vol.56 no.3 september 2022 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 oncologic imaging, interventional radiology, nuclear medicine, radiotherapy, clinical and experimental oncology, radiobiology, medical physics, and radiation protection. Papers on more general aspects of interest to the radiologists and oncologists are also published (no case reports). Editor-in-Chief Gregor Serša, Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia (Subject Area: Experimental Oncology) Executive Editor Viljem Kovac, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia (Subject Areas: Clinical Oncology, Radiotherapy) Editorial Board Subject Areas: Radiology and Nuclear Medicine Sotirios Bisdas, University College London, Department of Neuroradiology, London, UK Boris Brkljacic, University Hospital “Dubrava”, Department of Diagnostic and Interventional Radiology, Zagreb, Croatia Maria Godény, National Institute of Oncology, Budapest, Hungary Gordana Ivanac, University Hospital Dubrava, Department of Diagnostic and Interventional Radiology, Zagreb, Croatia Luka Ležaic, University Medical Centre Ljubljana, Department for Nuclear Medicine, Ljubljana, Slovenia Katarina Šurlan Popovic, University Medical Center Ljubljana, Clinical Institute of Radiology, Ljubljana, Slovenia Jernej Vidmar, University Medical Center Ljubljana, Clinical Institute of Radiology, Ljubljana, Slovenia Advisory Committee Tullio Giraldi, University of Trieste, Faculty of Medicine and Psyhology, Department of Life Sciences, Trieste, Italy Vassil Hadjidekov, Medical University, Department of Diagnostic Imaging, Sofia, Bulgaria Marko Hocevar, Institute of Oncology Ljubljana, Department of Surgical Oncology, Ljubljana, Slovenia Deputy Editors Andrej C, University of Primorska, Faculty of Health Science, Izola, Slovenia (Subject Areas: Clinical Oncology, Experimental Oncology) Božidar Casar, Institute of Oncology Ljubljana, Department for Dosimetry and Quality of Radiological Procedures, Ljubljana (Subject Area: Medical Physics) Maja Cemažar, Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia (Subject Area: Experimental Oncology) Subject Areas: Clinical Oncology and Radiotherapy Serena Bonin, University of Trieste, Department of Medical Sciences, Cattinara Hospital, Surgical Pathology Blg, Molecular Biology Lab, Trieste, Italy Luca Campana, Veneto Institute of Oncology (IOV-IRCCS), Padova, Italy Christian Dittrich, Kaiser Franz Josef - Spital, Vienna, Austria Blaž Grošelj, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana Luka Milas, UT M. D. Anderson Cancer Center, Houston, USA Miha Oražem, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana Gaber Plavc, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana Csaba Polgar, National Institute of Oncology, Budapest, Hungary Dirk Rades, University of Lubeck, Department of Radiation Oncology, Lubeck, Germany Luis Souhami, McGill University, Montreal, Canada Borut Štabuc, University Medical Center Ljubljana, Division of Internal Medicine, Department of Gastroenterology, Ljubljana, Slovenia Andrea Veronesi, Centro di Riferimento Oncologico- Aviano, Division of Medical Oncology, Aviano, Italy Branko Zakotnik, Institute of Oncology Ljubljana, Department of Medical Oncology, Ljubljana, Slovenia Mikl Kásler, National Institute of Oncology, Budapest, Hungary Maja Osmak, Ruder Boškovic Institute, Department of Molecular Biology, Zagreb, Croatia September 2022 Vol. 56 No. 3 Pages 267-408 ISSN 1318-2099 UDC 616-006 CODEN: RONCEM Igor Kocijancic, University Medical Center Ljubljana, Institute of Radiology, Ljubljana, Slovenia (Subject Areas: Radiology, Nuclear Medicine) Karmen Stanic, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia (Subject Areas: Radiotherapy; Clinical Oncology) Primož Strojan, Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia (Subject Areas: Radiotherapy, Clinical Oncology) Subject Area: Experimental Oncology Metka Filipic, National Institute of Biology, Department of Genetic Toxicology and Cancer Biology, Ljubljana, Slovenia Janko Kos, University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia Tamara Lah Turnšek, National Institute of Biology, Ljubljana, Slovenia Damijan Miklavcic, University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia 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 Nystr, Skandionkliniken, Uppsala, Sweden Ervin B. Podgoršak, McGill University, Medical Physics Unit, Montreal, Canada Matthew Podgorsak, Roswell Park Cancer Institute, Departments of Biophysics and Radiation Medicine, Buffalo, NY ,USA Radiol Oncol 2022; 56(3): A. Editorial office Radiology and Oncology Zaloška cesta 2 P. O. Box 2217SI-1000 Ljubljana Slovenia Phone: +386 1 5879 369Phone/Fax: +386 1 5879 434 E-mail: gsersa@onko-i.si Copyright © Radiology and Oncology. All rights reserved. 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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 This journal is printed on acid- free paper On the web: ISSN 1581-3207 https://content.sciendo.com/raon http://www.radioloncol.com • Microsoft Academic • Naviga (Softweco) • Primo Central (ExLibris) • ProQuest (relevant databases) • Publons • PubMed • PubMed Central • PubsHub • QOAM (Quality Open Access Market) • ReadCube • Reaxys • SCImago (SJR) • SCOPUS • Sherpa/RoMEO • Summon (Serials Solutions/ProQuest) • TDNet • Ulrich's Periodicals Directory/ulrichsweb • WanFang Data • Web of Science - Current Contents/Clinical Medicine • Web of Science - Science Citation Index Expanded • WorldCat (OCLC) Radiol Oncol 2022; 56(3): B. contents contents review 267 Radiation therapy for melanoma brain metastases: a systematic review John F. Thompson, Gabrielle J. Williams, Angela M. Hong 285 Electrochemotherapy for solid tumors: literature review and presentation of a novel endoscopic approach Francesca Matilde Schipilliti, Maurizio Onorato, Giulia Arrivi, Martina Panebianco, Debora Lerin Annalisa Milano, Michela Roberto, Carlo Capalbo, Federica Mazzuca radiology 292 Portal hypertension may influence the registration of hypointensity of small hepatocellular carcinoma in the hepatobiliary phase in gadoxetic acid MR Carla Caparroz, Alejandro Forner, Jordi Rimola, Anna Darnell, Ángeles García-Criado, Juan Ram Ayuso, María Reig, Jordi Bruix, Carmen Ayuso 303 Early isolated subarachnoid hemorrhage versus hemorrhagic infarction in cerebral venous thrombosis Jan Kobal, Ksenija Cankar, Kristijan Ivanusic, Borna Vudrag, Katarina Surlan Popovic 311 Safety and efficacy of drug-eluting microspheres chemoembolization under cone beam computed tomography control in patients with early and intermediate stage hepatocellular carcinoma Spela Korsic, Nastja Levasic, Rok Dezman, Lara Anja Lesnik Zupan, Blaz Trotovsek, Rado Jansa, Alojz Smid, Peter Popovic 319 Cone-beam computed tomography guided nusinersen administrations in adult spinal muscular atrophy patients with challenging access: a single-center experience Vladka Salapura, Ziga Snoj, Lea Leonardis, Blaz Koritnik, Viktorija Kostadinova experimental oncology 326 Nanosecond electric pulses are equally effective in electrochemotherapy with cisplatin as microsecond pulses Angelika Vizintin, Stefan Markovic, Janez Scancar, Jerneja Kladnik, Iztok Turel, Damijan Miklavcic clinical oncology 336 Impact of AKT1 polymorphism on DNA damage, BTG2 expression, and risk of colorectal cancer development Hina Zubair, Zahid Khan, Muhammad Imran Radiol Oncol 2022; 56(3): C. contents 346 Real-life long-term outcomes of upfront surgery in patients with resectable stage I-IIIA non-small cell lung cancer Marko Bitenc, Tanja Cufer, Izidor Kern, Martina Miklavcic, Sabrina Petrovic, Vida Groznik, Aleksander Sadikov 355 Identification of women with high grade histopathology results after conisation by artificial neural networks Marko Mlinaric, Miljenko Krizmaric, Iztok Takac, Alenka Repse Fokter 365 Advancements in the radiooncological treatment of high-risk prostate cancer: a quarter century of achievements Matthias Moll, Harald Herrmann, Alexandru Zaharie, Gregor Goldner 371 Real-world outcomes, treatment patterns and T790M testing rates in non-small cell lung cancer patients treated with first-line first- or second-generation epidermal growth factor receptor tyrosine kinase inhibitors from the Slovenian cohort of the REFLECT study Nina Turnsek, Rok Devjak, Natalija Edelbaher, Ilonka Osrajnik, Mojca Unk, Dusanka Vidovic, Tina Jeric, Urska Janzic 380 Trends in treatment of childhood cancer and subsequent primary neoplasm risk Maja Cesen Mazic, Raoul C. Reulen, Janez Jazbec, Lorna Zadravec Zaletel 390 Abbreviated 13C-mixed triglyceride breath test for detection of pancreatic exocrine insufficiency performs equally as standard 5-hour test in patients after gastrectomy performed for gastric cancer Darko Siuka, Kristina Kumer, Borut Stabuc, David Stubljar, David Drobne, Rado Jansa 398 Treatment of skin tumors with intratumoral interleukin 12 gene electrotransfer in the head and neck region: a first-in-human clinical trial protocol Ales Groselj, Masa Bosnjak, Tanja Jesenko, Maja Cemazar, Bostjan Markelc, Primoz Strojan, Gregor Sersa I slovenian abstracts Radiol Oncol 2022; 56(3): D. 267 review Radiation therapy for melanoma brain metastases: a systematic review John F. Thompson1,2,3, Gabrielle J. Williams1,2, Angela M. Hong1,2,4 1 Melanoma Institute Australia, The University of Sydney, North Sydney, NSW, Australia 2 Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia 3 Royal Prince Alfred Hospital, Sydney, NSW, Australia 4 Chris O’Brien Lifehouse, Sydney, NSW, Australia Radiol Oncol 2022; 56(3): 267-284. Received 25 May 2022 Accepted 17 June 2022 Correspondence to: Prof. John Thompson, Melanoma Institute Australia, The University of Sydney, North Sydney, NSW, Australia. E-mail: John.thompson@melanoma.org.au Disclosure: JFT is the recipient of an Australian National Health and Medical Research Council Program Grant (APP1093017). He has received honoraria for advisory board participation from BMS Australia, MSD Australia, GSK and Provectus Inc, and travel and conference support from GSK, Provectus Inc and Novartis. AMH has received honoraria for advisory board participation from Bayer and Oncobeta. GJW has nothing to disclose. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Background. Radiation therapy (RT) for melanoma brain metastases, delivered either as whole brain radiation therapy (WBRT) or as stereotactic radiosurgery (SRS), is an established component of treatment for this condition. However, evidence allowing comparison of the outcomes, advantages and disadvantages of the two RT modalities is scant, with very few randomised controlled trials having been conducted. This has led to considerable uncertainty and inconsistent guideline recommendations. The present systematic review identified 112 studies reporting outcomes for patients with melanoma brain metastases treated with RT. Three were randomised controlled trials but only one was of sufficient size to be considered informative. Most of the evidence was from non-randomised studies, either specific treatment series or disease cohorts. Criteria for determining treatment choice were reported in only 32 studies and the quality of these studies was variable. From the time of diagnosis of brain metastasis, the median survival after WBRT alone was 3.5 months (IQR 2.4–4.0 months) and for SRS alone it was 7.5 months (IQR 6.7–9.0 months). Overall patient survival increased over time (pre-1989 to 2015) but this was not apparent within specific treatment groups. Conclusions. These survival estimates provide a baseline for determining the incremental benefits of recently intro­duced systemic treatments using targeted therapy or immunotherapy for melanoma brain metastases. Key words: radiation therapy; stereotactic radiosurgery; melanoma; brain metastases Introduction Brain metastases are common in patients with advanced-stage melanoma, with a 20%–30% inci­dence in the first year after diagnosis of Stage IV disease, a 30%–40% incidence by 3-years, and an incidence of up to 73% in autopsy series.1-3 For pa­tients with untreated, symptomatic brain metasta­ses, the reported average survival times range from several weeks to a few months.4,5 Patients who have melanoma brain metastases have a worse progno­sis than patients who have brain metastases from other solid tumours.6 The two main radiation therapy (RT) tech­niques used to treat melanoma brain metastases are whole brain radiotherapy (WBRT) and stereo-tactic radiosurgery (SRS). WBRT has largely fallen out of favour in recent times due to its apparently limited benefits while SRS has gained favour, es­pecially as modern imaging has enabled earlier identification of smaller lesions before they be­come symptomatic. Other treatment options for melanoma brain metastases include surgery and systemic therapy. Newer systemic therapies with immune check­point inhibitors and BRAF-targeted agents have shown considerable benefit in patients with meta­static melanoma7-9 and evidence is accumulating that they can effectively treat brain metastases.10,11 Combinations of surgery, RT and systemic therapy are now often used, sometimes sequentially, some­times concurrently. Many contemporary clinical management guidelines suggest multidisciplinary advice tailored for individual patients, given the complexity of treatment options and sequenc­ ing.12-15 Surgery can provide rapid symptomatic relief and may be the treatment of choice for sin­gle or few2-3 lesions or larger symptomatic metas­tases in surgically-accessible sites. SRS can be an alternative to surgical resection as a local therapy in patients with smaller metastases, multiple le­sions or surgically-inaccessible ones.16-18 Although WBRT was a common treatment in the past, it is used much less frequently today19,20, and is often reserved for patients whose brain metastases pro­gress during systemic therapy and who are not suitable for further surgery or SRS.21,22 The evidence base for assessing the efficacy of RT to treat melanoma brain metastases has been weak because few well-designed randomised con­trolled trials have been conducted. Clinical prac­tice guidelines have therefore been based largely on low-level evidence or consensus opinion and, as a result, recommendations vary considerably. Guidelines in the USA23 suggest that some patients should receive systemic therapy as their sole initial treatment modality with no need for brain-directed local therapy unless there is intracranial progres­sion, and advise that many patients will require a combined modality approach. European guide­lines24 recommend combination immunotherapy or targeted therapy as the preferred initial option and their consensus-based recommendations are to treat melanoma brain metastases with SRS, but with surgery when SRS is not possible, restricting WBRT to patients without systemic therapy or lo­cal therapy options. Australian guidelines13 pro­vide a practice point that concurs with European opinion about the use of systemic drug therapy and suggest that this be considered as first-line treatment in asymptomatic patients; the evidence-based recommendation, however, is for SRS to be considered in patients with single or few brain me­tastases, while WBRT may be used for palliation. Another practice point states that surgical resec­tion of brain metastases is recommended for me­tastases >1 cm in diameter in non-eloquent areas or for symptomatic metastases. The aim of this systematic review was to analyse the results of all published studies documenting the results of RT, without systemic immunothera­py or targeted therapy, as treatment for melanoma brain metastases. The review was prompted by the need to provide a benchmark for assessing the outcomes of upfront systemic therapy for patients with melanoma brain metastases. Materials and methods Terms covering melanoma, brain metastases and RT (WBRT or SRS) were used in the search strat­egy of Medline (1947 – 24 September 2020), Embase (1947 – 25 September 2020), the Cochrane Database of systematic reviews and the Cochrane Central Trials Registry (to 30 September 2020). Full details and results are provided in Supplementary Table 1. No language restrictions were used. Included stud­ies were those reporting outcomes in patients with melanoma brain metastases treated with RT. Studies reporting patients with a mixture of cancer types including melanoma were excluded, as were stud­ies of melanoma in which not all patients had brain metastases. Single case reports were also excluded, as were studies in which all patients received a com­bination of radiation and some form of contempo­rary systemic therapy (immune checkpoint inhibi­tors, BRAF –directed targeted therapies) without a radiation-only cohort.Non-contemporary systemic immunotherapies included interferon, interleu­kin, BCG vaccine, and non-contemporary systemic chemotherapies included temolozolmide, fotemus-tine, dacarbazine, razoxane, cisplatin and lomustine. Complete search results were imported into Endnote, duplicates were removed and references were coded for inclusion/exclusion with reasons. Those included in the review had their data extract­ed by one author (GW). Reference lists of identified studies and review articles were examined to iden­tify additional studies. Extracted data included ar­ticle identifiers, design features, inclusion criteria, method of diagnosis, patient characteristics, treat­ment details, follow-up duration, deaths, adverse events, survival data and details of recurrences or new intracranial lesions. Quality assessment was performed using a specific tool for cohort studies25 and the Cochrane collaboration risk of bias assess­ment for randomised controlled trials (RCTs).26 Descriptive statistics were generated using SPSS v2527 with medians and interquartile ranges (IQR), 269 TABLE 1. Studies of radiation treatment in patients with melanoma brain metastases Katz51 1981 US 1971–1980 63 • Treatment cohort • • • • Vlock52 1982 US 1970–1980 46 • Treatment cohort • • • • Byrne43 1983 US 1978–1980 80 • Treatment cohort • • • • Stridsklev53 1984 Norway 1973–1980 39 • Treatment cohort • • • • Choi (A)54 1985 US 1972–1977 194 • Treatment cohort • • • • Choi (B)55 1985 US 1972–1977 59 • Treatment cohort • • • • Ziegler56 1986 US 1972–1984 72 • Treatment cohort • • • • Rate44 1988 US 1980–1987 77 • Treatment cohort • • • • Hagen57 1990 US 1972–1987 35 • Treatment cohort • • • • Stevens38 1992 Australia 1982–1990 129 • Treatment cohort • • • • Somaza58 1993 US 1988–1992 23 • Treatment cohort • • • • Willner59 1995 Germany 1985–1993 30 • Disease cohort • • • • Isokangas40 1996 Finland 1980–1994 60 • Treatment cohort • • • • Skibber60 1996 US 1979–1991 34 • Treatment cohort • • • • Gieger36 1997 US 1992–1994 12 • Treatment cohort • • • • Gupta61 1997 UK 1991–1996 31 • Treatment cohort • • • • Grob62 1998 France 1993–1996 35 • Treatment cohort • • • • Sampson63 1998 US past 20 years 670 • Disease cohort • • • • Seung64 1998 US 1991–1995 55 • Treatment cohort • • • • Lavine65 1999 US 1994–1997 45 • Treatment cohort • • • • Kontsadoulakis66 2000 US 1970–1992 136 • Disease cohort • • • • Ellerhorst67 2001 US 1992–1995 87 • Treatment cohort • • • • Buchsbaum68 2002 US 1994–1998 74 • Disease cohort • • • • • Gonzalez­Martinez69 2002 US 1996–NS 24 • Treatment cohort • • • • Mingione70 2002 US 1989–1999 45 • Treatment cohort • • • • Noel71 2002 France 1994–2001 25 • Treatment cohort • • • • Yu72 2002 US 1994–1999 122 • Treatment cohort • • • • Zacest73 2002 Australia 1979–1999 147 • Treatment cohort • • • • Harrison74 2003 US 1990–1997 65 • Treatment cohort • • • • Conill75 2004 Spain 1997–2002 26 • Treatment cohort • • • • Fife37 2004 Australia 1985–2000 (also 1952– 1984) 686 (+ 451) • Disease cohort • • • • Meier76 2004 Switzerland 1966–2002 100 • Disease cohort • • • • • Morris77 2004 UK 1998–2003 102 • Treatment cohort • • ? ? • Radbill78 2004 US 1996–2001 51 • Treatment cohort • • • • Selek79 2004 US 1991–2001 103 • Treatment cohort • • • • Stone80 2004 US 1989–1999 83 • Disease cohort • • • • Koc81 2005 US 1999–2003 26 • Treatment cohort • • • • Panagiotou82 2005 Greece 1986–2001 64 • Disease cohort • • • Rhomberg83 2005 Austria 1982–2002 19 • Treatment cohort • • • • Christopoulou84 2006 UK 1998–2004 29 • Treatment cohort • • • • Gaudy­Marquesta85 2006 France 1997–2003 106 • Treatment cohort • • • • Conill86 2007 Spain 1997–2004 37 • Treatment cohort • • • • Mathieu87 2007 US 1987–2005 245 • Treatment cohort • • • • Samlowski32 2007 US 1999–2004 44 • Treatment cohort • • • • Raizer5 2008 US 1991–2001 355 • Disease cohort • • ? ? • Redmond88 2008 US 1998–2007 59 • Treatment cohort • • • • Carrubba89 2009 US 2002–2007 37 • Disease cohort • • ? ? • Ahmad90 2010 UK 2001–2009 65 • Treatment cohort • • • • Rades91 2010 Germany 1989–2008 51 • Treatment cohort • • • • Schild92 2010 US NS 7 (+ 53) Y+N Treatment cohort • • • • Staudt93 2010 Germany 1986–2003 265 • Disease cohort • • • • Davies34 2011 US 1986–2004 330 • Disease cohort • • ? ? • Eigentler94 2011 Germany 1986–2007 672 • Disease cohort • • ? ? • Liew95 2011 US 1987–2008 333 • Treatment cohort • • • • Skeie96 2011 Norway 1996–2006 77 • Treatment cohort • • • • Zakrzewski97 2011 US 2002–2008 89 • Disease cohort • • ? ? • Bernard98 2012 US 2004–2010 54 • Treatment cohort • • • • Hauswald33 2012 Germany 2000–2011 87 • Treatment cohort • • ? ? • Knisely35 2012 US 2002–2010 77 • Treatment cohort • • • • Koay99 2012 US 2005–2011 296 • Disease cohort • • ? ? • Lo100 2012 US 2000–2007 28 • Treatment cohort • • • • Salvati101 2012 Italy 1997–2007 84 • Treatment cohort • • ? ? • 271 Mathew102 2013 US 2008–2011 58 • Treatment cohort • • • • Miller103 2013 Germany 2000–2010 34 • Treatment cohort • • • • Partl31 2013 Austria 1988–2009 87 • Treatment cohort • • ? ? • Silk41 2013 US 2005–2012 70 • Treatment cohort • • ? ? • Zukauskaite104 2013 Denmark 1995–2009 80 • Treatment cohort • • ? ? • Dyer105 2014 US 2000–2010 147 • Treatment cohort • • • • Marcus106 2014 US 1998–2010 135 • Treatment cohort • • ? ? • Neal107 2014 US 2000–2009 129 • Treatment cohort • • • • Rades 108 2014 Germany 2000–2013 54 • Treatment cohort • • • • • Vecchio109 2014 Italy 1994–2010 115 • Disease cohort • • ? ? • Christ110 2015 US 2005–2011 103 • Treatment cohort • • • • Frakes111 2015 US 2008–2012 28 • Treatment cohort • • • • Hauswald112 2015 Germany 1990–2011 84 • Treatment cohort • • • • Ivanov113 2015 Russia 2009–2013 95 • Treatment cohort • • • • Ly114 2015 US 2009–2012 52 • Treatment cohort • • • • Ostheimer115 2015 Germany 1992–2011 100 • Treatment cohort • • ? ? • Gallaher116 2016 US since 2006 19 • Treatment cohort • • • • Gupta29 2016 UK NS 18 Yes RCT • • • • 2007–2014 Patel117 2016 US (abstract says 2005– 2013) 87 • Treatment cohort • • • • Rades118 2016 Germany 2000–2015 23 • Treatment cohort • • • • Szyszka-Chare39 2016 Poland 1985–2012 110 • Disease cohort • • ? ? • Wolf119 2016 US 2012–2015 80 • Treatment cohort • • • • Acharya120 2017 US 2006–2016 72 • Treatment cohort • • • • All121 2017 US 2008–2016 58 • Treatment cohort • • ? ? • Feng122 2017 US 2007–2014 87 • Treatment cohort • • • • • Kaidar-Person123 2017 US 2007–2015 58 • Treatment cohort • • • • Minniti124 2017 Italy 2008–2015 120 • Treatment cohort • • • • Patel125 2017 US 2009–2013 54 • Treatment cohort • • • • Pessina126 2017 Italy 2011–2015 53 • Treatment cohort • • ? ? • Sperduto127 2017 US 2006–2013 823/481 • Disease cohort • • ? ? • Xu128 2017 US 2010–2014 65 • Treatment cohort • • • • Diao(A) 129 2018 US 2006–2015 72 • Treatment cohort • • • • Diao(B) 130 2018 US 2006–2015 91 • Treatment cohort • • • • Fang131 2018 US 2005–2011 235 • Disease cohort • • ? ? • Gabani132 2018 US 2011–2013 1104 • Treatment cohort • • ? ? • Kano133 2018 US 1988–2012 422 • Treatment cohort • • • • Kotecha134 2018 US 1987–2014 366 • Disease cohort • • • • • Ladwa135 2018 Australia 2009–2016 142 • Disease cohort • • ? ? • Matsunaga136 2018 Japan 1991–2015 177 • Treatment cohort • • • • Tio137 2018 Australia 2011–2014 355 • Disease cohort • • ? ? • Zubatkina138 2018 Russia 2009–2014 78 • Treatment cohort • • • • Hauswald28 2019 Germany 2013–2017 7 Yes RCT • • • • Hong30 2019 Australia 2009–2017 215 Yes RCT • • ? ? • Jardim139 2019 Australia 2015–2017 43 • Treatment cohort • • • • Mastorakos140 2019 US 2011–2015 198 • Treatment cohort • • • • Phillips42 2019 Canada 2000–2018 277 NS Disease cohort • • ? ? • Tjong141 2019 Canada 2008–2017 97 • Treatment cohort • • ? ? • McHugh142 2020 New Zealand 2005–2017 110 • Treatment cohort • • ? ? • Pomeranz­Krumme143 2020 US 2010–2018 25 • Treatment cohort • • • • • GK = Gamma Knife methods; Non-contemp = non-contemporary systemic therapy; LA = linear accelerator; SRS = stereotactic radiosurgery; WBRT = whole brain radiation therapy stereotactic radiosurgery as data were not normally distributed. Medians were tested for difference using the non-para­metric median test, Fisher’s exact (2-sided). When three or more studies reported the same outcome for the same treatment group, data were pooled and analysed. Results Search results Search results and exclusions are shown in Figure 1. There were 142 publications between 1980–2020, 112 of which were unique studies or were the primary publication of a series of publica­tions, and 30 were duplicates or non-primary pub­lications (Table 1.) Seven studies were published only as abstracts. 57.1% (64/112) of the publica­tions were from the USA, 30.4% from Europe, and 11.6% from other countries. Sample sizes ranged from 7–1304 patients (median 77). While our focus was on RT, most articles (96/112) included patients who had received a variety of other therapies for their brain metastases. Surgery was reported in 79 studies, WBRT in 95 studies, SRS in 84 studies and 64 reports included subsets of patients who received some form of systemic therapy as well as RT. Outcomes for the subsets of patients treated with both RT and any form of contemporary sys­temic therapy were not analysed. Amongst studies reporting the use of SRS, five reported using both linear accelerator and Gamma Knife methods, 32 used Gamma Knife only, 18 used linear accelerator only and 29 did not report which was used. Only three studies were prospectively-conduct­ed RCTs; the remainder were retrospective studies of patient cohorts of specific treatment/s (n = 85) or cohorts of patients treated for melanoma brain me­tastases (n = 24). A comparison of outcomes for dif­ferent treatment regimens was reported in 97 stud­ies, while 15 studies were non-comparative, report­ing outcome data for a single treatment group. 273 Risk of bias assessment Details of the risk of bias assessment for each study are provided in Supplementary Table 2 and summarised in Figure 2. Of the three RCTs, none reported how their randomisation sequence was generated but all reported complete outcome data and clinically-relevant outcomes. Two of the three trials28,29 were closed early due to poor accrual and had sample sizes of 7 and 18, greatly limiting the reliability of their results. Baseline characteristics for the different treatment arms were reported and similar for the trial of 18 patients29 but were not re­ported for the trial of 7 patients.28 The largest trial30 had 215 patients, and while not stratifying for pre­vious treatments, randomisation was effective as previous treatments were well balanced between the two study arms, as were baseline characteris­tics. This trial provided the most reliable data for identifying the effects of adjuvant WBRT in con­junction with surgery and/or SRS for patients with 1–3 brain metastases. For the 109 cohort studies, selection bias was a significant concern, as 77 studies (71%) did not pro­vide information specifying how a treatment choice was made. Sixteen studies reported that there were no significant differences in patient characteris­tics such as age, number of brain metastases and tumour volume between treatment groups, while 11 studies reported significant differences between patients treated using different modalities. The remaining 82 studies did not report similarities or differences in patient characteristics, although in 21 studies the patient characteristics were pro­vided. Fifty-six studies did not report if or how the diagnosis of melanoma brain metastases was veri­fied; this may have resulted in misclassification of disease, although this is probably a relatively in­consequential source of bias. Many studies reported analyses of one treatment without considering prior and subsequent forms of treatment (e.g. SRS preceded by surgical resection). Our analysis was based on grouping data based on all treatments received for brain metastases. Treatment decisions Five studies (651 patients) included only asympto­matic patients, nine studies (532 patients) included only symptomatic patients, 30 studies (5906 pa­tients) had a mixture of asymptomatic and symp­tomatic patients and 57 studies (5452 patients) did not report this detail. Seven of the 95 studies provided specific crite­ria for choosing WBRT in their patients; four stated that it was used for multiple brain metastases31-34, three reported its use for progression of brain me­tastases31,35,36, one its use for single, large metasta­ses36 and one its use for symptomatic metastases.37 Nineteen studies reported criteria for choos­ing SRS. Twelve stated that it was used for small metastases, often <30mm in diameter, nine re­quired good performance status as measured by Karnofsky performance score (KPS), with four of these using a cut-off of KPS = 70. Seven studies used SRS for a small number of brain metastases (usually 1–3). Five studies used SRS when metas­tases were inaccessible for surgery, four used it in for multiple metastases, but only one specified a number (= 9), and three stated that it was used for asymptomatic lesions. Other infrequently used cri­teria were; expected survival > 3 months, non-life threatening lesions, high risk for surgery, includ­ing proximity to the brain stem or optic nerve. A single study32 reported criteria for using a combi­nation of SRS and WBRT, stating that this was used for = 5 lesions. Fourteen studies provided criteria for surgery; a single metastasis (5 studies), few or <3 metastases FIGURE 2. (A) Risk of bias assessments for randomised controlled trials evaluating radiation therapies in patients with melanoma brain metastases. (B) Quality assessment of cohort studies of patients with melanoma brain metastases treated with radiation therapy. (2 studies), accessible metastases (8 studies), symp­tomatic metastases (4 studies), stable extracranial disease (4 studies), good KPS (1 study), life expec­tancy > 3 months (1 study), and 2–3 brain metasta­ses if one was life-threatening (2 studies). Treatment groupings Many treatment groupings that included RT were reported but outcomes were not reported for all groups. Given the limited amount of data for clear­ly-defined treatment groups, we re-grouped data into two additional treatment options; (i) patients treated with WBRT and any of SRS, surgery or non-contemporary systemic therapy, and (ii) pa­tients treated with SRS and any of WBRT, surgery, or non-contemporary systemic therapy. Patient characteristics within treatment groups Patient characteristics within different treatment groups are summarised in Table 2. For all treat­ment types, there was a predominance of males. Patients treated with WBRT alone were somewhat younger than those receiving SRS and patients un­dergoing WBRT were less likely to have a single brain metastasis. While the data were sparse, there was considerable overlap in patient characteristics across different treatment modalities, indicating that the choice of treatment was not consistently determined by age, presence of symptoms, num­ber of metastases or control of primary disease. Median survival Ninety-six studies reported median survival for all patients or subsets of patients and there were 49 different treatment groupings. Within-study comparisons were possible for six treatment groupings (Table 3.). Eleven stud­ies reported median survival for patients treated with WBRT alone or with WBRT and surgery. The median survival in the WBRT alone group was 4.0 months (IQR 3.0–4.0 months), significantly less than for those treated with surgery and WBRT (11.0 months, IQR 8.8–11.8 months) (p = 0.002). Expressing these findings as a median difference between treatments, patients who had WBRT and surgery had a 5.4 month (IQR 4.6–8.0 months) longer survival compared with those treated with WBRT alone. In this group of 11 studies, three re­ported using surgery in patients with a single brain metastasis37-39, and two studies reported features for treatment with WBRT, this being good perfor­mance status alone in one study40 and multiple le­sions, good performance and symptoms in the sec­ond study.37 Significant differences in median sur­vival were also apparent between WBRT alone and surgery alone (6.0 months longer for surgery) and median survival for patients treated with WBRT plus SRS was 3.4 months longer than with WBRT alone. In the five studies with groups treated with WBRT alone or surgery alone, three reported that surgery was used for a single or few brain metas­tases34,37,38 and WBRT was used for patients with more than one brain metastasis (1 study)38 and for patients with good performance status (1 study37). None of the four studies reporting patients treated with WBRT alone or WBRT+SRS described features leading to these treatment choices. There were no significant differences in median survival between WBRT alone and SRS alone, or between WBRT alone and WBRT with chemotherapy, or SRS alone compared to WBRT with SRS. Summarised findings for median survival in all studies are detailed in Table 3 and for other group­ings in Supplementary Table 4. The group treated with WBRT alone had the shortest survival; 3.5 months (IQR 2.4–4.0 months). For the group treat­ed with surgery and WBRT, the median survival was 11.0 months (IQR 7.8–12.0 months). Adding chemotherapy to WBRT appeared to provide lit­ 275 TABLE 2. Patient characteristics within treatment group for the 51 studies that reported baseline characteristics WBRT alone SRS alone WBRT and any of SRS, surgery, chemotherapy, non-contemp SRS and any of; surgery, WBRT, chemotherapy, non-contemp Median IQR N studies(pts) Median IQR N studies(pts) Median IQR N studies(pts) Median IQR N studies(pts) 53.0 49.0–55.8 10 (295) 60.2 56.25–62.25 9 (444) 53.0 47.00–58.75 5 (243) 56.9 52.5 – 59.25 17 (1127) 63.8% 42.5–73.7 % 13 (496) 66.7% 54.0–76.3% 12 (822) 63.4% 51.64–73.88% 6 (266) 59.1% 54.82 – 68.04% 20 (1838) i11.5%, i32.2% NA 2 (85) 66.8% 59.65–78.79% 4 (359) --0 65.4% 51.28 – 66.38% 5 (953) 26.6% 18.5–48.6% 7 (339) 54.8% 41.19–61.22 9 (706) 51.7% 40.72–72.91% 4 (223) 38.8% 30.11 – 51.74% 16 (1697) 29.0% 13.9–47.8% 10 (329) 32.2% 26.16–36.32% 8 (669) 45.9% 30.18–68.06% 6 (262) 24.0% 17.56 – 39.70% 16 (1660) GK = Gamma Knife methods; non-contemp = non-contemporary systemic therapy; i = individual study data; IQR = interquartile range; NA = not applicable; SRS = stereotactic radiosurgery; WBRT = whole brain radiation therapy stereotactic radiosurgery tle benefit, with six studies of 137 patients report­ing a median survival of 4.3 months (IQR 2.8–6.0 months) (Supplementary Table 4). The compiled grouping of WBRT with any of SRS, surgery, or non-contemporary systemic therapy had a median survival of 7.2 months (IQR 4.6–9.4 months) across 47 studies with 2230 patients. Median survival after SRS treatment alone was reported in eight studies, giving a median survival of 7.5 months (IQR 6.7–9.0 months). The median survival for patients treated with Gamma Knife SRS (5 studies, 208 patients) was 7.0 months (IQR 5.6–7.8 months) and for