vol.54 no.3 september 2020 Skrajšan povzetek glavnih znacilnosti zdravila (kabozantinib) TERAPEVTSKE INDIKACIJE Zdravljenje napredovalega karcinoma ledvicnih celic (KLC) pri predhodno nezdravljenih odraslih bolnikih s srednje ugodnim ali slabim prognosticnim 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 NACIN UPORABE Pri bolnikih s KLC in HCK je priporoceni odmerek 60 mg enkrat na dan. Zdravljenje je treba nadaljevati tako dolgo, dokler bolnik vec nima klinicnih koristi od terapije ali do pojava nesprejemljive toksicnosti. Pri sumu na neželene reakcije bo morda treba zdravljenje zacasno prekiniti in/ali zmanjšati odmerek. Ce je treba odmerek zmanjšati, se priporoca zmanjšanje na 40 mg/dan in nato na 20 mg/dan. Prekinitev odmerka se priporoca pri obravnavi toksicnosti 3. ali višje stopnje po CTCAE (common terminology criteria for adverse events) ali nevzdržni toksicnosti 2. stopnje. Zmanjšanje odmerka se priporoca za dogodke, ki bi lahko cez cas postali resni ali nevzdržni. Za priporocila glede prilagoditve odmerka ob pojavu neželenih ucinkov glejte celoten povzetek glavnih znacilnosti zdravila. Pri blagi ali zmerni ledvicni okvari je treba kabozantinib uporabljati previdno. Uporaba se ne priporoca pri hudi ledvicni okvari. Pri blagi okvari jeter odmerka ni treba prilagajati. Pri zmerni okvari jeter (Child Pugh B) je priporocljivo skrbno spremljanje celokupne varnosti. Pri bolnikih s hudo okvaro jeter (Child Pugh C) uporaba kabozantiniba ni priporocljiva. Nacin uporabe: Tablete je treba pogoltniti cele in jih ni dovoljeno drobiti. Bolnikom je treba narociti, naj vsaj 2 uri pred uporabo zdravila in 1 uro po tem nicesar ne jedo. KONTRAINDIKACIJE Preobcutljivost na ucinkovino ali katero koli pomožno snov. POSEBNA OPOZORILA IN PREVIDNOSTNI UKREPI Vecina 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 obicajno pojavijo zgodaj, vkljucujejo hipokalciemijo, hipokaliemijo, trombocitopenijo, hipertenzijo, sindrom palmarno-plantarne eritrodisestezije (PPES), proteinurijo in GI dogodke (bolecine v trebuhu, vnetje sluznice, zaprtje, driska, bruhanje). Pred uvedbo zdravljenja s kabozantinibom je priporocljivo 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 crevesja, ki imajo tumorsko in.ltracijo prebavil ali so imeli pred posegom na prebavilih zaplete, je treba pred uvedbo zdravljenja skrbno oceniti, nato pa natancno spremljati za pojav simptomov GI perforacij in .stul, vkljucno z abscesi in sepso. Z uporabo kabozantiniba je treba pri bolnikih, pri katerih se pojavi GI perforacija ali .stula, ki je ni možno ustrezno obravnavati, prenehati. Driska, navzea/bruhanje, zmanjšanje apetita in vnetje ustne sluznice/bolecina v ustni votlini so nekateri od najpogosteje porocanih neželenih ucinkov na prebavila. Nemudoma je treba uvesti ustrezne medicinske ukrepe, vkljucno s podpornim zdravljenjem z antiemetiki, antidiaroiki ali antacidi. Ce pomembni neželeni ucinki na prebavila vztrajajo ali se ponavljajo, je treba presoditi o prekinitvi odmerjanja, zmanjšanju odmerka ali trajni ukinitvi zdravljenja s kabozantinibom. Za to zdravilo se izvaja dodatno spremljanje varnosti. Tako bodo hitreje na voljo nove informacije o njegovi varnosti. Zdravstvene delavce naprošamo, da porocajo o katerem koli domnevnem neželenem ucinku zdravila. Kabozantinib je treba uporabljati previdno pri bolnikih, pri katerih obstaja tveganje za pojav venske trombembolije, vkljucno s pljucno 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 klinicno 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. Med zdravljenjem s kabozantinibom je treba spremljati vrednosti trombocitov in odmerek prilagoditi glede na resnost trombocitopenije. Vsaj 28 dni pred nacrtovanim kirurškim posegom je treba zdravljenje ustaviti, ce je mogoce. Kabozantinib je treba ukiniti pri bolnikih z zapleti s celjenjem rane, zaradi katerih je potrebna zdravniška pomoc. 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 oz. prenehati z zdravljenjem. V primeru hipertenzijske krize je treba zdravljenje ukiniti. Pred uvedbo kabozantiniba je treba opraviti pregled ustne votline in le­tega v casu zdravljenja periodicno ponavljati. Ob pojavu osteonekroze celjusti, je treba prenehati z uporabo kabozantiniba. Pri resni PPES je treba razmisliti o prekinitvi zdravljenja. Nadaljevanje zdravljenja naj se zacne z nižjim odmerkom, ko se PPES umiri do 1. stopnje. V casu zdravljenja je treba redno spremljati beljakovine v urinu. Ce se pri bolniku razvije nefroticni sindrom, je treba z uporabo kabozantiniba prenehati. Pri uporabi kabozantiniba so opazili sindrom posteriorne reverzibilne encefalopatije (PRES). Pri bolnikih s PRES je treba zdravljenje ukiniti. Kabozantinib je treba uporabljati previdno pri bolnikih s podaljšanjem intervala QT v anamnezi, pri bolnikih, ki jemljejo antiaritmike, in pri bolnikih z relevantno obstojeco boleznijo srca, bradikardijo ali elektrolitskimi motnjami. Uporaba kabozantiniba je bila povezana z vecjo pojavnostjo elektrolitskih nepravilnosti, zato je priporocljivo spremljati biokemijske parametre in po potrebi uvesti ustrezno nadomestno zdravljenje v skladu s standardno klinicno prakso. Bolniki z redko dedno intoleranco za galaktozo, laponsko obliko zmanjšane aktivnosti laktaze ali malabsorpcijo glukoze/ galaktoze ne smejo jemati tega zdravila. Plodnost, nosecnost in dojenje: Ženskam v rodni dobi je treba svetovati, da v casu zdravljenja s kabozantinibom ne smejo zanositi. Zanositev morajo prepreciti tudi ženske partnerice moških bolnikov, ki uporabljajo kabozantinib. Med zdravljenjem in še vsaj 4 mesece po koncanju terapije je treba uporabljati zanesljiv nacin kontracepcije. Kabozantiniba se ne sme uporabljati med nosecnostjo, razen ce zdravljenje ni nujno potrebno zaradi klinicnega stanja ženske. Matere med zdravljenjem in še 4 mesece po koncanju terapije ne smejo dojiti. Kabozantinib lahko predstavlja tveganje za plodnost pri moških in ženskah. INTERAKCIJE Kabozantinib je substrat za CYP3A4. Pri socasni uporabi mocnih zaviralcev CYP3A4 (npr. ritonavirja, itrakonazola, eritromicina, klaritromicina, soka grenivke) je potrebna previdnost. Kronicni socasni uporabi mocnih induktorjev CYP3A4 (npr. fenitoina, karbamazepina, rifampicina, fenobarbitala ali pripravkov zelišcnega izvora iz šentjanževke) se je treba izogibati. Razmisliti je treba o socasni uporabi alternativnih zdravil, ki CYP3A4 ne inducirajo in ne zavirajo ali pa inducirajo in zavirajo le neznatno. Pri socasni uporabi zaviralcev MRP2 (npr. ciklosporina, efavirenza, emtricitabina) je potrebna previdnost, saj lahko povzrocijo povecanje koncentracij kabozantiniba v plazmi. Ucinka kabozantiniba na farmakokinetiko kontraceptivnih steroidov niso preucili, vendar pa se priporoca 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 lahko poveca koncentracije socasno uporabljenih substratov P-gp v plazmi. Bolnike je treba opozoriti na uporabo substratov P-gp (npr. feksofenadina, aliskirena, ambrisentana, dabigatran eteksilata, digoksina, kolhicina, maraviroka, posakonazola, ranolazina, saksagliptina, sitagliptina, talinolola, tolvaptana) socasno s kabozantinibom. NEŽELENI UCINKI Za popolno informacijo o neželenih ucinkih, prosimo, preberite celoten povzetek glavnih znacilnosti zdravila Cabometyx. Najpogostejši resni neželeni ucinki zdravila v populaciji bolnikov s KLC so bili bolecine v trebuhu, driska, navzea, hipertenzija, embolija, hiponatriemija, pljucna embolija, bruhanje, dehidracija, utrujenost, astenija, zmanjšanje apetita, globoka venska tromboza, omotica, hipomagneziemija in PPES. Najpogostejši resni neželeni ucinki zdravila v populaciji bolnikov s HCK so bili jetrna encefalopatija, astenija, utrujenost, PPES, driska, hiponatriemija, bruhanje, bolecine v trebuhu in trombocitopenija. Zelo pogosti: anemija, trombocitopenija, hipotiroidizem, zmanjšanje apetita, hipomagneziemija, hipokaliemija, hipoalbuminemija, paragevzija, glavobol, omotica, hipertenzija, krvavitev, disfonija, dispneja, kašelj, driska, navzea, bruhanje, stomatitis, obstipacija, bolecine v trebuhu, dispepsija, bolecina v zgornjem predelu trebuha, PPES, izpušcaj, bolecine v okoncinah, utrujenost, vnetje sluznice, astenija, periferni edem, zmanjšanje telesne mase, zvišanje ALT v serumu, zvišanje AST. Pogosti: absces, nevtropenija, limfopenija, dehidracija, hipofosfatemija, hiponatriemija, hipokalciemija, hiperkaliemija, hiperbilirubinemija, hiperglikemija, hipoglikemija, periferna nevropatija (vkljucno s senzoricno), tinitus, globoka venska tromboza, venska tromboza, arterijska tromboza, pljucna embolija, GI perforacija, .stula, GERB, hemoroidi, bolecina v ustni votlini, suha usta, disfagija, glosodinija, jetrna encefalopatija, pruritus, alopecija, suha koža, akneiformni dermatitis, sprememba barve las oz. dlak, hiperkeratoza, mišicni krci, artralgija, proteinurija, zvišanje ALP v krvi, GGT, kreatinina v krvi, amilaze, lipaze, holesterola v krvi, trigliceridov v krvi. Obcasni: konvulzije, pankreatitis, holestaticni hepatitis, osteonekroza celjusti, zapleti z ranami. Neznana pogostnost: možganska kap, miokardni infarkt, anevrizme in disekcije arterij. Vrsta ovojnine in vsebina: Plastenka vsebuje 30 .lmsko 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 znacilnosti zdravila! CAB-300420 SAMO ZA STROKOVNO JAVNOST PharmaSwiss d.o.o., Brodišce 32, 1236 Trzin CAB0720-03, julij 2020 telefon: +386 1 236 47 00, faks: +386 1 236 47 05 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 Serša, 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) 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 Godény, National Institute of Oncology, Budapest, Hungary Gordana Ivanac, University Hospital Dubrava, Department of Diagnostic and Interventional Radiology, Zagreb, Croatia Luka Ležaic, University Medical Centre Ljubljana, Department for Nuclear Medicine, Ljubljana, Slovenia Katarina Šurlan Popovic, University Medical Center Ljubljana, Clinical Institute of Radiology, Ljubljana, Slovenia Jernej Vidmar, University Medical Center Ljubljana, Clinical Institute of Radiology, Ljubljana, Slovenia 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 Deputy Editors Andrej C, 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 Cemažar, Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia (Subject Area: Experimental Oncology) 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 Mikl Kásler, National Institute of Oncology, Budapest, Hungary Maja Osmak, Ruder Boškovic Institute, Department of Molecular Biology, Zagreb, Croatia September 2020 Vol. 54 No. 3 Pages 253-370 ISSN 1318-2099 UDC 616-006 CODEN: RONCEM Igor Kocijancic, University Medical Center Ljubljana, Institute of Radiology, Ljubljana, Slovenia (Subject Areas: Radiology, Nuclear Medicine) Karmen Stanic, 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) 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 Turnšek, National Institute of Biology, Ljubljana, Slovenia Damijan Miklavcic, University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia Justin Teissié, 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 Häkan Nystr, Skandionkliniken, Uppsala, Sweden Ervin B. Podgoršak, McGill University, Medical Physics Unit, Montreal, Canada Matthew Podgorsak, Roswell Park Cancer Institute, Departments of Biophysics and Radiation Medicine, Buffalo, NY ,USA Editorial office Radiology and Oncology Zaloška cesta 2 P. O. Box 2217SI-1000 Ljubljana Slovenia Phone: +386 1 5879 369Phone/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 KlemencicZvezdana Vukmirovic Design Monika Fink-Serša, Samo Rovan, Ivana Ljubanovic Layout Matjaž Lužar Printed by Tiskarna Ozimek, Slovenia Published quarterly in 400 copies Beneficiary name: DRUŠTVO RADIOLOGIJE IN ONKOLOGIJE Zaloška cesta 2 1000 Ljubljana Slovenia Beneficiary bank account number: SI56 02010-0090006751 IBAN: SI56 0201 0009 0006 751 Our bank name: Nova Ljubljanska banka, d.d., Ljubljana, Trg republike 2, 1520 Ljubljana; Slovenia SWIFT: LJBASI2X Subscription fee for institutions EUR 100, individuals EUR 50 The publication of this journal is subsidized by the Slovenian Research Agency. Indexed and abstracted by: • 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 This journal is printed on acid- free paper On the web: ISSN 1581-3207 https://content.sciendo.com/raon http://www.radioloncol.com • 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) contents review 253 Transarterial embolization of the external carotid artery in the treatment of life-threatening hemorrhage following blunt maxillofacial trauma Crt Langel, Dimitrij Lovric, Ursa Zabret, Tomislav Mirkovic, Primoz Gradisek, Anita Mrvar-Brecko, Katarina Surlan Popovic 263 Current management of intrahepatic cholangiocarcinoma: from resection to palliative treatments Ilenia Bartolini, Matteo Risaliti, Laura Fortuna, Carlotta Agostini, Maria Novella Ringressi, Antonio Taddei, Paolo Muiesan 272 Consensus molecular subtypes (CMS) in metastatic colorectal cancer ­personalized medicine decision Martina Rebersek nuclear medicine 278 Prognostic role of positron emission tomography and computed tomography parameters in stage I lung adenocarcinoma Angelo Carretta, Alessandro Bandiera, Piergiorgio Muriana, Stefano Viscardi, Paola Ciriaco, Ana Maria Samanes Gajate, Gianluigi Arrigoni, Chiara Lazzari, Vanesa Gregorc, Giampiero Negri 285 [18F]FDG PET immunotherapy radiomics signature (iRADIOMICS) predicts response of non-small-cell lung cancer patients treated with pembrolizumab Damijan Valentinuzzi, Martina Vrankar, Nina Boc, Valentina Ahac, Ziga Zupancic, Mojca Unk, Katja Skalic, Ivana Zagar, Andrej Studen, Urban Simoncic, Jens Eickhoff, Robert Jeraj radiology 295 Improvement of the primary efficacy of microwave ablation of malignant liver tumors by using a robotic navigation system Jan Schaible, Benedikt Pregler, Niklas Verloh, Ingo Einspieler, Wolf Bäumler, Florian Zeman, Andreas Schreyer, Christian Stroszczynski, Lukas Beyer 301 Simplified perfusion fraction from diffusion-weighted imaging in preoperative prediction of IDH1 mutation in WHO grade II–III gliomas: comparison with dynamic contrast-enhanced and intravoxel incoherent motion MRI: SPF, DCE and IVIM for IDH1 mutation Xiaoqing Wang, Mengqiu Cao, Hongjin Chen, Jianwei Ge, Shiteng Suo, Yan Zhou 311 The feasibility of ultrasound-guided vacuum-assisted evacuation of large breast hematomas” Sa’ed Almasarweh, Mazen Sudah, Sarianna Joukainen, Hidemi Okuma, Ritva Vanninen, Amro Masarwah experimental oncology 317 Analysis of damage-associated molecular pattern molecules due to electroporation of cells in vitro Tamara Polajzer, Tomaž Jarm, Damijan Miklavcic clinical oncology 329 Impact of COVID-19 on cancer diagnosis and management in Slovenia – preliminary results Vesna Zadnik, Ana Mihor, Sonja Tomsic, Tina Zagar, Nika Bric, Katarina Lokar, Irena Oblak 335 Breast cancer risk based on adapted IBIS prediction model in Slovenian women aged 40–49 years - could it be better? Tjasa Oblak, Vesna Zadnik, Mateja Krajc, Katarina Lokar, Janez Zgajnar 341 Standard and multivisceral colectomy in locally advanced colon cancer Artur M. Sahakyan, Andranik Aleksanyan, Hovhannes Batikyan, Hmayak Petrosyan, Mushegh .. Sahakyan 347 Percutaneous image guided electrochemotherapy of hepatocellular carcinoma: technological advancement Mihajlo Djokic, Rok Dezman, Maja Cemazar, Miha Stabuc, Miha Petric, Lojze M. Smid, Rado Jansa, Bostjan Plesnik, Masa Bosnjak, Ursa Lampreht Tratar, Blaz Trotovsek, Bor Kos, Damijan Miklavcic, Gregor Sersa, Peter Popovic 353 Consolidation radiotherapy for patients with extended disease small cell lung cancer in a single tertiary institution: impact of dose and perspectives in the era of immunotherapy Karmen Stanic, Martina Vrankar, Jasna But-Hadzic radiophysics 364 Assessment of set-up errors in the radiotherapy of patients with head and neck cancer: standard vs. individual head support Sabina Androjna, Valerija Zager Marcius, Primoz Peterlin, Primoz Strojan I slovenian abstracts 253 review Transarterial embolization of the external carotid artery in the treatment of life-threatening haemorrhage following blunt maxillofacial trauma Crt Langel1, Dimitrij Lovric1, Ursa Zabret1, Tomislav Mirkovic2, Primoz Gradisek2, Anita Mrvar-Brecko2, Katarina Surlan Popovic1 1 Institute of Radiology, University Medical Centre Ljubljana, Ljubljana, Slovenia 2 Department of Anaesthesiology and Surgical Intensive Care, Division of Surgery, University Medical Centre Ljubljana, Ljubljana, Slovenia Radiol Oncol 2020; 54(3): 253-262. Received 7 February 2020 Accepted 22 April 2020 Correspondence to: Assoc. Prof. Katarina Šurlan Popovic, M.D., Ph.D., Clinical Institute of Radiology, University Medical Centre Ljubljana, Zaloška c 7, 1000 Ljubljana, Slovenia. E-mail: katarina.surlan-popovic@mf.uni-lj.si. Disclosure: No potential conflicts of interest were disclosed. Background. Severe bleeding after blunt maxillofacial trauma is a rare but life-threatening event. Non-responders to conventional treatment options with surgically inaccessible bleeding points can be treated by transarterial emboliza­tion (TAE) of the external carotid artery (ECA) or its branches. Case series on such embolizations are small; considering the relatively high incidence of maxillofacial trauma, the ECA TAE procedure has been hypothesized either underused or underreported. In addition, the literature on the ECA TAE using novel non-adhesive liquid embolization agents is remarkably scarce. Patients and methods. PubMed review was performed to identify the ECA TAE literature in the context of blunt maxillofacial trauma. If available, the location of the ECA injury, the location of embolization, the chosen embolization agent, and efficacy and safety of the TAE were noted for each case. Survival prognostic factors were also reviewed. Additionally, we present an illustrative TAE case using a precipitating hydrophobic injectable liquid (PHIL) to safely and effectively control a massive bleeding originating bilaterally in the ECA territories. Results and conclusions. Based on a review of 205 cases, the efficacy of TAE was 79.4–100%, while the rate of ma­jor complications was about 2–4%. Successful TAE haemostasis, Glasgow Coma Scale score = 8 at presentation, injury severity score = 32, shock index = 1.1 before TAE and = 0.8 after TAE were significantly correlated with higher survival rate. PHIL allowed for fast yet punctilious application, thus saving invaluable time in life-threatening situations while si­multaneously diminishing the possibility of inadvertent injection into the ECA-internal carotid artery (ICA) anastomoses. Key words: blunt maxillofacial trauma; external carotid artery injury; intractable bleeding; non-adhesive liquid embo­lization agent; precipitating hydrophobic injectable liquid, neurointervention Introduction Maxillofacial trauma comprises roughly 10% of all trauma cases.1 It is associated with a wide range of problems, including airway compromise, cervi­cal spine injuries and bleeding.2 Life-threatening haemorrhage secondary to blunt maxillofacial trauma is considered rare, occurring in 1.2%–4.5% of trauma-related maxillofacial fracture cases.2-5 The most common origins of haemorrhage in max-illofacial trauma are the internal maxillary artery (IMA), the IMA’s distal branches, and the main trunk of the external carotid artery (ECA).6 A diverse range of imaging manifestations can present in the setting of blunt carotid artery trau­ma, including various dissection subtypes (mini- 254 mal intimal injury, raised intimal flap, dissection with an intramural hematoma, occlusion), pseu­doaneurysm, transection with an active haemor­rhage, and arteriovenous fistula.7,8 In treating severe maxillofacial injuries, airway, breathing and circulation management precede all other procedures. Special care is aimed towards the protection of the airway as the tongue and the soft tissues of the lower face can move backward and obstruct the pharynx due to the decreased level of consciousness, bilateral mandibular fractures, soft tissue swelling and expanding hematomas.2 Further treatment includes manual compression, nasal packing, coagulopathy correction, cauteriza­tion, reduction of the fractures and local vascular control to stop the bleeding from the intra-osseous branches near the fracture lines.9 Open surgical li­gation or transarterial embolization (TAE) of the ECA are available as the most definite options. Advantages of TAE over ligation include rapid access, not necessarily requiring general anaes­thesia, superior haemorrhage origin localization using angiography, the ability to control multiple local bleeding points, the ability to perform super-selective therapeutic vessel occlusion by cannulat­ing the smaller vessel branches not amenable to open surgical repair, and short procedure time.10 In addition, ECA ligation oftentimes requires repeat ligation or subsequent TAE to effectively stop the bleeding, whereas TAE is usually efficacious in a single session.2,11 Furthermore, TAE offers the op­tion to embolize the bleeding origins of a possible concomitant abdominal or other internal haemor­rhage in the same session.12 In certain guidelines, surgical ECA ligation has been completely re­placed by TAE for maxillofacial bleeding control.2 The initial search data for the review part of this manuscript was processed following a simpli­fied variant of the Preferred Reporting Items for Systematic Reviews and MetaAnalyses (PRISMA) guidelines showing the number of cases identified, included and excluded, plus the reasons for exclu­sions (Figure 1).13 The included cases were then ana­lysed in order to obtain the data regarding the use of embolization agents, and the efficacy and safety of the TAE procedure in the context of blunt maxillofa­cial trauma with bleeding originating from the ECA. Transarterial embolization of the ECA Embolization therapy aims at controlling an active bleeding by occluding the feeding artery with an embolization agent (EA).14 The TAE technique was first suggested by Brooks et al. in the 1930s. In the early 1970s, Rosch and Dotter used it for the first time to treat a traumatic vascular injury.15 It also became an accepted treatment option for a variety of vascular lesions unrelated to trauma, including arteriovenous malformations, glomus tumours, juvenile angiofibromas, and intracranial meningi­omas.16 A pioneer case describing a successful IMA TAE in the treatment of intractable epistaxis was reported by Sokoloff et al. in 1974.17 Decades of innovation brought about major ad­vances in embolization materials, however, to this day, the principles of the TAE procedure remain largely unaltered.18 Initially, a vascular access is established by a standard transfemoral approach using the Seldinger technique.19 Alternatively, bra-chial or axillary arterial access might be required in case of severe bilateral lower extremity injury.20 Diagnostic angiography of the whole circulation at risk is then performed, including bilateral common carotid arteries (CCAs), internal carotid arteries (ICAs), vertebral arteries (VAs), and ECAs. If the 255 preliminary computed tomography angiography (CTA) or clinical findings suggest an injury to a spe­cific smaller vessel, e.g. the IMA, the lingual artery or the superficial temporal artery, further micro-catheter angiography of these territories is carried out. The purpose is to obtain a general overview of the complete vasculature and to exclude a possible concomitant injury to the CCA, ICA or VA.21 The vessel that is the source of an active haemorrhage is then therapeutically occluded. The aim of the embolization is to stop the active bleeding, prevent any subsequent rebleeding events, and preserve as much perfusion to the nearby structures as possi­ble, thereby reducing the chance of unnecessary tis­sue damage. This means embolization is attempted as close to the lesion as possible to avoid occluding the vessels branching off proximally to the embo­lization site. Circumstances permitting, the culprit vessel should ideally be embolized both distally and proximally to the lesion in order to prevent rebleeding via the collateral circulation.21 The de­livery of the EA is performed under fluoroscopic visualization to the point of contrast medium stasis within the embolized vessel. Embolization agents Depending on the vessel calibre, type of vessel in­jury and other factors, a variety of EAs with dif­fering inherent properties and behaviour may be used. Historically, the first EA was autologous tissue (including blood clots, subcutaneous tissue and muscle) followed by silk threads.22 Their use gradually declined with the advances in newer EAs, imaging and (micro)catheter technologies.14,23 Data gathered in Table 1 indicates coils and gel-atine foam (Gelfoam) are the EAs most frequently used for TAE in the context of blunt maxillofacial trauma. There have also been numerous instanc­es of polyvinyl alcohol (PVA), microspheres, and N-butyl-2 cyanoacrylate (NBCA) use. One case of silastic spheres, two cases of Onyx, and a single case of precipitating hydrophobic injectable liquid (PHIL) use have been reported. Coils are made from platinum or steel, measure 0.2–1.3 mm in diameter and can be supplied in a variety of lengths, shapes and levels of stiffness. They may be bare or fibered with materials such as wool, silk, nylon fibres, polyester, Dacron or PVA.18 Coils embolize a vessel by physically slowing down the local blood flow, by providing a thrombogenic locus, and by damaging the vessel wall, thus induc­ing the release of thrombogenic factors. Time to oc­clusion is typically 5 minutes or less after coil inser­tion, depending on the type of coil used, the rate of blood flow through the target vessel and the blood’s coagulation properties.18 Larger diameter platinum coils offer good radiopacity, while smaller diameter coils (microcoils) provide for more targeted distal deployment. The possible complications of TAE us­ing coils are non-target vessel occlusion, vessel in­jury, coil migration, and infection.18,24 Coils prohibit any future endovascular access distal to the occlu­sion point, which is particularly relevant in rebleed­ing events following collateralization.21 Gelatine foam (Gelfoam) (Pfizer, Kalamazoo, MI, USA) is a porous material with haemostatic properties prepared from purified porcine skin gelatine. It usually supplied as a block of sponge that needs to be cut into smaller cube- or torpedo-shaped particles prior to embolization. Gelfoam induces foreign body reaction and necrotizing ar­teritis, resulting in the formation of a thrombus.25 Gelfoam as a standalone EA provides a temporary vessel occlusion; recanalization typically occurs within 3 weeks to 3 months, but the exact time and the extent cannot reliably be predicted.23 A com­bined coils-Gelfoam embolization may be particu­larly well suited for coagulopathic patients as such vessel occlusion is precise, fast, and permanent.23 The most significant disadvantage of Gelfoam-only embolization is the reliance on manual prepara­tion of the particles, limiting the reproducibility and predictability of the exact embolization site. Furthermore, air bubbles typically form in the Gelfoam-contrast mixture, presenting a potential risk for an aerobic infection.18 Polyvinyl alcohol (PVA) particles (Boston Scientific, Cork, Ireland; Cordis J&J Endovascular, Miami, FL, USA) are irregularly-shaped permanent embolic agents ranging from 100 to 1100 µm in size. The PVA’s mechanism of action includes adherence to the vessel wall, induction of an inflammatory re­action, focal angionecrosis and the resulting vessel fibrosis. There is a considerable variability in parti­cle size because fragments smaller than the stated size range are allowed to enter the particulate mix­ture during production. This in turn increases the risk of distal, non-target embolization as particles tend to lodge in the smallest vessel they can fit in. On the other hand, the PVA particles are also prone to aggregation; this can lead to more proximal ves­sel occlusion than expected based on the stated size range of the particles.18 Microspheres (Embosphere and EmboGold, Merit Medical Systems, South Jordan, UT, USA; Contour SE, Boston Scientific, Natick, MA, USA; 256 TABLE 1. Data from studies, case series and case reports pertaining to TAE of the ECA or its branches in the treatment of haemorrhage caused by blunt maxillofacial trauma 1 none identified RL ECA partial tongue necrosis 2 R IMA R IMA none 3 R IMA RL IMA none 4 none identified RL ECA above LA none Bynoe2 2003 5 6 none identified none identified RL ECA above LA RL ECA above LA C GF PVA 100% groin hematoma none 7 L IMA L IMA none 8 9 L IMA L IMA L IMA R ECA above LA, L IMA groin hematoma none 10 none identified R ECA above LA none 11 12 13 R IMA, R STA R IMA L IMA not specified not specified not specified C GF C GF GF could not be assessed none could not be assessed Chen12 2009 14 15 16 17 R IMA L IMA RL IMA L ECA not specified not specified not specified not specified GF GF GF C GF 100% none none could not be assessed none Cogbill48 2008 18 19–39 L IMA not discernible due to the merging of blunt and penetrating trauma patients' data not specified not discernible due to the merging of blunt and penetrating trauma patients' data GF C GF 85% none none Kim49 2011 40 not specified L IMA PVA success none 41 not specified R SPA none 42 not specified RL SPA, IOA, FA none Komiyama50 1998 43 44 45 46 not specified not specified not specified not specified L SPA RL SPA, LPA, ADTA6, IAA7, FA, LA RL SPA, FA BL SPA, AAA8 C GF PVA 100% none none none could not be assessed 47 not specified R STA, FA none 48 not specified RL GPA9, EA none 49 not specified RL SPA, SPAA10 none 12 x IMA 6 x FA 6 x LA Kuan47 2015 50–76 5 x MMA11 3 x ECA 1 x APA12 not specified C GF PVA NBCA 92.3%; data includes one penetrating injury no serious systemic or neurologic complications 5x other vessels Note: this statistics also includes one penetrating trauma. 257 Langel 2020 77 L IMA + R SPA 25 x IMA L ECA + R SPA C PHIL 25 success none 5 x MMA 4 x ECA 4 x SPA 4 x STA Liao40 2007 78–112 2 x APA 1 x FA not specified not specified 79.4% none reported 1 x SALA 1 x IALA Liu3 2008 113 1 x DPA 5 x other (observed contrast pooling) L STA + L IMA not specified GF success none reported Maiorello51 2011 114 L FA L FA Onyx 18 success none Mauldin52 Mehringer52 Mehrotra6 Noy38 Remonda54 Thiex33 1989 1982 1984 2007 2000 2011 115 116–194 195–196 197 198 199 R ECA not discernible due to the merging of blunt and penetrating trauma patients' data R IMA L IMA L IMA, RL FA RL IMA L FA R ECA not discernible due to the merging of blunt and penetrating trauma patients' data R IMA L IMA L IMA, RL FA not specified not specified C GF PVA silastic spheres GF PVA GF MS NBCA C PVA NBCA Onyx success 100% success success success success success none reported 1x cerebral infarction 2x transient occulomotor nerve palsy none reported none reported none Transient trismus none 200 L STA not specified C none 201 L FA not specified C none Wang55 2015 202 R STA not specified C 100% none 203 R STA not specified C none 204 L STA not specified C none Wong56 2013 205 R IMA R IMA NBCA success none Anatomical abbreviations: AAA = anterior auricular artery; ADTA = anterior deep temporal artery; APA = ascending pharyngeal artery; DPA = descending palatine artery; ECA = external carotid artery; FA = facial artery; GPA = greater palatine artery; IAA = inferior alveolar artery; IALA = inferior alveolar artery; IMA = internal maxillary artery; IOA = infraorbital artery; L = left; LPA = lesser palatine artery; MMA = middle meningeal artery; R = right; SALA = superior alveolar artery; SPA = sphenopalatine artery; SPAA = superior posterior alveolar artery; STA = superficial temporal artery Embolization agent abbreviations: C = coils; GF = Gelfoam; MS = microspheres; PHIL = precipitating hydrophobic injectable liquid; PVA = polyvinyl alcohol particles Embozene, Celenova, San Antonio, TX, USA; impregnated with porcine gelatine. They are hy-Quadrasphere, Merit Medical Systems, South drophilic, non-resorbable, non-aggregating, non-Jordan, UT, USA; Bead Block and LC Bead, fragmenting spheres sized 40 - 1200 µm. Sizing Biocompatibles, Farnham, UK) are smooth globu-inside a particular stated size range follows the lar structures made from an acrylic polymer matrix Gaussian distribution and is thus more predictable 258 than is the case with PVA particles. After lodging in vessels, microspheres induce a histological reaction similar to PVA particles. The most notable down­side of using microspheres is the necessity to in­termittently agitate the particle-saline suspension prior to application in order to prevent sedimenta­tion.18,25,26 Also of note is the fact that microspheres of different manufacturers vary in elasticity and, as a consequence, particles of identical size range but different composition occlude vessels at different levels of the vascular tree.25,27 NBCA (TruFill, Cordis, Miami Lakes, FL; Histoacryl, B. Braun Aesculap, Tokyo, Japan; Glubran 2, Gem, Viareggio, Lucca, Italy) is a syn­thetic adhesive liquid EA (glue) that is accompa­nied by a separately packed tantalum powder, act­ing as a radiographic opacifier, and ethiodized oil, functioning as a polimerization retardant. All three components are mixed just before deployment. Once the solution is exposed to anionic environ­ment such as blood, polymerization takes place at a rate dependent on the NBCA concentration. NBCA forms a permanent cast obstructing the vessel lu­men. This occurs independently of the endogenous coagulation system - an important characteristic when dealing with exsanguinating trauma cases.18 NBCA glue also induces vessel wall inflamma­tion reaction resulting in fibrosis. The downside of NBCA use is that the catheter may become glued to the vessel wall if not pulled back quickly enough following glue injection. Glue polymerisation can also occur both distally or proximally to the in­tended occlusion location. NBCA deployment thus requires a skilled operator.28 Silastic spheres are the oldest non-absorbable particulate EA, introduced in 1964, and have since been replaced by more modern EAs.29 Onyx (Medtronic, Dublin, Ireland) is an ethyl­ene vinyl alcohol (EVOH) copolymer-based non­adhesive liquid EA dissolved in dimethyl sulfox­ide (DMSO) with added radiopaque tantalum powder. It was introduced in 1990. As is the case with other DMSO-based non-adhesive liquid EAs, once injected, the DMSO component dissolves in­to the blood and the copolymer component starts gradually precipitating in a centripetal fashion. The non-adhesive nature allows for long injec­tion times and mid-injection control angiography, if needed. Onyx’s final solidification occurs in 5 minutes.30 There are several drawbacks to this EA, including long pre-injection preparation time (20 minutes of mixer shaking are required to achieve homogenization), a self-hiding effect when used in larger amounts due to high radiopacity, plenty of artefacts in a postinterventional imaging, and the potential to combust or produce sparks during monopolar surgical cauterization.31,32 In addition, Onyx’s dark colour may result in a black discol­oration of the skin after superficial embolization or subcutaneous extravasation of the EA.33,34 Precipitating hydrophobic injectable liquid (PHIL) (MicroVention, Tustin, CA, USA) is a non-EVOH copolymer-based non-adhesive liquid EA suspended in DMSO with iodine covalently bond­ed to copolymer to provide radiopacity. It was in­troduced in 2015. In comparison to Onyx, PHIL is supplied ready-to-use (no shaking is necessary), requires lower volume to achieve the same extent of embolization, is faster to fully precipitate (3 min­utes), does not suffer from the self-hiding effect, pro­duces fewer artefacts in postinterventional imaging, is not hazardous to surgical cauterization, and is not dark coloured which diminishes the possibility of skin discoloration.35,36 PHIL is also more homog­enous on fluoroscopy during prolonged injections, but less radiopaque than Onyx once injected.23 According to the literature, no cases of blunt trauma-related ECA-territory embolization using other modern non-adhesive liquid EAs, such as Squid (Emboflu, Gland, Switzerland), have so far been reported. In the future, other cutting-edge EAs, such as homogenous microparticles or biode­gradable drug-bearing microspheres produced by droplet microfluidics technology, are expected to see regular clinical use.37 Efficacy and safety Various studies and case reports have shown the ECA TAE to be a safe and efficacious method in maxillofacial blunt-trauma related haemorrhage control, although direct comparison of the re­viewed literature is rendered difficult by the vari­ations in reporting. Two studies authored by Noy et al. and by Hayes et al. investigating intractable maxillofacial bleeding of various aetiologies, in­cluding but not limited to trauma, enrolling 74 pa­tients in total, found TAE to be efficacious in 89.1 - 90.0%.38,39 A study by Liao et al. focusing exclu­sively on trauma-related oronasal bleeding enroll­ing 34 patients discovered TAE to be efficacious in 79.4%.40 The data collected in Table 1 show that the efficacy of TAE ECA ranges from 79.4% to 100%, with the largest series attaining the perfect success rate comprising 10 cases. These results are similar to the efficacy of non-trauma-related TAE proce­dures involving the ECA (80%–97%).41-43 259 The complications among the 205 cases re­viewed in Table 1 include groin hematoma (2 cas­es), cerebral infarction (1 case), partial tongue ne­crosis (1 case), transient occulomotor nerve palsy (1 case), and transient trismus (1 case), indicating an overall complication rate of 3%. However, there is high variability in reporting styles regarding the complications. For example, certain authors lim­ited the reporting only to serious neurologic or systemic complications without further expound­ing on the exact criterion that delimited the serious complications from the minor ones. Furthermore, complications that could not be assessed might have occurred, e.g. due to patient dying or entering a vegetative state. In addition, it was not possible to determine the risk of publication bias or selective reporting. It is thus safe to assume the overall com­plication rate to be higher than directly indicated by Table 1 data. Duncan et al. found the rate of major complications (comprising cerebral vascular insult only) to be 2% but also reported a rate of mi­nor complications (comprising headache, transient facial pain, paraesthesia, and local groin complica­tions) of 25% in their series of 57 embolizations, of these 3 trauma-related.44 Cullen and Tami re­viewed the literature of 264 cases of IMA emboli­zations for the treatment of posterior epistaxis and found the rate of major complications (hemiplegia, facial nerve paralysis, cheek necrosis, ICA intimal injury, catheter stuck in a vessel, myocardial infarc­tion) to be 4%, and the rate of minor complications (IMA spasm, hypotension, hematoma, groin bleed, oedema, trismus, paraesthesia, persistent pain, skin slough, palate ulceration, aspiration pneumo­nia, hepatitis) to be 10%.45 The relatively low rate of major complications might be in part due to the rich collateral flow between the ipsilateral and contralateral ECA branches distal to the lingual artery that ensure ad­equate tissue perfusion in case of one-sided ECA embolization.46,2 Duncan et al. discovered that the complication rate tends to drop with the use of mi-crocatheter techniques. They have also observed a decrease in complication rate in the more recent studies that could be ascribed to factors such as im­provement in catheter and guidewire design and increased operator experience.44 Survival prognostic factors Liao et al. examined a series of 34 cases of crani­ofacial trauma requiring TAE and discovered that there was a significant contribution of successful TAE haemostasis to patient survival (p = 0.001). Glasgow Coma Scale score (GCS) = 8 at presenta­tion, injury severity score (ISS) = 32, and shock in­dex (SI; heart rate divided by systolic blood pres­sure) = 1.1 before TAE and = 0.8 after TAE were also significantly correlated with the patients’ higher survival rate (p < 0.05). The need to treat a second­ary abdominal bleeding origin by laparotomy sig­nificantly decreased the rate of survival (p = 0.023). The patients’ age, the need to perform craniotomy, the bilateral distribution of the bleeding vessels, and the number of the haemorrhaging vessels per patient were not correlated with the patient sur­vival (p > 0.05).40 Kuan et al. confirmed some of the findings by Liao et al. and further discovered that patients with initial haemoglobin level lower than 10 g/dL and patients with brain midline shift observed by com­puted tomography (CT) had statistically higher odds ratios predicting mortality than their coun­terparts as estimated by univariate logistic regres­sion.47 An illustrative case report A 20-year-old, previously healthy male was brought to the emergency department in 2019 after an acci­dental 20 m (65 ft), head-first fall to the ground. He had been cleaning windows of his 7th floor dorm room when he lost balance and fell. Eyewitnesses reported the patient had been lying on his stomach after impacting the ground but later managed to roll on his back by himself. A physician-led emer­gency medical service arrived on the scene in 10 minutes, finding the patient verbally responsive and making an effort to get up. Initial Glasgow Coma Scale (GCS) was an estimated 13. Severe fa­cial trauma compromising the airway and, within a few minutes, cessation of spontaneous respira­tion necessitated rapid sequence intubation which proved challenging with several failed attempts. Asystole was observed on ECG prompting resus­citation efforts that resulted in the return of spon­taneous circulation and sinus rhythm 20 minutes later. Upon arrival at a Level I trauma centre, the patient presented with GCS 5, blood pressure 70/40 mmHg, heart rate 100/min., a multifragment facial fracture (Figure 2A), a ruptured right eye, massive bleeding from the nose and the left ear, a fractured right 4th rib, a fractured left radius, a displaced left femoral fracture and a fractured left tibia. Adhering to our institution’s standard trauma pro­tocol, the possible abdominal and thoracic sources 260 FIGURE 3. (A) A lateral left ECA angiogram showing ECA laceration with 3 × 4 cm pseudoaneurysm continuing into the proximal part of the left IMA. Contrast extravasation can be observed in the vicinity of the pseudoaneurysm. (B) A fluoroscopic view showing left ECA embolization using coils (black arrow) and PHIL 25 liquid embolization agent (white arrow) under balloon flow control (empty arrow). Also of note area small number of stray coils anchored in the vessel in the region of PHIL application (white arrow). (C) A post-embolization lateral left ECA angiogram showing complete exclusion of the ECA distally to the facial artery (black arrow). FIGURE 4. (A) A lateral right ECA angiogram showing two pseudoaneurysms in the region of the right pterygopalatine fossa and nasal cavity (black arrows). (B) Fluoroscopy showing microcatether proximally to the hematoma in the right pterygopalatine fossa (black arrow) prior to PHIL 25 application. Also visible is the embolized contralateral ECA (empty arrow). (C) A post-embolization right ECA angiogram showing complete sphenopalatine artery occlusion (black arrow). Also visible are the patent vessels proximally to the embolization. of major blood loss were excluded. Astonishingly, US, XR, CT and CTA imaging indicated no signifi­cant damage to the neurocranium, parenchymal organs or major thoracic or abdominal vessels. Aorto-cervical CTA was then performed, reveal­ing contrast extravasation from the left IMA and the right sphenopalatine artery (SPA) (Figure 2B). This was consistent with the clinical presentation of severe antero-posterior epistaxis and pulsatile bleeding from the left ear. Nasal packing using bal­loon catheter inserted through the nares into the nasopharynx was performed by an ear, nose and throat (ENT) specialist to successfully control the nose bleeding. Tamponade of the left ear, however, proved to be inadequate with profound bleeding still persisting. Surgical treatment to control the haemorrhage by ligating the left ECA was decid­ed against due to lesion inaccessibility caused by extensive soft tissue damage and swelling. Blood pressure remained low (60/40 mmHg) despite hav­ing hitherto administered a total of 5 litres of flu­ids, including blood transfusion. Tranexamic acid and vasopressors were also applied, to little avail. In these life-threatening circumstances, TAE of the bleeding origins was considered the only remain­ing option. The patient was transferred to the neurointer­ventional suite and a standard right transfemoral vascular access was established. Digital substrac­tion angiography (DSA) showed a laceration of the left ECA with an ensuing 3 × 4 cm pseudoaneurysm continuing into the proximal part of the left IMA (Figure 3A). DSA also showed a right SPA lacera­tion with two small accompanying pseudoaneu­rysms (Figure 4A). The two culprit arteries were then superselectively catheterized and embolized. ECA embolization was performed using platinum coils and PHIL 25, while the SPA was embolized with PHIL 25 only (Figures 3B and 4B). In the case of the ECA embolization, coils created a mesh scaf­fold acting as a thrombogenic locus, and PHIL was then added to form a coagulopathy-independent lumen-obliterating cast. PHIL was chosen over other available liquid EAs for its ready-to-use char­acteristics, saving precious time in an emergency setting. In addition, its lava-like polymerisation properties ensured a well-controlled application, helping prevent any inadvertent injection into the dangerous ECA-internal carotid artery and ECA-vertebral artery anastomoses. Effort was made to embolize at or just proximal to the laceration point in order to preserve proxi­mal arterial territories. Embolization was contin­ued to the point of arterial stasis. Due to the well- 261 developed left ECA and the resulting high blood flow to the pseudoaneurysm, the ECA emboliza­tion was performed under flow control provided by temporary proximal balloon occlusion. No flow control was needed for the SPA embolization. The embolizations of both lesions were immediately followed by complete cessation of the ear bleeding. Postembolization imaging showed total exclu­sion of the lacerated vessels (Figures 3C and 4C), complete patency of all proximal vessels, no collat­eral pathways to the pseudoaneurysm and no oth­er origins of bleeding. There were no procedure-related complications. To our knowledge, this case is the very first published report of PHIL 25 use for a safe and ef­ficacious management of a massive bleeding origi­nating in the ECA territory. Informed consent was obtained from the patient included in this case re­port as well as the consent for publication of the individual person’s data. Conclusions Severe bleeding secondary to blunt maxillofacial trauma is a rare but life-threatening occurrence. Conventional treatment options include manual compression, nasal packing, coagulopathy correc­tion, cauterization, fracture reduction and local vascular control. Open surgical ligation or TAE of the ECA are available as the most definite ap­proaches. Both are similarly efficacious and safe, but TAE offers many advantages, including short­er procedure time, more precise haemorrhage lo­calization, vessel occlusion superselectivity, and the ability to embolize a possible concomitant abdominal or other bleeding during the same ses­sion. Major complications of TAE are rare owing to rich collateral blood supply in the ECA territory, with the exception of the lingual artery. The diver­sion of embolization material into the ICA territo­ries via the ECA-ICA anastomoses is a potentially hazardous complication warranting thorough pre­embolization angiographic overview of the whole vasculature at risk. There are several known fac­tors positively affecting the survival of the maxillo-facial trauma patients undergoing ECA TAE, most notably a successful TAE haemostasis, higher GCS, lower ISS, lower SI and higher haemoglobin level on arrival. Novel non-adhesive liquid EAs such as PHIL al­low for faster, more punctilious and coagulopathy-independent application, thus saving invaluable time in life-threatening situations while simultane­ously diminishing the possibility of inadvertent in­jection into the ECA-ICA anastomoses. Prospective randomized clinical studies comparing EAs are warranted to further evaluate their efficacy, safety and feasibility. References 1. American College of Surgeons, Committee on Trauma. Advanced trauma life support manual. Chicago: American College of Surgeons; 1994. 2. Bynoe RP, Kerwin AJ, Parker HH 3rd, Nottingham JM, Bell RM, Yost MJ, et al. Maxillofacial injuries and life-threatening hemorrhage: treatment with tran­scatheter arterial embolization. J Trauma 2003; 55: 74-79. doi: 10.1097/01. TA.0000026494.22774.A0 3. Liu WH, Chen YH, Hsieh CT, Lin EY, Chung TT, Ju DT. Transarterial emboliza­tion in the management of life-threatening hemorrhage after maxillofacial trauma: a case report and review of literature. Am J Emerg Med 2008; 26: 516. e3-5. doi: 10.1016/j.ajem.2007.07.036 4. Thaller SR, Beal SL. Maxillofacial trauma: a potentially fatal injury. Ann Plast Surg 1991; 27: 281-3. doi: 10.1097/00000637-199109000-00015 5. Buchanan RT, Holtmann B. Severe epistaxis in facial fractures. Plast Reconstr Surg 1983 71: 768-71. doi: 10.1097/00006534-198306000-00003 6. Mehrotra ON, Brown GE, Widdowson WP, Wilson JP. Arteriography and selective embolisation in the control of life-threatening haemorrhage fol­lowing facial fractures. Br J Plast Surg 1984; 37: 482-5. doi: 10.1016/0007­1226(84)90135-8 7. Sliker CW. Blunt cerebrovascular injuries: imaging with multidetector CT an­giography. RadioGraphics 2008; 28: 1689-708. doi: 10.1148/rg.286085521 8. Biffl WL, Moore EE, Offner PJ, Brega KE, Franciose RJ, Burch JM. Blunt carotid arterial injuries: implications of a new grading scale. J Trauma 1999; 47: 845­53. doi: 10.1097/00005373-199911000-00004 9. Ardekian L, Samet N, Shoshani Y, Taicher S. Life-threatening bleeding fol­lowing maxillofacial trauma. J Craniomaxillofac Surg 1993; 21: 336-8. doi: 10.1016/s1010-5182(05)80493-7 10. Radvany MG, Gailloud P. Endovascular management of neurovascular arte­rial injuries in the face and neck. Semin Intervent Radiol 2010; 27: 44-54. doi: 10.1055/s-0030-1247888 11. Mahmood S, Lowe T. Management of epistaxis in the oral and maxillofacial surgery setting: an update on current practice. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003; 95: 23-9. 10.1067/moe.2003.10 12. Chen YF, Tzeng IH, Li YH, Lo YC, Lin WC, Chiang HJ, et al. Transcatheter arterial embolization in the treatment of maxillofacial trauma induced life-threatening hemorrhages. J Trauma 2009; 66: 1425-30. doi: 10.1097/ TA.0b013e3181842046 13. Moher D, Liberati A, Tetzlaff J, Altman DG; PRISMA Group. Preferred report­ing items for systematic reviews and meta-analyses: the PRISMA statement. PLoS Med 2009; 6: e1000097. doi: 10.1371/journal.pmed.1000097 14. Hayes SB, Johnson JN, Most Z, Elhammady MS, Yavagal D, Aziz-Sultan MA. Transarterial embolization of intractable nasal and oropharyngeal hemor­rhage using liquid embolic agents. J Neurointerv Surg 2015; 7: 537-41. doi: 10.1136/neurintsurg-2014-011101 15. Rosch J, Dotter C T, Brown M J. Selective arterial embolization: a new method for control of acute gastrointestinal bleeding. Radiology 1972; 102: 303-6. doi: 10.1148/102.2.303 16. Brooks B. The treatment of traumatic arteriovenous fistula. Med J 1930; 23: 100-6. 17. Sokoloff J, Wickbom I, McDonald D, Brahme F, Goergen TC, Goldberger LE. Therapeutic percutaneous embolization in intractable epistaxis. Radiology 1974; 111: 285-7. doi: 10.1148/111.2.285 18. Vaidya S, Tozer KR, Chen J. An overview of embolic agents. Semin Intervent Radiol 2008; 25: 204-15. doi: 10.1055/s-0028-1085930 262 19. Seldinger SI. Catheter replacement of the needle in percutaneous ar­teriography; a new technique. Acta Radiol 1953; 39: 368-76. doi: 10.3109/00016925309136722 20. Bauer JR, Ray CE. Transcatheter arterial embolization in the trauma patient: a review. Semin Intervent Radiol 2004; 21: 11-22. doi: 10.1055/s-2004­831401 21. Mangla S, Sclafani SJ. External carotid arterial injury. Injury 2008; 39: 1249­56. doi: 10.1016/j.injury.2008.06.012 22. Kunstlinger F, Brunelle F, Chaumont P, Doyon D. Vascular occlusive agents. AJR Am J Roentgenol 1981; 136: 151-6. doi: 10.2214/ajr.136.1.151 23. Lubarsky M, Ray CE, Funaki B. Embolization agents-which one should be used when? Part 1: large-vessel embolization. Semin Intervent Radiol 2009; 26: 352-7. doi: 10.1055/s-0029-1242206 24. Vrachliotis TG, Falagas ME. Infections after endovascular coil embolization. J Endovasc Ther 2007; 14: 805-6. 10.1583/07-2219C.1 25. Sheth RA, Sabir S, Krishnamurthy S, Avery RK, Zhang YS, Khademhosseini A, et al. Endovascular embolization by transcatheter delivery of particles: past, present, and future. J Funct Biomater 2017; 8: E12. doi: 10.3390/jfb8020012 26. Lewis AL, Adams C, Busby W, Jones SA, Wolfenden LC, Leppard SW, et al. Comparative in vitro evaluation of microspherical embolisation agents. J Mater Sci Mater Med 2006; 17: 1193-204. doi: 10.1007/s10856-006-0592-x 27. Liang B, Xiong F, Wu H, Wang Y, Dong X, Cheng S, et al. Effect of tran­scatheter intraarterial therapies on the distribution of Doxorubicin in liver cancer in a rabbit model. PLoS One 2013; 8: e76388. doi: 10.1371/journal. pone.0076388 28. Niimi Y, Berenstein A, Setton A. Complications and their management dur­ing NBCA embolization of craniospinal lesions. Interv Neuroradiol 2003; 9: 157-64. doi: 10.1177/15910199030090S122 29. Berenstein A, Lasjaunias P, Brugge KG. Surgical neuroangiography. Vol. 2: Clinical and endovascular treatment aspects in adults. Berlin: Springer-Verlag; 2004. 30. Vollherbst DF, Sommer CM, Ulfert C, Pfaff J, Bendszus M, Mohlenbruch MA. Liquid embolic agents for endovascular embolization: evaluation of an es­tablished (Onyx) and a novel (PHIL) embolic agent in an in vitro AVM model. Am J Neuroradiol 2017; 38: 1377-82. doi: 10.3174/ajnr.A5203 31. Siekmann R. Basics and principles in the application of Onyx LD Liquid Embolic System in the endovascular treatment of cerebral ar­teriovenous malformations. Interv Neuroradiol 2005; 11: 131-40. doi: 10.1177/15910199050110S117 32. Schirmer CM, Zerris V, Malek AM. Electrocautery-induced ignition of spark showers and self-sustained combustion of onyx ethylene-vinyl al­cohol copolymer. Neurosurgery 2006; 59: ONS413-8. doi: 10.1227/01. NEU.0000240683.15391.99 33. Thiex R, Wu I, Mulliken JB, Greene AK, Rahbar R, Orbach DB. Safety and clini­cal efficacy of Onyx for embolization of extracranial head and neck vascular anomalies. AJNR Am J Neuroradiol 2011; 32: 1082-6. doi: 10.3174/ajnr. A2439. 34. Koo HW, Lee JJ. Forehead pigmentation after Onyx embolization for dural arteriovenous fistula presenting with parkinsonism. Interdiscip Neurosurg 2020; 19: 100616. doi: 10.1016/j.inat.2019.100616 35. Leyon JJ, Chavda S, Thomas A, Lamin S. Preliminary experience with the liquid embolic material agent PHIL (Precipitating Hydrophobic Injectable Liquid) in treating cranial and spinal dural arteriovenous fistulas: techni­cal note. J Neurointerv Surg 2016; 8: 596-602. doi: 10.1136/neurint­surg-2015-011684 36. Koçer N, Hanmoglu H, Batur S, Kandemirli SG, Kzlklç O, Sanus Z. Preliminary experience with precipitating hydrophobic injectable liquid in brain arteriovenous malformations. Diagn Interv Radiol 2016; 22: 184-9. doi: 10.5152/dir.2015.15283 37. Li W, Zhang L, Ge X, Xu B, Zhang W, Qu L, et al. Microfluidic fabrication of microparticles for biomedical applications. ChemSoc Rev 2018; 47: 5646-83. doi: 10.1039/c7cs00263g 38. Noy D, Rachmiel A, Emodi O, Amsalem Y, Israel Y, Nagler RM. Transarterial embolization in maxillofacial intractable potentially life-threatening hemorrhage. J Oral Maxillofac Surg 2017; 75: 1223-31. doi: 10.1016/j. joms.2017.01.033 39. Hayes SB, Johnson JN, Most Z, Elhammady MS, Yavagal D, Aziz-Sultan MA. Transarterial embolization of intractable nasal and oropharyngeal hemor­rhage using liquid embolic agents. J Neurointerv Surg 2015; 7: 537-41. doi: 10.1136/neurintsurg-2014-011101 40. Liao CC, Hsu YP, Chen CT, Tseng YY. Transarterial embolization for intrac­table oronasal hemorrhage associated with craniofacial trauma: evalu­ation of prognostic factors. J Trauma 2007; 63: 827-30. doi: 10.1097/ TA.0b013e31814b9466 41. Roberson G, Rearden E. Angiography and embolization of the internal maxil­lary artery for posterior epistaxis. Arch Otolaryngol1979; 105: 333-7. doi: 10.1001/archotol.1979.00790180031006 42. Vitek J. Idiopathic intractable epistaxis: endovascular therapy. Radiology. 1991; 181: 113-6. doi: 10.1148/radiology.181.1.1887018 43. Elahi M, Parnes LS, Fox AJ, Pelz DM, Lee DH. Therapeutic embolization in the treatment of intractable epistaxis. Arch Otolaryngol Head Neck Surg1995; 121: 65-9. doi: 10.1001/archotol.1995.01890010051009 44. Duncan IC, Fourie PA, le Grange CE, van der Walt HA. Endovascular treat­ment of intractable epistaxis - results of a 4-year local audit. S Afr Med J 2004; 94: 373-8. doi: 10.4102/sajr.v8i3.118 45. Cullen MM, Tami TA. Comparison of internal maxillary artery ligation versus embolization for refractory posterior epistaxis. Otolaryngol Head Neck Surg 1998; 118: 636-42. doi: 10.1177/019459989811800512 46. Burdick TR, Hoffer EK, Kooy T, Ghodke B, Starnes BW, Valji K, et al. Which ar­teries are expendable? The practice and pitfalls of embolization throughout the body. Semin Intervent Radiol 2008; 25:191-203. doi: 10.1055/s-0028­1085925. 47. Kuan CH, Lin CY, Hsaio JK, Chen JS, Han YY. Prognostic factors of survival from intractable oronasal bleeding after successful transarterial embolization. J Oral Maxillofac Surg 2015; 73: 1790-4. doi: 10.1016/j.joms.2015.03.028 48. Cogbill TH, Cothren CC, Ahearn MK, Cullinane DC, Kaups KL, Scalea TM, et al. Management of maxillofacial injuries with severe oronasal hemor­rhage: a multicenter perspective. J Trauma 2008; 65: 994-9. doi: 10.1097/ TA.0b013e318184ce12. 49. Kim DY, Dong DK, Park H, Chung J. Endovascular treatment of life-threaten­ing bleeding of bilateral maxillary arteries in a patient with multiple facial bone fractures - a case report. J Korean Neutrotraumatol Soc 2011; 7: 108­111. doi: 10.13004/jknts.2011.7.2.108 50. Komiyama M, Nishikawa M, Kan M, Shigemoto T, Kaji A. Endovascular treat­ment of intractable oronasal bleeding associated with severe craniofacial injury. J Trauma 1998; 44: 330-4. doi: 10.1097/00005373-199802000-00017 51. Maioriello AV, Stanley DJ, Ho T, Ashley WW. Successful Onyx emboliza­tion of life threatening traumatic posterior jugal artery fistula following mandibular fracture. J Neurointerv Surg 2012; 4: e15. doi: 10.1136/neurint­surg-2011-010071 52. Mauldin FW, Cornay WJ 3rd, Mahaley MS Jr, Hicks JN. Severe epistaxis from a false aneurysm of the external carotid artery. Otolaryngol Head Neck Surg 1989; 101: 588-90. doi: 10.1177/019459988910100515 53. Mehringer CM, Hieshima GB, Grinnell VS, Tsai FY, Bentson JR, Hasso AN, et al. Therapeutic embolization for vascular trauma of the head and neck. AJNR Am J Neuroradiol 1983; 4: 137-42. PMID: 6405591 54. Remonda L, Schroth G, Caversaccio M, Lädrach K, Lblad KO, Zbären P, et al. Endovascular treatment of acute and subacute hemorrhage in the head and neck. Arch Otolaryngol Head Neck Surg 2000; 126: 1255-62. doi: 10.1001/archotol.126.10.1255 55. Wang D, Su L, Han Y, Fan X. Embolization treatment of pseudoaneurysms originating from the external carotid artery. J Vasc Surg 2015; 61: 920-6. doi: 10.1016/j.jvs.2014.10.093. 