R a d io lo g y a n d O n c o lo g y I V o lu m e 5 6 I N u m b e r 1 I P a g e s 1 -1 2 8 I M a r c h 2 0 2 2 march 2022 vol.56 no.1 aksitinib Pfi zer Luxembourg SARL, GRAND DUCHY OF LUXEMBOURG, 51, Avenue J.F. Kennedy, L – 1855, Pfi zer, podružnica Ljubljana, Letališka cesta 29a, 1000 Ljubljana Samo za strokovno javnost. • Datum priprave: januar 2022 • PP-INL-EEP-0040 BISTVENI PODATKI IZ POVZETKA GLAVNIH ZNAČILNOSTI ZDRAVILA Inlyta 1 mg/3 mg/5 mg/7 mg fi lmsko obložene tablete Sestava in oblika zdravila: Ena tableta vsebuje 1 mg, 3 mg, 5 mg oz. 7 mg aksitiniba. Indikacije: Zdravljenje napredovalega karcinoma ledvičnih celic (RCC) pri odraslih bolnikih, pri katerih predhodno zdravljenje s sunitinibom ali citokinom ni bilo uspešno. Odmerjanje in način uporabe: Zdravljenje mora izvajati zdravnik, ki ima izkušnje z uporabo zdravil za zdravljenje rakavih bolezni. Priporočeni odmerek je 5 mg dvakrat na dan. Zdravljenje naj traja, dokler je mogoče opaziti klinično korist oz. do pojava nesprejemljive toksičnosti, ki je ni mogoče obvladovati s sočasno uporabljanimi zdravili ali prilagajanjem odmerka. Če bolnik bruha ali izpusti odmerek, ne sme vzeti dodatnega odmerka; naslednji predpisan odmerek je treba vzeti ob običajnem času. Prilagajanja odmerka: Pri bolnikih, ki aksitinib v začetnem odmerku 5 mg dvakrat na dan prenašajo brez neželenih učinkov > 2. stopnje dva tedna zapored, je odmerek mogoče zvečati na 7 mg dvakrat na dan, razen če je krvni tlak pri bolniku > 150/90 mmHg ali če jemlje antihipertenzive. Kasneje je z uporabo enakih meril pri bolnikih, ki prenašajo 7 mg dvakrat na dan, odmerek mogoče zvečati na največ 10 mg dvakrat na dan. Za obvladovanje nekaterih neželenih učinkov bo morda treba začasno ali trajno prekiniti zdravljenje in/ali zmanjšati odmerek na 3 mg dvakrat na dan in nato na 2 mg dvakrat na dan. Prilagajanje odmerka glede na bolnikovo starost, raso, spol ali telesno maso ni potrebno. Sočasno zdravljenje z močnimi zaviralci CYP3A4/5: Lahko zveča plazemske koncentracije aksitiniba. V primeru sočasne uporabe močnega zaviralca CYP3A4/5, je odmerek aksitiniba priporočljivo zmanjšati na približno polovico odmerka; morda bo potrebna začasna ali trajna prekinitev zdravljenja z aksitinibom. Če prekinemo sočasno uporabo močnega zaviralca, je treba razmisliti o vrnitvi na odmerek aksitiniba, ki je bil uporabljen pred uvedbo močnega zaviralca CYP3A4/5. Sočasno zdravljenje z močnimi induktorji CYP3A4/5: Lahko zmanjša plazemske koncentracije aksitiniba. V primeru sočasne uporabe močnega induktorja CYP3A4/5 je odmerek aksitiniba priporočljivo postopoma zvečati in bolnika skrbno nadzorovati glede pojava toksičnosti. Morda bo treba začasno ali trajno prekiniti zdravljenje in/ali zmanjšati odmerek aksitiniba. Če prekinemo sočasno uporabo močnega induktorja, je treba takoj začeti uporabljati odmerek aksitiniba, ki je bil uporabljen pred uvedbo močnega induktorja CYP3A4/5. Okvara ledvic: Prilagajanje odmerka ni potrebno; o uporabi pri bolnikih z očistkom kreatinina < 15 ml/min ni podatkov. Okvara jeter: Prilagajanje odmerka ni potrebno pri bolnikih z blago okvaro jeter (razred A po Child-Pughu). Zmanjšanje odmerka je priporočljivo pri bolnikih z zmerno okvaro jeter (razred B). Zdravila se ne sme uporabljati pri bolnikih s hudo okvaro jeter (razred C). Pediatrična populacija: Varnost in učinkovitost pri otrocih < 18 let nista bili dokazani; podatkov ni na voljo. Način uporabe: Peroralna uporaba. Tablete je treba pogoltniti cele, s kozarcem vode, dvakrat na dan, v približno 12-urnih časovnih presledkih, s hrano ali brez nje. Kontraindikacije: Preobčutljivost na aksitinib ali katerokoli pomožno snov. Posebna opozorila in previdnostni ukrepi: Dogodki srčnega popuščanja: Poročali so o dogodkih srčnega popuščanja. Med zdravljenjem je treba redno spremljati znake ali simptome srčnega popuščanja. Obravnava dogodkov srčnega popuščanja lahko zahteva začasno ali stalno prekinitev zdravljenja z aksitinibom in/ali zmanjšanje odmerka. Hipertenzija: O hipertenziji so poročali zelo pogosto. Pred začetkom zdravljenja mora biti krvni tlak ustrezno urejen; bolnike je treba spremljati in po potrebi uporabiti standardno antihipertenzivno zdravljenje. V primeru trdovratne hipertenzije (kljub uporabi antihipertenzivov) je treba odmerek aksitiniba zmanjšati, pri hudi hipertenziji pa zdravljenje začasno prekiniti in ga ponovno uvesti z manjšim odmerkom, ko se krvni tlak normalizira. Pri hudi ali trdovratni arterijski hipertenziji in simptomih sindroma posteriorne reverzibilne encefalopatije je treba razmisliti o diagnostičnem slikanju možganov z uporabo magnetne resonance. Motnje delovanja ščitnice: Poročali so o primerih hipotiroidizma in, v manjšem obsegu, hipertiroidizma. Delovanje ščitnice je treba spremljati pred začetkom zdravljenja in v rednih časovnih presledkih med zdravljenjem. Venski in arterijski embolični in trombotični dogodki: Poročali so o venskih in arterijskih emboličnih in trombotičnih dogodkih. Previdna uporaba pri bolnikih s tveganjem za pojav teh dogodkov ali anamnezo teh dogodkov. Zvišanje ravni hemoglobina ali hematokrita: Med zdravljenjem lahko pride do zvišanj ravni hemoglobina ali hematokrita, njuno raven je treba spremljati pred začetkom zdravljenja in v rednih časovnih presledkih med zdravljenjem. Krvavitve: Poročali so o pojavu krvavitev. Pri bolnikih z znaki nezdravljenih možganskih metastaz ali nedavne aktivne krvavitve v prebavilih se zdravila ne sme uporabljati. Če je pri krvavitvi potreben zdravniški poseg, je treba z odmerjanjem aksitiniba začasno prekiniti. Anevrizme in arterijske disekcije: Uporaba zaviralcev poti VEGF pri bolnikih s hipertenzijo ali brez nje lahko spodbudi nastanek anevrizem in/ali disekcij arterij. Pred uvedbo aksitiniba je treba to tveganje skrbno preučiti pri bolnikih z dejavniki tveganja, kot sta hipertenzija ali anamneza anevrizme. Perforacija prebavil in nastanek fi stule: Poročali so o pojavu perforacij prebavil in fi stul. Med zdravljenjem je potrebno redno spremljanje glede morebitnega pojava simptomov perforacije prebavil ali nastanka fi stule. Zapleti pri celjenju ran: Zdravljenje z aksitinibom je treba prekiniti najmanj 24 ur pred načrtovanim kirurškim posegom; odločitev glede ponovne uvedbe zdravljenja po posegu mora temeljiti na klinični presoji ustreznosti celjenja rane. Sindrom posteriorne reverzibilne encefalopatije (PRES): Poročali so o primerih PRES. Pri bolnikih z znaki ali simptomi PRES je treba zdravljenje začasno ali trajno prekiniti. Varnost ponovne uvedbe zdravljenja pri bolnikih, pri katerih je v preteklosti prišlo do PRES, ni znana. Proteinurija: Poročali so o proteinuriji, vključno s proteinurijo 3. in 4. stopnje izraženosti. Pred začetkom zdravljenja in v rednih časovnih presledkih med zdravljenjem je priporočljivo spremljanje glede pojava proteinurije; ob pojavu zmerne do hude proteinurije je treba zmanjšati odmerek ali začasno prekiniti zdravljenje. Zdravljenje je treba trajno prekiniti, če se pri bolniku pojavi nefrotski sindrom. Neželeni učinki na jetra: Zvišanja ravni ALT, AST in bilirubina v krvi. Pred začetkom zdravljenja in v rednih časovnih presledkih med njim je treba spremljati rezultate preiskav delovanja jeter. Zdravilo vsebuje laktozo: Bolniki z redko dedno intoleranco za galaktozo, odsotnostjo encima laktaze ali malabsorpcijo glukoze/galaktoze ne smejo jemati tega zdravila. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Zaviralci CYP3A4/5: Sočasna uporaba z močnimi zaviralci (npr. ketokonazol, itrakonazol, klaritromicin, eritomicin, atazanavir, indinavir, nefazodon, nelfi navir, ritonavir, sakvinavir in telitromicin) ter uživanje grenivk lahko zveča plazemske koncentracije aksitiniba. Priporočljiva je izbira sočasno uporabljanih zdravil, ki ne zavirajo ali minimalno zavirajo CYP3A4/5. Če je treba sočasno uporabljati močan zaviralec CYP3A4/5, je odmerek aksitiniba priporočljivo prilagoditi. Zaviralci CYP1A2 in CYP2C19: Zaradi tveganja, da se plazemske koncentracije aksitiniba povečajo, je potrebna previdnost. Induktorji CYP3A4/5: Sočasna uporaba aksitiniba z močnimi induktorji (npr. rifampicin, deksametazon, fenitoin, karbamazepin, rifabutin, rifapentin, fenobarbital in šentjanževka) lahko zmanjša plazemske koncentracije aksitiniba. Priporočljiva je izbira sočasno uporabljanih zdravil, ki ne inducirajo ali minimalno inducirajo CYP3A4/5. Če je treba sočasno uporabljati močan induktor CYP3A4/5, je odmerek aksitiniba priporočljivo prilagoditi. Plodnost, nosečnost in dojenje: Ne sme se uporabljati med nosečnostjo, razen če klinično stanje ženske zahteva zdravljenje s tem zdravilom. Ženske v rodni dobi morajo uporabljati kontracepcijo med zdravljenjem in še en teden po njem. V obdobju dojenja se ne sme uporabljati. Lahko neugodno vpliva na sposobnost razmnoževanja in plodnost pri ljudeh. Vpliv na sposobnost vožnje in upravljanja strojev: Ima blag vpliv na sposobnost vožnje in upravljanja strojev. Med zdravljenjem se lahko pojavijo učinki, kot je npr. omotica in/ali utrujenost. Neželeni učinki: Najpogostejši (≥ 20 %) neželeni učinki so bili driska, hipertenzija, utrujenost, zmanjšan apetit, navzea, zmanjšana telesna masa, hripavost, sindrom palmarno-plantarne eritrodisestezije (sindrom dlani-podplati), krvavitev, hipotiroidizem, bruhanje, proteinurija, kašelj in zaprtje. Ostali zelo pogosti (≥ 1/10 bolnikov) neželeni učinki so: glavobol, disgevzija, dispneja, bolečine v trebuhu, stomatitis, dispepsija, izpuščaj, suha koža, artralgija, bolečine v okončinah, astenija, vnetje sluznice. Način in režim izdaje: Predpisovanje in izdaja zdravila je le na recept, zdravilo pa se uporablja samo v bolnišnicah. Izjemoma se lahko uporablja pri nadaljevanju zdravljenja na domu ob odpustu iz bolnišnice in nadaljnjem zdravljenju. Imetnik dovoljenja za promet: Pfi zer Europe MA EEIG, Boulevard de la Plaine 17, 1050 Bruxelles, Belgija. Datum zadnje revizije besedila: 29.07.2021 Pred predpisovanjem se seznanite s celotnim povzetkom glavnih značilnosti zdravila. Literatura: 1. Melichar B, Poprach A, Kubackova K, et al. Effi cacy and tolerability of axitinib in metastatic renal cell carcinoma (mRCC): Comparison of Czech clinical registry and AXIS trial data. ECC. 25–29 September 2015. Vienna, Austria. Poster: 2615. 2. Matias M, Le Teuff G, Albiges L, et al. Real world prospective experience of axitinib in metastatic renal cell carcinoma in a large comprehensive cancer centre. Eur J Cancer. 2017;79:185–192. 3. Rossetti S, Romano FJ, D‘Aniello C, et al. Activity of second line axitinib in metastatic renal cell carcinom (mRCC) patients treated with sunitinib: Results from SAX Italian real world trial. J Clin Oncol. 2017;35(15_suppl):e16054. 4. Povzetek glavnih značilnosti zdravila Inlyta, 29.7.2021. Zdravilo Inlyta je indicirano za zdravljenje napredovalega karcinoma ledvičnih celic pri odraslih bolnikih, pri katerih predhodno zdravljenje s sunitinibom ali citokinom ni bilo uspešno.4 Zagotovite svojim bolnikom z metastatskim karcinomom ledvičnih celic v drugi liniji zdravljenja vsakodnevne zmage z zdravilom Inlyta®.1–3 1-3 Radiol Oncol 2021; 56(1): A. March 2022 Vol. 56 No. 1 Pages 1-128 ISSN 1318-2099 UDC 616-006 CODEN: RONCEM Publisher Association of Radiology and Oncology Aims and Scope Radiology and Oncology is a multidisciplinary journal devoted to the publishing original and high quality scientific papers and review articles, pertinent to diagnostic and interventional radiology, computerized tomography, magnetic resonance, ultrasound, nuclear medicine, radiotherapy, clinical and experimental oncology, radiobiology, medical physics and radiation protection. Therefore, the scope of the journal is to cover beside radiology the diagnostic and therapeutic aspects in oncology, which distinguishes it from other journals in the field. Editor-in-Chief Gregor Serša, Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia (Subject Area: Experimental Oncology) Executive Editor Viljem Kovač, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia (Subject Areas: Clinical Oncology, Radiotherapy) Deputy Editors Andrej Cör, University of Primorska, Faculty of Health Science, Izola, Slovenia (Subject Areas: Clinical Oncology, Experimental Oncology) Božidar Casar, Institute of Oncology Ljubljana, Department for Dosimetry and Quality of Radiological Procedures, Ljubljana (Subject Area: Medical Physics) Maja Čemažar, Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia (Subject Area: Experimental Oncology) Igor Kocijančič, University Medical Center Ljubljana, Institute of Radiology, Ljubljana, Slovenia (Subject Areas: Radiology, Nuclear Medicine) Karmen Stanič, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia (Subject Areas: Radiotherapy; Clinical Oncology) Primož Strojan, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia (Subject Areas: Radiotherapy, Clinical Oncology) Editorial Board Subject Areas: Radiology and Nuclear Medicine Sotirios Bisdas, University College London, Department of Neuroradiology, London, UK Boris Brkljačić, University Hospital “Dubrava”, Department of Diagnostic and Interventional Radiology, Zagreb, Croatia Maria Gődény, National Institute of Oncology, Budapest, Hungary Gordana Ivanac, University Hospital Dubrava, Department of Diagnostic and Interventional Radiology, Zagreb, Croatia Luka Ležaić, University Medical Centre Ljubljana, Department for Nuclear Medicine, Ljubljana, Slovenia Katarina Šurlan Popovič, University Medical Center Ljubljana, Clinical Institute of Radiology, Ljubljana, Slovenia Jernej Vidmar, University Medical Center Ljubljana, Clinical Institute of Radiology, Ljubljana, Slovenia Subject Areas: Clinical Oncology and Radiotherapy Serena Bonin, University of Trieste, Department of Medical Sciences, Cattinara Hospital, Surgical Pathology Blg, Molecular Biology Lab, Trieste, Italy Luca Campana, Veneto Institute of Oncology (IOV-IRCCS), Padova, Italy Christian Dittrich, Kaiser Franz Josef - Spital, Vienna, Austria Blaž Grošelj, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana Luka Milas, UT M. D. Anderson Cancer Center, Houston, USA Miha Oražem, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana Gaber Plavc, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana Csaba Polgar, National Institute of Oncology, Budapest, Hungary Dirk Rades, University of Lubeck, Department of Radiation Oncology, Lubeck, Germany Luis Souhami, McGill University, Montreal, Canada Borut Štabuc, University Medical Center Ljubljana, Division of Internal Medicine, Department of Gastroenterology, Ljubljana, Slovenia Andrea Veronesi, Centro di Riferimento Oncologico- Aviano, Division of Medical Oncology, Aviano, Italy Branko Zakotnik, Institute of Oncology Ljubljana, Department of Medical Oncology, Ljubljana, Slovenia Subject Area: Experimental Oncology Metka Filipič, 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 Miklavčič, University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia Ida Ira Skvortsova, EXTRO-lab, Dept. of Therapeutic Radiology and Oncology, Medical University of Innsbruck, Tyrolean Cancer Research Institute, Innsbruck, Austria 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 Nyström, 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 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 Hočevar, Institute of Oncology Ljubljana, Department of Surgical Oncology, Ljubljana, Slovenia Miklós Kásler, National Institute of Oncology, Budapest, Hungary Maja Osmak, Ruder Bošković Institute, Department of Molecular Biology, Zagreb, Croatia Radiol Oncol 2021; 56(1): B. Editorial office Radiology and Oncology Zaloška cesta 2 P. O. Box 2217 SI-1000 Ljubljana Slovenia Phone: +386 1 5879 369 Phone/Fax: +386 1 5879 434 E-mail: gsersa@onko-i.si Copyright © Radiology and Oncology. All rights reserved. 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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 • Microsoft Academic • Naviga (Softweco) • Primo Central (ExLibris) • ProQuest (relevant databases) • Publons • PubMed • PubMed Central • PubsHub • QOAM (Quality Open Access Market) • ReadCube • Reaxys • SCImago (SJR) • SCOPUS • Sherpa/RoMEO • Summon (Serials Solutions/ProQuest) • TDNet • Ulrich's Periodicals Directory/ulrichsweb • WanFang Data • Web of Science - Current Contents/Clinical Medicine • Web of Science - Science Citation Index Expanded • WorldCat (OCLC) This journal is printed on acid- free paper On the web: ISSN 1581-3207 https://content.sciendo.com/raon http://www.radioloncol.com Radiol Oncol 2021; 56(1): C. review Cancer gene therapy goes viral: viral vector platforms come of age Urban Bezeljak Ultrasound-guided carpal tunnel injections Tilen Tumpaj, Vesna Potocnik Tumpaj, Domenico Albano, Ziga Snoj nuclear medicine Lack of association between cortical amyloid deposition and glucose metabolism in early stage Alzheimeŕ s disease patients Daniela Ehrlich, Andreas Dunzinger, Gertraud Malsiner-Walli, Bettina Grün, Raffi Topakian, Marina Hodolic, Elmar Kainz, Robert Pichler radiology Reliability of new radiographic measurement techniques for elbow bony impingement Uros Meglic, Oskar Zupanc Efficacy of transvaginal ultrasound versus magnetic resonance imaging for preoperative assessment of myometrial invasion in patients with endometrioid endometrial cancer: a prospective comparative study Anis Cerovac, Dzenita Ljuca, Lejla Arnautalic, Dubravko Habek, Gordana Bogdanovic, Jasminka Mustedanagic-Mujanovic, Gordana Grgic Bladder paraganglioma: CT and MR imaging characteristics in 16 patients Jing Zhang, Xu Bai, Jing Yuan, Xiaojing Zhang, Wei Xu , Huiyi Ye, Haiyi Wang Diagnostic performance of apparent diffusion coefficient values for the differentiation of intrahepatic cholangiocarcinoma from gastrointestinal adenocarcinoma liver metastases Temel Fatih Yilmaz, Mehmet Ali Gultekin, Hacı Mehmet Turk, Mehmet Besiroglu, Dilek Hacer Cesme, Melih Simsek, Alpay Alkan, Huseyin Toprak Assessment of hyperbaric oxygenation treatment response in parotid glands by T2 mapping following radiotherapy for head and neck tumours Jernej Vidmar, Ksenija Cankar, Maja Groselj, Zarko Finderle, Igor Sersa Comparison of local recurrence in transcatheter arterial chemoembolization of hepatocellular carcinoma with or without accumulation of iodized oil beyond corona enhancement area: short-term results Yukinobu Watanabe, Masahiro Ogawa, Masahiro Kaneko, Mariko Kumagawa, Midori Hirayama, Naoki Matsumoto, Hiroshi Nakagawara, Toshiki Yamamoto, Mitsuhiko Moriyama contents contents Radiol Oncol 2021; 56(1): D. clinical oncology Pre-treatment risk assessment of women with endometrial cancer: differences in outcomes of molecular and clinical classifications in the Slovenian patient cohort Jure Knez, Monika Sobocan, Urska Belak, Rajko Kavalar, Mateja Zupin, Tomaz Büdefeld, Uros Potocnik, Iztok Takac Cystatin C and cystatin SN as possible soluble tumor markers in malignant uveal melanoma Maria A. Dikovskaya, Galina S. Russkikh, Konstantin V. Loktev, Thomas P. Johnston, Margarita M. Gevorgyan, Natalya P. Voronina, Valery V. Chernykh, Alexander N. Trunov, Tatiana A. Korolenko Clinical impacts of copy number variations in B-cell differentiation and cell cycle control genes in pediatric B-cell acute lymphoblastic leukemia: a single centre experience Klementina Crepinsek, Gasper Marinsek, Marko Kavcic, Tomaž Prelog, Lidija Kitanovski, Janez Jazbec, Marusa Debeljak Percutaneous electrochemotherapy in primary and secondary liver malignancies – local tumor control and impact on overall survival Hannah Spallek, Peter Bischoff, Willi Zhou, Francesca de Terlizzi, Fabian Jakob, Attila Kovàcs The learning curve of laparoscopic liver resection utilising a difficulty score Arpad Ivanecz, Irena Plahuta, Matej Mencinger, Iztok Perus, Tomislav Magdalenic, Spela Turk, Stojan Potrc In vitro maturation of immature oocytes for fertility preservation in cancer patients compared to control patients with fertility problems in an in vitro fertilization program Irma Virant-Klun, Jure Bedenk, Nina Jancar slovenian abstracts contents Radiol Oncol 2022; 56(1): 1-13. doi: 10.2478/raon-2022-0002 1 review Cancer gene therapy goes viral: viral vector platforms come of age Urban Bezeljak COBIK, Ajdovščina, Slovenia Radiol Oncol 2022; 56(1): 1-13. Received 25 November 2021 Accepted 4 January 2022 Correspondence to: Urban Bezeljak, Ph.D., COBIK, Mirce 21, 5270 Ajdovščina, Slovenia. E-mail: urban.bezeljak@cobik.si Disclosure: No potential conflicts of interest were disclosed. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Background. Since the advent of viral vector gene therapy in 1990s, cancer treatment with viral vectors promised to revolutionize the field of oncology. Notably, viral vectors offer a unique combination of efficient gene delivery and engagement of the immune system for anti-tumour response. Despite the early potential, viral vector-based cancer treatments are only recently making a big impact, most prominently as gene delivery devices in approved CAR-T cell therapies, cancer vaccines and targeted oncolytic therapeutics. To reach this broad spectrum of applications, a number of challenges have been overcome – from our understanding of cancer biology to vector design, manu- facture and engineering. Here, we take an overview of viral vector usage in cancer therapy and discuss the latest advancements. We also consider production platforms that enable mainstream adoption of viral vectors for cancer gene therapy. Conclusions. Viral vectors offer numerous opportunities in cancer therapy. Recent advances in vector production platforms open new avenues in safe and efficient viral therapeutic strategies, streamlining the transition from lab bench to bedside. As viral vectors come of age, they could become a standard tool in the cancer treatment arsenal. Key words: viral vector; gene therapy; oncolytic virus; immunotherapy; bioprocess platform Introduction Cancer remains a major public health concern worldwide and is the second leading cause of death in Europe and the United States, with one-in- two to one-in-three chance to develop an invasive cancer during individual’s lifetime.1 In Slovenia, cancer caused over 35 % deaths in males in 2016, which is the highest share in European Union.2 As a result of positive lifestyle changes and advances in tumour detection and treatment, we can observe a continuous drop in mortality rates in the last 20 years.3 Despite, current treatments like chemother- apy, surgery and radiation commonly have debili- tating side effects. Consequently, new therapeutic options are becoming available to curb the tremen- dous death toll and increase the quality of life for cancer survivors.4 In this review we will focus on cancer therapeutics in form of viral gene therapy vectors and oncolytic viruses (OVs). Viral vectors are an attractive drug delivery op- tion due to their evolved efficiency to transduce human cells. Compared to other delivery methods, viruses are also easier to use in targeted transfer of genetic cargo. That is why modified viruses are used as reliable and safe gene therapy vectors in cancer and hereditary disease treatment.5 However, early attempts at viral gene therapy came too soon for the budding technology, which lead to contro- versy and poor public image. For example, the ini- tial trials for severe combined immunodeficiency (SCID) saw only limited improvement and adeno- virus vector-associated complications lead to tragic death of Jesse Gelsinger in 1999.6 The tides turned in later years, when viral vectors were successfully used as ex vivo hematopoietic gene delivery de- Radiol Oncol 2022; 56(1): 1-13. Bezeljak U / Cancer gene therapy goes viral2 vices for severe β-thalassemia, SCID and Wiskott– Aldrich syndrome. In 2003, Gendicine was the first approved adenoviral cancer gene therapeutic in China. It took almost another decade to see the first gene therapy approval in Europe, where the ade- no-associated virus (AAV) alipogene tiparvovec (Glybera, uniQure) received authorisation for lipo- protein lipase deficiency (LPLD) treatment in 2012. Since then, many other therapies reached regulato- ry approval for in- or ex vivo gene delivery.7,8 More recently, novel vector-based vaccines are key in battling the coronavirus disease 2019 (COVID-19) pandemic on an unprecedented scale.9,10 This was made possible by the constant development of vi- ral vector production and purification platforms, which had their roots in viral vector gene therapy. As the technology matures, the rapid turnaround of vector design and scalable particle production capacities hold promise to equally revolutionize cancer gene therapy. Indeed, over two thirds of gene therapy clinical trials are focused on cancer treatment, with many drug candidates in late de- velopment stages.11 Oncolytic therapy represents another use of viral vectors. It was sparked by serendipitous ob- servations of transient remissions when cancer patients contracted viral infections.12 This led to experimentation with natural pathogens to help cure tumours, mostly with little success. Although initial attempts of viral oncolytic therapy were ineffective, the genetic engineering revolution enabled development of effective OVs in 1990s.13,14 Thirty years later, three oncolytic therapeutics are approved for use, with many more entering the clinics. Overall, oncolytic and viral gene delivery vectors have great potential to complement estab- lished (immuno)therapy approaches. These ad- vanced nanotherapeutics are armed with a wide variety of genetic elements that take advantage of essential hallmarks of cancer, harnessing the accu- mulated knowledge in cancer biology, immunolo- gy and virology. Examples of therapeutic viral vec- tor platforms, their opportunities and challenges are discussed below. Viral vectors At present, over 1000 clinical trials for cancer ther- apy with viral vectors are underway worldwide (Figure 1).11 The use of virus particles in cancer treatment can be broadly classified in two groups: as gene delivery vehicles and OVs.15,16 For gene de- livery, lentiviral, adenoviral and AAV vector chas- sis are used – depending on the specific application and targeting specificity. OVs encompass many viral families and are often additionally armed to eradicate the tumour and induce anti-cancer im- mune response.4 The main difference between vec- tors in gene- and oncolytic therapy is their replica- tive potential. Gene therapy vectors are specifically engineered to prevent replication. Consequently, they function as nanoparticle drug delivery vehi- cles and cannot actively infect host cells. In con- trast, OVs are less attenuated and can replicate in infected tissues. Each of these vector designs has its own set of advantages and disadvantages, which also depend on the clinical indication. Below, we overview some of the most widely used viral vec- tors for cancer treatment that were either approved for clinical use or introduce exciting new concepts for future therapies. Gene delivery Gene delivery vectors are used to transfer thera- peutic genetic material to target tissues. In cancer therapy this includes tumour suppressor genes, tumour-associated antigens (TAAs), pro-inflam- matory factors, immune checkpoint inhibitors, anti-angiogenic proteins, small interference RNA (siRNA), cancer stroma-degrading enzymes and cytotoxic convertases.17 In addition, vector gene de- livery is used to reprogramme therapeutic cells ex vivo for adoptive cell therapy like chimeric antigen receptor (CAR) T and natural killer (NK) cells.18,19 Here, we present the most well-known viral vec- Retrovirus 43.1 % Adenovirus 27.1 % Poxvirus 18.1 % Herpes simplex virus 7.4 % AAV 1.6 % Other n = 1129 2.7 % FIGURE 1. Use of viral vectors in clinical trials to treat cancer. Overall, retrovirus viral family vectors are the most widespread. These include lenti- and gammaretroviruses, which are used in adoptive cell therapy. Other popular vectors for cancer treatment are adenovirus, poxvirus like vaccinia, herpes simplex virus (HSV) and adeno-associated virus (AAV). Measles virus, vesicular stomatitis virus (VSV) and poliovirus are some of the other vectors that are not explicitly depicted. Data on all open cancer trials are from Wiley Journal of Gene Medicine Gene Therapy Clinical Trials Worldwide database (retrieved October 2021).11 Radiol Oncol 2022; 56(1): 1-13. Bezeljak U / Cancer gene therapy goes viral 3 tor platforms: adenovirus, which is used in in vivo gene therapy and retroviruses that are used for ex vivo gene delivery. In addition, we discuss AAVs, which are currently the most exciting vector plat- form and will likely set trends in cancer gene ther- apy in the future. Adenovirus vectors Native adenoviruses are 90 nm icosahedral parti- cles (Figure 2A) that commonly cause respiratory, gastrointestinal, urinary and keratoconjunctivi- tis infections in humans. Their ubiquity results in high proportion of life-long immunity in human population towards the most common serotypes.20 Adenoviruses have 36 kilobase pair (kbp) linear double-stranded DNA (dsDNA) genome consist- ing of over 30 genes that are flanked by inverted terminal repeats (ITRs) and a capsid-packing sig- nal sequence ψ. The adenoviral genes are divided into early (E) and late (L) genes, depending on their expression pattern. Early expressed regula- tory proteins interact with the host cell and initiate viral genome replication. On the other hand, late genes encode structural proteins that form the vi- rion.21 Adenoviral vectors were developed by de- leting key regulatory genes, which depend on the desired transgene size and application (Figure 2B). Replication-competent adenovirus vectors are used in oncolytic cancer therapy, while non-replicative deletion mutants are gene delivery vehicles. In the first generation of adenovirus vectors, the essential early E1A and E1B genes are replaced by constitu- tive expression cassette with transgene for gene de- livery. Additionally, E2, E3 and E4 genes can also be deleted to accommodate larger therapeutic in- serts of 10 kbp and improve performance. For viral vector assembly, the modified adenovirus genome is expressed from plasmid DNA in human embry- onic kidney (HEK) 293 cell line that complements for deleted E1, E2 and E4 genes. Lastly, as much as 36 kbp inserts can be accommodated in helper- dependent adenovirus vectors that retain only ITR and genome packaging sequence ψ, rest is filled with one or several transgene expression cassettes. All adenoviral proteins needed for vector replica- tion, packaging and assembly are provided by the replication-competent helper virus, which has its packaging signal flanked by loxP recombination sites. The helper-dependent vector production takes place in cell lines expressing Cre recombinase that specifically excises the loxP-flanked helper ψ sequence. This ensures only the transgene vector retains the ψ packaging signal and is incorporated in the budding viral particles. Remaining helper virus contaminants are eliminated in the following chromatography purification process. Adenoviral vectors have broad tropism and do not integrate Adenovirus 90 nm fibre capsid proteins ITR E1A E1B E2 E3 E4 ITR dsDNA Wild type genome Adenovirus vector <6.5 kbp transgene cassette Helper-dependent adenovirus vector <36 kbp transgene cassette pDNA pDNA + E1A and E1B are complemented by the HEK 293 producer cell + deleted genes are complemented by the helper virusA B FIGURE 2. Overview of adenovirus vector design. (A) Schematic representation of adenovirus structure. Adenoviruses are non- enveloped 90 nm particles with pointing fibre rods. (B) Outline of wild type adenovirus genome, the first-generation adenovirus vector plasmid and helper-dependent adenoviral vector plasmid with the transgene expression cassette. The wild type genome highlights key early genes, while other genetic elements are omitted for clarity. The first-generation adenovirus vector particles are assembled in HEK 293 cell line by transgene vector plasmid transfection. Additionally, the helper-dependent vector assembly also requires infection with a helper virus. ITR = inverted terminal repeat; dsDNA = double-stranded DNA; pDNA = plasmid DNA. Radiol Oncol 2022; 56(1): 1-13. Bezeljak U / Cancer gene therapy goes viral4 into target cell genome. Instead, the delivered ge- netic material remains episomal.15,22 In gene delivery, acquired and innate immunity towards adenovirus vectors is hindering their ap- plication. For example, the most widely used Ad5 serotype has 50% seroprevalence in North America and over 90% in Côte d’Ivoire.15 Additionally, the adenoviral capsid and nucleic acid stimulates components of the complement system and Toll- like receptors (TLR). This raises safety concerns and efficacy issues for systemic adenoviral gene delivery in vivo. However, the intrinsic vector im- munogenicity can also be harnessed in local cancer therapy by engaging the immune system and pro- moting anti-tumour responses.20 In 2003, the adenovirus-based Gendicine became the first registered cancer gene therapy treatment. Gendicine is an E1- and E3-deletion Ad5 viral vec- tor, which encodes tumour suppressor p53 under Rous sarcoma virus (RSV) promoter regulation. The loss of p53 protective function is associated with at least half of cancers.23 Once the Ad5 vec- tor delivers p53 transgene, it resumes anti-tumour function by promoting cancer cell apoptosis and stimulating the immune response. It received ap- proval in China for advanced head and neck cancer treatment.23,24 Adenovirus vectors are also used to deliver can- cer suicide genes that convert prodrugs to cytotoxic compounds. Examples include 5-fluorouridine (5-FU)-producing cytosine deaminase, purine nu- cleoside phosphorylase (PNP) that converts fludara- bine phosphate (F-ara-AMP) to toxic 2-fluoro- adenine, and ganciclovir-converting thymidine kinase (TK).15 Sitimagene ceradenovec (Cerepro, Ark Therapeutics) is a first-generation Ad5 vector that expresses convertase from herpes virus HSV- TK. In 2005 it entered phase 3 trial for treatment of glioblastoma. In the trial, 1·1012 Ad5 vector particles were applied locally into the resected tumour and ganciclovir was administered intravenously. The study found no effect on survival, while the viral vector treatment improved time to re-intervention or death after resection – the primary trial end- points.25 Despite this, the Cerepro marketing appli- cation in Europe was withdrawn in 2010.26 Similarly, immunostimulatory adenovirus can- cer gene therapy was used to promote interferon alpha and beta (IFNα, -β), interleukin 2 (IL-2) and Fms-like tyrosine kinase 3 ligand (Flt3L) ex- pression.20 A phase 3 trial for bacille Calmette- Guérin (BCG)-unresponsive bladder cancer with nadofaragene firadenovec, a replication defi- cient vector expressing IFNα, recently reported favourable results.27 The non-muscle-invasive and BCG-unresponsive bladder cancer currently does not have efficient non-surgical treatments, which are often the only option for many patients. Adenoviral gene therapy is a promising alterna- tive, since local administration led to 60- and 30% complete response rate after 3 and 12 months, re- spectively.28 Lastly, the engineered chimpanzee ChAdOx1 vector vaccine platform – also used by the AZD1222 COVID-19 vaccine (Vaxzevria, Oxford-AstraZeneca) – is aimed at prostate cancer treatment in combination with checkpoint inhibi- tors.10,29,30 The cancer vaccine treatment consists of ChAdOx1 immunization against 5T4 tumour anti- gen and a Modified Vaccinia Ankara (MVA) vector boost. The phase 1 trial confirmed vaccine safety and immunogenicity, while phase 1/2 efficacy trial was expected to complete in 2021.31 Overall, ade- noviral gene delivery remains a promising venue for cancer therapy, either alone or in combination with radiotherapy, chemotherapy or checkpoint inhibitors.32 Also, the ease of industrial scale-up and established Good Manufacturing Practice (GMP) processes will further promote adenoviral platform for in vivo patient gene delivery.33 Adeno-associated virus vectors Adeno-associated viruses (AAVs) hold great prom- ise in the gene therapy field. AAVs do not cause any human disease, are non-replicative and have broad tissue tropism. AAVs are 25 nm icosahedral viruses (Figure 3A) with single-stranded DNA (ssDNA) ge- nome, which naturally lacks many key regulatory genes for replication and expression. The missing genes are instead complemented with adenoviral co-infection of the host cell. Alternatively, herpes simplex and baculovirus can also provide the help- er function. For gene delivery, the AAV genome is “gutted”—devoid of all viral genes – and replaced with transgene expression cassette (Figure 3B). The major AAV vector downside is its relatively low ca- pacity for transgene inserts – it can accommodate 4.7 kbp of genetic cargo, which can be limiting for many applications.15,34 The therapeutic AAV parti- cles are commonly produced from three plasmid constructs in transfected HEK 293 cells, which al- ready encode the adenoviral E1 helper gene. The vector plasmids contain the ITR-flanked transgene, AAV rep and cap genes and the adenoviral E2, E4 and VA genes, respectively. A more scalable so- lution is possible with Sf9 insect cells, which are co-infected with ITR-transgene and AAV cap/rep baculoviruses, respectively.35 Radiol Oncol 2022; 56(1): 1-13. Bezeljak U / Cancer gene therapy goes viral 5 Different AAV serotypes display distinct tro- pism, but they generally require AAV receptor (AAVR) expression, heparin sulphate peptidogly- cans, sialic acid or galactose with several co-re- ceptors for cell transduction.36 Once the vector en- ters the cell, it travels to nucleus and uncoats the transgene DNA, which persists as concatemerized episomal circle for many years.35 Currently, AAV vectors are the most successful in treatment of mo- nogenic diseases like spinal muscle atrophy (SMA) with onasemnogene abeparvovec (Zolgensma, Novartis).37 In contrast, AAV-based cancer thera- pies are still in early development. However, the modular vector design enables new promising ap- proaches to targeted gene delivery.38 For instance, AAVs that cross the blood-brain barrier and are specific for central nervous system could be used for treatment of invasive glioblasto- ma.39 To improve cancer specificity, wild-type AAV capsids can also be engineered to target cell surface tumour antigens.40,41 For example, AAV2 was modi- fied to bind HER2 receptor by inserting designed ankyrin repeat proteins (DARPins) into the AAV capsid.42 The researches later used these Her2-AAVs to specifically deliver checkpoint inhibitors against programmed cell death protein-1 (PD-1) and HSV- TK suicide gene in a mice xenograft model.43,44 A sin- gle systemic injection of Her2-AAV vector, armed with HSV-TK, lead to considerable tumour mass reduction in combination with ganciclovir.44 On the other hand, PD-1 inhibition lead to only marginal tumour clearance in combination with chemothera- py.43 In an ex vivo application, an AAV6 vector was used to prepare allogenic CAR-T cells by replacing the endogenous T cell receptor (TCR) with CD19 CAR through targeted cleavage and homologous repair.45,46 In the future, AAVs could also be used for CAR-T cell generation in vivo, replacing the challenging retroviral T cell modification. This con- cept of “AAV delivering CAR gene therapy” (ACG) was proved on a T-cell leukaemia animal model, where murine immune cells were reprogrammed to express CD4 CAR.47 Finally, therapeutic AAVs are developed to include CRISPR/Cas gene editing components. This combination of powerful biotech- nology platforms promises highly efficient tumour delivery and precise oncogene knock-out or silenc- ing.48,49 To this end, a sub-4.7 kbp CRISPR/Cas13a that distinguishes between wild type and oncogenic KRAS G12D was constructed and tested in cell cul- ture. A similar AAV vector with oncogene-specific Cas13a could someday induce tumour eradication through mRNA silencing.50 Overall, AAV particles are less immunogenic compared to other vector types, although majority of adults have pre-existing neutralizing antibodies that can affect AAV-based gene therapy efficency.51 Also, AAVs are regarded as very safe due to their non-toxic nature and expected lack of genome in- tegration. However, a recent long-term study of AAV-treated dogs with haemophilia raised con- cerns as numerous vector integration events were surprisingly discovered in vicinity of cancer-asso- ciated genes.52 Nonetheless, the superior versatility makes AAVs currently the up-and-coming method for gene delivery in vivo.53 AAV capsid proteins ITR rep cap ITR ssDNA Wild type genome AAV vector pDNA + deleted rep/cap and viral regulatory genes are complemented by the helper plasmid (E2, E4 and VA), a rep/cap-expressing plasmid and HEK 293 producer cell 25 nm <4.7 kbp transgene cassette FIGURE 3. Overview of AAV vector design. (A) Schematic representation of AAV structure. AAV virions are non-enveloped 25 nm icosahedral particles. (B) Outline of wild type AAV genome and AAV vector plasmid with the transgene expression cassette. AAV vector particles are assembled in adenoviral E1-expressing HEK 293 cell line, which is co-transfected with transgene AAV vector plasmid, a helper plasmid and a rep/cap plasmid. Alternatively, AAV vectors can be produced in insect cells, which are co- infected with ITR-flanked transgene and rep/cap recombinant baculoviruses. ITR = inverted terminal repeat; pDNA = plasmid DNA; ssDNA = single-stranded DNA A B Radiol Oncol 2022; 56(1): 1-13. Bezeljak U / Cancer gene therapy goes viral6 Lenti- and gammaretrovirus vectors While adenovirus and AAV vectors are predomi- nantly used to deliver gene drugs in vivo, retrovirus vectors like lenti- and gammaretroviruses are the most common choice for transformation of isolated patient cells ex vivo. Another distinction is their structure: retroviruses are enveloped 100 nm par- ticles (Figure 4A), while adenoviruses and AAVs have smaller and more rigid proteinaceous shells. Lentivirus family include human- (HIV-1, HIV-2), simian- (SIV) and feline immunodeficiency viruses (FIV). In fact, the widely used lentiviral vectors are derived from HIV-1 that have been modified not to cause disease and to express vesicular stoma- titis virus G glycoprotein (VSV-G) instead of the native envelope (env) protein. Pseudotyping the lentivirus particles with VSV-G increased the vec- tor productivity, stability and infectivity, as well as broadened its tropism for different cell types and tissues.54–56 To guarantee vector safety, the majority of HIV-1 RNA genome is deleted. Only three key structural and regulatory genes remain: gag, pol and rev. Deletion of viral accessory proteins ren- ders the lentiviral vector harmless. What is more, gag/pol, rev, VSV-G and the transgene (< 9 kbp) are divided on separate plasmids in the producing cell lines, preventing assembly of replication-com- petent virus through recombination (Figure 4B).57 This way only the transgene is included in the len- tivirus particles, while other genetic elements re- main behind. The produced lentivirus vectors can- not replicate and can transfer the therapeutic gene with high efficiency.58 The delivered transgene RNA sequence is flanked by modified long ter- minal repeats (LTR), promoter, packaging and re- verse-transcription elements. This expression cas- sette is integrated into genome of transduced cells, ensuring long-term expression in dividing and non-dividing cells. Alternatively, non-integrating lentiviral vectors (NILVs) were developed that per- sist as episomal DNA. NILVs circumvent the safety concerns regarding oncogenic potential of integra- tion mutagenesis and offer prolonged transgene expression.59,60 In cancer therapy, lentiviral vectors are most used for ex vivo modification of T and NK cells. Particularly, CAR-T cells are successful in treating relapsed and refractory non-Hodgkin lymphoma and acute lymphoblastic leukaemia (ALL), result- ing in the first FDA-approved therapy tisagenle- cleucel (Kymriah, Novartis) in 2017.61,62 There, the patient T cells are harvested by leukapheresis, acti- vated and transduced with lentivirus vector, which encodes CD19 CAR. The lentivirus is produced under biosafety level 2 (BSL-2) GMP conditions in transfected HEK 293T cells, purified and sterile fil- tered before T cell transduction.63,64 A dendritic cell (DC)-specific lentiviral cancer vaccine LV305 was also used to promote expression and immune pres- entation of New York Esophageal Squamous Cell Carcinoma-1 (NY-ESO-1) cancer antigen. The non- integrating vector is pseudotyped with Sindbis virus envelope glycoprotein that binds CD209 re- ceptor on DC. In the first-in-human phase 1 study, LV305 vaccination induced CD4+ and CD8+ T cell Lentivirus 100 nm membrane envelope glycoprotein LTR gag pol LTR ssRNA Wild type genome Lentivirus vector <9 kb transgene cassette pDNA env rev + gag/pol, rev and VSV-G instead of env are co-transfected on separate plasmids FIGURE 4. Overview of lentivirus vector design. (A) Schematic representation of lentivirus structure. Lentiviruses are 100 nm enveloped particles with exposed glycoprotein that defines the virus and vector tropism.55 (B) Outline of wild type lentivirus genome and lentivirus vector plasmid with the transgene expression cassette. Only key genetic elements are highlighted in the genome structure, rest are omitted for clarity. Lentivirus particles are assembled in mammalian cell culture by co-transfection of four plasmids: the transgene plasmid, gag/pol and rev packaging plasmids and VSV-G expression plasmid. pDNA = plasmid DNA; ssRNA = single-stranded RNA; LTR= long terminal repeat A B Radiol Oncol 2022; 56(1): 1-13. Bezeljak U / Cancer gene therapy goes viral 7 responses against NY-ESO-1-expressing tumours. Based on these results, further LV305 combination therapies are planned.65 Gammaretrovirus vectors, which are derived from murine leukaemia virus (MLV), are another type of retroviral vectors. In contrast to lentivi- rus, gammaretrovirus vector infects only actively dividing cells and is prone to integrate into gene regulatory regions, raising concerns for insertional oncogenesis.56 Nevertheless, axicabtagen ciloleucel (Yescarta, Gilead) is a CD19 CAR-T cell therapy for diffuse large B-cell lymphoma, which utilizes gammaretroviral vector for chimeric receptor gene delivery.66 Another gammaretroviral therapeutic is vocimagene amiretrorepvec (Toca 511), which en- codes yeast cytosine deaminase that converts prod- rug 5-fluorocytosine to toxic 5-FU in glioma cells.67 Despite promising results from mouse brain tu- mour models, a phase 2/3 did not show improved patient survival compared to standard-of-care after Toca 511 injection.68 Lenti- and gammaretro- viral vectors will remain the method of choice for ex vivo stable cell transduction. Their use in cancer therapy will focus on next-generation CAR-T and -NK cells with improved potency and solid tumour treatment.69 Oncolytic viruses In contrast to viral vectors for gene therapy, engi- neered OVs are replication-competent and more closely resemble their natural counterparts. In fact, tumour regressions after natural viral infections have been reported since the end of 19th century.13 The OV therapy takes advantage of rapidly divid- ing cancer cells, which often lack antiviral defence mechanisms present in normal cells. For example, misregulation of interferon, Wnt, Ras/MAPK, p53 and pRb signalling pathways leaves cancer vulner- able for viral infection.70,71 Consequently, OVs pref- erentially replicate in cancerous cells, resulting in lysis, tumour eradication and immune system en- gagement. Besides the viral tumour debulking, the immunostimulatory effect is especially important in “cold” tumours with few infiltrating lympho- cytes, inhibitory tumour microenvironment (TME) and impaired antigen presentation. There, viral antigens, danger-associated molecular patterns (DAMPs), TAAs and neoantigens are released to TME during infected tumour cell lysis. Released factors are then presented to the innate and adap- tive immune system, acting as self-adjuvating in si- tu cancer vaccine. This leads to localized inflamma- tion, recruiting immune cells into TME and mount- ing response towards distant metastatic lesions.16,72 The multifaceted oncolytic virotherapy is potenti- ated with immune checkpoint inhibitors or CAR-T cells, which is reflected in multiple combination therapy approaches.73–75 Similar to gene delivery vectors, OVs are often armed with transgenes that additionally modulate the TME or the immune system, including matrix-degrading enzymes, cy- tokines, checkpoint inhibitors, therapeutic anti-/ nanobodies and bispecific T cell engagers (BiTEs).17 The OV therapy can even be custom-made for each individual patient. Personalized therapeutic vec- tors can be designed by inserting patient-specific antigens directly into viral envelope or by encod- ing the neoantigen sequences for gene delivery.76,77 A broad range of oncolytic vectors are used for can- cer therapy, representing different viral families. Below we mention some examples, which have been extensively tested and reached late stages of clinical trials. Replication-competent adenovirus vectors were the first oncolytic viruses to reach clinical trials in 1998 with ONYX-015.78 This adenoviral vector harbours E1B-55K deletion, which attenuates vi- ral replication in normal cells. While ONYX-015 presented a remarkable safety profile, it conferred only limited responses in combination with chem- otherapy for many different cancers.22 In 2005, a similar vector H101 (Oncorine) became the first approved oncolytic virotherapy for late-stage na- sopharyngeal carcinoma treatment in China. The objective response rate of Oncorine in combination with chemotherapy was 76% versus 59% for chem- otherapy alone in phase 3 trial.79 Altogether, hun- dreds of patients received Oncorine, which was well tolerated and without adverse side effects.22 It took 10 years for another OV to be approved by the FDA and EMA in 2015. T-VEC or talimo- gene laherparepvec (Imlgyc, Amgen) is an onco- lytic herpesvirus that expresses immunomodula- tory granulocyte macrophage colony-stimulating factor (GM-CSF) for advanced melanoma treat- ment.80 Non-modified herpes simplex virus type 1 (HSV-1) is a neurotropic human pathogen with 152 kbp dsDNA genome and about 90 genes. For cancer therapy with T-VEC vector, HSV-1 from a clinical isolate was modified with infected cell protein 34.5 (ICP34.5) deletions, preventing virus replication in neurons and other slowly replicat- ing cells. Conversely, this deletion increases HSV-1 replication specificity for tumour cells, while an- other deletion in ICP47 increases viral and tumour antigen presentation. Lastly, T-VEC is armed with two copies of GM-CSF to further promote activa- Radiol Oncol 2022; 56(1): 1-13. Bezeljak U / Cancer gene therapy goes viral8 tion of local antigen-presenting cells.72,81 The vi- rotherapy proved effective in phase 3 trial where 16% of patients showed durable response com- pared to 2% for GM-CSF treatment. The overall response rate was 26% for T-VEC and 6% for GM- CSF alone. Furthermore, 64% of injected lesions more than halved in size, together with 34% and 15% of distal uninjected regional and visceral le- sions, respectively.82 A similar antitumour T-VEC activity was recently reported for primary cutane- ous B cell lymphoma. Interestingly, this phase 1 study also showed therapeutic virus replication in non-malignant cells and determined that induced immunological responses are more important in cancer eradication than selective viral cell lysis.83,84 Currently, combination therapy studies with T-VEC are underway for different cancers.16,85,86 A similar set of HSV-1 attenuations is also present in G47∆ or teserpaturev (Delytact, Daiichi Sankyo), which is derived from a different parental strain. In 2021, it received conditional authorization for malignant glioma therapy in Japan.87 In contrast to T-VEC, which is armed with GM-CSF, Delytact does not encode any transgenes.88 The GM-CSF cytokine sequence is also loaded in oncolytic vaccinia virus pexastimogene devacire- pvec (JX-594, Pexa-Vec) to promote in situ vaccine activity. Pexa-Vec is a TK-deleted poxvirus that demonstrated specificity for tumour cells, which often have increased TK levels that compensate the OV attenuation. In contrast to T-VEC, Pexa-Vec is administered systemically by intravenous injec- tion for hepatocellular carcinoma (HCC), renal cell cancer and colorectal cancer treatment.74,89 In early HCC trials, Pexa-Vec replication was detected in cancer and tumour-associated endothelial cells, triggering specific immune response and destruc- tion of tumour blood vessels.90,91 However, a phase 3 HCC trial with Pexa-Vec and a protein kinase inhibitor sorafenib was prematurely stopped due to lack of interim efficacy.92 A similar fate faced Prostvac-VF, a vaccinia and fowlpox prime-boost vector combination that delivers prostate cancer- specific antigen PSA and three additional immu- nostimulatory factors. The dual vector combina- tion prevents antibody neutralization of the repli- cating viral particles, which resulted in increased survival for prostate cancer patients in phase 2 study. In contrast, a multicentre phase 3 trial did not confirm these findings, ending the study pre- maturely. Prostvac-VF combination therapies are still evaluated in phase 1 and 2 clinical trials.93–95 The complexity of viral vector genomes still pos- es a potential risk of recombination and acquired pathogenicity during vector production and ther- apy. Indeed, with an increasing number of effec- tive OVs reaching late clinical trials and regulatory approvals, a particular care is given to biosafety monitoring and interaction of replication-compe- tent vectors with the host and environment. Based on gathered experience, engineered oncolytic viral vectors remain a safe and promising venue for can- cer treatment. The most common reported side ef- fects are mild flu-like symptoms, while there was no documented uncontrolled transmission of the oncolytic virus.14 It is becoming clear that OVs offer unique benefits in tumour immunotherapy, par- ticularly in combination with advanced cell thera- pies, chemotherapy and checkpoint inhibitors.96 Production platforms Reliable production platforms are key to success- ful translation of viral vector therapies into clinics. Indeed, the development of robust manufacturing capabilities enabled the widespread adoption of recombinant biotherapeutics like monoclonal an- tibodies. Compared to proteins, viral vectors are orders of magnitude more complex, where minute changes between serotypes can affect production and purification strategies. What is more, conven- tional protein purification methods are often not appropriate for shear-sensitive viral particle isola- tion. Enveloped vectors are easily ruptured during the purification process, which decreases the ratio of functional infectious particles in the final prod- uct. Consequently, it is no surprise that viral vec- tor production platforms are continuously being optimized and are yet to reach their full potential on the industrial scale.6,97 Moreover, viral vectors for gene delivery and oncolysis span several viral families and are further genetically engineered to ensure safety and anti-tumoral potencies, adding to the diversity. This offers exciting opportunities for tailored therapies, but also raises challenges in manufacturing and regulation. Thus, paths to viral vector platform success and adoption are specific for each therapeutic. In order to support consistent production and reliable scale-up to clinical-grade drug manufac- ture, an extra care has to be taken in initial selection of viral vector production platforms. Established mammalian cell cultures are most widely used for vector propagation. This makes sense since therapeutic viral vectors originate from natural viruses that co-evolved with vertebrate hosts. The producer cell lines include African green monkey Radiol Oncol 2022; 56(1): 1-13. Bezeljak U / Cancer gene therapy goes viral 9 Vero cells, human HEK 293 and HeLa cells and baby hamster kidney (BHK) line.98 They are han- dled under BSL-2 regulations to limit vector dis- semination and contamination with adventitious agents.99 Mammalian cell lines are primarily ad- herent – growing attached to a solid support. For production, adherent cells are grown on microcar- riers in bioreactors, on multilayer tissue plates and roller bottles. However, suspension cell cultures are preferred for easier scale-up. Luckily, many ad- herent cell lines were successfully adapted for sus- pension growth in stirred-tank and rocking Wave bioreactors.97,98,100 For example, the adenovirus- transformed HEK 293 cells are easily adapted for suspension culture and are the most widely used cell line for adenoviral and AAV vector production. Vector components and helper constructs are delivered to the selected producer cells with tran- sient transfection or infection, which represent a bottleneck and a significant expense in viral vec- tor production for clinical trials.101 In principle, stable cell lines overcome transfection issues in large scale production. However, assembled vec- tor components are often toxic for the host cell.5 This is why stable lentiviral production lines rely on inducible vector expression using TetON/OFF system.102 Alternatively, the cytotoxic VSV-G enve- lope protein and HIV-1 protease can be swapped with Ampho MLV 4070A glycoprotein and T26S modified protease to generate stable lentiviral vec- tor producers LentiPro26 from HEK 293T cells.103 Besides mammalian cell cultures, insect cells are also increasingly used for vector assembly. Most commonly, Sf9 or HighFiveTM insect cell lines are grown in suspension where therapeutic vector backbones are introduced through infection with recombinant baculoviruses. Insect cell expres- sion system is fast to implement, scalable and has superior safety profile since insects are poor hosts for human pathogens. So far, insect cells are used to produce helper-free AAV vectors for gene therapy.104,105 Like with mammalian viral vector systems, insect cells are grown in single-use cell culture flasks and bioreactors to ensure reproduc- ibility and sterility during manufacture for clinical use.101,106 Viral vector purification platforms encompass purification steps to generate highly pure vector particles that comply with stringent quality, safety and efficacy standards. Historically, purification relied on ultracentrifugation to separate the large viral particles from smaller producer cell contami- nants. Nonetheless, this approach is not scalable and does not guarantee elimination of biophysi- cally similar particles.107 Instead, chromatography- based purification processes are taking the centre stage.108 Due to diversity of viral vectors – ranging from small 25 nm AAV particles to large enveloped vaccinia vector, which exceeds 300 nm in size – a universal purification process does not exist and has to optimized for each application. Generally, the purification process starts with vector particle harvesting, where secreted vectors like lentivirus- es, herpes and poxviruses are separated from cel- lular debris with centrifugation or filtration, while intracellular adenoviral and AAV particles often require cell lysis for release. Then, the collected harvest is extensively purified over a series of dif- ferent chromatography columns and tangential- flow filtration (TFF) cassettes to obtain pure ther- apeutic vector particles. In the end, the purified cancer drug is exchanged to the final formulation solution and sterile filtered through 0.2 μm pores, which can be problematic for some larger vectors. Instead, the entire production can be operated un- der controlled sterile conditions.97,108 The final dose of vector particles varies from 109 for ex vivo gene delivery up to 1014/kg for AAV-based gene thera- py.108,109 However, all purification steps are asso- ciated with loss of infectious particles, which in- creases the cost of manufacture. The complexity of viral vector production and low yields result in ex- ceedingly high price of advanced therapeutics. For example, CAR-T therapies Kymriah and Yescarta cost $373,000, while Zolgensma gene therapy was marketed at $2.125 million at launch in 2019.15,110 The constant improvements in production technol- ogy will make the viral vector therapeutics more accessible to the patients.111 With many viral vector therapeutics reaching final clinical stage and regulatory approvals, the attention is focused on vector particle production platforms that support scalable industrial scale production. This is essential to bring down the cost of therapies, which is often prohibitive. New bio- technological solutions like gene editing, bioreac- tor cultivation and multimodal chromatography are boosting cell-based productivity and improv- ing particle purity. Novel analytical methods are also improving the quality monitoring of the final product. In gene therapy, great emphasis is given to ensuring a high ratio of functional infectious vector particles versus defective and empty cap- sid contaminants.112 These exciting developments are enabling the viral vector platforms to produce safe and potent drugs to combat cancer – as mono- therapies or in combination with other therapeutic venues. Radiol Oncol 2022; 56(1): 1-13. Bezeljak U / Cancer gene therapy goes viral10 Conclusions Viral vectors represent 100% of approved gene therapeutics and over 60% of delivery devices in gene therapy trials, including oncolytic viro- therapy.11 This accumulation of knowledge helps us identify where vector particles can provide the most benefits. Based on recent success and rapid advances in the field, the clinical viral vector use will continue to increase. Particularly, combina- tion therapy with complementary radiotherapy, chemotherapy and immunotherapies like CAR-T and checkpoint inhibitors hold great promise. Due to modular vector genome design, novel biothera- peutics like BiTEs, cytokines and CRISPR/Cas can be encoded in the genetic cargo to expand the rep- ertoire of anti-tumour potency.113 In addition to new vector development, approved viral drugs like T-VEC are being tested to treat several other solid tumours beyond melanoma. Finally, oncolytic vi- ruses and non-replicative vectors can be used in prime-boost cancer vaccine regimens, covering the full spectrum of the discussed vector platforms.114 With obvious benefits to the viral vector onco- therapy, these engineered nanotherapeutics will continue to expand the cancer treatment repertoire. It seems like viral vector platforms are finally living up the high expectations thanks to the advances in biopharmaceutical manufacturing and our under- standing of cancer and viral biology. In the future, many more vector particles will enter the clinics. 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Mol Ther 2009; 17: 1814-21. doi: 10.1038/mt.2009.154 Radiol Oncol 2022; 56(1): 14-22. doi: 10.2478/raon-2022-0004 14 review Ultrasound-guided carpal tunnel injections Tilen Tumpaj1, Vesna Potocnik Tumpaj2, Domenico Albano3, Ziga Snoj2,4 1 Department of Anaesthesiology and Intensive Care Medicine, University Medical Centre Ljubljana, Slovenia 2 Institute of Radiology, University Medical Centre Ljubljana, Slovenia 3 Istituto Ortopedico Galeazzi, IRCCS Milano “Galeazzi”, Unit of Diagnostic and Interventional Radiology Milan, Italy 4 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2022; 56(1): 14-22. Received 10 Avgust 2021 Accepted 15 December 2021 Correspondence to: Tilen Tumpaj, M.D., Department of Anaesthesiology and Intensive Care Medicine, University Medical Centre Ljubljana, Zaloška 7, SI-1000 Ljubljana, Slovenia. E-mail: tilen.tumpaj@gmail.com Disclosure: No potential conflicts of interest were disclosed. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Background. Carpal tunnel syndrome (CTS), one of the most common entrapment neuropathies, can, in fact, be considered as a socio-economic issue that reduces work productivity, increases disability, and requires prolonged rehabilitation. The imaging modality of choice in CTS imaging is the ultrasound (US), as several morphological pa- rameters can be used in CTS diagnosis and follow-up. In recent years, US-guided CTS injection therapy has become an established treatment option for mild to moderate CTS. The authors of this review performed a literature search that revealed several differences in US-guided carpal tunnel injection in an attempt to unify individual stages of CTS injections protocol for future guidance: patient preparation, injection approach, needle positioning, injected medica- tions, and injectate volume. The three approaches to carpal tunnel injections described in the literature, that is, the ulnar, radial, and longitudinal, can be implemented with single or multiple deposits and different injection volumes. Medications used for injections are corticosteroids, local anaesthetics, dextrose, saline, platelet-rich plasma, and progesterone. Conclusions. Although no consensus has yet been reached as to which protocol should be used, the ulnar ap- proach with a single deposit injected in large volumes should be considered as the first choice, while dextrose should be the first-line medication option. Furthermore, as terminological differences make it difficult to draw a uniform comparison the presented steps for US-guided carpal tunnel injection might serve as a guideline for future studies. Key words: carpal tunnel syndrome; ultrasound-guided injections; injection approach; needle positioning; corticoster- oid; local anaesthetics Introduction Carpal tunnel syndrome (CTS), one of the most common entrapment neuropathies, can, in fact, be considered as a socio-economic issue that re- duces work productivity, increases disability, and requires prolonged rehabilitation.1 The diagnos- tic workup of CTS must include a comprehensive evaluation including clinical findings, nerve con- duction studies, and ultrasound imaging (US)2,3, which represents the modality of choice due to the superficial course of the median nerve (MN).3,4 Nerve US examination is performed with high- frequency probes that provide a detailed depic- tion of nerve echotexture and fascicles.5,6 US-based morphological parameters for detecting CTS are an increased cross-section area (CSA) of the MN at the carpal tunnel inlet or outlet, flattening of the MN, and bowing of the transverse carpal liga- ment (TCL) at the level of the carpal tunnel outlet.7-9 Elastography has been proven as a useful adjunct US method in CTS evaluation.10 Therapeutic recommendations for CTS depend on disease severity and may include anything from a conservative approach to surgical intervention.2,3 US-guided injections have become increasingly Radiol Oncol 2022; 56(1): 14-22. Tumpaj T et al. / Ultrasound-guided carpal tunnel injections 15 important in the treatment of mild and moderate CTS2,3 and, as landmark-guided injections have been proven to be less effective and cause more complications, US imaging has become the founda- tion of therapeutic recommendations.2,3 US-guided carpal tunnel injections are the most effective mini- mally invasive treatment method with minimum side effects that can remarkably improve the symp- toms and functional status.11 The authors of this review performed a literature search that revealed several differences in US-guided carpal tunnel in- jections and summarized the differences in an at- tempt to unify individual stages of CTS injections protocol for future guidance. Carpal tunnel injections Literature search For our narrative review paper, we conducted an Ovid MEDLINE and PubMed search in which we included the papers published from 2002, when the first paper on US-guided injection was published12, to 2021 with the keywords »carpal tunnel injection« and »ultrasound-guided carpal tunnel injection«. The search results included case-control studies, systematic reviews, and meta-analyses that con- tained data on US-guided carpal tunnel injections. References in these papers were carefully reviewed and were included in our review if they met our criteria (Figure 1). After we reviewed the papers, we used the differences between them to define the steps of US-guided carpal tunnel injections: patient preparation, approaches, needle positioning, inject- ed medications and injected volumes. Patient preparation The patient can be either seated or lying supine.13 When seated, the elbow is resting on the examina- tion table in a 90° flexion position.14 When lying supine, the arm is abducted to 90° with the elbow extended.11,15,16 In both positions the forearm is su- pinated and the wrist is in a 15–35° dorsiflexion position.11,15,16 Careful US examination is important for injection planning. The wrist is examined with a linear probe along the carpal tunnel and special attention is given to the MN anatomy (size, posi- tion, aberrant variants) and the course of blood vessels.13,17,18 Authors reported two different procedures for disinfecting the skin11,16,17,19: some clean the punc- ture area with an alcohol swab and use a sterile ultrasound gel11, while others perform surgical disinfection and use a sterile probe sleeve.16,17,19 Even though there is no clear consensus on patient preparation, in recent years some authors (e.g. Guo, Green, Chianca, etc.) have reported that surgical disinfection of the area can help avoid infections of the puncture site or the deep tissues16,17,19, which can also be avoided if a sleeve is used for the US probe. A few authors have reportedly used a short- acting local anaesthetic to numb the dermal and subdermal area before performing US-guided car- pal tunnel injection15,20, but in the majority of cases, the local anaesthetic is added to the injected mix- ture.21-23 Needles of different sizes (22 to 30 Gauges) have been used in US-guided carpal tunnel injec- tions.11,14-16,24 Needles with a small diameter cause less pain upon insertion and are less likely to cause nerve damage during the procedure, but are not FIGURE 1. Depicting a flowchart of paper search and selection with exclusion criteria. Radiol Oncol 2022; 56(1): 14-22. Tumpaj T et al. / Ultrasound-guided carpal tunnel injections16 suitable for injecting protein-rich plasma, because they damage the platelets due to the small diam- eter and platelet size.11,14-16,24,25 Approaches Three approaches to carpal tunnel injections can be found in the literature and all of them are per- formed with a linear transducer of varying fre- quencies (5–17 MHz).11,13,14,26-29 Ulnar approach The transducer is positioned at the distal wrist crease perpendicularly to the course of the MN (Figure 2A).13 The probe is then moved ulnarly keeping the MN in view until the pisiform bone, ulnar nerve, and artery are brought into view. On the ulnar side, the pisiform is seen as a hyperechoic structure and the honeycomb appearance of the ulnar nerve may be differentiated radially to the pulsating ulnar artery (Figure 2B).13 The needle is introduced in plane in an ulnar to radial direc- tion, then passes the ulnar nerve and ulnar artery superficially, and punctures the TCL so that the needle tip can be advanced adjacently to the MN (Figure 2C).15,26-28 Radial approach In the radial approach, the transducer is positioned at the distal wrist crease perpendicularly to the course of the MN (Figure 3A).14 The probe is moved radially keeping the MN in view until the scaphoid and flexor carpi radialis tendon are brought into view. On the radial side, the scaphoid is seen as a hyperechoic structure and the flexor carpi radialis tendon lies inferolateral to the MN (Figure 3B). The needle is introduced in plane in a radial to ulnar direction, then proceeds above the flexor carpi ra- dialis tendon, and punctures the TCL so that the needle tip can be advanced adjacently to the MN (Figure 3C).14 Longitudinal approach In the longitudinal approach, the transducer is po- sitioned parallel to the MN at the distal wrist so that the MN is seen along the TCL from the carpal tun- nel inlet to the carpal tunnel outlet (Figures 4A,B). FIGURE 2. (A), (B), and (C) showing the ulnar approach. (A) wrist and needle positioning for carpal tunnel injection, (B) ultrasound anatomy of the carpal tunnel shown in the short axis, (C) penetrating transverse carpal ligament positioning the needle tip above the median nerve. Ultrasound of the carpal tunnel after the needle penetrates the transverse carpal ligament. Comparing B and C note the expansion of the perineural space marked with a white cross. A = ulnar artery; black arrows = needle; MN = median nerve; RAD = radial; T = flexor tendons; Tr = trapezium; ULN = ulnar; white arrows = transverse carpal ligament; white cross = perineural injectate; white star = ulnar nerve A B C Radiol Oncol 2022; 56(1): 14-22. Tumpaj T et al. / Ultrasound-guided carpal tunnel injections 17 The probe is then moved laterally to the MN ap- proximately 0.5 cm until the nerve disappears.11 Two modifications can be found in the literature, namely the proximal to the distal and the distal to the proximal.11,29 In the proximal to distal, the needle is inserted at the distal wrist crease and is advanced distally.29 In the distal to proximal, the needle is inserted approximately 2 cm distally to the distal wrist crease and is advanced proxi- mally.11 In both modifications, the needle is intro- duced in plane and punctures the TCL so that the needle tip can be advanced adjacently to the MN (Figures 4C,D).11,29 Needle positioning The aim of carpal tunnel injection is to position the needle tip adjacent to the MN without inducing nerve or vascular injury. When the needle is po- sitioned perineurally, the injection volume can be injected as a single or multiple deposit. In a single deposit, the injection is deposited at a single loca- tion11,14,29, whereas in a multiple deposit, the needle is repositioned to deposit the injection volume on multiple locations.16 Injected medications Medications used for carpal tunnel injections are corticosteroids30-35, local anaesthetics (LA)35, dex- trose36,37, platelet-rich plasma (PRP)25,38, progester- one23,39 and saline.40 The most widely used medi- cations for carpal tunnel injections are corticoster- oids30-35, which ameliorate MN compression due to their anti-inflammatory properties.3 Particulate (e.g. methylprednisolone) or nonparticulate (e.g. dexa- methasone) corticosteroids can be injected in doses of 40–80 mg.30-35 The adverse effects of corticoster- oids are rare and range from skin discoloration and irritation at the injection site to neurotoxicity and atrophy of thenar muscles.35 Another medication commonly used in the treatment of CTS are LAs35, predominantly short-acting LAs (e.g. lidocaine 2%) that can be injected as a single compound or in conjunction with corticosteroids.28,35 These offer immediate pain relief and may give long-lasting FIGURE 3. (A), (B), and (C) showing the radial approach. (A) wrist and needle positioning for carpal tunnel injection, (B) ultrasound anatomy of the carpal tunnel shown in the short axis, (C) penetrating transverse carpal ligament positioning the needle tip below the median nerve. Ultrasound of the carpal tunnel after the needle penetrates the transverse carpal ligament. Comparing figures B and C note the expansion of the perineural space marked with a white cross. A = ulnar artery; black arrows = needle; H = hamate; RAD = radial; S = scaphoid; MN = median nerve; T = flexor tendons; ULN = ulnar; white arrows = transverse carpal ligament; white cross = perineural injectate A B C Radiol Oncol 2022; 56(1): 14-22. Tumpaj T et al. / Ultrasound-guided carpal tunnel injections18 effects, which are speculated to be caused by the blockage of the sympathetic reflex arc, suppression of nociceptive discharge, blockade of sensitization, and anti-inflammatory effects.35 Adverse effects of LAs are rare and range from common allergic reac- tions to inadvertent intravascular injection, a very serious complication that can lead to seizures and heart conduction blocks.35 More recently, dextrose has also been used as a medication.36,37 The exact mechanism of action of dextrose is not known, but it is thought that it stimulates an anti-inflammatory response through the inhibition of capsaicin, caus- ing sensitive receptors to prevent the release of substance P and calcitonin gene-related peptide, both of which are known to cause swelling of the nerve and induce pain.37 The main advantage of dextrose is that no serious adverse effects due to biochemical properties were reported.37 Saline is widely used either as a single compound in hydro- dissection or as a diluting substance for corticos- teroids or LAs (Figure 5A,B,C).30-35,40,41 There are no serious adverse effects of saline; however, pain up- on injection has been reported when no LA is add- ed.22,28,30,40 A promising type of injectate is PRP25,38 that triggers a neuroregenerative response by re- leasing several hormones and growth factors, such as platelet-derived growth factor, transforming growth factor, epidermal growth factor, vascular endothelial growth factor, and insulin-like growth factor-1. These stimulate healing by reducing the FIGURE 4. (A), (B), (C), and (D) showing the longitudinal approach. (A) Wrist and needle positioning for carpal tunnel injection for the proximal to distal approach, (B) Wrist and needle positioning for carpal tunnel injection for the distal to proximal approach, (C) Ultrasound of the carpal tunnel shown in the long axis after the needle penetrates the transverse carpal ligament and positioning the needle tip parallel to the median nerve, (D) ultrasound of the carpal tunnel shown in the short axis after the needle penetrates the transverse carpal ligament positioning the needle tip parallel to the median nerve. Note in C and D the expanded perineural space marked with a white cross. A = ulnar artery; black arrows = needle; D = distal; MN = median nerve; P = proximal; RAD = radial; T = flexor tendons; ULN = ulnar; white arrows = transverse carpal ligament; white cross = perineural injectate A B C D Radiol Oncol 2022; 56(1): 14-22. Tumpaj T et al. / Ultrasound-guided carpal tunnel injections 19 inflammatory response.38 Lastly, progesterone, which has anti-inflammatory and neuroprotective effects on nerves, has also been used recently as an injectate.23,39 Injection volume There is no consensus on the optimal volume in- jected into the carpal tunnel and injection volumes anywhere between 1 ml to 10 ml can be found in the literature.30-35,37,40,42-46 The injectate can be a manufactured single-compound solution or a mul- ti-compound preparation. The latter is a mixture of active compounds with either saline or LA or both.30-35,40,42-46 Corticosteroids are predominantly injected as a multi-compound solution of 1–2 ml corticosteroids with 1–2 ml of LA or 1–2 ml of sa- line.28,30-34,45,46 LAs are usually added to multi-com- pound solutions as an anaesthetic during the injec- tion and are rarely injected as a single compound, but when they are, the volume ranges from 0.5 to 4 ml.35,47 Saline is mostly used as a mixture substance for other medications as a part of multi-compound solutions.30-37,40,44-46 In hydrodissection, saline is used as a single-compound solution of 3–10 ml in volume.40,48 Dextrose is injected as a single-com- pound solution of 3–10 ml in volume.37,40,48 PRP is injected as a single-compound solution of 1–3 ml in volume.25,38 Progesterone is injected as a multi- compound solution, a mixture of 0.5 ml of LA and 0.5 ml of progesterone.23,39 Discussion Even though US-guided injection therapy has be- come an established treatment option for CTS2, no consensus has yet been achieved on what steps should be taken to achieve the best results. Carpal tunnel injections may be performed with the land- mark-guided approach, but several complications have been noted, such as nerve injury, intravascu- lar application of medication, failure to perforate TCL, etc.18 When the US is used to guide the injec- tions, the risk of these complications is reduced.18 Three approaches to carpal tunnel injections can be found in the literature. The ulnar approach is the most frequently used, as it helps better visual- ize the carpal tunnel content and thus enables ac- curate perineural injection by avoiding neurovas- cular structures.13 This approach is also easier to learn in comparison to the other two approaches and provides good needle control.13 Reports on ra- dial and longitudinal approaches are scarce.14,27,29,49 The reviewed authors do not offer any personal perspective on the benefits and difficulties of the radial approach.14 Jurbala and Burbank have come to the conclusion that the ulnar approach carries a higher risk of inadvertent penetration of the neu- rovascular structures because the needle is direct- ed toward and not tangential to the MN.11 It has to be noted that scanning in the long axis can be challenging because it is difficult to differentiate between swollen nerve fascicles, muscles, and in- flamed tendons.11 Only a single study was found FIGURE 5. (A), (B), and (C) showing the carpal tunnel in the long axis. The effect of injectate volume on perineural space expansion and subsequent hydrodissection. Note the expansion of the perineural space (white arrow) around the median nerve before (A), during (B) and after injecting 6 ml of the injectate and subsequent hydrodissection (C). black arrows = needle; D = distal; MN = median nerve; P = proximal; white cross = transverse carpal ligament A B C Radiol Oncol 2022; 56(1): 14-22. Tumpaj T et al. / Ultrasound-guided carpal tunnel injections20 where different approaches – radial and ulnar, to be precise – were compared to one another, but the results showed no difference in patient outcome or measured US parameters.14 Even so, the ulnar ap- proach should be considered as the first choice be- cause it is backed by the largest amount of evidence and is easier to learn than other approaches.13 Only a few papers included a detailed report on needle positioning, making it difficult to discern the benefits of different approaches.11,16,29 A rand- omized controlled study on US-guided single-de- posit injections of corticosteroids showed no differ- ence in patient outcome, electrodiagnostic, and US findings in terms of deposition between the MN and TCL or deposition between the MN and flexor tendons.14 The authors emphasized that placing the needle below the MN is technically less demand- ing and is, therefore, the better option for less ex- perienced practitioners.14 A study by Nwawka et al. where the spread of US-guided injections was ob- served in different anatomical positions concluded that a single-deposit injection offers circumferen- tial coverage using injection volumes of 2 ml, sug- gesting that it is unnecessary to place the needle between the MN and TCL at multiple sites along the nerve.49 These two studies suggest that a sin- gle deposit offers circumferential coverage of the MN.14,49 Further studies are warranted to discern the potential superiority of multiple deposit versus single deposit; however, a single deposit should be considered as the first choice as a multiple deposit is harder to perform and has not yet shown any superior benefits. Another aspect to be taken into consideration in US-guided CTS injection treatment is the choice of medication. Several medications can be used for carpal tunnel injections, of which the most widely utilized are corticosteroids.30-35 Although there is no consensus on what type or dose of corticoster- oids achieves the best outcome, it is thought that the effect of particulate corticosteroids lasts longer due to the quick uptake of nonparticulate corti- costeroids; however, recent studies did not prove the superiority of either type of corticosteroids.30-35 Salman Roghani et al. compared the effects of 40 mg to 80 mg of methylprednisolone for carpal tun- nel injections and found no significant differences in patient outcome.33 Similarly, Karimzadeh et al. found no significant differences in patient outcome between 40 mg of methylprednisolone and 80 mg triamcinolone, but on the other hand, Habib et al. emphasized that a lower dose could be beneficial due to fewer side effects such as pain upon injection, glucose control after the procedure, and potential neurotoxicity.31,32 The majority of US-guided carpal tunnel injections are performed with LAs as part of the multi-compound solution, but these can also be used as a single-compound solution. The effects of LAs as a single compound are poorly researched and only a few studies have been published on this topic.35,47 Karadas et al. compared the effects of LAs and corticosteroids in CTS treatment and found no significant difference in patient outcome.47 In re- cent years, dextrose and PRP have also been used in the treatment of CTS. The effects of dextrose as a single-compound solution have been widely re- searched36,37,43,50, but it is still unclear whether they are caused by the release of anti-inflammatory tis- sue mediators after the injection or better median nerve gliding due to hydrodissection. Comparison between dextrose, saline, and corticosteroids has shown that dextrose is superior in terms of patient outcome.37,43 Due to improved patient outcomes, authors have even proposed that dextrose should be a first-line medication option for patients with CTS.37,43 Injections of PRP have also given prom- ising results in CTS treatment. In a meta-analysis by Lin et al. PRP was ranked second to dextrose in the terms of clinical effects.43 The downside of PRP is the difference in the preparation protocol, which can produce different clinical outcomes due to compositional differences.43 There are also the issues of higher costs, more complex organiza- tion, and limited availability of machines needed to prepare PRP.43 Progesterone has also been pro- posed as a possible choice of injectate and its effect has been compared to that of corticosteroids due to their similar molecular structure.23,39 According to the proposed theory, progesterone receptors are located on the transverse ligament lining cells and wrist synovial tissue.39 Bahrami et al. concluded that progesterone is equal to corticosteroids in pa- tient outcomes, while Raeissadat et al. reported that progesterone is equal or even superior to corticos- teroids in symptom relief.23,39 Several medications are used in clinical practice with no clinical consen- sus on medication of choice; however, the papers with the highest level of evidence suggest that dex- trose should be the first-line medication option.37,43 Injection volume also remains a question of de- bate as only a few studies compared the effects of different volumes. In most studies, relatively low volumes of injectate were used (1–3 ml).42 In the study by Lin et al., the authors compared different injection volumes of dextrose (1, 2, and 4 ml) and concluded that the injection of 4 ml provided the best outcome.43 However, Schrier et al. were unable to prove the superiority of a 5 ml versus 2 ml in- Radiol Oncol 2022; 56(1): 14-22. Tumpaj T et al. / Ultrasound-guided carpal tunnel injections 21 jection of corticosteroids and LA.28 It is speculated that larger injected volumes (> 5 ml) yield better results due to the conjoined effect of hydrodis- section and better injection distribution.30,43 With hydrodissection, adhesiolysis can be achieved by separating TCL from the MN and enabling normal tendon gliding.28,41 A prospective randomized con- trol trial on the effects of hydrodissection showed a significant improvement of the intervention group at a 3-month follow-up in comparison to the con- trol group.37 In the intervention group, a multi- deposit injection was performed to detach the MN from the TCL and separate the MN from the flexor tendons, whereas in the control group saline was injected subcutaneously.37 It has been suggested that a cumulative effect of hydrodissection may be expected if injections are repeated.37,40 Although only a few studies have been published on this topic, there appears to be a tendency of better out- comes with larger injected volumes. Some limitations of our review need to be not- ed. The majority of papers reported a short-term follow-up and only a few reported a follow-up of up to 12 months. Furthermore, all the reviewed papers provided very little information on the optimal protocol of US-guided carpal tunnel in- jection. Even though we retrieved a large number of papers, only a few of them focused on a spe- cific question related to the proposed steps in US- guided carpal tunnel injection. Further studies are required to fully assess the contribution and effi- cacy of US-guided injection therapy for CTS, and this paper should serve as a reference to determine which study aims are important. Conclusions In recent years, US-guided injection therapy has become an established treatment option in mild to moderate CTS. Although no consensus has yet been reached as to which protocol gives the best results, the ulnar approach with a single deposit should be considered as the first choice and dextrose as the first-line medication option injected in larger vol- umes. Furthermore, as terminological differences make it difficult to draw a uniform comparison be- tween the reviewed papers, the presented steps of US-guided carpal tunnel injection might serve as a guideline for future studies. References 1. Petrover D, Richette P. Treatment of carpal tunnel syndrome: from ultra- sonography to ultrasound-guided carpal tunnel release. Joint Bone Spine 2018; 85: 545-52. doi: 10.1016/j.jbspin.2017.11.003 2. Padua L, Coraci D, Erra C, Pazzaglia C, Paolasso I, Loreti C, et al. Carpal tun- nel syndrome: clinical features, diagnosis, and management. Lancet Neurol 2016; 15: 1273-84. doi: 10.1016/S1474-4422(16)30231-9 3. Sconfienza LM, Adriaensen M, Albano D, Allen G, Aparisi Gómez MP, Bazzocchi A, et al; Ultrasound and Interventional Subcommittees of the European Society of Musculoskeletal Radiology (ESSR). Clinical indica- tions for image-guided interventional procedures in the musculoskeletal system: a Delphi-based consensus paper from the European Society of Musculoskeletal Radiology (ESSR)-part III, nerves of the upper limb. Eur Radiol 2020; 30: 1498-506. doi: 10.1007/s00330-019-06479-z 4. Sconfienza LM, Albano D, Allen G, Bazzocchi A, Bignotti B, Chianca V, et al. Clinical indications for musculoskeletal ultrasound updated in 2017 by European Society of Musculoskeletal Radiology (ESSR) consensus. Eur Radiol 2018; 28: 5338-51. doi: 10.1007/s00330-018-5474-3 5. Albano D, Aringhieri G, Messina C, De Flaviis L, Sconfienza LM. High- frequency and ultra-high frequency ultrasound: musculoskeletal maging up to 70 MHz. Semin Musculoskelet Radiol 2020; 24: 125-34. doi: 10.1055/s- 0039-3401042 6. 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Ultrasound-guided percutaneous injection, hydrodissection, and fenestration for carpal tunnel syndrome: description of a new technique. J Appl Res 2010; 10: 107-14. 23. Raeissadat SA, Shahraeeni S, Sedighipour L, Vahdatpour B. Randomized controlled trial of local progesterone vs corticosteroid injection for carpal tunnel syndrome. Acta Neurol Scand 2017; 136: 365-71. doi: 10.1111/ ane.12739 24. Lam KHS, Hung CY, Chiang YP, Onishi K, Su DCJ, Clark TB, et al. Ultrasound- guided nerve hydrodissection for pain management: rationale, methods, current literature, and theoretical mechanisms. J Pain Res 2020; 13: 1957- 68. doi: 10.2147/JPR.S247208 25. Uzun H, Bitik O, Uzun Ö, Ersoy US, Aktaş E. Platelet-rich plasma versus corticosteroid injections for carpal tunnel syndrome. J Plast Surg Hand Surg 2017; 51: 301-5. doi: 10.1080/2000656X.2016.1260025 26. Kim DH, Jang JE, Park BK. Anatomical basis of ulnar approach in carpal tun- nel injection. Pain Physician 2013; 16: E191-8. PMID: 23703418 27. Lee JY, Park Y, Park KD, Lee JK, Lim OK. Effectiveness of ultrasound-guided carpal tunnel injection using in-plane ulnar approach: a prospective, randomized, single-blinded study. Medicine 2014; 93: e350. doi: 10.1097/ MD.0000000000000350 28. Schrier VJMM, Brault JS, Amadio PC. Ultrasound-guided hydrodissection with corticosteroid injection in the treatment of carpal tunnel syndrome: a pilot study. J Ultrasound Med 2020; 39: 1759-68. doi: 10.1002/jum.15279 29. Ustün N, Tok F, Yagz AE, Kizil N, Korkmaz I, Karazincir S, et al. Ultrasound- guided vs. blind steroid injections in carpal tunnel syndrome: a single-blind randomized prospective study. Am J Phys Med Rehabil 2013; 92: 999-1004. doi: 10.1097/PHM.0b013e31829b4d72 30. DeLea SL, Chavez-Chiang NR, Poole JL, Norton HE, Sibbitt WL Jr, Bankhurst AD. Sonographically guided hydrodissection and corticosteroid injection for scleroderma hand. Clin Rheumatol 2011; 30: 805-13. doi: 10.1007/ s10067-010-1653-6 31. Habib GS, Badarny S, Rawashdeh H. A novel approach of local corticosteroid injection for the treatment of carpal tunnel syndrome. Clin Rheumatol 2006; 25: 338-40. doi: 10.1007/s10067-005-0002-7 32. Karimzadeh A, Bagheri S, Raeissadat SA, Bagheri S, Rayegani SM, Rahimi- Dehgolan S, et al. The comparison of the effectiveness between different doses of local methylprednisolone injection versus triamcinolone in carpal tunnel syndrome: a double-blind clinical trial. J Pain Res 2019; 12: 579-84. doi: 10.2147/JPR.S190652 33. Salman Roghani R, Holisaz MT, Tarkashvand M, Delbari A, Gohari F, Boon AJ, et al. Different doses of steroid injection in elderly patients with carpal tun- nel syndrome: a triple-blind, randomized, controlled trial. Clin Interv Aging 2018; 13: 117-24. doi: 10.2147/CIA.S151290 34. Stark H, Amirfeyz R. Cochrane corner: local corticosteroid injection for carpal tunnel syndrome. J Hand Surg Eur 2013; 38: 911-4. doi: 10.1177/1753193413490848 35. MacMahon PJ, Eustace SJ, Kavanagh EC. Injectable corticosteroid and local anesthetic preparations: a review for radiologists. Radiology 2009; 252: 647- 61. doi: 10.1148/radiol.2523081929 36. Li TY, Chen SR, Shen YP, Chang CY, Su YC, Chen LC, et al. Long-term outcome after perineural injection with 5% dextrose for carpal tunnel syndrome: a retrospective follow-up study. Rheumatology 2021; 60: 881-7. doi: 10.1093/ rheumatology/keaa361 37. Wu YT, Ho TY, Chou YC, Ke MJ, Li TY, Tsai CK, et al. Six-month efficacy of perineural dextrose for carpal tunnel syndrome: a prospective, randomized, double-blind, controlled trial. Mayo Clin Proc 2017; 92: 1179-89. doi: 10.1016/j.mayocp.2017.05.025 38. Catapano M, Catapano J, Borschel G, Alavinia SM, Robinson LR, Mittal N. Effectiveness of platelet-rich plasma injections for nonsurgical management of carpal tunnel syndrome: a systematic review and meta-analysis of rand- omized controlled trials. Arch Phys Med Rehabil 2020; 101: 897-906. doi: 10.1016/j.apmr.2019.10.193 39. Bahrami MH, Shahraeeni S, Raeissadat SA. Comparison between the effects of progesterone versus corticosteroid local injections in mild and moderate carpal tunnel syndrome: a randomized clinical trial. BMC Musculoskelet Disord 2015; 16: 322. doi: 10.1186/s12891-015-0752-6 40. Wu YT, Chen SR, Li TY, Ho TY, Shen YP, Tsai CK, et al. Nerve hydrodissection for carpal tunnel syndrome: a prospective, randomized, double-blind, controlled trial. Muscle Nerve 2019; 59: 174-80. doi: 10.1002/mus.26358 41. Evers S, Thoreson AR, Smith J, Zhao C, Geske JR, Amadio PC. Ultrasound- guided hydrodissection decreases the gliding resistance of the median nerve within the carpal tunnel. Muscle Nerve 2018; 57: 25-32. doi: 10.1002/ mus.25723 42. Evers S, Bryan AJ, Sanders TL, Gunderson T, Gelfman R, Amadio PC. Influence of injection volume on rate of subsequent intervention in carpal tunnel syndrome over 1-year follow-up. J Hand Surg Am 2018; 43: 537-44. doi: 10.1016/j.jhsa.2018.02.024 43. Lin MT, Liao CL, Hsiao MY, Hsueh HW, Chao CC, Wu CH. Volume matters in ultrasound-guided perineural dextrose injection for carpal tunnel syn- drome: a randomized, double-blinded, three-arm trial. Front Pharmacol 2020; 11: 625830. doi: 10.3389/fphar.2020.625830 44. Armstrong T, Devor W, Borschel L, Contreras R. Intracarpal steroid injection is safe and effective for short-term management of carpal tunnel syndrome. Muscle Nerve 2004; 29: 82-8. doi: 10.1002/mus.10512 45. Atroshi I, Flondell M, Hofer M, Ranstam J. Methylprednisolone injections for the carpal tunnel syndrome: a randomized, placebo-controlled trial. Ann Intern Med 2013; 159: 309-17. doi: 10.7326/0003-4819-159-5-201309030- 00004 46. Cass SP. Ultrasound-guided nerve hydrodissection. Current Sports Medicine Reports 2016; 15: 20-2. doi: 10.1249/jsr.0000000000000226 47. Karadaş Ö, Tok F, Akarsu S, Tekin L, Balaban B. Triamcinolone acetonide vs procaine hydrochloride injection in the management of carpal tunnel syndrome: a randomized placebo-controlled study. J Rehabil Med 2012; 44: 601-4. doi: 10.2340/16501977-0990 48. Elawamy A, Hassanien M, Hamed A, Roushdy ASI, Abass NA, Mohammed G, et al. Efficacy of hyalase hydrodissection in the treatment of carpal tun- nel syndrome: a randomized, double-blind, controlled, clinical trial. Pain Physician 2020; 23: E175-83. PMID: 32214296 49. Nwawka OK, Miller TT, Jawetz ST, Saboeiro GR. Ultrasound-guided peri- neural injection for nerve blockade: does a single-sided injection produce circumferential nerve coverage? J Clin Ultrasound 2016; 44: 465-9. doi: 10.1002/jcu.22364 50. Chen LC, Ho TY, Shen YP, Su YC, Li TY, Tsai CK, et al. Perineural dextrose and corticosteroid injections for ulnar neuropathy at the elbow: a randomized double-blind trial. Arch Phys Med Rehabil 2020; 101: 1296-303. doi: 10.1016/j.apmr.2020.03.016 Radiol Oncol 2022; 56(1): 23-31. doi: 10.2478/raon-2021-0051 23 research article Lack of association between cortical amyloid deposition and glucose metabolism in early stage Alzheimeŕ s disease patients Daniela Ehrlich1, Andreas Dunzinger2, Gertraud Malsiner-Walli3, Bettina Grün4, Raffi Topakian5, Marina Hodolic6,7, Elmar Kainz1, Robert Pichler2,8,9 1 Department of Gerontology, Kepler University Hospital, Neuromed Campus, Linz, Austria 2 Institute of Nuclear Medicine, Kepler University Hospital, Neuromed Campus, Linz, Austria 3 Institute for Applied Statistics, Johannes Kepler University, Linz, Austria 4 Institute for Statistics and Mathematics, WU University of Economics and Business, Vienna, Austria 5 Department of Neurology, Klinikum Wels-Grieskirchen, Wels, Austria 6 Nuclear Medicine Research Department, IASON, Graz, Austria 7 Department of Nuclear Medicine, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic 8 Institute of Nuclear Medicine, Steyr Hospital, Steyr, Austria 9 Department of Radiology, Clinic of Nuclear Medicine, Medical University Graz, Graz, Austria Radiol Oncol 2022; 56(1): 23-31. Received 12 October 2021 Accepted 9 November 2021 Correspondence to: Prof. Marina Hodolic, M.D., Ph.D, Nuclear Medicine Research Department, IASON GmbH, Feldkirchner Straße 4, A-8054 Graz Seiersberg, Austria. Phone: + 43 664 830 9492; E-mail: marina.hodolic@gmail.com Disclosure: No potential conflicts of interest were disclosed. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Background. Beta amyloid (Aβ) causes synaptic dysfunction leading to neuronal death. It is still controversial if the magnitude of Aβ deposition correlates with the degree of cognitive impairment. Diagnostic imaging may lead to a better understanding the role of Aβ in development of cognitive deficits. The aim of the present study was to investi- gate if Aβ deposition in the corresponding brain region of early stage Alzheimer´s disease (AD) patients, directly cor- relates to neuronal dysfunction and cognitive impairment indicated by reduced glucose metabolism. Patients and methods. In 30 patients with a clinical phenotype of AD and amyloid positive brain imaging, 2-[18F] fluoro-2-deoxy-d-glucose (FDG) PET/CT was performed. We extracted the average [18F] flutemetamol (Vizamyl) up- take for each of the 16 regions of interest in both hemispheres and computed the standardized uptake value ratio (SUVR) by dividing the Vimazyl intensities by the mean signal of positive and negative control regions. Data were analysed using the R environment for statistical computing and graphics. Results. Any negative correlation between Aβ deposition and glucose metabolism in 32 dementia related and cor- responding brain regions in AD patients was not found. None of the correlation coefficient values were statistically significant different from zero based on two-sided p- value. Conclusions. Regional Aβ deposition did not correlate negatively with local glucose metabolism in early stage AD patients. Our findings support the role of Aβ as a valid biomarker, but does not permit to conclude that Aβ is a direct cause for an aberrant brain glucose metabolism and neuronal dysfunction. Key words: Alzheimer disease, PET, tau, FDG Introduction Alzheimer’s disease (AD) is the most common cause of dementia in the elderly people. The hall- mark pathologies include beta-amyloid (Aβ) depo- sitions in plaques and brain vessels, tau pathology, microglia activation, and inflammation. The causes of AD are unknown; however, Aβ may play an im- Radiol Oncol 2022; 56(1): 23-31. Ehrlich D et al. / Alzheimers disease, amyloid deposition and glucose metabolism24 portant role in development of AD. According to the amyloid cascade hypothesis, Aβ causes synap- tic dysfunction and neuronal death leading to cog- nitive impairment.1,2 However, several studies suggested only a mod- est correlation between Aβ pathology and cogni- tion. There is little correlation between amyloid plaques location at autopsy and affected brain re- gions according to patient’s clinical symptoms.3 A large burden of amyloid plaques is found in sub- jects without cognitive deficits.4 Amyloid plaque formation does occur late in hippocampus, al- though this structure is the first to fail clinically.5 Indeed, the reduction of plaque load in the brain in therapeutic trials has not yielded to cognitive ben- efit in AD patients.6 In contrast, the burden of neurofibrillary tan- gles, consisting of hyperphosphorylated tau, cor- relates with the degree of cognitive impairment in AD.7 In vivo studies using PET tracers for tau replicated these findings showing that more ad- vanced Braak stages are associated with decreased global cognitive status.8,9 Tau pathology leads to a reduced glucose metabolism correlating with cog- nitive decline.10 Thus, tau pathology rather than amyloid accumulation, may contribute to cognitive dysfunction in AD patients. Imaging may lead to a better understanding the role of Aβ in the development of cognitive deficits. Techniques such as 2-[18F]fluoro-2-deoxy-d-glu- cose (FDG) Positron Emission Tomography (PET) or amyloid PET are widely used for supporting the diagnosis of dementia. To exclude cerebral pathol- ogies, such as tumors, subdural hematoma or nor- mal pressure hydrocephalus, magnetic resonance imaging (MRI) is used. Morphologically, MRI may detect a hippocampal volume reduction in AD [11]. Amyloid PET allows in vivo detection of amyloid plaques indicating the pathophysiologic state of dementia. Several tracers, such as 18[F] florbeta- pir, 18[F] flutemetamol and 18[F] florteban are ap- proved by the US Food and Drug Administration (FDA). Timing of amyloid accumulation is at a pre- clinical stage of AD.12 However, healthy individuals without cogni- tive symptoms can have a positive amyloid PET scan. Thus, amyloid imaging may be helpful for differential diagnosis in early onset dementia, particularly to rule out AD dementia. FDG PET is an important tool to detect early neurodegen- erative dementia, differentiate neurodegenerative dementia or comorbidity of other neurodegenera- tive disease. FDG PET is described as a neuronal injury biomarker in AD and neuronal dysfunction is indicated by reduced glucose metabolism.13 In AD patients, FDG PET demonstrates a glucose metabolic reduction in the parietotemporal asso- ciation cortices, posterior cingulate and precuneus regions.14 In the later stages of AD, hypometabolic regions spread to the frontal association cortices.15 In patients with mild cognitive impairment (MCI), the transitional stage between aging and AD, FDG PET appears to add the greatest prognostic infor- mation.16 According to the amyloid cascade hypothesis, in brain regions with amyloid deposition, neuronal injury and an altered FDG metabolism may be ex- pected. However, several studies showed contro- versial findings regarding the correlation of amy- loid deposition and glucose metabolism.17-19 Thus, it is still not clear if amyloid deposition comparably to tau pathology is a leading cause of cognitive im- pairment. In this study, we included patients with a clini- cal phenotype of AD with cognitive dysfunction and amyloid deposition in cortical brain areas as detected by Aβ binding PET tracer. The aim was to investigate if Aβ deposition in the corresponding brain region directly correlates to neuronal dysfunction indicated by reduced glu- cose metabolism. Such a correlation should be easy and convenient to recognize when clinically AD is at a relatively early stage and impaired glucose me- tabolism is still restricted to certain areas and not globalized. Therefore, our cohort consists mostly of patients at the early stage of disease. Patients and methods Patients Ninety patients underwent amyloid PET analy- sis at the Institute of Nuclear Medicine, Kepler University Hospital, Neuromed Campus, Linz, between January 2016 and November 2017. These patients were assigned from Departments of Gerontology, Neurology or Psychiatry of various hospital institutions located in upper Austria. At least all amyloid positive patients (n = 30) under- went a comprehensive clinical and neuropsycho- logical evaluation. Probable AD was diagnosed clinically and according to the S3 guidelines for dementia by experienced clinicians.20 For screen- ing, the mini mental state examination (MMSE) according to Folstein was performed.21 Each sub- ject underwent computed tomography or MRI to rule out any structural abnormalities, such as brain tumours, hematomas, hydrocephalus or ischemia, Radiol Oncol 2022; 56(1): 23-31. Ehrlich D et al. / Alzheimers disease, amyloid deposition and glucose metabolism 25 as the cause for dementia. Vitamin deficiencies and thyroid abnormalities were excluded by blood analysis. FDG PET, amyloid PET and neuropsy- chological testing were acquired within 60 days. Brain Aβ deposition was quantified by performing PET scans using the tracer 18[F] flutemetamolm (Vizamyl). In a standardized procedure patients were rated as amyloid positive (n = 30, age 65.0 ± 14.3 years) or amyloid negative (n = 60, age 64.6 ± 8.7 years). FDG PET was used to evaluate brain glucose metabolism. It was rated in clinical routine by ex- perienced clinicians blinded to clinical symptoms using the Neuro Q, Version 3.5, 2007-analysis sys- tem, a schematic summary, comparing the patients scan to the scan of an asymptomatic control group. For our investigation, we selected 32 dementia re- lated brain regions as regions of interest (Table 1). The regions of interest were based on brain areas, which are suggested by the Neuro Q program and include all dementia related brain regions. For each region NeuroQ compares the FDG metabolism to that of a cohort of normal persons, numbers indi- cate extent of standard deviation in comparison to a normal situation. Negative numbers represent relative hypometabolism. For correlation, we manually defined cortical ar- eas of representative gyri in dementia related brain regions of amyloid positive PET scans and exclud- ed non cortical areas and sulci to avoid bias. Pons and the cerebellum represent the reference regions for positive and negative control within each brain. Further, we evaluated the Vizamyl uptake value ratio in corresponding brain regions. We extracted the average Vizamyl uptake for each of the 16 re- gions of interest in both hemispheres and comput- ed the standardized uptake value ratio (SUVR) by dividing the Vimazyl intensities by the mean signal of the individual positive and negative control re- gions as mentioned above. Positron emission tomography All PET scans were obtained with a Philips Gemini GXL PET/CT. For amyloid imaging patients re- ceived 185 MBq of 18F-Flutemetamol i.v. The tracer was distributed by GE healthcare Austria. PET/CT images were obtained 60 min after tracer injection. FDG PET had been scheduled on a different day. Patients fasted for a minimum of 6 h before FDG injection to ensure standardized metabolic conditions. Blood glucose level was measured and had to be < 160 mg % in all patients. 185 MBq of FDG was injected i.v.. PET images were acquired 30 min post injection (3D acquisition). The scanner acquires transaxial planes, simultaneously cover- ing an 18 cm axial field of view. Eliminating the sub-sampling required in conventional techniques, line-of-response (LOR) removes averaging and consequent image degradation. Detector material is gadolinium oxyorthosilicate (GSO) with a crystal size of 4 x 6 x 30 mm. A 6-slice helical CT – Philips brilliance air 6 – was used for attenuation correc- tion. For further evaluation, data were transferred to a Hermes Medical Solutions, Sweden work sta- tion (HERMES). PET interpretation was done visually by two experienced nuclear medicine specialists in knowl- edge of clinical data of the patient and consensual- ly. The procedure and technical data are described in detail by Pichler et al.22,23 Statistics Data were analysed using the R environment for statistical computing and graphics.24 Scatter plots of the amyloid mean score versus the FDG mean score visualize separately for each region and side the association. To test for association between these two scores, the Pearson correlation coefficient was determined for each region and side. Two- sided p-values were calculated assuming normally distributed data. P-values were for each side cor- rected for multiple testing using Holm’s method.25 TABLE 1. Regions of interest superior frontal cortex middle frontal cortex Inferior frontal cortex anterior cingulate cortex posterior cingulate cortex sensorimotoric cortex superior lateral temporal cortex medial anterior temporal cortex medial posterior temporal cortex inferior lateral anterior temporal cortex inferior lateral posterior temporal cortex superior parietal cortex inferior parietal cortex parietotemporal cortex primary visual cortex associative visual cortex Radiol Oncol 2022; 56(1): 23-31. Ehrlich D et al. / Alzheimers disease, amyloid deposition and glucose metabolism26 The study was done in accord with ethical stand- ards and in accord with the Helsinki Declaration of 1975. Results In clinical routine, 90 amyloid PET scans for diag- nosis of dementia were performed. In detail, AD was diagnosed in 30 patients (65.0 ± 14.3 years), 16 male and 14 female. All patients with amyloid pos- itive PET scan underwent also PET scanning with FDG and computed tomography (CT) or MRI, as well as neuropsychological and clinical examina- tion. In the AD group mean score MMSE was 23 ± 5 (n = 30). Figures 1 and 2 demonstrate FDG PET and amy- loid positive PET images of patients with the clini- cal suspected diagnosis of AD. In 43 subjects with amyloid negative PET scans a MMSE was performed. In the non AD group the mean score MMSE was 26 ± 3 (n = 43). Subjects with negative amyloid PET (n = 60, 24 female, 36 male, age 64.6 ± 8.7) received the following diag- nosis according to ICD-10: affective disorders (n = 33), Parkinson’s disease (n = 3), psychoorganic syn- drom (POS, n = 3), Hashimoto’s encephalopathy (n = 1), hepatic encephalopathy (n = 1), frontotempo- ral dementia (FTD, n = 8), vascular dementia (vaD, n = 8) and β-amyloid associated angiopathy (n = 3). As shown in Figure (3) and Figure (4) we did not find any significant negative correlation be- tween amyloid deposition and glucose metabolism in 32 dementia related and corresponding brain regions in AD patients. The estimated correlation coefficient values differed between 0.48 and -0.32. None of the correlation coefficient values were sta- tistically significant different from zero based on two-sided p- value at significance level 0.05 after correcting for multiple testing for each side using Holm´s method. No statistical evidence was found to confirm a negative correlation between amyloid deposition and glucose metabolism in general or specifically for some brain regions. Discussion There is an ongoing debate to which extent amy- loid is related to AD pathology. The concept of a direct mechanism leading to clinical manifestation lead to various trials of vaccination therapies. As therapeutic success was disappointing the patho- physiological role of amyloid in AD had to be re- discussed. In AD pathology, the amyloid cascade hypoth- esis may play a fundamental role.2 Plaques in AD brains consist of insoluble Aβ peptides cleaved by different secretases from the amyloid precur- sor protein (APP).26 The cleavage results in Aβ-40 with a length of 40 amino acids and Aβ-42 with a length of 42 amino acids, which is the plaque pron- ing form. Distinct plaque subtypes with low (dif- fuse plaques) and high (cored or neurotic plaques) A B FIGURE 1. 2-[18F] fluoro-2-deoxy-d-glucose (FDG) and amyloid brain PET/CT of 59-year-old woman. (A) FDG brain PET/CT of a 59-year-old woman with a history of fluctuating cognitive impairment (mini mental state examination [MMSE] = 14/30). Glucose hypometabolism was demonstrated in the parietal dorsolateral and temporolateral, and occipatal cortical areas. The glucose metabolism in the left temporomesial area is weak. The other cortical structures show a slight attenuation of FDG metabolism. Basal ganglia show more intense uptake compared to the cortical areas. This FDG brain PET study shows the typical picture of abnormal glucose metabolism that occurs in Alzheimer´s disease (AD) and is additionally compatible with pronounced microvascular changes. (B) On the amyloid PET a non-specific tracer accumulation from the pons to the basal ganglia is evident. PET images of the white matter demonstrate individual non-specific enrichments. In the frontal and temporal cortices as well as sporadically in the parietal cortical areas, a pathological tracer accumulation occurs. This global cortical tracer uptake is consistent with the neuropathology of AD. Radiol Oncol 2022; 56(1): 23-31. Ehrlich D et al. / Alzheimers disease, amyloid deposition and glucose metabolism 27 proportion of fibrillar components have been iden- tified.27 In fact, insoluble Aβ exceeds soluble forms of Aβ by a factor of about 100-fold in AD brain.28 However, Aβ does not correlate well with clinical symptoms and anti-amyloid pharmaceuticals have failed to improve significantly patient’s symp- toms3,6, even when amyloid deposits are efficiently reduced.29 As a possible exception, the updated analysis of the EMERGE trial showed a significant reduction in decline of global functions for the pa- tients treated with a high dose of aducanumab.30 Thus, it is still not clear if Aβ directly leads to neuronal dysfunction. High levels of Aβ may sub- sequently lead to a downstream of pathological events, including tau pathology, inflammation, oxidative stress, excitotoxicity, loss of synaptic con- nections, and cell death, causing the clinical symp- toms of AD. Levels of prefibrillar Aβ forms, such as soluble oligomers and protofibrils, correlate better than plaques with disease severity.31 This may in- dicate that soluble species are the neurotoxic form of Aβ leading to neurodegeneration.31 Aβ deposition is a valid biomarker to sup- port AD diagnostic.32 Available PET radioligands visualizing Aβ bind to insoluble fibrils, such as Aβ plaques. Recently, several 18F-labeled tracers were designed including flobetapir ([18F]AV-45), flutemetamol ([18F]GE067), florbetaben ([18F] BAY94–9172) [28, 33-36]. We used [18F]flutemeta- mol PET as a surrogate marker for brain amyloid deposition. Several studies suggested a high cor- relation between [18F]flutemetamol retention and neuropathologic findings.37-40 However, amyloid specific tracers may not be able to provide an ac- curate measurement of Aβ. In fact, there is a lack of data of in vivo Aβ specificity.41 Amyloid PET scan is able to rule out an AD diagnosis. Amyloid PET may have an additive, but primarily confirmatory role as a diagnostic marker in patients suspected of early-onset AD. An amyloid-positive PET scan often supports or changes diagnosis into AD.42 Amyloid pathology is also present in other forms of dementia and interpreted as mixed or copathol- ogy and not always the primary cause of the clini- cal manifestation of dementia. FDG PET, a neuroimaging tool in AD, plays an important role in discriminating different forms of dementia.12,34 Decreased glucose metabolism in temporal and parietal cortex indicates synaptic dysfunction and in contrast to amyloid deposition, this occurs mainly in the symptomatic phase of AD.28 In the present study, we investigated a pos- sible correlation between local amyloid deposition and glucose metabolism in dementia related corre- sponding brain regions in AD patients. In an early stage of AD impaired glucose metabolism is still restricted to certain areas and not globalized.43 Our cohort consists mostly of patients at the early stage of disease, which is a result of reasonable referrals. Clinical impact for different diagnosis of dementia in an advanced state of disease is questionable, be- cause all therapeutical strategies are more helpful at an early phase of the disease. The availability of both PET modalities in 30 patients diagnosed for AD in the present study allowed investigating the correlation of amyloid deposition and glucose me- tabolism, retrospectively. A B FIGURE 2. FDG and amyloid brain PET/CT of a 70-year-old woman. (A) 2-[18F] fluoro- 2-deoxy-d-glucose (FDG) brain PET/CT of a 70-year-old woman, who presented with a history of cognitive decline (mini mental state examination [MMSE] = 15/30). The glucose metabolism in the cerebral cortex is inhomogenous and moderately attenuated. In the cerebellum, normal glucose metabolism was demonstrated. This FDG brain PET study does not show the typical picture of abnormal glucose metabolism that occurs in Alzheimer´s disease (AD), but temporomesial and temporolateral some decreased tracer uptake can be observed. Additionally the images are compatible with pronounced microvascular changes. (B) Amyloid PET images demonstrate pathologically increased tracer accumulation in the entire brain, more pronounced in the frontal and temporal cortical areas. This is compatible with the diagnosis of AD. Radiol Oncol 2022; 56(1): 23-31. Ehrlich D et al. / Alzheimers disease, amyloid deposition and glucose metabolism28 To avoid bias due to computer assisted meas- urement we manually designated the cortical ar- eas of dementia related brain regions and evalu- ated the SUVR of [18F]flutemetamol. Compared to previous investigations17-19 we included a higher number of AD patients. Engler et al. found a nega- tive correlation with metabolism in parietal cortex in 16 AD patients.17 Another author showed that a higher amyloid tracer uptake correlated with lower regional glucose metabolism in 19 AD pa- tients.18 We did not find any negative correlation of amyloid tracer uptake and glucose metabolism in corresponding brain areas in 30 AD patients. Our data supports the concept that amyloid deposi- tions may not be the direct cause of dysfunctional metabolism. One limitation of this study is that measurement of Aβ40, Aβ 42 and phosphorylated tau in the cer- ebrospinal fluid (CSF) was not performed44 and thus, the correlation between CSF amyloid levels FIGURE 3. Scatter plots of the amyloid standardized uptake value (SUV) (on the x- axis) and the 2-[18F] fluoro-2-deoxy-d-glucose (FDG) values represented by NeuroQ (on the y-axis) for the 30 amyloid positive patient for the left side. Each panel represents a brain regions with the name indicated in the strip. Least-squares regression lines with slope proportional to the Pearson correlation coefficient indicate the association. The Pearson correlation (r) and the associated p-value (P) are shown in the label on the top of each panel. Radiol Oncol 2022; 56(1): 23-31. Ehrlich D et al. / Alzheimers disease, amyloid deposition and glucose metabolism 29 and amyloid tracer uptake could not be shown. If Aβ deposition plays a role in the development of cognitive deficits in AD, the lack of direct correla- tions requires other involved mechanisms such as tau pathology.2,45,46 The hyperphosphorylation and abnormal ag- gregation of tau, a microtubule-associated protein essential to neuronal stability and functioning, is a hallmark in AD pathology. Tau imaging revealed that neurofibrillary tangels are mainly located in the hippocampus and associative cortical regions.47 Tau PET imaging may serve as a valuable and early biomarker for the localization of neuronal injury. In contrast to Aβ accumulation, tau may cause cogni- tive decline mediated by glucose hypometabolism. Indeed, tau pathology was observed in brain re- gions related to clinical symptoms and overlapped with areas of hypometabolism.48.49 Exactly what we were not able to show for the relation of amyloid and glucose metabolism. Aβ may play an indirect FIGURE 4. Scatter plots of the amyloid standardized uptake value (SUV) (on the x-axis) and the 2-[18F] fluoro-2-deoxy-d-glucose (FDG) values represented by NeuroQ (on the y-axis) for the 30 amyloid positive patient for the right side. Each panel represents a brain regions with the name indicated in the strip. Least-squares regression lines with slope proportional to the Pearson correlation coefficient indicate the association. The Pearson correlation (r) and the associated p-value (P) are shown in the label on the top of each panel. Radiol Oncol 2022; 56(1): 23-31. Ehrlich D et al. / Alzheimers disease, amyloid deposition and glucose metabolism30 role in AD pathology in the development of NFTs. It is likely, that Aβ indirectly promotes tau phos- phorylation through upregulation of kinases such as GSK-3β and CDK5, which phosphorylate tau.50 Conclusios The focus of the study was to investigate the cor- relation between local amyloid deposition and glucose metabolism in vivo at corresponding brain areas. Therefore, we manually designated the ar- eas of interest in an early stage of disease, which in that manner has not been performed before. We showed that regional amyloid deposition did not correlate negatively with local glucose metabolism. Our findings support the role of Aβ as a valid bio- marker, but does not permit to conclude that Aβ is a direct cause for an aberrant glucose metabolism and neuronal dysfunction. 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J Alzheimers Dis 2018; 61: 265-81. doi: 10.3233/JAD-170490 Radiol Oncol 2022; 56(1): 32-36. doi: 10.2478/raon-2021-0056 32 research article Reliability of new radiographic measurement techniques for elbow bony impingement Uros Meglic1,2, Oskar Zupanc 1,2 1 Department of Orthopaedic Surgery, University Medical Centre Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2022; 56(1): 32-36. Received 19 October 2021 Accepted 10 November 2021 Correspondence to: Prof. Oskar Zupanc, M.D., Ph.D, Department of Orthopedic Surgery, University Medical Centre Ljubljana, Slovenia. E-mail: oskarzupanc@gmail.com Disclosure: No potential conflicts of interest were disclosed. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Background. Identifying the location and scale of radiographic changes in elbow bony impingement (EBI) is critical in formulating an appropriate diagnosis and treatment plan for such patients. The purpose of present study was to evaluate the intra-rater and inter-rater reliability of the new radiographic parameters, Anterior Impingement angle (AIa) and Posterior Impingement angle (PIa), for EBI. In addition, to determine if there was a relationship between radiographic parameters and clinical evaluation. Patients and methods. Three raters of different levels of training evaluated the radiographs of 60 patients (30 in EBI group and 30 in normal group) twice, at least 2 weeks apart. Intra-rater and inter-rater reliabilities were calculated by Intraclass Correlation Coefficients (ICC) with 95% confidence intervals. Correlation between radiographic parameters and clinical evaluation was calculated by Pearson correlation coefficient. Results. In both groups, intra-rater and inter-rater reliabilities were substantial. There were no significant differences in reliability between upper-hand expert surgeons and resident for either measurement. Good correlation was observed between impingement arcs and range of motion values. Conclusions. Both AIa and PIa measurements demonstrated substantial intra-rater and inter-rater reliability for nor- mal radiographs and in EBI patients. Good reliability, for either expert surgeons or residents in training, and good cor- relation between radiographic measurements and manual testing, appoints this method may be easily and reliably used in every day practice. Key words: elbow; osteoarthritis; impingement; classification; reliability Introduction Bony impingement of the elbow (EBI) is an early radiographic sign of the elbow degenerative dis- ease.1 Although being rare in general population, with prevalence up to 2%, it can be noticed with increased prevalence, up to 10%, in professional overhead athletes and manual laborers.2 As a re- sult of excessive and repetitive motions, bony os- teophytes occur in anterior and posterior compart- ment of the elbow.3 Changing elbow geometry, it causes flexion and extension deficit of the elbow motion.4 Although a flexion-extension range of motion (fROM) between 30 degrees and 130 degrees of flexion is enough to achieve 90% of the daily living activities, in professional athletes or manual labor- ers even a smaller loss of fROM can be devastating, with huge impact on their quality of life.5 Thus it has to be recognized early and treated properly. To date, there is no consensus among orthope- dic surgeons as to when in the course of the disease and how much to treat EBI, to provide symptomat- ic relief for a given patient. One reason for the lack of consensus is an inability to predict success based on preoperative assessment. A paucity of informa- Radiol Oncol 2022; 56(1): 32-36. Meglic U and Zupanc O / New radiographic measurements for elbow bony impingement 33 tion regarding specific radiographic parameters is a significant cause in these cases. The purpose of present study was, first, to eval- uate the intra-rater and inter-rater reliability of the new radiographic parameters for EBI. The second goal was to determine if there was a relationship between radiographic parameters and clinical eval- uation. Patients and methods Slovenia National Medical Ethics Committee ap- proval (No. 1650513) was obtained for this inves- tigation. A total 60 subjects were enrolled. Among them 30 subjects with fROM deficit and diagnosed, not yet treated, EBI were recruited as the EBI group. Remaining 30 subjects with other elbow patholo- gies (epicondylitis, ulnar neuritis etc.), but with a normal fROM and no clinical signs of EBI were recruited as the NORMAL group. A brief clinical history was obtained in order to rule out previous injury or upper extremity abnormality. Focused physical examination was performed with manual fROM testing. Standard antero-posterior and lateral x-ray views were obtained. Unsatisfactory films were re- peated in order to maintain consistency. Digital ra- diograph images were analyzed using Agfa IMPAX 6 software (Agfa HealthCare, Belgium). Broberg and Morrey (BM) as well as Hasting and Rettig (HR) classifications of elbow osteoarthritis were used to assess elbow joint’s degenerative chang- es.6,7 Sigmoid notch coverage (SNC) measurement was performed as described by Goldfarb et al., as a line connecting the center of the circle, fitted to sigmoid notch, to both the tip of the olecranon and coronoid (Figure 1C).8 Measurements of the Anterior Impingement angle (AIa) and Posterior Impingement angle (PIa) were obtained on lateral x-ray images as previously described by Meglic and Zupanc.9 The angle between the centralized ulnar direction line and the line between the center of rotation (COR) and the tip of the coronoid pre- sents AIa. The angle between the centralized ulnar direction line and the line between the COR and the tip of the olecranon presents PIa (Figure 1A, B).9 For reliability evaluation, two upper extrem- ity surgeons (OZ, UM) and one resident after ra- diological training (NK), independently evaluated each radiograph for radiographic measurements of AIa and PIa. Each evaluator re-measured both parameters after an interval of at least 2 weeks, a period used in other reliability studies in upper extremity.1,10 The examiners were blinded to their previous measurements. Subjects were excluded from participation if there was evidence of: an upper extremity injury history, a growth or congenital abnormality, mod- erate or severe grade on BM or HR classification (grade II and III). Statistical Package for Social Sciences version 21.0 (SPSS Inc, Chicago, IL, USA) was used for all statistical analyses. Student t test was used for group comparisons when normality was accepted, and a Mann-Whitney U test was used if normal- ity was rejected. The Fisher exact test was used for categorical data between groups. Original data from all 3 raters were used to assess reliability of measurements. Inter- and intra-rater reliability were calculated using intraclass correlation coef- ficients (ICC), ICC 2.1 for inter-rater, ICC 3.1 for intra-rater.11 Pearson correlation coefficient was used to measure correlation between impingement arcs and fROM. Correlation coefficient values less than 0.5 are indicative of poor reliability, values between 0.5 and 0.74 indicate moderate reliability, values between 0.75 and 0.89 indicate good reliabil- ity, and values greater than 0.90 indicate excellent reliability.11 All tests were 2-tailed, with p < 0.05 considered significant. All ICC values were calcu- lated with 95% confidence interval (95% CI). Results All patients included in the study were analyzed (60 patients, 100%). There were 20 males (67%), 10 females (33%) in EBI group and 12 males (40%), 18 females (60%) in NORMAL group (p = 0.07). The average age was 44 years (range 21–64 years) in EBI group and 33 years (range 18–60 years) in NORMAL group (p = 0.02). Pathology was pre- sented on dominant hand in 22 cases (73%) in EBI group and in 26 cases (87%) in NORMAL group (p = 0.33). In EBI group in all cases BM and HR classi- fication was graded stage I and in NORMAL group in all cases, no radiographic signs of osteoarthrosis were reported. Table 1 summarizes manual and radiographic measurements, comparing both groups. In all measurements, the differences between groups were statistically significant. ICCs for AIa and Pia measurements demon- strated good to excellent intra-rater and inter-rater reliability in both groups. Almost all ICCs were in 0.75–0.89 class, except intra-rater ICC in AIa in Radiol Oncol 2022; 56(1): 32-36. Meglic U and Zupanc O / New radiographic measurements for elbow bony impingement34 NORMAL group and inter-rater ICC in AIa be- tween Surgeon 1 and Resident in NORMAL group being in > 0.90 class. AIa measurements ranged from 34˚ to 60˚ (average 44˚) in EBI group and from 10˚ to 25˚ (average 20˚) in NORMAL group. PIa measurements ranged from 148˚ to 202˚ (average 173˚) in EBI group and from 140˚ to 160˚ (average 150˚) in NORMAL group. Inter-rater reliability and intra-rater reliabilities for all 3 raters are summa- rized in Table 2. There were no significant differences in reli- ability between two upper-hand expert surgeons and one resident for either measurement. Both sur- geons and a resident demonstrated a substantial agreement in all measurements. Inter-rater reliabil- ities between all raters are summarized in Table 3. Statistically significant correlations were ob- served between fROM measurements and ac- cording Impingement arc. Correlation coefficient showed good negative correlation between flex- ion and AIa measurements, measured 0.76, (95% CI = 0.86–0.51; p < 0.05) (Figure 2A). A good posi- tive correlation between extension deficit and PIa measurements was observed, measured 0.79, (95% CI = 0.59–0.89; p < 0.05) (Figure 2B). Discussion Treating EBI remains a challenging problem. A fac- tor influencing heavily in determining the appro- priate treatment choice is a lack of a reliable and accurate measurement technique. Very little infor- mation exists describing pathologic radiographic anatomy of EBI, thus making diagnosis and treat- ment difficult for both clinical and research pur- poses. The results of this study revealed substantial intra-rater and inter-rater reliability of both AIa and PIa measurements on EBI and normal elbow radiographs. For selecting and reporting ICC reli- ability we used Koo et al. guidelines.11 Following Koo’s guidelines, we used 3 raters with blinded re-measurements, used ICC 3.1 for intra-rater and ICC 2.1 for inter-rater reliability and reported all A B C FIGURE 1. Radiographic measurements: (A) Anterior Impingement angle (AIa) and Posterior Impingement angle (PIa) in normal group, (B) AIa and Pia in EBI group, (C) sigmoid notch coverage angle (SNC) in normal group. F IGURE 2. Manual flexion-extension range of motion (fROM) and radiographic measurement correlations between (A) f lexion and Anterior Impingement angle AIa, (B) extension deficit and Posterior Impingement angle (Pia). TABLE 1. Manual and radiographic measurements EBI groupa NORMAL groupa p Flexion (º) 115 ± 8 139 ± 5 < 0.05 Extension deficit (º) 19 ± 12 0 ± 0 < 0.05 fROM (º) 96 ± 14 139 ± 5 < 0.05 SNC (º) 217 ± 10 170 ± 6 < 0.05 AIa (º) 44 ± 5 20 ± 4 < 0.05 Pia (º) 173 ± 10 150 ± 5 < 0.05 a Mean ± SD; AIa = Anterior Impingement angle; EBI = elbow bony impingement; SNC = sigmoid notch coverage; PIa = Posterior Impingement angle; fROM = flexion-extension range of motion Radiol Oncol 2022; 56(1): 32-36. Meglic U and Zupanc O / New radiographic measurements for elbow bony impingement 35 results with 95% CI. Therefore, we can conclude that our results, with good to excellent reliability, are valid. Furthermore, our results suggest that training level does not affect the reliability of both measure- ment techniques, which is largely substantial for both surgeons and trainees before applying them to practice. Lastly, correlation of manual testing and radio- graphic measurements is necessary before routine use of these methods. Our results showed a good correlation of AIa with flexion measurement, and PIa with extension deficit. Thus, these radiograph- ic measurements are valid for diagnosing and clini- cal evaluation of EBI. In up to date literature, previous studies focus mainly on normal radiographic anatomy, ossifica- tion patterns, gender differences and fracture out- comes.8,12-14 Most of those described parameters are not usable in EBI evaluations, as it is an early sign of elbow degeneration. For elbow osteoarthrosis HR classification is usually used.7 Yet, no specific radiographic parameter in HR is described that can be used for EBI classification, only staging of the disease. Without a specific method of measure- ment, that is objectively verifiable, measurements and conclusions can vary markedly from evaluator to evaluator. To our knowledge, ours is the first study ex- amining reliability and clinical correlation of Impingement angles for EBI evaluation. Only pa- rameter in the literature being slightly associated with EBI is the SNC described by Goldfarb et al..8 Although, they report SNC being only a moder- ately reliable parameter, we decided to use it in our measurements for group comparison. SNC was significantly greater in EBI group compared to NORMAL group. We believe this reflects the com- mon characteristics seen in degenerative elbow disease, such as osteophyte formation. However, SNC does not specify the origin and extent of EBI, as impingement can be mainly in anterior or in posterior compartment of the elbow joint. For that reason, we decided not to use it in the reliability measurements. Our study has a few limitations. It demonstrat- ed substantial reliability that may be partially due to the smaller number of raters, all from a single institution. Also, the strong reliability between upper-hand expert surgeons and a resident may be because the resident-rater was under the tute- lage of the surgeon-rater. However, our study was TABLE 2. Inter-rate r and intra-rater reliabilitiesa Inter-rater reliabilitya Intra-rater reliability for rater 1a Intra-rater reliability for rater 2a Intra-rater reliability for rater 3a EBI group AIa 0.85 (0.75–0.94) 0.87 (0.75–0.94) 0.87 (0.74–0.94) 0.87 (0.75–0.95) PIa 0.84 (0.69–0.92) 0.83 (0.66–0.91) 0.88 (0.76–0.94) 0.85 (0.75–0.93) NORMAL group AIa 0.87 (0.76–0.94) 0.90 (0.81–0.95) 0.87 (0.74–0.94) 0.87 (0.75–0.95) PIa 0.85 (0.75–0.93) 0.86 (0.73–0.93) 0.86 (0.73–0.93) 0.86 (0.74–0.94) a ICC value (95% CI); AIa = Anterior Impingement angle; CI = confidence interval; EBI = elbow bony impingement; ICC = intraclass correlation coefficients; PIa = Posterior Impingement angle TABLE 3. Inter-rater reliability between two upper-hand expert surgeons and one resident Surgeon 1 vs. surgeon 2a Surgeon 1 vs. residenta Surgeon 2 vs. residenta EBI group AIa 0.85 (0.71–0.93) 0.85 (0.74–0.94) 0.86 (078–0.95) PIa 0.84 (0.69–0.92) 0.89 (0.79–0.95) 0.87 (0.74–0.94) NORMAL group AIa 0.87 (0.74–0.94) 0.90 (0.80–0.95) 0.88 (0.77–0.94) PIa 0.85 (0.71–0.93) 0.89 (0.77–0.94) 0.87 (0.760–0.94) a ICC value (95% CI); AIa = Anterior Impingement angle; CI = confidence interval; EBI = elbow bony impingement; ICC = intraclass correlation coefficients; PIa = Posterior Impingement angle Radiol Oncol 2022; 56(1): 32-36. Meglic U and Zupanc O / New radiographic measurements for elbow bony impingement36 designed with Koo’s ICC guidelines and can be treated as valid. Another limitation is that radiographic assess- ment was performed only on plain radiographs. In clinical practice a computer tomography (CT) scan is often used for evaluating EBI in cases to be surgi- cally treated.15 A 3D CT study by Lim et al. showed osteophytes predominating in the humeroulnar compartment, specifically in the anterior coronoid area (in 95%) and posteromedial compartment (in 86%).16 A CT scan helps visualizing osteophytes and asses their relationship to normal joint sur- faces. Nonetheless, given the fact that most clinical assessments are based on plain radiographs and obtaining a CT scan in most cases means a trans- fer of patient to another department, we feel our techniques are reasonable in a way to be easy ac- cessible. Lastly, as a study limitation, elbow motion in- cludes pronation and supination, which was not assessed. Clinically, patients with EBI have limited flexion-extension ROM and pain in terminal exten- sion and forced flexion. Characteristically, these patients (grade I on HR) do not have pronation- supination limitations.7 Pronation-supination limi- tations are associated with radio-capitellar joint degeneration and subluxation, estimated grade II or III on HR, which was an exclusion parameter in our study.7 In summary, the findings in our study support the use of AIa and PIa measurements on plain ra- diographs of elbow joint in patients with suspected EBI. This may prove helpful in future studies by allowing comparison of function, treatment choice and outcomes according to radiographic measure- ments. Conclusions I dentifying the location and scale of radiographic changes in EBI is critical in formulating an appro- priate diagnosis and treatment plan for such pa- tients. B oth AIa and PIa measurement demonstrat- ed substantial intra-rater and inter-rater reliability. In this study both measurements were reliably ap- plied by expert surgeons and resident, with good correlation to manual testing of the elbow function. Acknowledgments The authors thank Nerma Kulasic for the support and contribution in this study. References 1. Amini MH, Sykes JB, Olson ST, Smith RA, Mauck BM, Azar FM, et al. Reliability testing of two classification systems for osteoarthritis and post- traumatic arthritis of the elbow. J Shoulder Elbow Surg 2015; 24: 353-7. doi: 10.1016/j.jse.2014.10.015 2. Adla DN, Stanley D. Primary elbow osteoarthritis: an updated review. Shoulder Elbow 2011; 3: 41-8. doi: 10.1111/j.1758-5740.2010.00089.x 3. Adams JE, Wolff LH 3rd, Merten SM, Steinmann SP. Osteoarthritis of the elbow: results of arthroscopic osteophyte resection and capsulectomy. J Shoulder Elbow Surg 2008; 17: 126-31. doi: 10.1016/j.jse.2007.04.005 4. Wilson V. Upper extremity injuries in the throwing athlete. Mo Med 2011; 108: 170-2. PMID: 21736074 5. Blonna D, Bellato E, Marini E, Scelsi M, Castoldi F. Arthroscopic treatment of stiff elbow. ISRN Surg 2011; 2011: 378135. doi: 10.5402/2011/378135 6. Broberg MA, Morrey BF. Results of delayed excision of the radial head after fracture. J Bone Joint Surg Am 1986; 68: 669-74. PMID: 3722222 7. Rettig LA, Hastings H 2nd, Feinberg JR. Primary osteoarthritis of the elbow: lack of radiographic evidence for morphologic predisposition, results of operative debridement at intermediate follow-up, and basis for a new radiographic classification system. J Shoulder Elbow Surg 2008; 17: 97-105. doi: 10.1016/j.jse.2007.03.014 8. Goldfarb CA, Patterson JM, Sutter M, Krauss M, Steffen JA, Galatz L. Elbow radiographic anatomy: measurement techniques and normative data. J Shoulder Elbow Surg 2012; 21: 1236-46. doi: 10.1016/j.jse.2011.10.026 9. Meglic U, Zupanc O. Significance of radiographic parameters in the diagno- sis and treatment of a bony impingement of the elbow. Mater Technol 2019; 53: 747-50. doi: 10.17222/mit.2018.232 10. Blonna D, Zarkadas PC, Fitzsimmons JS, O’Driscoll SW. Validation of a photography-based goniometry method for measuring joint range of mo- tion. J Shoulder Elbow Surg 2012; 21: 29-35. doi: 10.1016/j.jse.2011.06.018 11. Koo TK, Li MY. A Guideline of selecting and reporting intraclass correlation coefficients for reliability research. J Chiropr Med 2016; 15: 155-63. doi: 10.1016/j.jcm.2016.02.012 12. Cheng JC, Wing-Man K, Shen WY, Yurianto H, Xia G, Lau JT, et al. A new look at the sequential development of elbow-ossification centers in children. J Pediatr Orthop 1998; 18: 161-7. PMID: 9531396 13. Keenan WN, Clegg J. Variation of Baumann’s angle with age, sex, and side: implications for its use in radiological monitoring of supracondylar fracture of the humerus in children. J Pediatr Orthop 1996; 16: 97-8. doi: 10.1097/01241398-199601000-00019 14. Zarezadeh A, Mamelson K, Thomas WC, Schoch BS, Wright TW, King JJ. Outcomes of distal humerus fractures: what are we measuring? Orthop Traumatol Surg Res 2018; 104: 1253-1258. doi: 10.1016/j.otsr.2018.08.017 15. Ko CC, Tai MH, Lin CH, Tzeng WS, Chen JH, Shu G, et al. Posteromedial olec- ranon impingement of the pitching elbow: additional findings provided by CT. Eur J Radiol 2016; 85: 211-217. doi: 10.1016/j.ejrad.2015.11.022 16. Lim YW, van Riet RP, Mittal R, Bain GI. Pattern of osteophyte distribution in primary osteoarthritis of the elbow. J Shoulder Elbow Surg 2008; 17: 963-6. doi: 10.1016/j.jse.2008.03.012 Radiol Oncol 2022; 56(1): 37-45. doi: 10.2478/raon-2022-0005 37 research article Efficacy of transvaginal ultrasound versus magnetic resonance imaging for preoperative assessment of myometrial invasion in patients with endometrioid endometrial cancer: a prospective comparative study Anis Cerovac1,2, Dzenita Ljuca2, Lejla Arnautalic3, Dubravko Habek4, Gordana Bogdanovic2,5, Jasminka Mustedanagic-Mujanovic2,6, Gordana Grgic2,5 1 Department of Gynaecology and Obstetrics, General Hospital Tešanj, Tešanj, Bosnia and Herzegovina 2 School of Medicine, University of Tuzla, Tuzla, Bosnia and Herzegovina 3 Clinic for Radiology and Nuclear Medicine, University Clinical Centre Tuzla, Tuzla, Bosnia and Herzegovina 4 University Department of Gynaecology and Obstetrics Clinical Hospital “Sveti Duh”, Zagreb, School of Medicine, Catholic University of Croatia, Zagreb, Croatia 5 Clinic for Gynaecology and Obstetrics, University Clinical Centre Tuzla, Tuzla, Bosnia and Herzegovina 6 Department for Pathology, Policlinic for Laboratory Diagnostic, University Clinical Center Tuzla, Tuzla, Bosnia and Herzegovina Radiol Oncol 2022; 56(1): 37-45. Received 03 09 2021 Accepted 13 12 2021 Correspondence to: Anis Cerovac, M.D., Department of Gynaecology and Obstetrics, General Hospital Tešanj, Tešanj, Bosnia and Herzegovina. E-mail: Cerovac.anis@gmail.com Disclosure: No potential conflicts of interest were disclosed. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Background. We compared the accuracy of preoperative transvaginal ultrasound (TVUS) versus magnetic reso- nance imaging (MRI) for the assessment of myometrial invasion (MI) in patients with endometrial cancer (EC), while definitive histopathological diagnosis served as a reference method. Patients and methods. Study performed at a single tertiary centre from 2019 to 2021, included women with a histopathological proven EC, hospitalized for scheduled surgery. TVUS and MRI were performed prior to surgical stag- ing for assessment MI, which was estimated using two objective TVUS methods (Gordon’s and Karlsson’s) and MRI. Patients were divided into two groups, after surgery and histopathological assessment of MI: superficial (≤ 50%) and deep (> 50%). Results. Sixty patients were eligible for the study. According to the reference method, there were 34 (56.7%) cases in the study with MI < 50%, and 26 (43.3%) with MI > 50%. Both objective TVUS methods and MRI showed no statistical significant differences in overall diagnostic performance for the preoperative assessment of MI. The concordance coefficient between both TVUS methods, MRI and histopathology was statistically significant (p < 0.001). Gordon’s method calculating MI reached a positive predictive value (PPV) of 83%, negative predictive value (NPV) of 83%, 77% sensitivity, 88% specificity, and 83% overall accuracy. Karlsson’s method reached PPV of 82%, NPV of 79%, 69% sensitiv- ity, 88% specificity, and 80% overall accuracy. Accordingly, MRI calculating MI reached PPV of 83%, NPV of 97%, 97% sensitivity, 85% specificity, and 90% overall accuracy. Conclusions. We found that objective TVUS assessment of myometrial invasion was performed with a diagnostic accuracy comparable to that of MRI in women with endometrial cancer. Key words: endometrial neoplasms; radiology; oncology; cancer staging Radiol Oncol 2022; 56(1): 37-45. Cerovac A et al. / Myometrial invasion in endometrial cancer38 Introduction Endometrial cancer (EC) is the most common ma- lignancy in the female genital tract in developed countries and its incidence is increasing.1-3 Due to the early occurrence of abnormal uterine bleed- ing, most cases are diagnosed at first stage, when prognosis is very good, with a 5-year survival rate of 90%.4 The EC prognosis is determined by the FIGO (International Federation of Obstetrics and Gynecology) stage of disease, the histological type and grade of tumour, the depth of myome- trial invasion (MI), cervical stromal invasion, and lymph nodes involvement.1-3 The depth of MI > 50%, which is considered to be one of the most im- portant prognostic factors, highly correlates with lymph node metastases.1-3 The preoperative assess- ment of the depth of MI is crucial to determining the most effective therapeutic approach and to decide whether the patient should be referred for hysterectomy with bilateral adnexectomy or pelvic lymphadenectomy is need.1-3 The depth of MI can be evaluated by a number of imaging methods. These are magnetic reso- nance imaging (MRI), computer tomography (CT) and transvaginal ultrasonography (TVUS), among which the best results can be achieved by the MRI.1,3 TVUS has been used extensively to assess depth of MI by EC.3 TVUS when carried out by experienced hands has been shown to perform equally well as MRI, in the preoperative staging of EC.4 Several studies, systematic review and meta- analyses compared subjective assessment and ob- jective TVUS measurements techniques2,5,6, TVUS (subjective assessment) and MRI3,7-11, three-dimen- sional TVUS and MRI12, and different MRI tech- niques.13-15 However, to the best of our knowledge this is the first study that compared two objective TVUS measurements techniques (Gordon’s and Karlsson’s method) with MRI on the same set of patients. The aim of our study was to determine the ac- curacy of preoperative TVUS versus MRI for the assessment of MI depth in EC patients, while de- finitive histopathological diagnosis served as a ref- erence method. Patients and methods Patients This prospective cohort study included 60 wom- en with a histopathological proven endometrioid EC by dilatation and curettage, hospitalized for scheduled surgery during the period between July 2019 and April 2021 at the Clinic for Gynaecology and Obstetrics, University Clinical Centre Tuzla. Inclusion criteria were women with a histopatho- logical proven endometrioid EC. Exclusion criteria were women with another malignant disease, who previously have surgery for EC or other malignant disease, who have previously received chemother- apy and/or radiotherapy due to a malignant dis- ease, women with a histopathological proven EC who preoperatively have made pelvic CT, cases that were diagnosed incidentally after hysterec- tomy. Patients were divided into two groups, after surgery and histopathological assessment of depth of MI: invasion to less or equal and to more than half the thickness of the myometrium. The sur- vey was approved by the Ethics Committee of the University Clinical Centre (No 02-09/2-2/20) Tuzla and signed informed consent of patients was ob- tained. FIGURE 1. Assessment of myometrial invasion in patients with transvaginal ultrasonography (TVUS). Stage IA endometrial cancer in a 61-year-old postmenopausal woman, correctly diagnosed by TVUS and MRI. Karlsson’s method indicating infiltration of superficial muscle (29%) (A). Stage IA endometrial cancer in a 53-year-old postmenopausal woman. Gordon’s method indicating infiltration of superficial muscle (35%) (B). A B Radiol Oncol 2022; 56(1): 37-45. Cerovac A et al. / Myometrial invasion in endometrial cancer 39 Methods Transvaginal ultrasonography All TVUS examinations were performed by a single ultrasound examiner (CA), certified for ultrasound diagnostics, on standardized study protocol con- taining all investigated sonographic parameters defined before the beginning of the study. TVUS was made using a Voluson E8® equipped with a 5–9-MHz two-dimensional transducer, within ten days before surgery, without insight into the MRI finding. We assessed of MI by two TVUS meth- ods, those proposed by Gordon et al. and Karlsson et al.2,5 Transvaginally, the whole uterus was ob- served in sagittal and transversal section.5 Depth of MI was measured as the ratio between the maximum AP diameter of the endometrial tu- mor (B) and the uterine AP diameter (A) in sagittal plane, with B/A > 50% indicating deep MI intro- duced by Karlsson et al. (Figure 1A, Figure 2A).2,5 In other objective method, investigated by Gordon et al., depth of MI was measured as the ratio of the distance between the maximum tumor depth (B) and the total myometrial thickness (A) in sag- ittal plane, with B/A > 50% indicating deep MI (Figure 1B, Figure 2B).2,5 The selected cut-off limit for the extent of MI (50%) followed FIGO staging classification from 2009.5 Static images with all measurements were col- lected for each patient and examination protocols were noted immediately during image acquisi- tion. Magnetic resonance imaging Abdominal and pelvic MRI examination was com- posed of T2 and T1-weighted images and dynamic contrast-enhanced fat-suppressed images at 1.5 Tesla Avanto Siemens Medical Systems® device, according to the dedicated MRI protocol of the European Society of Uro-Genital Radiologists since 2009 for the diagnosis of EC.11,16 All study partici- pants underwent abdominal and pelvic MRI ex- amination within 10 days prior to surgery. MRI protocol for accurate assessment of MI, based on T2-weighted images in three orthogonal planes oriented perpendicular and parallel to the uterine cavity (sagittal, axial, coronal and oblique axial), in axial and sagittal plane in T1-weighted images and T1-weighted contrast-enhanced fat- suppressed images.14,16 For optimal assessment of MI dynamic contrast-enhanced fat-suppressed MRI was done after intravenous bolus injection of 1 mg/kg of a paramagnetic contrast agent is admin- istered.14,16 The criteria for MRI MI diagnosis was disrup- tion and irregularity of the endomyometrial junc- tional zone.12 The ratio of the tumor to the endo- myometric junctional zone and the depth of MI to the junctional zone were determined. If the signal intensity of the tumour on T2W was greater than half, it was regarded as a deep MI.12 In order to measure the depth of MI on MRI, the line must be drawn along the expected inner edge of the myometrium (corresponding to the endomyometric junctional zone) on axial oblique plane obtained perpendicular to the endometrium; then, two measures should be taken: one repre- sents the thickness of the entire myometrium; the second is measuring the maximum range of tumor within the myometrium. The ratio of these meas- ures represents the percentage of MI. The ratio of the thickness of the tumor from the uterine cavity to the outer border and the total thickness of the FIGURE 2. Assessment of myometrial invasion in patients with transvaginal ultrasonography (TVUS). Stage IB endometrial cancer in a 78-year-old postmenopausal woman, correctly diagnosed by TVUS and MRI. Karlsson’s method indicating infiltration of deep muscle (58%) (A). Stage IB endometrial cancer in an 83-year-old postmenopausal woman. Gordon’s method indicating infiltration of deep muscle (73%) (B). A B Radiol Oncol 2022; 56(1): 37-45. Cerovac A et al. / Myometrial invasion in endometrial cancer40 myometrial wall on the side on which the tumor is located was calculated (Figure 3). The radiologist was not blinded to the diagnosis of EC but remained unaware of the TVUS results. All analyses have been performed by the same radiologist (LA), with experience in analysing ab- dominal and pelvic MRI. Surgical procedure Primary surgery was performed by an gynaeco- logical oncological surgeon with experience in gy- naecologic-oncological surgery in median ten days after MRI, and ten day after TVUS. Scheduled surgery was indicated based on preoperative histopathological diagnosis with tu- mour type and grade, TVUS and MRI. The sur- geon was not blinded towards the histopathologi- cal diagnosis, TVUS and MRI results.11 Surgery was performed by an open abdominal approach and according to the decision of the gynaecologic- oncological interdisciplinary tumour board. The surgical approach was planned based on the FIGO classification derived from MRI.11 The depth of MI obtained by TVUS and MRI has been correlated to help shed light on the dilemma of whether classical hysterectomy with bilateral adnexectomy is suffi- cient or pelvic lymphadenectomy is necessary for oncological treatment to be adequate. Histopathological diagnosis Surgical specimens were examined by patholo- gists with experience in gynaecologic oncology using a predetermined protocol regarding: histo- logical subtype, grade, lymphovascular invasion, tumour size, depth of myometrial invasion, mini- mal tumour-free myometrium, presence of cervical stromal invasion, presence, location and number of fibroids.5 The FIGO 2009 criteria were applied for clinical staging. The “gold standard” was based on final histology of the specimen obtained by hyster- ectomy.5 Statistical analysis Basic tests of descriptive statistics were made, showing the measures of central tendency and dispersion (Table 1). Sensitivity, specificity, posi- tive predictive value (PPV) and negative predictive value (NPV), overall accuracy, likelihood ratio of a positive test, likelihood ratio of a negative test, as well as receiver operating characteristics (ROC) FIGURE 3. Assessment of myometrial invasion in patients with MRI, sagittal T2- weighted. Stage IA endometrial cancer in a 68-year-old postmenopausal woman, correctly diagnosed by TVUS and MRI. Arrow shows blurring of the junctional zone and the infiltration of superficial muscle (20%) (A). Stage IB endometrial cancer in a 58-year-old postmenopausal woman, correctly diagnosed by TVUS and MRI. Arrows refers to deep myometrial infiltration (60%) (B). TABLE 1. Demographic and clinical characteristics of 60 women with histologically confirmed endometrial cancer Characteristics Value (%) Age (years) 60 (40–83) Body mass index (kg/m²) 32.05 (21.6–49.2) Postmenopausal 45 (75) Myometrial invasion Superficial (≤ 50 %) 34 (56.7) Deep (> 50%) 26 (43.3) Hystological grade Grade 1 18 (30) Grade 2 35 (58) Grade 3 7 (12) Data are given as median (5th percentile; 95th percentile) for continuous variables; n (%) for categorical variables. A B Radiol Oncol 2022; 56(1): 37-45. Cerovac A et al. / Myometrial invasion in endometrial cancer 41 curves assessing MI were calculated for each stag- ing method (TVUS; Gordon’s and Karlsson’s meth- od and MRI) in comparison to the final histology. Receiver operating characteristic (ROC) curves were used to evaluate the accuracy of the tests. Statistical processing was done in the software package SPSS 24.0 (Chicago, IL, USA). All statisti- cal tests were performed with a statistical probabil- ity level of 95% (p < 0.05). Results Of 72 cases diagnosed with endometrioid EC dur- ing the study period and who underwent preoper- ative TVUS and MRI, 60 cases were enrolled in the study, of mean (SD) age 60 (10) in range from 40–83 years. Forty-five (75%) out of the 60 were postmen- opausal. Median body mass index was 32.05 kg/m2 (range 21.6-49.2). The most frequently encountered histological grade was grade 2 (58%, 35/60). Twelve cases (16.6%) evaluated during the study period were excluded from the database, because of preoperatively made CT (morbid obesity and/or claustrophobia). The patient demographics and tumour charac- teristics are summarized in Table 1. Regarding final surgical procedure, 60 cases (100%) underwent open surgery. Hysterectomy with bilateral adnexectomy was performed in 60 cases (100%). Pelvic lymphadenectomy was per- formed in 32 cases out of 60 (53.3%). Pelvic lym- phadenectomy was performed in eight cases out of 36 (22.2%) with MI ≤ 50%, in 24 cases out of 26 (92.3%) with MI > 50%, that is statistically signifi- cant frequently in group with MI > 50% (z = -5.29, p < 0.00001). According to the gold standard, histopathologi- cal diagnostics, there were 34 (56.7%) cases in the study with MI ≤ 50%, and 26 (43.3%) with MI > 50%. The depth of MI was correctly assessed by Gordon’s method in 50 (83.3 %) cases, overestimat- ed in four (6.6%) and underestimated in six (10%) (Table 2). The concordance coefficient between TVUS and histopathology was also statistically significant (p < 0.001) and kappa was 0.658. These data cor- responded to a PPV of 83% and NPV of 83%, 77% sensitivity, 88% specificity, and 83 % overall accu- racy (Table 3). TABLE 2. Myometrial invasion in endometrial cancer according to histopathology, transvaginal ultrasonography and magnetic resonance imaging Histopathology TVUS (Gordon) TVUS (Karlsson) MRI Total N(%)≤ 50% N(%) > 50% N(%) ≤ 50% N(%) > 50% N(%) ≤ 50% N(%) > 50% N(%) ≤ 50% N (%) 30 (88.2) 4 (11.8) 30 (88.2) 4 (11.8) 29 (85.3) 5 (14.7) 34 (100) > 50% N(%) 6 (23.1) 20 (76.9) 8 (30.8) 18 (69.2) 1 (3.8) 25 (96.2) 26 (100) Total 36 (60) 24 (40) 38 (63.3) 22 (36.7) 30 (50) 30 (50) 60 (100) TVUS = transvaginal ultrasound TABLE 3. Diagnostic performance of transvaginal ultrasonography and magnetic resonance imaging in predicting myometrial invasion in endometrial cancer Diagnostic test measure TVUS (Gordon) TVUS (Karlsson) MRI %, (95% CI) %, (95% CI) %, (95% CI) Accuracy 83 (70–92) 80 (67–88) 90 (78–93) Sensitivity 77 (62–87) 69 (54–79) 97 (83–99) Specificity 88 (77–96) 88 (77–96) 85 (75–88) Positive predictive value 83 (67–94) 82 (64–93) 83 (72–87) Negative predictive value 83 (72–90) 79 (69–86) 97 (85–99) Likelihood Ratio of a Positive Test 6.54 (2.65–19.00) 5.89 (2.30–18.17) 6.54 (3.31–8.37) Likelihood Ratio of a Negative Test 0.26 (0.14–0.50) 0.35 (0.22–0.60) 0,05 (0.002–0.23) The estimates are stated along with the 95% confidence intervals (95% CI); TVUS = transvaginal ultrasound Radiol Oncol 2022; 56(1): 37-45. Cerovac A et al. / Myometrial invasion in endometrial cancer42 The depth of MI was correctly assessed by Karlsson’s method in 48 (80%) cases, overestimated in four (6.6%) and underestimated in eight (13.3%) (Table 2). The concordance coefficient between TVUS and histopathology was statistically signifi- cant (p < 0.001) and kappa was 0.585. Accordingly, Karlsson’s method calculating MI reached PPV of 82% and NPV of 79%, 69% sensitivity, 88% speci- ficity, and 80% overall accuracy (Table 3). MRI correctly assessed MI in 54 (90%) cases, overestimated it in five (8.3%) cases and under- estimated it in one (1.6%) (Table 2). The concord- ance coefficient between MRI and histopathology was statistically significant (p < 0.001) and kappa was 0.80. Accordingly, MRI calculating MI reached PPV of 83% and NPV of 97%, 97% sensitivity, 85% specificity, and 90% overall accuracy (Table 3). The diagnostic performance of two objective transvaginal ultrasonography methods assessment and MRI calculations in predicting deep MI as well as the statistical comparison of ultrasonography to MRI method are introduced in Table 3. Differences in the performance of the two TVUS imaging modalities (Karlsson vs. Gordon) were not statistically significant (p = 0.867). Differences in the performance between the Gordon’s meth- od and MRI were not statistically significant (p = 0.417). Differences in the performance between of the Karlsson’s method and MRI were not statisti- cally significant (p = 0.464). The prevalence of myometrial pathology was the same in the incorrectly classified patients in TVUS and MRI methods of assessment of MI. Of the 10 patients in whom infiltration was misclassi- fied on TVUS and of the 6 patients misclassified on MRI, 5 (50%) and three (50%) had benign myome- trial pathologies (adenomyosis and leiomyoma), respectively. TVUS and MRI correctly estimated MI in 45 (75%) patients, underestimated it in one (1.6%) and overestimated it in two (3.3%). If we consider only the cases in which the two techniques were in agreement (48 cases), the concordance with histol- ogy was 80%. Evaluation of diagnostic accuracy was per- formed through Receiver Operating Characteristics (ROC) analysis and for percentage assessment of MI with TVUS by Gordon’s and Karlsson’s meth- od, and with MRI. A graphical representation of this analysis is given in Figure 4, and a tabelar representation of the areas below the ROC curve (AUC) in the Table 4. As can be seen, the best diagnostic accuracy ac- cording to the ROC analysis had MRI with an AUC of 0.911 (total accuracy 91.1%), which is in correla- tion with the already performed diagnostic accu- racy analyses. Discussion In this prospective, comparative, ultrasonogra- pher-blinded study on patients with EC two ob- jective TVUS methods (Gordon’s and Karlsson’s) and MRI were compared for the MI assessment in the same cohort of patients. All three tested ap- proaches were found to be statistically significant predictors of the MI, exceeding AUC value of 0.85 and reaching final p value < 0.001. We found that MRI assessment of MI is better than any objective TVUS measurement technique in all measures of the diagnostic tests, but without statistically sig- nificance. FIGURE 4. Receiver operating characteristics (ROC) analysis for percentage assessment of myometrial invasion (MI) with transvaginal ultrasound (TVUS) by Gordon’s and Karlsson’s, and with MRI TABLE 4. Representation of the areas below the receiver operating characteristics (ROC) curve (AUC) for percentage assessment of MI with transvaginal ultrasound (TVUS) by Gordon and Karlsson, and with MRI Method of assessment AUC P 95% CI for AUC Lower limit Upper limit TVUS (Gordon) 0.872 < 0.001 0.769 0.965 TVUS (Karlsson) 0.865 < 0.001 0.766 0.964 MRI 0.911 < 0.001 0.833 0.989 Radiol Oncol 2022; 56(1): 37-45. Cerovac A et al. / Myometrial invasion in endometrial cancer 43 Median age in our patients is similar to Pineda et al. study17, 60 vs. 60.9, respectively, however there are studies with higher1,4-6,8, and lower median age.12,18 Postmenopausal in recent study were 75% pa- tients which correlate with other studies, where postmenopausal patients were in the range from 70.2% to 92%.5-10,15,17,19 EC is disease of older and postmenopausal women, that was also confirmed by our study.3 Patients in recent study were obese with an me- dian body mass index (BMI) of 32.05 which is simi- lar to median BMI of 31 in Rei et al. study8, however there are studies with higher7,15 and lower median BMI.4-6,17,19 Obesity is a proven risk factor for de- veloping EC, what was also confirmed by recent study.⁸ The most frequently encountered histological grade in recent study was grade 2 (58%) which is the same percentage as in Karatasli et al. study.15 In most of the reviewed studies, histological grade 1 is more common than other grades which agrees with the fact that endometrial cancer is usually a well-differentiated.1,5-7,10,14,17 In current study according to the gold standard, histopathological diagnostics, it is more common superficial MI (≤ 50%), which correlate with most reviewed studies and with fact that EC is detected at an early stage in most cases.4-8,10-12,14,15,18,22,23 Several studies evaluated objective measure- ments such as those proposed by Gordon et al. and Karlsson et al.2,3 Alcazar et al. in systematic review and meta- analysis found that the overall diagnostic perfor- mance of TVUS for Karlsson’s and Gordon’s meth- od in detecting deep MI in women with EC gave a pooled sensitivity of 84 % and 85 %, pooled speci- ficity of 82% and 80% which is higher sensitivity and lower specificity than in our study for both methods.2 They observed that both methods were similar, without statistical differences, in terms of diagnostic performance, similar as in our study.2 In reviewed studies diagnostic performance for Gordon’s method reached sensitivity from 69.6% to 92.3%, specificity from 65.9% to 79.2%, PPV from 56.7% to 61%, NPV from 77.1% to 96.1% and over- all accuracy from 67.3% to 82.6%.1,5 Recent studies reported for Karlsson’s method sensitivity from 56.3% to 86.8%, specificity from 64.4% to 76.4%, PPV from 62.8% to 83.6%, NPV from 70% to 71.2% and overall accuracy 68.1%.5,6,19,21 Besides, current study did not find statisti- cal differences between Gordon’s and Karlsson’s method. Although both TVUS objective calcula- tions, Gordon’s and Karlsson’s method, had simi- lar accuracy, the approach published by Gordon et al. have better sensitivity and accuracy in preop- erative assessment of MI in EC. However, in our opinion Gordon’s method might be more difficult for assessment of MI.5 On the other side, under or overestimation of MI by Karlsson’s method is often caused by large polypoid EC, submucosal leiomyomas and adenomyosis which make longer anterio-posterior uterine diameter.5 In 2017 Alcazar et al. published a systematic re- view and meta-analysis based on preoperative de- tection of deep MI comparing TVUS and MRI on the same set of women.3 However, they found out that sensitivity and specificity for diagnosing deep MI were 75% and 82% for TVUS, and 83% and 82% for MRI, respectively. MRI showed a better sensitivity than TVUS for detecting depth of MI in women with EC, but without statistical differences, as in our study.3 However, none of these studies compared the three imaging methods altogether in one cohort of patients as is the case in our compara- tive study. In analysed studies diagnostic performance for MRI reached sensitivity from 70% to 92.6%, speci- ficity from 71% to 95%, PPV from 65% to 92.2%, NPV from 70% to 98%, and overall accuracy from 74% to 89%.7-10,12-15,20-22 We have shown that two imaging modali- ties (contrast-enhanced MRI and TVUS) perform equally well in the assessment of MI, differences in the performance were not statistically significant. Cubo-Abert et al. obtained similar results as in our study, when it comes to the diagnostic accu- racy of TVUS versus MRI in estimating the depth of MI, with the difference that they used the TVUS method based on the measurement of the minimal distance to the uterine serosa.24 Costas et al. in systematic review and meta- analysis have not found a satisfying number of studies about the comparison of 2D-TVUS and 3D-TVUS methods for MI assessment in EC.25 They have identified lack of knowledge and studies re- garding the objective methods used for 3D-TVUS assessment and their comparison with subjective 3D-TVUS methods.25 Although the definitive staging of endometrial cancer is based on histopathology, an accurate pre- operative assessment of MI by TVUS and/or MRI provides the opportunity for surgical planning to provide an adequate type of surgery, the need of a multidisciplinary team, time management in the operating room, and avoid morbidity associated with unnecessary lymphadenectomy.8,9,23 Radiol Oncol 2022; 56(1): 37-45. Cerovac A et al. / Myometrial invasion in endometrial cancer44 Causes of over or understaging were similar for the TVUS and MRI: a polypoid EC, large exo- phytic tumors with distension and thinning of the myometrium with regular endometrial junction and without MI, fibromatosis, adenomyosis, leio- myomata, deep MI, small isolated glandular foci, uterine anomalies, uterine prolapse or retrover- sion, short time after previous endometrial biopsy, poorly defined endometrial borders on TVUS, and disappearance of the junctional zone on MRI.1,6,7,9,19 Considering that there are no statistically sig- nificant differences in diagnostic performance be- tween TVUS and MRI and the cost and availability of MRI, TVUS may have a role as the first imaging technique for assessing MI in women with EC, es- pecially as it is implemented in the everyday prac- tice of gynecologists.1,2,4,8 As Miklos et al. concluded the diagnostic ac- curacy of the TVUS depends more on the indi- vidual experience and professional potential of the examiner than diagnostic accuracy of the MRI.1 Examiner experience, technological advances and different protocols for assessing MI among the studies, for both TVUS and MRI can contribute to the heterogeneity of published results of TVUS and MRI in the assessment of MI.1,3,8 Expert TVUS and MRI were comparable and superior to non-expert TVUS for assessing MI in EC.26 MRI, which is more expensive, time consuming and difficult to access, could be employed as a sec- ond-line imaging technique in patients in whom TVUS gives images of poor quality in case of obe- sity and factors above mentioned that may cause over or understaging.9 MRI is currently recommended for preoperative imaging in some guidelines, as the imaging mo- dality of choice and most appropriate for the as- sessment of disease extent in patients with newly diagnosed EC.3,15,20 Iitsuka et al. in their study and review of litera- ture found that their data and the pooled analysis with previous studies indicate that the frozen sec- tion diagnosis is sensitive as MRI assessment in predicting deep MI, and has a higher specificity compared with MRI.27 Contraindications for MRI should also be kept in mind, when MRI cannot be performed such as metal foreign body, pathologic obesity, contrast al- lergies and claustrophobia. The main strength of our study is that, to the best of our knowledge, this is the first study that com- pares prospectively the two objective TVUS meth- ods (Gordon’s and Karlsson’s) and objective MRI method, for assessing MI in patients with endome- trioid EC in the same set of patients. A strength of our study are the prospective design and the fact that the gynaecologists performing TVUS and the radiologists performing MRI were blinded to each other’s results. Another strength is that all TVUS assessment of MI were made by the same physi- cian, MRI assessment of MI by the same radiolo- gist, and all surgeries and pathologic examinations were done at the same center. Strength of our study is also that we compared the three imaging meth- ods altogether in one cohort of patients which is ideally, for comparing the diagnostic performance of different approaches to assess MI, and this is rar- ity in reviewed studies. Limitations of our study are the relatively small number and short time of collection of participants, but this can be justified by the prospective design of the study, the strict inclusion and exclusion cri- teria, and in fact that the study was performed in the largest tertiary center in the country. Conclusions In conclusion, we found that objective TVUS as- sessment of myometrial invasion was performed with a diagnostic accuracy comparable to that of MRI in women with endometrial cancer. Further multi-centric studies with prospective designs and standardized protocols are needed to investigate which objective measurement techniques and MRI have the highest reproducibility, and how well they perform in the hands of examiners with more or less experience. In addition, studies evaluating inter-observer agreement as well as the impact of TVUS training would be of great interest. Acknowledgments This article is a part of research conducted within the doctoral dissertation of Anis Cerovac. We are thankful to personnel of the Clinic for Gynecology and Obstetrics, Clinic for Radiology and Nuclear Medicine and Policlinic for Laboratory Diagnostics, University Clinical Center Tuzla for their cooperation. We give our deep appreciation to all women who participated in this trial because with their participation they can help women with endome- trial cancer, but also offer new diagnostic approach and opportunities for future generations. Radiol Oncol 2022; 56(1): 37-45. Cerovac A et al. / Myometrial invasion in endometrial cancer 45 References 1. Miklos P, Klacko M, Babala P, Masak L, Ondrus D, Waczulikova I. Transvaginal ultrasound examination of myometrial infiltration by endometrial cancer. Bratisl Lek Listy 2014; 115: 14-18. doi: 10.4149/bll_2014_003 2. Alcázar JL, Orozco R, Martinez-Astorquiza Corral T, Juez L, Utrilla-Layna J, Mínguez JA, et al. 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Received 3 August 2021 Accepted 24 November 2021 Correspondence to: Haiyi Wang M.D., Department of Radiology, First Medical Centre, Chinese PLA General Hospital, No. 28 Fuxing Road, Haidian District, Beijing 100853, China. E-mail: wanghaiyi301@outlook.com Jing Zhang and Xu Bai contributed equally. Disclosure: No potential conflicts of interest were disclosed. This is an open access article under the CC BY-NC-ND license (http://creativeco mmons.org/licenses/by-nc-nd/4.0/). Background. Bladder paraganglioma (BPG) is a rare extra-adrenal pheochromocytoma with variable symptoms and easy to be misdiagnosed and mishandled. The aim of the study was to document the imaging features of BPG using computed tomography (CT) and magnetic resonance imaging (MRI). Patients and methods. We retrospectively enrolled consecutive patients with pathology-proven BPG, who under- went CT or MRI examinations before surgery between October 2009 and October 2017. The clinical characteristics, CT, and MRI features of the patients were described and analysed. Results. A total of 16 patients with 16 bladder tumours (median age 51 years, 9 females) were included. Among them, 13 patients underwent CT examinations and eight patients underwent MRI examinations preoperatively. Tumour diameters ranged from 1.6−5.4 cm. M ost of the tumours grew into the bladder cavity (n = 11) with oval shapes (n = 10) and well-defined margins (n = 14). Intratumour cystic degeneration or necrosis (n = 2) was observed. Two lesions showed peripheral tissue invasion, suggesting malignant BPGs. All 13 lesions imaged with CT exhibited slight hypoat- tenuation and moderate to marked enhancement. Compared to the gluteus maximus, all lesions showed slight h yperintensity in T2-weighted images, hyperintensity on diffusion-weighted images (DWI), hypointensity on apparent diffusion coefficient maps, hyperintensity on T1-weighted images and a “fast in and slow out” enhanced pattern on contrast-enhanced MRI images. Conclusions. BPGs are mostly oval-shaped, broadly-based and hypervascular bladder tumours with hypoattenua- tion on non-contrast CT, T2 hyperintensity, slight T1 hyperintensity compared to the muscle, marked restricted diffusion on DWI. Peripheral tissue invasion can suggest malignancy of the BPGs. All of these features contribute to preoperative decision-making. Key words: p araganglioma; urinary bladder; computed tomography; magnetic resonance imaging Introduction Paragangliomas are rare neoplasms of extra-ad- renal chromaffin cells that belong to the family of neuroendocrine tumours.1 They account for 15– 20% of pheochromocytomas and occur in a variety of locations such as the head and neck, paraspinal region, chest, abdomen, and pelvis. The bladder is the most common site of paragangliomas in the genitourinary system (79.2%).2 B ladder paragangliomas (BPGs) are exceed- ingly rare and constitute only 0.06% of bladder tu- mours and 1% of all paragangliomas.3 BPG is an extra-adrenal pheochromocytoma that arises from t he chromaffin tissue of the sympathetic nervous system associated with the bladder wall and has Radiol Oncol 2022; 56(1): 46-53. Zhang J et al. / CT and MR imaging features of bladder paraganglioma 47 the potential to secrete catecholamines (norepi- nephrine, epinephrine, and dopamine).2 The surgi- cal preparation and procedure therefore needs to be effectively formulated to avoid life-threatening malignant cardiovascular events caused by a burst release of catecholamines.4 D ue to its rarity and symptomatic variability5, the disorder can easily be misdiagnosed and mishandled. Thus, accurate preoperative diagnosis is crucial. In the present study, a series of 16 patients with BPG treated in our institution over an eight-year period were retrospectively reviewed and the com- puted tomography (CT) and magnetic resonance imaging (MRI) characteristics of BPG were ana- lysed. Patients and methods The study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of The First Medical Center of Chinese PLA General Hospital. Written informed consent was obtained from all patients. A computerized search of the hospital’s pathology databases for BPGs from October 2009 to October 2017 was performed. During this period, there were 29 adult patients (age > 18 years) with 29 pathology-proven BPGs. This list of patients was then cross referenced with the institutional radiol- ogy database to identify the patients who under- went preoperative pelvic CT and/or MRI examina- tions. This yielded 16 patients with BPGs, which comprised our study population. Among them, 13 underwent contrast-enhanced CT examinations, eight underwent non-enhanced (n = 1) and dynam- ic contrast-enhanced (n = 7) MRI examinations, and five underwent both CT and MR preoperative ex- aminations. C T examinations were performed with GE Light Speed 16-row scanner (Milwaukee, WI, USA) (n = 3), Siemens Somatom 64-row scanner (Erlangen, Germany) (n = 5), Siemens Sensation Cardiac 64-row scanner (Erlangen, Germany) (n = 3) and GE Optima CT660 128-row spiral scan- ner (Milwaukee, WI, USA) (n = 2). All examina- tions were performed using similar scanning pa- rameters with a slice thickness of 5 mm, 1.5 mm reconstruction, 120 kVp, and 500 mA. Nonionic contrast agents (U ltravist, Bayer HealthCare, Berlin, Germany; I ohexol, Beilu, Beijing, China) were used, dose 90–100 ml, injection rate 3.5 ml/s. Contrast-enhanced CT images were only acquired in the arterial phase (30~35s after contrast agent injection). The patients fasted for 4–6 h and were FIGURE 1. CT and MR images of a 61-year-old male patient with bladder paraganglioma. The tumour was located in the posterior bladder wall, oval, well-defined margin, protruding into the bladder cavity with broad-base attachment to the bladder wall (short arrow). The lesion showed slight hypodensity and obvious enhancement on axial pre- and post-contrast-enhanced CT images (A, B), homogenous slight hyperintensity on T2-weighted images (T2WI) (C), marked hyperintensity on diffusion-weighted images (DWI) (D), hypointensity on apparent diffusion coefficient (ADC) maps (mean ADC value, 0.870 × 10-3 mm2/s) (E), hyperintensity compared to the gluteus maximus on T1-weighted images (T1WI) (F) and “fast in and slow out” on dynamic contrast-enhanced MRI (G, H). A B C G H D E F Radiol Oncol 2022; 56(1): 46-53. Zhang J et al. / CT and MR imaging features of bladder paraganglioma48 given water 30 min before the examination. The acquisition was performed with the bladder full of urine, from the inferior symphysis pubis to the apex of the bladder. MRI examinations were performed with GE Signa Excite 1.5T and 3.0T MR imaging system (Milwaukee, WI, USA) (n = 5), and GE Signa HDxt 3.0T MR imaging system (Milwaukee, WI, USA) (n = 3). Patients were imaged in the supine posi- tion using an eight-channel surface phased-array coil. Pelvic MRI sequences and parameters were as follows: axial, sagittal and coronal fast spin echo (FSE) T2-weighted images (T2WI): repetition time (TR) / echo time (TE), infinite / 90–105 ms; field of view (FOV), 36×44cm; slice thickness / interlayer distance, 5.0 mm / 0.5 mm; and matrix, 320 × 224. Diffusion-weighted images (DWI) using single- shot echo planar imaging (SE-EPI): b values, 0 / 800 s/mm2; TR / TE, 2500–5000 / 60–65 ms; FOV, 36×44 cm; slice thickness / interlayer distance, 5.0 mm / 0.5 mm; and matrix, 128×128. Axial gradient recalled echo (GRE) T1-weighted images (T1WI): TR / TE, 3.2–4.5 / 1.5–2.2 ms; FOV, 36×44 cm; slice thickness / interlayer distance, 5.0 mm / −2.5 mm; and matrix, 288 × 224. Dynamic contrast enhanced T1WI was performed in the axial plane during the arterial phase, venous phase, and delayed phase. Gadobenate dimeglumine (MultiHance; Bracco TABLE 1. Clinical characteristics of patients with bladder paraganglioma Characteristics Number (%)of patients Median age in years (interquartile range) 51 (40, 63) Sex Male 7 (43.8) Female 9 (56.2) Clinical manifestations Postmicturition syndrome* 6 (37.5) Hypertension 3 (18.8) Hematuria or progressive dysuria 3 (18.7) 24-h urinary VMA and CA level Not measured 11 (68.8) Normal 4 (25.0) Elevated 1 (6.2) Tumor number Single 14 (87.5) Multiple 2 (12.5) Surgical approach Partial cystectomy 7 (43.8) Local resection of bladder tumor** 9 (56.2) Imaging methods Computerized tomography 13 (81.3) Magnetic resonance imaging 8 (50.0) CA = catecholamine; VMA = vanillylmandelic acid; * postmicturition syndrome includes symptoms of catecholamine release such as sweating, palpitations, headaches, hypertension and syncope; ** local resections include transurethral laser and electric resection of bladder tumor FIGURE 2. CT and MR images of a 47-year-old female with malignant bladder paraganglioma. The bladder tumour was located in the inferior bladder wall with an irregular shape and ill-defined margin, invading the adjacent tissues (short arrow). The tumour showed iso-density and moderate enhancement on sagittal pre- and post-contrast enhanced CT images (A, B), inhomogenous hyperintensity on sagittal T2-weighted images (T2WI) (C), hyperintensity on diffusion-weighted images (DWI) (D), hypointensity on apparent diffusion coefficient (ADC) maps (mean ADC value, 0.852 × 10-3 mm2/s) (E), inhomogenous slight hyperintensity compared to the gluteus maximus on T1-weighted images (T1WI) (long arrow) (F), heterogenous marked enhancement on arterial phase (G) and coronal contrast-enhanced images (arrowhead) (H). In addition, a uterine fibroid on the posterior wall of the uterus was also found (asterisk on sagittal T2WI). A B C G H D E F Radiol Oncol 2022; 56(1): 46-53. Zhang J et al. / CT and MR imaging features of bladder paraganglioma 49 Sine, Shanghai, China) was injected intravenously at a dosage of 0.1 mmol/kg as a rapid bolus in- jection at a rate of 2 mL/s with a power injector (Spectris Solaris EP, Medrad, Indianola, PA, USA), followed by a 20 mL saline flush. Two experienced abdominal radiologists (Huiyi Ye and Haiyi Wang, with 30 and 20 years of experi- ence, respectively) at our institution reviewed the CT or MR image characteristics of each lesion, and reached a consensus on imaging analysis. The im- aging parameters observed included the location, size, shape, margin, density / signal intensity, en- hancement pattern, cystic degeneration/necrosis, haemorrhage, and calcification. On pre- / post-con- trast enhanced CT and MR images, the density and signal intensity relative to the gluteus maximus at the same layer were measured. The area of interest (ROI) was determined based on avoiding cystic de- generation and necrosis in the lesion. The mean ap- parent diffusion coefficient (ADC) value was meas- ured by drawing a ROI over the most hypointense area within the tumour on the ADC maps Blind to the image findings, all specimens were reviewed by an experienced uropathologist (Jing Yuan with 15 years of experience) according to the World Health Organization Classification of Tumours of Endocrine Organs.6 TABLE 2. Location and morphological characteristics of bladder paraganglioma Characteristics Number (%)of patients Mean maximum diameter of tumour (cm)* 2. 6 ± 1.0 Location Anterior wall 3 (18.7) Posterior wall 4 (25.0) Left wall 1 (6.3) Right wall 3 (18.7) Dome 2 (12.5) Bottom 3 (18.8) Spatial relationship with the bladder wall Protruding into the bladder cavity 11 (68.7) Protruding into the pelvic cavity 1 (6.3) Protruding into the bladder and pelvic cavities 4 (25.0) Morphological characteristics Oval 10 (62.5) Lobulated 4 (25.0) Fusiform 2 (12.5) Tumor margin Well-defined 14 (87.5) Ill-defined 2 (12.5) * Data are means ± standard deviations FIGURE 3. CT and MR images of a 25-year-old female with malignant bladder paraganglioma. The tumour was located in the left bladder wall with irregular shape, presenting heterogenous hypodensity (short arrow) and obvious enhancement on axial pre- and post-contrast enhanced CT images (A, B), heterogenous slight or marked hyperintensity on T2-weighted images (T2WI) (C), heterogenous hyperintensity on diffusion-weighted images (DWI) (long arrow) (D), hypointensity on apparent diffusion coefficient (ADC) maps (mean ADC value, 0.997×10-3 mm2/s) (E), slight hyperintensity compared to the gluteus maximus on T1WI (F) and early marked enhancement on arterial phase images (G). Coronal enhanced MRI showed the lesion encased the left iliac artery branch (arrowhead); a similar enhanced lesion was located next to the left iliac vessels (asterisk), suggesting multiple paraganglioma (H). A B C G H D E F Radiol Oncol 2022; 56(1): 46-53. Zhang J et al. / CT and MR imaging features of bladder paraganglioma50 Results Among the 16 patients enrolled (median age 51 years, interquartile range 40–63; 9 female), 12 pa- tients had preoperative clinical symptoms, includ- ing six with typical symptoms related to micturi- tion before surgery (increased blood pressure, headache, dizziness, pale complexion, chest tight- ness, palpitations, hyperhidrosis, abdominal pain), three had a long history of hypertension, two had haematuria and one had a progressive dysuria (tumour close to the trigone). The 24 h urinary vanillylmandelic acid (VMA) and urinary catecho- lamine concentrations were measured for five of the patients with typical symptoms, and only one patient had an elevated VMA concentration (429 nmol / 24 h; standard range, 59.1–266 nmol / 24 h). Twelve patients were accurately diagnosed as BPG FIGURE 4. Enhancement trend charts of bladder paragangliomas on CT and MR images. (A) Broken line graph of enhancement trend on CT images; (B) Box plot of density distribution on pre- and post-contrast enhanced CT images (arterial phase); (C) Broken line graph of dynamic enhancement trend on MR images; (D) Box plot of signal distribution on unenhanced and dynamic contrast- enhanced MR images. (five of them took phenoxybenzamines for 1 to 2 weeks before surgery), and the remaining four pa- tients were misdiagnosed preoperatively because of negative hormonal activity or atypical manifes- tation. Based on pathological confirmation, mul- tiple paraganglioma was found in two cases (one with two lesions located in the bladder wall and the left adrenal gland and the other with two le- sions located in the bladder wall and next to the left iliac vessels). (Table 1) The maximum diameters of the BPGs ranged from 1.6 to 5.4 cm. The lesions were distributed in different locations of the bladder wall, and most of them grew into the bladder cavity (11/16) with broad-based attachment to the wall. The tumours mostly exhibited oval in shape (10/16) and well- defined margins (14/16) (Table 2). One of the two BPGs with ill-defined margins invaded the upper middle urethra and the anterior wall of the vagina A B C D Radiol Oncol 2022; 56(1): 46-53. Zhang J et al. / CT and MR imaging features of bladder paraganglioma 51 while the other encased the left iliac vein branch. (Figure 1–3) On non-contrast CT images, the lesions mainly demonstrated homogeneous and soft-tissue den- sity, with CT values ranging from 19.9 to 55.2 Hounsfield Units (HU). Intra-tumoural cystic de- generation or necrosis was rare (2/16). All lesions showed moderate to marked enhancement in the arterial phase of contrast-enhanced CT images (Table 3), with CT values of 64.3–117.9 HU, which were about 2.3 times that of the CT value on pre- contrast enhanced images (Figure 4A–B). In the two patients with multiple paragangliomas, the density and enhancement pattern of lesions in the non-bladder sites were similar to those in the blad- der. On T2WI, the lesions demonstrated homoge- nous hyperintensity (10/16), higher than the gluteus maximus and lower than the urine in the bladder, without typical “pepper and salt” sign. Due to the restricted diffusion, the lesions showed hyperin- tensity on DWI and hypointensity on ADC maps (mean ADC value ± standard deviation, 0.883 ± 0.126×10-3 mm2/s). On T1WI, the lesions showed hyperintensity and averaged 1.4 times higher than that of the gluteus maximus at the same layer. Following MRI enhancement, the BPGs all had ob- vious enhancement in the arterial phase (an aver- age of 2.5 times higher than the tumour signal in- tensity on T1WI), slightly decreased enhancement in the venous phase and the delayed phase (an av- erage of 2.4 and 2.0 times higher than the tumour signal intensity on T1WI, respectively), exhibiting a “fast in and slow out” enhanced pattern (Table 3, Figure 4C–D). Similar to CT findings, the two pa- tients with multiple paragangliomas showed com- parable MRI findings between the bladder and non-bladder lesions. Pathological examination revealed that the tumours were of varying sizes, nodular, with a greyish-tan cut surface and medium hardness. Microscopic examination showed polygonal or ov- al tumour cells that were rich in basophil granular cytoplasm and arranged in a sheet or organoid ar- rangement, which had abundant sinuses and capil- laries. Immunohistochemical staining showed that the tumour cells were positive for chromogranin A (Figure 5). Discussion BPG is a rare ne uroendocrine neoplasm with po- tential hormonal activity derived from the medul- lary tissue of the bladder wall sympathetic nervous system.7 Better surgical preparation and effective perioperative anaesthetics management is required to avoid potentially fatal consequences, such as hy- pertensive crisis. In this study, the clinical and im- aging characteristics of 16 patients with BPG were documented and analysed. Several typical BPG symptoms caused by cat- echolamine release, such as hypertension, head- ache, palpitations, and perspiration, were present in 83% of BPG patients.8 Because of the special location of BPGs, the above symptoms may be caused by over distension of the bladder, micturi- tion or defecation.7 In this study, 75% of patients had typical symptoms, which was consistent with previous literature. Similar to other paraganglio- mas, BPGs can usually be diagnosed by biochemi- cal tests, such as 24 h urine measurements of cat- echolamines or metabolites. However, some BPGs showed no biochemical abnormalities, as were the lesions in this study, which may be due to the small size of the tumour at diagnosis or the transient hor- mone-release during micturition.2 BPGs have the predilection age of 30–50 years with no gender difference. Primary lesions can occur in any part of the bladder wall and mainly have cavity growth with a size ranging from 1.2– 5.0 cm.9 The ages and tumour sizes of the 16 pa- tients were compatible with the reported informa- tion. BPGs are prone to attach to the bladder wall with a broad base. This morphological character- istic was observed in all 16 lesions, which may be TABLE 3. CT and MR image characteristics* Computerized tomography (n = 13) Density Moderate or slightly lower density Enhancement characteristics Moderate to marked enhancement Calcification None Cystic degeneration or necrosis Rare (n = 2) Haemorrhage None Magnetic resonance imaging (n = 8) T2-weighted imaging Slight hyperintensity Diffusion-weighted imaging Hyperintensity Apparent diffusion coefficient (ADC) map Hypointensity(ADC value, 0.883±0.126×10-3 mm2/s)** T1-weighted imaging Slight hyperintensity Enhancement characteristics “Fast in and slow out” pattern Cystic degeneration or necrosis Rare (n = 2) Haemorrhage None * The density and signal intensity of the tumours were reported with reference to those of the gluteus maximus in the same layer; ** data are means ± standard deviations Radiol Oncol 2022; 56(1): 46-53. Zhang J et al. / CT and MR imaging features of bladder paraganglioma52 pathologically based on the tendency of BPGs to infiltrate and grow along the muscularis.10 Lu et al. suggested that intramural (located in the muscular layer) and subserosal (protruding from the serosa) tumours often had systemic symptoms caused by catecholamine release. Furthermore, the tumours with typical manifestations were larger than those without typical manifestations.7 In this study, 50% and 100% of the intramural and subserosal tumours showed systemic manifestations, respec- tively, but the correlation between symptoms and sizes was not observed. Imaging methods are primarily used for locali- zation of the paraganglioma.11 In the absence of typical clinical manifestations and negative bio- chemical tests, imaging can also be used as a com- plementary approach for qualitative diagnosis. CT has a high sensitivity (82%) in detecting extra- adrenal pheochromocytoma.9 We demonstrated that most BPGs are typically present as a solitary lesion protruding into or out of the bladder cav- ity, with an oval shape, soft tissue density, well- defined margin and a broad-base attachment to the bladder wall. The tumour exhibits slightly lower density and early marked enhancement on the con- trast-enhanced CT images. Contrary to the previ- ous literature, T2WI showed that BPGs are mostly homogeneous and high-intensity, without the typi- cal “pepper and salt” appearance.12,13 The homog- enous nature may be due to the fact that the lesion was still small when it was detected, and intratu- moural degeneration has not yet occurred. Similar to bladder cancer, BPGs show marked hyperin- tensity on DWI and hypointensity on ADC maps due to restricted diffusion of water molecules.14 The tumours presented slight hyperintensity on T1WI and “fast in and slow out” enhanced pat- tern on contrast-enhanced images, which may be distinctive MRI features of BPGs.10,15 The potential pathological bases for the above imaging findings are that the tumour cells are large with abundant cytoplasm and the intercellular stromata are rich in blood vessels showing fissure or haemangioma- like dilatation.16 The malignant rate of BPGs is estimated to be 10–15%.9 Because no reliable pathological evi- dence exists for early differentiation between be- nign and malignant tumours, direct invasion of adjacent tissues or distant metastases are consid- ered to represent potential malignancy.17 In this study, two lesions showed features of peripheral tissue invasion, suggesting malignancy. At pre- sent, dynamic contrast-enhanced MRI incorpo- rating non-fat suppression T2WI with small FOV and high resolution, by means of the natural con- trast between pelvic fat and urine in bladder, is the preferred imaging method for recognition of possible malignant tumours.18 In addition to CT and MRI, functional imaging is recommended to detect multi-focal and metastatic disease, such as 123I-metaiodobenzylguanidine (123I-MIBG) SPECT and 68Gallium-labeled somatostatin analogues (68Ga-DOTA-SSA) PET/CT. Depending on specific ligands that target specific cell membrane trans- porters or vesicular catecholamine transport sys- tems, this modality can provide greater diagnostic specificity.12,19 Due to their rarity and symptomatic variabil- ity, BPGs, especially malignant BPGs, should be differentiated from bladder carcinoma, the most FIGURE 5. Microscopic examination of paraganglioma. (A) Polygonal or oval tumour cells were rich in basophil granular cytoplasm and arranged in a sheet or organoid arrangement. (Hema toxylin and eosin staining, × 400). (B) Immunohistochemical staining of tumours cells was positive for chromogranin A (CgA) (EnVision × 200). A B Radiol Oncol 2022; 56(1): 46-53. Zhang J et al. / CT and MR imaging features of bladder paraganglioma 53 common malignant tumour of the bladder. The latter is more likely to occur in older men, related to smoking, and includes common symptoms of painless gross haematuria and progressive dysu- ria. Bladder carcinoma mostly shows hypointensi- ty on T1WI compared to the appearance of muscle and no early marked enhancement. This may be the crucial difference between these two kinds of tumours. Other infrequent tumours of the bladder, such as lymphoma leiomyoma and haemangioma, cannot be ignored and they can be distinguished from BPGs by comprehensive analysis of CT and MRI characteristics. This study has limitations. It is a retrospective series of a small study population, imaging with CT and MRI was not available in all patients, and contrast-enhanced CT contained only plain and arterial phase images. Although we attempted to objectively describe the image characteristics by calculating tumour-muscle signal ratio, due to the different scanners, inconsistent image acquisition time and subjective observation, the features ob- served require further validation in more cases. In conclusion, on CT and MRI, BPGs are mostly oval-shaped, well-defined and broadly-based blad- der tumours with hypoattenuation on non-contrast CT, T2 hyperintensity, slight T1 hyperintensity compared to the muscle, marked restricted diffu- sion on DWI and “fast in and slow out” enhanced pattern on contrast-enhanced images. Preoperative CT and MRI can be used to determine the location of the tumours, but can also assist in qualitative di- agnosis, malignant risk assessment and operative proposal formulation. Acknowledgement We acknowledge financial support from the National Natural Science Foundations of China (Grant No. 81971580 and No. 81471641-JT); the Medical Big Data Research and Development Project supported by Chinese PLA General Hospital (Grant No. 2018MBD-023). Reference 1. Lenders JW, Duh QY, Eisenhofer G, Gimenez-Roqueplo AP, Grebe SK, Murad MH, et al. Pheochromocytoma and paraganglioma: an endocrine society clinical practice guideline. J Clin Endocrinol Metab 2014; 99: 1915-42. doi: 10.1210/jc.2014-1498 2. Beilan JA, Lawton A, Hajdenberg J, Rosser CJ. Pheochromocytoma of the urinary bladder: a systematic review of the contemporary literature. BMC Urol 2013; 13: 22. doi: 10.1186/1471-2490-13-22 3. Qin J, Zhou G, Chen X. Imaging manifestations of bladder paraganglioma. Ann Palliat Med 2020; 9: 346-51. doi: 10.21037/apm.2020.03.09 4. Sharma AP, Bora GS, Mavuduru RS, Panwar VK, Mittal BR, Singh SK. Management of bladder pheochromocytoma by transurethral resection. Asian J Urol 2019; 6: 298-301. doi: 10.1016/j.ajur.2018.05.010 5. Tevosian SG, Ghayee HK. Pheochromocytomas and paragangliomas. Endocrinol Metab Clin North Am 2019; 48: 727-50. doi: 10.1016/j. ecl.2019.08.006 6. International Agency for Research on Cancer. World Health Organization. WHO classification of tumours of endocrine organs. 4th edition. Lloyd RV, Osamura RY, Klöppel G, Rosai J, editors. Lyon, France: IARC; 2017. p. 78-80. 7. Lu H, Male M, Jiang K, Ye Z, Song D, Xia D. Clinical significance of functional and anatomical classifications in paraganglioma of the urinary bladder. Urol Oncol 2019; 37: 354.e9-17. doi: 10.1016/j.urolonc.2019.01.027 8. Jain A, Baracco R, Kapur G. Pheochromocytoma and paraganglioma-an update on diagnosis, evaluation, and management. Pediatr Nephrol 2020; 35: 581-94. doi: 10.1007/s00467-018-4181-2 9. Henderson SJ, Kearns PJ, Tong CM, Reddy M, Khurgin J, Bickell M, et al. 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Al Johi RS, Seifeldein GS, Moeen AM, Aboulhagag NA, Moussa EM, Hameed DA, et al. Diffusion weighted magnetic resonance imaging in bladder can- cer, is it time to replace biopsy? Cent European J Urol 2018; 71: 31-7. doi: 10.5173/ceju.2017.1427 15. Wang H, Ye H, Guo A, Wei Z, Zhang X, Zhong Y, et al. B ladder paraganglioma in adults: MR appearance in four patients. Eur J Radiol 2011; 80: e217-20. doi: 10.1016/j.ejrad.2010.09.020 16. Fan DG, Wu CL, Huang HJ, Wu L, Lin SY. [Paraganglioma of urinary bladder: a clinicopathological features analysis of 23 cases]. [Chinese]. Zhonghua Bing Li Xue Za Zhi 2020; 49: 311-6. doi: 10.3760/cma.j.cn112151-20190928-00535 17. Quist EE, Javadzadeh BM, Johannesen E, Johansson SL, Lele SM, Kozel JA. Malignant paraganglioma of the bladder: a case report and review of the lit- erature. Pathol Res Pract 2015; 211: 183-88. doi: 10.1016/j.prp.2014.10.009 18. Raza SA, Jhaveri KS. MR imaging of urinary bladder carcinoma and beyond. Radiol Clin North Am 2012; 50: 1085-110. doi: 10.1016/j.rcl.2012.08.011 19. Lenders JWM, Eisenhofer G. Update on modern management of pheochro- mocytoma and paraganglioma. Endocrinol Metab (Seoul) 2017; 32: 152-61. doi: 10.3803/EnM.2017.32.2.152 Radiol Oncol 2022; 56(1): 54-59. doi: 10.2478/raon-2021-0052 54 research article Diagnostic performance of apparent diffusion coefficient values for the differentiation of intrahepatic cholangiocarcinoma from gastrointestinal adenocarcinoma liver metastases Temel Fatih Yilmaz1, Mehmet Ali Gultekin1, Hacı Mehmet Turk2, Mehmet Besiroglu2, Dilek Hacer Cesme1, Melih Simsek2, Alpay Alkan1, Huseyin Toprak1 1 Departments of Radiology, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey 2 Departments of Medical Oncology, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey Radiol Oncol 2022; 56(1): 54-59. Received 29 July 2021 Accepted 4 November 2021 Correspondence to: Asist. Prof. Temel Fatih Yilmaz, M.D., Department of Radiology, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey. Phone: 90 212 4531700; Fax: 90 212 6217580; E-mail: temelfatihyilmaz@gmail.com Disclosure: No potential conflicts of interest were disclosed. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Background. We aimed to investigate whether there is a difference between intrahepatic cholangiocarcinoma (IHCC) and liver metastases of gastrointestinal system (GIS) adenocarcinoma in terms of apparent diffusion coefficient (ADC) values. Patients and methods. From January 2018 to January 2020, we retrospectively examined 64 consecutive patients with liver metastases due to gastrointestinal system adenocarcinomas and 13 consecutive IHCC in our hospital’s medical records. After exclusions, fifty-three patients with 53 liver metastases and 10 IHCC were included in our study. We divided the patients into two groups as IHCC and liver metastases of GIS adenocarcinoma. For mean apparent diffusion coefficient (ADCmean) values, the region of interests (ROI) was placed in solid portions of the lesions. ADCmean values of groups were compared. Results. The mean age of IHCC group was 62.50 ± 13.49 and mean age of metastases group was 61.15 ± 9.18. ADCmean values were significantly higher in the IHCC group compared to the metastatic group (p < 0.001). ROC curves method showed high diagnostic accuracy (AUC = 0.879) with cut-off value of < 1178 x 10-6 mm2/s for ADCmean (Sensitivity = 90.57, Specificity = 70.0, positive predictive value [PPV] = 94.1, negative predictive value [NPV] = 58.3) in differentiating adenocarcinoma metastases from IHCC. Conclusions. The present study results suggest that ADC values have a potential role for differentiation between IHCC and GIS adenocarcinoma liver metastases which may be valuable for patient management. Key words: cholangiocarcinoma, gatrointestinal system; liver metastases; apparent diffusion coefficient; diffusion weighted image; MRI Introduction Intrahepatic cholangiocarcinoma (IHCC) is the sec- ond most common primary malignant lesion of the liver after hepatocellular cancer and estimated 15% of primary liver cancer worldwide. The incidence of IHCC has been increasing recently.1 According to macroscopic appearance, cholangiocarcinoma (CC) is divided into three types: mass forming type, periductal infiltrative type and intraductal grow- Radiol Oncol 2022; 56(1): 54-59. Yilmaz TF et al. / Apparent diffusion coefficient value in differentiation of intrahepatic colangiocarcinoma from liver metastases 55 ing type.1-2 Mass forming type CC is the most com- mon type with a rate of 60%.3 Diffusion weighted image (DWI) usually is added to standard abdo- men protocols, because it is a rapid technique and talented of detecting most liver masses in patients with supposed malignant disease. It may be diffi- cult to differentiate between IHCC and gastrointes- tinal system (GIS)-derived adenocarcinoma even when contrast is given, and DWI can be helpful in differential diagnosis in these cases.4 DWI provides diagnostic value in differentia- tion of benign and malignant liver masses. It gives information about cellularity of tissues and integri- ty of cell membranes. DWI increases the sensitivity of detection for liver metastases when combined with dynamic contrast enhanced upper abdomen MRI. DWI has been used in characterization of metastatic and primary liver tumors.5 The sensitiv- ity of using DWI in addition to routine imaging in detecting malignancy was reported as 94.9% and the specificity as 97.8%.6 To avoid unnecessary di- agnostic and therapeutic interventions, differentia- tion between IHCC and metastases of adenocarci- nomas is very significant, because they have differ- ent treatment options and prognosis. It would be valuable to precisely differentiate the liver metasta- ses of GIS from IHCC based on apparent diffusion coefficient (ADC) values. Because it’s sometimes hard to differentiate even based on the histologi- cal analysis since all these tumors are adenocarci- nomas. We aimed to investigate whether there is a difference between IHCC’s and adenocarcinoma liver metastases from GIS origin in terms of ADC values. The goal of this study was to evaluate the value of DWI, using the mean ADC value for distinguish- ing IHCC from liver metastases of adenocarcino- mas originating in the gastrointestinal tract. Patients and methods Patients We consecutively reviewed medical records of pa- tients who had a diagnosis or an imaging study showing IHCC or metastasis between January 2018 to January 2020. Inclusion criteria were determined as histopathological confirmation of the primary or metastatic lesion GIS adenocarcinoma arising from stomach, colon, and pancreas with liver metastases at the initial diagnosis, and CC with no history of chemotherapy at initial imaging and the presence of pretreatment MRI of the abdomen with a proper DWI. Exclusion criteria were determined as pre-exam- ination neoadjuvant chemotherapy for the primary tumor (n = 3), lack of DWI sequence (n = 2), heavy image artefacts and technical reasons (n = 1), and lesions smaller than 10 mm with difficult to meas- ure ADC values (n = 5). We only included one ma- jor metastatic lesion from each patient to provide study homogeneity. Finally, a total of 53 patients with 53 liver metastases and 10 patients with IHCC were included in our study (Figure 1). We divided the patients into two groups as IHCC’s (n = 10 pa- tients) and liver metastases of GIS adenocarcino- ma (n = 53 patients with 53 lesions). In our study group, there were no underlying chronic liver dis- eases and no metastases contained mucinous com- ponents in it. The study protocol was approved by our institu- tion’s ethics committee. All procedures performed in the studies involving human participants were in accordance with the ethical standards of the in- stitutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study. Histopathologic analysis Diagnosis of all IHCCs and primary GIS adeno- carcinomas were confirmed histopathologically. For IHCC’s, primary tumor sites were right he- patic lobe (n = 4), and left hepatic lobe (n = 6), Histopathological diagnosis was obtained by per- cutaneous tru-cut biopsy. For the metastasis group, primary tumor sites were colorectal (n = 32), gastric (n = 9), and pan- creas (n = 12). Tissue samples of colorectal and gastric adenocarcinomas were obtained through endoscopic biopsy samples. For pancreatic adeno- carcinomas, endosonographic-guided fine needle From January 2016 to January 2020 74 (<10mm) FIGURE 1. Flow-chart of the study showing the exclusion criteria of the patients. Radiol Oncol 2022; 56(1): 54-59. Yilmaz TF et al. / Apparent diffusion coefficient value in differentiation of intrahepatic colangiocarcinoma from liver metastases56 aspiration biopsy (n = 7), percutaneous tru-cut bi- opsy of metastatic liver lesions (n = 3), and surgical biopsy (n = 2) were done. MRI protocol All patients underwent MRI using a 1.5-T system (Siemens, Avanto, Erlangen, Germany). T1-W in- and out-of-phase (TR/TE, 128/4.90 and 128/2.37; NEX, 1; the FOV of 38 to 50 cm; 5 mm thickness and 2 mm intersection gap), T2-weighted axial (TR/TE, 2000/120; NEX, 1; the FOV of 38 to 50 cm; 5 mm thickness and 2 mm intersection gap), fast spin echo T2-W axial (TR/TE, 2000/117; NEX, 1; the FOV of 38 to 50 cm; 5 mm section thickness and 2 mm intersection gap) and T2-W coronal images were performed (TR/TE, 1400/106; NEX, 1; the FOV of 38 to 50 cm; 5 mm section thickness and 1 mm in- tersection gap). The DWI images were obtained at b-values of 50, 400, and 800 s/mm2 (TR/TE, 7300/78; NEX, 2; FOV 38-50 cm; slice thickness 5 mm and no intersection gap). Pre and postcontrast fat-satu- rated T1-W axial (VIBE) (TR/TE, 4.90/2.39; NEX, 1; and the FOV of 38 to 50 cm; 3 mm section thickness and no intersection gap) were performed. Dynamic imaging was performed after a rapid bolus of gad- olinium-diethylenetriamine pentaacetic acid, 0.1 mmol/kg body weight, intravenously at a rate of 1.5 mL/s, followed by a 30 mL saline flush using a power injector. Contrast-enhanced dynamic imag- es were performed in arterial phase, portal venous phase, and interstitial phase in the axial plane and in interstitial phase in the coronal plane. Image analysis Image analysis and region of interests (ROI) place- ment was made by two abdominal radiologists who had ten and eleven years’ experience of abdominal radiology. The morphological features were evalu- ated as follows: 1- lesion size, measurements were made in the axial plane on contrast enhanced T1- weighted (T1W) images at the largest diameter. 2-lesion localization (right or left lobe of the liver). 3-ADC values of the metastases and IHCC’s. For ADCmean values, the ROIs were placed over the three different enhancing solid portions of the le- sions on contrast enhanced T1W images blinded to ADC maps. Conventional T2-weighted (T2W) and contrast enhanced T1W images were used as refer- ence to determine the enhanced portions of the le- sion areas and to avoid the cystic or necrotic parts of the lesions. Final ADCmean values were calculated as the average of the ADC values obtained from 3 different ROIs (Figure 2–3). We used synapse 3D® (Fujifilm Medical, Tokyo, Japan) and Leonardo console (software version 2.0, Siemens) to evaluate metastatic liver lesions and to calculate ADC val- ues of metastatic liver lesions. Statistical analysis Statistical analysis was performed using the IBM SPSS Statistics 26.0 statistical software (Armonk, NY: IBM Corp.). The Kolmogorov-Smirnov test was used for normality. non-parametric Mann- Whitney-U test was used in the comparison of the ADCmean values of the IHCC and adenocarci- noma groups from the enhanced solid metastases and the lesion sizes. Descriptive statistics are pre- sented as median (50%) and interquartile range (IQR = Q3(75%)-Q1(25%)) values. p value below 0.05 was accepted as statistically significant. ROC curves were evaluated to determine the cut-off value to differentiate between ADCmean values of IHCC and adenocarcinoma metastases. FIGURE 2. 