the three studies (980 pa­tients) that did not report which SRS technology was used the median survival was 8.8 months. Eighteen studies reported using linear accelera­tor SRS but none reported the median survival for patients treated with SRS alone. Adding non-contemporary systemic therapy to SRS treatment did not improve the median survival (7.9 months, IQR 6.1–9.9 months) but the addition of surgery was associated with an increase in median sur­vival of around 5 months (13.0 months, IQR 9.4, 13.5 months). Compiled grouping of SRS with any of WBRT, surgery, or non-contemporary systemic therapy gave a median survival of 8.0 months (IQR 6.2–10.9 months) over 42 studies involving 2702 patients. In the only completed RCT in patients with mel­anoma brain metastases30, median survival in the group treated with adjuvant WBRT after definitive local treatment of 1–3 metastases was 16.5 months (95% CI 13–24 months) compared to 13.0 months (95% CI 10–19 months) for those that did not re­ceive adjuvant WBRT (p=0.86). No studies reported median survival within treatment groups separately for asymptomatic and symptomatic patients. For the 5 studies that in­cluded only asymptomatic patients, there were no common treatment groups. In 4 studies of 89 symp­tomatic patients, the median survival for the treat­ment group WBRT with surgery was 9.2 months (IQR 5.4–12.8 months) and for WBRT with any other treatment; 5 months (IQR 2.5–10.0 months, 7 studies of 245 patients). Median survival in different time periods Median survival over time was explored by group­ing the data into three time periods based on the first year of patient recruitment within each study (Table 4). Eighty-one studies reported median sur­vival for their whole cohort irrespective of treat­ment, and these showed increasing survival over the years. There were significant differences in median survival between the pre-1989 group com­pared with the 1990–2002 (p = 0.017) and 2003–2015 groups (p = 0.002) and also between the groups first treated in 1990–2002 compared with 2003–2015 (p = 0.021). Median survival within treatment groups over the three time periods showed a trend toward slightly increased survival in more recent years, but none of the differences was statistically signifi­cant. One-year survival Fifty-six studies reported 1-year survival for all patients or subsets of patients (Table 3). Pooled 276 TABLE 3. Pooled outcome results for studies of radiation treatments WBRT vs. WBRT + Surgery Non-random, comparative WBRT WBRT+Surg 11 (980) 11 (439) 4.0 (3.0, 4.0) 11.0 (8.8,11.8) 0 0 -- 0 0 -- 0 0 -- Not reported All studies WBRT WBRT+Surg 26 (2185) 16 (619) 3.5 (2.4, 4.0) 11.0 (7.8, 12.0) 7 (189) 1 (19) 9.0 (0.0, 22.5) i41.0 1 (74) 0 5.5 (0.0, 12.0) 0 0 -- Post-op death; 2% (1 study), Hemorrhage; 3/72 lesions (1 Study) WBRT vs. Surgery Non-random, comparative All studies WBRT Surgery Surgery 5 (699) 5 (234) 9 (359 3.9 (3.6, 5.0) 9.8 (7.6, 16.5) 8.7 (6.2, 10.4) 0 0 1 (16) --i36.0 0 0 0 --- 0 0 0 --- -Gr3 tox; 3/39 (1 study) Post-op death; 2% (1 Study) WBRT vs. SRS Non-random, comparative All studies SRS Type WBRT SRS SRS SRS-GK SRS-LA SRS-NS 3 (931) 3 (980) 8 (1188) 5 (208) 0 3 (980) 4.1 (3.2, 5.6) 8.8 (7.2, 11.4) 7.5 (6.7, 9.0) 7.0 (5.6, 7.8) -8.8 (7.2, 11.4) 0 0 6 (330) 1 (83) 0 0 --35.5 (20.8, 47.8) i26.0 -- 0 0 4 (260) 0 0 0 --76.0 (62.8, 88.5) --- 0 0 0 0 0 0 ------ -Hemorrhage; 4/56 lesions (1 study) (SRS-GK) WBRT vs. WBRT + Chemotherapy Non-random, comparative All studies WBRT WBRT +Chemo WBRT+Chemo 4 (148) 4 (62) 6 (137) 2.5 (1.0, 4.2) 5.5 (4.0, 6.0) 4.3 (2.8, 6.0) 0 1 (7) 2 (15) -i0.0 i0.0, i37.0 0 0 0 --- 0 0 0 --- Gr3 tox; 3/39 (1 study) Leukopenia; 2/8 (1 study), Toxicity; 9/14 (1 study) SRS vs. WBRT+SRS Non-random, comparative All studies SRS WBRT+SRS WBRT+SRS 5 (881) 5 (344) 12 (516) 7.0 (6.0, 8.1) 6.5 (5.7, 6.5) 7.0 (6.0, 8.0) 1 (83) 1 (39) 3 (58) i26.0 i23.0 36.0 (29.5, 37.0) 0 0 0 --- 0 0 0 --- Swelling requiring surgical decompression; 3/77 pts (1 study) - WBRT vs. WBRT+SRS Non-random, comparative WBRT WBRT+SRS 4 (337) 4 (197) 3.6 (2.7, 5.0) 7.4 (6.5, 10.7) 1 (59) 1 (8) i10.0 i38.0 0 0 -- 0 0 -- -- SRS+ Chemotherapy Al studies SRS+Chemo SRS+/- Chemo 1 (23) 7 (580) i6.5 7.9 (6.1, 9.9) 0 2 (358) -i13.2, i27.9 0 1 (106) -i69.0 0 1 (106) -i12.0 -Hemorrhage; 1/106 pt (1 study), 4/56 lesions (1 study). Radiation necrosis; 1/106 pts (1 study) SRS + Surgery All studies SRS+Surg 4 (200) 13 (9.4, 13.5) 1 (60) i58.0 1 (34) i52.0 1 (34) i32.0 Hemorrhage; 18% (1 study) WBRT+ other treatments All studies WBRT and any of surgery, SRS, non-contemp 47 (2230) 7.2 (4.6, 9.4) 19 (827) 21.4 (13.6, 37.0) 5 (208) 1.0 (0.0, 16.0) 8 (986) 46.5 (39.8, 55.5) WBRT specific; Deaths;6/194 (1 study), headache; 12/26 (1 study), Toxicity > Gr3; 3/7 (1 study) LeukopeniaGr1-2; 2/9 (1 study) Hemorrhage; 1/20 (1 study) SRS+ other treatments All studies SRS and any of surgery, WBRT, non-contemp 42 (2702) 8.0 (6.2, 10.9) 35 (2644) 31.0 (25.0, 39.0) 16 (1043) 69.0 (60.0, 82.0) 10 (1261) 49.0 (42.0, 56.0) SRS specific; Hemorrhage; 14% (4 studies, 441 patients) Radiation necrosis; 6.6% (4 studies, 241 patients Seizure-edema- death; 1/55 (1 study) Complications; 6/106 (1 study) Chemo = chemotherapy; GK = Gamma Knife methods; Gr = grade; i = individual study data; IQR = interquartile range; LA = linear accelerator; non-contemp = non contemporary systemic therapy; SRS = stereotactic radiosurgery; Surg = surgery; WBRT = whole brain radiation therapy 277 TABLE 4. Median survival within treatment groups and grouped by the first year of patient recruitment All patients 25 4.8 3.25–8.05 33 6.0 4.35–8.00 22 9.2 6.90–11.43 1 i3.0 NA WBRT alone 17 3.6 2.49–4.0 7 2.5 2.3–4.0 6 4.2 2.75–4.80 3 4.3 3.40–6.40 SRS alone 2 i6.4, i7.7 4 7.3 5.78–7.88 2 i10.0, i11.9 0 WBRT and any of surgery, SRS, non-contemporary 43 7.4 4.00–9.20 23 7.3 5.50–10.00 4 8.0 5.73–10.50 2 i3.6, i4.3 systemic therapy SRS and any of surgery, WBRT, non-contemporary 17 8.3 5.90–9.65 29 7.9 5.85–10.04 18 9.0 6.90-13.00 0 systemic therapy i = individual study data; IQR = interquartile range; NA = not applicable; SRS = stereotactic radiosurgery; WBRT = whole brain radiation therapy data from 7 studies of 189 patients treated with WBRT alone gave a 1-year survival rate of 9.0% (IQR 0.0–22.5%) while for SRS alone the 1-year survival was 35.5% (IQR 20.8–47.8%, p = 0.041) in six studies of 330 patients. In the compiled group­ing of WBRT with or without any other therapy, the 1-year survival was 21.4% (IQR 13.6%–37.0%). For the SRS grouping with or without any other therapy (WBRT, surgery, non-contemporary sys­temic therapy), the 1-year survival was 31.0% (IQR 25.0–39.0%) across 35 studies of 2644 patients. In the only completed RCT30, 1-year survival in the group treated with adjuvant WBRT was 58.4% (95%CI 49.6%–68.9%) compared with 54% (95%CI 45.3%–64.3%, p = 0.89) for those treated without WBRT. Local control Most studies defined local control as a reduction in metastasis size or stability of metastasis size, as de­termined by follow-up imaging. Fifty-three studies reported local control data, 21 without a defined time frame and for almost all it was reported for the total patient group, not separately for differ­ent treatment groupings. The 1-year local control rate was highest for those treated with SRS; 76% (IQR 62.8%–88.5%). The 1-year local control rate after WBRT was 5.5% (IQR 0.0%–12.0%, 1 study, 74 patients). For the 550 patients in 11 studies that treated patients with any combination of WBRT, SRS, surgery and non-contemporary systemic ther­apy, the 1-year local control rate was 68.0% (IQR 66.072.0%). There was no difference in the 1-year local control rate between Gamma Knife SRS and linear accelerator-based SRS (69% vs. 72.0%). New brain lesions Thirty-six studies reported rates of new brain le­sions developing during the follow-up period. For the 23 studies that reported new brain lesions at 6-months the median rate was 44% (IQR 32.0%– 53.0%) and at 12-months 67% (IQR 62.3%–71.5%, 14 studies). Three studies (189 patients) reported a 6-month new brain lesion rate in patients treated with WBRT and other treatment, giving a median rate of 39% (IQR 34.0%–44.5%) and for SRS and other treatment a rate of 47% (IQR 34.5%–55.5%, 11 studies, 1306 patients). At 12 months, the RCT of patients with 1–3 brain metastases reported a new brain lesion rate of 42% in the adjuvant WBRT group and 50.5% in those who did not receive ad-juvant WBRT (p = 0.22). For patients treated with SRS, the proportion who developed a new brain le­sion by 12 months was 67% (IQR 57.0%–75.0%; 11 studies, 1278 patients). Neurologic deaths Neurologic death was reported in 40 studies but only 11 reported this for a defined treatment group. The definition of neurologic death was variable. Only one study provided a definition that combined an objective measurement with ra­diological and clinical neurologic changes.41 Other studies used brain lesion progression and/or recur­rence (18 studies), brain hemorrhage alone (4 stud­ies), neurologic dysfunction alone (4 studies) or other features (3 studies) as criteria for designating a death as neurologic. The reported proportion of patients with a neurologic cause of death ranged from 0% to 90%. Three studies reported the propor­tion of patients treated with WBRT with or without surgery who experienced neurologic death (0%, 24%, 83%). Two studies reported neurologic deaths for patients treated with WBRT alone (14%, 88%), two studies reported on SRS alone (41%, 90%), two studies reported neurologic deaths in those treated with SRS and WBRT (50%, 58%) and two studies reported neurologic death in patients treated with WBRT with or without systemic therapy (57%, 75%). Re-grouping the data into SRS with any other treatment (6 studies), gave a median neuro-logic death rate of 53% (IQR 44.8%–69.4%), while for WBRT and any other treatment (10 studies), the median was 50% (IQR 19.3%–62.3%). Effect of number of brain metastases on survival Seventy-six studies assessed whether the number of brain metastases present at diagnosis impacted survival, with 58 reporting significantly improved survival for patients with single lesions while 18 reported no impact. In the RCT30, the number of brain metastases (1 vs. 2–3) did not influence over­all survival. Only three studies reported these data within specific treatment groups.42-44 Two stud­ ies43,44 reported survival in patients treated with WBRT alone comparing those with one metasta­sis to those with = 2 brain metastases; one study44 reported better survival in the single metastasis group (16 weeks) compared to the multiple metas­tases group (12 weeks) while the other study43 did not (9 weeks for a single metastases and 11 weeks for multiple metastases). Adverse effects of radiation therapy Adverse effects of RT were reported in 41 studies, but only 17 reported events within treatment groups and these were primarily studies that included sys­temic therapy. Radiation necrosis (with various ra­diological and/or pathological definitions) was re­ported in 13 studies, 11 of which focussed on SRS. The median rate was 8.1% (IQR 3.4%–22.2%). In the four studies using Gamma Knife SRS, the median radiation necrosis rate was 3.4% (IQR 0.47%–5.49%) and for the 4 studies using linear accelerator SRS it was 22.2% (IQR 15.59%–25.66%). Two studies re­ported this for WBRT, with rates of 1.9% and 3.6%. Eight studies reported intracranial haemorrhage in their patients, with seven studies focussed on SRS, giving a median rate of 14.7% (IQR 0.94–18.8%). Three studies using Gamma Knife SRS reported brain haemorrhage rates with a median rate of 18.8% (IQR 9.86–24.01%) and two studies used lin­ear accelerator SRS, with brain haemorrhage rates of 15% and 16%. Other reported adverse effects in­cluded headaches, seizures, skin reactions, fatigue, nausea, alopecia and confusion but because data were sparse and pooled analysis was not possible. Discussion For unbiased comparisons of an intervention, pro­spective randomised controlled trials are required. Although RT has long been used in the manage­ment of patients with melanoma brain metastases, there have been only three randomised trials of RT for this condition, and only one of these30 re­cruited sufficient patients for meaningful analysis. However, a large number of non-randomised stud­ies (n = 109) have published outcomes for patients with melanoma brain metastases treated with vari­ous RT regimens. The number of patients in each study varied, but most (86%) had fewer than 200 patients and medians of 20–30 for different treat­ment groups. This low number of patients per treatment group reduces the precision of estimates of survival duration within each study but when pooled over many studies, greater precision can be achieved. These non-randomised studies were of variable quality with multiple study design fea­tures poorly reported, hindering our understand­ing of how patients were selected for the stud­ies and how representative they were. Over the 40-year period encompassed by this review there was a consistent trend towards improvement in the median survival of patients with melanoma brain metastases. This is likely due to earlier diagnosis of small brain metastases using newer imaging tech­nologies, as well as a general improvement in treat­ment. However, we were unable to demonstrate an improvement in median survival within treatment groups over time, possibly due to a paucity of data for individual treatment groups. Within-study comparisons were possible for on­ly six treatment groupings. These analyses demon­strated significantly longer median survival times for patients who were treated with surgery alone (+6 months), WBRT and surgery (+7 months) and WBRT and SRS (+4 months) compared to those treated with WBRT alone (4 months). The better survival after surgery or SRS than after WBRT is al­most certainly due mainly to selection issues since patients with fewer lesions, better performance sta­tus and a lower burden of extracranial disease were more likely to receive surgery or SRS and these fea­ 279 tures are associated with improved survival. The benefit of within-study comparisons is the pres­ence of a “control” group in the same study, mean­ing that treatment decisions, management and out­come assessment were likely to be more consistent than comparisons with studies performed at differ­ent institutions and at different times. Many treatment groups were not represented in the within-study comparisons and were therefore reviewed across studies to provide estimates of median and 1-year survival rates for major treat­ment groupings. Patients treated with WBRT alone had a median survival of only 3.5 months, while those treated with SRS had a median survival of 7.5 months. Data were somewhat limited but sug­gest that linear accelerator-based SRS resulted in similar local control rates as Gamma Knife-based SRS. This is a reassuring finding as there is no ran-domised comparison of different SRS techniques for brain metastases. The combination of surgical removal of the lesion/s and WBRT was associated with substantially improved median survival, ap­parently adding 7.5 months of life, with median survival 11.0 months. These across-study median survival estimates are reassuringly consistent with the within-study findings. These findings, however, conflict with those of the randomised controlled trial that showed no survival gain and no improvement in intracranial control or perfor­mance status with adjuvant WBRT after adequate local treatment of 1–3 brain metastases.30 This may be because about one third of the patients in the RCT also received SRS, which may have enhanced survival and limits our ability to compare their out­comes with those of patients treated with WBRT and surgery but no SRS. Median survival for patients treated with sur­gery and SRS also showed benefit (+5.5 months), with a median survival of 13 months. Again, this is likely attributable to selection of patients with few­er metastases for surgery and SRS. Importantly, the data confirmed a lack of any survival benefit from the addition of non-contemporary systemic chemotherapy or non-contemporary forms of im­munotherapy. Limitations Risk of bias assessment for these studies showed that many of the non-randomised studies included patients who were treated without explanation of how treatment choices were made. In the 30% of studies that did report treatment selection criteria there was considerable variation, reflecting the di­versity of clinical practice between and even within individual centres and over the 40-year study pe­riod. This selection bias limited our ability to apply results to specific patient groups as we could not be sure in many instances which types of patients received particular treatments. Also, important prognostic factors such as performance status and extent of extracranial disease were rarely reported within treatment groups. Compiling the rather limited patient characteristics data for the differ­ent treatment groups showed that there was con­siderable overlap in the types of patients receiving WBRT and SRS. There was a degree of consistency in offering surgery to patients with a single or few brain metastases, as almost half of the studies that reported criteria for surgery stated this. However, it was not possible to determine survival outcomes for patients who underwent surgery for a single brain metastasis followed by RT as this was not reported. Most studies that analysed the effect on survival of having a single versus multiple brain metastases, irrespective of other treatments, re­ported improved survival with a single metastasis. This suggests that patients who undergo surgery have a greater likelihood of increased survival at baseline. A valid comparison of different RT mo­dalities should consider or control for factors that have a major impact on survival, an issue not pos­sible to evaluate using the current evidence. Further difficulties arose in relation to the multi­tude of different outcomes reported that could not be easily combined. For example, median, 6-month, 1 and 2-year survival rates were often reported but recurrence/regrowth at a treated site versus new le­sions at new sites were often not clearly specified within treatment groups or time frames. Similarly, the definitions of neurologic death varied between studies. Only one study provided a robust, meas­urable definition of this while others relied on less precise features. Definitions of radiation necrosis were also variable, provided in only eight stud­ies, each of which was different; three relied solely on various imaging features, one solely on clinical signs of bleeding and four on combined imaging features and clinical signs. Radiation necrosis and neurologic death are important endpoints being measured in current clinical trials and an assur­ance of similar definitions and measurements will greatly aid interpretation of these outcomes across studies. A possible solution to the diverse and var-iably-defined outcomes in studies would be for cli­nicians, researchers and patients to agree on a min­imum required and consistently-defined outcome reporting set, as has been done for other diseases 280 such as rheumatoid arthritis, ulcerative colitis, and lung cancer.45-47 Researchers have developed a pro­cess for selecting outcomes of interest to clinicians and patients, and deciding how these can be im­plemented in their respective settings.48,49 A similar strategy for future studies of patients with mela­noma brain metastases would be feasible. Few studies reported whether the treatments resulted in relief of symptoms for symptomatic patients. Australian guidelines suggest that WBRT may be considered in a palliative setting for relief of symptoms, and there are many anecdotal re­ports of its value in this situation, but we found lit­tle reported evidence to support the effectiveness of this option. Use of treatment groupings was a substan­tial limitation to interpretation, as many studies grouped together patients who received different treatment combinations. Ideally more uniform treatment groups should be used but this would require studies of much greater size to achieve ad­equate numbers within each group. Conclusions One randomised trial and many observational studies have reported survival outcomes for pa­tients treated with RT for melanoma brain metasta­ses. WBRT alone and SRS alone resulted in median survival times of about 4 and 8 months respective­ly. For patients who were selected to have surgery in addition to RT, there was a 5–7-month improve­ment in survival, however, this likely reflects the tendency to select patients with a better baseline prognosis relative to patients not offered surgery. While most studies included in this review were not optimal for determining the efficacy of an inter­vention, they provide the only evidence currently available. Given the improved efficacy of newer systemic therapies in the treatment of metastatic melanoma, RT alone today has a diminished role in the management of melanoma brain metastases, and large-scale trials or cohort studies of RT alone would be considered unethical. Therefore, this sys­tematic review of the various forms of RT with or without surgery provides baseline estimates for measuring the incremental benefits of contempo­rary systemic therapies over RT with or without surgery in the treatment of patients with melano­ma brain metastases. Acknowledgments Financial support was provided by an Australian National Health and Medical Research Council Program Grant (APP1093017) to JFT and by Melanoma Institute Australia. References 1. de la Monte SD, Moore GW, Hutchins GM. Patterned distribution of metas­tases from malignant melanoma in humans. Cancer Res 1983; 43: 3427-33. PMID: 6850649 2. Amer MH, Al-Sarraf M, Baker LH, Vaitkevicius VK. 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Potential role for LINAC-based stereotactic radiosurgery for the treatment of 5 or more radioresistant melanoma brain metastases. J Neurosurg 2015; 123: 1261-7. doi: 10.3171/2014.12.JNS141919 112. Hauswald H, Stenke A, Debus J, Combs SE. Linear accelerator-based ste­reotactic radiosurgery in 140 brain metastases from malignant melanoma. BMC Cancer 2015; 15: 537. doi: 10.1186/s12885-015-1517-1 113. Ivanov PI, Zubatkina IS, Tumarova DI, Andreev GI, Volkov NM. [Analysis of results of Gamma knife radiosurgery for patients with melanoma brain metastases]. [Russian]. Voprosy onkologii 2015; 61: 586-91. doi: ...616.5­616-831-006;615.47.616-072.7 114. Ly D, Bagshaw HP, Anker CJ, Tward JD, Grossmann KF, Jensen RL, et al. Local control after stereotactic radiosurgery for brain metastases in patients with melanoma with and without BRAF mutation and treatment. J Neurosurg 2015; 123: 395-401. doi: 10.3171/2014.9.JNS141425 115. Ostheimer C, Bormann C, Fiedler E, Marsch W, Vordermark D. Malignant melanoma brain metastases: treatment results and prognostic factors - a single-center retrospective study. Int J Oncol 2015; 46: 2439-48. doi: 10.3892/ijo.2015.2970 116. Gallaher IS, Watanabe Y, DeFor TE, Dusenbery KE, Lee CK, Hunt MA, et al. BRAF mutation is associated with improved local control of melanoma brain metastases treated with Gamma knife radiosurgery. Front Oncol 2016; 6: 107. doi: 10.3389/fonc.2016.00107 117. Patel BG, Ahmed KA, Johnstone PA, Yu HH, Etame AB. Initial experience with combined BRAF and MEK inhibition with stereotactic radiosurgery for BRAF mutant melanoma brain metastases. Melanoma Res 2016; 26: 382-6. doi: 10.1097/CMR.0000000000000250 118. Rades D, Sehmisch L, Bajrovic A, Janssen S, Schild SE. Comparison of 20x2 Gy and 12x3 Gy for whole-brain irradiation of multiple brain metastases from malignant melanoma. In Vivo 2016; 30: 917-9. doi: 10.21873/in­vivo.11013 119. Wolf A, Zia S, Verma R, Pavlick A, Wilson M, Golfinos JG, et al. Impact on overall survival of the combination of BRAF inhibitors and stereotactic radiosurgery in patients with melanoma brain metastases. J Neurooncol 2016; 127: 607-15. doi: 10.1007/s11060-016-2072-6 120. Acharya S, Mahmood M, Mullen D, Yang D, Tsien CI, Huang J, et al. Distant intracranial failure in melanoma brain metastases treated with stereotactic radiosurgery in the era of immunotherapy and targeted agents. Adv Radiat Oncol 2017; 2: 572-80. doi: 10.1016/j.adro.2017.07.003 121. All S, Patel H, Keller A, Ramakrishna NR. Initial volumetric response and local control following stereotactic radiosurgery for melanoma brain metastases. Int J Radiat Oncol Biol Phys 2017; 99 (2 Suppl 1): E62-3. doi: 10.1016/j.ijrobp.2017.06.739 122. Feng R, Oermann EK, Shrivastava R, Gold A, Collins BT, Kondziolka D, et al. Stereotactic radiosurgery for melanoma brain metastases: a compre­hensive clinical case series. World Neurosurg 2017; 100: 297-304. doi: 10.1016/j.wneu.2017.01.014 284 123. Kaidar-Person O, Zagar TM, Deal A, Moschos SJ, Ewend MG, Sasaki-Adams D, et al. The incidence of radiation necrosis following stereotac-tic radiotherapy for melanoma brain metastases: the potential impact of immunotherapy. Anticancer Drugs 2017; 28: 669-75. doi: 10.1097/ CAD.0000000000000497 124. Minniti G, Paolini S, D’Andrea G, Lanzetta G, Cicone F, Confaloni V, et al. Outcomes of postoperative stereotactic radiosurgery to the resection cav­ity versus stereotactic radiosurgery alone for melanoma brain metastases. J Neurooncol 2017; 132: 455-62. doi: 10.1007/s11060-017-2394-z 125. Patel KR, Shoukat S, Oliver DE, Chowdhary M, Rizzo M, Lawson DH, et al. Ipilimumab and stereotactic radiosurgery versus stereotactic radiosurgery alone for newly diagnosed melanoma brain metastases. Am J Clin Oncol 2017; 40: 444-50. doi: 10.1097/COC.0000000000000199 126. Pessina F, Navarria P, Tomatis S, Cozzi L, Franzese C, Di Guardo L, et al. Outcome evaluation of patients with limited brain metastasis from malignant melanoma, treated with surgery, radiation therapy, and tar­geted therapy. World Neurosurg 2017; 105: 184-90. doi: 10.1016/j. wneu.2017.05.131 127. Sperduto PW, Jiang W, Brown PD, Braunstein S, Sneed P, Wattson DA, et al. The prognostic value of BRAF, C-KIT, and NRAS mutations in melanoma patients with brain metastases. Int J Radiat Oncol Biol Phys 2017; 98: 1069­77. doi: 10.1016/j.ijrobp.2017.03.030 128. Xu Z, Lee CC, Ramesh A, Mueller AC, Schlesinger D, Cohen-Inbar O, et al. BRAF V600E mutation and BRAF kinase inhibitors in conjunction with ste­reotactic radiosurgery for intracranial melanoma metastases. J Neurosurg 2017; 126: 726-34. doi: 10.3171/2016.2.JNS1633 129. Diao K, Bian SX, Routman DM, Yu C, Kim PE, Wagle NA, et al. Combination ipilimumab and radiosurgery for brain metastases: tumor, edema, and adverse radiation effects. J Neurosurg 2018; 129: 1397-406. doi: 10.3171/2017.7.JNS171286 130. Diao K, Bian SX, Routman DM, Yu C, Ye JC, Wagle NA, et al. Stereotactic radiosurgery and ipilimumab for patients with melanoma brain metas­tases: clinical outcomes and toxicity. J Neurooncol 2018; 139: 421-9. doi: 10.1007/s11060-018-2880-y 131. Fang P, Boehling NS, Koay EJ, Bucheit AD, Jakob JA, Settle SH, et al. Melanoma brain metastases harboring BRAF V600K or NRAS mutations are associated with an increased local failure rate following conventional therapy. J Neurooncol 2018; 137: 67-75. doi: 10.1007/s11060-017-2695-2 132. Gabani P, Fischer-Valuck BW, Johanns TM, Hernandez-Aya LF, Keller JW, Rich KM, et al. Stereotactic radiosurgery and immunotherapy in melanoma brain metastases: patterns of care and treatment outcomes. Radiother Oncol 2018; 128: 266-73. doi: 10.1016/j.radonc.2018.06.017 133. Kano H, Morales-Restrepo A, Iyer A, Weiner GM, Mousavi SH, Kirkwood JM, et al. Comparison of prognostic indices in patients who undergo mela­noma brain metastasis radiosurgery. J Neurosurg 2018; 128: 14-22. doi: 10.3171/2016.9.JNS161011 134. Kotecha R, Miller JA, Venur VA, Mohammadi AM, Chao ST, Suh JH, et al. Melanoma brain metastasis: the impact of stereotactic radio-surgery, BRAF mutational status, and targeted and/or immune-based therapies on treatment outcome. J Neurosurg 2018; 129: 50-9. doi: 10.3171/2017.1.JNS162797 135. Ladwa R, Atkinson V. The changing paradigm of management in melanoma brain metastases. Asia Pac J Clin Oncol 2018; 14: 453-8. doi: 10.1111/ ajco.12998 136. Matsunaga S, Shuto T, Yamamoto M, Yomo S, Kondoh T, Kobayashi T, et al. Gamma Knife radiosurgery for metastatic brain tumors from malignant melanomas: a Japanese multi-institutional cooperative and retrospective cohort study (JLGK1501). Stereotac Funct Neurosurg 2018; 96: 162-71. doi: 10.1159/000489948 137. Tio M, Wang X, Carlino MS, Shivalingam B, Fogarty GB, Guminski AD, et al. Survival and prognostic factors for patients with melanoma brain metas­tases in the era of modern systemic therapy. Pigment Cell Melanoma Res 2018; 31: 509-15. doi: 10.1111/pcmr.12682 138. Zubatkina I, Ivanov P. Early imaging radioresponsiveness of melanoma brain metastases as a predictor of patient prognosis. J Neurosurg 2018; 129: 354-65. doi: 10.3171/2017.1.JNS162075 139. Jardim A, Scott J, Drew Z, Foote MC, Sadasivan AP, Hall B, et al. Extent of surrounding edema does not correlate with acute complications after ra­diosurgery for melanoma brain metastases. Neurooncol 2019; 145: 581-5. doi: 10.1007/s11060-019-03330-9 140. Mastorakos P, Xu Z, Yu J, Hess J, Qian J, Chatrath A, et al. BRAF V600 muta­tion and BRAF kinase inhibitors in conjunction with Stereotactic radiosur­gery for intracranial melanoma metastases: a multicenter retrospective study. Neurosurgery 2019; 84: 868-80. doi: 10.1093/neuros/nyy203 141. Tjong MC, Moraes FY, Shultz D. Intracranial control and radionecrosis in melanoma patients with brain metastases treated with stereotactic radiosurgery. Radiother Oncol 2019; 139(Suppl 1): S59-60. doi: 10.1016/ S0167-8140%2819%2933190-1 142. McHugh FA, Kow CY, Falkov A, Heppner P, Law A, Bok A, et al. Metastatic melanoma: surgical treatment of brain metastases - Analysis of 110 pa­tients. J Clin Neurosci 2020; 73: 144-9. doi: 10.1016/j.jocn.2019.12.063 143. Pomeranz Krummel DA, Nasti TH, Izar B, Press RH, Xu M, Lowder L, et al. Impact of sequencing radiation therapy and immune checkpoint inhibitors in the treatment of melanoma brain metastases. Int J Radiat Oncol Biol Phys 2020; 108: 157-63. doi: 10.1016/j.ijrobp.2020.01.043 285 review Electrochemotherapy for solid tumors: literature review and presentation of a novel endoscopic approach Francesca Matilde Schipilliti1, Maurizio Onorato1, Giulia Arrivi1, Martina Panebianco1, Debora Lerin1, Annalisa Milano1, Michela Roberto2, Carlo Capalbo1, Federica Mazzuca1 1 Oncological Department, Sant’Andrea Hospital, University Sapienza in Rome, Rome, Italy 2 Oncological Department, University Sapienza in Rome, Rome, Italy Radiol Oncol 2022; 56(3): 285-291. Received 25 March 2022 Accepted 13 April 2022 Correspondence to: Francesca Matilde Schipilliti, University Sapienza in Rome, Sant’Andrea Hospital, Oncological Department, Rome, Italy. E-mail: francescaschipilliti@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. Electrochemotherapy (ECT) is a minimally invasive and safe treatment gaining positive and long-lasting antitumoral results that are receiving the attention of the scientific community. It is a local treatment that combines the use of electroporation and the administration of cytotoxic drugs to induce cell death in the target tissue. ECT is largely used for the treatment of cutaneous and subcutaneous lesions, and good results have been reported for the treatment of deep visceral tumors. The latest literature review is provided. Moreover, in line with its development for the treatment of visceral tumors in this article, we describe a novel approach of ECT: endoscopic treatment of colo-rectal cancer. Endoscopic ECT application was combined with systemic chemotherapy in the treatment of obstruct­ing rectal cancer without prospective surgery. A good response after ECT was described: concentric involvement of the rectum was reduced, and no stenosing lesions were detected. Conclusions. Clinical studies have demonstrated that ECT is a very effective treatment for tumors of different histo-logic types and localizations. Endoscopic treatment for gastrointestinal cancer is an innovative application of ECT. The combination of systemic treatment and ECT was safe and highly effective in the treatment of colorectal cancer, especially when obstructive, giving the patient a significant gain in quality of life. Key words: electrochemotherapy; colorectal cancer; endoscopy Introduction Electrochemotherapy (ECT) is a local ablative ther­apy based on electroporation, a physical approach for enhanced delivery of cytotoxic drugs into tu­mors. Electroporation is the application of short-intensity high-voltage electric pulses to tumors, where it creates transient pores in the cell mem­brane of tumor cells and consequently increases the permeability of the cell membrane to allow hy­drophilic drugs to enter the cytoplasm and induce cell death.1 Electroporation can be reversible or irrevers­ible. Irreversible electroporation is a nonthermal ablation procedure employing multiple short-term electrical pulses that irreversibly destroy cells in the application area; reversible electroporation, on the other hand, can induce apoptosis instead of necrotic cell death due to the action of cytotoxic drugs, which can result in a superior immune ef­fect. The reversibility or irreversibility of elec­troporation mainly depends on two electric pulse characteristics: electric field strength and time pulse length. For reversible electroporation, pulses of 1000 V/cm intensity and 100 µsec duration are used. In irreversible electroporation, higher electric field strengths or time pulse lengths are commonly used.2 Several clinically approved drugs have been tested in preclinical studies, but bleomycin and cisplatin are currently the most suitable and most commonly used chemotherapeutic drugs in associ­ation with ECT. In ECT after drug administration, a short time interval is needed for anticancer drugs, either administered intravenously or intratumor-ally, to distribute in the tumors. Bleomycin causes multiple DNA breaks and cisplatin intra- and in-terstrand DNA bonds in tumor cells. The cytotoxic effect of antitumoral drugs in ECT is increased 1000-fold for bleomycin and 80-fold for cisplatin.3 For several years, ECT has received attention in the scientific community because it is a minimally invasive and safe treatment, gaining positive and long-lasting antitumoral results. Great experience has been collected in the literature in the treatment of cutaneous and subcutaneous lesions, and in recent years, the first experiences with ECT treat­ment in deep-seated tumors have been reported with promising results.4 This article aims to describe the technique and clinical applications of ECT as a review of the latest publications. The review focuses on the treatment results and indications of this interesting treatment option. Furthermore, a new technical advancement is reported in a case of an obstructing rectal can­cer not indicated for radiotherapy, treated with systemic chemotherapy combined with ECT, gain­ing systemic and local response after 3 months of FOLFOXIRI and bevacizumab chemotherapy and a single ECT treatment conducted endoscopically. ECT indications, contraindications The indication for ECT should be made by clini­cians after a multidisciplinary team discussion to better evaluate the localization, size, characteris­tics of the lesions, general conditions and expected survival of the patient to define the purpose of the treatment. The technique can be applied in a vari­ety of malignant lesions; fields of applications of ECT may be classified into three groups: treatment of cutaneous and subcutaneous