56. Wong CW, Tan WC, Yeh YT, Chou MC, Yeh CB. Transarterial embolization for traumatic intractable oronasal hemorrhage. J Emerg Med 2013; 44: 1088­91. doi: 10.1016/j.jemermed.2012.06.029 263 review Current management of intrahepatic cholangiocarcinoma: from resection to palliative treatments Ilenia Bartolini1, Matteo Risaliti1, Laura Fortuna1, Carlotta Agostini1, Maria Novella Ringressi1, Antonio Taddei1, Paolo Muiesan1,2 1 Hepatobiliary Unit, Department of Clinical and Experimental Medicine, University of Florence, AOU Careggi, Florence, Italy 2 Liver Unit, Queen Elizabeth Hospital, Birmingham, UK Radiol Oncol 2020; 54(3): 263-271. Received 10 May 2020 Accepted 29 June 2020 Correspondence to: Paolo Muiesan, MD, FRCS, FEBS, Department of Clinical and Experimental Medicine, University of Florence, AOU Careggi, Largo Brambilla 3, 50134, Florence, Italy. E-mail: paolo.muiesan@uhb.nhs.uk Disclosure: No potential conflicts of interest were disclosed. Background. Intrahepatic cholangiocarcinoma (ICC) is the second most common liver primary tumour after hepa­tocellular carcinoma and represents 20% of all the cholangiocarcinomas. Its incidence is increasing and mortality rates are rising. Surgical resection is the only option to cure the disease, despite the high recurrence rates reported to be up to 80%. Intrahepatic recurrences may be still treated with curative intent in a small percentage of the patients. Unfortunately, due to lack of specific symptoms, most patients are diagnosed in a late stage of disease and often unsuitable for resection. Liver transplantation for ICC is still controversial. After the first published poor results, improving outcomes have been reported in highly selected cases, including locally advanced ICC treated with neoadjuvant chemotherapy, when successful in controlling tumour progression. Thus, liver transplantation should be considered a possible option within study protocols. When surgical management is not possible, palliative treatments include chemotherapy, radiotherapy and loco-regional treatments such as radiofrequency ablation, trans-arterial chemoem­bolization or radioembolization. Conclusions. This update on the management of ICC focusses on surgical treatments. Known and potential prog­nostic factors are highlighted in order to assist in treatment selection. Key words: intrahepatic cholangiocarcinoma; liver resection; liver transplantation Introduction Epidemiology Cholangiocarcinoma is a rare tumour originating from the biliary epithelium. It can arise from the distal biliary tract, at the hepatic hilum or from intrahepatic ducts, beyond second-order biliary ducts. The classification based on the site of origin identifies three entities requiring different treat­ments and prognoses.1 With an incidence of 0.85 per 100,000 world­wide2, intrahepatic cholangiocarcinoma (ICC) represents up to 20% of all the cholangiocarcino-mas. It is the second primary liver tumour fol­lowing hepatocellular carcinoma (HCC)1 account­ing for 5-30% of all primary liver malignancies.3,4 Although reports in literature are scarce, its inci­dence has been rising all over the world in the last three decades.1,5 Such increase may be associated with a greater prevalence of risk factors but also to improvements in diagnostic tools.6 Intrahepatic cholangiocarcinoma is a highly invasive tumour, it is frequently multifocal and it is scarcely respon­sive to treatments. Thus, its mortality rate is about 0.69 per 100,000 and it is increasing along with tu­mour incidence.2 264 Well-known risk factors include liver disease and chronic inflammation including cirrhosis, hep­atitis B (mostly in Asian countries) and C (mostly in the Western countries), primary sclerosing cholan­gitis (PSC), biliary tract cysts, intrahepatic biliary stones, toxins, infection with hepatobiliary flukes (frequently in East Asia), metabolic syndrome and obesity.1,7,8 Presentation and diagnosis Intrahepatic cholangiocarcinoma is often clinically silent and there are no specific symptoms in the early stages. Diagnosis is therefore incidental in at least 20-25% of the patients.1 Symptoms include ab­dominal pain and, in more advanced cases, weight loss, malaise and asthenia.1 Jaundice is rarely pre­sent (about 15% of the cases) and it can be caused by both external compression and infiltration of the hepatic hilum.7 Macroscopically, ICC may present as a mass-forming tumour, with periductal or intraductal growth, or with a combination of these patterns.9 The mass-forming pattern is the most frequent and it spreads mostly via portal system. Instead, the periductal forms grow mostly through lymphatic vessels.10 Microscopically, it is composed of bile duct cells with stromal fibrosis and collagen fibres.1 Diagnosis can be difficult, clinical suspicion and laboratory exams need to be confirmed by radio­logic findings.11 Laboratory investigations compre­hend serum tumour markers including CA19-9 and CEA. The CA 19-9 sensitivity is 62% and its specificity is 63%.10 However, tumour markers may be elevated also in presence of tumours different from ICC or in case of benign conditions including cholangitis or cholestasis.6 Therefore, they are not sensitive enough to be utilised for screening purposes. Recently, some effort has been placed in the pro-teomic evaluation of organic fluids and in search­ing products of cancer cells (including cytokines, enzymes and growth factors) trying to find better biomarkers.12 Potential serum, urinary and bil­iary biomarkers have been investigated over the years. Serum markers include trypsinogen-2, IL-6, MUC5AC, cytocheratin-19 fragment (CYFRA 21­ 1) and progranulin while some of the biliary bio-markers are insulin-like growth factor 1 (IGF1) and microRNA-laden vesicles. However, none of these is currently used in clinical practice.1,12 The Ultrasound Sonography (US) is the first imaging test that usually identifies an abdominal mass, but its sensitivity and specificity are opera-tor-dependent. Tumour markers may improve sig­nificantly the sensitivity of US. Furthermore, the colour Doppler mode may show portal venous and parenchymal involvement.13 Unlike HCC, there are no specific radiologi­cal patterns for an imaging-based diagnosis.3 On Computed Tomography (CT), ICC presents as a predominantly hypodense mass with irregular margins, with a peripheral rim enhancement in the arterial phase. Contrast uptake is progressive on the venous and late phases.3 The hyper-enhancing pattern on delayed phase reflects stromal fibrosis of interstitial space. Therefore, hyper-attenuating ICCs are more aggressive. Other characteristics of advanced tumours include bile ducts thickening and dilatation, retraction of liver capsule, enlarged regional lymph nodes, vascular invasion and dis­tant metastases.13 On contrast-enhanced Magnetic Resonance Imaging (MRI), the ICC is a hypo-intense lesion on T1-weighted images and hyper-intense on T2­weighted images. Central hypo-intensity, on de­layed pictures, reflects the presence of fibrosis.13 The contrast medium uptake in MRI is similar to CT scan. The typical HCC “wash-in and wash-out” pattern is never present, even in case of small tu­mours.1,10 The MRI with cholangiopancreatogra­phy (MRCP) is the gold standard in the imaging of the biliary tree without the need of invasive techniques (i.e. percutaneous transhepatic cholan­giography). The MRI is a powerful tool to evaluate tumour extent and resectability with an accuracy of up to 95%.13 However, both CT scan and MRI have low spec­ificity and the diagnosis of small or rare forms of tumour, including mixed HCC-ICC or in presence of PSC, may be difficult by imaging only.10,14 Positron emission tomography with 18-fluoro­deoxyglucose (FDG-PET) scan is not recommend­ed as a routine staging exam.14 However, it could be a great tool to discover occult primary tumours, distant or nodal metastasis with a sensitivity and specificity of about 40-55% and 80-87%, respec­tively.13 Furthermore, a modification in patient management has been reported in up to 15% of the cases after diagnosis of nodal involvement with FDG-PET.14,15 The role of biopsy is still controversial when di­agnostic doubt persists after imaging techniques. When a nodule is suitable for resection, most au­thors suggest that liver biopsy should not be per­formed because of the risk of seeding.7,11 Anyhow, there is no strong evidence supporting this risk.1 Moreover, histological analysis on biopsy is not al- 265 ways able to differentiate a primary from a second­ary adenocarcinoma.16 On the contrary, since dis­tant nodal metastases are a contraindication to liver resection, a biopsy of suspect distant lymph nodes should be performed via endoscopic ultrasound (EUS) with fine-needle aspiration, eventually.17 Treatment and prognosis When technically feasible, surgical resection is the best treatment that can be offered to the patients. In the great majority of them, a major hepatec­tomy will be necessary to achieve a R0 resection. Nevertheless, reported 5-years survival rates range from 22% and 45%,5,11,18 mostly due to high recur­rence rates (up to 80%).19-21 Unfortunately, 60 to 88% of the patients with ICC have unresectable tu­mours due to a late diagnosis.22,23 Indications for liver transplantation (LT) for ICC are still controversial. Outcomes of liver transplan­tation have been changed over the years. In the '90s, a 5-year survival rate of less than 25% was reported.24-26 Recent papers reported an acceptable 5-years overall survival rate, up to 83%, in highly selected patients.5,27 The most important prognostic factors after re­section include: tumor-related features (e.g. size and number, vascular and nodal involvement, perineural and periductal invasion and tumour bi-ology)17, margin status2, and time-to-recurrence.21 Palliative treatments include chemotherapy, radi­ation therapy or locoregional therapies but all these strategies provide only a modest improvement in prognosis with a median survival inferior to 1 year 5,23,28 and a 5-years survival of less than 10%.29 The focus of this paper is on surgical manage­ment of ICC with an assessment of prognostic fac­tors for recurrence, which may assist to better select the appropriate treatment for each patient. A brief overview of palliative treatments is also provided. Surgical management Surgical resection Currently, surgical resection is the only accepted treatment for potential cure.1 Due to the improve­ments in surgical techniques and advances in perioperative care, surgical indications have been extended in recent years. However, only a minor­ity of patients, 12-40%, are resectable at the time of diagnosis.11,30 Surgery aims to achieve complete resection of the tumour with adequate free margins, and, at the same time, leaving a sufficient functional liver rem­nant. The assessment of resectability is associated with a variety of factors including tumour location and extension, liver function and underlying liver disease and, last but not least, performance status. In case of involvement of major vascular struc­tures or of first- and second-order biliary branches, a liver resection should be carefully assessed and planned. Up to half of patients have multifocal disease at presentation.31 Resection of multifocal ICC is con­troversial since it usually requires a more demoli­tive liver resection and it is associated with poorer survival rates. However, multifocality itself should not prevent surgery according to current published evidence.18,31,32 On the other hand, distant metastases are a con­traindication to surgery. Similarly, metastases of distant lymph nodes are considered a reason for unresectability.17 In case of suspected infiltration of regional lymph nodes, surgical resection should be assessed carefully given that lymph nodes positiv­ity is one of the most important factors linked to poor prognosis.29 In 10-20% of the patients, locally advanced tumours may be downstaged and recon­sidered for liver resection after neoadjuvant treat­ments including chemotherapy (based on gemcit­abine, cisplatin and paclitaxel) and locoregional procedures.32 Conversion rate varies between 0% and 53% and up to half of the patients present with stabilized disease.32 In presence of cirrhosis, portal pressure should be assessed since clinically significant portal hy­pertension, defined as an hepatic vein pressure gradient (HVPG) = 10 mmHg33, is a relative con­traindication to major resections.17,20 Further tests to reduce at a minimum the risk of postoperative liver failure include liver function tests, calculation of future liver remnant volume, and evaluation of the presence of fibrosis.17 Small tumours or peripherally located lesions can be treated with an atypical or anatomical mi­nor resection. However, in most cases (70-80%), the lesion is multisegmental and a major hepatectomy may be needed.19 Similarly to HCC, anatomical resections of ICC seem to have better outcomes in terms of survival and recurrence when compared with non-anatomical resections.34 A biliary resection and reconstruction is re­quired in about 20-30% of the cases.19 The neces­sity of a vascular reconstruction to achieve an R0 resection should not prevent surgery in selected patients. Reames et al., in a multicentric analysis evaluating a total of 1087 patients, reported similar 266 results between patients requiring a caval or portal resection and those who did not.35 In case of a predicted small future liver remnant, portal vein embolization can be performed prior to surgery. Liver hypertrophy develops in approxi­mately 40% of patients within 4 weeks. However, 20-30% of these patients will never undergo resec­tion because of tumour progression or inadequate future liver remnant hypertrophy.17 The use of ALPPS (Associating Liver Partition and Portal vein ligation for Staged hepatectomy) has been reported for ICC. Liver hypertrophy is achieved faster when compared with PVE and the rate of achievement of second stage is higher with ALPPS compared to other staged procedures, but the cost in terms of morbidity and mortality is sig­nificant.36 Similarly to other cancers, lymphadenectomy has an undisputed role in staging the disease cor­rectly.37 For this purpose, a minimum of 6 lymph nodes are required as suggested in the 8th edition of the American Joint Committee on Cancer (AJCC) manual.38 However, the impact of lymphadenecto-my has been previously questioned and only about 50% of patients have been reported to receive a lymphadenectomy.39 In particular, the therapeutic role of lymphadenectomy is debated although sev­eral more recent papers reported a survival ben­efit.39 A complete lymphadenectomy is routinely performed by some authors to try to reduce local recurrence but in the subgroup of cirrhotic patients the related high morbidity rates may exceed the benefits.17 Minimally invasive surgery for ICC is feasible and safe in selected cases, with the advantages of laparoscopic and robotic techniques and similar oncological outcomes to open surgery.40,41 Adjuvant therapy is not fully standardized yet due to the rarity of this disease. However, chemo­therapy should be offered to patients with positive nodes at histology though it seems to offer only partial control on lymph node metastatic disease. Post-operative mortality is less than 5% in high-volume centres.1 Five-years OS rates after curative surgery range between 22 and 45%. Median sur­vival is reported to be 40 months.5,11,21,42 Recurrence rates are still very high, ranging from 53% to 80%.5,21 The reported 1-, 3-, and 5-year disease-free survival is 44%, 18%, and 11%, respectively.18 The great majority of patients experience disease recur­rence within 2 years5 with a median time to recur­rence ranging from 9 to 26 months.43 However, recurrence has been reported up to 9.5 years after liver resection.18 Despite improvements in pre- and intra-opera­tive imaging21,44, local tumour control may result incomplete, possibly due to unidentified small metastatic lesions at surgery.5,21 Common extrahe­patic sites of recurrence include lungs, abdominal lymph nodes and peritoneum.21 In case of intrahepatic only recurrence, further treatments with curative intent may still be pos­sible if an R0 treatment is achievable by surgical resection or radiofrequency ablation.2,21 However, disease recurrence is the major cause of death in these patients with a disease-specific mortality rate of about 90%.21 Liver transplantation Currently, ICC is a controversial indication for LT.5,27 The main reasons of such controversy in­clude the shortage of deceased donor organs, the potential of tumour progression whilst waiting for LT after chemotherapy, the high recurrence rates of ICC and the fact that immunosuppression may facilitate recurrence. Papers from the ‘90s reported a poor prognosis after liver transplant for ICC with 5-years survival rates of 10-25%.24,25,45 Furthermore, in most LTs ICC was an incidental diagnosis on the resected speci­men, thus patients had not received preoperative adjuvant treatments.26 Recently, Sapisochin et al. retrospectively looked at 48 patients transplanted for presumed HCC or decompensated cirrhosis but diagnosed as ICC at post-transplant pathology.27 Fifteen had very early ICC (<2 cm) and 33 had advanced ICC (>2 cm or multifocal). The 5-years OS rate was 65% and 45% for very early and advanced ICC, respec­tively. Tumour recurrence occurred in 13% of the very early group and in 54.5% of the advanced group, being the main cause of death of the latter. Therefore, LT could be a possible treatment only for cirrhotic patients with very early ICC.27 However, the role of neoadjuvant chemotherapy has remained unclear for a long time. Good results in terms of survival (5-years OS rate up to 76%) have been reported in highly selected patients with hilar cholangiocarcinoma who received LT after neoadjuvant chemotherapy or chemo-radiothera­py with good disease control.46-48 These results en­couraged further studies. In their well-designed prospective case-series, Lunsford et al. reported a 1-, 3- and 5-years overall survival rate of 100%, 83.3% and 83.3%, respective­ly. One-, 3- and 5-years recurrence-free survival was 50% with a median time of recurrence of 7.6 months. 267 TABLE 1. A comparison between the prognostic factors of the three main recognized staging systems (the Liver Cancer Study Group of Japan [LCSGJ]9, the National Cancer Center of Japan [NCCJ]53, and the American Joint Committee on Cancer [AJCC, 8th edition38], Wang et al.49 and Hyder et al.43 nomograms LCSGJ9 Cut-off: 2 cm Yes Invasion of the serosa Yes Yes Yes NCCJ53 Yes Yes Yes Symptoms AJCC38 Cut-off: 5 cm Yes Yes Yes Yes Wang et al.49 Yes Yes Yes Yes Yes CEA CA19.9 Hyder et al.43 Yes Yes Yes Yes Age Cirrhosis CA19.9= Carbohydrate Antigen 19.9; CEA = Carcinoembryonic Antigen Inclusion criteria included: the presence of a locally advanced ICC (> 2 cm or multifocal, confirmed with biopsy or cytology), deemed unresectable after the evaluation of the multidisciplinary team; absence of distant metastasis or major vascular structures involvement; absence of tumour progression after a minimum of 6 months of neoadjuvant chemo­therapy or radio-chemotherapy (gemcitabine-based regimens). Prior to proceeding to LT, sampling and frozen section of the hepatic hilum lymph nodes was performed to exclude malignancy.5 Despite promising results, the main issue with this paper was related to the highly selected pa­tients and such good prognosis could be a conse­quence of the indolent behaviour of the disease. Responsiveness to chemotherapy for at least 6 months is a “test of time” and excludes patients with aggressive disease from transplantation. Furthermore, this paper included a small sample size (six patients in 8 years received LT) and the short median follow-up of 36 months. Further prospective clinical trials taking into account tumour morphology and biology are still needed to draft definite conclusions. Prognostic factors for recurrence There are many recognized prognostic factors re­lated to the tumour and to liver resection as well as the previous history of PSC. Tumour-related factors include size and num­ber, vascular and nodal involvement, perineural and periductal invasion and tumour biology.17 Serum biomarkers have a controversial role in prognosis establishment.49 Most authors recognize tumour size as a prog­nostic factor for recurrence.42 Different cut-offs have been reported: 2-3 cm27, 5 cm18,19 or 8 cm.50 In particular, Sapisochin et al. stratified the patients into three groups according to tumour size: smaller than 2 cm, between 2 and 3 cm, larger than 3 cm. They found a 5-years OS of 80%, 61% and 42%, re­spectively.27 However, some other authors found alternative factors with a stronger prediction po­tential after resection10 or LT including not receiv­ing neoadjuvant therapies.51 Since multifocality and vascular invasion have a prognostic impact, the American Joint Committee on Cancer (AJCC) classifies both multifocal and single tumours in presence of vascular invasion as T2.38 Furthermore, multifocality has been reported to significantly correlate with tumour size and dif­ferentiation, nodal metastasis and vascular infiltra­tion. Satellitosis seems to confer a worse prognosis when compared with bilateral tumour location al­though this result may suffer from a selection bias of the patients.31 On the contrary, the previously cited paper of Lunsford reported that both volume and number of lesions do not impact on recurrence after LT.5 However, these different findings may suffer from bias related to the small sample group evaluated. Node metastasis is an important prognostic factor. Lymph nodes positivity resulted in about 45% of resections and even N0 patients may har­bour nodal micrometastases in about 10-20% of the cases.52 There are three main recognized staging sys­tems: the Liver Cancer Study Group of Japan (LCSGJ)9, the American Joint Committee on Cancer (AJCC), 8th edition38 and the National Cancer Center of Japan (NCCJ)53,54 (Table 1). Principal prognostic factors for LCSGJ are tu­mour diameter with a cut-off of 2 cm, number of lesions, vascular infiltration and invasion of the serosa.9 268 The AJCC, 8th edition38, applied a cut-off o 5 cm to divide T1 into T1a and T1b. The NCCJ, Okabayashi and Nathan system53,54, do not consider tumour diameter as an independ­ent prognostic factor while presence of symptoms, nodal invasion, lesion number and vascular inva­sion have a prognostic impact. These three systems displayed lack of accuracy in predicting progno­sis10, thus several nomograms have been proposed to predict survival.43,49 Spolverato et al. developed a model to specifically predict cure rate and time necessary to define the patient cured. This cure model included tumour number, size and differ­entiation, vascular and periductal invasion, nodal positivity and it is easily accessible on internet.18 Similar results have been previously published.43,49 Tumour biology is another fundamental aspect. Grading has been reported to be significantly re­lated with tumour recurrence.27,29 A high grade of diversity in ICC molecular profile has been report­ed.1 Several genetic modifications, epigenetic al­terations, gene fusions products (including FGFR2 gene fusion), hormone influences (including evalu­ation of tumour estrogen sensitivity) and growth factors effects have been assessed and are still un­der continuous evaluation.1 The whole-genome analysis helped in understanding two potential altered pathways related with ICC development: activation of the inflammatory response pathway and cellular proliferation pathway, the latter being related with a worse prognosis.1,55 However, a com­plete knowledge at the cellular level together with the microenvironment in which tumours develop is far from being achieved.1 This effort may wid­en the perspectives in different aspects of tumour management: diagnosis (with the discovery of new circulating biomarkers), treatment allocation (including personalized targeted therapies) and prognosis prediction. For example, while KRAS mutation has been found in patients experiencing recurrence, FGFR gene fusion seems related with an indolent disease.5,56 Obviously, patients with in­dolent tumours will have a better prognosis despite the treatments received and they will benefit more from each treatment. The previously reported ab­sence of disease progression during chemotherapy is strictly related with tumour biology.2,5 The most important prognostic factor after sur­gical resection is the state of the margins. Tumour-free margins are related with a significantly better prognosis when compared with infiltrated mar­gins.2,57 Finally, time-to-recurrence after surgery with a curative intent has been reported to be itself a prognostic factor.21 Using a cut-off of 24 months, Zhang et al. showed a significantly worse progno­sis for patients experiencing early recurrence when compared with those with late recurrence (median OS of 10 and 18 months, respectively).21 Although recurrence was mainly in the liver, the frequency of extrahepatic localization was higher in the early recurrence group.21 Furthermore, they found that the size and number of tumours, vascular invasion, presence of satellitosis or surgical margins of less than 1 cm were all associated with the early recur­rence pattern at univariate analysis. On the contra­ry, adjuvant treatments and presence of cirrhosis resulted significantly linked to late recurrence.21 Interestingly, when further treatments with a cura­tive intent were possible, OS rates resulted similar between the two groups.21 Further studies are needed to evaluate with greater detail potential prognostic factors and their weight. Palliative treatments Unfortunately, a great majority of the patients pre­sent with unresectable disease due to major vas­cular or bile duct involvement, metastasis or huge burden of disease leading to a potential insufficient future liver remnant.11 The 5-years survival rate of these patients is less than 10%.29 Although ICC tends to develop chemoresistance, systemic therapy is the main treatment in the sub­set of palliative cures. Chemotherapy could be con­sidered to treat patients with macroscopic residual tumour after surgery, locally advanced or meta­static unresectable tumours or recurrent ICCs. The National Comprehensive Cancer Network guide­lines recommend gemcitabine/cisplatin therapy as first-line treatment.37 In alternative, fluoropyrimi­dine-based or other gemcitabine-based chemother­apy regimens could be considered.37 However, the optimal second-line therapy is still controversial.58 The role of targeted therapy is still under evalua­tion.1 Furthermore, a better understanding of the mechanisms that are behind chemoresistance may widen and improve treatment options. The addition of radiation to chemotherapy is as­sociated with better outcomes in terms of disease-free and overall survival.59,60 On the contrary, the role of radiotherapy alone for ICC is controversial. Different approaches of radiotherapy are available such as external beam irradiation, brachytherapy with iridium-192, stereotactic body radiotherapy and proton beam irradiation.59 Technical advances 269 now allow a selective delivery of radiation to the lesion, sparing adjacent tissue. To date there are no randomized trials comparing new techniques with the more conventional ones. While distant metastasis is a less frequent cause of death, many of these patients die of liver failure caused by tumour-related vascular involvement or biliary obstruction. It is thus important to try to achieve local control of the tumour to improve quality of life.32 There are no randomized data showing a single optimal local treatment, so a tai­lored therapy is required. The choice of the best lo-coregional treatment must consider factors related to the patient (comorbidity, liver function, previ­ous treatments) and to the tumour, such as size, vascularity and its involvement of bile ducts, blood vessels, bowel and chest wall.32 Data concerning the use of transarterial em-bolization therapies for ICC are scarce. These treatments include transarterial chemoemboliza­tion (TACE), bland embolization, chemoinfusion (TACI) and radioembolization (TARE, known also as selective internal radiation therapy, SIRT). These therapies are indicated in case of hypervascular le­sions and in absence of complete portal vein throm­bosis61 with the exception of TARE that can be used in cases of neoplastic thrombosis. Unfortunately, ICC is typically hypovascular and characterized by fibrous content.62 In a retrospective multi-institutional analysis evaluating 198 patients with ICC, partial/complete response or stability of disease was found in 26% and 62% of patients, respectively. Median OS was 13.2 months. Outcomes did not differ on the type of intra-arterial treatment.63 These results were confirmed by Yang who performed a systematic review including 926 patients.64 Mean complete radiological response was 10% while partial radio­logical response was 22.2%. One third of patients suffered from acute toxicity, 30-day mortality was less than 1% and median OS was 13 months. These data showed that transarterial embolization thera­pies could be safely and effectively used in unre­sectable cholangiocarcinoma, conferring a survival benefit.64 Percutaneous ablation techniques such as radi­ofrequency or microwave ablation are effective for small lesions (4-5 cm), not located close to major bile ducts or blood vessels or on the liver surface.65 Irreversible electroporation is a new ablation tech­nique with similar results for small lesions but with no limitations in terms of distance from bile ducts and vessels.61 Photodynamic therapy is another palliative treatment that may have a small beneficial effect on survival.66 Conclusions Intrahepatic cholangiocarcinoma is a rare tumour but with an increasing incidence over the years. Unfortunately, mortality rates are rising consensu­ally despite improvements in surgical techniques and perioperative care. When technically feasible and patients are fit, surgical resection is the best option that can be offered. However, survival rates are still discouraging and recurrence rates are high. Liver transplantation may be considered in highly selected patients including those with a very early tumour and cirrhosis or in locally advanced unre­sectable ICC but stable after neoadjuvant therapy. Unfortunately, the majority of patients present with unresectable disease. Palliative treatments may confer an improvement in survival. However, we should aim at an improved stratification of patients using the known prognostic factors and, hopefully, at a better understanding of biologic cancer profiling. This stratification, together with standardization and improvements in neoadjuvant and adjuvant therapies, may allow a better alloca­tion of treatments and, possibly, an expansion of the indications for surgery in a subset of patients. References 1. Banales JM, Cardinale V, Carpino G, Marzioni M, Andersen JB, Invernizzi P, et al. Expert consensus document: cholangiocarcinoma: current knowledge and future perspectives consensus statement from the European Network for the Study of Cholangiocarcinoma (ENS-CCA). Nat Rev Gastroenterol Hepatol 2016; 13: 261-80. doi: 10.1038/nrgastro.2016.51 2. Spolverato G, Kim Y, Alexandrescu S, Marques HP, Lamelas J, Aldrighetti L, et al. Management and outcomes of patients with recurrent intrahepatic cholangiocarcinoma following previous curative-intent surgical resection. Ann Surg Oncol 2016; 23: 235-43. doi: 10.1245/s10434-015-4642-9 3. Valls C, Gumŕ A, Puig I, Sanchez A, Andía E, Serrano T, et al. 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Radiofrequency ablation in the treatment of unresectable intrahepatic cholangiocarcinoma: system­atic review and meta-analysis. J Vasc Interv Radiol 2015; 26: 943-8. doi: 10.1016/j.jvir.2015.02.024 66. Lu Y, Liu L, Wu JC, Bie LK, Gong B. Efficacy and safety of photodynamic therapy for unresectable cholangiocarcinoma: a meta-analysis. Clin Res Hepatol Gastroenterol 2015; 39: 718-24. doi: 10.1016/j.clinre.2014.10.015 272 review Consensus molecular subtypes (CMS) in metastatic colorectal cancer - personalized medicine decision Martina Rebersek1,2 1 Department of Medical Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2020; 54(3): 272-277. Received 13 March 2020 Accepted 29 April 2020 Correspondence to: Assist. Prof. Martina Reberšek, M.D., Ph.D., Department of Medical Oncology, Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. E-mail: mrebersek@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. Colorectal cancer (CRC) is one of the most common types of cancer in the world. Metastatic disease is still incurable in most of these patients, but the survival rate has improved by treatment with novel systemic chemo­therapy and targeted therapy in combination with surgery. New knowledge of its complex heterogeneity in terms of genetics, epigenetics, transcriptomics and microenvironment, including prognostic and clinical characteristics, led to its classification into various molecular subtypes of metastatic CRC, called consensus molecular subtypes (CMS). The CMS classification thus enables the medical oncologists to adjust the treatment from case to case. They can deter­mine which type of systemic chemotherapy or targeted therapy is best suited to a specific patient, what dosages are needed and in what order. Conclusions. CMS in metastatic CRC are the new tool to include the knowledge of molecular factors, tumour stroma and signalling pathways for personalized, patient-orientated systemic treatment in precision medicine. Key words: metastatic colorectal cancer; heterogeneity; biomarkers; consensus molecular subtypes; CMS1; CMS2; CMS3; CMS4 Introduction Colorectal cancer (CRC) is still one of the most common types of cancer and one of the lead caus­es of cancer- related deaths worldwide, as well as in Slovenia. According to the Cancer Registry of Slovenia, there were 1467 new cases of CRC in 2016, of which 871 men and 596 women.1 The prognosis of these patients has improved signifi­cantly over the last decade because of successful preventive screening programme, improved sur­gical techniques, radiation therapy and systemic treatment for both early and advanced stages. In Slovenia, the incidence of CRC has been declin­ing in the last few years, mainly due to increased awareness and preventive screening programme called SVIT, which has been implemented in Slovenia in 2009. According to the National Cancer Control Program Slovenia, the incidence of CRC has been declining annually. In the last official report from 2015, there were about 400 cases less from 2010 to 2015 (from 1729 cases in 2010 to 1357 cases in 2015).2 Metastatic CRC is still an incurable disease for most of the patients, with most commonly liver, lung or lymph nodes and peritoneal metastases. In the past, 15 years ago, median overall survival (mOS) was approximately 12 months and the 5-year survival rate was 13%. However, the sur­vival rate of these patients has increased, mainly due to the combined treatment of metastases with surgery and systemic therapy.3-5 Long-term sur­vival or even cure can be attained in 20%–50% of the patients who undergo complete R0 resection of liver or lung metastases, and around 70% 5-year survival of these patients can be achieved.3,4 273 However, in the field of systemic therapy there has been a significant progress with new drugs in the recent years. There are more options of initial systemic chemotherapy, oxaliplatin, irinotecan, and fluoropyrimidines, in combination with tar­geted therapy with anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (cetuxi­mab, panitumumab) in case of KRAS wild type tumours or anti-vascular endothelial growth factor (VEGF) inhibitors (monoclonal antibodies bevaci­zumab, aflibercept, ramucirumab, regorafenib as per oral tyrosine kinase inhibitor).3-5 The combi­nation of these novel chemotherapy and targeted therapy now extends the mOS up to 40 months.3-5 Additionally, testing for new biomarkers ena­bles the usage of new targeted treatment in met­astatic CRC patients, such as human epidermal growth factor receptor 2 (HER2/new) amplifica­tions for double HER2 blockade, immunotherapy with anti-programmed cell death protein 1 (PD-1) monoclonal antibodies in high microsatellite insta­ble (MSI) tumours, and neurotrophic tyrosine ki­nase receptor (NTRK) inhibitors in case of NTRK gene fusions.3-5 BRAF V600E mutation is associated with poor prognosis under standard treatment of mOS less than 1 year and the responses to targeted therapy of combinations with anti- EGFR, BRAF and MEK inhibitors are promising with longer mOS.3-5 Pharmacogenomics’ biomarkers such as dihy­dropyrimidine dehydrogenase, uridine diphos­phate glucuronosyltransferase 1A1, excision re­pair cross complementing rodent repair deficiency complementation group 1, VEGF and thymidylate synthase are also important when planning the treatment and deciding on the type (to choose the alternative systemic therapy), appropriate combi­nation (less toxic) and dosages (to adjust the dose to lower the frequency and grade of the adverse ef­fects) of systemic therapy.6 New knowledge about the molecular heteroge­neity of CRC, the discovery of biomarkers as pre­dictive factors for disease prognosis and response to systemic treatment, and thus personalized medi­cine in this field, have also significantly contributed to the prolonged survival rates of patients. Besides gene mutations, tumour stroma and immunity also play a very important role in response to the sys­temic treatment and the prognosis of the disease. In 2015, Guinney et al. first published the clas­sification of consensus molecular subtypes (CMS), namely MSI immune CMS1, canonical CMS2, met­abolic CMS3 and mesenchymal CMS4.7 The CMS classification includes clinical factors, all patholog­ical and molecular features of the tumour, signal-ling pathways and immunity. However, it still cur­rently has not translated into regular clinical prac­tice, which could guide the clinicians in their more personalized treatment decisions. At present, the CMSs do not have an impact on clinical decisions, because we do not yet have approved algorithms available for everyday clinical practice The clinical implications of CMS Colorectal cancer is genetically and transcriptomi­cally heterogeneous disease. In adjuvant setting for early-stage CRC, there are several gene expression signatures such as ColoPrint, Oncotype DX and others, but they are still not recommended in eve­ryday clinical practice by international guidelines for CRC.2,3 In metastatic setting, MSI, RAS and BRAF mutational statuses are routinely tested for prognosis and predictions for systemic treatment. KRAS mutational status was the first biomarker in metastatic CRC to predict the response to anti-EG­FR inhibitors since 2008. Additionally, mutational status testing in RAS gene (KRAS and NRAS genes) is used in daily clinical practice since 2013. In the past, BRAF mutation was a negative prognostic biomarker for a shorter median OS of 12 months. This was also confirmed in our prospective clini­cal trial, conducted at the Institute of Oncology Ljubljana between 2010 and 2013, in which we ana­lysed the impact of the molecular biomarkers and histological parameters on survival and response to the first- line systemic therapy of metastatic colorectal cancer patients.8 Median OS of wild type wtBRAF patients was significantly longer than in mutated mtBRAF patients, with 59.2 and 27.6 months, respectively, p = 0.05. Today, targeted therapy combining BRAF in­hibitors and MEK inhibitors in combination with anti-EGFR inhibitors with mOS of 24 months is approved by FDA, but not by EMA in Europe for the BRAF mutated patients.2,9 However, metastatic CRC is not a simple disease but rather a hetero­geneous one, with different treatment responses and outcomes. Thus, these routinely identified biomarkers provide only some information about tumour biology. In 2015 Guinney et al. in the CRC Subtyping Consortium established four consensus molecu­lar subtypes 1 (CMS1), 2 (CMS2), 3 (CMS3) and 4 (CMS4), based on six independent CRC classifica­tion systems.7 They analysed tumour characteris­tics of more than 4000 patients, including not only 274 TABLE 1. Classification of consensus molecular subtypes (CMS). Adopted by Guiney et al.7 Frequency 14% 37% 13% 23% MSI, CIMP high, hypermutation SCNA high Mixed MSI status, SCNA low, CIMP low SCNA high BRAF mutation KRAS mutation Characteristics Immune infiltration and activation WNT and MYC activation Metabolic deregulation Stromal infiltration, TGF-ß activation, angiogenesis Worse survival after relapse Worse relapse-free and overall survival CIMP = CpG island methylator phenotype; MSI = microsatellite instable; SCNA = somatic copy number alterations; TGF-ß = transforming growth factor beta their genetic alterations, but also their immune system, cellular metabolism, epithelium, signal-ling activation, immune tumour infiltration, tu­mour microenvironment and angiogenesis. The CMS are characterized and named by their main distinguishing features. CMS1 is denoted as MSI immune, presented in 14% of the cases, hypermu­tated, microsatellite unstable and with strong im­mune cell infiltration and activation. CMS2 is ca­nonical, presented in 37% of the cases, with marked WNT and MYC signalling activation. CMS3 is called metabolic, presented in 13% of the cases, with epithelial and evident metabolic dysregula­tion, with KRAS mutations and mixed MSI status, low somatic copy number alterations (SCNA) and CpG island methylator phenotype (CIMP). CMS4 is called mesenchymal, presented in 23% of the cases, with prominent transforming growth factor ß activation, stromal infiltration and angiogenesis. The main features of CMS subtypes are presented in Table 1. The CMS subtypes are not classified only by molecular features, but also by clinical features, with prognosis included in its classification.10-13 Sidedness of the primary tumour is also included. Right-sided tumours, including cecum, ascend­ing colon or transverse colon are characterized by mucinous, signet ring histology, microsatellite instability, hypermethylation, poor differentia­tion, higher mutation rates of PI3KCA, KRAS and BRAF. They are more frequent in older patients and female patients. Left-sided tumours, includ­ing descending colon, sigmoid colon and rectum are characterized by chromosomal aberrations, 18q loss and 20q gain, aneuploidy, p53 mutation, EGFR and HER2 gain, high VEGF-1 mRNA, cyclooxyge­nase 2 (COX2), high EGFR ligand epiregulin and amphiregulin expression.10-13 However, tumour location inside the intestine is even more important than sidedness.12,14 Namely, CMS1 is more often present in the proximal colon (the cecum, the ascending colon, the transverse colon), CMS2 in the distal colon (the descending colon, the sigmoid colon) and the rectum, CMS3 in the sigmoid colon and the rectum and CMS4 in the distal colon (the descending colon, the sigmoid colon) and the rectum. Tumours of distal colon and rectum appear unique and tumours of the trans­verse colon appears distinct from other tumours of the right colon.14 Because of this tumour heteroge­neity of different parts of colon and the differences between tumours of colon and rectum, and also in­tra- tumour heterogeneity of the primary tumour, Fontana et al. highlighted the importance of the careful sampling from biopsies or resected primary tumour for each patient to get the right informa­tion about his biomarkers.12 Since secondary acquired resistance can develop during specific systemic therapy with anti EGFR inhibitors, because of tumour heterogeneity and clonal selection process, it is important to include circulating tumour DNA analyses in evaluation of effectiveness of systemic therapy. This technique can detect genomic alterations in RAS and other genes to help adjust systemic therapy before clini­cal and radiological progression.11,15-17 Two recently published papers explain the impact of CMS subtypes on the survival of meta­static CRC patients and the differences to the re­sponse to systemic treatment according to CMS subtypes.18,19 Patients from two phase III clinical trials, the CALBG/SWOG 80405 and the FIRE-3, were included in this analysis. Both clinical trials assessed the combination of anti-VEGFR inhibitor bevacizumab or anti- EGFR inhibitor cetuksimab with different types of chemotherapy - oxalipl- 275 atin with 5-FU (FOLFOX) in 75% of the patients in CALGB/SWOG 80405 and irinotecan with 5-FU (FOLFIRI) in all patients in the FIRE- 3.18-20 Both studies showed that left-sided colorectal cancer re­sponded better to cetuximab-based in combination with irinotecan therapy in case of CMS2 and CMS4 compared to bevacizumab-based therapy, whereas for right-sided tumours this possibility has to be further explored. Lenz et al. have retrospectively analysed the im­pact of the CMSs on survival of KRAS wild type metastatic CRC patients from CALGB/SWOG 80405 clinical study.18 For the CMS classification, the NanoString panel for the CALBG/SWOG 80405 cohort and the official CMS classifier software were used. Based on the CALGB study results, CMSs are predictive biomarkers for bevacizumab and cetuxi­mab in terms of OS and progression-free survival (PFS). In the CMS2 cohort, patients who received cetuximab had significantly longer OS and slightly improved PFS compared to those who received bevacizumab, although this was not statistically significant. In the CMS1 cohort, patients who re­ceived bevacizumab had significantly longer OS and longer PFS compared to the patients who re­ceived cetuximab. They concluded that CMS clas­sification is an independent prognostic marker for metastatic CRC patients in the first-line systemic therapy with a combination of chemotherapy with bevacizumab or cetuximab. Patients with CMS1 had the shortest OS and PFS, whereas patients with CMS2 had the longest OS with the lowest risk of death and PFS. They also emphasized the limita­tions of their analysis to the KRAS wild-type meta­static patients and stated that it was not possible to do a more detailed exploration of the interactions between a specific chemotherapy and targeted therapy. However, in 2019, Aderka et al. published a research, studying this topic.19 The responses of the patients with different CMS subtypes to sys­temic chemotherapy with oxaliplatin or irinotecan in combination with different targeted therapy, anti- VEGFR inhibitor bevacizumab or anti- EGFR inhibitor cetuximab were analysed. They found that both cytostatics have synergistic effect in com­bination with cetuximab. Irinotecan upregulates EGFR and promotes the binding of cetuximab and so promotes its antibody-dependent cell-mediat­ed cytotoxicity (ADCC), stimulates the release of IFN-. and activates dendritic cells, macrophages, T cells and encourages the apoptosis of cancer cells. Furthermore, cetuximab inhibits the tumour’s mul­tidrug resistance mechanism for the active metabo­lite of irinotecan - SN-38 - which accumulates in the cells and thus improves its antitumour effect. The oxaliplatin acts in two ways, as oxaliplatin – DNA adducts and causes DNA oxidative damage. EGRF activation upregulates nucleotide excision repair proteins and base excision repair proteins (ERCC1) and in this way neutralises effects of oxaliplatin. The combination of oxaliplatin and anti- EGFR in­hibitor cetuximab has a synergistic effect in terms of cetuximab downregulation of ERCC1 and, which could further improve oxaliplatin activity.19 The tumour microenvironment is also an impor­tant factor in resistance of CRCs to specific com­bination of chemotherapy and targeted therapy. The CMS1 and CMS4 tumours have a fibroblast-rich microenvironment.19 In that case of CMS1 and CMS4 oxaliplatin has an antagonistic action to anti-EGFR inhibitors cetuximab and panitumumab, in­ducing the release of interleukin 17A from fibro­blasts promoting proliferation of cancer stem cells and antagonising the growth suppression and ap­optosis of cancer stem cells induced by cetuximab. Activated cancer-associated fibroblasts also secrete transforming growth factor beta (TGF-ß) and me­diate tumour resistance to anti-EGFR inhibitors by providing an intrinsic EGFR-independent survival pathway to cancer cells. TGF-ß also prolongs inhib­itory effect on the cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC), inhibits activation of immune cells, natural killer cells, den-dritic cells and macrophages.19 In both articles, of Aderka and Lenz, the authors also explained why such differences occur.18,19 The first significant factor is the previously described synergistic or antagonistic action of the combina­tion of chemotherapy and the biological drug. The second important factor is the sequence of biologi­cals, bevacizumab and cetuximab, in terms of CMS, which is supported by both studies. If anti VEGFR inhibitor bevacizumab is administrated in first-line systemic treatment, before cetuximab, it reduces the permeability of blood vessels and consequently diffusion and tumour cell binding of cetuximab. The third factor is the half-life of bevacizumab compared to cetuximab, which is also important concerning the sequence of. With a half-life of 21 days, bevacizumab is still active for a period when initiating a second line of cetuximab treatment, re­ducing the permeability to tumour stroma and the anti-EGFR effect after the first line of bevacizumab. Lastly, in the FIRE-3 study, chemotherapy with on­ly irinotecan hydrochloride (CPT 11) with 5-fluoro-uracil (5-FU) was used in combination with beva­cizumab or cetuksimab; and oxaliplatin with 5-FU was used in 75% in combination with bevacizumab 276 or cetuximab in CALGB study. Thus, researchers concluded that both studies are complementary and not opposing in terms to relevant conclusions from retrospective analyses.19 Based on all clinical and molecular knowledge, the mOS for 16 different combinations of oxalipl­atin, irinotecan and targeted therapy in first-line treatment was calculated for each CMS subtype. The most effective first- line combination is oxali­platin with bevacizumab, irinotecan or oxaliplatin with cetuximab, oxaliplatin with cetuximab and irinotecan with cetuximab, in CMS1, CMS2, CMS3 and CMS4 respectively.19 Additionally, Stintzing et al. conducted an anal­ysis according to CMS classification in terms of objective responses (OR) and PFS from the FIRE-3 clinical trial, in which the first-line therapy was FOLFIRI (irinotecan plus 5-FU) with bevacizumab or cetuximab in KRAS wild-type metastatic CRC patients.20 The retrospective analysis was carried out for RAS wild-type metastatic CRC patients. They confirmed the prognostic role of CMS classi­fication in CMS3 and CMS4 subtypes and the pre­dictive role for a better outcome in CMS4 subtype in RAS wild-type patients, treated with FOLFIRI and cetuximab. Significantly higher overall re­sponse rate (ORR) were seen in CMS2 subtype in the same regimen. OS of patients with CRC sub­type CMS4 was significantly longer in treatment with FOLFIRI cetuximab compared to that with FOLFIRI bevacizumab. In patients with CMS3, OS was in favour of FOLFIRI and cetuximab, OS in CMS1 and CMS2 were comparable and independ­ent of targeted therapy. Lastly, gut microbiome is probably another im­portant biomarker to consider in future studies in treating metastatic CRC patients.10,21 Gut mi-crobiomes are associated with CMS1 and CMS2 subtypes. It is known that gut microbiome has an important role in carcinogenesis of CRC, show­ing initial inflammation and modulation of dif­ferent signalling pathways. Each part of the colon and rectum is characterized by different strains of bacteria. The most important and studied strains were Fusobcterium nucleatum, Escherichia coli and Bacteroides fragilis. Gut microbiome also varies geo­graphically, seven strains are the most important for carcinogenesis, B. fragilis, four oral as F. nu-cleatum, Parvimonas micra, Porphyromonas asaccha­rolytica and Prevotella intermedia, Alistipes finegoldii and Thermanaerovibrio acidaminovorans.21 Bacterial biomarkers have potential to detect CRC, predict clinical outcome and have a prognostic value.21 Gut microbiome also mediates the response to chemotherapy, especially of irinotecan, oxalipl­atin and 5-flurouracil, prescribed in treatment of metastatic CRC. There are several ways like immu­nomodulation, metabolism regulation, resistance to chemotherapy, microbial translocation, reduced ecological diversity and others. It also plays an important role in effectiveness of immunotherapy with checkpoint inhibitors in terms of to enhance the action of it. It can be also associated with the adverse effects of immunotherapy, especially with immune-related colitis, depending of the present­ed strains of bacteria in the gut.21 Therefore; the knowledge about gut microbiome will have clini­cal implications for CRC prevention, improvement of treatment responses and reduction of the ad­verse effects. Conclusions and future directions Predictive and prognostic biomarkers are impor­tant for personalized medicine and treatment of patients with metastatic CRC and therefore en­able better optimization and tailoring of treatment. Pharmacogenomics biomarkers will allow us to adjust and determine the optimum effective dose of the drug for each patient. Gut microbiome is an­other important biomarker predicting the progno­sis of disease and the response to the specific sys­temic therapy. CMS subtypes, including molecular heterogene­ity at different levels of genetics, epigenetics, tran­scriptomic, clinical features and more important tumour microenvironment will enable us to esti­mate the prognosis and make precision medicine individualized for each patient. In the future, it is important to develop algo­rithms for everyday clinical practice to determine the CMS subtype for each patient individually, based on patient and tumour characteristics. This will result in the most optimal, patient-tailored treatment to maximize the response, prolong sur­vival, minimize the treatment cost and avoid po­tential unwanted adverse effects of ineffective therapy. Acknowledgement The research was financially supported by The Slovenian Research Agency (ARRS), grant number P3-0321. 277 References 1. Cancer in Slovenia 2016. Ljubljana: Institute of Oncology Ljubljana, Epidemiology and Cancer Registry, Cancer Registry of Republic of Slovenia; 2019. 2. 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Nat Rev Gastroenterol Hepatol 2019; 16: 690-704. doi: 10.1038/s41575-019-0209-8 278 research article Prognostic role of positron emission tomography and computed tomography parameters in stage I lung adenocarcinoma Angelo Carretta1,5, Alessandro Bandiera1, Piergiorgio Muriana1, Stefano Viscardi1, Paola Ciriaco1, Ana Maria Samanes Gajate2, Gianluigi Arrigoni3, Chiara Lazzari4, Vanesa Gregorc4, Giampiero Negri1,5 1 Department of Thoracic Surgery, San Raffaele Hospital, Milan, Italy 2 Department of Nuclear Medicine, San Raffaele Hospital, Milan, Italy 3 Department of Pathology, San Raffaele Hospital, Milan, Italy 4 Department of Oncology, San Raffaele Hospital, Milan, Italy 5 School of Medicine, Vita Salute San Raffaele University, Milan, Italy Radiol Oncol 2020; 54(3): 278-284. Received 4 February 2020 Accepted 4 May 2020 Correspondence to: Angelo Carretta, M.D., Department of Thoracic Surgery, San Raffaele Hospital, School of Medicine, Vita Salute San Raffaele University, Via Olgettina, 60 – 20132, Milan, Italy. E-mail angelo.carretta@hsr.it Disclosure: No potential conflicts of interest were disclosed. Background. According to the current pathological classification, lung adenocarcinoma includes histological subtypes with significantly different prognoses, which may require specific surgical approaches. The aim of the study was to assess the role of CT and PET parameters in stratifying patients with stage I adenocarcinoma according to prognosis. Patients and methods. Fifty-eight patients with pathological stage I lung adenocarcinoma who underwent surgi­cal treatment were retrospectively reviewed. Adenocarcinoma in situ and minimally-invasive adenocarcinoma were grouped as non-invasive adenocarcinoma. Other histotypes were referred as invasive adenocarcinoma. CT scan assessed parameters were: ground glass opacity (GGO) ratio, tumour disappearance rate (TDR) and consolidation diameter. The prognostic role of the following PET parameters was also assessed: standardized uptake value (SUV) max, SUVindex (SUVmax to liver SUVratio), metabolic tumour volume (MTV), total lesion glycolysis (TLG). Results. Seven patients had a non-invasive adenocarcinoma and 51 an invasive adenocarcinoma. Five-year dis-ease-free survival (DFS) and cancer-specific survival (CSS) for non-invasive and invasive adenocarcinoma were 100% and 100%, 70% and 91%, respectively. Univariate analysis showed a significant difference in SUVmax, SUVindex, GGO ratio and TDR ratio values between non-invasive and invasive adenocarcinoma groups. Optimal SUVmax, SUVindex, GGO ratio and TDR cut-off ratios to predict invasive tumours were 2.6, 0.9, 40% and 56%, respectively. TLG, SUVmax, SUVindex significantly correlated with cancer specific survival. Conclusions. CT and PET scan parameters may differentiate between non-invasive and invasive stage I adenocar­cinomas. If these data are confirmed in larger series, surgical strategy may be selected on the basis of preoperative imaging. Key words: adenocarcinoma; lung; surgery; computed tomography; PET Introduction cal subtypes with different tumour invasiveness and prognosis. In this classification the former The current IASLC/ATS/ERS pathological classifi-term bronchoalveolar carcinoma (BAC) is no long-cation of lung adenocarcinoma includes histologi-er included and a distinction between adenocarci- 279 noma in situ, minimally invasive adenocarcinoma and invasive adenocarcinoma with its variants has been established.1 Patients with adenocarcinoma in situ and minimally invasive adenocarcinoma have extremely high survival rates after surgery. Invasive stage I adenocarcinoma is on the other side associated with a relatively high risk of recur­rence. Different surgical approaches have there­fore been proposed according to the histological features of the tumour, with sublobar resection as a possible treatment option for adenocarcinoma in situ and minimally-invasive adenocarcinoma.2,3 Conversely, major resection is still considered the treatment of choice of early-stage invasive adeno­carcinomas.4 Hence, the identification of pre-op­erative parameters that allow differentiating neo­plastic lesions according to tumour invasiveness is crucial for the planning of surgical treatment. This point is even more important considering the relatively low accuracy in the definition of tumour invasion of the histological analysis obtained after needle biopsy or with intraoperative frozen sec­tion.5,6 At Computed Tomography (CT), tumours with lepidic growth pattern appear as ground-glass opacities (GGO), which may represent a variable part of the neoplastic lesion, while on the other hand the solid part of the tumour is mainly an ex­pression of invasive adenocarcinoma.7,8 CT scan derived parameters as GGO ratio, tumour disap­pearance rate (TDR) and consolidation diameter are an expression of the proportion of ground-glass and solid features of the tumour, and may correlate with histology and clinical behaviour. Previous reports have analysed the correlation of radiologic parameters with tumour invasive­ness, but the prognostic role of these factors still has to be completely clarified.9 Positron emission tomography (PET) derived parameters have also been progressively used in the differential diagno­sis and as prognostic factors in patients with ad-enocarcinoma, the most used of which being the maximum standardized uptake value (SUVmax) of the tumour.10,11 Moreover, a prognostic role of other PET derived parameters as SUVindex, meta­bolic tumour value (MTV) and total lesion glycoly-sis (TLG) was also demonstrated, and some studies showed a better predictive performance of these parameters in comparison with SUVmax.12,13 The aim of the current study was to assess the role of CT and PET parameters in the differentia­tion of non-invasive and invasive adenocarcino-mas and in stratifying patients with stage I adeno-carcinoma according to their prognosis. Patients and methods Patients with pathological stage I lung adeno-carcinoma who underwent surgical treatment at our Institution following CT and PET scan evalu­ation between August 2006 and July 2011 were reviewed. The study was approved by the local Ethics Committee and registered on Clinicaltrials. gov (NCT04202614). Histological specimens were classified ac­cording to the current IASLC/ATS/ERS patho­logical classification of lung adenocarcinoma.1 Adenocarcinoma in situ and minimally invasive adenocarcinoma were grouped as non-invasive adenocarcinoma. Other histotypes were referred as invasive adenocarcinoma. Tumours were re-staged according to the current 8th edition of the TNM staging system.14 Pre-operative imaging work-up included CT scan and whole body PET scan. Nodal involve­ment in patients with clinical N2/N3 disease was preoperatively excluded by invasive mediastinal assessment (EBUS-TBNA or mediastinoscopy). Major resections were considered the treatment of choice in patients with invasive adenocarcinoma. Wedge resections were performed in the treatment of adenocarcinoma in situ and minimally-invasive tumours, and in patients with invasive adenocarci­noma with a functional contraindication to major resection. The features analysed for all patients were: age, sex, smoking habit, type of surgical resection, tu­mour histology, stage of disease, morbidity, mor­tality, overall survival, cancer specific survival and disease free survival, PET-derived and CT scan pa­rameters. CT scan parameters CT images were obtained using a commercial­ly available scanner (Toshiba X-press, Toshiba Medical Systems, Tokio, Japan). After infusion of intravenous contrast material spiral acquisition was obtained during breath-hold at the end of in­spiration. The chest region was scanned with a de­tector configuration of 120 kVp, 200 mAs, 1 mm sec­tion thickness. The images were assessed using the mediastinal window setting (level, 40 Hounsfield units [HU]; width, 350 HU) and the lung window setting (level, 600 HU; width, 1500 HU). CT scan assessed parameters were: ground glass opacity (GGO) ratio, tumour disappearance rate (TDR) and consolidation diameter. GGO ratio was defined as the percentage of the tumour with GGO 280 TABLE 1. Characteristics of 58 surgically-treated patients with stage I adenocarcinoma Gender Female Male Age (median;range) Type of surgery Wedge resection Lobectomy Bilobectomy TNM Tis T1aN0 T1bN0 T1cN0 T2aN0 4 3 67 (46-75) 3 4 0 1 3 1 2 0 38 0.178 13 65 (48-85) 0.530 10 40 0.188 1 0 9 17 0.056 16 9 appearance (1-[maximum dimension of consolida­tion on lung windows/maximum dimension of tu­mour on lung windows]) x 100, TDR% was defined as the ratio between the area of consolidation on mediastinal windows and the area of consolidation on lung window (1-[maximum area of consolida­tion on mediastinal windows/maximum area of tumour on lung windows]) x 100, consolidation diameter was defined as the maximum diameter of consolidation on lung window. PET scan parameters The prognostic role of the following PET-derived parameters was also assessed: standardized up­take value (SUV)max, SUVindex (SUVmax to liver SUVratio), MTV, TLG. PET-derived parameters (SUVmax, SUVindex, MTV and TLG) were calcu­lated with a dedicated software (GE Advantage workstation - GEMS) developed for biomedical images. A volume of interest (VOI) was created for each lesion around the area of FDG uptake enclosing the tumour and SUVmax was obtained. SUVmean and MTV were measured using an auto­matic isocontour threshold method based on 50% of tumour SUVmax. SUVindex for each neoplastic lesion was calculated according to the method de­fined by Shiono et al.12 A 6-cm circular region of interest (ROI) was drawn on three consecutive PET slices on the liver parenchyma. Liver SUVmean was defined as the mean of the SUVmax values of the three PET slices. SUVindex was calculated as the ratio of tumour SUVmax to liver SUVmean. TLG was calculated by multiplying MTV by tu­mour SUVmean . Statistical analysis Analysis was performed by SPSS Statistics soft­ware, version 18.0 (SPSS Inc., Chicago, IL, USA). Differences between classes of patients were tested for significance with the X2 or Fisher’s exact test for discrete variables and with the Student’s t test for continuous variables. Receiver-operating char­acteristic (ROC) curves for PET and CT derived parameters were generated to define the cut-off values to differentiate non-invasive and invasive tumours and dichotomize patients on the basis of cancer-specific survival. Survival curves were reconstructed according to the Kaplan and Meier method. Differences in survival rates of patients grouped according to selected variables were es­timated by means of the log-rank test. The Cox regression analysis was performed to assess the independent value of the significant variables at univariate analysis. Results were considered significant when p-values less than 0.05 were ob­served. Confidence intervals were calculated at the 95% level. Results Fifty-eight patients (41 males, 17 females, mean age 66, range 46 to 85 years) with pathological stage I lung adenocarcinoma entered the study. The characteristics of the patients are depicted in Table 1. Forty-four patients underwent a lobec­tomy, one patient a bilobectomy and 13 a wedge resection. Seven patients had a non-invasive and 51 an invasive adenocarcinoma. The pathological staging was as follows: Tis in one patient, T1aN0 in 12 cases, T1bN0 in 18 cases, T1cN0 in 18 cases and T2aN0 in 9 cases. The follow-up was complete for all 58 patients. The median follow-up was 60 months (range 3–126). At the end of follow-up thir­ty-nine patients are alive without evidence of can­cer recurrence, 10 patients are alive with evidence of relapse, 4 patients died of cancer recurrence and 5 patients died due to other causes. Five-year disease-free survival (DFS) and can-cer-specific survival (CSS) was 100% and 100% for non-invasive and 70% and 91% for invasive adenocarcinoma, respectively (Figures 1 and 2) (p = 0.115, p = 0.46). Significant differences in GGO ratio, TDR ratio, SUVmax and SUVindex values were observed between non-invasive and invasive adenocarcinoma groups. Mean GGO ratio was 42% in non-invasive and 19% in invasive adenocarcino-ma (p = 0.011); mean TDR ratio was 53% in non- 281 invasive and 24% in invasive adenocarcinoma (p = TABLE 2. Differences in CT and PET scan parameters according to histology 0.001); mean SUVmax was 2.75 in non-invasive and 7.16 in invasive adenocarcinoma (p = 0.033); mean SUVindex was 0.98 in non-invasive and 3.12 in in­vasive adenocarcinoma (p = 0.037) (Table 2). TDR% 53±9.31 24±2.89 < 0.001 According to the ROC curve analysis optimal Consolidation diameter 13±2.19 21±1.44 0.07 GGO ratio and TDR cut-off ratios to distinguish non-invasive from invasive adenocarcinoma were SUVmax 2.75±0.91 7.16±0.73 0.033 40% (area under the curve [AUC] 82%, sensitivity SUVindex 0.98±0.25 3.12±0.36 0.037 67%, specificity 81%) and 56% (AUC 85%, sensitiv-MTV 3.6±1.74 5.3±0.49 0.293 ity 67%, specificity 96%), respectively; SUVmax TLG 12±7.31 19.5±4.34 0.541 and SUVindex cut-off ratios were 2.6 (AUC 81.5%, sensitivity 84%, specificity 71%) and 0.9 (AUC GGO = gound-glass opacity; MTV = metabolic tumour volume; SUV = standardized uptake value; TDR = tumour disappearance rate; TLG = total lesion glycolysis 84%, sensitivity 90%, specificity 71%), respective­ly. Patients with higher SUVmax and SUVindex TABLE 3. Characteristics of patient population grouped by standardized uptake values had a significantly higher incidence of less value (SUV)max and SUVindex differentiated and larger tumours (Table 3). CSS significantly correlated with SUVmax, SUVindex and TLG. The statistical analysis with ROC curves identified the following best cut-off values to dif­ferentiate the patients according to prognosis: Histology SUVmax 8.6, SUVindex 4.08, TLG 9.38. Five-year NIA (7)43 52 CSS was 97% in patients with a SUVmax < 8.6 and IA (51) 8 43 0.028 5 46 0.001 81% in patients with a SUVmax > 8.6 (p = 0.036) Gender(Figure 3). Five-year CSS was 97% in patients with male 9 32 6 35 female 1.00 0.458 a SUVindex < 4.08 and 76% in patients with a 3 14 4 13 SUVindex > 4.08 (p = 0.01) (Figure 4). Five-year CSS Smoke Yes1037 7 40 was 100% in patients with a TLG < 9.38 and 82% in 1.00 0.381 No 2938 patients with a TLG > 9.38 (p = 0.02) (Figure 5). The T type of surgical resection did not have a prognos- Tis–T1a6 7 67 T1b513 3 15 0.014 0.011 tic role (Five-year CSS 89% in patients submitted T1c117 1 17 to wedge resection and 93% in patients submitted T2a 09 09 Grading to major resection, p = 0.822). In particular, in pa­ G131 31 tients submitted to wedge resection a correlation G29 38 0.011 7 40 0.004 G3 07 07 of DFS and CSS with CT and PET parameters was not observed, although patients with a TLG value IA = invasive adenocarcinoma; NIA – Non-invasive adenocarcinoma FIGURE 1. Kaplan-Meier disease free survival (DFS) plot for FIGURE 2. Kaplan-Meier cancer specific survival (CSS) plot for non-invasive and invasive adenocarcinoma (Invasive non-invasive and invasive adenocarcinoma. Five-year CSS was adenocarcinoma). Five-year DFS (disease free survival) was 100% for non-invasive and 91% for invasive adenocarcinoma 100% for non-invasive and 70% for invasive adenocarcinoma (p = 0.46). (p = 0.115). 