59-year-old male with liver metastases due to colorectal adenocarcinoma. Contrast-enhanced axial T1-weighted (T1W) (A), diffusion weighted image (DWI) obtained at b value of 800 s/mm2 (B) and apparent diffusion coefficient (ADC) maps (C) with free hand ROI placement technique. A B C Radiol Oncol 2022; 56(1): 54-59. Yilmaz TF et al. / Apparent diffusion coefficient value in differentiation of intrahepatic colangiocarcinoma from liver metastases 57 Results IHCC was diagnosed in 10 patients with 10 lesions and adenocarcinoma was diagnosed in 53 patients with 53 liver metastases. There was no statistically significant difference between IHCC and adeno- carcinoma in terms of age. Demographic features were summarized at Table 1. ADCmean values were significantly higher in the IHCC group compared to the adenocarcinoma group (p < 0.001) (Figure 4). These results are sum- marized in Table 2. ROC curves method showed diagnostic accura- cy for ADCmean (AUC = 0.879). Cut-off value was < 1178 x 10-6 mm2/s for ADCmean (Sensitivity = 90.57%, Specificity = 70.0%, positive predictive val- ue [PPV] = 94.1, negative predictive value [NPV] = 58.3) in differentiating adenocarcinoma metastases from IHCC’s with the 95% confidence interval (Figure 5). The AUC rates showed that ADCmean values were statistically significant in dif- ferentiating the two groups (p < 0.0001). Discussion Gastrointestinal cancer is from the most common malignant tumors worldwide with rising inci- dence.7 Surgery is now the primary treatment for gastrointestinal cancer. Liver metastasis occurs in approximately 45% of patients.8 IHCC originates from small intrahepatic bile ducts and grows through adjacent liver parenchy- ma. IHCC typically occurs as a large mass, which is difficult to differentiate from a metastatic focus of adenocarcinoma. The only curative treatment of IHCC is surgery and even with surgery its 5-year survival rates remain at 39–41%.9 In contrast there is no surgical treatment option for some metastatic GIS tumors.10 IHCC is hypointense on T1-weighted (T1W), and hyperintense on T2W imaging relative to liver parenchyma. The grade of hyperintensity on T2W imaging frequently depends on the quantity of fibrosis, necrosis, and mucin within the tumor.11 The imaging features of IHCC have been further described, with the targetoid appearance being one of the most common characteristics.12 Magnetic resonance (MR) DWI is a technique that provides image contrast by free water mol- FIGURE 3. 58-year-old female with an expansile liver mass with central hypovascular fibrous stroma and peripheral contrast enhancement on contrast- enhanced axial T1-weighted (T1W) (A) images. diffusion weighted image (DWI) obtained at b value of 800 s/mm2 (B) and apparent diffusion coefficient (ADC) maps (C) with region of interests (ROI) placement with three different contrast enhancing area for calculating ADCmean values. TABLE 1. Univariate analysis of patient characteristics for gastrointestinal system (GIS) liver metastases and intrahepatic cholangiocarcinoma (IHCC) Patient characteristics Number of patients (n = 63) IHCC (n = 10) Liver metastases (n = 53) P value Age (years) 61.4 ± 9.93 62.50 ± 13.49 61.15 ± 9.18 0.679 Gender < 0.001 Male 35 2 33 Female 28 8 20 Diameter (mm) Location 47 ± 31.27 82.70 ± 28.58 40.26 ± 27 < 0.001 < 0.001 Right lobe 24 2 38 Left lobe 43 8 15 TABLE 2. Apparent diffusion coefficient (ADC) values of intrahepatic cholangiocarcinoma (IHCC) and gastrointestinal system (GIS) liver metastases Patient Groups ADC x 10-6mm/sn2 (median, IQR) IHCC (1293.0), (1422.0–951.75) GIS metastases (861.0), (1053.0–695.0) IQR = interquartile range A B C Radiol Oncol 2022; 56(1): 54-59. Yilmaz TF et al. / Apparent diffusion coefficient value in differentiation of intrahepatic colangiocarcinoma from liver metastases58 ecule movement within tissue and the ADC (ex- pressed in mm2/s), is a numerical parameter of DWI. ADC values give information about cellu- larity of tissues and integrity of cell membranes.12 Biopsy is required before surgical or oncological treatment in cases where IHCC is considered by imaging. It is sometimes difficult to distinguish liver me- tastases from IHCC with routine abdominal MRI. It is not possible to differentiate especially in soli- tary or hypovascular metastases. DWI must be added to routine abdominal MR imaging since it is very sensitive to detect liver malignancies. ADC is a measure of the magnitude of diffusion within tissue and is commonly clinically calculated using DWI. ADC values reflect the structure of masses and vary in different tumor types. Lower ADC values may reflect the hypercellularity of these tu- mors, and decreased intra and extracellular space. Mungai et al. reported mean ADC values of CC as 0.970 x 10-3mm/sn2 and metastases as 0.947 x 10-3mm2/sn and these results were not statistically significant.4 This may be a result of evaluation of metastases arising from any origin and the evalu- ation of all subtypes of CC. In our study we have found median ADC value of IHCC 1293 x 10-6mm2/ sn and mean ADC value of metastases of GIS 861 x 10-6mm2/sn and this was statistically significant (p < 0.0001). Decreased ADC values in GIS liver metastases may be attributed to hypercellularity. However increased ADC values in IHCC may be the result of decreased cellularity due to fibrotic changes compared to metastases. Drevelegas et al. demonstrated that mean ADC value in 12 patients with liver CC was 1.34 ± 0.27 x 10-3mm2/s and 1.11 ± 0.295 x 10-3mm2/s in 51 pa- tients with secondary liver malignancy.13 They only concluded that primary liver tumors have higher ADC values than secondary ones. The result of this study supports our outcomes. Namimoto et al. found the mean ADC value of 1.51 ± 0.47 x 10-3mm2/s in IHCC and 1.23 ± 0.32 x 10-3mm2/s in metastatic liver lesions. These results are in accordance with the results in our study.6 Lee et al. in their study on 91 patients, stated good prognosis and survival of IHCCs when areas with diffusion restriction are dominant.14 But they do not provide ADC values of masses in their study. Yamada et al. notified that low ADC value is associ- ated with poor differentiation and prognosis which has rich fibrotic stroma. The researchers, who di- vided the patients into two groups as high or low ADC, showed that the prognosis was poor in the tumors with decreased ADC. This result conflicts with the results of previous studies by Lee et al.15 In our study, we did not make prognostic grouping of our IHCC patients. In our study, we found significantly higher ADC values in IHCC patients compared to GIS adenocarcinoma liver metastases. We distinguished these tumors with a cut off ADCmean value of 1178 x 10–6 mm2/s and 90.57% sensitivity. We speculated that the rich des- moplastic stroma of IHCC is effective in revealing the ADC difference in the differentiation from liver metastases of GIS. FIGURE 4. Mean apparent diffusion coefficient (ADCmean) values of liver metastases of gastrointestinal system adenocarcinomas and intrahepatic cholangiocarcinoma (IHCC). FIGURE 5. Receiver operating characteristic curve of the apparent diffusion coefficient (ADC) mean values. Radiol Oncol 2022; 56(1): 54-59. Yilmaz TF et al. / Apparent diffusion coefficient value in differentiation of intrahepatic colangiocarcinoma from liver metastases 59 In a recent study, Kovac et al. investigated the contribution of ADC values in the differentiation between solitary hypovascular liver metastases and IHCC.16 They reported lower ADC values in the peripheral enhancing areas and higher in the central parts in IHCC compared to solitary hypo- vascular metastases. Authors explained that high ADC values in the central part of IHCC are related to rich fibrous tissue.16 In our study, ADC values were obtained from enhancing solid area and we found higher ADC values compared to GIS metas- tases. High ADC values in IHCC could be attribut- ed to desmoplastic stromal changes and decreased cellularity within the tumor. We thought that the reproducibility of ADC measurement may be limited or needs more effort in clinical practice, but it provides significant diag- nostic clues in differential diagnosis of such liver malignancies. There were several limitations in the current study. First this is a retrospective study and has relatively small sample size, especially for the IHCC group because of the rarity of these tumors. Second, the fibrous intensity and differentiation levels of tumors was not assessed by histopatho- logically. Furthermore, the manual placement of ROI, as a known limitation in all ROI-based stud- ies, may have led to biased results. In addition, the histopathological confirmation of liver metastases was made from primary tumor localization. But this situation was ignored because of low probabil- ity of synchronous IHCC and GIS metastases. Conclusions As far as we know, this is the first study that par- ticularly demonstrates the utility of ADC values in differentiation of IHCC from GIS adenocarcinoma liver metastases. Our study results suggest that ADC values have a potential role for differentia- tion between IHCC and GIS liver metastases which may be valuable for patient management. Further larger-scale studies are needed to establish the re- lationship between IHCC and different tumor ori- gins in terms of ADC values. References 1. Shin HR, Oh JK, Masuyer E, Curado MP, Bouvard V, Fang YY, et al. Epidemiology of cholangiocarcinoma: an update focusing on risk factors. Cancer Sci 2010; 101: 579-85. doi: 10.1111/j.1349-7006.2009.01458.x 2. Zhou Y, Zhou G, Gao X, Xu C, Wang X, Xu P. Apparent diffusion coefficient value of mass-forming intrahepatic cholangiocarcinoma: a potential imag- ing biomarker for prediction of lymph node metastasis. Abdom Radiol 2020; 45: 3109-18. doi: 10.1007/s00261-020-02458-x. 3. Lim JH. Cholangiocarcinoma: morphologic classification according to growth pattern and imaging findings. AJR Am J Roentgenol 2003; 181: 819-27. doi: 10.2214/ajr.181.3.1810819 4. Mungai F, Morone M, Villanacci A, Bondioni MP, Mazzoni LN, Grazioli L, et al. Diffusion weighted MR and apparent diffusion coefficient measurement in classification and characterization of noncystic focal liver lesions: does a clinical role exist? Medicine (Baltimore) 2014; 93: e40. doi: 10.1097/ MD.0000000000000040 5. Shenoy-Bhangle A, Baliyan V, Kordbacheh H, Guimaraes AR, Kambadakone A. Diffusion weighted magnetic resonance imaging of liver: Principles, clini- cal applications and recent updates. World J Hepatol 2017; 9: 1081-91. doi: 10.4254/wjh.v9.i26.1081 6. Namimoto T, Nakagawa M, Kizaki Y, Itatani R, Kidoh M, Utsunomiya D, et al. Characterization of liver tumors by diffusion-weighted imaging: Comparison of diagnostic performance using the mean and minimum apparent diffu- sion coefficient. J Comput Assist Tomogr 2015; 39: 453-61. doi: 10.1097/ RCT.0000000000000228 7. Rawla P, Barsouk A. Epidemiology of gastric cancer: global trends, risk factors and prevention. Prz Gastroenterol 2019; 14: 26-38. doi: 10.5114/ pg.2018.80001 8. Zheng DX, Ning AY, Levoska MA, Xiang L, Wong C, Scott JF, et al. Predicting liver metastasis of gastrointestinal tract cancer by diffusion-weighted imag- ing of apparent diffusion coefficient values. World J Gastroenterol 2016; 22: 3031-7. doi: 10.3748/wjg.v22.i10.3031 9. Wang K, Zhang H, Xia Y, Liu J, Shen F. Surgical options for intrahepatic chol- angiocarcinoma. Hepatobiliary Surg Nutr 2017; 6: 79-90. doi: 10.21037/ hbsn.2017.01.06 10. Vanderveen KA, Hussain HK. Magnetic resonance imaging of cholangiocarci- noma. Cancer Imaging 2004; 4: 104-15. doi: 10.1102/1470-7330.2004.0018 11. Maetani Y, Itoh K, Watanabe C, Shibata T, Ametani F, Yamabe H, et al. MR imaging of intrahepatic cholangiocarcinoma with pathologic correlation. AJR Am J Roentgenol 2001; 176: 1499-507. doi: 10.2214/ajr.176.6.1761499 12. Kele PG, van der Jagt EJ. Diffusion weighted imaging in the liver. World J Gastroenterol 2010; 16: 1567-76. doi: 10.3748/wjg.v16.i13.1567 13. Drevelegas K, Nikiforaki K, Constantinides M, Papanikolaou N, Papalavrentios L, Stoikou I, et al. Apparent diffusion coefficient quantification in determin- ing the histological diagnosis of malignant liver lesions. J Cancer 2016; 7: 730-5. doi: 10.7150/jca.14197 14. Lee J, Kim SH, Kang TW, Song KD, Choi D, Jang KT. Mass-forming intrahepatic cholangiocarcinoma: diffusion-weighted imaging as a preoperative prognos- tic marker. Radiology 2016; 281: 119-28. doi: 10.1148/radiol.2016151781 15. Yamada S, Morine Y, Imura S, Ikemoto T, Arakawa Y, Saito Y, et al. Prognostic prediction of apparent diffusion coefficient obtained by diffusion-weighted MRI in mass-forming intrahepatic cholangiocarcinoma. J Hepatobiliary Pancreat Sci 2020; 27: 388-95. doi: 10.1002/jhbp.732 16. Kovač JD, Galun D, Đurić-Stefanović A, Lilić G, Vasin D, Lazić L, et al. Intrahepatic mass-forming cholangiocarcinoma and solitary hypovascular liver metastases: is the differential diagnosis using diffusion-weighted MRI possible? Acta Radiol 2017; 58: 1417-26. doi: 10.1177/0284185117695666 Radiol Oncol 2022; 56(1): 60-68. doi: 10.2478/raon-2022-0001 60 research article Assessment of hyperbaric oxygenation treatment response in parotid glands by T2 mapping following radiotherapy for head and neck tumours Jernej Vidmar1,2,3, Ksenija Cankar1, Maja Groselj4, Zarko Finderle1, Igor Sersa1,2 1 University of Ljubljana, Faculty of medicine Institute of physiology, Ljubljana, Slovenia 2 Jožef Stefan Institute, Ljubljana, Slovenia 3 Institute of Radiology, University Medical Center Ljubljana, Ljubljana, Slovenia 4 University of Ljubljana, Faculty of medicine Department of Dental Diseases and Normal Dental Morphology, Ljubljana, Slovenia Radiol Oncol 2022; 56(1): 60-68. Received 23 September 2021 Accepted 10 December 2021 Correspondence to: Assist. Prof. Jernej Vidmar, M.D., Ph.D., Institute of Physiology, Faculty of Medicine, University of Ljubljana, Zaloška 4, SI-1000 Ljubljana, Slovenia. E-mail: jernej.vidmar@mf.uni-lj.si Disclosure: No potential conflicts of interest were disclosed. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Background. The study was designed to evaluate the influence of hyperbaric oxygenation therapy (HBOT) on the parotid gland in patients following radiotherapy for head and neck tumours. Patients and methods. HBOT response was monitored by 3T magnetic resonance imaging (MRI) using T2 mapping and subsequent measurement of mean T2 and T2 variability as well as by salivary tests (salivary flow, buffer capacity, and pH). Eighteen patients previously treated with irradiation doses between 50 and 80 Gy as well as 18 healthy gen- der and age matched controls were enrolled. MRI was performed prior to HBOT (40.2 ± 20 months after radiotherapy) and after 20 daily HBOT at 2.5 ATA (absolute atmosphere). Each HBOT consisted of breathing 100% oxygen for 90 minutes. Results. Significant differences in mean T2 prior to HBOT were observed between the ipsilateral irradiated (121 ± 20 ms), contralateral parotids (107 ± 21) and control group (96 ± 12 ms). A positive correlation in patients between T2 variability and irradiation dose was detected in contralateral parotids before HBOT (R = 0.489, p = 0.0287). In addition, negative correlations were observed between mean T2 in the ipsilateral as well as the contralateral gland and salivary flow before and after HBOT. Negative correlations between mean T2, T2 variability and pH of unstimulated saliva were also observed in the sides of parotid before and after HBOT. Conclusions. The study confirmed that T2 mapping had a potential for monitoring the differences between irradiated and normal parotid glands. It could also be useful in the assessment of the glandular tissue response to HBOT. Key words: salivary glands; MRI; T2 mapping; hyperbaric oxygenation therapy Introduction The main method of treatment of the malignant head and neck tumours is surgical removal and/ or radiation therapy with therapeutic doses be- tween 50 and 70 Gy.1,2 Doses above 40 Gy results in irreversible changes in the salivary glands – at- rophy and necrosis3,4 – which leads to the reduc- tion of the flow of saliva and the development of xerostomia when salivary glands are in the field of irradiation4-6; the latter is probably due to the apoptosis of salivary tissue, as observed in other Radiol Oncol 2022; 56(1): 60-68. Vidmar J et al. / Hyperbaric oxygenation in post-radiation salivary glands and T2 mapping 61 tissues under radiation therapy.7-9 Consequently, both stimulated and unstimulated salivary flow, salivary pH and buffer capacity are reduced6,10 and the ion composition of saliva is also changed.11,12 These changes cause the accumulation of plaque and an increased number of microorganisms in the saliva13-15 which may result in rapidly progressing radiation caries.16,17 In addition, the reduced excre- tion of saliva causes complaints associated with oral dryness in patients. It effects the use of oral prostheses as well as speech and taste. The qual- ity of life is also compromised.16,17 Effective treat- ments of radiation-induced xerostomia are war- ranted.18,19 Hyperbaric oxygenation therapy (HBOT) is an acceptable method of treatment for the preven- tion of osteoradionecrosis and soft tissue necrosis in the oral cavity.20 It improves blood circulation in post-ischemic tissues21, reduces oedema forma- tion22, increases diffusion of oxygen in the tissues23, and accelerates activation of stem cells.24 The likely cause of the beneficial effect of HBOT against the long-term negative effects of radiotherapy is the accelerated angiogenesis and revascularization of tissues.25 Until now, there has been no studies that objectively measured the impact of HBOT on sali- vary gland tissue. Multiparametric MRI is already a common di- agnostic tool for the parotid gland tumours26, 27, since it enables the optimization of contrast among various soft tissues based on the values of T1 and T2 relaxation times of various tissues and organs. T2 mapping is specifically a magnetic resonance im- aging technique used to calculate the T2 relaxation times of the specific tissues and displaying them voxel-vice on a parametric map. It has been used for tissue characterization in various types of tissue (e.g. myocardium).28 The T2 relaxation time, also referred to as the spin-spin or transverse relaxa- tion, is a time constant for the decay of transverse magnetization and is tissue-specific with regards to its ability to differentiate the abnormal tissues from the normal ones. T2 values reflect water con- tent in the respective tissue and are mainly used for the evaluation of oedema, e.g., in myocardial inflammation or infarction as in other pathologies. In addition, T2 mapping and mDIXON Quant im- aging proved to be useful for non-invasive evalu- ation of the radiation-induced parotid damage.29 Consequently, the mapping of the transversal re- laxation time (T2-mapping) enables good differ- entiation among different stages of soft tissue in- flammation without the need of contrast medium and would be an appropriate method for the early detection of salivary gland tissue changes due to radiation and HBOT. In the present in vivo study, the patients with head and neck tumours who had undergone radia- tion therapy were scanned by the T2 mapping MRI technique before and after HBOT. The obtained T2 maps were further correlated with the standard clinical salivary tests (salivary flow, pH, and buff- ering capacity). Patients and methods Patient group The study was carried out on 18 patients (2 females and 16 males) with the head and neck tumours pre- viously treated with radiotherapy. The mean age of the patients was 60.9 ± 11.7 years. Patients had been diagnosed with different types of tumours, the majority of which were located in the oral cav- ity. In all patients, the salivary glands were in the radiation field. The patients received irradiation doses from 50 to 80 Gy (mean irradiation dose 64.3 ± 6.3 Gy). Each patient included in the study received 20 daily HBOT in a hyperbaric chamber at 2.5 ATA (absolute atmosphere) where patients breathed 100% oxygen for 90 minutes each day. Patients were examined by MRI as well as salivary function testing twice; the first MRI examination was performed at baseline before the first HBO therapy (40.2 ± 20 months after radiation therapy) and the second 3 to 7 days after the last HBOT to avoid possible acute effects of higher oxygen lev- els. All patients were able to perform routine daily activities prior to each MRI examination. Contraindications to MRI such as an implant- ed pacemaker constituted exclusion criteria. All participants provided written informed con- sent approved by the Ethical Committee of the National Ministry of Health (Approval number 0120-41/2017/13) to participate in this protocol and conformed to the STROBE guidelines. The clinical study was undertaken with the understanding and written consent of each subject according to the Declaration of Helsinki (version 2008). Control group The control group was composed of 18 healthy gender- and age-matched participants (mean age: 56.7 ± 11.8 years; 2 females and 16 males), who were enrolled in the study as volunteers and did not re- ceive any HBOT. All the measurements in the con- trol group (MRI measurements as well as salivary Radiol Oncol 2022; 56(1): 60-68. Vidmar J et al. / Hyperbaric oxygenation in post-radiation salivary glands and T2 mapping62 function testing) were performed only once in the same manner as in the patient group. Specifically, analysis of the MRI measurements was performed in both parotid glands. The results of the control group were used as a reference. MR image acquisition The MR imaging of the parotid glands was per- formed on a 3T MRI system (TX Achieva, Philips, Netherlands) with a maximum gradient strength of 80 mT/m and use of a 32-channel receive head coil. The MR images were acquired using a multi-spin- echo (MSE) MRI sequence with parameters: TR = 2000 ms; TE = 7.8, 16, 24, 32, 40, 47 ms; field of view (FOV) 160 × 160 mm2; slice thickness 2 mm; image acquisition/reconstruction matrix 380 × 311/560 × 560; acquisition/reconstruction voxel size 0.42 × 0.51 × 2.5/0.29 × 0.29 × 2.5 mm3; single slice; band- width 290 Hz/pixel; no signal acquisition accelera- tion; and acquisition time for all 6 echoes was equal to 10 min 24 s. The imaging plane was oriented so that it contained most of the parotid glad, i.e., in the transversal orientation. MR data analysis A central slice in the transversal plane, covering the largest area of the parotid gland tissue, was used for T2 mapping analysis. T2 map was calculated using pixel-wise least-square fitting analysis of a set of T2-weighted images with TE values as speci- fied above. The fitting analysis implemented in the MRI for the calculation of T2 maps used Analysis Calculator plugin (ImageJ, National Institutes of Health, USA) that utilizes a mono-exponential T2 signal decay function as the model function for the analysis and the pair (i,j) denotes the pixel coordinate. From the calculat- ed T2 maps, mean and variability of T2 values in the region of interest (encircled in Figure 1) were de- termined in the ipsilateral as well as the contralat- eral parotid gland of the subjects in the study. The variability of T2 values was used for a quantitative assessment of tissue heterogeneity. Salivary function testing Tests for the evaluation of the functioning of the salivary glands were always performed between 11-12 a.m. The patients were instructed to clean their teeth in the morning and not to drink, eat or smoke for two hours before the measurements. Unstimulated salivary flow was determined by a 5-minute saliva collection. Saliva production was then stimulated with a 5-minute chewing of a par- affin block. The paraffin blocks (each weighting 1g and with a melting point of 48 °C) were part of the CRT buffer test provided by Ivoclar Vivadent (Liechtenstein). During this time, patients were not allowed to swallow saliva. After the stimu- lation, the salivary flow was determined. The buffering capacity was determined only in stimu- lated saliva with a CRT buffer (Ivoclar Vivadent, Liechtenstein) due to the negligible amount of un- stimulated saliva. After five minutes, the colour of the pad was compared with the colour chart. The salivary pH was determined by a pH-meter (Iskra, Slovenia). In order to exclude the influence of sa- liva enhancement and disinfection procedures on 0 100 200 300 T 2 (ms) A B C FIGURE 1. Representative T2 maps of parotid glands in a single transversal slice in a patient following radiotherapy for head and neck tumour before (A) and after hyperbaric oxygenation therapy (HBOT) (B) and in healthy control (C). Region of interest (ROI) on the ipsilateral side is encircled by green and on the contralateral side by the white-blue colour. Retromandibular veins (white arrows in B) were always carefully omitted from the ROI. Radiol Oncol 2022; 56(1): 60-68. Vidmar J et al. / Hyperbaric oxygenation in post-radiation salivary glands and T2 mapping 63 the results of the study, instructions regarding oral hygiene and saliva flow enhancement were also provided after the saliva evaluation at the end of HBOT. Protocol for HBOT Patients were treated in a multi-place hyperbaric chamber (Kovinarska P&P, Slovenia). For each patient, 20 dives were held consecutively on each working day of the week. Each individual dive in the hyperbaric chamber filled with air at a pressure of 2.5 ATA (absolute atmosphere) lasted 90 min. The patients breathed 100-percent oxygen through a mask at a pressure of 2.5 ATA. For each patient, MRI as well as saliva tests before the start and 3 to 7 days after the last HBOT were performed. Statistical analysis The results were expressed as mean and standard deviation in the case of a passed Shapiro-Wilk test or as the median value and the interquartile range (IQR) in the case of a failed Shapiro-Wilk test, both with the criterion of significance at p < 0.05. The mean T2and T2 variability values of the pa- tients’ parotid glands were compared by Analysis of Variance (ANOVA) with repeated measures us- ing 19 degrees of freedom and a Bonferroni’s post- hoc test. The obtained mean T2 and T2 variability values in the patient group (ipsilateral and con- tralateral gland respectively) were compared with values obtained in healthy controls with a Student t-test. The values of the salivary flow and pH of un- stimulated as well as stimulated saliva in patients before and after the end of HBOT were compared with a paired t-test. To assess the magnitude and direction of change in the buffer capacity at the beginning and at the end of HBOT, the Wilcoxon signed-rank test was used. Correlation tests between mean T2 and T2 vari- ability values from the ipsilateral as well as the contralateral gland and saliva test parameters (i.e. salivary flow, pH and buffering capacity) and with irradiation doses in patients were made by a linear regression (Pearson correlation coefficient). Results MRI analysis At the beginning of HBO therapy, significantly higher mean T2 value was observed in the exam- ined slice of the ipsilateral parotid when compared to the mean T2 value on the contralateral gland (p = 0.007, Table 1). Furthermore, significant differ- ence was observed between mean T2 values in the parotid glands of healthy controls and in ipsilateral parotid glands of patients before HBO therapy (p = 0.0004). In contrast, no significant difference was observed between mean T2 in the contralateral pa- rotid glands of patients and healthy controls. In addition, no significant differences in T2 variabil- ity of parotid glands of patients before HBOT and healthy controls were observed. A significant higher mean T2 on the ipsilateral gland was found in patients after the end of HBOT when compared to healthy controls (p = 0.002). In contrast, no difference was observed on the con- tralateral side. On the ipsilateral side, statistically significant decrease in mean T2 and T2 variability was observed in patient as a response to HBOT. In contrast, no significant change in mean T2 and T2 variability of contralateral parotid glands was ob- served in patients in response to HBOT. Analysis of salivary tests The salivary flow and pH value of unstimulated and stimulated saliva were significantly lower in TABLE 1. Mean T2 and T2 variability values of parotid glands in patients following radiotherapy for head and neck tumours before and after hyperbaric oxygenation therapy (HBOT) and in healthy controls Ipsilateral side (N = 18) Contralateral side (N = 18) Controls (N = 18) before HBOT after HBOT before HBOT after HBOT MEAN T2 (ms) 121 ± 20† 113 ± 16†* 107 ± 21** 103 ± 14 96 ± 12 T2 VARIABILITY (ms) 30 ± 8 25 ± 8* 21 ± 8 19 ± 6 16 ± 4 †-statistically significant difference with healthy controls *-statistically significant change in response to HBOT **-statistically significant difference between ipsilateral and contralateral side Radiol Oncol 2022; 56(1): 60-68. Vidmar J et al. / Hyperbaric oxygenation in post-radiation salivary glands and T2 mapping64 patients prior to and after the end of HBOT when compared to the values obtained in healthy con- trols (Table 2, p < 0.01). In contrast, no significant difference in the buffering capacity of stimulated saliva was observed between patients and controls. Statistically significant increase in the unstimulat- ed salivary flow as well as in the buffering capacity of stimulated saliva was observed in patients in re- sponse to HBOT. In contrast, no significant change was found in other measured salivary parameters. Correlation between MRI parameters, irradiation dose and salivary tests A significant positive correlation between T2 variability of the contralateral parotid gland and the irradiation dose was observed before HBOT (Figure 2). In contrast, no significant correlation be- tween mean T2 on either ipsilateral or contralateral gland or T2 variability in ipsilateral parotid glands before HBOT and the irradiation dose was found. On the ipsilateral side, a significant negative cor- relation was observed between mean T2 and stimu- lated salivary flow before HBOT (Figure 3A) and between mean T2 and unstimulated salivary flow after HBOT (Figure 3B). On the contralateral side a negative correlation between mean T2 and unstim- ulated (Figure 4A) as well as stimulated salivary flow (Figure 4B) was observed after the HBOT. In addition, significant negative correlations between mean T2 and T2 variability and pH of unstimulated saliva were observed in ipsilateral parotid glands in patients before and after HBOT (Table 3). On the contralateral side negative corre- lations were also observed except for the correla- tion between T2 variability and pH of unstimulated saliva before HBOT. No correlations were found between mean T2 or T2 variability and pH of stimu- lated saliva. Discussion In the present study, the structural and functional response to HBOT in parotid glands of the patients after the radiotherapy of head and neck tumours was monitored by T2 mapping and functional salivary test. Two of the MRI parameters obtained from the T2 maps, i.e. mean T2 and T2 variability were used for the assessment of tissue structure af- ter radiotherapy, prior to and after HBOT. Mean T2 was used to assess tissue oedema and T2 variability TABLE 2. Salivary flow, pH, and buffer capacity in patients following radiotherapy for head and neck tumours before and after hyperbaric oxygenation therapy (HBOT) and in healthy controls before HBOT (N = 18) after HBOT (N = 18) Controls (N = 18) Unstimulated salivary flow (mL/min) (median and IQR) 0.22 (0.04-0.54) † 0.32 (0.08-0.70)*† 0.61 (0.49-0.99) pH of unstimulated saliva (mean ± SD) 6.61± 0.69† 6.72 ± 0.71† 7.56 ± 0.53 Stimulated salivary flow (mL/min) (mean ± SD) 0.82 ± 0.60† 0.90 ± 0.64† 2.04 ± 0.91 pH of stimulated saliva (mean ± SD) 7.38 ± 0.74† 7.48 ± 0.51† 8.00 ± 0.28 Buffering capacity of stimulated saliva (median and IQR) 2.00 (1.75-3.00) 3.00 (2.00-3.00)* 3.00 (2.00-3.00) †-statistically significant difference with healthy controls *-statistically significant change in response to HBOT IQR = interquartile range; SD = standard deviation R = 0.489, p = 0.0287 Irradiation dose (Gy) 50 60 70 80 T 2 v ar ia bi lit y in c ot ra la te ra l g la nd (m s) 0 10 20 30 40 50 60 FIGURE 2. A correlation between an irradiation dose and variability of T2 values in contralateral parotid glands before hyperbaric oxygenation therapy (HBOT). Radiol Oncol 2022; 56(1): 60-68. Vidmar J et al. / Hyperbaric oxygenation in post-radiation salivary glands and T2 mapping 65 R = - 0.592, p = 0.0096 R= - 0.615, p = 0.0067 Stimulated salivary flow before HBO (mL/min) 0.0 0.5 1.0 1.5 2.0 2.5 M ea n T 2 in ip si la te ra l g la nd (m s) 80 90 100 110 120 130 140 150 160 Unstimulated salivary flow after HBO (mL/min) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 M ea n T 2 in ip si la te ra l g la nd (m s) 80 90 100 110 120 130 140 150 160 affected tissues. Tissue oedema is probably im- proved through an osmotic effect of oxygen while the onset of a steep oxygen gradient across an ir- radiated tissue margin is a powerful stimulus for TABLE 3. Correlations between mean T2 or T2variability and pH of unstimulated saliva before and after hyperbaric oxygenation therapy (HBOT) (R-correlation coefficients and p-values) Ipsilateral side (N = 18) Contralateral side (N = 18) before HBOT after HBOT before HBOT after HBOT R p R p R p R p Mean T2 (ms) -0.647 0.0037 -0.571 0.0133 -0.557 0.0164 -0.675 0.0021 T2 variability (ms) -0.595 0.0092 -0.506 0.0323 -0.130 0.607 -0.588 0.0133 for the assessment of structural changes in the tis- sue, e.g., tissue heterogeneity. Mean T2 is strongly dependent on the free water content and its mobil- ity in the tissue; however, it lacks more detailed in- formation about the tissue structure heterogeneity, otherwise visualized on the T2 maps of the exam- ined slice. Therefore, mean T2 values were comple- mented with T2 variability. Prior to HBOT, consistent high mean T2 values were observed in the examined slices with the pa- rotid gland on the ipsilateral side compared to the values obtained from the glands on the contralater- al side of the same patients as well as healthy con- trols. The most plausible explanation for this phe- nomenon is the onset of radiation induced paren- chymal changes in the affected parotid glands in patients following radiotherapy.26,29 Therefore, the augmented T2 values in the parotid glands on the side of radiation can be explained by the prolonged effect of the inflammation along with glandular oedema. T2 variability values in parotid glands are also the highest in the ipsilateral irradiated parotid glands and somewhat lower on the contralateral side in patients before HBOT when compared to the control group. This can be explained by a dif- ferent structural tolerance of parotid glands to the received radiation. Due to the proximity of the ipsi- lateral side to the radiation source, a relatively high radiation dose was accumulated, causing scarring and narrowing of the blood vessels with more se- vere parenchymal atrophy.30 The latter resulted in a more heterogeneous structure, as seen in T2 maps of the parotid glands on the ipsilateral side, and consequently in a relatively high T2 variability. It should be emphasized that the MRI assessment of parotid glands performed at the late time prior to HBOT might represent late radiation effects (LRE) resulting in tissue oedema as well as chronic tissue changes.31 After the HBOT, a decrease in mean T2 and T2 variability in the patients’ ipsilateral gland was observed. This can be explained by HBOT effects on LRE through a complex series of changes in the FIGURE 3. A correlation between the mean T2 values in ipsilateral parotid glands and stimulated salivary flow before hyperbaric oxygenation therapy (HBOT) (A) and unstimulated salivary flow after HBOT (B). A B Radiol Oncol 2022; 56(1): 60-68. Vidmar J et al. / Hyperbaric oxygenation in post-radiation salivary glands and T2 mapping66 the growth of new blood vessels and subsequent tissue neovascularisation. In addition, an increase in oxygen levels, improves white cell and fibroblast function, thus enabling further enhancement of wound healing and tissue quality improvement.32 The effect of HBOT was slightly more pronounced on the ipsilateral side due to the more severe struc- tural changes. A positive correlation between T2 variability and the irradiation dose was observed only in contralat- eral parotid glands prior to HBOT. This confirms that the contralateral side could also be affected with higher doses of radiation. In contrast, absence of any significant correlation on the ipsilateral side could be attributed to rather comparable cumula- tive radiation doses between patients, i.e., most of them were exposed to doses between 60 to 70 Gy. Furthermore, mean T2 and T2 variability were also relatively high on the ipsilateral side and would probably require enrolment of more patients with doses ranging from relatively low (~50 Gy) to rela- tively high (~80 Gy) to obtain a significant correla- tion. Previous studies on the effects of radiation on the function of salivary glands have shown that the reduction of salivary gland activity depends on the dose of radiation and the volume of irradiated tis- sues.33 Namely, doses above 60 Gy result in a dra- matic decrease in salivary flow rate.4 The latter is in agreement with the results of functional salivary tests in our patient group prior to HBOT. Since the radiation doses were nearly the same in all pa- tients, we could not find any correlation between the radiation doses received and salivary flow. As a response to HBOT, a significant improvement of salivary gland function was observed in all meas- ured salivary parameters. The results confirm the findings of previous studies demonstrating a sub- jective reduction of problems related to swallow- ing, taste sensation and saliva quantity.34 A negative correlation between mean T2 and T2 variability in the examined slice of ipsilateral pa- rotid glands and unstimulated saliva pH as well as stimulated salivary flow was observed in patients prior to and after HBOT. These correlations can be attributed to the fact that structural changes in glandular tissues influence the function of all gland and subsequent cumulative saliva secretion. In ad- dition, these results are also in agreement with an- other MRI study showing an increase in apparent diffusion coefficient (ADC) of salivary glands due to radiation injury as well as a correlation between ADC, stimulated salivary flow and xerostomia questionnaire scores.35 The present study has several limitations, main- ly due to MRI scanning time as well as the com- parison of MRI results with the functional salivary tests. Firstly, the achievable resolution in our ex- perimental setup was limited by a reasonable MRI scanning time, e.g., approximately ten minutes per scan for T2 mapping, therefore allowing only T2 mapping in the central slice of the parotid gland. Consequently, only a T2 map of the slice with the largest proportion of glandular tissue was meas- ured and analysed. For the purpose of more in- depth analysis of the parotid structure, the whole area of the parotid gland should be scanned for T2 mapping; however, this would require unrea- sonably prolonged scanning time. Secondly, we R = -0.610, p = 0.0072 R = -0.608, p = 0.0074 Stimulated salivary flow after HBOT (mL/min) 0.0 0.5 1.0 1.5 2.0 2.5 M ea n T 2 in c on tr al at er al g la nd (m s) 70 80 90 100 110 120 130 140 Unstimulated salivary flow after HBOT (mL/min) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 M ea n T 2 in c on tr al at er al g la nd (m s) 70 80 90 100 110 120 130 140 FIGURE 4. A correlation between the mean T2 values in contralateral parotid glands and unstimulated (A) and stimulated salivary flow (B) after hyperbaric oxygenation therapy (HBOT). A B Radiol Oncol 2022; 56(1): 60-68. Vidmar J et al. / Hyperbaric oxygenation in post-radiation salivary glands and T2 mapping 67 analysed parotid glands on both sides. Because of limitations in the experimental setup, allowing only single slice T2 mapping and clinically applica- ble salivary tests, only single-sided T2 values from ipsilateral and contralateral parotid gland were correlated with the salivary tests in each patient. Such analysis does not enable accurate correlation between a single-sided T2 values with functional salivary testing, which includes the cumulative function of all glands. Namely, in the case of hy- pofunction of one gland, its function deficit may be compensated by the glands on the contralateral side. Since structural changes observed in T2 maps on both glandular sides were proportional prior to and after HBOT, this is less likely, suggesting that the function of both glands was affected to some extent and our approach seems still reasonable. Ideally, this could be avoided by using advanced MRI methods, combing T2 mapping or even ADC mapping with MR functional salivary flow imag- ing (MR dynamic sialography). However, such complex scanning results in excessively long scan- ning time.36 Conclusions The results of the present study confirm that T2 mapping has a potential for the evaluation of the differences between irradiated and normal parotid glandular tissue. In this study, it is shown that T2 mapping could also be useful in the evaluation of the glandular tissue response to HBOT. Acknowledgement This work was supported by Grant No.: P3- 0019, Ministry of Higher Education, Science and Technology, Slovenia. References 1. Palme CE, Gullane PJ, Gilbert RW. Current treatment options in squamous cell carcinoma of the oral cavity. Surg Oncol Clin N Am 2004; 13: 47-70. doi: 10.1016/S1055-3207(03)00123-6 2. Lefebvre JL. Current clinical outcomes demand new treatment options for SCCHN. Ann Oncol 2005; 16(Suppl 6): vi7-vi12. doi: 10.1093/annonc/ mdi452 3. Han P, Lakshminarayanan P, Jiang W, Shpitser I, Hui X, Lee SH, et al. 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Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008; 106: 115-23. doi: 10.1016/j. tripleo.2008.03.012 Radiol Oncol 2022; 56(1): 69-75. doi: 10.2478/raon-2021-0047 69 research article Comparison of local recurrence in transcatheter arterial chemoembolization of hepatocellular carcinoma with or without accumulation of iodized oil beyond corona enhancement area: Short-term results Yukinobu Watanabe, Masahiro Ogawa, Masahiro Kaneko, Mariko Kumagawa, Midori Hirayama, Naoki Matsumoto, Hiroshi Nakagawara, Toshiki Yamamoto, Mitsuhiko Moriyama Department of Gastroenterology and Hepatology, Nihon University School of Medicine, Tokyo, Japan Radiol Oncol 2022; 56(1): 69-75. Received 12 August 2021 Accepted 1 October 2021 Correspondence to: Prof. Yukinobu Watanabe, Department of Gastroenterology and Hepatology, Nihon University Hospital, 1-6 Kanda, Surugadai, Chiyoda-ku, Tokyo 101-8309, Japan. E-mail: koushin0809@yahoo.co.jp Disclosure: No potential conflicts of interest were disclosed. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Background. Local tumor recurrence of hepatocellular carcinoma (HCC) often occurs in blood drainage areas. Corona enhancement is determined by computed tomography during hepatic arteriography (CTHA) and is consid- ered to represent the blood drainage area. This study aimed to investigate the relationship between embolization of corona enhancement area and local tumor recurrence of patients with HCC who underwent transcatheter arterial chemoembolization (TACE). Patients and methods. The study retrospectively selected 53 patients with 60 HCC nodules that showed corona enhancement area on late-phase CTHA and showed homogenous accumulation of iodized oil throughout the nodule on non-contrast-enhanced CT performed immediately after TACE. We divided the nodules into two groups, according to whether the accumulation of iodized oil covered the entire corona enhancement area (group A) or not (group B). Local tumor recurrence was compared between the two groups. Results. The cumulative local tumor recurrence rates for group A (n = 36) were 2.8%, 2.8%, 8.3% at 3, 6, and 12 months, respectively, whereas the recurrence rates for group B (n = 24) were 20.8%, 45.8%, 75% at 3, 6, and 12 months, respectively. The cumulative local tumor recurrence rates for group A were significantly lower than those for group B (hazard ratio, 0.079; 95% confidence interval, 0.026–0.24; p < 0.001). Conclusions. The results of the study suggest that the corona enhancement area may be an accurate safety margin in TACE which should be performed until the embolic area covers the entire corona enhancement area. Key words: corona enhancement; transcatheter arterial chemoembolization; computed tomography during he- patic arteriography; hepatocellular carcinoma Introduction Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and is the third leading cause of cancer death worldwide.1 Transcatheter arterial chemoembolization (TACE) has been widely used to treat unresectable HCC.2- 5 According to the Barcelona Clinic Liver Cancer staging system, TACE is the first-line treatment for patients with intermediate-stage HCC6, underlin- ing the importance of TACE in the treatment of HCC. Radiol Oncol 2022; 56(1): 69-75. Watanabe Y et al. / TACE using corona enhancement70 HCC often has satellite lesions that cannot be diagnosed by imaging modalities, and the local tumor recurrences may occur because of untreated satellite lesion.7,8 Therefore, it is necessary to treat not only the tumor itself but also the area around the tumor in the treatment of HCC. Previous stud- ies reported that the adequate safety margin of io- dized oil in TACE also affects microsatellite lesions around the tumor, which resulted in a lower rate of local tumor recurrence.9-11 Corona enhancement is one of the characteris- tic findings of hypervascular HCC, and it results in perinodular enhancement with bright branching structures depicted on late-phase computed tomog- raphy during hepatic arteriography (CTHA).12-16 The corona enhancement area has been proposed as the blood drainage area of HCC and a previous study reported that satellite lesions of HCC existed in the blood drainage area.17 The corona enhance- ment area is expected to be an accurate safety mar- gin in the treatment of HCC. However, there have been no studies that have evaluated the association between embolization of corona enhancement area and local tumor recurrence following TACE. The aim of this retrospective study was to inves- tigate the relationship between local recurrence fol- lowing TACE and embolization of corona enhance- ment area. Patients and methods This study was conducted in accordance with the guidelines of the Declaration of Helsinki and was approved by our institutional ethics committee (protocol number 20200502). Informed consent was waived by the committee because this study was a retrospective study. Study population Between April 2016 and October 2019, TACE pro- cedures were performed on 345 patients with HCC at our hospital. TACE was done because patients were ineligible for surgery or they refused to un- dergo surgical resection. Radiofrequency ablation (RFA) procedures were not performed due to the location of the tumor, where ablation may cause insufficient therapeutic effects and/or adverse ef- fects on adjacent organs. The diagnosis of HCC was confirmed by at least 2 of the following modalities: contrast-enhanced computed tomography (CECT), contrast-enhanced ultrasonography, and/or gad- olinium-ethoxybenzyl-diethylenetriamine pen- taacetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI). Eligibility criteria for this study were as follows: (a) the presence of fewer than five nodules; (b) nodules treated with balloon- occluded TACE (B-TACE); (c) nodules that showed corona enhancement on late-phase CTHA; and (d) nodules showing dense accumulation of iodized oil throughout the tumor on non-contrast-enhanced CT performed immediately after TACE. The exclu- sion criteria were as follows: (a) the nodules with prior treatment; (b) impaired renal function, a con- traindication for CECT; and (c) extrahepatic metas- tases. Fifty-three patients with a total of 60 nodules were enrolled in this study. A flow diagram of pa- tient and nodule selection is shown in Figure 1. The nodules were retrospectively divided into two groups based on the degree of iodized oil ac- cumulation identified on non-contrast-enhanced CT performed immediately after TACE. In group A, a dense accumulation of iodized oil covered the whole tumor and the entire corona enhancement area. In group B, a dense accumulation of iodized oil covered the whole tumor but did not cover the entire corona enhancement area. CTHA procedure Double-phase CTHA was performed in all patients prior to TACE. After left brachial artery puncture, A 4-Fr catheter (FNSAC IV, Angiomaster; Terumo, FIGURE 1. Flow diagram of patients’ and nodules’ selection. CTHA = computed tomography during hepatic arteriography; DEB-TACE = drug-eluting beads transcatheter arterial chemoembolization; RFA = radiofrequency ablation; TACE = transcatheter arterial chemoembolization Radiol Oncol 2022; 56(1): 69-75. Watanabe Y et al. / TACE using corona enhancement 71 Tokyo, Japan) was selectively inserted into the proper, common, or replaced hepatic artery and CTHA was performed to identify tumor staining, corona enhancement, and the feeding arteries of the tumor. Examinations were performed using a 64 multidetector-row CT scanner (Aquilion CX; Canon, Tokyo, Japan). The imaging CT parameters were rotation time 0.5 s, beam collimation 64 × 0.5 mm, 0.5 mm slice thickness at 0.4 mm intervals, a tube voltage 120 kV, and volume EC. Helical scan- ning was initiated 6 or 7 s after the infusion of 13 ml iomeprol (iodine concentration 350 mg/mL; Iomeron, Eisai, Tokyo, Japan) diluted with 26 ml of saline into the common, proper, or replaced he- patic artery at a rate of 3 or 3.5 ml/s. The late-phase scanning was performed 40 s after the initiation of the infusion. TACE procedure All patients had a B-TACE using a 1.8-Fr microb- alloon catheter (Attendant Delta, Terumo, Tokyo, Japan) through a 4-Fr catheter. The microballoon catheter was placed as close to the tumor feed- ing artery as possible, and miriplatin (MIRIPLA; Dainippon-Sumitomo Pharmaceutical, Tokyo, Japan) suspended in iodized oil (Ultra-Fluid; Dainippon-Sumitomo Pharmaceutical) was inject- ed into the tumor feeding artery under balloon oc- clusion. The doses of miriplatin were determined on the basis of tumor size, however. injections of miriplatin were stopped immediately before the flow ceased completely. Th e total amount of miri- platin per session was limited to 120 mg. This stage was followed by embolization with 1-mm gelatin sponge particles (Gerpart; Nippon Kayaku, Tokyo, Japan) crushed by pumping ten times with two 2.5- mL syringes and a three-way stopcock. If several tumor feeding arteries were confirmed, B-TACE was also performed through each artery. Image evaluation Double-phase CTHA images were evaluated to determine whether corona enhancement was de- picted around the hypervascular tumor. When intranodular enhancement was observed on the early-phase CTHA and subsequently perinodular enhancement appeared on the late phase of CTHA, we determined that corona enhancement was posi- tive in this study. we also measured the thickness of corona enhancement. Based on previous re- port16, perinodular enhancement with or without irregular protrusions that was greater than 2 mm thickness was classified as thick corona enhance- ment, and flat perinodular enhancement that was less than or equal to 2 mm thick was classified as thin corona enhancement. In all patients, an un- enhanced CT was performed immediately after TACE to check for iodized oil accumulation in the target nodules and the nodules were divided into group A or B based on the degree of iodized oil ac- cumulation. Contrast-enhanced CT was performed 4 weeks after TACE for evaluation of the treatment effect. If no local tumor recurrence was identified, then contrast-enhanced CT or Gd-EOB-DTPA- enhanced MRI was performed every 3–4 months thereafter. Local tumor recurrence was judged when identifiable nodular enhancement in the ar- terial phase was seen in or adjacent to the treated tumor. In this study, two board-certified hepatolo- gists (> 10 years of experience in abdominal CT) independently assessed the images of unenhanced CT, contrast-enhanced CT, Gd-EOB-DTPA en- hanced MRI, and CTHA. If two hepatologists had different assessments, the final result of that par- ticular investigation was obtained by consensus through discussion of them. Statistical analysis Data analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). The significance of differences in background parameters was evaluated by the Mann-Whitney U test and Fisher exact test. Univariate and multivari- ate logistic regression analyses were performed to identify the factors correlated with local tumor recurrence. The main objective of this study was to investigate the relationship between emboliza- tion including corona enhancement area and local tumor recurrence in TACE, and thus, tumor recur- rence rates were compared between group A and group B. The cumulative local recurrence rate was calculated using the Kaplan-Meier method and evaluated using the log-rank test. p values of less than 0.05 were considered to be a statistically sig- nificant difference. Results Group A had 36 cases and group B had 24 cases. The clinical characteristics of nodules are summa- rized in Table 1. Except for des-gamma-carboxy Radiol Oncol 2022; 56(1): 69-75. Watanabe Y et al. / TACE using corona enhancement72 TABLE 1. Clinical characteristics of nodules in group A and B Clinical characteristics Group A (n = 36) Group B (n = 24) p value Age, years* 74 (70–79) 71 (64–79) 0.149 Gender, male/female 8/28 6/18 0.999 Etiology (HCV/HBV/Alcohol/Unknown) 16/8/8/4 9/5/6/4 0.912 Child-Pugh classification (A/B) 31/5 19/5 0.569 BCLC stage(0/A/B) 11/20/5 5/17/2 0.545 Size of tumor, mm* 14.5 (12–21.3) 15.5 (11–22) 0.757 Thickness of corona enhancement(≤ 2mm/>2mm) 17/19 10/14 0.793 AFP, ng/ml* 4.9 (2.6–10.6) 5.9 (2.7–14.1) 0.419 DCP, mAU/mL* 26.4 (17.0–49.2) 48.8 (25.3–153.9) 0.021 Usage of miriplatin, mg* 28 (20–38.5) 30 (22.4–51.5) 0.310 AFP = alpha-fetoprotein; BCLC = The Barcelona Clinic Liver Cancer Classification; DCP = des-gamma-carboxy prothrombin; HBV = hepatitis B virus; HCV = hepatitis C virus; * = data are the median (interquartile range) FIGURE 2. Local tumor recurrence after transcatheter arterial chemoembolization (TACE) in an 82-year-old patient with a hepatocellular carcinoma. (A) Early-phase computed tomography during hepatic arteriography (CTHA) demonstrated a hypervascular nodule in S5. (B) Late-phase CTHA demonstrated the corona enhancement around the tumor (white arrow). (C) Non-contrast-enhanced computed tomography performed immediately after TACE showed dense accumulation of iodized oil throughout the tumor, but not in the entire corona enhancement area. (D) In contrast enhanced magnetic resonance images obtained 4 months after TACE, local recurrence developed around the tumor (black arrow). FIGURE 3. Complete response after transcatheter arterial chemoembolization (TACE) in a 77-year-old patient with a hepatocellular carcinoma. (A) Early-phase computed tomography during hepatic arteriography (CTHA) demonstrated a hypervacular nodule in S4. (B) Late-phase CTHA demonstrated the corona enhancement around the tumor (white arrow). (C) Non-contrast-enhanced computed tomography performed immediately after TACE showed dense accumulation of iodized oil beyond corona enhancement area. (D) Contrast-enhanced computed tomography performed 15 months after TACE showed no enhancement around the tumor. prothrombin, the baseline characteristics did not differ significantly between groups A and B. In the total sample, local tumor recurrence was observed in 22 out of 60 cases (36.7%) and the cumulative 3-, 6-, 12-month tumor local recur- rence rates for all cases were 10%, 20%, and 35% respectively. The median time to local recurrence was 167 days (range, 32–375 days). In group B, lo- cal tumor recurrence was observed in 18 out of 24 cases (75%) (Figure 2). Meanwhile, in group A, lo- A AB B C CD D Radiol Oncol 2022; 56(1): 69-75. Watanabe Y et al. / TACE using corona enhancement 73 cal tumor recurrence was observed in only 4 out of 36 cases (11.1%), and most nodules had no local tumor recurrence (Figure 3). The cumulative 3-, 6-, 12-month tumor local recurrence rates were 2.8%, 2.8%, 8.3%, respectively for group A, and 20.8%, 45.8%, 75%, respectively for group B. The cumula- tive local tumor recurrence rates in group A were significantly lower than those in group B (hazard ratio [HR], 0.079; 95% confidence interval [CI], 0.026–0.24; p < 0.001) (Figure 4). Univariate logistic regression analysis for prog- nostic factors affecting local tumor recurrence was performed for the following factors: the size of tumors, serum alpha-fetoprotein level, serum des-gamma-carboxy prothrombin level, usage of miriplatin, thickness of corona enhancement, and the degree of iodized oil accumulation (Group A or B). The degree of iodized oil accumulation only showed p values < 0.05 in univariate analy- sis. Multivariate analysis identified the degree of iodized oil accumulation as independent prog- nostic factors of local tumor recurrence (p < 0.001) (Table 2). Discussion In this study, we investigated whether the embo- lization of not only the tumor but also the corona enhancement area could provide a better therapeu- tic effect for TACE. Our findings showed that the group with a dense embolization of HCC including the corona enhancement area had a significantly lower local tumor recurrence rate and a higher ther- apeutic effect than the group with a dense emboli- zation of HCC itself but not including the corona enhancement area. Our study demonstrated a rela- tionship between local recurrence following TACE and embolization of corona enhancement area. HCC often has satellite lesions that cannot be diagnosed by imaging modalities.7,8 Previous his- topathological research reported that microsatellite lesions found in 46% of HCCs were smaller than 5 cm, and in 29% of HCCs, the lesions were smaller than 2.5 cm.18 Another study reported that 28 of 149 resected specimens exhibited microsatellite le- sions.7 These reports indicate that it is important to embolize the tumor and the surrounding tissues by TACE. Miyayama et al. reported that local tumor recurrence developed in 29.1% of the sufficient margin group and 66.7% of the insufficient margin group after TACE, and embolization of the safety margin was important for local tumor control by TABLE 2. Univariate and multivariate logistic regression analysis for factor affecting local tumor recurrence Factor Univariate analysis Multivariate analysis Odds ratio (95% CI) p value Odds ratio (95% CI) p value Size of tumor (< 20 vs. ≥ 20 mm) 0.67 (0.20–2.32) 0.577 AFP (< 10 vs. ≥ 10 ng/mL) 0.45 (0.13–1.61) 0.242 DCP (< 40 vs. ≥ 40 mAU/mL) 0.59 (0.18–1.92) 0.417 Usage of miriplatin (< 30 vs. ≥ 30 mg) 0.70 (0.17–1.89) 0.420 Thickness of corona enhancement (≤ 2mm/ > 2mm) 0.97 (0.30–3.20) 0.999 Degree of iodized oil accumulation (Group A vs. B) 0.045 (0.0080–0.20) < 0.001 0.042 (0.010–0.17) < 0.001 AFP = alpha-fetoprotein; CI = confidence interval; DCP = des-gamma-carboxy prothrombin FIGURE 4. Local tumor recurrence-free rate according to the range of the embolization for transcatheter arterial chemoembolization (TACE). The cumulative 3-, 6-, 12-month tumor local recurrence rates were 2.8%, 2.8%, 8.3%, respectively, for cases with embolization of the entire corona enhancement area, and 20.8%, 45.8%, 75%, respectively, for cases without embolization of the entire corona enhancement area (hazard ratio [HR], 0.079; 95% confidence interval [CI], 0.026–0.24; p < 0.001). Radiol Oncol 2022; 56(1): 69-75. Watanabe Y et al. / TACE using corona enhancement74 TACE.9 Several other studies also reported that the safety margin of iodized oil during TACE result- ed in a lower rate of local tumor recurrence.10,11,19 Corona enhancement area determined by CTHA was reported to reflect the blood drainage area of HCC16,20, and this finding served as the basis for this study. We considered that the corona enhance- ment area would be an accurate safety margin for TACE. Hirooka et al. reported that the cumulative local recurrence rate was significantly lower in the group that ablated the entire blood drainage area, depicted as the area of corona enhancement on CTHA, than in the group that did not by RFA (0, 0, and 1.5% vs. 3.8, 17.0, and 22.8% at 1, 3, and 5 years).21 Although there are differences in the pro- cedures of RFA and TACE, the results of this previ- ous study are similar to those of our study, indicat- ing that the corona enhancement area can be an ac- curate margin for HCC treatment. The thicker the corona enhancement, the larger the drainage area that requires embolization, but thickness of corona enhancement wasn’t identified as independent prognostic factors of local tumor recurrence in this study. Therefore, we consider that the size of the drainage area cannot be a factor that makes embo- lization of the entire drainage. The usefulness of CTHA, including cone-beam CTHA during the TACE procedure, has been re- ported because of its ability to offer relevant in- formation for tumor identification, assessment of tumor feeding artery, and navigation guidance.22-24 Moreover, it has become possible for the accurate identification of tumor feeding arteries in combi- nation with guidance software19, suggesting that CTHA will be performed more frequently during TACE. By performing not only the early phase of CTHA but also the late phase of CTHA and de- picting the corona-like deep stain, CTHA can be used not only for tumor vessel identification and navigation guidance but also for determination of safety margin. As it requires two CT scans, in the early phase and in the delayed phase, it has the disadvantage of increasing the radiation exposure. However, since the therapeutic effect of TACE can be expected to increase, we consider that the ben- efits outweigh the risks for the patient. If conven- tional CT or cone-beam CT is performed before the end of the TACE procedure and accumulation of iodized oil to the corona enhancement area is in- sufficient, we believe that additional embolization should be performed considering the possibility of inadequate drug administration or the presence of the feeding artery. Whether the additional emboli- zation will improve the treatment effect is an issue for further study. Our study had several limitations. First, our study population was small and the study was ret- rospective. This limitation could have led to some patient selection bias. Moreover, as many tumors with relatively small diameters have been enrolled and only four tumors were larger than 30 mm in diameter in this study, it is unclear whether the same results as this study apply to large diameter tumors. Therefore, confirmation of our findings would require additional studies on large numbers of patients, including patients with large diameter tumors. Second, the observation period after TACE was relatively short. Longer-term observations may increase local recurrence rates. However, there was a large difference in local recurrence rates between the two groups, and it is consid- ered that there is a significant difference between the two groups even after long-term observation. Third, as late-phase CTHA was not performed at a single-slice level, assessment of corona enhance- ment area may be slightly incorrect. In the nodules in which local tumor recurrence was recognized despite the assessment that the embolization was obtained throughout the corona enhancement ar- ea, the corona enhancement area may have been underestimated. In conclusion, local tumor recurrence was sig- nificantly lower when embolizing not only the tu- mor itself but also the corona enhancement area, and corona enhancement area may be an accurate safety margin in TACE. 