lesions, treatment of deep metastases located in liver, bone, renal can­cer, soft tissues and treatment of primary tumors.3 During patient selection, it is important to evaluate laboratory tests (number of platelets, coagulation, creatinine), cardiac, renal and lung function, body surface, and performance status (PS). The choice of treatment modality is standard­ized for use in cutaneous and subcutaneous le­sions.6 Physicians have to accurately select, on the basis of current recommendations, the employed drug (bleomycin or cisplatin), delivery route (intra­tumoral or intravenous), type of anesthesia (local or general) and type of electrodes. The ECT procedure may vary depending on the size and location of the lesions. A correlation between tumor size and effectiveness of ECT has been evaluated for cutaneous and subcutaneous tumors by Mali et al. on cutaneous tumors: ECT was less effective on tumors larger than 3 cm. In the case of large lesions, the multiple application and repositioning of electrodes needs to be considered.7 As a standardized procedure in the treatment of deep-seated tumors is lacking, it is difficult to de­sign a general ECT protocol. The time of response may vary depending on tumor characteristics and adopted techniques: for cutaneous tumors, the 2-month follow-up time is used as a reference for response, but for deep-seated tumors, it is difficult to establish a specific follow-up scheduled time after ECT treatment. ECT is not indicated in pregnancy or lactation or in cases of allergy or hypersensitivity to bleomycin or cisplatin. Literature review The earliest in vivo studies on the efficacy of ECT were carried out in the early 1990s: ECT was proven to be very efficient in animal studies, and safety and dosage were assessed in phase I and II studies.8,9 Several drugs commonly used in clini­cal settings were experimentally investigated, and the data supported the role of bleomycin, a hydro­philic molecule, as the drug of choice for ECT and demonstrated an increase in carboplatin and cispl­atin cytotoxicity when used in ECT.10 Treatment of cutaneous and subcutaneous tu­mor nodules was one of the first effective applica­tions of ECT.5 The management of this tumor lo­cation often represents a challenge for clinicians, particularly when surgical excision is not possible or when lesions are radiotherapy-resistant. Several trials confirmed the efficacy of ECT in the treatment of cutaneous and subcutaneous primary tumors or metastases from different malignancies (melanoma, basal cell carcinoma, squamous cell carcinoma, breast cancer, sarcomas): both bleomy­cin and cisplatin ECT were proven to be safe and well tolerated. Moreover, efficacy was excellent, reaching high objective response rates (ORs) and a high percentage of long-lasting responses. ECT was effective regardless of the histological type of tumor.11 287 The analysis of recent literature showed a wide variability according to the number, size and histo­logical type of treated cutaneous and subcutaneous metastases.12,13 Benevento et al. reported a 75.3% complete response (CR) and a 92.3% objective re­sponse rate (ORR) without intolerable side effects14 in breast cancer patients. Campana et al. identi­fied a subgroup of elderly breast cancer patients as the most responsive to ECT.15 A recent review of the literature by a Swiss group confirmed ECT as a feasible, safe and easy-to-perform procedure; moreover, their analysis identified an OR rate for ECT across all the selected studies of 84.0%.16 Borgognoni et al. found similar results in the treat­ment of cutaneous melanoma metastases and rare nonmelanoma skin cancer: the OR rate was 88.6% in melanoma and 91.7% in nonmelanoma tumors.17 Caracet al. reported a long-lasting response after ECT, with a mean duration of follow-up of 27.5 months for melanoma patients.18 Other authors an­alyzed data on ECT treatment of cutaneous metas­tasis from head and neck (HN) cancer and noticed a significant correlation between response to ECT and tumor size: ECT seems more effective in small tumor nodules (< 3 cm).19,20 Because of the results obtained with ECT in cu­taneous and lesions, many efforts have recently been made to translate the application of ECT into the treatment of non-superficial tumors. Preclinical in vitro studies were carried out on colorectal car­cinoma cells, showing an increased cytotoxicity of bleomycin combined with electroporation.21 Bianchi et al. performed a prospective phase II trial to evaluate the safety and efficacy of ECT in the treatment of bone metastases.22 Twenty-nine patients affected by painful bone metastases from various malignancies in different skeleton sites were treated: 27 patients obtained a partial re­sponse (PR) or stable disease (SD) after 3 months, 20 patients reported improvement of pain condi­tion (>50%) and reduction of consumption of anal­gesic drugs, and no complications were observed during and after ECT treatment. These results demonstrate that ECT might be safe and feasible for the treatment of painful bone metastases. Furthermore, in 2014, a pilot study was per­formed on 16 patients to evaluate the feasibility, safety and efficacy of intraoperative ECT in the treatment of colorectal liver metastasis: the tech­nique was proven to be safe (no serious adverse events were observed) and effective (85% of pa­tients with complete response and 15% of those with partial response).23 A similar Italian study conducted in 2017 obtained comparable results.24 The long-term safety and effectiveness of ECT was recently confirmed in the treatment of colorec­tal liver metastases in the vicinity of major hepatic vessels and was thus unsuitable for surgery, radi­ofrequency or microwave ablation.25,26 Based on the encouraging clinical results ob­tained in the treatment of colorectal liver metas­tases, Djokic et al. conducted a prospective pilot study to evaluate the role of ECT in the treatment of hepatocellular carcinoma (HCC).27 Ten patients resistant or not suitable for surgery or local ablative technique (TACE/RFA) were enrolled; they were treated with ECT during open surgery; bleomy­cin was the injected drug. Treatment was safe and well tolerated; at the first radiologic follow-up after 1 month, 88% of the lesions achieved a complete response (CR), and the others achieved a PR. The treatment was safe and effective even in tumors lo­cated in proximity to the hepatic or portal vessels and for tumors larger than 3 cm. ECT was shown to be a safe and effective treatment; it is minimally in­vasive with short hospitalization and good patient compliance; moreover, in selected cases, it may be considered a technique with curative intent; more studies need to be carried out to confirm these re­sults. Recently, the first percutaneous ECT of HCC was performed.28 In 2018, Tarantino et al. published the results of the first study evaluating the safety and effective­ness of percutaneous ECT in the treatment of unre­sectable perihilar-cholangiocarcinoma (PHCCA).29 Five patients were enrolled in the study: two pa­tients had CR, and in one patient, a complete res­toration ad integrum of liver parenchyma and high local efficacy without hepatic recurrence during follow-up were observed. These results confirmed the efficacy of ECT, even in combination with sys­temic chemotherapy. An Italian group of works re­cently obtained similar results.30 In the last 10 years, several studies have been conducted to evaluate the potential adjunctive role of ECT and IRE in patients affected by lo­cally advanced pancreatic cancer, who currently have a poor prognosis, with a 5-year survival rate of approximately 12%.31 Preclinical studies were performed to evaluate ECT’s role: the results of an animal study conducted on 10 mice with ortho-tropic human pancreatic carcinoma suggested that ECT might allow increased drug delivery in tumor cells, increasing gemcitabine efficacy and poten­tially reducing local tumor recurrence.32 IRE’s role has been evaluated in clinical studies: results of a multicenter prospective trial published in 2014 conducted on 200 patients with pancreatic can­cer demonstrated that the addition of irreversible electroporation (IRE) to systemic chemotherapy could prolong survival.33 Irreversible laparotomic and laparoscopic electroporation in addition to systemic chemotherapy was performed in a total of 70 patients with locally advanced pancreatic cancer enrolled in a multicenter Asian study.34 The results suggested that adding IRE to chemotherapy might provide a survival advantage. The necessity to evaluate the combination of IRE and multidrug systemic chemotherapy for the treatment of pan­creatic cancer led to the creation of the American Hepato-Pancreato-Biliary Association (AHPBA) Pancreatic Registry. Holland et al. reported initial outcomes of the AHPBA registry on 152 patients. The median overall survival (OS) was 30.7 months, the median progression-free survival (PFS) was 22.8 months, and the median time to tumor pro­gression (TTP) was 27.3 months. The combination of ECT and systemic chemotherapy for pancreatic cancer is a safe treatment with encouraging sur­vival.35 The efficacy of ECT was evaluated in the treat­ment of refractory cases of vulvar cancer in a mul­ticenter observational study carried out in five Italian centers.36 Sixty-one patients were treated: a clinical response rate was obtained in 83.6% of cases, and the procedure was safe and well toler­ated. Corrado et al. observed similar results in the palliative treatment of primary or recurrent vulvar cancer.37 The overall response rate (ORR) was 80% after 1 month, 61.5% of patients were alive at the 6-month follow-up and 50% at 1 year. ECT may have a role in the management of vulvar cancer in palliative setting. ECT with bleomycin may be con­sidered also for vulvar metastasis to reduce pain and bleeding.38 ECT may be used for the treatment of different histological types of cancer. Andresciani et al. de­scribed the application of ECT as a treatment op­tion in local recurrences of renal cell carcinoma 2 years after radical nephrectomy: ECT was effective, without evidence of residual disease 6 months af­ter the procedure.1 Endoscopic treatment for gastrointestinal can­cer is a novel application of ECT. The first human phase I study in patients affected by advanced es­ophageal cancer was conducted in 2018: ECT treat­ment was well tolerated, and tumor regression was gained and endoscopically confirmed in all patients.39 A similar phase I clinical study was conducted by Hansen et al. to investigate the safety and effi­cacy of endoscopically delivered ECT in patients with colorectal tumors.40 The results were encour­aging: all the treated patients, elderly and with multiple comorbidities, were successfully treated and gained local complete or partial tumor re­sponse after one treatment; the procedure was well tolerated without relevant side effects. Case report A 61-year-old male patient affected by metastatic colorectal cancer was treated by ECT in January 2021. The study was conducted in accordance with the Declaration of Helsinki and informed consent was obtained, also for the publication of the results. The patient had no known significant comorbidity; he was overweight and suffered from hypertension controlled with therapies. The diagnosis of cancer was obtained in November 2020. During the colo­noscopy, a substenosing, ulcerated bleeding lesion was observed from 20 cm to 10 cm before the anal edge. The lesion was histologically proven to be poorly differentiated adenocarcinoma. At the basal CT scan, multiple hepatic lesions and a single lung metastasis were described. The low abdominal MRI, performed in December 2020 (Figure 1A), described a bulky pathological cancer mass extending from the distal part of the sigma to the rectum until 8 centimeters from the anal border. The rectum was concentri­cally involved by the tumor, and local nodes were increased in volume. The radiotherapist rejected the possibility of combined chemoradiotherapy: local radiotherapy was not indicated because of the high risk of colo-rectal obstruction. First-line systemic chemotherapy with fluo­rouracil, leucovorin, irinotecan and oxaliplatin (FOLFOXIRI) plus BEVACIZUMAB was started in December 2020. Four cycles of chemotherapy were led between December 2020 and January 2021; therapy was well tolerated, and a dose reduction of 25% of IRINOTECAN was required for IV cycles of gas-troenteric G3 toxicity therapy. At the first abdominal MRI evaluation (Figure 1B), a good response was described. Concentrical involvement of the rectum was re­duced, there were no stenosing lesions, and patho­logical local nodes were reduced in volume and number. Local treatment with ECT was endoscopically conducted in the region of colorectal cancer by employing colonoscopy. One day before the treat­ 289 ment, physical examination, laboratory tests and cardiologic evaluation were carried out. Treatment was performed under general anesthesia (deep se­dation with Propofol) with standard hemodynam­ic monitoring. This procedure was conducted in the presence of a multidisciplinary team constituted by an oncolo­gist, gastroenterologist, anesthetist and specialized nurse. The Cliniporator EPS-02 produced by IGEA was used. Software was used to optimize the place­ment of electrodes in the predefined area. Electric pulses were delivered by linear needle electrodes. The electrode used (Stinger E_L2_00_S4) has been developed by IGEA and is dedicated to laparo­scopic/endoscopic approach, being equipped with a long connector cable (20 cm) and 4 expandable needle electrodes positioned at a fixed distance of 0.4 cm in a square geometry and parallel one to each other. Total needle electrode length is 4 cm, with an active part at the 2 cm extremity (Figure 2) In accordance with European Standard Operating Procedures of Electrochemotherapy (ESOPE) guidelines, ECT was performed 8 min­utes after the end of slow bleomycin intravenous infusion (bleomycin 15000 IU/m2 of body surface area diluted in 100 cc of physiologic solution in 10 minutes).5 Rectosigmoidoscopy was performed, and then the electrode was endoscopically introduced to perform electroporation of the lesion (Figure 3). Five applications of the electric pulses were per­formed with repositioning of the electrode, in or­der to completely cover the lesion. The duration of the procedure was 30 minutes. Antibiotic prophylaxis with cephalosporin was employed. The hospitalization time was 2 days. 290 The procedure was well tolerated, without side effects. At the first colonoscopy performed 4 days af­ter ECT treatment, tumor downstaging was con­firmed, the endoscopic device could easily pass through the lesions, and all the colon tubes were studied. Moreover, at the total body CT performed 4 weeks after ECT treatment, lung and hepatic me­tastasis were reduced in volume (Figure 1B, 2C). Systemic chemotherapy treatment was carried out after the procedure. After one month, no ad­verse events were reported; moreover, the patient reported a reduction in gastroenteric symptoma­tology and a subjective improvement in his well­being and quality of life. Conclusions ECT was the main topic of the present article. Literature data show the role of ECT as both cura­tive and palliative treatment, improving quality of life of patients with different tumour types. Moreover, the present article described our cent­er’s preliminary experience with ECT combined with standard systemic chemotherapy in the treat­ment of advanced colorectal adenocarcinoma not indicated for surgery or radiotherapy. The combination of systemic treatment and ECT was highly effective and safe in the treatment of this tumor: a good local response was observed with a resolution of the local stenosis caused by cancer and an improvement in patient quality of life because of a reduction in gastroenteric symp­toms. To the best of our knowledge, at the time of writ­ing this paper, no data concerning colorectal endo­scopic ECT treatment are available. Preliminary results may be considered a proof of concept for future prospective studies that are needed to con­firm these data. References 1. 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 2. Ritter A, Bruners P, Isfort P, Barabasch A, Pfeffer J, Schmitz J, et al. Electroporation of the liver: more than 2 concurrently active, curved electrodes allow new concepts for irreversible electroporation and electro-chemotherapy. Technol Cancer Res Treat 2018; 17: 153303381880999. doi: 10.1177/1533033818809994 3. Probst U, Fuhrmann I, Beyer L, Wiggermann P. Electrochemotherapy as a new modality in interventional oncology: a review. Technol Cancer Res Treat 2018; 17: 153303381878532. doi: 10.1177/1533033818785329 4. Leiter U, Gutzmer R, Alter M, Ulrich C, Meiwes A, Heppt MV, et al. [Cutaneous squamous cell carcinoma]. [German]. Hautarzt 2020; 71: 597­606. doi: 10.1007/s00105-020-04620-4 5. Marty M, Sersa G, Garbay JR, Gehl J, Collins CG, Snoj M, et al. Electrochemotherapy – an easy, highly effective and safe treatment of cuta­neous and subcutaneous metastases: results of ESOPE (European Standard Operating Procedures of Electrochemotherapy) study. Eur J Cancer Suppl 2006; 4: 3-13. doi: 10.1016/j.ejcsup.2006.08.002 6. Gehl J, Sersa G, Matthiessen LW, Muir T, Soden D, Occhini A, et al. Updated standard operating procedures for electrochemotherapy of cutaneous tumours and skin metastases. Acta Oncologica 2018; 57: 874-82. doi: 10.1080/0284186X.2018.1454602 7. Mali B, Miklavcic D, Campana LG, Cemazar M, Sersa G, Snoj M, et al. Tumor size and effectiveness of electrochemotherapy. Radiol Oncol 2013; 47: 32­41. doi: 10.2478/raon-2013-0002 8. Mir LM, Orlowski S, Belehradek J, Paoletti C. Electrochemotherapy potentia­tion of antitumour effect of bleomycin by local electric pulses. Eur J Cancer Clin Oncol 1991; 27: 68-72. doi: 10.1016/0277-5379(91)90064-K 291 9. Belehradek M, Domenge C, Luboinski B, Orlowski S, Belehradek J, Mir LM. Electrochemotherapy, a new antitumor treatment. First clinical phase I-II trial. Cancer 1993; 72: 3694-700. doi: 10.1002/1097-0142(19931215) 72:12<3694::aid-cncr2820721222>3.0.co;2-2 10. Gehl J, Skovsgaard T, Mir LM. Enhancement of cytotoxicity by electroper­meabilization: an improved method for screening drugs. Anticancer Drugs 1998; 9: 319-25. doi: 10.1097/00001813-199804000-00005 11. Mir LM, Glass LF, Sersa G, Teissié J, Domenge C, Miklavcic D, et al. Effective treatment of cutaneous and subcutaneous malignant tumours by electro-chemotherapy. Br J Cancer 1998; 77: 2336-42. doi: 10.1038/bjc.1998.388 12. Ferioli M, Perrone AM, Buwenge M, Arcelli A, Zamagni A, Macchia G, et al. Electrochemotherapy of skin metastases from breast cancer: a system­atic review. Clin Exp Metastasis 2021; 38: 1-10. doi: 10.1007/s10585-020­10063-x 13. Crimini E, Roberto M, Degli Effetti V, Marchetti P, Botticelli A, Schipilliti FM, et al. Electrochemotherapy as promising treatment option in rare recurrent cutaneous neoplasm of the scalp: case report of an elderly patient. Case Rep Oncol Med 2019; 2019: 2507642. doi: 10.1155/2019/2507642 14. Benevento R, Santoriello A, Perna G, Canonico S. Electrochemotherapy of cutaneous metastasis from breast cancer in elderly patients: a preliminary report. BMC Surg 2012; 12(Suppl 1): S6. doi: 10.1186/1471-2482-12-S1-S6 15. Campana LG, Galuppo S, Valpione S, Brunello A, Ghiotto C, Ongaro A, et al. Bleomycin electrochemotherapy in elderly metastatic breast cancer patients: clinical outcome and management considerations. J Cancer Res Clin Oncol 2014; 140: 1557-65. doi: 10.1007/s00432-014-1691-6 16. Seyed Jafari SM, Jabbary Lak F, Gazdhar A, Shafighi M, Borradori L, Hunger RE. Application of electrochemotherapy in the management of primary and metastatic cutaneous malignant tumours: a systematic review and meta-analysis. Eur J Dermatol 2018; 28: 287-313. doi: 10.1684/ejd.2018.3291 17. Borgognoni L, Pescitelli L, Gerlini G, Brandani P, Gelli R, Giannotti V, et al. Efficacy of electrochemotherapy in the treatment of cutaneous melanoma metastases and rare non-melanoma skin cancer. Anticancer Res 2020; 40: 6485-92. doi: 10.21873/anticanres.14670 18. CaracC, Mozzillo N, Marone U, Simeone E, Benedetto L, Di Monta G, et al. Long-lasting response to electrochemotherapy in melanoma patients with cutaneous metastasis. BMC Cancer 2013; 13: 564. doi: 10.1186/1471-2407­13-564 19. Bertino G, Sersa G, De Terlizzi F, Occhini A, Plaschke CC, Groselj A, et al. European Research on Electrochemotherapy in Head and Neck Cancer (EURECA) project: results of the treatment of skin cancer. Eur J Cancer 2016; 63: 41-52. doi: 10.1016/j.ejca.2016.05.001 20. Clover AJP, de Terlizzi F, Bertino G, Curatolo P, Odili J, Campana LG, et al. Electrochemotherapy in the treatment of cutaneous malignancy: outcomes and subgroup analysis from the cumulative results from the pan-European International Network for Sharing Practice in Electrochemotherapy data­base for 2482 lesions in 987 patients (2008-2019). Eur J Cancer 2020; 138: 30-40. doi: 10.1016/j.ejca.2020.06.020 21. Todorovic V, Sersa G, Flisar K, Cemazar M. Enhanced cytotoxicity of bleomy­cin and cisplatin after electroporation in murine colorectal carcinoma cells. Radiol Oncol 2009; 43: 264-73. doi: 10.2478/v10019-009-0037-5 22. Bianchi G, Campanacci L, Ronchetti M, Donati D. Electrochemotherapy in the treatment of bone metastases: a phase II trial. World J Surg 2016; 40: 3088-94. doi: 10.1007/s00268-016-3627-6. 23. 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 24. Coletti L, Battaglia V, De Simone P, Turturici L, Bartolozzi C, Filipponi F. Safety and feasibility of electrochemotherapy in patients with unresectable colo-rectal liver metastases: a pilot study. Internat J Surg 2017; 44: 26-32. doi: 10.1016/j.ijsu.2017.06.033 25. Edhemovic I, Brecelj E, Cemazar M, Boc N, Trotovsek B, Djokic M, et al. Intraoperative electrochemotherapy of colorectal liver metastases: a pro­spective phase II study. Eur J Surg Oncol 2020; 46: 1628-33. doi: 10.1016/j. ejso.2020.04.037 26. Edhemovic I, Gadzijev EM, Brecelj E, Miklavcic D, Kos B, Zupanic A, et al. Electrochemotherapy: a new technological approach in treatment of metas­tases in the liver. Technol Cancer Res Treat 2011; 10: 475-85. doi: 10.7785/ tcrt.2012.500224 27. Djokic M, Cemazar M, Popovic P, Kos B, Dezman R, Bosnjak M, et al. Electrochemotherapy as treatment option for hepatocellular carcinoma, a prospective pilot study. Eur J Surg Oncol 2018; 44: 651-7. doi: 10.1016/j. ejso.2018.01.090 28. Djokic M, Dezman R, Cemazar M, Stabuc M, Petric M, Smid LM, et al. Percutaneous image guided electrochemotherapy of hepatocellular car­cinoma: technological advancement. Radiol Oncol 2020; 54: 347-52. doi: 10.2478/raon-2020-0038 29. Tarantino L, Busto G, Nasto A, Nasto RA, Tarantino P, Fristachi R, et al. Electrochemotherapy of cholangiocellular carcinoma at hepatic hilum: a feasibility study. Eur J Surg Oncol 2018; 44: 1603-9. doi: 10.1016/j. ejso.2018.06.025 30. 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 Review Med Pharmacol Sci 2020; 24: 7051-7. doi: 10.26355/eurrev_202006_21698 31. Bhutiani N, Li Y, Zheng Q, Pandit H, Shi X, Chen Y, et al. Electrochemotherapy with irreversible electroporation and FOLFIRINOX improves survival in murine models of pancreatic adenocarcinoma. Ann Surg Oncol 2020; 27: 4348-59. doi: 10.1245/s10434-020-08782-2 32. Bhutiani N, Agle S, Li Y, Li S, Martin RCG. Irreversible electroporation en­hances delivery of gemcitabine to pancreatic adenocarcinoma. J Surg Oncol 2016; 114: 181-6. doi: 10.1002/jso.24288 33. Martin RCG, Kwon D, Chalikonda S, Sellers M, Kotz E, Scoggins C, et al. Treatment of 200 locally advanced (stage III) pancreatic adenocarcinoma pa­tients with irreversible electroporation: safety and efficacy. Ann Surg 2015; 262: 486-94; discussion 492-494. doi: 10.1097/SLA.0000000000001441 34. Huang K-W, Yang P-C, Pua U, Kim M-D, Li S-P, Qiu Y-D, et al. The efficacy of combination of induction chemotherapy and irreversible electroporation ablation for patients with locally advanced pancreatic adenocarcinoma. J Surg Oncol 2018; 118: 31-6. doi: 10.1002/jso.25110 35. Holland MM, Bhutiani N, Kruse EJ, Weiss MJ, Christein JD, White RR, et al. A prospective, multi-institution assessment of irreversible electroporation for treatment of locally advanced pancreatic adenocarcinoma: initial outcomes from the AHPBA pancreatic registry. HPB (Oxford) 2019; 21: 1024-31. doi: 10.1016/j.hpb.2018.12.004 36. Perrone AM, Cima S, Pozzati F, Frakulli R, Cammelli S, Tesei M, et al. Palliative electro-chemotherapy in elderly patients with vulvar cancer: a phase II trial: electro-chemotherapy in vulvar cancer. J Surg Oncol 2015; 112: 529-32. doi: 10.1002/jso.24036 37. 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 38. Merlo S, Vivod G, Bebar S, Bošnjak M, Cemažar M, Serša G, et al. Literature review and our experience with bleomycin-based electrochemotherapy for cutaneous vulvar metastases from endometrial cancer. Technol Cancer Res Treat 2021; 20: 15330338211010134. doi: 10.1177/15330338211010134 39. Egeland C, Baeksgaard L, Johannesen HH, Lgren J, Plaschke CC, Svendsen LB, et al. Endoscopic electrochemotherapy for esophageal cancer: a phase I clinical study. Endosc Int Open 2018; 6: E727-34. doi: 10.1055/a-0590-4053 40. Falk Hansen H, Bourke M, Stigaard T, Clover J, Buckley M, O’Riordain M, et al. Electrochemotherapy for colorectal cancer using endoscopic elec­troporation: a phase 1 clinical study. Endosc Int Open 2020; 8: E124-32. doi: 10.1055/a-1027-6735 research article Portal hypertension may influence the registration of hypointensity of small hepatocellular carcinoma in the hepatobiliary phase in gadoxetic acid MR Carla Caparroz1, Alejandro Forner2,3, Jordi Rimola1, Anna Darnell1, Ángeles García-Criado1, Juan Ram Ayuso1, María Reig2,3, Jordi Bruix2,3, Carmen Ayuso1,3 1 Radiology Department, Barcelona Clinic Liver Cancer (BCLC) Group, Hospital Clinic Barcelona, University of Barcelona, Spain 2 Liver Unit, Barcelona Clinic Liver Cancer (BCLC) Group, Hospital Clinic Barcelona. IDIBAPS, University of Barcelona, Spain 3 Centro de Investigaci Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain Radiol Oncol 2022; 56(3): 292-302. Recived 4 February 2022 Accepted 24 April 2022 Correspondence to: Prof. Carmen Ayuso, M.D., Radiology Department. BCLC group. Hospital Clínic, c/ Villarroel, 170. Escala 3, Planta 1. 08036. Barcelona. Spain. E-mail: cayuso@clinic.cat Disclosure: Carla Caparroz and Juan Ram Ayuso report no conflict of interest; Alejandro Forner: Lecture fees from Bayer, Gilead, Boston Science and MSD. Consultancy fees from Bayer, AstraZeneca, Roche, SIRTEX, AB Exact Science and Guerbert; Anna Darnell: speaker fees and travel grants from Bayer; Jordi Rimola: speaker fees and travel grants from Bayer and BTG and Terumo, consultancy fees from Roche andCOR2ED; Ángeles García-Criado: speaker fees from BTG and Terumo; María Reig: Lecture fees from Gilead, BMS, BTG, Eisai, Lilly, and Bayer. Consultancy fees from Bayer, BMS, AstraZeneca, ROCHE, Lilly, Ipsen and Boston Scientific. Research grants from Bayer and Ipsen; Jordi Bruix: Consultancy fees from Arqule, Bayer-Shering Pharma, Novartis, BMS, BTG-Biocomptatibles, Eisai, Kowa, Terumo, Gilead, Bio-Alliance, Roche, AbbVie, MSD, Sirtex, Ipsen, Astra-Medimmune, Incyte, Quirem, Adaptimmune, Lilly, Basilea, Nerviano, Sanofi. Research grants from Bayer. Educational grants from Bayer. Lecture fees from Bayer, BTG, AstraZeneca Eisai, Terumo, Sirtex, and Ipsen. Carla Caparroz and Alejandro Forner contributed equally and share the first authorship. 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 analyze the association between the liver uptake of Gadolinium-ethoxybenzyl-diethylenetriamine penta-acetic acid (Gd-EOB-DTPA) in the hepatobiliary phase (HBP) in cirrhotic patients and the presence of clinically significant portal hypertension (CSPH), and how these features impact on hepatocellular carcinoma (HCC) detection in the HBP. Patients and methods. Post-hoc analysis of a prospective cohort of 62 cirrhotic patients with newly US-detected nodule between 1–2 cm (study group). Twenty healthy subjects were used as control group. Qualitative and quantita­tive analysis of the liver contrast uptake in the HBP assessed by Relative Liver-Enhancement (RLE), Liver-Spleen (LSCR), Liver-Muscle (LMCR), and Liver-Kidney Contrast-Ratio (LKCR), Contrast Enhancement Index (CEI), and Hepatic Uptake (HUI), and biliary excretion, were registered. CSPH was confirmed invasively (HVPG > 10 mmHg) or by indirect param­eters. The appearance of HCC at the HBP was analyzed. Results. Nineteen patients (30.6%) did not have CSPH. In 41 patients (66.1%) the final diagnosis was HCC. All indices were significantly higher in the control group, indicating a more intense HBP liver signal intensity compared to patients with cirrhosis, even if the comparison was restricted to patients with no CSPH. CSPH was associated to a lower rate of HCC hypointensity in the HBP (51.9% vs. 85.7% without CSPH, p = 0.004). Conclusions. Liver uptake of Gd-EOB-DTPA at the HBP is decreased in cirrhosis even if the liver function is minimally impaired and it falls down significantly in patients with CSPH compromising the recognition of hypointense lesions. This fact may represent a limitation for the detection of small HCC in patients with cirrhosis and CSPH. Key words: liver cirrhosis; magnetic resonance imaging; hepatocellular carcinoma 293 Introduction Portal hypertension (PH) is a clinical syndrome that often complicates cirrhosis. It is related to the increased hepatic resistance to portal blood flow through the liver because of the architectural dis­ruption of the liver vascular anatomy caused by fibrosis and nodule formation.1 An important step in the pathophysiology of PH is the dysfunction at the hepatic sinusoidal cells in response to different liver injuries in the early stages of the cirrhosis de­velopment.1 PH is defined as clinically significant (CSPH) when the portal pressure increases above a critical threshold value of 10 mmHg.2 Although under this value there are no complications related to PH (such as ascites or variceal bleeding), sig­nificant changes in the hepatic sinusoidal system are already present in cirrhotic patients with he­patic venous pressure gradient below 10 mmHg. Interestingly, development of CSPH is the well-es­tablished key event defining a clinically significant risk of hepatocellular carcinoma development.3-5 Gadolinium-ethoxybenzyl-diethylenetriamine penta-acetic acid (Gd-EOB-DTPA; Primovist/ Eovist®), also known as gadoxetic acid, has an early phase with distribution into the extracellular space similar to other gadolinium-based contrast agents, followed by the uptake into hepatocytes and excretion into bile during the hepatobiliary phase (HPB).6-8 The uptake of Gd-EOB-DTPA by the hepatocytes during this delayed phase is im­paired in chronic liver diseases and in recent years, there have been several attempts to evaluate the potential of magnetic resonance (MR) with Gd-EOB-DTPA as a reliable tool for liver dysfunction assessment.9 Increased model for end-stage liver disease (MELD), bilirubin and indocyanine green clearance ratio at 15 minutes, and decreased choles­terol have been associated with suboptimal HBP10­14, and the parenchymal enhancement among dif­ferent HBP phases (at 5, 10, 15 and 20 minutes) is lower in Child-Pugh (CP) B and C compared to CP-A patients.15,16 Also, MR with Gd-EOB-DTPA has been tested for preoperative identification of those patients with major contraindications for he­patectomy.17-20 A recent experimental study in animals evalu­ating the impact of PH in the pharmacokinetics of gadobenate dimeglumine (Gd-BOPTA) has shown that the clearance across sinusoidal membranes of contrast agents is modified by changes in portal flow rates and as a result, at a given perfused con­centration, portal flow rates modified Gd-BOPTA hepatocyte concentrations.21 However, the impact of these changes related to PH in the contrast up­take in the HBP and in the diagnostic accuracy of the MR with Gd-EOB-DTPA has not been exten­sively studied. Accordingly, the aim of the present study is to evaluate the impact of CSPH and liver function impairment on the liver uptake of Gd-EOB-DTPA during the HBP, and consequently, how they may impact on hepatocellular carcinoma (HCC) detec­tion in the HBP. Patients and methods Between July 2012 and October 2015, we prospec­tively included consecutive asymptomatic patients with Child-Pugh A-B cirrhosis with no previ­ous history of hepatocellular carcinoma (HCC), in whom a new solitary well defined solid nod­ule between 1 and 2 cm was detected by screen­ing ultrasonography (US). The Institutional Ethics Committee for Clinical Research approved the study and all patients provided written informed consent before enrollment. These patients were included in a prospective study conducted in our Unit.22 Hepatic extracellular-contrast-enhanced MR (EC-MR) followed by Gd-EOB-DTPA MR were obtained in less than 1-month interval. The final HCC diagnosis was established by EC-MR accord­ing to the accepted non-invasive criteria5, or by bi­opsy in lesions with atypical vascular profile. All these patients (n = 62) were considered the study group. We also included patients with healthy liver who were submitted to Gd-EOB-DTPA MR for the study of a solitary hepatocellular liver lesion (focal nodular hyperplasia, or hepatocellular adenoma) during the same period, and they were included as a control group for evaluating the liver uptake of Gd-EOB-DTPA during HBP (control group). Diagnosis of CSPH in patients in the study group was established based on Baveno recom­mendations2 including 1) hepatic venous pressure gradient (HVPG) greater than 10 mm Hg, or 2) by indirect clinical findings when: a) the hepatic elastography (Fibroscan®-Echosens, Paris, France) registered a value greater than 21 Kpa,23 b) pres­ence of venous shunts, and/or ascites at imaging techniques and/or 3) presence of gastroesophageal varices by upper endoscopy. MR imaging Two 1.5-T MR units were used: SIGNA HDxt, GE Healthcare and Magnetom AERA, Siemens Medical Solutions. The sequence protocol for Gd-EOB-DTPA MR is detailed in supplemental materi­al. Dynamic images were acquired after IV injection of gadoxetic acid 0,25 mmol/ml (Primovist; Bayer) at a dose of 0.1 ml/kg body weight at a rate of 1,5 ml/s followed by a 20 ml saline flush at the same rate. The arterial phase was acquired 7 s after the arrival of contrast medium in the aortic arch. Portal and transitional and HB phases were acquired 50 s, 90 s, 10 min and 20 min thereafter respectively. The 20 minutes HBP was not acquired in those patients without underlying chronic liver disease. Evaluation of liver uptake of Gd-EOB­DTPA in the hepatobiliary phase Qualitative analysis All 10 and 20 minutes HBPs were reviewed by two independent radiologists (A.D. and J.R). The qual­ity of the HBP was classified as 1) adequate, when the liver parenchyma showed signal intensity (SI), higher than the SI of the intrahepatic vessels, or 2) Non-adequate quality, when the SI in the liver parenchyma was non-superior to the SI in intrahe­patic vessels (Figure 1).24 Also, the biliary contrast excretion was evaluated qualitatively according to the extension (intrahepatic only and extrahepatic). Inadequate hepatobiliary contrast excretion was defined as the lack of contrast agent in the extra-hepatic bile ducts in the hepatobiliary phase at 20 minutes Quantitative analysis Different MR-derived parameters focused to esti­mate the amount of Gd-EOB-DTPA liver uptake were calculated in the 10 and 20 minutes HBP. In all these indices, greater values mean more liver uptake of Gd-EOB-DTPA in the HBP. The quantita­ 295 tive assessments were done by C.C. (Figure 2). All formulas are summarized in Table 1 and described in detail in supplemental material. Relative Liver Enhancement (RLE): RLE10 and RLE20 establish the relationship between the SI of the liver parenchyma in the 10 minutes (LSI10) and the 20 minutes HBP (LSI20), and the liver SI before contrast injection (LSIpre).25 Liver to Spleen/muscle/Kidney Contrast Ratios: These indices determine the relationship between the SI of the liver and the SI of the spleen (LSCR), muscle (LMCR) and kidney (LKCR). To estimate the LSCR, an additional ROI was drawn on the spleen, over the same three images selected previ­ously.26 (Figure 3A) For LMCR, an additional ROI with an average area of 100 mm2 was drawn on the right paraspinal muscle. (Figure 3B). Finally, for LKCR an additional ROI with an average area of 0,5 to 1 cm2 was drawn on the upper pole of the right kidney (Figure 3C). Contrast Enhancement Index (CEI): The CEI10 and CEI20 were calculated as a ratio between the liver-to-muscle SI ratio 10 and 20 minutes after contrast injection (LMCR10 and LMCR20) respectively, and the liver-to muscle SI ratio before contrast injection (LMCRpre).27 FIGURE 2. Location of the 6 regions of interest (ROIs) in the liver parenchyma, to calculate the liver signal intensity (LSI) in the pre-contrast sequence (LSIpre) (A), at 10 minutes (LSI10) (B) and at 20 minutes (LSI20) hepatobiliary phase (C). Four of the ROIs were located in the anterior and posterior segments respectively of the right hepatic lobe, and two more were placed in the lateral and medial segments of the left lobe respectively. ROIs were drawn avoiding the inclusion of vascular structures and possible focal liver lesions. Hepatic Uptake index (HUI): The HUI provides a functional information of the liver volume. The in­dex takes into account the value of the entire liver volume (VolLiver), and the liver and spleen signal intensity and the formula is described in Table 1. was calculated in the late venous T1WI se- VolLiver quence obtained 5 minutes after contrast injection. For this purpose, a free hand irregular-ROI was drawn delineating the liver contour in every one of the images (ALMA 3D Workstation®), defining a liver area by liver plane. The VolLiver, expressed in cm3 was the sum of all the measured liver areas. Analysis of the focal liver lesions The imaging characteristics of the target lesion (TL) were independently registered in an electronic case report form by A.D. and J.R. They were blinded to final diagnosis, and imaging findings registered by each other. Any discrepancies during image analy­sis were solved by consensus discussion between the two investigators. Qualitative appearance of the lesion on delayed post-contrast sequences were registered as hypo, hyper or isointense lesions re­spect to the surrounding liver parenchyma. Statistical analysis Baseline characteristics of the patients were ex­pressed as median and range or count and propor­tion. Comparisons were done by using the Student t test or the Mann-Whitney test for continuous variables and the chi-square test or Fisher-exact test for categorical variables. A p value of less than 0.05 was considered significant. Calculations were done with the SPSS package version 20 (SPSS, Inc., Chicago, IL) Results A total of 62 cirrhotic patients were included in the study group and their characteristics are summa­rized in supplemental Table 1. Fifty-three out of 62 (85.5%) were CP-A patients [A-5 points (n = 44) and A-6 points (n = 9)] and 9 (14.5%) were CP-B [B-7 points (n = 4), B-8 points (n = 4) and B-9 points (n = 1)]. Forty-three (69.4%) had CSPH: 11 confirmed by HVPG > 10 mm Hg (n = 11) and 32 by indirect signs. All patients with Child-Pugh score = 6 (n = 18) had CSPH. The control group included 20 pa­tients without chronic liver disease and normal liver function. Final diagnosis of the 62 target lesions in the study group patients was: HCC (n = 41; 66.1%), intrahepatic cholangiocarcinoma (ICC) (n = 2; FIGURE 3. Location of the region of interest in the 20 minutes hepatobiliary phase in the spleen (A), in the right paravertebral muscle (B), and in the upper pole of the right kidney (C) to calculate the different quantitative parameters of contrast liver uptake: spleen-liver intensity (SLI) and liver-spleen contrast ratio (LSCR), muscle-liver intensity (MLI) and the liver-muscle contrast ratio (LMCR) and kidney-liver intensity (KLI) and the liver-kidney contrast ratio (LKCR), respectively. 