282 under the 9.38 cut-off value tended to have a better survival (p = 0.061). No significant correlation with outcome was identified at multivariate analysis. Discussion The current classification of lung adenocarcinoma identifies different histologic subtypes with a clear differentiation between non-invasive and invasive tumours, due to their significantly different prog­nosis.1 Preoperative assessment of the invasiveness of stage I adenocarcinoma has become increasingly important for the definition of the ideal surgical treatment. In fact, the standard of care of stage I adenocarcinoma is at present lobectomy with me-diastinal lymphadenectomy.4,15 Conversely, non­invasive lesions may benefit of lung-sparing lim­ited resections. Sublobar resections have in fact been reported as being oncologically equivalent to major anatomical resections in non-invasive and minimally invasive tumours.2,3 However, tumour invasiveness is hard to be determined at preopera­tive or intraoperative assessment, since significant limitations exist in the definition of tumour inva­siveness in histological specimens obtained by needle biopsy and with intraoperative frozen sec­tion.5,6 Thus, the identification of CT and PET fea­tures of non-invasive and invasive tumours may be essential to differentiate invasive and non-invasive lesions, in order to select the optimal surgical treat­ment. Previous studies have investigated the role of imaging techniques in distinguishing different adenocarcinoma subtypes. In particular, the pro­portion of GGO, which reflects the presence of a lepidic pattern, may predict adenocarcinoma inva­siveness and prognosis.7,8,9 In the present retrospec­tive analysis a significant difference in GGO ratio, TDR, SUVmax and SUVindex was observed be­tween non-invasive and invasive adenocarcinoma. These data confirm that CT and PET parameters reflect tumour invasiveness and may be useful for the preoperative differentiation between invasive and non-invasive lesions. Moreover, the combina­tion of PET and CT scan parameters may increase the accuracy of such evaluation. In our study ROC analysis identified a cut-off value of 40% for GGO ratio to differentiate be­tween invasive and non-invasive adenocarcinoma. These findings are similar to those of a previous study performed by Takahashi et al., who identi­fied a GGO ratio of > 50% to differentiate between non-invasive and invasive adenocarcinoma, data 283 confirmed by Honda et al.9,15 More recently, Huang et al. have on the other hand observed that a GGO ratio = 75% is a favourable prognostic factor in re-sected lung adenocarcinoma.16 Another CT feature analysed in our study which allowed to differenti­ate between non-invasive and invasive adenocar­cinoma was TDR. In our series a TDR value > 56% was more frequently associated with non-invasive adenocarcinoma. In previous studies Takahashi et al. reported a TDR cut-off between non-inva­sive and invasive adenocarcinoma of 75%, while Nakayama et al. observed that a TDR > 50% was a favourable prognostic factor in resected pulmo­nary adenocarcinoma.9,17 The results of our analy­sis confirm the role of these CT scan derived pa­rameters in the definition of tumour invasiveness. We also analysed the role of PET derived pa­rameters in predicting invasive tumour features in resected stage I adenocarcinomas. SUV is the most widely used parameter in the diagnosis and prog­nostic analysis of lung cancer.10,11,18 However, de­spite its usefulness in diagnosis, staging and prog­nostic assessment, the role of SUV in predicting tumour invasiveness in adenocarcinoma has not been completely investigated. Furthermore, the use of SUV is impaired by two major factors: it de­pends on biologic and technological variables that limit its reproducibility, and is not representative of the neoplastic volume.19 Shiono et al. therefore proposed to correct the value of lung cancer SUV using the liver as internal control (SUVindex).12 The present study demonstrated that SUVindex was also a predictive factor for recurrence in stage I adenocarcinoma. The cut-off values of SUVmax and SUVindex which allowed to differentiate be­tween invasive and non-invasive adenocarcinomas were 2.6 and 0.9, respectively. In a previous study Hattori et al. identified a SUVmax < 1 as a cut-off value to predict adenocarcinoma in situ.20 Considering cancer specific survival, the uni­variate statistical analysis in our series demon­strated that SUVmax, SUVindex and TLG could be identified as prognostic factors. The best cut­off values to differentiate the patients according to prognosis were: SUVmax 8.6, SUVindex 4.08, TLG 9.38. Similar results concerning the SUVmax value were observed in a previous study by Lee et al., where patients with a SUVmax = 9.5 had a significantly higher overall and disease-free sur­vival.18 Dichotomizing the patients according to the cut-off values of SUVmax, SUVindex and TLG it was therefore possible to stratify the groups of patients according to their prognosis. Patients with parameters over the cut-off value of SUVmax, SUVindex and TLG had in fact a worse CSS. These data confirm the prognostic role of these PET de­rived parameters. Besides considering the advan­tages of SUVindex in terms of reproducibility, it is also important to highlight the role of TLG, which seems to be a promising prognostic factor as it is representative of both tracer uptake and metabolic tumour burden. Considering the results of our study and previ­ous data of the literature, it is reasonable to try to discriminate preoperatively between non-invasive and invasive adenocarcinoma by integrating CT and PET parameters. The association of CT and PET parameters could in fact allow improving the preoperative differential diagnosis of invasive and non-invasive tumours in order to differentiate the surgical approach. Moreover, PET derived param­eters as SUVmax, SUVindex and TLG may play an additional role to that of histology in the definition of the prognosis of patients with stage I adenocar­cinoma. The present study, aiming at focusing the atten­tion on both CT and PET parameters in providing prognostic information in stage I adenocarcinoma, has some limitations, being a retrospective and sin-gle-institution study based on a relatively limited series of patients. In particular, the two groups of patients (non-invasive and invasive tumours) were relatively unbalanced, a point which could have limited the results. Even so, the advantage of a sin-gle-institution study is that the methodology to as­sess CT and PET parameters could be homogene­ous and clinical data were uniform. Further studies with a larger cohort of patients are nevertheless required to confirm the results of our analysis. References 1. Travis WD, Brambilla E, Nicholson AG, Yatabe Y, Austin JHM, Beasley MB, et al. The 2015 World Health Organization classification of lung tumors. Impact of genetic, clinical and radiologic advances since the 2004 classification. J Thoracic Oncol 2015; 10: 1243-60. doi: 10.1097/JTO.0000000000000630 2. Tsutani Y, Miyata Y, Nakayama H, Okumura S, Adachi S, Yoshimura M, et al. Appropriate sublobar resection choice for ground glass opacity-dominant clinical stage Ia lung adenocarcinoma. Chest 2014; 145: 66-71. doi: 10.1378/ chest.13-1094 3. Moon Y, Lee KY, Moon SW, Park JK. Sublobar resection margin width does not affect recurrence of clinical N0 non-small cell lung cancer presenting as GGO-predominant nodule of 3 cm or less. World J Surg 2017; 41: 472-9. doi: 10.1007/s00268-016-3743-3 4. Khullar OV, Liu Y, Gillespie T, Higgins KA, Ramalingam S, Lipscomb J, et al. Survival after sublobar resection versus lobectomy for clinical stage IA lung cancer: an analysis from the National Cancer Data Base. J Thorac Oncol 2015; 10: 1625-33. doi: 10.1097/JTO.0000000000000664 5. He P, Yao G, Guan Y, Lin Y, He J. Diagnosis of lung adenocarcinoma in situ and minimally invasive adenocarcinoma from intraoperative frozen sections: an analysis of 136 cases. J Clin Pathol 2016; 69: 1076-80. doi: 10.1136/ jclinpath-2016-203619 284 6. Walt AE, Marchevsky AM. Root cause analysis of problems in the frozen sec­tion diagnosis of in situ, minimally invasive, and invasive adenocarcinoma of the lung. Arch Pathol Lab Med 2012; 136: 1515-21. doi: 10.5858/arpa.2012­0042-OA 7. Lee HY, Lee KS. Ground-glass opacity nodules: histopathology, imaging evaluation, and clinical implications. J Thorac Imaging 2011; 26: 106-18. doi: 10.1097/RTI.0b013e3181fbaa64 8. Liu Y, Sun H, Zhou F, Su C, Gao G, Ren S, et al. Imaging features of TSCT predict the classification of pulmonary preinvasive lesion, minimally and invasive adenocarcinoma presented as ground glass nodules. Lung Cancer 2017; 108: 192-7. doi: 10.1016/j.lungcan.2017.03.011 9. Takahashi M, Shigematsu Y, Ohta M, Tokumasu H, Matsukura T, Hirai T. Tumor invasiveness as defined by the newly proposed IASCL/ATS/ERS clas­sification has prognostic significance for pathologic stage Ia lung adenocar­cinoma and can be predicted by radiologic parameters. J Thorac Cardiovasc Surg 2014; 147: 54-9. doi: 10.1016/j.jtcvs.2013.08.058 10. Zhou J, Li Y, Zhang Y, Liu G, Tan H, Hu Y, et al. Solitary ground-glass opacity nodules of stage Ia pulmonary adenocarcinoma: combination of 18F-FDG PET/CT and high resolution computed tomography features to predict invasive adenocarcinoma. Oncotarget 2017; 8: 23312-21. doi: 10.18632/ oncotarget.15577 11. Uehara H, Tsutani Y, Okumura S, Nakayama H, Adachi S, Yoshimura M. Prognostic role of positron emission tomography and high-resolution com­puted tomography in clinical stage Ia lung adenocarcinoma. Ann Thorac Surg 2013; 96: 1958-65. doi: 10.1016/j.athoracsur.2013.06.086 12. Shiono S, Abiko M, Okazaki T, Chiba M, Yabuki H, Sato T. Positron emis­sion tomography for predicting recurrence in stage I lung adenocarci­noma: standardized uptake value corrected by mean liver standardized uptake value. Eur J CardioThorac Surg 2011; 40: 1165-9. doi: 10.1016/j. ejcts.2011.02.041 13. Melloni G, Gajate AMS, Sestini S, Gallivanone F, Bandiera A, Landoni C, et al. New positron emission tomography derived parameters as predictive factor for recurrence in resected stage I non-small cell lung cancer. Eur J Surg Oncol 2013; 39: 1254-61. doi: 10.1016/j.ejso.2013.07.092 14. Brierley JD, Gospodarowicz MK, Wittekind C. UICC TNM classification of malignant Tumours. 8th Edition. Oxford: Wiley-Blackwell; 2017. p. 105-12. 15. Honda T, Kondo T, Murakami S, Saito H, Oshita F, Ito H, et al. Radiographic and pathological analysis of small lung adenocarcinoma using the new IASLC classification. Clin Radiol 2013; 68: e21-6 doi: 10.1016/j.crad.2012.09.002 16. Huang TW, Lin KH, Huang HK, Chen YI, Ko KH, Chang CK, et al. The role of the ground-glass opacity ratio in resected lung adenocarcinoma. Eur J Cardiothorac Surg 2018; 54: 229-34. doi: 10.1093/ejcts/ezy040 17. Nakayama H, Yamada K, Saito H, Oshita F, Ito H, Kameda Y, et al. Sublobar resection for patients with peripheral small adenocarcinomas of the lung: surgical outcome is associated with features on computed tomographic imaging. Ann Thorac Surg 2007; 84: 1675-79. doi: 10.1016/j.athorac­sur.2007.03.015 18. Lee HY, Lee SW, Lee KS, Jeong JY, Choi JY, Kwon OJ, et al. Role of CT and PET Imaging in predicting tumor recurrence and survival in patients with lung adenocarcinoma: a comparison with the International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification of lung adenocarcinoma. J Thorac Oncol 2015; 10: 1785-94 doi: 10.1097/JTO.0000000000000689 19. Nair VS, Krupitskaya Y, Gould MK. Positron emission tomography 18F-Fluorodeoxyglucose uptake and prognosis in patients with surgically treated, stage I non-small cell lung cancer: a systematic review. J Thorac Oncol 2009; 4: 1473-9. doi: 10.1097/JTO.0b013e3181bccbc6 20. Hattori A, Suzuki K, Matsunaga T, Fukui M, Tsushima Y, Takamochi K, et al. Tumor standardized uptake value on positron emission tomography is a novel predictor of adenocarcinoma in situ for c-Stage IA lung cancer pa­tients with a part-solid nodule on thin-section computed tomography scan. Interact Cardiovasc Thorac Surg 2014; 18: 329-34. doi: 10.1093/icvts/ivt500 285 research article [18F]FDG PET immunotherapy radiomics signature (iRADIOMICS) predicts response of non-small-cell lung cancer patients treated with pembrolizumab Damijan Valentinuzzi1,2, Martina Vrankar3,4, Nina Boc3, Valentina Ahac3, Ziga Zupancic3, Mojca Unk3, Katja Skalic3, Ivana Zagar3, Andrej Studen1,2, Urban Simoncic1,2, Jens Eickhoff5, Robert Jeraj1,2,6 1 Jožef Stefan Institute, Ljubljana, Slovenia 2 Faculty of Mathematics and Physics, University of Ljubljana, Ljubljana, Slovenia 3 Institute of Oncology Ljubljana, Ljubljana, Slovenia 4 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 5 Department of Biostatistics and Medical Informatics, University of Wisconsin, Madison, WI, USA 6 Department of Medical Physics, University of Wisconsin, Madison, WI, USA Radiol Oncol 2020; 54(3): 285-294. Received 30 April 2020 Accepted 5 June 2020 Correspondence to: Prof. Robert Jeraj, Ph.D., Department of Medical Physics, University of Wisconsin, 1111 Highland Avenue, Madison, WI 53705, USA. Phone: +1 608 263 8619; E-mail: rjeraj@wisc.edu Disclosure: No potential conflicts of interest were disclosed. Background. Immune checkpoint inhibitors have changed the paradigm of cancer treatment; however, non­invasive biomarkers of response are still needed to identify candidates for non-responders. We aimed to investigate whether immunotherapy [18F]FDG PET radiomics signature (iRADIOMICS) predicts response of metastatic non-small-cell lung cancer (NSCLC) patients to pembrolizumab better than the current clinical standards. Patients and methods. Thirty patients receiving pembrolizumab were scanned with [18F]FDG PET/CT at baseline, month 1 and 4. Associations of six robust primary tumour radiomics features with overall survival were analysed with Mann-Whitney U-test (MWU), Cox proportional hazards regression analysis, and ROC curve analysis. iRADIOMICS was constructed using univariate and multivariate logistic models of the most promising feature(s). Its predictive power was compared to PD-L1 tumour proportion score (TPS) and iRECIST using ROC curve analysis. Prediction accuracies were assessed with 5-fold cross validation. Results. The most predictive were baseline radiomics features, e.g. Small Run Emphasis (MWU, p = 0.001; hazard ratio = 0.46, p = 0.007; AUC = 0.85 (95% CI 0.69–1.00)). Multivariate iRADIOMICS was found superior to the current standards in terms of predictive power and timewise with the following AUC (95% CI) and accuracy (standard deviation): iRADI-OMICS (baseline), 0.90 (0.78–1.00), 78% (18%); PD-L1 TPS (baseline), 0.60 (0.37–0.83), 53% (18%); iRECIST (month 1), 0.79 (0.62–0.95), 76% (16%); iRECIST (month 4), 0.86 (0.72–1.00), 76% (17%). Conclusions. Multivariate iRADIOMICS was identified as a promising imaging biomarker, which could improve man­agement of metastatic NSCLC patients treated with pembrolizumab. The predicted non-responders could be offered other treatment options to improve their overall survival. Key words: anti-PD-1; [18F]FDG PET/CT; non-small-cell lung cancer; radiomics analysis; iRADIOMICS Introduction survival rate around 15%.1 A new hope has come with renaissance of immunotherapy, such as pro-In spite of the advances in lung cancer treatment, grammed death-1 antibodies (anti-PD-1), which prognosis for patients has been poor with a 5-year invigorate a patient’s immune system to fight 286 against malignant cells.2 In non-small-cell lung cancer (NSCLC), which represents 85% of all lung cancer cases, treatment outcomes of anti-PD-1 im­munotherapy are significantly better compared to conventional cytotoxic therapies. In selected pa­tient population, response rates can be over 40%.3 The responding patients usually achieve durable benefit and prolonged survival. Occasionally, even complete remissions of metastatic disease are ob­served, but such complete responses are still in mi­nority. Due to possible unusual response patterns (e.g. pseudoprogression), treatment response assess­ment in immunotherapy is challenging.4 The most routinely used methods are Response Evaluation Criteria in Solid Tumours (RECIST) and its modi­fication for use in immunotherapy (iRECIST), among others.5 Although iRECIST was found supe­rior to RECIST in identifying pseudoprogression, iRECIST is a late response assessment method, be­cause anatomical changes observed on computed tomography are usually delayed, and the suspicion of progressive disease needs to be confirmed with an additional scan 1–2 months after the first assess­ment.6 Importantly, studies have shown that none of the RECIST-based endpoints could be used as valid surrogates for overall survival (OS) in anti­PD-1 trials, while the correlation of iRECIST-based endpoints with OS is yet to be explored.7,8 Since the molecular and functional tumour changes are known to appear faster compared to anatomical changes, several immunotherapy response assess­ment methods, based on 2-deoxy-2-[fluorine-18] fluoro-D-glucose positron emission tomography/ computed tomography ([18F]FDG PET/CT), have been proposed.9-12 However, there is still a lack of sufficient evidence to infer, which method, if any, might be the most appropriate for the routine clini­cal use.13-15 Recently, research into the identification of new biomarkers for use in immunotherapy has also increased. Various predictive and prognostic biomarkers of response have been identified, in­cluding tumour PD-1 ligand (PD-L1) expression, tumour mutation burden, tumour infiltrating lymphocytes density, mismatch repair deficiency, microsatellite instability, and gut microbiota.16,17 However, the reports from different studies some­times oppose each other, therefore the current biomarkers need further validation.18 Moreover, most of them require invasive biopsies, and are impractical or too expensive for a routine clini­cal use. On the other hand, few immunotherapy clinical studies examined possible non-invasive imaging biomarkers, but there is still a lack of re­search performed in NSCLC patients.14 Three ret­rospective anti-PD-1 studies showed associations of pre-treatment sum of maximum standardized )19 uptake values (SUVmax) of all lesions (SUVmaxwb, SUVmax of the most avid lesion20, and volumetric parameters (metabolic tumour volume [MTV], and total lesion glycolysis [TLG])21, with NSCLC patient response as defined by RECIST. However, significant correlations of these features with OS were not observed. There is also a lack of clinical studies in immunotherapy investigating more so­phisticated image analysis methods such as radi­omics analysis. Radiomics analysis harnesses the full power of medical imaging by extracting nu­merous quantitative features, hypothesized to re­flect more deeply the tumour phenotype, as well as the genotype.22,23 Recent anti-PD-(L)1 radiomics studies have shown associations of CT radiom­ics signatures with tumour immune phenotype24, hyperprogression25, and progression-free surviv­al (PFS)26. Moreover, two studies also examined the predictive value of PET radiomics features. Polverari et al. observed significant differences in tumour heterogeneity (as defined by kurtosis and skewness) between patients with progressive dis­ease (PD) and non-PD21, while the study by Mu et al. proposed a combined PET and CT radiomics signature for predicting patient PFS and OS.27 In these studies (except Polverari et al. ), data min­ing using vast number of features (up to 1160) was performed in order to build multivariate radi­omics signatures containing up to eight features. Although on one hand, such approach might al­low for a more precise quantification of tumour characteristics, on the other hand, the so obtained predictive models could be prone to overfitting, and probably too complex and non-intuitive for a successful clinical translation. Moreover, it is also well known that a lot of radiomics features are not suitable candidates for biomarkers, for example due to an excessive test-retest variability.28 The primary aim of our prospective study was to determine whether immunotherapy [18F]FDG PET radiomics signature (iRADIOMICS) predicts response of stage IV NSCLC patients to pem­brolizumab better than the current routinely used clinical standards (PD-L1 immunohistochemistry, and iRECIST). To overcome the aforementioned pitfalls, we deliberately analysed only a small sub­set of radiomics features, which were previously proven to be robust and reliable according to test-retest variability28, and built iRADIOMICS with minimum number of features. 287 Patients and methods Patients Thirty consecutive patients who met the follow­ing inclusion criteria were enrolled from January 2017 – March 2019 at the Institute of Oncology Ljubljana (Slovenia): = 18 years old, cytologically or histologically confirmed stage IV NSCLC (8th TNM classification of the International Association for the Study of Lung Cancer), no history of oth­er malignancies, PD-L1 tumour proportion score (TPS) > 1% (assessed by a validated immunohisto-chemistry assay), Eastern Cooperative Oncology Group criteria (ECOG) performance status 0–2. Enrolment required approval of the multidiscipli­nary tumour board that the patient was a candi­date for treatment with pembrolizumab. The study (NCT04007068) was approved by the institutional review board committee and the National Ethics Committee (KME 117/02/17). All patients gave in­formed consent to participate. Study protocol All patients underwent standard diagnostic pro­cedures including clinical examination and blood tests. Baseline [18F]FDG PET/CT was performed = 4 weeks before treatment, and follow-up [18F] FDG PET/CTs were performed 1 month (± 5 days) and 4 months (± 14 days) after treatment initia­tion. Patients were treated with pembrolizumab until progression, clinical benefit, or unaccepted toxicities. Pembrolizumab dosage was 2 mg/kg or 200 mg/patient (depending on the guidelines at the time of treatment), intravenously, every three weeks (q3w). Patients could also receive palliative radiotherapy in case of symptomatic lesions. Such treatment intervention required approval of the multidisciplinary tumour board. Imaging acquisition and analysis Patients fasted for at least 6 hours before intrave­nous application of 3.7 MBq/kg [18F]FDG and re­mained seated or recumbent for 60 minutes. Data acquisition was performed on a Biograph 40 mCT (Siemens Healthcare, Erlangen, Germany) with the following parameters: CT (tube current 100 kV, tube voltage 80 mAs, Care dose 4D and Care kV dose modulation, collimation 16×1.2 mm, pitch 1.2, reconstruction using 3 mm slice thickness in 2 mm increment, abdominal window, B40f kernel), [18F] FDG PET (acquired from skull base to mid-thigh, 2 minutes per bed position, reconstruction using TruX+TOF (UltraHD-PET) algorithm, 2 iterations per 21 subsets, matrix size 200×200, 3 mm slice thickness, 2.5 mm pixel size). Two physicians seg­mented the lesions semi-automatically in 3D Slicer using SUV > 4.0 g/ml as the threshold. The seg­mentations were then examined by an experienced radiologist and, if necessary, manually edited. The radiologist also performed iRECIST assessment. All researchers involved in tumour segmentations were blinded to the outcome of the study. Feature extraction At first, eight [18F]FDG radiomics features were extracted from primary tumours, including three volume-based features (volume, maximum standardized uptake value (SUVmax), total SUV )) and five texture-based heterogeneity (SUVtotal features, derived from Grey-Level Co-occurrence Matrix (GLCM) (Sum Entropy, Entropy-GLCM, Difference Entropy) and Grey-Level Run Length Matrix (GLRLM) (Small Run Emphasis (SRE), Run Percentage).29,30 Importantly, these five texture-based features were deliberately chosen, because they were identified as very robust and reliable, based on test-retest variability in a prospective multicentre study of NSCLC tumours imaged with [18F]FDG PET/CT.28 Feature definitions and their intuitive explanations are summarized in Table S1. Feature extraction was performed using an in-house software, see references.31-33 Briefly, features were extracted using a voxel-based method. The image was discretized into 256 grey levels. For each voxel, the feature was calculated over a 5 × 5 voxel patch in axial, coronal, and sagittal planes, and averaged over the three planes for each voxel. The final feature was calculated by averaging over all voxels. After examining the correlation between features using Pearson correlation coefficient, we excluded SUVtotal and Run Percentage from further analysis, because they were too closely correlated with other features (Figure S1). Statistical analysis Response was defined based on overall survival (OS), the gold standard end-point in immunother­apy8, therefore OS was the primary outcome meas­ure in our study. OS was defined as the time from initiation of pembrolizumab until death from any cause. Patients with OS > 14.9 months were defined as responders. The selected threshold was median OS in the multicentre KEYNOTE-10 study (sub­group of NSCLC patients with PD-L1 TPS > 50%, 288 treated with pembrolizumab dose 2 mg/kg)).34 Although the inclusion criteria in our study was PD-L1 TPS > 1%, the majority of patients (26/30, 87%) had PD-L1 TPS > 50%, resulting in compara­ble median OS (15.95 months). Mann-Whitney U-test and Fisher exact test were used to investigate the differences in radiomics features and demographic data between the re­sponders and non-responders. Receiver operating characteristic (ROC) curve analysis was used to assess the predictive power of each radiomics fea­ture. Univariate and multivariate Cox proportional hazards (Cox PH) regression analyses were used to study the relationship between the radiomics fea­tures and OS. A multivariate Cox PH model was FIGURE 1. Baseline radiomics features of primary tumours – Receiver operating characteristic curve (ROC) analysis. For each radiomics feature, the area under the ROC curve (AUC) with the corresponding 95% confidence interval (CI) is reported. AUC of 0.8 or above indicates a high level of predictive power, while an AUC of 0.6 or less indicates poor level of predictive power. constructed utilizing forward selection, consid­ering univariate predictors of level p < 0.05. The results of the variable selection procedure were confirmed using backward selection based on the Akaike Information Criterion (AIC). Since the hazard ratio depends on the unit of the measure­ment, all radiomics features were normalized into z-scores.35 Probability of OS as a function of time was analysed with Kaplan-Meier diagrams, and the difference between survival curves was tested with the log-rank test. iRADIOMICS, iRECIST, and PD-L1 signatures were constructed using univariate or multivariate logistic regression analyses. The iRADIOMICS sig­natures consisted of the most promising radiomics features. The iRECIST signature consisted of one categorical variable with five ordered iRECIST re­sponse categories.5 The predictive power of each model was assessed by calculating the area under the curve (AUC) of the corresponding ROC anal­ysis. The accuracy of each model (percentage of correctly classified patients) was assessed with re­peated (10×) 5-fold cross validation, so that the pa­tients were randomly split into five groups: at each validation step, four unique groups were chosen to train the model and the remaining group was used to validate accuracy of model predictions. A planned sample size of 30 evaluable patients was deemed to be sufficient for evaluating the pre­dictive power of each model. Specifically, assum­ing an anticipated response rate of 50%, a sample size of 30 evaluable patients provided >85% power to detect an AUC of at least 0.80 (high predictive power) at the two-sided 0.05 significance level un­der the null hypothesis that the AUC is at most 0.5. All analyses were performed in R (3.5.3.) and were considered statistically significant if p < 0.05. Results Patient demographic and clinical data Thirty patients were enrolled in the study. Median follow-up time (time to censoring) was 21.4 months. A full list of demographic characteristics is presented in Table 1. The examination of demo­graphic data did not reveal any significant differ­ences between the responders and non-responders. Individual radiomics features as predictors of overall survival (OS) We analysed radiomics features extracted from primary tumours at baseline, month 1, and month 289 TABLE 1. Patient demographic and clinical data. The data is presented for all patients, responders (overall survival [OS] > 14.9 months), and non-responders (OS < 14.9 months). The reported p-value is the result of Mann-Whitney U-test (MWU) (continuous variables) and Fisher exact test (categorical variables) comparing differences between responders and non-responders Number of patients 30 16 14 Age [years] 65 (46–77) 67 (48–76) 61 (46–77) 0.298 Female 15 9 6 Male 157 8 Adenocarcinoma 17 8 9 Squamous cell carcinoma 8 4 4 Other 5 4 1 Never 1 0 1 Former > 3 years ago 12 7 5 Former < 3 years ago 5 3 2 Until current disease 8 3 5 Current smoker 4 3 1 0 8 2 6 1 18 12 6 2 4 2 2 1st 15 10 5 2nd 13 4 9 3rd 2 2 0 No 241212 Yes 642 ECOG PS = Eastern Cooperative Oncology Group performance status; RT = radiotherapy; TPS = tumour proportion score (TPS) 4. Two patients did not have primary tumours, excluding them from this analysis (N = 28). The analysis of the features extracted at baseline is pre­sented in Table 2 and Figure 1. Neither standard volume-based features (volume, SUVmax) were able to discriminate responders from non-responders. Among the texture-based features, Entropy-GLCM (p = 0.046) and Small Run Emphasis (SRE) (p = 0.001) were found to be significantly different between the two groups. ROC curve analysis re­vealed SRE having high level of predictive power (AUC = 0.85 (95% CI 0.69–1.00)), while the predic­tive power of other features was moderate (0.6 < AUC < 0.8). At month 1, only volume was significantly dif­ferent between the responders and non-responders (p = 0.035, AUC = 0.75 (0.55-0.95)), while none of the radiomics features reached high level of pre­dictive power (AUC < 0.8). At month 4, none of the features were significantly different between responders and non-responders, and all radiomics features had AUC < 0.7. To further explore the impact of baseline radiom­ics features on OS, we performed Cox proportional 290 TABLE 2. Baseline radiomics features of primary tumours – Mann-Whitney U-test (MWU) and receiver operating characteristic (ROC) curve analysis. Patients were dichotomized into 2 groups: responders (OS > 14.9 months) and non-responders (OS < 14.9 months). For each radiomics feature median value, range, p-value of MWU, and the area under the ROC curve (AUC) with the corresponding 95% confidence interval (CI), are reported. See also Figure 1 Volume [cm3] 27.9 (2.64–351) 44.4 (7.81–792) 0.098 0.69 (0.49–0.89) SUVmax [g/ml] 20.6 (5.21–32.1) 15.6 (9.54–37.0) 0.185 0.65 (0.43–0.87) Sum entropy 3.69 (3.53–3.77) 3.7 (3.54–3.76) 0.387 0.60 (0.38–0.82) Entropy-GLCM 4.07 (3.99–4.15) 4.11 (4.03–4.14) 0.046 0.72 (0.52–0.92) Difference entropy 2.98 (2.74–3.07) 2.89 (2.74–3.06) 0.080 0.70 (0.49–0.90) Small Run Emphasis (SRE) 0.0382 (0.00962–0.0615) 0.0163 (0.00854–0.0303) 0.001 0.85 (0.69–1.00) GLCM = Grey-Level Co-occurrence Matrix; SUVmax = maximum standardized uptake value TABLE 3. Baseline radiomics features of primary tumours – univariate and multivariate Cox proportional hazards regression analysis (Cox PH). For each radiomics feature, the hazard ratio (HR), corresponding 95% confidence interval (CI), and p-value of univariate analysis are reported. The 2-variable multivariate regression model was chosen based on the Akaike information criterion (AIC). In order to achieve comparable HRs, all radiomics features were normalized into z-scores Volume 1.6 (1.1–2.4) 0.015 SUVmax 0.77 (0.46–1.3) 0.320 Sum Entropy 0.96 (0.60–1.5) 0.860 Entropy-GLCM 1.4 (0.82–2.3) 0.230 Difference entropy 0.62 (0.40–0.97) 0.037 0.54 (0.31–0.93) 0.026 Small Run Emphasis (SRE) 0.46 (0.26–0.81) 0.007 0.39 (0.20–0.76) 0.006 GLCM = Grey-Level Co-occurrence Matrix; SUVmax = maximum standardized uptake value hazards (Cox PH) regression analysis (Table 3). In univariate analysis, volume (hazard ratio (HR) = 1.6, p = 0.015), Difference Entropy (HR = 0.62, p = 0.037), and SRE (HR = 0.46, p = 0.007) showed sta­tistically significant relationship with patient OS. Multivariate Cox PH regression model with the lowest AIC consisted of Difference Entropy (HR = 0.54, p = 0.026) and SRE (HR = 0.39, p = 0.006). As shown in Figure S1, SRE and Difference Entropy also exhibited low correlation (. = 0.20), confirming that these two features were independent predic­tors of survival. For the feature SRE, which was found to be the most informative in all statistical tests, we per­formed Kaplan-Meier survival analysis for base­line SRE where patients were dichotomized by the median (Figure 2). Survival probability was signifi­cantly different between groups (p = 0.015). Median OS of the patients with SRE < SREmedian was 10.4 months (95% CI 6.0 months–not reached), while median OS of the patients with SRE = SREmedian was not reached (95% CI 15.9 months–not reached). Ability of iRADIOMICS, iRECIST, and PD-L1 signatures to predict patient overall survival Finally, we examined the predictive power of iRA-DIOMICS (baseline), iRECIST (month 1 and 4), and PD-L1 (baseline) signatures. 25 patients, which had both baseline and month 1 scans available, were suitable for this analysis. Two patients were excluded because they had no primary tumours (impossible to extract iRADIOMICS), and three other patients had no month 1 scans (impossible to assess iRECIST). For the three additional patients, who died before the scheduled month 4 scanning, we used month 1 iRECIST assessment for the con­struction of month 4 iRECIST signature. Otherwise, the statistics of month 4 iRECIST signature could 291 FIGURE 2. Kaplan-Meier diagram – Small Run Emphasis (SRE). Blue: patients with SRE = SREmedian, yellow: patients with SRE < SREmedian. The reported p-value is the result of log-rank test. be biased due to the exclusion of hyperprogres­sive patients. The results are presented in Figure 3. PD-L1 TPS showed poor predictive power (AUC = 0.60 (0.37-0.83)). The AUC of iRECIST signatures were 0.79 (0.62–0.95) and 0.86 (0.72–1.00) for month 1 and month 4, respectively. On the other hand, the AUC of the univariate iRADIOMICS at base­line was 0.81 (0.62–0.99), which was comparable to iRECIST at month 1. The highest predictive power was achieved by the multivariate baseline iRADI-OMICS (consisting of SRE and Difference Entropy) with AUC = 0.90 (0.78–1.00). Model coefficients of iRADIOMICS are summarized in Table S2. To further validate the predictive ability of all models, the accuracy of predictions was calculated using 5-fold cross validation. PD-L1 TPS achieved poor accuracy of only 53% (standard deviation SD = 18%). iRECIST signatures at month 1 and month 4 correctly classified 76% (16%) and 76% (17%) of patients, respectively. The accuracy of univariate iRADIOMICS at baseline was slightly lower, 73% (18%). The highest accuracy was achieved by mul­tivariate baseline iRADIOMICS, which correctly classified 78% (18%) of patients. Additionally, we performed a sensitivity study by repeating the same analyses either with a sub­set of 22 patients, who were scanned at all three time-points (excluding hyperprogressive patients who died before month 4), or by using all avail­able data at each specific time-point (resulting in different number of analysed patients at baseline, month 1 and month 4), but the change of the results was negligible. In each scenario, multivariate iRA-DIOMICS reached AUC around 0.90 with accuracy up to 80%, and always performed better than the other models. Discussion New biomarkers of response to immunotherapy are urgently needed. In NSCLC, PD-L1 TPS is still the only predictive biomarker routinely used in clinics, in spite of the growing evidence sug- 292 \ FIGURE 3. Receiver operating characteristic (ROC) curve analysis. Blue: baseline iRADIOMICS multivariate logistic model (independent variables: Small Run Emphasis [SRE], Difference Entropy), yellow: baseline iRADIOMICS univariate logistic model (independent variable: SRE), grey: month 1 iRECIST univariate logistic model (independent variable: iRECIST response category), red: month 4 iRECIST univariate logistic model (independent variable: iRECIST response category). For each model, area under curve (AUC) and 95% confidence interval (CI) are reported. gesting that it is far from optimal.36 Among the reasons for its questionable predictive power are inconsistent measurement methodologies, intra-tumour PD-L1 expression heterogeneity, and the fact that immune cells infiltrating the tumour can express PD-L1.37 Even in our study, the survival predictions based on PD-L1 TPS performed poorly. Additionally, because it is not clear to what extent the current standards for treatment response as­sessment (RECIST, iRECIST) correlate with overall survival (OS), the duration of treatment, as well as the decision about cessation of anti-PD-1 im­munotherapy, rely on the subjective judgment of the treating physician, which is mainly based on the observed immune-related adverse events and achieved clinical benefit. We aimed to address these issues with the use of [18F]FDG PET/CT imaging, since it is widely used, affordable, and non-invasive. When we examined the predictive ability of individual radiomics fea­tures, we found that some of the features showed high predictive power at baseline, while at month 1 and month 4 their informative value decreased significantly. This is consistent with a number of studies suggesting that intrinsic tumour charac­teristics, such as tumour histopathology, tumour microenvironment, and immune contexture, most likely have a major impact on response to immu­notherapy.16,17,38 The most dominant feature was Small Run Emphasis (SRE), which was able to discriminate responders from non-responders to anti-PD-1 therapy, it had a significant relation­ship with patient OS, and high predictive power. In patients with SRE > SREmedian, the probability of survival by Kaplan-Meier analysis was also sig­nificantly higher. Although studies have shown that texture-based features might reflect tumour heterogeneity on macroscopic, cellular, or even molecular or genomic level39, their clear relation­ship with the underlying biology still needs to be elucidated. However, from the definitions of tex­ture features used in our study we can infer that at baseline, primary tumours of responders have fin­er and more homogeneous metabolic structure, as reflected by higher SRE and lower Entropy-GLCM, respectively. See Table S1 for formal mathematical definitions, as well as intuitive descriptions of the studied texture features. In terms of underlying bi­ology we could speculate that these findings might reflect tumours with spatially more homogene­ous clonal structure, more homogeneous intrinsic infiltration of immune cells, more homogeneous tumour microenvironment, or fewer hypoxic or necrotic regions. Interestingly, this finding is in agreement with the study by Polverari et al., where patients with progressive disease (PD) exhibited higher tumour heterogeneity at baseline (reflected by higher kurtosis and skewness), compared to non-PD patients. On the other hand, the finding is at odds with the study by Mu et al., where hetero­geneous tumours presumably had a higher chance to achieve durable clinical benefit.27 However, het­erogeneous tumour phenotype in this study was inferred from two components of eight-variable radiomics signature, making intuitive conclusions about the underlying tumour biology even more difficult compared to our study. In agreement with the study by Takada et al., we also observed the trend of higher SUVmax among the responding pa­tients, although it was not statistically significant.20 A similar lack of statistical significance of SUVmax, or even the opposite trend, was observed by other groups, therefore the predicitve value of SUVmax should be considered highly questionable.13,19,21 We analysed only primary tumours, yet ne­glected lymph nodes (LN) and distant metastases (DM). The main reason for this approach is that radiomics analyses might not accurately quantify intra-tumour heterogeneity of small lesions due 293 to the partial volume effects, which could be even more pronounced in PET imaging with limited spatial resolution.40 However, inclusion of LN and DM in future predictive models could addition­ally improve their predictive power and accuracy. Especially an [18F]FDG PET signal of LN might be connected with the cancer immunity cycle, possi­bly capturing the processes that occur in LN after the initiation of anti-PD-1 therapy, including T cell priming and activation.41 The analysis of the predictive ability of iRECIST, PD-L1, and iRADIOMICS signatures revealed some interesting aspects. First, the response to anti-PD-1 therapy seems to occur fast, as iRECIST signature was able to predict the response of 76% of patients already at month 1, while the predictive ability at month 4 had not improved. These results suggest that treatment response assessment could be performed as soon as 1 month after treatment initiation. Moreover, its satisfactory ability to pre­dict OS indicates that clinical decisions about (dis) continuation of anti-PD-1 therapy could (at least in part) rely on iRECIST assessment rather than purely on the observed clinical benefit. However, the correlation of other iRECIST-based endpoints with patient survival should be further explored. Lastly, the iRADIOMICS was found superior to PD-L1 and iRECIST both in terms of predictive power and, importantly, timing. From the clinical point of view, each additional month (or day) of an ineffective therapy can be crucial for metastatic NSCLC patients. The fact that the iRADIOMICS was able to correctly predict the response of al­most 80% of patients before therapy, could have an important clinical impact. The predicted non-re­sponders to pembrolizumab could be offered other treatment options to improve their OS. However, the predictive ability of iRADIOMICS needs to be confirmed in future independent studies with a higher number of patients. Our study compared the predictive power of baseline biomarkers (iRADIOMICS and PD-L1) to the early treatment response assessment method (iRECIST) – single point vs. multiple point assess­ment. However, from the practical standpoint, the baseline prediction is desirable to the treatment response assessment as it is earlier and allows more time for favourable clinical decision making. Potentially the two approaches could be combined, but such study would require higher number of patients to secure clinical significance because of more degrees of freedom (variables). Acknowledgments The authors acknowledge the financial support from the Slovenian Research Agency (research core funding P1-0389), and the University of Wisconsin Carbone Cancer Center (support grant P30 CA014520). Trial registration: ClinicalTrials.gov NCT04007068. 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Comput Graph Image Process 1975; 4: 172-9. doi: 10.1016/s0146-664x(75)80008-6 295 research article Improvement of the primary efficacy of microwave ablation of malignant liver tumors by using a robotic navigation system Jan Schaible1, Benedikt Pregler1, Niklas Verloh1, Ingo Einspieler1, Wolf Bäumler1, Florian Zeman2, Andreas Schreyer3, Christian Stroszczynski1, Lukas Beyer1 1 Department of Radiology, University Medical Center Regensburg, Regensburg, Germany 2 Center for Clinical Studies, University Medical Center Regensburg, Regensburg, Germany 3 Department of Radiology, Hospital Brandenburg, Brandenburg, Germany Radiol Oncol 2020; 54(3): 295-300. Received 17 February 2020 Accepted 3 May 2020 Correspondence to: Dr. Jan Schaible, Department of Radiology, University Hospital Regensburg, 93053 Regensburg, Germany. E-mail: jan.schaible@ukr.de Disclosure: No potential conflicts of interest were disclosed. Background. The aim of the study was to assess the primary efficacy of robot-assisted microwave ablation and compare it to manually guided microwave ablation for percutaneous ablation of liver malignancies. Patients and methods. We performed a retrospective single center evaluation of microwave ablations of 368 liver tumors in 192 patients (36 female, 156 male, mean age 63 years). One hundred and nineteen ablations were performed between 08/2011 and 03/2014 with manual guidance, whereas 249 ablations were performed between 04/2014 and 11/2018 using robotic guidance. A 6-week follow-up (ultrasound, computed tomography and magnetic resonance imaging) was performed on all patients. Results. The primary technique efficacy outcome of the group treated by robotic guidance was significantly higher than that of the manually guided group (88% vs. 76%; p = 0.013). Multiple logistic regression analysis indicated that a small tumor size (= 3 cm) and robotic guidance were significant favorable prognostic factors for complete ablation. Conclusions. In addition to a small tumor size, robotic navigation was a major positive prognostic factor for primary technique efficacy. Key words: interventional radiology; robotic assistance; microwave ablation; liver tumor Introduction Local ablation therapy has been established as a suitable alternative to resection for the treatment of tumors in the liver, lung, kidney and bone. It is considered a curative treatment for hepatocellular carcinoma (HCC) and can prolong the survival of patients with unresectable colorectal liver metasta­ses.1,2 In recent years, microwave ablation (MWA), which is a thermal ablation method, has increas­ingly been used as an alternative to radiofrequen­cy ablation (RFA). Although there are only a few studies that have compared MWA and RFA, MWA seems to have an advantage for the treatment of large tumors and tumors near vessels owing to its higher energy output.3-5 Although the local recur­rence rate has decreased owing to technological advances such as the development of multi-appli­cator systems for MWA and increasing application experience, surgical resection still seems superior with respect to local tumor control.6-9 To achieve optimal therapy results with the best possible local tumor control, it is extremely impor­tant to obtain complete ablation while maintain­ing a sufficient safety distance.10 Although there 296 are still no uniform guidelines for the minimum safety margin (distance between treated tumor and ablation margin), most operators assume a safety margin of approximately 0.5–1 cm.11-13 To achieve this, the microwave applicator (antenna) must be positioned with millimeter precision, which can be very challenging, especially in the case of several overlapping ablation areas. In addition to the com­mon freehand placement, modern navigation sys­tems have been introduced to allow 3D planning and precise antenna placement.14,15 Unfortunately, there are only a few studies of the use of navigation systems, and often only with a small number of patients. Although it has been shown that modern navigation systems en­able very accurate antenna placement, it is not yet clear whether this also leads to improved primary efficacy of the technique.14,16,17 Therefore, the aim of this study was to compare the primary efficacy of robotic-guided ablation with that of manually guided ablation, as evidenced by magnetic reso­nance imaging (MRI) follow-up after 6 weeks. Patients and methods Study design and participant selection The indications for percutaneous tumor ablation were established by a multi-disciplinary tumor board. The following exclusion criteria were ap­plied: coagulation disorders not amendable to sub­stitution; portal vein, hepatic vein or inferior vena cava invasion; extrahepatic metastases; and mul­tifocal hepatic disease not amenable to complete ablation. A total of 192 patients underwent either free­hand or robotic guided microwave ablation from 08/2011 to 11/2018, inclusive. All procedures were performed by the same three experienced interven­tional radiologists (blinded). Ethical approval This single-center retrospective observational study was approved by the local ethics commit­tee. All procedures performed in studies involv­ing human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments and the guidelines for Good Clinical Practice from the International Conference on Harmonization. Informed consent was obtained from all individual participants included in the study. Navigation system and thermoablation procedure All microwave ablations were performed under general anesthesia. During CT scans and anten­na positioning, control of the respiratory move­ment was performed by temporary tube discon­nection. Arterial and portal venous helical CT scans (Somatom 16 or Definition Egde, Siemens Healthcare, Forchheim, Germany) with a slice thickness of 1 mm were acquired. CT fluoroscopy was used for ablations without navigation support, an acquisition mode that al­lows continuous image update using in-room table control. After the initial 2-phase planning CT, the antenna was placed during repeated temporary breath holds. To verify the correct antenna place­ment, one unenhanced CT was obtained before starting the ablation. If necessary, the antenna was repositioned until the whole tumor volume was covered. When using robot navigation, the initial CT data was sent to the navigation system (Maxio, Perfint Healthcare, Chennai, India).18,19 The desired abla­tion area and the antenna entry point were defined using the planning software, and the trajectory was visualized. If necessary, multiple antenna positions were planned with overlapping ablation zones. After approval of the plan, the robotic arm was automatically positioned over the patient and the antenna was positioned using the targeting device during breath hold. The probes were pushed for­ward manually along the preplanned path while held by the robotic needle holder. Before ablation, a CT scan was performed and the antenna posi­tion was verified by overlaying it with the planned trajectory. Consistent docking and absolute regis­tration of the robotic device was performed using a base plate fixed on the ground. The navigation system is connected to the local PACS as a DICOM node. The images are automatically pushed to the navigation system by an auto transfer task after successful reconstruction of the 1mm images in the CT scanner. For ablation, either the Acculis Microwave Tissue Ablation (MTA) System (AngioDynamics, Latham, NY, USA; Accu2i pMTA Applicator 1.8 mm diameter in 14 or 19 cm length) or the Emprint Ablation System (Medtronic, Minneapolis, USA; Emprint™ Percutaneous Antennas 1.8 mm diam­eter in 15 or 20 cm length) was used, depending on tumor configuration and relationship to the sur­rounding tissue. By comparison of the expected ablation zone in the unenhanced scan (typically 297 TABLE 1. Patient characteristics 1583 9 (n = 192) (23.56) (57.75, 72.00) (81) (19) (1.00, 2.00) (72) (15) (4) (9) CCC = cholangiocellular carcinoma; CRC = colorectal liver metastasis; HCC = hepatocellular carcinoma; IQR = interquartile range; SD = standard deviation TABLE 2. Tumors treated using freehand and robotic guidance Freehand 19.79 1 101616 9 131715226554 9 28 4 85 (n = 119) (12.42) (1) (8) (13) (13) (8) (11) (14) (13) (18) (55) (45) (76) (24) Robotic 18.78 6 24 20 26 16 38 36 36 47 143106 219 30 guidance 3 64 (10.78) (2) (10) (8) (10) (6) (15) (14) (14) (19) (57) (43) (88) (12) (n = 249) SD = standard deviation hypodense) to the initial tumor in the planning scan. If there was suspicion of insufficient ablation margin repositioning was performed. After ablation and track ablation, all patients underwent a noncontrast multislice CT scan of the liver to detect complications. Imaging follow-up All patients underwent our standard follow-up scheme after 6 weeks including CT, MRI with hepatospecific contrast agent and ultrasound. Further follow-up investigations were only carried out using MRI and ultrasound for radiation protec­tion. The radiographic adjudication/visual assess­ment of the complete success of the ablation was retrospectively determined in consensus by two experienced radiologists (blinded). The primary technique efficacy was defined as the percentage of the target tumors that were successfully eradicated following the initial procedure as evidenced in the 6-week follow up according to the standardization of terminology by Ahmed et al. 20 Statistical analysis R 3.51 was used to perform all statistical calcula­tions. A p-value of p = 0.05 was considered the cut­off point of statistical significance. For multivari­ate analysis of primary efficacy using nested data (multiple ablations per patient in some cases), we applied generalized estimation equations (GEEs). Results Patient characteristics A total of 192 patients (156 male) were included in the study (Table 1). The median age was 64 years (range: 57–72). In total, 264 ablation sessions were performed with a median number of treatment sessions per patient of 1 (range: 1–4). 