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J Vasc Intervent Radiol 2018; 29: 425-31. doi: 10.1016/j.jvir.2017.10.025 Radiol Oncol 2022; 56(1): 76-82. doi: 10.2478/raon-2021-0036 76 research article Pre-treatment risk assessment of women with endometrial cancer: differences in outcomes of molecular and clinical classifications in the Slovenian patient cohort Jure Knez1,2, Monika Sobocan1,2, Urska Belak1, Rajko Kavalar3, Mateja Zupin4, Tomaz Büdefeld4, Uros Potocnik4,5, Iztok Takac1,2 1 Divison of Gynecology and Perinatology, University Medical Centre Maribor, Maribor, Slovenia 2 Department of Obstetrics and Gynecology, Faculty of Medicine, University of Maribor, Maribor, Slovenia 3 Department of Pathology, University Medical Centre Maribor, Maribor, Slovenia 4 Centre for Human Molecular Genetics and Pharmacogenomics, Faculty of Medicine, University of Maribor, Maribor, Slovenia 5 Faculty of Chemistry and Chemical Engineering, University of Maribor, Maribor, Slovenia Radiol Oncol 2022; 56(1): 76-82. Received 13 May 2021 Accepted 20 August 2021 Correspondence to: Assist. Monika Sobočan, M.D., Division for Gynecology and Perinatology, University Medical Centre Maribor, Ljubljanska ulica 5, SI-2000 Maribor, Slovenia. E-mail: monika.sobocan@gmail.com Disclosure: No potential conflicts of interest were disclosed. Background. The aim of this study was to evaluate changes in prognostic risk profiles of women with endometrial cancer by comparing the clinical risk assessment with the integrated molecular risk assessment profiling. Patients and methods. This prospective study recruited patients with biopsy proven endometrial cancer treated at the University Medical Centre Maribor between January 2020 to February 2021. Patient clinical data was assessed and categorized according to the currently valid European Society of Gynaecological Oncology, European SocieTy for Radiotherapy and Oncology, and European Society of Pathology (ESGO/ESTRO/ESP) guidelines on endometrial can- cer. Molecular tumour characterization included determination of exonuclease domain of DNA polymerase-epsilon (POLE) mutational status by Sanger sequencing and imunohistochemical specimen evaluation on the presence of mismatch repair deficiencies (MMRd) and p53 abnormalities (p53abn). Results. Fourty-five women were included in the study. Twenty-two tumours were of non-specific mutational profile (NSMP) (56.4%), 13 were classified as MMRd (33.3%), 3 were classified as p53abn (7.7%) and 1 was classified as POLE mutated (2.6%). Six tumours (15.4%) had multiple molecular classifiers, these were studied separately and were not included in the risk assessment. The clinical risk-assessment classified 21 women (53.8%) as low-risk, 5 women (12.8%) as intermediate risk, 2 women as high-intermediate risk (5.1%), 10 women (25.6%) as high risk and 1 patient as advanced metastatic (2.6%). The integrated molecular classification changed risk for 4 women (10.3%). Conclusions. Integrated molecular risk improves personalized risk assessment in endometrial cancer and could potentially improve therapeutic precision. Further molecular stratification with biomarkers is especially needed in the NSMP group to improve personalized risk-assessment. Key words: endometrial cancer; molecular classification; risk assessment Introduction Endometrial cancer is the most common gynaeco- logical malignancy, with an increasing incidence in the developed world.1 Most endometrial carcinoma occur in post-menopausal women, however in rare cases they can also affect young women.2 In most cases it is diagnosed in early disease stages. With current therapeutic approaches, patients achieve an overall survival (OS) from 74% to 91%.1 Recently Radiol Oncol 2022; 56(1): 76-82. Knez J et al. / Integrated molecular risk assessment in endometrial cancer 77 published data3 on 5-year OS of women with endo- metrial cancer in Slovenia shows a modest increase in survival between the years 2012–2016 (80.6%) as compared to between the years 1997–2001 (79.8%).3 Endometrial carcinoma are divided, based on their histopathological characteristics, into Type I and Type II carcinoma. Type I carcinomas represent the majority of EC and are of endometrioid sub- type. Type II carcinomas are a more hetereogenous group of histopathological subtypes and include clear-cell, serous, mixed histology tumours and carcinosarcomas.4 There is a constant need to improve the risk as- sessment for improved precision in endometrial cancer treatment. An important aim is to identify patients that experience disease reccurrence re- gardless of the primary early stage endometrial cancer diagnosis. In the last decade, the molecular classification of endometrial cancer has emerged as a feasible possibility to stratify risk in women with endometrial cancer.5 The evaluated molecu- lar classification showed, that determining the status of endometrial carcinoma tumours for: i) pathogenic variants of the exonuclease domain of DNA polymerase-epsilon (POLE), ii) mismatch repair deficiency (MMRd) and iii) copy-number high TP53 mutations, enables the determination of specific molecular endometrial cancer subtypes6 that could be used in the risk of recurrence assess- ment.4 Clinical trials showed that patients with POLE ultramutated (POLEmut) tumours had a 100% 10-year recurrence free survival (RFS) ver- sus 80.1% in POLE wild type (POLEwt) patients.7 The recent individual patient meta-analysis has shown that the outcome of POLEmut tumours is good regardless of traditional risk classifiers.8 The prognosis is intermediate in MMRd tumours and significantly worse in p53 aberrant tumours.9 This led to the incorporation of molecular classi- fication to the recently updated European Society of Gynaecological Oncology, European SocieTy for Radiotherapy and Oncology, and European Society of Pathology (ESGO/ESTRO/ESP) guide- lines for endometrial cancer. The current guide- lines recommend the use of clinical risk assessment or an integrated clinico-molecular risk assessment, if available.4 The molecular risk assessment has been ap- plied for interventions and assessments of adju- vant therapy management decisions.9 Based on the current ESGO-ESTRO-ESP guidelines, women with low-risk EC do not need additional adjuvant therapy. For intermediate risk, radiotherapy has been suggested as the optimal course of treatment. In high-intermediate or some cases of intermediate risk, chemotherapy can be considered in addition to radiotherapy. The guidelines recommend for women with high risk EC to undergo radiotherapy with concurrent chemotherapy.4 Abdulfatah et al., reported also the use of molecular classification on biopsy specimens which showed a high level of concordance to hysterectomy specimens and out- performed pre-treatment risk assessment based on histological specimen sample and grade.10 Based on the currently available guidelines we aimed to assess the changes that clinical and in- tegrated molecular risk assessment represent in terms of primary patient management. Patients and methods Patients This single centre study prospectively recruited consecutive women treated at the University Medical Centre Maribor (UMC Maribor), Slovenia. Participants were recruited from February 2020 to February 2021. Women were eligible to participate in this study if they had a biopsy-proven EC diag- nosis and were candidates for surgical treatment. Informed consent was obtained prior to surgical treatment from all study participants. This study was approved by the Slovenian Ethics Committee for Research in Medicine under the registration number 0120-40/2020/4 and was carried out in ac- cordance to the Declaration of Helsinki. Management plan and risk assessment All included women underwent complete diag- nostic work-up at our centre, which routinely in- cludes a comprehensive transvaginal ultrasound (TVUS) scan to assess for disease extent. Based on the clinical assessment of myometrial involve- ment and disease extent, as well as histopathologi- cal tumour assessment, a clinical risk prediction was made. Afterwards, they were presented to the interdisciplinary tumour board to plan the opti- mal treatment. Standard treatment of early stage endometrial cancer is surgical and is most com- monly performed by minimally invasive surgery, but open surgical approach is also an option. This depends on the tumour type and patient charac- teristics. Surgical treatment usually consists of total hysterectomy with bilateral salpingo-oophorecto- my. This is most commonly combined with senti- nel lymph node biopsy (SLN) or pelvic/paraaortic lymph node dissection (LND). Radiol Oncol 2022; 56(1): 76-82. Knez J et al. / Integrated molecular risk assessment in endometrial cancer78 Molecular classification of endometrial cancer The molecular risk-profile was determined accord- ing to current guidelines on endometrial cancer4, based on determining the tumour POLE status, the mismatch repair decifiency status (MMRd) of the tumour as well as the p53 tumour expression status. For determining the POLE status of the tumour, DNA was isolated from tissue samples procured from the resected uterus by a patholo- gist. Tumor DNA was extracted from fresh frozen tissue using a QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manu- facturer’s protocol. DNA purity and concentration were determined using a Synergy™ 2 spectropho- tometer (Biotek, Winooski, VT, USA). Primers used for PCR amplification of selected exons 9, 12 and 13 of POLE were designed as described previously.11 For amplification of target sequences, we used PCR technique which was performed using DreamTaq DNA Polymerase (Thermo Scientific, Vilnius, Lithuania). Briefly, 2 μL of 7 ng/ μL DNA was am- plified using 8 μL mix of 1.0 μL DreamTaq buffer, 0.5 μL of each primer (10 μM), 0.2 μL dNTPs (10 mM each), 0.05 μL DreamTaq DNA polymerase (5 U/μL) and water in a final volume of 10 μL. Samples were subjected to incubation at 95˚C for 5 min, then 38 amplification cycles of 95˚C for 30 sec, 62˚C for 30 sec and 72˚C for 30 sec, and a final incubation at 72˚C for 7 min using The TProfessional Basic Thermocycler (Biometra, Analytik Jena, Jena, Germany). The PCR products were visualised on 2% agarose gel electrophoresis excised from the gel and purified using a MinElute Gel Extraction Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol. Sanger sequencing was performed by Eurofins Genomics (Germany), and nucleotide sequences were manually analysed for the most frequent somatic mutations P286R, V411L, and S459F.12 MMRd status was determined by evaluating the immunohistochemical (IHC) markers (MLH1, MSH2, MSH6, PMS2). Previous research showed13, that IHC markers represent an appropriate sur- rogate for screening for MMRd. The p53 protein expression status was evaluated using IHC expres- sion according to current recommendations.14 Based on genetic and IHC data, women were grouped, according to the ESGO/ESTRO/ESP guidelines5 into the following molecular classifica- tion groups: POLE mutated (POLEmut), MMRd, p53 abnormal (p53abn) or non-specific mutational profile (NSMP). If there were multiple molecular classifiers available for a patient, they were ana- lysed separately. Statistical analysis Descriptive statistics were used to evaluate the dis- tribution of each variable. Continuous variables were expressed as mean values with standard de- viations. Categorical variables were reported as frequency or percentage. All statistical analyses were performed using SPSS software version 23.0 (IBM Corp., Armonk, NY, USA). Results Molecular characterisation was performed in all 45 women enrolled. Among them, 39 women (86.7%) were classified according to the current recom- mendations in one of the four molecular classifica- tion groups. Their characteristics are presented in Table 1. Six women (13.3%) had multiple molecular classifiers. The characteristics of these women are presented in Table 2. Following the ESGO/ESTRO/ESP guidelines on risk assessment, women were classified according to the clinical risk assessment and the integrated molecular risk (Table 3). Most remained classified as low-risk endometrial cancer, reclassification changed for 4 women. The risk profiles of three women decreased after integrated molecular risk classification: one was re- classified from high risk to high-intermediate risk and two from intermediate to low-risk. The risk profile of women changed from: i) intermediate to low risk as one woman had a POLEmut tumour, ii) from intermediate to low risk since it was of NSMP and iii) from high to high-intermediate (HIR) risk due to the MMRd classification. In one case, the risk profile increased from low to intermediate risk due to the detected p53abn mutation. Primary surgical treatment Surgical treatment was performed in all patients included in the study. The specifics of the treat- ment are presented in Table 4. Thirty women (77%) underwent a laparoscopic procedure and 9 women (23%) an open surgical procedure. The multidisci- plinary (MDT) recommendation in cases of pre-op- eratively persumed low-risk disease is to perform sentinel lymph node biopsy (SLN). If the lymph nodes are not visualized, further LND is generally omitted. In cases of persumed intermediate risk, Radiol Oncol 2022; 56(1): 76-82. Knez J et al. / Integrated molecular risk assessment in endometrial cancer 79 the recommendation in case of SLN visualization failure is to perform LND. The impact of molecular classification on treatment decisions In the post-operative period two women (5.1%) died. Two women (5.1%) rejected the recommend- ed adjuvant therapy. The final analysis was per- formed for 37 women as depicted in Figure 1. Our study was observational and the molecu- lar classification did not impact the MDT decision making. Two women which were re-classified from intermediate to low risk by the molecular classifi- cation received radiation therapy and one women that has been reclassified from high risk to HIR received radiotherapy and chemotherapy. One pa- tient for whom the risk assesment increased from low to intermediate risk has been recommended no adjuvant treatment. In one patient of the inter- mediate risk group, the presence of co-morbidities led the MDT to suggest follow-up as the optimal strategy. Discussion In this study we assessed the risk of endometrial cancer for women using the clinical classification and the integrated molecular classification as sug- gested by the recent ESGO/ESTRO/ESP guide- TABLE 1. Patient characteristics Age at time of diagnosis (n = 39) 65.2 years (min 32 – max 86) Body Mass Index at time of diagnosis (n = 36) 31 (17–43) Reproductive history Parity (median, range) 2 (0–5) Spontaneuos abortion (median, range) 0 (0–2) Menopausal status Pre-menopausal 5 (12.8%) Post-menopausal 34 (87.2%) Tumour marker levels CA125 (n = 32) 136.3 (min 2 – max 2084) CEA (n = 32) 3.4 (min 2 – max 17) FIGO stage (n = 39) IA 21 (54%) IB 8 (20.5%) II 1 (2.6%) IIIA 2 (5.1%) IIIB 2 (5.1%) IIIC1 3 (7.7%) IIIC2 1 (2.6%) IV 1 (2.6%) Tumour type Type 1 36 (92.3%) Type 2 3 (7.7 %) Molecular tumour classification POLEmut 1 (2.6%) MMRd 13 (33.3%) NSMP 22 (56.4%) p53abn 3 (7.7%) MMRd = mismatch repair deficient tumour; NSMP = non-specific molecular profile tumour; p53abn = p53 expression abnormal tumour; POLE = DNA polymerase-epsilon; POLEmut = POLE ultramutated tumour FIGURE 1. Potential impact of risk shifts on adjuvant therapy. Depicted in circles are the absolute numbers of women with endometrial cancer and their adjuvant therapy recommendations. Arrows point to a potential risk shift impacting therapy with the use of the molecular classification. CT = chemotherapy; RT = radiotherapy Radiol Oncol 2022; 56(1): 76-82. Knez J et al. / Integrated molecular risk assessment in endometrial cancer80 lines. Out of 45 women enrolled in our study, the risk evaluation through integrated molecular risk groups was possible for 39 women. Six women had multiple molecular classifiers, which precluded further risk assessment using the current molecu- lar classification. In the remaining group, assess- ment of molecular risk decreased in three and in- creased in one woman. TABLE 2. Characteristics of patients with multiple molecular classifiers Age at time of diagnosis Multiple-classifier EC POLE variant Tumor type FIGO stage Lymphovascular invasion Clinical risk assessment Patient 1 76 POLEmut and p53abn P286R endometrioid IIIC2 Yes High Patient 2 75 POLEmut and p53abn P286R carcinosarcoma IB Yes High Patient 3 70 MMRd and p53abn wild-type endometrioid IA No Low Patient 4 87 MMRd and p53abn wild-type endometrioid IIIB Yes High Patient 5 53 POLEmut and p53abn P286R endometrioid IA No Low Patient 6 52 POLEmut and p53abn P286R endometrioid IA No Intermediate MMRd = mismatch repair deficient tumour; p53abn = p53 expression abnormal tumour; POLE = DNA polymerase-epsilon; POLEmut = POLE ultramutated tumour TABLE 3. Risk assessment Number of patients (%) ESGO Clinical Risk Group Low risk 21 (53.8%) Intermediate risk 5 (12.8%) High-intermediate risk 2 (5.1%) High risk 10 (25.6%) Advanced metastatic 1 (2.6%) Integrated molecular risk Low risk 22 (56.4%) Intermediate risk 4 (10.3%) High-intermediate risk 3 (7.7%) High risk 9 (23.1%) Advanced metastatic 1 (2.6%) ESGO = European Society of Gynaecological Oncology TABLE 4: Analysis of surgical treatment of women based on ESGO Clinical Risk Group assesment. SLN – sentinel lymph node biopsy, LND - lymphadenectomy ESGO integrated molecular risk Total number of women Open surgery Laparoscopic SLN LND Unilateral SNB and contralateral LND No LN treatment Low risk 22 (56.4%) 2 (5.1%) 20 (51.3%) 18 (46.2%) 0 0 4 (10.3%) Intermediate 4 (10.3%) 0 4 (10.3%) 3 (7.7%) 1 (2.6%) 0 0 HIR 3 (7.7%) 1 (2.6%) 2 (5.1%) 0 2 (5.1%) 1 (2.6%) 0 High 9 (23.1%) 5 (12.8%) 4 (10.3%) 2 (5.1%) 5 (12.8%) 2 (5.1%) 1 (2.6%) Advanced 1 (2.6%) 1 (2.6%) 0 0 0 0 1 (2.6%) HIR = high-intermediate risk; LND = lymphadenectomy; SLN = sentinel lymph node biopsy The classification of women with endometrial cancer into risk groups based on clinical and mo- lecular data aims to improve individualised treat- ment according to the tumour biological potential. According to the currently valid guidelines4, his- topathological characteristics, including lymph- vascular space invasion (LVSI) are the cornerstone of risk stratification. This is one of the reasons that shifts in risk groups between clinical and integrated molecular groups were present, as LVSI has been identified as an important marker of prognosis in low-risk endometrial cancer and is associated with adverse outcomes.15 In comparison with a study of Oberndorfer et al.16 which compared molecular risk with the guidelines on clinical risk assessment in endometrial cancer published in 201617, there were less shifts in the clinical risk assessment in our study. Therefore, by using the newly implemented clinical or integrated molecular risk assessment, comparable risk assessment is achieved. Our study is the first to compare the clinical and molecular risk stratification in the cohort of Slovenian pa- tients. The clinical value of this approach needs to be verified in further prospective studies. The nature of our study was observational and the molecular subtype was not taken into consid- eration for decision-making in regards to surgical Radiol Oncol 2022; 56(1): 76-82. Knez J et al. / Integrated molecular risk assessment in endometrial cancer 81 or adjuvant therapy. Table 4 represents the surgi- cal approach taken for women based on the pre- treatment assessment and the final ESGO Risk Group classification. The body of knowledge on using the integrated molecular classification for decision making on surgical treatment is scarce. Histopathological pre-operative evaluation might not always be concordant with post-operative di- agnosis18,19 and could potentially impact the MDT to suggest less invasive surgical procedures. This is especially true in recognizing serous EC. In these instances based on the molecular characteristics such as TP53 mutations20, integrated molecular evaluation could potentially enable individualized therapy already in the primary surgical setting. Considering the changes in risk assessment for patients within our study, if using the integrated molecular assessment, two patients would most likely been recommended follow-up instead of radiotherapy, thus de-escalating their therapy based on their individual biological features. One patient would have been, based on her biological characteristics upgraded from low to intermedi- ate risk. Especially in the low-intermediate and intermediate-high risk group, it is important to identify the patients correctly. Without appropri- ate adjuvant therapy, these patients were found to be at a approximately 30% higher risk than if adju- vant therapy was offered.21 One of our patients was downgraded from high risk to the HIR risk group. While this individual has received radiotherapy and chemotherapy, this is also a valid strategy of treatment in the HIR risk group4 and the decision process most likely would have not changed for her. Participants in our study have been mostly classified as NSMP (56.4%). NSMP tumours are currently classified in the low-risk or intermedi- ate risk4 based on a combination with other histo- pathological characteristics. As NSMP represents the largest group of currently classified tumours this shows the need for further refinements of mo- lecular risk stratification.22,23 A potential additional marker which has shown potential prognostic value, especially in the intermediate risk groups, is L1CAM expression23,24 and in low risk endometrial cancer also mutation of CTNNB1.25 Further evalua- tion of these and other novel markers could enable us to further individualise and stratify risk assess- ment in this large and clinically diverse group.26 An important consideration in our study is how to evaluate and incorporate risk assessment for women with multiple classifiers. There were six women with multiple classifiers in which four tu- mours were p53abn and POLEmut. Multiple classi- fiers MMRd and p53abn were present in two wom- en. Recently, Leon-Castilo et al.27 published data on their cohort of patients with multiple-classifier endometrial cancer. They supported the bioinfor- matic clustering of TCGA data which stated that MMRd-p53abn tumours mostly clustered around MMRd tumours and p53-POLEmut tumours most- ly clustered around POLEmut tumours. Clinical outcomes showed that patients with MMRd- p53abn had a 5-year recurrence free survival (RFS) of 92.2% and patients with POLEmut-p53abn en- dometrial cancer a 5-year RFS of 94.1%.27 This also supports the hypothesis that these tumours are biologically less aggressive than single classifier p53abn tumors, which had a 5-year RFS of 48%.28 Further research and biomarker development is therefore needed to evaluate the appropriate ap- proaches for patient treatment in cases of non-spe- cific mutational profiles and multiple-classifiers. The strength of our study is that it is the first pro- spectively designed study to evaluate the imple- mentation of molecular risk stratification to endo- metrial cancer patients in Slovenia. The limitation is the low number of patients included. The follow- up data is not yet available and the clinical implica- tions of this approach are yet to be determined. Conclusions The introduction of molecular risk stratification in the management of women with endometrial cancer represents a significant shift from the es- tablished clinical practice. Several adjustments to the routine workflow and significant additional resources are necessary in order to implement this approach to the clinics. Our data shows that in comparison to the current clinical risk stratifica- tion based on clinical and histopathological data, this may lead to change in management in a small proportion of women. The clinical value of this re- mains to be proven in further prospective studies. It is also important to note that the molecular risk stratification is not applicable to all women and refinements of the current classification with addi- tional biomarkers are likely to improve and further de-escalate treatment in certain subtypes of endo- metrial cancer in the future. Acknowledgement The project was funded by the Institutional Research funding of UMC Maribor, reg. no IRP- Radiol Oncol 2022; 56(1): 76-82. Knez J et al. / Integrated molecular risk assessment in endometrial cancer82 2019/02-13 and by the Slovenian Research Agency (research core funding P3-0067). References 1. Morice P, Leary A, Creutzberg C, Abu-Rustum N, Darai E. Endometrial cancer. Lancet 2016; 387:1094-108. doi: 10.1016/S0140-6736(15)00130-0 2. Repše-Fokter A. Endometrial cancer in young woman. Acta Medico– Biotechnica 2020; 13: 60-4. 3. Zadnik V, Zagar T, Lokar K, Tomsic S, Konjevic AD, Zakotnik B. Trends in population-based cancer survival in Slovenia. Radiol Oncol 2021; 55: 42-9. doi: 10.2478/raon-2021-0003 4. Concin N, Matias-guiu X, Vergote I, Cibula D, Mirza MR, Marnitz S, et al. ESGO / ESTRO / ESP Guidelines for the management of patients with en- dometrial carcinoma. Int J Gynecol Cancer 2021; 31: 12-39. doi: 10.1136/ ijgc-2020-002230 5. Urick ME, Bell DW. Clinical actionability of molecular targets in endometrial cancer. Nat Rev Cancer 2019; 19: 510-21. doi: 10.1038/s41568-019-0177-x 6. Cancer Genome Atlas Research Network, Kandoth C, Schultz N, et al. Integrated genomic characterization of endometrial carcinoma. Nature 2013; 497: 67-73. doi: 10.1038/nature12113 7. Wortman BG, Creutzberg CL, Putter H, Jürgenliemk-Schulz IM, Jobsen JJ, Lutgens LCHW, et al. Ten-year results of the PORTEC-2 trial for high-interme- diate risk endometrial carcinoma: improving patient selection for adjuvant therapy. Br J Cancer 2018; 119: 1067-74. doi: 10.1038/s41416-018-0310-8 8. McAlpine JN, Chiu DS, Nout RA, Church DN, Schmidt P, Lam S, et al. Evaluation of treatment effects in patients with endometrial cancer and POLE mutations: An individual patient data meta-analysis. Cancer 2021; 26: 5400-10. doi: 10.1002/cncr.33516 9. Vermij L, Smit V, Nout R, Bosse T. Incorporation of molecular characteristics into endometrial cancer management. Histopathology 2020; 76: 52-63. doi: 10.1111/his.14015 10. Abdulfatah E, Wakeling E, Sakr S, Al-Obaidy K, Bandyopadhyay S, Morris R, et al. Molecular classification of endometrial carcinoma applied to endome- trial biopsy specimens: Towards early personalized patient management. Gynecol Oncol 2019; 154: 467-74. doi: 10.1016/j.ygyno.2019.06.012 11. Malentacchi F, Turrini I, Sorbi F, Projetto E, Castiglione F, Vergoni F, et al. Identification of a gene panel for endometrioid endometrial cancer: a pos- sible prognostic value? Reprod Sci 2020; 27: 592-8. doi: 10.1007/s43032- 019-00059-8 12. McAlpine J, Leon-Castillo A, Bosse T. The rise of a novel classification system for endometrial carcinoma; integration of molecular subclasses. J Pathol 2018; 244: 538-49. doi: 10.1002/path.5034 13. Raffone A, Travaglino A, Cerbone M, Guida M, Insabato L, Zannoni GF, et al. Diagnostic accuracy of immunohistochemistry for mismatch repair proteins as surrogate of microsatellite instability molecular testing in endome- trial cancer. Pathol Oncol Res 2020; 26: 1417-27. doi: 10.1007/s12253-020- 00811-5 14. Köbel M, Ronnett BM, Singh N, Soslow RA, Gilks CB, McCluggage WG. Interpretation of P53 immunohistochemistry in endometrial carcinomas. Int J Gynecol Pathol 2019; 38: S123-S131. doi: 10.1097/PGP.0000000000000488 15. Ayhan A, Şahin H, Sari ME, Yalçin I, Haberal A, Meydanli MM. Prognostic sig- nificance of lymphovascular space invasion in low-risk endometrial cancer. Int J Gynecol Cancer 2019; 29: 505-12. doi: 10.1136/ijgc-2018-000069 16. Oberndorfer F, Moling S, Hagelkruys LA, Grimm C, Polterauer S, Sturdza A, et al. Risk reclassification of patients with endometrial cancer based on tumor molecular profiling: First real world data. J Pers Med 2021; 11: 1-11. doi: 10.3390/jpm11010048 17. Colombo N, Creutzberg C, Amant F, Cibula D, Mirza MR, Marnitz S, et al. ESMO-ESGO-ESTRO consensus conference on endometrial cancer. Int J Gynecol Cancer 2016; 26: 2-30. doi: 10.1097/igc.0000000000000609 18. Han G, Sidhu D, Duggan MA, Arseneau J, Cesari M, Clement PB, et al. Reproducibility of histological cell type in high-grade endometrial carci- noma. Mod Pathol 2013; 26: 1594-604. doi: 10.1038/modpathol.2013.102 19. Sobočan M, Ogrizek AM, Ledinek T, Takač I, Knez J. Importance of pre- operative ultrasound examination and pathological tumour evaluation in the management of women with endometrial cancer. Eur J Obstet Gynecol Reprod Biol 2021; 257: 121-126. doi: 10.1016/j.ejogrb.2020.12.029 20. Bogani G, Ray-Coquard I, Concin N, Ngoi NYL, Morice P, Enomoto T, et al. Uterine serous carcinoma. Gynecol Oncol 2021; 162: 226-34. doi: 10.1016/j. ygyno.2021.04.029 21. Van Den Heerik ASVM, Horeweg N, De Boer SM, Bosse T, Creutzberg CL. Adjuvant therapy for endometrial cancer in the era of molecular classifi- cation: radiotherapy, chemoradiation and novel targets for therapy. Int J Gynecol Cancer 2021; 31: 594-604. doi: 10.1136/ijgc-2020-001822 22. Talhouk A, McConechy MK, Leung S, Li-Chang HH, Kwon JS, Melnyk N, et al. A clinically applicable molecular-based classification for endometrial cancers. Br J Cancer 2015; 113: 299-310. doi: 10.1038/bjc.2015.190 23. Kommoss FKF, Karnezis AN, Kommoss F, Talhouk A, Taran FA, Staebler A, et al. L1cam further stratifies endometrial carcinoma patients with no specific molecular risk profile. Br J Cancer 2018; 119: 480-6. doi: 10.1038/s41416- 018-0187-6 24. Weinberger V, Bednarikova M, Hausnerova J, Ovesna P, Vinklerova P, Minar L, et al. A novel approach to preoperative risk stratification in endometrial cancer: The added value of immunohistochemical markers. Front Oncol 2019; 9: 1-13. doi: 10.3389/fonc.2019.00265 25. Imboden S, Tapia C, Scheiwiller N, Kocbek V, Altermatt HJ, Janzen J et al. Early-stage endometrial cancer, CTNNB1 mutations, and the relation be- tween lymphovascular space invasion and recurrence. Acta Obstet Gynecol Scand 2020; 99: 196-203. doi: 10.1111/aogs.13740 26. Kolehmainen A, Pasanen A, Tuomi T, Koivisto-Korander R, Butzow R, Loukovaara M. Clinical factors as prognostic variables among molecular subgroups of endometrial cancer. PLoS One 2020; 15:1-12. doi: 10.1371/ journal.pone.0242733 27. León-Castillo A, Gilvazquez E, Nout R, Smit VT, McAlpine JN, McConechy M, et al. Clinicopathological and molecular characterisation of ‘multiple-clas- sifier’ endometrial carcinomas. J Pathol 2020; 250: 312-22. doi: 10.1002/ path.5373 28. Leon-Castillo A, De Boer SM, Powell ME, Mileshkin LR, Mackay HJ, Leary A, et al. Molecular classification of the PORTEC-3 trial for high-risk endometrial cancer: Impact on prognosis and benefit from adjuvant therapy. J Clin Oncol 2020; 38: 3388-97. doi: 10.1200/JCO.20.00549 Radiol Oncol 2022; 56(1): 83-91. doi: 10.2478/raon-2021-0049 83 research article Cystatin C and cystatin SN as possible soluble tumor markers in malignant uveal melanoma Maria A. Dikovskaya1,2, Galina S. Russkikh3, Konstantin V. Loktev4, Thomas P. Johnston5, Margarita M. Gevorgyan1, Natalya P. Voronina1, Valery V. Chernykh2, Alexander N. Trunov2,4, Tatiana A. Korolenko1 1 Scientific Research Institute of Neurosciences and Medicine, Novosibirsk, Russia 2 The S. Fyodorov Eye Microsurgery Federal State Institution, Novosibirsk Branch. Novosibirsk, Russia 3 Federal Research Center of Fundamental and Translational Medicine, Institute of Biochemistry, Novosibirsk, Russia 4 Federal State Budget Scientific Institution, Federal Research Center of Fundamental and Translational Medicine, Novosibirsk, Russia 5 Division of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri, USA Radiol Oncol 2022; 56(1): 83-91. Received 16 August 2021 Accepted 21 October 2021 Correspondence to: Prof. Tatiana A. Korolenko, Ph.D., Scientific Research Institute of Neurosciences and Medicine. Timakov St., 4, 630117 Novosibirsk, Russia. E-mail: t.a.korolenko@physiol.ru Disclosure: No potential conflicts of interest were disclosed. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Background. The aim of the study was to determine the concentration of endogenous cystatin C and cystatin SN, as potential tumor biomarkers, in the serum and biological fluids of the eye in both healthy controls and patients with uveal melanoma. Patients and methods. The concentration of both cystatins was determined in the intraocular fluid (IOF), tear fluid, and serum of patients with uveal melanoma and compared to baseline measurements in IOF, tears, serum, cerebral spinal fluid, saliva and urine of healthy controls. Results. The concentration of cystatin C in all the biological matrices obtained from healthy controls significantly exceeded the concentration of cystatin SN and was independent of gender. Cystatin C concentrations in the tear fluid of patients with uveal melanoma (both the eye with the malignancy, as well as the contralateral, non-affected eye), were significantly greater than cystatin C concentrations in the tear fluid of healthy controls and was independ- ent of tumor size. The concentration of cystatin SN in IOF of patients with uveal melanoma was significantly less than the corresponding concentration of cystatin SN in healthy controls. Conclusions. The ratio of cystatins (CysC:CysSN) in both the serum and tear fluid, as well as the concentration of cystatin SN in IOF, would appear to strongly suggest the presence of uveal melanoma. It is further suggested that multiple diagnostic criteria be utilized if a patient is suspected of having uveal melanoma, such as determination of the cystatin C and cystatin SN concentrations in serum, tears, and IOF, ocular fundus and ultrasound imaging, and biopsy with histopathological evaluation. Key words: uveal melanoma; cystatins; biomarkers; diagnosis Introduction Uveal melanoma frequently leads to progression of the malignancy and subsequent metastasis, which often results in death in patients with metastatic disease. The methods developed for the treatment of uveal melanoma consist of either removing the affected eye (enucleation), or complex ocular thera- pies (brachytherapy, laser photocoagulation, heat therapy, proton therapy, etc.).1,2 Radiol Oncol 2022; 56(1): 83-91. Dikovskaya MA et al. / Cystatins as biomarkers in uveal melanoma84 Despite advances in the diagnosis and treatment of uveal melanoma, mortality 5 years after enuclea- tion of the eye is 16.5%; after 10 years, it is 58%. Extra-scleral germination significantly worsens the prognosis; in fact, in these patients, the mortal- ity reaches 69–73% after 10 years.3,4 According to recent results of large-scale studies in the United States with more than 7500 patients with uveal melanoma, the risk of metastasis and death in- creases significantly with each stage of cancer di- agnosis. For example, Stage T1 (2 times), Stage T2 (4 times), and Stage T3 (8 times).5,6 Uveal melanoma, arising from melanocytes in the stroma, is the most common primary intraocu- lar tumor in adults.7.8 Detection of specific proteins allows for the identification of possible molecular markers of malignancy in several eye diseases. Specific tear proteins (~ 64 of 491 proteins), studied by proteome analysis and gel electrophoresis, are classified as proteases and protease inhibitors and carry special significance in the context of eye ma- lignancies. Mammalian cystatins (to date, there are 12 known human cystatins) include a large family of proteins that have the ability to inhibit cysteine proteases9,10, which are further divided into three types based on their molecular structure and dis- tribution in the body.11 As just mentioned, cystatins can be categorized into three types. The first type (e.g., cystatins A and B) are intracellular cystatins (stefins). The second type (e.g., cystatins C, D, E/M, F, G, S, SN, SA) are extracellular cystatins. Finally, the third type of cystatins (e.g., L-kininogen, H-kininogen) are intra- vascular proteins.12 Cystatin SN has not been thor- oughly studied to date13, whereas, the most well- known cystatin, cystatin C, was the first to be iden- tified and its amino acid sequence determined.14,15 Subsequently, the functions of cystatin C, as an inhibitor of cysteine proteases, was investigated, as well as its role in cell proliferation, migration, ag- ing, and cell death.16,17 Cystatins are endogenous and reversible inhibi- tors of cysteine peptidases that are important play- ers in cancer progression.18-20 Importantly, cysta- tin C plays a significant role in the physiological functions of eye fluids12, as well as in the patho- logical processes associated with a number of eye tumors.21-23 As an example, in 2009, Paraoan et al. reported that for one particular lysosomal cysteine protease (cathepsin S), there was an increase in the active form of this protease that was not counter- balanced by the expression of its strongest endoge- nous inhibitor (cystatin C) in an aggressive, highly- metastatic form of uveal melanoma.24 The imbal- ance in cathepsin S and its inhibitor (cystatin C) is both relevant and important in the context of uveal melanoma, because it may provide a link to thera- peutic anti-cancer strategies based on targeting the elastolytic and collagenolytic activity of cysteine cathepsins, as well as add to our understanding of the dysregulation in proteolytic activity that occurs in uveal melanoma.24 As previously mentioned, a large number of pro- teases and protease inhibitors have been identified among the 491 proteins in the tear fluid proteome.25 Changes in the composition of tear proteins are as- sociated with a number of inflammatory, degen- erative, and malignant eye diseases.26,27 In fact, the balance between proteases and protease inhibitors is important for controlling the rates of cellular metabolism and the barrier function of the eye cor- nea.25 Furthermore, changes in the biological fluids of the eye are related to the ratio of proteases and protease inhibitors27-29, which can affect the compo- sition of proteins and peptides in the lacrimal fluid. Thus, it would seem reasonable to assume that the identification of specific proteins in the biological fluids of the eye may make it possible to identify new molecular markers for several eye diseases. The precise role of cysteine protease inhibitors in the development of eye tumors has not been ful- ly elucidated to date.30-32 This is significant to oph- thalmology, because some of these inhibitors may be of therapeutic benefit for the treatment of eye tumors. Thus, the aim of this study was to inves- tigate the concentration of endogenous inhibitors of cysteine proteases; namely, cystatin C and cys- tatin SN, in the serum and the biological fluids of the eye in both healthy controls and patients with uveal melanoma. Patients and methods Patients and the collection of various biological matrices/fluids All studies were carried out with informed con- sent of patients and in accordance with the ethi- cal norms of the Helsinki Declaration (2000) and local regulations (Russian Council of Medical Research). The protocols were approved by the Institutional Review Board of biomedical eth- ics of the S. Fyodorov Eye Microsurgery Federal State Institution, Novosibirsk Branch (Protocol N4, 15.11.2017). Lastly, all the patients gave their informed consent for laboratory tests, as well as consent to process their personal data for scientific purposes. Radiol Oncol 2022; 56(1): 83-91. Dikovskaya MA et al. / Cystatins as biomarkers in uveal melanoma 85 Fifty-seven patients (mean age = 56.6 ± 2.4 years) with a diagnosis of choroidal melanoma in the Novosibirsk Branch of the S. Fyodorov Eye Microsurgery Federal State Institution were in- cluded in this investigation. The control group con- sisted of 37 healthy individuals (medical staff of the clinic and students of the Medical University, with a mean age = 31.0 ± 4.1 years for the subjects ≤ 60 years old [i.e., n = 28 (n = 13 healthy controls ≤ 40 years old + n = 15 healthy controls 41–60 years old)]. Of the 37 healthy controls, 20 were men and 17 were women, with 9 control subjects over 60 years old. Since the literature indicates that the serum levels of cystatin C increase with age in normal healthy indi- viduals, and especially after the age of 60 years33-36, we selected a subgroup of the healthy individuals (41–60 years; mean age = 53.1 ± 3.4 years; n = 15) as an age-matched control group to facilitate appro- priate statistical comparisons with choroidal mela- noma patients. Intraocular fluid (IOF) was obtained from 7 control patients (3 men and 4 women) close to, but not exceeding 60 years of age, undergoing an uncomplicated cataract removal procedure and submitted for biochemical analysis. Tears from the conjunctival sac were collected by microcannulas and blood from the ulnar vein. Specifically, the tear fluid was obtained from the lower conjunctival arch of the eye and placed into a dry, sealed tube of 300–500 microliters. To evaluate the IOF in patients with ocular melanoma, mois- ture in the anterior chamber of the enucleated eye was obtained during the operation. Samples of cerebrospinal fluid were obtained from 8 additional patients in the Federal State Budget Institute (“Federal Neurosurgical Center”, Novosibirsk, Russia) as part of a standard exami- nation for neurosurgical patients without tumors. An exclusion-criteria relevant to this study was the value of the estimated glomerular filtration rate (eGFR). Since the levels of cystatin C in various bio- logical fluids could potentially be affected by over- all kidney function, patients with an eGFR value less than 90 mL/min/1.73 m2 were excluded from the present study to control for this variable. Analysis of cystatins in biological fluids The concentration of cystatin C in biological fluids was evaluated using ELISA kits for human cystatin C (BioVendor, Czechia). The measurements were performed using a biochemical analyzer AU 480 (Beckman Coulter, USA). The concentration of cystatin SN was also de- termined using commercial ELISA kits for human cystatin SN (Cusabio, China). The measurements were conducted using a Stat Fax 2100 microplate reader for enzyme immunoassay (Awareness Technology Inc., USA) at 450 nm. Statistical analysis All acquired data were reported as the mean ± the standard deviation (s.d.). Mean values were ana- lyzed for statistically significant differences with the software program STATISTICA 10.0 using a one-way, analysis-of-variance (ANOVA). Post-hoc analysis of ANOVA testing was performed using the Least Significant Difference (LSD) test. When comparing only two mean values, we used the Student’s t-test to identify a difference that was statistically significant. All statistical results using either ANOVA, or the Student’s t-test, in which p < 0.05 were deemed statistically significantly dif- ferent and were noted in the Figures, as well as in Table 1. Results Eye imaging Figure 1A shows a typical image of the ocular fun- dus of a normal eye as compared to an eye with a choroidal melanoma having a thickness of 2.4 mm and a diameter of 8 mm. The thickness of the choroidal melanoma was also determined from an ultrasound image as shown in Figure 1B and was determined to be 3.1 mm. Concentration of cystatin C and cystatin SN in various biological matrices in healthy individuals (controls) Figure 2 shows the concentration of cystatin C in various biological matrices (cerebral spinal TABLE 1. Concentrations of cystatin C and cystatin SN in biological fluids of healthy individuals as a function of age (Mean ± s.d.) Groups Inhibitor Serum(ng/mL) Tears (ng/mL) IOF (ng/mL) Healthy (≤ 40 years) [n = 13] Cystatin C Cystatin SN 561 ± 10.0 2.24 ± 0.20 296 ± 11.1 0.49 ± 0.30 - Healthy (41–60 years) [n = 15] Cystatin C Cystatin SN 539 ± 111 2.96 ± 0.70 256 ± 82.3 0.6 ± 0.35 414 ± 28 2.7 ± 1.40 Healthy (61–80 years) [n = 9] Cystatin C Cystatin SN a1,341 ± 177 b4.77 ± 0.10 382 ± 116 0.75 ± 0.14 a844 ± 113 2.18 ± 0.20 a = significantly greater (p < 0.01) cystatin C concentration in serum and IOF compared to individuals ≤ 60 years; b = significantly greater (p < 0.01) cystatin SN concentration in serum compared to individuals ≤ 60 years; IOF = Intraocular fluid; s.d. = standard deviation Radiol Oncol 2022; 56(1): 83-91. Dikovskaya MA et al. / Cystatins as biomarkers in uveal melanoma86 concentration of cystatin C in all the biological matrices significantly exceeded the concentration of cystatin SN in the same matrices (Figure 2 vs. Figure 3). Additionally, the rank order of cystatin C concentrations in the various biological fluids followed the order CSF > saliva > serum > IOF > tears > urine (Figure 2), whereas, for cystatin SN, the rank order was saliva > urine > CSF > serum > IOF > tears (Figure 3). The concentration of cystatin C and cysta- tin SN in three relevant biological fluids (serum, tears, and IOF) was determined for three different age groups to assess whether there was an age- dependent variation in the concentration of these two inhibitors (Table 1). As was dete rmined with the concentrations of cystatin C and cystatin SN in the six biological fluids of healthy individuals (Figure 2 and 3), there was a significantly greater concentration of cystatin C relative to cystatin SN in the serum, tears, and IOF (Table 1). While we de- termined that there was no gender difference ob- served between the concentration of each inhibitor in each age group for each of the three biological fluids mentioned above, there was a significant (p < 0.01) elevation in the serum concentration of both cystatins in healthy individuals (age 61–80 years) when compared to individuals less than or equal to 60 years of age (Table 1). This finding was also observed for IOF, but only for cystatin C and not cystatin SN (Table 1). Cystatin C and cystatin SN concentrations and their ratio in the serum and tear fluid of patients with uveal melanoma Cystatin C levels were significantly (p < 0.01) greater in both serum and tear fluid in patients with uveal melanoma when compared to healthy controls (Figure 4A). However, with regard to the concentration of cystatin SN in these same two biological fluids, there was only a significant (p < 0.01) decrease in the concentration of cystatin SN in the serum of patients with uveal melanoma com- pared to healthy controls (Figure 4B). Importantly, the ratio of cystatin C to cystatin SN (CysC:CysSN) in both serum and tear fluid was significantly (p < 0.001) increased in patients with uveal melanoma when compared to corresponding mean values of this ratio in healthy controls (Figure 4C). As an aside, we also determined the inhibitor with higher concentrations in all biological flu- ids tested in this study; namely, cystatin C, for its prevalence in the tear fluid of patients with dif- FIGURE 1. Ocular fundus of a normal eye, and eye with choroidal melanoma. (A) Ocular fundus. Normal healthy eye (left) versus eye with choroidal melanoma (right; dia = 8 mm and thickness = 2.4 mm). (B) Ultrasound image of the choroidal melanoma (thickness = 3.1 mm). A B 0 500 1000 1500 2000 2500 3000 3500 4000 CSF Saliva Serum IOF Tears Urine C on ce nt ra tio n of C ys ta tin C (n g/ m L) a a,b a,b,c a,b,d a,b,c,d,e a,b,c,d,e FIGURE 2. Cystatin C concentration in various biological matrices in healthy individuals. Concentration of cystatin C in each biological fluid (mean ± standard deviation [s.d.]; n = 28 [n = 13 healthy controls ≤ 40 years old + n = 15 healthy controls 41–60 years old]), since Table 1 shows a significant increase in cystatin C concentrations in serum and intraocular fluid (IOF) in 61–80 year old healthy controls, and thus, this age group was not included; cerebral spinal fluid (CSF) was obtained from an additional and separate group of cancer-free neurosurgical patients (n = 8) for the determination of the cystatin C concentration as described in the Materials and methods section. a Significant difference (p < 0.001) from mean values indicated with the same letter. b Significant difference (p < 0.001) from mean values indicated with the same letter. c Significant difference (p < 0.01) from mean values indicated with the same letter. d Significant difference (p < 0.01) from mean values indicated with the same letter. e Significant difference (p < 0.01) from the mean value indicated with the same letter. fluid [CSF], saliva, serum, IOF, tears, and urine) in healthy individuals, while Figure 3 depicts the concentration of cystatin SN in these same bio- logical matrices. In general, it was found that the Radiol Oncol 2022; 56(1): 83-91. Dikovskaya MA et al. / Cystatins as biomarkers in uveal melanoma 87 ferent size uveal melanoma tumors. The cystatin C concentrations in the tear fluid of patients with uveal melanoma (both the eye with the malignan- cy, as well as the contralateral, non-affected eye), were significantly (p < 0.05) greater than cystatin C concentrations determined in the tear fluid of healthy controls (i.e., range of 450–500 ± 60 ng/mL in the diseased eye with uveal melanoma vs. 250 ± 25 ng/mL in both eyes of healthy controls) and was independent of tumor size. Moreover, at the time of clinical presentation, there was no significant difference between the cystatin C concentration in the tear fluid of the malignant eye versus the cor- responding concentration of cystatin C in the tear fluid of the contralateral, non-affected eye regard- less of tumor size (data not shown). Concentration of cystatin C and cystatin SN in IOF of patients with uveal melanoma versus healthy controls Figure 5A shows the concentrations of cystatin C in IOF in healthy controls and patients with uveal melanoma, while Figure 5B shows the concentra- tion of cystatin SN in these same two patient co- horts. There was no significant difference between the concentration of cystatin C in IOF of healthy controls and patients with uveal melanoma, but there was a significant (p < 0.001) reduction in the concentration of cystatin SN in IOF of patients with uveal melanoma when compared to this same pa- rameter in healthy controls. Discussion The present study has addressed the question as to whether cystatin C and/or cystatin SN may poten- 0 1 2 3 4 5 6 7 8 9 10 CSF Saliva Serum IOF Tears Urine C on ce nt ra tio n of C ys ta tin S N (n g/ m L) a,b b,c b,d a,c,d,e e FIGURE 3. Cystatin SN concentration in various biological matrices in healthy individuals. Concentration of cystatin SN in each biological fluid (mean ± standard deviation [s.d.]; n = 28 [n = 13 healthy controls ≤ 40 years old + n = 15 healthy controls 41–60 years old]), since Table 1 shows a significant increase in the cystatin SN concentration in the serum of 61–80 year old healthy controls, and thus, this age group was not included; cerebral spinal fluid (CSF) was obtained from an additional and separate group of cancer-free neurosurgical patients (n = 8) for the determination of the cystatin SN concentration as described in the Materials and methods section. a Significant difference (p < 0.001) from the mean value indicated with the same letter. b Significant difference (p < 0.05) from mean values indicated with the same letter. c Significant difference (p < 0.001) from the mean value indicated with the same letter. d Significant difference (p < 0.01) from the mean value indicated with the same letter. e Significant difference (p < 0.001) from the mean value indicated with the same letter. 0 200 400 600 800 1000 1200 1400 C on c. o f C ys ta tin C (n g/ m L) Healthy Controls Patients with Uveal Melanoma Serum Tear Fluid * * 0 200 400 600 800 1000 1200 1400 1600 C ys C :C ys S N R at io Healthy Controls Patients with Uveal Melanoma Serum Tear Fluid * * 0 1 2 3 4 5 C on c. o f C ys ta tin S N (n g/ m L) Healthy Controls Patients with Uveal Melanoma Serum Tear Fluid * tially function as biomarkers in uveal melanoma. Clearly, our work has shown that the concentra- tion of each cysteine proteinase inhibitor (cystatin C and cystatin SN) is perturbed in uveal melanoma in various biological fluids. We first briefly de- scribe the role of each cystatin, and then their use as potential biomarkers in cancer. Cysteine proteinase inhibitors, cystatins, are involved in mechanisms controlling intracellular and extracellular protein degradation.11,37 Cystatin FIGURE 4. Cystatin C (A) and cystatin SN (B) concentrations and their ratio (C) in the serum and tear fluid of patients with uveal melanoma. (A) Values represent the mean ± standard deviation (s.d.) of n = 15 healthy controls (mean age = 53.1 ± 3.4 years) and n = 51 of 57 total patients with uveal melanoma (mean age = 51.7 ± 2.8 years; 6 patients were > 60 years old and were therefore not included). (B) Values represent the mean ± s.d. of n = 15 healthy controls (mean age = 53.1 ± 3.4 years) and n = 51 of 57 total patients with uveal melanoma (mean age = 51.7 ± 2.8 years; 6 patients were > 60 years old and were therefore not included). (C) Values represent the mean ± s.d. of n = 15 healthy controls (mean age = 53.1 ± 3.4 years) and n = 51 of 57 total patients with uveal melanoma (mean age = 51.7 ± 2.8 years; 6 patients were > 60 years old and were therefore not included). * = significant difference (p < 0.001) from the mean value for healthy controls in each biological matrix A B C Radiol Oncol 2022; 56(1): 83-91. Dikovskaya MA et al. / Cystatins as biomarkers in uveal melanoma88 C is a secreted cysteine protease inhibitor, which is abundantly expressed in body fluids and pos- sibly regulated at both the transcriptional and post-translational levels.38,39 Production of cysta- tin C from hematopoietic cell lineages contributes significantly to the overall systemic pool of cysta- tin C.40 This particular cystatin is the most abun- dant and potent member21,27 of the cystatin family, which is important due to the fact that the activity of various cysteine proteases, both inside and out- side of cells, requires careful regulation or control by endogenous inhibitors such as cystatin C. The levels of cystatin C in the systemic circulation (se- rum) are typically different from the concentration of cystatin C in biological fluids of the eye, such as IOF and tears.29,41 In fact, according to our data, the concentration of cystatin C in serum was sig- nificantly greater than in both IOF and tears. It has been suggested that tears may function as a pool, or reservoir, for biomarkers of various pathological eye conditions, as well as for diseases beyond just ocular disorders.42 Cystatin C is involved in numerous diseases, including atherosclerosis and cancer, as well as the aging process.17,29,41 Importantly, cystatin C is believed to prevent tumor progression by inhibit- ing the activities of a family of lysosomal cysteine cathepsins. Using cystatin C-deficient animals, Huh et al. reported that cystatin C concentrations in vivo might influence tumor metastasis in some tissues.43 Interestingly, cystatin C is downregu- lated in prostate cancer and may prevent tumor progression by inhibiting the activities of a fam- ily of lysosomal cysteine proteases.44 However, Hammouda et al.45 suggested that although serum cystatin C levels may potentially represent a novel biomarker that reflects tumor burden (based on the fact that cystatin C levels were significantly more elevated in diffuse large B-cell lymphoma patients than in controls), there was no prognostic value regarding overall survival. Jiang et al. have recent- ly reported that both serum and urine cystatin C levels are elevated, and the cystatin C gene is up- regulated nearly 50-fold, in patients with multiple myeloma, which suggests it use as a diagnostic bio- marker in multiple myeloma.31 Additionally, Leto and Sepporta32 suggested the use of cystatin C as a predictive biomarker for breast cancer. Lastly, it is worth noting that Kos et al. considers cystatin C to be a potential anticancer agent.41 Next, we turn to the other cysteine protease inhibitor evaluated in the present study; namely, cystatin SN. Cystatin SN, along with cystatins S and SA, belongs to the second type of extracellu- lar cystatins (in this case, salivary cystatins), which has not been as thoroughly studied as cystatin C. While cystatin SN is not as prevalent as cystatin C in normal mammalian tissues46, it is a member of the cystatin family that inhibits the proteolytic ac- tivity of cysteine proteases. In fact, univariate and multivariate analyses have indicated that cystatin SN possibly acts as a marker for cancer prognosis.13 For example, cystatin SN has been shown to be a tumor biomarker that provides useful information for the diagnosis of esophageal47-49, gastric, pancre- atic, and colorectal cancers30,50, as well as neuro- blastomas and melanomas.12 As it pertains to cancer progression, cystatin SN is thought to be involved in several malignant tu- mors.51 For instance, it was recently reported by Cui et al. that upregulation of this inhibitor promoted the progression of hepatocellular carcinoma.51 Of note, knockdown of cystatin SN significantly re- duced the expression of proliferation-related pro- teins p-AKT and PCNA10, which indicates a more complex role of cystatins in tumor growth and pro- gression beyond their role as inhibitors of cysteine cathepsins. Lastly, a survival study in patients with surgically-resected, non-small cell lung cancer re- vealed an association between elevated expres- sion levels of cystatin SN and poor prognosis.46 Specifically, the study indicated that significantly increased expression of cystatin SN was directly correlated with a higher rate of cancer recurrence, metastatic risk, and poor overall survival.46 The present study focused on the use of cys- tatin C and cystatin SN as potential biomarkers 0 100 200 300 400 500 600 700 800 C on c. o f C ys ta tin C In IO F (n g/ m L) Healthy Controls Patients with Uveal Melanoma 0 1 2 3 4 5 C on c. o f C ys ta tin S N In IO F (n g/ m L) Healthy Controls Patients with Uveal Melanoma * A B FIGURE 5. Concentration of cystatin C (A) and cystatin SN (B) in intraocular fluid of patients with uveal melanoma versus healthy controls. (A) Values represent the mean ± standard deviation (s.d.) of n = 7 healthy controls (mean age = 57.5 ± 1.9 years) and n = 18 of 57 total uveal melanoma patients selected based on an age close to the mean age of the 7 healthy controls (mean age = 55.9 ± 3.2 years; n = 18). (B) Values represent the mean ± s.d. of n = 7 healthy controls (mean age = 57.5 ± 1.9 years) and n = 18 of 57 total uveal melanoma patients selected based on an age close to the mean age of the 7 healthy controls (mean age = 55.9 ± 3.2 years; n = 18). * = significant difference (p < 0.001) from mean value for healthy controls; IOF = intraocular fluid Radiol Oncol 2022; 56(1): 83-91. Dikovskaya MA et al. / Cystatins as biomarkers in uveal melanoma 89 in the context of uveal melanoma. We success- fully showed that the concentrations of cystatin C and cystatin SN were significantly elevated, and reduced, respectively, in the serum of pa- tients with uveal melanoma compared to healthy controls. While there was a significant increase in the concentration of cystatin C in the tear fluid of patients with uveal melanoma when compared to healthy controls, there was no significant dif- ference in the concentration of cystatin SN in the tear fluid between these same two patient cohorts. However, we would suggest that the value of the CysC:CysSN ratio in both biological matrices (i.e., serum and tears) may potentially be a better indi- cator of uveal melanoma than either inhibitor (cys- tatin) alone, since the ratio was very significantly increased in both matrices. The change in the concentration of cystatin SN in another ocular fluid; specifically, IOF, may serve as further evidence to suggest the presence of uveal melanoma, since the concentration of this cystatin was significantly reduced in patients with uveal melanoma when compared to corresponding con- centrations of cystatin SN in healthy controls. This finding may argue for a combined determination of the concentrations of both cystatin C and cysta- tin SN in serum, tear fluid, and IOF to assist oph- thalmologists that have a preliminary suspicion concerning the presence of uveal melanoma, es- pecially when combined with both ocular fundus and ultrasound imaging. However, it is important to mention that there are other ocular disorders (e.g., AMD) that may perturb the concentrations of cystatins in various biological fluids, which is why it is important to have multiple diagnostic cri- teria to confirm the presence of uveal melanoma. For example, a variant of cystatin C (i.e., variant B, cystatin C) differs from the wild-type protein by a single amino acid (A25T) and is associated with de- creased plasma cystatin C levels and an increased risk of developing AMD, which potentially raises the prospect of cystatin C replacement therapy for patients homozygous for variant B.52 In conclusion, the present investigation has documented changes in the concentrations of cys- tatin C and cystatin SN in various biological fluids in both healthy controls and patients with uveal melanoma, which may possibly serve as potential biomarkers of uveal melanoma, especially when the value of the CysC:CysSN ratio is determined in both the serum and tear fluid. That is, the value of the CysC:CysSN ratio may be a better indica- tor of the possibility of uveal melanoma than ei- ther cystatin alone. We would also suggest that the profound reduction in the concentration of cysta- tin SN in IOF may provide further support for the possible presence of uveal melanoma. However, it is imperative for ophthalmologists to utilize multi- ple diagnostic criteria if they suspect that a patient has uveal melanoma, including, but not limited to, the concentrations of cystatin C and cystatin SN in serum, tears, and IOF, together with ocular fundus and ultrasound imaging. Lastly, as it pertains to the present findings de- scribed herein, we further suggest that the concen- trations of cystatin C and cystatin SN in serum, tears, and IOF, as well as diagnostic ocular imaging studies, be combined with tissue biopsy and sub- sequent evaluation by surgical pathology to differ- entiate between malignant and benign eye tumors, since Dikovskaya et al. reported that the level of cystatin C in tears was significantly elevated (rela- tive to the concentration of cystatin C in the tears of healthy controls) in both malignant and benign eye tumors.27 That is, the concentration of cystatin C in the tear fluid of patients with both malignant and benign eye tumors was significantly elevated relative to this same measurement in healthy con- trol patients, but was not significantly different (p > 0.05) between patients with either a malignant, or benign, eye tumor.27 It is for the latter reason that a tissue biopsy with histopathological evaluation is absolutely necessary to distinguish a malignant eye tumor from one that is benign, although, as mentioned directly above and verified in the pre- sent study, cystatin C levels in the serum and tears of patients with uveal melanoma are profoundly elevated relative to cystatin C levels in these same two biological fluids in healthy controls, and thus, is certainly suggestive of possible uveal melanoma. Reliance on multiple diagnostic criteria is critically important for uveal melanoma, since surgery to re- move the melanoma and a small area of healthy tis- sue is reserved for small melanomas, whereas enu- cleation is typically required for large eye tumors. Acknowledgements The authors would like to gratefully acknowledge the assistance of Prof. Kuleshova Olga Nikolaevna (S. Fyodorov Eye Microsurgery Federal State Institution, Novosibirsk, Russia) with the clini- cal portion of this work. We are also grateful to Professor J. Kos (Slovenia) for support and Dr. I.N. Ignatik (AquaTest, St. Petersburg) for providing as- sistance with the determination of human cystatin C in biological samples. Radiol Oncol 2022; 56(1): 83-91. Dikovskaya MA et al. / Cystatins as biomarkers in uveal melanoma90 The present study was supported with funding provided by the Institute of Neurosciences and Medicine, Novosibirsk, Russia (2017–2021) for ba- sic scientific research. References 1. 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Sci Rep 2015; 5: 8230. doi: 10.1038/srep08230 47. Chen YF, Ma G, Cao X, Luo RZ, He LR, He JH, et al. Overexpression of cystatin SN positively affects survival of patients with surgically resected esophageal squamous cell carcinoma. BMC Surg 2013; 13: 15. doi: 10.1186/1471-2482- 13-15 48. Oh SS, Park S, Lee KW, Madhi H, Park SG, Lee HG, et al. Extracellular cystatin SN and cathepsin B prevent cellular senescence by inhibiting abnormal glycogen accumulation. Cell Death Dis 2017; 8: e2729. doi: 10.1038/cd- dis.2017.153 49. Oh BM, Lee SJ, Cho HJ, Park YS, Kim JT, Yoon SR, et al. Cystatin SN inhibits auranofin-induced cell death by autophagic induction and ROS regulation via glutathione reductase activity in colorectal cancer. Cell Death Dis 2017; 8: e2682. doi: 10.1038/cddis.2017.100 50. Yoneda K, Iida H, Endo H, Hosono K, Akiyama T, Takahashi H, et al. Identification of cystatin SN as a novel tumor marker for colorectal cancer. Int J Oncol 2009; 35: 33-40. doi: 10.3892/ijo_00000310 51. 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Vision Research 2010; 50: 737-42. doi: 10.1016/j.visres.2009.10.022 Radiol Oncol 2022; 56(1): 92-101. doi: 10.2478/raon-2021-0050 92 research article Clinical impacts of copy number variations in B-cell differentiation and cell cycle control genes in pediatric B-cell acute lymphoblastic leukemia: a single centre experience Klementina Crepinsek1,2, Gasper Marinsek1, Marko Kavcic2,3, Tomaž Prelog3, Lidija Kitanovski3, Janez Jazbec2,3, Marusa Debeljak1,2 1 Clinical Institute for Special Laboratory Diagnostics, University Children’s Hospital, University Medical Centre Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Department of Oncology and Haematology, University Children’s Hospital, University Medical Centre Ljubljana, Ljubljana, Slovenia Radiol Oncol 2022; 56(1): 92-101. Received 7 September 2021 Accepted 5 November 2021 Correspondence to: Assist. Prof. Maruša Debeljak, Ph.D., Clinical Institute for Special Laboratory Diagnostics, University Children’s Hospital, University Medical Centre Ljubljana, Vrazov trg 1, 1000 Ljubljana, Slovenia. E-mail: marusa.debeljak@kclj.si Disclosure: No potential conflicts of interest were disclosed. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Background. IKZF1 gene deletions have been identified as a poor prognostic factor in pediatric B-cell acute lympho- blastic leukemia (B-ALL), especially in the presence of co-occurring deletions (IKZF1plus profile). This study aimed to determine the frequency of IKZF1 deletions and deletions in other B-cell differentiation and cell cycle control genes, and their prognostic impact in Slovenian pediatric B-ALL patients. Patients and methods. We studied a cohort of 99 patients diagnosed with B-ALL from January 2012 to December 2020 and treated according to the ALL IC-BFM 2009 protocol. Eighty-eight bone marrow or peripheral blood samples were analysed for copy number variations (CNVs) using the SALSA MLPA P335 ALL-IKZF1 probemix. Results. At least one CNV was detected in more than 65% of analysed samples. The most frequently altered genes were PAX5 and CDKN2A/B (30.7%, 26.1%, and 25.0%, respectively). Deletions in IKZF1 were present in 18.2% of analysed samples and were associated with an inferior 5-year event-free survival (EFS; 54.8% vs. 85.9%, p = 0.016). The IKZF1plus profile was identified in 12.5% of the analysed samples, and these patients had an inferior 5-year EFS than those with deletions in IKZF1 only and those without deletions (50.8% vs. 75.0% vs. 85.9%, respectively, p = 0.049). Overall survival (OS) was also worse in patients with the IKZF1plus profile than those with deletions in IKZF1 only and those without dele- tions (5-year OS 76.2% vs. 100% vs. 93.0%, respectively). However, the difference between the groups was not statisti- cally significant. Conclusions. Our results are in concordance with the results obtained in larger cooperative clinical trials. Copy num- ber variations analysis using the SALSA MLPA kit is a reliable tool for initial diagnostic approach in children with B-ALL, even in smaller institutions in low- and middle-income countries. Key words: B-acute lymphoblastic leukemia; IKZF1 deletions; IKZF1plus; MLPA; pediatric; copy number variations (CNVs) Introduction Improvements in risk stratification and new thera- peutic approaches have dramatically improved treatment outcomes in pediatric B-cell acute lymphoblastic leukemia (B-ALL). In developed countries, the overall survival for these patients is approaching 90%.1,2 Nevertheless, some genetic Radiol Oncol 2022; 56(1): 92-101. Crepinsek K et al. / Copy number variations in pediatric B-ALL 93 subtypes still imply poor outcomes, and 10–20% of patients experience a relapse that is often ac- companied by treatment resistance and failure.3,4 Therefore, the need for new diagnostic and prog- nostic markers remains of paramount importance. In the previous years, deletions in the IKZF1 gene have been identified as an important predic- tor of relapse in B-ALL.5-9 IKZF1 gene is located on chromosome 7p12.2 and consists of 8 exons, and of those, exons 2–8 are protein-coding. The gene codes for the transcription factor IKAROS, which regulates the expression of genes that control cell cycle progression and cell survival. It is involved in the development of all lymphoid lineages, especial- ly in the differentiation of B-progenitor cells. Exons 4–6 encode four zinc finger DNA-binding domains that are essential for the tumour-suppressive func- tion of IKAROS, and exon 8 encodes two zinc fin- gers that are responsible for the homo- or heter- odimerization of IKAROS.10,11 Genomic deletions in IKZF1 occur in around 15% of pediatric B-ALL cases.5,6,8,12-14 Their occurrence is exceptionally high in BCR-ABL1-positive (70%)15,16 and BCR-ABL1-like (40%)13,17 B-ALL, and have been associated with poor treatment response and an increased risk of relapse.5–9 Deletions are most common in exons 4–7 or affect the whole gene, however, other less com- mon lesions may also be present (e. g. deletions in exons 2–8, 2–7), and they are all associated with an unfavourable outcome in pediatric B-ALL.6,18 Therefore, some study groups on B-ALL treatment have decided to include IKZF1 deletion status into their risk stratification protocols. Others, however, did not, as there was hesitation on whether the prognostic impact of these deletions was strong enough to justify treatment intensification.14,19,20 Recently, another, minimal residual disease (MRD) dependent prognostic profile IKZF1plus with an immensely poor prognostic value was identified. This profile is defined by additional de- letions in genes involved in cell differentiation and cell cycle regulation. IKZF1 deletions that co-occur with deletions in CDKN2A, CDKN2B, PAX5, or the PAR1 region (deletions of CSF2RA and IL3RA, but not CRLF2) in the absence of ERG deletions are as- sociated with the worst event-free and overall sur- vival.21 This profile is already being used in the cur- rent AIEOP-BFM ALL 2017 trial as a high-risk cri- terion.20 Copy number variations (CNVs) in some aforementioned genes (PAX5, CDKN2A, CDKN2B) also seem to be independently associated with poor prognosis, however, the results remain con- flicting.19,22-25 The inclusion of the CNV status of these genes may significantly improve risk strati- fication in B-ALL, but more studies are needed to elucidate their true prognostic effect. Deletions in IKZF1 are mostly observed in high- risk pediatric ALL subtypes. Many studies have confirmed the association of IKZF1 deletions and IKZF1plus profile with poor treatment outcomes. In Slovenia, no studies have been done yet to deter- mine the frequency of IKZF1 deletions and CNVs in other cell differentiation and cell cycle regula- tion genes in pediatric B-ALL patients and treat- ment outcomes for these patients. Due to the im- portance of these alterations in the prognosis and choosing the best treatment approach, it is of great importance to determine their presence. Therefore, the study aimed to analyze bone marrow samples from Slovenian pediatric patients diagnosed with B-ALL from January 2012 to December 2020 for the presence of these CNVs and to determine their prognostic value. Patients and methods Patients and samples In total, 99 children with B-ALL that were treated at the University Children’s Hospital, University Medical Centre Ljubljana between January 2012 and December 2020 according to the ALL IC-BFM 2009 protocol were included in this study. Diagnoses were established following standard clinical, cyto- morphological, and immunological criteria. We obtained bone marrow samples for 92 pa- tients as part of the diagnostic procedure before starting treatment. For 7 patients, bone marrow samples were not available, therefore, peripheral blood samples were obtained for the analysis. For four patients, there was no sufficient material avail- able to perform the multiplex ligation-dependent probe amplification (MLPA) assay, 3 samples con- tained less than 40% of blasts and were excluded from analysis, and for an additional four, the assay failed due to poor sample quality. Therefore, data analysis was performed on 88 patient samples (82 bone marrow and 6 peripheral blood). The bone marrow samples contained 77.4 ± 16.7% of blast in average, and for the peripheral blood samples this value was 77.8 ± 16.0%. For survival analysis, patient samples from the year 2020 were excluded, due to the short follow-up period. Therefore, the survival analysis was carried out on 72 patients diagnosed between January 2012 and December 2019. Informed consent was obtained from all sub- jects involved in the study, or their parents. The Radiol Oncol 2022; 56(1): 92-101. Crepinsek K et al. / Copy number variations in pediatric B-ALL94 study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the Republic of Slovenia (ref- erence number KME 51/03/11). DNA extraction Genomic DNA was extracted from bone marrow or peripheral blood samples using the FlexiGene DNA Kit 250 (QIAGEN®, Hilden, Germany) according to the manufacturer’s instructions. All samples were quantified using DS-11 FX+ Spectrophotometer (DeNovix, Wilmington, USA), and stored at 4°C. Before analysis, the DNA con- centration was established at 25 ± 1 ng/μL for the MLPA assay. Analysis of copy number alterations DNA was analysed for copy number alterations us- ing the SALSA MLPA P335 ALL-IKZF1 probemix, according to the manufacturer’s instructions (MRC Holland, Amsterdam, the Netherlands). The P335 probemix allows for the detection of deletions and duplications in B-cell differentiation and cell cycle control genes (IKZF1, CDKN2A/B, PAX5, EBF1, ETV6, BTG1, and RB1), as well as in genes from the X/Y PAR1 region (CRLF2, CSF2RA, SHOX, IL3RA, and P2RY8). It also contains 13 reference probes that function as internal controls. Additionally, analysis for copy number alterations for the deter- mination of ERG status was carried out on samples that carried deletions in IKZF1 and at least one ad- ditional gene (namely CDKN2A, CDKN2B, PAX5, CSF2RA, and IL3RA) with the SALSA MLPA P327 iAMP21-ERG probemix. The P327 probemix is used for the detection of deletions, duplications or amplifications of specific sequences on chromo- some 21, including intragenic deletions of ERG. It contains 59 MLPA probes that bind to several regions on chromosome 21, including the ERG gene, and 13 reference probes. DNA samples from healthy donors were used as controls. MLPA reactions were carried out on a 96-well PCR thermocycler SimpliAmp Thermal Cycler (Applied Biosystems, Thermo Fisher, Massachusetts, USA), and the products were sepa- rated by capillary electrophoresis on an ABI-3500 genetic analyser (Applied Biosystems, Thermo Fisher, Massachusetts, USA). The resulting peak in- tensities were analysed using Coffalyser software (MRC-Holland) which performed the intrasample and intersample normalization of the peaks with the manufacturer’s reference probes and normal control DNA, respectively. Values above 1.3 were considered as gain, between 1.3 and 0.75 normal, between 0.75 and 0.25 heterozygous loss, and be- low 0.25 homozygous loss. Statistical analysis All statistical analyses were performed using SPSS 22.0 software (IBM Corp., Armonk, NY, USA). TABLE 1. The demographic and clinical characteristics of Slovenian B-ALL patients included in the study Characteristic Nr. of patients 99 Sex Male 54 (54.5%) Female 45 (45.5%) Primary genetic abnormalities ETV6-RUNX1 28 (28.3%) BCR-ABL1 7 (7.1%) KMT2A rearrangements 4 (4.0%) TCF3-PBX1 4 (4.0%) Hyperdiploidy 27 (27.3%) Hypodiploidy 3 (3.0%) iAMP21 2 (2.0%) No recurrent abnormalities 24 (24.2%) Age at diagnosis < 1 3 (3.0%) 1–5 57 (57.6%) ≥ 6 39 (39.4%) Risk group Standard risk 17 (17.2%) Intermediate risk 59 (59.6%) High risk 23 (23.2%) FC- minimal residual disease Day 15 < 0.1% 35 (35.4%) 0.1–10% 48 (48.5%) > 10% 13 (13.1%) Unknown 3 (3.0%) Day 33 < 0.01% 73 (73.7%) 0.01–1% 20 (20.2%) > 1% 3 (3.0%) Unknown 3 (3.0%) Radiol Oncol 2022; 56(1): 92-101. Crepinsek K et al. / Copy number variations in pediatric B-ALL 95 carried additional deletions in