali o hudih z infuzijo povezanih reakcijah, vključno s preobčutljivostjo in anafilaksijo. Pembrolizumab se iz obtoka odstrani s katabolizmom, zato presnovnih medsebojnih delovanj zdravil ni pričakovati. Uporabi sistemskih kortikosteroidov ali imunosupresivov pred uvedbo pembrolizumaba se je treba izogibati, ker lahko vplivajo na farmakodinamično aktivnost in učinkovitost pembrolizumaba. Vendar pa je kortikosteroide ali druge imunosup-resive mogoče uporabiti za zdravljenje imunsko pogojenih neželenih učinkov. Kortikosteroide je mogoče uporabiti tudi kot premedikacijo, če je pembrolizumab uporabljen v kombinaciji s kemoterapijo, kot antiemetično profilakso in/ali za ublažitev neželenih učinkov, povezanih s kemoterapijo. Ženske v rodni dobi morajo med zdravljenjem s pembrolizumabom in vsaj še 4 mesece po zadnjem odmerku pembrolizumaba uporabljati učinkovito kontracepcijo, med nosečnostjo in dojenjem se ga ne sme uporabljati. Varnost pembrolizumaba pri samostojnem zdravljenju so v kliničnih študijah ocenili pri 5.884 bolnikih z napredovalim mela-nomom, kirurško odstranjenim melanomom v stadiju III (adjuvantno zdravljenje), NSCLC, cHL, urotelijskim rakom ali HNSCC s štirimi odmerki (2 mg/kg na 3 tedne, 200 mg na 3 tedne in 10 mg/kg na 2 ali 3 tedne). V tej populaciji bolnikov je mediani čas opazovanja znašal 7,3 mesece (v razponu od 1 dneva do 31 mesecev), najpogostejši neželeni učinki zdravljenja s pembrolizumabom so bili utrujenost (32 %), navzea (20 %) in diareja (20 %). Večina poročanih neželenih učinkov pri samostojnem zdravljenju je bila po izrazitosti 1. ali 2. stopnje. Najresnejši neželeni učinki so bili imunsko pogojeni neželeni učinki in hude z infuzijo povezane reakcije. Varnost pembrolizumaba pri kombiniranem zdravljenju s kemoterapijo so ocenili pri 1.067 bolnikih NSCLC ali HNSCC, ki so v kliničnih študijah prejemali pembrolizumab v odmerkih 200 mg, 2 mg/kg ali 10 mg/kg na vsake 3 tedne. V tej populaciji bolnikov so bili najpogostejši neželeni učinki naslednji: anemija (50 %), navzea (50 %), utrujenost (37 %), zaprtost (35%), diareja (30 %), nevtropenija (30 %), zmanjšanje apetita (28 %) in bruhanje (25 %). Pri kombiniranem zdravljenju s pembrolizumabom je pri bolnikih z NSCLC pojavnost neželenih učinkov 3. do 5. stopnje znašala 67 %, pri zdravljenju samo s kemoterapijo pa 66 %, pri kombiniranem zdravljenju s pembrolizumabom pri bolnikih s HNSCC 85 % in pri zdravljenju s kemoterapijo v kombinaciji s cetuksimabom 84 %. Varnost pembrolizumaba v kombinaciji z aksitinibom so ocenili v klinični študiji pri 429 bolnikih z napredovalim rakom ledvičnih celic, ki so prejemali 200 mg pembrolizumaba na 3 tedne in 5 mg aksitiniba dvakrat na dan. V tej populaciji bolnikov so bili najpogostejši neželeni učinki diareja (54 %), hipertenzija (45 %), utrujenost (38 %), hipotiroidizem (35 %), zmanjšan apetit (30 %), sindrom palmarno-plantarne eritrodisestezije (28 %), navzea (28 %), zvišanje vrednosti ALT (27 %), zvišanje vrednosti AST (26 %), disfo-nija (25 %), kašelj (21 %) in zaprtost (21 %). Pojavnost neželenih učinkov 3. do 5. stopnje je bila med kombiniranim zdravljenjem s pembrolizumabom 76 % in pri zdravljenju s sunitinibom samim 71 %. Za celoten seznam neželenih učinkov, prosimo, glejte celoten Povzetek glavnih značilnosti zdravila. Način in režim izdaje zdravila: H - Predpisovanje in izdaja zdravila je le na recept, zdravilo se uporablja samo v bolnišnicah. Imetnik dovoljenja za promet z zdravilom: Merck Sharp & Dohme B.V. , Waarderweg 39, 2031 BN Haarlem, Nizozemska. Datum zadnje revizije besedila: 24. marec 2020. ^MSD Merck Sharp & Dohme inovativna zdravila d.o.o., Šmartinska cesta 140, 1000 Ljubljana tel: +386 1/ 520 42 01, fax: +386 1/ 520 43 50 Vse pravice pridržane Pripravljeno v Sloveniji, april 2020; SI-KEY-00091 EXP: 04/2022 Samo za strokovno javnost. H - Predpisovanje in izdaja zdravila je le na recept, zdravilo pa se uporablja samo v bolnišnicah. Pred predpisovanjem, prosimo, preberite celoten Povzetek glavnih značilnosti zdravila Keytruda, ki je na voljo pri naših strokovnih sodelavcih ali na lokalnem sedežu družbe. <(Ročhe> Za bolnike s HER2 pozitivnim zgodnjim rakom dojk z ostankom invazivne bolezni po neoadjuvantnem zdravljenju s taksani in proti HER2 usmerjenim zdravljenjem1 PODALJŠAMO PREŽIVETJE* BOLNIKOM Z ZGODNJIM HER2 POZITIVNIM RAKOM DOJK. Kadcyla trastuzumab emtanzin SKRAJSAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA Kadcyla 100 mg in 160 mg prašek za koncentrat za raztopino za infundiranje Ime zdravila: Kadcyla 100 mg in 160 mg prašek za koncentrat za raztopino za infundiranje. Kakovostna in količinska sestava: Kadcyla l0o mg: Ena viala praška za koncentrat