3.2%), colorectal cancer metastases (n = 1; 1.6%), and benign conditions (angioma, n = 2; Dysplastic/ regenerative nodules, n = 4 and unspecific benign lesions, n = 12).22 The HBP at 20 minutes was not available in one patient with a final diagnosis of HCC. Thirty-one out of 41 HCC were diagnosed by non-invasive criteria (in 8 cases, pathology con­firmation was also available) and by pathology in the remaining 10 cases. Impact of liver function on the Gd-EOB­DTPA uptake in the HBP Table 2 describes the different quantitative param­eters evaluating the contrast liver uptake in the 10 and 20 minutes HBP considering the degree of liver function impairment according to Child-Pugh classification. All quantitative indices were significantly higher in CP-A patients compared to CP-B. We also focused our analysis in those CP-A patients comparing between 5 and 6 points: Except in LMCR, all indices were significantly higher in CP-A 5 points patients. Impact of CSPH on the Gd-EOB-DTPA liver uptake in the HBP We further evaluated the impact of CSPH on the liver enhancement in the HBPs (Table 3). All indi­ces except RLE at 20 minutes and CEI were signifi­ 297 TABLE 1. Formulas used for qualitative assessment of liver uptake of Gadolinium-ethoxybenzyl-diethylenetriamine penta-acetic acid (Gd-EOB-DTPA) in the hepatobiliary phase (HPB) Relative Liver Enhancement (RLE) Liver to Spleen Contrast Ratio (LSCR) Liver to muscle Contrast Ratio (LMCR) Liver to Kidney Contrast Ratios (LKCR) Contrast Enhancement Index (CEI) Hepatic Uptake index (HUI) RLE10 and RLE20: RLE at 10- and 20-min HBP LSIpre, LSI10 and LSI20: Liver signal intensity pre-contrast, at 10- and 20-min HBP, respectively LSCRpre, LSCR10 and LSCR20r: LSCR pre-contrast, at 10- and 20- min HBP, respectively LSIpre, LSI10 and LSI20: Liver signal intensity pre-contrast, at 10- and 20- min HBP, respectively SSIpre, SSI10 and SSI20: Spleen signal intensity pre-contrast, at 10- and 20- min HBP, respectively LMCRpre, LMCR10 and LMCR20r: LMCR pre-contrast, at 10- and 20- min HBP, respectively LSIpre, LSI10 and LSI20: Liver signal intensity pre-contrast, at 10- and 20- min HBP, respectively MSIpre, MSI10 and MSI20: Muscle signal intensity pre-contrast, at 10- and 20- min HBP, respectively LKCRpre, LKCR10 and LKCR20r: LKCR pre-contrast, at 10- and 20- min HBP, respectively LSIpre, LSI10 and LSI20: Liver signal intensity pre-contrast, at 10- and 20- min HBP, respectively KSIpre, KSI10 and KSI20: Kidney signal intensity pre-contrast, at 10- and 20- min HBP, respectively - LMCRpre, LMCR10 and LMCR20r: LMCR pre-contrast, at 10- and 20- min HBP, respectively - CEI10 and CEI20: Contrast Enhancement Index at 10- and 20- min HBP, respectively - HUI10 and HUI20: Hepatic Uptake index at 10- and 20- min HBP, respectively LSI10 and LSI20: Liver signal intensity at 10- and 20- min HBP, respectively SSI10 and SSI20: Spleen signal intensity at 10- and 20- min HBP, respectively cantly higher in absence of CSPH. To confirm the impact of CSPH irrespectively of liver function, we evaluated those patients with well-preserved liver function (CP-A 5 points) and in them, CSPH impacted in the liver Gd-EOB-DTPA uptake since LSCR, LMCR and LKCR were significantly higher in those patients without CSPH. Finally, we com­pared the liver contrast uptake in patients with very well-preserved liver function (all CP-A 5 points and those without CSPH) and patients with normal liver (control group B) and all scores were significantly higher in patients with healthy liver (Table 4). Gd-EOB-DTPA liver uptake through the different HBP (10 and 20 minutes) The quantitative assessment is exposed in supple­mental Table 2. All parameters that quantify the liver contrast uptake were significantly higher at 20 minutes compared to at 10 minutes. These differ­ences were maintained in CP-A and CP-A 5 points patients. The calculations were not done in Child-Pugh B due to low number of patients. Impact of CSPH over the biliary excretion of Gd-EOB-DTPA in the HBP 20 minutes In the 49 patients with adequate HBP at 20 min­utes, the biliary excretion of the contrast media arrived to the extrahepatic bile duct (n = 20) and the intestinal lumen (n = 28). Contrarily, only in 6 out 12 cases with poor HBP quality, the biliary excretion was present in the extrahepatic bile duct (p < 0.001). In all patients without CSPH (n = 18), the biliary excretion arrived to extrahepatic bile duct. Contrarily, in 7 out of 43 patients with CSPH (16.3%), the biliary excretion was not identified in the extrahepatic biliary tree. Impact of the quality of the HBP and the presence of CSPH in the registration of hypointense HCC lesions in HBP In 49 out of 62 MR studies (79%), the HBP was cat­egorized as adequate, in 12 (19.4%) non-adequate an in one patient (1.6%), the 20 minutes HBP was not available. Six out of 12 patients with non-ade­ TABLE 2. Quantitative assessment of liver uptake of Gadolinium-ethoxybenzyl-diethylenetriamine penta-acetic acid (Gd-EOB­DTPA) during the hepatobiliary phase (HPB) at 10 and 20 minutes considering the degree of liver function impairment according to Child-Pugh classification. Variables described as median and interquartile range N 62 53 9 44 18 RLE10 0.65 [0.49-0.75] 0.68 [0.51-0.81] 0.41 [0.30-0.52] <0.001 0.68 [0.52-0.82] 0.50 [0.40-0.58] 0.005 RLE20 0.63 [0.51-0.78] 0.67 [0.55-0.82] 0.41 [0.30-0.52] <0.001 0.68 [0.56-0.83] 0.52 [0.40-0.61] 0.006 LSCR10 1.32 [1.17-1.55] 1.35 [1.11-1.59] 1.20 [0.99-1.28] 0.016 1.43 [1.19-1.61] 1.20 [1,11-1.28] 0.001 LSCR20 1.48 [1.23-1.71] 1.51 [1.30-1.80] 1.20 [1.01-1.43] 0.006 1.54 [1.32-1.81] 1.25 [1.17-1.53] 0.008 LMCR10 2.14 [1.78-2.51] 2.16 [1.81-2.62] 1.78 [1.52-2.13] 0.030 2.26 [1.81-2.71] 2.08 [1.73-2.16] NS LMCR20 2.25 [1.86-2.63] 2.29 [1.89-2.69] 1.79 [1.52-2.23] 0.030 2.34 [1.88-2.70] 2.05 [1.75-2.31] NS LKCR10 1.02 [0.85-1.17] 1.04 [0.89-1.21] 0.83 [0.74-0.90] 0.005 1.06 [0.91-1.25] 0.87 [0.76-1.05] 0.008 LKCR20 1.14 [0.90-1.13] 1.19 [0.96-1.33] 0.89 [0.80-0.96] 0.009 1.20 [0.96-1.38] 0.91 [0.81-1.22] 0.012 CEI10 1.39 [1.27-1.57] 1.43 [1.34-1.1.58] 1.26 [1.22-1.32] 0.006 1.47 [1.34-1.58] 1.33 [1.25-1.38] 0.003 CEI20 1.43 [1.30-1.61] 1.45 [1.35-1.63] 1.32 [1.19-1.37] 0.007 1.50 [1.39-1.66] 1.35 [1.19-1.42] 0.001 HUI10 407.3 [223.7-640.9] 486.2 [235.3-845.4] 312.4 [-3.31­400] 0.022 522.4 [284.9-1036.4] 265.7 [130.5-383.7] <0.001 HUI20 660.5 [301.5-956.5] 697.8 [367.1-1028.9] 265 [72.5-620.4] 0.009 720.1 [450.5-1062.4] 327.2 [198.1-772.2] 0.004 CEI10 and CEI20 = contrast enhancement index at 10-20 min; HUI = hepatic uptake index; LKCR10 and LKCR20 = liver-kidney contrast ratio at 10-20 min; LMCR10 and LMCR20 = liver-muscle contrast ratio at 10-20 min; LSCR10 and LSCR20 = liver-spleen contrast ratio at 10-20 min; N = number; NS = non-significant; RLE10 and RLE20 = relative liver enhancement at 10-20 quate HBP at 20 minutes were CP-B patients and all of them had CSPH. Contrarily, in all 19 patients without CSPH, the HBP was classified as adequate. Table 5 describes the different quantitative param­eters evaluating the Gd-EOB-DTPA liver uptake in the 20 minutes HBP considering the quality of the HBP according to the subjective assessment. All indices were significantly higher in those studies classified as having an adequate HBP compared to those categorized as non-adequate. We further evaluated the impact of the quality of the HBP in the registration of the signal intensity of HCC le­sions related to the surrounding liver parenchyma. At the 20 minutes HBP, 26 out of 41 HCC nodules (63.4%) were hypointense, 8 (19.5%) isointense, 6 hyperintense (14,6%) and 1 HCC (2.4%) was heter­ogenous. In the 49 patients with an adequate Gd-EOB-DTPA liver uptake, 25 out of 32 HCC nodules (78.1%) were hypointense. Contrarily, in the 12 patients with non-adequate Gd-EOB-DTPA liver uptake, one of the 9 nodules diagnosed as HCC were hypointense (p<0.001). We further compared the appearance of HCC in the HBP according to the presence of CSPH. In the 19 patients without CSPH, 12 out of 14 HCCs (85.7%) were hypoin-tense. On the other hand, only 14 out of 27 HCCs (51.9%) in the 43 patients with CSPH appeared as hypointense (p = 0.044). Discussion The results of our study show that the presence of CSPH in cirrhotic patients determines an impair­ment of the liver Gd-EOB-DTPA uptake reflected by significant lower values by almost all quanti­tative indices evaluated. In addition, the patients with healthy liver displayed more intense liver contrast uptake during the 10 minutes HBP, even when they were compared with cirrhotic patients with very well-preserved liver function defined as Child-Pugh A 5 points and absence of CSPH. Very interestingly, the rate of hypointense HCC in the HBP is significantly lower in those patients with CSPH, having an impact on HCC detection by MR with Gd-EOB-DTPA. Our findings are clinically relevant since cirrhotic patients who present CSPH and impaired liver function are those at high risk of HCC development in whom an early diagnosis and accurate tumor staging are critical. This fact represents a severe limitation for the detection of new HCC nodules in cirrhotic patients when dy­namic sequences are skipped in the abbreviated MRI protocol for the HCC screening.28-31 There are several hypotheses that could justify the suboptimal HBP in the cirrhotic liver. The po­tential decrease in the number of hepatocytes due to the increase of fibrous tissue in the cirrhotic 299 TABLE 3. Quantitative assessment of liver uptake of Gadolinium-ethoxybenzyl-diethylenetriamine penta-acetic acid (Gd-EOB­DTPA) during hepatobiliary phase (HPB) at 10 and 20 minutes in the study group considering the presence of clinically significant portal hypertension (CSPH). On the left, including all study cohort and on the right, considering only cirrhotic patients with preserved liver function (Child-Pugh A 5 points). Variables described as median and interquartile range N 19 43 19 25 RLE10 0.73 [0.52-0.89] 0.57 [0.47-0.69] 0.027 0.73 [0.52-0.89] 0.65 [0.51-0.77] NS RLE20 0.73 [0.56-0.85] 0.59 [0.47-0.74] NS 0.73 [0.56-0.85] 0.66 [0.54-0.77] NS LSCR10 1.54 [1.37-1.67] 1.23 [1.15-1.39] < 0.001 1.54 [1.37-1.67] 1.29 [1,15-1.51] 0.014 LSCR20 1.68 [1.53-1.84] 1.35 [1.20-1.57] 0.003 1.68 [1.53-1.84] 1.37 [1.26-1.74] 0.036 LMCR10 2.41 [2.12-2.83] 2.06 [1.75-2.45] 0.006 2.41 [2.12-2.83] 1.88 [1.76-2.50] 0.034 LMCR20 2.49 [2.24-2.96] 1.95 [1.77-2.39] 0.005 2.49 [2.24-2.96] 1.92 [1.78-2.6] 0.036 LKCR10 1.15 [0.98-1.34] 0.93 [0.81-1.13] 0.001 1.15 [0.98-1.34] 1.01 [0.84-1.21] 0.032 LKCR20 1.25 [1.16-1.43] 0.98 [0.88-1.28] 0.001 1.25 [1.16-1.43] 1.05 [0.88-1.03] 0.017 CEI10 1.46 [1.34-1.59] 1.36 [1.26-1.52] NS 1.46 [1.34-1.59] 1.47 [1.31-1.58] NS CEI20 1.50 [1.38-1.71] 1.41 [1.24-1.54] NS 1.50 [1.38-1.71] 1.48 [1.33-1.65] NS HUI10 609.7 [501.4-970.3] 327.8 [191.7-511.8] 0.004 609.7 [501.4-970.3] 411.4 [223.4-1210.7] NS HUI20 803.6 [678.7-1091.2] 450.5 [274.8-864.4] 0.033 803.6 [678.7-1091.2] 569.0 [341.8-987.1] NS CEI10 and CEI20 = contrast enhancement index at 10-20 min; HUI = hepatic uptake index; LKCR10 and LKCR20 = liver-kidney contrast ratio at 10-20 min; LMCR10 and LMCR20 = liver-muscle contrast ratio at 10-20 min; LSCR10 and LSCR20 = liver-spleen contrast ratio at 10-20 min; N = number; NS = non-significant; RLE10 and RLE20 = relative liver enhancement at 10-20 min TABLE 4. Quantitative assessment of liver uptake of Gadolinium-ethoxybenzyl-diethylenetriamine penta-acetic acid (Gd-EOB­DTPA) during hepatobiliary phase (HPB) at 10 minutes in patients with normal liver (control group) compared with all Child-Pugh (CP) A 5 points patients (left panel) and with patients Child-Pugh A 5 points patients without clinically significant portal hypertension (CSPH). Variables described as median and interquartile range N 20 44 20 19 RLE10 1.06 [0.82-2.16] 0.68 [0.52-0.82] < 0.001 1.06 [0.82-2.16] 0.73 [0.52-0.89] < 0.001 LSCR10 2,27 [2.06-2.88] 1.43 [1.19-1.61] < 0.001 2,27 [2.06-2.88] 1.54 [1.37-1.67] < 0.001 LMCR10 3.11 [2.90-3.55] 2.26 [1.81-2.71] < 0.001 3.11 [2.90-3.55] 2.41 [2.12-2.83] < 0.001 LKCR10 1.82 [1.51-1.99] 1.06 [0.91-1.25] < 0.001 1.82 [1.51-1.99] 1.15 [0.98-1.34] < 0.001 CEI10 1.71 [1.55-1.85] 1.47 [1.34-1.58] 0.001 1.71 [1.55-1.85] 1.46 [1.34-1.59] 0.007 HUI10 1449.6 [1259-1717.7] 522.4 [284.9-1036.4] < 0.001 1449.6 [1259-1717.7] 609.7 [501.4-970.3] < 0.001 CEI10 = contrast enhancement index at 10 min; HUI = hepatic uptake index; LKCR10 = liver-kidney contrast ratio at 10 min; LMCR10 = liver-muscle contrast ratio at 10 min; LSCR10 = liver-spleen contrast ratio at 10 min; N = number; RLE10 = relative liver enhancement at 10 min liver may have a role10,32 and also, the eventual with Gd-EOB-DTPA, as shown in the sinusoidal alteration of the Gd-EOB-DTPA transport system obstruction syndrome (SOS), characterized by si-in the hepatocellular membrane, with decrease nusoidal congestion and dilatation due to detach-of the expression of the organic anion transport-ment of the cellular endothelium that obstructs the ing polypeptides (OATP1B1 and OATP1B3) and/ sinusoidal fenestrations in the centrilobular space, or the increase of the multidrug resistance protein associated with hepatocellular necrosis and perisi­MRP2 expression.6,32,33 Furthermore, structural and nusoidal fibrosis.34,35 biochemical changes at the sinusoidal system may To our knowledge, there are few studies evalu-also contribute to a suboptimal HBP in the MR ating the impact of PH in the Gd-EOB-DTPA MR. TABLE 5. Quantitative parameters evaluating the liver uptake in the 20 minutes hepatobiliary phase (HPB) of Gadolinium-ethoxybenzyl-diethylenetriamine penta-acetic acid (Gd-EOB-DTPA) according to the quality of the HBP. In one patient the HBP at 20 minutes was not available (n = 61). Variables described as median and interquartile range N 49 12 RLE20 0.68 [0.57-0.83] 0.40 [0.31-0.51] < 0.001 LSCR20 1.53 [1.34-1.78] 1.19 [1.06-1.22] < 0.001 LMCR20 2.34 [1.88-2.78] 1.91 [1.50-2.11] 0.002 LKCR20 1.20 [0.95-1.35] 0.89 [0.78-0.98] 0.001 CEI20 1.23 [1.09-1.32] 1.00 [0.90-1.10] 0.001 HUI20 744.1 [444.8-1024.1] 251.5 [65-4-331.0] < 0.001 CEI10 and CEI20 = contrast enhancement index at 10-20 min; HUI = hepatic uptake index; LKCR10 and LKCR20 = liver-kidney contrast ratio at 10-20 min; LMCR10 and LMCR20 = liver-muscle contrast ratio at 10-20 min; LSCR10 and LSCR20 = liver-spleen contrast ratio at 10-20 min; N = number; RLE10 and RLE20 = relative liver enhancement at 10-20 min Asenbaum et al.36 included in a retrospective study 178 patients with chronic liver disease without superimposed HCC, 109 (61.2%) with CSPH. The authors demonstrated an inverse correlation be­tween HVPG and RLE (r2 = 0.18, p< 0.0001), find­ings in line with our results. Regrettably, in this study the authors did not include a control group and thus, were not able to demonstrate the worse contrast uptake in the HBP in cirrhotics without CSPH compared with healthy liver patients. In ad­dition, in our study the quantitative analysis of the contrast uptake was done not only by the meas­urement of RLE, but also by the determination of contrast enhancement index, hepatic uptake index, and liver to spleen, muscle and kidney contrast in­dices, thus confirming the impact of CSPH in the Gd-EOB-DTPA hepatocyte uptake. More recently, Hectors et al. conducted a prospective study with 35 patients with chronic liver disease who under­went HVPG measurements and dynamic Gd-EOB­DTPA MR. Twenty-one (60%) patients had PH, of whom 9 had CSPH, and the authors report a statis­tically significant decrease of liver contrast uptake in presence of CSPH.37 A very relevant finding of our study is the unex­pected high rate of HCC nodules that were hyper or isointense (36.6%) compared to the surround­ing liver parenchyma in the HBP, since previous studies have described that less than 15% of HCC nodules were not hypointense in the HBP.8,28,38­40 Interestingly, this rate is significantly higher in those patients with CSPH (48.1%) compared to those without (14.3%). This finding is supported by the poorer contrast uptake of the non-tumoral liver parenchyma in those patients with CSPH. Consequently, the diagnostic capacity of Gd-EOB­DTPA is significantly impaired in those patients at higher risk of HCC and thus, in higher need of properly establishing if a hepatic nodule corre­sponds to a malignant focus or not. According to our results, portal pressure determines the target population for the optimal use of Gd-EOB-DTPA MR in patients with chronic liver disease, and those patients with no CSPH potentially candidates to resection in case an early HCC is diagnosed may benefit most from Gd-EOB-DTPA MR. Our study has some limitations. First, it includes a small number of patients and the number of pa­tients with no CSPH or with impaired liver func­tion was relatively low. Finally, it could be argued that the determination of CSPH was done inva­sively in 11 out of 43 cases. However, we applied a very stringent, internationally validated non-in­vasive criteria based on available evidence, which minimizes a potential misclassification. Finally, we did not conduct T1 relaxation time measurements at HBP, which has been suggested as an accurate approach for evaluating liver function.41,42 In conclusion, our study shows that the liver uptake of Gd-EOB-DTPA at the HBP is impaired in cirrhosis compared to healthy livers regardless the degree of liver function impairment. Even in patients with compensated cirrhosis categorized as CP-A 5 points, the liver contrast uptake is im­paired when CSPH is present. This limits the abil­ity to register hypointensity in the HBP and thus, hampers the detection capacity of HCC when us­ing MR with organ-specific contrast and the dy­namic sequences are skipped of the MRI protocol for HCC screening. Acknowledgments This is a post-hoc analysis from an investigator-initiated study partially funded by an unrestricted grant by Bayer Healthcare. CIBEREHD is funded by Instituto de Salud Carlos III. 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Invest Radiol 2017; 52: 462-9. doi: 10.1097/RLI.0000000000000366 37. Hectors SJ, Bane O, Kennedy P, Cuevas J, Thung S, Fischman A, et al. Noninvasive diagnosis of portal hypertension using gadoxetate DCE-MRI of the liver and spleen. Eur Radiol 2021; 31: 4804-12. doi: 10.1007/s00330­020-07495-0 38. Choi JY, Lee JM, Sirlin CB. CT and MR imaging diagnosis and staging of hepatocellular carcinoma. Part II. Extracellular agents, hepatobiliary agents, and ancillary imaging features. Radiology 2014; 273: 30-50. doi: 1148/ radiol.14132362 39. Renzulli M, Golfieri R, Bologna Liver Oncology Group (BLOG). Proposal of a new diagnostic algorithm for hepatocellular carcinoma based on the Japanese guidelines but adapted to the Western world for patients under surveillance for chronic liver disease. J Gastroenterol Hepatol 2016; 31: 69­80. doi: 10.1111/jgh.13150 40. Renzulli M, Biselli M, Brocchi S, Granito A, Vasuri F, Tovoli F, et al. New hallmark of hepatocellular carcinoma, early hepatocellular carcinoma and high-grade dysplastic nodules on Gd-EOB-DTPA MRI in patients with cir­rhosis: a new diagnostic algorithm. Gut 2018; 67: 1674-82. doi: 10.1136/ gutjnl-2017-315384 41. Ding Y, Rao SX, Chen C, Li R, Zeng MS. Assessing liver function in patients with HBV-related HCC: a comparison of T1 mapping on Gd-EOB-DTPA-enhanced MR imaging with DWI. Eur Radiol 2015; 25: 1392-8. doi: 10.1007/ s00330-014-3542-x 42. Haimerl M, Verloh N, Zeman F, Fellner C, Mler-Wille R, Schreyer AG, et al. Assessment of clinical signs of liver cirrhosis using T1 mapping on Gd-EOB­DTPA-enhanced 3T MRI. PLoS One 2013; 8: e85658 doi: 10.1371/JOURNAL. PONE.0085658 303 research article Early isolated subarachnoid hemorrhage versus hemorrhagic infarction in cerebral venous thrombosis Jan Kobal1, Ksenija Cankar2, Kristijan Ivanusic3, Borna Vudrag4, Katarina Surlan Popovic3,5 1 Department of Neurology, University Medical Centre Ljubljana, Ljubljana, Slovenia 2 Institute of Physiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Department of Neuroradiology, University Medical Centre Ljubljana, Ljubljana, Slovenia 4 Service of Neurology, Izola General Hospital, Izola, Slovenia 5 Department of Radiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2022; 56(3): 303-310. Received 25 February 2022 Accepted 14 June 2022 Correspondence to: Jan Kobal, M.D., Department of Neurology, University Medical Centre Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. E-mail: jan.kobal@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. Cerebral venous thrombosis (CVT) is a rare cerebral vascular disease, the presentation of which is highly variable clinically and radiologically. A recent study demonstrated that isolated subarachnoid hemorrhage (iSAH) in CVT is not as rare as thought previously and may have a good prognostic significance. Hemorrhagic venous infarction, however, is an indicator of an unfavorable outcome. We therefore hypothesized that patients who initially suffered iSAH would have a better clinical outcome than those who suffered hemorrhagic cerebral infarction. Patients and methods. We selected patients hospitalized due to CVT, who presented either with isolated SAH or cerebral hemorrhagic infarction at admission or during the following 24 hours: 23 (10 men) aged 22–73 years. The data were extracted from hospital admission records, our computer data system, and the hospital radiological database. Results. The iSAH group consisted of 8 (6 men) aged 49.3 ± 16.2 and the hemorrhagic infarction group included 15 (4 men) aged 47.9 ± 16.8. Despite having a significantly greater number of thrombosed venous sinuses/deep veins (Mann-Whitney Rank Sum Test, p = 0.002), the isolated SAH group had a significantly better outcome on its modified Rankin Score (mRs) than the hemorrhagic infarction group (Mann-Whitney Rank Sum Test, p = 0.026). Additional vari­ables of significant impact were edema formation (p = 0.004) and sulcal obliteration (p = 0.014). Conclusions. The patients who suffer iSAH initially had a significantly better outcome prognosis than the hemorrhagic infarction patients, despite the greater number of thrombosed sinuses/veins in the iSAH group. A possible explanation might include patent superficial cerebral communicating veins. Key words; cerebral venous thrombosis; subarachnoid hemorrhage; hemorrhagic brain infarction; superficial com­municating veins Introduction Cerebral venous thrombosis (CVT) is a rare cerebral vascular disease that represents a minor proportion of all strokes. A recent Dutch multicentric study re­vealed an incidence of 1.3 per 100,000 adults.1 The presentation of CVT is highly variable, not only clinically but also radiologically.2,3 Improvement in imaging techniques has enabled the identification of less obvious CVT cases; the incidence of CVT is increasing.4 Symptoms and signs depend not only on the location but also on the rate of thrombus pro­gression. Involvement of multiple venous sinuses/ veins may cause a wide variety of symptoms, e.g., headache, seizures, focal deficits, and disturbed consciousness, which may even proceed to coma. The wide spectrum of possible radiological pres­entations ranges from brain edema accompanying venous sinus or cortical vein thrombosis to venous infarction, which may be hemorrhagic and accom­panied by SAH and hematocephalus. Isolated SAH (iSAH) may also appear without venous infarc­tion.5,6 Superficial CVT manifestations such as cor­tical or perimesencephalic SAH secondary to cer­ebral venous thrombosis are considered to be very rare.7,8 A recent study, however, demonstrated that 33 CVT patients in a series of 332 presented with SAH and 22 of those with iSAH mostly accompa­nied by thrombosis in cortical veins, lateral sinus, and/or superior sagittal sinus. The outcome was favorable in all but one patient, who died of pul­monary embolism.9 In contrast, a prospective and retrospective Pakistani and Middle East study re­vealed hemorrhagic infarction to be the most signif­icant feature of a long-term unfavorable outcome.10 Another study from Pakistan revealed hemorrhagic brain infarction to be usually associated with mul­tiple venous sinus/vein occlusions. Superior sagit­tal, transverse, and sigmoid sinuses were most of­ten occluded, as well as the internal jugular vein, straight sinus, cortical and deep cerebral veins.11 Hemorrhagic infarction as a factor in an unfavora­ble outcome, therefore, seems to be associated with multiple venous sinus, superficial and deep vein occlusions. Nevertheless, unsolved dilemmas about hemodynamics and the therapeutic approach still exist.12,13 Multiple cerebral sinuses/veins may be oc­cluded in hemorrhagic infarction patients as also iSAH patients. An interesting recent hypothesis suggests that isolated SAH in CVT may be a conse­quence of blood leakage from fragile dilated bridg­ing cortical veins, which have no valves or muscu­lar layer.7,14,15 Bridging cortical veins are abundant near the tentorial and dural venous sinuses.16,17 According to a previous experimental study, cortical SAH and perimesencephalic SAH in CVT patients indicates an increased blood flow in the superficial communicating veins from which the bridging veins originate.18 We, therefore, propose that early iSAH, either cortical or perimesencephal, indicates persistent collateral venous flow and is a good prognostic sign in CVT patients. To find out what features may influence the outcome in CVT patients, we decided to analyze retrospectively files of CVT patients hospitalized at our clinical ward for vascular neurology in the past 11 years. We hypothesized that patients with a better clinical outcome might have retained patent superficial communicating veins and consequently suffer iSAH rather than hemorrhagic infarct. They would therefore be spared from the mass effect of a hemorrhagic infarct. We consequently decided to examine by hand clinical and radiological details in files of our CVT patients who presented with iSAH or hemorrhagic infarction within the first 24 hours after admission; we sought to identify radiological features that might explain their clinical outcome. Patients and methods Patients and methods Sixty-three CVT patients were admitted to the Department of Neurology, UMC Ljubljana, be­tween January 1, 2008, and December 31, 2018.19 The patients were diagnosed and treated in our hospital, and only those in whom CVT was prov­en clinically and radiologically were identified as such. Among those, we chose patients who pre­sented either with iSAH or cerebral hemorrhagic infarction at admission or within the next 24 hours. Twenty-three patients from our inventory were enrolled in our retrospective observational study. Clinical and radiological data were analyzed. The patients were 13 women and 10 men aged 22–73 years. The data were reviewed by an experi­enced neurologist working in the vascular neurol­ogy ward of the Department of Neurology, UMC Ljubljana. At admission, all the patients presented with either hemorrhagic venous infarction or iSAH due to CVT. Patients presenting with other intrac­ranial and/or systemic pathology that can cause hemorrhagic lesions and/or isolated SAH (e.g., ruptured aneurysms, arteriovenous malforma­tion, amyloidosis, PRES syndrome) were not in­cluded.20,21 The data were extracted from hospital admission records, our computer data system, and the AGFA radiological database, all in accord with the Helsinki Declaration. Reports and scans were de-identified and coded before evaluation. The study was approved by the Slovenian National Medical Ethics Committee (163/02/09). CVT was diagnosed by clinical examination, fol­lowed by brain CT, CT venography (CTV), brain MR and MR venography (MRV), whenever nec­essary, and laboratory findings, (e.g., d-dimer, C-reactive protein, coagulation screening, along with a complete blood count and biochemical pro­file). The following sinuses/deep veins were found to be obstructed in the iSAH group: the transverse sinus in 8 (bilaterally in 4 patients), the sigmoid si­nus in 7 (bilaterally in 1), the superior sagittal sinus in 5, the jugular vein bulb in 5, the confluence of sinuses in 3, the straight sinus in 2, and the vein 305 of Galen in 1 patient. In the hemorrhagic infarc­tion group, we found the superior sagittal sinus obstructed in 8 patients, the straight and sigmoid sinus in 6, the jugular vein bulb in 3, the internal cerebral vein in 2 (in 1 bilaterally), the basilar vein in 1 (bilaterally), the straight sinus in 1, the pet-rosal sinus in 1, and the vein of Galen in 1 patient. We also searched for environmental precipitating factors for CVT (e.g., trauma) as well as known intrinsic and acquired predisposing/precipitating factors (e.g., infections, contraceptive abuse, malig­nant disease, hematologic conditions, noninfective inflammatory disease, intracranial hypotension, acquired and genetic prothrombotic states).21 All the patients received anticoagulant treatment im­mediately after the diagnostic procedures were completed. They were started on low molecular heparin in a therapeutic dose and switched to war-farin before discharge. Average discharge time was about 3 weeks from admission, and a control clini­cal examination was typically performed 3–4 but not more than 6 months after discharge. Radiological analysis The type/location of venous infarct, intracerebral hemorrhage, and/or subarachnoid hemorrhage was determined by CT and/or MR whenever need­ed to confirm the presence of a venous hemorrhagic infarct in the perfusion area of cerebral veins and/ or blood in the subarachnoid space and/or throm­bosed sinuses/veins. The thrombus location in cer­ebral veins and major cerebral venous sinuses was determined by CTV and/or MRV. An initial CT was typically used as an accurate and fast method to detect hemorrhagic lesions and CTV as a fast and reliable method to investigate the structure of deep cerebral sinuses/veins.22 Brain CT was performed on a CT 40-slice mul-ti-detector row CT scan (SIEMENS SOMATOM Sensation Open 40). CT imaging was obtained with a 3-mm section thickness through the posterior fos­sa and basal brain structures and a 4.8-mm section thickness through the supratentorial hemispheres. CTV, as a fast thin-section volumetric helical CT ex­amination, was performed with a time-optimized bolus of contrast medium to enhance the cerebral venous system. A 75–100 mL non-ionic contrast medium (iodine, 300 mg/mL) was administered at a rate of 3 mL/sec with a 45-second pre-scanning delay. Helical scanning was performed on the cra­nial region, from the first vertebral body to the calvaria vertex. Post-processing included two-di­mensional (2D) and sometimes three-dimensional (3D) multiplanar images, slice thickness 3mm with 1 mm overlap. MRI was performed on a 1.5T unit (Philips Achieva 1.5T MRI system) using a standardized protocol for brain examination, including the fol­lowing sequences: axial T1, axial T2, axial fluid-attenuated inversion recovery (FLAIR), T2*, diffu­sion-weighted imaging (DWI) sequences, apparent diffusion coefficient (ADC) map and axial, sagittal and coronal T1 after the application of paramag­netic contrast agent. MR images were not used for statistics and were therefore not described in fur­ther detail. Data analysis A