CDKN2A, CDKN2B and PAX5, three in CDKN2A and CDKN2B, one in only CDKN2B, and one had deletions in the PAR1 region. ERG deletions were not found in any of these 11 samples. The comparison of patient characteristics de- pending on IKZF1 deletion is summarized in FIGURE 2. Primary genetic alterations in patients with IKZF1 deletions. FIGURE 1. Prevalence of ALL subtypes in the Slovenian pediatric B-ALL cohort. FIGURE 3. The number of CNVs present in Slovenian B-ALL samples. Event-free survival (EFS; defined as the time be- tween diagnosis and relapse or death) and overall survival (OS; defined as time between diagnosis and death or last follow-up) were analysed using the Kaplan-Meier method and the differences be- tween multiple groups were analysed using the log- rank test. Multivariate analysis was performed us- ing a Cox regression model, which was adjusted for other risk factors, namely sex, age at diagnosis, and risk group (HR vs. non-HR). Comparisons of cat- egorical values were carried out using the Fischer’s exact test, and for the comparison of numerical val- ues, the Mann–Whitney U-test was used. The sig- nificance level for all the tests was 5% (p < 0.05 was considered to be statistically significant). Results Study group The cohort included 54 males and 45 females (N = 99), with median age 4 years (range from 1 day to 23 years). Of these, 75 harboured recurrent ge- netic abnormalities (28 ETV6-RUNX1, 27 hyperdip- loid karyotype, 7 BCR-ABL1, 4 KMT2A rearrange- ments, 4 TCF3-PBX1, 3 hypodiploid karyotype and 2 iAMP21), while no genetic alterations were identified in 24 patients (Figure 1). Based on age, white blood cell (WBC) count at diagnosis, blast cell counts on day 8, genetic abnormalities, and MRD at days 15 and 33, 17 patients were classified as standard risk (SR), 59 as intermediate risk (IR), and 23 as high risk (HR). The main patient charac- teristics are summed up in Table 1. Detection and analysis of IKZF1 deletions by MLPA Altogether, 92 patient samples and 5 controls were analysed by MLPA using the SALSA MLPA P335 ALL-IKZF1 probemix. Four patient samples failed the MLPA analysis. Out of the remaining 88 sam- ples, IKZF1 deletions were found in 16 (18.2%). The most common was the deletion of the whole gene, which was observed in eight patients (50%), oth- ers were focal deletions. The second most common deletion was the deletion of exons 4–8, which was found in four patients (25%). This deletion was de- scribed as rare in other studies. Other deletions that were also detected in our cohort were the deletion of exons 2–8 (two patients; 12.5%), 4–7 and 5 (both found in one patient; 6.3%). Eleven patients with IKZF1 deletion had additional deletions present, which put them in the IKZF1plus group. Of these, six Radiol Oncol 2022; 56(1): 92-101. Crepinsek K et al. / Copy number variations in pediatric B-ALL96 Table 2. Of the 16 patients that harboured IKZF1 deletions, five patients had no recurrent genetic alterations. The presence of primary recurrent ge- netic abnormalities found in patients with IKZF1 deletions is shown in Figure 2. IKZF1 deletions were significantly more common in Ph+ patients than in Ph– patients (6/7, 85.7% vs. 10/81, 12.3%, p TABLE 2. Patients’ characteristics and response to treatment according to IKZF1 deletion status in 91 Slovenian pediatric B-ALL patients Characteristic IKZF1 status No IKZF1 deletion IKZF1 deletion only IKZF1 plus Nr. of patients 72 5 11 Sex Male 34 (47.2%) 5 (100%) 9 (81.8%) Female 38 (52.8%) 0 (0.0%) 2 (18.2%) Primary genetic abnormalities ETV6-RUNX1 24 (33.3%) 0 (0.0%) 1 (9.1%) BCR-ABL1 1 (1.4%) 2 (40.0%) 4 (36.4%) KMT2A rearrangements 4 (5.6%) 0 (0.0%) 0 (0.0%) TCF3-PBX1 3 (4.2%) 0 (0.0%) 1 (9.1%) Hyperdiploidy 20 (27.8%) 2 (40.0%) 1 (9.1%) Hypodiploidy 2 (2.8%) 0 (0.0%) 1 (9.1%) iAMP21 1 (1.4%) 0 (0.0%) 0 (0.0%) No recurrent abnormalities 18 (25.0%) 1 (20.0%) 3 (27.3%) Age at diagnosis < 1 3 (4.2%) 0 (0.0%) 0 (0.0%) 1–5 41 (56.9%) 2 (40.0%) 5 (45.5%) ≥ 6 28 (38.9%) 3 (60.0%) 6 (54.5%) Risk group Standard risk 13 (18.1%) 0 (0.0%) 0 (0.0%) Intermediate risk 46 (63.9%) 2 (40.0%) 4 (36.4%) High risk 13 (18.1%) 3 (60.0%) 7 (63.6%) FC- minimal residual disease Day 15 < 0.1% 30 (41.7%) 0 (0.0%) 1 (9.1%) 0.1–10% 33 (45.8%) 2 (40.0%) 6 (54.5%) > 10% 6 (8.3%) 3 (60.0%) 4 (36.4%) Unknown 3 (4.2%) 0 (0.0%) 0 (0.0%) Day 33 < 0.01% 56 (77.8%) 0 (0.0%) 8 (72.7%) 0.01–1% 13 (18.1%) 3 (60.0%) 2 (18.2%) > 1% 2 (2.8%) 1 (20.0%) 0 (0.0%) Unknown 1 (1.4%) 1 (20.0%) 1 (9.1%) = 0.0001), and so was the presence of the IKZF1plus profile (4/7, 57.1% vs. 7/81, 8.6%, p = 0.004). The IKZF1 deletions were significantly more common in males than in females (p = 0.0045), how- ever, the presence of the IKZF1plus profile did not significantly differ between the sexes (p = 0.0607). Patients with IKZF1 deletions were also older at diagnosis than those without deletions (median age 6 years, interquartile range (IQR) = 8 years vs. median age 4 years, IQR = 3.25 years, p = 0.052), and so were the patients with IKZF1plus profile in comparison to those who did not have this profile (median age 6 years, IQR = 8 years vs. median age 4 years, IQR = 4, p = 0.136), however the differenc- es were not statistically significant. Patients with IKZF1 deletions showed higher blast count values on the 8th day of chemotherapy treatment (medi- an blast count 252 blasts/μL, IQR = 2018.5 blasts/ μL vs. median blast cell count 17 blasts/μL, IQR = 192,5 blasts/μL, p = 0.0005), higher values of MRD on day 15 (median MRD15 6.15% IQR = 30.67% vs. median MRD15 0.184%, IQR = 1.24%, p = 0.0005), and 33 of treatment (median MRD33 0.001%, IQR = 0.15% vs. median MRD33 0.000%, IQR = 0.01%, p = 0.033) compared to those without deletions. Similarly, blast cell count and MRD15 values of patients with the IKZF1plus profile were higher compared to patients without the profile (median blast cell counts 224 blasts/μL, IQR = 2112 blasts/ μL vs. median blast cell count 24 blasts/μL, IQR = 220 blasts/μL, p = 0.006; median MRD15 3.8%, IQR = 17.6% vs. median MRD15 0.24%, IQR = 2.23%, p = 0.030), while the difference was not significant for MRD33. The deletions were also more common in the HR group than in the IR (10/23, 43.5% vs. 6/52, 11.5%, p = 0.0032) and the SR group (10/23, 43.5% vs. 0/13, 0%, p = 0.0045). Patients in the HR group also exhibited the IKZF1plus profile more of- ten than those in the IR (7/23, 30.4% vs. 4/52, 7.7%, p = 0.0160) and the SR group (7/23, 30.4% vs. 0/13, 0%, p = 0.0294). In our cohort, 13 patients (14.8%) experienced an event (either relapse or death). Among these, four patients had no recurrent genetic alterations, three patients carried the ETV6-RUNX1 fusion gene, two the BCR-ABL1 fusion gene, two were hyper- diploid, one had a KMT2A rearrangement and one carried the TCF3-PBX1 fusion gene. One patient was classified as SR, eight as IR and four as HR. In this group, five patients (5/13, 38.5%) carried an IKZF1 deletion, and of those, four had additional deletions, but lacked the ERG deletions, which met the criteria for the IKZF1plus profile. The presence of IKZF1 deletions and the IKZF1plus profile was Radiol Oncol 2022; 56(1): 92-101. Crepinsek K et al. / Copy number variations in pediatric B-ALL 97 higher in the group of patients who experienced an event in comparison to those who did not, however the difference did not reach statistical significance (5/13 vs. 11/75, p = 0.055 and 4/13 vs. 7/75, p = 0.053, respectively). Detection and analysis of other gene deletions and duplications by MLPA The SALSA MLPA P335 ALL-IKZF1 probemix can detect deletions or duplications in the following B-cell differentiation and cell cycle control genes: IKZF1, EBF1, CDKN2A/B, PAX5, ETV6, BTG1, RB1, and in the PAR1 region (SHOX area, CRLF2, CSF2RA, IL3RA and P2RY8 genes). At least one CNV was detected in 60 patient samples (68.2%). Of those, 60% carried three or more CNVs, 28.3% carried only one CNV, and 11.7% carried two CNVs (Figure 3). The most common CNVs were those in the PAX5 gene that were present in 30.7% of analysed samples, followed by CDKN2A and CDKN2B that were altered in 26.1% and 25.0% of analysed sam- ples, respectively. In these genes, deletions were more common than amplifications. CNVs were also very common in the PAR1 region, 22.7% of analysed samples had at least one CNV present in this region, and in these genes, amplifications were observed more often than deletions. Detailed information about CNVs in all analysed genes are shown in Figure 4. Prognostic significance of IKZF1 deletions First, the patients were divided into two groups, a group with and a group without IKZF1 deletions. The 5-year EFS was significantly worse for patients harbouring IKZF1 deletions, compared to those without the deletions (54.8% vs. 85.9%, p = 0.016) (Figure 5A). The 5-year OS was slightly worse as well for these patients, although the difference was not statistically significant (81.5% vs. 93.0%, p = 0.295) (Figure 5B). The 5-year EFS and OS were also compared be- tween groups with no IKZF1 deletions, with dele- tions in the IKZF1 gene only and with the IKZF1plus profile. The difference in EFS between groups was statistically significant (p = 0.049). Pairwise com- parison showed that patients in group IKZF1plus had significantly poorer EFS in comparison to those in group with no IKZF1 deletions (5-year EFS 50.8% vs. 85.9%, p = 0.016), while the difference was not significant between other groups (Figure 6A). The OS analysis between groups showed no differ- ences between the groups (Figure 6B). A multivariate Cox regression model was ap- plied to this data to see, whether after adjusting for other relevant risk factors, IKZF1 deletions profile retained a prognostic impact on event-free sur- vival. We included the following variables in the model: sex, age at diagnosis, risk group (HR vs. nonHR) and the presence of IKZF1 deletions. The overall model showed borderline significance (p = 0.05). When each separate variable was inspected, it was observed that both sex and the presence of the IKZF1 deletions showed a certain trend (males having poorer survival than females (HR = 1.44), and those with the IKZF1 deletions having poorer survival than those without the deletions (HR = 1.15)), however, they did not reach significance (p = 0.072 and p = 0.078, respectively). Similar results were obtained when this analysis was applied for the IKZF1plus profile. This can most likely be attrib- uted to the small sample size of our sample for sur- vival analysis. FIGURE 4. Frequency of copy number variations: (A) Gene deletions in the cohort. (B) Gene amplifications in the cohort. BTG1 = BTG anti-proliferation factor 1; CDKN2A/2B = cyclin dependent kinase inhibitor 2A/2B; CRLF2 = cytokine receptor-like factor 2; CSF2RA = colony-stimulating factor 2 receptor α subunit; EBF1 = early B-cell factor 1; ETV6 = ETS variant 6; IKZF1 = IKAROS family zinc finger 1; IL3RA = interleukin 3 receptor subunit α; JAK2 = Janus kinase 2; PAX5 = paired box 5; P2RY8 = purinergic receptor P2Y8; SHOX = short-stature homeobox gene; RB1 = RB transcriptional corepressor 1 A B Radiol Oncol 2022; 56(1): 92-101. Crepinsek K et al. / Copy number variations in pediatric B-ALL98 We further looked at the group of patients, clas- sified as non-high risk (either SR or IR). In this group, six patients carried the IKZF1 deletions, and of those, two patients experienced an event (2/6, 33.3%), and among the patients without the deletions, seven experienced an event (7/59, 11.9%) (p = 0.1907). The 5-year EFS for those without the deletions was 83.6%, while it was only 50% for those with the deletion. Once again, there is a cer- tain trend to be seen, however, the difference was not statistically significant (p = 0.114). We also examined patients with the IKZF1plus profile in the non-HR group. The frequency of events was higher amongst the patients with the profile com- pared to those without it (2/4, 50% vs. 7/61, 11.5%, p = 0.0890), and their EFS was poorer, although not significantly (50.0% vs. 83.7%, p = 0.087). FIGURE 5. (A) Event-free survival in patients with or without IKZF1 deletions (5-year event-free survival [EFS] 54.8% vs. 85.9%, p = 0.016). (B) Overall survival in patients with or without IKZF1 deletions (5-year overall survival [OS] 81.5% vs. 93.0%, p = 0.295). FIGURE 6. (A) Event-free survival in patients without IKZF1 deletions, with IKZF1 deletions only, and those with the IKZF1plus profile (5-year EFS 85.9% vs. 75.0% vs. 50.8%, p = 0.049). (B) Overall survival in patients without IKZF1 deletions, with IKZF1 deletions only and those with the IKZF1plus profile (5-year OS 93.0% vs. 100% vs. 76.2%, p = 0.290). A A B B Radiol Oncol 2022; 56(1): 92-101. Crepinsek K et al. / Copy number variations in pediatric B-ALL 99 Discussion Various alterations in genes involved in cell dif- ferentiation and cell cycle regulation are a hall- mark of B-ALL. The role of deletions in the IKZF1 gene in B-ALL has previously been described as very prognostically revealing and predictive for relapse.8,21 Due to their association with poorer treatment outcomes, it is important to detect them to adjust the treatment protocol accordingly. Moreover, alterations in other genes, such as PAX5 and CDKN2A/2B, are also often present. However, information regarding their prognostic impact is still rather ambiguous19,22,23,26, therefore, more stud- ies need to be carried out to define their true value for patient risk stratification. Our study was carried out on a smaller (88 patients), yet consecutive, unselected, and well- controlled population of pediatric patients with B-ALL. This is the first report about CNVs in cell differentiation and cell cycle regulation genes in Slovenian pediatric B-ALL patients. In our co- hort, IKZF1 deletions were identified in 18.2% of analysed samples. This is in concordance with reports of IKZF1 deletions ranging from 10.7 to 15.9% in other studies that analysed unselected co- horts.6,8,12–14,21 The IKZF1 deletions were more com- mon in the HR group when compared to the IR and SR groups, as was also shown in the study done by Dörge et al.6 Six out of seven (86%) patients with the BCR-ABL1 translocation also carried deletions in the IKZF1 gene, four of those had the IKZF1plus profile. As it was previously described, these dele- tions are very commonly, however not exclusively, present in BCR-ABL1 ALL.12,15,16,21 Some studies ex- cluded patients with the BCR-ABL1 translocation and determined the IKZF1 status only in the BCR- ABL1-negative patients. These studies reported fre- quencies of IKZF1 deletions from 9.4 to 16%.7,27-30 Our results show that 12.3% of patients without BCR-ABL1 in this cohort carried IKZF1 deletions, which is again similar to previously published re- sults. The most common IKZF1 deletion in our cohort was, as was also reported by other studies, the de- letion of the whole gene (50%). Interestingly, how- ever, the second most common deletion was that of exons 4–8 (25%), whereas this deletion was de- scribed as rare in other studies. The second most common deletion reported by other groups was that of exons 4–7 6,8,18,31 which results in a dominant- negative isoform (IK6). This deletion occurred in only one patient in our cohort. Nevertheless, all IKZF1 deletions, including the rare ones, have an important prognostic impact.18 Therefore, all should be considered when selecting the appropri- ate treatment. In our cohort, IKZF1 deletions and the IKZF1plus profile were more common amongst males and were associated with higher blast values on day 8 of chemotherapy, higher values of MRD15 and MRD33, and were more often found in the HR group. The presence of IKZF1 deletions and the IKZF1plus profile was higher amongst the patients who experienced an event (relapse or death) than those who did not, albeit this difference was not significant. Two of the patients who carried these deletions and experience a relapse also had the BCR-ABL1 fusion gene, which placed them in the HR group. However, three others with these dele- tions and an event did not exhibit genetic altera- tions that would predict a poorer outcome – one patient carried the ETV6-RUNX1 fusion, one was hyperdiploid and one had no recurrent genetic abnormalities. This shows that alterations in cell differentiation and cell cycle regulation genes may play a crucial role in disease development even in patients with primary genetic alterations that are thought to be favourable, which is in concordance with reports of these deletions being an independ- ent prognostic factor.6 Previously published studies have shown a poorer event-free and overall survival of patients with IKZF1 deletions in comparison to those with- out such aberrations6,8,12,14,16,30, and Stanulla et al. discovered that patients with additional muta- tions in certain genes that define the IKZF1plus pro- file have even more dismal outcomes.21 Our study similarly confirmed the inferior event-free survival for patients with IKZF1 deletions and the IKZF1plus profile, however, the overall survival did not sig- nificantly differ between the groups. After adjust- ing for confounding factors, our data did not con- firm the independent prognostic role of the IKZF1 deletions, as well as IKZF1plus, on EFS. However, the trend of poorer EFS in patients with the IKZF1 deletions and the IKZF1plus profile was observed. These discrepancies, as well as the high proportion of males presenting with the IKZF1 mutations, are most likely the result our small sample size and short follow-up duration. When inspecting non- high-risk patients with the IKZF1plus profile, the relative frequency of events among the patients with the profile was higher, and there was a trend of poorer EFS for these patients. The dismal out- comes for patients with the IKZF1plus profile have previously been confirmed by other studies, and this profile is currently already being used in cer- Radiol Oncol 2022; 56(1): 92-101. Crepinsek K et al. / Copy number variations in pediatric B-ALL100 tain treatment protocols20, and more are likely to follow. The CNV analysis of other genes showed that the most common alterations were in the PAX5 gene (30.7%), CDKN2A, and CDKN2B (26.1% and 25.0%, respectively). However, deletions in CDKN2A/2B were more common than deletions in PAX5 which was also observed by Öfverholm et al.31 and Mullighan et al.9 Deletions in PAX5 have previ- ously been reported as much more common than intragenic amplifications in this gene, and the latter have mostly been reported only in isolated cases.31-34 However, more recently, Schwab et al.23 showed that PAX5 amplifications occur in around 1% of B-ALL cases, out of which 40% experienced a relapse, sug- gesting that these alterations may play an important role in leukemogenesis. Interestingly, in our cohort, the amplifications were even more common, as they occurred in 10.2% of all B-ALL cases. Out of 27 patients with CNVs in PAX5, 9 (33.3%) carried amplifications. As already suggested by Schwab et al.23, more studies need to be conducted to evalu- ate the prognostic impact of PAX5 amplifications. The PAX5 deletions were also more frequent in our cohort (20.5%) than previously described (10%).35,36 Amongst the samples with PAX5 deletion, a sizable amount (61.1%) also carried the CDKN2A/2B dele- tions. The frequent co-occurrence of these deletions has previously been described by Kim et al.37 In our cohort, amplifications in the PAR1 region were observed quite frequently. Altogether, 17 pa- tients (19.3%) had at least one gene amplification in this region. This is due to the fact that our cohort is unselected, and therefore also includes patients with hyperdiploidy. In hyperdiploidy, gains in the X chromosome are very common (present in 70% of hyperdiploid childhood B-ALL cases)38, and indeed, 14 out of our 17 patients with amplifica- tions in PAR1 had a hyperdiploid karyotype. This karyotype is associated with a favourable outcome. However, the hyperdiploid patients in our cohort did not have a significantly better 5-year EFS, and the same was seen for the patients with amplifica- tions in the PAR1 region that were identified with MLPA. Two patients carried deletions in this re- gion (namely in CSF2RA, IL3RA and P2RY8 genes), which resulted in the formation of the P2RY8- CRLF2 fusion gene. This was confirmed with fluo- rescence in situ hybridization. While the P2RY8- CRLF2 fusion is associated with a poorer treatment response and outcomes39, the two patients in our cohort did not experience an event. Despite the limitations of our study due to a lower number of analyzed samples and relatively short follow-up period, it produced results that are in concordance with the results obtained in larger co- operative clinical trials. We have shown that it is possible to provide comparable results regarding the presence of certain CNVs and their prognostic value in pediatric B-ALL patients even within a single-center experience. This study is only a start- ing point for the more comprehensive screening of patients diagnosed with B-ALL in Slovenia that we have planned for the future and will enable us to better evaluate and treat these patients. Acknowledgments The research was supported by the Slovenian Research Agency (ARRS) grant number P3-0343. References 1. Roberts KG, Reshmi SC, Harvey RC, Chen IM, Patel K, Stonerock E, et al. Genomic and outcome analyses of Ph-like ALL in NCI standard-risk patients: a report from the children’s oncology group. Blood 2018; 132: 815-24. doi: 10.1182/blood-2018-04-841676 2. Hunger SP, Lu X, Devidas M, Camitta BM, Gaynon PS, Winick NJ, et al. 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Poor prognosis for P2RY8-CRLF2 fusion but not for CRLF2 over-expression in chil- dren with intermediate risk B-cell precursor acute lymphoblastic leukemia. Leukemia 2012; 26: 2245-53. doi: 10.1038/leu.2012.101 Radiol Oncol 2022; 56(1): 102-110. doi: 10.2478/raon-2022-0003 102 research article Percutaneous electrochemotherapy in primary and secondary liver malignancies – local tumor control and impact on overall survival Hannah Spallek1, Peter Bischoff1, Willi Zhou1, Francesca de Terlizzi2, Fabian Jakob3, Attila Kovàcs1 1 Clinic for Diagnostic and Interventional Radiology and Neuroradiology, MediClin Robert Janker Klinik, Bonn, Germany 2 IGEA Clinical Biophysics, Laboratory Carpy, Modena, Italy 3 Clinic for Radiology and Nuclear Medicine, University Hospital Schleswig Holstein, Campus Luebeck, Luebeck, Germany Radiol Oncol 2022; 56(1): 102-110. Received 19 11 2021 Accepted 24 12 2021 Correspondence to: Attila Kovàcs, Clinic for Diagnostic and Interventional Radiology and Neuroradiology, MediClin Robert Janker Klinik, Bonn, Germany. E-mail: Attila.Kovacs@mediclin.de Disclosure: No potential conflicts of interest were disclosed. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Background. Local nonsurgical tumor ablation currently represents a further option for the treatment of patients with liver tumors or metastases. Electrochemotherapy (ECT) is a welcome addition to the portfolio of local therapies. A retrospective analysis of patients with liver tumors or metastases treated with ECT is reported. Attention is given to the safety and efficacy of the treatment over time. Patients and methods. Eighteen consecutive patients were recruited with measurable liver tumors of different histopatologic origins, mainly colorectal cancer, breast cancer, and hepatocellular cancer. They were treated with percutaneous ECT following the standard operating procedures (SOP) for ECT under general anaesthesia and mus- cle relaxation. Treatment planning was performed based on MRI preoperative images. The follow-up assessment included contrast-enhanced MR within at least 1–3 months after treatment and then after 5, 7, 9, 12, and 18 months until progression of the disease or death. Results. Only mild or moderate side effects were observed after ECT. The objective response rate was 85.7% (com- plete response 61.9%, partial 23.8%), the mean progression-free survival (PFS) was 9.0 ± 8.2 months, and the overall survival (OS) was 11.3 ± 8.6 months. ECT performed best (PFS and OS) in lesions within 3 and 6 cm diameters (p = 0.0242, p = 0.0297). The effectiveness of ECT was independent of the localization of the lesions: distant, close or adjacent to vital structures. Progression-free survival and overall survival were independent of the primary histology considered. Conclusions. Electrochemotherapy provides an effective valuable option for the treatment of unresectable liver metastases not amenable to other ablative techniques. Key words: electrochemotherapy; liver metastases Introduction Globally, liver cancer ranks sixth for cancer inci- dence and fourth for cancer deaths, being the sec- ond leading cause of cancer-related years of life lost. During the next decade, a further increase in the number of new cases of primary liver cancer is predicted each year in most countries as a result of changes in risk factors.1,2 Globally, colon and rectal cancer ranks third for cancer incidence and second for cancer deaths.1 Population-based studies have shown that 25–30% of patients diagnosed with colorectal cancer (CRC) develop liver metastases during the course of their Radiol Oncol 2022; 56(1): 102-110. Spallek H et al. / Electrochemotherapy in liver tumors or metastases 103 disease. Indications for curative-intended treat- ment of CRC liver metastases have expanded in re- cent years. Unfortunately, despite oncological and surgical advances, only 25% of patients affected are amenable to resection.3,4 Local nonsurgical interventional tumor abla- tion currently represents a further option for the treatment of cancer patients. Local treatments can be divided into thermal (radiofrequency or micro- wave ablation and cryoablation) and nonthermal treatments (high precision radiotherapy, brachy- therapy and electroporation). The European Society of Medical Oncology (ESMO) included local ablation procedures in the current consensus guidelines on the treatment of metastatic colorectal cancer (mCRC).5 The choice of therapy is determined by the num- ber, size, configuration and location or environ- ment of the target lesion. Thermal ablation tech- niques are emerging as alternative treatment op- tions to open surgery for both primary and second- ary hepatic tumors.6 A disadvantage of RFA is the therapeutic limitation to smaller target lesions up to 3.5 cm in diameter; in the case of microwave and cryoablation, the ablation zone can be enlarged up to 5 cm in tumor diameter. However, hyperther- mia-based technologies have some limitations, in- cluding heat sink effects in the proximity of large blood vessels, the risk of causing cholestasis when treating lesions close to the thermosensitive bile ducts or damaging critical structures if proximal to the hepatic portal Glisson’s capsule or diaphragm, or if located on the intra-abdominal free surface. A special form of local nonthermal ablation is in- ternal radiation (interstitial brachytherapy), which uses radiation with a very limited range because it is limited in the proximity of organs vulnerable to radiation; it cannot be repeated, and furthermore, some tumor entities are not radiosensitive. In such cases, chemoablation, such as electrochemothera- py (ECT), is a welcome addition to the portfolio of local therapies. ECT is a local ablative technique that utilizes electroporation for enhanced drug (bleomycin or cisplatin) delivery to cells by generating transient permeation structures in the cell membrane.7,8 Over the past 20 years, ECT has been shown to have proven effectiveness in the treatment of cutaneous, subcutaneous, mucosal, or deep-seated tumors of various histologies and in different body sites.9-11 The international, multicenter clinical study European Standard Operating Procedures for Electrochemotherapy (ESOPE) developed the Standard Operating Procedures for ECT on cutane- ous tumors with the Cliniporator™ Device (IGEA S.p.A., Carpi, Italy).8 Based on its effectiveness for cutaneous tumors, ECT is now being developed and has been shown to be feasible, safe, and effec- tive for deep-seated tumors, such as liver tumors. ECT can be used near collagenous structures such as vessels and bile ducts12, and it is repeatable and suitable as a local therapy between chemotherapy cycles. ECT has the potential to close relevant gaps in the spectrum of local ablative therapies, ena- bling the treatment of i) lesions that are too large for thermal ablation, ii) nonradiation-sensitive tu- mors or iii) lesions located in the immediate vicin- ity of radiation- or temperature-vulnerable organs. ECT is specifically suitable for the treatment of liver metastases located centrally, close to the cap- sule or in proximity of the major vessels, which are not resectable and not suitable for radiofrequency ablation or microwave ablation due to the heat sink effect. The safety of ECT in the treatment of metastases located near large liver vessels was also proven in animal models.13,14 In cancer patients, ECT is well tolerated, with few side effects and no relevant pain, nausea or systemic side effects.15-18 In this study, we present a retrospective analysis of patients with liver tumors or metastases treated with ECT at our institution. This is the first real- world clinical experience on percutaneous applica- tion of ECT in the liver in a large cohort of patients. Attention is given to the safety and efficacy of the treatment over time. Patients and methods In this cohort study, 18 patients with measurable liver tumors of different histopathologic origins were recruited: colorectal cancer, breast cancer, hepatocellular cancer, ovarian cancer, anal cancer, non-small cell lung cancer (NSCLC) and cancer of unknown primary origin (CUP). They were treated with ECT between June 2018 and June 2020. The study was conducted according to the Helsinki dec- laration. The patients signed an informed consent form. The study was approved by the Committee for Medical Ethics of the Institution. Imaging Standard pretreatment evaluation of patients with liver tumors included liver MRI with a hepatospe- cific contrast agent and CT of the thorax and abdo- men, including the pelvis at least 1 month before ECT. MRI was performed using a GE Signa Hdxt Radiol Oncol 2022; 56(1): 102-110. Spallek H et al. / Electrochemotherapy in liver tumors or metastases104 1.5T using standard imaging sequences for the liver: tra T2w fs, tra DWI, tra T1w nat, tra and cor ce T1w. The patients were reviewed by a multidis- ciplinary team. The follow-up assessment included contrast-enhanced MRI of the liver within at least 1–3 months after treatment and then after 5, 7, 9, 12, 18 months and/or until progression of the disease or death. Electrochemotherapy Freehand electrodes are used for the percutaneous therapy of parenchymatous organs or for intraop- erative positioning. Depending on the size, con- figuration and localization of the target region, dif- ferent active electrode tips, shaft lengths and thick- nesses can be selected. A maximum of 6 probes can be operated synchronously. Therapy plan- ning is software-based: the parallel positioning of the probes at a defined distance from each other plays an essential role in the optimal therapeutic coverage of the target lesion. The aim is to achieve a distance of 2.0 to 2.5 cm from each other and a voltage to be applied of approx. 1000 volts per cm. Treatment planning was performed based on MRI preoperative images. During ECT treatment, nee- dle electrodes were percutaneously inserted based on the CT-fluoro guided planning images, and a distance of at least 0.5 and maximally 3.0 cm be- tween the electrodes was ensured. Electrodes were freely positionable single needle probes with a di- ameter of 1.2 mm; a minimum of 2 and a maximum of 8 electrodes (median 6) were used per treatment (Table 2). In particular: in 8 patients electrodes 16 cm long and with an active part of 4 cm, in 6 pa- tients electrodes 16 cm long with an active part of 3 cm, in 4 patients electrodes 20 cm long with 3 cm active part and in 1 patient electrodes 20 cm long with active part of 4 cm (IGEA, Carpi, Italy). The direction of electrode access was determined by the performing surgeon. Since the pulses are delivered, among pairs of electrodes, the computation of the voltage to be ap- plied was performed by the device for each pair of electrodes separately, and the appropriate electric field for each pair combined assured the complete coverage of a tumor. By sequentially activating the electrodes in this way, a larger tumor volume can be covered with sufficiently strong electric fields.19 The goal was to ensure 100% coverage of the clini- cal target volume with an electric field above 400 V/cm and to limit the maximum current delivered to the tissue to be below 50 A (hardware limit of the IGEA Cliniporator Vitae pulse generator).20 ECT was performed using the same treatment protocol as defined by the SOP for ECT of cutane- ous tumors regarding the drug dosage and electri- cal parameters (i.e., pulse duration and number of FIGURE 1. (A) Solitary liver metastasis from a breast carcinoma in a challenging location between the left and right lobes of the liver, not amenable to surgical resection and progressive under various lines of systemic chemotherapy. The dimensions of the metastasis in segment IVa/b adjacent to segment VIII were 4 x 7 x 5 cm (volume 70 cc). (B) Position of the electrodes in the coronary reconstruction. The aim is to achieve the most uniform coverage of the target lesion by the electrodes. (C) Position of the electrodes in axial cross-sectional imaging. This image shows another essential requirement for the therapeutic success of ECT – the parallelism of the electrodes. (D) The most recent imaging control, complete two years after the ECT procedure, shows complete chemoablation of the entire metastasis, thus formally complete remission of the target lesion without residual or marginal recurrence. A B C D Radiol Oncol 2022; 56(1): 102-110. Spallek H et al. / Electrochemotherapy in liver tumors or metastases 105 pulses) of electroporation.8,21 The needle electrodes were percutaneously inserted into the lesions un- der CT-fluoro guidance. The electrodes were con- nected to an electric pulse generator (Cliniporator VITAE, IGEA SpA, Carpi, Italy). Thereafter, the patients were given 15,000 U/m2 bleomycin intra- venously in bolus. Eight electric pulses of 100 μs duration were delivered between pairs of elec- trodes 8 min after the bleomycin injection, when the maximal pharmacological peak of bleomycin in the tumors was expected. Complete coverage of the tumor by repositioning of the electrodes should possibly be completed by 40 minutes thereafter. Care is also taken to ensure that the electrical puls- es are delivered only in the refractory phase of the heart by automatic ECG synchronization to avoid interferences with the heart rhythm. ECT treat- ment takes place under general anaesthesia and muscle relaxation as a minimally invasive, usually percutaneous, procedure (Figure 1). Statistical analysis Descriptive statistics are reported as the mean, standard deviation, median and range for con- tinuous variables and absolute numbers and per- centage for categorical variables. Comparisons between groups were performed by ANOVA (con- tinuous variables) and contingency tables and chi square tests (for categorical variables). A P value lower than 0.05 was considered statistically signifi- cant. Statistical analysis was performed with NCSS 9 (NCSS 9 Statistical Software (2013)). NCSS, LLC. Kaysville, Utah, USA, ncss.com/software/ncss). Results Eighteen patients were treated with ECT in the pe- riod June 2018 – June 2020 and were followed for a median time of 9 months (mean 11.3 ± 8.6 months). One patient was lost to follow-up. Three patients were treated with 2 lesions, all the others in a single lesion of the liver. The characteristics of the cohort of patients are reported in Table 1. The mean age was 64.3 ± 11.1 years (median 64, range 41–83 years). Lesion sizes were 5.9 ± 2.5 cm in the LA (long axis) direction (median 4.6, range 1.5–11.2 cm) and 5.4 ± 2.1 cm in the SA (short axis) direction (median 5.5, range 1.5–10 cm). The overall mean volume was 129.6 ± 137.3 cm3 (median 57, range 23–475 cm3). Table 2 shows other characteristics of the lesions and the technical parameters of ECT treatment. Safety/toxicity Only mild or moderate side effects were observed after ECT: in 16 of 21 patients, temporary (1st day) mild pain at the treated site; in 1 patient, CRP (C-reactive protein) elevation and leucocytosis were successfully treated with i.v. antibiotics; and in 1 patient, moderate pain due to a liver capsu- lar hematoma, w.o. hemoglobin drop, successfully treated with ibuprofen/pantoprazole self-resolving after 10 days. TABLE 1. Demographic N % PATIENTS 18 GENDER M F 8 10 44.4% 55.6% DIAGNOSIS Colorectal cancer Breast cancer Hepatocellular cancer Ovarian cancer Anal cancer Cancer of unknown primary origin (CUP) Non-small cell lung cancer (NSCLC) 7 4 2 2 1 1 1 38.9% 22.2% 11.1% 11.1% 5.6% 5.6% 5.6% TUMOURS TREATED 21 LIVER METASTASES Synchronous Metachronous No 8 8 2 44.4% 44.4% 11.2% METASTASES LOCATION Liver only Liver + lung Liver + bone Liver + kidney Liver + lung + bone + brain Liver + bone + peritoneum Liver + pleural + bone Liver + retroperitoneal 7 3 1 1 1 1 1 1 43.7% 21.5% 6.2% 6.2% 6.2% 6.2% 6.2% 6.2% PREVIOUS TREATMENTS Systemic therapy 16 88.8% Liver surgery 4 22.2% TACE TACE + RFA TACE + CP CRYOTH NO 8 1 1 1 7 44.4% 5.6% 5.6% 5.6% 38.9% COMORBIDITIES* Cardiac diseases Pulmonary diseases Liver diseases 6 3 9 33.3% 16.7% 50.0% * Cardiac diseases were cardiomyopathies, status post coronary bypass, status post aortocoronary venous bypass operation, valvular disease, pericardial effusion; pulmonary diseases were chronic obstructive pulmonary diseases; liver diseases were hematomas, ascites, cholestasis, hemochromatosis CP = chemoperfusion; CRYOTH = cryotherapy; F = female; M = male; N = number; RFA = radiofrequency ablation; TACE = hepatic artery chemoembolization Radiol Oncol 2022; 56(1): 102-110. Spallek H et al. / Electrochemotherapy in liver tumors or metastases106 Response to treatment The response to treatment was evaluated between 1 and 3 months after the ECT session; the overall response is reported in Table 3. Objective response rate was 85.7%. Mean progre ssion-free survival (PFS) was 9.0 ± 8.2 months. Three patients progressed during follow-up after 3, 5, and 7 months. The first un- derwent TACE and was in complete response (CR) after 6 months; the second underwent interstitial brachytherapy and was in CR after 2 months; the last underwent TACE + CRYO and was in CR after 11 months. Mean overall survival (OS) was 11.3 ± 8.6 months. Three patients died for reasons related to liver metastases (14.3%), 11 patients (52.4%) died for other reasons, and 7 (33.3%) were still alive. Furthermore, response to treatment, PFS and OS were evaluated according to lesion size, histology of the primary tumor and location of the liver le- sions. The results are shown in Table 4. Discussion Interventional oncology is the fastest developing area of interventional radiology. Minimally in- vasive, image-guided procedures are playing an increasingly important role in multimodal cancer therapy.22-24 In this study, we evaluated the effect of percuta- neous ECT on liver tumors of different pathologic origins. Percutaneous ECT treatments of 21 lesions in 18 patients were included in the analysis. The lesions were close to the capsule or in proximity of the major vessels, not suitable for radiofrequency or microwave ablation due to the heat sink effect, and not surgically resectable. In 90.5% of cases, the lesions were in a challenging location (liver dome, near portal vein main trunk, near main bile duct). The mean volume of treated lesions was 129.59 ± 137.31 cm3, which is definitely larger than the vol- umes usually accessible for other minimally inva- sive procedures. The most important advantage of minimally invasive technologies is that, in combination with standard therapies, they significantly increase overall survival compared to standard treatment alone. This has been proven in 2 different studies: the CLOCC22 and the SABR-COMET25 trials. In the CLOCC trial, patients with nonresectable colorec- tal cancer liver metastases were randomized either to receive systemic chemotherapy or a combination of systemic and minimally invasive therapies; the overall survival at 8 years was significantly im- proved in the combined arm versus the standard therapies arm (36% vs. 8%). In the same way, the SABR-COMET study analyzed the impact of ste- reotactic ablative radiotherapy (SABR) in combina- tion with standard of care in the treatment of differ- ent oligometastatic patients from various cancers (breast, lung, colorectal, prostate) in comparison with standard of care alone. The first arm showed a superior 5-year survival (42.3% vs. 17.7%). Other advantages of minimally invasive technologies are good tolerability, less impact on quality of life, fewer systemic side effects and tissue preservation when compared to classical surgery. Furthermore, except for radiotherapy, minimally invasive tech- nologies are also repeatable. Thermo and radioablative techniques are limit- ed in some situations, for example, when target le- sions have a size that exceeds the safe ablation zone of thermal procedures, usually estimated at 3.5 cm in diameter.26 On the other hand, radioablation is limited near radiation-sensitive organs. In these cases, ECT can truly be a valuable option, as it is TABLE 2. Lesions and treatment description N % LESIONS 21 100% PLANNING MRI 21 100% TYPE Hypervascular Intermediate Hypovascular 2 14 5 9.5% 71.4% 19.0% CHALLENGING LOCATION* Yes No 19 2 90.5% 9.5% VESSELS OR BILE DUCTS SURROUNDING THE METASTASES Distant (> 10 mm) Close (1 mm to 10 mm) Adjacent (< 1 mm) 4 6 11 19.0% 28.6% 52.4% PREVIOUS LOCAL TREATMENT ON THE LESION Local ablative therapy (LAT) Transarterial chemoembolization (TACE) Chemoperfusion (CP) Treatment-naive 0 6 1 14 0% 28.6% 4.8% 66.7% TECHNICAL SUCCESS Yes No 20 1 95.2% 4.8% # ELECTRODES PER TREATMENT 2 3 4 6 8 1 2 1 16 1 4.8% 9.5% 4.8% 76.2% 4.8% * Challenging location represented in liver were liver dome, vicinity of portal vein main trunk, vicinity of main bile duct Radiol Oncol 2022; 56(1): 102-110. Spallek H et al. / Electrochemotherapy in liver tumors or metastases 107 a combined tumor therapy that enhances the local effect of a systemically administered chemothera- peutic drug by reversible electroporation.26 The most commonly used chemotherapeutic agent is bleomycin, which, when combined with electropo- ration, has the major advantage of being cytotoxic regardless of the tumor’s histology. The aim of minimally invasive, local ablative procedures is to destroy primary and secondary malignancies efficiently and gently at the same time using image guidance. These novel techniques can be used for diverse applications ranging from curative intent for small localized tumors, down staging of large tumors for resection, or locoregion- al control and palliation of advanced disease. The choice of therapy is determined by the parameters “number, size and location” of the target lesions. Given that all standard local ablative procedures were available at the hospital that conducted the present study, a proactive decision was made to perform ECT in each individual case. Due to the aforementioned limitations of each procedure, it was most often the size, location and immediate environment of the target lesions that contraindi- cated thermal ablation or compromised its efficacy in the first place. In most decisions, from the thera- pist’s point of view, ECT seemed to us to be the only alternative. This technique has already been shown to be ef- fective on cutaneous, subcutaneous and mucosal lesions10,11, with a response rate of 70–80%, and to be particularly effective on basal cell carcinoma, with a complete remission of treated lesions up to 91%.11 ECT has also shown convincing results with deep-seated tumors. In a prospective multicenter study27, Campanacci et al. evaluated ECT in the treatment of symptomatic bone metastases. The results on 102 patients from 11 European cent- ers demonstrated that ECT is a safe and effective treatment for painful bone metastases resistant to other local treatments. Furthermore, a significant decrease in pain intensity and significantly better quality of life were observed after the ECT session and at later follow-up. Several pilot studies on intraoperative ECT on liver metastases16,28-30, hepatocellular carcinoma31,32, perihilar cholangiocarcinoma33,34, vulvar cancer35–37, and renal cancer38 are available in the literature. In a recently published phase II study on ECT in the treatment of colorectal liver metastases, the objective response rate (OR) per lesion was 75%, with 63% complete response (CR) and 12% partial response (PR), while OR, CR and PR per patient were 59%, 44% and 15%, respectively. The median response time was 20.8 months for metastases in CR and 9.8 months for metastases in PR. There was no difference in treatment response with re- gard to the location of the metastases, e.g., me- tastases in the central vs. peripheral location. The median overall survival of patients after ECT was 29.0 months.16 The histopathological assessment of some of these colorectal liver metastases after ECT treatment showed that most vessels (> 5 mm) and biliary structures remained intact, while smaller blood vessels were damaged. This study shows that ECT can be safely applied to treat metastases in the immediate vicinity of the large blood vessels in the liver.12 Regarding hepatocellular carcinoma (HCC), a prospective phase II study was recently published on 24 patients with 32 HCC lesions not suitable for other curative treatments according to the BCLC classification or refractory to previous surgery and different local ablative techniques. In this study, the treatment was proven to be equally effective for tumors located centrally and peripherally, with a median response rate per patient of 95.8% (79.2% CR and 16.6% PR). The overall survival over 5 years of observation was 72.0%.31 In these studies, ECT treatment was performed intraoperatively as part of an open procedure. To the best of our knowledge, the present study is the largest case series about ECT on liver tumors with a percutaneous approach. To date, only a few studies with a percutane- ous approach to liver tumors using ECT have been published.15 Tarantino et al. 2017 demonstrated the efficiency and effectiveness of ECT in the treat- ment of six patients with portal vein thrombosis at the hepatic hilum, resulting in two patients with regained complete patency of the portal vein and three patients with a persistent avascular nontu- moral shrinked thrombus; none of these patients developed a local recurrence.39 The same group TABLE 3. Response of target lesions evaluated between 1 and 3 months RESPONSE N % Complete response (CR) 13 61.9% Partial response (PR) 5 23.8% Stable disease (SD) 1 4.8% Progressive disease (PD) 0 0% Lost to follow-up 2 9.5% Radiol Oncol 2022; 56(1): 102-110. Spallek H et al. / Electrochemotherapy in liver tumors or metastases108 also successfully treated patients with perihilar cholangiocarcinoma with minimally invasive ECT treatment.33 In our study, we showed that ECT performed best (in terms of progression-free survival and overall survival) in lesions < 6 cm in diameter compared to lesions > 6 cm in diameter (p = 0.0209 and p = 0.0322, respectively). These results demon- strate that the technique is effective even in lesions of large size, up to 6 cm, significantly larger than lesions addressable with thermal ablation tech- niques. Furthermore, we proved that the effectiveness of ECT is independent of the localization of the le- sions: distant, close or adjacent to vital structures, with the latter being devoid of therapeutic options. We observed that progression-free survival and overall survival in our cohort of patients were simi- lar for all primary histologies considered: colorec- tal metastases, breast cancer metastases, and hepa- tocellular cancer (p = 0.8781 and p = 0.8379, respec- tively). This result demonstrates the effectiveness of ECT and bleomycin independent of the histol- ogy of the treated lesions. Apart from its effectiveness, one of the most im- portant advantages of ECT is that it spares colla- genous healthy structures such as vessels and bile ducts.26 ECT is repeatable and suitable as a local therapy even if performed between chemotherapy cycles. In conclusion, ECT has the potential to close relevant gaps in the local ablative therapy field: ECT allows the treatment of lesions too large for thermal ablation, nonradiation-sensitive tumors, and lesions adjacent to radiation-vulnerable or- gans. The various thermo, radio- and chemoablative procedures as well as endovascular and percutane- ous therapies do not compete with each other but complement each other and are used supplementa- rily in the hands of experienced interventionalists. Radiological-interventional expertise implies that a broad spectrum of procedures and technologies must be mastered. Too many evidence-based rec- ommendations for sequencing minimally invasive procedures do not yet exist. The scarce knowledge is limited to the combination of TACE and RFA in larger HCCs, where thermal ablation alone is limit- ed.40 It can be considered an imperative task of the interventional community to generate this same evidence as soon as possible. In our study, we demonstrated that ECT was well tolerated by the patients, and no serious ad- TABLE 4. Response to treatment, progression-free survival and overall survival according to different subgroups of analysis CR PR SD PD NE PFS (mo) OS (mo) N (%) N (%) N (%) N (%) N (%) Mean ± s.d. Mean ± s.d. SIZE < 6 cm 9 (90.0%) 0 0 0 1 (10.0%) 12.0 ± 9.2 15.1 ± 8.0 > 6 cm 4 (36.4%) 5 (45.4%) 1 (9.1%) 0 1 (9.1%) 4.7 ± 5.4 7.9 ± 7.9 P value 0.0483 0.0209 0.0322 HISTOLOGY Colorectal cancer 4 (50.0%) 2 (25.0%) 0 0 2 (25.0%) 7.3 ± 12.1 12.1 ± 12.1 Breast cancer 4 (80.0%) 1 (20.0%) 0 0 0 9.8 ± 7.5 10.6 ± 6.9 Hepatocellular cancer 1 (33.3%) 2 (66.7%) 0 0 0 10.3±10.1 15.0 ± 7.2 P-value 0.3615 0.8781 0.8379 LOCATION Distant (> 10 mm) 2 (100%) 0 0 0 0 6.5 ± 3.5 8.5 ± 0.7 Close (> 1 mm) 5 (62.5%) 1 (12.5%) 1 (12.5%) 0 1 (12.5%) 8.0 ± 7.4 10.7 ± 7.3 Adjacent (< 1 mm) 6 (54.5%) 4 (36.3%) 0 0 1 (9.2%) 8.8 ± 9.8 10.2 ± 10.5 P-value 0.6643 0.9364 0.9539 CR = comlete response; NE = no evidence, lost to follow up; OS = overall survival; PFS = progression-free survival; PR = partial response; s.d. = standard deviation; SD = stable disease Radiol Oncol 2022; 56(1): 102-110. Spallek H et al. / Electrochemotherapy in liver tumors or metastases 109 verse events were observed during the procedure or in the follow-up; side effects were limited in number and intensity, and no relevant pain or sys- temic side effects were observed. This study has several limitations: a retrospec- tive design, the limited number of cases and their heterogeneity in terms of diagnosis of primary tu- mor, localization, and previous treatments. This is a first experience conducted in our center and therefore includes all patients treated in the obser- vation period. Moreover, this study well represents the variety of the cohort of patients, which can ben- efit from the application of ECT in clinical practice. In one case, where 100% technical success could not be achieved, a plastic bile duct stent was placed at the margin of the target lesion. The generator reported inadequate discharges at the two elec- trodes closest to the biliary stent. Although several discharge cycles were performed, residual contrast uptake in this area was documented in the postint- erventional MR control after 48 hours. However, it cannot be concluded from this single case observa- tion that bile duct stents per se represent a limita- tion to ECT. In addition to these limitations, our study pro- vides further evidence on the effectiveness of ECT in the treatment of liver metastases of different ori- gins, different sizes and locations. This treatment provides long-term local tumor control as well as long progression-free survival (mean progression- free survival of 9.0 ± 8.2 months). ECT, therefore, provides an effective valuable option for the treat- ment of unresectable liver metastases not amena- ble to other ablative techniques. References 1. Global Burden of Disease Cancer Collaboration; Fitzmaurice C, Allen C, Barber RM, Barregard L, Bhutta ZA, Brenner H, et al. 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Radiol Oncol 2018; 52: 383-91. doi: 10.2478/raon-2018-0041 20. Bertacchini C. Cliniporator: medical electroporation of tumors. In: Miklavcic D, editor. Handbook of Electroporation. Cham: Springer International Publishing; 2017. p. 1-36. 21. Gehl J, Sersa G, Matthiessen LW, Muir T, Soden D, Occhini A, et al. Updated standard operating procedures for electrochemotherapy of cu- taneous tumours and skin metastases. Acta Oncol 2018; 57: 874-82. doi: 10.1080/0284186X.2018.1454602 22. 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. J Natl Cancer I 2017; 109: djx015. doi: 10.1093/ jnci/djx015 Radiol Oncol 2022; 56(1): 102-110. Spallek H et al. / Electrochemotherapy in liver tumors or metastases110 23. Slovak R, Ludwig JM, Gettinger SN, Herbst RS, Kim HS. Immuno-thermal ablations - boosting the anticancer immune response. J Immunother Cancer 2017; 5: 78. doi: 10.1186/s40425-017-0284-8 24. Camacho JC, Petre EN, Sofocleous CT. Thermal ablation of metastatic colon cancer to the liver. Semin Intervent Radiol 2019; 36: 310-8. doi: 10.1055/s- 0039-1698754 25. Olson R, Mathews L, Liu M, Schellenberg D, Mou B, Berrang T, et al. Stereotactic ablative radiotherapy for the comprehensive treatment of 1–3 Oligometastatic tumors (SABR-COMET-3): study protocol for a randomized phase III trial. BMC Cancer 2020; 20: 380. doi: 10.1186/s12885-020-06876-4 26. Kovács A, Bischoff P, Haddad H, Kovács G, Schaefer A, Zhou W, et al. Personalized image-guided therapies for local malignencies: interdiscipli- nary options for interventional radiology and interventional radiotherapy. Front Oncol 2021; 11: 616058. doi: 10.3389/fonc.2021.616058 27. Campanacci L, Bianchi G, Cevolani L, Errani C, Ciani G, Facchini G, et al. Operating procedures for electrochemotherapy in bone metastases: results from a multicenter prospective study on 102 patients. Eur J Surg Oncol 2021; 47: 2609-17. doi: 10.1016/j.ejso.2021.05.004 28. Edhemovic I, Gadzijev EM, Brecelj E, Miklavcic D, Kos B, Zupanic A, et al. Electrochemotherapy: a new technological approach in treatment of metas- tases in the liver. Technol Cancer Res Treat 2011; 10: 475-85. doi: 10.7785/ tcrt.2012.500224 29. Edhemovic I, Brecelj E, Gasljevic G, Marolt Music M, Gorjup V, Mali B, et al. Intraoperative electrochemotherapy of colorectal liver metastases. J Surg Oncol 2014; 110: 320-7. doi: 10.1002/jso.23625 30. Coletti L, Battaglia V, De Simone P, Turturici L, Bartolozzi C, Filipponi F. Safety and feasibility of electrochemotherapy in patients with unresectable colorectal liver metastases: a pilot study. Int J Surg 2017; 44: 26-32. doi: 10.1016/j.ijsu.2017.06.033 31. Djokic M, Cemazar M, Popovic P, Kos B, Dezman R, Bosnjak M, et al. Electrochemotherapy as treatment option for hepatocellular carci- noma, a prospective pilot study. EJSO 2018; 44: 651-7. doi: 10.1016/j. ejso.2018.01.090 32. Djokic M, Dezman R, Cemazar M, Stabuc M, Petric M, Smid LM, et al. Percutaneous image guided electrochemotherapy of hepatocellular car- cinoma: technological advancement. Radiol Oncol 2020; 54: 347-52. doi: 10.2478/raon-2020-0038 33. Tarantino L, Busto G, Nasto A, Nasto RA, Tarantino P, Fristachi R, et al. Electrochemotherapy of cholangiocellular carcinoma at hepatic hilum: a feasibility study. EJSO 2018; 44: 1603-9. doi: 10.1016/j.ejso.2018.06.025 34. Granata V, Palaia R, Albino V, Piccirillo M, Venanzio Setola S, Petrillo A, et al. Electrochemotherapy of cholangiocellular carcinoma at hepatic hilum: a case report. Eur Rev Med Pharmacol Sci 2020; 24: 7051-7. doi: 10.26355/ eurrev_202006_21698 35. Corrado G, Cutillo G, Fragomeni SM, Bruno V, Tagliaferri L, Mancini E, et al. Palliative electrochemotherapy in primary or recurrent vulvar cancer. Int J Gynecol Cancer 2020; 30: 927-31. doi: 10.1136/ijgc-2019-001178 36. Perrone AM, Galuppi A, Pirovano C, Borghese G, Covarelli P, De Terlizzi F, et al. Palliative Electrochemotherapy in vulvar carcinoma: preliminary results of the ELECHTRA (Electrochemotherapy Vulvar Cancer) multicenter study. Cancers 2019; 11: 657. doi: 10.3390/cancers11050657 37. Perrone AM, Ferioli M, Argnani L, De Terlizzi F, Pirovano C, Covarelli P, et al. Quality of life with vulvar carcinoma treated with palliative electrochemo- therapy: The ELECHTRA (ELEctroCHemoTherapy vulvaR cAncer) Study. Cancers 2021; 13: 1622. doi: 10.3390/cancers13071622 38. Andresciani F, Faiella E, Altomare C, Pacella G, Beomonte Zobel B, Grasso RF. Reversible electrochemotherapy (ECT) as a treatment option for local RCC recurrence in solitary kidney. Cardiovasc Intervent Radiol 2020; 43: 1091-4. doi: 10.1007/s00270-020-02498-2 39. Tarantino L, Busto G, Nasto A, Fristachi R, Cacace L, Talamo M, et al. Percutaneous electrochemotherapy in the treatment of portal vein tumor thrombosis at hepatic hilum in patients with hepatocellular carcinoma in cirrhosis: A feasibility study. WJG 2017; 23: 906. doi: 10.3748/wjg.v23.i5.906 40. Liu F, Chen M, Mei J, Xu L, Guo R, Lin X, et al. Transarterial chemoembo- lization combined with radiofrequency ablation in the treatment of stage B1 intermediate hepatocellular carcinoma. J Oncol 2019; 2019: 1-7. doi: 10.1155/2019/6298502 Radiol Oncol 2022; 56(1): 111-118. doi: 10.2478/raon-2021-0035 111 research article The learning curve of laparoscopic liver resection utilising a difficulty score Arpad Ivanecz1,2, Irena Plahuta1, Matej Mencinger3,4,5, Iztok Perus2,6, Tomislav Magdalenic1, Spela Turk1, Stojan Potrc1,2 1 Clinical Department of Abdominal and General Surgery, University Medical Centre Maribor, Maribor, Slovenia 2 Department of Surgery, Faculty of Medicine, University of Maribor, Maribor, Slovenia 3 Faculty of Civil Engineering, Transportation Engineering and Architecture, University of Maribor, Maribor, Slovenia 4 Centre of Applied Mathematics and Theoretical Physics, University of Maribor, Maribor, Slovenia 5 Institute of Mathematics, Physics and Mechanics, Ljubljana, Slovenia 6 Faculty of Natural Science and Engineering, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2022; 56(1): 111-118. Received 2 June 2021 Accepted 16 July 2021 Correspondence to: Assist. Prof. Arpad Ivanecz, M.D., Ph.D., Clinical Department of Abdominal and General Surgery, University Medical Centre Maribor, Ljubljanska ulica 5, 2000 Maribor, Slovenia. E-mail: arpad.ivanecz@ukc-mb.si Disclosure: No potential conflicts of interest were disclosed. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Background. This study aimed to quantitatively evaluate the learning curve of laparoscopic liver resection (LLR) of a single surgeon. Patients and methods. A retrospective review of a prospectively maintained database of liver resections was conducted. 171 patients undergoing pure LLRs between April 2008 and April 2021 were analysed. The Halls difficulty score (HDS) for theoretical predictions of intraoperative complications (IOC) during LLR was applied. IOC was defined as blood loss over 775 mL, unintentional damage to the surrounding structures, and conversion to an open approach. Theoretical association between HDS and the predicted probability of IOC was utilised to objectify the shape of the learning curve. Results. The obtained learning curve has resulted from thirteen years of surgical effort of a single surgeon. It consists of an absolute and a relative part in the mathematical description of the additive function described by the loga- rithmic function (absolute complexity) and fifth-degree regression curve (relative complexity). The obtained learning curve determines the functional dependency of the learning outcome versus time and indicates several local ex- treme values (peaks and valleys) in the learning process until proficiency is achieved. Conclusions. This learning curve indicates an ongoing learning process for LLR. The proposed mathematical model can be applied for any surgical procedure with an existing difficulty score and a known theoretically predicted as- sociation between the difficulty score and given outcome (for example, IOC). Key words: learning curve; difficulty score; laparoscopy; hepatectomy; intraoperative complication Introduction Interest in laparoscopic liver resection (LLR) has grown since the publication of the International Louisville Statement on laparoscopic liver sur- gery.1 Since then, the number of LLRs performed worldwide has increased exponentially.2 The laparoscopic approach must not compro- mise the technical quality of the liver resection. The message from the second Morioka consen- sus conference in 2014 was the need for a formal structure of education for those interested in per- forming LLR.3 The need for the organisation of LLR was achieved by the establishment of the Radiol Oncol 2022; 56(1): 111-118. Ivanecz A et al. / Learning curve of laparoscopic liver resection112 International Laparoscopic Liver Society in 2016.4 In Southampton, 2017, the third consensus meeting has produced a set of clinical practice guidelines to direct the speciality’s continued safe progres- sion and dissemination.5 A few difficulty scoring systems have been proposed to rate the difficulty of LLR, and the need for validating the existing tools before the clinical application has been high- lighted.6-9 Halls et al.10 developed and internally validated a difficulty score estimating the risk of intraoperative complications (IOC) during LLR, which was externally validated by the authors of the present study.11 Along with the evolution of LLR, its learning curves (LCs) have received increased attention.12-14 The idealised model of the LC has been described, demonstrating continuous result improvement along with experience.15 Recently, the LC has been reported to resemble a true model, in which alter- nating periods of progression and regression oc- curred until mastery was achieved.16 The present study was based on a thirteen-years single-centre experience and was designed to ana- lyse the real LC of LLR. To the best of our knowl- edge, it is the only study quantitatively presenting the LC of LLR. Patients and methods Patients Study subjects were identified from a prospective- ly maintained database of patients who underwent liver resections at the Department of Abdominal and General Surgery, University Medical Centre Maribor, Slovenia. This institution has been a ter- tiary referral centre specialised in hepato-pancre- ato-biliary surgery, where the first LLR was per- formed in April 2008. The study included all the patients in whom a pure laparoscopic liver proce- dure was performed (intention-to-treat analysis) until 31st March 2021. For the present study, pa- tients who underwent laparoscopic cyst fenestra- tion, liver biopsies, and radiofrequency ablation were excluded. Only pure LLR were performed; no hand-assist- ed or hybrid procedures were used. All patients were operated by the same surgeon (AI). He had expertise in open hepato-pancreatico-biliary and laparoscopic surgery but no experience in LLR be- fore this series. Perioperative definitions were pro- vided elsewhere.11 The surgical technique for LLR has been extensively described by others17 and per- formed as reported previously.18-20 At the time of the operation all patients had given their written consent that anonymous data can be used for research purposes. Patient records were anonymized and de-identified before analy- sis. Ethical approval for this study was obtained from the local institutional review board. Statistical analysis IBM SPSS for Windows Version 26.0 (IBM Corp., Armonk, NY, USA) and Wolfram Mathematica for Windows Version 10.4 (Wolfram Research, Inc., Champaign, IL, USA) were used for statistical com- putations. Categorical variables were reported as fre- quency (percentages). Continuous variables were reported as mean and standard deviation when data distribution was normal; otherwise, they were reported as median (minimum-maximum, interquartile range). The chi-square and the paired samples t-test were used. Percentages were list- ed to one decimal place, and a difference in the P-value of <0.05 was considered statistically sig- nificant. Mathematical modelling of the learning curve The Halls difficulty score (HDS)10 was applied. Its parameters (neoadjuvant chemotherapy, previous open liver resection, benign or malignant lesion, lesion size, and classification of resection) were captured from the institutional database. Each LLR was retrospectively scored from 0 to 15. In the proposed model, IOC was used as a sen- sible measure of the complexity of the resection.10 IOC’s key markers were blood loss over 775 mL, unintentional damage to the surrounding struc- tures and conversion to open approach.10 The con- version was defined as the requirement for lapa- rotomy at any time of the procedure, except for the extraction of the resected specimen.10 In11, the authors searched for functorial depend- ence between IOC and HDS using the first 128 pa- tients of the observed cohort. The best-fit-depend- ency was found to be the Weibull cumulative dis- tribution function21 of the form with and . Here x represents the HDS, and represents the predicted probability of IOC occurrence. This functional de- pendence will be referred as the theoretical prob- ability of IOC11 and is graphically represented in Radiol Oncol 2022; 56(1): 111-118. Ivanecz A et al. / Learning curve of laparoscopic liver resection 113 Figure 1. This figure is rendered here for the self- sufficiency of this article. The Weibull curve in Figure 1 is monotonically increasing. Regarding the LC, we assume that a procedure with a higher difficulty score must be graded better than a procedure with a lower dif- ficulty score if the resection is done without IOC. Therefore, the difference between the theoretically predicted probability of IOC and obtained IOC is greater if the difficulty score is higher (if IOC = 0). On the other hand, if IOC was detected (if IOC = 1), the difference between the theoretically predicted probability of IOC and obtained IOC is negative (implying a lower grade for a surgeon) if the dif- ficulty score is low. Thus, the learning outcome is proportional to the share of IOC caused by the sur- geon obtained in each of the ten classes. We wanted to test if the time dependency of HDS is (on average) an ascending function. Therefore, resections were divided into three (time) sequen- tial classes (each consisting of 57 patients), and the number of obtained IOC in each class was counted. HDS10 was used in the analysis of LC. Its de- pendency was proven to be (on average) an in- creasing function (Figure 1). The proposed mathematical model of a learning curve The probability (the share of IOC in the time-de- pendent class) of IOC depends on HDS. The share of IOC in a time-dependent class measures the complexity of resections. Therefore, a novel model for presenting the learning outcome in the case of LLR with existing theoretical dependence between HDS and (the probability of) IOC was introduced. We assume that the learning outcome consists of two additive components. The first represents the absolute complexity of the resection according to time (which is proportional to effort). The sec- ond (additive) component is obtained by compar- ing the share of IOC to the theoretically predicted (probability of) IOC depending on the HDS of the patient. Components share the same physical units; therefore, the addition is justified. The sum of com- ponents results in the learning outcome for any patient and finally in the LC. The first component reflects the absolute complexity of the resections within the same class, while the second one reflects the relative complexity (comparing to the theoreti- cally predicted HDS), which can be interpreted as the surgeon’s efficiency. At this point, we mathematically define the ob- jectives determining the learning outcomes, and consequently, the LC. The cohort of 171 patients is divided into ten sequential classes (the last class contains 17+1 patient). By , we denote the sequen- tial number of the patient. By , we denote the sequential number of the class (for every class, its cardinality is equal to 17). Our main assumptions and proposals are the following: 1. Since the resections were listed chronological- ly, we may assume that the sequential num- ber of the patient corresponds to the effort of the surgeon (the correspondence is monotoni- cally increasing). 