za raztopino za infundiranje vsebuje 100 mg trastuzumaba emtanzina. Po rekonstituciji vsebuje ena viala s 5 ml raztopine 20 mg/ml trastuzumaba emtanzina. Kadcyla 160 mg: Ena viala praška za koncentrat za raztopino za infundiranje vsebuje 160 mg trastuzumaba emtanzina. Po rekonstituciji vsebuje ena viala z 8 ml raztopine 20 mg/ml trastuzumaba emtanzina. Trastuzumab emtanzin je konjugirano zdravilo iz protitelesa trastuzumaba in zaviralca mikrotubulov DM1. Terapevtske indikacije: Zgodnji rak dojk: Zdravilo Kadcyla je kot monoterapija indicirano za adjuvantno zdravljenje odraslih bolnikov s HER2-pozitivnim zgodnjim rakom dojk, ki imajo rezidualno invazivno bolezen v dojki in/ali bezgavkah, po neoadjuvantnem zdravljenju na osnovi taksanov in proti HER2 usmerjene terapije. Razsejani rak dojk: Zdravilo Kadcyla je kot monoterapija indicirano za zdravljenje odraslih bolnikov s HER2-pozitivnim, neoperabilnim, lokalno napredovalim ali razsejanim rakom dojk, predhodno zdravljenih s trastuzumabom in taksanom, samostojno ali v kombinaciji. Bolniki so pred tem: prejemali predhodno zdravljenje za lokalno napredovalo ali razsejano bolezen ali se jim je bolezen ponovila med adjuvantnim zdravljenjem ali v šestih mesecih po koncu adjuvantnega zdravljenja. Odmerjanje in način uporabe: Zdravilo Kadcyla sme predpisati le zdravnik in se ga sme v obliki intravenske infuzije uporabiti le pod nadzorom zdravnika, ki ima izkušnje z zdravljenjem onkoloških bolnikov. Bolniki, zdravljeni s trastuzumabom emtanzinom, morajo imeti HER2-pozitivni tumor, imunohistokemično opredeljen kot 3+ ali razmerje pri in situ hibridizaciji (ISH) ali fluorescentni in situ hibridizaciji (FISH) > 2,0, določeno z validiranim testom (z diagnostičnim medicinskim pripomočkom in vitro z oznako CE). Če takšnega pripomočka z oznako CE ni na voljo, je treba stanje HER2 oceniti z drugim validiranim testom. Za preprečitev napak pri dajanju zdravila je pomembno preveriti nalepke na vialah in tako zagotoviti, da je pripravljeno in uporabljeno zdravilo res zdravilo Kadcyla (trastuzumab emtanzin) in ne zdravilo Herceptin (trastuzumab). Odmerjanje: Priporočeni odmerek trastuzumaba emtanzina je 3,6 mg/kg telesne mase v intravenski infuziji na 3 tedne. Začetni odmerek je treba dati kot 90-minutno intravensko infuzijo. Bolnike je treba med infundiranjem in vsaj še 90 minut po prvem odmerku opazovati zaradi možnosti zvišanja telesne temperature, pojava mrzlice in drugih z infundiranjem povezanih reakcij. Mesto infundiranja je treba skrbno kontrolirati zaradi možne subkutane infiltracije med dajanjem zdravila. Če je bolnik predhodno infuzijo dobro prenesel, je mogoče poznejše odmerke trastuzumaba emtanzina dati kot 30-minutno infuzijo. Bolnike je treba opazovati med infundiranjem in vsaj še 30 minut po njem. Če se pojavijo z infundiranjem povezani simptomi, je treba hitrost infundiranja trastuzumaba emtanzina upočasniti ali infundiranje prekiniti. Pri življenje ogrožujočih infuzijskih reakcijah je treba trastuzumab emtanzin prenehati uporabljati. Trajanje zdravljenja: Zgodnji rak dojk (EBC): Bolniki naj prejmejo 14 ciklov zdravljenja v celoti, razen v primeru ponovitve bolezni ali neobvladljive toksičnosti. Razsejani rak dojk (MBC): Bolniki naj prejemajo zdravljenje do napredovanja bolezni ali neobvladljive toksičnosti. Prilagoditev odmerka: Obvladovanje simptomatskih neželenih učinkov lahko zahteva začasno prekinitev zdravljenja, zmanjšanje odmerka ali prenehanje zdravljenja trastuzumabom emtanzinom v skladu s smernicami (za podrobnejša navodila glede prilagoditve odmerka prosimo glejte SmPC zdravila). Odmerka trastuzumaba emtanzina se po zmanjšanju ne sme več povečati. Zapoznel ali izpuščen odmerek: Če je bolnik načrtovani odmerek izpustil, mu ga je treba dati čim prej brez čakanja do naslednjega načrtovanega cikla. Urnik uporabe je treba prilagoditi tako, da se ohrani 3-tedenski presledek med odmerki. Naslednji odmerek je treba uporabiti v skladu z zgornjimi priporočili za odmerjanje. Periferna nevropatija: Uporabo trastuzumaba emtanzina je treba prehodno prekiniti pri bolnikih s periferno nevropatijo 3. ali 4. stopnje, in sicer za toliko časa, da se zmanjša na < 2. stopnjo. Posebne populacije: Starejši bolniki: Bolnikom, starim > 65 let, odmerka ni treba prilagoditi. Za ugotovitev varnosti in učinkovitosti pri bolnikih, starih > 75 let, ni dovolj podatkov. Ledvična okvara: Bolnikom z blago ali zmerno ledvično okvaro začetnega odmerka ni treba prilagoditi. Bolnike s hudo ledvično okvaro je zato treba natančno kontrolirati. Jetrna okvara: Bolnikom z blago ali zmerno jetrno okvaro začetnega odmerka ni treba prilagoditi. Pri bolnikih s hudo jetrno okvaro