multiple linear regression was performed to test the effects upon clinical outcome of age, gender, predisposing/precipitating factors (e.g. genetic or acquired thrombophilia, steroid hormonal ther­apy, autoimmune disorders, malignancy, preg­nancy), clinical signs/symptoms (e.g. headache, seizures, focal signs, nausea/vomiting, disturbed consciousness), and CT diagnostics (e.g herniation, brain edema, sulcal effacement, ventricular com­pression). The impact of any pattern and burden of venous sinus/deep vein thrombosis on venous stroke was tested. A Spearman rank correlation co­efficient was determined. The patients were divided into 2 groups. The first group consisted of patients who were diag­nosed at admission as having isolated SAH asso­ciated with CVST, and the second group included patients who initially suffered from hemorrhagic venous brain infarction. The outcomes were clini­cally ranked according to the modified Rankin score of 0 to 6.23 The clinical outcome results were revised by a neurologist experienced in cerebro-vascular pathology. After comparing the baseline and demographic data of the groups, the laboratory and radiologi­cal data of the 2 groups were compared using the Mann-Whitney Rank sum test and/or Spearman rank-order correlation, as appropriate. The statisti­cal analyses were performed using the Sigma plot statistic package. Results With the multiple linear regression test, a positive correlation between CT diagnostic scores at admis­sion and mRS outcome scores at discharge was observed (Spearman rank correlation coefficient, R TABLE 1. The basic data and predisposing/precipitating factors in isolated subarachnoid hemorrhage (iSAH) and haemorrhagic infarction groups of patients Age (mean ± SD) 49.3 ± 16.2 47.9 ± 16.8 Gender 6 M, 2 W 4 M, 11 W* Genetic thrombophilia (%) 4 (50.0%) 2 (13.3%) Acquired thrombophilia (%) 0 (0%) 4 (26.7%) Autoimmune disorder (%) 4 (50.0%) 4 (26.7%) Hypothyroid disorder (%) 1 (12.5%) 1 (6.7%) Venous sinuses injury (%) 1 (12.5%) 0 (0%) Malignancy (%) 1 (12.5%) 1 (6.7%) Pregnancy (%) 0 (0%) 1 (6.7%) Glucocorticoid/sex steroid therapy (%) 1 (12.5%) 6 (40.0%) * statistically significant difference between the two at p < 0.05; Hem. inf. group = haemorrhagic infarction groups; M = men; N = number; W = women = 0.850; p < 0.001). A positive correlation was also found between CT diagnostic scores at admission and mRS outcome scores at a 6-month control (R = 0.911; p < 0.001). There was no correlation between age, gender, predisposing/precipitating factors or clinical signs/symptoms at admission and the mRS outcome at discharge or at the 6-month control. The iSAH group consisted of 8 patients (6 men) aged 49.3 ± 16.2 years. In 7 of them, we identified isolated cortical SAH and in 1, perimesencephal. The hemorrhagic infarction group was composed of 15 patients (4 men) aged 47.9 ± 16.8 years. We observed significant gender differences (p = 0.032), with men significantly predominating in the iSAH group and women in the hemorrhagic infarction group. Genetic thrombophilia and autoimmune disorders prevailed among the risk/provoking fac­tors in the iSAH group. In the hemorrhagic infarc­tion group, the results were more dispersed; how- TABLE 2. Clinical signs on admission in isolated subarachnoid hemorrhage (iSAH) and haemorrhagic infarction groups Headache (%) 6 (75.0%) 9 (60.0%) Seizure (%) 3 (37.5%) 8 (53.3%) Focal signs (%) 2 (25.0%) 5 (33.3%) Nausea/vomiting (%) 2 (25.0%) 3 (20.0%) Confusion (%) 0 (0%) 2 (13.3%) Disturbed consciousness(%) 0 (0%) 4 (26.7%) Hem. inf. group = haemorrhagic infarction groups; N = number ever, glucocorticoid/sex steroid therapy was most frequently observed (Table 1). The most frequently reported symptoms/signs on admission in both groups are presented in Table 2. Headache and seizures predominated in both groups. There was a statistically significant difference between the groups (Mann-Whitney Rank Sum Test, p = 0.026; Table 2) in the mRS outcome score at the control examination but not at discharge. The iSAH group had a significantly better outcome than the hemorrhagic infarction group. Nevertheless, the number of thrombosed venous sinuses/deep veins in the iSAH group was significantly greater (p = 0.002). In this group, we also observed signifi­cantly more occlusions of a confluence of sinuses (p = 0.015), transverse sinuses (p = 0.015), sigmoid sinuses (p = 0.023), and jugular vein bulbs (p = 0.013) than in the hemorrhagic infarction group. In contrast, there was a larger number of sulcal oblit­eration (p = 0.014) and edema formation (p = 0.004) in the hemorrhagic infarction group. There was no statistically significant difference between the two groups of patients (p = 0.128) in the number of her­niations. However, herniation was observed in all 3 patients with a fatal outcome in the hemorrhagic infarction group (mRS 6); minor subfalcine hernia­tion was observed in another patient in this group (Table 3). Discussion In this retrospective observational study, we found that patients with CVT who suffered initially from iSAH had a better clinical outcome than patients suffering from hemorrhagic brain infarct. Isolated SAH patients had significantly more venous si­nuses and deep veins obstructed than those in the hemorrhagic infarction group. Due to the CT ve­nography that was routinely performed, however, the detection of cortical vein thrombosis was not ac­curate enough to perform statistics.24 Nevertheless, we did observe a specific pattern of occluded si­nuses in this group. The confluence of sinuses, transverse sinuses, sigmoid sinuses, and jugular vein bulbs were occluded significantly more often than in the hemorrhagic infarction group. Despite more sinus/deep vein obstructions in the iSAH group, all the fatal cases occurred in the hemorrhagic infarction group as also signifi­cantly more edema formation/sulcal effacement. We observed gender differences, with men signifi­cantly predominating in the isolated SAH group 307 FIGURE 1. A 23-year old woman with headache followed by seizure and focal neurological deficit MRI on admission showed no focal lesions/oedema (A); contrast material-enhanced (CE) T1 and T2 showed occlusion of the left sigmoid sinus (B) and left transverse (C). Despite immediate anticoagulant treatment (fractioned heparin), the next day the patient became drowsy. CT revealed hemorrhagic infarction; in addition to the transverse sinus (arrow), the Labbe vein was suspected to be occluded due to the infarction territory (D). Decompressive craniotomy failed to prevent progression to irreversible coma (E,F). and women in the hemorrhagic infarction group. favorable outcome10, especially when it presents Significant differences in predisposing/precipitat-as a space-occupying lesion.25 In our patients who ing factors were not found. There were also no sig-presented with hemorrhagic infarction accompa­nificant differences regarding clinical symptoms/ nied by edema formation/sulcal obliteration, the signs between the groups. clinical outcome at the control examination was Previous experience has indicated that hemor-significantly worse than in isolated SAH patients. rhagic infarction is a major long-term factor of un-There were also 3 lethal outcomes (mRS 6) among TABLE 3. Comparison of thrombosed veins/sinuses (CVS), oedema formation, herniation, sulcal obliteration, modified Rankin Scores (mRs) at discharge and control examination in both groups of patients Average No. of thrombosed CVS (median, 25%, 75% percentiles) 4 (25% 3.25, 75% 5.75) 2 (25% 1, 75% 3)* Sulcal obliteration 0 (0.0%) 13 (86.7%)* Subfalcine/uncal herniation 0 (0.0%) 4 (26.7%) Oedema formation 2 (25.0%) 8 (53.3%)* Average mRS at discharge (median, 25% , 75% percentiles) 1 (25% 0, 75% 1.75) 2 (25% 0, 75% 3) Average mRS at control (median, 25% , 75% percentiles) 0 (25% 0, 75% 0) 1 (25% 0, 75% 3)* *statistically significant difference between the two groups at p < 0.05; Hem. inf. group = haemorrhagic infarction groups; iSAH = isolated subarachnoid hemorrhage; N = number hemorrhagic infarction patients. In each of those patients, we observed brain herniation within 24 hours of admission. Brain edema and sulcal oblit­eration were also present in each; brain edema of predominately the infratentorial region due to deep venous system obliteration was found in 1 of them. Edema formation, sulcal obliteration, and herniation are indicators of a space-occupying le­sion and a worse clinical outcome.25 Venous infarc­tion formation may be due to venous reflux in the cerebral veins, which have no valves; or perhaps, similarly to superficial communicating veins, due to increased venous and capillary tissue pressure leading to diapedesis of erythrocytes, blood-brain barrier disruption, blood vessel damage and blood leak, all of which further lead to hemorrhagic in­farction formation26. Given a rigid skull and me­ninges, the brain cannot distend, which leads to in­creased intracranial pressure, as also reduced cer­ebral perfusion pressure, cerebral blood flow, and oxygenation.27 However, given the experimental study by Ungersbk K. et al.18, which revealed the progression of thrombosis from the venous sinus to the bridging and cortical communicating veins completing an obstruction of venous collaterals, we presume this same progression in our patients and its eventuating a fatal outcome. The occlusion of the venous sinus alone seems to be not enough to cause cerebral infarction (Figure 1).18 In addi­tion, it seems that iSAH in CVT might be connected to a specific pattern of sinuses being occluded. The patients with iSAH, either cortical or in the posterior fossa (e.g., perimesencephal), in our study had an excellent outcome in that they were practically free of functional disability at the con­trol examination. None of them showed sulcal obliteration, although 3 of them experienced su­pratentorial edema. Isolated SAH patients were also found to have a good outcome in previous studies and reports.9,14,15,28 In the present study, we focused on clinical and radiological features that may influence different presentations of CVT. The previous studies examined patients experiencing either iSAH 9,15 or hemorrhagic brain infarct.10 We found no clinical studies focusing on the manner of iSAH and hemorrhagic venous brain infarct forma­tion after CVT. Venous blood flows along veins by the pressure gradient to the nearest venous sinus.16,29 If there is no communication through which blood flows, then venous stasis, edema, blood leakage, and in­farction develop.26 The leakage and formation of iSAH presumably evolve from congested super­ficial communicating veins and/or overstretched thin-walled bridging veins.7,27 According to a previous experimental study, cortical SAH/perimesencephalic SAH in CVT pa­tients indicate increased blood flow in the super­ficial communicating veins from which the bridg­ing veins originate.18 Persistent communication through the communicating/bridging venous sys­tem may reduce venous stasis and attenuate brain edema when the venous sinus is occluded. We suggest that iSAH is an indicator of that process. It seems that in patients who initially suffer iSAH, venous blood flow is partly transferred from the occluded veins/sinuses to superficial anastomotic veins, which carry venous blood towards venous 309 sinuses that are not occluded; blood flow is also partly transferred by thin superficial communicat­ing veins leading blood towards meningeal veins and perhaps diploic veins (Figure 2).7,30 The findings in our patients are consistent with a recent neuroradiological study demonstrating that occlusion of the Labbe’s vein significantly correlates with occlusion of the ipsilateral transversal sinus.31 Predisposing/precipitating factors in the iSAH and hemorrhagic infarction groups were not sig­nificantly different. Five women were receiving sex steroid therapy, 1 of which was in the isolated SAH group. One man in the hemorrhagic infarc­tion group was receiving corticosteroid therapy. Hence, gender differences between the iSAH and hemorrhagic infarction groups cannot be ex­plained by the effect of women-specific risk factors. Men predominated in our iSAH group, similar to a study from India.7 In contrast, a French study of 22 of such patients included only 4 men.9 In each of the case series, sampling was relatively small, pos­sibly due to the rarity of the pathology; hence, bias is possible. Anticoagulant therapy was introduced in all the patients as recommended.32 In patients experiencing large hemispheric le­sions, a decompressive craniotomy was found to be effective.33 Decompressive craniotomy relieves pressure on patent venous pathways, although it does not open the occluded ones. We propose that craniotomy should be attempted soon enough to prevent large edema/sulcal displacement, which is followed by compression and thrombosis of super­ficial communicating veins (e.g., Labbe’s vein), as in line with recent updates/neuroradiologic stud­ ies.31,33 Among the limitations of the present study are the retrospective and observational methods, since such methods may involve bias due to differences in the approach of various clinicians/radiologists. The examinations were likewise not performed ac­cording to a standardized protocol. This is a limita­tion in the value of the results. The available clini­cal results and radiological images were, however, reviewed and interpreted by an experienced clini­cal neurologist and neuroradiologist. At the same time, an observational method might be a strength, since it originates from real life and real clinical problems, possibly providing insights on how to perform future clinical and neuroradiological evaluations. Another limitation is the small num­ber of patients in the iSAH/hemorrhagic infarction groups, which can be explained by the rarity of the pathology and the strict inclusion criteria. In short, this retrospective study has shown that patients with CVT who have suffered from cortical subarachnoid hemorrhage have an excellent clini­cal outcome, despite a higher number of occluded deep cerebral veins/sinuses. Further, a specific pat­tern of occluded venous sinuses was found, a clue to which might be patent communicant superfi­cial venous pathways, e.g., vein of Labbe, vein of Trolard, and other less defined communicating cor­tical veins that drain to the nearest patent venous sinus. The pattern of sinuses that are occluded may have a role. Patients who suffered an early hemor­rhagic venous infarction had a worse outcome – a mass effect leading to brain edema and superficial vein obliteration may be the explanation. References 1. Coutinho JM, Zuurbier SM, Aramideh M, Stam J. The incidence of cerebral venous thrombosis: a cross-sectional study. Stroke 2012; 43: 3375-77. doi: 10.1161/STROKEAHA.112.671453 2. Stam J. Thrombosis of the cerebral veins and sinuses. N Engl J Med 2005; 352: 1791-98. doi: 10.1056/NEJMra042354 3. Bousser MG, Ferro JM. Cerebral venous thrombosis: an update. Lancet Neurol 2007; 6: 162-70. doi: 10.1016/S1474-4422(07)70029-7 4. Coutinho JM. Cerebral venous thrombosis. J Thromb Haemost 2015; 13 (Suppl 1): S238-44. doi: 10.1111/jth.12945 5. Einhaupl KM, Masuhr F. Cerebral venous and sinus thrombosis - an update. Eur J Neurol 1994; 1: 109-26. doi: 10.1111/j.1468-1331.1994.tb00059.x 6. Ghoneim A, Straiton J, Pollard C, Macdonald K, Jampana R. Imaging of cerebral venous thrombosis. Clin Radiol 2020; 75: 254-64. doi: 10.1016/j. crad.2019.12.009 7. Panda S, Prashatha DK, Shankar SR, Nagaraja D. Localized convexity suba­rachnoid hemorrhage-a sign of early cerebral venous sinus thrombosis. Eur J Neurol 2010; 17: 1249-58. doi: 10.1111/j.1468-1331.2010.03001.x 8. Sahin N, Solak A, Genc B, Bilgic N. Cerebral venous thrombosis as a rare cause of subarachnoidal hemorrhage; case report and literature review. Clin Imaging 2014; 38: 373-9. doi: 10.1016/j.clinimag.2014.03.005 9. Boukobza M, Crassard I, Bousser MG, Chabriat H. Radiological findings in cerebral venous thrombosis presenting as subarachnoid hemorrhage: a series of 22 cases. Neuroradiology 2016; 58: 11-16. doi: 10.1007/s00234­015-1594-5 10. Khealani BA, Wasay M, Saadah M, Sultana E, Shahid M, Shohab Khan F, et al. Cerebral venous thrombosis a descriptive multicenter study of patients in Pakistan and Middle East. Stroke 2008; 39: 2707-11. doi: 10.1161/ STROKEAHA.107.512814 11. Azeemuddin M, Awais M, Mubarak F, Rehman A, Baloch NU. Prevalence of subarachnoid hemorrhage among patients with cranial venous sinus thrombosis in the presence and absence of venous infarcts. Neuroradiol J 2018; 31: 496-503. doi: 10.1177/1971400918783060 12. Pizzi MA, Alejos DA, Siegel JL, Kim BYS, Miller DA, Freedman WD. Cerebral venous thrombosis associated with intracranial hemorrhage. J Stroke Cerebrovasc Dis 2016; 25: 2312-16. doi: 10.1016/j.jstrokecerebrovas­dis.2016.05.025 13. Sun J, He Z, Nan G. Cerebral venous sinus thrombosis presenting with multifocal intracerebral hemorrhage and subarachnoid hemorrhage: a case report. Medicine 2018; 97: e13476. doi: 10.1097/MD.0000000000013476 14. Sztajzel R, Coeytaux A, Dehdashti AR, Delavelle J, Sinnreich M. Subarachnoid hemorrhage: a rare presentation of cerebral venous thrombosis. Headache 2001; 41: 889-92. doi: 10.1111/j.1526-4610.2001.01161.x 310 15. Oda S, Shimoda M, Hoshikawa K, Osada T, Yoshiyama M, Matsumae M. Cortical subarachnoid hemorrhage caused by cerebral venous thrombosis. Neurol Med Chir 2011; 51: 30-6. doi: 10.2176/nmc.51.30 16. Kiliç T, Akakin A. Anatomy of cerebral veins and sinuses. Front Neurol Neurosci 2008; 23: 4-15. doi: 10.1159/000111256 17. Tsutsumi S, Ono H, Ishii H. Cortical and bridging veins of the upper cerebral convexity: a magnetic resonance imaging study. Surg Radiol Anat 2021; 43: 235-42. doi: 10.1007/s00276-020-02579-4 18. Ungersbk K, Heimann A, Kempski O. Cerebral blood flow alterations in a rat model of cerebral sinus thrombosis. Stroke 1993; 24: 563-9; discussion 569-70. doi: 10.1161/01.str.24.4.563 19. Vudrag B, Kobal J. Cerebral venous sinus thrombosis: an 11-year experience. [Slovenian]. In: Rot U, Horvat Ledinek A, Rakusa M, editors. Proceedings of the Annual Conference of Slovenian Neurologists. Ljubljana: Association of Neurologists of Slovenia; 2019. p. 55-6. 20. Cuvinciuc V, Viguier A, Calviere L, Raposo N, Larrue V, Cognard C, Bonneville F. Isolated acute nontraumatic cortical subarachnoid hemorrhage. Am J Neuroradiol 2010; 31: 1355-62. doi: 10.3174/ajnr.A1986 21. de Freitas GR, Bogousslavsky J. Risk factors of cerebral vein and sinus throm­bosis. Front Neurol Neurosci 2008; 23: 23-54. doi: 10.1159/000111259 22. Rodallec MH, Krainik A, Feydy A, He´lias A, Colombani JM, Julle`s MC, et al. Cerebral venous thrombosis and multidetector CT angiography: tips and tricks. RadioGraphics 2006; 26: S5-18. doi: 10.1148/rg.26si065505 23. Saver JL, Filip B, Hamilton S, Yanes A, Craig S, Cho M, et al. FAST-MAG investi­gators and coordinators. Improving the reliability of stroke disability grading in clinical trials and clinical practice: the Rankin Focused Assessment (RFA). Stroke 2010; 41: 992-5. doi: 10.1161/STROKEAHA.109.571364 24. Coutinho JM, Gerritsma JJ, Zuurbier SM, Stam J. Isolated cortical vein throm­bosis: systematic review of case reports and case series. Stroke 2014; 45: 1836-8. doi: 10.1161/STROKEAHA.113.004414 25. Kowoll CM, Kaminski J, Weiß V, Bel J, Dietrich W, Jtler E, e tal. Severe cerebral venous and sinus thrombosis: clinical course, imaging correlates, and prognosis. Neurocrit Care 2016; 25: 392-99. doi: 10.1007/s12028-016­0256-8 26. Schaller B, Graf R. Cerebral venous infarction: the pathophysiological con­cept. Cerebrovasc Dis 2004; 18: 179-88. doi: 10.1159/000079939 27. Wilson MH. Monro-Kellie 2.0: The dynamic vascular and venous patho-physiological components of intracranial pressure. JCBFM 2016; 16: 1338­50. doi: 10.1177/0271678X16648711 28. Fu FW, Rao J, Zheng YY, Song L, Chen W, Zhou QH, et al. Perimesencephalic nonaneurysmal subarachnoid hemorrhage caused by transverse sinus thrombosis: a case report and review of literature. Medicine 2017; 96: e7374. doi: 10.1097/MD.0000000000007374 29. Uddin MA, Haq TU, Rafique MZ. Cerebral venous system anatomy. J Pak Med Assoc 2006; 56: 516-9. PMID: 17183980 30. Andeweg J. The anatomy of collateral venous flow from the brain and its value in aetiological interpretation of intracranial pathology. Neuroradiology 1999; 38: 621-8. doi: 10.1007/s002340050321 31. Boukobza M, Crassard I, Bousser MG, Chabriat H. Labbé vein thrombosis. Neuroradiology 2020; 62: 935-45. doi: 10.1007/s00234-020-02396-x 32. Ferro JM, Coutinho JM, Dentali F, Kobayashi A, Alasheev A, Canhão P, et al; RE-SPECT CVT Study Group. Safety and efficacy of dabigatran etexilate vs dose-adjusted warfarin in patients with cerebral venous thrombosis: a randomized clinical trial. JAMA Neurol 2019; 76: 1457–65. doi: 10.1001/ jamaneurol.2019.2764 33. Ferro JM, Aguiar de Sousa D. Cerebral venous thrombosis: an update. Curr Neurol Neurosci Rep 2019; 19: 74. doi: 10.1007/s11910-019-0988-x 311 research article Safety and efficacy of drug-eluting microspheres chemoembolization under cone beam computed tomography control in patients with early and intermediate stage hepatocellular carcinoma Spela Korsic1,2, Nastja Levasic3, Rok Dezman1,2, Lara Anja Lesnik Zupan1, Blaz Trotovsek2,4, Rado Jansa2,5, Alojz Smid2,5, Peter Popovic1,2 1 Clinical Institute of Radiology, University Medical Centre Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 General Hospital Izola, Izola, Slovenia 4 Department of Abdominal Surgery, University Medical Centre Ljubljana, Ljubljana, Slovenia 5 Department of Gastroenterology and Hepatology, University Medical Centre Ljubljana, Ljubljana, Slovenia Radiol Oncol 2022; 56(3): 311-318. Received 19 December 2021 Accepted 31 March 2022 Correspondence to: Assoc. Prof. Peter Popovic, M.D., Ph.D., Clinical Institute of Radiology, University Medical Centre Ljubljana, Zaloška cesta 7, SI-1000 Ljubljana, Slovenia. E-mail: peter.popovic@kclj.si This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Background. Drug-eluting microsphere transarterial chemoembolization (DEM-TACE) is the standard of care in pa­tients with intermediate-stage hepatocellular carcinoma and ensures targeted and controlled cytotoxic and ischemic effects. Proper patient selection and optimized treatment techniques are associated with longer median survival. The aim of this single-institution retrospective study was to evaluate safety and efficacy of DEM-TACE under cone beam computed tomography (CBCT) control in patients with early and intermediate stage hepatocellular carcinoma. Patients and methods. A total of 144 patients (mean age 67.9 ± 8.0 years, 127 males and 17 females) between February 2010 and December 2018 were studied. Microparticles of different dimensions according to two manufac­ turers (diameter of 70–150 µm, 100–300 µm or 300–500 µm and 40-µm, 75-µm or 100-µm) were used and loaded with 50–150 mg of doxorubicin. The objective tumour response according to the modified Response Evaluation Criteria in Solid Tumours (mRECIST), the time to progression, adverse events and overall survival were (OS) evaluated. Results. In total, 452 procedures were performed (median, 3 per patient). Four (0.9% of all procedures) major com­plications were noted. Postembolization syndrome occurred after 35% of procedures. At the first imaging follow-up 2–3 months after first treatment, 91% of patients achieved an objective response. The median time to progression was 10.2 months (95% CI: 8.3-12.1 months). OS rates at 1, 2, 3, 4, and 5 years were 85%, 53%, 33%, 20% and 14%, respectively. The median survival time was 25.8 months (95% CI: 22.1–29.5 months). Conclusions. DEM-TACE under CBCT control in patients with early and intermediate stage hepatocellular carcinoma is a safe and effective method of treatment with high objective tumour response and survival rates. Key words: hepatocellular carcinoma; drug-eluting microspheres; doxorubicin; transarterial chemoembolization; cone beam computed tomography; safety; efficacy Introduction mon cause of cancer-related death in the world.1 The prognosis of the disease correlates strongly Hepatocellular carcinoma (HCC) accounts for most with liver function (defined by Child-Pugh’s class, primary liver tumours and is the fourth most com-bilirubin, albumin, clinically relevant portal hyper­tension, ascites), tumour status (defined by num­ber and size of nodules, presence of vascular in­vasion, extrahepatic spread) and general tumour-related health status.2 The Barcelona Clinic of Liver Cancer (BCLC) staging system divides patients in­to five groups to facilitate treatment selection and prognosis prediction.2 According to the guidelines of the European Association for the Study of the Liver (EASL) and the European Society of Medical Oncology (ESMO), transarterial chemoemboliza­tion (TACE) is the standard treatment for HCC in patients with intermediate-stage disease, i.e., BCLC stage B.2,3 Furthermore, a clinical situation known as “treatment stage migration” has been introduced since not all patients with early-stage disease can be treated with surgery or ablation but may benefit from TACE.4 The intention of treat­ment stage migration is to offer the next most suit­able option within the same stage or the next prog­nostic stage to patients who do not respond to the recommended treatment or do not qualify for it, thus improving their outcomes.4–6 The principle of TACE is selective delivery of a high local concentration of a chemotherapeutic drug mixed with embolic material, which results in strong cytotoxic and ischemic effects.2 Two tech­niques are currently recommended: conventional TACE (cTACE) and drug-eluting microsphere transarterial chemoembolization (DEM-TACE).2,3 Conventional TACE (cTACE) uses a mixture of Lipiodol and chemotherapeutics, while DEM­TACE uses microparticles that can be loaded with a chemotherapeutic drug that is released in the tar­get tissue slowly and in a controlled manner. The most frequently used drug in DEM-TACE is doxo­rubicin.7 Untreated patients with BCLC stage B disease have a median OS of 10 to 16 months after HCC di­agnosis.2,8 The median OS for patients treated with TACE is approximately 20 months after treatment initiation.5,9 In well-selected patients, median OS can be prolonged to 30–50 months.2,3 A difference in OS between patients treated with doxorubicin-loaded DEM-TACE and those treated with cTACE has not been observed.10,11 A further improvement in TACE was the in­troduction of cone beam CT (CBCT) control dur­ing the procedure. CBCT is mounted on a C-arm fluoroscopy unit in the Institute of Radiology suite, allowing enhanced visibility in soft-tissue and vas­cular procedures. CBCT enables more precise im­plementation of TACE from planning to microcath­eter positioning, which ensures visualization of the tumour-feeding vessels and parenchymal staining during TACE, achieving a detection accuracy sig­nificantly superior to that of standard 2D angiog­raphy.12,13 The aim of this single-institution retrospective study was to evaluate the safety and effectiveness of doxorubicin drug-eluting microsphere chem-oembolization under CBCT control in patients with early and intermediate stage HCC. Patients and methods Patient selection The present retrospective study included 144 pa­tients with early and intermediate stage HCC who underwent doxorubicin-loaded DEM-TACE at our interventional oncology centre between February 2010 and December 2018. The last date for follow­up evaluation was 31 January 2020. Clinical ex­amination, laboratory tests and contrast-enhanced four-phase computed tomography (CT) or mag­netic resonance (MR) imaging with hepatobiliary contrast media (Primovist; Bayer HealthCare, Germany) were performed for each patient at baseline. A decision in favour of treatment with doxorubicin-loaded DEM-TACE was reached by consensus at a multidisciplinary hepatopancrea­tobilliary tumour board (MTB) at our institution, consisting of abdominal and interventional radiol­ogist, gastroenterologist, hepatic surgeon, nuclear medicine physician, oncologist, and pathologist. The inclusion and exclusion criteria for the study are presented in Table 1. Written informed consent for the procedure was obtained from all patients before each treatment. The need for informed consent for publication was waived by the national ethics committee due to the retrospective, anonymized study design. The study was performed in accordance with the Helsinki Declaration ethical standards for biomedical stud­ies on humans and was approved by the Republic of Slovenia National Medical Ethics Committee on the 18th of April 2017 (decision number 60/04/17). All data were collected from patient charts held at the Clinical Institute of Radiology, Clinical Department of Gastroenterology and Clinical Department of Abdominal Surgery at University Medical Centre Ljubljana. Treatment The first treatment cycle was defined by at least two doxorubicin-loaded DEM-TACE procedures at intervals of 4–6 weeks (i.e. TACE 1a and 1b). 313 Additional procedures prior to the first dynamic contrast enhanced CT or MR evaluation were performed if the multifocality of the disease did not allow complete targeting of the tumours. All procedures were carried out under CBCT con­trol (using Allura Xper FD20; Philips Healthcare and Artis Zee floor with DynaCT; Siemens, Forchheim, Germany). Typically, a 2.4-French microcatheter (Progreat®, Terumo Europe N. V, Belgium) was advanced into either a subseg-mental or a segmental tumour-feeding artery de­pending on the location of the targeted tumour. CBCT was performed with the administration of a nonionic contrast agent (Ultravist 370®, Bayer HealthCare, Germany; Visipaque 320, GE Healthtcare, Norway) through a power injector (Avanta®, Medrad, Bayer HealthCare, Germany). The injection rate for the initial lesion visualiza­tion with catheter placed in main hepatic artery was typically 1 mL/s with a total injected volume of contrast agent of 10 mL and a delay time of 8–10 seconds.13 For each CBCT scan, the area of interest was positioned in the system isocenter, and, over approximately 10 seconds, 310–321 projection im­ages were acquired with the motorized C-arm. X-ray parameters of 51–120 kV and 101–125 mA, covering approximately 180–200° clockwise arc at a rotation speed of 20° per second were used. Multiple two-dimensional projections were ac­quired and reconstructed by using Feldkamp al­gorithm to generate three-dimensional volumetric images. The matrix size was 512 x 512, and the field of view (FOV) 38 x 38 cm. Selective or superselective chemoemboliza­tion was performed with microparticles with varying diameters from different manufactur­ers loaded with 50–150 mg of doxorubicin: DC Beads (DC Bead®, Boston Scientific, Marlborough, Massachusetts) with a diameter of 70–150 µm, 100–300 µm or 300–500 µm; Tandem (Tandem®, Boston Scientific, Marlborough, Massachusetts) 2 ml or 3 ml of 40-µm, 75-µm and 100-µm, LifePearl microspheres (LifePearl®, Terumo Europe N. V, Belgium). The same size of the microparticles were then used for the following procedures in each pa­tient. Since 2013, we have been using small micro-particles (i.e. 70–150 µm) in all patients. In patients with multifocal tumours, the posi­tion of the microcatheter was changed within the same session to ensure superselective delivery to each lesion.13 Prior to microparticle delivery, CBCT was repeated to confirm the catheter position in feeding artery and complete coverage of the tar­geted lesions. TABLE 1. Inclusion and exclusion criteria (Child-Pugh; CP) early-stage HCC patients ineligible for resection, transplantation or ablation; intermediate-stage HCC Inclusion criteria patients with a CP score of A or B (up to 7 points); treatment with DEM-TACE under cone beam CT control. DEM-TACE prior to liver transplantation; Exclusion criteria inability of regular follow-up. DEM-TACE = drug-eluting microsphere transarterial chemoembolization; HCC = hepatocellular carcinoma Treatment complications Procedure-related complications were classified as complications occurring during the procedure or complications detected up to 1 month after the procedure. The complications were classified as minor and major according to the CIRSE Quality-Improvement Guidelines for Hepatic Transarterial Chemoembolization.14 Postembolization syndrome (PES) was defined as fever, pain, nausea, elevation of liver transaminases (i.e. doubling of baseline value of aspartate aminotransferase (AST)) and an increased white blood cell (WBC) count that oc­cur 24–72 hours after the procedure and was not considered a complication in accordance with the CIRSE guidelines. Treatment response Tumour treatment responses were evaluated with dynamic contrast-enhanced CT or dynamic MR imaging with hepatobiliary contrast media 2–3 months after the last doxorubicin-loaded DEM-TACE procedure according to the modified Response Evaluation Criteria in Solid Tumours (mRECIST).15 Patients with a complete response or a partial response were classified as having an ob­jective response to treatment. In cases of an objec­tive response to treatment, a radiological follow-up was performed every 3 months for the first 2 years, mostly with MRI of the liver. If no progression oc­curred after 2 years, follow-up imaging was then scheduled every 6 months. Doxorubicin-loaded DEM-TACE treatment was repeated when neces­sary in patients with residual or additional tu­mours observed on imaging, i.e. retreatment “on demand”.13 A decision for retreatment was again reached by MTB. Statistical analysis Categorical variables were expressed as frequen­cies and percentages. Continuous variables were TABLE 2. Patient characteristics at baseline The follow-up time was determined as the num­ber of months from the first doxorubicin-loaded Gender, number of patients (%) Male/Female 127 (88.2)/17 (11.8) Age, years Mean ± SD 67.9 ± 8 Imaging characteristics Number of lesions per patient, 3 (1–10) median (range) Bilobar, n. (%) 52 (36.1) Unilobar, n. (%) 92 (63.9) Right lobe, n. (%) 71 (49.3) Left lobe, n. (%) 21 (14.6) Signs of portal hypertension, n. (%) Yes/No 76 (52.8)/68 (47.2) Ascites, n. (%) Yes/No 34 (23.6)/110 (76.4) Cirrhosis, n. (%) Yes/No 120 (83.3)/24 (16.7) Cirrhosis aetiology, n. (%) Alcohol 63 (52.5) HBV 16 (13.3) HCV 14 (11.6) Primary biliary cirrhosis 2 (1.8) Other 25 (20.8) Child-Pugh score (avg. points ± SD) 5.7 ± 0.8 Child-Pugh class, n. (%) A/B 91 (75.8)/29 (24.2) Barcelona Clinic of Liver Cancer stage, n. (%) A/B 50(34.7)/94 (65.3) Laboratory characteristics, median (range) Albumin, [g/l] 39.5 (28–50) Total bilirubin, [µmol/l] 18 (5–83) AFP, [ng/ml] 14.4 (1.1–12809.8) AST, [µkat/l] 0.82 (0.35–3.29) GGT, [µkat/l] 1.77 (0.25–24.95) Creatinine, [µmol/l] 78 (39.0–148.0) AFP = alpha fetoprotein; AST = aspartate aminotransferase; GGT = gamma-glutamyl transferase); HBV = hepatitis B virus; HCV = hepatitis C virus; SD = standard deviation expressed as the mean ± standard deviation or the median and range in case of skewed distributions. Survival rates and the time to progression (TTP) were calculated using the Kaplan–Meier method and compared using the log rank test. The limit of statistical significance was set at p < 0.05. DEM-TACE procedure until death or until 31 January 2020. The time to progression was calcu­lated as the time until the date of imaging control showing progression or, in censored cases, until 31 January 2020, or patient death. The analysis was performed using IBM® SPSS® Statistics 22 (International Business Machines Corp., Armonk, NY) for Windows. Results Patient characteristics The baseline demographic, clinical, laboratory and imaging characteristics of 144 patients included in the analysis are summarized in Table 2. The mean patient age was 67.9 ± 8.0 years, and most patients were male (88.2%). The median number of lesions per patient was 3 (range, 1–10), and the median size of the largest lesion was 3.8 cm. Ninety-one patients were classified as Child-Pugh class A (75.8%), and the remaining 29 were classified as class B (24.2%). Twenty-four of the 144 (16.7%) pa­tients were not cirrhotic. The most common aetiol­ogy of cirrhosis was alcohol abuse (52.5%, n = 63), followed by other (20.8%, n = 25), hepatitis B (13.3%, n = 16) and hepatitis C (11.6%, n = 14). Ninety-two (63.9%) patients had unilobar, predominantly right lobe, disease. Clinical signs of portal hypertension were observed in 76 (52.8%) patients, and ascites was observed in 34 (23.6%) patients. Sixty-eight of 101 (67.3%) patients with available AFP data had an elevated AFP level (> 7.5 ng/mL). Procedure Overall, 452 doxorubicin-loaded DEM-TACE procedures were performed in 144 patients. The median number of procedures per patient was 3 (range, 1–8). In 136 (94.4%) patients, at least two doxorubicin-loaded DEM-TACE procedures were performed. The remaining 8 (5.6%) patients had only one procedure at the time of their follow-up. Large microparticles (300–500 µm) were used in 16% of patients (n = 23), intermediate-size micro-particles (100–300 µm) in 19.4% of patients (n = 28) and small microparticles (40–100 µm) in 64.6% of patients (n = 93) in the first doxorubicin-loaded DEM-TACE procedure. The maximum cumula­tive dose of doxorubicin per procedure was 150 mg (range 50–150 mg). Doxorubicin-loaded DEM­TACE was the primary treatment for 120 patients (83.0%). Other treatments for HCC preceding the first doxorubicin-loaded DEM-TACE are presented in Table 3. Procedure complications PES occurred in 158 procedures (35.0%). All PES cases were managed medically and did not prolong hospitalization. In addition to PES, eleven (2.4% of all procedures) minor complications occurred and are presented in Table 4. Gastric erosions in symp­tomatic patients were detected by gastroscopy and treated medically. Intraprocedural small arte­rial branch rupture was immediately successfully treated with coil embolization. Four (0.9% of all procedures) major complica­tions were noted: ischemic cerebrovascular insult to the cerebellum, radial artery thrombosis follow­ing a transradial approach, variceal bleeding re­sulting from emesis after the procedure and infec­tion of the necrotic tumour, which resolved after antibiotic treatment. Overall survival After a median follow-up of 23.8 months (range, 5.6–94.6), 115 patients had died. The one-, two-, three-, four- and five-year survival rates were 85%, 53%, 33%, 20% and 14%, respectively (Figure 1). The median OS was 25.8 months (95% CI: 22.1–29.5 months). 