2. For every class , the absolute complexity of the tasks in the class is proportional to the ratio of the IOC cases. The non-smooth dependency was fitted to smooth logarithmic function . Additionally, it was modified to absolute complexity for each sequential patient 3. For every class , the relative complexity or efficiency of the surgeon is proportional to the sum of differences between the theoreti- cally predicted probabilities of IOC and the obtained probabilities of IOC FIGURE 1. The continuous mean risk curve of intraoperative complication (IOC) as a function of the Halls difficulty score: the theoretical probability of intraoperative complication.11 Radiol Oncol 2022; 56(1): 111-118. Ivanecz A et al. / Learning curve of laparoscopic liver resection114 Additionally, was finally modi- fied to relative complexity for each se- quential patient 4. Adding both components, we get the learning curve of the surgeon: Results The presentation of the cohort Between April 2008 and April 2021, 171 patients underwent pure LLR. Their baseline characteristics are presented in Table 1. Perioperative outcomes are given in Table 2. Two patients (1.2%) suffered from unintentional laceration of the transverse colon, sutured laparo- scopically. The procedure was completed laparo- scopically in 147 (86.0%) patients. The reasons for conversion to laparotomy in 24 (14.0%) patients were diffuse parenchymal bleeding (N = 3), inabil- ity to proceed due to the large liver or dense adhe- sions (N = 6), and oncological concern (N = 15). The decision to proceed to conversion was not made upon life-threatening bleeding. The indication for liver resection in converted cases was malignant tumours. Three (1.8%) patients died – one bled out from ruptured oesophageal varices, and two died of liver failure; they all had hepatocellular carci- noma and liver cirrhosis Child-Pugh B. Learning curve analysis results The analysis of the learning curve was motivat- ed by the increasing time dependency of HDS. Therefore, resections were divided into three se- quential classes of 57 resections, and the number of obtained IOC in each class was counted. The re- sults are graphically presented in Figure 2. On significance level the p-value for Chi Square test is slightly above 5% (p = 0.055). However, for linear-by-linear (Mantel Haenszel) test for trend, the p-value is < 0.05. HDS10 was used in the analysis of LC. The risk- of-IOC dependency was proven to be (on average) an increasing function in terms of HDS (Figure 1). A time-dependent and increasing trend can also be seen in Figure 3 (see the red linear trend-line for HDS; the blue chart represents actual data). The sequential number of the patient corre- sponds to the effort of the surgeon (the correspond- ence is monotonically increasing). In the first class, TABLE 1. Baseline characteristics of 171 patients who underwent laparoscopic liver resection Baseline characteristics Na,b Male sexa 104 (60.8%) Age (years)b 64 (20-86, 15) BMI (kg/m2)b 27 (18-50, 4.8) ASA scorea 1 44 (25.7%) 2 73 (42.7%) 3 51 (29.8%) 4 3 (1.8%) Liver cirrhosis Child-Pugh (22)a A 33 (19.3%) B 4 (2.3%) Previous abdominal surgerya 41 (24.0%) Previous liver resectiona 8 (4.6%) Malignant tumoura 128 (74.9%) Neoadjuvant chemotherapya 25 (14.6%) Max. diameter (mm)b 38 (2-160, 33) Number of tumoursa 1 (1-10, 0). Deep location within livera 50 (29.2%) Posterosuperior liver segmentsa 49 (28.7%) a = categorical variables; b = continuous variables have been reported as median (minimum- maximum, interquartile range); ASA = American Society of Anaesthesiologists; BMI = body mass index FIGURE 2. Histogramic time classes dependency of intraoperative complication (IOC) (yes/no) on the observed cohort. Interpolating the data to a polynomial of degree five, one gets a smooth function obtained by Mathematica command SplineFit using option Cubic. Radiol Oncol 2022; 56(1): 111-118. Ivanecz A et al. / Learning curve of laparoscopic liver resection 115 the average time difference between sequential surgeries was 117 days (with a standard deviation of 132 days), while in the last class, the time differ- ence was 13 days with a standard deviation of 12 days. The paired samples t-test shows that (at the level of confidence of 95%) the two means are not equal (p < 0.05). The final result of our LC data analysis is pre- sented in Figure 4. Ten consecutive classes of 17 patients are given on abscissa. The height of the columns represents the share of the IOC in the time class. Two types of LCs for the observed cohort and the surgeon under consideration are given. The orange line represents the logarithmic regres- sion curve based on absolute complexity for data . The green line represents the sum of the orange curve and the quintic regression line of relative complexity for data . This green line represents our LC. Discussion Like any other human activity, where individu- als perform more difficult and intricated tasks over time, surgeons have been interested in their LC when performing LLR.16 The obtained learn- ing curve has resulted from thirteen years of sur- gical effort of a single surgeon. It consists of an absolute and a relative part in the mathematical description of the additive function described by the logarithmic function (absolute complexity) and fifth-degree regression curve (relative complexity). The obtained LC determines the functional de- pendency of the learning outcome versus time and indicates several local extreme values (peaks and valleys) in the learning process until proficiency is achieved. A typical LC graphically represents the relation- ship between the learning effort and achievement. LC consists of a measure of learning which usually lies on the ordinate (y-axis), a measure of effort, which usually lies on the abscissa (x-axis) and a mathematical linking function. The shapes of this mathematical (functorial) dependence can vary de- pending on the nature and difficulty of the learn- ing outcomes and difficulty of the task.26,27 It may be assumed that LC should be increasing in time (i.e., with effort). There are several typical LCs for learning different skills whose shape de- pends on the complexity of the task. When learning simple skills, S-shaped or logistic curves appear. The logistic curve admits a single inflexion point (indicating the point when half of the knowledge TABLE 2. Perioperative outcomes of 171 patients who underwent laparoscopic liver resection Intraoperative details and postoperative course Na,b Anatomic resection (23) a 101 (59.1%) Anatomically major resection (23) a 27 (15.8%) Technically major resection (24)a 29 (17.0%) Operation time (min)b 160 (25-450, 90) Blood loss (mL)b 150 (0-2200, 180) Intraoperative complication (10)c 34 (19.9%) Conversion to open approacha 24 (14.0%) Blood loss > 775 mLa 12 (7.0%) Unintentional damage to the surrounding structuresa 2 (1.2%) Hepatic pedicle clampinga 45 (26.3%) Total hepatic pedicle clamping time (min)b 8 (0-75, 10) Transfusion requireda 20 (11.7%) Pathohistological diagnosis Colorectal liver metastases 53 (31%) Hepatocellular carcinoma 46 (29.6%) Intrahepatic cholangiocarcinoma 14 (8.2%) Other metastases 11 (6.4%) Hepatic cysts 10 (5.8%) Hepatic adenoma 6 (4.7%) Focal nodular hyperplasia 8 (4.7%) Haemangioma 6 (3.5%) Other pathology 15 (8.8%) R0 resection 163 (95.3%) Major morbidity CD 3a–4b (25)a 21 (12.3%) Hospital stay (days)b 6 (2-79, 4) a = categorical variables; b = continuous variables have been reported as median (minimum- maximum, interquartile range); c = intraoperative complication was defined as blood loss over 775 mL, unintentional damage to the surrounding structures and conversion to open approach was acquired) and a horizontal asymptote (repre- senting the cap to be acquired). In surgical proce- dures for more complex skills, often a logarithmic LC without a cap appears. However, when inter- preting a paediatric ankle radiograph, the LC turns out to be logarithmic.27 The zig-zag shape can ap- pear as well.27 A steep LC is rare in medicine since the skills are associated with difficult and complex procedures.26,27 We have considered the LC of a single surgeon in a technically demanding LLR. When implement- ing a new surgical procedure, a surgeon already has some fundamental knowledge. The learning outcome is assumed to be proportional to the share of IOC made by the surgeon, i.e. we learn from our Radiol Oncol 2022; 56(1): 111-118. Ivanecz A et al. / Learning curve of laparoscopic liver resection116 It is assumed that a higher level of (the sum of) theoretically predicted probability of IOC (within a particular class) reflects a higher level of gained knowledge (higher grade for the LC). This may be justified because the average HDS is also increas- ing with time (Figure 4). Therefore, HDS affects the relative complexity of the case. The orange line represents the basic LC. The relative complexity depends on the subjective decision made by the surgeon according to previously successfully fin- ished cases with no IOC. LLR has been encompass- ing different procedures, each with its own ana- tomic and procedural considerations. Komatsu et al.13 demonstrated an ideal learning curve effect for the left lateral sectionectomy and left hepatectomy, but it was not observed for the right hepatectomy. The more successive cases with no IOC encouraged the surgeon to do more cases with increased HDS. When analysing IOC, the conversion rate of 14% was consistent with the reported ones, counting from 1% to 17%.15,29 An increased risk of conver- sion has been associated with neoadjuvant chemo- therapy, previous open liver resection, malignant tumours, their size, anatomically major and techni- cally major resection.30 Patients who had an elec- tive conversion for an unfavourable intraoperative finding had better outcomes than patients who had an emergency conversion secondary to an adverse intraoperative event.30 All our converted cases oc- curred in malignant tumours. None of the cases was related to life-threatening bleeding. The most common indications for conversion were the in- ability to proceed and oncological concern, respec- tively. A chosen method does not change the prin- ciple of the surgery. Therefore, an oncologically uncompromised resection has been more crucial than the laparoscopic completion of the procedure. The overall major morbidity and mortality rates of 12.3% and 1.8% followed reports in the litera- ture.13,14,16 To sum up, this conversion rate reflected the surgeon’s reliance on the open method when dealing with adverse intraoperative findings.20 Although the first anatomical LLR was per- formed in 199631, the first difficulty score was pub- lished not earlier than 2014.32 Our first LLR was performed in 2008, and the surgeon had to lean on his experience from open liver surgery. It would be riveting to study the results of the surgeon’s trainees who could benefit from the evolution of techniques, learning modules12,16,33, and difficulty scores.6-8,10 The main shortcoming of the presented research is a relatively low number of patients. Therefore, in future research, a larger number of patients should FIGURE 3. Time dependency of the Halls difficulty score on the observed cohort (blue points) and its regression (trend) line (red line). FIGURE 4. Two types of learning curves for observed cohort and the surgeon under consideration. The orange line (AC) represents the logarithmic regression curve based on absolute complexity. The green line (LC) represents the sum of the orange curve and the quintic regression line of relative complexity. This line represents our learning curve. AC = absolute complexity; ac (N) = absolute complexity expressed by the number of intraoperative complications; LC = learning curve mistakes (IOC). However, LLR has not been a sin- gle procedure, and the complexity of operations varies from wedge resections to extended major hepatectomies. This fact contributes to the difficul- ties during learning and assessing the LC.12-16 In the beginning, solitary and peripherally located symp- tomatic benign tumours in anterolateral segments were resected.28 With growing experience, the laparoscopic approach was implemented regard- less of tumour location and its characteristics.1,5 The time difference between sequential surgeries in time classes shortened from 117 days to 13 days. Therefore, one could reasonably assume this was a part of the learning strategy. Radiol Oncol 2022; 56(1): 111-118. Ivanecz A et al. / Learning curve of laparoscopic liver resection 117 be involved to show the robustness of the present- ed LC. Furthermore, its retrospective manner is an- other limitation. The propose d LC and used methodology could guide the trainee surgeons and monitor their per- formance. In this sense, practitioners should be provided with a statistically independent set of pa- tients with a constant increase (i.e., a constant gra- dient) of HDS over time. Thus, more difficult cases would be taken over by more qualified surgeons. A newly created application would randomly select patients with the appropriate HDS for each prac- titioner. It would enable control of the (accidental) variability in HDS and its consequences on IOC, which could not be completely avoided in practice. Under the supervision of a qualified operator, the objective evaluation of the LC would avoid deeper valleys in it (higher number of IOCs than theoreti- cally expected) and thus ensure the most optimal learning. Given the basic assumption that we learn from our mistakes (see section A mathematical mod- elling of a learning curve), the maximal acceptable number and type of mistakes in the learning pro- cess should be objectively evaluated through fur- ther research. To conclude, our LC is closer to a true model in which alternating periods of progression and regression occurred until mastery was achieved.16 Furthermore , the method presented in this paper can be applied to any (surgical) procedure with a difficulty score and given outcome (for example IOC), if a theoretically predicted probability de- pendence for the given outcome is available. From this point of view, the method is novel. Acknowledgement This work was supported by University Medical Centre Maribor (grant number IRP-2019/01-03). The funding source has no role in the design, prac- tice, or analysis of this study. References 1. Buell JF, Cherqui D, Geller DA, O’Rourke N, Iannitti D, Dagher I, et al. The in- ternational position on laparoscopic liver surgery: The Louisville Statement, 2008. Ann Surg 2009; 250: 825-30. doi: 10.1097/sla.0b013e3181b3b2d8 2. Ciria R, Cherqui D, Geller DA, Briceno J, Wakabayashi G. Comparative short- term benefits of laparoscopic liver resection: 9000 cases and climbing. Ann Surg 2016; 263: 761-77. doi: 10.1097/sla.0000000000001413 3. Wakabayashi G, Cherqui D, Geller DA, Buell JF, Kaneko H, Han HS, et al. Recommendations for laparoscopic liver resection: a report from the sec- ond international consensus conference held in Morioka. Ann Surg 2015; 261: 619-29. doi: 10.1097/sla.0000000000001184 4. Cherqui D, Wakabayashi G, Geller DA, Buell JF, Han HS, Soubrane O, et al. The need for organization of laparoscopic liver resection. J Hepatobiliary Pancreat Sci 2016; 23: 665-67. doi: 10.1002/jhbp.401 5. Abu Hilal M, Aldrighetti L, Dagher I, Edwin B, Troisi RI, Alikhanov R, et al. The Southampton consensus guidelines for laparoscopic liver surgery: from indication to implementation. Ann Surg 2018; 268: 11-8. doi: 10.1097/ sla.0000000000002524 6. Wakabayashi G. What has changed after the Morioka consensus conference 2014 on laparoscopic liver resection? Hepatobiliary Surg Nutr 2016; 5: 281- 9. doi: 10.21037/hbsn.2016.03.03 7. Hasegawa Y, Wakabayashi G, Nitta H, Takahara T, Katagiri H, Umemura A, et al. A novel model for prediction of pure laparoscopic liver resection surgical difficulty. Surg Endosc 2017; 31: 5356-63. doi: 10.1007/s00464-017-5616-8 8. Kawaguchi Y, Fuks D, Kokudo N, Gayet B. Difficulty of laparoscopic liver resection: proposal for a new classification. Ann Surg 2018; 267: 13-7. doi: 10.1097/sla.0000000000002176 9. Hallet J, Pessaux P, Beyfuss KA, Jayaraman S, Serrano PE, Martel G, et al. Critical appraisal of predictive tools to assess the difficulty of laparoscopic liver resection: a systematic review. Surg Endosc 2019; 33: 366-76. doi: 10.1007/s00464-018-6479-3 10. Halls MC, Berardi G, Cipriani F, Barkhatov L, Lainas P, Harris S, et al. Development and validation of a difficulty score to predict intraoperative complications during laparoscopic liver resection. Br J Surg 2018; 105: 1182- 91. doi: 10.1002/bjs.10821 11. Ivanecz A, Plahuta I, Magdalenić T, Mencinger M, Peruš I, Potrč S, et al. The external validation of a difficulty scoring system for predicting the risk of intraoperative complications during laparoscopic liver resection. BMC Surg 2019; 19: 179. doi: 10.1186/s12893-019-0645-y 12. Guilbaud T, Birnbaum DJ, Berdah S, Farges O, Beyer Berjot L. Learning curve in laparoscopic liver resection, educational value of simulation and train- ing programmes: a systematic review. World J Surg 2019; 43: 2710-9. doi: 10.1007/s00268-019-05111-x 13. Komatsu S, Scatton O, Goumard C, Sepulveda A, Brustia R, Perdigao F, et al. Development process and technical aspects of laparoscopic hepatectomy: learning curve based on 15 years of experience. J Am Coll Surg 2017; 224: 841-50. doi: 10.1016/j.jamcollsurg.2016.12.037 14. van der Poel MJ, Besselink MG, Cipriani F, Armstrong T, Takhar AS, van Dieren S, et al. Outcome and learning curve in 159 consecutive patients undergoing total laparoscopic hemihepatectomy. JAMA Surg 2016; 151: 923-28. doi: 10.1001/jamasurg.2016.1655 15. Vigano L, Laurent A, Tayar C, Tomatis M, Ponti A, Cherqui D. The learning curve in laparoscopic liver resection: improved feasibility and reproducibil- ity. Ann Surg 2009; 250: 772-82. doi: 10.1097/SLA.0b013e3181bd93b2 16. Villani V, Bohnen JD, Torabi R, Sabbatino F, Chang DC, Ferrone CR. “Idealized” vs. “True” learning curves: the case of laparoscopic liver resec- tion. HPB (Oxford) 2016; 18: 504-9. doi: 10.1016/j.hpb.2016.03.610 17. Han HS, Cho JY, Yoon YS. Techniques for performing laparoscopic liver resec- tion in various hepatic locations. J Hepatobiliary Pancreat Surg 2009; 16: 427-32. doi: 10.1007/s00534-009-0118-2 18. Ivanecz A, Krebs B, Stozer A, Jagric T, Plahuta I, Potrc S. Simultaneous pure laparoscopic resection of primary colorectal cancer and synchronous liver metastases: a single institution experience with propensity score matching analysis. Radiol Oncol 2018; 52: 42-53. doi: 10.1515/raon-2017-0047 19. Ivanecz A, Pivec V, Ilijevec B, Rudolf S, Potrč S. Laparoscopic anatomical liver resection after complex blunt liver trauma: a case report. Surg Case Rep 2018; 4: 25. doi: 10.1186/s40792-018-0432-5 20. Ivanecz A, Plahuta I, Magdalenić T, Ilijevec B, Mencinger M, Peruš I, et al. Evaluation of the Iwate model for predicting the difficulty of laparoscopic liver resection: does tumor size matter? J Gastrointest Surg 2021; 25: 1451- 60. doi: 10.1007/s11605-020-04657-9 21. Weibull W. A statistical distribution function of wide applicability. J Appl Mech 1951; 18: 293-97. 22. Pugh RN, Murray-Lyon IM, Dawson JL, Pietroni MC, Williams R. Transection of the oesophagus for bleeding oesophageal varices. Br J Surg 1973; 60: 646-9. doi: 10.1002/bjs.1800600817 23. Strasberg SM, Belghiti J, Clavien PA, Gadzijev E, Garden JO, Lau WY, et al. The Brisbane 2000 terminology of liver anatomy and resections. HPB 2000; 2: 333-39. doi: 10.1016/S1365-182X(17)30755-4 Radiol Oncol 2022; 56(1): 111-118. Ivanecz A et al. / Learning curve of laparoscopic liver resection118 24. Kazaryan AM, Røsok BI, Marangos IP, Rosseland AR, Edwin B. Comparative evaluation of laparoscopic liver resection for posterosuperior and antero- lateral segments. Surg Endosc 2011; 25: 3881-9. doi: 10.1007/s00464-011- 1815-x 25. Clavien PA, Barkun J, de Oliveira ML, Vauthey JN, Dindo D, Schulick RD, et al. The Clavien-Dindo classification of surgical complications: five-year experi- ence. Ann Surg 2009; 250: 187-96. doi: 10.1097/SLA.0b013e3181b13ca2 26. Hopper AN, Jamison MH, Lewis WG. Learning curves in surgical practice. Postgrad Med J 2007; 83: 777-9. doi: 10.1136/pgmj.2007.057190 27. Pusic MV, Boutis K, Hatala R, Cook DA. Learning curves in health pro- fessions education. Acad Med 2015; 90: 1034-42. doi: 10.1097/ acm.0000000000000681 28. Nguyen KT, Gamblin TC, Geller DA. World review of laparoscopic liver resection-2,804 patients. Ann Surg 2009; 250: 831-41. doi: 10.1097/ SLA.0b013e3181b0c4df 29. Costi R, Scatton O, Haddad L, Randone B, Andraus W, Massault PP, et al. Lessons learned from the first 100 laparoscopic liver resections: not delaying conversion may allow reduced blood loss and operative time. J Laparoendosc Adv Surg Tech A 2012; 22: 425-31. doi: 10.1089/ lap.2011.0334 30. Halls MC, Cipriani F, Berardi G, Barkhatov L, Lainas P, Alzoubi M, et al. Conversion for unfavorable intraoperative events results in significantly worse outcomes during laparoscopic liver resection: lessons learned from a multicenter review of 2861 cases. Ann Surg 2018; 268: 1051-57. doi: 10.1097/sla.0000000000002332 31. Azagra JS, Goergen M, Gilbart E, Jacobs D. Laparoscopic anatomical (he- patic) left lateral segmentectomy-technical aspects. Surg Endosc 1996; 10: 758-61. doi: 10.1007/bf00193052 32. Ban D, Tanabe M, Ito H, Otsuka Y, Nitta H, Abe Y, et al. A novel difficulty scoring system for laparoscopic liver resection. J Hepatobiliary Pancreat Sci 2014; 21: 745-53. doi: 10.1002/jhbp.166 33. Goh BKP, Prieto M, Syn N, Koh YX, Lim KI. Critical appraisal of the learning curve of minimally invasive hepatectomy: experience with the first 200 cases of a Southeast Asian early adopter. ANZ J Surg 2020; 90: 1092-98. doi: 10.1111/ans.15683 Radiol Oncol 2022; 56(1): 119-128. doi: 10.2478/raon-2021-0053 119 research article In vitro maturation of immature oocytes for fertility preservation in cancer patients compared to control patients with fertility problems in an in vitro fertilization program Irma Virant-Klun1, Jure Bedenk2, Nina Jancar2 1 Clinical Research Centre, University Medical Centre Ljubljana, Ljubljana, Slovenia 2 Division of Obstetrics and Gynecology, University Medical Centre Ljubljana, Ljubljana, Slovenia Radiol Oncol 2022; 56(1): 119-128. Received 5 August 2021 Accepted 25 November 2021 Correspondence to: Prof. Irma Virant-Klun, Ph.D., Clinical Research Centre, University Medical Centre Ljubljana, Zaloška cesta 2, SI 1000 Ljubljana, Slovenia. E-mail: irma.virant@kclj.si Disclosure: No potential conflicts of interest were disclosed. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Background. The aim of this study was to determine whether in vitro maturation (IVM) of immature oocytes after controlled hormonal stimulation of the ovaries could be important in cancer patients to improve their chances of conception in the future. Patients and methods. After ovarian stimulation in cancer patients, the number of oocytes and their quality and maturity were compared to control patients with fertility problems in the in vitro fertilization (IVF) program. In both groups of patients, immature oocytes at the developmental stage of germinal vesicle were matured in vitro and the proportion of oocytes that matured in vitro was compared between groups. In a subset of women with fertility problems, intracytoplasmic sperm injection (ICSI) was performed on IVM oocytes to assess their ability to be fertilized and develop into an embryo compared to vivo matured oocytes in the same cycles and consider the procedure in cancer patients. Results. In patients with different cancers, the disease did not affect the number and quality of retrieved oocytes. In cancer patients, there was even a significantly lower proportion of immature oocytes than in patients with fertility problems (30.0% vs. 43.6%; P < 0.05). However, in patients with cancer, fewer oocytes per patient matured in vitro than in patients with fertility problems (1.39 ± 1.04 vs. 2.48 ± 1.83; P < 0.05). After ICSI, the proportions of fertilized oocytes and fertilized oocytes developing into an embryo did not differ between oocytes matured in vitro and in vivo in the same cycles. Conclusions. Oocyte IVM is proving to be a reliable procedure for resolving immature oocytes after controlled ovarian stimulation in cancer patients. Key words: cancer; fertility preservation; oocyte; in vitro maturation; vitrification Introduction Many young women in the reproductive period of life who do not yet have children or would like to have another child suffer from cancer. Today, cancer therapies are successful, but unfortunately, they can negatively affect the ovarian function (in- cluding oocyte quality) and fertility. At a median of 5.0 years from initial breast cancer diagnosis, 49% patients after adjuvant chemotherapy with anthracyclines and taxanes and 11% after therapy with tamoxifen had become post- and peri-meno- pausal.1 Decreased ovarian follicle reserve occurs in more than one-third of patients after breast can- Radiol Oncol 2022; 56(1): 119-128. Virant-Klun I et al. / In vitro maturation of immature oocytes for fertility preservation120 cer treatment resulting in permanent infertility.2 In long-term female survivors of pediatric hema- tologic malignancies 26.7% experienced premature ovarian insufficiency and face infertility after can- cer treatment.3 The situation is similar with other cancers; cancer therapy is the cause of premature ovarian failure in 25% of women with this diagno- sis.4 Therefore, it is very important to consider the preservation of female fertility before oncotherapy. An improvement in the survival rates of cancer pa- tients and recent advances in assisted reproductive technologies have led to significant progress in fer- tility preservation treatments. One option is vitrification and long-term storage of the patient's oocytes for later in vitro fertilization (IVF) with partner's sperm and transfer of embryos into the uterus. This program is established in many health care institutions around the world for a va- riety of cancers, including breast cancer.5-12 Oocyte cryopreservation is an effective approach13-15, but it is still thought that further studies are needed in cancer patients to ensure the excellent outcomes obtained in women without cancer.16 After con- trolled hormonal stimulation of the ovaries, in vitro maturation (IVM) of immature oocytes before vitri- fication is recommended and not after vitrification/ devitrification procedure.17 Even if there are no dif- ferences in survival rates between oocytes vitrified before or after IVM procedure, decreased matura- tion rates of immature oocytes vitrified before IVM may be explained by underlying ultrastructural and biomolecular alterations.17 Human oocyte cryopreservation may offer some advantages compared to embryo freezing in can- cer patients and also eliminates some ethical, legal, and moral concerns of embryo freezing17, and is an option in young cancer patients who are single.9,18 However, the chance of success depends primarily on the number of oocytes that have been vitrified in the patient15 and some breast cancer patients may have contraindications to exogenous gonadotropin administration for controlled ovarian stimulation.19 Some recent data show that ovarian stimulation for oocyte vitrification does not modify disease-free survival and overall survival rates in patients with early breast cancer20 and the safety of pregnancy after an established diagnosis of breast cancer has been confirmed in numerous studies.21 In the case of vitrification and storage of oocytes, controlled hormonal stimulation of the ovaries is required to obtain oocytes. Despite careful hor- monal stimulation of the ovaries, the significant proportion of oocytes obtained by ultrasound- guided aspiration of ovarian follicles is immature as metaphase I (MI) oocytes or prophase I oocytes with germinal vesicle (GV). Immature MI oocytes mostly mature spontaneously in vitro and are vit- rified, while immature GV oocytes do not mature spontaneously and are incapable of fertilization. Therefore, GV oocytes are not vitrified and stored in liquid nitrogen in clinical practice and are dis- carded and lost to the patient. The important ques- tion is whether the maturation of these oocytes in vitro makes sense. There is a lack of data regarding the outcome of in vitro matured oocytes cryopre- served in cancer patients.22 Recently, the first birth achieved after fertility preservation using vitrifica- tion of in vitro matured oocytes in a patient with breast cancer has been reported.23 The purpose of this study was to investigate the effectiveness of maturation of immature GV oo- cytes of cancer patients in laboratory conditions (in maturation medium and co-culture with cu- mulus cells from mature oocytes of the same pa- tients) compared to control women involved in the IVF program due to fertility problems. Because all oocytes of cancer patients are still frozen, we tried to elucidate the success of IVF procedure, actual- ly intracytoplasmic sperm injection (ICSI), on the in vitro matured oocytes of patients with fertility problems as a model for cancer patients. Patients and methods This research was approved by the Slovenian National Medical Ethical Committee (No. 0120- 222/2016-2; KME 115/04/16). In this prospective research the immature (germinal vesicle-GV, pro- phase I) oocytes of two groups of patients were included: i) 45 oocytes of 18 cancer patients with predominating breast cancer (Figure 1) and ii) 74 oocytes of 21 healthy (non-cancer) patients (control) with fertility problems (partners of infertile men with impaired semen quality: oligozoospermia with less than 15 million spermatozoa/ml or tera- tozoospermia with less than 4% morphologically normal spermatozoa according to the World Health Organization (WHO) Criteria 201024 who were in- cluded in the IVF program. All patients were in the reproductive period of life, aged 18 to 43 years. Oocytes after controlled hormonal stimulation of the ovaries In both groups of patients, both immature and mature oocytes were together retrieved after con- trolled hormonal stimulation of the ovaries using Radiol Oncol 2022; 56(1): 119-128. Virant-Klun I et al. / In vitro maturation of immature oocytes for fertility preservation 121 the same, antagonist protocol and ultrasound- guided aspiration of ovarian follicles. In patients with fertility problems, the stimulation was started on day 2 of the menstrual cycle with 150 to 300 I.U. of recombinant follicle-stimulating hormone (rFSH) daily. In cancer patients, the stimulation was initiated immediately after they have been sent to our department, no matter of the cycle phase. The ovarian stimulation was started with 225 to 300 I.U. of rFSH. In breast cancer patients, an aromatase inhibitor – letrozole (2.5 mg every 12 hours) was added to prevent estradiol rise and its possible detrimental effect on breast cancer. In all patients, the gonadotropin-releasing hormone (GnRH) antagonist was added, when dominant follicle measured 14 mm in a diameter. In patients with fertility problems, the oocyte maturation was triggered with choriogonadotropin alfa – Ovitrelle, when follicles measured 18 mm or more. If there were more than 15 follicles in both ovaries, the maturation triggering was performed with GnRH agonist. In majority of cancer patients, GnRH ag- onist was used for oocyte maturation to prevent ovarian hyperstimulation (ovarian hyperstimula- tion syndrome; OHSS), but some of them, if there were less than 10 follicles in both ovaries, were also treated by Ovitrelle. All follicles with a diameter of 16 mm or more were aspirated in all patients. A constant aspiration pressure of 180 mm Hg was used to aspirate the oocytes from the follicles. Mature oocytes with expressed polar body were immediately vitrified by soaking in a mixture of cryoprotectants, direct plunging into liquid nitro- gen (-196oC), and stored in it, as described else- where.25 In vitro maturation of immature oocytes Immature GV oocytes were exposed to the pro- cedure of IVM in a seria of media of the IVM Maturation System (MediCult IVM®System, Origio/CooperSurgical, Denmark). For IVM, each GV oocyte was first exposed for two hours in the LAG Medium for conditioning and then for 24 to 28 hours to the maturation medi- um of this system containing the reproductive hor- mones: follicle stimulating hormone (FSH; 75 mIU/ ml) and human chorionic gonadotropin (HCG; 100 mIU/ml) and in a co-culture with cumulus cells denuded from mature oocytes of the same patient, as described elsewhere (Figure 2).26 During incuba- tion in these media, oocytes were cultured in a CO2- incubator at 37oC and 6% CO2 in air. Oocytes were supposed to be mature (in a metaphase II) when they extruded a polar body. All oocytes of cancer patients and the majority of oocytes of women with fertility problems that matured in vitro were vitri- fied and stored in liquid nitrogen for later clini- cal use (IVF). Patients with ≥ 31% GV oocytes had increased oocyte immaturity. A subset of in vitro matured oocytes of women with fertility problems was fertilized in vitro. FIGURE 1. Types of disease in cancer patients included in this study. Breast cancer patients predominated. FIGURE 2. In vitro maturation of the oocyte in the maturation medium and in coculture with the cumulus cells of mature oocytes in the same patient at magnification 40 X (A) and magnification 100 X (B). CC = cumulus (granulosa) cells; O = oocyte; Red bar = A) 100 μm in B) 50 μm A B Radiol Oncol 2022; 56(1): 119-128. Virant-Klun I et al. / In vitro maturation of immature oocytes for fertility preservation122 In vitro fertilization by ICSI In a subgroup of 17 in vitro matured oocytes from 17 patients (1 oocyte per patient) with fertility problems (female partners of infertile men with oligozoospermia or teratozospermia) ICSI was per- formed with partner's sperm one day later after oocyte and sperm retrieval from a couple. Oocytes were denuded by hyaluronidase to remove the cumulus cells and microinjection of one spermato- zoon per oocyte was performed, as described else- where.27 Only motile spermatozoa were used for ICSI. The next day, fertilization (presence of two pronuclei and extruded second polar body) was checked and embryo cleavage one day later. Good quality embryos were vitrified and stored in liquid nitrogen (-196oC) for later clinical use (transfer to the uterus). The rates of fertilization and embryo cleavage were compared between oocytes that ma- tured in vitro and in vivo (in the ovaries; aspirated as mature) in the same patients after controlled ovarian hormonal stimulation. Statistics Both groups of patients, cancer and infertile pa- tients, were compared in terms of the number of oocytes obtained after controlled hormonal stimu- lation of their ovaries, the proportion of immature and degenerated oocytes, and the proportion of immature (GV) oocytes that matured in vitro. Due to the relatively small number of patients/oocytes included and the abnormal distribution of data, tested by Shapiro-Wilk normality test, non-para- metric tests (Fisher's exact and Mann-Whitney U tests) were performed; statistical significance was set at P < 0.05. After ICSI, the fertilization and em- bryo cleavage rates of oocytes that matured in vitro were compared with oocytes of the same patients that matured in vivo and were aspirated from the ovaries as mature oocytes in the same cycle of controlled ovarian hormonal stimulation using Fisher's exact and Wilcoxon tests; statistical signifi- cance was set at P < 0.05. Results Cancer patients had different types of cancer, but breast cancer was predominant (Figure 1). The av- erage age of cancer patients was 30.3 ± 6.3 years and of women with fertility problems 33.4 ± 5.0 years. The two groups of women did not differ sig- nificantly in their age. There was also no significant difference in the age of patients with breast cancer and patients with other cancers (32.0 ± 6.2 vs. 29.0 ± 6.0 years). Numbers, quality and immaturity of oocytes after controlled hormonal stimulation of the ovaries After controlled hormonal stimulation of the ova- ries, 198 oocytes were retrieved in cancer patients and 259 oocytes in infertile women. Cancer pa- tients and patients with fertility problems did not differ significantly in the number of retrieved oo- cytes (11.0 ± 9.0 oocytes/patient vs. 12.3 ± 9.2 oo- cytes/patient), as revealed by Mann-Whitney U test. In cancer patients, the proportion of immature GV and MI oocytes was significantly lower than in patients with fertility problems (30.0% vs. 43.6%; P < 0.05), as revealed by Fisher's exact test (Table 1). The groups did not differ significantly in the pro- portion of GV oocytes (23.0% vs. 28.6%) (Table 1); among patients with immature oocytes, 50% of cancer patients and 48.0% of patients with fertility problems had increased proportion (≥ 31%) of GV oocytes with a germinal vesicle, which did not dif- fer significantly. There was also no difference in the proportion of degenerated oocytes between cancer patients and patients with fertility problems (8.6 vs. 6.5%) (Table 1). If we considered the type of cancer, we found that there was no significant difference in the number of all immature (MI and GV) oocytes in patients with breast cancer compared to patients with fertility problems or patients with other can- cers (4.17 ± 3.25 vs. 5.29 ± 3.76 and 2.92 ± 2.47 im- mature oocytes/patient, respectively), as revealed by Mann-Whitney U test. There was also no sta- tistically significant difference in the proportion of all immature (GV and MI) oocytes in breast cancer patients compared to patients with other cancers or patients with fertility problems (36.0% vs. 27.0% and 43.6%), as found using the Fisher's exact test. Nevertheless, there was a tendency for a signifi- cantly higher proportion of immature, GV oocytes in breast cancer patients compared to patients with other cancers (31.43% vs. 18.75%; P = 0.0531, Fisher's exact test). In vitro matured oocytes Forty-five GV oocytes in cancer patients and 74 GV oocytes in patients with fertility problems un- derwent IVM procedure (Table 2); the proportion of oocytes (15.5% in cancer patients and 12.2% in Radiol Oncol 2022; 56(1): 119-128. Virant-Klun I et al. / In vitro maturation of immature oocytes for fertility preservation 123 women with fertility problems) degenerated just before or during conditioning in LAG medium. We found that a lower proportion of oocytes ma- tured in vitro in cancer patients compared to pa- tients with fertility problems (66.0 vs. 80.0%), how- ever, the difference was not statistically significant, as revealed by Fisher's exact test. In spite of that, number of oocytes that matured in vitro per patient was significantly lower in cancer patients than in patients with fertility problems (1.39 ± 1.04 vs. 2.48 ± 1.83 oocytes/patient; P < 0.05, Mann-Whitney U test), as shown in Table 2. In cancer patients, there was also a lower pro- portion of oocytes that matured in vitro in patients with breast cancer than in patients with other can- cers and patients with fertility problems (54.5% vs. 81.2% and 80.0%) (Table 2). The difference between patients with breast cancer and women with fertil- ity problems tended to be statistically significant (P = 0.0862; Fisher's exact test) and was probably not significant due to the relatively low number of oocytes included. Overall, 198 oocytes were retrieved in cancer pa- tients, of which 139 were mature. Following IVM, the number of total mature oocytes increased to 164 (13.0% increase in mature oocyte yield). In patients with fertility problems, 259 oocytes were retrieved, of which 146 were mature. After IVM, the number of total mature oocytes increased to 198, which means 20.1% increase in mature oocyte yield. Thus, there was no significant difference in the yield of mature oocytes after IVM between cancer patients and patients with fertility problems. 15.5% (7/45) GV oocytes in cancer patients and 12.2% (9/74) GV oocytes in patients with fertility problems degener- ated before in vitro maturation procedure. Results of ICSI of in vitro matured oocytes in patients with fertility problems In vitro fertilization of 49 oocytes in 17 patients with fertility problems (average age 34.3 ± 4.4 years) was performed by ICSI with partner's semen (in 2 men oligozoospermia and 15 men teratozoospermia). After performing this method, 27 (55.1%) oocytes were fertilized (expressing two pronuclei and two polar bodies) and 23 (85.2%) fertilized oocytes (zy- gotes) further developed into an embryo, as shown in Table 3; four zygotes did not cleave further. Good quality embryos were vitrified and stored in liquid nitrogen for future clinical use in patients (transfer into the uterus). TABLE 1. Differences in the number, quality and immaturity of oocytes after controlled hormonal stimulation of the ovaries in cancer patients compared to patients with fertility problems Cancer patients (n = 18) Patients with fertility problems (n = 21) Age (years) 30.3 ± 6.3 33.4 ± 5.0 Number of retrieved oocytes 198 259 Oocytes per patient 11.0 ± 9.0 12.3 ± 9.2 Number of degenerated oocytes 17 (8.6%) 17 (6.5%) Number of immature (MI + GV) oocytes 59 (30.0%)* 113 (43.6%) * Number of immature GV oocytes 45 (23.0%) 74 (28.6%) * = statistically significant difference (P = 0.0064) revealed by Fisher's exact test; significance was set at P < 0.05; GV = germinal vesicle; MI = metaphase I (oocyte meiosis) TABLE 2. Numbers and percentages of oocytes that matured in vitro in patients with different cancers compared to patients with fertility problems Number of oocytes that underwent in vitro maturation Number of oocytes that matured in vitro All cancer patients (n = 18) 38 / 45 25 (1.39 ± 1.04 per patient)* (66.0%) Patients with breast cancer (n = 8) 22 12 (54.5%) Patients with other cancers (n = 10) 16 13 (81.2%) Patients with fertility problems (n = 21) 65 / 74 52 (2.48 ± 1.83 per patient)* (80.0%) * = statistically significant difference (P < 0.05; Mann-Whitney U test) After ICSI, the fertilization and cleavage rates of 49 oocytes that matured in vitro were compared with 121 oocytes of the same patients that matured in vivo and were aspirated from their ovaries as mature oocytes (metaphase II [MII] oocyte meiosis) in the same cycle of controlled ovarian hormonal stimulation. Of the 121 oocytes obtained as mature oocytes, 69 oocytes were fertilized, representing a fertilization rate of 57.0%. Sixty-one fertilized oo- cytes further developed into an embryo (Table 3). Fisher's exact test revealed no statistical differ- ences in the proportions of fertilized oocytes, non- cleaved zygotes, and embryos obtained by ICSI on in vitro and in vivo matured oocytes (Table 3). Radiol Oncol 2022; 56(1): 119-128. Virant-Klun I et al. / In vitro maturation of immature oocytes for fertility preservation124 In patients with an increased proportion of im- mature (GV) oocytes (≥ 31%), there was a tendency for a lower proportion of fertilized oocytes and a higher proportion of non-cleaved zygotes, but the differences were not statistically significant (Table 4). Discussion The results of this research show that cancer and control healthy patients with fertility problems did not differ in the number and quality of oocytes after controlled hormonal stimulation of their ova- ries, which is positive. In cancer patients, there was even a significantly lower proportion of immature oocytes than in patients with fertility problems. However, in patients with cancer, fewer oocytes per patient matured in vitro than in patients with fertility problems (1.39 ± 1.04 vs. 2.48 ± 1.83, P < 0.05). Following ICSI of oocytes in patients with fertility problems, the fertilization and embryo cleavage rates were approximately the same in oo- cytes that matured in vitro and in vivo in the same patients, in the same cycles of controlled hormonal stimulation of the ovaries. This is also to be expect- ed in cancer patients. The proportion of mature, MII oocytes in the patients with fertility problems included in this research was relatively low (56.4%) compared to the internationally accepted reference value of 70- 80%28, because we included mainly patients with a higher proportion of immature oocytes which did not reflect the average condition; in cancer pa- tients, the proportion of mature oocytes was higher (70%) and within the reference value.28 The num- ber and quality of oocytes in cancer patients did not differ between different cancers and from con- trol patients with fertility problems. The same has been found in other studies for different types of cancer such as breast cancer, lymphoma, gliomas and other cancers.29,30 For breast cancer, the results of various studies are otherwise contradictory. In a study by Malacarne et al., as in our study, the aver- age number of oocytes obtained per breast cancer patient after ovarian stimulation did not differ sig- nificantly from healthy control women including oocyte donors, women with fertility preservation for non-medical reasons, and female partners of in- fertile men in an IVF program31; it was concluded that patients with breast cancer undergoing con- trolled ovarian hormonal stimulation for fertility preservation can expect the ovarian response pre- dicted for their age. The results obtained by differ- ent studies do not support the notion of a negative impact of the breast cancer gene 1/2 (BRCA1/2) mutation on the ovarian response of women with breast cancer.31-33 Nevertheless, the results of some other studies suggest the reduced number and ma- turity of oocytes obtained for cryostorage in pa- tients with breast cancer34,which may be attributed to the higher grade of cancer35 or different expres- sion of hormonal receptors.36 There is little data in the literature on how dif- ferent cancers affect the oocyte IVM in cancer pa- tients. The oocyte IVM rate in breast cancer patients was found to be approximately 53.2 to 64.2% in the TABLE 3. Non-significant differences in fertilized oocytes, non-cleaved zygotes, and cleavage embryos obtained by intracytoplasmic sperm injection (ICSI) on in vitro matured and in vivo matured oocytes of patients with fertility problems (in the same cycles) ICSI cycles (n = 17) In vitro matured oocytes In vivo matured oocytes* Number of microinjected oocytes 49 121 Fertilized oocytes 27 (55.1%) 69 (57.0%) Non-cleaved zygotes 4 (15.0%) 8 (11.6%) Cleavage embryos 23 (85.2%) 61 (88.4%) * = non-significant differences, as revealed by Fisher's exact test TABLE 4. Non-significant differences in results of intracytoplasmic sperm injection (ICSI) cycles (fertilized oocytes, non-cleaved zygotes, cleavage embryos) on in vitro matured oocytes of patients with fertility problems regarding the number of immature (germinal vesicle [GV]) oocytes ICSI cycles (n = 17) ≤ 30% GV oocytes ≥ 31% GV oocytes Female age (years) 34.4 ± 3.0 34.3 ± 5.6 Number of microinjected oocytes 16 33 Fertilized oocytes 11 (69.0%) 16 (48.5%) Non-cleaved zygotes 0 (0%) 4 (25.0%) Cleavage embryos 11 (100%) 12 (75%) Non-significant differences, as revealed by Fisher's exact test Radiol Oncol 2022; 56(1): 119-128. Virant-Klun I et al. / In vitro maturation of immature oocytes for fertility preservation 125 study of Shalom Paz et al.37, which is very similar to our study (54.5%), or slightly higher – 62.0%, 66.0% or 66.7% in some other studies.22,32 In this study, the proportion of immature oocytes matured in vitro tended to be lower in breast cancer patients (54.5%) than in patients with fertility problems (80.0%), while this was not observed in patients with other cancers (81.2%). Although, the differ- ence was not significant, possibly due to relatively low number of patients and oocytes included. In a study conducted by Liu et al., 811 genes were iden- tified that were expressed differently in malignant breast tissue compared to healthy breast tissue38; among the up-regulated genes was also a group of genes involved in the cell cycle and progesterone- mediated oocyte maturation. For cancer patients, Cohen et al. found that the mean oocyte maturation rate in stimulated IVF cycles was 38%39, which is significantly lower than in our study (66.0%; 54.5% in breast cancer and 81.0% in other cancers). In our study, IVM of oocytes in coculture with cumulus cells from mature oocytes in the same patients may have been beneficial, at least in part providing an ovarian niche.26 Also Chatroudi et al. found that cu- mulus cell supplementation in IVM culture media enhances the viability of human embryos (blas- tocysts) after IVF.40 The rates of in vitro matured oocytes in cancer patients and patients with fertil- ity problems in our study were very similar to the published rates of IVM of immature oocytes in the usual IVF program, where 65.0%, 68.7%, 68.9% and 69.7% maturation rates were obtained in different maturation media.41-43 Oktay et al. reported the 45% increase in mature oocyte yield after IVM of imma- ture oocytes after controlled hormonal stimulation of the ovaries in breast cancer patients and a high fertilization rate of these oocytes.44 Moreover, an IVM of oocytes retrieved without hormonal stim- ulation of the ovaries was considered for fertility preservation in breast cancer patients to avoid the ovarian stimulation, shorten the time to oocyte re- trieval, and not to increase both the serum estradiol level and delay in cancer treatment.45-47 It should be noted that our study was limited to a relatively small number of patients involved and a small number of oocytes. In cancer patients and patients with fertility problems, we tried to per- form as comparable controlled hormonal stimula- tion of the ovaries as possible using a GnRH an- tagonist. Nevertheless, we also had to take certain safety precautions in cancer patients. In these pa- tients, the ovarian hormonal stimulation was initi- ated immediately after they have been sent to our department, no matter of the cycle phase to be fast and prevent further progression of disease. The oo- cyte maturation in cancer patients was initiated by GnRH analogue to prevent hyperstimulation, but more patients with less than 10 follicles were also treated with Ovitrelle similar to patients with fer- tility problems. Thus, in most patients, oocyte mat- uration was triggered by Ovitrelle. If we used ex- actly the same method of hormonal ovarian stimu- lation and triggering oocyte maturation in cancer patients and patients with fertility problems, there might be more immature oocytes in cancer pa- tients, but this was not possible for cancer-related safety reasons. In addition, for safety reasons, breast cancer patients were also treated with an aromatase in- hibitor, letrozole, to prevent an increase in estra- diol and worsening of the disease. Thus, based on our own experience and literature48,49, we believe that both random start of hormonal stimulation in cancer patients and use of aromatase inhibitor in patients with breast cancer do not affect the num- ber, maturity and in vitro maturation of oocytes ob- tained in these patients. Letrozole treatment may also increase the in- traovarian androgen levels, which have a negative impact on granulosa cells (apoptosis) in the late an- tral and pre-ovulatory follicles.50 In this research, granulosa (cumulus) cells were used in co-culture for oocyte maturation, which may lower the matu- ration rate. However, we performed in vitro matu- ration of oocytes with cumulus cells of mature oo- cytes because our previous work showed that co- culture with cumulus cells does not affect the pro- portion of in vitro matured oocytes, but improves the molecular status of oocytes (gene expression profile) compared to oocytes matured in vivo.26 In patients with fertility problems, we deter- mined the FSH, LH and AMH levels in early fol- licular phase of the cycle as well as the number of antral follicles. For cancer patients, we have no such data. Only informative ovarian scan with an- tral follicle estimation was performed at the begin- ning of ovarian stimulation. In our study, approximately the same propor- tion of oocytes were fertilized and further cleaved into an embryo after ICSI of in vitro and in vivo ma- tured oocytes. Some studies have shown poorer embryo development and live birth rates with in vitro matured oocytes51,52, which could be linked to structural and morphologic differences in human oocytes after IVM53 due to suboptimal maturation medium and lack of ovarian niche. It needs to be point out that in our study, oocyte IVM was per- formed in coculture with cumulus cells from ma- Radiol Oncol 2022; 56(1): 119-128. Virant-Klun I et al. / In vitro maturation of immature oocytes for fertility preservation126 ture oocytes of the same patients thus providing a degree of ovarian niche. In spite of a lower rate of good-quality embryos and different developmen- tal dynamics of embryos, pregnancy rates as well as live births did not necessarily differ after oocyte IVM, as found by Roesner et al.54 Moreover, in a matched setting between IVM and IVF babies born from women with polycystic ovaries, no significant increased risk associated with IVM has been iden- tified in 2-year-old singletons born after IVM and after a mean follow-up up to 7.5 years.55,56 In gen- eral, more studies are urgently required to improve IVM –vitrification method to successfully preserve oocytes collected from cancer patients.57,58 Conclusions We may conclude that ‘rescue’ of immature oo- cytes with IVM is a useful strategy to improve the mature oocyte yield of fertility preservation cycles in cancer patients. Immature oocytes retrieved dur- ing oocyte and also embryo cryopreservation cy- cles in cancer patients should not be discarded in order to improve the future potential of pregnancy in these patients. Their immature oocytes can ma- ture in vitro comparable to healthy controls. After ICSI, approximately the same proportion of in vitro matured oocytes could be fertilized and developed into an embryo as in oocytes matured in vivo. Acknowledgments We would like to thank all colleagues, gynecolo- gists, embryologists, and nurses at the Department of Gynecology and Obstetrics, Reproductive Unit (IVF), who in any way helped in this work. We also thank all the patients who kindly donated imma- ture oocytes for this research. Last but not least, we would also like to thank our institution, University Medical Centre Ljubljana, which funded this re- search as part of a tertiary research project. References 1. Yeo W, Pang E, Liem GS, Suen JJS, Ng RYW, Yip CCH, et al. 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Front Endocrinol 2019; 10: 464. doi: 10.3389/ fendo.2019.00464 58. Shirasawa H, Terada Y. In vitro maturation of human immature oocytes for fertility preservation and research material. Reprod Med Biol 2017; 16: 258- 67. doi: 10.1002/rmb2.12042 Slovenian abstracts Radiol Oncol 2022; 56(1): I-XIV. I Radiol Oncol 2022; 56(1): 1-13. doi: 10.2478/raon-2022-0002 Genska terapija raka postane viralna. Vzpon platform virusnih vektorjev Bezeljak U Izhodišča. Zdravljenje raka z virusnimi vektorji je obetalo revolucijo v onkologiji vse od vzpona vektorske genske terapije v 90. letih prejšnjega stoletja. Vektorji, ki temeljijo na virusnih delcih, ponujajo edinstveno kombinacijo učinkovite transdukcije tumorja in stimulacijo imunskega sistema za zdravljenje raka. Kljub začetni obetavnosti se zdravljenje z virusnimi vektorji šele sedaj prebija v ospredje. Virusne vektorje upo- rabljamo kot gensko orodje za pripravo celičnih terapevtikov CAR-T, kot cepiva za raka in kot ciljana onkolitična zdravila. Da bi dosegli tako širok nabor področij uporabe smo premagali vrsto preprek – od razumevanja temeljne biologije raka do procesov priprave in načrtovanja vektorjev. Objavljamo pre- gled najnovejših dognanj in uporabe virusnih vektorjev pri zdravljenju raka. Izpostavljamo platforme za proizvodnjo virusnih delcev, ki omogočajo splošno uporabo virusnih vektorjev pri genski terapiji raka. Zaključki. Virusni vektorji ponujajo številne priložnosti pri zdravljenju raka. Nedavni napredek v proi- zvodnih platformah vektorjev odpira nove možnosti za varno in učinkovito zdravljenje ter poenostavlja prenos tehnologije iz laboratorija v kliniko. Tehnologija virusnih vektorjev je v zadnjih letih napredovala do stopnje, da lahko postane standardno orodje pri zdravljenju raka. Radiol Oncol 2022; 56(1): 14-22. doi: 10.2478/raon-2022-0004 Ultrazvočno vodeno injiciranje v karpalni kanal Tumpaj T, Potočnik Tumpaj V, Albano D, Snoj Ž Izhodišča. Sindrom karpalnega kanala je ena izmed najpogostejših utesnitvenih mononevropatij, ki predstavlja velik socialno-ekonomski problem zaradi nezmožnosti dela, invalidnosti in podaljšane rehabi- litacije. Ultrazvok je slikovna metoda izbora za potrditev diagnoze ter sledenje bolnikov s sindromom kar- palnega kanala. V zadnjih letih se je za zdravljenje blage do zmerne oblike sindroma karpalnega kanala uveljavilo ultrazvočno vodeno injiciranje v karpalni kanal. S pregledom literature smo strnili zabeležene razlike pri naslednjih korakih: priprava bolnika, pristop vboda, položaj igle, vrsta in količina injiciranih učin- kovin. V literaturi opisujejo tri pristope: ulnarni, radialni in longitudinalni. Vse tri pristope lahko izvedemo z enkratnim ali večkratnim injiciranjem z različnimi količinami učinkovin. Učinkovine, ki jih uporabljamo za izvedbo postopka so kortikosteroidi, lokalni anestetiki, dekstroza, fiziološka raztopina, plazma obogatena s trombociti in progesteron. Zaključki. Soglasja glede optimalnega protokola še ni. Pri pregledu literature se za najbolj najbolj prime- ren pristop kaže ulnarni pristop z enkratnim vbrizganjem, najboljši rezultati pa so bili doseženi pri uporabi dekstroze v večjih količinah. Zaradi terminoloških razlik v literaturi je primerjava protokolov težavna , zato lahko predstavljeni koraki ultrazvočno vodenih injekcij služijo kot vodilo nadaljnjim raziskavam. Slovenian abstracts Radiol Oncol 2022; 56(1): I-XIV. II Radiol Oncol 2022; 56(1): 23-31. doi: 10.2478/raon-2021-0051 Kopičenje amiloida beta ni povezano z lokalnim metabolizmom glukoze pri bolnikih z zgodnjim stadijem Alzheimerjeve bolezni Ehrlich D, Dunzinger A, Malsiner-Walli G, Grün B, Topakian R, Hodolic M, Kainz E, Pichler R Izhodišča. Pri bolnikih z Alzheimerjevo boleznijo so ugotovili nabiranje amiloida beta, kar povzroča disfunkcijo sinaps in nato odmiranje nevronov. Ni še znano, ali količina kopičenja amiloida beta korelira s stopnjo kognitivne prizadetosti. Slikovna diagnostika bi lahko vodila k boljšemu razumevanju vloge amiloida beta pri razvoju kognitivne prizadetosti. Namen pričujoče raziskave je bil preučiti, ali nabiranje amiloida beta v posameznih predelih možganov pri bolnikih z zgodnjo Alzheimerjevo boleznijo sovpada z disfunkcijo nevronov in kognitivno prizadetostjo, kar bi lahko videli z znižanim metabolizmom glukoze. Bolniki in metode. Pri 30 bolnikih z Alzheimerjevo boleznijo in dokazanim nabiranjem amiloida smo izvedli 2-(18F)Fluoro-2-deoksi-d-glukozni (FDG) PET/CT. Izračunali smo povprečno kopičenje (18F)fluteme- tamola (Vizamyal) za 16 področij v vsaki možganski hemisferi in izračunali razmerje vrednosti standardizi- ranega privzema (angl. standardised uptake value ratio; SUVR), tako da smo delili intenziteto nabiranja (18F)flutemetamola z srednjim kopičenjem pozitivnih in negativnih kontrolnih področij. Podatke smo analizirali z okoljem R za statistični in grafični izračun. Rezultati. Nismo ugotovili nobene negativne korelacije med kopičenjem amiloida beta in metabo- lizmom glukoze pri 32 področjih možganov ob obravnavi bolnikov z Alzheimerjevo boleznijo in demen- co. Noben koeficient korelacije ni bil statistično značilno višji od 0, izračunano s pomočjo dvostranske p vrednosti. Zaključki. Regionalno kopičenje amiloida beta ni koreliralo z lokalnim metabolizmom glu koze pri bol- nikih z zgodnjim stadijem Alzheimerjeve bolezni. Rezultati raziskave podpirajo vlogo amiloida beta kot pomembnega biološkega označevalca, vendar ne moremo zaključiti, da je amiloid beta vzrok spreme- njenega metabolizma glukoze in nevronske disfunkcije. Slovenian abstracts Radiol Oncol 2022; 56(1): I-XIV. III Radiol Oncol 2022; 56(1): 32-36. doi: 10.2478/raon-2021-0056 Zanesljivost novih rentgenskih meritvenih tehnik pri kostni utesnitvi komolca Meglič U, Zupanc O Izhodišča. Ugotavljanje lokacije in obsega rentgenskih sprememb pri kostni utesnitvi komolca je ključnega pomena pri oblikovanju ustrezne diagnoze in načrta zdravljenja za tovrstne bolnike. Namen pričujoče raziskave je bil oceniti zanesljivost novih rentgenskih parametrov, sprednjega utesnitvenega kota in zadnjega utesnitvenega kota, pri kostni utesnitvi komolca in ugotoviti ali obstaja povezava med rentgenskimi parametri in klinično oceno. Bolniki in metode. Rentgenske posnetke 60 bolnikov (30 v skupini s kostno utesnitvijo komolca in 30 v skupini brez bolezni) so z 2-tedenskim razmakom dvakrat ocenjevali trije ocenjevalci različnih stopenj usposobljenosti. Zanesljivost med ocenjevalci je bila izračunana s koeficienti korelacije (ICC) s 95-od- stotnim intervalom zaupanja. Korelacijo med rentgenskimi parametri in klinično oceno smo izračunali s Pearsonovim korelacijskim koeficientom. Rezultati. V obeh skupinah je bila zanesljivost merjenja med ocenjevalci dobra. Za obe meritvi ni bilo pomembnih razlik v zanesljivosti meritve med kirurgi specialisti in specializanti. Ugotovljena je bila dobra korelacija med rentgenskimi meritvami in obsegom giba. Zaključki. Meritve sprednjega utesnitvenega kota in zadnjega utesnitvenega kota so pokazale dobro zanesljivost merjenja med ocenjevalci, tako pri slikah bolnikov s kostno utesnitvijo komolca kot pri normal- nih rentgenskih slikah. Dobra zanesljivost ocenjevanja, ki smo jo ugotovili pri specialistih kirurgih in tudi pri specializantih na usposabljanju ter dobra korelacija med rentgenskimi meritvami in kliničnim testiranjem, kaže, da se nove rentgenske meritvene tehnike lahko enostavno in zanesljivo uporabljajo v vsakodnevni praksi. Slovenian abstracts Radiol Oncol 2022; 56(1): I-XIV. IV Radiol Oncol 2022; 56(1): 37-45. doi: 10.2478/raon-2022-0005 Učinkovitost transvaginalnega ultrazvoka v primerjavi z magnetno resonančnim slikanjem za predoperativno oceno invazije miometrija pri bolnicah z endometrioidnim rakom endometrija. Prospektivna primerjalna raziskava Cerovac A, Ljuca D, Arnautalić L, Habek D, Bogdanović G, Mustedanagić-Mujanović J, Grgić G Izhodišča. Primerjali smo natančnost predoperativnega transvaginalnega ultrazvoka (TVUS) in slikanja z magnetno resonanco (MRI) za oceno invazije miometrija pri bolnicah z rakom endometrija. Dokončna histopatološka diagnoza je služila kot referenčna metoda. Bolniki in metode. Raziskavo smo izvedli v terciarnem centru v obdobju 2019 do 2021 in vanjo vključili bolnice s histopatološko dokazanim rakom endometrija, ki smo jih hospitalizirali zaradi načrtovane opera- cije. Invazijo miometrija smo ocenili s TVUS in MRI pred kirurško določitvijo stadija. Uporabili smo dve objek- tivni metodi TVUS (Gordonovo in Karlssonovo metodo) in MRI. Bolnike smo po operaciji in histopatološki oceni invazije miometrija razdelili v dve skupini: površinsko (≤ 50 % invazije) in globoko (> 50 % invazije). Rezultati. Za študijsko obravnavo je bilo primernih 60 bolnic. Histopatološko je bilo v raziskavi 34 (56,7 %) bolnic z invazije miometrija ≤ 50 % in 26 (43,3 %) bolnic z invazije miometrija > 50 %. Obe slikovni metodi, TVUS in MRI, nista pokazali statistično pomembnih razlik pri predoperativni oceni invazije mio- metrija. Koeficient skladnosti med metodami TVUS in MRI ter histopatološko je bil statistično pomemben (p < 0,001). Gordonova metoda za izračun invazije miometrija je dosegla pozitivno napovedno vrednost (PNV) 83 %, negativno napovedno vrednost (NNV) 83 %, občutljivost 77 %, specifičnost 88 % in splošno natančnost 83 %; Karlssonova metoda je dosegla PNV 82 %, NNV 79 %, občutljivost 69 %, specifičnost 88 % in splošno natančnost 80 %. MRI pa je dosegla PNV 83 %, NNV 97 %, občutljivost 97 %, specifičnost 85 % in splošno natančnost 90 %. Zaključki. V raziskavi je bila ocena invazije miometrija izvedena s TVUS primerljiva z natančnostjo MRI pri bolnicah z rakom endometrija. Slovenian abstracts Radiol Oncol 2022; 56(1): I-XIV. V Radiol Oncol 2022; 56(1): 46-53. doi: 10.2478/raon-2021-0055 Paragangliom sečnega mehurja. Značilnosti računalniškotomografskega in magnetnoresonančnega slikanja pri 16 bolnikih Zhang J, Bai X, Yuan J, Zhang X, Xu W, Ye H, Wang H Izhodišča. Paragangliom sečnega mehurja je redek feokromociton izven adrenalne žleze. Pogosto povzroča različne simptome, ki jih lahko napačno interpretiramo in zato neustrezno zdravimo. Namen raziskave je bil, opredeliti radiološke značilnosti paraganglioma z uporabo računalniškotomografskega (CT) in magnetnoresonančnega (MR) slikanja. Bolniki in metode. Retrospektivno smo proučevali bolnike, ki smo jim diagnosticirali paragangliom seč- nega mehurja v obdobju od oktobra 2009 do oktobra 2017 in smo jim pred kirurškim posegom naredili CT ali MR slikanje. Analizirali smo klinične značilnosti, ter značilnosti njihove CT in MR diagnostike. Rezultati. V raziskavo smo vključili 16 bolnikov s 16 tumorji sečnega mehurja (srednja starost 51 let, 9 žensk). Pri 13 bolnikih smo naredili slikanje s CT in pri 8 z MR. Velikost tumorjev je bila 1,6–5,4 cm. Večina tumorjev je vraščala v mehur (n = 11) z ovalno obliko (n = 10) in dobro omejenim robom (n = 14). Pri dveh bolnikih smo opazili intratumorsko cistično degeneracijo ali nekrozo in tudi periferno tkivno invazijo, kar je nakazovalo na maligni paragangliom. Vseh 13 lezij, diagnosticiranih s CT slikanjem, je kazalo rahlo hipoatenuacijo signala in srednjo do izrazito ojačenje signala. V primerjavi z m. gluteus maximus, so vse lezije imele rahlo ojačitev T2 poudarjenega slikanja, hiperintenzivnost difuzijsko poudarjenega slikanja in hipointenzivnost na različnih slikah difuzijske konstante, hiperintenzivnost na T1 poudarjenih slikah in ‚‘hitro in upočasnjeno‘‘ (angl. ‚‘fast and slow out‘‘) ojačenega signala na MR slikah. Zaključki. Paragangliomi so največkrat ovalne oblike, široko razraščeni in dobro ožiljeni tumorji z hipo- atenuacijo na slikah CT-ja brez kontrasta, s hiperintenzivnostjo na T2 poudarjenih slikah, rahlo hiperinten- zivni na T1 poudarjenih slikah in v primerjavi z mišičnim tkivom izrazito omejen difuziji poudarek slikanja. Periferna tkivna invazija tumorja je zelo sumljiva za maligni paragangliom. Vse te karakteristike lahko pomagajo pri predoperativnem odločanju in načrtovanju operacije. Slovenian abstracts Radiol Oncol 2022; 56(1): I-XIV. VI Radiol Oncol 2022; 56(1): 54-59. doi: 10.2478/raon-2021-0052 Diagnostična učinkovitost vrednosti dejanskega difuzijskega koeficienta za diferenciacijo med intrahepatičnim holangiokarcinomom in jetrnimi metastazami adenokarcinoma prebavil Yilmaz TF, Gultekin MA, Turk HM, Besiroglu M, Cesme DH, Simsek M, Alkan A, Toprak H Izhodišča. Želeli smo raziskati, ali obstaja razlika med intrahepatičnim holangiokarcinomom in jetrnimi metastazami adenokarcinoma prebavil glede na vrednosti dejanskega difuzijskega koeficienta (angl. apparent diffusion coefficient, ADC). Bolniki in metode. Med januarjem 2018 in januarjem 2020 smo retrospektivno pregledali zdravstveno dokumentacijo 13 bolnikov z intrahepatičnim holangiokarcinomom in 64 bolnikov z metastazami v jetrih zaradi adenokarcinoma prebavil. Po upoštevanju izključitvenih kriterijev smo v raziskavo vključili 10 bol- nikov z intrahepatičnim holangiokarcinomom in 53 bolnikov z metastazami adenokarcinoma prebavil (53 metastaz) ter na ta način hkrati bolnike razdelili v dve skupini. Za povprečno vrednost dejanskega difuzijskega koeficienta (angl. ADCmean) smo področje zanimanja (angl. region of interes, ROI) postavili v solidni del lezij. Primerjali smo povprečne vrednosti ADC obeh skupin. Rezultati. Povprečna starost skupine z intrahepatičnim holangiokarcinomom je bila 62,50 ± 13,49 let, skupine z metastazami pa 61,15 ± 9,18 let. Povprečne vrednosti ADC so bile v skupini z intrahepatičnim holangiokarcinomom značilno višje v primerjavi z skupino z metastazami (p < 0,001). Metoda s krivuljami ROC je pokazala visoko diagnostično natančnost (območje pod krivuljo [AUC] = 0,879), z mejno vredno- stjo < 1178 x 10–6 mm2/s za povprečno vrednost ADC (senzitivnost = 90,57; specifičnost = 70,0; pozitivna napovedna vrednost [PPV] = 94,1; negativna napovedna vrednost [NPV] = 58,3) pri razlikovanju intrahe- patičnega holangiokarcinoma od metastaz adenokarcinoma prebavil. Zaključki. Rezultati pričujoče raziskave kažejo, da imajo vrednosti ADC potencialno vlogo pri diferen- ciaciji med intrahepatičnim holangiokarcinomom in jetrnimi metastazami adenokarcinoma prebavil, kar je lahko pomembno pri nadaljnji obravnavi bolnikov. Slovenian abstracts Radiol Oncol 2022; 56(1): I-XIV. VII Radiol Oncol 2022; 56(1): 60-68. doi: 10.2478/raon-2022-0001 Ocena učinkovitosti zdravljenja žlez slinavk z beleženjem T2 pri bolnikih, ki so bili zdravljeni s hiperbarično oksigenacijo in so bili predhodno obsevani zaradi tumorjev glave in vratu Vidmar J, Cankar K, Grošelj M, Finderle Ž, Serša I Izhodišča. V raziskavi smo analizirali vpliv terapevtske hiperbarične oksigenacije (HBO) na funkcijo žlez slinavk po zaključeni radioterapiji pri bolnikih s tumorji glave in vratu. Bolniki in metode. Učinek terapije HBO smo vrednotili s pomočjo slikanja z magnetno resonanco (MRI) žlez slinavk z uporabo metode kartiranja karakterističnega časa transverzalne relaksacije (relaksacijske- ga časa) T2 na kliničnem sistemu jakosti 3T. V raziskavo smo vključili 18 bolnikov s prejeto dozo sevanja od 50 do 80 Gy ter 18 kontrolnih oseb, uravnoteženih po spolu in starosti. Izhodiščne meritve so bile opravlje- ne pred prvo terapijo HBO (40,2 ± 20 mesecev po radioterapiji), nato pa po prejetih 20 dnevnih terapijah HBO pri 2,5 absolutne atmosfere (ATA), pri katerih so bolniki vsak dan 90 minut vdihovali 100% kisik. Poleg meritev MRI smo zabeležili tudi pretok, kapaciteto pufra in pH sline. Rezultati. Povprečne vrednosti T2 na obsevani strani so se ob koncu zdravljenja s HBO zmanjšale s 121 ± 20 ms na 113 ± 16 msec (p = 0,002), medtem ko na kontralateralni strani ni bilo opaziti statistično značilnih sprememb. Analiza je dodatno pokazala negativen Pearsonov korelacijski koeficient med povprečno vrednostjo izmerjenih T2 vrednosti v parotidni žlezi na obsevani strani in nestimuliranim pH sline (R = -0,647, p = 0,004) ter stimuliranim pretokom sline (R = -0,592, p = 0,01). Zaključki. Z raziskavo smo potrdili hipotezo, da je s kartiranjem relaksacijskega časa T2 žlez slinavk mo- goče precej zanesljivo kvantitativno in indirektno opredeliti odziv žlez slinavk na zdravljenje s HBO ter je zato takšna metoda lahko komplementarno diagnostično orodje za oceno funkcije žlez slinavk pri po- obsevanih bolnikih s hiposalivacijo. Slovenian abstracts Radiol Oncol 2022; 56(1): I-XIV. VIII Radiol Oncol 2022; 56(1): 69-75. doi: 10.2478/raon-2021-0047 Primerjava lokalnih recidivov jeternoceličnega raka zdravljenega s kemoembolizacijo in kopičenja jodiranega olja v koronskem delu tumorja v kratkotrajnem opazovanem obdobju Watanabe Y, Ogawa M, Kaneko M, Kumagawa M, Hirayama M, Matsumoto N, Nakagawara H, Yamamoto Y, Moriyama M Izhodišča. Lokalne ponovitve jeternoceličnega raka so pogoste v žilnem povirju jeter. Sij okoli tumorja (koronski del tumorja) vidimo pri računalniški tomografiji med arteriografijo in naj bi predstavljal področje drenaže tumorja. Namen raziskave je bil raziskati povezavo med embolizacijo tega sija okoli tumorja in ponovitvami bolezni jetnoceličnega raka, ki smo ga zdravili z transkatetersko arterijsko kemoembolizacijo (TACE). Bolniki in metode. V retrospektivno raziskavo smo vključili 52 bolnikov s 60 lezijami jeternoceličnega raka, ki so kazali koronski sij okoli tumorja v pozni fazi računalniško tomografske arteriografije in so homo- geno kopičili jodirano olje . Opazovane lezije so bile vidne na slikah računalniške tomografije brez kontra- sta takoj po TACE. Razdelili smo jih v dve skupini: (A) tiste, kjer se je jodirano olje kopičilo in (B) tiste, kjer se jodirano olje ni kopičilo po celotnem predelu koronskega sija. Ocenjevali smo število lokalnih ponovitev bolezni. Rezultati. Stopnja ponovitev bolezni je bila v skupini A (n = 36) 2,8 %, 2,8 % in 8,3 % po 3, 6 in 12 mesecih po posegu, medtem ko je bila v skupini B 20,8 %, 45,8 % in 75 % po 3, 6, in 12 mesecih po posegu. Stopnja ponovitev bolezni je tako bila v skupini A statistično značilno nižja kot v skupini B (razmerje obetov [HR] 0,079; 95 % interval zaupanja [CI] 0,026–0,24; p < 0.001). Zaključki. Rezultati nakazujejo, da je koronski sij natančen varnostni rob pri TACE ter ga je potrebno upoštevati in embolizirati celotno njegovo površino. Slovenian abstracts Radiol Oncol 2022; 56(1): I-XIV. IX Radiol Oncol 2022; 56(1): 76-82. doi: 10.2478/raon-2021-0036 Ocena tveganja za ponovitev bolezni pri bolnicah z rakom endometrija. Razlike v izidih molekularnih in kliničnih klasifikacij pri