137 patients required one session, 41 patients required two ses­sions and the remaining patients required three or more sessions. The median number of tumors treated per patient was 1 (range: 1–9). Tumor characteristics A total of 368 tumors spread across all liver seg­ments were treated using MWA and either robot-ic-assistance or CT fluoroscopy (Table 2). The two most frequent tumor entities were hepatocellular carcinoma (n = 271) and liver metastasis of colorec­tal carcinoma (n = 54), followed by cholangiocellu­lar carcinoma (n = 18). The median tumor size was 16 mm, with 59 tumors larger than 30 mm. Primary technique efficacy and prognostic factors The primary efficacy rate using robotic guidance was 88%, i.e., 219 of the 249 tumors were covered completely by the ablation volume. Needle reposi­tioning was necessary in 92 of 249 ablations (37%). In contrast, the primary efficacy rate for freehand 298 TABLE 3. Influence of tumor characteristics on primary efficacy Adverse events 141 (80.11%) of the robotic-guided and 62 (70.45%) of the CT-fluoroscopy-guided procedures were = 30 Reference Long axis, mm performed without any adverse events. Grade I > 30 - 0.8717 0.3657 5.68 0.0171 (mild), II (moderate) and III (severe) adverse events Freehand Reference occurred in 9 (5.11%), 6 (3.41%) and 1 (0.57%) of Guidance Robotic 0.8064 0.3256 6.13 0.0133 the robotic-guided procedures, respectively, and 3 HCC Reference (3.41%), 3 (3.41%) and 1 (1.14%) of the freehand-guided procedures, respectively. CRC 0.2922 0.4483 0.42 0.5145 Tumor entity Grade IV (life-threatening) adverse events oc- CCC 0.0665 0.6524 0.01 0.9188 curred in 1 (0.57%) of the robotic-guided proce- Other 0.0832 0.5228 0.03 0.8736 dures and 2 (2.27%) of the freehand-guided pro- I Reference cedures. The patient in the robotic-guided group II - 0.3956 1.0798 0.13 0.7141 suffered an injury to the 10th intercostal artery during ablation, which led to persistent bleeding III - 0.0449 1.1025 0.00 0.9675 and had to be treated with embolization. One of IVa 0.2688 1.1180 0.06 0.8100 the patients in the freehand group, who had previ- Liver segment IVb - 0.2539 1.1597 0.05 0.8267 ously undergone partial liver resection and a con-V 0.5961 1.1285 0.28 0.5974 secutive Chilaiditi situation, had a perforation of a VI 0.9036 1.1736 0.59 0.4413 prolapsed intestinal loop that had to be surgically VII 0.0656 1.1090 0.00 0.9528 overstitched. The other patient in the freehand group suffered from bleeding from the 7th and 8th VIII - 0.1069 1.0929 0.01 0.9221 intercostal artery after ablation, which had to be CCC = cholangiocellular carcinoma; CRC = colorectal liver metastasis; HCC = hepatocellular closed by embolization. carcinoma; Wald = .2 test for the coefficients Treatment-related patient death (Grade V) oc­ curred in 1 (0.57%) of the robotic-guided proce­ dures and 0 (0.00%) of the freehand guided pro- TABLE 4. Monte Carlo simulation of primary technique efficacy cedures. A patient that had a previous liver and rate depending on tumor size and antenna guidance kidney transplant developed severe cholangitis two days after ablation and subsequent liver and kidney failure with lactate acidosis, which could = 30 Freehand 0.60 0.75 0.86 not be controlled despite ultima ratio crush hepa­tectomy. > 30 Freehand 0.34 0.55 0.76 There was no significant difference in the fre- = 30 Robotic 0.78 0.87 0.92 quency of adverse events (p = 0.07) between the > 30 Robotic 0.55 0.74 0.87 two groups. CI-2.5% and CI-97.5% = the central interval bounds at the lower 2.5 and upper 97.5 percentiles, respectively; Median = the simulated distribution’s median Discussion ablation was 76% (91 of 119 tumors). Logistic re-In recent years, the importance of local ablative gression was performed to investigate whether tu-procedures for the treatment of liver tumors has mor characteristics (size, entity and location) and steadily increased. It is well-known that an initial the type of guidance (robotic or freehand) can im-complete response is associated with improved pact primary technique efficacy (Table 3). survival from hepatocellular carcinoma and colo- Compared with tumor size = 3 cm, tumor size > rectal liver metastasis.10,21 Therefore, the exact 3 cm was a significantly unfavorable prognostica-placement of the antenna is critically important to tor of primary technique efficacy (odds ratio 0.42; achieve complete ablation with a sufficient safety p = 0.02). Compared with freehand antenna place-margin. ment, robotic guidance was a significant favorable Navigation procedures are increasingly used to prognostic factor (odds ratio 2.24; p = 0.01). Table 4 assist with accurate antenna placement. We have shows estimations of the primary technique effi-also switched from manual guidance to navigation cacy for robotic and freehand guidance. in almost all cases. Only in very few cases (tumor 299 right below diaphragm or right next to stomach) we switched to manual placement for better con­trol. Although a very high accuracy of the robot-supported placement has already been shown14,18 until now, it has not been clear whether this im­proves the primary efficacy, i.e., the percentage of target tumors successful eradicated. Studies have shown that the robotic-guided approach improves the accuracy of targeting the tumor, reduces patient radiation dose and in­creases procedural performance when compared with conventional non-navigated antenna place­ment.14,22-24 Other studies claim that there is no sta­tistically significant reduction in the dose between the robotic-assisted and conventional method.25 In one of our earlier studies, we showed that robotic assistance for liver tumor ablation reduces the pa­tient radiation dose and allows a fast positioning of the microwave applicator with high accuracy.18 Due to the small number of patients (n = 46) we could not show any significant difference in the primary efficacy rate. In one of our previous stud­ies, we were able to show that additional overhead does not save time in the case of only one tumor, and that savings can only be expected in complex procedures.26 Although these previous studies have shown that antenna placement is highly accurate when us­ing a robotic-guided navigation system, the impact of higher accuracy on the technical efficacy has not been investigated. In this study, we show for the first time in a large patient population (249 tu­mors ablated using robotic assistance) that robotic guidance is associated with a significantly higher technical success rate (primary efficacy rate using robotic guidance was 88%, primary efficacy rate for freehand ablation was 76%). From our point of view, this difference is very remarkable, because we had many years of expertise in manual guid­ance and still managed to achieve this improve­ment with the new type of navigation. Although the large patient population indicates a high significance, some limitations have to be dis­cussed. One aspect that needs to be considered is that interindividual differences could play a role. However, from our point of view, the high experi­ence and the large number of ablations of each in-terventionalist speak against great interindividual differences. In addition, the learning curve also plays a role, which undoubtedly occurs over time, as Beermann et al. also stated.27 In summary, our study was the first to show that robotic-guided antenna placement goes hand in hand with a higher primary efficacy. References 1. Kokudo N, Hasegawa K, Akahane M, Igaki H, Izumi N, Ichida T, et al. Evidence-based clinical practice guidelines for hepatocellular carcinoma: The Japan Society of Hepatology 2013 update (3rd JSH-HCC Guidelines). Hepatol Res 2015; 45: n/a-n/a. doi: 10.1111/hepr.12464 2. Ruers T, Van Coevorden F, Punt CJ, Pierie JE, Borel-Rinkes I, Ledermann JA, et al. Local treatment of unresectable colorectal liver metastases: results of a randomized Phase II trial. JNCI J Natl Cancer Inst 2017; 109: 9. doi: 10.1093/ jnci/djx015 3. Facciorusso A, Di Maso M, Muscatiello N. Microwave ablation versus radiofrequency ablation for the treatment of hepatocellular carcinoma: a systematic review and meta-analysis. Int J Hyperth 2016; 32: 339-44. doi: 10.3109/02656736.2015.1127434 4. Vogl TJ, Farshid P, Naguib NN, Zangos S, Bodelle B, Paul J, et al. Ablation therapy of hepatocellular carcinoma: a comparative study between radiof­requency and microwave ablation. Abdom Imaging 2015; 40: 1829-37. doi: 10.1007/s00261-015-0355-6 5. Abdelaziz AO, Nabeel MM, Elbaz TM, Shousha HI, Hassan EM, Mahmoud SH, et al. Microwave ablation versus transarterial chemoembolization in large hepatocellular carcinoma: prospective analysis. Scand J Gastroenterol 2015; 50: 479-84. doi: 10.3109/00365521.2014.1003397 6. Chu KF, Dupuy DE. Thermal ablation of tumours: biological mechanisms and advances in therapy. Nat Rev Cancer 2014; 14: 199-208. doi: 10.1038/ nrc3672 7. van Amerongen MJ, Jenniskens SFM, van den Boezem PB, Fterer JJ, de Wilt JHW. Radiofrequency ablation compared with surgical resection for curative treatment of patients with colorectal liver metastases – a meta-analysis. HPB 2017; 19: 749-56. doi: 10.1016/j.hpb.2017.05.011 8. Wang Y, Luo Q, Li Y, Deng S, Wei S, Li X. Radiofrequency ablation versus hepatic resection for small hepatocellular carcinomas: a meta-analysis of randomized and nonrandomized controlled trials. PLoS One 2014; 9: e84484. doi: 10.1371/journal.pone.0084484 9. Xu Q, Kobayashi S, Ye X, Meng X. Comparison of hepatic resection and radi­ofrequency ablation for small hepatocellular carcinoma: a meta-analysis of 16,103 patients. Sci Rep 2015; 4: 7252. doi: 10.1038/srep07252 10. Sala M, Llovet JM, Vilana R, Bianchi L, Solé M, Ayuso C, et al. Initial response to percutaneous ablation predicts survival in patients with hepatocellular carcinoma. Hepatology 2004; 40: 1352-60. doi: 10.1002/hep.20465 11. Kurilova I, Gonzalez-Aguirre A, Beets-Tan RG, Erinjeri J, Petre EN, Gonen M, et al. Microwave ablation in the management of colorectal cancer pulmonary metastases. Cardiovasc Intervent Radiol 2018; 41: 1530-44. doi: 10.1007/s00270-018-2000-6 12. Ke S, Ding XM, Qian XJ, Zhou YM, Cao BX, Gao K, et al. Radiofrequency abla­tion of hepatocellular carcinoma sized > 3 and = 5 cm: is ablative margin of more than 1 cm justified? World J Gastroenterol 2013; 19: 7389-98. doi: 10.3748/wjg.v19.i42.7389 13. Schaible J, Pregler B, Baler W, Einspieler I, Jung EM, Stroszczynski C, et al. Safety margin assessment after microwave ablation of liver tumors: inter-and intrareader variability. Radiol Oncol 2020; 54(3): 295-300.; 54: 57-61. doi: 10.2478/raon-2020-0004 14. Mbalisike EC, Vogl TJ, Zangos S, Eichler K, Balakrishnan P, Paul J. Image-guided microwave thermoablation of hepatic tumours using novel robotic guidance: an early experience. Eur Radiol 2015; 25: 454-62. doi: 10.1007/ s00330-014-3398-0 15. Bale R, Widmann G, Schullian P, Haidu M, Pall G, Klaus A, et al. Percutaneous stereotactic radiofrequency ablation of colorectal liver metastases. Eur Radiol 2012; 22: 930-37. doi: 10.1007/s00330-011-2314-0 16. Beyer LP, Michalik K, Niessen C, Platz Batista da Silva N, Wiesinger I, Stroszczynski C, et al. Evaluation of a robotic assistance-system for per­cutaneous computed tomography-guided (CT-guided) facet joint injec­tion: a phantom study. Med Sci Monit 2016; 22: 3334-9. doi: 10.12659/ MSM.900686 17. Solomon SB, Patriciu A, Bohlman ME, Kavoussi LR, Stoianovici D. Robotically driven interventions: a method of using CT fluoroscopy without radiation exposure to the physician. Radiology 2002; 225: 277-82. doi: 10.1148/ radiol.2251011133 300 18. Beyer LP, Pregler B, Niessen C, Dollinger M, Graf BM, Mler M, et al. Robot-assisted microwave thermoablation of liver tumors: a single-center experience. Int J Comput Assist Radiol Surg 2016; 11: 253-9. doi: 10.1007/ s11548-015-1286-y 19. Beyer LP, Pregler B, Michalik K, Niessen C, Dollinger M, Mler M, et al. Evaluation of a robotic system for irreversible electroporation (IRE) of ma­lignant liver tumors: initial results. Int J Comput Assist Radiol Surg 2017; 12: 803-9. doi: 10.1007/s11548-016-1485-1 20. Ahmed M, Solbiati L, Brace CL, Breen DJ, Callstrom MR, Charboneau JW, et al Image-guided tumor ablation: standardization of terminology and report­ing criteria - 10-year update. Radiology 2014; 273: 241-60. doi: 10.1148/ radiol.14132958 21. Solbiati L, Ahmed M, Cova L, Ierace T, Brioschi M, Goldberg SN. Small liver colorectal metastases treated with percutaneous radiofrequency ablation: local response rate and long-term survival with up to 10-year follow-up. Radiology 2012; 265: 958-68. doi: 10.1148/radiol.12111851 22. Koethe Y, Xu S, Velusamy G, Wood BJ, Venkatesan AM. Accuracy and ef­ficacy of percutaneous biopsy and ablation using robotic assistance under computed tomography guidance: a phantom study. Eur Radiol 2014; 24: 723-30. doi: 10.1007/s00330-013-3056-y 23. Hiraki T, Matsuno T, Kamegawa T, Komaki T, Sakurai J, Matsuura R, et al. Robotic insertion of various ablation needles under computed tomography guidance: accuracy in animal experiments. Eur J Radiol 2018; 105: 162-7. doi: 10.1016/j.ejrad.2018.06.006 24. Heerink WJ, Ruiter SJS, Pennings JP, Lansdorp B, Vliegenthart R, Oudkerk M, et al. Robotic versus freehand needle positioning in CT-guided ablation of liver tumors: a randomized controlled trial. Radiology 2019; 290: 826-32. doi: 10.1148/radiol.2018181698 25. Abdullah BJJ, Yeong CH, Goh KL, Yoong BK, Ho GF, Yim CCW, et al. Robotic-assisted thermal ablation of liver tumours. Eur Radiol 2015; 25: 246-57. doi: 10.1007/s00330-014-3391-7 26. Beyer LP, Lken L, Verloh N, Haimerl M, Michalik K, Schaible J, et al. Stereotactically navigated percutaneous microwave ablation (MWA) com­pared to conventional MWA: a matched pair analysis. Int J Comput Assist Radiol Surg 2018; 13: 1991-7. doi: 10.1007/s11548-018-1778-7 27. Beermann M, Lindeberg J, Engstrand J, Galmén K, Karlgren S, Stillstr D, et al. 1000 consecutive ablation sessions in the era of computer assisted image guidance – lessons learned. Eur J Radiol Open 2019; 6: 1-8. doi: 10.1016/j. ejro.2018.11.002 301 research article Simplified perfusion fraction from diffusion-weighted imaging in preoperative prediction of IDH1 mutation in WHO grade II–III gliomas: comparison with dynamic contrast-enhanced and intravoxel incoherent motion MRI Xiaoqing Wang1, Mengqiu Cao1, Hongjin Chen2, Jianwei Ge2, Shiteng Suo1, Yan Zhou1 1 Department of Radiology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China, 2 Department of Neurosurgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China Radiol Oncol 2020; 54(3): 301-310. Received 23 March 2020 Accepted 13 May 2020 Correspondence to: Yan Zhou, Department of Radiology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Pudong New District, Shanghai, China. E-mail: clare1475@hotmail.com Disclosure: No potential conflicts of interest were disclosed. Background. Effect of isocitr ate dehydrogenase 1 (IDH1) mutation in neovascularization might be linked with tissue perfusion in gliomas. At present, the need of injection of contrast agent and the increasing scanning time limit the ap­plication of perfusion techniques. We used a simplified intravoxel incoherent motion (IVIM)-derived perfusion fraction (SPF) calculated from diffusion-weighted imaging (DWI) using only three b-values to quantitatively assess IDH1-linked tissue perfusion changes in WHO grade II-III gliomas (LGGs). Additionally, by comparing accuracy with dynamic con-trast-enhanced (DCE) and full IVIM MRI, we tried to find the optimal imaging markers to predict IDH1 mutation status. Patients and methods. Thirty patients were prospectively examined using DCE and multi-b-value DWI. All param­eters were compared between the IDH1 mutant and wild-type LGGs using the Mann–Whitney U test, including the DCE MRI-derived Ktrans, ve and vp, the conventional apparen t diffusion coefficient (ADC0,1000), IVIM-de rived perfusion fraction (f), di ffusion coefficient (D) and pseudo-diffusion coefficient (D*), SPF. We evaluated the diagnostic perfor­mance by receive r operating characteristic (ROC) analysis. Results. Significant differences were detected between WHO grade II-III gliomas for all perfusion and diffusion pa­rameters (P < 0.05). When compared to IDH1 mutant LGGs, IDH1 wild-type LGGs exhibited significantly higher perfu­sion metrics (P < 0.05) and lower diffusion metrics (P < 0.05). Among all parameters, SPF showed a higher diagnostic performance (area under the curve 0.861), with 94.4% sensitivity and 75% specificity. Conclusions. DWI, DCE and IVIM MRI may noninvasively help discriminate IDH1 mutation statuses in LGGs. Specifically, simplified DWI-derived SPF showed a superior diagnostic performance. Key words: IDH1 mutation; glioma perfusion; diffusion-weighted MRI; dynamic contrast-enhanced MRI; intravoxel incoherent motion; 2016 WHO CNS tumor classification Introduction Gliomas, the most common primary intracranial neoplasms in humans, are classified as grade I–IV based on histopathological criteria. Different from grade IV, also known as glioblastoma, the outcome of grade II-III gliomas (lower-grade gliomas, LGGs) are highly variable. Published survival duration of LGGs ranged from 1 to over 15 years, reflect­ing molecular heterogeneity of these tumors.1-5 The 2016 revised fourth edition of the World Health Organization (WHO) classification of tumors of 302 the central nervous system defines a large subset of gliomas based on molecular alterations, among which mutation of isocitrate dehydrogenase (IDH1) has shown to be the most important, for this muta­tion is thought to be a predictor of early steps in gliomag enesis. It has been shown that 70%–90% of LGGs- carry IDH1 mutations, and that IDH1 mu­tant glioma have a survival benefit associated with the maximal surgical resection, and the use of ra­diation and chemical therapy.6-8 Hence, assessing grade II and III gliomas by genetic alteration, which might be helpful for patient prognosis and clinical treatment, is now a common clinical practice. The IDH1 gene plays an important role in tu­mor angiogenesis and vasculogenesis, which have been recognized as hallmarks of histopathological growth and progression of gliomas.9-11 Therefore, preoperative assessment of tumor perfusion by MRI may give insight into the IDH1 mutation sta­tus, thus aiding in clinical decision making. Several MR perfusion techniques have been developed to evaluate the degree of tissue vascularization. Dynamic contrast-enhanced (DCE) MRI and intra-vo xel incoherent motion (IVIM) MRI are two com­mon MR perfusion techniques with distinct imag­ing mechanisms.11-15 Using rapid T1-weighted imaging to measure the changes resulting from gadolinium contrast agent leakage in and out of the extracellular ex-travascular space, DCE MRI enables the determi­nation of several hemodynamic parameters, in­cluding the vo lume transfer constant (Ktrans), the extravascular extracellular volume fraction (ve), ).11,16 and the vascular plasma volume fraction (vpPrevious studies have demonstrated the clinical potential of DCE MRI in glioma grading and dif­ferential diagnosis.17,18 However, the need for an intravenous injection of contrast agent limits its clinical application in patients with renal dysfunc­tion or individuals who are allergic to gadolinium. IVIM MRI is a variant of conventional diffusi on-weighted imaging (DWI) in that images at multi­ple b-values are required to fit the two-component mathematical model. In this model, the effect of microcirculation of blood in the capillary network (characterized by the pseudo-diffusion coefficient D*) is separated from the pure water diffusion component (characterized by the diffusion coef­ficient D). More than eight b-values are typically needed to fully characterize biexponential signal attenuation, thus increasing the scanning time. Some simplified models based on IVIM theory with fewer b-values have been proposed. Both the full and simplified IVIM models have shown their abilities in characterizing tumor perfusion and as­sessing the glioma grade.19-21 The purpose of our study, therefore, was to de­termine the association of the three b-value DWI-derived simplif ied perfusion fraction (SPF) with tumor perfusion and to compare the performance with DC E and IVIM MRI-derived parameters in the preoperative prediction of IDH1 mutation sta­tus in LGGs using surgical and histopathological findings as a standard of reference. Patient s and methods Patient enrollment This prospective single-center study was per­formed in accordance with the principle of the Declaration of Helsinki and was approved by the local ethics committee. Written informed consent was obtained from all subjects prior to study en­rollment. The flowchart of the study design is dem­onstrated in Figure 1. From April 2018 to March 2019, 55 patients who were suspected of primary brain tumors were prospectively enrolled in the study. All patients underwent initial MRI at the same unit and were then underwent neurosurgical resection at our hospital. Excluded from the study were 17 patients with pathological diagnosis other than LGGs, five patients without complete DCE MRI or IVIM data, and three patients due to poor image quality as­sociated with head movement. Finally, a total of 30 patients (13 women, 17 men; average age, 44.73 years; age range, 19–78 years) with histopatho-logically confirmed LGGs (WHO II glioma, n = 22; WHO III glioma, n = 8) were enrolled. The descrip­tive statistics are shown in Table 1. 303 MRI ac quisition protocols MRI of all patients was performed on a 3.0-T MRI unit (Signa HDxt; GE Medical Systems, Milwaukee, WI, USA) using a standard 8-channel head coil. The advanced MRI protocol included DCE MRI and DWI with 10 b-values (0–1000 s/mm2). Conventional protocol—T1- and T2-weighted im­aging with fast spin-echo sequences (T1WI, T2WI), T2 fluid-attenuated inversion recovery (FLAIR) se­quence, and contrast-enhanced T1WI— were per­formed during the same examination. Three-dimensional DCE MRI of head was per­formed after intravenous administration of a gadopentetate dimeglumine (Magnevist; Bayer Healthcare, Berlin, Germany, 0.1 mmol per kilo-gramof body weight) at a rate of 4 ml/s via a power injector (Spectris; Medrad, Pittsburgh, PA, USA). Precont rast scans with four dynamics were col­lected before gadopentetate dimeglumine was injected. The detailed parameters of the pre- and postcontrast scans were as follows: repetition time (TR)/echo time (TE), 3.3 ms/1.3 ms, flip angle, 15°; matrix, 256 × 160; field of view (FOV), 220 × 220 mm; section thickness, 2 mm; number of sections, 40; and total scanning time, 4 min. DWI was acquired before contrast injection. Ten b-values (0, 20, 50, 80, 150, 200, 300, 500, 800, and 1000 s/mm2) were applied with a fat-suppressed single-shot echo-planar sequence in three orthog­onal directions sequentially, they were averaged two times, and then trace images were generated. The other imaging parameters were: TR/TE, 3000 ms/106 ms; matrix, 192 × 192; FOV, 260 × 260 mm; section thickness/gap, 5/1 mm; number of signal averages, 2; number of sections, 15. The multi-b-value DWI was acquired at 5 min and 36 s, and if separately, 2 min and 11 s for three-b-value DWI. MR image ana lysis DCE MRI analysis Pharmacokinetic parameters (Ktrans, ve, vp) were calculated off-line by using commercially avail­able software (MIStar; Apollo Medical Imaging, Melbourne, VIC, Australia) according to the two-compartment Tofts model.22 Preprocessing for the perfusion data included semiautomatic selection of arterial input function (AIF). The AIF was obtained independently for every patient from the intrac­ranial internal carotid artery. Parametric maps of Ktrans, v e, and vp were generated on a pixel-by-pixel basis. TABLE 1. Patient characteristics Mean age (y)a 42.8 (22–67) 47.9 (19–78) Sex distribution (M/F)b 10/8 7/5 WHO grade II 15 7 III 3 5 Histologic type Astrocytoma 12 5 Oligodendroglioma 3 0 Oligoastrocytoma 0 1 Anaplastic astrocytoma 1 3 Anaplastic oligodendroglioma 1 2 Anaplastic oligoastrocytoma 1 1 * Mean (range) or count is reported; a = significant difference in age was noted between isocitrate dehydrogenase 1 (IDH1) mutant and wild-type groups (P = 0.020); b = no significant difference in sex distribution was noted between IDH1 mutant and wild-type groups (P = 0.769) F = female; M = male DWI analysi s DWI data were performed with a program in MATLAB (MATLAB 2017a; MathWorks, Natick, MA, USA) programming tool. Full IVIM features - the diffusion coefficient (D), pseudo-diffusion co­efficient (D*), and the perfusion fraction (f)—were extracted by fitting the biexponential model using all b-values as follows: Sb = S0[f exp(-bD*) + (1 - f) exp(-bD)], where Sb stands for the signal intensity in present b-value and S0 stands for the signal intensity in the absence of diffusion gradient. The monoexponential DWI model used in cal­culating the ADC value can be written as follows: ADClow,high = -ln (Slow/Shigh)/ (blow - bhigh), where Shigh is signal intensity at bhigh and Slow is sig­nal intensity at blow, respectively. As the b-value has a differential sensitivity to Brownian motion of water protons, ADC0,200 represents mixed diffusion and perfusion effects and ADC200,1000 is almost pure­ly related to diffusion.23,24 The b-value scheme was chosen following previous recommendations25-27 which indicated that the effects of diffusion and microcapillary perfusion are both reflected within low b-values (b < 200 s/mm2), while for higher b-values (b > 200 s/mm2), a large proportion of meas­ured signal in each imaging voxel was caused by tissue diffusion. When a typical b-value (1000 s/ mm2) was used, the contribution of perfusion has 304 FIGURE 2. Images obtained in a 44-year-old man with astrocytoma (isocitrate dehydrogenase 1 [IDH1] mutant glioma). (A) Fluid-attenuated inversion recovery (FLAIR) image shows a heterogeneous hyperintense lesion in the right frontal lobe. (B) Apparent diffusion coefficient (ADC)0,1000 map shows increased ADC value in the lesion. (C, D) Intravoxel incoherent motion (IVIM) perfusion fraction (f) and simplified perfusion fraction (SPF) maps show no increased values in the corresponding area of the hyperintense lesion as shown in (A). (E) On contrast-enhanced T1-weighted image, the lesion is non-enhancing. (F–H) Dynamic contrast-enhanced (DCE) MRI parametric maps of volume transfer constant (Ktrans), extravascular extracellular volume fraction (ve) and vascular plasma volume fraction (vp) show no increased values in the lesion. Regions of interest are marked on parametric maps. TABLE 2. Parameters derived from dynamic contrast-enhanced (DCE) MRI and diffusion-weighted imaging (DWI) between WHO grade II and III gliomas Ktrans (min-1) 0.067 ± 0.048 0.116 ± 0.064 0.013 ve 0.071 ± 0.057 0.401 ± 0.344 0.018 vp 0.036 ± 0.020 0.051 ± 0.018 0.035 ADC0,1000 (×10-3 mm2/s) 1.093 ± 0.203 0.904 ± 0.184 0.028 SPF (%) 10.78 ± 4.378 16.391 ± 5.471 0.012 D (×10-3 mm2/s) 1.194 ± 0.261 0.949 ± 0.169 0.021 D* (×10-3 mm2/s) 6.692 ± 1.564 8.618 ± 2.215 0.037 f (%) 3.315 ± 1.536 6.380 ± 3.419 0.020 * P-values are considered significant at P < 0.05. ADC = apparent diffusion coefficient; D = diffusion coefficient; D* = pseudo-diffusion coefficient; f = perfusion fraction; Ktrans = volume transfer constant; ve = extravascular extracellular volume fraction; vp = vascular plasma volume fraction; SPF = simplified perfusion fraction faded away entirely. The ADC thus appears to be a sensitive index of diffusion component. On the other hand, since a b-value of 1000 s/mm2 is small enough, high image quality may be guaranteed and the kurtosis effect may be avoided. As the contribution of kurtosis is greater when b-value is beyond 1000 s/mm2.28,29 Therefore, the relative pro­portion of the perfusion component in the whole diffusion pool, named SPF, can be determined as follows (20): SPF = (ADC0,200 – ADC200,1000)/ADC0,200. Region of in terest analysis The regions o f interest (ROIs) were drawn by two readers who have 6(M.C.) and 19(Y.Z.) years of ex­perience in neuroradiology, respectively, and con- 305 FIGURE 3. Images obtained in a 72-year-old woman with astrocytoma (isocitrate dehydrogenase 1 [IDH1] wildtype glioma). (A) FLAIR shows a heterogeneous hyperintense lesion in the right hemisphere. (B) Apparent diffusion coefficient (ADC)0,1000 map shows a mixed pattern of high and intermediate ADC values in the lesion. (C, D) Intravoxel incoherent motion (IVIM) perfusion fraction (f) and simplified perfusion fraction (SPF) maps show markedly increased f and SPF values in the corresponding area of the contrast-enhanced lesion as shown in (E). (E) On contrast-enhanced T1-weighted image, the lesion is vividly enhanced. (F–H) Dynamic contrast-enhanced (DCE) MRI parametric maps of volume transfer constant (Ktrans), extravascular extracellular volume fraction (ve) and vascular plasma volume fraction (vp) show obviously increased values in the corresponding area of the contrast-enhanced lesion. Regions of interest are marked on parametric maps. TABLE 3. Parameters derived from dynamic contrast-enhanced (DCE) MRI and sensus was researched. Both readers were blinded diffusion-weighted imaging (DWI) between isocitrate dehydrogenase 1 (IDH1) to the histopathological results and other clinical mutant and wild-type gliomas data, including age and gender. Following pre vi-ous studies30,31, an elliptical ROI (20–340 mm2) was placed by each doctor on parametric ma ps of the solid tumor area as much as possible to include the Ktrans (min-1) 0.054 ± 0.024 0.123 ± 0.073 0.007 portion with the minimum values of diffusion (D ve 0.052 ± 0.035 0.121 ± 0.080 0.007 and ADC0,1000) and maximum values of perfusion vp 0.032 ± 0.015 0.051 ± 0.022 0.015 (SPF, f, D*, Ktrans, vp, and ve). For correla tion analy­ ADC0,1000 (×10-3 mm2/s) 1.123 ± 0.185 0.923 ± 0.199 0.009 sis between SPF and other perfusion parameters, SPF (%) 9.572 ± 3.437 16.332 ± 4.925 < 0.001 the similar-sized ROIs used for SPF images were D (×10-3 mm2/s) 1.108 ± 0.245 0.959 ± 0.146 0.047 placed in the corresponding area of DCE images D* (×10-3 mm2/s) 6.546 ± 1.757 8.196 ± 1.794 0.020 and IVIM images. T1-weighted contrast-enhanced images where contrast agent leakage in tumors f (%) 3.080 ± 1.581 5.712 ± 2.924 0.005 was observed were used as a reference to define the * P-values are considered significant at P < 0.05 ROIs on parametric maps.32,33 The study used ADC images combined with T1-weighted, T2-weighted, ADC = apparent diffusion coefficient; D = diffusion coefficient; D* = pseudo-diffusion coefficient; Ktrans = volume transfer constant; f = perfusion fraction; SPF = simplified perfusion fraction; v = and FLAIR images to determine the ROI of tumor extravascular extracellular volume fraction; vp = vascular plasma volume fraction e FIGURE 4. Receiver operating characteristic (ROC) curves and corresponding area under the curve values for (A) diffusion-weighted imaging (DWI) parameters (simplified perfusion fraction [SPF], perfusion fraction [f], apparent diffusion coefficient [ADC]0,1000) and (B) dynamic contrast-enhanced (DCE) MRI parameters (transfer constant [Ktrans], extravascular extracellular volume fraction [ve] and vascular plasma volume fraction [vp]) in the differentiation of isocitrate dehydrogenase 1 (IDH1) mutant and wildtype gliomas. SPF showed the highest diagnostic performance with the area under the curve value of 0.86. area in nonenhancing lesion. Special care was tak-considered correlation coefficients < 0.4, 0.4–0.6, en to exclude necrosis, cys ts, hemorrhage, calcifica-0.6–0.8, and > 0.8 to indicate week, moderate, tion, and intralesional macrovessels. strong, and very strong correlation, respectively. The unpaired t-test and Mann–Whitney U test were used to determine the difference in DWI, Statistical analysis DCE and IVIM MRI parameters between WHO Statistical analysis was performed using commer-grade II and III gliomas, as well as between IDH1 cial software (SPSS version 22, IBM Corporation, mutant and wild-type gliomas, according to the Armonk, NY, USA and MedCalc, version 11.4.2.0, data normality (Kolmogorov-Smirnov test). ROC MedCalc Software, Mariakerke, Belgium). The curves were constructed to evaluate the ability to relations hip between perfusion parameters was identify different IDH1 mutation statuses. Area analyzed with Spearman rank correlation. We under the curve (AUC) values of < 0.7, 0.7–0.9, and TABLE 4. Diagnostic performance of parameters for differentiation between isocitrate dehydrogenase 1 (IDH1) mutant and wild-type gliomas Ktrans (min-1) 0.773 (0.563–0.983) 77.8 75.0 > 0.062 ve 0.760 (0.569–0.951) 94.4 58.3 > 0.119 vp 0.680 (0.451–0.909) 55.6 91.7 > 0.029 ADC0,1000 (×10-3 mm2/s) 0.718 (0.531–0.904) 83.3 75.0 = 1.002 SPF (%) 0.861 (0.686–0.959) 94.4 75.0 > 14.500 D (×10-3 mm2/s) 0.727 (0.541–0.913) 72.2 83.3 > 1.065 D* (×10-3 mm2/s) 0.690 (0.493–0.886) 44.4 91.4 = 5.959 f (%) 0.810 (0.658–0.963) 72.2 83.3 > 3.617 ADC = apparent diffusion coefficient; D = diffusion coefficient; D* = pseudo-diffusion coefficient; f = perfusion fraction;*Ktrans = volume transfer constant; SPF = simplified perfusion fraction; ve = extravascular extracellular volume fraction; vp = vascular plasma volume fraction Radiol Oncol 2020; 54(3): 301-310. 307 > 0.9 were considered to indicate low, medium, and high diagnostic performance, respectively. Differences between AUC values were analyzed by using the Delong method (34). Optimal thresh­olds were determined by maximizing the Youden index ((specificity + sensitivity) - 1). A P-value less than 0.05 was considered to indicate statistical sig­nificance. Results In terms of histology, 16 patients had astrocytomas, three had oligodendrogliomas, one had an oligoas­trocytoma, four had anaplastic astrocytomas, three had anaplastic oligodendrogliomas, and three had anaplastic oligoastrocytomas. Intercorrelation analysis between perfusion parameters revealed a significant association for SPF and f (. = 0.768, P < 0.001). The study also found a moderate correla­tion between SPF and ve (. = 0.548, P = 0.002) and between SPF and Ktrans (. = 0.535, P = 0.002). The statistical data of DCE MRI and DWI-derived parameters in differentiating WHO grade II and III gliomas are summarized in Table 2. Perfusion-related parameters including Ktrans, ve, vp, f, D *, and SPF were all significantly higher in WHO grade III gliomas than in WHO grade II gliomas (all P < 0.05), while ADC and D values were both significantly lower in WHO grade III gliomas (both P < 0.05). Representative cases of IDH1 mutant and wild-type LGGs are shown in Figures 2 and 3. The mean values ± standard deviations of DCE MRI and DWI-derived parameters for the IDH1 mutant and wild-type tumors in the whole LGGs group, are summarized in Table 3. Compared with IDH1 mutant LGGs, IDH1 wild-type LGGs exhibited sig­nificantly higher perfusion values, that is, Ktrans, ve, vp, f, D*, and SPF (all P < 0.05), and significantly lower diffusion values, that is, ADC and D (both P < 0.05). In the WHO grade II subgroup, vp and SPF differed significantly between IDH1 mutant and wild-type tumors (P = 0.018 and P = 0.049, respec­tively), whereas in the WHO grade III subgroup, only f showed a significant difference (P = 0.014). The results of ROC curve analysis are presented in Figure 4 and Table 4. For differentiation between IDH1 mutant and wild-type LGGs, the ROC curve analysis showed that among all parameters, SPF gave the highest AUC value (0.86), followed by f (0.81) and ADC (0.80), though no significant dif­ference in AUC values was found (P > 0.05). The optimal SPF threshold for IDH1 mutation discrimi­nation was 14.5%, with a sensitivity and specificity of 94.4% and 75.0%, respectively. Discussion In this study, an analysis of DWI, DCE, and IVIM MRI was performed to evaluate the tissue diffu­sion and perfusion characteristics to identify histo­logical and molecular profiles of LGGs. Our results showed that diffusion and perfusion metrics exhib­ited substantial differences between WHO grade II and III gliomas, as well as between IDH1 mutant and wild-type LGGs. Among all parameters, the simplified DWI-derived perfusion fraction showed higher efficacy in IDH1 mutation detection, indi­cating that this recently developed three-b-value DWI approach may serve as a surrogate method for LGGs molecular diagnosis. DWI, DCE, and IVIM MRI-derived parameters showed significant differences between grade II and III gliomas. Diffusion-related parameters, in­cluding ADC and D values, were significantly low­er in WHO grade III gliomas; this result is in line with those of previous studies.19,35 It is now well es­tablished that ADC is strongly correlated with cel­lularity and the nuclear cytoplasmic ratio in tumor tissue36-38, both of which are important criteria in the histopathological grading of gliomas. Notably, perfusion-related parameters, espe­cially SPF, f, and Ktrans, showed a relatively good performance for glioma grading compared with diffusion parameters. This is most likely due to the increased perfusion feature in higher grade glio-mas; Ktrans reflects the volume transfer constant of a contrast agent from the plasma space to the ex-travascular extracellular space.39,40 In higher-grade gliomas, active angiogenesis and incomplete base­ment membrane of tumor neovasculature lead to an increment in microvascular permeability, thus a high Ktrans value. A previous study13 showed that SPF and IVIM-derived f correlated well with DCE MRI-derived Ktrans and were useful in differentiat­ing high- from low-grade gliomas. Our results fur­ther show that f and SPF also exhibited significant differences between WHO grade II and III gliomas. Over the last decade, studies have shown that gliomas with IDH mutation are less aggressive and more sensitive to chemotherapy, contribut­ing to a longer overall survival.41-44 Therefore, IDH plays a key role in the determination of the glioma molecular phenotype. Zhao et al.45 have shown that compared with IDH1 mutant gliomas, IDH1 wild-type gliomas are characterized by increased 308 neoangiogenesis and a higher nuclear cytoplasmic ratio due to the infiltrative nature. Higher vascu­lar proliferation leads to stronger perfusion effects. In this study, DWI, DCE, and IVIM MRI-derived perfusion parameters all showed significant differ­ences between IDH1 mutant and wild-type LGGs. Elevated perfusion was observed in IDH1 wild-type LGGs, which is in agreement with several previous reports using other perfusion imaging techniques.46-48 For example, Kickingereder et al.46 and Brendle et al.48 performed dynamic suscepti­bility contrast and arterial spin labeling perfusion-weighted imaging on patients with LGGs, respec­tively, and both found significantly higher cerebral blood flow values in IDH1 wild-type LGGs. This could be explained by considering the molecular function of IDH1. Cui et al.49 and Rei s et al.50 sug­gested that IDH1 mutation is associated with de­creased invasiveness and reduced angiogenesis via downregulation of the Wnt/ß-catenin signaling pathway. Furthermore, the accum ulation of 2-hy­droxyglutarate, an oncometabolite produced upon IDH1 mutation, has been shown to affect hypoxia-inducible factor (HIF) levels and the HIF response and may, consequently, reduce hypoxia-induced neovascularization.51 According to our ROC curve analysis, the sim­plified DWI-derived perfusion fraction showed a superior diagnostic accuracy as a predictor for IDH1 mutation in LGGs compared to the full IVIM-derived f. This result suggests that the three-b-value simplified DWI approach could save sub­stantial scanning time compared with the full IVIM approach, with no loss of diagnostic efficiency. Additionally, both simplified and full IVIM perfu­sion performed better than DCE MRI. These two perfusion methods represent different aspects of vasculature. IVIM measures microscopic transla­tional motions associated with microcirculation of blood in the capillary network, while DCE MRI measures capillary leakage of gadolinium contrast agent based on pharmacokinetic modeling. When WHO grade II and III gliomas were analyzed sepa­rately, we found SPF exhibited a statistically signif­icant difference in assessing IDH1 mutation status of WHO grade II tumors, whereas f helped assess WHO grade III tumors. However, these prelimi­nary results must be interpreted with caution due to the small sample size. Besides perfusion, diffu­sion parameters like ADC0,1000 were also predictive of IDH1 mutation in LGGs, with a lower diffusion coefficient found in IDH1 wild-type tumors. Our findings are in agreement with the existing litera­ture regarding their association.47,52 Our study has several limitations. First, the co­hort was relatively small, especially that of WHO grade III LGGs (n = 8). Therefore, we may have un­derestimated some associations, such as the associ­ation between perfusion-related metrics and IDH1 mutation status, in WHO grade III gliomas. A fur­ther prospective study with a larger cohort should be performed to validate our results. Second, es­timation bias may occur as a result of different cutoff b-values for IVIM analysis. Therefore, the set of b-values needs to be further optimized for brain tumors. Finally, the placement of ROIs was subjective and specific to a limited area on MRI. Automatic segmentation and image analysis of the entire tumor volume may improve preoperative risk stratification. In conclusion, DWI, DCE, and IVIM MRI can be used as quantitative perfusion methods in preoperative IDH1 mutation prediction in LGGs. Specifically, the simplified DWI-derived perfu­sion fraction showed a superior diagnostic per­formance, which holds the potential to serve as a contrast-free and time-saving alternative in the clinical setting. However, further validation in a large patient population is warranted. Acknowledgements YZ, STS, MQC, XQW contributed to the conception and design of the study. Data collection and evalu­ation were carried out by HJC, MQC, XQW as well as JWG. Statistical analyses and visualization were performed by XQW and STS. The manuscript was written by XQW, STS, YZ and MQC. All authors critically reviewed and approved the manuscript. This work was supported by the National Natural Science Foundation of China [grant num­bers 81501458, 81701642, 81571650, and 81901693); Shanghai Science and Technology Committee Medical Guide Project (western medicine) (grant number17411964300); Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (grant number 20172013); Medical Engineering Cross Research Foundation of Shanghai Jiao Tong University (grant number YG2015QN37, YG2017QN47) and Incubating Program for Clinical Research and Innovation of Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University (PYIII-17-027). 309 References 1. Cancer Genome Atlas Research N, Brat DJ, Verhaak RGW, Aldape KD, Yung WKA, Salama SR, et al. Comprehensive, integrative genomic analysis of diffuse lower-grade gliomas. The N Engl J Med 2015; 372: 2481-98. doi: 10.1056/NEJMoa1402121 2. Chang EF, Clark A, Jensen RL, Bernstein M, Guha A, Carrabba G, et al. 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Glioma-derived mutations in IDH1 dominantly inhibit IDH1 catalytic activity and induce HIF-1alpha. Science 2009; 324: 261-5. doi: 10.1126/science.1170944 46. Kickingereder P, Sahm F, Radbruch A, Wick W, Heiland S, Deimling Av, et al. IDH mutation status is associated with a distinct hypoxia/angiogenesis tran­scriptome signature which is non-invasively predictable with rCBV imaging in human glioma. Sci Rep 2015; 5: 16238. doi: 10.1038/srep16238 47. Leu K, Ott GA, Lai A, Nghiemphu PL, Pope WB, Yong WH, et al. Perfusion and diffusion MRI signatures in histologic and genetic subtypes of WHO grade II–III diffuse gliomas. J Neurooncol 2017; 134: 177-88. doi: 10.1007/s11060­017-2506-9 48. Brendle C, Hempel JM, Schittenhelm J, Skardelly M, Tabatabai G, Bender B, et al. Glioma grading and determination of IDH mutation status and ATRX loss by DCE and ASL perfusion. Clin Neuroradiol 2018; 28: 421-8. doi: 10.1007/s00062-017-0590-z 49. Cui D, Ren J, Shi J, Feng L, Wang K, Zeng T, et al. 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AJR Am J Roentgenol 2018; 210: 621-8. doi: 10.2214/AJR.17.18457 311 research article The feasibility of ultrasound-guided vacuum-assisted evacuation of large breast hematomas Sa’ed Almasarweh1, Mazen Sudah1, Sarianna Joukainen2, Hidemi Okuma1, Ritva Vanninen1,3, Amro Masarwah1 1 Kuopio University Hospital, Diagnostic Imaging Center, Department of Clinical Radiology, Kuopio, Finland 2 Kuopio University Hospital, Department of Plastic Surgery, Division Surgery, Kuopio, Finland 3 University of Eastern Finland, Cancer Center of Eastern Finland, Kuopio, Finland Radiol Oncol 2020; 54(3): 311-316. Received 3 May 2020 Accepted 27 May 2020 Correspondence to: Sa’ed Almasarweh, Department of Clinical Radiology, Kuopio University Hospital, Puijonlaaksontie 2, P.O. Box 100, FI-70029 Kuopio, Finland. E-mail: saadn.masarweh@gmail.com Disclosure: No potential conflicts of interest were disclosed. Background. Breast hematoma is an often underrated and disregarded post-procedural complication in the litera­ture. Current treatment modalities are comprised of either surgical or expectant therapy, while percutaneous proce­dures play a smaller role in their treatment. We aimed to examine the efficacy of vacuum-assisted evacuation (VAE) in the treatment of clinically significant large breast hematomas as an alternative to surgery. Patients and methods. We retrospectively analysed patients that underwent breast interventions (surgical and per­cutaneous), who later developed clinically significant large hematomas and underwent a trial of VAE of hematoma in our hospital within the period of four years. Patient and procedure characteristics were acquired before and after VAE. Success of intervention was based on = 50% clearance of hematoma volume and patients’ subjective resolution of symptoms. All patients were followed clinically and by ultrasound if needed at different intervals depending on the severity of presenting symptoms. Results. Eleven patients were included in the study. The mean largest diameter of hematomas was 7.9 cm and mean surface area was 32.4 cm2. The mean duration of the procedure was 40.5 min. In all patients VAE of hematoma was implemented successfully with no complications. Control visits showed no major residual hematoma or seroma formation. Conclusions. Our results show that VAE of hematoma can be implemented as a safe alternative to surgery in large, clinically significant hematomas, regardless of aetiology or duration. The procedure carries less risk, stress and cost with the added benefit of outpatient treatment when compared to surgical treatment. Key words: breast hematoma; vacuum assisted breast biopsy; hematoma evacuation; breast Introduction Complications following therapeutic, reconstruc­tive, or aesthetic breast surgeries as well as percu­taneous procedures, both biopsies and excisions, are important considerations for women undergo­ing or pursuing these options. In general, the most common local complications following routine breast interventions are inherent to the surgery itself e.g. infection, pain, hematoma, delayed heal­ing, and abnormal scarring. Risk factors for com­plications include smoking, obesity, larger breasts, anticoagulant treatments, and older age.1 Early clinically significant postoperative he-matomas typically develop within the first 12 to 48 hours after surgery.2 Immediate reoperation is usually indicated in expanding hematomas, hemodynamic instability, and jeopardized flap viability.3 Less commonly, a hematoma appears days or weeks after surgery and may be associated 312 with minor injury or trauma to the breast, with the majority identified within the first 14 days. Late hematomas can also occur and are thought to be related to direct trauma, clotting disorders, overac­tivity, and use of intraoperative corticosteroids.4,5 Postprocedural hematomas are not uncommon, yet most of them are small and resolve spontane­ously. Large, clinically more significant hemato-mas in the late postoperative period are infrequent. Symptomatic, painful, or infected hematomas are treated surgically since hematomas with dense contents or clots do not drain with needle aspira­tions or drains are blocked immediately.6 If a clinically significant hematoma does oc­cur, an evacuation is advised. Expectant manage­ment is not favoured due to the lengthy nature of spontaneous liquefaction and discomfort that patients report, which eventually leads to fibrosis and distortion of breast tissue.7 Surgical manage­ment is aimed at the rapid decompression of the closed wound space through exploration, drainage and establishing haemostasis. After evacuation the wound is thoroughly irrigated and closed in or­der to preserve the cosmetic aspect of the breast.8 Percutaneous drainage of the hematoma during the first 24 hours of hematoma formation might be challenging, on the assumptions that an organized clot would have already been formed. Partial lique­faction occurs 6 to 7 days after the formation of the hematoma, which is considered as the best interval for percutaneous evacuation.9 Breast imaging-guided interventions are widely used in daily practice e.g. core biopsy and vacuum assisted breast biopsy and excision (VABB and VABE) to diagnose different types of imaging find­ings and remove benign or risk lesions. The larger the needle used for biopsy and the number of cores obtained, the more likely complications will ap­pear.10 Significant vascular damage is more prob­able in VABB or VABE procedures.11 Recently it was reported that VABB can be used as a treatment mo­dality for clinically significant hematoma in patients with small hematomas less than 4cm in size.6 The effectiveness of vacuum-assisted evacuation of large breast hematomas has not been previously reported. In this study we aimed to investigate ultra-sound-guided vacuum-assisted evacuation (VAE) of breast hematoma as a safe, viable, time and resource-sparing treatment modality for larger (> 5 cm) breast hematomas irrespective of aetiology. This technique could decrease the rate of multiple operations and eliminate added morbidity of sur­gery and anaesthesia while yielding satisfactory therapeutic results. Patients and methods Patients All VAE of hematomas performed in our institu­tion between February 2016 and February 2020 were retrospectively retrieved from the regional picture archiving and communication system (PACS) and the clinical data of these patients were also retrieved from the local digital archives. In our institution, hematomas that do not fulfil the crite­ria for immediate surgery, cause discomfort and unsettling symptoms for the patients (considerable pain, pressure symptoms, local infection and pro­longed healing) or patients who refuse surgical in­tervention are offered a trial of VAE of hematoma. The Chair of the Hospital District waived the need for written informed consent from the patients due to the retrospective nature of this study. All clini­cal investigations were conducted according to the relevant guidelines and the principles expressed in the Declaration of Helsinki. Data collection The total amount of breast surgeries, VABB and VABE as well as postoperative haemorrhagic com­plications requiring surgical intervention were retrieved from the hospital’s digital information systems. All medical records of patient undergoing VAE procedures were also reviewed and the following parameters were recorded and included in a data­base: Age, type of breast procedure, time interval between previous procedure and VAE of hemato-ma, symptoms exhibited pre- as well as post-VAE, medications, comorbidities, size of hematoma and the estimated residual volume after the procedure, echogenicity of hematoma at time of VAE, gauge of the needle used, complications during or after the procedure and findings at control. The total dura­tion of the procedure was measured from the time the patient has entered the ultrasound room until discharge. Procedure In Kuopio University Hospital (KUH), automated VABB procedures were introduced in 2015. With experience in VABB, VABE was gradually intro­duced and consequently evacuation of large he-matomas was offered as an alternative to surgery. The procedures were carried out with EnC or™ Breast Biopsy System (BD Bard, Tempe, AZ, USA). US-guided interventions were performed using 313 FIGURE 1. Illustration of a vacuum assisted evacuation of hematoma in patient number 9. Vacuum assisted excision of a discordant lesion (A) at core biopsy resulted in a palpable painful hematoma. Ultrasound image of the 5.5x4.0 cm hematoma (B). Complete evacuation of the hematoma with sparing of hematoma wall (C); Large arrows indicate needle’s shaft and small white arrows indicate hematoma wall a Logiq E9 class US scanner (GE, Wauwatosa, Wisconsin, USA) equipped with a 5–15 MHz linear array transducer. All procedures were performed by, or under the supervision of, a breast radiologist with over 25 years of experience in multimodality breast imaging and interventions. No change in pa­tients’ medications was required. After thorough local disinfection, application of aseptic measures as well as the injection of local anaesthetic through the insertion channel (lidocaine with adrenaline; max 10 ml) a small skin incision was made. An EnCor™ 7/10G vacuum needle was then inserted into the base of the hematoma. The needle’s cut­ting blade was opened, and continuous suction was applied until the hematoma emptied and its walls collapsed (Figure 1). Residual hematomas in side-pockets were ignored. In the case of incom­plete hematoma aspiration (less than 50%) due to large, blocking or hard clots, the blade was used to fragment the fibrotic tissue through multiple bi­opsy samples. Sample container was continuously flushed with saline during the aspiration-fragmen­tation procedure to avoid blockage, and the con­tainer would be changed if filled with accumulated material as needed. The walls of the hematoma were carefully avoided during any fragmentation procedure to avoid possible rebleeding. After the procedure, the area of the breast with hematoma was manually compressed for at least 10 minutes, longer, if the patient received anti-coagulants. The use of a tight sports-brassiere was recommended for a minimum of 24 hours with compression pads over the area of the hematoma to prevent rebleed­ing or major seroma formation. Since 2019 we continued to provide fully adjustable and flexible breast compression wraps to all patients. Patients were then discharged. Some hematomas were longer than the shaft of the vacuum needle, thus separate insertions from opposite sides were implemented to complete the procedure. Otherwise, procedures were completed through single insertion. Success of intervention was based on clearance of a targeted = 50% of hematoma’s volume, visu­ally estimated by the operator, patients’ subjective assessment of symptom resolution and the reso­lution of hematoma without the need for surgery during follow-up. All patients were followed clini­cally and by US if needed at different intervals de­pending on the severity of presenting symptoms. Results During the recruitment period, a total of 1208 breast operations and 358 VABB or lesion excision procedures were performed. We detected a total of 44 clinically large hematomas as complications. Of the 1208 operative patients, 33 had early post­operative bleeding and had to undergo surgical evacuation while 8 patients suffered from delayed hematoma formation. Therefore, the reoperation rate for early postoperative bleeding was 2.7% (33/1208) and the rate of late hematomas treated with VBE was 0.7% (8/1208). On the other hand, of the 358 patients that have undergone VABB and ex­cision procedures, 3 patients were later diagnosed to have clinically relevant hematomas with an inci­dence rate of 0.84% (3/358). Altogether 11 consecutive patients who have been diagnosed with breast hematoma and treated with the VAE system were included in the analysis. Patients had a mean age of 59 years (range 38-85) and their characteristics are presented in Table 1. 314 TABLE 1. Characteristics of patients with hematoma 1 42 14 BLES Anti-Coagulant 6 x 4 2 67 1 VABB None 8 x 4 3 38 36 Surgery None 6 x 3 4 48 78 Surgery None 6 x 5 5 49 21 Surgery None 5.5 x 3 6 51 34 Surgery Anti-Platelet and hydrocortisone 7 x 6.5 7 84 15 Surgery Anti-Platelet 12 x 2.5 8 71 597 Surgery None 5.5 x 3.5 9 85 51 VABE Anti-Coagulant 5.5 x 4 10 60 29 Surgery None 20 x 5 11 53 22 Surgery None 5.5 x 5 * Wait time = number of days between surgical intervention/biopsy and VAE of hematoma; BLES = breast lesion excision system; VABB = vacuum assisted breast biopsy; VABE = vacuum assisted breast-lesion excision TABLE 2. Hematoma characteristics pre- and post-vacuum assisted evacuation (VAE) 1 6 x 4 > 50% Resolution 2 8 x 4 > 50% Resolution 3 6 x 3 100% Resolution 4 6 x 5 100% Resolution 5 5.5 x 3 100% Resolution 6 7 x 6.5 80% Resolution 7 12 x 2.5 70% Resolution 8 5.5 x 3.5 90% Resolution 9 5.5 x 4 100% Resolution 10 20 x 5 80% Resolution 11 5.5 x 5 100% Resolution Of the 11 participants, 3 patients had hematomas as complications after percutaneous interventional procedures and 8 patients after surgeries, of which 5 were reduction mammoplasties. The mean num­ber of days between the initial intervention and VAE of hematoma of 10 patients was 30 days with an outlier of 597 days due to an idiopathic late de­veloping complicated hematoma in a mastectomy site after radiotherapy. One of these patients had a slowly progressive hematoma after VABB and refused surgical evacuation. Regarding symptoms prior to evacuation, all patients reported pain, 45.5% prolonged healing (n = 5), 45.5% mass effect (n = 5), 18.2% infection (n = 2). Of the ultrasound imaging of the hematomas taken prior to VAE of hematoma, 7/11 were hypo-echoic, 1/11 was hyperechoic and 3/11 had mixed echogenicity. All hematomas underwent unsuc­cessful aspiration trials with fine needles (G18–23). The mean duration of the VAE procedure was 40.5 min (range 19–62). One patient was taking aspirin alone, one aspirin and hydrocortisone, one patient was taking Warfarin, one patient taking Dabigatran and one patient taking Apixaban. One patient had a massive two-sided communi­cating 20 x 5 and 12 x 4 cm hematoma, therefore in this analysis, we included only the largest portion. The mean maximum diameter of the evacuated he-matomas was 7.9 cm and an average surface area of 32.4cm2. The gauge of the VAE probes was a choice between 7G or 10G. Most of the procedures were performed using 7G-sized needles (n = 8) owing to the larger size of the treated hematomas. Four patients underwent ultrasound-guided aspiration of the hematoma cavity due to post-evacuation se­roma formation 1–7 days after VAE procedure. No complications were reported post-evacuation or aspiration procedures. The parameters before as well as after VAE of hematoma are depicted in Table 2. All patients underwent regular follow-up after evacuation. Upon follow-up, all cases were deemed success­fully treated with no major hematoma residue or seroma formation. Discussion One of the most common complications in breast interventions is hematoma formation, which re­mains grossly underrated and disregarded in the literature, especially when its frequency is taken into consideration. Breast hematomas can range from small mammographically-detected hemato-mas to large clinically significant hematomas that can cause severe discomfort to patients. Our results suggest that Vacuum-assisted evacuation of hema­toma is a time-sparing, cost-effective and success­ful method of evacuation for small as well as large breast hematomas regardless of aetiology. Our patient population was comprised of both post-biopsy and post-surgical patients, thus ex­panding the aetiological factors to not only include biopsy-induced hematomas. In our study we in­cluded all consecutive patients treated in our in­stitution presenting with hematomas of different 315 FIGURE 2. Patient number 10 with massive two-sided communicating hematoma treated through separate punctures. Image (A) represents a panoramic view of the cranial aspect of the 12 cm long hematoma and image (B) represents a panoramic view after the evacuation. Image (C) represents the caudal 20 cm long hematoma and correspondingly image (D) shows the view after treatment. sizes. This goes to prove that VAE can be imple­mented in clinically large significant hematomas. It is stipulated that percutaneous drainage of acute hematomas should be attempted between days 7–14 after formation of hematoma, in order to al­low time for hematoma liquefaction. Moreover, delayed hematomas, i.e. hematomas developing 6 months or more post-intervention, are always evacuated surgically.7 In our study the wait time was variable, in that the procedure timing was not set on a set-point schedule, rather on different time-intervals regarding date of hematoma forma­tion. Furthermore, one large delayed hematoma was evacuated successfully after 597 days (1.64 years), proving that even delayed hematomas can be successfully treated with VAE of hematoma ir­respective of duration. While the true incidence of large clinically sig­nificant hematomas remains unknown, findings from this study show that it is relatively uncom­mon. The treatment of breast hematomas in the literature is suggested as either surgical or expect­ant. Both treatment modalities impose certain risks and added morbidity for the patient. Patients face problems such as added costs, the ordeal of going through surgery or stress due to the aesthetic and psychological impact of the procedure. Moreover, expectant therapies may pose future diagnostic difficulties.9 A recent report evaluated the efficacy of the VABB system in evacuating symptomatic hemato-mas after VABB excision of benign breast lesions in 8 patients. Evacuation was successful in all the cases and no technique-related complications were observed.6 However, the inclusion criteria were restricted to hematomas observed post-VABB or VABE and not post-surgical complications, which limits the patient population on which this tech­nique can be used. Moreover, 75% (6/8) of hema­tomas were smaller than 4 cm with a largest maxi­mum diameter of 5.6 cm. This means that most clinically significant breast hematomas that are difficult to handle conservatively were excluded. The study failed to address whether this technique could be attempted on larger and more difficult to evacuate hematomas, which would otherwise need to be evacuated surgically. Vacuum-assisted biopsies are also currently im­plemented as a treatment modality for small palpa­ble or non-palpable benign or risk lesions, by assur­ing rapid and complete excision of these lesions to be better histopathologically evaluated and there­fore obviating the need for therapeutic surgery or continuous follow-up.12 Minimally invasive man­agement of many B3 lesions with VABE continues to be a suitable alternative to first-line surgical excision in most cases.13 In this study, we wanted to study the efficacy of automatic VABE system in removing symptomatic clinically significant he-matomas. Not only are VABE systems faster with less implications on patients, but the endogenous vacuum capability, the large bore size as well as the slicing mechanism of the probe could be used to evacuate organized hematomas, which otherwise would be difficult to aspirate percutaneously and would need surgical drainage and evacuation. Evacuations were performed immediately up­on request and without prior scheduling as our 316 patients presented with acute symptoms and the procedure offered immediate relief. Furthermore, most of these patients were discharged immedi­ately after the procedure. No change in anticoagu­lant medications were required as the procedures were quite straightforward without any excision of fibroglandular breast tissues. The tip of the blade is very sharp and penetrates even denser tissues easily, hence special care should be applied in han­dling the needle inside the hematoma cavity in or­der not to induce any damage to the cavity walls. Due to the large size of the needles, we regularly chose the shortest insertion pathway and used a combination of local anaesthetic with adrenaline to reduce any possible bleeding consequences. The obvious limitations of the study are the small number of patients and the retrospective nature. This study needs to be validated on a larger scale to include more patients with a more controlled inclusion and outcome criteria. Furthermore, the volume of the evacuated part of the hematoma was not measured during the procedure due to the con­tinuous saline flush used in our practice and could not be retrospectively accurately verified. To conclude, this study shows that VAE proce­dure is a successful, time-conserving, easily im­plemented interventional treatment modality for both small and large breast hematomas that would decrease the morbidity, costs and inconvenience of repeated surgery. References 1. Araco A, Gravante G, Araco F, Delogu D, Cervelli V, Walgenbach K. 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A long-term study of out­comes, complications, and patient satisfaction with breast implants. Plast Reconstr Surg 2006; 117: 757-67; discussion 768-72. doi: 10.1097/01. prs.0000201457.00772.1d. 6. Guzman-Aroca F, Berna-Serna JD, Garcia-Ortega AA, Hernandez-Gomez D, Berna-Mestre JD. A new management technique for symptomatic haema­tomas following therapeutic vacuum-assisted biopsy. J Clin Med 2019; 8: 1493. doi: 10.3390/jcm8091493 7. Smith B. Complications of Breast Surgery. In: Cance WG, editor. Breast sur­gery Amsterdam: IOS Press; 2001. p. 95-102. 8. Vitug AF, Newman LA. Emergencies in breast surgery. Surg Clin North Am 2007; 87: 431-51. doi: 10.1016/j.suc.2007.01.005 9. Polverini A, Kruper L. Surgical Emergencies in Breast Surgery. In: Fong Y, editor. Surgical Emergencies in the Cancer Patient. New York: Springer International Publishing; 2017. p. 431-51. 10. Zagouri F, Gounaris A, Liakou P, Chrysikos D, Flessas I, Bletsa G, et al. 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Breast Cancer Res Treat 2019; 174: 279-96. doi: 10.1007/s10549-018-05071-1. 