trastuzumaba emtanzina niso preučevali. Pri zdravljenju bolnikov z jetrno okvaro moramo biti previdni zaradi znane hepatotoksičnosti, opažene pri uporabi trastuzumaba emtanzina. Pediatrična populacija: Varnost in učinkovitost pri otrocih in mladostnikih do 18. leta starosti nista ugotovljeni. Za indikacijo rak dojk pri pediatrični populaciji ni relevantne uporabe. Način uporabe: Zdravilo Kadcyla je namenjeno intravenski uporabi. Trastuzumab emtanzin mora pripraviti in razredčiti zdravstveni delavec. Zdravilo je treba dati kot intravensko infuzijo. Ne sme se ga dajati kot hitro intravensko infuzijo ali bolus. Kontraindikacije: Preobčutljivost na zdravilno učinkovino ali katero koli pomožno snov. Posebna opozorila in previdnostni ukrepi: Trombocitopenija: Stevilo trombocitov je priporočljivo kontrolirati pred vsakim odmerkom trastuzumaba emtanzina. Bolnike s trombocitopenijo in bolnike, ki prejemajo antikoagulante, je treba med zdravljenjem s trastuzumabom emtanzinom natančno kontrolirati. Krvavitve: V nekaterih od opaženih primerov so imeli bolniki trombocitopenijo ali pa so prejemali tudi antikoagulantno ali antitrombotično zdravljenje, pri drugih pa ni bilo znanih dodatnih dejavnikov tveganja. Pri uporabi teh zdravil je potrebna previdnost in razmisliti je treba o dodatnem nadzoru, kadar je sočasna uporaba klinično potrebna. Hepatotoksičnost: Delovanje jeter je treba kontrolirati pred uvedbo zdravljenja in pred vsakim odmerkom. Bolniki z izhodiščnim zvišanjem ALT imajo lahko večje tveganje za poškodbo jeter z večjim tveganjem za jetrne neželene dogodke stopnje 3 do 5 ali zvišanje jetrnih testov. Trastuzumab emtanzin ni bil raziskan pri bolnikih, ki imajo pred uvedbo zdravljenja serumske transaminaze > 2,5 ' ZNM ali celokupni bilirubin > 1,5 ' ZNM. Pri bolnikih, ki imajo serumske transaminaze > 3 ' ZNM in obenem celokupni bilirubin > 2 ' ZNM, je treba zdravljenje s trastuzumabom emtanzinom dokončno ukiniti. Pri zdravljenju bolnikov z jetrno okvaro moramo biti previdni. Nevrotoksični učinki: Bolnike je treba stalno klinično kontrolirati zaradi možnih znakov ali simptomov nevrotoksičnosti. Disfunkcija levega prekata: Bolniki, zdravljeni s trastuzumabom emtanzinom, imajo večje tveganje za pojav disfunkcije levega prekata. Pri bolnikih, zdravljenih s trastuzumabom emtanzinom, so opažali iztisni delež levega prekata (LVEF) < 40 %, zato je možno simptomatsko kongestivno srčno popuščanje. Pred uvedbo zdravljenja in tudi ob rednih intervalih med zdravljenjem je treba opraviti standardno preiskavo delovanja srca. V primerih disfunkcije levega prekata je treba odmerek preložiti ali zdravljenje prenehati, če je to potrebno. Pljučna toksičnost: Pri bolnikih z ugotovljeno intersticijsko boleznijo pljuč ali pnevmonitisom je priporočljivo zdravljenje s trastuzumabom emtanzinom dokončno ukiniti, razen v primeru pnevmonitisa zaradi obsevanja med adjuvantnim zdravljenjem, kjer je treba trastuzumab emtanzin dokončno ukiniti pri pnevmonitisu 3 3. stopnje ali 2. stopnje brez odziva na standardno zdravljenje. Bolniki z dispnejo v mirovanju zaradi zapletov napredovale maligne bolezni, sočasnih bolezni in sočasnega obsevanja pljuč imajo lahko večje tveganje za pljučne neželene dogodke. Z infundiranjem povezane reakcije: Trastuzumab emtanzin ni raziskan pri bolnikih, pri katerih so predhodno zdravljenje s trastuzumabom trajno ukinili zaradi reakcij, povezanih z infundiranjem. Zdravljenje takšnih bolnikov s tem zdravilom ni priporočljivo. Pri bolnikih s hudimi reakcijami, povezanimi z infundiranjem, je treba zdravljenje prekiniti, dokler znaki in simptomi ne minejo. O ponovnem zdravljenju je treba presoditi glede na klinično oceno tega, kako huda je bila reakcija. Zdravljenje je treba dokončno ukiniti pri pojavu smrtno nevarnih reakcij, povezanih z infundiranjem. Preobčutljivostne reakcije: Trastuzumab emtanzin ni raziskan pri bolnikih, pri katerih so zdravljenje s trastuzumabom dokončno ukinili zaradi preobčutljivosti. Zdravljenje takšnih bolnikov s trastuzumabom emtanzinom ni priporočljivo. Bolnike je treba skrbno opazovati zaradi možnosti pojava preobčutljivostnih/alergijskih reakcij, ki imajo lahko enako klinično sliko kot reakcije, povezane z infundiranjem. V kliničnih študijah s trastuzumabom emtanzinom so zabeležili resne anafilaktične reakcije. Za takojšnjo uporabo morajo biti na voljo zdravila za zdravljenje takšnih reakcij in oprema za nujne primere. Pri dejanski preobčutljivostni reakciji je treba zdravljenje s trastuzumabom emtanzinom dokončno ukiniti. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Formalnih študij medsebojnega delovanja niso izvedli. Studije presnove in vitro v človeških jetrnih mikrosomih kažejo, da se DM1 presnovi v glavnem s CYP3A4 in v manjši meri s CYP3A5. Izogniti se je treba sočasni uporabi močnih zaviralcev CYP3A4 in trastuzumaba emtanzina, ker obstaja možnost večje izpostavljenosti DM1 in toksičnih učinkov. Razmisliti je treba o uporabi drugega zdravila, ki nima potenciala za zavrtje CYP3A4 ali pa je ta majhen. Če je sočasna uporaba močnih zaviralcev CYP3A4 neizogibna, pride v poštev odložitev zdravljenja s trastuzumabom emtanzinom, dokler se močni zaviralec CYP3A4 ne odstrani iz obtoka, če je to mogoče. Če zdravljenja s trastuzumabom emtanzinom med sočasno uporabo močnega zaviralca CYP3A4 ni mogoče odložiti, je treba bolnike natančno kontrolirati zaradi možnih neželenih učinkov. Neželeni učinki: Neželeni učinki, ki so se pojavili pri bolnikih, zdravljenih z trastuzumabom emtanzinom: Zelo pogosti: okužba sečil, trombocitopenija, anemija, nespečnost, periferna nevropatija, glavobol, krvavitev, epistaksa, kašelj, dispneja, stomatitis, driska, bruhanje, navzea, zaprtost, suhost ust, bolečine v trebuhu, zvišanje transaminaz, mišično-skeletne bolečine, artralgija, mialgija, utrujenost, pireksija inastenija. Pogosti: nevtropenija, levkopenija, preobčutljivost na zdravilo, hipokaliemija, omotica, spremenjen okus, okvara spomina, suho oko, konjunktivitis, zamegljen vid, močnejše solzenje, disfunkcija levega prekata, hipertenzija, dispepsija, krvavitev iz dlesni, zvišanje alkalne fosfataze v krvi, zvišanje bilirubina v krvi, izpuščaj, srbenje, alopecija, bolezni nohtov, sindrom palmarno-plantarne eritrodizestezije, urtikarija, periferni edemi, mrzlica, in z infundiranjem povezane reakcije. Poročanje o domnevnih neželenih učinkih: Poročanje o domnevnih neželenih učinkih zdravila po izdaji dovoljenja za promet je pomembno. Omogoča namreč stalno spremljanje razmerja med koristmi in tveganji zdravila. Od zdravstvenih delavcev se zahteva, da poročajo o katerem koli domnevnem neželenem učinku zdravila na: Javna agencija Republike Slovenije za zdravila in medicinske pripomočke, Sektor za farmakovigilanco, Nacionalni center za farmakovigilanco, Slovenčeva ulica 22, SI-1000 Ljubljana, Tel: +386 (0)8 2000 500, Faks: +386 (0)8 2000 510, e-pošta: h-farmakovigilanca@jazmp.si, spletna stran: www.jazmp.si. Za zagotavljanje sledljivosti zdravila je pomembno, da pri izpolnjevanju obrazca o domnevnih neželenih učinkih zdravila navedete številko serije biološkega zdravila. Režim izdaje zdravila: H. Imetnik dovoljenja za promet: Roche Registration GmbH, Em il-Barel l-Strasse 1, 79639 Grenzach-Wyhlen, Nemčija. Verzija: 3.0/19. Vir: 1. Povzetek glavnih značilnosti zdravila Kadcyla, dostopano maja 2020 na: https://www.ema.europa.eu/en/documents/ product-information/kadcyla-epar-product-information_sl.pdf. * preživetje brez invazivne bolezni SAMO ZA STROKOVNO JAVNOST • M-SI-00000008 • Informacija pripravljena: maj 2020 Dodatne informacije so na voljo pri: Roche farmacevtska družba d.o.o., Stegne 13G, 1000 Ljubljana. trifluridin/tipiracil ^¿r Več časa za trenutke, ki štejejo Kolorektalni rak Zdravilo Lonsurf je indicirano v monoterapiji za zdravljenje odraslih bolnikov z metastatskim kolorektalnim rakom (KRR), ki so bili predhodno že zdravljeni ali niso primerni za zdravljenja, ki so na voljo. Ta vključujejo kemoterapijo na osnovi fluoropirimidina, oksaliplatina in irinotekana, zdravljenje z zaviralci žilnega endotelijskega rastnega dejavnika (VEGF - Vascular Endothelial Growth Factor) in zaviralci receptorjev za epidermalni rastni dejavnik (EGFR - Epidermal Growth Factor Receptor). Rak želodca Zdravilo Lonsurf je indicirano v monoterapiji za zdravljenje odraslih bolnikov z metastatskim rakom želodca vključno z adenokarcinomom gastro-ezofagealnega prehoda, ki so bili predhodno že zdravljeni z najmanj dvema sistemskima režimoma zdravljenja za napredovalo bolezen. Družba Servier ima licenco družbe Taiho za zdravilo Lonsurf®. Pri globalnem razvoju zdravila sodelujeta obe družbi in ga tržita na svojih določenih področjih. ^^ SERVIER Skrajšan povzetek glavnih značilnosti zdravila: Lonsurf 15 mg/6,14 mg filmsko obložene tablete in Lonsurf 20 mg/8,19 mg filmsko obložene tablete ▼ 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*: Lonsurf 15 mg/6,14 mg: Ena filmsko obložena tableta vsebuje 15 mg trifluridina in 6,14 mg tipiracila (v obliki klorida). Lonsurf 20 mg/8,19 mg: Ena filmsko obložena tableta vsebuje 20 mg trifluridina in 8,19 mg tipiracila (v obliki klorida). TERAPEVTSKE INDIKACIJE*: Kolorektalni rak - zdravilo Lonsurf je indicirano v monoterapiji za zdravljenje odraslih bolnikov z metastatskim kolorektalnim rakom, ki so bili predhodno že zdravljeni ali niso primerni za zdravljenja, ki