slovenski skupini bolnic Knez J, Sobočan M, Belak U, Kavalar R, Zupin M, Büdefeld T, Potočnik U, Takač I Izhodišča. Cilj raziskave je bil primerjati klinično oceno tveganja za ponovitev raka endometrija z inte- griranim profiliranjem molekularnega tveganja. Bolniki in metode. V prospektivno raziskavo smo k sodelovanju povabili bolnice s histološko dokazanim rakom endometrija, ki so bile zdravljene v UKC Maribor med januarjem 2020 in februarjem 2021. Klinične podatke smo ocenili in razvrstili v skladu s trenutno veljavnimi smernicami o zdravljenju raka endometrija Evropskega združenja za ginekološko onkologijo, Evropskega združenja za radioterapijo in onkologijo ter Evropskega združenja za patologijo (ESGO/ESTRO/ESP). Molekularna karakterizacija tumorjev je vključe- vala določitev statusa mutacij DNA polimeraze epsilon (POLE) s sekvenciranjem po Sangerju ter imuno- histokemično oceno prisotnosti pomanjkljivosti neujemanja proteinov popravljanja DNA (MMRd) in p53 izraženosti (p53abn). Rezultati. V raziskavo smo vključili 45 bolnic z rakom endometrija. V skupino z nespecifičnim mutacijskim profilom (NSMP) smo uvrstili 22 bolnic (56,4 %), v skupino z visoko verjetnostjo MMRd pa 13 (33,3 %), v skupino p53abn smo uvrstili tri (7,7 %) in v skupino z mutacijo POLE eno bolnico (2,6 %). Več molekularnih označevalcev je imelo šest bolnic (15,4 %). To skupino bolnic smo proučevali ločeno in niso bile vključene v primarno oceno tveganja. Glede na klinično ocena smo med nizko tvegane uvrstili 21 bolnic (53,8 %), v skupino z zmernim tveganjem pa pet (12,8 %), v skupino z zmerno-visokim tveganjem 2 (5,1 %), visoko tveganih smo opredelili 10 (25,6 %) in v skupino napredovalih metastatskih bolezni smo uvrstili eno bolnico (2,6 %). Integrirana molekularna klasifikacija je spremenila tveganje pri 4 bolnicah (10,3 %). Zaključki. Integrirana molekularna ocena tveganja za ponovitev bolezni izboljša invidualizirano oceno tveganja pri raku endometrija in bi lahko izboljšala terapevtsko natančnost. Večjo pozornost je potrebno dodatno nameniti skupini NSMP pri prepoznavi dodatnih molekularnih bioloških označevalcev za boljšo individualizacijo zdravljenja. Slovenian abstracts Radiol Oncol 2022; 56(1): I-XIV. X Radiol Oncol 2022; 56(1): 83-91. doi: 10.2478/raon-2021-0049 Cistatin C in cistatin SN kot možna tumorska označevalca za uvealni maligni melanom Dikovskaya MA, Russkikh GS, Loktev, Johnston TP, Gevorgyan MM, Voronina NP, Chernykh VV, Trunov AN, Korolenko TA Izhodišča. Namen raziskave je bil opredeliti endogene koncentracije cistatinov C in SN kot potencialne tumorske označevalce v serumu in bioloških tekočinah očesa pri bolnikih z uvealnim melanomom in pri zdravih ljudeh. Bolniki in metode. Koncentracije obeh statinov smo določali v intraokularni tekočini, solzah in serumu bolnikov z uvealnim melanomom ter jih primerjali s kontrolnimi vrednostmi v intraokularni tekočini, solzah, serumu, cerebralni tekočini, slini in urinu zdravih ljudi. Results. Koncentracije cistatina C so bile veliko višje kot cistatina SN pri zdravih ljudeh ter so bile neod- visne od spola bolnikov. Prav tako so bile koncentracije cistatina C statistično značilno povišane v solzah prizadetega očesa, kot tudi kontralateralnega očesa pri bolnikih v primerjavi z zdravimi ljudmi ter neod- visne od velikosti tumorja. Koncentracije cistatina SN pa so bile znižane v intraokularni tekočini bolnikov z uvealnim melanomom v primerjavi z zdravimi ljudmi. Zaključki. Rezultati nakazujejo, da razmerje med cistatinoma (CysC : CysSN), tako v serumu, kot v sol- zah ter tudi koncentracija cistatina SN v intraokularni tekočini kažejo na prisotnost uvelanega melanoma. To nakazuje na potrebo po večplastni diagnostiki bolnikov s sumom na uvelani melanom, kot so določe- vanje cistatina C in cistatina SN v serumu, solzah, intraokularni tekočini, bazi očesa kot tudi opredelitev sprememb z ultrazvokom in histopatološko. Slovenian abstracts Radiol Oncol 2022; 56(1): I-XIV. XI Radiol Oncol 2022; 56(1): 92-101. doi: 10.2478/raon-2021-0050 Kliničen pomen sprememb v številu kopij genov, povezanih z diferenciacijo celic B in uravnavanjem celičnega cikla, pri pediatrični B-celični akutni limfoblastni levkemiji. Izkušnje terciarnega centra Črepinšek K, Marinšek G, Kavčič M, Prelog T, Kitanovski L, Jazbec J, Debeljak M Izhodišča. Delecije v genu IKZF1 predstavljajo slab napovedni dejavnik pri pediatričnih bolnikih z B-celično akutno limfoblastno levkemijo (B-ALL), zlasti ob prisotnosti dodatnih delecij v drugih genih (profil IKZF1plus). Namen raziskave je bil določiti pri slovenskih pediatričnih bolnikih z B-ALL pogostost delecij v genu IKZF1 ter delecij v drugih genih, povezanih z diferenciacijo celic B in z nadzorovanjem celičnega cikla. Zato smo raziskovali tudi gene PAX5, ETV6, RB1, BTG1, EBF1, CDKN2A/2B in pet genov v regiji PAR1. Želeli smo ugotoviti njihov napovedni vpliv na potek bolezni. Bolniki in metode. Preučili smo kohorto 99 bolnikov, ki smo jim ugotovili B-ALL med januarjem 2012 in decembrom 2020, ter jih zdravili po protokolu ALL IC-BFM 2009. Analizirali smo 88 vzorcev kostnega mozga ali periferne krvi in ugotavljali prisotnosti sprememb števila kopij genov z uporabo reagenčnega kompleta SALSA MLPA P335 ALL-IKZF1. Rezultati. Pri več kot 65 % analiziranih vzorcev smo odkrili vsaj eno spremembo v številu kopij genov. Najpogostejše so bile spremembe v genih PAX5 in CDKN2A/2B (30,7 %, 26,1 % in 25,0 %). Delecije v genu IKZF1 so bile prisotne pri 18,2 % analiziranih vzorcev in so bile povezane s slabšim petletnim preživetjem brez dogodka (54,8 % proti 85,9 %, p = 0,016). Profil IKZF1plus smo prepoznali pri 12,5 % analiziranih vzorcev, ti bolniki pa so imeli slabše petletno preživetje brez dogodka kakor tisti, ki so imeli prisotne delecije le v genu IKZF1, in tisti brez delecij (50,8 % proti 75,0 % proti 85,9 %, p = 0,049). Celokupno preživetje je bilo prav tako slabše pri bolnikih s profilom IKZF1plus, kakor pri bolnikih, ki so imeli prisotne delecije le v genu IKZF1, in tistih brez delecij (petletno celokupno preživetje 76,2 % proti 100 % proti 93,0 %), vendar razlike med skupinami niso bile statistično značilne. Zaključki. Rezultati pričujoče raziskave so v skladu z rezultati, pridobljenimi v obsežnejših kliničnih raziska- vah, v katerih je sodelovalo več inštitucij. Analiza sprememb v številu kopij genov z reagenčnim komple- tom SALSA MLPA je zanesljivo orodje za začetno diagnostiko pri otrocih z B-ALL, tudi v manjših ustanovah v državah z nizkim in srednjim dohodkom. Slovenian abstracts Radiol Oncol 2022; 56(1): I-XIV. XII Radiol Oncol 2022; 56(1): 102-110. doi: 10.2478/raon-2022-0003 Perkutana elektrokemoterapija primarnih in sekundarnih jetrnih tumorjev. Lokalna kontrola in vpliv na preživetje bolnikov Spallek H, Bischoff P, Zhou W, de Terlizzi F, Jakob F, Kovàcs A Izhodišča. Lokalne ablativne tehnike so terapija izbora za zdravljenje bolnikov z jetrnimi tumorji ali jetr- nimi metastazami. Ena novejših ablativnih tehnik je elektrokemoterapija. Retrospektivno smo pregledali lokalno učinkovitost in varnost zdravljenja ter vpliv na preživetje elektrokemoterapije pri bolnikih z jetrnimi tumorji ali metastazami. Bolniki in metode. V raziskavo smo vključili 18 bolnikov, ki smo jih zdravili z elektrokemoterapijo in so imeli izmerljive jetrne lezije tumorjev različnega izvora, predvsem kolorektalnega raka, raka dojke in jetr- noceličnega raka. Elktrokemoterapijo smo izvedli s perkutanim pristopom, po standardiziranih postopkih, v splošni anesteziji z miorelaksacijo. Plan zdravljenja smo naredili na osnovi predoperativnih slik magnetne resonance (MR). Učinek zdravljenja smo sledili s slikanjem MR, narejenim s kontrastnim sredstvom 1–3 mesece po končanem zdravljenju in za tem 5., 7., 9., 12., 18. mesec, vse do napredovanja bolezni ali smrti bolnika. Rezultati. Po elektrokemoterapiji smo zaznali le blage ali zmerne stranske učinke. Zabeležili smo 85,7 % objektivnih odgovorov tumorjev na elektrokemoterapijo (popolnih odgovorov 61,9 %; delnih odgovorov 23,8 %). Srednji čas preživetja brez napredovanja bolezni je bil 9,0 ± 8,2 mesecev, celokupno preživetje pa 11,3 ± 8,6 mesecev. Najboljši odgovor na zdravljenje je bil pri tumorjih velikosti 3 in 6 cm v premeru (p = 0,0242, p = 0,0297). Učinek elektrokemoterapije je bil neodvisen od lege tumorjev, ali so bili oddaljeni ali blizu vitalnih jetrnih struktur , ali pa so jih obraščali. Preživetje brez napredovanja bolezni in celokupno preživetje sta bila neodvisni od primarne histološke diagnoze tumorjev. Zaključki. Elektrokemoterapija je pomembna pri zdravljenje neresektabilnih jetrnih tumorjev in jetrnih metastaz, ki niso primerni za druge ablativne tehnike. Slovenian abstracts Radiol Oncol 2022; 56(1): I-XIV. XIII Radiol Oncol 2022; 56(1): 111-118. doi: 10.2478/raon-2021-0035 Učna krivulja laparoskopske resekcije jeter, ki upošteva točkovni sistem težavnosti Ivanecz A, Plahuta I, Mencinger M, Peruš I, Magdalenić T, Turk Š, Potrč S Izhodišče. Namen raziskave je bil kvantitativno oceniti kirurgovo učno krivuljo laparoskopske resekcije jeter. Bolniki in metode. Opravili smo retrospektivni pregled prospektivno vodene podatkovne baze laparoskopskih resekcij jeter. Med aprilom 2008 in aprilom 2021 smo s to metodo operirali 171 bolnikov. Za teoretično napoved medoperativnega zapleta smo uporabili točkovni sistem težavnosti, ki so ga vpeljali Halls in sodelavci. Medoperativni zaplet nastopi, če bolnik izgubi kot več kot 775 mL krvi, če so nenamerno poškodovani okolni organi ali če je po- trebno nadaljevati operacijo z odprto metodo. Teoretično zvezo med Hallsovim točkovnim sistemom težavnosti in napovedano verjetnostjo medoperativnega zapleta smo uporabili kot objektivno oceno učnega izida z namenom pridobitve učne krivulje. Rezultati. Pridobljena učna krivulja je bila rezultat trinajstletnega prizadevanja kirurga (AI). V mate- matičnem modelu je bila privzeta aditivna funkcija, sestavljena iz absolutnega in relativnega prispevka (kompleksnosti) k učni krivulji. Absolutni prispevek predstavlja logaritemska funkcija, relativnega pa regresijski polinom pete stopnje. Pridobljena učna krivulja predstavlja funkcijsko odvisnost učnega izida v odvisnosti od časa. Lokalni ekstremi predstavljajo vrhove in doline v učnem procesu kirurga, dokler (končno) ne doseže odličnosti. Zaključki. Učna krivulja prikazuje dolgotrajen učni proces laparoskopske resekcije jeter. Predlagani matematični model je mogoče uporabiti za katerikoli (kirurški) postopek, ki premore točkovni sistem tveganja in teoretično napovedan odnos med njim in objektivnim učnim izidom (npr. medoperativnim zapletom). Slovenian abstracts Radiol Oncol 2022; 56(1): I-XIV. XIV Radiol Oncol 2022; 56(1): 119-128. doi: 10.2478/raon-2021-0053 Zorenje nezrelih jajčnih celic in vitro za ohranjanje plodnosti pri bolnicah z rakom v primerjavi s kontrolnimi bolnicami, ki so imele težave s plodnostjo v programu zunajtelesne oploditve Virant-Klun I, Jure Bedenk J, Jančar N Izhodišča. Namen raziskave je bil ugotoviti, ali je lahko in vitro zorenje nezrelih jajčnih celic, ki smo ga izvedli po nadzorovanem hormonskem spodbujanju jajčnikov, pomembno za izboljšanje možnosti zano- sitve pri bolnicah, ki so zbolele za rakom. Bolniki in metode. Obravnavali smo bolnice, ki so zbolele za rakom in so želele v prihodnosti zano- siti. Po spodbujanju jajčnikov smo primerjali število jajčnih celic, njihovo kakovost in zrelost s kontrolnimi bolnicami, ki so imele težave s plodnostjo in so bile vključene v program zunajtelesne oploditve. V obeh skupinah bolnic smo in vitro dozorevali njihove nezrele jajčne celice v razvojni fazi germinalnega vezikla in primerjali delež jajčnih celic, ki so in vitro dozorele. Pri podskupini bolnic s težavami s plodnostjo smo ne- posredno vnesli semenčice v citoplazmo jajčne celice (angl. intracytoplasmic sperm injection, ICSI) na in vitro dozorelih jajčnih celicah, da bi ocenili njihovo sposobnost oploditve in razvoja v zarodek v primerjavi z in vivo dozorelimi jajčnimi celicami v istih postopkih in ocenili primernost postopka pri bolnicah z rakom. Rezultati. Pri bolnicah z različnimi raki onkološka bolezen ni vplivala na število in kakovost pridobljenih jajčnih celic. Pri bolnicah z rakom je bil delež nezrelih jajčnih celic celo značilno manjši kot pri bolnicah s težavami s plodnostjo (30,0 % proti 43,6 %; P < 0,05). Vendar pa je pri bolnicah z rakom in vitro dozorelo značilno manj jajčnih celic na bolnico kot pri bolnicah s težavami s plodnostjo (1,39 ± 1,04 vs. 2,48 ± 1,83; P < 0,05). Po ICSI se deleži oplojenih jajčnih celic in oplojenih jajčnih celic, ki so se razvile v zarodek, niso razlikovali med jajčnimi celicami, ki so dozorele in vitro ali in vivo v istih postopkih. Zaključki. Metoda in vitro zorenja nezrelih jajčnih celic je zanesljiv postopek za dozorevanje jajčnih celic, ki smo jih pridobili z nadzorovanim spodbujanjem jajčnikov pri bolnicah z rakom. 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 Za lajšanje bolečine in oteklin v ustni votlini in žrelu, ki so posledica radiomukozitisa PR /B SI /B EN /2 02 2/ 01 3 Bistvene informacije iz Povzetka glavnih značilnosti zdravila Tantum Verde 1,5 mg/ml oralno pršilo, raztopina Tantum Verde 3 mg/ml oralno pršilo, raztopina Sestava 1,5 mg/ml: 1 ml raztopine vsebuje 1,5 mg benzidaminijevega klorida, kar ustreza 1,34 mg benzidamina. V enem razpršku je 0,17 ml raztopine. En razpršek vsebuje 0,255 mg benzidaminijevega klorida, kar ustreza 0,2278 mg benzidamina. Sestava 3 mg/ml: 1 ml raztopine vsebuje 3 mg benzidaminijevega klorida, kar ustreza 2,68 mg benzidamina. V enem razpršku je 0,17 ml raztopine. En razpršek vsebuje 0,51 mg benzidaminijevega klorida, kar ustreza 0,4556 mg benzidamina. Terapevtske indikacije: Samozdravljenje: Lajšanje bolečine in oteklin pri vnetju v ustni votlini in žrelu, ki so lahko posledica okužb in stanj po operaciji. Po nasvetu in navodilu zdravnika: Lajšanje bolečine in oteklin v ustni votlini in žrelu, ki so posledica radiomukozitisa. Odmerjanje in način uporabe: Odmerjanje 1,5 mg/ml: Odrasli: 4 do 8 razprškov 2- do 6-krat na dan (vsake 1,5 do 3 ure). Pediatrična populacija: Mladostniki, stari od 12 do 18 let: 4-8 razprškov 2- do 6-krat na dan. Otroci od 6 do 12 let: 4 razprški 2- do 6-krat na dan. Otroci, mlajši od 6 let: 1 razpršek na 4 kg telesne mase; do največ 4 razprške 2- do 6-krat na dan. Odmerjanje 3 mg/ml: Uporaba 2- do 6-krat na dan (vsake 1,5 do 3 ure). Odrasli: 2 do 4 razprški 2- do 6-krat na dan. Pediatrična populacija: Mladostniki, stari od 12 do 18 let: 2 do 4 razprški 2- do 6-krat na dan. Otroci od 6 do 12 let: 2 razprška 2- do 6-krat na dan. Otroci, mlajši od 6 let: 1 razpršek na 8 kg telesne mase; do največ 2 razprška 2- do 6-krat na dan. Starejši bolniki, bolniki z jetrno okvaro in bolniki z ledvično okvaro: Uporabo oralnega pršila z benzidaminijevim kloridom se svetuje pod nadzorom zdravnika. Način uporabe: Za orofaringealno uporabo. Zdravilo se razprši v usta in žrelo. Kontraindikacije: Preobčutljivost na učinkovino ali katero koli pomožno snov. Posebna opozo- rila in previdnostni ukrepi: Če se simptomi v treh dneh ne izboljšajo, se mora bolnik posvetovati z zdravnikom ali zobozdravnikom, kot je primerno. Benzidamin ni priporočljiv za bolnike s preobčutljivostjo nasalicilno kislino ali druga nesteroidna protivnetna zdravila. Pri bolnikih, ki imajo ali so imeli bronhialno astmo, lahko pride do bronhospazma, zato je potrebna previdnost. To zdravilo vsebuje majhne količine etanola (alkohola), in sicer manj kot 100 mg na odmerek. To zdravilo vsebuje metilpar- ahidroksibenzoat (E218). Lahko povzroči alergijske reakcije (lahko zapoznele). Zdravilo z jakostjo 3 mg/ml vsebuje makrogolglicerol hidroksistearat 40. Lahko povzroči želodčne težave in drisko. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Študij medsebojnega delovanja niso izvedli. Nosečnost in dojenje: O uporabi benzidamina pri nosečnicah in doječih ženskah ni zadostnih podatkov. Uporaba zdravila med nosečnostjo in dojenjem ni priporočljiva. Vpliv na sposobnost vožnje in upravljanja strojev: Zdravilo v priporočenem odmerku nima vpliva na sposobnost vožnje in upravljanja strojev. Neželeni učinki: Neznana pogostnost (ni mogoče oceniti iz razpoložljivih podatkov): anafilaktične reakcije, preobčutljivostne reakcije, odrevenelost, laringospazem, suha usta, navzea in bruhanje, angioedem, fotosenzitivnost, pekoč občutek v ustih. Neposredno po uporabi se lahko pojavi občutek odrevenelosti v ustih in v žrelu. Ta učinek se pojavi zaradi načina delovanja zdravila in po kratkem času izgine. Način in režim izdaje zdravila: BRp-Izdaja zdravila je brez recepta v lekarnah in specializiranih prodajalnah. Imetnik dovoljenja za promet: Aziende Chimiche Riunite Angelini Francesco – A.C.R.A.F. S.p.A., Viale Amelia 70, 00181 Rim, Italija Datum zadnje revizije besedila: 14. 10. 2019 Pred svetovanjem ali izdajo preberite celoten Povzetek glavnih značilnosti zdravila. Samo za strokovno javnost. Datum priprave informacije: oktober 2021 Odgovoren za trženje: Bonifar d.o.o. C M Y CM MY CY CMY K Podaljšajmo, kar lahko. Dokazano podaljša celokupno preživetje (OS) na več kot 1 leto (12,6 mesecev VARGATEF® + docetaksel v primerjavi z 10,3 mesecev placebo + docetaksel; HR: 0,83 [95% CI 0,70 – 0,99]; P = 0,0359) pri bolnikih, ki ga prejemajo v kombinaciji z docetakselom, z lokalno napredovalim, metastatskim ali lokalno ponovljivim nedrobnoceličnim pljučnim rakom (non-small cell lung cancer – NSCLC) s histologijo adenokarcinoma po kemoterapiji prve izbire.1,2 NINTEDANIB Boehringer Ingelheim RCV podružnica Ljubljana Šlandrova 4b, 1231 Ljubljana Črnuče Vargatef 100 mg mehke kapsule, Vargatef 150 mg mehke kapsule Sestava: ena mehka kapsula vsebuje 100 mg nintedaniba oz. 150 mg nintedaniba (v obliki esilata). Vsebuje 1,2 mg oz. 1,8 mg sojinega lecitina .Terapevtske indikacije: indicirano v kombinaciji z docetakselom za zdravljenje odraslih bolnikov z lokalno napredovalim, metastatskim ali lokalno ponovljivim nedrobnoceličnim pljučnim rakom (NSCLC) s histologijo adenokarcinoma po kemoterapiji prve izbire. Odmerjanje in način uporabe: zdravljenje mora uvesti in nadzirati zdravnik, ki ima izkušnje z uporabo onkoloških zdravil. Priporočeni odmerek nintedaniba je 200 mg 2x/dan, ki ga je treba jemati v približno 12-urnem razmiku, od 2. do 21. dne standardnega 21-dnevnega cikla zdravljenja z docetakselom. Bolnik ne sme vzeti Vargatefa istega dne, ko prejme kemoterapijo z docetakselom (to je 1. dne). Če bolnik pozabi vzeti priporočeni odmerek nintedaniba, naj ga začne ponovno jemati ob naslednjem načrtovanem času. Posameznih dnevnih priporočenih odmerkov nintedaniba ni dovoljeno povečati, zato da bi nadomestili pozabljene odmerke. Ne smete prekoračiti niti največjega priporočenega dnevnega odmerka 400 mg. Bolniki lahko z zdravljenjem z nintedanibom nadaljujejo po prekinitvi docetaksela, dokler so vidne klinične koristi ali do pojava nesprejemljive toksičnosti. Prilagajanje odmerka: začetni ukrep za obravnavo neželenih učinkov je začasna prekinitev zdravljenja z nintedanibom, dokler specifi čni neželeni učinek ne bo izzvenel do ravni, ki omogoča nadaljevanje zdravljenja (do 1. stopnje ali izhodiščnega stanja). Zdravljenje lahko nadaljujete z zmanjšanim odmerkom; priporočljivo je postopno prilagajanje odmerka po 100 mg na dan (to je zmanjšanje za 50 mg na odmerek) na podlagi individualne varnosti in prenašanja. Kadar neželeni učinki ne izginejo, tj. če bolnik ne prenaša odmerka po 100 mg 2x/dan, je treba zdravljenje trajno ukiniti. V primeru specifi čnih povišanih vrednosti AST/ ALT na > 3 x ULN v povezavi s povečanjem celokupnega bilirubina na ≥ 2 x ULN in ALKP < 2 x ULN je treba zdravljenje prekiniti. Če ni ugotovljen drug razlog, je treba zdravljenje trajno ukiniti. Posebne skupine bolnikov: varnost in učinkovitost pri otrocih, starih 0 do 18 let, še nista dokazani. Pri starejših bolnikih (≥ 65 let) pa na splošno niso opazili razlike. Začetnega odmerka ni treba prilagajati bolnikovi starosti. Podatki o varnosti za črnce in Afroameričane so omejeni. Bolnikom z blago do zmerno ledvično okvaro ali z blago jetrno okvaro začetnega odmerka ni treba prilagajati. Začetnega odmerka pri bolnikih z blago jetrno okvaro (Child Pugh A) na podlagi kliničnih podatkov ni treba prilagajati. Zdravljenje bolnikov z zmerno (Child Pugh B) in hudo (Child Pugh C) jetrno okvaro z Vargatefom ni priporočeno. Kapsule Vargatefa je treba zaužiti cele z vodo, najbolje s hrano; ne sme se jih žvečiti. Kapsule se ne sme odpreti ali drobiti. V primeru stika z vsebino kapsule, je potrebno takoj umiti roke z veliko vode. Kontraindikacije: preobčutljivost za nintedanib, arašide ali sojo ali katerokoli pomožno snov. Previdnostni ukrepi in opozorila: bolezni prebavil (driska, ki tesno sovpada z dajanjem docetaksela; resni primeri driske s posledično dehidracijo in elektrolistkimi motnjami, navzea in bruhanje; zdravljenje je zato včasih treba prekiniti, zmanjšati odmerek ali trajno ukiniti), nevtropenija in sepsa (tudi smrtni primeri; zato je zlasti v kombinaciji s docetakselom potrebno spremljati krvno sliko), delovanje jeter (večja izpostavljenost pri Child Pugh A, zdravljenje pri Child Pugh B ali C pa ni priporočeno, opažene poškodbe jeter (vključno s hudo poškodbo jeter s smrtnim izidom), tveganje za povečanje ravni jetrnih encimov), delovanje ledvic (pozornost ob ledvični okvari/odpovedi), krvavitev (blaga do zmerna epistaksa, večina usodnih krvavitev je bila povezanih s tumorjem. Poročali so o resnih in neresnih krvavitvah (tudi smrtni izid), ki vključujejo prebavila, dihala in organe osrednjega živčnega sistema, najbolj pogoste pa so krvavitve v dihalih. V primeru krvavitve je treba razmisliti o prilagoditvi odmerka, prekinitvi ali trajni ukinitvi zdravljenja na podlagi klinične ocene), terapevtska antikoagulacija, metastaza v možganih (stabilne in aktivne metastaze v možganih), venska trombembolija (povečano tveganje za vensko trombembolijo, vključno s pljučno embolijo in globoko vensko trombozo), arterijski trombembolični dogodki (pri bolnikih z IPF, z večjim srčnožilnim tveganjem, vključno z znano koronarno arterijsko boleznijo), anevrizme in disekcije arterij (pred uvedbo Vargatefa je treba tveganje spodbude nastanka anevrizme in/ali disekcij arterij skrbno preučiti pri bolnikih z hipertenzijo ali anamnezo anevrizme), predrtje prebavil, zelo redko so poročali o primerih nefrotske proteinurije, zapleti s celjenjem ran, vpliv na interval QT, alergijska reakcija (alergija na sojo in arašidove beljakovine), posebne populacije (izpostavljenost se veča z bolnikovo starostjo in obratno korelira s telesno maso, večja pri bolnikih azijske rase). Nosečnice nintedaniba ne smejo uporabljati, razen če je zdravljenje potrebno zaradi njihovega kliničnega stanja. Ženskam je treba svetovati, naj obvestijo zdravnika ali farmacevta, če med zdravljenjem z Vargatefom zanosijo. Interakcije: močni zaviralci P-gp (ketokonazol, eritromicin), močni induktorji P-gp (rifampicin, karbamazepin, fenitoin in šentjanževka), encimi citokroma (CYP). Sočasno dajanje nintedaniba z docetakselom ni spremenilo farmakokinetike nobenega od zdravil v pomembnem obsegu. Sočasno dajanje nintedaniba s peroralnimi hormonskimi kontraceptivi ni pomembno spremenilo farmakokinetike peroralnih hormonskih kontraceptivov. Neželeni učinki: Zelo pogosti: nevtropenija (vključno s febrilno nevtropenijo), zmanjšan apetit, neravnovesje elektrolitov, periferna nevropatija, krvavitev, driska, bruhanje, navzea, trebušna bolečina, povečana vrednost ALT, AST in ALKP, mukozitis (vključno s stomatitisom), izpuščaj in alopecija. Pogosti: febrilna nevtropenija, abscesi, sepsa, trombocitopenija, dehidracija, zmanjšanje telesne mase, glavobol, venska trombembolija, hipertenzija, hiperbilirubinemija, povečana vrednost GGT, pruritus in proteinurija. Občasni: miokardni infarkt, perforacija, pankreatitis, z zdravilom povzročena poškodba jeter in ledvična odpoved. Neznana pogostnost: anevrizme in disekcije arterij, kolitis. Imetnik dovoljenja za promet: Boehringer Ingelheim International GmbH, Binger Str. 173, D-55216 Ingelheim am Rhein, Nemčija. Način in režim izdaje: Rp. Za podrobnejše informacije glejte SPC, z dne 10/2021. Literatura: 1. VARGATEF® Povzetek glavnih značilnosti zdravila 2021 2. Reck M et al. Lancet Oncol. 2014;15:143-55. PC-SL-100772 Samo za strokovno javnost. Datum priprave informacije: februar 2022 V kolikor imate medicinsko vprašanje v povezavi z zdravilom podjetja Boehringer Ingelheim, Podružnica Ljubljana, Vas prosimo, da pokličete na telefonsko številko 01/5864-000 ali pošljete vaše vprašanje na elektronski naslov: medinfo@boehringer-ingelheim.com. Referenca: 1. Keytruda EU SmPC Ime zdravila: KEYTRUDA 25 mg/ml koncentrat za raztopino za infundiranje vsebuje pembrolizumab. Terapevtske indikacije: Zdravilo KEYTRUDA je kot samostojno zdravljenje indicirano za zdravljenje: napredovalega (neoperabilnega ali metastatskega) melanoma pri odraslih; za adjuvantno zdravljenje odraslih z melanomom v stadiju III, ki se je razširil na bezgavke, po popolni kirurški odstranitvi; metastatskega nedrobnoceličnega pljučnega raka (NSCLC) v prvi liniji zdravljenja pri odraslih, ki imajo tumorje z ≥ 50 % izraženostjo PD-L1 (TPS) in brez pozitivnih tumorskih mutacij EGFR ali ALK; lokalno napredovalega ali metastatskega NSCLC pri odraslih, ki imajo tumorje z ≥ 1 % izraženostjo PD-L1 (TPS) in so bili predhodno zdravljeni z vsaj eno shemo kemoterapije, bolniki s pozitivnimi tumorskimi mutacijami EGFR ali ALK so pred prejemom zdravila KEYTRUDA morali prejeti tudi tarčno zdravljenje; odraslih in pediatričnih bolnikov, starih 3 leta ali več, s ponovljenim ali neodzivnim klasičnim Hodgkinovim limfomom (cHL), pri katerih avtologna presaditev matičnih celic (ASCT) ni bila uspešna, ali po najmanj dveh predhodnih zdravljenjih kadar ASCT ne pride v poštev kot možnost zdravljenja; lokalno napredovalega ali metastatskega urotelijskega raka pri odraslih, predhodno zdravljenih s kemoterapijo, ki je vključevala platino; lokalno napredovalega ali metastatskega urotelijskega raka pri odraslih, ki niso primerni za zdravljenje s kemoterapijo, ki vsebuje cisplatin in imajo tumorje z izraženostjo PD-L1 ≥ 10, ocenjeno s kombinirano pozitivno oceno (CPS); ponovljenega ali metastatskega ploščatoceličnega raka glave in vratu (HNSCC) pri odraslih, ki imajo tumorje z ≥ 50 % izraženostjo PD-L1 (TPS), in pri katerih je bolezen napredovala med zdravljenjem ali po zdravljenju s kemoterapijo, ki je vključevala platino; za adjuvantno zdravljenje odraslih z rakom ledvičnih celic s povišanim tveganjem za ponovitev bolezni po nefrektomiji, ali po nefrektomiji in kirurški odstranitvi metastatskih lezij in za prvo linijo zdravljenja metastatskega kolorektalnega raka z visoko mikrosatelitsko nestabilnostjo (MSI-H – microsatellite instability-high) ali s pomanjkljivim popravljanjem neujemanja pri podvojevanju DNA (dMMR - mismatch repair de cient) pri odraslih. Zdravilo KEYTRUDA je kot samostojno zdravljenje ali v kombinaciji s kemoterapijo s platino in 5- uorouracilom (5-FU) indicirano za prvo linijo zdravljenja metastatskega ali neoperabilnega ponovljenega ploščatoceličnega raka glave in vratu pri odraslih, ki imajo tumorje z izraženostjo PD-L1 s CPS ≥ 1. Zdravilo KEYTRUDA je v kombinaciji s pemetreksedom in kemoterapijo na osnovi platine indicirano za prvo linijo zdravljenja metastatskega neploščatoceličnega NSCLC pri odraslih, pri katerih tumorji nimajo pozitivnih mutacij EGFR ali ALK; v kombinaciji s karboplatinom in bodisi paklitakselom bodisi nab- paklitakselom je indicirano za prvo linijo zdravljenja metastatskega ploščatoceličnega NSCLC pri odraslih; v kombinaciji z aksitinibom ali v kombinaciji z lenvatinibom je indicirano za prvo linijo zdravljenja napredovalega raka ledvičnih celic (RCC) pri odraslih; v kombinaciji s kemoterapijo s platino in  uoropirimidinom je indicirano za prvo linijo zdravljenja lokalno napredovalega neoperabilnega ali metastatskega raka požiralnika ali HER-2 negativnega adenokarcinoma gastroezofagealnega prehoda pri odraslih, ki imajo tumorje z izraženostjo PD-L1 s CPS ≥ 10; v kombinaciji s kemoterapijo je indicirano za zdravljenje lokalno ponovljenega neoperabilnega ali metastatskega trojno negativnega raka dojk pri odraslih, ki imajo tumorje z izraženostjo PD-L1 s CPS ≥ 10 in predhodno niso prejeli kemoterapije za metastatsko bolezen; v kombinaciji z lenvatinibom je indicirano za zdravljenje napredovalega ali ponovljenega raka endometrija (EC) pri odraslih z napredovalo boleznijo med ali po predhodnem zdravljenju s kemoterapijo, ki je vključevala platino, v katerih koli terapevtskih okoliščinah, in ki niso kandidati za kurativno operacijo ali obsevanje. Odmerjanje in način uporabe: Testiranje PD-L1: Če je navedeno v indikaciji, je treba izbiro bolnika za zdravljenje z zdravilom KEYTRUDA na podlagi izraženosti PD-L1 tumorja potrditi z validirano preiskavo. Testiranje MSI-H/dMMR pri bolnikih s CRC: Za samostojno zdravljenje z zdravilom KEYTRUDA je priporočljivo opraviti testiranje MSI-H/dMMR statusa tumorja z validirano preiskavo, da se izbere bolnike s CRC. Odmerjanje: Priporočeni odmerek zdravila KEYTRUDA pri odraslih je bodisi 200 mg na 3 tedne ali 400 mg na 6 tednov, apliciran z intravensko infuzijo v 30 minutah. Priporočeni odmerek zdravila KEYTRUDA za samostojno zdravljenje pri pediatričnih bolnikih s cHL, starih 3 leta ali več, je 2 mg/kg telesne mase (do največ 200 mg) na 3 tedne, apliciran z intravensko infuzijo v 30 minutah. Za uporabo v kombinaciji glejte povzetke glavnih značilnosti zdravil sočasno uporabljenih zdravil. Če se uporablja kot del kombiniranega zdravljenja skupaj z intravensko kemoterapijo, je treba zdravilo KEYTRUDA aplicirati prvo. Bolnike je treba zdraviti do napredovanja bolezni ali nesprejemljivih toksičnih učinkov (in do maksimalnega trajanja zdravljenja, če je le to določeno za indikacijo). Pri adjuvantnem zdravljenju melanoma ali RCC je treba zdravilo uporabljati do ponovitve bolezni, pojava nesprejemljivih toksičnih učinkov oziroma mora zdravljenje trajati do enega leta. Če je aksitinib uporabljen v kombinaciji s pembrolizumabom, se lahko razmisli o povečanju odmerka aksitiniba nad začetnih 5 mg v presledkih šest tednov ali več. V primeru uporabe v kombinaciji z lenvatinibom je treba zdravljenje z enim ali obema zdraviloma prekiniti, kot je primerno. Uporabo lenvatiniba je treba zadržati, odmerek zmanjšati ali prenehati z uporabo, v skladu z navodili v povzetku glavnih značilnosti zdravila za lenvatinib, in sicer za kombinacijo s pembrolizumabom. Pri bolnikih starih ≥ 65 let, bolnikih z blago do zmerno okvaro ledvic, bolnikih z blago okvaro jeter prilagoditev odmerka ni potrebna. Odložitev odmerka ali ukinitev zdravljenja: Zmanjšanje odmerka zdravila KEYTRUDA ni priporočljivo. Za obvladovanje neželenih učinkov je treba uporabo zdravila KEYTRUDA zadržati ali ukiniti, prosimo, glejte celoten Povzetek glavnih značilnosti zdravila. Kontraindikacije: Preobčutljivost na učinkovino ali katero koli pomožno snov. Povzetek posebnih opozoril, previdnostnih ukrepov, interakcij in neželenih učinkov: Imunsko pogojeni neželeni učinki (pnevmonitis, kolitis, hepatitis, nefritis, endokrinopatije, neželeni učinki na kožo in drugi): Pri bolnikih, ki so prejemali pembrolizumab, so se pojavili imunsko pogojeni neželeni učinki, vključno s hudimi in smrtnimi primeri. Večina imunsko pogojenih neželenih učinkov, ki so se pojavili med zdravljenjem s pembrolizumabom, je bila reverzibilnih in so jih obvladali s prekinitvami uporabe pembrolizumaba, uporabo kortikosteroidov in/ali podporno oskrbo. Pojavijo se lahko tudi po zadnjem odmerku pembrolizumaba in hkrati prizadanejo več organskih sistemov. V primeru suma na imunsko pogojene neželene učinke je treba poskrbeti za ustrezno oceno za potrditev etiologije oziroma izključitev drugih vzrokov. Glede na izrazitost neželenega učinka je treba zadržati uporabo pembrolizumaba in uporabiti kortikosteroide – za natančna navodila, prosimo, glejte Povzetek glavnih značilnosti zdravila Keytruda. Zdravljenje s pembrolizumabom lahko poveča tveganje za zavrnitev pri prejemnikih presadkov čvrstih organov. Pri bolnikih, ki so prejemali pembrolizumab, so poročali o hudih z infuzijo povezanih reakcijah, vključno s preobčutljivostjo in ana laksijo. Pembrolizumab se iz obtoka odstrani s katabolizmom, zato presnovnih medsebojnih delovanj zdravil ni pričakovati. Uporabi sistemskih kortikosteroidov ali imunosupresivov pred uvedbo pembrolizumaba se je treba izogibati, ker lahko vplivajo na farmakodinamično aktivnost in učinkovitost pembrolizumaba. Vendar pa je kortikosteroide ali druge imunosupresive mogoče uporabiti za zdravljenje imunsko pogojenih neželenih učinkov. Kortikosteroide je mogoče uporabiti tudi kot premedikacijo, če je pembrolizumab uporabljen v kombinaciji s kemoterapijo, kot antiemetično pro lakso in/ali za ublažitev neželenih učinkov, povezanih s kemoterapijo. Ženske v rodni dobi morajo med zdravljenjem s pembrolizumabom in vsaj še 4 mesece po zadnjem odmerku pembrolizumaba uporabljati učinkovito kontracepcijo, med nosečnostjo in dojenjem se ga ne sme uporabljati. Varnost pembrolizumaba pri samostojnem zdravljenju so v kliničnih študijah ocenili pri 7.148 bolnikih z napredovalim melanomom, kirurško odstranjenim melanomom v stadiju III (adjuvantno zdravljenje), NSCLC, cHL, urotelijskim rakom, HNSCC, CRC, rakom endometrija, želodca, tankega črevesa, žolčnika, trebušne slinavke ali adjuvantnim zdravljenjem RCC s štirimi odmerki (2 mg/kg telesne mase na 3 tedne, 200 mg na 3 tedne in 10 mg/kg telesne mase na 2 ali 3 tedne). V tej populaciji bolnikov je mediani čas opazovanja znašal 7,9 meseca (v razponu od 1 dneva do 39 mesecev), najpogostejši neželeni učinki zdravljenja s pembrolizumabom pa so bili utrujenost (31 %), diareja (22 %) in navzea (21 %). Večina poročanih neželenih učinkov pri samostojnem zdravljenju je bila po izrazitosti 1. ali 2. stopnje. Najresnejši neželeni učinki so bili imunsko pogojeni neželeni učinki in hude z infuzijo povezane reakcije. Pojavnost imunsko pogojenih neželenih učinkov pri uporabi pembrolizumaba samega za adjuvantno zdravljenje (n = 1.480) je znašala 36,1 % za vse stopnje in 8,9 % od 3. do 5. stopnje, pri metastatski bolezni (n = 5.375) pa 24,2 % za vse stopnje in 6,4 % od 3. do 5. stopnje. Pri adjuvantnem zdravljenju niso zaznali nobenih novih imunsko pogojenih neželenih učinkov. Varnost pembrolizumaba pri kombiniranem zdravljenju s kemoterapijo so ocenili pri 2.033 bolnikih z NSCLC, HNSCC, rakom požiralnika ali TNBC, ki so v kliničnih študijah prejemali pembrolizumab v odmerkih 200 mg, 2 mg/kg telesne mase ali 10 mg/kg telesne mase na vsake 3 tedne. V tej populaciji bolnikov so bili najpogostejši neželeni učinki naslednji: anemija (52 %), navzea (52 %), utrujenost (37 %), zaprtost (34 %), nevtropenija (33 %), diareja (32 %), zmanjšanje apetita (30 %) in bruhanje (28 %). Pojavnost neželenih učinkov 3. do 5. stopnje je pri bolnikih z NSCLC pri kombiniranem zdravljenju s pembrolizumabom znašala 67 % in pri zdravljenju samo s kemoterapijo 66 %, pri bolnikih s HNSCC pri kombiniranem zdravljenju s pembrolizumabom 85 % in pri zdravljenju s kemoterapijo v kombinaciji s cetuksimabom 84 %, pri bolnikih z rakom požiralnika pri kombiniranem zdravljenju s pembrolizumabom 86 % in pri zdravljenju samo s kemoterapijo 83 % ter pri bolnikih s TNBC pri kombiniranem zdravljenju s pembrolizumabom 78 % in pri zdravljenju samo s kemoterapijo 74 %. Varnost pembrolizumaba v kombinaciji z aksitinibom ali lenvatinibom pri napredovalem RCC in v kombinaciji z lenvatinibom pri napredovalem EC so ocenili pri skupno 1.456 bolnikih z napredovalim RCC ali napredovalim EC, ki so v kliničnih študijah prejemali 200 mg pembrolizumaba na 3 tedne skupaj s 5 mg aksitiniba dvakrat na dan ali z 20 mg lenvatiniba enkrat na dan, kot je bilo ustrezno. V teh populacijah bolnikov so bili najpogostejši neželeni učinki diareja (58 %), hipertenzija (54 %), hipotiroidizem (46 %), utrujenost (41 %), zmanjšan apetit (40 %), navzea (40 %), artralgija (30 %), bruhanje (28 %), zmanjšanje telesne mase (28 %), disfonija (28 %), bolečine v trebuhu (28 %), proteinurija (27 %), sindrom palmarno-plantarne eritrodizestezije (26 %), izpuščaj (26 %), stomatitis (25 %), zaprtost (25 %), mišično-skeletna bolečina (23 %), glavobol (23 %) in kašelj (21 %). Neželenih učinkov od 3. do 5. stopnje je bilo pri bolnikih z RCC med uporabo pembrolizumaba v kombinaciji z aksitinibom ali lenvatinibom 80 % in med uporabo sunitiniba samega 71 %. Pri bolnicah z EC je bilo neželenih učinkov od 3. do 5. stopnje med uporabo pembrolizumaba v kombinaciji z lenvatinibom 89 % in med uporabo kemoterapije same 73 %. Za celoten seznam neželenih učinkov, prosimo, glejte celoten Povzetek glavnih značilnosti zdravila. Za dodatne informacije o varnosti v primeru uporabe pembrolizumaba v kombinaciji glejte povzetke glavnih značilnosti zdravila za posamezne komponente kombiniranega zdravljenja. Način in režim izdaje zdravila: H – Predpisovanje in izdaja zdravila je le na recept, zdravilo se uporablja samo v bolnišnicah. Imetnik dovoljenja za promet z zdravilom: Merck Sharp & Dohme B.V. , Waarderweg 39, 2031 BN Haarlem, Nizozemska. Merck Sharp & Dohme inovativna zdravila d.o.o., Ameriška ulica 2, 1000 Ljubljana, tel: +386 1/ 520 42 01, fax: +386 1/ 520 43 50; Pripravljeno v Sloveniji, 01/2022; SI-KEY-00404 EXP: 01/2024 Samo za strokovno javnost. H - Predpisovanje in izdaja zdravila je le na recept, zdravilo pa se uporablja samo v bolnišnicah. Pred predpisovanjem, prosimo, preberite celoten Povzetek glavnih značilnosti zdravila Keytruda, ki je na voljo pri naših strokovnih sodelavcih ali na lokalnem sedežu družbe. (pembrolizumab, MSD) KLJUČ ZA VEČ PRILOŽNOSTI PRI ZDRAVLJENJU VAŠIH BOLNIKOV KEYTRUDA je odobrena za zdravljenje 18 indikacij rakavih obolenj1 M-SI-00000413(v1.0) Datum priprave informacije: februar 2022. Vir: 1. Povzetek glavnih značilnosti zdravila Gavreto je dosegljiv na povezavi: https://www.ema.europa.eu/en/ documents/product-information/gavreto-epar-product-information_sl.pdf DODATNE INFORMACIJE SO NA VOLJO PRI: Roche farmacevtska družba d.o.o., Stegne 13G, 1000 Ljubljana Samo za strokovno javnost. Monoterapija za zdravljenje odraslih bolnikov z napredovalim nedrobnoceličnim rakom pljuč (NDRP) s preureditvijo gena RET (rearranged during transfection), ki predhodno še niso bili zdravljeni z zaviralcem RET.1 GAVRETO® Ime zdravila: Gavreto 100 mg trde kapsule. Kakovostna in količinska sestava: Ena trda kapsula vsebuje 100 mg pralsetiniba. Terapevtske indikacije: Zdravilo Gavreto je indicirano kot monoterapija za zdravljenje odraslih bolnikov z napredovalim nedrobnoceličnim rakom pljuč (NDRP) s preureditvijo gena RET (rearranged during transfection), ki predhodno še niso bili zdravljeni z zaviralcem RET. Odmerjanje in način uporabe: Izbira bolnikov za zdravljenje napredovalega NDRP s preureditvijo gena RET mora temeljiti na validirani testni metodi. Odmerjanje: Priporočeni odmerek pralsetiniba je 400 mg enkrat na dan na prazen želodec. Zdravljenje je treba nadaljevati do napredovanja bolezni ali nesprejemljivih neželenih učinkov. Prilagoditve odmerka v primeru neželenih učinkov: Za obvladanje neželenih učinkov pride v poštev prekinitev zdravljenja z zmanjšanjem odmerka ali brez njega. Bolnikom je mogoče odmerek zmanjševati po 100 mg do najmanjšega odmerka 100 mg enkrat na dan. Način uporabe: Zdravilo Gavreto je namenjeno za peroralno uporabo. Trde kapsule morajo bolniki pogoltniti cele s kozarcem vode na prazen želodec. Bolniki ne smejo jesti vsaj dve uri pred jemanjem pralsetiniba in vsaj eno uro po njem. Kontraindikacije: Preobčutljivost na učinkovino ali katero koli pomožno snov. Posebna opozorila in previdnostni ukrepi: Pnevmonitis/intersticijska bolezen pljuč: Pri bolnikih, ki so v kliničnih preskušanjih prejemali pralsetinib, so poročali o hudih, življenje ogrožajočih ali smrtnih primerih pnevmonitisa/intersticijske bolezni pljuč. Bolnikom je treba naročiti, da morajo zdravnika nemudoma obvestiti o novonastalih dihalnih simptomih ali poslabšanju takšnih simptomov. Pri bolnikih z akutnimi dihalnimi simptomi ali poslabšanjem dihalnih simptomov s sumom na pnevmonitis/intersticijsko bolezen pljuč je treba opraviti ustrezno diagnostiko, da se izključijo drugi možni vzroki. Če je ocenjeno, da je pnevmonitis/intersticijska bolezen pljuč povezana s pralsetinibom, je treba glede na izrazitost potrjenega pnevmonitisa/intersticijske bolezni pljuč odmerjanje zdravila Gavreto prekiniti, zmanjšati ali dokončno ukiniti. Hipertenzija: V kliničnih preskušanjih so pri bolnikih, zdravljenih s pralsetinibom, opažali hipertenzijo. Z zdravljenjem povezano hipertenzijo so najpogosteje obvladovali z antihipertenzivnimi zdravili. Bolnikom z neurejeno hipertenzijo se ne sme uvesti zdravljenja z zdravilom Gavreto. Že obstoječo hipertenzijo je treba pred začetkom zdravljenja z zdravilom Gavreto ustrezno urediti. Krvni tlak je priporočljivo preverjati po 1 tednu, nato pa vsaj enkrat na mesec in kot je klinično indicirano. Uvesti je treba antihipertenzivno zdravljenje oziroma ga prilagoditi, kot je primerno. Glede na to, kako izrazita je hipertenzija, opažena med zdravljenjem z zdravilom Gavreto, je treba odmerjanje zdravila prekiniti, zmanjšati ali dokončno ukiniti. Zvišanja transaminaz: Pri bolnikih, ki so v kliničnih preskušanjih prejemali pralsetinib, so poročali o hudih primerih zvišanja transaminaz. Vrednosti ALT in AST je treba preveriti pred uvedbo zdravila Gavreto, nato na 2 tedna prve 3 mesece, potem pa enkrat na mesec in kot je klinično indicirano. Glede na to, kako izrazito je zvišanje transaminaz, opaženo med zdravljenjem z zdravilom Gavreto, je treba odmerjenje zdravila prekiniti, zmanjšati ali dokončno ukiniti. Krvavitve: Pri zdravljenju z zdravilom Gavreto se lahko pojavijo hude krvavitve, vključno s smrtnimi. Pri bolnikih z življenje ogrožajočimi ali ponovljenimi hudimi krvavitvami je treba zdravljenje z zdravilom Gavreto dokončno ukiniti. Podaljšanje intervala QT: Pri bolnikih, ki so prejemali zdravilo Gavreto v kliničnih preskušanjih, so opažali podaljšanje intervala QT. Zato morajo imeti bolniki pred začetkom zdravljenja z zdravilom Gavreto interval QTc ≤ 470 ms in serumske elektrolite v normalnem območju. Hipokaliemijo, hipomagneziemijo in hipokalciemijo je treba korigirati tako pred zdravljenjem z zdravilom Gavreto kot med zdravljenjem z njim. Elektrokardiogram in elektrolite v serumu je treba kontrolirati ob koncu prvega tedna in prvega meseca zdravljenja z zdravilom Gavreto, nato pa občasno, kot je klinično indicirano, odvisno tudi od prisotnosti drugih dejavnikov tveganja. Pralsetinib je treba previdno uporabljati pri bolnikih z anamnezo motenj srčnega ritma ali podaljšanja intervala QT, prav tako tudi pri bolnikih, ki prejemajo močne zaviralce CYP3A4 ali zdravila, za katera je znano, da podaljšajo interval QT/QTc. Morda bo treba prekiniti zdravljenje z zdravilom Gavreto, prilagoditi odmerek ali zdravljenje ukiniti. Plodnost in nosečnost: Moški, ki imajo partnerke v rodni dobi, morajo med zdravljenjem z zdravilom Gavreto in vsaj še 1 teden po njegovem zadnjem odmerku uporabljati učinkovito kontracepcijo. Ženskam v rodni dobi je treba povedati, da med zdravljenjem z zdravilom Gavreto ne smejo zanositi. Ženske morajo med zdravljenjem s pralsetinibom uporabljati visoko učinkovito nehormonsko kontracepcijo, kajti pralsetinib lahko povzroči neučinkovitost hormonske kontracepcije. Uporabo učinkovite kontracepcije je treba nadaljevati vsaj 2 tedna po zadnjem odmerku zdravila. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Farmakokinetično medsebojno delovanje: Podatki in vitro kažejo, da se pralsetinib presnovi predvsem s CYP3A4 in prenaša s P-gp. Zato lahko spodbujevalci in zaviralci CYP3A4 in P-gp spremenijo koncentracijo pralsetiniba v plazmi. Učinkovine, ki lahko vplivajo na pralsetinib: Močni zaviralci CYP3A4 ali kombinacija zaviralcev P-gp in močnih zaviralcev CYP3A4: Sočasna uporaba pralsetiniba z močnim zaviralcem CYP3A4 ali kombinacijo zaviralca P-gp in močnega zaviralca CYP3A4 lahko poveča koncentracijo pralsetiniba v plazmi, to pa lahko poveča pojavnost in izrazitost neželenih učinkov pralsetiniba. Zato se je treba izogibati sočasni uporabi pralsetiniba z močnimi zaviralci CYP3A4 ali kombinaciji zaviralcev P-gp in močnih zaviralcev CYP3A4. Če se sočasni uporabi z močnim zaviralcem CYP3A4 ali kombinaciji z zaviralci P-gp in močnimi zaviralci CYP3A4 ni mogoče izogniti, zmanjšajte trenutni odmerek pralsetiniba. Močni spodbujevalci CYP3A4: Sočasna uporaba pralsetiniba z močnim spodbujevalcem CYP3A4 lahko zmanjša koncentracijo pralsetiniba v plazmi, to pa lahko zmanjša učinkovitost pralsetiniba. Zato se je treba izogibati sočasni uporabi pralsetiniba z močnimi spodbujevalci CYP3A4. Če se sočasni uporabi ni mogoče izogniti, povečajte odmerek pralsetiniba. Občutljivi substrati CYP3A4, CYP2C8, CYP2C9, P-gp, BCRP, OATP1B1, OATP1B3, OAT1, MATE1 in MATE2-K z ozkim terapevtskim indeksom: Sočasno dajanje pralsetiniba lahko spremeni izpostavljenost občutljivim substratom encimov CYP in zgoraj naštetim prenašalcem. Zdravilom, ki so substrati omenjenih encimov CYP in prenašalci z ozkim terapevtskim indeksom, se je treba izogibati. Neželeni učinki: Najpogostejši neželeni učinki so bili anemija, zvišana aspartataminotransferaza, nevtropenija, zaprtje, mišično-skeletna bolečina, utrujenost, levkopenija, zvišana alanin-aminotransferaza in hipertenzija. Najpogostejši resni neželeni učinki so bili pljučnica, pnevmonitis in anemija. Poročanje o domnevnih neželenih učinkih: Poročanje o domnevnih neželenih učinkih zdravila po izdaji dovoljenja za promet je pomembno. Omogoča namreč stalno spremljanje razmerja med koristmi in tveganji zdravila. Od zdravstvenih delavcev se zahteva, da poročajo o katerem koli domnevnem neželenem učinku zdravila na: Javna agencija Republike Slovenije za zdravila in medicinske pripomočke, Sektor za farmakovigilanco, Nacionalni center za farmakovigilanco, Slovenčeva ulica 22, SI-1000 Ljubljana, Tel: +386 (0)8 2000 500, Faks: +386 (0)8 2000 510, e-pošta: h-farmakovigilanca@jazmp.si, spletna stran: www.jazmp.si. Režim izdaje zdravila: Rp/Spec. Imetnik dovoljenja za promet: Roche Registration GmbH, Emil-Barell- Strasse 1, 79639 Grenzach-Wyhlen, Nemčija. Verzija: 1.0/22. Za to zdravilo se izvaja dodatno spremljanje varnosti. Tako bodo hitreje na voljo nove informacije o njegovi varnosti. Zdravstvene delavce naprošamo, da poročajo o katerem koli domnevnem neželenem učinku zdravila. Kako poročati o neželenih učinkih, si poglejte skrajšani povzetek glavnih značilnosti zdravila pod ‘‘Poročanje o domnevnih neželenih učinkih‘‘. Skrajšan povzetek glavnih značilnosti zdravila Gavreto ONIVYDE: IZDELAN POSEBEJ ZA BOJ PROTI RAKU TREBUŠNE SLINAVKE ONIVYDE VSEBUJE PEGILIRANE LIPOSOME Z IRINOTEKANOM IN JE IZDELAN POSEBEJ ZA UČINKOVITO ZDRAVLJENJE METASTATSKEGA RAKA TREBUŠNE SLINAVKE2–5 KLINIČNI PODATKI ŠTUDIJE 3. FAZE POTRJUJEJO EDINSTVENO KLINIČNO VREDNOST ZDRAVILA ONIVYDE V KOMBINACIJI S 5-FU/LV: ∞ skladni podatki o učinkovitosti pri vseh opazovanih dogodkih: pomembno podaljšanje preživetja in povečana stopnja odziva6–8 ∞ ohranjena kakovost življenja6,9 ∞ dobro poznan varnostni profi l1,6,7 POMEMBNA UČINKOVITOST ONIVYDE + 5-FU/LV JE POTRJENA V KLINIČNI PRAKSI10–12 ONIVYDE + 5-FU/LV PRIPOROČAJO VSE GLAVNE MEDNARODNE SMERNICE13–16 LITERATURA: 1. Povzetek glavnih značilnosti zdravila ONIVYDE pegylated liposomal. 2. Lamb YN, Scott LJ. Drugs. 2017;77:785– 792. 3. Drummond DC et al. Cancer Res. 2006;66:3271–3277. 4. Kalra AV et al. Cancer Res. 2014;74:7003–7013. 5. Carnevale J, Ko AH. Future Oncol. 2016;12:453–464. 6. Wang-Gillam A et al. Lancet. 2016;387:545–557. 7. Wang-Gillam A et al. Eur J Cancer. 2019;108:78–87. 8. Chen LT et al. Eur J Cancer. 2018;105:71–78. 9. Hubner RA et al. Eur J Cancer. 2019;106:24–33. 10. Kieler M et al. Ther Adv Med Oncol. 2019;11:1–13. 11. Yoo C et al. Ther Adv Med Oncol. 2019;11:1–9. 12. Pellino A et al. ESMO. 2019;P660. 13. Ducreux M et al. Ann Oncol. 2015;25(suppl 5):v56–v68. 14. eUpdate Cancer of the Pancreas Treatment Recommendations. Objavljeno 20. junija, 2019. ESMO Guidelines Committee. 15. Okusaka T et al. Pancreas. 2020;49(3):326–335. 16. NCCN Guidelines Version 2, 2021. Pancreatic Adenocarcinoma. Objavljeno 25. februarja, 2021. Samo za strokovno javnost. Datum priprave informacije: september 2021. ONI AD1 C1 2021-22. ONIVYDE pegylated liposomal je odobren za zdravljenje metastatskega adenokarcinoma trebušne slinavke v kombinaciji s 5-fl uorouracilom (5-FU) in levkovorinom (LV) pri odraslih bolnikih, pri katerih je bolezen po zdravljenju na osnovi gemcitabina napredovala.1 SKRAJŠAN POVZETEK GLAVNIH ZNAČILNOSTI ZDRAVILA Onivyde pegylated liposomal 4,3 mg/ml SESTAVA*: Onivyde pegylated liposomal 4,3 mg/ml koncentrat za disperzijo za infundiranje: ena viala z 10 ml koncentrata vsebuje 43 mg brezvodnega irinotekana (v obliki irinotekanijeve soli saharoznega oktasulfata v pegilirani liposomski formulaciji). TERAPEVTSKE INDIKACIJE*: Zdravljenje metastatskega adenokarcinoma trebušne slinavke v kombinaciji s 5fl uorouracilom (5-FU) in levkovorinom (LV) pri odraslih bolnikih, pri katerih je bolezen po zdravljenju na osnovi gemcitabina napredovala. ODMERJANJE IN NAČIN UPORABE*: Onivyde pegylated liposomalsmejo bolnikom predpisati in dajati samo zdravstveni delavci, ki imajo izkušnje pri uporabi zdravil za zdravljenje raka. Zdravilo Onivyde pegylated liposomal ni enakovredno drugim neliposomskim formulacijam irinotekana, zato jih ne smemo zamenjevati. Priporočeni odmerek in režim odmerjanja zdravila Onivyde pegylated liposomal je 70 mg/m2  intravensko 90 minut, čemur sledi LV 400 mg/m2 intravensko 30 minut in nato 5FU 2400 mg/m2 intravensko 46 ur, vsaka 2 tedna. Zdravilo Onivyde pegylated liposomal se ne daje kot samostojno zdravilo. Pri bolnikih z znano homozigotnostjo za alel UGT1A1*28 je treba razmisliti o manjšem začetnem odmerku zdravila Onivyde pegylated liposomal 50 mg/m2. Če zdravilo bolniki dobro prenašajo, lahko v naslednjih ciklih razmislimo o odmerku zdravila Onivyde pegylated liposomal 70 mg/m2. Prilagajanje odmerka se priporoča za obvladovanje toksičnosti 3. ali 4. stopnje, povezane z zdravilom Onivyde pegylated liposomal. KONTRAINDIKACIJE*: Anamneza hude preobčutljivosti na irinotekan ali katero koli pomožno snov. Dojenje. OPOZORILA*: Zdravilo Onivyde pegylated liposomal ni enakovredno drugim neliposomskim formulacijam irinotekana, zato jih ne smemo zamenjevati. Mielosupresija/nevtropenija: Med zdravljenjem z zdravilom Onivyde pegylated liposomal se priporoča nadziranje celotne krvne slike. Bolniki se morajo zavedati tveganja za nevtropenijo in pomena povišane telesne temperature. Febrilno nevtropenijo je treba nujno zdraviti v bolnišnici s širokospektralnimi intravenskimi antibiotiki. Pri bolnikih, ki doživijo hude hematološke neželene učinke, se priporoča zmanjšanje odmerka ali prekinitev zdravljenja. Bolnikov s hudo odpovedjo kostnega mozga ne smemo zdraviti z zdravilom Onivyde pegylated liposomal. Anamneza predhodnega obsevanja trebuha poveča tveganje za hudo nevtropenijo in febrilno nevtropenijo po zdravljenju z zdravilom Onivyde pegylated liposomal. Pri bolnikih, ki hkrati prejemajo zdravilo Onivyde pegylated liposomal in so obsevani, je potrebna previdnost. Bolniki s pomanjkljivo glukuronidacijo bilirubina, kot so bolniki z Gilbertovim sindromom, imajo med zdravljenjem z zdravilom Onivyde pegylated liposomal lahko večje tveganje za mielosupresijo. Bolniki azijskega porekla imajo večje tveganje za hudo in febrilno nevtropenijo. Posamezniki s homozigotnostjo 7/7 za alel UGT1A1*28 imajo povečano tveganje za nevtropenijo. Imunosupresivni učinki in cepiva: Dajanje živih ali atenuiranih cepiv bolnikom z oslabljenim imunskim sistemom lahko povzroči resne ali smrtne okužbe. Interakcije z močnimi induktorji encima CYP3A4, močnimi zaviralci encima CYP3A4 in močnimi zaviralci encima UGT1A1: Zdravila Onivyde pegylated liposomal ne smemo dajati skupaj z močnimi induktorji encima CYP3A4, močnimi zaviralci encima CYP3A4 ali z močnimi zaviralci encima UGT1A1, razen če ni drugih terapevtskih možnosti. Zdravljenje z močnimi zaviralci encima CYP3A4 moramo prekiniti vsaj 1 teden pred začetkom zdravljenja z zdravilom Onivyde pegylated liposomal. Driska: Driska se lahko pojavi zgodaj (v ≤ 24 urah po začetku zdravljenja z zdravilom Onivyde pegylated liposomal) ali pozno (> 24 ur). Pri bolnikih, ki doživijo zgodnji pojav driske (v ≤ 24 urah po začetku zdravljenja z zdravilom Onivyde pegylated liposomal), je treba razmisliti o terapevtskem in profi laktičnem zdravljenju z atropinom, razen če je kontraindicirano. Bolnike je treba opozoriti na tveganje za zapoznelo drisko (> 24 ur), ki je izčrpavajoča in v redkih primerih tudi življenjsko nevarna. Loperamid je treba uvesti ob prvem pojavu neoblikovanega ali mehkega blata ali takoj, ko odvajanje blata postane pogostejše kot običajno. Loperamid je treba dajati, dokler bolnik ni brez driske vsaj 12 ur. Če driska traja tudi, ko bolnik prejema loperamid več kot 24 ur, je treba razmisliti o dodatni peroralni antibiotični podpori. Loperamida zaradi tveganja za paralitični ileus ne smemo uporabljati več kot 48 ur zaporedoma. Zdravljenje z zdravilom Onivyde pegylated liposomal je treba odložiti, dokler se driska ne umiri do ≤ 1. stopnje (2–3 odvajanja/ dan več kot pred zdravljenjem). Zdravila Onivyde pegylated liposomal ne smemo dajati bolnikom z zaporo črevesja ali kronično vnetno črevesno boleznijo, dokler se ta ne pozdravi. Holinergične reakcije: Zgodnjo drisko lahko spremljajo rinitis, povečano slinjenje, zardevanje, diaforeza, bradikardija, mioza in hiperperistaltika. Uporabiti je treba atropin. Akutne infuzijske in povezane reakcije: V primeru hudih preobčutljivostnih reakcij je treba zdravljenje z zdravilom Onivyde pegylated liposomal prekiniti. Predhodna Whipplova operacija: Večje tveganje za resne okužbe. Bolnike je treba spremljati glede znakov okužbe. Žilne bolezni: Zdravilo Onivyde pegylated liposomal je bilo povezano s trombemboličnimi dogodki, kot so pljučna embolija, venska tromboza in arterijska trombembolija. Treba je pridobiti podrobno zdravstveno anamnezo, da bi prepoznali bolnike z več dejavniki tveganja poleg osnovne neoplazme. Bolnike je treba obvestiti o znakih in simptomih trombembolije in jim svetovati, da se v primeru katerega od teh znakov ali simptomov takoj obrnejo na svojega zdravnika ali medicinsko sestro. Pljučna toksičnost: Pri bolnikih, ki so prejemali neliposomski irinotekan, so se pojavili dogodki, podobni intersticijski pljučni bolezni (IPB), ki so vodili do smrtnih primerov. Pri bolnikih z dejavniki tveganja (obstoječo pljučno boleznijo, uporabo pnevmotoksičnih zdravil, kolonije stimulirajočimi dejavniki ali predhodnim zdravljenjem z obsevanjem) je treba pred zdravljenjem z zdravilom Onivyde pegylated liposomal in po njem skrbno nadzirati respiratorne simptome. Dokler ni opravljena diagnostična ocena, je treba ob pojavu nove ali napredovale dispneje, kašlja in povišane telesne temperature zdravljenje z zdravilom Onivyde pegylated liposomal začasno prekiniti. Pri bolnikih s potrjeno diagnozo IPB moramo zdravljenje z zdravilom Onivyde pegylated liposomal dokončno prekiniti. Jetrna okvara: Bolniki s hiperbilirubinemijo so imeli povišane koncentracije skupnega SN-38, zato je tveganje za nevtropenijo povečano. Pri bolnikih z vrednostjo skupnega bilirubina 1,0–2,0 mg/dl je treba redno nadzirati celotno krvno sliko. Previdnost je potrebna pri bolnikih z jetrno okvaro (bilirubin > 2-kratna zgornja meja normalnih vrednosti [ULN]; aminotransferaze > 5-kratna ULN). Previdnost je potrebna, če zdravilo Onivyde pegylated liposomal dajemo v kombinaciji z drugimi hepatotoksičnimi zdravili. Ledvična okvara: Uporaba zdravila Onivyde pegylated liposomal pri bolnikih s pomembno ledvično okvaro ni bila ocenjena. Bolniki s premajhno telesno maso (indeks telesne mase < 18,5 kg/ m2): Potrebna je previdnost. Pomožne snovi: To zdravilo vsebuje 33,1 mg natrija na vialo, kar je enako 1,65 % največjega dnevnega vnosa natrija za odrasle osebe, ki ga priporoča SZO in znaša 2 g. En mililiter zdravila Onivyde pegylated liposomal vsebuje 0,144  mmol (3,31 mg) natrija. INTERAKCIJE*: Previdnostni ukrepi: Sočasno dajanje z induktorji encima CYP3A4 (npr. antikonvulzivi, rifampicin, rifabutin in šentjanževka) lahko zmanjša sistemsko izpostavljenost zdravilu Onivyde pegylated liposomal. Sočasno dajanje z zaviralci encima CYP3A4 (npr. grenivkinim sokom, klaritromicinom, indinavirjem, itrakonazolom, lopinavirjem, nefazodonom, nelfi navirjem, ritonavirjem, sakvinavirjem, telaprevirjem, vorikonazolom) ali encima UGT1A1 (npr. atazanavirja, gemfi brozila, indinavirja, regorafeniba) lahko poveča sistemsko izpostavljenost zdravilu Onivyde pegylated liposomal. PLODNOST*. NOSEČNOST*: Uporaba ni priporočljiva. DOJENJE*: Zdravilo je kontraindicirano. KONTRACEPCIJA*: Ženske v rodni dobi morajo med zdravljenjem in še 1 mesec po zdravljenju z zdravilom Onivyde pegylated liposomal uporabljati učinkovito kontracepcijo. Moški morajo med zdravljenjem z zdravilom Onivyde pegylated liposomal in 4 mesece po zdravljenju uporabljati kondome. VPLIV NA SPOSOBNOST VOŽNJE IN UPRAVLJANJA STROJEV*: Bolniki morajo biti med zdravljenjem pri vožnji in upravljanju strojev previdni. NEŽELENI UČINKI*: Zelo pogosti: nevtropenija, levkopenija, anemija, trombocitopenija, hipokaliemija, hipomagneziemija, dehidracija, zmanjšan apetit, omotica, driska, bruhanje, navzea, bolečine v trebuhu, stomatitis, alopecija, pireksija, periferni edem, vnetje sluznic, utrujenost, astenija, zmanjšana telesna masa. Pogosti: septični šok, sepsa, pljučnica, febrilna nevtropenija, gastroenteritis, oralna kandidoza, limfopenija, hipoglikemija, hiponatriemija, hipofosfatemija, nespečnost, holinergični sindrom, dizgevzija, hipotenzija, pljučna embolija, embolija, globoka venska tromboza, dispneja, disfonija, kolitis, hemoroidi, hipoalbuminemija, akutna ledvična odpoved, z infuzijo povezana reakcija, edem, zvišana raven bilirubina, zvišana raven alanin- aminotransferaze, zvišana raven aspartat-aminotransferaze, zvišano mednarodno umerjeno razmerje. Občasni: biliarna sepsa, preobčutljivost, tromboza, hipoksija, ezofagitis, proktitis, makulopapulozni izpuščaj, obarvanje nohtov. PREVELIKO ODMERJANJE*: Za preveliko odmerjanje zdravila ni znanega antidota. Treba je uvesti maksimalno podporno nego, s katero preprečimo dehidracijo zaradi driske in zdravimo zaplete zaradi okužb. FARMAKODINAMIČNE LASTNOSTI*: Irinotekan (zaviralec topoizomeraze I), inkapsuliran v vezikel z lipidnim dvoslojem oziroma liposom. Irinotekan je derivat kamptotecina. Kamptotecini delujejo kot specifi čni zaviralci encima DNA-topoizomeraza I. Irinotekan in njegov aktivni presnovek SN-38 se reverzibilno vežeta na kompleks topoizomeraze I in DNA ter sprožita poškodbe v enoverižni DNA, kar zaustavi replikacijske vilice pri podvajanju DNA in povzroča citotoksičnost. Irinotekan se presnavlja s karboksilesterazo do SN-38. SN-38 je približno 1.000-krat močnejši kot irinotekan kot zaviralec topoizomeraze I, očiščene iz tumorskih celičnih linij človeka in glodavcev. PAKIRANJE*: Pakiranje vsebuje eno vialo z 10 ml koncentrata. NAČIN PREDPISOVANJA IN IZDAJE ZDRAVILA: H - Predpisovanje in izdaja zdravila je le na recept, zdravilo pa se uporablja samo v bolnišnicah. DATUM ZADNJE REVIZIJE BESEDILA: september 2021. Imetnik dovoljenja za promet: Les Laboratoires Servier, 50, rue Carnot, 92284 Suresnes cedex, Francija. *Pred predpisovanjem preberite celoten povzetek glavnih značilnosti zdravila. Celoten povzetek glavnih značilnosti zdravila in podrobnejše informacije so na voljo pri: Servier Pharma d.o.o., Podmilščakova ulica 24, 1000 Ljubljana, www.servier.si. Zdravilo je na slovenskem trgu na voljo v tuji ovojnini. Za uporabnika so informacije v slovenskem jeziku dostopne na uradni spletni strani www.cbz.si. Navodila za uporabo v slovenskem jeziku so na voljo tudi na www.servier.si. 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The statement of disclosure must be in the Cover letter accompany- ing 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 ac- ceptable 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. instructions aksitinib Pfi zer Luxembourg SARL, GRAND DUCHY OF LUXEMBOURG, 51, Avenue J.F. Kennedy, L – 1855, Pfi zer, podružnica Ljubljana, Letališka cesta 29a, 1000 Ljubljana Samo za strokovno javnost. • Datum priprave: januar 2022 • PP-INL-EEP-0040 BISTVENI PODATKI IZ POVZETKA GLAVNIH ZNAČILNOSTI ZDRAVILA Inlyta 1 mg/3 mg/5 mg/7 mg fi lmsko obložene tablete Sestava in oblika zdravila: Ena tableta vsebuje 1 mg, 3 mg, 5 mg oz. 7 mg aksitiniba. Indikacije: Zdravljenje napredovalega karcinoma ledvičnih celic (RCC) pri odraslih bolnikih, pri katerih predhodno zdravljenje s sunitinibom ali citokinom ni bilo uspešno. Odmerjanje in način uporabe: Zdravljenje mora izvajati zdravnik, ki ima izkušnje z uporabo zdravil za zdravljenje rakavih bolezni. Priporočeni odmerek je 5 mg dvakrat na dan. Zdravljenje naj traja, dokler je mogoče opaziti klinično korist oz. do pojava nesprejemljive toksičnosti, ki je ni mogoče obvladovati s sočasno uporabljanimi zdravili ali prilagajanjem odmerka. Če bolnik bruha ali izpusti odmerek, ne sme vzeti dodatnega odmerka; naslednji predpisan odmerek je treba vzeti ob običajnem času. Prilagajanja odmerka: Pri bolnikih, ki aksitinib v začetnem odmerku 5 mg dvakrat na dan prenašajo brez neželenih učinkov > 2. stopnje dva tedna zapored, je odmerek mogoče zvečati na 7 mg dvakrat na dan, razen če je krvni tlak pri bolniku > 150/90 mmHg ali če jemlje antihipertenzive. Kasneje je z uporabo enakih meril pri bolnikih, ki prenašajo 7 mg dvakrat na dan, odmerek mogoče zvečati na največ 10 mg dvakrat na dan. Za obvladovanje nekaterih neželenih učinkov bo morda treba začasno ali trajno prekiniti zdravljenje in/ali zmanjšati odmerek na 3 mg dvakrat na dan in nato na 2 mg dvakrat na dan. Prilagajanje odmerka glede na bolnikovo starost, raso, spol ali telesno maso ni potrebno. Sočasno zdravljenje z močnimi zaviralci CYP3A4/5: Lahko zveča plazemske koncentracije aksitiniba. V primeru sočasne uporabe močnega zaviralca CYP3A4/5, je odmerek aksitiniba priporočljivo zmanjšati na približno polovico odmerka; morda bo potrebna začasna ali trajna prekinitev zdravljenja z aksitinibom. Če prekinemo sočasno uporabo močnega zaviralca, je treba razmisliti o vrnitvi na odmerek aksitiniba, ki je bil uporabljen pred uvedbo močnega zaviralca CYP3A4/5. Sočasno zdravljenje z močnimi induktorji CYP3A4/5: Lahko zmanjša plazemske koncentracije aksitiniba. V primeru sočasne uporabe močnega induktorja CYP3A4/5 je odmerek aksitiniba priporočljivo postopoma zvečati in bolnika skrbno nadzorovati glede pojava toksičnosti. Morda bo treba začasno ali trajno prekiniti zdravljenje in/ali zmanjšati odmerek aksitiniba. Če prekinemo sočasno uporabo močnega induktorja, je treba takoj začeti uporabljati odmerek aksitiniba, ki je bil uporabljen pred uvedbo močnega induktorja CYP3A4/5. Okvara ledvic: Prilagajanje odmerka ni potrebno; o uporabi pri bolnikih z očistkom kreatinina < 15 ml/min ni podatkov. Okvara jeter: Prilagajanje odmerka ni potrebno pri bolnikih z blago okvaro jeter (razred A po Child-Pughu). Zmanjšanje odmerka je priporočljivo pri bolnikih z zmerno okvaro jeter (razred B). Zdravila se ne sme uporabljati pri bolnikih s hudo okvaro jeter (razred C). Pediatrična populacija: Varnost in učinkovitost pri otrocih < 18 let nista bili dokazani; podatkov ni na voljo. Način uporabe: Peroralna uporaba. Tablete je treba pogoltniti cele, s kozarcem vode, dvakrat na dan, v približno 12-urnih časovnih presledkih, s hrano ali brez nje. Kontraindikacije: Preobčutljivost na aksitinib ali katerokoli pomožno snov. Posebna opozorila in previdnostni ukrepi: Dogodki srčnega popuščanja: Poročali so o dogodkih srčnega popuščanja. Med zdravljenjem je treba redno spremljati znake ali simptome srčnega popuščanja. Obravnava dogodkov srčnega popuščanja lahko zahteva začasno ali stalno prekinitev zdravljenja z aksitinibom in/ali zmanjšanje odmerka. Hipertenzija: O hipertenziji so poročali zelo pogosto. Pred začetkom zdravljenja mora biti krvni tlak ustrezno urejen; bolnike je treba spremljati in po potrebi uporabiti standardno antihipertenzivno zdravljenje. V primeru trdovratne hipertenzije (kljub uporabi antihipertenzivov) je treba odmerek aksitiniba zmanjšati, pri hudi hipertenziji pa zdravljenje začasno prekiniti in ga ponovno uvesti z manjšim odmerkom, ko se krvni tlak normalizira. Pri hudi ali trdovratni arterijski hipertenziji in simptomih sindroma posteriorne reverzibilne encefalopatije je treba razmisliti o diagnostičnem slikanju možganov z uporabo magnetne resonance. Motnje delovanja ščitnice: Poročali so o primerih hipotiroidizma in, v manjšem obsegu, hipertiroidizma. Delovanje ščitnice je treba spremljati pred začetkom zdravljenja in v rednih časovnih presledkih med zdravljenjem. Venski in arterijski embolični in trombotični dogodki: Poročali so o venskih in arterijskih emboličnih in trombotičnih dogodkih. Previdna uporaba pri bolnikih s tveganjem za pojav teh dogodkov ali anamnezo teh dogodkov. Zvišanje ravni hemoglobina ali hematokrita: Med zdravljenjem lahko pride do zvišanj ravni hemoglobina ali hematokrita, njuno raven je treba spremljati pred začetkom zdravljenja in v rednih časovnih presledkih med zdravljenjem. Krvavitve: Poročali so o pojavu krvavitev. Pri bolnikih z znaki nezdravljenih možganskih metastaz ali nedavne aktivne krvavitve v prebavilih se zdravila ne sme uporabljati. Če je pri krvavitvi potreben zdravniški poseg, je treba z odmerjanjem aksitiniba začasno prekiniti. Anevrizme in arterijske disekcije: Uporaba zaviralcev poti VEGF pri bolnikih s hipertenzijo ali brez nje lahko spodbudi nastanek anevrizem in/ali disekcij arterij. Pred uvedbo aksitiniba je treba to tveganje skrbno preučiti pri bolnikih z dejavniki tveganja, kot sta hipertenzija ali anamneza anevrizme. Perforacija prebavil in nastanek fi stule: Poročali so o pojavu perforacij prebavil in fi stul. Med zdravljenjem je potrebno redno spremljanje glede morebitnega pojava simptomov perforacije prebavil ali nastanka fi stule. Zapleti pri celjenju ran: Zdravljenje z aksitinibom je treba prekiniti najmanj 24 ur pred načrtovanim kirurškim posegom; odločitev glede ponovne uvedbe zdravljenja po posegu mora temeljiti na klinični presoji ustreznosti celjenja rane. Sindrom posteriorne reverzibilne encefalopatije (PRES): Poročali so o primerih PRES. Pri bolnikih z znaki ali simptomi PRES je treba zdravljenje začasno ali trajno prekiniti. Varnost ponovne uvedbe zdravljenja pri bolnikih, pri katerih je v preteklosti prišlo do PRES, ni znana. Proteinurija: Poročali so o proteinuriji, vključno s proteinurijo 3. in 4. stopnje izraženosti. Pred začetkom zdravljenja in v rednih časovnih presledkih med zdravljenjem je priporočljivo spremljanje glede pojava proteinurije; ob pojavu zmerne do hude proteinurije je treba zmanjšati odmerek ali začasno prekiniti zdravljenje. Zdravljenje je treba trajno prekiniti, če se pri bolniku pojavi nefrotski sindrom. Neželeni učinki na jetra: Zvišanja ravni ALT, AST in bilirubina v krvi. Pred začetkom zdravljenja in v rednih časovnih presledkih med njim je treba spremljati rezultate preiskav delovanja jeter. Zdravilo vsebuje laktozo: Bolniki z redko dedno intoleranco za galaktozo, odsotnostjo encima laktaze ali malabsorpcijo glukoze/galaktoze ne smejo jemati tega zdravila. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Zaviralci CYP3A4/5: Sočasna uporaba z močnimi zaviralci (npr. ketokonazol, itrakonazol, klaritromicin, eritomicin, atazanavir, indinavir, nefazodon, nelfi navir, ritonavir, sakvinavir in telitromicin) ter uživanje grenivk lahko zveča plazemske koncentracije aksitiniba. Priporočljiva je izbira sočasno uporabljanih zdravil, ki ne zavirajo ali minimalno zavirajo CYP3A4/5. Če je treba sočasno uporabljati močan zaviralec CYP3A4/5, je odmerek aksitiniba priporočljivo prilagoditi. Zaviralci CYP1A2 in CYP2C19: Zaradi tveganja, da se plazemske koncentracije aksitiniba povečajo, je potrebna previdnost. Induktorji CYP3A4/5: Sočasna uporaba aksitiniba z močnimi induktorji (npr. rifampicin, deksametazon, fenitoin, karbamazepin, rifabutin, rifapentin, fenobarbital in šentjanževka) lahko zmanjša plazemske koncentracije aksitiniba. Priporočljiva je izbira sočasno uporabljanih zdravil, ki ne inducirajo ali minimalno inducirajo CYP3A4/5. Če je treba sočasno uporabljati močan induktor CYP3A4/5, je odmerek aksitiniba priporočljivo prilagoditi. Plodnost, nosečnost in dojenje: Ne sme se uporabljati med nosečnostjo, razen če klinično stanje ženske zahteva zdravljenje s tem zdravilom. Ženske v rodni dobi morajo uporabljati kontracepcijo med zdravljenjem in še en teden po njem. V obdobju dojenja se ne sme uporabljati. Lahko neugodno vpliva na sposobnost razmnoževanja in plodnost pri ljudeh. Vpliv na sposobnost vožnje in upravljanja strojev: Ima blag vpliv na sposobnost vožnje in upravljanja strojev. Med zdravljenjem se lahko pojavijo učinki, kot je npr. omotica in/ali utrujenost. Neželeni učinki: Najpogostejši (≥ 20 %) neželeni učinki so bili driska, hipertenzija, utrujenost, zmanjšan apetit, navzea, zmanjšana telesna masa, hripavost, sindrom palmarno-plantarne eritrodisestezije (sindrom dlani-podplati), krvavitev, hipotiroidizem, bruhanje, proteinurija, kašelj in zaprtje. Ostali zelo pogosti (≥ 1/10 bolnikov) neželeni učinki so: glavobol, disgevzija, dispneja, bolečine v trebuhu, stomatitis, dispepsija, izpuščaj, suha koža, artralgija, bolečine v okončinah, astenija, vnetje sluznice. Način in režim izdaje: Predpisovanje in izdaja zdravila je le na recept, zdravilo pa se uporablja samo v bolnišnicah. Izjemoma se lahko uporablja pri nadaljevanju zdravljenja na domu ob odpustu iz bolnišnice in nadaljnjem zdravljenju. Imetnik dovoljenja za promet: Pfi zer Europe MA EEIG, Boulevard de la Plaine 17, 1050 Bruxelles, Belgija. Datum zadnje revizije besedila: 29.07.2021 Pred predpisovanjem se seznanite s celotnim povzetkom glavnih značilnosti zdravila. Literatura: 1. Melichar B, Poprach A, Kubackova K, et al. Effi cacy and tolerability of axitinib in metastatic renal cell carcinoma (mRCC): Comparison of Czech clinical registry and AXIS trial data. ECC. 25–29 September 2015. Vienna, Austria. Poster: 2615. 2. Matias M, Le Teuff G, Albiges L, et al. Real world prospective experience of axitinib in metastatic renal cell carcinoma in a large comprehensive cancer centre. Eur J Cancer. 2017;79:185–192. 3. Rossetti S, Romano FJ, D‘Aniello C, et al. Activity of second line axitinib in metastatic renal cell carcinom (mRCC) patients treated with sunitinib: Results from SAX Italian real world trial. J Clin Oncol. 2017;35(15_suppl):e16054. 4. Povzetek glavnih značilnosti zdravila Inlyta, 29.7.2021. Zdravilo Inlyta je indicirano za zdravljenje napredovalega karcinoma ledvičnih celic pri odraslih bolnikih, pri katerih predhodno zdravljenje s sunitinibom ali citokinom ni bilo uspešno.4 Zagotovite svojim bolnikom z metastatskim karcinomom ledvičnih celic v drugi liniji zdravljenja vsakodnevne zmage z zdravilom Inlyta®.1–3 1-3 R a d io lo g y a n d O n c o lo g y I V o lu m e 5 6 I N u m b e r 1 I P a g e s 1 -1 2 8 I M a r c h 2 0 2 2 march 2022 vol.56 no.1