317 research article Analysis of damage-associated molecular pattern molecules due to electroporation of cells in vitro Tamara Polajzer1, Tomaz Jarm1, Damijan Miklavcic1 1 Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2020; 54(3): 317-328. Received 18 June 2020 Accepted 7 July 2020 Correspondence to: Prof. Damijan Miklavcic, Ph.D., Faculty of Electrical Engineering, University of Ljubljana, Tržaška 25, SI-1000 Ljubljana, Slovenia. E-mail: damijan.miklavcic@fe.uni-lj.si Disclosure: No potential conflicts of interest were disclosed. Background. Tumor cells can die via immunogenic cell death pathway, in which damage-associated molecu­lar pattern molecules (DAMPs) are released from the cells. These molecules activate cells involved in the immune response. Both innate and adaptive immune response can be activated, causing a destruction of the remaining infected cells. Activation of immune response is also an important component of tumor treatment with electrochemo-therapy (ECT) and irreversible electroporation (IRE). We thus explored, if and when specific DAMPs are released as a consequence of electroporation in vitro. Materials and methods. In this in vitro study, 100 µs long electric pulses were applied to a suspension of Chinese hamster ovary cells. The release of DAMPs – specifically: adenosine triphosphate (ATP), calreticulin, nucleic acids and uric acid was investigated at different time points after exposing the cells to electric pulses of different amplitudes. The release of DAMPs was statistically correlated with cell permeabilization and cell survival, e.g. reversible and irreversible electroporation. Results. In general, the release of DAMPs increases with increasing pulse amplitude. Concentration of DAMPs de­pend on the time interval between exposure of the cells to pulses and the analysis. Concentrations of most DAMPs correlate strongly with cell death. However, we detected no uric acid in the investigated samples. Conclusions. Release of DAMPs can serve as a marker for prediction of cell death. Since the stability of certain DAMPs is time dependent, this should be considered when designing protocols for detecting DAMPs after electric pulse treatment. Key words: electroporation; pulsed electric field treatment; damage-associated molecular pattern molecules; im­munogenic cell death; electrochemotherapy Introduction Electroporation or pulsed electric field (PEF) treat­ment can cause changes in membrane perme­ability, which allows molecules, that are other­wise membrane impermeable, to cross the plasma membrane. In reversible electroporation the dam­age to cell membrane is repaired, enabling the cell to reestablish its metabolism and survive. This type of electroporation is used in multiple thera­pies. Electrochemotherapy (ECT) is one of such widely used therapies in which the increased cell membrane permeability enables chemotherapeutic drug to enter the cell and thus potentiates the cy­totoxicity of the drug.1,2 In irreversible electropora­tion (IRE) the damage to the cells however is too severe for the cells to recover which leads to cell death. While the cells are destroyed, the integrity of tissue like vessels, nerves and extracellular ma­trix remains preserved3,4, making this therapy very appealing for ablation of tumor and other tissues, otherwise unsuitable for surgical removal or ther­mal ablation such as radiofrequency ablation or cryo-ablation.5,6 318 In ECT eight square 100 µs electrical pulses, with an amplitude of 100-1000 V are usually used to induce a reversible membrane permeabilization. For IRE, more pulses (80-100 pulses) at higher am­plitude (up to 3000 V) are required, to overwhelm the reparative capacity of the cells which leads to cell death.7 From morphological, biochemical, and functional perspectives, different cell death path-ways/types can be activated.8 Historically, based on morphological changes, three different forms of cell death were defined: apoptosis (cell shrinkage, chromatin condensation, formation of apoptotic bodies); autophagy (cytoplasmic vacuolization); and necrosis (loss of plasma membrane integrity).8,9 Such classification is still employed, but in newer classification based on genetic, biochemical, phar­macological and functional differences, cell death is either accidental (uncontrollable death caused by disassembly of the plasma membrane) or regu­lated (activation of signal transduction). Depending on signaling pathways different types of regulated cell death are being characterized, e.g. intrinsic and extrinsic apoptosis, necroptosis, ferroptosis, pyroptosis, immunogenic cell death, lysosome-de­pendent cell death, mitochondrial permeably tran­sition driven necrosis and many others gathered and described by Galluzi et al..10 In electroporation studies, cell death has been most extensively ex­plored in the range of nanosecond pulse treatment, where the majority of studies confirmed cell death by apoptosis (intrinsic and extrinsic) and only few studies indicated necrosis.11,12 Both pathways were confirmed also in microsecond pulse treatment.13-9 Nevertheless, in recent studies new cell death types were also detected like pyroptosis20, necroptosis20,21 and immunogenic cell death.22-29 In IRE 18,30-34 and ECT with either bleomycin or cisplatin26,35-37 used for cancer treatment, involve­ment and importance of host immune response was demonstrated, counteracting tumor escape mechanisms.29,38,39 After these therapies, dying tu­mor cells can release specific molecules, which are being recognized by the cells of immune system. These molecules can activate the innate and adap­tive immune response, leading to the destruction of the remaining tumor cells in the body40 and induc­ing long-lasting protective antitumor immunity.41 Some studies even suggest that immunogenic effect of IRE is more pronounced than in other ablation therapies like radiofrequency ablation31 and cryoa­blation.32 Evidence suggests that administration of immune-stimulating molecules can even enhance the local effectiveness of ECT35 and IRE29,42-44 allow­ing simultaneous treatment of distant tumors. Our immune system consists of two comple­mentary and closely collaborative systems, an innate (non-specific) and an adaptive (antigen-specific) system. Activation of immune system is essential for our survival, as it distinguishes and eliminates potentially harmful molecules, even the ones that derive from the host/our own tissues. Well known are the pathogen-associated molecules (PAMPs), which are present on microbes and are being recognized by cells of the innate immune system when they bind to pattern recognition re­ceptors (PRRs). The same pathways are activated by the host’s damage-associated molecular pat­tern molecules (DAMPs), which act as endogenous damage signal in case of cell death or response to stress, leading to inflammatory response.45-47 Release of DAMPs characterizes immunogenic cell death (ICD). Most of DAMPs are normally lo­cated intracellularly48, where under normal physi­ological conditions have an important intracellular role. When a cell is damaged or dies, DAMPs are actively or passively exposed or released to extra­cellular space.49-51 The release of DAMPs is often accompanied by cytokines, chemokines and other inflammatory mediators.52 In extracellular space DAMPs have a completely different function, as they are being recognized by pattern recognition receptors (PRRs), such as TRLs, NOD-like, PRLs and RAGE receptors on immune cells.50,53 Binding of DAMPs to these receptors stimulates innate im­mune response through promoting the release of pro-inflammatory mediators and recruiting im­mune cells (dendritic cells, macrophages, T cells and neutrophils). Usually, the exposure of differ­ent DAMPs depends on endoplasmic reticulum stress, followed by reactive oxygen species (ROS) production.41 Release of DAMPs correlates with the degree of trauma.54 Some DAMPs can even be involved in tissue repair pathway.55-57 It depends on DAMPs and their triggered pathways, together with cytokines and growth factor to determine, if mild acute inflammation and wound healing 56,58 or severe inflammation and fibrosis will follow.59 Electroporation causes an increase in membrane permeability and allows molecules, for which the membrane is usually impermeable, including DAMPs, to cross it. ATP, one of the main DAMPs, was even used as an indicator of cell membrane permeabilization in the first electroporation stud­ies.60 In recent years reports on electroporation studies have started to emerge investigating the immunogenic cell death caused by electroporation. Studies detected DAMPs, like ATP, high-mobility group box 1 protein (HMGB1) release and calreti- 319 culin externalization, as they are the gold standard for predicting the ICD in cancer cells.41 So far most­ly single (or a small subset of) DAMPs were stud­ied. Most studies involved nanosecond pulse treat­ment22-25, whereas studies using microsecond26,29, millisecond28 and H-FIRE pulse treatments27 are even more scarce. For now, different DAMPs were investigated at different intervals after electropora­tion ranging from 30 min to 72 hours, using differ­ent types of cancer cells. Because in both ECT and IRE the immune sys­tem response is essential for successful and com­plete tumor eradication, we decided to explore if and when specific DAMPs are released in response to electroporation in vitro. The experiments were performed using 100 µs long pulses, as they are most commonly used in ECT treatment and in IRE for soft tissue ablation. Materials and methods Cell preparation Chinese hamster ovary (CHO-K1) from European MS, differential probe HVD3206A and the current Collection of Authenticated Cell Cultures were probe CP031A, all from LeCroy, USA, were used grown in culture flasks (TPP, Switzerland) filled to monitor the delivered pulses, i.e. voltage and with HAM F-12 growth medium (PAA, Austria) at current. The delivered voltage was approximately 37°C with a humidified 5% CO2. The growth me-10-15% lower than the value set on the pulse gen-dium was enriched with 10% fetal bovine serum erator and the current was in the range of 3-21 A. (FBS) (Sigma-Aldrich, Germany), L-glutamine When pulses with high amplitudes were applied (StemCell, Canada) and antibiotics penicillin/ (Figure 1), current decreased slightly during the streptomycin (PAA, Austria) and gentamycin pulse, presumingly due to electrochemistry at elec-(Sigma-Aldrich, Germany). At 70% confluency, trode-electrolyte interface reducing the available cells were detached with trypsin solution (10x interface area for ion exchange between the metal trypsin-EDTA (PAA, Austria) 1:9 diluted in Hank’s electrode and the electrolyte and possibly also due basal salt solution (StemCell, Canada), which was to ion depletion at the said interface. inactivated after 3 minutes by the growth medium. After 5 minutes of centrifugation at 180 g and 22°C supernatant was removed. Cell were mixed with Results the growth medium to obtain cell density at 2x106 cells/ml. First, the permeabilization and the survival curves were obtained to determine experimental points for the studies on release of DAMPs. Permeabilization Electric pulse generation and survival curves are presented in all figures Laboratory prototype pulse generator (University showing the concentration of various DAMPs to of Ljubljana), based on H-bridge digital amplifier visualize how the presence of DAMPs is related to with 1 kV MOSFETs (DE275-102N06A, IXYS, USA), changes in permeabilization and cell viability. In described in 61 was used. Eight 100 µs long mo-figures the permeabilization and survival curves nopolar electric pulses with repetition frequency 1 are shown only at pulse amplitudes tested for the Hz and amplitude of 0–600 V (0-3 kV/cm; voltage presence of DAMPs; in steps of 50 V in the range to distance ratio) with increments of 100 V (and of pulses where changes in permeabilization occur additional increments in the permeabilization as-and in steps of 100 V above 200 V. say) were applied between stainless steel 304 plate The concentration of ATP in supernatant was first electrodes (d = 2 mm). Oscilloscope HDO6104A-measured with fluorescent method 30 minutes and 320 FIGURE 2. Release of adenosine triphosphate (ATP) as a function of electric pulse amplitude determined by fluorescent assay. Two-time points after electroporation were assessed. Permeabilization and survival curves are also presented. Black and green asterisks (*) indicate statistically significant differences between the samples at different voltages and the corresponding control at 0 V (one-way analysis of variance [ANOVA] followed by Holm-Sidak post-hoc test, (p < 0.05) and within the pair of samples at different voltages (t-test, p < 0.05), respectively). 24 hours after electroporation (Figure 2). At 30 min­utes the concentration of ATP in supernatant was detected at 200 V. However, statistical difference between the control and the treatment groups (ob­tained by one-way analysis of variance [ANOVA] followed by the post-hoc test) was only detected at 500 V and above. The concentration of ATP in su- FIGURE 3. Release of adenosine triphosphate (ATP), as a function of electric pulse amplitude determined by luminescence assay. Two-time points after electroporation were assessed. Permeabilization and survival curves are also presented. Black and green asterisks (*) indicate statistically significant differences between the samples at different voltages and the corresponding control at 0 V (one-way analysis of variance [ANOVA] followed by Holm-Sidak post-hoc test, (p < 0.05) and within the pair of samples at different voltages (t-test, p < 0.05), respectively). pernatant grew with increasing pulse amplitude, which after 24 hours led to decreased cell viability; e.g. correlation between the cell survival and ATP concentration in supernatant detected after 30 min is quite strong and negative; R = -0.864. Also, weak correlation between cell permeabilization and ATP concentration in supernatant (R = 0.594) confirms, that ATP presence in supernatant is more strongly correlated with the irreversible than the reversible electroporation. It may indicate that strong ATP re­lease from cells leads to cell death. After 24 hours (Figure 2) the lowest ATP con­centration was achieved at the pulse amplitude resulting in death of most cells (500, 600 V). The concentration of ATP at these points is statistically different to the results obtained 30 minutes after pulse treatment. After 24 hours the concentration of ATP had decreased with the lower viability, but statistical differences between the control and the treatment groups were present from 400 to 600 V. At 24 hours there is a positive statistical correlation between the cell survival and concentration of ATP in supernatant (R = 0.888), which is stronger than the correlation to permeabilization (R = -0.695). Since no ATP was detected in supernatant with­in the range of reversible electroporation after 30 minutes using the fluorescent method (Figure 2), we also used a more sensitive luminescent method (Figure 3). Furthermore, since ATP analysis showed that 24 h after treatment ATP is not detected in all samples, we were also interested in how fast ATP was degraded. Scuderi et at. showed complete re­sealing of plasma membrane 10 minutes after pulse treatment using 8 x 100 µs pulses.63 Thus, another time point for ATP measurement was chosen, i.e. 15 minutes after (Figure 3). With more sensitive luminescent detection assay, ATP was detected in supernatant already at 100 V, however statistically significant difference to control was only detected at 300 V and higher. These results are more reliable due to higher assay sensitivity however even with this method statistically significant amount of ATP in supernatant is detected in the range of irrevers­ible electroporation, as increased electric field/volt­age kills more cells more ATP is present in the ex­tracellular space. This is also confirmed by a strong correlation between the survival and the amount of ATP in supernatant, R = -0.947 for 15 min and R = -0.964 for 30 min, and much weaker correlation between the permeabilization and the amount of ATP (R = 0.704 for 15 min and R = 0.728 for 30 min). In our results, only one significant difference was found in detected ATP amount between 15 and 30 minutes after pulse treatment at 500 V. Since this 321 difference was not detected for all the experimental points (voltages), we believe that ATP in extracel­lular space is not degraded in this first 30 minutes after pulse treatment. Calreticulin (CRT) is an endoplasmic reticulum protein which needs to be transferred to the outer leaflet of the plasma membrane in order to act as a DAMP. Externalization of calreticulin to outer membrane in an active process involving also its transport across the cell. Due to this active and time demanding process the externalization of calreticulin was investigated 4 and 24 hours (also used in previous studies23-25,28) after pulse treat­ment (Figure 4) on viable cells (determined by pro-pidium iodide [PI] staining). Calreticulin was first detected at 300 V and its fluorescence increased with increasing voltage of pulses. Furthermore, FIGURE 4. Externalization of calreticulin as a function of electric pulse amplitude. the lowest viability at 600 V with < 5% of viable Two-time points after electroporation were assessed. Permeabilization and survival cell has the strongest signal of calreticulin after 4 (MTS) curves are also presented. Survival detected by propidium iodide (PI) protocol and 24 hours. This could indicate the amount of is normalized to control and presented with red (4 hours after pulse treatment) and orange (24 hours after pulse treatment) line. Approximate baseline of calreticulin externalized calreticulin per viable cell increases is presented with -----. Black and green asterisks (*) indicate statistically significant with the level of stress (amplitude of applied elec- differences between the samples at different voltages and the corresponding tric pulses). Furthermore, analysis shows a strong control at 0 V (one-way analysis of variance [ANOVA] followed by Holm-Sidak post-correlation between survival determined by MTS hoc test, (p < 0.05) and within the pair of samples at different voltages (t-test, p < test and externalization of calreticulin, as survival 0.05), respectively). decreased, the detection of calreticulin increased (R = -0.801 for 4h and R = -0.946 for 24h) and weak correlation between permeabilization and exter-release of nucleic acids, which after 24 h resulted nalization of calreticulin was observed (R = 0.535 in lower cell viability. This is confirmed also by a for 4h and R = 0.556 for 24h). Since calreticulin was strong negative correlation between survival and detected only in viable cells (determined by PI) ad­ditional information on viability was obtained, and results were normalized to control (0 V) for each investigated time point separately. Except for the results at 300 V, no statistically significant differ­ence between 4 and 24 hours was detected at any other experimental point, suggesting that calreti­culin can be detected 4 hours after pulse treatment and that expression of the protein remains stable for the next 20 hours. Until now, nucleic acids (in the role of DAMPs) have not been investigated in relation to electropo-ration. Most of RNA (except fresh transcripted mRNA) is located in cytoplasm, while DNA is lo­cated in the cell nucleus. The concentration of RNA and DNA in supernatant has been detected 15, 30 minutes and 24 hours after electroporation like in ATP assay (Figure 5 and 6). Concentration of DNA/ RNA started to rise from 400 V up (Figure 5 and 6). FIGURE 5. Release of RNA as a function of electric pulse amplitude. Three-time This happened at the same pulse amplitudes where points after electroporation were assessed. Permeabilization and survival curves are also presented. Approximate baseline of RNA is presented with -----. Black and after 24 hours cell viability was affected, indicating green asterisks (*) indicate statistically significant differences between the samples the amount of nucleic acid occurs in the range of cell at different voltages and the corresponding control at 0 V (one-way analysis of death, i.e. irreversible electroporation. Exposure variance [ANOVA] followed by Holm-Sidak post-hoc test, (p < 0.05) and within the of cells to higher pulse amplitudes caused higher pair of samples at different voltages (t-test, p < 0.05), respectively). 322 FIGURE 6. Release of DNA as a function of electric pulse amplitude. Three-time points after electroporation were assessed. Permeabilization and survival curves are also presented. Approximate baseline of DNA is presented with -----. Black and green asterisks (*) indicate statistically significant differences between the samples at different voltages and the corresponding control at 0 V (one-way analysis of variance [ANOVA] followed by Holm-Sidak post-hoc test, (p < 0.05) and within the pair of samples at different voltages (t-test, p < 0.05) respectively. release of RNA (R = -0.909 for 15 min, R = -0.909 for 30 min, R = -0.919 for 24 h) and weak correlation between permeabilization and release of RNA (R = 0.584 for 15 min, R = 0.696 for 30 min, R is not sig­nificant for 24 h), respectively. Similarly, for DNA, a strong correlation between survival and release of DNA (R = -0.935 for 15 min, R = -0.919 for 30 min, R = -0.928 for 24 h) and a weak correlation between permeabilization and release of DNA (R = 0.571 for 15 min, R = 0.689 for 30 min, R is not significant for 24 h) was found. This correlation may indicate that loss of nucleic acids results in cell death. Comparison of the concentration of RNA de­tected in supernatant 15, 30 minutes and 24 hours after pulse treatment did not show any significant differences. Comparison of the concentration of DNA detected in supernatant 15, 30 minutes and 24 hours after pulse treatment showed only signifi­cant differences between 15 and 30 minutes at 600 V, yet interestingly this was not the case between 15/30 minutes and 24 hours after pulse treatment. According to our results, the released nucleic acids in vitro are stable and are not degraded within 24 hours after pulse treatment. We also tried to detect uric acid, another well know DAMP molecule. The release of uric acid in supernatant was analyzed 24 hours after pulse treatment (Figure 7). In our experiments we were however unable to detect any uric acid in superna­tant after pulse treatment. Initial experiments were also performed after 30 minutes, but results were the same (data not shown). Discussion Besides the induced membrane permeabilization, followed by cell death, activation of the immune response seems to be an important component in effectiveness of ECT26,35-37 and IRE18,30-34 treatment in vivo. Activation of the immune system can be trig­gered by a special type of cell death, called immu­nogenic cell death (ICD) in which DAMPs are the key mediators.64 Presence of DAMPs after pulse treatment has been detected in different types of cancer cells and normal tissues.22-29 However, only one study was performed with 100 us pulses, which are predominantly used in ECT and IRE.26 The au­thors investigated release of ATP, calreticulin and HMGB1 due to electroporation pulses alone, bleo­mycin alone and combination of electroporation pulses and bleomycin. Their study demonstrated the release of ATP and calreticulin after pulse treat­ment, but how this correlates to reversible and/or irreversible electroporation remained elusive. This question is addressed in our study, were release/ detection of different DAMPs was correlated with permeabilization and survival curve in vitro. Detected amounts of DAMPs were correlated to cell membrane permeabilization determined by PI assay immediately after pulse treatment and cell survival, analyzed 24 hours after treatment by 323 MTS test, i.e. to reversible and irreversible elec­troporation, respectively. It is generally believed that membrane permeabilization and cell survival after pulse treatment are causally related, i.e. in IRE cell death occurs due to membrane permeabiliza­tion and loss of cell homeostasis (in this study cor­relation coefficient between the two is -0.680) and in ECT increased accumulation of drug leads to in­creased cell cytotoxicity. In ECT and IRE it was also demonstrated that the immune response plays an important role in achieving therapeutic effect.33-36 We have therefore determined different DAMPs at different times after exposing the cells to electric pulses and de­termined whether the concentrations of extracel­lular DAMPs were better correlated to cell death or to membrane permeabilization. While activation inflammatory response and activation of immune system is desired in cancer therapies, on the other hand it can be a wanted or an unwanted effect in gene therapy.68,69 In DNA vaccination therapies, changed permeability of cell membrane enhances the introduction of DNA vaccine inside of cell and the presence of DAMPs additionally activates in­flammatory response, which leads to enhanced production of antibodies, thus enhancing the ef­ficiency of vaccination.65-67 Nevertheless, in most cases of gene therapy, the immune response in un­wanted, as it may destroy the transfected cells and prevent transgenic protein expression.68,69 By now many DAMPs have been identified and the number is still increasing. Most known are the HMGB1, nu­cleic acids, proteins like heat-shock proteins, S100 and calreticulin, purine metabolites like ATP and uric acid and saccharides. A list of know DAMPs and their receptors is given by Roh and Sohn.48 ATP is a well-known molecule in biology and biochemistry for being a universal energy source in the cell and necessary for multiple cell processes and cell metabolism. Interestingly, first studies of electroporation and increase in plasma membrane permeability involved adenosine triphosphate ATP detection in electroporation buffer.70 The released ATP is also considered a DAMP. In our study two types of ATP detection methods with different sen­sitivities were used.71,72 In a previous study per­formed by Calvet et al.26 using the same pulses as in our study, the release of ATP was detected 30 min­utes after the treatment. We were able to confirm their observations with the fluorescence and the luminescence method (Figures 2 and 3 respective­ly). Furthermore, the investigation of the effects at different pulse amplitudes showed that the release of ATP increases with increasing amplitude. This was expected, as ATP release was previously used as a permeability marker after electroporation.70 However, in our results statistical differences be­tween the control and the treatment groups were not detected in the range where the permeabiliza­tion curve is ascending, but was detected only in the range of pulse amplitudes at which all cells were already permeabilized and many were dead. Since ANOVA analysis is less sensitive when large amount of samples with big differences between them are analyzed, additional ANOVA was per­formed, taking into account only the results from 0 to 300 V (e.g. where permeabilization changes from 0 to 100%). Now additional analysis showed that statistical differences between control and the treatment groups are present at 200 V and above in luminescent method, suggesting ATP release as possible membrane permeabilization detection method. In the fluorescence method, this statisti­cal difference was obtained only at 300 V. Such dif­ference in analysis and also a bigger ratio of ATP between the control and the treatment groups indi­cates that the luminescence method is more sensi­tive method than the fluorescence method. Taken into consideration ATP release at all investigated voltages (also the one leading to cell death after 24 hours) the release of ATP is more strongly corre­lated to cell death/irreversible electroporation (R = 0.888) than permeability/reversible electroporation (R = -0.695). 24 hours after pulse treatment (Figure 2) the highest amount of ATP was detected in the super­natant of control sample and the amount of ATP decreased with increasing pulse amplitude. This can be explained by homeostasis of ATP in living cells. In a living homeostatic cell most of the ATP is located intracellularly, however in considerably lower concentration ATP is also present in extracel­lular space.73 When cells are damaged, considerable release of ATP molecule affects ATP pumps, caus­ing depletion of intracellular K+ and accumulation of intracellular NA+ and Ca2+ and leading to cell death.74 A previous study showed that electropo-ration pulses cause ATP depletion, which in 24 re­sults in lower viability, presumingly by affecting Ca2+-ATPase.75 In our study the effect on survival was also confirmed by very strong positive corre­lation between survival/irreversible electropora­tion and amount of ATP detected in supernatant (R = 0.888). Nevertheless, we need to consider, that some of the ATP detected in supernatant could be from the cells damaged due to cell handling during experiment. In extracellular space ATP is degraded by nucleotides like CD39 and CD37, which convert 324 ATP through ADP and AMP to adenosine73, which explains why ATP was detected 30 minutes at very high voltages (500, 600 V), but was no longer de­tected after 24 hours (Figure 2). This can also ex­plain why Calvet et al.26, was unable to detect ATP 30 hours after pulse treatment alone, however it does not explain, why ATP was still detected when bleomycin alone or in combination with electropo-ration pulses was used. How fast ATP degrades in extracellular space, remains unknown. Our results do not indicate that ATP degrades within the first 30 minutes after pulse treatment, since no differ­ence between 15 and 30 minutes after pulse treat­ment was detected. In a different study76 the results for ATP 4 hours after pulse treatment was lower in samples exposed to pulse treatment than in the control. If this is taken into consideration together with our results, then ATP degradation in vitro oc­curs somewhere between 30 minutes and 4 hours after pulse treatment. Calreticulin was another molecule of interest in our study. This highly conserved protein has major functions in lumen of the endoplasmic reticulum (ER). It is involved in correct folding of proteins that are produced in endoplasmic reticulum77 and in regulation of calcium metabolism, as it affects Ca2+ capacity of the ER stores.78 In the early phase of cell death, activated ER stress leads to transloca­tion of calreticulin to cells surface trough ER-Golgi pathway or lysosome exocytosis.79,80 Calreticulin, as DAMP, was investigated previously in electropo-ration studies.22-26,28 In Calvets’s study26, which used the same pulses as in our study (eight 100 µs pulses), calreticulin was determined 30 hours after treatment using different treatments. Calreticulin was detected on the plasma membrane after elec­troporation pulses alone or in combination with bleomycin (ECT), yet no externalization was de­tected in cells threated with bleomycin alone. Since only calreticulin, exposed on the cell surface acts as a DAMP, only viable cells (determined by PI) were taken into analysis. The presence of calreti­culin on the cell surface was previously detected already 4 hours after electroporation with milli­second pulses28, thus we assumed 4 hours is suf­ficient time for calreticulin to transfer to cells sur­face. Additionally, calreticulin was detected also 24 after pulse treatment. In our study calreticulin was investigated 4 and 24 hours after treatment (Figure 4), which is after the resealing of cell mem­brane.63 Even though calreticulin was detected on the surface of live cells, it was detected only in the range of irreversible electroporation. Additionally, calreticulin detection increased with decreasing cell viability (less viable cells), implicating that big­ger stress or in this case pulse amplitude causes more calreticulin molecules to be externalized to cell surface. Nevertheless, since it is believed that externalization of calreticulin occurs in early phase of cell death79,80, it is possible that cell determined as viable would die within next hours. In comparison to ATP, calreticulin is more sta­ble. Only at 300 V the difference between 4 and 24 hours was statistically significant. Stability of externalized calreticulin was previously also con­firmed in another in vitro study.24 Nevertheless, in vivo study shows expression is the strongest be­tween four and six hours, and diminishes 24 h after the treatment.28 So far studies investigating DAMPs, released by the electroporation treatment, included ATP, calreticulin and HMGB1. In addition to ATP and calreticulin we also included other known DAMPs in our study, namely nucleic acids and uric acid, which so far have not been investigated as DAMPs after electroporation. Inside the cells nucleic acids are the source of genetic information. As DAMPs in extracellular space nucleic acids bind to TLR receptors. Bound DNA can even attract HMGB1 (a non-histone nuclear protein, which can be ac­tively or passively released into extracellular space, where it acts as a DAMP81) and together they form complexes stimulating dendritic cells to produce type 1 interferon (non-specific immune response), which can lead to anti-DNA autoan­tibody production (specific immune response).82 Nucleic acids (RNA and DNA) can be detected in supernatant already within minutes after pulse treatment (Figure 5,6). Nevertheless, we need to consider – based on the control, 0 V in Figures 5 and 6, that some of the nucleic acids detected in supernatant could be from the cells damaged due to cell handling during experiment. Since RNA is more abundantly present in cells than DNA83,84, the same was expected to be the case in the super­natant after pulse treatment. However, the amount of detected DNA in our samples was bigger than that of RNA. Since RNA is more prone to degra­dation than DNA84, it is possible that some of the RNA was destroyed during the process of analy­sis. Nevertheless, the amount of released nucleic acids increases with increasing voltage of electric pulses to which the cells were exposed. Our results indicate that the release of nucleic acids (RNA and DNA) occurs in the range of irreversible electropo-ration; i.e. pulse amplitudes that lead to cell death as determined by MTS test at 24 h post treatment. This was confirmed also by very strong negative 325 correlation between the cell survival and the re­lease for RNA and DNA. Uric acid is a product of purine metabolism within the cell, like degradation of nucleic acids, and is released from injured and dying cells.85 A molecule that is soluble inside the cell, accumu­lates in extracellular space, where it is transformed in insoluble crystal of monosodium urate, stimu­lating the maturation of dendritic cells and T-cell response.85,86 Here, the presence of uric acid after electroporation was investigated for the first time. Presence of uric acid in supernatant was inves­tigated 24 hours after electroporation treatment (Figure 7). We expected uric acid to show a simi­lar behavior in pulse parameter dependency as other DAMPs. However, we did not detect uric acid in supernatant after pulse treatment. Standard curve was obtained, therefore Uric Acid Assay Kit worked. Maybe uric acid production did not hap­pen or uric acid was still inside of cells and not yet in supernatant as predicted. Furthermore, we found no existing data on CHO cells and uric acid in the literature, so maybe formation of uric acid in ovarian cells does not occur. With respect to the results obtained, detection of DAMPs and its correlation to cell membrane per-meabilization and cell survival seems to be more complex than initially thought. Even a DAMP like ATP, which can be released due to electropo-ration alone is better correlated to cell survival than membrane permeabilization (Tables 1, 2). A recent study performed by Ringel-Scaia et al.27, in which multiple signaling pathways were ana­lyzed, showed that the cell and cell population is a dynamic system which changes with time. Two hours after pulse treatment RNA analyses showed activation of immunosuppressive pathway, cell in­jury and apoptosis. With time these genes became less pronounces and after 24 hours change in gene expression indicated proinflammatory response, cell repair and necrosis/pyroptosis. This explains changes in DAMPs detection hours after pulse treatment, including the presence and absence of different DAMPs and its correlation with cell sur­vival. Since statistical correlations between DAMP release and cell survival is much stronger than with membrane permeabilization, involvement of immune system in IRE can be explained. However, activation of immune system was demonstrated also in ECT treatments, were reversible electropo-ration is used. How can that be, if correlation be­tween membrane permeabilization and released DAMPs is weak or does not even exist? Modeling87 and in vivo experiments88 show that application of TABLE 1. Correlation (R) between survival and release of damage-associated molecular pattern molecules (DAMPs) after pulse treatment. Investigated time points for each molecule are presented in the bottom row. Correlation was evaluated with Pearson correlation coefficient and survival was analyzed via MTS assay 24 hours after pulse treatment PI -0.680 ATP -0.947 (L) -0.964 (L)/-0.864 (F) 0.888 (F) DNA -0.935 -0.919 -0.928 RNA -0.909 -0.909 -0.919 CRT -0.801 -0.946 uric acid NS time points after EP 3 min 15 min 30 min 4 h 24 h ATP = adenosine triphosphate; CRT = calreticulin; (F) = fluorescence assay; (L) = luminescence assay; NS = no statistical significance; PI = propidium iodide TABLE 2. Correlation (R) between permeabilization and release of damage-associated molecular pattern molecules (DAMPs) after pulse treatment. Investigated time points for each molecule are present in the bottom row. Correlation was evaluated with Pearson correlation coefficient and permeabilization was analyzed by propidium iodide (PI) assay 3 minutes after pulse treatment MTS -0.680 ATP 0.704 0.728 (L)/0.594 (F) -0.695 (F) DNA 0.571 0.689 NS RNA 0.584 0.696 NS CRT 0.535 0.556 uric acid NS time points after EP 3 min 15 min 30 min 4 h 24 h ATP = adenosine triphosphate; CRT = calreticulin; (F) = fluorescence assay; (L) = luminescence assay; NS = no statistical significance nominally reversible electroporation pulses such as those used for ECT of tumors still causes some cell death by means of irreversible electroporation in tissue close to the electrodes, due to inhomoge­neous electric field distribution, which can thus lead to the release of DAMPs and activation of the immune system. The aim of this study was to explore, if and when specific DAMPs are released as a conse­quence of electroporation and if the release of DAMPs can be correlated to reversible and/or ir­reversible electroporation. Even though detection of certain DAMPs remains uncertain, others show strong correlation to cell survival/irreversible elec- 326 troporation and much weaker correlation to mem­brane permeabilization/reversible electroporation. Release of DAMPs could perhaps serve as a pre­dictor of cell death. In addition, it may indicates that the stability of certain DAMPs is questionable and thus their presence and detectability is time dependent. This needs to be taken into considera­tion when designing protocols to detect DAMPs after electroporation treatment. Finally, to obtain a better insight of DAMP release with respect to electroporation treatment other cell types includ­ing also cancer cell types should be investigated. Acknowledgements Authors would like to thank L. Vukanovic and D. Hodžic for their help in the cell culture laboratory and Dr. Matej Reberšek for help with pulse record­ing and image production. 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Sci Rep 2019; 9: 3649. doi: 10.1038/s41598-019-40395-y 329 research article Impact of COVID-19 on cancer diagnosis and management in Slovenia – preliminary results Vesna Zadnik1,3, Ana Mihor1, Sonja Tomsic1, Tina Zagar1, Nika Bric1, Katarina Lokar1, Irena Oblak2,3 1 Epidemiology and Cancer Registry, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Department of Radiation Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 3 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2020; 54(3): 329-334. Received 8 July 2020 Accepted 15 July 2020 Correspondence to: Prof. Vesna Zadnik, M.D., Ph.D., Epidemiology and Cancer Registry, Institute of Oncology Ljubljana, Zaloška cesta 5, SI-1000 Ljubljana, Slovenia. E-mail: vzadnik@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. The COVID-19 pandemic has disrupted the provision and use of healthcare services throughout the world. In Slovenia, an epidemic was officially declared between mid-March and mid-May 2020. Although all non-essential health care services were put on hold by government decree, oncological services were listed as an exception. Nevertheless, as cancer control depends also on other health services and additionally major changes in people’s behaviour likely occurred, we aimed to analyse whether cancer diagnosis and management were affected during the COVID-19 epidemic in Slovenia. Methods. We analysed routine data for the period November 2019 through May 2020 from three sources: (1) from the Slovenian Cancer Registry we analysed data on pathohistological and clinical practice cancer notifications from two major cancer centres in Ljubljana and Maribor; (2) from the e-referral system we analysed data on all referrals in Slovenia issued for oncological services, stratified by type of referral; and (3) from the administrative data of the Institute of Oncology Ljubljana we analysed data on outpatient visits by type as well as on diagnostic imaging per­formed. Results. Compared to the November 2019 – February 2020 average, the decrease in April 2020 was about 43% and 29% for pathohistological and clinical cancer notifications; 33%, 46% and 85% for first, control and genetic counselling referrals; 19% (53%), 43% (72%) and 20% (21%) for first (and control) outpatient visits at the radiotherapy, surgery and medical oncology sectors at the Institute of Oncology Ljubljana, and 48%, 76%, and 42% for X-rays, mammograms and ultrasounds performed at the Institute, respectively. The number of CT and MRI scans performed was not affected. Conclusions. Significant drops in first referrals for oncological services, first visits and imaging studies performed at the Institute, as well as cancer notifications in April 2020 point to a possibility of a delayed cancer diagnosis for some patients during the first surge of SARS-CoV-2 cases in Slovenia. The reasons for the delay cannot be ascertained with certainty and could be linked to health-seeking behaviour of the patients, the beliefs and practices of doctors and/ or the health system management during the epidemic. Drops in control referrals and control visits were expected and are most likely due to the Institute of Oncology Ljubljana postponing non-essential follow-ups through May 2020. Key words: cancer; COVID; delay in diagnosis; referral Introduction Many cancer experts have highlighted the prob­lem of access to and utilisation of cancer care ser­vices during and after the COVID-19 pandemic.1–3 Control measures are effective at containing the spread of disease, and once extensive community transmission of the virus occurs they undoubtedly contribute to preserving cancer services through protecting the health system from collapsing, al­though they are expected to also have negative effects for cancer control. In Slovenia, a middle 330 European country of approximately two million inhabitants with universal health care, the response to COVID-19 epidemic was swift and included changes in the functioning of the health care sys­tem that potentially affected cancer diagnosis and management. An overview of the COVID-19 epidemic in Slovenia The first confirmed COVID-19 patient in Slovenia was registered on the 4th of March 2020. The first cases were imported, though soon, secondary, tertiary and quaternary transmissions of the nov­el virus were detected and on the 12th of March, the Health Minister following the advice of the National Institute of Public Health (NIPH) de­clared an epidemic, which meant the activation of the Slovenian Pandemic Plan. Control measures implemented thereafter were strict and introduced rapidly with the aim of mitigating the spread of COVID-19. On the 16th of March, all schools and educational institutions were closed, all public transport services stopped and all non-essential services shut. Soon after, all gatherings of people were prohibited, with the exception of members of the same household, working from home was encouraged and restrictions on movement of peo­ple were put in place limiting movement to within their municipality (lock-down). Measures concerning the provision of health care services were enacted through the Ordinance on temporary measures in health care to contain and con­trol the COVID-19 epidemic4 from the 20th of March, which stipulated that all non-essential ambulatory visits (those not referred as needing urgent or very fast management) and elective surgery appoint­ments be put on hold. Oncological services were listed as an exception, though all preventive care activities were also put on hold by decree, mean­ing all three cancer screening programmes (cervi­cal, breast and colorectal cancer) were temporar­ily stopped. Screening was stopped also in other countries.5,6 At the Institute of Oncology Ljubljana, the only tertiary comprehensive cancer centre, COVID-19 preventive measures were being con­tinually introduced and adapted starting on 26th of February. A triage, at first only physical and later also via telephone, was set up to screen patients for COVID-19 symptoms, relative escorts of patients to the hospital and visits of hospitalised patients were not allowed, except for dying patients, while non-essential follow-up visits and surgeries were postponed through May. Despite this, work at the Institute continued almost uninterrupted. Similar measures were taken by oncology departments across Europe7,8 and many highlighted the need for stricter measures and more testing with the aim of keeping cancer clinics COVID-free given reports of the higher risk COVID-19 poses to people with cancer9 and in order to maintain the provision of oncological services.10,11 Towards the end of March, the epidemic peaked with daily cases starting to decrease. In the second half of April, easing of control measures in the country started and on the 9th of May, the govern­ment lifted restrictions on provision of healthcare services. Following this, on the 15th of May Slovenia declared an end to the epidemic. During this time, the Institute of Oncology Ljubljana continued with normal follow-up and surgeries, also introduc­ing working Saturdays to make up for the delay in these services. Furthermore, cancer screening programmes gradually began sending invitations again and were operating close to or at full capacity in June 2020. Aim of the study In light of severe restrictions in movement of in­dividuals, cancellation of non-essential health care services and ensuing behavioural responses among the population, there might be collateral conse­quences of COVID-19 related measures for cancer control, despite the Institute of Oncology Ljubljana having retained almost normal functioning. In or­der to gain a quick and timely understanding of how cancer care in Slovenia has been affected by the COVID-19 epidemic, we carried out an analysis on readily available, up-to-date and reliable data sources. Methods We carried out an analysis of data from the Slovenian Cancer Registry, the e-referral system of Slovenia, managed by the NIPH, and the admin­istrative hospital data of the Institute of Oncology Ljubljana. Using this data, we evaluated referrals for first and control oncological examination and treatment from all levels of healthcare, as well as cancer diagnosis and treatment at tertiary level on­ly. The observed period was from November 2019 through May 2020. The Slovenian Cancer Registry is one of the old­est cancer registries in Europe, operating since 1950. In 2018, the transition from passive to active regis- 331 tration started, which allows for up-to-date data on 600 4500 cancer notifications. This is an important feature, considering the need for real-time analysis of data to be able to inform decision-makers regarding the measures for COVID-19 control. From the Cancer Registry, we extracted data on monthly cancer no­ tifications from the two major oncological centres in Slovenia, the Institute of Oncology Ljubljana and the University Medical Centre Maribor which are included in the active registration. The Ljubljana and Maribor oncological centres cover a major part of newly diagnosed cancers in Slovenia. Two types of cancer notifications were evaluated: those from No. of referrals (first and gen. counselling) 500 800 nificant reduction in the number of referrals can be seen in April, with a somewhat smaller reduction 600 in March. The reduction was seen for all types of 400 300 200 1500 1000 100 pathohistological departments and those from clinical setting. The second source was the NIPH e-referral sys­tem. We accessed the data from the e-referral sys­tem on all monthly referrals issued in Slovenia for selected types of oncological health services as cod­ed in the Codebook of healthcare services, namely the Oncological examination – first, Oncological examination – control and Oncological genetic test­ing and counselling. As Slovenia has a gate-keep­ing system in place, where secondary and tertiary care is only possible through referrals, this means the number of referrals is an accurate reflection of demand for specialist oncological care. Finally, from the administrative data of the Institute of Oncology Ljubljana we analysed data on monthly patient visits, stratified according to first and control outpatient visits, and data on can­cer diagnostic imaging, namely the monthly num­ber of X-rays, mammograms, ultrasounds, CT and MRI scans performed. Results and discussion Referral for oncological examination and treatment Figure 1 shows the time trend of monthly referrals during November 2019 – May 2020 where a sig­ 0 0 Nov 19 Dec 19 Jan 20 Feb 20 Mar 20 Apr 20 May 20 First Referral Genetic Counselling Referral Control Referral FIGURE 1. Referrals for oncological services stratified by first referral, control referral and referral for genetic counselling in Slovenian health-care system between November 2019 and May 2020. The drop in control referrals can most likely be explained as a consequence of the cancer insti­tutes’ policies to defer non-essential control visits. All patients were notified about their deferral and thus there was probably lower demand for control referrals from patients though other reasons could also play a role. Oncological genetic testing and counselling is a preventive service, meaning that doctors were probably less likely to refer patients for this type of care, since the decree on health care stipulated these services are temporarily dis­ 1600 1400 1200 No. of cancer notifications 1000 referrals, though significantly more pronounced for control referrals compared to first referrals, whereas referring for oncological genetic test­ing and counselling stopped almost completely. Compared to the November – February average, the decrease in April was about 33%, 46% and 85% for first, control and genetic counselling referrals, respectively. In May, the number of all types of re­ferrals started rising again. 400 200 0 Nov 19 Dec 19 Jan 20 Feb 20 Mar 20 Apr 20 May 20 Pathohistological Clinical FIGURE 2. All cancer notifications from pathohistological and clinical departments at the Institute of Oncology Ljubljana and University Medical Centre Maribor between November 2019 and May 2020. 332 No. of first outpatient visits 400 300 200 100 0 Nov 19 Dec 19 Jan 20 Feb 20 Mar 20 Apr 20 May 20 Radiotherapy Surgery Medical Oncology FIGURE 3. First outpatient visits to the Institute of Oncology Ljubljana stratified by type of sector (radiotherapy, surgery, and medical oncology) between November 2019 600 doctors in order to complain about their issues. Another factor could be that primary level doctors 500 were less likely to refer symptomatic patients for secondary and tertiary diagnostics because these services were not freely available and most of the first symptoms of cancer are rather unspecific. A combination of these factors was likely at play. As a result, we fear that fewer cancers were diagnosed in early stages. Other countries have reported similar findings. In the UK, a dermatology service found a reduc­tion in referrals for skin cancer of more than 50% in April 2020 compared to April 2019. Additionally, they analysed referrals for other types of cancer in their hospital and found similar reductions for a wide range of cancers, most pronounced for colo-rectal cancer.12 Also in the UK, others have shown that in the whole country, urgent referrals for can- and May 2020. cer from GPs fell by 60% in April.13 In Italy, the re­ferrals for BRCA testing to a genetic laboratory had 4.000 decreased by about 60%.14 3.500 Delay in diagnosis No. of control outpatient visits 3.000 2.500 2.000 1.500 1.000 500 0 Nov 19 Dec 19 Jan 20 Feb 20 Mar 20 Apr 20 May 20 Radiotherapy Surgery Medical Oncology FIGURE 4. Control outpatient visits to the Institute of Oncology Ljubljana stratified by Figure 2 shows the trend in the number of cancer notifications from pathohistological and clinical departments sent to the Cancer Registry from the two main cancer centres, Institute of Oncology Ljubljana and University Medical Centre Maribor. Again, the same pattern can be observed with the largest decrease observed for April and an upward trend in May. Compared to the November 2019 – February 2020 average, the decrease in April was about 43% and 29% for pathohistological and clini­cal cancer notifications, respectively. The absolute number of new notifications is not equivalent to the number of newly diagnosed cancers because a cancer case can be reported to the Cancer Registry more than once from differ- type of sector (radiotherapy, surgery, and medical oncology) between November 2019 and May 2020. ent healthcare providers that come into contact continued, even though oncological services were clearly listed as an exception. Patients themselves were also less likely to seek services for non-urgent care during lock-down. The reasons behind the drop in first referrals are difficult to determine. It is possible that, compared to pre-epidemic period, during lock-down, people were less likely to seek medical care even if they experienced symptoms of disease. On the other hand, access to primary and secondary level care could have been so disrupted that some patients could not get through to their with a patient with a cancer, while on the other hand, a small part of notifications turn out not to be malignant cases after additional investigations by the Cancer Registry. Despite this, the relative decline in new notifications can be interpreted as a decrease in newly diagnosed cancers. Roughly, this means in April there were about a third fewer cancers diagnosed in Slovenia compared to the average pre-epidemic period. The reasons for the lower number of cancer notifications are likely re­lated to the drop in referrals. It is not surprising therefore, that the maximum drop in referrals and the maximum drop in newly diagnosed cancers are concurrent. Perhaps the time shift might have 333 been visible if we stratified the data into weeks in-1.600 stead of months, because it takes a week or two for patients who are referred for oncological exam to 1.400 be diagnosed with cancer. No doubt, another fac­ tor for the drop in notifications was the temporary two-month long complete cessation of all three cancer screening programmes, though at the mo­ ment it is not possible to quantify what proportion of delayed cancer diagnoses could be attributed to lack of access to cancer screening. Our results are in line with a study from the Netherlands, where the Netherlands Cancer Registry recorded a decrease in weekly patho- No. of diagnostic imaging procedures 1.200 1.000 800 600 400 logical cancer notifications between the end of February and start of April 2020. The decrease was observed for all age groups and all cancer groups but was largest (max. 60%) for skin cancer (exclud­ing basal cell carcinoma), followed by breast can­cer (max. 50%). The largest weekly decrease for all cancers excluding skin cancer was approximately a quarter.15 Fewer cancer diagnoses were reported also in Italy. A Pathologic Anatomy Unit in the province of Macerata recorded a decrease in patho-histological diagnoses of cancer during weeks 11­20 (March and April) of 2020 compared to the same period in 2018 and 2019. Unlike in the Netherlands, they did not observe a decrease for malignant mel­anoma but observed the highest drops for prostate (75%), bladder (66%) and colorectal cancer (62%). Clinically relevant delay was considered only for colorectal cancer. Interestingly, screening for colo-rectal cancer was disrupted in Italy but was more preserved for breast cancer, which saw a reduction of (only) 25%.16 Diagnostics and treatment Administrative data from the Institute of Oncology Ljubljana are presented in Figures 3–5. Compared to the November 2019 – February 2020 average, the decrease in first outpatient visits in April 2020 was 19%, 43% and 20% at the radiotherapy, sur­gery and medical oncology sectors of the Institute, respectively, whereas for control outpatient visits, these numbers were 53%, 72% and 21%. Visits to the medical oncology department, where patients receive active chemotherapy treat­ment, were least affected. The largest drop in both first and control visits can be observed for the surgical department. These results are expected, as all truly elective surgeries at the Institute were postponed, though we cannot say if a part of the decrease in the first visits could also to a minor degree reflect less patients having been diagnosed 200 0 Nov 19 Dec 19 Jan 20 Feb 20 Mar 20 Apr 20 May 20 X-rays Mammograms Ultrasounds CT MRI FIGURE 5. The number of X-rays, mammograms, ultrasounds, CT and MRI scans performed at the Institute of Oncology Ljubljana between November 2019 and May 2020. with cancer and planned for surgery as part of their primary treatment. The decline in first visits to ra­diotherapy and medical oncology departments was small but could also point to fewer (newly di­agnosed) patients being treated. In general, reduc­tions in control outpatient visits were expected due to either postponing non-essential follow-up visits or carrying them out as telehealth visits. For radio­therapy, it might be also indicative of the rationali­sation in radiotherapy regimes (such as fewer frac­tions of radiotherapy). Outpatient visits to oncological centres must have declined across Europe, though we could not find any already published European study which reported on the number of cancer outpatient visits. A report from the US shows that oncology outpa­tient visits had fallen by as much as 47% in April 2020.17 Regarding diagnostic imaging, in April 2020 compared to the November 2019 – February 2020 average, there were also significant reductions in X-rays (48%), mammograms (76%) and ultrasounds (42%) performed at the Institute. This could again point to fewer patients being in the diagnostic pro­cess though there were changes in the functioning of the Institute that could also have contributed to this result. The numbers of CT and MRI scans were not affected. The reduction in diagnostic imaging was thus most pronounced for mammography, which is only in part linked to the suspension of 334 breast cancer screening, as mammograms within the screening programme are tallied separately. Conclusions Significant drops in first oncological referrals, first outpatient visits, x-rays, mammograms and ultra­sounds as well as cancer notifications from the two major cancer centres all point to a delay in diag­nosis and treatment of cancer for some patients during the COVID-19 epidemic in Slovenia. The reasons that lead to this decline cannot be assessed in our study but are presumed to be a combina­tion of COVID-19 related factors on the side of the patients and doctors as well as the health care sys­tem and its management during the peak of the crisis. To what extent the pausing of screening pro-grammes influenced cancer diagnosis should be evaluated at least after six months of restarting the programmes. The drop in control referrals and vis­its is not as relevant clinically and was an expected outcome in light of the decision to postpone non-urgent care. Long-term studies are needed in order to evaluate what the effects of the perceived delay in diagnosis and treatment during the COVID-19 epidemic will be in terms of classical cancer bur­den indicators, such as poorer survival or a shift to­ward more advanced stage at diagnosis for specific cancer types. For example, projections for the US show that cumulative excess deaths from colorec­tal and breast cancers between 2020 and 2030 could be around 1%18, highlighting the need for extreme caution when deciding on what measures to adopt if/when subsequent surges in COVID-19 cases oc­cur so as to not significantly disrupt cancer control services also in the future. References 1. Amit M, Tam S, Bader T, Sorkin A, Benov A. Pausing cancer screening during the severe acute respiratory syndrome coronavirus 2 pandemic: should we revisit the recommendations? Eur J Cancer 2020; 134: 86-9. doi: 10.1016/j. ejca.2020.04.016 2. Vanni G, Pellicciaro M, Materazzo M, Palombi L, Buonomo OC. Breast cancer diagnosis in coronavirus-era: alert from italy. Frontiers Oncol 2020; 10: 938. doi: 10.3389/fonc.2020.00938 3. Vrdoljak E, Sullivan R, Lawler M. Cancer and coronavirus disease 2019; how do we manage cancer optimally through a public health crisis? Eur J Cancer 2020; 132: 98-9. doi: 10.1016/j.ejca.2020.04.001 4. Ordinance on interim measures in the field of health activities to contain and control the COVID-19 epidemic.[Slovenian]. Uradni list Republike Slovenije; 32/20; Ljubljana; 2020. [cited 2020 Jun 30]. Available at https:// www.uradni-list.si/glasilo-uradni-list-rs/vsebina/2020-01-0645/odlok-o­zacasnih-ukrepih-na-podrocju-zdravstvene-dejavnosti-zaradi-zajezitve-in­obvladovanja-epidemije-covid-19 5. Del Vecchio Blanco G, Calabrese E, Biancone L, Monteleone G, Paoluzi OA. The impact of COVID-19 pandemic in the colorectal cancer prevention. Int J Colorectal Dis 2020; [Ahead of print] 4 Jun 2020. doi:10.1007/s00384-020­03635-6 6. World Health Organization. Rapid assessment of service delivery for NCDs during COVID-19 pandemic. Geneva; 2020. 