so na voljo. Ta vključujejo kemoterapijo na osnovi fluoropirimidina, oksaliplatina in irinotekana, zdravljenje z zaviralci žilnega endotelijskega rastnega dejavnika (VEGF - Vascular Endothelial Growth Factor) in zaviralci receptorjev za epidermalni rastni dejavnik (EGFR - Epidermal Growth Factor Receptor). Rak želodca - zdravilo Lonsurf je indicirano v monoterapiji za zdravljenje odraslih bolnikov z metastatskim rakom želodca vključno z adenokarcinomom gastro-ezofagealnega prehoda, ki so bili predhodno že zdravljeni z najmanj dvema sistemskima režimoma zdravljenja za napredovalo bolezen. ODMERJANJE IN NAČIN UPORABE*: Priporočeni začetni odmerek zdravila Lonsurf pri odraslih je 35 mg/m2/odmerek peroralno dvakrat dnevno na 1. do 5. dan in 8. do 12. dan vsakega 28-dnevnega cikla zdravljenja, najpozneje 1 uro po zaključku jutranjega in večernega obroka (20 mg/m2/odmerek dvakrat dnevno pri bolnikih s hudo ledvično okvaro). Odmerjanje, izračunano glede na telesno površino, ne sme preseči 80 mg/odmerek. Možne prilagoditve odmerka glede na varnost in prenašanje zdravila: dovoljena so zmanjšanja odmerka na najmanjši odmerek 20 mg/m2 dvakrat dnevno (oz. 15 mg/m2 dvakrat dnevno pri bolnikih s hudo ledvično okvaro). Potem ko je bil odmerek zmanjšan, povečanje ni dovoljeno. KONTRAINDIKACIJE*: Preobčutljivost na zdravilni učinkovini ali katero koli pomožno snov. OPOZORILA IN PREVIDNOSTNI UKREPI*: Supresiia kostnega mozga: Pred uvedbo zdravljenja in po potrebi za spremljanje toksičnosti zdravila, najmanj pred vsakim ciklom zdravljenja, je treba pregledati celotno krvno sliko. Zdravljenja ne smete začeti, če je absolutno število nevtrofilcev < 1,5 x 109/l, če je število trombocitov < 75 x 109/l ali če se je pri bolniku zaradi predhodnih zdravljenj pojavila klinično pomembna nehematološka toksičnost 3. ali 4. stopnje, ki še traja. Bolnike je treba skrbno spremljati zaradi morebitnih okužb, uvesti je treba ustrezne ukrepe, kot je klinično indicirano. Toksičnost za prebavila: Potrebna je uporaba antiemetikov, antidiaroikov ter drugih ukrepov, kot je klinično indicirano. Če je potrebno, prilagodite odmerke. Ledvična okvara: Zdravilo Lonsurf ni primerno za uporabo pri bolnikih s končno stopnjo ledvične okvare. Bolnike z ledvično okvaro je potrebno med zdravljenjem skrbno spremljati; bolnike z zmerno ali hudo ledvično okvaro je treba zaradi hematološke toksičnosti bolj pogosto spremljati. Jetrna okvara: Uporaba zdravila Lonsurf pri bolnikih z obstoječo zmerno ali hudo jetrno okvaro ni priporočljiva. Proteinurija: Pred začetkom zdravljenja in med njim je priporočljivo spremljanje proteinurije z urinskimi testnimi lističi. Pomožne snovi: Zdravilo vsebuje laktozo. INTERAKCIJE*: Zdravila, ki medsebojno delujejo z nukleozidnimi prenašalci CNT1, ENT1 in ENT2, zaviralci OCT2 ali MATE1, substrati humane timidin-kinaze (npr. zido-vudinom), hormonskimi kontraceptivi. PLODNOST*, NOSEČNOST IN DOJENJE*: Ni priporočljivo. KONTRACEPCIJA*: Ženske in moški morajo uporabljati učinkovito metodo kontracepcije med zdravljenjem in do 6 mesecev po zaključku zdravljenja. VPLIV NA SPOSOBNOST VOŽNJE IN UPRAVLJANJA STROJEV*: Med zdravljenjem se lahko pojavijo utrujenost, omotica ali splošno slabo počutje. NEŽELENI UČINKI*: Zelo pogosti: nevtropenija, levkopenija, anemija, trombocitopenija, zmanjšan apetit, diareja, navzea, bruhanje, utrujenost. Pogosti: okužba spodnjih dihal, febrilna nevtropenija, limfopenija, hipoalbuminemija, disgevzija, periferna nevropatija, dispneja, bolečina v trebuhu, zaprtje, stomatitis, bolezni ustne votline, hiperbilirubinemija, sindrom palmarne plantarne eritrodisestezije, izpuščaj, alopecija, pruritus, suha koža, proteinurija, pireksija, edem, vnetje sluznice, splošno slabo počutje, zvišanje jetrnih encimov, zvišanje alkalne fosfataze v krvi, zmanjšanje telesne mase. Občasni: septični šok, infekcijski enteritis, pljučnica, okužba žolčevoda, gripa, okužba sečil, gingivitis, herpes zoster, tinea pedis, okužba s kandido, bakterijska okužba, okužba, nevtropenična sepsa, okužba zgornjih dihal, konjunktivitis, bolečina zaradi raka, pancitopenija, granuloci-topenija, monocitopenija, eritropenija, levkocitoza, monocitoza, dehidracija, hiperglikemija, hiperkaliemija, hipokaliemija, hipofosfatemija, hipernatriemija, hiponatriemija, hipokalciemija, protin, anksioznost, nespečnost, nevrotoksičnost, disestezija, hipereste-zija, hipoestezija, sinkopa, parestezija, pekoč občutek, letargija, omotica, glavobol, zmanjšana ostrina vida, zamegljen vid, diplopija, katarakta, suho oko, vrtoglavica, neugodje v ušesu, angina pektoris, aritmija, palpitacije, embolija, hipertenzija, hipotenzija, vročinski oblivi, pljučna