315 TABLE 3. Hepatocellular carcinoma (HCC) treatment prior to initial doxorubicin-loaded drug-eluting microsphere transarterial chemoembolization (DEM-TACE) No previous treatment 120 (83.0) cTACE 2 (1.4) Surgical resection 13 (9.3) Transplantation 1 (0.7) Surgical resection and RFA 1 (0.7) Surgical resection and cTACE 1 (0.7) RFA 2 (1.4) MWA 1 (0.7) ECT 3 (2.1) cTACE = conventional TACE; ECT = electrochemotherapy; MWA = microwave ablation; RFA = radiofrequency ablation TABLE 4. Minor complications after drug-eluting microsphere transarterial chemoembolization (DEM-TACE) in the study population Chest pain 4 (0.9) Pain in the right shoulder 1 (0.2) Hematoma at the 3 (0.7) puncture site Gastric erosions 2 (0.4) Intraprocedural small arterial 1 (0.2) branch rupture Child-Pugh class, ascites and portal hyperten­sion showed statistically significant differences with respect to OS on univariate analysis (Table 5) (p = 0.008; p = 0.001; p = 0.016). No statistically significant difference was ob­served between unilobar and bilobar disease (p = 0.609). Treatment response and time to progression An objective response was achieved in 131 (91%) of 144 patients at the first dynamic contrast en­hanced imaging follow-up. A complete response was achieved in 72 of 131 patients (55.0%) patients, and a partial response was achieved in 59 (45.0%) patients. Progression was observed at the first imaging follow-up in 13 of 144 patients. Eight patients had target lesion progression, 3 patients had a new TABLE 5. Overall survival after DEM-TACE in the study population Senci polja med vrsticami Child-Pugh A 91 26.6 17.7–35.5 0.008 Child-Pugh B 29 19.6 18.4–20.8 Portal hypertension 76 20.2 14.7–25.7 0.001 No portal hypertension 68 32.4 24.0–40.8 Ascites 34 19.6 15.3–23.9 0.016 No ascites 110 29 23.0–35.0 Unilobar disease 92 24.9 21.0–28.8 0.609 Bilobar disease 52 26.3 21.5–31.1 intrahepatic lesion, and 2 developed extrahepatic lesions. Further decisions were based on clinical evaluations, laboratory values and imaging data. Patients with target lesion progression were fur­ther treated with doxorubicin-loaded DEM-TACE (37.5%, n = 3) or systemic chemotherapy (25%, n = 2) or received best supportive care (37.5%, n = 3). Patients with new intrahepatic lesions were further treated with doxorubicin-loaded DEM-TACE (n = 1), systemic chemotherapy (n = 1), or both (n = 1). Both patients with extrahepatic lesions received systemic chemotherapy. During the follow-up, progression was ob­served in 115 patients (79.9%). Of these, 27 patients (23.5%) had target lesion progression. The medi­an time to progression was 10.2 months (95% CI: 8.3–12.1 months). Overall, further HCC treatment after progression was performed in 93 patients. Doxorubicin-loaded DEM-TACE was performed in 66 patients, MWA in 2 patients and both in 1 patient. Twenty-one patients were treated with systemic therapy alone, and 3 were treated with systemic therapy and doxorubicin-loaded DEM­TACE. The remaining 22 patients received best supportive care. Discussion The purpose of this retrospective study was to eval­uate the safety and effectiveness of DEM-TACE in patients with early and intermediate stage HCC. TACE is the standard of care for HCC patients with intermediate-stage disease. Moreover, for patients who are ineligible for surgical resection or percutaneous ablation, TACE is also considered a first-line treatment option in the early stage.16,17 However, intermediate HCC corresponds to a highly heterogeneous group of patients with sig­nificant variations in number and size of tumour, patient performance status and liver function, re­sulting in variable survival rates.18 The median OS for patients treated with TACE is reported to range from approximately 19 months to 40–50 months in well-selected patients2,3,9, which is consistent with our results. The median OS in our study was 25.8 months, with a range from 5.6 months to more than 7 years. A multicentric study by Han et al. in 2019 showed a median OS of 19.9 months with a range from 7 months to more than 4 years.19 A study by Burrel et al. showed that the median OS can be pro­longed up to 48.6 months with careful patient se­lection.4 Several prognostic factors, such as Child Pugh class and tumour number, have been linked to higher survival rates and can be used to select ideal candidates for TACE.19,20 The Child Pugh score is a valuable tool for assessing liver function, and our results support its correlation with sur­vival. However, this score does not consider some events that may indicate end-stage liver disease (e.g., renal failure, spontaneous bacterial peritoni­tis, hyponatremia, recurrent encephalopathy, and malnutrition) and may be replaced by other se­lection criteria in the future.16 Another factor that may lead to better survival rates in some studies is treatment stage migration, as the survival of BCLC-A patients is expected to be better than that of BCLC-B patients.6 Treatment stage migration al­so applies to BCLC stage B. Although most patients achieve an objective response after treatment, they can present with new tumour sites during their follow-up and thus qualify as having disease pro­gression. Intrahepatic treatable progression can be treated with repeat TACE. On the other hand, the untreatable progression may necessitate initiation of systemic therapy. In this study, migration to sys­temic therapy was recorded in 24 patients. Higher OS may be related to the high objec­tive response rates achievable with TACE, which is supported by the fact that an objective response to treatment measured by mRECIST correlates with prolonged OS.21,22 According to the European Association for the Study of the Liver mRECIST represents the gold standard for radiologically evaluating tumour response during HCC locore­gional treatment.2 Previously used EASL criteria express the change in the two dimensions of hy­perenhancing tumour and therefore also reflect the extent of necrosis caused by the treatment. mRECIST criteria adopted a single long-axis meas­urement and are simplified objective measure of treatment response, especially with the use of su­ 317 perselective approach where little or no viable tu­mour is expected. An objective response to TACE is achieved in approximately 50% of patients, with the lowest rate reported to be approximately 16% and the highest reported to be approximately 85.6%.16,17,23 A prospective, randomized phase II study comparing doxorubicin-loaded DEM-TACE with cTACE showed higher objective response rates in the doxorubicin-loaded DEM-TACE group – 51.6% vs. 43.5%, respectively.11 In a study by Suk et al. assessing CBCT after doxorubicin-loaded DEM-TACE, an objective response was achieved in 85.6% of patients (63.8% complete response, 21.8% partial response).23 Our results exceed those of the above studies, showing a 91% rate of an objective response defined according to the mRECIST. The benefits of CBCT for intra-arterial liver pro­cedures are now well established and documented. The detection accuracy of CBCT for HCC lesions is equivalent to those of multidetector CT and MRI and superior to that of angiography. CBCT is the most accurate imaging technique to identify tu-mour-feeding arteries and can be used to rule out nontarget embolization of nontumour-feeding ex-trahepatic arteries. These advantages of CBCT can result in better treatment efficacy of DEM-TACE and other intra-arterial therapies.13 DEM-TACE has been demonstrated to result in fewer complications than cTACE.24 In a systemat­ic review of cTACE by Lencioni et al., 274 articles with a total of 34,137 patients were analyzed and adverse events were observed in 15,351 patients in a total of 217 selected studies.9 The most common procedure-related complication was PES, with the incidence of 47.7%. Other studies describe an even higher incidence of PES, with an estimated rate of 48%.9 According to the CIRSE guidelines, PES by itself is considered an expected outcome of the procedure rather than a complication.14,25 Procedural complications, including intraproce­dural small arterial branch rupture, hematoma at the puncture site, cerebellar stroke and radial ar­tery thrombosis following a transradial approach, occurred in 1.1% of our cases. Eosophageal variceal bleeding was managed with endoscopic variceal banding. To our knowledge there is no known mechanism through which TACE could facilitate variceal bleeding. Since the bleeding occurred on­ly once in otherwise large cohort of patients with high risk for variceal bleeding, we believe that this was probably a coincidental event. Other compli­cations are all recognized although extremely rare following TACE procedures and all of them were one-time events.26-28 Intraprocedural small arterial branch rupture was managed with endovascular coil embolization and this complication didn’t pro­long the patient’s hospital stay. Ischemic stroke is an extremely rare, but recognized event.27 A recent literature review describes twelve cases in patients undergoing DEB-TACE or cTACE with mecha­nisms including intrahepatic arteriovenous shunts, hepatopulmonary shunts and intracardiac shunts. The presence of arteriovenous shunting to hepatic or pulmonary veins is routinely checked for at in-traprocedural angiography and is a well-known contraindication to performing TACE, while check­ing for intracardiac shunts is not routinely per­formed in these patients. The patient in our cohort had a cerebellar stroke, which occurred after the procedure being performed via the right internal mamarian artery. Due to the cerebellar location, we hypothesize this non-target embolization was the result of the reflux of the embolic agent into the vertebral artery. In our patient the symptoms of ischemic insult resolved spontaneously, and no specific treatment was applied. Thrombosis of the radial artery was a result of transradial approach in a patient with a severe stenosis of iliac arteries pre­venting transfemoral approach. Radial artery oc­clusion is not a TACE specific complication and it occurs in 1 – 10% of patients following transradial interventions with lower numbers of complications occurring in experienced centers.26 Our patient was managed conservatively while hospitalized but was then lost to angiological follow-up. Our study has some limitations. This was a ret­rospective study including only a limited number of patients and no control group. A standard treat­ment methodology and patient selection criteria for TACE are lacking due to the heterogeneity of the HCC patient population with BCLC stages A and B. Therefore, patient selection should be care­fully considered to achieve the best outcomes. Subsequent studies should include a larger group of patients. Other medical centres should also be included since the results of this single-centre study may not be applicable to other centres and geographic regions. Furthermore, although four interventional radiologists performed DEM-TACE according to uniform protocols, minor variations, such as in catheterization selectivity, were inevi­table. Finally, the treatment effect of DEM-TACE may also be significantly influenced by multimo­dality treatment; thus, the long-term outcomes might not be representative. In conclusion, DEM-TACE under CBCT control in patients with early and intermediate stage HCC is a safe and effective method of care. The results 318 of this study are consistent with that in our study from 2016, confirming that proper patient selection, routine utilization of CBCT control for superselec­tive TACE guidance, regular treatment response evaluation and liver function tests, together with retreatment “on demand” results in high objective tumour response and survival rates. References 1. Global Burden of Disease Liver Cancer Collaboration, Akinyemiju T, Abera S, Ahmed M, Alam N, Alemayohu MA, et al. The burden of primary liver cancer and underlying etiologies from 1990 to 2015 at the global, regional, and national level: results from the Global Burden of Disease Study 2015. JAMA Oncol 2017; 3: 1683-91. doi: 10.1001/jamaoncol.2017.3055 2. European Association for the Study of the Liver. EASL Clinical Practice Guidelines: management of hepatocellular carcinoma. J Hepatol 2018; 69: 182-236. doi: 10.1016/j.jhep.2018.03.019 3. Vogel A, Cervantes A, Chau I, Daniele B, Llovet JM, Meyer T, et al. Hepatocellular carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2018; 29: iv238-55. doi: 10.1093/an­nonc/mdy308 4. Burrel M, Reig M, Forner A, Barrufet M, de Lope CR, Tremosini S, et al. Survival of patients with hepatocellular carcinoma treated by transarte-rial chemoembolisation (TACE) using drug eluting beads. Implications for clinical practice and trial design. J Hepatol 2012; 56: 1330-5. doi: 10.1016/j. jhep.2012.01.008 5. European Association for the Study of the Liver, European Organisation for Research and Treatement of Cancer. EASL-EORTC Clinical Practice Guidelines: management of hepatocellular carcinoma. J hepatol 2012; 56: 908-43. doi: 10.1016/j.jhep.2011.12.001 6. Bargellini I, Sacco R, Bozzi E, Bertini M, Ginanni B, Romano A, et al. Transarterial chemoembolization in very early and early-stage hepatocellu­lar carcinoma patients excluded from curative treatment: a prospective co­hort study. Eur J Radiol 2012; 81: 1173-8. doi: 10.1016/j.ejrad.2011.03.046 7. Varela M, Real MI, Burrel M, Forner A, Sala M, Brunet M, et al. Chemoembolization of hepatocellular carcinoma with drug eluting beads: efficacy and doxorubicin pharmacokinetics. J Hepatol 2007; 46: 474-81. doi: 10.1016/j.jhep.2006.10.020 8. Giannini EG, Farinati F, Ciccarese F, Pecorelli A, Rapaccini GL, Di Marco M, et al. Prognosis of untreated hepatocellular carcinoma. Hepatology 2015; 61: 184-90. doi: 10.1002/hep.27443 9. Lencioni R, de Baere T, Soulen MC, Rilling WS, Geschwind JF. Lipiodol tran­sarterial chemoembolization for hepatocellular carcinoma: a systematic re­view of efficacy and safety data. Hepatology 2016; 64: 106-16. doi: 10.1002/ hep.28453 10. Facciorusso A, Di Maso M, Muscatiello N. Drug-eluting beads versus conventional chemoembolization for the treatment of unresectable hepa­tocellular carcinoma: a meta-analysis. Dig Liver Dis 2016; 48: 571-7. doi: 10.1016/j.dld.2016.02.005 11. Lammer J, Malagari K, Vogl T, Pilleul F, Denys A, Watkinson A, et al. Prospective randomized study of doxorubicin-eluting-bead embolization in the treatment of hepatocellular carcinoma: results of the PRECISION V study. Cardiovasc Intervent Radiol 2010; 33: 41-52. doi: 10.1007/s00270­009-9711-7 12. Floridi C, Radaelli A, Abi-Jaoudeh N, Grass M, Lin M, Chiaradia M, et al. C-arm cone-beam computed tomography in interventional oncology: tech­nical aspects and clinical applications. Radiol Med 2014; 119: 521-32. doi: 10.1007/s11547-014-0429-5 13. Popovic P, Stabuc B, Jansa R, Garbajs M. Survival of patients with intermedi­ate stage hepatocellular carcinoma treated with superselective transarte-rial chemoembolization using doxorubicin-loaded DC Bead under cone-beam computed tomography control. Radiol Oncol 2016; 50: 418-26. doi: 10.1515/raon-2015-0045 14. Basile A, Carrafiello G, Ierardi AM, Tsetis D, Brountzos E. Quality-improvement guidelines for hepatic transarterial chemoembolization. Cardiovasc Intervent Radiol 2012; 35: 765-74. doi: 10.1007/s00270-012­0423-z 15. Lencioni R, Llovet J. Modified RECIST (mRECIST) Assessment for hepatocel­lular carcinoma. Semin Liver Dis 2010; 30: 52-60. doi: 10.1055/s-0030­1247132 16. Forner A, Reig M, Bruix J. Hepatocellular carcinoma. Lancet 2018; 391: 1301-14. doi: 10.1016/S0140-6736(11)61347-0 17. Llovet J, Bruix J. Systematic review of randomized trials for unresect-able hepatocellular carcinoma: chemoembolization improves survival. Hepatology 2003; 37: 429-42. doi: 10.1053/jhep.2003.50047 18. Piscaglia F, Bolondi L. The intermediate hepatocellular carcinoma stage: should treatment be expanded? Dig Liver Dis 2010; 42(Suppl 3): S258-63. doi: 10.4254/wjh.v7.i9.1184 19. Han G, Berhane S, Toyoda H, Bettinger D, Elshaarawy O, Chan AWH, et al. Prediction of survival among patients receiving transarterial chem-oembolization for hepatocellular carcinoma: a response-based approach. Hepatology 2020; 72: 198-212. doi: 10.1002/hep.31022 20. Xiao J, Li G, Lin S, He K, Lai H, Mo X, et al. Prognostic factors of hepatocellular carcinoma patients treated by transarterial chemoembolization. Int J Clin Exp Pathol 2014; 7: 1114-23. PMID: 24696728 21. Chen S, Peng Z, Zhang Y, Chen M, Li J, Guo R, et al. Lack of response to transarterial chemoembolization for intermediate-stage hepatocellular carcinoma: Abandon or repeat? Radiology 2021; 298: 680-92. doi: 10.1148/ radiol.2021202289 22. Olivo M, Valenza F, Buccellato A, Scala L, Virdone R, Sciarrino E, et al. Transcatheter arterial chemoembolisation for hepatocellular carcinoma in cirrhosis: survival rate and prognostic factors. Dig Liver Dis 2010; 42: 515-9. doi: 10.1016/j.dld.2009.09.012 23. Suk Oh J, Jong Chun H, Gil Choi B, Giu Lee H. Transarterial chemoemboliza­tion with drug-eluting beads in hepatocellular carcinoma: usefulness of contrast saturation features on cone-beam computed tomography imaging for predicting short-term tumor response. J Vasc Interv Radiol 2013; 24: 483-9. doi: 10.1016/j.jvir.2013.01.001 24. Ikeda M, Inaba Y, Tanaka T, Sugawara S, Kodama Y, Aramaki T, et al. A prospective randomized controlled trial of selective transarterial chemoem­bolization using drug-eluting beads loaded with epirubicin versus selective conventional transarterial chemoembolization using epirubicin-lipiodol for hepatocellular carcinoma: The JIVROSG-1302 PRESIDENT study. [Abstract]. JCO 2020; 38(15 Suppl): 4518. doi: 10.1200/jco.2020.38.15_suppl.4518 25. Dhand S, Gupta R. Hepatic transcatheter arterial chemoembolization com­plicated by postembolization syndrome. Semin Intervent Radiol 201; 28: 207-11. doi: 10.1055/s-0031-1280666 26. Rashid M, Kwok CS, Pancholy S, Chugh S, Kedev SA, Bernat I, et al. Radial artery occlusion after transradial interventions: a systematic review and meta-analysis. J Am Heart Assoc 2016; 5: e002686. doi: 10.1161/ JAHA.115.002686 27. Zach V, Rapaport B, Yoo JY, Goldfeder L, Weinberger J. Multiple ischemic strokes after transcatheter arterial chemoembolization for hepatocel­lular carcinoma with a radiographic and pathological correlate. J Stroke Cerebrovasc Dis 2012; 21: 217-24. doi: 10.1016/j.jstrokecerebrovas­dis.2010.08.001 28. Onizuka H, Sueyoshi E, Ishimaru H, Sakamoto I, Uetani M. Arterial injury dur­ing transcatheter arterial chemoembolization for hepatocellular carcinoma: predictors of risk and outcome. Abdom Radiol (NY) 2017; 42: 2544-50. doi: 10.1007/s00261-017-1168-6 319 research article Cone-beam computed tomography guided nusinersen administrations in adult spinal muscular atrophy patients with challenging access: a single- center experience Vladka Salapura1,2, Ziga Snoj1,2, Lea Leonardis2,3, Blaz Koritnik2,3, Viktorija Kostadinova1 1 Clinical Institute of Radiology, University Medical Centre Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Institute of Clinical Neurophysiology, University Medical Centre Ljubljana, Ljubljana, Slovenia Radiol Oncol 2022; 56(3): 319-325. Received 19 April 2022 Accepted 12 June 2022 Correspondence to: Assoc. Prof. Vladka Salapura, M.D., Ph.D., Clinical Institute of Radiology, University Medical Centre Ljubljana, Zaloška cesta 7, SI-1000 Ljubljana, Slovenia. E-mail: salapura@siol.net 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 challenging anatomic predispositions in adult patients with spinal muscular atrophy (SMA) pre­clude the conventional lumbar punctures. Consequently, an introduction of alternative method for intrathecal deliv­ery of nusinersen is required. Cone-beam CT (CBCT) allows volumetric display of the area of interest, pre-procedural planning and real time needle guidance which results in accurate anatomic navigation. The aim of the study was to evaluate technical success, safety, and feasibility of CBCT lumbar intrathecal delivery of nusinersen in the adult SMA patients with challenging anatomical access. Patients and methods. Thirty-eight adult SMA patients were treated in our institution. Patients with challenging ac­cess were selected by multidisciplinary board for image guided administration of nusinersen either due to implanta­tion of the posterior fusion instrumentation, severe scoliosis defined as Cobb’s angle > 40º or body mass index over 35. Technical success, radiation exposure and occurrence of adverse events were assessed. Results. Twenty patients were selected, and 108 CBCT-guided procedures were performed. Each patient underwent at least 4 administrations. Transforaminal approach was performed in 82% of patients. The technical success was 100%, with primary success of 93.5%. The median radiation effective dose of the administrations was 5 mSv, the mean value equalled 10 mSv. Only mild adverse events were reported in the study. Conclusions. CBCT-guided lumbar intrathecal administrations of nusinersen in an adult SMA population with chal­lenging access was feasible and safe image guided method. Key words: nusinersen; cone-beam CT; lumbar puncture Introduction Antisense-oligonucleotide nusinersen (Spinraza, Biogen Netherlands, Netherlands) was the first ap­proved intrathecal drug for treatment of 5q spinal muscular atrophy (SMA) experienced as safe and clinically beneficial for lifelong treatment of infants and children.1,2 Growing evidence indicate safety and efficacy even in some subgroups of adult pa­tients.3-5 Intrathecal administrations of nusinersen require lumbar puncture. In the natural progression of SMA, particularly in type 2 and 3, patients may develop severe scoliosis.6 In the most severe, type 1 SMA the patients rarely survive to adulthood, where as in type 4 being the mildest, involvement of the spine is rare.6 Debilitating scoliosis which requires surgery may not develop up to the onset of puberty and dur­ing childhood a conventional lumbar puncture is usually feasible.6 In older patients factors such as al­tered spine anatomy, obliterated interlaminar space and obesity are commonly present and preclude the conventional lumbar puncture.6-8 Consequentially, the need for reproducible and safe image-guided method for intrathecal administrations in adult pa­tients has been accentuated.9 Several studies described successful techniques for administration of nusinersen in patients with challenging access. In these patients, successful interlaminar (IL) or transforaminal (TF) lumbar accesses have been performed under CT-, fluoro­scopic- or ultrasound-guidance.7,10-14 However, the possibility of spine deformity progression and the need for repetitive injections require a method with satisfactory deep soft tissue resolution and follow­ing the “as low as reasonable achievable” radiation principle.10,15,16 The cone-beam CT (CBCT) allows volumetric display of the area of interest, pre-pro­cedural planning and real time needle guidance which results in accurate anatomic navigation.17 So far, only three studies described the use of CBCT for intrathecal delivery of nusinersen in children and adults.11,18,19 However, up to our knowledge there are no larger studies on CBCT guided in-trathecal nusinersen delivery that would present data only on adult SMA patients. The purpose of this prospective study was to present a single-center experience on implemen­tation of lumbar spine CBCT-guided intrathecal nusinersen delivery in consecutive adult SMA patients with challenging access to investigate the technical success, feasibility, and safety. Patients and methods Patients The treatment with nusinersen has been available to the adult Slovenian patients with SMA in the University Medical Centre Ljubljana since the be­ginning of 2019. Thirty-eight adult SMA patients have undergone the repetitive treatment with in-trathecal administrations in our institution from April 2019 to May 2021. Patients with challenging access were selected for CBCT-guided intrathecal nusinersen delivery. The study was approved by the National Ethics Committee. An informed written consent from the patients was obtained before the beginning of the study. Inclusion criteria The criteria for the group eligible for CBCT-guided lumbar punctures were determined by a multidis­ciplinary board team of neurologists and interven­tional radiologists. Patients with history of scoliosis corrective surgery with implantation of posterior fusion instrumentation, severe scoliosis defined as Cobb’s angle > 40° or patients with body mass in­dex (BMI) over 35 were included in CBCT-guided group.8,20,21 The remaining patients underwent con­ventional lumbar puncture. Procedure All procedures were performed on Siemens Artis Q (Siemens Healthineers, Erlangen, Germany) CBCT system with C-arm and navigational overlay in outpatient setting in the interventional radiology suite by interventional radiologists with more than 10-year experience in image-guided procedures. Upon arrival to the interventional suite, the patients with implanted posterior instrumenta­tion were placed in half lateral position to expose the curvature of the spine for TF approach. For IL approach either prone or half-lateral position was used. Following optimal patient positioning, a sin­gle rotation of a low dose CBCT was performed. Using the navigational computer program, the en­try and target point were defined by the performing radiologist considering the safest and most feasible approach. For patients with history of spine sur­gery and posterior fusion instrumentation a TF ap­proach was used as it was the only feasible option.20 In this approach the trajectory line was positioned via the inferior portion of the intervertebral fora-men to evade the exiting neurovascular bundle. IL approach was selected for patients without spinal instrumentation and visible interlaminar space.12 Lumbar punctures were performed under sterile conditions, local anesthesia was used. For the pro­cedures 20 G spinal needles were used, the length of needle based on the distance from the skin to the target point. Integrated laser of the C-arm marked the en­try position of the needle on the skin. The target point position was visualized under intermittent fluoroscopic guidance using two orthogonal views (Figures 1, 2, 3). Once the needle tip reached the target point, an aspiration of cerebrospinal fluid (CSF) was performed to confirm the intrathecal po­sition. Afterwards, 5 ml of CSF was aspirated, and 5 ml nusinersen solution was intrathecally deliv­ered according to the manufacturer’s instructions. 321 After the procedure the patients were surveilled for 4-6 hours before being discharged. Variables and data collection Patient age, sex, BMI, type of SMA were recorded and spine anatomy was evaluated. For each pro­cedure the type of the approach (TF or IL) with level of injection, total duration of the procedure from the arrival in the interventional suite to exit­ing, the technical success and peri-procedural ad­verse events (AE) were noted. Radiation exposure was calculated as effective dose (ED) as a product from dose-area product (DAP) and theoretical coefficient. The theoretical coefficient used in the equation was 0.0012 mSv/µGy*m2. The data was obtained through patient databases and systematic questionnaire. Technical success The technical success was defined by extraction of a macroscopically clear, no-blood-contaminated cerebrospinal fluid (CSF) with successful intrathe-cal application of 12 mg nusinersen.11 Primary suc­cess was determined when a macroscopically clear CSF was extracted at first attempt without further repositioning of the needle. Secondary success was defined for procedures that required additional attempts for secure access at the same or another level during the same procedure. Adverse events Peri-procedural AEs that occurred during the first 24 hours were recorded in accordance with the Society of Interventional Radiology guidelines.22 AEs were noted during the peri-procedural sur­veillance period and reported by the patients upon their next regular clinical visit at the outpatient department. The patients assessed the overall dis­comfort and pain level during the CBCT-guided nusinersen intrathecal delivery using the Visual Analogue Scale (VAS). Statistical analysis Calculations were performed with statistical spreadsheet computer program SPSS Inc. (SPSS for Windows, Version 22.0. Chicago, SPSS Inc). The normality of variable distribution was obtained us­ing the Shapiro-Wilk test. Statistical analyses were performed using t-test for independent variables and Mann-Whitney test. Results Patients 20 patients (53%) were found eligible for CBCT-guided intrathecal nusinersen delivery. Seventeen patients had severe scoliosis, ten patients had pos­terior fusion instrumentation and two patients were obese. In 18 patients (47%) intrathecal ad­ministrations were possible by conventional lum­bar puncture and were not included in our study. Patient characteristics are presented in Table 1. None of the patients withdrew from the CBCT-guided intrathecal nusinersen delivery. Procedure During the study 108 CBCT-guided procedures were performed. Each patient underwent at least 4 administrations. The patient with most adminis­trations had 8 administrations. The predominant approach to the spine was TF with L2-L3 or L3-L4 being the most frequent levels of lumbar puncture. For IL approach L3-L4 level was most frequently chosen (Table 2). Total procedure time was approx­imately 1 hour (Table 2). TABLE 1. Patient characteristics for cone-beam CT (CBCT)-guided intrathecal nusinersen delivery patients and classical lumbar puncture patients Male sex (%) 10 (50) 12 (67) Age at first administration, median (range) year 33.5 (20–62) 44.5 (19–69) 0.62 BMI, median (range) kg/m2 23.4 (14.2–41.8) 24.7 (14.3–33.6) 0.08 SMA type 2 (%) 13 (65) 0 SMA type 3 (%) 7 (35) 15 (83) SMA type 4 (%) 0 3 (17) Posterior fusion instrumentation due to scoliosis (%) 10 (50) 0 Severe scoliosis (%) 17 (85) N/A BMI = body mass index; N/A = not available; SD = standard deviation; SMA = spinal muscle atrophy TABLE 2. Procedure summary Technical success All CBCT-guided procedures were technically successful. Primary success was achieved in 101 (94%) procedures. In others secondary success was achieved; 2 IL and 5 TF approach. Effective dose The median ED for all administrations was 5.5 mSv (interquartile range 2.7–13 mSv) and mean 10 mSv (standard deviation 11 mSv). There was no differ­ence in median ED between patients with poste­rior fusion instrumentation in comparison to the patients without (5 mSv vs. 5.8 mSv). There was a statistically important difference in median ED for obese patients in comparison to other patients (12 mSv vs. 5 mSv, p = 0.004). ED for every application is presented in Figure 4. Adverse events L1-L2 (%) L2-L3 (%) L3-L4 (%) L4-L5 (%) Number of procedures (%) Duration per procedure, mean ± SD min 0 (0) 4 (4) 4 (4) 7 (6) 48 (44) 55 (50) 10 (9) 35 (32) 45 (41) 2 (2) 3 (3) 4 (5) 19 (18) 89 (82) 108 (100) 63 ± 21 60 ± 26 62 ± 25 SD = standard deviation Median value of patients’ subjective assessment of pain level on the VAS scale for CBCT-guided pro­cedures was 4. After the CBCT-guided procedures, twelve patients (60 %) at least once experienced headaches or low back pain (Table 3). Two patients (10 %) additionally experienced pain in the upper extremity due to positioning during the procedure. One patient (5%) reported radiating pain in the leg. AEs were labeled as mild, since no or nominal therapy was required.22 There were no AEs in the remaining 5 patients (25%). Discussion We report our experience on implementation of lumbar spine CBCT-guided intrathecal nusinersen delivery in consecutive adult SMA patients with challenging access. Evidence gained support good technical success, safety, and feasibility of CBCT-guided intrathecal nusinersen delivery. The patient selection in our study was multidis­ciplinary, prospective, and based on inclusion crite­ria which considered the anatomy of the adult SMA patients. Additionally, to criteria regarding sco­liosis and history of corrective surgery, we also ac­knowledged the patients’ general constitution. This problem was so far addressed in only one study in a patient with BMI of 28, in which the intrathecal ad­ministration was performed under CT-guidance.10 Only few studies have reported their experi­ence in CBCT-guided intrathecal administrations 323 of nusinersen.11,18,19 An early study utilized CBCT for needle positioning in three TF procedures in children.18 The authors reported later switch to fluoroscopy guidance as the performing physi­cians gained confidence with the intrathecal deliv­eries.18 A later study analyzed lumbar punctures with TF approach in seven adult patients.19 The latest study by Weaver et al. presented the largest group of 28 patients for CBCT-guided intrathecal nusinersen delivery.11 In these studies the CBCT-guided lumbar intrathecal delivery was performed