7. Fong D, Rauch S, Petter C, Haspinger E, Alber M, Mitterer M. Infection rate and clinical management of cancer patients during the COVID-19 pandemic: experience from a tertiary care hospital in northern Italy. ESMO Open 2020; 5: e000810. doi: 10.1136/esmoopen-2020-000810 8. van de Haar J, Hoes LR, Coles CE, Seamon K, Frling S, Jäger D, et al. Caring for patients with cancer in the COVID-19 era. Nat Med 2020; 26: 665-71. doi: 10.1038/s41591-020-0874-8 9. Liang W, Guan W, Chen R, Wang W, Li J, Xu K, et al. Cancer patients in SARS­CoV-2 infection: a nationwide analysis in China. Lancet Oncol 2020; 21: 335-7. doi: 10.1016/S1470-2045(20)30096-6 10. Restivo A, De Luca R, Spolverato G, Delrio P, Lorenzon L, D’Ugo D, et al. The need of COVID19 free hospitals to maintain cancer care. Eur J Surg Oncol 2020; 46: 1186-7. doi: 10.1016/j.ejso.2020.04.003 11. Mahase E. Covid-19: cancer research urges mass testing to enable care to continue during pandemic. BMJ 2020; 369: m1561. doi: 10.1136/bmj. m1561 12. Earnshaw CH, Hunter HJA, McMullen E, Griffiths CEM, Warren RB. Reduction in skin cancer diagnosis, and overall cancer referrals, during the COVID-19 pandemic. Br J Dermatol 2020; [Ahead of print] 4 Jun 2020. doi:10.1111/ bjd.19267 13. Mahase E. Covid-19: Urgent cancer referrals fall by 60%, showing “brutal” impact of pandemic. BMJ 2020; 369: m2386. doi: 10.1136/bmj.m2386 14. Minucci A, Scambia G, Santonocito C, Concolino P, Urbani A. BRCA testing in a genomic diagnostics referral center during the COVID-19 pandemic. Mol Biol Rep 2020; 47: 4857-60. doi: 10.1007/s11033-020-05479-3 15. Dinmohamed AG, Visser O, Verhoeven RHA, Louwman MWJ, van Nederveen FH, Willems SM, et al. Fewer cancer diagnoses during the COVID-19 epidemic in the Netherlands. Lancet Oncol 2020; 21: 750-1. doi: 10.1016/S1470-2045(20)30265-5 16. De Vincentiis L, Carr RA, Mariani MP, Ferrara G. Cancer diagnostic rates during the 2020 ‘lockdown’, due to COVID-19 pandemic, compared with the 2018–2019: an audit study from cellular pathology. J Clin Pathol 2020; [Ahead of print] 19 June 2020. doi: 10.1136/jclinpath-2020-206833 17. Mehrotra A, Chernew M, Linetsky D, Hatch H, Cutler D. The impact of the COVID-19 pandemic on outpatient visits: a rebound emerges. To the Point (blog), Commonwealth Fund 2020; Published Online First: 19 May 2020. [cited 2020 Jun 30]. Available at: https://www.commonwealthfund.org/ publications/2020/apr/impact-covid-19-outpatient-visits. doi: https://doi. org/10.26099/ds9e-jm36 18. Sharpless NE. COVID-19 and cancer. Science 2020; 368: 1290. doi: 10.1126/ science.abd3377 335 research article Breast cancer risk based on adapted IBIS prediction model in Slovenian women aged 40–49 years - could it be better? Tjasa Oblak4, Vesna Zadnik1,2, Mateja Krajc3,4, Katarina Lokar1, Janez Zgajnar2,5 1 Epidemiology and Cancer Registry, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia 3 Cancer Genetic Clinic, Institute of Oncology Ljubljana, Ljubljana, Slovenia 4 Faculty of Health Sciences, University of Primorska, Izola, Slovenia 5 Department of Surgical Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia Radiol Oncol 2020; 54(3): 335-340. Received 25 March 2020 Accepted 7 May 2020 Correspondence to: Prof. Janez Žgajnar, M.D, Ph.D., Department of Surgical Oncology, Institute of Oncology Ljubljana, Zaloška cesta 2, SI-1000 Ljubljana, Slovenia. E-mail: jzgajnar@onko-i.si Disclosure: No potential conflict of interest were disclosed. Background. The aim of the study was to assess the proportion of women that would be classified as at above-average risk of breast cancer based on the 10 year-risk prediction of the Slovenian breast cancer incidence rate (S-IBIS) program in two presumably above-average breast cancer risk populations in age group 40-49 years: (i) women referred for any reason to diagnostic breast centres and (ii) women who were diagnosed with breast cancer aged 40–49 years. Breast cancer is the commonest female cancer in Slovenia, with an incidence rate below European av­erage. The Tyrer-Cuzick breast cancer risk assessment algorithm was recently adapted to S-IBIS. In Slovenia a tailored mammographic screening for women at above average risk in age group 40–49 years is considered in the future. S-IBIS is a possible tool to select population at above-average risk of breast cancer for tailored screening. Patients and methods. In 357 healthy women aged 40–49 years referred for any reason to diagnostic breast cen­tres and in 367 female breast cancer patients aged 40–49 years at time of diagnosis 10-years breast cancer risk was calculated using the S-IBIS software. The proportion of women classified as above-average risk of breast cancer was calculated for each subgroup of the study population. Results. 48.7% of women in the Breast centre group and 39.2% of patients in the breast cancer group had above-average 10-year breast cancer risk. Positive family history of breast cancer was more prevalent in the Breast centre group (p < 0.05). Conclusions. Inclusion of additional risk factors into the S-IBIS is warranted in the populations with breast cancer incidence below European average to reliably stratify women into breast cancer risk groups. Key words: breast cancer; early detection; risk prediction model; tailored screening Introduction Breast cancer is the most common cancer in wom­en with more than 2 million new cases diagnosed worldwide in the year 2018 and therefore represents a major public health problem. In Europe alone, the number of women diagnosed with breast cancer in 2018 was approximately 523 000 with an estimated age-standardized incidence rate of 100.9/100 000.1 Breast cancer is the most common cancer in women in Slovenia as well and in 2016 there were 1386 new cases diagnosed. However, the age-standardized incidence rate in Slovenia is lower than European average rate (68.5/100 000 women).2,3 Mammographic screening is one of the estab­lished strategies to deal with the breast cancer problem in public healthcare. The Slovenian na­tional mammographic screening program offers biennial screening mammography to all women in the age group 50–69 years.4 336 However, approximately one sixth of breast cancer patients are diagnosed at age 40 to 49 years, on average with a more advanced stage at the time of diagnosis compared to patients diagnosed in the age group 50–69 years.2 Despite this fact, in Slovenia there is no organized mammographic screening program in this age group of women due to lack of convincing evidence that population mammographic screening reduces breast cancer mortality in women aged 40–49 years. Namely, ac­cording to European guidelines there is condition­al recommendation against breast cancer screening for women aged 40 to 45 years, and only condition­al recommendation for the screening for the age group 45 to 49 years.5 In Slovenia all breast cancer in the age group 40–49 years are diagnosed in regional diagnostic breast clinics whether due to symptomatic disease or as a result of opportunistic screening. Women can be referred to opportunistic screening by their gynaecologists or general practitioners based on family history of breast cancer or other risk factors. Women with high breast cancer risk (i.e. genetic predisposition) may opt for surveillance separately in a dedicated centre. To overcome the limitations of the population screening in younger women, tailored breast can­cer screening limited to women with an above-average breast cancer risk is one of the research options today. Based on the 2018 Slovenian recom­mendations on breast cancer prevention and treat­ment a tool is needed to stratify women according to 10-years breast cancer risk in three groups: pop­ulation risk, moderately increased risk and high-risk group, respectively.6 Only women at above the population risk should be offered screening before the age of 50. To improve identification of women at above-average risk of breast cancer, many breast cancer prediction models have been developed in the last three decades.7 The IBIS software, based on the Tyrer-Cuzick algorithm, is one of the most consist­ent, both in the general population and in familial setting.8-10 IBIS calculates breast cancer risk based on classical risk factors including age, family histo­ry of breast or ovarian cancer in first- and second-degree relatives, age at menarche and menopause, parity and age at first childbirth.8 Recently mam-mographic density and polygenic risk score were added as additional risk factors to be taken into account in the calculation of breast cancer risk.11,12 However, these two risk factors are very seldom available in routine clinical setting. The IBIS pro­gram was developed with breast cancer incidence rates of the United Kingdom and was recently separately adapted for the Swedish and Slovenian populations (S-IBIS software).13 The IBIS program was validated on several populations, varying both in age and geographic location.7,9,10,14,15 However, the performance of the recently adapted S-IBIS in Slovenian population is still unknown. We were particularly interested in S-IBIS performance in two presumably above the average breast cancer risk populations: (i) women aged 40 to 49 years referred for any reason to di­agnostic breast centres and (ii) women who were diagnosed with breast cancer between the ages of 40 to 49. The aim of our study was to conduct S-IBIS calculations in the two aforementioned groups of patients and determine the proportions of three 10-years breast cancer risk groups (population risk, moderately increased and high risk) in both group of patients. Patients and methods In this study two groups of patients were included: 1. 357 women aged 40–49 years attending oppor­tunistic screening in 5 diagnostic breast centres in central Slovenia in year 2014; 2. 367 women aged 40–49 at time of breast cancer diagnosis, treated at the Institute of Oncology Ljubljana between 2014 and 2019. Patients are regularly followed up in outpatient clinics of the Institute of Oncology Ljubljana. All women were asked to answer a question­ naire about established risk factors for breast cancer according to the IBIS requirements and family his­tory of breast and ovarian cancer concerning first- and second-degree relatives (Table 1). Women in the breast cancer group were specifically asked to provide data available at their age of 40. Personal history of breast cancer diagnosis was not included in the risk calculation for the breast cancer group. Mammographic density and polygenic risk score could not be included in the risk calculation due to unavailable data, therefore these fields were left blank. Results of genetic testing were also not in­cluded as the testing was usually performed after the diagnosis of breast cancer in the breast cancer group. Women from the Breast centre group did not fill the criteria for genetic testing and the test­ing was therefore not performed. Women with known genetic predisposition (i.e. BRCA and other mutations) were not included in the study. The majority of women who are carriers 337 TABLE 1. Breast cancer risk factors used for 10-year breast cancer risk calculation with S-IBIS software Risk factor Age (years) Height Weight Age at menarche (years) Age at first childbirth Menopausal status Hormone replacement therapy use Benign breast disorder Family history of breast cancer (breast cancer in first- and second-degree relatives and age at presentation) Family history of ovarian cancer (ovarian cancer in first-degree relatives and age at presentation) of a hereditary breast cancer related mutations are already followed up in a dedicated centre and they would not benefit from an improved population screening but may ultimately alter the proportion of women in the low and high-risk groups. The participants were informed about the mean­ing and use of the provided data and signed an in­formed consent. Based on the acquired data 10-year risk of breast cancer for each woman with the S-IBIS software was calculated. For the purpose of a separate sub analysis the patients were divided in two subgroups, age 40–44 and age 45–49. In the breast cancer group there were 125 participants in the 40–44 years age group and 242 participants in the 45–49 years group, while in the breast centre group there were 153 participants in the 40–44 years age group and 204 participants in the 45–49 years group. In breast cancer patient group the personal di­agnosis of breast cancer was not included in the calculation of risk. Breast cancer risk thresholds for the Slovenian population as described in the literature (popula­tion risk: below 2%, moderately increased risk: 2–6.5%, high risk: above 6.5%) were taken into ac­count for assessment of performance of the S-IBIS software.13 IBM SPSS Statistics v25 was used to generate data analysis. Mann-Whitney and Chi-square tests was used to assess statistically significant differ­ences in baseline data; p < 0.05 was considered sta­tistically significant. The study was approved by the National Ethics Committee. TABLE 2. Baseline characteristics of participants Age (yaars, mean)* 45.6 44.8 BMI, mean (kg/m2)* 24.3 24.8 Age at menarche (years, mean) 13.0 13.0 Nulliparity 10.5% 11.1% Age at first childbirth (years, mean) 23.0 23.4 Positive family history for breast and/or ovarian cancer* 48.8% 56.6% * statistically significant difference was observed between the two groups (p < 0.05); BMI = Body mass index TABLE 3. Risk stratification for all participants and for age subgroups 40–44 years and 45–49 years based on S-IBIS calculation for breast cancer patients and women screened in Breast centre; risk categories for women aged 40 to 49 as in 2018 Slovenian guidelines Breast cancer group - 10-year 60.8 % 37.8 % 1.4 % breast cancer risk (age 40–49) Breast centre group - 10-year 51.3 % 47.6 % 1.1 % breast cancer risk (age 40–49) Breast cancer group - 10-year 64.0 % 34.4 % 1.6% breast cancer risk (age 40–44) Breast centre group - 10-year 58.2% 41.8 % 0.0% breast cancer risk (age 40–44) Breast cancer group - 10-year 59.1 % 39.7 % 1.2 % breast cancer risk (age 45–49) Breast centre group - 10-year 46.1 % 51.9% 2.0% breast cancer risk (age 45–49) Results The baseline characteristics of the two groups re­garding the breast cancer risk factors are report­ed in Table 2. Statistically significant differences where noticed between the two groups while an-alysing age, body mass index (BMI) and positive family history for breast and/or ovarian cancer, with participants in the Breast centre group being younger, with higher BMI and positive family his­tory in more cases. The risk calculations for the whole popula­tion and within each age subgroup are shown in Table 3. Discussion S-IBIS risk calculation based on the included par­ticipants’ data identifies only 48.7% of women re­ferred to Breast centres as above population breast 338 cancer risk (10 years risk > 2%). Furthermore S-IBIS as used in our study identifies as above the popu­lation risk 39.2% of women, who were diagnosed with breast cancer. It should be once again noted that some data such as mammographic density and polygenic risk score (PRS) that could be included in the risk calculation, could not be retrieved for the participants of our study. Still, the identification of almost 40% breast cancer patients as at above-aver­age risk is a promising result, that is comparable to results of other studies.10, 16-19 However it is still wor­risome that as much as 60% of patients diagnosed with breast cancer in age group 40–49 would be di­agnosed outside the screening program if women were invited to breast cancer screening based on S-IBIS risk calculation as it could be widely avail­able at the present moment (that is, without data about mammographic density and PRS). Therefore our study showed that tailored mammographic screening in the age group 40–49 in Slovenian population cannot be organized based on this form of S-IBIS alone. Assuming the expected less than 100% attendance rate of the invited population and lower mammography sensitivity in this age group, the proportion of diagnosed cancers would be even lower. These data are in clear contrast to current Slovenian screening program in age group 50–69 in which 70% of all cancers in this age group are diagnosed within the screening program with an average 75% attendance rate.2 Interestingly, a higher proportion of women were identified as above population risk in healthy women referred to breast centres for opportunis­tic screening compared to breast cancer patients, 48.7% vs. 39.7%, respectively. One of the reasons could be the higher proportion of women with positive family history and higher BMI in the breast centre group. The reason for relatively poor performance of the S-IBIS could be caused also by some personal characteristics of Slovene women that differ from other European populations where IBIS was validated, e.g. the age at first childbirth in Slovenia is lower than European average.20 When analysing the S-IBIS performance sepa­rately in the 40–44 year and 45–49 year age sub­groups, we found that S-IBIS performed slightly better in the age group 45–49 years compared to younger age group (40–44 years). The difference was not big enough however to allow to draw dif­ferent conclusions between the subgroups studied. Extension of mammographic screening to wom­en younger than 50 is a matter of debate, although several studies have confirmed that the harms of early screening do not outweighs the benefits. Over-diagnosis and false positive recalls in women younger than 50 years and non-significant lower breast cancer mortality between younger and older breast cancer patients make early breast cancer screening unreliable and unadvisable in the gen­eral population.21,22 However, the problem of early detection of breast cancer in women younger than 50 persists and as previously stated, screening of women at higher-than-average risk of breast can­cer seems one of the most feasible solutions. Based on data presented, further steps in refining a breast cancer risk calculation tool will have to be done before a tailored screening is implemented, as the inclusion of more breast cancer risk factors like mammographic density. Mammographic density is considered as a strong risk factor for breast can­cer and, as already mentioned, can be included in the S-IBIS calculation.23-25 Another promising risk factor is the polygenic risk score (PRS) based on the presence of single nucleotide polymorphisms (SNPs) related to breast cancer risk and which can be also included in the S-IBIS calculation.26-28 At the present moment, there are numerous differ­ent sets of SNPs being studied worldwide, none of them yet approved to routine use. Of note, several studies in European populations with higher than Slovenian breast cancer incidence have shown that both factors independently increase the sensitivity of IBIS.27-30 Due to technical limitations both mam-mographic density and PRS are not routinely in­cluded in S-IBIS calculations throughout Slovenia, therefore currently no data on value of mammo-graphic density and PRS in Slovenian population is available. Data are available for selected breast centres and are yet to be analysed at the time writ­ing this article. Studies with S-IBIS risk calcula­tions that include these risk factors are necessary and will have to be performed to further improve the stratification of women in the breast cancer risk groups and reveal the true potential of the S-IBIS program. Our study had several limitations. Since it is a cross-sectional study, it lacks follow up and we could not observe the eventual crossover between the two groups. Only follow-up of the Breast cen­tre group until the age of 50 would reveal the per­cent of overlap between the two groups and the true quality of risk stratification based on risks calculated by S-IBIS. Due to inability to assess the proportion of women undergoing early screening that would develop breast cancer before the age of 50, statistical comparison between the two groups was not performed, as it would lead to false as­sumptions. Furthermore, the non-systematic ac- 339 crual of women referred to opportunistic screening in Breast centres can result in high proportion of women at average breast cancer risk in the Breast centre group. Despite these limitations however, our study demonstrated the inability of the S-IBIS alone to reliably stratify women between the breast cancer risk groups. We acknowledge that a pro­spective study would give clearer and more reli­able data, but in the given settings only a retrospec­tive analysis was possible and perhaps necessary to plan a valid perspective study. Conclusions In conclusion, risk stratification based on S-IBIS calculation confirmed that at least half of women referred to regional Breast centres have above-average 10-year breast cancer risk and are entitled to regular screening prior to age 50 according to Slovenian guidelines. However, more than half of breast cancer patients aged 40–49 would not be se­lected for early tailored screening based on S-IBIS calculations with the chosen risk factors. Inclusion of additional risk factors (as mammographic breast density or PRS) into the S-IBIS is warranted in the populations with breast cancer incidence below European average to reliably stratify women into breast cancer risk groups. Tailored mammography screening in age group 40–49 based on S-IBIS alone can not be organized. Acknowledgment The study was supported by the research program of the Slovenian research agency P3-0352. References 1. Ferlay J, Colombet M, Soerjomataram I, Gavin A, Visser O, Bray F, 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. Zadnik V, Žagar T. SLORA: Slovenia and Cancer. Epidemiology and Cancer Registry. Institute of Oncology Ljubljana. [cited: 2019 Dec 20 ]. Available from: www.slora.si 3. Cancer in Slovenia 2016. Ljubljana: Institute of Oncology Ljubljana, Epidemiology and Cancer Registry, Slovenian Cancer Registry; 2019. 4. Krajc M. National breast cancer screening programme DORA. Residential public health thesis. [Slovenian]. Ljubljana: Institute of Oncology Ljubljana; 2009. 202 p. 5. Schemann HJ, Lerda D, Quinn C, Follmann M, Alonso-Coello P, Giorgi Rossi P, et al. Breast cancer screening and diagnosis: a synopsis of the European Breast Guidelines. Ann Intern Med 2020; 172: 46-56. doi: 10.7326/M19-2125 6. Borštnar S, Blatnik A, Perhavec A, Gazic B, Vidergar-Kralj B, Matos E, et al. Recommendations for diagnosis and treatment of patients with breast cancer (Part 1). [Slovenian]. Onkologija 2019; 23: 40-53. doi: 10.25670/ oi2019-006on 7. Amir E, Freedman OC, Seruga B, Evans DG. Assessing women at high risk of breast cancer: a review of risk assessment models. 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Allman R, Dite GS, Hopper JL, Gordon O, Starlard-Davenport A, Chlebowski R, et al. SNPs and breast cancer risk prediction for African American and Hispanic women. Breast Cancer Res Treat 2015; 154: 583-9. doi: 10.1007/ s10549-015-3641-7 20. Mean age of women at birth of first child, 2017. Eurostat. [cited: 2019 Dec 20]. Available at: https://ec.europa.eu/eurostat/web/products-eurostat-news/-/DDN-20190318-1?fbclid=IwAR0j_8KDUqMFY-Tca_wqFn3qHVxk­g4lPSeZb2Vg2zGrBh_B5K8r4hOI-Hys 21. Bucchi L, Ravaioli A, Baldacchini F, Giuliani O, Mancini S, Vattiato R, et al. Annual mammography at age 45-49 years and biennial mammography at age 50-69 years: comparing performance measures in an organised screening setting. Eur Radiol 2019; 29: 5517-27. doi: 10.1007/s00330-019­06050-w 22. van den Ende C, Oordt-Speets AM, Vroling H, van Agt HME. Benefits and harms of breast cancer screening with mammography in women aged 40-49 years: a systematic review. 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Rudolph A, Song M, Brook MN, Milne RL, Mavaddat N, Michailidou K, et al. Joint associations of a polygenic risk score and environmental risk factors for breast cancer in the Breast Cancer Association Consortium. Int J Epidemiol 2018; 47: 526-36. doi: 10.1093/ije/dyx242 28. Brentnall AR, Evans DG, Cuzick J. Distribution of breast cancer risk from SNPs and classical risk factors in women of routine screening age in the UK. Br J Cancer 2014; 110: 827-8. doi: 10.1038/bjc.2013.747 29. Vachon CM, Scott CG, Tamimi RM, Thompson DJ, Fasching PA, Stone J, et al. Joint association of mammographic density adjusted for age and body mass index and polygenic risk score with breast cancer risk. Breast Cancer Res 2019; 21: 68. doi: 10.1186/s13058-019-1138-8 30. Zhang X, Rice M, Tworoger SS, Rosner BA, Eliassen AH, Tamimi RM, et al. Addition of a polygenic risk score, mammographic density, and endog­enous hormones to existing breast cancer risk prediction models: A nested case-control study. PLoS Med 2018; 15: e1002644. doi: 10.1371/journal. pmed.1002644 31. Brentnall AR, van Veen EM, Harkness EF, Rafiq S, Byers H, Astley SM, et al. A case-control evaluation of 143 single nucleotide polymorphisms for breast cancer risk stratification with classical factors and mammographic density. Int J Cancer 2020; 146: 2122-9. doi: 10.1002/ijc.32541 341 research article Standard and multivisceral colectomy in locally advanced colon cancer Artur M. Sahakyan1,2, Andranik Aleksanyan1,3, Hovhannes Batikyan1, Hmayak Petrosyan2, Mushegh .. Sahakyan1,4 1 Department of Surgery ,N1, Yerevan State Medical University after M.Heratsi, Yerevan, Armenia 2 Department of General and Abdominal Surgery, ArtMed MRC, Yerevan, Armenia 3 Clinic of Surgery, Mickaelyan Institute of Surgery, Yerevan, Armenia 4 The Intervention Center, Oslo University Hospital Rikshospitalet, Oslo, Norway Radiol Oncol 2020; 54(3): 341-346. Received 21 February 2020 Accepted 30 April 2020 Correspondence to: Mushegh A. Sahakyan, M.D., Ph.D., The Intervention Center, Oslo University Hospital Rikshospitalet, Sognsvannsveien 20, 0424 Oslo, Norway; E-mail: sahakyan.mushegh@gmail.com Disclosure: No potential conflicts of interest were disclosed. Background. Management of locally advanced colon cancer (LACC) is challenging. Surgery is the mainstay of the treatment, yet its outcomes remain unclear, especially in the setting of multivisceral resections. The aim of the study was to examine the outcomes of standard and multivisceral colectomy in patients with LACC. Patients and methods. Patients demographics, clinical and perioperative data of patients operated within study period 2004–2018 were collected. LACC was defined as stage T4 colon cancer including tumor invasion either through the visceral peritoneum or to the adjacent organs/structures. Accordingly, either standard or multivisceral colectomy (SC and MVC) was performed. Results. Two hundred and three patients underwent colectomy for LACC. Of those, 112 had SC (55.2%) and 91 (44.8%) had MVC. Severe morbidity and mortality rates were 5.9% and 2.5%, respectively. MVC was associated with an increased blood loss (200 ml vs. 100 ml, p = 0.01), blood transfusion (22% vs. 8.9%, p = 0.01), longer operative time (180 minutes vs. 140 minutes, p < 0.01) and postoperative hospital stay (11 days vs. 10 days, p < 0.01) compared with SC. The complication-associated parameters were similar. Male gender, presence of = 3 comorbidities, tumor location in the left colon and perioperative blood transfusion were associated with complications in the univariable analysis. In the multivariable model, the presence of = 3 comorbidities was the only independent predictor of complications. Conclusions. Colectomy with or without multivisceral resection is a safe procedure in LACC. In experienced hands, the postoperative outcomes are similar for SC and MVC. Given the complexity of the latter, these procedures should be reserved to qualified expert centers. Key words: colectomy; colon cancer; locally advanced; multivisceral; morbidity Introduction Colon is the second most common site for cancer both in men and women.1,2 Significant advances in its treatment have been achieved over the last decades due to the improvements in surgical tech­nique, chemotherapy, targeted therapy and ex­amination of tumor biomarkers.3,4 Unfortunately, many of these patients are diagnosed relatively late presenting with locally advanced colon cancer (LACC), which is challenging to manage.5 The defi­nition of LACC is controversial as no uniform ap­proach exists in the literature to date. Some authors qualify stage T3 cancer with extramural invasion = 5 mm and T4 as locally advanced, while others define LACC as stage T4 cancer solely.6,7 Multimodal treatment is the standard approach for LACC, although surgery remains its corner­stone. Microscopically complete resection pro­vides satisfactory oncologic outcomes, however, 342 complex multiorgan resection may be required in these patients.8 The latter is associated with post­operative complications, prolonged hospital stay, increased treatment-associated costs and later start of adjuvant chemotherapy, which may ultimately increase the risk of tumor recurrence.9 Thus, po- TABLE 1. Patient characteristics and perioperative outcomes in patients with T4 colon cancer undergoing colectomy Age, years, mean (± SD) 63.1 (11.6) Body mass index, kg/m2, mean (± SD) 27 (4.9) Female gender, n (%) 87 (42.9%) Comorbidities, n (%) 21 (10.3%) ASA score > III, n (%) 5 (2.4%) Colonic obstruction, n (%) 82 (40.4%) Hemoglobin, mean (± SD) 111 (29) Total protein, mean (± SD) 72 (6) Albumin, mean (± SD) 40 (6) Tumor location, n (%) Right 68 (33.5%) Left 118 (58.1%) Transverse colon 17 (8.4%) T stage, n (%) T4a 79 (38.9%) T4b 124 (61.1%) N stage, n (%) N0 83 (40.9%) N1 46 (22.7%) = N2 74 (36.4%) M stage, n (%) M0 145 (71.4%) M1 58 (28.6%) Tumor size = 6cm, n (%) 169 (83.3%) Operative time, min, median (range) 160 (60–480) Estimated blood loss, ml, median (range) 175 (50–900) Red blood cell transfusion, n (%) 30 (14.8%) Morbidity (= II C–D), n (%) 25 (12.3%) Severe morbidity (= IIIa C–D), n (%) 12 (5.9%) Anastomosis leakage, n (%) 10 (4.9%) Relaparotomy, n (%) 10 (4.9%) Mortality, n (%) 5 (2.5%) Postoperative stay, days, median (range) 11 (5–44) ASA = American Society of Anesthesiologists; SD = standard deviation tential improvement in survival after surgery for LACC should outweigh the aforementioned risks. This study explores the outcomes of colectomy in patients with LACC. The impact of surgical ap­proaches on postoperative results as well as poten­tial risk factors for complications were evaluated. Patients and methods Study design and patients This retrospective observational study included pa­tients with LACC operated at a single surgical unit between 2004 and 2018. Retrospective study was approved by the accredited Institutional Review Board for medical Ethics. LACC was defined as stage T4 (T4a and T4b) colon cancer confirmed on final pathology. Tumor stage was determined and classified based on the criteria suggested by the 8th edition of American Joint Committee on Cancer.10 Given the diversity of T4a and T4b colon cancers, patients underwent either standard colectomy (SC) or multivisceral colectomy (MVC). Patient demo­graphics, clinical characteristics, imaging findings, lab tests, perioperative and pathology work-up data were prospectively collected and registered in the database. Surgical outcomes and risk factors for postoperative complications were examined. Comparisons were drawn between the outcomes of SC and MVC. Patients diagnosed with tumors other than adenocarcinoma were excluded from the analysis. The selection criteria for surgery did not change throughout the study period. Normally, all func­tionally fit patients with no preoperative signs of distant metastases were referred to surgery. However, some patients with metastatic LACC were operated due to life-threatening conditions, such as TABLE 2. Organs and structures resected during multivisceral colectomies (n = 91) for locally advanced colon cancer Small bowel 18 Stomach 13 Uterus and/or ovaries 13 Kidney/Ureter/Urinary bladder 11 Liver 11 Gallbladder 3 Pancreas 1 > 1 organ/structure 21 Total 91 343 colon obstruction or bleeding. Of note, colon stent-TABLE 3. Outcomes of colectomy for locally advanced colon cancer depending on the type of resection (standard and multivisceral) ing was not available at our institution during the study period, hence surgery was the only available option. None of the patients had received neoadju­vant chemotherapy. D2 lymphadenectomy was per-Age, years, mean (±SD) 63.5 (11.7) 62.5 (11.3) 0.57 formed routinely. All anastomoses were performed Body mass index, kg/m2, mean (±SD) 27.5 (5.3) 26.2 (4.2) 0.41 using a hand-sewn uninterrupted suture. Following Gender, n (%) 0.78surgery, the patients were mobilized on a next day. Male 63 (56.2%) 53 (58.2%) Nasogastric tube was removed on postoperative Female 49 (43.8%) 38 (41.8%) day 3 and the enteral feeding was started. Comorbidities, n (%) 98 (87.5%) 84 (92.3%) 0.26 Definitions Number of comorbidities, mean (±SD) 2.6 (0.9) 2.6 (1.0) 0.62 0.55 Tumor size was defined as the largest dimension of Type of comorbidities, n (%) the tumor measured microscopically at the pathol- Cardiovascular 66 (58.9%) 56 (61.5%)ogy work-up. Resection radicality was regarded as Diabetes mellitus 9 (8 %) 3 (3.3%)R1 if microscopic presence of tumor or tumor in­ Thrombophlebitis 13 (11.6%) 9 (9.9%) volved lymph node was found within 1mm of the ASA score > III, n (%) 1 (0.9%) 4 (4.4%) 0.07 resection margin. R2 resection included at least one Colonic obstruction, n (%) 54 (48.2%) 28 (30.8%) 0.16 of the following: macroscopic tumor at the resec- Hemoglobin, g/dl, mean (±SD) 114 (28) 107 (29) 0.11 tion margin, distant metastases or peritoneal car- Total protein, g/dl, mean (±SD) 69 (14.9) 72.8 (5.9) 0.045 cinomatosis. Postoperative complications were defined and Albumin, g/dl, mean (±SD) 40.1 (6.2) 39.2 (6.2) 0.6 graded according to the classification system sug-CEA, ng/ml, median (range) 3.0 (0.7–267) 7.5 (0.8–1155) 0.52 gested by Clavien and Dindo.11 Complications that CA 19-9, U/ml, median (range) 8.6 (0.6–13444) 7.7 (2–2147) 0.96 were grade II and higher were registered. Grade Tumor location, n (%) 0.07 = III complications were defined as severe. The Right 45 (40.2%) 23 (25.3%) Comprehensive Complication index was used for Left 60 (53.6%) 58 (63.7%) a comprehensive assessment of postoperative com­ Transverse colon 7 (6.2%) 10 (11%) plications.12,13 Mortality included all cases of death within 30 days of surgery. T stage, n (%) < 0.01 T4a 74 (66.1%) 5 (5.5%) T4b 38 (33.9%) 86 (94.5%) Statistics N stage, n (%) 0.2 Continuous data were presented as mean (± stand­ N0 45 (40.2%) 38 (41.8%) ard deviation) or median (range) depending on N1 21 (18.8%) 25 (27.5%) data distribution. The two-sample T-test was used = N2 46 (41.1%) 28 (30.8%) to compare means, and the Mann-Whitney U test was used for medians. The categorical variables M1 stage, n (%) 35 (31.2%) 23 (25.3%) 0.35 were presented as frequencies (percentages). The Tumor size = 6cm, n (%) 91 (81.2%) 78 (85.7%) 0.4 Chi-square test or Fisher’s exact test were used to Operative time, min, median (range) 140 (60–480) 180 (85–390) < 0.01 compare the categorical data. P-value < 0.05 was Estimated blood loss, ml, median 100 (50-900) 200 (100–600) 0.01 (range) considered statistically significant. The aforemen- Red blood cell transfusion, n (%) 10 (8.9%) 20 (22.0%) 0.01 tioned tests were used in the univariable analysis of risk factors for postoperative complications. Morbidity (= II C-D), n (%) 15 (13.4%) 10 (11%) 0.6 Variables significant at p-value < 0.1 were added to Severe morbidity (= IIIa C-D), n (%) 8 (7.1%) 4 (4.4%) 0.41 the binary logistic regression model to determine CCI, median (range) 33.7 (20.9–100) 29.6 (20.9–100) 0.33 the independent predictors of complications. Anastomosis leakage, n (%) 5 (4.5%) 5 (5.5%) 0.76 Relaparotomy, n (%) 5 (4.5%) 5 (5.5%) 0.76 Mortality, n (%) 2 (1.8%) 3 (3.3%) 0.66 Results Postoperative stay, days, median 10 (5–44) 11 (7–44) 0.04 (range) A total number of 474 patients with colon cancer underwent surgery throughout the study period. ASA = American Society of Anesthesiologists; CA 19-9 = Carbohydrate antigen 19-9; CEA = Carcinoembryonic antigen; CCI = Comprehensive Complication Index Of those, 203 (42.8%) were operated for LACC. 344 TABLE 4. Univariable analysis of factors associated with postoperative complications tients with non-metastatic LACC (n = 145), the rate of curative resections (R0) was 97%. Morbidity and mortality rates were 12.3% and 2.5%, respectively. Median length of stay was 11 (5–44) days. Age = 65 years, n (%) 15 (60%) 98 (55.1%) 0.68 SC was performed in 112 (55.2%) patients and Body mass index = 30 kg/m2, n (%) 2 (8%) 36 (20.2%) 0.44 MVC in 91 (44.8%). The latter included resections of one (n = 70) or = 2 organs/structures (n = 21) Male gender, n (%) 18 (72%) 98 (55.1%) 0.09 (Table 2). In patients with = 2 organs/structures Comorbidities, n (%) 1 (4%) 20 (11.2%) 0.48 resected the resection of the small bowel, stomach Number of comorbidities = 3, n (%) 7 (28%) 18 (10.1%) 0.02 and pancreas was most often carried out. Cardiovascular disease n (%) 15 (60%) 107 (60.1%) 0.85 A comparative analysis between the outcomes of ASA score > III, n (%) 1 (4%) 4 (2.2%) 0.51 MVC and SC was performed (Table 3). Preoperative parameters were similar except the significantly Colon obstruction, n (%) 12 (48%) 70 (39.3%) 0.78 higher total protein levels in the MVC group. The Hemoglobin, g/dl, mean (±SD) 111 (27) 111 (28) 0.94 latter was almost always performed in patients Total protein, g/dl, mean (±SD) 73.3 (5.5) 72.1 (6.2) 0.27 with T4b adenocarcinoma (94.5% vs. 33.9%, p < Albumin, g/dl, mean (±SD) 38.5 (4.7) 39.8 (6.5) 0.55 0.01). Operative time was significantly longer for 0.05MVC (180 minutes vs. 140 minutes, p<0.01) and so Right 5 (20%) 63 (35.4%)was the median blood loss (200 ml vs. 100 ml, p = 0.01). The red blood cell transfusion rate was sig- Tumor location, n (%) Left 20 (80%) 98 (55.1%) nificantly higher for MVC - 22% vs. 8.9%, p = 0.01. T stage, n (%) 0.23 Microscopically complete resection was achieved T4a 7 (28%) 72 (40.4%) in a similar number of patients (98.5% vs. 95.3%, T4b 18 (72%) 106 (59.6%) p = 0.56). Proportions of postoperative complica-N+ disease, n (%) 17 (68%) 103 (57.9%) 0.6 tions and their types were comparable between the groups. The length of stay after surgery was longer M1 stage, n (%) 9 (36%) 49 (27.5%) 0.38 following MVC (11 days vs. 10 days, p = 0.04). Tumor size = 6cm, n (%) 21 (84%) 148 (83.1%) 1.0 Univariable analysis of factors associated with Estimated blood loss, ml, median (range) 200 (50–900) 100 (100–500) 0.12 postoperative complications was performed (Table 4). Male gender, presence of = 3 comorbidi- Red blood cell transfusion, n (%) 7 (28%) 23 (12.9%) 0.07 ties, tumor location in the left colon and red blood = 2 organs resected, n (%) 3 (12%) 18 (10.1%) 0.73 cell transfusion were associated with grade = II Single-layer anastomosis suture, n (%) 15 (60%) 136 (76.4%) 0.13 complications. These factors were analyzed togeth- End-to-end anastomosis, n (%) 9 (36%) 53 (29.8%) 0.57 er in the multivariable model (Table 5). The latter demonstrated that only presence of = 3 comor- ASA = American Society of Anesthesiologists bidities was associated with grade = II morbidity. Specifically, their risk increased more than three times in these patients - OR 3.1 (1.1–9.2), p = 0.038. TABLE 5. Multivariable analysis of risk factors for postoperative complications Male gender 1.88 (0.7–5.04) 0.21 Our findings indicate that colectomy in patients Number of comorbidities = 3 3.1 (1.1–9.2) 0.04 with LACC is a safe procedure providing satisfac­ Left-sided tumor 2.2 (0.74–6.48) 0.16 tory surgical outcomes when performed in a spe-Red blood cell transfusion 1.94 (0.67–5.64) 0.22 cialized surgical unit. This is applicable to both SC and MVC. In the literature, postoperative mor­bidity and mortality rates following surgery for Patient characteristics, perioperative data and LACC are 25–38% and 3.3–6.9%, respectively.5,14-17 pathology findings are presented in Table 1. The Hoffman et al., demonstrated that morbidity rate most common location of LACC was the left co-may increase with the use of multiorgan resec­lon (58.1%), while 82 (40.4%) patients had colon tions.18 In this series, postoperative morbidity rate obstruction. More than one-fourth of the patients was 12.8% including 7.4% severe complications. (28.6%) had distant metastases at surgery. In pa-Although MVC was associated with an increased 345 blood loss, need for blood transfusion, as well as with longer operative time and hospital stay, post­operative morbidity-associated parameters and mortality were comparable to those of SC. Severe complications were mostly caused by the anastomotic leakage (4.9%), which is consist­ent with the data in the literature.14,19 Multiple pa­rameters are found to be associated with the risk of leakage including patient-specific variables, intraoperative complications, surgeon- and tech-nique-related factors. In this report, such analysis was not possible due to the small number of cases. However, when analyzing risk factors for compli­cations technical parameters such as single-layer anastomosis suture or its end-to-end type did not increase the rate of complications. We believe that single-layer suture is a simple technique that sig­nificantly expedites the procedure, while the end-to-end anastomosis avoids the need for additional closure of the intestinal stumps on the proximal and/or distal loops. The effectiveness of this tech­nique was reported also by Liu et al.20 Our results do not confirm the association between the postop­erative results and blood transfusion suggested by Marinello et al.14 Despite being a significant predic­tor in the univariable analysis, blood transfusion was not an independent predictor of complications in the multivariable model. According to the literature, MVC is performed in only 1.2–12% of patients with colon cancer.14,15,21,22 In our study, MVC was performed in 44.8% of pa­tients with LACC, which accounts for 19.2% of a total number of colon resections for cancer. Higher incidence of MVC in our series can be attributed to strict selection criteria in the aforementioned stud­ies, as well as to a significantly higher proportion of late diagnosed patients in our population. This is confirmed also by the fact that nearly 40% of our patients presented with partial or total colon ob­struction prior to surgery. Intraoperatively, it is not always possible to as­sess whether or not colectomy will be curative, thus the main goal is to achieve a complete resec­tion of the primary tumor and suspicious adjacent tissues if these are found at surgery.9 According to the literature, the most common invasion sites for T4b colon cancer are the small bowel, urinary bladder and abdominal wall.15-17 In this series, most often these patients had tumor ingrowth into = 2 organs, predominantly to the small bowel, distal pancreas and stomach. Tumor invasion into ad­jacent structure(s) was verified by the pathology examination in about 95% of patients who had un­dergone MVC. Given that this parameter ranges from 44% to 72.5% in the literature5,9,15,16, the choice of surgical approach was adequate in this series. This report has several limitations, includ­ing retrospective design with its inherent biases. Furthermore, we did not register grade I complica­tions (according to Clavien-Dindo)11, which some­what limits the information on postoperative re­sults of colectomy for LACC. It is also worth men­tioning that our data are based on an experience of a specialized center of colorectal surgery, thus the reproducibility of our results is limited and surgi­cal outcomes should be interpreted with caution. Conclusions In conclusion, colectomy including MVC is a safe procedure in the setting of LACC. In experienced hands, the postoperative outcomes are acceptable showing no differences between the SC and MVC. However, their oncologic benefits require further investigation. Given the complexity of MVC, these procedures should be reserved to qualified expert centers that are familiar with colorectal procedures as well as with the surgery of other organ systems. References 1. Teufel A, Gerken M, Hartl J, Itzel T, Fichtner-Feigl S, Stroszczynski C. Benefit of adjuvant chemotherapy in patients with T4 UICC II colon cancer. BMC Cancer 2015; 15: 419. doi: 10.1186/s12885-015-1404-9 2. Sokolov M. Surgical approach in locally advanced colorectal cancer - com­bined, extended and compound surgery. Khirurgiia (Sofiia) 2013; 4: 29-50. PMID: 24800318 3. Rousseau B, Chibaudel B, Bachet JB, Larsen AK, Tournigand C, Louvet C, et al. Stage II and stage III colon cancer: treatment advances and future direc­tions. Cancer J 2010; 16: 202-9. doi: 10.1097/PPO.0b013e3181ddc5bf 4. Akagi T, Inomata M. Essential advances in surgical and adjuvant therapies for colorectal cancer 2018-2019. Ann Gastroenterol Surg 2020; 4: 39-46. doi: 10.1002/ags3.12307 5. Rosander E, Nordenvall C, Sjall A, Hjern F, Holm T. Management and outcome after Mmltivisceral resections in patients with locally advanced primary colon cancer. Dis Colon Rectum 2018; 61: 454-60. doi: 10.1097/ DCR.0000000000001046 6. Ngaard A, Dam C, Jakobsen A, Pln J, Lindebjerg J, Rafaelsen SR. Selection of colon cancer patients for neoadjuvant chemotherapy by preoperative CT scan. Scand J Gastroenterol 2014; 49: 202-8. doi: 10.3109/00365521.2013.862294 7. Ludmir EB, Arya R, Wu Y, Palta M, Willett CG, Czito BG. Role of adjuvant radiotherapy in locally advanced colonic carcinoma in the modern chemo­therapy era. Ann Surg Oncol 2016; 23: 856-62. doi: 10.1245/s10434-015­4907-3 8. Gezen C, Kement M, Altuntas YE, Okkabaz N, Seker M, Vural S, et al. Results after multivisceral resections of locally advanced colorectal cancers: an analysis on clinical and pathological T4 tumors. World J Surg Oncol 2012; 10: 39. doi: 10.1186/1477-7819-10-39 9. Lehnert T, Methner M, Pollok A, Schaible A, Hinz U, Herfarth C. Multivisceral resection for locally advanced primary colon and rectal cancer: an analysis of prognostic factors in 201 patients. Ann Surg 2002; 235: 217-25. doi: 10.1097/00000658-200202000-00009 346 10. Weiser MR. AJCC 8th Edition: Colorectal cancer. Ann Surg Oncol 2018; 25: 1454-5. doi: 10.1245/s10434-018-6462-1 11. Dindo D, Demartines N, Clavien P-A. Classification of surgical complications. Ann of Surg 2004; 240: 205-13. doi: 10.1245/s10434-018-6462-1 12. Slankamenac K, Nederlof N, Pessaux P, de Jonge J, Wijnhoven BP, Breitenstein S, et al. The comprehensive complication index: a novel and more sensitive endpoint for assessing outcome and reducing sample size in randomized controlled trials. Ann Surg 2014; 260: 757-63. doi: 10.1097/ SLA.0000000000000948 13. Slankamenac K, Graf R, Barkun J, Puhan MA, Clavien PA. The comprehensive complication index: a novel continuous scale to measure surgical morbidity. Ann Surg 2013; 258: 1-7. doi: 10.1097/SLA.0000000000002132 14. Marinello FG, Baguena G, Lucas E, Frasson M, Hervás D, Flor-Lorente B, et al. Anastomotic leakage after colon cancer resection: does the individual surgeon matter? Colorectal Dis 2016; 18: 562-9. doi: 10.1111/codi.13212 15. Croner RS, Merkel S, Papadopoulos T, Schellerer V, Hohenberger W, Goehl J.Multivisceral resection for colon carcinoma. Dis Colon Rectum 2009; 52: 1381-6. doi: 10.1007/DCR.0b013e3181ab580b 16. Luna-Pérez P, Rodríguez-Ramírez SE, De la Barrera MG, Zeferino M, Labastida S. Multivisceral resection for colon cancer. J Surg Oncol 2002; 80: 100-4. doi: 10.1002/jso.10105 17. Wasmann KATGM, Klaver CEL, van der Bilt JDW, Nagtegaal ID, Wolthuis AM, van Santvoort HC et al. Subclassification of multivisceral resections for T4b colon cancer with relevance for postoperative complications and oncological risks. J Gastrointest Surg 2019; [Ahead of print]. doi: 10.1007/ s11605-019-04426-3 18. Hoffmann M, Phillips C, Oevermann E, Killaitis C, Roblick UJ, Hildebrand P, et al. Multivisceral and standard resections in colorectal cancer. Langenbecks Arch Surg 2012; 397: 75-84. doi: 10.1007/s00423-011-0854-z 19. Frasson M, Flor-Lorente B, Rodríguez JL, Granero-Castro P, Hervás D, Alvarez Rico MA, et al. Risk factors for anastomotic leak after colon resection for cancer: multivariate analysis and nomogram from a multicentric, prospec­tive, national study with 3193 patients. Ann Surg 2015; 262: 321-30. doi: 10.1097/SLA.0000000000000973 20. Liu Z, Wang G, Yang M, Chen Y, Miao D, Muhammad S et al. Ileocolonic anas­tomosis after right hemicolectomy for colon cancer: functional end-to-end or end-to-side? World J Surg Oncol 2014; 12: 306. doi: 10.1186/1477-7819­12-306 21. Leijssen LGJ, Dinaux AM, Amri R, Kunitake H, Bordeianou LG, Berger DL. The impact of a multivisceral resection and adjuvant therapy in locally advanced colon cancer. J Gastrointest Surg 2019; 23: 357-66. doi: 10.1002/jso.25610 22. Zhao YZ, Han GS, Lu CM, Ren YK, Li J, Ma PF, et al. Right hemicolectomy and multivisceral resection of right colon cancer: a report of 21 cases. J Huazhong Univ Sci Technolog Med Sci 2015; 35: 255-58. doi: 10.1007/ s11596-015-1420-7 347 research article Percutaneous image guided electrochemotherapy of hepatocellular carcinoma: technological advancement Mihajlo Djokic1, Rok Dezman2, Maja Cemazar3,4, Miha Stabuc2, Miha Petric1, Lojze M. Smid5, Rado Jansa5, Bostjan Plesnik1, Masa Bosnjak3, Ursa Lampreht Tratar3, Blaz Trotovsek1, Bor Kos6, Damijan Miklavcic6, Gregor Sersa3,7, Peter Popovic2 1 University Medical Centre Ljubljana, Clinical Department of Abdominal Surgery, Ljubljana, Slovenia 2 University Medical Centre Ljubljana, Clinical Institute of Radiology, Ljubljana, Slovenia 3 Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia 4 University of Primorska, Faculty of Health Sciences, Izola, Slovenia 5 University Medical Centre Ljubljana, Clinical Department of Gastroenterology, Ljubljana, Slovenia 6 University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia 7 University of Ljubljana, Faculty of Health Sciences, Ljubljana, Slovenia Radiol Oncol 2020; 54(3): 347-352. Received 14 Maj 2020 Accepted 1 June 2020 Correspondence to: Prof. Gregor Serša, Ph.D., Institute of Oncology Ljubljana, Department of Experimental Oncology, Zaloška 2, SI-1000 Ljubljana, Slovenia. E-mail: gsersa@onko-i.si and Prof. Peter Popovic, M.D., Ph.D., University Medical Centre Ljubljana, Clinical Institute of Radiology, Zaloška 7, SI-1000 Ljubljana, Slovenia. E-mail: peter.popovic@kclj.si Mihajlo Djokic and Rok Dezman contributed equally. Disclosure: Damijan Miklavcic holds patents on electrochemotherapy that have been licensed to IGEA S.p.a (Carpi, Italy) and is also a consultant to various companies with an interest in electroporation-based technologies and treatments. The other authors have no competing interests. Background. Electrochemotherapy is an effective treatment of colorectal liver metastases and hepatocellular carcinoma (HCC) during open surgery. The minimally invasive percutaneous approach of electrochemotherapy has already been performed but not on HCC. The aim of this study was to demonstrate the feasibility, safety and effective­ness of electrochemotherapy with percutaneous approach on HCC. Patient and methods. The patient had undergone the transarterial chemoembolization and microwave ablation of multifocal HCC in segments III, V and VI. In follow-up a new lesion was identified in segment III, and recognized by multidisciplinary team to be suitable for minimally invasive percutaneous electrochemotherapy. The treatment was performed with long needle electrodes inserted by the aid of image guidance. Results. The insertion of electrodes was feasible, and the treatment proved safe and effective, as demonstrated by control magnetic resonance imaging. Conclusions. Minimally invasive, image guided percutaneous electrochemotherapy is feasible, safe and effective in treatment of HCC. Key words: electrochemotherapy; hepatocellular carcinoma; percutaneous; minimally invasive; bleomycin Introduction Electrochemotherapy is safe and effective treat­ment of cutaneous tumors and metastases, its ap­plication is described in the published Standard Operating procedures, and clinical indications defined in NICE, and several other national guide­lines.1-3 Electrochemotherapy in treatment of deep-seated tumors, like liver metastases and hepatocel­lular carcinoma (HCC) proved to be safe and ef­fective.4-6 The three published studies were done 348 using electrochemotherapy during open surgery. The surveillance of high-risk population using ultrasound permits to diagnose HCC at an early stage, at which curative treatments can be em­ployed. According to European Association for the Study of Liver (EASL) recommendations, thermal ablation with radiofrequency is the standard of care for patients with Barcelona clinic liver cancer (BCLC) 0 and A, tumors not suitable for surgery. However, in patients with very early stage HCC (BCLC-0) radiofrequency ablation (RFA) in favora­ble locations can be adopted as first-line therapy even in patients amenable to surgical procedure. Electrochemotherapy is local therapy with simi­lar modes of action as local ablative therapies, e.g. RFA, microwave ablation (MWA) and in particular irreversible electroporation (IRE).7-9 However, the main difference between electrochemotherapy and other local ablative therapies is that electrochemo-therapy combines two modalities, chemotherapy and the application of electric pulses. Thus, the tumor cells are dying not directly due to the ap­plication of physical energy, such as in the case of other local thermal ablative therapies or IRE, but due to the action of chemotherapeutic drug, which in the case of bleomycin means that the cells are dying by mitotic cell death.10 Therefore, electro-chemotherapy is effective and safe in treatment of tumors located in close proximity to major hepatic vessels11-13 and can be performed by image guided percutaneous approach.14 Percutaneous approach of electrode insertion is well established in IRE. Several studies demon­strate the feasibility and safety of percutaneous approach of IRE in treatment of liver tumors, in­cluding HCC.9,15-17 Some reports describe percuta­neous approach also for electrochemotherapy of cholangiocarcinoma, spine metastases18,19, lysis of portal vein thrombosis in hepatic hilum, and me­tastasis from renal cell cancer, however not in treat­ment of HCC.20-23 In this report we therefore tested the feasibility, safety and effectiveness of electro-chemotherapy with image guided percutaneous approach, in a patient with HCC. Patient and methods Sixty six-year old male patient was presented at multidisciplinary team meeting with multifocal HCC in segments III, V, VI in September 2017. At the time that patient was presented he had Child A liver cirrhosis - ethylic etiology, arterial hyperten­sion and diabetes type 2. He was a former smoker and had a history of excessive alcohol consump­tion. In 2018 he had undergone 1a and 1b drug-eluting bead doxorubicin transarterial chemoem­bolization (DEBDOX TACE) treatment of hepatic lesions. Two months after the treatment, control computed tomography (CT) showed complete response of the target lesions in segments III and VI and stable disease of the lesion in segment V. Therefore, his documentation was reviewed on hepatopancreaticobiliary (HPB) multidisciplinary team meeting, which concluded that the patient is a candidate for MWA of the lesion in segment V.On control CT scan 1 month after MWA, lesion in segment V was completely avital (complete re­sponse), but new lesion, 14 mm in diameter, in seg­ment III was identified. On CT scan 3 months later 349 hypervascular lesion in segment III appeared to be larger - 18 mm in diameter (Figure 1). No signs of extrahepatic disease were found. According to HPB multidisciplinary team meeting, the patient was eligible candidate for percutaneous electro-chemotherapy. The patient signed informed con­sent and was treated in the frame of the clinical study (NCT02291133) approved by the National Ethics Committee (21k/02/14) of the Republic of Slovenia. Electrochemotherapy was performed according to the standard operating procedures for electro­chemotherapy2 and as described in previous study on electrochemotherapy of HCC5, performed dur­ing the open surgery using cone-beam computed tomography (CBCT) guided percutaneous ap­proach. Results Treatment was performed under general anesthe­sia and deep muscle relaxation. The patient was positioned in supine position. Because the tumor was not visible on ultrasound and CBCT with a contrast agent, we decided for angiography to visualize the lesion. Coeliac truncus was reached through the punction of common femoral artery and left hepatic artery was selectively catheterized. CBCT (Siemens Medical Solutions, Forchheim, Germany) was performed with the administration of non-ionic contrast agent (Ultravist 370®, Bayer HealthCare) through a power injector (Avanta®, Medrad, Bayer HealthCare). CBCT after contrast injection through 2.4 F microcatheter (Progreat®, Terumo Europe N.V.) into segmental branches for liver segment III confirm 18 mm large tumor (Figure 2A). Four electrodes with 3 cm active length were placed percutaneously around the tumor in the form of pseudo-square under stereotactic CBCT guidance according to European Standard Operating Procedures on Electrochemotherapy (ESOPE) recommendations (Figure 2B,C).2 The distance between the electrodes ranged from 18 to 23 mm (Figure 2B, Figure 3A). Then, bleomycin (Bleomycin medac, Medac, Germany) 30.000 IU in 20 ml of physiological saline; 15 000 IU/m2, was administered intravenously in bolus lasting 2 min­utes. Two trains of 4 electric pulses (duration 100 µs, pulse repetition frequency 1 kHz) of opposite polarity with voltage-to-distance ratio of 1000 V/ cm and were delivered between all electrode pairs starting 8 minutes after the bleomycin injection (to­tal number of pulses = 48). The voltages and me- TABLE 1. VOLTAGES and currents delivered in the treatment 2 3 2800 38.0 4 1 2800 36.5 1 3 2300 34.5 1 2 2000 29.4 3 4 1800 27.7 2 4 1800 26.5 dian currents delivered to each electrode pair are listed in Table 1. Delivery of the electric pulses was synchronized with the ECG, triggered during the refractory phase of the heart.24 The maximal cur­rent amplitude measured during electroporation of the tumor was 40 A. During the treatment, no changes in cardiologic (ECG, pulse rate) and hemo-dynamic parameters were noticed. After electrode extraction, control CBCT with contrast injection through microcatheter showed area of avital lesion (Figure 2D). The whole procedure from the induc­tion of anesthesia until the end of the application of electric pulses lasted 1 h and 10 minutes. A numeric reconstruction of the performed treatment, prepared using the treatment planning 350 methods presented in previous work showed that whole tumor area with safety margin (range: 6.2 to 39 mm) was covered, comprising a total vol­ume of 78 cm3 (Figure 3A).25 A numerical analysis showed, that a successful treatment would also be possible with a 3 electrode (Figure 3B) and 2 elec­trode (Figure 3C) configuration. The volumes of obtained lesion are smaller than the actual treat­ment (26 and 23 cm3 for the 3 and 2 electrode setup, respectively), but they still achieved a good safety margin (range 3.6 mm to 21.5 mm for 2 electrodes and 5.1 mm to 20.9 mm for 3 electrodes). Postprocedural course was uneventful, abdomi­nal ultrasound 24 hours post-electrochemotherapy showed normal postinterventional finding - no bleeding, hematoma or fluid collections. Therefore, patient was discharged the day after the procedure with analgesics and antithrombotic prophylaxis. Two months after percutaneous electrochemo-therapy, control magnetic resonance (MRI) of liver showed 36 mm large non enhancing area of abla­tion necrosis within the treated area - complete response of targeted lesion according to modified Response Evaluation Criteria In Solid Tumors (mRECIST) (Figure 4A). The patient was feeling well, in good physical condition and pain-free. On the second follow-up, 6 months after the procedure control liver MRI showed complete response of the treated lesion with ablated area decreasing in size, which is in line with expected necrosis resolution dynamics and formation of fi­brosis. The lesion was in complete response also 18 months after the treatment, however new HCC foci occurred in other locations. Discussion We describe the first case of percutaneous electro-chemotherapy of HCC. Minimally invasive, image guide percutaneous electrochemotherapy proved feasible, safe and effective treatment modality, which can be used in selected group of patients with HCC. The management of HCC has changed in re­cent years. Percutaneous local ablation is currently considered to be viable treatment for patients with very early HCC, as defined by the BCLC staging system. Indications for percutaneous local ablation include: HCC in BCLC stage A with Child-Pugh class A/B cirrhosis; ECOG performance status of 0-1; ideal tumor size of less than 3 cm and solitary or multiple lesions (up to three lesions). RFA has been the most widely investigated modality of per­cutaneous ablation. It has been shown that RFA is a safe method with potential drawback due to the heat sink effect. It is believed that 10-25% of pa­tients with HCC may not be eligible for RFA due to this effect.26 MWA offers all the benefits of RFA as well as some substantial advantages. Promising results of MWA for HCC have been demonstrated in sev­eral studies.27–29 The advantages of MWA include a larger volume of cellular necrosis, reduction in procedure times, greater temperatures delivered to the target lesion and greater efficacy in lesions in proximity to vascular structures with a reduc­tion in the heat-sink effect compared to RFA.29 Due to the higher delivered energy a vessel thrombosis 351 as potential complication can occur when tumors adjacent to major vessels are treated. Although extremely rare, these complications have been de­scribed.29 Electrochemotherapy has already proven effec­tive in treatment of HCC in a series of 17 lesions in 10 patients treated by electrochemotherapy during the open surgery with median tumor size of 24 mm (range 8–41 mm). No treatment related adverse ef­fects or major post-operative complications were observed. The complete response rate at last follow up ranging from 12 to 31 months was 80% per pa­tient and 88% per treated lesion.5 This response rate of electrochemotherapy is comparable although lower than the response rate achieved by RFA and MWA.30 Newer studies report the response rate in HCC smaller than 30 mm above 98% for RFA and MWA with low percentage of local recurrence.31 The advantage of the electrochemotherapy is that it is effective in treatment of tumors also locat­ed in close proximity of the major hepatic vessels. In comparison to RFA electrochemotherapy is not affected by heat sink effect, and this indication was not proven only in the clinical study treating HCC with electrochemotherapy5, but also in the study treating liver metastases of colorectal cancer by IRE.32 The safety of treating tumors close to major liver vessels was demonstrated also in the recent study in healthy pigs, where no significant vascu­lar damage/abnormalities were observed in liver vessels, even when the electrodes were inserted through the hepatic or portal vessels.11 IRE as an ablation method has also been dem­onstrated to be effective for treatment of HCC.15,33 Similar observations were reported for electro-chemotherapy, without major complications. IRE, though, is executed percutaneously in many cancer centers, with the aid of image guidance.34 Due to similar technological approach, electrochemother­apy can also be performed percutaneously. Same principles must be followed - careful pre-treatment planning, image guided electrode insertion and safe delivery of electric pulses with ECG synchro­nization.24,35,36 Electrochemotherapy however may offer additional advantages over IRE: shorter treat­ment duration due to a lower number of pulses re­quired (e.g. 8 vs. 90), the possibility of achieving larger volumes with fewer electrodes and without electrode repositioning. The advantage of electrochemotherapy in com­parison to IRE is its different mode of action. IRE is an ablative technique that by delivering sets of pulses disrupts cell’s homeostasis due to cell membrane electroporation leading to cell death. Therefore, the tumor is ablated in the confined area and no selective action on tumor cells is present. IRE being nonthermal ablative technology also elicits strong local immune response and preserves critical structures which is also well established in electrochemotherapy. Electrochemotherapy how­ever acts through three mechanisms. First one is selective cellular cytotoxicity by drug delivered to cells, and cell death due to mitotic catastrophe.37 In that case tumor safety margins can be wider due to predominantly tumor cell death and sparing of normal tissue. Electrochemotherapy, can therefore be employed also in tumors that are bigger than 3 cm in diameter, which is currently the limit for IRE. The second mode of action is vascular disruption that is well established in electrochemotherapy38, but not well explored in IRE. And the third is the elicitation of local immune response39 that could be exploited in combination with immunothera­ pies.40,41 Using percutaneous approach will provide elec­trochemotherapy broader clinical application in treatment of HCC and other liver tumors/metasta­ses, being minimally invasive, with short hospitali­zation and good patient’s compliance. Acknowledgement This work was financially supported by the Slovenian Research Agency (ARRS), grant No. P3­0003 and P2-0249 and grant of University Clinical Center Ljubljana #20180061 References 1. Campana LG, Clover AJ, Valpione S, Quaglino P, Gehl J, Kunte C, et al. Recommendations for improving the quality of reporting clinical electro-chemotherapy studies based on qualitative systematic review. Radiol Oncol 2016; 50: 1-13. doi: 10.1515/raon-2016-0006 2. Gehl J, Sersa G, Matthiessen LW, Muir T, Soden D, Occhini A, et al. Updated standard operating procedures for electrochemotherapy of cutaneous tumours and skin metastases. Acta Oncol 2018; 57: 874-82. doi:10.1080/02 84186X.2018.1454602 3. Campana LG, Miklavcic D, Bertino G, Marconato R, Valpione S, Imarisio I, et al. Electrochemotherapy of superficial tumors - current status: basic princi­ples, operating procedures, shared indications, and emerging applications. Semin Oncol 2019; 46: 173-91. doi: 10.1053/j.seminoncol.2019.04.002 4. 