embolija, plevralni izliv, izcedek iz nosu, disfonija, orofaringealna bolečina, epistaksa, kašelj, hemoragični enterokolitis, krvavitev v prebavilih, akutni pankreatitis, ascites, ileus, subileus, kolitis, gastritis, refluksni gastritis, ezofagitis, moteno praznjenje želodca, abdominalna distenzija, analno vnetje, razjede v ustih, dispepsija, gastroezofagealna refluksna bolezen, proktalgija, bukalni polip, krvavitev dlesni, glositis, parodontalna bolezen, bolezen zob, siljenje na bruhanje, flatulenca, slab zadah, hepatotoksičnost, razširitev žolčnih vodov, luščenje kože, urtikarija, preobčutljivostne reakcije na svetlobo, eritem, akne, hiperhidroza, žulj, bolezni nohtov, otekanje sklepov, artralgija, bolečina v kosteh, mialgija, mišično-skeletna bolečina, mišična oslabelost, mišični krči, bolečina v okončinah, ledvična odpoved, neinfektivni cistitis, motnje mikcije, hematurija, levkociturija, motnje menstruacije, poslabšanje splošnega zdravstvenega stanja, bolečina, občutek spremembe telesne temperature, kseroza, nelagodje, zvišanje kreatinina v krvi, podaljšanje intervala QT na elektrokardiogramu, povečanje mednarodnega umerjenega razmerja (|Nr), podaljšanje aktiviranega parcialnega tromboplastinskega časa (aPTČ), zvišanje sečnine v krvi, zvišanje laktatne dehidrogenaze v krvi, znižanje celokupnih proteinov, zvišanje C-reaktivnega proteina, zmanjšan hematokrit. Post-marketinške izkušnje: intersticijska bolezen pljuč. PREVELIKO ODMERJANJE*: Neželeni učinki, o katerih so poročali v povezavi s prevelikim odmerjanjem, so bili v skladu z uveljavljenim varnostnim profilom. Glavni pričakovani zaplet prevelikega odmerjanja je supresija kostnega mozga. FARMAKODINAMIČNE LASTNOSTI*: Farmakoterapevtska skupina: zdravila z delovanjem na novotvorbe, antimetabolite oznaka ATC: L01BC59. Zdravilo Lonsurf sestavljata antineoplastični timidinski nukleozidni analog, trifluridin, in zaviralec timidin-fosforilaze (TPaze), tipiracilijev klorid. Po privzemu v rakave celice timidin-kinaza fosforilira trifluridin. Ta se v celicah nato presnovi v substrat deoksiribonukleinske kisline (DNA), ki se vgradi neposredno v DNA ter tako preprečuje celično proliferacijo. TPaza hitro razgradi trifluridin in njegova presnova po peroralni uporabi je hitra zaradi učinka prvega prehoda, zato je v zdravilo vključen zaviralec TPaze, tipiracilijev klorid. PAKIRANJE*: 20 filmsko obloženih tablet. NAČIN PREDPISOVANJA IN IZDAJE ZDRAVILA: Rp/Spec. Imetnik dovoljenja za promet: Les Laboratoires Servier, 50, rue Carnot, 92284 Suresnes cedex, Francija. Številka dovoljenja za promet z zdravilom: EU/1/16/1096/001 (Lonsurf 15 mg/6,14 mg), EU/1/16/1096/004 (Lonsurf 20 mg/8,19 mg). Datum zadnje revizije besedila: april 2020.*Pred predpisovanjem preberite celoten povzetek glavnih značilnosti zdravila. Celoten povzetek glavnih značilnosti zdravila in podrobnejše informacije so na voljo pri: Servier Pharma d.o.o., Podmilščakova ulica 24, 1000 Ljubljana, tel: 01 563 48 11, www.servier.si. Datum priprave informacije: april 2020. 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The statement of disclosure must be in the Cover letter accompanying the manuscript or submitted on the form available on www.icmje.org/coi_disclosure.pdf Page proofs Page proofs will be sent by E-mail to the corresponding author. It is their responsibility to check the proofs carefully and return a list of essential corrections to the editorial office within three days of receipt. Only grammatical corrections are acceptable at that time. Open access Papers are published electronically as open access on https://content.sciendo.com/raon, also papers accepted for publication as E-ahead of print. TAI > 1 i ^ Va ET J-L. -L-L niHKg ■ ■ i XALKORI® - 1. linija zdravljenja napredovalega, ALK pozitivnega nedrobnoceličnega pljučnega raka1 BISTVENI PODATKI IZ POVZETKA GLAVNIH ZNAČILNOSTI ZDRAVILA XALKORI 200 mg, 250 mg trde kapsule Sestava in oblika zdravila: Ena kapsula vsebuje 200 mg ali 250 mg krizotiniba. Indikacije: Monoterapija za: - prvo linijo zdravljenja odraslih bolnikov z napredovalim nedrobnoceličnim pljučnim rakom (NSCLC - Non-Small Cell Lung Cancer), ki je ALK (anaplastična limfomska kinaza) pozitiven; - zdravljenje odraslih bolnikov s predhodno zdravljenim, napredovalim NSCLC, ki je ALK pozitiven; -zdravljenje odraslih bolnikov z napredovalim NSCLC, ki je ROSI pozitiven. Odmerjanje in način uporabe: Zdravljenje mora uvesti in nadzorovati zdravnik z izkušnjami z uporabo zdravil za zdravljenje rakavih bolezni. Preverjanje prisotnosti ALK in ROSI: Pri izbiri bolnikov za zdravljenje je treba pred zdravljenjem opraviti točno in validirano preverjanje prisotnosti ALK ali