in both children and adult patients, whereas our study focused only on adult patients.11,18,19 In our study CBCT-guided procedures were predomi­nantly performed by the TF approach, a similar experience previously described by Weaver et al.11 IL approach was not possible either due to severe scoliosis or no visible interlaminar space on CT af­ter the posterior fusion instrumentation. Therefore, our data is in line with previous study, which ac­knowledges that a growing population of SMA pa­tients requires alternative to the IL approach.18 Technical success of the CBCT-guided adminis­trations was achieved in all patients. Only in few procedures secondary success was noted. This is in accordance with the study by Weaver et al. which reported high primary success rate for TF approach performed by both CBCT and fluoros­copy.11 Similarly, high technical success is reported in studies utilizing CT as image-guidance.7,12,20,23 However, while we specifically determined the technical success according to successful approach to the intrathecal space, other studies defined tech­nical successes ambiguously; namely a primary success in most of the studies was not defined.7,12,20,23 Two CT-guided studies reported a high (95% and 96.2%) single puncture attempt, which was compa­rable to our primary technical success.12,23 In contrast to other CBCT studies, only mild peri-procedural AEs were reported in ours. Weaver et al. reported 4% occurrence of mild AEs such as radicular pain and headaches as well as 0.5% of severe AE such as meningitis.11 Shokuhfar et al. re­corded one case (10%) of bilateral radiculopathy.19 Although no severe AEs were noted in our study, the aforementioned studies raise attention to the potential risks one must take into consideration be­fore the procedure. In the study we also report AEs from the patients that underwent conventional lumbar punctures. In comparison to patients after the conventional lumbar puncture, patients after the CBCT-guided punctures reported lower inten­sity and duration of low back pain. This finding is contrary to the findings by Carrera-Garcia et al.8 A TABLE 3. Adverse events for cone-beam CT (CBCT)-guided intrathecal nusinersen delivery patients and classical lumbar puncture patients Headache occurence (%) 18 (17) 42 (37) Headaches VAS, median (range) 2 (0–10) 4.5 (0–10) 0.12 Headaches duration day, median (range) 0.05 (0–5) 2 (0–6) 0.05 Low back pain occurrence (%) 11 (10) 40 (36) Low back pain VAS, median (range) 0 (0–2) 2.75 (6) < 0.01 Low back pain duration day, median (range) 0 (0–4) 2.45 (0–14) < 0.01 VAS = visual analogue scale possible explanation might be the high proportion of primary technical success which minimized the trauma to the spinal meninges and the peridural Effective dose [mSv] 70 60 50 40 30 20 10 0 Number of successive nusinersen administrations FIGURE 4. Scatter plot presenting effective dose for each cone-beam CT-guided procedure (blue dots) and calculated average trend line with orange dots for every ten procedures. mSv = milli sievert membrane which are abundant in nociceptors and other sensory receptors.24 Direct comparison with other CBCT-guided studies regarding radiation was not possible due to differences in reported units.11,19 In comparison to our study, three CT-guided studies performed pre­dominantly on adult patients reported lower ED of around 2.5 mSv, whereas Spiliopolus et al. report­ed higher average ED of 12.7 mSv.3,7,23,25 Plausible rationale for these findings may be differences in population characteristics. Patients with posterior spinal instrumentation are expected to receive higher radiation exposure than the patients with­out fusion instrumentation.25,26 Contrary to this ob­servation our data did not reveal any ED differenc­es regarding spine instrumentation. However, the EDs for the two obese patients proved to be higher in comparison to the other patients. There was a decline in the average ED over the course of the study time, a finding that is in line with other studies.12,26 The following factors need to be accounted for the initial high ED. The highest contributing factor to the initial high ED was pa­tients’ repositioning with CBCT reacquisition. The patients were repositioned as they either found the initial position uncomfortable or the target region was insufficiently depicted on acquired CBCT or the most comfortable patients’ position did not allow optimal CBCT acquisition. Physicians’ ex­perience to adjust for specific anatomical consid­erations was also important in ED reduction. Thus, good cooperation between radiographers and per­forming physicians plays a key role in radiation exposure reduction. It is important to understand that each adult SMA patient is specific with differ­ences in anatomical considerations, thus patient-tailored approach needs to be implemented. There are few limitations that need to be noted. One might argue that this is only a single-center study. A multi-center comparison is not realizable since we are the only institution performing these procedures in our country. Furthermore, it would be difficult to standardize protocol in between or­ganizations due to specifics of such a group. Here we report consecutive patients with standard pro­tocol, which provides important insight in clinical work. Additionally, only partial comparison be­tween other studies with CBCT-guidance was pos­sible since there were differences in patient charac­teristics and methodology.11,18,19 Conclusions This single-center prospective study supported the use of CBCT-guided lumbar intrathecal adminis­trations of nusinersen in an adult SMA population with challenging access as feasible, technically suc­cessful, and safe image guided method. References 1. Finkel RS, Mercuri E, Darras BT, Connolly AM, Kuntz NL, Kirschner J, et al. Nusinersen versus Sham Control in Infantile-Onset Spinal Muscular Atrophy. N Engl J Med 2017; 377: 1723-32. doi: 10.1056/NEJMoa1702752 2. Mercuri E, Darras BT, Chiriboga CA, Day JW, Campbell C, Connolly AM, et al. Nusinersen versus sham control in later-onset spinal muscular atrophy. N Engl J Med 2018; 378: 625-35. doi: 10.1056/NEJMoa1710504 3. Stolte B, Nonnemacher M, Kizina K, Bolz S, Totzeck A, Thimm A, et al. Nusinersen treatment in adult patients with spinal muscular atrophy: a safety analysis of laboratory parameters. J Neurol 2021, 268: 4667-79. doi: 10.1007/s00415-021-10569-8 4. Maggi L, Bello L, Bonanno S, Govoni A, Caponnetto C, Passamano L, et al. Nusinersen safety and effects on motor function in adult spinal muscular atrophy type 2 and 3. J Neurol Neurosurg Psychiatry 2020; 91: 1166-74. doi: 10.1136/jnnp-2020-323822 5. Hagenacker T, Wurster CD, Gther R, Schreiber-Katz O, Osmanovic A, Petri S, et al. Nusinersen in adults with 5q spinal muscular atrophy: a non-interventional, multicentre, observational cohort study. Lancet Neurol 2020; 19: 317-25. doi: 10.1016/s1474-4422(20)30037-5 6. Wijngaarde CA, Brink RC, de Kort FAS, Stam M, Otto LAM, Asselman FL, et al. Natural course of scoliosis and lifetime risk of scoliosis surgery in spinal muscular atrophy. Neurology 2019; 93: e149-e58. doi: 10.1212/ wnl.0000000000007742 7. Bortolani S, Stura G, Ventilii G, Vercelli L, Rolle E, Ricci F, et al. Intrathecal ad­ministration of nusinersen in adult and adolescent patients with spinal mus­cular atrophy and scoliosis: Transforaminal versus conventional approach. Neuromuscul Disord 2019; 29: 742-6. doi: 10.1016/j.nmd.2019.08.007 8. Carrera-García L, Muchart J, Lazaro JJ, Expito-Escudero J, Cuadras D, Medina J, et al. Pediatric SMA patients with complex spinal anatomy: Implementation and evaluation of a decision-tree algorithm for administra­tion of nusinersen. Eur J Paediatr Neurol 2021; 31: 92-101. doi: 10.1016/j. ejpn.2021.02.009 325 9. Weaver JJ, Natarajan N, Shaw DWW, Apkon SD, Koo KSH, Shivaram GM, et al. Transforaminal intrathecal delivery of nusinersen using cone-beam computed tomography for children with spinal muscular atrophy and exten­sive surgical instrumentation: early results of technical success and safety. Pediatr Radiol 2018; 48: 392-7. doi: 10.1007/s00247-017-4031-6 10. Wurster CD, Winter B, Wollinsky K, Ludolph AC, Uzelac Z, Witzel S, et al. Intrathecal administration of nusinersen in adolescent and adult SMA type 2 and 3 patients. J Neurol 2019; 266: 183-94. doi: 10.1007/s00415-018-9124­ 11. Weaver JJ, Hallam DK, Chick JFB, Vaidya S, Shin DS, Natarajan N, et al. Transforaminal intrathecal delivery of nusinersen for older children and adults with spinal muscular atrophy and complex spinal anatomy: an analy­sis of 200 consecutive injections. J Neurointerv Surg 2021; 13: 75-8. doi: 10.1136/neurintsurg-2020-016058 12. Cordts I, Lingor P, Friedrich B, Pernpeintner V, Zimmer C, Deschauer M, et al. Intrathecal nusinersen administration in adult spinal muscular atrophy patients with complex spinal anatomy. Ther Adv Neurol Disord 2020; 13: 1756286419887616. doi: 10.1177/1756286419887616 13. Mendonça RH, Fernandes HDS, Pinto RBS, Matsui Jior C, Polido GJ, Silva A, et al. Managing intrathecal administration of nusinersen in adolescents and adults with 5q-spinal muscular atrophy and previous spinal surgery. Arq Neuropsiquiatr 2021; 79: 127-32. doi: 10.1590/0004-282x-anp-2020-0200 14. Snoj Z, Salapura V. Ultrasound-guided transforaminal approach for nusin­ersen delivery in adult spinal muscle atrophy patients with challenging access. Muscle Nerve 2022; 65: 585-9. doi: 10.1002/mus.27518 15. Hansson SO. Improvement principles. J Safety Res 2019; 69: 33-41. doi: 10.1016/j.jsr.2019.02.001 16. Cordts I, Deschauer M, Lingor P, Burian E, Baum T, Zimmer C, et al. Radiation dose reduction for CT-guided intrathecal nusinersen administration in adult patients with spinal muscular atrophy. Sci Rep 2020; 10: 3406. doi: 10.1038/ s41598-020-60240-x 17. Racadio JM, Babic D, Homan R, Rampton JW, Patel MN, Johnson ND. Live 3D guidance in the interventional radiology suite. AJR Am J Roentgenol 2007; 189: W357-64. doi: 10.2214/ajr.07.2469 18. Towbin R, Schaefer C, Kaye R, Abruzzo T, Aria DJ. The complex spine in children with spinal muscular atrophy: The transforaminal approach-A transformative technique. AJNR Am J Neuroradiol 2019; 40: 1422-6. doi: 10.3174/ajnr.A6131 19. Shokuhfar T, Abdalla RN, Hurley MC, Nazari P, Ansari SA, Ajroud-Driss S, et al. Transforaminal intrathecal access for injection of nusinersen in adult and pediatric patients with spinal muscular atrophy. J Neurointerv Surg 2020; 18: 88-94. doi: 10.1055/s-0039-1697583 20. Nascene DR, Ozutemiz C, Estby H, McKinney AM, Rykken JB. Transforaminal lumbar puncture: an alternative technique in patients with challenging ac­cess. AJNR Am J Neuroradiol 2018; 39: 986-91. doi: 10.3174/ajnr.A5596 21. Kuznia AL, Hernandez AK, Lee LU. Adolescent idiopathic scoliosis: com­mon questions and answers. Am Fam Physician 2020; 101: 19-23. PMID: 31894928 22. Khalilzadeh O, Baerlocher MO, Shyn PB, Connolly BL, Devane AM, Morris CS, et al. Proposal of a new adverse event classification by the society of in-terventional radiology standards of practice committee. J Vasc Interv Radiol 2017; 28: 1432-7.e3. doi: 10.1016/j.jvir.2017.06.019 23. Spiliopoulos S, Reppas L, Zompola C, Palaiodimou L, Papadopoulou M, Filippiadis D, et al. Computed-tomography-guided transforaminal intrathe-cal nusinersen injection in adults with spinal muscular atrophy type 2 and severe spinal deformity. Feasibility, safety and radiation exposure considera­tions. Eur J Neurol 2020; 27: 1343-9. doi: 10.1111/ene.14245 24. Sehgal IJ. Anatomy, back, spinal meninges. Treasure Island (FL): StatPearls Publishing; 2022. 25. Oldenburg D, Guberina N, Stolte B, Kizina K, Stenzel E, Radbruch A, et al. Radiation exposure of image-guided intrathecal administration of nusin­ersen to adult patients with spinal muscular atrophy. Neuroradiology 2019; 61: 565-74. doi: 10.1007/s00234-019-02189-x 26. Kizina K, Stolte B, Totzeck A, Bolz S, Fleischer M, Mninghoff C, et al. Clinical implication of dosimetry of computed tomography- and fluoroscopy-guided intrathecal therapy with nusinersen in adult patients with spinal muscular atrophy. Front Neurol 2019; 10: 1166. doi: 10.3389/fneur.2019.01166 research article Nanosecond electric pulses are equally effective in electrochemotherapy with cisplatin as microsecond pulses Angelika Vizintin1, Stefan Markovic2, Janez Scancar2, Jerneja Kladnik3, Iztok Turel3, Damijan Miklavcic1 1 Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia 2 Department of Environmental Sciences, Jožef Stefan Institute, Ljubljana, Slovenia 3 Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2022; 56(3): 326-335. Received 7 June 2022 Accepted 19 June 2022 Correspondence to: Prof. Damijan Miklavcic, Ph.D., Faculty of Electrical Engineering, University of Ljubljana, Tržaška cesta 25, SI-1000 Ljubljana, Slovenia. E-mail: Damijan.Miklavcic@fe.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. Nanosecond electric pulses showed promising results in electrochemotherapy, but the underlying mechanisms of action are still unexplored. The aim of this work was to correlate cellular cisplatin amount with cell survival of cells electroporated with nanosecond or standardly used 8 × 100 µs pulses and to investigate the effects of electric pulses on cisplatin structure. Materials and methods. Chinese hamster ovary CHO and mouse melanoma B16F1 cells were exposed to 1 × 200 ns pulse at 12.6 kV/cm or 25 × 400 ns pulses at 3.9 kV/cm, 10 Hz repetition rate or 8 × 100 µs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz repetition rate at three cisplatin concentrations. Cell survival was determined by the clonogenic assay, cellular platinum was measured by inductively coupled plasma mass spectrometry. Effects on the structure of cisplatin were investigated by nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. Results. Nanosecond pulses equivalent to 8 × 100 µs pulses were established in vitro based on membrane permea­bilization and cell survival. Equivalent nanosecond pulses were equally efficient in decreasing the cell survival and ac­ cumulating cisplatin intracellularly as 8 × 100 µs pulses after electrochemotherapy. The number of intracellular cisplatin molecules strongly correlates with cell survival for B16F1 cells, but less for CHO cells, implying the possible involvement of other mechanisms in electrochemotherapy. The high-voltage electric pulses did not alter the structure of cisplatin. Conclusions. Equivalent nanosecond pulses are equally effective in electrochemotherapy as standardly used 8 × 100 µs pulses. Key words: electroporation; electrochemotherapy; nanosecond pulses; cisplatin Introduction Electrochemotherapy (ECT) is a local cancer treat­ment. The dominant mechanism of ECT is in­creased cellular uptake of impermeant or low permeant anticancer drugs with high intrinsic cytotoxicity - most commonly bleomycin and cis­diaminedichloroplatinum(II) (cisplatin) - due to transiently increased membrane permeability of cells/tumors after exposure to short high-voltage electric pulses.1 Over the past ten years, the number of ECT treatments performed for superficial tumors has increased dramatically and new indications have been added, such as treatment of skin metastases from visceral or hematological malignancies, vul-var cancer, deep-seated malignancies, and some noncancerous skin lesions.2 ECT has become 327 broadly accepted mainly because of its simplicity (it is easy to master) and versatility (it allows treat­ing a variety of cancers). Its efficacy, tolerability, and high patient satisfaction have been demon­strated in several studies, but also some side effects have been reported. According to the reports, the main side effects are unpleasant sensations, which can be painful, and muscle contractions triggered by applied high voltage electric pulses.3,4 Most commonly, electric pulses are administrated as trains of eight monophasic pulses with a duration of 100 µs at 1 Hz or 5 kHz pulse repetition rate. Nanosecond pulses have shown potential ad­vantages over micro- and millisecond pulses in electroporation-based applications. The use of pulses with high electric field strength, but very short duration (i.e., in the nanosecond range) re­sults in low energy transfer by the pulses to the treated volume, resulting in a low heating5,6 and thereby minimizing the possibility of thermal dam­age to the tissue, which is very important for spar­ing delicate structures in and around the treated area.7 In addition, nanosecond pulses limit elec­trochemical reactions at the electrode-electrolyte interface8 which may affect the treated medium or cells/tissues.9-11 Although a much higher electric field strength is required to achieve a comparable biological effect, excitation thresholds appear to be higher than the electroporation thresholds with na­nosecond pulses12-16, implying that shortening the pulse duration to nanosecond pulses could also reduce neuromuscular stimulation in electropora­tion-based applications. Recently, nanosecond pulses have been ex­plored in ECT and calcium electroporation and have shown promising results – either tumor re­gression in vivo or a decrease in cell survival in vit­ro.8,17-21 We have previously reported that nanosec­ond pulses of an appropriately chosen amplitude in combination with cisplatin decreased cell sur­vival in in vitro assays to the same extent as stand­ard 8 × 100 µs pulses.8 The aim of our present work was to investigate the underlying mechanisms of ECT with nanosecond pulses and cisplatin in vitro on Chinese hamster ovary CHO and mouse skin melanoma B16F1 cells. Two nanosecond pulse protocols (1 × 200 ns pulse at 12.6 kV/cm and 25 × 400 ns pulses at 3.9 kV/cm, 10 Hz repetition rate) were compared with 8 × 100 µs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz repetition rate stand­ardly used in ECT. Accumulation of cisplatin and cell survival after in vitro ECT were measured and effects of high voltage electric pulses on the cispl­atin molecular structure were investigated by nu­clear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HRMS). Materials and methods Cell culture of Chinese hamster ovary (CHO) cells and in vitro cell survival after ECT experiment protocols were described previously.8 Mouse skin melanoma cell line B16F1 (European Collection of Authenticated Cell Cultures, cat. no. 92101203, Sigma Aldrich, Germany, mycoplasma free) was cultured in the same way as CHO cells except that Dulbecco’s Modified Eagle Medium (DMEM, cat. no. D5671, Sigma-Aldrich, Missouri, United States) supplemented with 10% FBS (cat. no. F9665, Sigma-Aldrich), 2.0 mM L-glutamine, 1 U/ml penicillin/ streptomycin and 50 µg/ml gentamycin was used instead of Nutrient Mixture F-12 Ham. Briefly, cis­platin (Cisplatin Kabi, 1 mg/mL, Fresenius Kabi, Germany or Cisplatin Accord, 1 mg/ml, Accord, UK) diluted in saline was added to cells suspended in complete growth medium DMEM just before electroporation so that the final concentration was 4 × 106 cell/ml and 0, 10, 30 or 50 µM cisplatin. The cell suspension was exposed to monophasic rectan­gular pulses (1 × 200 ns pulse at 12.6 kV/cm or 25 × 400 ns at 3.9 kV/cm, 10 Hz repetition rate or 8 × 100 µs at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz pulse repetition rate) or no pulses (non-electropo-rated controls). Cell survival was determined by the clonogenic assay. For determination of cellular cisplatin, 125 µl of the treated cell suspension was diluted 40–100 times in complete growth medium Ham F-12 (CHO) or complete growth medium DMEM (B16F1) 25 min after electroporation (or addition of cisplatin/saline for non-electroporated con­trols) and centrifuged at 900 g for 5 min at 23°C in 15 ml centrifuge tubes. The supernatant was separated from the cell pellet and the pellet was washed with 2 ml saline and centrifuged again. After centrifugation, saline was discarded, and the cell pellet was kept at -20°C until digestion.For digestion, 0.1 ml H2O2 and 0.1 ml HNO3 (both from Merck, Germany) were added to the cell pel­lets, and the tubes were closed and sealed with Teflon tape and left overnight at 80°C. After diges­tion, 1.8 ml of Milli-Q water (18.2 MO obtainedfrom a Direct-Q 5 Ultrapure water system, Merck Millipore, Massachusetts, USA) was added and samples were measured by inductively coupled plasma mass spectrometry (7900 ICP-MS Agilent Technologies, Japan) with 193Ir used as an inter­nal standard during the measurement. The ex­periments were repeated 4–7 times. The number of cisplatin molecules per cell was calculated by first dividing the measured total mass of Pt in the cell pellet by the number of cells in the pellet, then subtracting the average mass of Pt per cell of non-electroporated cell pellets that were not incubated with cisplatin, and finally calculating the number of cisplatin molecules per cell from the difference of the mass of Pt per cell in samples (assuming 1 mol of Pt is equivalent to 1 mol of cisplatin). Cell survival and amount of Pt data (after outli­ers, defined using the interquartile range method, were removed) were analyzed using the Kruskal– Wallis test and p-values were adjusted with the post-hoc Holm method test (a = 0.05) because the Shapiro-Wilk normality test failed (a = 0.05). The Spearman correlation coefficient was calculated to test the correlation between the number of cisplatin molecules per cell and cell survival. The data were processed and visualized using Microsoft Excel 2016 and R 3.6.1.22 Potential structural changes of cisplatin in the so­lution treated with high voltage electric pulses were investigated by NMR spectroscopy and HRMS. For practical reasons, both microsecond and nanosec­ond pulses were delivered to electroporation cu-vettes with 2 mm gap with the laboratory proto­type pulse generator based on an H-bridge digital amplifier for this set of experiments. For microsec­ond pulses, 8 × 100 µs at 1.1 kV/cm at 1 Hz pulse repetition rate were delivered (same pulse protocol as in cellular electrochemotherapy experiments). For nanosecond pulses, 25 × 400 ns at 2.2 kV/cm at 10 Hz repetition rate were delivered – the elec­tric field strength for this pulse protocol was lower than in cellular electrochemotherapy experiments because of the technical limitations of the prototype pulse generator. 1 × 200 ns pulse was not applied because the pulse generator used is not capable of generating such short pulses. 1H NMR spectra were obtained on NMR Bruker AscendTM 600 MHz spectrometer at room temperature at 600 MHz. Chemical shifts, reported in ppm, are referenced to residual peaks of D2O at 4.79 ppm. Spectra were re­corded in D2O (with and without NaCl) as well as in 90% H2O/10% D2O (with or without NaCl) using water suppression (WATERGATE) method. NMR data were processed with MestReNova 11.0.4. To approximately 1–2 mg of cisplatin (Sigma Aldrich) 1 mL of a) D2O, b) D2O containing 154 mM NaCl, c) 90% H2O/10% D2O or d) 90% H2O/10% D2O con­taining 154 mM NaCl was added. The obtained suspension was filtered through Minisart NML Cellulose Acetate Syringe Filter (28 mm, 0.2 µL). 1H NMR spectra were recorded immediately after the filtration when not treated with any pulse pro­tocol or directly after microsecond or nanosecond pulse application. HRMS spectra were recorded on Agilent 6224 Accurate Mass Time of Flight (TOF) Liquid Chromatography-Mass Spectrometry (LC­MS) instrument using water-acetonitrile solution (80:20, v/v) as the mobile phase. Fragmentor volt­age was set to 150.0 V. To approximately 1–2 mg of cisplatin (Sigma Aldrich) 1 mL of distilled water or saline was added and obtained suspension was filtered through Minisart NML Cellulose Acetate Syringe Filter (28 mm, 0.2 µL). Filtered solutions underwent a) no pulses, b) microsecond pulses, or c) nanosecond pulses application as mentioned above, followed by immediate injection of such so­lutions into the LC-MS. Results CHO and B16F1 cells were electroporated in pres­ence of 10, 30 and 50 µM cisplatin with: 1 × 200 ns pulse at 12.6 kV/cm; 25 × 400 ns pulses at 3.9 kV/ cm, 10 Hz pulse repetition rate; or 8 × 100 µs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz pulse rep­etition rate. The electric field strengths for specific pulse parameters were selected based on survival-permeabilization curves (refer to Vižintin et al.8 for graphs for CHO cells and to Figure S1 in the Supplementary material for graphs for B16F1 cells). Cell survival results after ECT determined by the clonogenic assay are shown in Figure 1. Survival data of CHO cells were combined from the previ­ous8 (for non-electroporated cells and cells elec­troporated with 25 × 400 ns and 8 × 100 µs pulses) and the present study (additional non-electropo-rated cells and cells electroporated with 1 × 200 ns pulse). As intended, electroporation alone (i.e., in the absence of cisplatin) did not decrease cell survival in both cell lines compared with the non-electroporated control for any of the pulse proto­cols tested. For the non-electroporated cells treated with cisplatin, a statistically significant decrease in cell survival was observed only for CHO cells at the highest (50 µM) cisplatin concentration tested. On the other hand, electroporation in the presence of cisplatin decreased cell survival except for B16F1 cells treated with 1 × 200 ns pulse. For CHO cells, 1 × 200 ns, 25 × 400 ns, and 8 × 100 µs pulse pro­tocols were all equally effective at decreasing cell survival at all the three tested cisplatin concentra­tions (Figure 1A). In B16F1 cells, 25 × 400 ns and 8 329 FIGURE 1. Cell survival of (A) CHO and (B) B16F1 cells at different cisplatin concentrations determined by the clonogenic assay for non-electroporated (non-EP) cells (black circles) and cells electroporated with 25 x 400 ns pulses at 3.9 kV/cm, 10 Hz repetition rate (dark blue squares), 1 × 200 ns pulse at 12.6 kV/cm (light blue diamonds) or 8 × 100 µs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz pulse repetition rate (orange triangles). Bars represent standard deviation, asterisks (*) show statistically significant differences (p < 0.05) to the survival of non-electroporated cells without cisplatin. Survival data were combined from the previous8 (for non-electroporated cells and cells electroporated with 25 × 400 ns and 8 × 100 µs pulses) and the present study (for B16F1 cells, additional non-electroporated CHO cells and CHO cells electroporated with 1 × 200 ns pulse). × 100 µs pulses were equally effective, whereas 1 × 200 ns pulse protocol was less effective (Figure 1B). The amount of Pt in the cells was determined by measuring the total mass of Pt in the cell pellets by ICP-MS. Electroporation increased the cellular Pt amount. For both cell lines, there were no sta­tistically significant differences in the measured Pt amount in cells electroporated with 25 × 400 ns or 8 × 100 µs pulses at the same cisplatin concentration. For CHO cells, the amount of Pt in cells electropo-rated with 1 × 200 ns pulse was statistically signifi­cantly lower compared to the amount of Pt in cells electroporated with 25 × 400 ns and 8 × 100 µs pulse incubated only at 50 µM cisplatin (Figure 2A). For B16F1 cells, lower cellular Pt was measured after application of 1 × 200 ns pulse compared to 25 × 400 ns and 8 × 100 µs pulses at all tested cisplatin concentrations (Figure 2B). From the measured Pt content, the number of cisplatin molecules per cell was calculated and plotted against the cell survival data. The num­ber of cisplatin molecules per cell and cell sur­vival were more strongly correlated for B16F1 cells (Spearman’s correlation coefficient: . = -0. 85, p < 0.001 for CHO and . = -0. 92, p < 0.01 for B16F1). In the case of CHO cells, at the same number of cisplatin molecules per cell, notably lower cell sur­vival was measured for electroporated cells com­pared to non-electroporated cells (Figure 3A). For example, cell survival of 98% was achieved for non-electroporated cells with 9.4 × 106 cisplatin molecules per cell, whereas cell survival of 68.5% was measured for cells electroporated with 1 × 200 ns pulse with 8.2 × 106 cisplatin molecules per cell, cell survival of 54.8% was measured for cells elec­troporated with 25 × 400 ns pulses with 9.5 × 106 cisplatin molecules per cell, and cell survival of 33.7% was measured for cells electroporated with 8 × 100 µs pulses with 9.2 × 106 cisplatin molecules per cell. From the data acquired, it could not be concluded if also in B16F1 cells a lower number of cisplatin molecules per cell causes a larger decrease in cell survival because the range of the number of cisplatin molecules in electroporated and non-elec­troporated cells did not overlap and thus survival could not be compared at approximately the same number of cisplatin molecules per cells (Figure 3B). Cisplatin has been widely investigated for its biospeciation in aqueous solutions due to its di­verse stepwise ligand displacement reactions.23 Therefore, 1H NMR spectroscopy was applied to investigate potential structural changes of cis­platin due to high voltage electric pulses. First, spectra of cisplatin in D2O and D2O with 154 mM NaCl (corresponding to physiological saline 0.9% NaCl) not exposed to electric pulses were recorded (Figure 4A–B). Weak broadened peaks for hydro­gen atoms of amino ligands (NH3) were found at approximately 4.08 ppm. Similarly, also repre­sentative peaks of cisplatin after treatment with 8 × 100 µs pulses at 1.1 kV/cm at 1 Hz pulse repetition rate or 25 × 400 ns pulses at 2.2 kV/cm at 10 Hz repetition rate remained at the same shift. The only major difference was observed in the spectrum of cisplatin recorded in D2O with 154 mM NaCl af­ter treatment with microsecond pulses (Figure 4B), where the broad peak for hydrogens of cisplatin disappeared. This can be attributed to the fast hy­drogen-deuterium (H/D) exchange of deuterium from D2O with hydrogen atoms of NH3 ligands.24 However, when spectra of cisplatin were recorded in 90% H2O/10% D2O solution containing 154 mM NaCl acquiring water suppression (to minimize the intensity of water signal to obtain a stronger signal of the NH3 ligand) no such disappearance of the peak was observed (Figure 4D). Comparable spectra with peaks at 4.08 ppm were obtained al­so when no electric pulses or nanosecond pulses were applied. Similarly, the hydrogen peak of NH3 was observed in the samples recorded in a 90% H2O/10% D2O solution without NaCl (Figure 4C). It is also important to note that no new peaks ap­peared in other regions of the NMR spectra. High-resolution mass spectrometry (HRMS), which can also provide abundant information on molecular structure, was also performed to inves­tigate possible newly formed cisplatin species. In some reports, authors detected hydrolysis prod­ucts corresponding to mono-, di- and trimeric spe­cies, by mass spectrometry.25-28 Therefore, HRMS was used in our structural investigation of cisplatin in water and saline (0.9% NaCl) exposed to micro- and nanosecond pulses. First, cisplatin in H2O was investigated and on the full-scan positive-ion mass spec­trum (mass range of m/z 100–1100) presented in Figure S2 in Supplementary Material. It can be observed that the most abundant peaks occur in the mass range of m/z 280–330, where the fol­lowing fragments were observed: [Pt(NH3)2(N2) 331 Cl]+ (m/z 292.9909), [M+NH4]+ (M – indicates mo­lecular formula for cisplatin, i.e. [Pt(NH3)2Cl2]) (m/z 317.9872) (both Figure S3), [M+H]+ (m/z 300.9601) (Figure S4), [Pt(NH3)2(CH3CN)Cl]+ (m/z 306.0101) (Figure S5) and [M+Na]+ (m/z 322.9425) (Figure S6). Additionally, three lower abundant clusters can be found in the mass range of m/z 540–590. Two of them were identified as [Pt(NH3)2Cl2·Pt(NH3)Cl]+ (m/z 547.9121) and [Pt(NH3)2Cl2·Pt(NH3)2Cl]+ (m/z 564.9378) (Figure S7). Additionally, one cluster at m/z 610–630 with the main ion fragment at m/z 617.9408 belongs to [2M+NH4]+ (Figure S8). Similar fragments have been observed when the samples were treated with micro- and nanosecond pulses (Figure S9–10 and Figure S11–S12). The species ob­served are in agreement with those reported in the literature.26 Figure S19 represents the spectrum of water from the electroporation cuvette without the application of electric pulses. No differences were observed between the solutions treated with either nanosecond or microsecond pulses or untreated control. HRMS experiments have been further per­formed in saline, where more extensive fragmenta­tion was observed throughout the mass range of m/z 100–1100 (Figure S13). However, these peaks are comparable to the ones in the spectrum of sa­line from electroporation cuvette without the ap­plication of electric pulses (Figure S20). Similarly to spectra without NaCl, peaks of [Pt(NH3)2(N2)Cl]+ fragment and sodium [M+Na]+ adduct were identi­fied on zoom-scan spectrum (Figure S14). Again, spectra recorded in saline that was not treated with electric pulses are comparable with the spec­tra where cisplatin in saline solutions were treated with micro- and nanosecond pulses (Figures S15– 16 and Figures S17–18, respectively). Overall, NMR, as well as HRMS investigations, point to cisplatin remaining structurally compara­ble after the exposure to high voltage electric puls­es similar to those used in in vitro ECT experiments with respect to its aqueous solutions without elec­tric pulses. Discussion ECT has been shown to be a safe and effective can­cer treatment, requiring much lower doses of the chemotherapeutic agent than conventional chemo­therapy. However, pain and muscle contractions were reported as a drawback. Nanosecond pulses and high-frequency biphasic pulses of a few mi­crosecond duration (H-FIRE)29-31 were suggested to limit neuromuscular stimulation and contrac­tions.15,16 Additionally, with nanosecond pulses, the possibility of thermal damage to the tissue is minimized5,6 due to low energy being transferred to the treated area and electrochemical reactions are reduced.8 ECT with nanosecond pulses has shown promising results8,17-19, but the underlying mechanisms of the observed decrease in cell sur­vival and tumor regression remain to be explained. In this study, we measured cell survival and cisplatin accumulation after in vitro ECT with 8 × 100 µs pulses, which are standardly used in ECT procedures, and equivalent nanosecond pulses, i.e. pulse protocols that have an equivalent biological effect on cell survival and cell membrane permea­bilization. The electric field strength was chosen for each pulse protocol at a value that resulted in the highest permeabilization (determined as the percentage YO-PRO1 fluorescing cells) of the cell membrane without a decrease in cell survival (measured by the metabolic MTS assay). In the case of 8 × 100 µs pulses, 1.1 kV/cm was selected for CHO cells, but the survival for B16F1 cells was around 55% at this electric field strength, thus a lower (i.e. 0.9 kV/cm) electric field strength was used for electroporating B16F1 cells with this pulse proto­col. For 25 × 400 ns pulses, the same electric field strength (3.9 kV/cm) was determined to be optimal for both cell lines. For 1 × 200 ns pulse, we used the highest experimentally achievable electric field FIGURE 5. The mechanism of cisplatin uptake into cells is not completely elucidated. In non-electroporated cells, cisplatin enters partially through passive diffusion and facilitated diffusion through ion channels including LRRC8 volume-regulated anion channels (VRAC) and membrane transporters like copper transporter 1 (CTR1) and organic cation transporters (OCTs). In electroporated cells, more cisplatin can enter through the permeabilized cell membrane (pore is a symbolic presentation of increased membrane permeability even though the mechanisms behind electroporation are more complex – refer to34). strength (i.e. 12.6 kV/cm), which did not decrease the cell survival in either cell line. Electroporating both cell lines with 8 × 100 µs or 25 × 400 ns pulses at the selected electric field strengths resulted in > 95% permeabilization (optimal for ECT), while for the 1 × 200 ns pulse at 12.6 kV/cm the permeabiliza­tion was 85% for CHO and only 42% for B16F1 cells (suboptimal for ECT). However, 1 × 200 ns pulse protocol was also included in the study based on results of cell survival of CHO cells after ECT de­termined by the metabolic MTS assay that showed that this pulse protocol was as effective in decreas­ing cell survival in ECT with cisplatin as the 25 × 400 ns protocol at all cisplatin concentrations.8 The aim was to test whether the combination of permeabilizing electric pulses (that alone do not cause a decrease in cell survival) and cisplatin re­sults in increased cellular cisplatin accumulation (compared to non-electroporated cells) and wheth­er the amount of cellular cisplatin is correlated to cell survival due to the increase of intracellular accumulation of the chemotherapeutic agent be­ing one of the main mechanisms of action of ECT. To exert its cytotoxic effect, cisplatin must enter the cell. The exact mechanisms of cisplatin uptake have not been fully elucidated. Cisplatin is only slightly permeant; thus, it only partially enters the cell through passive diffusion across the cell mem­brane. Recent studies pointed out active transport mechanisms such as facilitated diffusion involved in cisplatin uptake - and LRRC8 volume-regulat­ed anion channels (VRAC), copper transporter 1 (CTR1), and organic cation transporters (OCTs) were shown to be involved in cisplatin uptake.32,33 Electroporation makes the cell membrane non-se­lectively permeable, allowing a larger quantity of cisplatin to enter the cell (Figure 5). As expected, the measured amount of Pt was higher in electroporated cells when compared to non-electroporated cells incubated at the same cisplatin concentration, although the differences were not always statistically significant (Figure 2). These results indicate that the application of elec­tric pulses indeed increases the intracellular accu­mulation of cisplatin. Overall, the amount of Pt in B16F1 was lower than in CHO cells exposed to the same cisplatin concentration, with or without elec­troporation, which also correlates with the higher cell survival of B16F1 cells (Figure 1). A compari­son of cell survival of CHO and B16F1 cells with a similar number of cisplatin molecules per cell (Figure 3) reveals that a higher number of cisplatin molecules is needed to decrease the cell survival of B16F1 cells compared to CHO. There were no statistically significant differ­ences in the cell survival and amount of cellular Pt obtained in cells electroporated with 25 × 400 ns and 8 × 100 µs pulses at the same cisplatin concen­tration when comparing within the same cell line. Thus, it can be assumed that by using equivalent nanosecond pulses, it is possible to achieve the same decrease in cell survival and same cisplatin accumulation in cells and the as with the standard 8 × 100 µs pulses; in other words, equivalent nano­second pulses are equally effective in ECT as 8 × 100 µs pulses. The 1 × 200 ns pulse in combination with cispl­atin did not decrease cell survival in B16F1 cells. This could be explained by the fact that 1 × 200 ns pulse permeabilizes less than half of the cell pop­ulation of B16F1 and is also consistent with the measured Pt amount which was not significantly higher as in non-electroporated cells (Figure 2B). Application of 1 × 200 ns pulse alone (i.e., in the absence of cisplatin) seemed to even slightly pro­mote cell growth (although the cell survival was not statistically significantly higher compared to the non-electroporated control). More interesting­ly, however, is that application of 1 × 200 ns pulse to CHO cells resulted in a lower amount of Pt in cells electroporated with 1 × 200 ns pulse as with 25 × 400 ns or 8 × 100 µs pulses, but the same decrease in cell survival was achieved with the 1 × 200 ns pulse as with 25 × 400 ns or 8 × 100 µs pulses. The lower amount of cisplatin in CHO cells electropo-rated with 1 × 200 ns could be explained per se by the fact that this pulse protocol achieved subop­timal cell membrane permeabilization compared to the 25 × 400 ns and 8 × 100 µs pulse protocols. 