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Normal and fibrotic liver parenchyma respond differently to irreversible electroporation. HPB 2019; 21: 1344-53. doi: 10.1016/j.hpb.2019.01.019 9. Geboers B, Scheffer HJ, Graybill PM, Ruarus AH, Nieuwenhuizen S, Puijk RS, et al. High-voltage electrical pulses in oncology: Irreversible elec­troporation, electrochemotherapy, gene electrotransfer, electrofusion, and electroimmunotherapy. Radiology 2020; 295: 254-72. doi: 10.1148/ra­diol.2020192190 10. Miklavcic D, Mali B, Kos B, Heller R, Serša G. Electrochemotherapy: from the drawing board into medical practice. Biomed Eng Online 2014; 13: 29. doi: 10.1186/1475-925X-13-29 11. Brloznik M, Boc N, Sersa G, Zmuc J, Gasljevic G, Seliskar A, et al. Radiological findings of porcine liver after electrochemotherapy with bleomycin. Radiol Oncol 2019; 53: 415-26. doi: 10.2478/raon-2019-0049 12. Zmuc J, Gasljevic G, Sersa G, Edhemovic I, Boc N, Seliskar A, et al. 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Irreversible electroporation for unresectable hepatocellular carcinoma: ini­tial experience. Cardiovasc Intervent Radiol 2019; 42: 584-90. doi: 10.1007/ s00270-019-02164-2 17. Kalra N. Locoregional treatment for hepatocellular carcinoma: the best is yet to come. World J Radiol 2015; 7: 306-18. doi: 10.4329/wjr.v7.i10.306 18. Gasbarrini A, Campos WK, Campanacci L, Boriani S. Electrochemotherapy to metastatic spinal melanoma: a novel treatment of spinal metastasis? Spine 2015; 40: E1340-6. doi: 10.1097/BRS.0000000000001125 19. Cornelis FH, Ben Ammar M, Nouri-Neuville M, Matton L, Benderra MA, Gligorov J, et al. Percutaneous image-guided electrochemotherapy of spine metastases: initial experience. Cardiovasc Intervent Radiol 2019; 42: 1806­9. doi: 10.1007/s00270-019-02316-4 20. Tarantino L, Busto G, Nasto A, Nasto RA, Tarantino P, Fristachi R, et al. Electrochemotherapy of cholangiocellular carcinoma at hepatic hilum: a feasibility study. 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J Gastrointest Oncol 2017; 8: 337-46. doi: 10.21037/jgo.2016.09.06 28. Wang T, Lu XJ, Chi JC, Ding M, Zhang Y, Tang XY, et al. Microwave ablation of hepatocellular carcinoma as first-line treatment: long term outcomes and prognostic factors in 221 patients. Sci Rep 2016; 6: 32728. doi: 10.1038/ srep32728 29. Ding J, Jing X, Liu J, Wang Y, Wang F, Wang Y, et al. Complications of thermal ablation of hepatic tumours: comparison of radiofrequency and micro­wave ablative techniques. Clin Radiol 2013; 68: 608-15. doi: 10.1016/j. crad.2012.12.008 30. Lucchina N, Tsetis D, Ierardi AM, Giorlando F, Macchi E, Kehagias E, et al. Current role of microwave ablation in the treatment of small hepatocel­lular carcinomas. Ann Gastroenterol 2016; 29: 460-5. doi: 10.20524/ aog.2016.0066 31. Ding J, Jing X, Liu J, Wang Y, Wang F, Wang Y, et al. Comparison of two differ­ent thermal techniques for the treatment of hepatocellular carcinoma. Eur J Radiol 2013; 82: 1379-84. doi: 10.1016/j.ejrad.2013.04.025 32. Distelmaier M, Barabasch A, Heil P, Kraemer NA, Isfort P, Keil S, et al. Midterm safety and efficacy of irreversible electroporation of malignant liver tumors located close to major portal or hepatic veins. Radiology 2017; 285: 1023-31. doi: 10.1148/radiol.2017161561 33. Pompili M, Francica G. Irreversible electroporation for hepatic tumors. J Ultrasound 2019; 22: 1-3. doi: 10.1007/s40477-019-00367-4 34. Mafeld S, Wong JJ, Kibriya N, Stenberg B, Manas D, Bassett P, et al. Percutaneous irreversible electroporation (IRE) of hepatic malignancy: a bi-institutional analysis of safety and outcomes. Cardiovasc Intervent Radiol 2019; 42: 577-83. doi: 10.1007/s00270-018-2120-z 35. Mali B, Jarm T, Corovic S, Paulin-Kosir MS, Cemazar M, Sersa G, et al. The effect of electroporation pulses on functioning of the heart. Med Biol Eng Comput 2008; 46: 745-57. https://doi.org/10.1007/s11517-008-0346-7 36. Ball C, Thomson KR, Kavnoudias H. Irreversible electroporation: a new chal­lenge in “out of operation theatre” anaesthesia. Anesth Analg 2010; 110: 1305-9. doi: 10.1213/ANE.0b013e3181d27b30 37. Mekid H, Tounekti O, Spatz A, Cemazar M, Kebir E, Mir LM. In vivo evolu­tion of tumour cells after the generation of double-strand DNA breaks. Br J Cancer 2003; 88: 1763-71. doi: 10.1038/sj.bjc.6600959 38. Markelc B, Sersa G, Cemazar M. Differential mechanisms associated with vascular disrupting action of electrochemotherapy: intravital microscopy on the level of single normal and tumor blood vessels. PLoS One 2013; 8: e59557. doi: 10.1371/journal.pone.0059557 39. Calvet CY, Famin D, André FM, Mir LM. Electrochemotherapy with bleomy­cin induces hallmarks of immunogenic cell death in murine colon cancer cells. Oncoimmunology 2014; 3: e28131. doi: 10.4161/onci.28131 40. Sersa G, Teissie J, Cemazar M, Signori E, Kamensek U, Marshall G et al. Electrochemotherapy of tumors as in situ vaccination boosted by immuno-gene electrotransfer. Cancer Immunol Immunother 2015; 64: 1315-27. doi: 10.1007/s00262-015-1724-2 41. Heppt MV, Eigentler TK, Kähler KC, Herbst RA, Gpner D, Gambichler T, et al. Immune checkpoint blockade with concurrent electrochemotherapy in advanced melanoma: a retrospective multicenter analysis. Cancer Immunol Immunother 2016; 65: 951-9. doi: 10.1007/s00262-016-1856-z 353 research article Consolidation radiotherapy for patients with extended disease small cell lung cancer in a single tertiary institution: impact of dose and perspectives in the era of immunotherapy Karmen Stanic1,2, Martina Vrankar1,2, Jasna But-Hadzic1,2 1 Department of Radiotherapy, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2020; 54(3): 353-363. Received 9 March 2020 Accepted 4 July 2020 Correspondence to: Assist. Prof. Jasna But-Hadžic, M.D. PhD., Institute of Oncology Ljubljana, Department of Radiotherapy, Zaloška 2, 1000 Ljubljana. E-mail: jbut@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. Consolidation radiotherapy (cRT) in extended disease small cell lung cancer (ED-SCLC) showed im­proved 2-year overall survival in patients who responded to chemotherapy (ChT) in CREST trial, however results of two meta - analysis were contradictive. Recently, immunotherapy was introduced to the treatment of ED-SCLC, making the role of cRT even more unclear. The aim of our study was to access if consolidation thoracic irradiation improves survival of ED-SCLC patients treated in a routine clinical practice and to study the impact of cRT dose on survival. We also discuss the future role of cRT in the era of immunotherapy. Patients and methods. We retrospectively reviewed 704 consecutive medical records of patients with small cell lung cancer treated at the Institute of Oncology Ljubljana from January 2010 to December 2014 with median follow up of 65 months. We analyzed median overall survival (mOS) of patients with ED-SCLC treated with ChT only and those treated with ChT and cRT. We also compared mOS of patients treated with different consolidation doses and performed univariate and multivariate analysis of prognostic factors. Results. Out of 412 patients with ED-SCLC, ChT with cRT was delivered to 74 patients and ChT only to 113 patients. Patients with cRT had significantly longer mOS compared to patients with ChT only, 11.1 months (CI 10.1–12.0) vs. 7.6 months (CI 6.9–8.5, p < 0.001) and longer 1-year OS (44% vs. 23%, p = 0.0025), while the difference in 2-year OS was not significantly different (10% vs. 5%, p = 0.19). The cRT dose was not uniform. Higher dose with 45 Gy (in 18 fractions) resulted in better mOS compared to lower doses 30–36 Gy (in 10–12 fractions), 17.2 months vs. 10.3 months (p = 0.03) and statistically significant difference was also seen for 1-year OS (68% vs. 30%, p = 0.01) but non significant for 2-year OS (18% vs. 5%, p = 0.11). Conclusions. Consolidation RT improved mOS and 1-year OS in ED-SCLC as compared to ChT alone. Higher dose of cRT resulted in better mOS and 1-year OS compared to lower dose. Consolidation RT, higher number of ChT cycles and prophylactic cranial irradiation (PCI) were independent prognostic factors for better survival in our analysis. For patients who received cRT, only higher doses and PCI had impact on survival regardless of number of ChT cycles received. Role of cRT in the era of immunotherapy is unknown and should be exploited in further trials. Key words: small cell lung cancer; ED-SCLC; radiotherapy; consolidation radiotherapy; immunotherapy Introduction ease and unfortunately diagnosed already in ad­ vanced stage in majority of patients.1 In Slovenia Small cell lung cancer (SCLC) represents only small 15.3% of lung cancer patients were diagnosed with proportion of lung cancer but is an aggressive dis-SCLC in 2014, and majority had metastatic dis- 354 ease.2 In recent years, percentage of patients with metastatic disease has slightly increased, but this might only be due to better staging with incorpora­tion of PET/CT and brain MRI.3,4 SCLC is highly chemo-sensitive disease and standard treatment for metastatic patients is platinum based chemotherapy (ChT), usually combined with etoposide or irinotecan.5,6 Almost 75% of the patients have persisting intra-thoracic disease after treatment with ChT and addition of chest radiotherapy (RT) aimed to improve progres­sion free survival (PFS) and overall survival (OS) in those patients.7 Prospective randomized CREST study suggested survival benefit of added thoracic RT in addition to PCI for ED-SCLC patients who respond to ChT; however, OS at 1-year, which was the primary endpoint of the study, was not signifi­cantly improved.8 Prospective RTOG 0937 study also failed to show 1-year survival benefit, though disease progression was delayed.9 On the other hand, some retrospective studies showed benefit of consolidation RT (cRT).10-13 None of the prospec­tive and only rare retrospective studies specifically researched the effect of radiation dose on survival. In addition, selective patients might benefit from prophylactic cranial irradiation (PCI), which riod studied, we were the only radiotherapy center showed increase in overall survival if added to ED-in the country and all patients that needed irradia-SCLC after ChT.14 In spite of that, the median over-tion, based on multidisciplinary tumor board deci-all survival (mOS) of metastatic disease remains sion, were treated at our institution. Only patients poor, ranging from 8 to13 months, with only 5% of who had at least stable disease or regression of dis-patients being alive at 2 years.15 Recently, immuno-ease after chemotherapy were eligible for thoracic therapy with atezolizumab or durvalumab added consolidation radiotherapy. The decision about the to ChT without chest irradiation has shown in-dose was at the discretion of radiation oncologist creased mOS in first line treatment of patients with and based on the volume of the tumor and perfor-metastatic SCLC, therefore in the future the role of mance status of the patient since during the time radiotherapy would need to be reconsidered.16,17 period studied there was no uniform dose suggest- The aim of our study was to access if cRT im-ed in any of the guidelines. proves survival of ED-SCLC patients treated in a Diagram in Figure 1 outlines the selection pro-routine clinical practice of tertiary single centre cess. During 5 year period 704 consecutive patients and to study the impact of cRT dose on survival. with SCLC were treated at the Institute of oncology We also discuss whether the cRT still has the role Ljubljana, 412 with extended disease and 292 with in the treatment of ED SCLC in the era of immu-locally advanced disease. Among all ED-SCLC pa-notherapy. tients, 59 (14.3%) were treated with BSC, 67 (16.2%) patients with RT only, 113 (27.4%) with ChT only and 173 (41.9%) with combined ChT and RT. RT Patients and methods was either consolidation RT (cRT), delivered to 74 patients or any other type of RT which included We retrospectively reviewed medical records of urgent RT, partly concurrent ChT or RT that was consecutive patients with SCLC treated at the prematurely closed due to any reason (99 patents). Institute of Oncology Ljubljana during the five The following parameters were recorded: demo-year period, from January 2010 to December 2014. graphic and clinical characteristics, date of diagno-Median follow up was 65 months. sis, TNM stage, treatment characteristics, including Not all metastatic SCLC patients were referred chemotherapy and radiation therapy details, meta-to our center for treatment; however, during the pe-static locations and date of death or last follow up. 355 Chemotherapy Of Majority (47.5%) of patients received all 6 planned cycles of chemotherapy, 66 patients (35.3%) received less than 4 cycles of ChT. Etoposide with platinum was the most frequent combination (83.8%), the rest received anthracycline based ChT. In the group with cRT were less patients who re­ceived 4 ChT cycles or less. Radiotherapy Radiotherapy with linear accelerators (photon beam 6-10MV), based on 3D CT-based conformal radiation therapy planning, started after ChT. There was no difference in frequency of patients who started before (29 patients) and after 4 weeks (25 patients) of ChT completion. Prophylactic cra­nial irradiation was delivered with two opposed lateral fields with the dose of 25 Gy in 10 fractions using 2D planning and 6MV photon beam energy. Statistical analysis The primary endpoints in this analysis were mOS, 1-year and 2-year OS of ED-SCLC patients treated with ChT only versus patients treated with ChT and cRT and those receiving higher vs. lower dose of cRT. Median OS was calculated from the time of diagnosis to the time of death due to any cause or last follow up visit. Kaplan-Meier (KM) method and log-rank test were used for comparison of sur­vival curves between different groups. Cox pro­portional hazards algorithm was used for univari­ate and multivariate analysis. Association between subgroups and clinico-pathological characteristics of patients were tested using chi-square method. All p values reported were based on 2 side hypoth­esis. The statistical analysis was computed using SPSS v.20 statistical package. Ethical consideration This survey was approved by Institutional Ethics Committee and Institutional Review Board in December 2017. Results We performed two analysis. In our first analysis we included 187 patients, 113 patients treated with Cht only were compared to 74 patients treated with ChT and cRT. Different fractionation schemes were TABLE 1. Patients’ characteristics: chemotherapy only vs. chemotherapy with consolidation radiotherapy Gender 187 (100) 113 (60.4) 74 (39.6) Male 126 (67.4) 81 (71.1) 45 (60.1) 0.12 Female 61 (32.6) 32 (28.9) 29 (39.9) Age median (range) 63 (42-80) 61 (42-80) 63 (47-80) 0.24 < 65 122 (65.2) 70 (61.9) 52 (70.3) > 65 65 (34.8) 43 (38.1) 22 (29.7) Number of ChT cycles* < 4 66 (35.3) 51 (47.2) 15 (20) <0.001 > 4 113 (60.4) 57 (52.8) 56 (80) T stage 0.23 T1–2 32 (17.1) 20(17.7) 12 (16.2) T3-4 122 (65.2) 69 (61) 53 (71.6) Tx 33 (17.7) 24 (21.3) 9 (12.2) N stage 0.56 N0–2 71 (38) 40 (35.4) 31(41.9) N3 91(48.7) 56 (49.6) 35 (47.3) Nx 25 (13.3) 17 (15) 8 (10.8) Metastases location** Brain 44 (23.5) 28 (24.8) 16 (21.6) 0.61 Liver 86 (46) 57 (50.4) 29 (39.2) 0.13 Bone 42 (22.5) 28 (24.8) 14 (18.9) 0.34 Adrenal gland 38 (20.3) 23 (20.4) 15 (20.3) 0.98 Other 92 (49.2) 62 (54.9) 30 (40.5) 0.06 Number of metastatic locations 1 105 (56.1) 55 (48.7) 50 (67.6) 0.01 > 2 82 (43.9) 58 (51.3) 24 (23.4) PCI Yes 41 (21.9) 20 (17.6) 21 (28.4) 0.08 no 146 (78.1) 93 (82.4) 53 (71.6) * for 8 patients we were not able to retrieve the exact number of cycles from medical records, percentage of patient is calculated only for those with known number of cycles (179); ** some patients had more than 1 metastatic location, percentages are calculated as part of all patients in a group; ChT = chemotherapy; cRT = consolidation radiotherapy; PCI = prophylactic cranial irradiation 356 TABLE 2. Patients’ characteristics: higher vs. lower dose of radiotherapy Gender 59 15 44 Male 35 (60) 6 (40) 29 (65.9) 0.078 Female 24 (40) 9 (60) 15 (34.1) Age median 62 (42–76) 60 (54–73) 62 (42–76) 0.12 < 65 42 (71.2) 13 (68.7) 29 ( 65.9) > 65 17 (28.8) 2 (13.3) 15 (34.1) Number of ChT cycles < 4 12 (20.3) 2 (13.3) 10 (22.7) 0.37 > 4 44 (74.6) 13 (68.7) 31 (70.5) unknown 3 (5.1) 0 (0) 3 (6.8) PS before RT 0.66 0-1 22 (37.3) 5 (33.3) 17 (38.6) 2–3 7 (11.8)) 1 (6.67) 6 (13.6) unknown 30 (50.9) 9 (0.6) 21 (47.8) T stage 0.15 T1–2 8 (13.6) 4 (26.7) 4 (9.1) T3–4 42 (71.2) 8 (53.3) 34 (77.3) Tx 9 (15.3) 3 (20) 6 (13.6) N stage 0.69 N0–2 24 (40.7) 7 (46.7) 17 (38.6) N3 29 (49.2) 6 (40) 23 (52.3) Nx 6 (10.1) 2 (13.3) 4 (9.1) Metastases location* Brain 14 (23.7) 5 (33.3) 9 (20.5) 0.31 Liver 27 (45.7) 6 (40) 21 (47.7) 0.60 Bone 13 (22) 3 (30) 10 ( 22.7) 0.82 Adrenal gland 15 (25.4) 3 (30) 12 (27.3) 0.57 Other 21 (35.6) 3 (30) 19 (43.2) 0.10 Number of metastatic locations 1 34 (57.6) 10 (66.7) 24 (54.5) 0.41 > 2 25 (42.4) 5 (33.3) 20 (45.4) Timing of RT** 0.15 < 4 weeks after ChT 17 (53.1) 6 (75) 11 (45.9) > 4 weeks after ChT 15 (46.9) 2 (25) 13 (54.1) PCI Yes 17 (28.8) 5 (33.3) 12 (27.3) 0.65 * some patients had more than one metastatic site; ** for 31 missing patients no reliable data of the completion chemotherapy date could be retrieved from the medical records; Ch =- chemotherapy; Gy = Gray; N = lymph nodes; PS = performance status; RT = radiotherapy; T = tumour used for cRT. The doses in cRT were not uniform, therefore we divided them into 3 groups: below 30 Gy, 30–36 Gy and 45 Gy. Only 59 patients with doses above 30 Gy were included in our second analysis of dose comparison. Patient characteristics Baseline characteristics of 187 patients, divided to those with ChT only and those who also received cRT are presented in Table 1. The two groups were balanced regarding gender, age, T and N stage and metastatic locations. However, lower number of patients received 4 or less cycles of ChT and had 2 or more metastases present at diagnosis in ChT plus cRT group. Table 2 present baseline characteristics of 59 patients who received > 30 Gy cRT, comparing those with higher dose (45 Gy) cRT and lower dose (30–36 Gy). In summary, median age was 63 years, more than half were men. Majority of patients were younger than 65 years. Unfortunately, reliable PS could not be retrieved from medical records for half of the patients and more than 10% of patients had PS 2-3 before cRT. Non-significantly more pa­tients had larger tumors (T3-4) and more extended lymph node disease (N3) in the group treated with lower dose RT. For more than 10% of patients with central tumors, the size of tumor (T) or nodal sta­tus could not be determined. Fifty-eight percent of patients had one metastatic site. The most frequent site of metastases were liver. Less than third of pa­tients had PCI. Survival data Median OS of patients who had either BSC or RT only was poor, 1.86 and 2.42 months, respectively. Patients who had any form of additional chest irra­diation (173 patients) had significantly better mOS than 113 patients with ChT only (9.9m vs. 7.6m, p = 0.002). Consolidation RT was delivered to 74 patients. Those patients had significantly longer mOS com­pared to 113 patients with ChT only as presented in Figure 2, 11.1 months (CI 10.1–12.0) vs. 7.6 months (CI 6.9–8.5), p < 0.001. They also had significantly longer 1-year OS (44% vs. 23%, p = 0.0025), but non significantly longer 2-year OS (10% vs. 5%, p = 0.19). Univariate survival analysis (UVA) for patients with or without cRT included the following vari­ables: cRT, gender, age, number of ChT cycles, T and N stage, metastatic location, number of meta- 357 static locations and PCI. Presence of cRT, female gender, number of ChT cycles (4 or less and more 1,0 than 4) and PCI were significant in univariate analysis and were tested in multivariate analysis (MVA) (Table 3). Except for gender, they were all 0,8 independent predictors of better survival. In the group of 59 patients irradiated with cRT Overall Survival 0,6 = 30 Gy patients irradiated with 45 Gy had better mOS compared to patients irradiated with doses 30–36 Gy, 17.2 months vs. 10.3 months, p = 0.03. (Figure 3) Patients with higher dose of consolida­tion RT had significantly longer 1-year OS (68%) than those with lower dose (30%), p = 0.01, but non­ 0,4 significantly longer 2-year OS (18% vs. 5%, p = 0.11). In the group of patients with cRT, we made an­other analysis. We included gender, age catego­ries, PS before RT, RT dose, T and N stage, meta­static locations, number of ChT cycles, number of metastatic lesions, PCI and timing of RT in UVA. Statistically significant predictors of longer mOS were PCI irradiation and higher RT dose. Both were analyzed in MVA (Table 4) and remained in­dependent predictors of improved survival. (PCI HR = 0.51, 95% CI 0.27–0.96; higher RT dose HR = 0.47, 95% CI 0.25–0.87). 0,2 0,0 Time (months) FIGURE 2. Overall survival of patients treated with chemotherapy (Cht only) vs. chemotherapy and consolidation radiotherapy (ChT + cRT). 1,0 Discussion 0,8 Thoracic irradiation has never been considered Overall Survival 0,6 0,4 such an important part of ED-SCLC treatment as chemotherapy. Since the pivotal study of Jeremic et al. two decades ago, who were the first to show importance of RT in ED SCLC, only lately intro­ duction of modern RT techniques with less toxicity rose interest again for the use of RT.18 Survival of patients with chemotherapy only and those who also had consolidation radiotherapy Our analysis showed that cRT significantly im­proved mOS compared to patients who had ChT only, 11.1 months vs. 7.6 months. Those patients also had significantly longer 1-year OS (44% vs. 23%) and non-significantly longer 2-year OS (10% vs. 5%). Apart from cRT, independent predictors of survival were also PCI and higher number of ChT cycles delivered. Unfortunately, the response to ChT could not be included to our analysis, as due to retrospective nature of this study the response to ChT was not uniformly evaluated. How results of our study compares to others is presented in Table 5. In a retrospective study of 119 0,2 0,0 Time (months) FIGURE 3. Overall survival of patients treated with higher (45 Gy) vs. lower (30-36 Gy) dose of irradiation. patients by Zhu et al., survival results were much better than in our analysis, with mOS of 17 months for patients in ChT plus cRT group and 9.3 months for those with ChT only, and 2-year OS of 35% and 17%, respectively. They delivered higher cRT dose (range 40–60 Gy) and had comparable mOS (17.2 months) as our group of patients irradiated with 45 358 TABLE 3. Univariate and multivariate analysis of overall survival for patients with cRT vs no cRT (n = 187) cRT < 0.001 1.73 (1.27–2.34) 0.01 1.52 (1.10–2.09) no yes Gender 0.042 1.17 (1.00–1.37) 0.68 1.03 (0.87–1.21) Male Female Age 0.25 1.19 (0.88–1.62) > 65 < 65 Number of cycles received < 0.001 3.23 (2.33–4.47) < 0.001 3.11 (2.22–4.35) < 4 > 4 T stage 0.98 1.00 (0.67–1.50) T1, 2 T3, 4 N stage 0.16 1.08 (0.96–1.22) N0-2 N3 Metastases location Brain no/yes 0.61 0.91 (0.64–1.29) Liver no/yes 0.40 1.13 (0.84–1.52) Bone no/yes 0.75 0.94 (0.67-1.33) Adrenal gland no/yes 0.62 1.09 (0.76–1.57) Other no/yes 0.18 1.21 (0.90–1.63) Number of metastatic locations 0.68 1.06 (0.79–1.42) 1 > 2 PCI < 0.001 0.49 (CI 0.32–0.76) 0.015 1.59 (1.09–2.32) No Yes cRT = consolidation radiotherapy; N = lymph nodes; PCI = prophylactic cranial irradiation; T = tumour Gy.10 Study by Yee et al. included only 33 patients, all with PCI and cRT (40 Gy), but their reported mOS of 8.3 months is lower than ours.11 Another small retrospective study of 19 patients with cRT 40 Gy in 15 fraction reported mOS 14 months with 1-year and 2-year OS 58% and 14%.12 Difference in the results of these studies show that survival benefit could not be attributed to RT only, but also to the increased chances of those patients who re­mained in a better shape and fitter at the time of disease progression to receive subsequent lines of chemotherapy. Data from SEER analysis on al­most 7000 patients also provide evidence that ra­diotherapy for thoracic lesion and any metastatic sites could significantly improve the OS, except for brain metastasis.13 Three prospective randomized trials researched impact of RT on survival in ED SCLC.8,9,18 Trial by Jeremic et al. differs in many ways from more recently reported studies. They used accelerated hyperfractionation (54 Gy in 36 fractions) with concomitant ChT after 3 cycles of induction ChT and additional 2 cycles after RT in one group or after 5 cycles of ChT in another, both groups also eligible for PCI. They studied combined modal­ity treatment rather than cRT. The reported mOS was excellent for those who received RT early (17 months) as compared to those who received late RT (6–8 months).18 Another concern regarding hy­perfractionated RT is that is delivered twice daily (BID) and is technically challenging for patients with bilateral mediastinal lesions, which repre­sented the majority in our population. Further, patients selected for combined modality treatment, which incorporates BID RT must have excellent performance status and baseline pulmonary func­tion. In our study more than 10% of patients had PS 2-3 before cRT and unfortunately in more than half of patients PS could not be reliably retrieved from medical records. Phase III EORTC study (CREST) included pat­ents with PS 0–2 without brain and pleural metas­tases. Responders after 4–6 cycles of ChT and re­sidual disease in the thorax were treated with irra­diation of 30 Gy in 10 fractions.8 Contrary to our re­sults, no benefit was shown for added RT after ChT regarding mOS, which reported to be 8 months in both groups and for 1-year OS (33% for ChT with cRT vs. 28% for ChT group only). However, they reported significant difference in 2-year OS 13% vs. 3% (p = 0.004). It should, however, be noted that mOS was calculated from the randomization while mOS from diagnosis (as calculated in our analysis) was 12 months. 359 More aggressive thoracic irradiation was given in RTOG 0937 trial with 45 Gy in 15 fractions.9 Reported median OS (15.8 months) was better than anticipated and much better than in CREST and our study. Unlike all other studies, they reported better mOS for ChT only group (15.8 months) than for ChT plus RT group (13.8 months), though the difference was not statistically significant. 1-year OS was similar, surprisingly higher for ChT only than for ChT plus cRT group (60.1% vs. 50.8%). Two meta-analyses were published. The first, published by Palma et al. in 2015 included 2 stud­ies with 604 patients, while the second published in 2019 by Rathod et al. added also 86 patients from prematurely closed RTOG 0937 data.19,20 First me-ta-analysis found increased OS (p = 0.01), while the second failed to show improvement in overall sur­vival by adding cRT to ChT, (p = 0.36). Effect of consolidation radiotherapy dose on survival We found that patients who had been irradiated with higher dose (45 Gy in 18 fractions) had bet­ter mOS compared to those who received lower doses 30–36 Gy (in 10–12 fractions), 17.2 months vs. 10.3 months. Patients with higher dose of cRT had better 1-year OS (68%) than those with lower dose (30%) and also better 2-year OS (18% vs. 5%). Not many studies looked into dose difference for cRT. In retrospective study including 306 pa­tients of whom 170 received cRT, those with higher RT dose (BED > 50 Gy) had longer 2y-OS, 32.3% vs. 17% (p < 0.001), respectively.21 In recently pub­lished retrospective analysis of National Cancer Database that included 3280 patients they also re­ported that patients treated with the dose at least 45 Gy had better survival; 1-year OS was 58.1% and 2-year OS was 25.2% compared to 43.8% and 15.1% for lower dose.22 Our results for 1-year OS compare favorable, but 2-year OS data are lower, suggesting our subsequent treatments were not as effective. In CREST study, cRT dose used was 30 Gy in 10 fractions. The relative high intrathoracic failure rate of 42% indicated that this dose might be insuf­ficient to eliminate all the residual disease. In addi­tional analysis from CREST study, for patients with complete intrathoracic response no benefit of TRT was observed. They concluded that TRT should be offered to patients with a good or partial response after chemotherapy, but not to those without resid­ual disease in the thorax. It appears that the greater the volume of the residual disease in the thorax is, the higher dose is needed to eliminate the tumor. TABLE 4. Univariate and multivariate analysis of overall survival for higher vs. lower dose of consolidation radiotherapy Dose 0.023 0.49 (0.27–0.90) 0.018 0.47 (0.25–0.87) 45 Gy 30-36 Gy Gender 0.17 1.4 (0.86–2.27) Male Female Age 0.38 1.25 (0.75–2.09) > 65 < 65 PS before RT 0.089 1.94 (0.90–4.18) 2–3 0–1 Number of ChT cycles 0.065 1.78 (0.96–3.31) < 4 > 4 T stage 0.34 0.72 (0.37–1.40) T1–2 T3–4 N stage 0.28 1.32 (0.79–2.20) N0–2 N3 Metastases location Brain da/ne 0.52 1.2 (0.68–2.11) Liver da/ne 0.39 1.22 (0.76–1.92) Bone da/ne 0.46 1.24 (0.70–2.21) Adrenal gland da/ne 0.98 0.99 (0.59–1.67) Other 0.84 0.95 (0.58–1.56) Number of metastatic 0.43 0.82 (0.51–1.33) locations 1 > 2 Timing of RT 0.71 1.13 (0.59–2.16) < 4 weeks after ChT > 4 weeks after ChT PCI 0.04 0.56 (CI 0.32–0.97) 0.037 0.51 (0.27–0.95) Yes No ChT = chemotherapy; cRT = consolidation radiotherapy; N = lymph nodes; PS = performance status; RT = radiatiotherapy; T = tumour 360 TABLE 5. Trials of consolidation radiotherapy (cRT) in extended disease small cell lung cancer (ED-SCLC) ED-SCLC with CR at Jeremic18 1999 P 1988–1993 109 metastatic sites and at 54 Gy in 36 fractions, BID 17 m vs. 11 m* P = 0.041 65% vs. 46% P = 0.05 38% vs. 28% P = 0.05 least PR in thorax Slotman (CREST)8 2015 P 2009–2012 495 ED-SCLC with any response to ChT 30 Gy in 10 fractions 8 m vs. 8 m 33% vs. 28% P = 0.066 13% vs. 3% P = 0.004 Gore (RTOG 0937)9 2017 P 2010–2016 97 ED-SCLC (1-4 extracranial m., any response to ChT 40 Gy in 15 fractions 15.8 m vs. 13.8 m P = 0.21 50.8% vs. 60.1% P = 0.21 NR Zhu10 2011 R 2003–2006 119 ED-SCLC 40–60 Gy 17 m vs. 9.3 m P = 0.014 NR 35% vs. 17% ED-SCLC Giuliani12 2011 R 2005–2009 19 with minimal metastatic 36–45 Gy 14 m 58% 14% disease Yee11 2012 R 2008–2009 32 ED-SCLC 40 Gy in 15 fractions 8.3 m NR NR 9 m vs. 7 m; Zhan13 (SEER database) 2018 R 2010–2012 6812 ED-SCLC from SEER database Different, not reported P < 0.001 8 m vs. 6 m for polymetastases P < 0.05 NR NR Stanic 2020 R 2010–2014 187 ED-SCLC 30–45 Gy 11.1 m vs. 7.6 m P < 0.001 44% vs. 23% P = 0.0025 10% vs. 5% P = 0.19 * group 1 CR/PR and RT vs. group 2 CR/PR, no RT; BID = twice daily; ChT = chemotherapy; CR = complete response; ED-SCLC = extended disease small cell lung cancer; m = months; mOS = median overall survival; NR-not reported; OS = overall survival; P = prospective; PR = partial response; R = retrospective However, dose restrictions to the organs at risk and consequent toxicity limit the actual received dose. Number of metastases was not predictive fac­tor for survival in our analysis. Contrary to that, in recent retrospective publications it was shown that tumor burden of metastatic disease should be taken into account when treating ED SCLC patients, since those with =2 metastases had significantly worse outcome than those with only one metastasis.23,24 No difference of timing was found in our sur­vival analysis if RT started before or after 4 weeks after ChT completion. In RTOG 0937 trial and one retrospective Chinese study also no difference was found in survival for patients who received RT ear­ly or late.9,25 On the contrary, meta-analysis for lim­ited SCLC, showed that earlier or shorter RT brings 7.7% advantage in 5-year survival.26 In our study PCI was independent predictor of better survival, although only 21.9% of patients received one. Our previous publication, focused on impact of PCI on survival in patients with LD­SCLC, also showed that only low number of pa­tients (6%) actually received PCI in routine clinical setting, nevertheless OS was improved with PCI.27 As our analysis is retrospective, this reflects real clinical situation. However, the reason why such a low number of patients actually received PCI is unclear. PCI as independent predictor of survival was reported also in retrospective study by Xu et al.21 PCI in ED-SCLC was studied in EORTC con­ducted prospective study that showed reduced incidence of symptomatic brain metastases and improved 1-year OS (27% vs. 13.3%, HR 0.68, p = 0.003). That study, however, was highly criticized due to the insufficient imaging prior to PCI.9 Japanese prospective study evaluated 224 patients with ED-SCLC who performed MRI prior to ran­domization to PCI or observation with MRI.28 The study was terminated prematurely due to lower rate of brain metastases in PCI arm (40%) vs. MRI observation only (64%), but they found no signifi­cant difference in 1-year OS. None of our patients had MRI prior to PCI and only one third had CT evaluation, indicating that imaging in routine 361 clinical practice should improve. In CREST study PCI dose was not uniform (20–30 Gy in 5–15 frac­tions) with unusual hypofractionated dose (20 Gy in 5 fraction) used in majority of patients (62%).8 It was delivered concurrently with thoracic irradia­tion in 88% of patients, while other studies used sequential approach and uniform dose of 25 Gy in 10 fractions.9,18 Difference in pre-PCI imaging and dose delivered as well as timing of PCI show diversified approach on this not fully researched area.29 Consolidation radiotherapy and immunotherapy Immunotherapy (IT) has been successfully incor­porated into the treatment of metastatic non-small cell lung cancer (NSCLC) either as combination of ChT and IT or as mono-IT and lately also in stage III as consolidation treatment after concomitant chemoradiotherapy.30-39 Recently, two randomized studies confirmed efficacy of IT also for the treatment in ED-SCLC. IMpower 133 study was the first to show improved OS in patients treated with atezolizumab combined with ChT (12.3 months) as compared to ChT plus placebo (10.3 months). 1-year OS rate was 51.7% in the atezolizumab group and 38.2% in the pla­cebo group.16 Consolidation RT was not permitted, while patients could have PCI. The same criteria about cRT and PCI were also applied in CASPIAN study with durvalumab.17 Again, IT combination showed increased results, mOS in ChT-IT arm was 13 months and 10.3 months in ChT only arm and 1-year OS was 54% vs. 40%, respectively. Though PCI was allowed in the non IT group, only 8% of patients received it. If the inclusion of immuno-therapy would prove to reduce the incidence of brain metastases in ES-SCLC considerably in fu­ture trials as suggested from present studies, then PCI and consequently neurotoxic sequels could be omitted in the future. The decision about skipping cRT might be more challenging. Survival data from current studies has not shown superior survival in first line treatment with ChT-IT in ED-SCLC com­pared to studies with ChT and cRT. Could cRT be combined with IT during the consolidation phase? First reported data indicate that the combination is tolerable, however trials are still ongoing and safety as well as survival results are expected in the future.40 As previously reported, the use of thoracic RT may enhance the effect of IT by influencing the immune system and its interactions with cancer cells and tumors, recruiting anti-tumor immune cells, increasing the exposure of tumor antigens, and improving cross-presentation of these anti­gens to the adaptive immune system.41-43 Beside retrospective nature of our analysis we should acknowledge several other limitations of our research. The irradiation dose was not speci­fied by the protocol or any other department regu­lation and the decision was under the discretion of treating physician. Larger tumors (T3-4, N3) were more frequently irradiated with lower dose, but this does not necessarily mean that larger tumors would not be feasible to the treatment with larger doses. Unfortunately, we were not able to retrieve reliable information about PS before RT in half of patients, reflecting real clinical practice. This would be valuable information as treatment decision in clinical practice is greatly influenced by PS and consequently might influence survival data. Due to the fact that not all patients were treated with ChT in our institution, PS before ChT could not be included in UVA and MVA. Also, the response to initial Cht as one of the main prognostic factors of cRT efficacy according to the published data, is missing, since not all the patients were treated at our institution. However, all the patients were dis­cussed at the MTB before the treatment which at least partially reduces this shortcoming. Conclusions Our analysis has shown that cRT improved mOS as compared to ChT alone of the ED-SCLC patients treated at our institution. Consolidation RT, higher number of ChT cycles and profilactic cranial irra­diation (PCI) were independent prognostic factors for better survival. For patients who received cRT, only higher doses and PCI had impact on surviv­al regardless of number of ChT cycles received. Whether cRT and PCI will still be players in the era of immunotherapy is unknown and will be shown in further trials. References 1. van Meerbeeck JP, Fennell DA, De Ruysscher DK. Small-cell lung cancer. Lancet 2011; 378: 1741-55. doi: 10.1016/S0140-6736(11)60165-7 2. Cancer in Slovenia 2014. Ljubljana: Institute of Oncology Ljubljana, Epidemiology and Cancer Registry, Cancer Registry of Republic of Slovenia; 2017. 3. Mitchell MD, Aggarwal C, Tsou AY, Torigian DA, Treadwell JR. Imaging for the pretreatment staging of small cell lung cancer: a systematic review. 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Lancet Oncol 2015; 16: e498-509. doi: 10.1016/S1470-2045(15)00007-8 364 research article Assessment of set-up errors in the radiotherapy of patients with head and neck cancer: standard vs. individual head support Sabina Androjna1, Valerija Zager Marcius1,2, Primoz Peterlin1, Primoz Strojan1,3 1 Department of Radiotherapy, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia 3 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2020; 54(3): 364-370. Received 17 February 2020 Accepted 3 May 2020 Correspondence to: Prof. Primož Strojan, M.D., Ph.D., Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. E-mail: pstrojan@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. The aim of the study was to (a) compare the accuracy of two different immobilization strategies for patients with head and neck tumors, and (b) compare the set-up errors on treatment units with different portal imag­ing systems. Patients and methods. Variations in the position of the isocenter (IC) relative to the reference point determined on the computed tomography simulator were measured in a vertical (anterior-posterior), longitudinal (superior-inferior), and lateral (medial-lateral) direction in 120 head and neck cancer patients irradiated with curative intent. Depending on the treatment unit (unit A - 2D/2D image previews; unit B - 2D image previews) and the time of irradiation, patients were divided into 6 groups of 20 patients. In patients irradiated in 2014, standard head supports were used (groups 1 and 2), whereas in those treated in 2015 and 2017 (groups 3–6) individual head supports were employed. The clinical-to-planning target volume safety margin was calculated according to the formula proposed by Van Herk. Results. In total, 2,454 portal images and 3,681 set-up errors were analysed. Implementation of individual head sup­ports in 2015 resulted in a statistically significant reduction in the average inter-fraction displacement in the vertical direction and in decreased number of IC displacements in the vertical and longitudinal direction (applies to both treatment units). The largest reduction of the safety margin was calculated in the longitudinal direction and the safety margins were larger for unit B than for unit A. Conclusions. The use of individual head supports and a more advanced imaging system were found to increase set-up precision. Key words: head and neck radiotherapy; immobilization; head support; set-up errors Introduction The basic tools for the immobilization of patients with head and neck (HN) tumors during radio­therapy are thermoplastic masks with a 5-point pinning system and supporting system for the head. These immobilization aids largely, but not completely, prevent major shifts during irradia­tion. Due to its regular use, the head support can shrink and deform over time, which leads to de­viations in the position of the HN compared to the reference position determined on the computed to­mography (CT) simulator (Figure 1). To overcome this problem, patient-specific head supports were introduced (i.e., customized head support that are moulded to the patient’s anatomy), which proved to effectively reduce systematic and random er­rors.1-4 At the Institute of Oncology Ljubljana, the ma­jority of HN cancer patients are irradiated on two similar treatment units with slightly different im­aging capabilities. Commercially available head 365 supports have been used since the 1990s (CIVCO, Coralville, Iowa, USA), whereas patient-specific head supports have never been introduced in rou­tine practice. Until 2015, all patients irradiated on a particular treatment unit shared the same set of head supports (i.e., standard head support). In order to reduce the set-up error, this policy was changed in 2015 and it was ensured that the same head support was used for a given patient from the CT simulator throughout the irradiation course (i.e., individual head support). Regular quality checks of head supports were performed: the dif­ference between the heights of used and unused supports was not allowed to exceed 3 mm. In the present study, two hypotheses were tested: (1) Deviations recorded by portal imaging system will be smaller in patients using individual head supports compared to those with standard ones; (2) The treatment unit with a more advanced portal imaging system will allow for a more accu­rate positioning of patients. Patients and methods Between January 2014 and October 2017, 120 HN cancer patients irradiated with curative intent were included in this retrospective non-interventional study. Patients were irradiated on either of the two low-energy linear accelerators equipped with MV imaging systems: unit A - Unique Performance Edition; and unit B - Clinac DBX (both: Varian Medical Systems, Palo Alto, California, USA). Depending on the treatment unit (A or B), the time of irradiation, and the type of head support used, patients were divided into 6 groups of 20 patients. Because individual head supports were introduced into routine practice in 2015, the consistency of re­sults related to their use over time was verified in two time periods (2015 and 2017) and, consequent­ly, in two independent groups of patients: - Group 1 – linear accelerator A, 2014 (standard head support) - Group 2 – linear accelerator B, 2014 (standard head support) - Group 3 – linear accelerator A, 2015 (individual head support) - Group 4 – linear accelerator B, 2015 (individual head support) - Group 5 – linear accelerator A, 2017 (individual head support) - Group 6 – linear accelerator B, 2017 (individual head support) Variations in the position of the isocenter (IC) relative to the reference point determined on the CT simulator (i.e., set-up errors) were measured in a vertical (anterior-posterior), longitudinal (supe­rior-inferior), and lateral (medial-lateral) direction. Simulation procedure At the CT simulator, the most appropriate head support was selected from the commercially avail­able set of items of various heights and contours, offering a comprehensive range of neck angula­tions (CIVCO, Coralville, Iowa, USA), according to the curvature of patient’s neck and occiput. The Kneefix™ and the Armaflex™ cushion were placed under the knees and the back and pelvis, respec­tively, and the head was additionally fixed with a thermoplastic 5-point Posicast® mask (all: CIVCO, Coralville, Iowa, USA). Radio-opaque markers (Beekley Medical, Bristol, Connecticut, USA) were used for three-point marking of the IC origin. CT scanning from 2 cm above the top of the head to the tracheal bifurcation (slice thickness: 2 mm) was accomplished using an intravenous administration of iodine contrast medium by power injector, fol­lowed by tattooing the thoracic skin for the central alignment of the patient. Geometric verification Patients on the treatment units were pre-positioned into the IC, based on the room lasers before the se t up imaging. Portal images were taken accord­ing to the Extended No Action Level (eNAL) pro­tocol.5 The PortalVision computer program with FIGURE 1. The example of shrinkage (right) of the head support. the AutoMatching registration procedure (Varian Medical Systems, Palo Alto, California, USA) was used to calculate the size and direction of the dis­placement. Electronic portal images acquired with gantry at 0° (anteroposterior projection) and 90° (or 270°, lateral projection) were compared with digitally reconstructed radiographs (DRRs). Portal images were obtained using the Varian’s EPID PortalVision using an amorphous silicon plane de­tector aS1000 (resolution of 1024 × 768 pixels, unit A)or aS500 (resolution 512 × 384 pixels, unit B). Whereas unit A allows simultaneous patient posi­tion alignment in all three directions using 2D/2D image matching, unit B requires the radiographers to combine the position corrections obtained from two separate orthogonal 2D image matchings. Statistical analysis The study protocol was approved by the Protocol Review Board of the Institute of Oncology Ljubljana on April 4, 2017. Testing set-up error distributions for normality was done using the Shapiro-Wilk test. As Shapiro-Wilk test did not support the normality hypothesis in any of the distribution, non-parametric Mann-Whitney U-test (two-sample rank-sum test) was employed for comparing median values in dis­tributions instead of two-sided Student’s t-test. For the same reason, non-parametric modified Levene’s test (using median instead of mean) was used to test the equality of variances in set-up er­ror distributions. Statistical calculations were per­formed using the GNU R statistical program.7 The sample size of 350 measurements per time period was calculated with the G*Power software, consid­ering a = 0.05, b = 0.8 and the effect size of 0.267, which was calculated on the basis of averages and standard deviations of similar studies.1,2,6,8 For comparison with published studies, the clinical tar­get volume (CTV) – planning target volume (PTV) safety margin was calculated according to the for­mula proposed by Van Herk.,9 The differences at p < 0.05 were considered statistically significant. Results In total, 2454 portal images and 3681 set-up errors were analysed: 828, 832, and 794 portal images ob­tained in 2014, 2015, and 2017, respectively. We first analysed the data sets for the presence of large set­up errors. The proportion of displacements smaller than 3 mm and smaller than 5 mm were 85% and 99.1%, respectively in the 2014 set, when standard head supports were employed. The introduction of individual head supports in 2015 increased these figures to 89% and 99.6%, respectively, and in a most recent data set from 2017 they were further increased to 90% and 99.6%, respectively. Inter-fraction set-up errors registered in units A and B at different periods are shown in Table 1 and Figure 2. The difference in distribution of inter-fraction displacements were tested using Mann-Whitney U-test. In four cases, the test showed that the distribution obtained in one year differ signifi­cantly (p < 0.05) from those obtained in the other two years. For unit A, set-up error distributions in the vertical direction obtained in 2014 and in the longitudinal direction obtained in 2017 differ from the other two years. For unit B, set-up error distribu­tions in the vertical direction in the year 2014 and in the lateral direction in the year 2014 differ from the other two years. Comparing the vertical shift dis­tribution for the unit A in the years 2014 and 2015 shows significant difference (Mann-Whitney U = 367 TABLE 1. Inter-fraction displacements recorded on units A and B at different periods UNIT A Average displacement – M [mm] - 0.86 0.05 0.05 - 0.47* - 0.24* - 0.05 - 0.16* 0.88 0.18 Systematic error – S [mm] 0.66 1.04 0.88 0.91 0.79 0.95 0.82 0.59 0.83 Random error – s [mm] 1.49 1.21 1.37 1.56 1.28 1.38 1.28 1.14 1.2 UNIT B Average displacement – M [mm] - 0.60 0.46 - 0.43 - 0.02* 0.51 0.25 - 0.18* 0.46 - 0.02 Systematic error – S [mm] 1.09 0.80 0.82 0.92 0.74 0.94 0.93 0.69 0.88 Random error – s [mm] 1.77 1.83 1.47 1.77 1.22 1.41 1.71 1.56 1.44 LAT = lateral (medial-lateral); LNG = longitudinal (superior-inferior); VRT = vertical (anterior-posterior) *p < 0.05 (2014 vs. 2015 or 2014 vs. 2017) 16293, n1 = 187, n2 = 201, p = 0.02, Hodges-Lehmann estimator (HL.) = -0.000012, 95% confidence inter­val (CI) is (-0.999974, -0.000010)). The difference in distributions is even more pronounced between the sets for the year 2014 and 2017 (U = 12918.5, n1 = 187, n2 = 175, p < 0.001, HL. = -0.999982, 95% CI (-0.999989, -0.000059). Comparing the distribu­tions for the years 2015 and 2017 did not show a significant difference. Comparing the longitudinal shift distributions for the unit A also doesn’t show a significant difference; however, comparing the dis­tributions for the years 2014 and 2017 does show a significant difference (U = 11230, n1 = 187, n2 = 175, p < 0.001, HL. = -0.999982, 95% CI (-1.000066, -0.999956)), and so does the comparison for the years 2015 and 2017 (U = 10171.5, n1 = 201, n2 = 175, p < 0.001, HL. = -1.000006, 95% CI (-1.000032, -0.999955)). Neither comparison for the lateral shifts for the unit A showed significance. Comparing the vertical shift distributions for the unit B also shows significant difference between the sets for the years 2014 and 2015 (U = 19637.5, n1 = 227, n2 = 215, p < 0.001, HL. = -0.999931, 95% CI (-1.000049, -0.000042)), as well as between the sets for the years 2014 and 2017 (U = 21674, n1 = 227, n2 = 222, p < 0.01, HL. = -0.000046, 95% CI (-0.999990, -0.000025)), while the difference between data sets for the years 2015 and 2017 is not significant. In unit B, none of the differences in the longitudinal shift distribution is considered significant. Comparing the lateral shift distributions shows significance between the data sets for the years 2014 and 2015 (U = 18998, n1 = 227, n2 = 215, p < 0.01, HL. = -0.999942, 95% CI (-0.999980, -0.000042)) and for the years 2014 and 2017 (U = 22030, n1 = 227, n2 = 222, p < 0.02, HL. = -0.000058, 95% CI (-0.999953, -0.000040)), while the distributions of lateral shifts between 2015 and 2017 is not considered significant. Comparing the variances of the distributions (which correspond to the systematic error S and the random error s com­bined) only shows significant differences in five cases: in unit A the vertical shift distributions for the years 2015 and 2017 differ significantly (Levene’s F = 6.3082, DF = 386, p < 0.02), as well as longitudinal TABLE 2. Number of IC displacements and of gross errors at treatment units in relation to time for both units. In the brackets, the most prevalent direction of applied movements is indicated 2014 – standard head support 11 / 15 (P) 5 / 15 (I) 4 / 5 (L) 2 / 5 2015 – individual head support 12 / 13 (P) 3 / 6 (I) 9 / 8 (R) 0 / 1 2017 – individual head support 5 / 14 (P) 7 / 10 (I) 6 / 7 (L) 0 / 1 I = inferior; L = left; LAT = lateral (medial-lateral); LNG = longitudinal (superior-inferior); P = posterior; R = right; VRT = vertical (anterior-posterior) 368 TABLE 3. Clinical target volume - planning target volume (CTV-PTV) safety margins for units A and B at different periods (calculated according to van Herk8) UNIT A 2014 – standard head support 2.7 3.4 3.2 2015 – individual head support 3.4 2.9 3.3 2017 – individual head support 2.9 2.3 2.9 UNIT B 2014 – standard head support 4.0 3.3 3.1 2015 – individual head support 3.5 2.7 3.3 2017 – individual head support 3.5 2.8 3.2 LAT = lateral (medial-lateral); LNG = longitudinal (superior-inferior); VRT = vertical (anterior-posterior) shift distributions for the years 2014 and 2017 (F = 9.2817, DF = 360, p < 0.01). In unit B, all three com­parisons of longitudinal shift distributions show significant difference: between the sets for 2014 and 2015 (F = 24.5077, DF = 440, p < 0.001), between the sets for 2014 and 2017 (F = 9.1372, DF = 447, p < 0.01), and between the sets for 2015 and 2017 (F = 4.5802, DF = 435, p = 0.03). In Table 2, the number of IC displacements for both units together at differ­ent periods are presented, as well as the number of recorded gross errors (> 5 mm). Most of the IC shifts were made in the posterior, inferior and in the left direction. With the implementation of individual head supports, their number decreased, except for the lateral direction, and the number of gross errors was also reduced. The CTV-PTV safety margins calculated from the population set-up errors for units A and B at different periods are shown in Table 3. In unit A, the largest reduction of the safety margin after im­plementation of individual head supports was cal­culated in the longitudinal direction (2014 vs. 2015, by 0.6; and 2014 vs. 2015, 1.2 mm), whereas in the lateral direction, the margin did not change sub­stantially. On the contrary, in the vertical direction the margin increased by 0.7 mm (2014 vs. 2015) and by 0.2 mm (2014 vs. 2017). In unit B, a general trend toward a reduction in the safety margins resulted from the employment of individual head supports. In addition, the average reduction of the safety margins was also larger in unit B. The most sig­nificant reductions (2014 vs. 2015 and 2017) were observed in the vertical (by 0.4 and 0.5 mm) and longitudinal directions (by 0.6 and 0.5 mm). In the lateral direction, the size of the safety margin did not increase substantially (by 0.3 and 0.1 mm). Discussion In the present study, individual head supports were found to significantly reduce inter-fraction displacements in the vertical direction, specifically in the posterior direction, compared to the stand­ard head supports. Reduction of average displace­ment in vertical direction recorded between 2014 and 2017 on units A and B was 0.70 mm and 0.42 mm, respectively. This observation pointed to the shrinkage of material, i.e. polyurethane foam, as a possible reason for the observed displacements due to the prolonged and frequent use of head sup­ports. Comparing the three periods, the systematic er­ror did not change significantly for either unit. In the vertical direction, the systematic error record­ed on unit A increased by an average of 0.15 mm, while on unit B it decreased by approximately the same extent. A negligible increase over the time was observed in the lateral direction, on average by less than 0.1 mm. It seems that the use of head supports and the shrinkage of the material they are made of influenced mainly the rotational set-up er­rors of the head in the sagittal plane, rather than the head displacements to the left or to the right.1 In the longitudinal direction, the systematic error was reduced over time on both treatment units. Similarly, by abolishing the standard head sup­ports, a statistically non-significant decrease in the size of the random error was recorded on unit A in all three directions. On unit B, the random error remained practically unchanged in two directions; in the longitudinal direction, its change was neg­ligible. Our observations are in line with those of other authors. A reduction of systematic and random er­rors in all directions was calculated by Van Lin et al.1 when customized and the standard head sup­ports were compared. A decrease was most notable in the longitudinal direction and least marked in the lateral direction, which is the pattern compa­rable to that found in our study. McKernan et al. showed a reduction in setup error by on average 1.3 mm with the use of customized head supports instead of standard ones.4 Similarly, Houweling et al. reported that the use of customized head sup­ports reduced systematic errors by at least 20% and random errors by at least 25%.2 They indicated a decrease in inter-fraction set-up errors by 40%; 369 most statistically significant displacements were recorded in the lateral direction. However, in this particular direction we recorded the smallest set­up errors. The observed discrepancy could be due to sample characteristics: this was significantly smaller (n = 22) in the study of Houweling et al. than in our study (n = 120). Thus, their results are less likely to adequately represent the characteris­tics of the population. To the contrary, in the study of Howlin et al., the difference in set-up errors be­tween patients with customized and standard head supports was not significant in any direction.6 Furthermore, our calculations of the estimated margins from CTV to PTV were also comparable to those reported in the literature. Humphreys et al. used a customized immobilization system: the estimated margins in lateral, longitudinal and ver­tical directions were 2.9, 2.6 and 3.3 mm, respec­tively.3 The authors used the same formula as we did.9 Similarly, Van Lin et al. suggested that with a customized head support and appropriate cor­rection protocol, suitable CTV-PTV margins would be 3 mm in the vertical and longitudinal directions and 4 mm in the lateral direction.1 However, we observed that the CTV-PTV safety margins were larger for unit B than for unit A, which confirms our second hypothesis that the treatment unit with a more advanced portal imaging system allows for more accurate positioning of patients. Humphreys et al.3 reported 94% of displace­ments smaller than 3 mm and 99% smaller than 5 mm, which is comparable to the results of this study. In addition, individual head supports re­duced the number of IC set-up errors in our pa­tients, particularly in the vertical direction, and also of gross errors by 66%; all but two of the latter were recorded in the posterior direction. In unit A, which was equipped with a more advanced portal imaging system, fewer IC displacements and fewer gross errors were documented than in unit B. In addition, there were some differences across the study groups recorded in the size of inter-frac­tion displacements (unit A: longitudinal displace­ment, 2014 vs. 2017), number of IC displacements (unit A: longitudinal axis, 2014 vs. 2015 vs. 2017), and in the size of CTV-PTV margin (unit A: vertical axis, 2014 vs. 2015 vs. 2017), which are not in line with the expected greater accuracy when using in­dividual head rests. However, these differences are small and, as such, seem to be of questionable im­portance for day-to-day clinical work. We are aware that there may be more causes for registered set-up errors that may also influence the calculation of the CTV-PTV margin; imprecision in daily set-up and patient movements when lying on the table of treat­ment unit are just two of the potential sources.10 As measurements within each of the six study groups were made within a relatively short time (i.e. 10–12 weeks) and with constant RTT teams, it can be ar­gued that the results of the group measurements were consistent. Of course, over the 2015–2017 pe­riod, there were changes in the composition of RTT teams, which could affect our calculations. Other causative factors for set-up errors would be differ­ent technical errors (inaccuracies in the in-room la­ser calibration or of the imaging IC, procedure of the matching process and its quality) or those origi­nated from the thermoplastic mask itself, changes in the patient anatomy (due to weight loss or vol­ume reduction/swelling of the tumor or specific organs-at-risk), or different physiological processes (swallowing respiration). However, we were able to account for these factors only in the context of a regular quality assurance program; their detailed analysis is beyond the scope of this study. The im­pact of eventual changes in the departmental pro­tocol used to position patients on irradiation units is negligible, since no significant protocol changes occurred during the study period. Conclusions When compared to standard head supports, the introduction of individual head supports reduced inter-fraction set-up errors in the vertical direction and the number of gross errors; in some directions, also the number of IC displacements and the size of the CTV-PTV safety margin were reduced. A more advanced imaging system with a better spatial res­olution contributed to a reduction in the systematic and random errors. Acknowledgement This work was financially supported by the Slovenian Research Agency (program no. P3-0307). References 1. Van Lin EN, van der Vight L, Huizenga H, Kaanders JHAM, Visser AG. Set-up improvement in head and neck radiotherapy using a 3D off-line EPID-based correctioon protocol and a customised head and neck support. Radiother Oncol 2003; 68: 137-48. doi: 10.1016/S0167-8140(03)00134-8 2. Houweling AC, van der Meer S, van der Wal E, Terhaard CHJ, Raaijmakers CPJ. Improved immobilization using an individual head support in head-and-neck cancer patients. Radiother Oncol 2010; 96: 100-3. doi: 10.1016/j. radonc.2010.04.014 370 3. Humphreys M, Guerrero Urbano MT, Mubata C, Miles E, Harrington KJ, Bidmead M, et al. Assessment of a customised immobilisation system for head and neck IMRT using electronic portal imaging. Radiother Oncol 2005; 77: 39-44. doi: 10.1016/j.radonc.2005.06.039 4. McKernan B, Bydder S, Ebert M, Waterhouse D, Joseph D. A simple and inexpensive method to routinely produce customized neck supports for patient immobilization during radiotherapy. J Med Imaging Radiat Oncol 2008; 52: 611-6. doi: 10.1111/j.1440-1673.2008.02024 5. De Boer HC, Heijmen BJ. eNAL: an extenstion of the NAL setup correction protocol for effective use of weekly follow-up measurements. Int J Radiat Oncol Biol Phys 2007; 67: 1586-95. doi: 10.1016/j.ijrobp.2006.11.050 6. Howlin C, O’Shea E, Dunne M, Mullaney L, McGarry M, Clayton-Lea A, et al. A randomized controlled trial comparing customized versus standard headrests for head and neck radiotherapy immobilization in terms of set-up errors, patient comfort and staff satisfaction. Radiography 2015; 21: 74-83. doi: 10.1016/j.radi.2014.07.009 7. R Core Team. R: a language and environment for statistical computing. Vienna: R Foundation for Statistical Computing; 2018. 