ROSI. Odmerjanje: Priporočeni odmerek je 250 mg dvakrat na dan (500 mg na dan), bolniki pa morajo zdravilo jemati brez prekinitev. Če bolnik pozabi vzeti odmerek, ga mora vzeti takoj, ko se spomni, razen če do naslednjega odmerka manjka manj kot 6 ur. V tem primeru bolnik pozabljenega odmerka ne sme vzeti. Prilagajanja odmerkov: Glede na varnost uporabe zdravila pri posameznem bolniku in kako bolnik zdravljenje prenaša, utegne biti potrebna prekinitev in/ali zmanjšanje odmerka pri bolnikih, ki se zdravijo s krizotinibom 250 mg peroralno dvakrat na dan (za režim zmanjševanja odmerka glejte poglavje 4.2 v povzetku glavnih značilnosti zdravila).Za prilagajanje odmerkov pri hematološki in nehematološki toksičnosti (povečanje vrednosti AST, ALT, bilirubina; ILD/pnevmonitis; podaljšanje intervala QTc, bradikardija, bolezni oči) glejte preglednici 1 in 2 v poglavju 4.2 povzetka glavnih značilnosti zdravila. Okvara jeter: Pri zdravljenju pri bolnikih z okvaro jeter je potrebna previdnost. Pri blagi okvari jeter prilagajanje začetnega odmerka ni priporočeno, pri zmerni okvari jeter je priporočeni začetni odmerek 200 mg dvakrat na dan, pri hudi okvari jeter pa 250 mg enkrat na dan (za merila glede klasifikacije okvare jeter glejte poglavje 4.2 v povzetku glavnih značilnosti zdravila). Okvara ledvic: Pri blagi in zmerni okvari prilagajanje začetnega odmerka ni priporočeno. Pri hudi okvari ledvic (ki ne zahteva peritonealne dialize ali hemodialize) je začetni odmerek 250 mg peroralno enkrat na dan; po vsaj 4 tednih zdravljenja se lahko poveča na 200 mg dvakrat na dan. Starejši bolniki (> 65let): Prilagajanje začetnega odmerka ni potrebno. Pediatrična populacija: Varnost in učinkovitost nista bili dokazani. Način uporabe: Kapsule je treba pogoltniti cele, z nekaj vode, s hrano ali brez nje. Ne sme se jih zdrobiti, raztopiti ali odpreti. Izogibati se je treba uživanju grenivk, grenivkinega soka ter uporabi šentjanževke. Kontraindikacije: Preobčutljivost na krizotinib ali katerokoli pomožno snov. Posebna opozorila in previdnostni ukrepi: Določanje statusa ALK in ROS1: Pomembno je izbrati dobro validirano in robustno metodologijo, da se izognemo lažno negativnim ali lažno pozitivnim rezultatom. Hepatotoksičnost: V kliničnih študijah so poročali o hepatotoksičnosti, ki jo je povzročilo zdravilo (vključno s primeri s smrtnim izidom). Delovanje jeter, vključno z ALT, AST in skupnim bilirubinom, je treba preveriti enkrat na teden v prvih 2 mesecih zdravljenja, nato pa enkrat na mesec in kot je klinično indicirano. Ponovitve preverjanj morajo biti pogostejše pri povečanjih vrednosti stopnje 2, 3 ali 4. Intersticijska bolezen pljuč (ILD)/pnevmonitis: Lahko se pojavi huda, življenjsko nevarna ali smrtna ILD/pnevmonitis. Bolnike s simptomi ILD/pnevmonitisa je treba spremljati, zdravljenje pa prekiniti ob sumu na ILD/pnevmonitis. Podaljšanje intervala QT: Opažali so podaljšanje intervala QTc. Pri bolnikih z obstoječo bradikardijo, podaljšanjem intervala QTc v anamnezi ali predispozicijo zanj, pri bolnikih, ki jemljejo antiaritmike ali druga zdravila, ki podaljšujejo interval QT, ter pri bolnikih s pomembno obstoječo srčno boleznijo in/ali motnjami elektrolitov je treba krizotinib uporabljati previdno; potrebno je redno spremljanje EKG, elektrolitov in delovanja ledvic; preiskavi EKG in elektrolitov je treba opraviti čimbližje uporabi prvega odmerka, potem se priporoča redno spremljanje. Če se interval QTc podaljša za 60 ms ali več, je treba zdravljenje s krizotinibom začasno prekiniti in se posvetovati s kardiologom. Bradikardija: Lahko se pojavi simptomatska bradikardija (lahko se razvije več tednov po začetku zdravljenja); izogibati se je treba uporabi krizotiniba v kombinaciji z drugimi zdravili, ki povzročajo bradikardijo; pri simptomatski bradikardiji je treba prilagoditi odmerek. Srčno popuščanje: Poročali so o hudih, življenjsko nevarnih ali smrtnih neželenih učinkih srčnega popuščanja. Bolnike je treba spremljati glede pojavov znakov in simptomov srčnega popuščanja in ob pojavu simptomov zmanjšati odmerjanje ali prekiniti zdravljenje. Nevtropenija in levkopenija: V kliničnih študijah so poročali o nevtropeniji, levkopeniji in febrilni nevtropeniji; spremljati je treba popolno krvno sliko (pogostejše preiskave, če se opazijo abnormalnosti stopnje 3 ali 4 ali če se pojavi povišana telesna temperatura ali okužba). Perforacija v prebavilih:V kliničnih študijah so poročali o perforacijah v prebavilih, v obdobju trženja pa o smrtnih primerih perforacij v prebavilih. Krizotinib je treba pri bolnikih s tveganjem za nastanek perforacije v prebavilih uporabljati previdno; bolniki, pri katerih se razvije perforacija v