8. Faul F, Erdfelder E, Lang AG, Buchner A. G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods 2007; 39: 175-91. doi: 10.3758/bf03193146 9. Van Herk M. Errors and margins in radiotherapy. Semin Radiat Oncol 2004; 14: 52-64. doi: 10.1053/j.semradonc.2003.10.003 10. International Atomic Energy Agency. Introduction of image guided radio­therapy into clinical practice. In: IAEA Human Health Reports No. 16. Vienna: International Atomic Energy Agency; 2019. Radiol Oncol 2020; 54(3): 253-262. doi: 10.2478/raon-2020-0035 Transarterijska embolizacija zunanje karotidne arterije pri zdravljenju življenje ogrožajoce krvavitve, nastale zaradi maksilofacialne poškodbe Langel C, Lovric D, Zabret U, Mirkovic T, Gradišek P, Mrvar-Brecko A, Šurlan Popovic K Izhodišca. Huda krvavitev zaradi maksilofacialne poškodbe je redka, a življenje ogrožajoca. Bolnike, pri katerih osnovne metode zdravljenja ne zadošcajo za zaustavitev krvavitve, lahko zdravimo s transarte­rijsko embolizacijo zunanje karotidne arterije ali njenih vej. Objavljeni tovrstni primeri niso pogosti, kar je presenetljivo glede na razmeroma visoko incidenco maksilofacialnih poškodb. Zato domnevamo, da o transarterijski embolizaciji bodisi premalo porocamo, bodisi jo premalo uporabljamo ali oboje. Ob tem je zelo malo strokovnih objav o uporabi novih neadhezivnih tekocih embolizacijskih sredstev za transarterij­sko embolizacijo v podrocju zunanje karotidne arterije. Bolniki in metode. S pomocjo pregleda zbirke PubMed smo zbrali objave o transarterijski emboliza­ciji v podrocju zunanje karotidne arterije v okviru tope (tj. nepenetrantne) maksilofacialne poškodbe. Zabeležili smo mesto poškodbe v povirju zunanje karotidne arterije, mesto embolizacije, izbrano em-bolizacijsko sredstvo in ucinkovitost ter varnost postopka transarterijske embolizacije. Naredili smo tudi pregled napovednih dejavnikov preživetja. Na koncu smo dodali prikaz primera iz slovenske terciarne ustanove, pri katerem smo za transarterijsko embolizacijo v podrocjih obeh karotidnih arterij uporabili novo embolizacijsko sredstvo obarjajoco hidrofobno tekocino za injiciranje PHIL (ang. precipitating hy­drophobic injectable liquid). Zakljucki. Pregled 205 primerov je pokazal, da je transarterijska embolizacija ucinkovita v 79,4–100 %; pomembni zapleti so se pojavljali v 2–4%. Napovedni dejavniki, statisticno znacilno povezani z višjo sto­pnjo preživetja, so bili: uspešna zaustavitev krvavitve, vrednost = 8 po Glasgowski lestvici kome, vrednost = 32 po tockovalniku resnosti poškodbe ISS (ang. injury severity score) ter indeks šoka = 1,1 pred transarterij­sko embolizacijo in = 0,8 po njej. PHIL dopušca hitro in hkrati filigransko vbrizganje, kar omogoca pomem­ben prihranek casa v življenje ogrožajocih okolišcinah in zmanjšuje verjetnost nenamernega injiciranja v potencialno nevarne anastomoze med zunanjo in notranjo karotidno arterijo. Radiol Oncol 2020; 54(3): 263-271. doi: 10.2478/raon-2020-0045 Zdravljenje intrahepaticnega holangiokarcinoma. Od resekcije do paliativnega zdravljenja Bartolini I, Risaliti M, Fortuna L, Agostini C, Novella Ringressi M, Taddei A, Muiesan P Izhodišca. Intrahepaticni holangiokarcinom je za hepatocelularnim karcinomom drugi najpogostejši primarni rak jeter. Intrahepaticni holangiokarcinom predstavlja 20 % rakov od vseh holangiokarcinomov. Njegova incidence in tudi mortaliteta narašcata. Kirurška resekcija je edina kurativna metoda zdravljena kljub visoki stopnji ponovitve bolezni, ki je do 80 %. Ponovitve te bolezni v intrahepaticnem prostoru je možno še enkrat operirati s kurativnim namenom, vendar v majhnem odstotku bolnikov. Žal je diagnoza pri vecini bolnikov pozna zaradi pomanjkanja specificnih simptomov in operacija ni možna. Indikacije za transplantacijo jeter pa so še vedno kontroverzne. V zadnjem casu je vec porocil o izboljšanju uspeha zdravljenja z neodjuvantnim zdravljenjem s kemoterapijo, vendar v zelo selektivnih primerih. Zato mora transplantacija jeter ostati kot možnost zdravljenja predvsem v klinicnih študijah. V paliativnih primerih, ko kirurški poseg ni mogoc, lahko uporabimo kemoterapijo, radioterapijo in loko-regionalne ablativne tehnike, kot so radiofrekvencna ablacija in trans-arterijska kemoembolizacija ali radioembolizacija. Zakljucki. Pricujoci pregled je osredotocen na kirurško zdravljenje intrahepaticnega holangiokarcino-ma. Pomembni so potencialni napovedni dejavniki, ki lahko pomagajo pri izbiri primernega zdravljenja. Radiol Oncol 2020; 54(3): 272-277. doi: 10.2478/raon-2020-0031 Konsenzusni molekularni podtipi (CMS) v personalizirani medicini metastatskega raka debelega crevesa in danke Reberšek M Izhodišca. Rak debelega crevesa in danke je eden najpogostejših rakov na svetu. Metastatski rak debelega crevesa in danke je še vedno neozdravljiva bolezen pri vecini bolnikov. Preživetje bolnikov se je izboljšalo, ko smo jih priceli zdraviti z novo sistemsko kemoterapijo v kombinaciji s tarcno terapijo. Pri nekaterih bolnikih lahko s kombiniranim zdravljenjem s sistemsko terapijo in kirurgijo dosežemo zazdravitve ali celo ozdravitve. Novo znanje o kompleksni heterogenosti raka debelega crevesa in danke z vidika genetike, epigenetike, transkriptomije in mikrookolja, kot tudi z vidika napovednih in klinicnih znacilnosti je privedlo do razvrstitve raka debelega crevesa in danke v razlicne molekularne podtipe. Imenujemo jih konsenzusni molekularni podtipi (CMS). Klasifikacija CMS bo v prihodnosti onkologom olajšala odlocitev, katero sistemsko kemoterapijo, tarcno terapijo, v kateri kombinaciji in v katerem zaporedju bodo izbrali za vsakega posameznega bolnika. Zakljucki. CMS so pri metastatskem raku debelega crevesa in danke novo orodje, ki vkljucuje znanja o klinicnih in molekularnih znacilnostih, tumorskem mikrookolju in signalnih poteh ter omogoca personali­zirano, bolniku prilagojeno zdravljenje. Radiol Oncol 2020; 54(3): 278-284. doi: 10.2478/raon-2020-0034 Napovedna vloga pozitronskoemisijske tomografije in racunalniške tomografije pri žleznem raku pljuc stadija I Carretta A, Bandiera A, Muriana P, Viscardi S, Ciriaco P, Samanes Gajate AM, Arrigoni G, Lazzari C, Gregorc V, Negri G Izhodišca. Veljavna patološka klasifikacija žleznega raka pljuc vkljucuje histološke podtipe z razlicnimi napovednimi dejavniki, ki lahko vplivajo na razlicne kirurške pristope. Namen raziskave je bil oceniti na­povedno vlogo parametrov racunalniške tomografije (CT) in pozitronskoemisijske tomografije (PET) pri razvršcanju bolnikov z žleznim rakom pljuc stadija I. Bolniki in metode. Retrospektivno smo pregledali 58 bolnikov s pljucnim rakom stadija I in s patološko diagnozo žlezni rak oz. adenokarcinom, ki smo jih kirurško zdravili. Adenokarcinom in situ in minimalno invazivni adenokarcinom smo opredelili kot neinvazivni žlezni rak. Drugi histološki tipi so bili oznaceni kot invazivni žlezni rak. Ocenili smo vlogo parametrov, ki smo jih pridobili s CT slikanjem: razmerje gostote mlecnega stekla, stopnjo izginotja tumorja in konsolidacijski premer. Ocenili smo tudi napovedno vloga naslednjih parametrov PET: maksimalno vrednost standardiziranega privzema (SUV)max, SUVindeks (raz­merje med SUVmax in jetrnim SUV), metabolicni volumen tumorja, skupno glikolizo lezije . Rezultati. Sedem bolnikov je imelo neinvazivni, 51 pa invazivni žlezni rak. Petletno preživetje brez bolezni za neinvazivni in invazivni žlezni rak sta bili 100% in 100%, preživetje, specificno za raka pa 70 % in 91 %. Univariatna analiza je pokazala pomembno razliko v vrednostih SUVmax, SUV indeks, razmerja gostote mlecnega stekla in razmerja stopnje izginotja tumorja med skupinami neinvazivnega in invazivnega žle­znega raka. Optimalne vrednosti za napoved invazivnih rakov so bile 2,6 za SUVmax, 0,9 za SUVindeks, 40 % za razmerje gostote mlecnega stekla in 56 % kot mejna vrednost za stopnjo izginotja tumorja. Skupna glikoliza lezije, SUVmax, SUVindeks so pomembno korelirali s preživetjem specificnim za raka. Zakljucki. Parametri slikovnih preiskav CT in PET se lahko razlikujejo med neinvazivnimi in invazivnimi žleznimi raki stadija I. Ce bi te izsledke lahko potrdili v vecjih raziskavah, bi lahko dodatno vplivali na izbiro najustreznejšega kirurškega zdravljenja. Radiol Oncol 2020; 54(3): 285-294. doi: 10.2478/raon-2020-0042 Radiomske znacilnosti slik [18F]FDG PET pri imunoterapiji (iRADIOMICS) napovejo odziv bolnikov z nedrobnocelicnim pljucnim rakom na zdravljenje s pembrolizumabom Valentinuzzi D, Vrankar M, Boc N, Ahac V, Zupancic Ž, Unk M, Škalic K, Žagar I, Studen A, Simoncic U, Eickhoff J, Jeraj R Izhodišca. Zaviralci imunskih kontrolnih tock so spremenili obravnavo bolnikov z rakom. Kljub temu pa še vedno obstaja potreba po neinvazivnih slikovnih bioloških oznacevalcih, s katerimi bi lahko napovedali, kateri bolniki ne bodo odgovorili na zdravljenje. Namen raziskave je bil prouciti, ali radiomske znacilnosti slik [18F]FDG PET pri imunoterapiji napovejo odgovor bolnikov z metastatskim nedrobnocelicnim pljucnim rakom na zdravljenje s pembrolizumabom bolje od trenutnih tumorskih oznacevalcev, ki jih uporabljamo v klinicni praksi. Bolniki in metode. V raziskavo smo vkljucili 30 bolnikov, ki smo jih zdravili s pembrolizumabom. Z [18F] FDG PET/CT smo jih slikali pred zacetkom zdravljenja, po enem mesecu in po štirih mesecih. Analizirali smo povezave šestih robustnih radiomskih metrik primarnih tumorjev s celokupnim preživetjem, za kar smo uporabili Mann-Whitney U-test, Coxovo regresijsko analizo sorazmernih tveganj in analizo krivulje ROC. iRADIOMICS smo oblikovali na podlagi univariatnega in multivariatnega logisticnega modela z najbolj obetajocimi metrikami. Napovedno moc iRADIOMICS smo primerjali z napovedno mocjo deleža tumor-skih celic (angl. tumour proportion score [TPS]) z izraženim PD-L1 ter z iRECIST, za kar smo uporabili analizo krivulje ROC. Natancnosti napovedi smo ocenili s petkratno navzkrižno validacijo. Rezultati. Najvecjo napovedno moc so imele radiomske metrike pred zdravljenjem, npr. poudarek na majhnem teku (angl. small run emphasis) (Mann-Whitney U-test, p = 0,001; razmerje tveganj [HR] = 0,46, p = 0,007; plošcina pod krivuljo [AUC] = 0.85 [95 % interval zaupanja [CI] 0.69–1.00). Multivariatni iRADI-OMICS se je izkazal kot boljši od trenutnih standardov tako s stališca napovedne moci kot casovno, ce primerjamo naslednje AUC (95 % CI) in natancnosti napovedi (standardne deviacije): iRADIOMICS (pred zdravljenjem), 0,90 (0,78–1,00), 78 % (18 %); TPS PD-L1 (pred terapijo) 0,60 (0,37–0,83), 53 % (18 %); iRECIST (1. mesec), 0,79 (0,62–0,95), 76 % (16 %); iRECIST (4. mesec), 0.86 (0.72–1.00), 76 % (17 %). Zakljucki. Multivariatni iRADIOMICS se je pokazal kot obetajoc slikovni biološki oznacevalec, ki bi lahko bolje napovedal odgovor na zdravljenje z imunoterapijo pri bolnikih z metastatskim nedrobnocelicnim pljucnim rakom. Bolnikom, katerim bi iRADIOMICS napovedal, da najverjetneje ne bodo odgovorili na zdravljenje s pembrolizumabom, bi lahko ponudili drugacno vrsto zdravljenja. Radiol Oncol 2020; 54(3): 295-300. doi: 10.2478/raon-2020-0033 Izboljšanje primarne ucinkovitosti mikrovalovne ablacije malignih tumorjev jeter s pomocjo robotskega navigacijskega sistema Schaible J, Pregler B, Verloh N, Einspieler I, Bäumler W, Zeman F, Schreyer A, Stroszczynski C, Beyer L Izhodišca. Namen raziskave je bil primerjati primarno ucinkovitost robotsko vodene mikrovalovne abla­cije jetrnih tumorjev z rocno vodeno. Bolniki in metode. Naredili smo retrospektivno analizo mikrovalovnih ablacij, ki smo jih izvedli v enem centru, na 368 tumorjih, pri 192 bolnikih (36 žensk, 156 moških, sredna starost 63 let). 119 ablacij smo opravili med 8/2011 in 03/2014 z rocno vodeno tehniko, 249 ablacij pa med 04/2014 in 11/2018 z robot-sko vodeno tehniko. Evaluacijo z ultrazvokom, kompjutersko tomografijo ali magnetno resonanco smo naredili 6 mesecev po posegu. Rezultati. Ucinkovitost robotsko vodene mikrovalovne ablacije je bila znacilno bolj ucinkovita v pri­merjavi z rocno vodeno ablacijo (88 % vs. 76 %; p = 0,013). Multipla logisticna regresija je pokazala, da je velikost tumorjev pod 3 cm premera pozitiven napovedni dejavnik za ucinek zdravljenja, prav tako je robotsko vodena ablacija pozitiven napovedni dejavnik za popolno ablacijo jetrnih tumorjev. Zakljucki. Mala velikost tumorjev in robotsko vodena mikrovalovna ablacija jetrnih tumorjev sta bila pozitivna napovedna dejavnika za primarno tehnicno ucinkovitost mikrovalovne ablacije . Radiol Oncol 2020; 54(3): 301-310. doi: 10.2478/raon-2020-0037 Gliomi stopnje II in III. Primerjava med dinamicnim magnetnoresonancnim slikanjem s kontrastom in magnetnoresonancnim slikanjem z znotraj­vokselnim nekoherentnih premikanjem Wang X, Cao M, Chen H, Ge J, Suo S, Zhou Y Izhodišca. Vpliv mutacije izocitrat dehidrogenaze 1 (IDH1) na tvorbo novih žil je lahko pri gliomih pove­zan s tkivno perfuzijo. Trenutno potreba po injiciranju kontrastnega sredstva in s tem podaljšanim casom slikanja omejujeta uporabo perfuzijskih tehnik. V raziskavi, smo uporabili izpeljano perfuzijsko frakcijo (ang. simplified perfusion fraction - SPF]) iz poenostavljenega nekoherentnega gibanja znotraj voksla (ang. intravoxel incoherent motion - IVIM), ki smo ga izracunali iz difuzijsko obteženega slikanja (ang: diffusion-weighted imaging - DWI) z uporabo le treh b-vrednosti, da bi kvantitativno ocenili spremembe tkivne perfuzije, povezane z IDH1, pri gliomih II–III stopnje po Svetovni zdravstveni organizaciji (WHO). Poleg tega smo s primerjavo natancnosti med dinamicnim magnetnoresonancni slikanjem s kontrastom (MRI DCE) in popolnim magnetno resonancnim slikanjem MRI IVIM poskušali najti optimalne slikovne oznacevalce za napoved statusa mutacije IDH1. Bolniki in metode. Prospektivno smo pregledali 30 bolnikov in uporabili DCE in DWI z vec vrednostmi b. Vse parametre smo primerjali med bolniki z gliomi stopnje II in III po WHO z mutiranim IDH1 in nemutiranim IDH1. Uporabili smo Mann-Whitney U test, vkljucno s Ktrans, ve in vp in konvencionlanim navideznim koefi­cientom (ADC0,1000) pridobljenim z MRI DCE, perfuzijsko frakcijo pridobljeno z IVIM (f), difuzijski koeficient (D), navidezni-difuzijski koeficient (D*), in SPF. Diagnosticno uspešnost smo ovrednotili z analizo lastnosti delovanja sprejemnika (ang. receiver operating characteristic - ROC). Rezultati. Med vsemi perfuzijskimi in difuzijskimi parametri smo ugotovili statisticno pomembne razlike med bolniki z gliomi stopnje II–III stopnje po WHO (P < 0,05). Gliomi z nemutiranim IDH1 so imeli obcutno višje perfuzijske vrednosti (P < 0,05) in nižje difuzijske vrednosti (P < 0,05). Med vsemi parametri je vecjo diagnosticno ucinkovitost pokazal SPF (obmocje pod krivuljo 0,861), z 94,4 % obcutljivostjo in 75,0 % spe­cificnostjo. Zakljucki. DWI, DCE in IVIM MRI lahko neinvazivno pomagajo pri razlikovanju statusov mutacije IDH1 pri bolnikih z gliomi stopnje II in II po WHO. Poenostavljeni SPF, ki smo ga izpeljali iz DWI, je pokazal zelo dobro diagnosticno ucinkovitost. Radiol Oncol 2020; 54(3): 311-316. doi: 10.2478/raon-2020-0041 Možnosti ultrazvocno vodene, vakumsko asistirane evakuacije velikih hematomov v dojki Almasarweh S, Sudah M, Joukainen S, Okuma H, Vanninen R, Masarwah A Izhodišca. V literaturi je hematom v dojki velikokrat podcenjena in zanemarjena post-proceduralna komplikacija. Sodoben nacin zdravljenja predstavljata kirurški ali konzervativni pristop, medtem ko imajo perkutani posegi manjšo vlogo. Preucevali smo ucinkovitost vakumsko asistirane evakuacije pri zdravlje­nju klinicno pomembnih velikih hematomih dojke kot alternativnim nacinom kirurškemu zdravljenju. Bolniki in metode. Retrospektivno smo analizirali bolnice iz zadnjih 4 let, ki so imele interventni poseg na dojki (kirurški ali perkutani) in so kasneje razvile klinicno pomemben velik hematom. Poizkusno smo jih zdravili z vakumsko asistirano evakuacijo hematoma . Interventno zdravljenje je bilo uspešno, ce smo odstranili vec kot 50 % volumna hematoma in dosegli zmanjšanje bolnicinih simptomov. Vse bolnice smo klinicno ali ultrazvocno sledili v razlicnih intervalih glede na resnost simptomov. Rezultati. V raziskavo smo vkljucili 11 bolnic. Povprecni najvecji diameter hematoma je bil 7,9 cm in povprecna površina hematoma je bila 32,4 cm2. Povprecno trajanje posega je bilo 40,5 min. Pri vseh bolnicah je bila vakumsko asistirana evakuacija hematoma uspešna in brez zapletov. Kontrolni pregledi niso pokazali vecjega hematoma ali formacije seroma. Zakljucki. Raziskava je pokazala, da je vakumsko asistirana evakuacija hematoma uspešen in varen nacin zdravljenja in zato lahko predstavlja alternativo kirurškemu zdravljenju pri velikih, klinicno pomemb­nih hematomih, ne glede na etiologijo ali trajanje. Poseg je manj tvegan, manj stresen in cenejši, lahko ga opravimo tudi ambulantno v primerjavi z kirurškim zdravljenjem. Radiol Oncol 2020; 54(3): 317-328. doi: 10.2478/raon-2020-0047 Analiza molekulskih vzorcev povezanih s celicnimi poškodbami po elektroporaciji celic in vitro Polajžer T, Jarm T, Miklavcic D Izhodišca. Imunogena celicna smrt je ena od oblik celicne smrti tumorskih celic, pri kateri se iz celic sprošcajo molekule imenovane molekulski vzorci povezani s poškodbami (DAMPs). Te molekule aktivirajo celice imunskega sistema. Posledicno se lahko aktivirata tako prirojeni kot pridobljeni imunski sistem, kar vodi v unicenje preostalih spremenjenih celic. Aktivacija imunskega sistema je pomembna komponenta zdravljenja tumorskih tkiv z elektrokemoterapijo in ireverzibilno elektroporacijo. V študiji smo raziskali, ce in kdaj po elektroporaciji celic in vitro se sprostijo specificne molekule DAMP. Materiali in metode. Suspenzijo hrckovih ovarijskih celic (CHO) smo izpostavili 100 µs dolgim elektricnim pulzom. Prisotnost molekul DAMP, kot so ATP, kalretikulin, nukleinske kisline in secna kislina, smo analizirali v razlicnih casovnih tockah po izpostavitvi elektricnim pulzom razlicnih amplitud. Za vrednotenje statisticne korelacije med kolicino molekul DAMP in deležem permeabiliziranih ter preživelih celic, oziroma reverzi­bilno ter ireverzibilno elektroporiranih celic smo uporabili Pearsonov korelacijski koeficient. Rezultati. Sprošcanje molekul DAMP se v splošnem povecuje z narašcajoco amplitudo pulzov, pri tem pa se sama koncentracija molekul lahko razlikuje v razlicnih casovnih tockah po izpostavitvi elektricnim pulzom. Vecinoma izlocanje molekul DAMP bolj korelira z deležem odmrlih celic, kot deležem permea­biliziranih celic. V analiziranih vzorcih nismo uspeli potrditi prisotnosti secne kisline. Zakljucki. Sprošcanje molekul DAMP lahko služi kot oznacevalec za napoved celicne smrti. Stabilnost nekaterih molekul DAMP je casovno odvisna, kar je potrebno upoštevati pri nacrtovanju poskusov ana­lize molekul DAMP po izpostavitvi elektricnim pulzom. Radiol Oncol 2020; 54(3): 329-334. doi: 10.2478/raon-2020-0048 Vpliv epidemije COVID-19 na diagnostiko in zdravljenje raka v Sloveniji. Prvi rezultati Zadnik V, Mihor A, Tomšic S, Žagar T, Bric N, Lokar K, Oblak I Izhodišca. Pandemija COVID-19 je otežila dostop do zdravstvenih storitev in zmanjšala njihovo uporabo po vsem svetu. V Sloveniji je bila epidemija uradno razglašena od sredine marca do sredine maja 2020. Z odlokom vlade so bile ustavljene vse nenujne zdravstvene storitve, vendar je bila onkološka dejavnost navedena kot izjema. Obvladovanje raka je odvisno tudi od drugih zdravstvenih storitev in ker se je med epidemijo vedenje ljudi predvidoma spremenilo, smo analizirati, ali je epidemija COVID-19 vplivala na diagnostiko in zdravljenje raka v Sloveniji. Metode. Iz treh virov smo analizirali rutinske podatke v obdobju od novembra 2019 do maja 2020: (1) iz podatkov Registra raka Republike Slovenije smo analizirali prijavnice rakavih bolezni iz patohistoloških izvidov in klinicnih obravnav, prispele iz Onkološkega inštituta Ljubljana in UKC Maribor; (2) iz sistema e­-Napotnica smo analizirali podatke o vseh napotnicah v Sloveniji, izdanih za onkološke storitve, stratifi­cirane po vrsti zdravstvene dejavnosti; in (3) iz administrativnih virov Onkološkega inštituta Ljubljana smo analizirali podatke o številu ambulantnih obiskov glede na vrsto obiskov in podatkov o številu opravljenih diagnosticnih slikovnih preiskav. Rezultati. V primerjavi s povprecjem v obdobju november 2019 – februar 2020 je bilo aprila 2020 43 % in 29 % manj registriranih prijavnic rakavih bolezni iz patohistoloških izvidov in klinicnih obravnav; 33 %, 46 % in 85 % manj napotitev na prve onkološke preglede, kontrolne onkološke preglede in genetska sve­tovanja; 19 % (53 %), 43 % (72 %) in 20 % (21 %) manj prvih (in kontrolnih) ambulantnih obiskov v sektorjih za radioterapijo, kirurgijo in internisticno onkologijo Onkološkega inštituta Ljubljana ter 48 %, 76 % in 42 % manj opravljenih rentgenskih, mamografskih in ultrazvocnih preiskav na Onkološkem inštitutu Ljubljana, v tem vrstnem redu. Število opravljenih preiskav CT in MRI ni bilo znižano. Zakljucki. Pomemben upad prvih napotitev na onkološke storitve, prvih ambulantnih obiskov in opravljenih slikovnih preiskav na Onkološkem inštitutu Ljubljana ter prijavnic rakave bolezni v aprilu 2020 nakazuje na možnost zamika pri diagnozi raka za nekatere bolnike v casu prvega porasta okužb s SARS­CoV-2 v Sloveniji. Vzrokov za zamik ne poznamo, verjetno so povezani s spremenjenim vedenjem bolni­kov (spremembe v iskanju zdravstvene pomoci), razmišljanji in praksami zdravnikov in/ali upravljanjem zdravstvenega sistema v casu epidemije. Upad kontrolnih napotitev in kontrolnih ambulantnih obiskov je najverjetneje posledica odlocitve Onkološkega inštituta Ljubljana, da do dva meseca prestavi nenujne preglede bolnikov, narocenih za spremljanje po zakljucenem zdravljenju. Radiol Oncol 2020; 54(3): 335-340. doi: 10.2478/raon-2020-0040 Ocena ogroženosti za raka dojk z napovednim modelom ogroženosti S-IBIS pri slovenskih ženskah v starostni skupini 40-49 let Oblak T, Zadnik V, Krajc M, Lokar K, Žgajnar J Izhodišca. Z izracunom 10-letne ogroženosti za raka dojk s programom S-IBIS smo želeli dolociti delež nadpovprecno ogroženih preiskovank starih 40 do 49 let v populaciji bolnic z rakom dojk ter žensk, obrav­navanih v Centrih za bolezni dojk (CBD). Rak dojk je najpogostejši rak pri ženskah v Sloveniji, njegova incidence pa je pod evropskim povprecjem. Napovedni model ogroženosti za raka dojk S-IBIS temelji na angleškem Tyrer-Cuzickovem algoritmu, vendar upošteva slovenske incidencne podatke za raka dojk. V Sloveniji razmišljamo o uvedbi presejalnega programa za raka dojk za nadpovprecno ogrožene ženske v starosti 40 do 49 let. S-IBIS je potencialno orodje za prepoznavo bolj ogroženih žensk, ki bi bile vabljene v prilagojeno presejanje. Bolniki in metode. Pri 367 bolnicah, ki so zbolele za rakom dojk v starosti od 40 do 49 let, in pri 357 zdravih ženskah, ki so bile obravnavane v CBD, smo izracunali 10-letno ogroženost za rakom dojk s pro-gramom S-IBIS. V posamezni podskupini smo izracunali delež žensk z nadpovprecno ogroženostjo za razvoj raka dojk. Rezultati. Pri 48,7 % žensk iz CBD ter 39,2 % bolnic smo izracunali nadpovprecno 10-letno ogroženost za razvoj raka dojk. Ženske iz CBD so imele pozitivno družinsko anamnezo za raka dojk v vecjem deležu kot bolnice (p < 0,05). Zakljucek. Za zanesljivo razvrstitev žensk v razrede ogroženosti za razvoj raka dojk, bo potrebno vkljuciti dodatne dejavnike ogroženosti v S-IBIS. Radiol Oncol 2020; 54(3): 341-346. doi: 10.2478/raon-2020-0032 Standardna in multivisceralna kolektomija pri lokalno napredovalem raku debelega crevesa Sahakyan AM, Aleksanyan A, Batikyan H, Petrosyan H, Sahakyan MA Izhodišca. Obravnava lokalno napredovalega raka debelega crevesa je še vedno izziv. Kirurško zdra­vljenje je temeljno zdravljenje, vendar so rezultati razmeroma nejasni, zlasti pri multivisceralnih resekcijah. Cilj raziskave je bil preuciti rezultate standardne in multivisceralne kolektomije pri bolnikih z lokalno napre­dovalim rakom debelega crevesa. Bolniki in metode. Zbrali smo demografske, klinicne in perioperativne podatke o bolnikih, operiranih v obdobju 2004–2018. Lokalno napredovali rak debelega crevesa smo opredelili rak v stadiju T4, kjer je tumor prerašcal visceralni peritonej ali sosednje organe oz. strukture. Glede na prerašcanje smo izvedli bodisi standardno bodisi multivisceralno kolektomijo. Rezultati. V raziskavo smo vkljucili 203 bolnikov, ki smo jim naredili kolektomijo zaradi lokalno napredo­valega raka debelega crevesa. Med njimi smo 112 bolnikom naredili standardno (55,2 %) in 91 (44,8 %) multivisceralno kolektomijo. Resno obolevnost in umrljivost smo ugotovili 5,9 % pri standardni kolektomiji in 2,5 % pri multivisceralni kolektomiji. Ta je bila povezana s povecano izgubo krvi (200 ml proti 100 ml, p = 0,01), z vec transfuzij krvi (22 % proti 8,9 %, p = 0,01), daljšim operativnim casom (180 minut proti 140 minut, p < 0,01) in z daljšo pooperativno hospitalizacijo (11 dni proti 10 dni, p <0,01) v primerjavi s stan­dardno kolektomijo. Parametri, povezani z zapleti so bili podobni v obeh skupinah. Univariatna analiza je pokazala, da so bili moški spol, prisotnost = 3 komorbidnosti, lokacija tumorja v levem debelem crevesu in perioperativna transfuzija krvi povezani s pooperativnimi zapleti. Multivariatna analiza pa je pokazala, da je bila prisotnost = 3 komorbidnosti edini neodvisni napovedni dejavnik zapletov. Zakljucki. Kolektomija z multivisceralno resekcijo ali brez nje je varen postopek pri obravnavi lokalno napredovalega raka debelega crevesa. Pooperativni rezultati med standardno in multivisceralno kolek­tomijo so podobni v primeru, da resekcijo naredi izkušen kirurški tim, zato bi morali multivisceralno kolek­tomijo opravljati zgolj za to strokovno usposobljeni centri. Radiol Oncol 2020; 54(3): 347-352. doi: 10.2478/raon-2020-0038 Perkutana slikovno vodena elektrokemoterapija hepatocelularnega raka Djokic M, Dežman R, Cemažar M, Štabuc M, Petric M, Šmid LM, Janša R, Plesnik B, Bošnjak M, Lampreht Tratar U, Trotovšek B, Kos B, Miklavcic D, Serša G, Popovic P Izhodišca. Elektrokemoterapija med operacijskim posegom je ucinkovita pri zdravljenju metastaz raka debelega crevesa in danke kot tudi hepatocelularnega raka. Slikovno vodeno elektrokemoterapijo s perkutanim pristopom so že izvedli, vendar ne pri hepatocelularnem raku. Zato je bil namen pricujoce raziskave preveriti izvedljivost, varnost in ucinkovitost elektrokemoterapije s perkutanim pristopom pri zdravljenju hepatocelularnega raka. Bolnik in metode. Bolniku smo naredili transarterijsko kemoembolizacijo, kot tudi mikrovalovno ablacijo vec lezij hepatocelularnega raka v III., V. in VI. jetrnem segmentu. Ob sledenju bolnika smo ugotovili novo lezijo v II. jetrnem segmentu. Na multidisciplinarnem timu smo ocenili, da je primerna za minimalno inva­zivni perkutani pristop elektrokemoterapije. Zdravljenje smo izvedli z dolgo igelnimi elektrodami, ki smo jih vstavili s kotrolo slikovne diagnostike. Rezultati. Zdravljenje smo izvedli brez zapletov in je bilo varno in ucinkovito, kar smo dokazali s kontrol­nim slikanjem z magnetno resonanco. Zakljucki. Minimalno invaziven perkutani pristop elektrokemoterapije s kotrolo slikovne diagnostike je izvedljiv, varen in ucinkovit pri zdravljenju hepatocelularnega raka. Radiol Oncol 2020; 54(3): 353-363. doi: 10.2478/raon-2020-0046 Konsolidacijska radioterapija pri bolnikih z razširjenim drobnocelicnim rakom pljuc v terciarni ustanovi. Vpliv sevalne doze in perspektive v dobi imunoterapije Stanic K, Vrankar M, But-Hadžic J Izhodišca. Konsolidacijska radioterapija pri razširjenem drobnocelicnem pljucnem raku (ED-SCLC) je bila povezana z boljšim dvoletnim celokupnim preživetjem pri bolnikih, ki so se v raziskavi CREST odzvali na kemoterapijo, rezultati dveh metaanaliz pa so bili nasprotujoci. Pred kratkim so uvedli imunoterapijo za zdravljenje ED-SCLC, zaradi cesar je vloga konsolidacijske radioterapije še bolj nejasna. Cilj študije je bil raziskati, ali konsolidacijsko obsevanje prsnega koša izboljša preživetje bolnikov z ED-SCLC, ki jih zdravimo v rutinski klinicni praksi, in preuciti vpliv odmerka konsolidacijske radioterapije na preživetje. Razpravljamo tudi o prihodnji vlogi konsolidacijske radioterapije v dobi imunoterapije. Bolniki in metode. Retrospektivno smo pregledali zdravstveno dokumentacijo 704 zaporednih bolni­kov z drobnocelicnim rakom pljuc, ki smo jih od januarja 2010 do decembra 2014 zdravili na Onkološkem inštitutu Ljubljana. Srednji cas spremljanja bolnikov je bil 65 mesecev. Analizirali smo srednje celokupno preživetje bolnikov z ED-SCLC, ki smo jih zdravili samo s kemoterapijo, ter tistih, ki smo jih zdravili s kemo­terapijo in konsolidacijsko radioterapijo. Primerjali smo tudi srednje preživetje bolnikov, ki smo jih zdravili z razlicnimi konsolidacijskimi dozami obsevanja in izvedli univariatno in multivariatno analizo napovednih dejavnikov. Rezultati. Od 412 bolnikov z ED-SCLC je prejelo kemoterapijo s konsolidacijsko radioterapijo 74 bolnikov, samo kemoterapijo pa 113 bolnikov. Bolniki s konsolidacijsko radioterapijo so imeli bistveno daljše srednje preživetje v primerjavi z bolniki zdravljenimi samo s kemoterapijo, 11,1 mesecev (interval zaupanja [CI] 10,1–12,0) v primerjavi s 7,6 meseca (CI 6,9–8,5, p < 0,001) in daljše enoletno celokupno preživetje (44 % v primerjavi s 23 %, p = 0,0025), medtem ko se razlika v dvoletnem celokupnem preživetju ni znacilno razlikovala (10 % v primerjavi s 5 %, p = 0,19). Doza konsolidacijske radioterapije ni bila enotna. Višja se­valna doza 45 Gy (v 18 frakcijah) je izboljšala srednje celokupno preživetje v primerjavi z nižjo dozo 30–36 Gy (v 10–12 frakcijah), 17,2 meseca v primerjavi z 10,3 meseca (p = 0,03). Statisticno znacilno razliko smo opazili tudi pri 1-letnem celokupnem preživetju (68 % v primerjavi s 3 0%, p = 0,01) in neznacilno razliko pri dvoletnem celokupnem preživetju (18 % v primerjavi s 5 %, p = 0,11). Zakljucki. Konsolidacijska radioterapija je izboljšala srednje celokupno preživetje in enoletno celokupno preživetje pri bolnikih z ED-SCLC v primerjavi s samo kemoterapijo. Višja sevalna doza konsolidacijske radioterapije je bila povezana z daljšim celokupnim preživetjem in enoletnim celokupnim preživetjem v primerjavi z nižjo dozo. Konsolidacijska radioterapija, vecje število krogov kemoterapije in profilakticno obsevanje glave so bili neodvisni napovedni dejavniki za boljše preživetje v naši analizi. Pri bolnikih, ki so prejeli konsolidacijsko radioterapijo, so na preživetje vplivali le višji odmerki in profilakticno obsevanje glave, ne glede na število prejetih krogov kemoterapije. Vloga konsolidacijske radioterapije v obdobju imunoterapije ni znana in bi jo veljalo raziskati. Radiol Oncol 2020; 54(3): 364-370. doi: 10.2478/raon-2020-0036 Ocena nastavitvenih napak pri obsevanju bolnikov z rakom glave in vratu. Standardne in individualne podlage za glavo Androjna S, Žager Marciuš V, Peterlin P, Strojan P Izhodišca. Cilj raziskave je bil (a) primerjati natancnost dveh razlicnih imobilizacijskih strategij pri bolnikih s tumorji glave in vratu; in (b) primerjati nastavitvene napake na terapevtskih enotah z razlicnimi sistemi za portalno slikanje. Bolniki in metode. Variacije v legi izocentra (IC) glede na referencno tocko, doloceno na CT simu­latorju smo merili v vertikalni (anteriorno-posteriorno), longitudinalni (superiorno-inferiorno) in lateralni (medialno-lateralno) smeri pri 120 bolnikih z rakom glave in vratu. Bolnike smo obsevali z namenom oz-dravitve in jih, odvisno od terapevtske enote (enota A – 2D/2D slikovni predogled; enota B – 2D slikovni predogled) in obdobje obsevanja razdelili v 6 skupin po 20 bolnikov. Pri tistih, ki smo jih obsevali v letu 2014, smo uporabljali standardne podlage za pod glavo in vrat (skupini 1 in 2), pri tistih, ki smo jih obsevali leta 2015 in 2017 (skupine 3-6) pa smo uporabljali individualne podlage za za pod glavo in vrat. Varnostni rob med klinicnim in planirnim tarcnim volumnom smo izracunali po formuli, ki jo je predlagal Van Herk. Rezultati. Analizirani smo skupaj 2.454 portalnih slik in 3.681 nastavitvenih napak. Z uvedbo individualnih podlag za za pod glavo in vrat v letu 2015 smo statisticno znacilno zmanjšali povprecni medfrakcijski odklon v vertikalni smeri in zmanjšali število odklonov izocentra v vertikalni in longitudinalni smeri (velja za obe terapevtski enoti). Najvecje zmanjšanje varnostnega robu smo izracunali v longitudinalni smeri, varnostni robovi pa so bili vecji za enoto B kot enoto A. Zakljucki. Ugotovili smo, da uporaba individualnih podlag za pod glavo in vrat in bolj naprednega slikovnega sistema povecuje natancnost nastavitve pri obsevanju bolnikov z rakom glave in vratu. Fundacija "Docent dr. J. Cholewa" je neprofitno, neinstitucionalno in nestrankarsko združenje posameznikov, ustanov in organizacij, ki želijo materialno spodbujati in poglabljati raziskovalno dejavnost v onkologiji. Dunajska 106 1000 Ljubljana IBAN: SI56 0203 3001 7879 431 Activity of "Dr. J. Cholewa" Foundation for Cancer Research and Education – a report for the third quarter of 2020 Dr. Josip Cholewa Foundation for cancer research and education continues with its planned activities in the third quarter of 2020. Its primary focus remains the provision of grants, 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. Borut Štabuc, M.D., Ph.D. Andrej Plesnicar, M.D., M.Sc. Viljem Kovac M.D., Ph.D. • pri metastatskem NSCLC *,1,2 • in napredovalem melanomu3 *NSCLC – non-small cell lung cancer Reference: 1. Gandhi L, Rodríguez-Abreu D, Gadgeel S, et. al.; for the KEYNOTE-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 me lanoma treated with pembrolizumab in KEYNOTE-001. Annals of Oncology 2019; 30: 582-588. SKRAJŠAN POVZETEK GLAVNIH ZNACILNOSTI ZDRAVILA Pred predpisovanjem, prosimo, preberite celoten Povzetek glavnih znacilnosti zdravi-la! Ime zdravila: KEYTRUDA 25 mg/ml koncentrat za raztopino za infundiranje vsebuje pem­brolizumab. Terapevtske indikacije: Zdravilo KEYTRUDA je kot samostojno zdravlje nje indicirano za zdravljenje: napredovalega (neoperabilnega ali metastatskega) melano ma pri odraslih; za adjuvantno zdravljenje odraslih z melanomom v stadiju III, ki se je razširil na bezgavke, po popolni kirurški odstranitvi; metastatskega nedrobnocelicnega pljucnega 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 predhod-no zdravljeni z vsaj eno shemo kemoterapije, bolniki s pozitivnimi tumorskimi mutacijami EGFR ali ALK so pred prejemom zdravila KEYTRUDA morali prejeti tudi tarcno zdravljenje; odraslih bolnikov s ponovljenim ali neodzivnim klasicnim Hodgkinovim limfomom (cHL), pri katerih avtologna presaditev maticnih celic (ASCT) in zdravljenje z brentuksimabom ve dotinom (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 vkljucevala 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 plošcatocelicnega 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 vkl-jucevala platino. Zdravilo KEYTRUDA je kot samostojno zdravljenje ali v kombinaciji s kemoterapijo s platino in 5-fluorouracilom (5-FU) indicirano za prvo linijo zdravljenja meta-statskega ali neoperabilnega ponovljenega plošcatocelicnega raka glave in vratu pri odraslih, ki imajo tumorje z izraženostjo PD-L1 s CPS = 1. Zdravilo KEYTRUDA je v kombinaciji s peme treksedom in kemoterapijo na osnovi platine indicirano za prvo linijo zdravljenja metastats­kega neplošcatocelicnega NSCLC pri odraslih, pri katerih tumorji nimajo pozitivnih mutacij EGFR ali ALK; v kombinaciji s karboplatinom in bodisi paklitakselom bodisi nab-paklitakselom je indicirano za prvo linijo zdravljenja metastatskega plošcatocelicnega NSCLC pri odraslih; v kombinaciji z aksitinibom je indicirano za prvo linijo zdravljenja napredovalega raka led-vicnih celic (RCC) pri odraslih. Odmerjanje in nacin uporabe: Testiranje PD-L1 pri bolnikih z NSCLC, urotelijskim rakom ali HNSCC: Za samostojno zdravljenje z zdravilom KEYTRUDA je priporocljivo opraviti testiranje izraženosti PD-L1 tumorja z validirano 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: Priporoceni 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. Pri­poroceni odmerek za kombinirano zdravljenje je 200 mg na 3 tedne, apliciran z intravensko infuzijo v 30 minutah. Za uporabo v kombinaciji glejte povzetke glavnih znacilnosti socasno uporabljenih zdravil. Ce se uporablja kot del kombiniranega zdravljenja skupaj z intravensko kemoterapijo, je treba zdravilo KEYTRUDA aplicirati prvo. Bolnike je treba zdraviti do napre dovanja bolezni ali nesprejemljivih toksicnih ucinkov. Pri adjuvantnem zdravljenju melano ma je treba zdravilo uporabljati do ponovitve bolezni, pojava nesprejemljivih toksicnih ucink ov oziroma mora zdravljenje trajati do enega leta. Ce je aksitinib uporabljen v kombinaciji s pembrolizumabom, se lahko razmisli o povecanju odmerka aksitiniba nad zacetnih 5 mg v presledkih šest tednov ali vec. 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 priporocljivo. Za obvlado vanje neželenih ucinkov je treba uporabo zdravila KEYTRUDA zadržati ali ukiniti, prosimo, glejte celoten Povzetek glavnih znacilnosti zdravila. Kontraindikacije: Preobcutljivost na ucinkovino ali katero koli pomožno snov. Povzetek posebnih opozoril, previdnostnih ukrepov, interakcij in neželenih ucinkov: Imunsko pogojeni neželeni ucinki (pnevmoni-tis, kolitis, hepatitis, nefritis, endokrinopatije, neželeni ucinki na kožo in drugi): Pri bolnikih, ki so prejemali pembrolizumab, so se pojavili imunsko pogojeni neželeni ucinki, vkljucno s hu­dimi in smrtnimi primeri. Vecina imunsko pogojenih neželenih ucinkov, ki so se pojavili med zdravljenjem s pembrolizumabom, je bila reverzibilnih in so jih obvladali s prekinitvami upo rabe pembrolizumaba, uporabo kortikosteroidov in/ali podporno oskrbo. Pojavijo se lahko tudi po zadnjem odmerku pembrolizumaba in hkrati prizadanejo vec organskih sistemov. V primeru suma na imunsko pogojene neželene ucinke je treba poskrbeti za ustrezno oceno za potrditev etiologije oziroma izkljucitev drugih vzrokov. Glede na izrazitost neželenega ucinka je treba zadržati uporabo pembrolizumaba in uporabiti kortikosteroide – za natancna na­vodila, prosimo, glejte Povzetek glavnih znacilnosti zdravila Keytruda. Zdravljenje s pembroli­zumabom lahko poveca tveganje za zavrnitev pri prejemnikih presadkov cvrstih organov. Pri bolnikih, ki so prejemali pembrolizumab, so porocali o hudih z infuzijo povezanih reakcijah, vkljucno s preobcutljivostjo in anafilaksijo. Pembrolizumab se iz obtoka odstrani s kataboliz-mom, zato presnovnih medsebojnih delovanj zdravil ni pricakovati. Uporabi sistemskih kor­tikosteroidov ali imunosupresivov pred uvedbo pembrolizumaba se je treba izogibati, ker lahko vplivajo na farmakodinamicno aktivnost in ucinkovitost pembrolizumaba. Vendar pa je kortikosteroide ali druge imunosupresive mogoce uporabiti za zdravljenje imunsko pogo jenih neželenih ucinkov. Kortikosteroide je mogoce uporabiti tudi kot premedikacijo, ce je pembrolizumab uporabljen v kombinaciji s kemoterapijo, kot antiemeticno profi lakso in/ali za ublažitev neželenih ucinkov, povezanih s kemoterapijo. Ženske v rodni dobi morajo med zdravljenjem s pembrolizumabom in vsaj še 4 mesece po zadnjem odmerku pembrolizuma­ba uporabljati ucinkovito kontracepcijo, med nosecnostjo in dojenjem se ga ne sme upora­bljati. Varnost pembrolizumaba pri samostojnem zdravljenju so v klinicnih študijah ocenili pri 5.884 bolnikih z napredovalim melanomom, 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 cas opazovanja znašal 7,3 mesece (v razponu od 1 dneva do 31 mesecev), najpo gostejši neželeni ucinki zdravljenja s pembrolizumabom so bili utrujenost (32 %), navzea (20 %) in diareja (20 %). Vecina porocanih neželenih ucinkov pri samostojnem zdravljenju je bila po izrazitosti 1. ali 2. stopnje. Najresnejši neželeni ucinki so bili imunsko pogojeni neželeni ucinki 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 klinicnih 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 ucinki 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 ucinkov 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 klinicni študiji pri 429 bolnikih z napre dovalim rakom ledvicnih 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 ucinki diare ja (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 %), disfonija (25 %), kašelj (21 %) in zaprtost (21 %). Po javnost neželenih ucinkov 3. do 5. stopnje je bila med kombiniranim zdravljenjem s pembroli­zumabom 76 % in pri zdravljenju s sunitinibom samim 71 %. Za celoten seznam neželenih ucinkov, prosimo, glejte celoten Povzetek glavnih znacilnosti zdravila. Nacin 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: 2. junij 2020. 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, junij 2020; SI-KEY-00118 EXP: 06/2022 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 znacilnosti zdravila Keytruda, ki je na voljo pri naših strokovnih sodelavcih ali na lokalnem sedežu družbe. Skrajšan povzetek glavnih znaËilnosti 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 poroËajo o katerem koli domnevnem neželenem uËinku 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 vkljuËujejo 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 vkljuËno z adenokarcinomom gastro-ezofagealnega prehoda, ki so bili predhodno že zdravljeni z najmanj dvema sistemskima režimoma zdravljenja za napredovalo bolezen. ODMERJANJE IN NA»IN UPORABE*: PriporoËeni zaËetni 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 zakljuËku jutranjega in veËernega obroka (20 mg/m2/odmerek dvakrat dnevno pri bolnikih s hudo ledviËno okvaro). Odmerjanje, izraËunano glede na telesno površino, ne sme preseËi 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 ledviËno okvaro). Potem ko je bil odmerek zmanjšan, poveËanje ni dovoljeno. KONTRAINDIKACIJE*: Pre­obËutljivost na zdravilni uËinkovini ali katero koli pomožno snov. OPOZORILA IN PREVIDNOSTNI UKREPI*: Supresija kostnega mozga: Pred uvedbo zdravljenja in po potrebi za spremljanje toksiËnosti zdravila, najmanj pred vsakim ciklom zdravljenja, je treba pregledati celotno krvno sliko. Zdravljenja ne smete zaËeti, Ëe je absolutno število nevtrofilcev < 1,5 x 109/l, Ëe je število trombocitov < 75 x 109/l ali Ëe se je pri bolniku zaradi predhodnih zdravljenj pojavila kliniËno pomembna nehematološka toksiËnost 3. ali 4. stopnje, ki še traja. Bolnike je treba skrbno spremljati zaradi morebitnih okužb, uvesti je treba ustrezne ukrepe, kot je kliniËno indicirano. ToksiËnost za prebavila: Potrebna je uporaba antiemetikov, antidiaroikov ter drugih ukrepov, kot je kliniËno indici­rano. »e je potrebno, prilagodite odmerke. LedviËna okvara: Zdravilo Lonsurf ni primerno za uporabo pri bolnikih s konËno stopnjo ledviËne okvare. Bolnike z ledviËno okvaro je potrebno med zdravljenjem skrbno spremljati; bolnike z zmerno ali hudo ledviËno okvaro je treba zaradi hematološke toksiËnosti bolj pogosto spremljati. Jetrna okvara: Uporaba zdravila Lonsurf pri bolnikih z obstojeËo zmerno ali hudo jetrno okvaro ni priporoËljiva. Proteinurija: Pred zaËetkom zdravljenja in med njim je priporoËljivo spreml­janje proteinurije z urinskimi testnimi listiËi. 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. zido­vudinom), hormonskimi kontraceptivi. PLODNOST*, NOSE»NOST IN DOJENJE*: Ni priporoËljivo. KONTRACEPCIJA*: Ženske in moški morajo uporabljati uËinkovito metodo kontracepcije med zdravljenjem in do 6 mesecev po zakljuËku zdravljenja. VPLIV NA SPOSOBNOST VOŽNJE IN UPRAVLJANJA STROJEV*: Med zdravljenjem se lahko pojavijo utrujenost, omotica ali splošno slabo poËutje. NEŽELENI U»INKI*: Zelo pogosti: nevtropenija, levkopenija, anemija, trombocitopenija, zmanjšan apetit, diareja, navzea, bruhanje, utrujenost. Pogosti: okužba spodnjih dihal, febrilna nevtropenija, limfopenija, hipoalbuminemija, disgevzija, periferna nevropatija, dispneja, boleËina v trebuhu, zaprtje, stomatitis, bolezni ustne votline, hiperbilirubinemija, sin-drom palmarne plantarne eritrodisestezije, izpušËaj, alopecija, pruritus, suha koža, proteinurija, pireksija, edem, vnetje sluznice, splošno slabo poËutje, zvišanje jetrnih encimov, zvišanje alkalne fosfataze v krvi, zmanjšanje telesne mase. ObËasni: septiËni šok, infekcijski enteritis, pljuËnica, okužba žolËevoda, gripa, okužba seËil, gingivitis, herpes zoster, tinea pedis, okužba s kandido, bakterijska okužba, okužba, nevtropeniËna sepsa, okužba zgornjih dihal, konjunktivitis, boleËina zaradi raka, pancitopenija, granuloci­topenija, monocitopenija, eritropenija, levkocitoza, monocitoza, dehidracija, hiperglikemija, hiperkaliemija, hipokaliemija, hipofosfatemija, hipernatriemija, hiponatriemija, hipokalciemija, protin, anksioznost, nespeËnost, nevrotoksiËnost, disestezija, hipereste­zija, hipoestezija, sinkopa, parestezija, pekoË obËutek, letargija, omotica, glavobol, zmanjšana ostrina vida, zamegljen vid, diplopija, katarakta, suho oko, vrtoglavica, neugodje v ušesu, angina pektoris, aritmija, palpitacije, embolija, hipertenzija, hipotenzija, vroËinski oblivi, pljuËna embolija, plevralni izliv, izcedek iz nosu, disfonija, orofaringealna boleËina, epistaksa, kašelj, hemoragiËni 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, hepatotoksiËnost, razširitev žolËnih vodov, lušËenje kože, urtikarija, preobËutljivostne reakcije na svetlobo, eritem, akne, hiperhidroza, žulj, bolezni nohtov, otekanje sklepov, artralgija, boleËina v kosteh, mialgija, mišiËno-skeletna boleËina, mišiËna oslabelost, mišiËni krËi, boleËina v okonËinah, ledviËna odpoved, neinfektivni cistitis, motnje mikcije, hematurija, levkociturija, motnje menstruacije, poslabšanje splošnega zdravstvenega stanja, boleËina, obËutek spremembe telesne temperature, kseroza, nelagodje, zvi­šanje kreatinina v krvi, podaljšanje intervala QT na elektrokardiogramu, poveËanje mednarodnega umerjenega razmerja (INR), podaljšanje aktiviranega parcialnega tromboplastinskega Ëasa (aPT»), zvišanje seËnine v krvi, zvišanje laktatne dehidrogenaze v krvi, znižanje celokupnih proteinov, zvišanje C-reaktivnega proteina, zmanjšan hematokrit. Post-marketinške izkušnje: intersticijska bolezen pljuË. PREVELIKO ODMERJANJE*: Neželeni uËinki, o katerih so poroËali v povezavi s prevelikim odmerjanjem, so bili v skladu z uveljavljenim varnostnim profilom. Glavni priËakovani zaplet prevelikega odmerjanja je supresija kostnega mozga. FARMAKODINAMI»NE LASTNOSTI*: Farmakoterapevtska skupina: zdravila z delovanjem na novotvorbe, antimetaboliti, oznaka ATC: L01BC59. Zdravilo Lonsurf sestavljata antineoplastiËni 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 prepreËuje celiËno proliferacijo. TPaza hitro razgradi trifluridin in njegova presnova po peroralni uporabi je hitra zaradi uËinka prvega prehoda, zato je v zdravilo vkljuËen zaviralec TPaze, tipiracilijev klorid. PAKIRANJE*: 20 filmsko obloženih tablet. NA»IN 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 znaËil­nosti zdravila. Celoten povzetek glavnih znaËilnosti zdravila in podrobnejše informacije so na voljo pri: Servier Pharma d.o.o., PodmilšËakova ulica 24, 1000 Ljubljana, tel: 01 563 48 11, www.servier.si. Datum priprave informacije: april 2020. LNF AD1 C2 19/20 Samo za strokovno javnost. Instructions for authors The editorial policy Radiology and Oncology is a multidisciplinary journal devoted to the publishing original and high quality scientific papers and review articles, pertinent to diagnostic and interventional radiology, computerized tomography, magnetic resonance, ultrasound, nuclear medicine, radiotherapy, clinical and experimental oncology, radiobiology, 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. The Editorial Board requires that the paper has not been published or submitted for publication elsewhere; the authors are responsible for all statements in their papers. Accepted articles become the property of the journal and, therefore cannot be published elsewhere without the written permission of the editors. 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The corresponding author is responsible for informing the coauthors of the manuscript status throughout the submission, review, and production process. Preparation of manuscripts Radiology and Oncology will consider manuscripts prepared according to the Uniform Requirements for Manuscripts Submitted to Biomedical Journals by International Committee of Medical Journal Editors (www.icmje.org). The manuscript should be written in grammatically and stylistically correct language. Abbreviations should be avoided. If their use is neces­sary, they should be explained at the first time mentioned. The technical data should conform to the SI system. The manu­script, excluding the references, tables, figures and figure legends, must not exceed 5000 words, and the number of figures and tables is limited to 8. Organize the text so that it includes: Introduction, Materials and methods, Results and Discussion. Exceptionally, the results and discussion can be combined in a single section. 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References to the Abstracts and Letters to the Editor must be identified as such. Citation of papers in preparation or submitted for publication, unpublished observations, and personal communications should not be included in the reference list. If essential, such material may be incorporated in the appropri­ate place in the text. References follow the style of Index Medicus, DOI number (if exists) should be included. All authors should be listed when their number does not exceed six; when there are seven or more authors, the first six listed are followed by “et al.”. The following are some examples of references from articles, books and book chapters: Dent RAG, Cole P. In vitro maturation of monocytes in squamous carcinoma of the lung. Br J Cancer 1981; 43: 486-95. doi: 10.1038/bjc.1981.71 Chapman S, Nakielny R. A guide to radiological procedures. London: Bailliere Tindall; 1986. Evans R, Alexander P. Mechanisms of extracellular killing of nucleated mammalian cells by macrophages. In: Nelson DS, editor. Immunobiology of macrophage. New York: Academic Press; 1976. p. 45-74. Authorization for the use of human subjects or experimental animals When reporting experiments on human subjects, authors should state whether the procedures followed the Helsinki Declaration. Patients have the right to privacy; therefore the identifying information (patient’s names, hospital unit numbers) should not be published unless it is essential. In such cases the patient’s informed consent for publication is needed, and should appear as an appropriate statement in the article. Institutional approval and Clinical Trial registration number is re­quired. Retrospective clinical studies must be approved by the accredited Institutional Review Board/Committee for Medical Ethics or other equivalent body. These statements should appear in the Materials and methods section. 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The non-commercial use of each article will be governed by the Creative Commons Attribution-NonCommercial-NoDerivs license. Conflict of interest When the manuscript is submitted for publication, the authors are expected to disclose any relationship that might pose real, apparent or potential conflict of interest with respect to the results reported in that manuscript. Potential conflicts of interest include not only financial relationships but also other, non-financial relationships. In the Acknowledgement section the source of funding support should be mentioned. The Editors will make effort to ensure that conflicts of interest will not compromise the evaluation process of the submitted manuscripts; potential editors and reviewers will exempt themselves from review process when such conflict of interest exists. 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. ALK = anaplastiína limfomska kinaza BISTVENI PODATKI IZ POVZETKA GLAVNIH ZNACILNOSTI 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 nedrobnocelicnim pljucnim rakom (NSCLC – Non-Small Cell Lung Cancer), ki je ALK (anaplasticna limfomska kinaza) pozitiven; - zdravljenje odraslih bolnikov s predhodno zdravljenim, napredovalim NSCLC, ki je ALK pozitiven; - zdravljenje odraslih bolnikov z napredovalim NSCLC, ki je ROS1 pozitiven. Odmerjanje in nacin uporabe: Zdravljenje mora uvesti in nadzorovati zdravnik z izkušnjami z uporabo zdravil za zdravljenje rakavih bolezni. Preverjanje prisotnosti ALK in ROS1: Pri izbiri bolnikov za zdravljenje je treba pred zdravljenjem opraviti tocno in validirano preverjanje prisotnosti ALK ali ROS1. Odmerjanje: Priporoceni odmerek je 250 mg dvakrat na dan (500 mg na dan), bolniki pa morajo zdravilo jemati brez prekinitev. Ce bolnik pozabi vzeti odmerek, ga mora vzeti takoj, ko se spomni, razen ce 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 znacilnosti zdravila). Za prilagajanje odmerkov pri hematološki in nehematološki toksicnosti (povecanje vrednosti AST, ALT, bilirubina; ILD/pnevmonitis; podaljšanje intervala QTc, bradikardija, bolezni oci) glejte preglednici 1 in 2 v poglavju 4.2 povzetka glavnih znacilnosti zdravila. Okvara jeter: Pri zdravljenju pri bolnikih z okvaro jeter je potrebna previdnost. Pri blagi okvari jeter prilagajanje zacetnega odmerka ni priporoceno, pri zmerni okvari jeter je priporoceni zacetni odmerek 200 mg dvakrat na dan, pri hudi okvari jeter pa 250 mg enkrat na dan (za merila glede klasi.kacije okvare jeter glejte poglavje 4.2 v povzetku glavnih znacilnosti zdravila). Okvara ledvic: Pri blagi in zmerni okvari prilagajanje zacetnega odmerka ni priporoceno. Pri hudi okvari ledvic (ki ne zahteva peritonealne dialize ali hemodialize) je zacetni odmerek 250 mg peroralno enkrat na dan; po vsaj 4 tednih zdravljenja se lahko poveca na 200 mg dvakrat na dan. Starejši bolniki (= 65 let): Prilagajanje zacetnega odmerka ni potrebno. Pediatricna populacija: Varnost in ucinkovitost nista bili dokazani. Nacin 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: Preobcutljivost na krizotinib ali katerokoli pomožno snov. Posebna opozorila in previdnostni ukrepi: Dolocanje statusa ALK in ROS1: Pomembno je izbrati dobro validirano in robustno metodologijo, da se izognemo lažno negativnim ali lažno pozitivnim rezultatom. Hepatotoksicnost:V klinicnih študijah so porocali o hepatotoksicnosti, ki jo je povzrocilo zdravilo (vkljucno s primeri s smrtnim izidom). Delovanje jeter, vkljucno 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 klinicno indicirano. Ponovitve preverjanj morajo biti pogostejše pri povecanjih vrednosti stopnje 2, 3 ali 4. Intersticijska bolezen pljuc (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 obstojeco 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 obstojeco srcno 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 cimbližje uporabi prvega odmerka, potem se priporoca redno spremljanje. Ce se interval QTc podaljša za 60 ms ali vec, je treba zdravljenje s krizotinibom zacasno prekiniti in se posvetovati s kardiologom. Bradikardija: Lahko se pojavi simptomatska bradikardija (lahko se razvije vec tednov po zacetku zdravljenja); izogibati se je treba uporabi krizotiniba v kombinaciji z drugimi zdravili, ki povzrocajo bradikardijo; pri simptomatski bradikardiji je treba prilagoditi odmerek. Srcno popušcanje: Porocali so o hudih, življenjsko nevarnih ali smrtnih neželenih ucinkih srcnega popušcanja. Bolnike je treba spremljati glede pojavov znakov in simptomov srcnega popušcanja in ob pojavu simptomov zmanjšati odmerjanje ali prekiniti zdravljenje. Nevtropenija in levkopenija:V klinicnih študijah so porocali o nevtropeniji, levkopeniji in febrilni nevtropeniji; spremljati je treba popolno krvno sliko (pogostejše preiskave, ce se opazijo abnormalnosti stopnje 3 ali 4 ali ce se pojavi povišana telesna temperatura ali okužba). Perforacija v prebavilih:V klinicnih študijah so porocali 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 pouciti o prvih znakih perforacije in jim svetovati, naj se nemudoma posvetujejo z zdravnikom. Vplivi na ledvice:V klinicnih študijah so opazili zvišanje ravni kreatinina v krvi in zmanjšanje ocistka kreatinina. V klinicnih študijah in v obdobju trženja so porocali tudi o odpovedi ledvic, akutni odpovedi ledvic, primerih s smrtnim izidom, primerih, ki so zahtevali hemodializo in hiperkaliemiji stopnje 4. Vplivi na vid: V klinicnih študijah so porocali o izpadu vidnega polja stopnje 4 z izgubo vida. Ce se na novo pojavi huda izguba vida, je treba zdravljenje prekiniti in opraviti oftalmološki pregled. Ce so motnje vida trdovratne ali se poslabšajo, je priporocljiv 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 znacilnosti, ki ne nakazujejo adenokarcinoma, vkljucno s plošcatocelicnim karcinomom (SCC). Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Izogibati se je treba socasni uporabi z mocnimi zaviralci CYP3A4, npr. atazanavir, ritonavir, kobicistat, itrakonazol, ketokonazol, posakonazol, vorikonazol, klaritromicin, telitromicin in eritromicin (razen ce morebitna korist za bolnika odtehta tveganje, v tem primeru je treba bolnike skrbno spremljati glede neželenih ucinkov krizotiniba), ter grenivko in grenivkinim sokom, saj lahko povecajo koncentracije krizotiniba v plazmi. Izogibati se je treba socasni uporabi z mocnimi induktorji CYP3A4, npr. karbamazepin, fenobarbital, fenitoin, rifampicin in šentjanževka, saj lahko zmanjšajo koncentracije krizotiniba v plazmi. Ucinek zmernih induktorjev CYP3A4, npr. efavirenz in rifabutin, še ni jasen, zato se je treba socasni 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: socasni uporabi s temi zdravili se je treba izogibati oziroma izvajati skrben klinicni nadzor; bupropion, efavirenz, peroralni kontraceptivi, raltegravir, irinotekan, mor.n, nalokson, metformin, prokainamid). Pfizer Luxembourg SARL, GRAND DUCHY OF LUXEMBOURG, 51, Avenue J.F. Kennedy, L-1855, Pfizer podružnica Ljubljana, Letališka cesta 29a, 1000 Ljubljana KRIZOTINIB Zdravila, ki podaljšujejo interval QT ali ki lahko povzrocijo Torsades de pointes (antiaritmiki skupine IA (kinidin, disopiramid), antiaritmiki skupine III (amiodaron, sotalol, dofetilid, ibutilid), metadon, cisaprid, moksi.oksacin, antipsihotiki) – v primeru socasne uporabe je potreben skrben nadzor intervala QT. Zdravila, ki povzrocajo bradikardijo (nedihidropiridinski zaviralci kalcijevih kanalckov (verapamil, diltiazem), antagonisti adrenergicnih receptorjev beta, klonidin, gvanfacin, digoksin, me.okin, antiholinesteraze, pilokarpin) – krizotinib je treba uporabljati previdno. Plodnost, nosecnost 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 nosecnostjo ne sme uporabljati, razen ce klinicno 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 ucinki: Najresnejši neželeni ucinki so bili hepatotoksicnost, ILD/ pnevmonitis, nevtropenija in podaljšanje intervala QT. Najpogostejši neželeni ucinki (= 25 %) so bili motnje vida, navzea, diareja, bruhanje, edem, zaprtje, povecane vrednosti transaminaz, utrujenost, pomanjkanje apetita, omotica in nevropatija. Ostali zelo pogosti (= 1/10 bolnikov) neželeni ucinki so: nevtropenija, anemija, levkopenija, disgevzija, bradikardija, bolecina v trebuhu in izpušcaj. Nacin in režim izdaje: Predpisovanje in izdaja zdravila je le na recept, zdravilo pa se uporablja samo v bolnišnicah. Izjemoma se lahko uporablja pri nadaljevanju zdravljenja na domu ob odpustu iz bolnišnice in nadaljnjem zdravljenju. Imetnik dovoljenja za promet: P.zer Europe MA EEIG, Boulevard de la Plaine 17, 1050 Bruxelles, Belgija. Datum zadnje revizije besedila: 31.10.2019. Pred predpisovanjem se seznanite s celotnim povzetkom glavnih znacilnosti zdravila. Vir: 1. Povzetek glavnih znacilnosti zdravila Xalkori, 31.10.2019 XAR-01-20 Samo za strokovno javnost