prebavilih, se morajo prenehati zdraviti s krizotinibom; bolnike je treba poučiti o prvih znakih perforacije in jim svetovati, naj se nemudoma posvetujejo z zdravnikom. Vplivi na ledvice: V kliničnih študijah so opazili zvišanje ravni kreatinina v krvi in zmanjšanje očistka kreatinina. V kliničnih študijah in v obdobju trženja so poročali tudi o odpovedi ledvic, akutni odpovedi ledvic, primerih s smrtnim izidom, primerih, ki so zahtevali hemodializo in hiperkaliemiji stopnje 4. Vplivi na vid: V kliničnih študijah so poročali o izpadu vidnega polja stopnje 4 z izgubo vida. Če se na novo pojavi huda izguba vida, je treba zdravljenje prekiniti in opraviti oftalmološki pregled. Če so motnje vida trdovratne ali se poslabšajo, je priporočljiv oftalmološki pregled. Histološka preiskava, ki ne nakazuje adenokarcinoma: Na voljo so le omejeni podatki pri NSCLC, ki je ALK in ROS1 pozitiven in ima histološke značilnosti, ki ne nakazujejo adenokarcinoma, vključno s ploščatoceličnim karcinomom (SCC). Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Izogibati se je treba sočasni uporabi z močnimi zaviralci CYP3A4, npr. atazanavir, ritonavir, kobicistat, itrakonazol, ketokonazol, posakonazol, vorikonazol, klaritromicin, telitromicin in eritromicin (razen če morebitna korist za bolnika odtehta tveganje, v tem primeru je treba bolnike skrbno spremljati glede neželenih učinkov krizotiniba), ter grenivko in grenivkinim sokom, saj lahko povečajo koncentracije krizotiniba v plazmi. Izogibati se je treba sočasni uporabi z močnimi induktorji CYP3A4, npr. karbamazepin, fenobarbital, fenitoin, rifampicin in šentjanževka, saj lahko zmanjšajo koncentracije krizotiniba v plazmi. Učinek zmernih induktorjev CYP3A4, npr. efavirenz in rifabutin, še ni jasen, zato se je treba sočasni uporabi s krizotinibom izogibati. Zdravila, katerih koncentracije v plazmi lahko krizotinib spremeni (midazolam, alfentanil, cisaprid, ciklosporin, derivati ergot alkaloidov, fentanil, pimozid, kinidin, sirolimus, takrolimus, digoksin, dabigatran, kolhicin, pravastatin: sočasni uporabi s temi zdravili se je treba izogibati oziroma izvajati skrben klinični nadzor; bupropion, efavirenz, peroralni kontraceptivi, raltegravir, irinotekan, morfin, nalokson, metformin, prokainamid). XALKORI KRIZOTINIB Zdravila, ki podaljšujejo Interval QT ali ki lahko povzročijo Torsades de pointes (antiaritmiki skupine IA (kinidin, disopiramid), antiaritmiki skupine III (amiodaron, sotalol, dofetilid, ibutilid), metadon, cisaprid, moksifloksacin, antipsihotiki) - v primeru sočasne uporabe je potreben skrben nadzor intervala QT. Zdravila, ki povzročajo bradikardijo (nedihidropiridinski zaviralci kalcijevih kanalčkov (verapamil, diltiazem), antagonisti adrenergičnih receptorjev beta, klonidin, gvanfacin, digoksin, meflokin, antiholinesteraze, pilokarpin) - krizotinib je treba uporabljati previdno. Plodnost, nosečnost in dojenje: Ženske v rodni dobi se morajo izogibati zanositvi. Med zdravljenjem in najmanj 90 dni po njem je treba uporabljati ustrezno kontracepcijo (velja tudi za moške). Zdravilo lahko škoduje plodu in se ga med nosečnostjo ne sme uporabljati, razen če klinično stanje matere ne zahteva takega zdravljenja. Matere naj se med jemanjem zdravila dojenju izogibajo. Zdravilo lahko zmanjša plodnost moških in žensk. Vpliv na sposobnost vožnje in upravljanja strojev: Lahko se pojavijo simptomatska bradikardija (npr. sinkopa, omotica, hipotenzija), motnje vida ali utrujenost; potrebna je previdnost. Neželeni učinki: Najresnejši neželeni učinki so bili hepatotoksičnost, ILD/ pnevmonitis, nevtropenija in podaljšanje intervala QT. Najpogostejši neželeni učinki (> 25 %) so bili motnje vida, navzea, diareja, bruhanje, edem, zaprtje, povečane vrednosti transaminaz, utrujenost, pomanjkanje apetita, omotica in nevropatija. Ostali zelo pogosti (> 1/10 bolnikov) neželeni učinki so: nevtropenija, anemija, levkopenija, disgevzija, bradikardija, bolečina v trebuhu in izpuščaj. Način in režim -k izdaje: Predpisovanje in izdaja zdravila je le na recept, zdravilo pa se uporablja ° samo v bolnišnicah. Izjemoma se lahko uporablja pri nadaljevanju zdravljenja na , j| domu ob odpustu iz bolnišnice in nadaljnjem zdravljenju. Imetnik dovoljenja za o promet: Pfizer Europe MA EEIG, Boulevard de la Plaine 17, 1050 Bruxelles, Belgija. g Datum zadnje revizije besedila: 31.10.2019. ~o Pred predpisovanjem se seznanite s celotnim povzetkom glavnih značilnosti tâ zdravila. n o Vir: 1. Povzetek glavnih značilnosti zdravila Xalkori, 31.10.2019 ™ Pfizer Luxembourg SARL, GRAND DUCHY OF LUXEMBOURG, 51, Avenue J.F. Kennedy, L-1855, Pfizer podružnica Ljubljana, Letališka cesta 29a, 1000 Ljubljana