333 Nevertheless, a comparable decrease in cell sur­vival was achieved, suggesting that increased accu­mulation of cisplatin into cells may not be the only cause of cell death in ECT. Figure 2A indicates that in electroporated CHO cells, a lower number of cis­platin molecules per cell is required to decrease cell survival to the same extent as in non-electroporated cells. Similar results have been reported previously in the literature35,36, but not discussed. There may be a synergistic effect of cisplatin and electropora­tion, i.e., the observed decrease in cell survival in ECT is not the sum of the decrease in cell survival caused by electric pulses and cisplatin alone, but electroporation appears to make cells more suscep­tible to cisplatin. The results of survival and number of internal­ized cisplatin molecules for B16F1 cells, however, do not show a similar synergistic effect of cisplatin and electroporation. Contrary to CHO cells, the number of cisplatin molecules per cell seems to lin­early correlate with the logarithm of cell survival for B16F1 cells (Figure 3). Nonetheless, as men­tioned above, lower cellular cisplatin was consist­ently measured for the B16F1 cell line and there is only one experimental point from the electroporat-ed cells (cells electroporated with 1 × 200 ns pulse at 10 µM cisplatin) that falls in the range of the num­ber of molecules of the non-electroporated cells. A similar number of internalized cisplatin molecules was measured for non-electroporated cells at 30 µM cisplatin and for cells electroporated with 1 × 200 ns pulse at 10 µM cisplatin, but the cell survival was even slightly higher for the latter. As discussed above, however, the 1 × 200 ns pulse protocol did not effectively permeabilize B16F1 cells. More data (from non-electroporated cells incubated at higher cisplatin concentrations) would thus be needed to determine if also in the case of B16F1 cells a lower number of internalized cisplatin molecules is need­ed to decrease cell survival in electroporated cells. To test whether electric pulses could affect cis­platin by modifying the structure of the molecule as proposed in theoretical studies37, we used NMR spectroscopy and HRMS spectrometry and found that the structure of cisplatin remains comparable after the application of electric pulses to either its saline or water solution (representing a simplified extra- and intracellular environment, respectively). Thus, high voltage electric pulses did not affect the structure of the studied complex under the condi­tions used in our experiments. Therefore, the rea­son for the observed increased susceptibility of the electroporated CHO cells to cisplatin is probably a consequence of the effect of electroporation on the cells. The cytotoxicity of cisplatin is thought to be mediated primarily by the formation of DNA ad­ducts and the resulting impairment of transcrip­tional and/or DNA replication mechanisms. It was shown that electroporation increases the amount of cisplatin bound to the DNA, which could increase cisplatin cytotoxicity in electroporated cells.35,38 However, additional mechanisms play an impor­tant role in exerting the toxic effects of cisplatin, including generation of ROS, mitochondrial dys­function, increase in intracellular Ca2+ concentra­tion, and activation of signal transduction path­ways.39 Electric pulses can also lead to generation of intracellular reactive oxygen species (ROS)40,41, damage mitochondria42,43, and disrupt calcium ho­meostasis through the entry of Ca2+ from the extra­cellular space or intracellular stores.44,45 It has been shown that an increase in ROS enhances the effica­cy of cisplatin and vice versa.46,47 Moreover, an in­crease in intracellular Ca2+ concentration enhances cisplatin-mediated ROS production and increases cisplatin cytotoxicity.48-50 This type of potentiation of cisplatin cytotoxicity may be responsible for the enhanced cisplatin cytotoxicity in electroporated cells, but it yet needs to be elucidated. Michel et al.51 observed an increased immunoreactivity with SOD-2 (an enzyme that clears mitochondrial ROS) in cells subjected to ECT with cisplatin. To the best of our knowledge, this is the only report that meas­ured ROS after ECT with cisplatin. Our study also has limitations. Two different pulse generators and electrode geometries (i.e., electroporation cuvettes with 2 or 4 mm gap) were used in the cell experiments because of the techni­cal limitations of the pulse generators used. Also in cell experiments, we did not directly measure the amount of cisplatin in cell pellets, but Pt was measured instead and assumed that cisplatin most likely accounts for the majority of the measured amount of Pt in cells incubated with cisplatin. This assumption is supported by the fact that the amount of Pt in non-electroporated cells that were not incubated with cisplatin was 2–3 orders of magnitude lower than in samples incubated with cisplatin or even below the detection limit. We also do not know whether the measured Pt was located inside the cells or was e.g. bound to the surface of the cell membrane. However, the for­mation of reactive hydrolyzed cisplatin products that would bind immediately and irreversibly to cell membrane phospholipids is not expected be­cause the electroporation medium used has a high concentration of chloride ions so cisplatin should be stable in it and the measured Pt most probably 334 comes from intracellular cisplatin.52 Additionally, in experiments investigating the effects of electric pulses on cisplatin structure, the conditions be­fore the measurements by NMR spectroscopy and HRMS spectrometry could not be fully matched with the conditions in the cell experiments due to several reasons. First, it was namely not possible to record spectra of cisplatin in growth media due to many species present in the growth medium which interfere with cisplatin signals; thus, pulses were delivered to cisplatin dissolved in water or saline for NMR spectroscopy and HRMS spectrometry. Second, because of the limitations of the pulse generator used for NMR spectroscopy and HRMS spectrometry experiments, 25 × 400 ns pulses were delivered at lower amplitudes than in the cell ex­periments. Third, because of the difference in con­ductivity, electric pulses delivered to H2O and D2O had a notably different shape than pulses delivered to saline or cells in growth medium; due to the low conductivity of the load, they resembled an expo­nentially decaying rather than a rectangular pulse shape. In conclusion, we have shown that by using equivalent nanosecond pulses in ECT, the same decrease in cell survival is achieved and the same amount of cisplatin accumulates in the cells as with the standard 8 × 100 µs pulses, i.e., that in ECT, equivalent nanosecond pulses are equally efficient as 8 × 100 µs pulses. By investigating the under­lying mechanisms in nanosecond pulse ECT, we discovered that electroporated CHO cells are more susceptible to cisplatin than non-electroporated cells (regardless of the pulse protocol). The electric pulses used for electroporation do not appear to alter the structure of the cisplatin molecule, so the observed increased susceptibility is likely a conse­quence of the effect of electroporation on the cells. The use of nanosecond pulses in ECT is promis­ing as it was demonstrated to be effective with the potential to mitigate muscle contractions. 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Biochim Biophys Acta -Biomembr 1996; 1283: 60-6. doi: 10.1016/0005-2736(96)00080-6 research article Impact of AKT1 polymorphism on DNA damage, BTG2 expression, and risk of colorectal cancer development Hina Zubair, Zahid Khan, Muhammad Imran Biochemistry Section, Institute of Chemical Sciences, University of Peshawar, Peshawar, Pakistan Radiol Oncol 2022; 56(3): 336-345. Received 27 February 2022 Accepted 3 July 2022 Correspondence to: Dr. Muhammad Imran, Ph.D., Biochemistry Section, Institute of Chemical Sciences, University of Peshawar, Peshawar-25120, KP, Pakistan; E-mail: imrancl@uop.edu.pk 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. AKT, also called protein kinase B, is a serine-threonine kinase that functions as a mediator of PI3K­Akt-mTOR signaling pathway and plays an important role in an array of cellular processes. Many single nucleotide polymorphisms (SNP) in AKT gene have been observed to be associated with various types of cancers. In the current research the association of a functional SNP rs1130233 in AKT, depicting G to A transition, was studied with AKT acti­vation, DNA damage, an early response B-cell translocation gene 2 (Btg2) expression and risk of colorectal cancer (CRC) development. Patients and methods. A total 197 population-based controls and 200 CRC patients were genotyped for SNP rs1130233. AKT expression, activation and BTG2 expression were determined in GG, AG and AA genotype carriers. DNA damage was determined through comet assay. Results. The heterozygous AG genotype (55.67%) was more prevalent in the local population compared to homozy­gous wild type GG (37.78%) and homozygous AA genotypes (6.55%). Moreover, AG and AA alleles were observed to be significant contributors (P = 0.01, OR = 1.80, CI = 1.18 to 2.74, and P = 0.001, OR = 5.00, CI = 1.90 to 13.18, respectively) in increasing the risk of CRC. The immunoblot analysis revealed that G to A transition decreased the expression and activation of AKT. Moreover, AG and AA genotypes of AKT1 rs1130233 showed a significant increase in DNA damage and Btg2 expression. Conclusions. The data concludes that G to A substitution is a risk factor for CRC development involving a decrease in AKT expression and activation and increase in DNA damage. Key words: AKT1; BTG2; colorectal cancer; DNA damage; rs1130233 Introduction Colorectal cancer is a multifactorial disease, and its life time risk in the general population increases ~5% with age.1 This may be caused by carcinogenic compounds ingestion through foods and impor­tantly individual differences in the metabolism of carcinogens as caused by both genetic and envi­ronmental risk factors which play essential roles in the development of colorectal cancer. Many genes sequence variations lead to the pathogenesis of in­herited and sporadic forms of colorectal cancer.2,3 AKT, also known as Protein Kinase B (PKB), is a serine/threonine protein kinase which was origi­nally discovered as an oncogene transduced by the acute transforming retrovirus (PKB-8/AKT8), isolated from mouse leukemia.4,5 AKT is the down­stream target of PI3K signaling that triggers a num­ber of biological processes including cell survival, cell growth, glucose metabolism, angiogenesis, cell cycle entry cell motility, and also stimulate malignant transformation of cells and tumor pro­gression. PI3K/AKT/mTOR pathway is one of the central nodes in many physiological abnormali­ 337 ties including cancer.6-10 Mammalian AKT gene has three isoforms; AKT1, AKT2, and AKT3. All these isoforms show broad tissue distribution and a broad range of functions. AKT1 also known as AKT kinase, is ubiquitously expressed isoform.8,11 Constitutive activation of AKT is mainly attrib­uted to the aberrant activation of upstream signal­ing such as mutation or hyperactivation of receptor tyrosine kinases (Src, Ras and PTEN proteins) and increased synthesis of growth factors, as has been observed in several types of cancers.12-14 Genetic variations in AKT gene (e.g. rs1130233, rs2498801, rs2494752) is linked with various types of cancers including liver, lungs and bladder cancers.15,16 These polymorphic forms of AKT differ widely in their role as oncogene and exert their actions by regulating a diverse array of genes including NF-kB, Btg2 etc. Human B-cell translocation gene 2 (BTG2), an ortholog of mouse TIS21, is a tumor suppressor gene that belongs to an antiproliferative gene fam­ily. BTG2 is implicated in a variety of physiological processes including cell differentiation, develop­ment, cell cycle arrest at G1/S and G2/M phases, cells death, DNA damage repair and antioxidant defenses. The downregulation of BTG2 thus has various physiological effects including cancer de­velopment.17-20 Genetic variability in AKT can affect an array of cellular processes including genes regulation, can­cer development or regression etc. Previous stud­ies have shown a strong correlation between AKT gene polymorphism and the prevalence of differ­ent types of cancers. The effect of genetic variabil­ity of this feedback loop hasn’t been worked out on colorectal cancer development. It was hypoth­esized that sequence variations in AKT may affect colorectal cancer development via BTG2 regula­tion and DNA damage. The study was designed to identify a risk factor for the prevalence of colorec­tal cancer development and the possible underly­ing mechanism of tumorigenesis. The data from CRC patients and control individuals revealed that AKT rs1130233 single nucleotide polymorphism increases the risk of CRC development through increased DNA damage and downregulation of a tumor suppressor BTG2. Patients and methods Patients This case-control study involved a total of 397 in­dividuals including both colorectal cancer patients (CRC; n = 200) and population-based controls (n = 197). CRC patients (n = 200) from both sex, hav­ ing age = 60 and with documentary evidence of pathologically confirmed adenocarcinoma of colorectal cancer were included in this study. Subjects with mixed ethnic background, comor­bidity, and patients who developed CRC at the age of above 60 years at the diagnosis were ex­cluded. Determination of tumor stages and types were done by experienced pathologist at Institute of radiation and nuclear medicine (IRNUM), Peshawar. All patients and their guardians were informed about the nature of the study and im­portant information of patients such as age, sex, ethnicity, medical records, pathology reports, drug history, family history, tumor size, tumor location and lymph node status etc. were ob­tained on a pre-designed proforma. Colorectal cancer risk factors such as taking red meat, veg­etables, fibers, fruits and cooking choices and smoking history were also obtained. For control blood samples were collected from healthy indi­viduals (n = 197) who had no sign of present or previous malignancy and no indication of CRC or nor any family history of cancer and had no blood relation with patients. Selection of control group of healthy donors was done on the basis of sex, age, smoking history and habits, residential, oc­cupational and food intake. Informed consent of all the enrolled subjects was obtained on a ques­tionnaire. The ethical approval was obtained from the institutional ethical board at Department of Biotechnology, University of Peshawar, Pakistan. Blood samples were collected both from colorectal cancer patients and controls at IRNUM Peshawar in 5 mL EDTA tubes and were stored at -20oC till further analysis. DNA extraction and genotyping DNA was extracted using DNA extraction kit (GeneJET Genomic DNA Purification Kit, Thermo Scientific, USA) and was quantified using UV-visible spectrophotometer (752 PC, China). Akt single nucleotide polymorphism was determined using polymerase chain reaction (PCR, Multigene Optimax, Labnet International, USA). PCR was performed in a 20 µL reaction mixture using allele specific primers. The sequences of primers and amplification condition are given in Table 1. The AA (379 bp), AG (245 and 379 bp) and GG (245 bp) genotypes were visualized with ethidium bro­mide and identified on agarose gel (2%) using UV transilluminator (Wealtec, USA). TABLE 1. Primers sequences and amplification conditions for genes BTG2 GAPDH AKT1 Forward Reverse Forward Reverse Forward Reverse Forward Reverse 5/-CCTGGGCAGAGAGTGAAAAG-3/ 5/-CCTTCCATCCTAACCCCAAT-3/ 5/-CCATGGAGAAGGCTGGGG-3/ 5/-CAAAGTTGTCATGGATGACC-3/ F1-5/-ATAGGGAGTCATGGAGGGTTTG-3/ R1-5/-CTTTACCAAATCCTGGTCACTGAA-3/ F2-5/-AAAAAATTGATTGATGGGAGGAAG-3/ R2-5/-TAATCCCTGGCCTGCTCAG-3/ 95°C for 5 min, followed by 30 cycles of 95°C for 30 s, 58°C for 45 s, 72°C for 45 s and 72°C for 10 min 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 60°C for 45 s, 72°C for 45 s and 72°C for 10 min Isolation of lymphocytes Lymphocytes were isolated from fresh blood as described previously.21 Briefly, blood containing EDTA was mixed with phosphate buffer saline (PBS; Ca+2 and Mg+2 free) and layered over 2 mL fi-coll / lymphocytes separation medium (LSMTM1077; Catalog Number: HiSep LSMTM 1077-LS001) in a 15 mL falcon tube. The mixture was centrifuged (2000 RPM for 30 min) that led to the formation of four distinct layers; the upper plasma layer, the second buffy coat layer containing lymphocyte and monocyte, the third ficoll layer (LSM) and the bot­tom layer of RBCs and cell debris. The buffy coat was isolated, mixed with 1 Ml PBS and centrifuged at 1500 RPM for 10 min. The pellet containing isolat­ed lymphocytes washed with PBS, gently suspend­ed in 1 ml PBS and used in subsequent experiments. Comet assay for DNA damage DNA damage in lymphocytes was assessed using comet assay, (also called single cell gel electropho­resis assay), as described previously.22 Briefly, cells were fixed in ethanol for 20 min, then hydrated in distilled water for 30 min followed by staining. The slides were washed with cold distilled water and mounted with the cover glass. For scoring of DNA comets, 100 stained nuclei were selected randomly from each group under the fluorescent microscope at 200x magnification and images were recorded. Total comet score was calculated as described pre­viously.21 Immunoblot analysis The isolated lymphocytes were lysed in a buffer con­taining Tris (50 mM, pH 7.4, Nacl (150 mM), EDTA (1.0 mM), phenylmethylsulphonyl fluoride (1.0 mM), aprotinin (1.0 µg/ml), leupeptin (1.0 µg/ml), NaF (1.0 mM, 1.0 mM) sodium orthovanadate, sodium deoxy­cholate (0.25 %) and Nonidet P-40 (1.0%). The extract­ed proteins were quantified and electrophoresed on SDS-PAGE, transferred onto a nitrocellulose mem­brane using immunoblotting kit. Membrane was incubated with anti-Akt, anti-pAkt and anti-tubulin and proteins were detected using immunoblotting detection kit Ab Signal™ (AbClon, Seoul, Republic of Korea). Antibodies for AKT and pAKT were purchased from Cell Signaling Technology while a-tubulin from Santa Cruz Biotechnology. a-Tubulin was used as a loading control. RNA extraction and polymerase chain reaction (PCR) Total RNA was extracted from purified PBMCs us­ing Trizol reagent. RNA was reverse transcribed to cDNA using reverse transcription kit (Invitrogen). BTG2 expression was determined using conven­tional PCR followed by agarose gel electrophore­sis. GAPDH was used as an endogenous control. The primer sequences and amplification condi­tions for BTG2 and GAPDH are given in Table 1. Statistical analysis Data was analysed using Minitab® 17 and was presented as Mean ± SD. Odds ratio (OR), 95% confidence interval (CI) were used to find out the association between AKT1 single nucleotide poly­ morphism and CRC risk. P = 0.05 was considered as statically significant. Results Association of Akt1 rs1130233 with risk of colorectal cancer development Frequency of selected demographic and risk factors in CRC cases and controls A total of 200 CRC patients and 197 age, and sex matched CRC free healthy subjects were enrolled 339 TABLE 2. Demographic and clinical information of control subjects and colorectal cancer patients Age* 40 = 131 (65.5) 120 (60.91) 40 > 69 (34.5) 77 (39.09) Sex* Male 119 (59.50) 112 (57.50) Female 81 (40.50) 85 (42.50) Food consumption* Mainly vegetables 106 (53.00) 97 (49.24) Mixed Food 94 (47) 100 (50.76) Smoking* Ever 36 (18.00%) 29 (14.72%) Never 164 (82.00%) 168 (85.28%) Cancer family history* Yes 27 (13.50) 17 (8.63) No 173 (86.50) 180 (91.37) Cancer Stages I 1 (0.50) II 33 (16.50) III 112 (56.00) IV 54 (27.00) *Non-significant (P > 0.05) difference between cases and control in this study. The data about the demographic in­formation is given in Table 2. Among 200 clinically diagnosed CRC cases, there were 81 (59.50%) fe­male and 119 (40.5%) male patients which shows that in female population of Khyber Pakhtunkhwa CRC frequency is relatively less than males as indi­cated by the higher incidence of CRC among males. There were 85 (42.50%) women and 112 (57.50%) men among the control group. The age and sex re­lated differences were non-significant between the CRC and control groups (P > 0.05). The smoking status indicated that most of the subjects, including both patients and control, were non-smokers and non-significantly different in case and control co­horts (P > 0.05). Food intake especially vegetables consumption plays an important role in maintain­ing proper health, however, with regard to vegeta­ble consumption the difference between control and patients was non-significant (P = 0.249). The family history data indicate that the prevalence of CRC was not linked with family history of any type of cancer as 173 CRC patients did not have fam- FIGURE 1. Akt rs1130233 single nucleotide polymorphism in (A) Control and (B) colorectal cancer patients. Representative images have been shown. AA genotype (379 bp band); AG genotype (245 and 379 bp bands); GG genotype (245 bp band). The number above the lanes indicate subjects identity. C = Control, P = CRC Patient, M = DNA Marker ily history of any type of cancer. All CRC patients were divided into four groups based on Tumor Node Metastasis (TNM) staging criteria; where pa­tients with stage I: 1 (0.50%); stag II: 33 (16.50%); stage III: 112 (56.00%), and stage IV: 54 (27.00%). Frequencies of Akt1 rs1130233 polymorphism and alleles distribution in colorectal cancer patients and control Overall 397 subjects including 200 colorectal can­cer patients (cases) and 197 healthy individuals (control) were enrolled in this study and genotyp­ing of AKT1 rs1130233 was performed to evaluate the status of rs1130233 polymorphism in case and control groups. Representative images of wild type GG, heterozygous mutatnt AG and homozygous mutatnt AA alleles for both control and CRC pa­tients are given in Figure 1. The data has been pre­sentated in Table 3. Among 200 colorectal cancer patients, 60 (30.0%) had wild type GG genotype, 120 (60.0%) had heterozygous mutatnt AG geno­type while remaining 20 (10.0%) had homozygous mutant AA genotype. In control population, 90 subjects (45.69%) had GG genotype, 101 (51.27%) had AG and 6 (3.04%) had AA genotypes. The presence of AG and AA alleles were assoiated with TABLE 3. Gene and allele frequencies of AKT1 rs1130233 polymorphism and its association with colorectal cancer GG Genotype Frequency AG AA GG Dominant Model AG+AA GG+AG Recessive Model AA G Allele Frequency A 60 (30.00) 120 (60.00) 20 (10.00) 60 (30.00) 140 (70.00) 180 (90.00) 20 (10.00) 0.6000 0.4000 colorectal cancer risk (OR = 1.80, CI = 1.18-2.74, P = 0.01 for AG and OR = 5.00, CI = 1.90-3.18, P = 0.001 for AA). The association between AKT1 rs1130233 polymorphism and colorectal cancer was also as­sessed using dominent and recessive models. For dominant model (GG vs AG+AA), homozygous wild type (GG) was present in 60 patients (30.0%) and 90 controls (45.69%) while heterozygous and homozygous mutatant alleles (AG+AA) were col­lectively present in 140 patients (70.00%) and 107 controls (54.31%). An increased risk for colorec­ tal cancer (OR = 1.96, CI = 1.30-2.96, P = 0.001) was observed for dominant model. For recessive model, (AA vs GG+AG), homozygous polymor­phism was observed in 20 patients (10.00%) and 6 controls (3.04%) while homozygous wild type and heterozygous polymorphism (GG+AG) was col­lectively observed in 180 patients (90.00%) and 191 controls (96.96%). An increased risk for colorectal cancer was also observed for recessive model (OR = 0.28, CI = 0.11-0.72, P = 0.01). Similarly, G and A allele frequencies were 0.60 and 0.40 respectively for cases and 0.7132 and 0.2868 respecively for con­trol and hence follows Hardy Weinberg equilib­rium. Overall genotype frequency of GG, AG and AA for both cases and control was 150 (37.78%), 221 (55.67%) and 26 (6.55%) respectively indicating that heterozygous AG genotype is more prevalent than GG and AA. Frequencies of AKT1 rs1130233 polymorphism and alleles in colon cancer cases and control The colorectal cancer patients were sub grouped into colon and rectum cancer pateints and their as­sociation AKT1 rs1130233 polymorphism was de­termined (Table 4). Among overall 200 colorectal cancer pateints, 102 (51%) were colon cancer pa­tients while 98 (49%) were rectum cancer patients. Among colon cancer patients, GG, AG and AA genoptes frequency were 29.41, 59.80 and 10.79% respectively. The AG and AA genotypes were as-soiated with higher risk for development of colon 90 (45.69) Reference 101 (51.27) 0.01 1.80 (1.18–2.74) 6 (3.04) 0.001 5.00 (1.90–13.18) 90 (45.69) 107 (54.31) 0.001 1.96 (1.30–2.96) 191 (96.96) 6 (3.04) 0.01 0.28 (0.11–0.72) 0.7132 0.2868 cancer (OR = 1.81, CI = 1.08-3.05; P = 0.02 for AG and OR = 5.50, CI = 1.87-16.15; P = 0.001 for AA). An increased risk for colon cancer was observed for dominant (OR = 2.02, CI = 1.21-3.36; P = 0.006) and recessive (OR = 3.85, CI = 1.38-10.73; P = 0.01) models. The association between AKT1 rs1130233 poly­morphism and rectum cancer was also evalutaed (Table 5). Among 98 rectum cancer patients, GG, AG and AA genotypes frequencies were 30.61, 60.20, and 9.19% respectively. Both AG (OR = 1.75, CI = 1.04-2.96; P = 0.04) and AA (OR = 4.50, CI = 1.48-13.69; P = 0.008) were assoiated with higher risk for development of recctum cancer. An in­creased risk for rectum cancer (OR = 1.91, CI = 1.14-3.18; P = 0.01) was observed for dominant and recessive models (OR = 3.22, CI = 1.11-9.32; P = 0.03). Association of AKT1 rs1130233 polymorphism with tumor location On the basis of tumor location, the colorectal cancer patients were separted as colon and rec­tum cancer pateints and the association of AKT1 polymorphism was assessed. Among overall 200 colorectal cancer pateints, 102 pateints (51%) had colon cancer while 98 patients (49%) were rectum cancer patients. Among 102 colon cancer patients, 32 pateints (31.37%) had GG, 67 patients (65.69%) had AG and 3 patients (2.94%) possessed AA genotypes. Among 98 rectum cancer patients, 36 341 TABLE 4. Frequencies of AKT1 rs1130233 polymorphism and alleles in colon cancer cases and control GG 30 (29.41) 90 (45.69) Reference Genotype frequency AG 61 (59.80) 101 (51.27) 0.02 1.81 (1.08–3.05) AA 11 (10.79) 6 (3.04) 0.001 5.50 (1.87–16.15) Dominant model GG AG+AA 30 (29.41) 72 (70.59) 90 (45.69) 107 (54.31) 0.006 2.02 (1.21–3.36) Recessive model GG+AG AA 91 (89.21) 11 (10.79) 191 (96.96) 6 (3.04) 0.01 3.85 (1.38–10.73) pateints (36.74%) had GG genotype, 55 patients (56.12%) had AG genotype while remaining 7 pa­tients (7.14%) had AA genotype. The AG and AA polymorphism had equaleffect on the prevalce of both colon and rectum cancer as both of them showed non-significant difference (P > 0.05) for AG and AA transition. Effect of rs1130233 on AKT protein expression and phosphorylation To find out whether rs1130233 G to A transition can have effect on AKT expression, lymphocytes were isolated from various subjects of different genotypes (GG = 21, AG = 25 and AA = 06) and their AKT and pAKT proteins levels were deter­mined using immunoblotting. The data indicated that G to A transition decreased AKT expression in both healthy in various individuals independ­ent of their age, sex and health status (Figure 2A). The densitometry analysis revealed GG genotype carriers had significantly (P < 0.05) higher level of AKT followed by heterozygous AG carriers while the AKT expression was lowest in AA genotypes (Figure 2B). pAKT represents the active kinase, therefore, the phosphorylation status of AKT was also determined in GG, AG and AA carriers. pAKT level also showed a decreased intensity in GG>AG>AA order (Figure 2C). The data shows that substitution of G by A have a significant im­pact on AKT expression and activation and hence could have an effect on colorectal cancer develop­ment in different ways. Association of AKT1 rs1130233 with DNA damage To find out the association of rs1130233 with ge­nome integrity, DNA damage was assessed by comet assay. Because cancer patients have multiple FIGURE 2. Association of AKT rs1130233 single nucleotide polymorphism with AKT expression and phosphorylation. (A) Representative images of immunoblotting showing expression of AKT and pAKT in lymphocytes of GG, AG and AA carriers. (B) Mean densitometry profile of AKT and pAkt expression of different subjects of various genotypes. P < 0.05 GG vs AG, and P < 0.01 GG vs AA. genes mutation leading to DNA damages, there­fore comet assay was performed only in control individuals carrying GG (n = 13), AG (n = 15) or AA (n = 4) alleles. Because age and life style can have an impact on DNA damage, therefore comet assay was performed in individuals of similar age groups, non-smoking subjects and subjects with similar dietary habits. Moreover, the frequency of AA genotype carriers in control individuals was very less, therefore, the combined total comet score was calculated for AG and AA genotype carriers (AG+AA) (Figure 3). The total comet score for indi­viduals carrying AA genotype was 190 ± 30.5, AG genotype (110 ± 20.54) and GG 63 ± 15.70. The total comet score of AA genotypes was significantly (P < 0.05) greater than AG and GG genotypes. Also, AG carriers had significantly higher (P < 0.05) comet score than GG genotype indicating a greater DNA damage. The data thus indicates that GG allele of AKT1 contributes to genome stability. TABLE 5. Frequencies of AKT1rs1130233 polymorphism and alleles in rectum cancer cases and control GG 30 (30.61) 90 (45.69) Reference Genotype frequency AG 59 (60.20) 101 (51.27) 0.04 1.75 (1.04–2.96) AA 9 (9.19) 6 (3.04) 0.008 4.50 (1.48–13.69) Dominant model GG AG+AA 30 (30.61) 68 (69.39) 90 (45.69) 107 (54.31) 0.01 1.91 (1.14–3.18) Recessive model GG+AG AA 89 (90.81) 9 (9.19) 191 (96.96) 6 (3.04) 0.03 3.22 (1.11–9.32) FIGURE 3. Association of AKT rs1130233 single nucleotide polymorphism BTG2 expression. Representative images have been shown. (A) BTG2 (360 bp) expression was determined in leukocytes of control individuals carrying GG, AG or AA genotypes of AKT1. GAPDH (158 bp) was used as a loading control. M = DNA Marker. (B) Mean densitometry profile of Btg2 mRNA expression of different subjects of various genotypes. P < 0.05 GG vs AG, and P < 0.01 GG vs AA. FIGURE 4. Association of AKT1 rs1130233 single nucleotide polymorphism with DNA damage. (A) Representative images of comet assay have been shown. (B) Comet tail was quantified in leukocytes of control individuals carrying GG, AG or AA genotypes of AKT1. P < 0.05 GG vs AG, and P < 0.01 GG vs AA. Association of AKT1 rs1130233 with BTG2 expression Previously we have reported that AKT downregu­lates BTG2 expression in various types of cells.20 Moreover, BTG2 has been shown to be involved in DNA damage repair, we therefore determined the expression of BTG2 in GG (n = 20), AG (n = 24) and AA (n = 6) carriers of AKT1 rs1130233 single nu­cleotide polymorphism. BTG2 expression was de­termined in lymphocytes of control individuals of similar age groups and life style. The BTG2 expres­sion profile and its densitometric analysis is given in Figure 4. The data shows that an inverse associa­tion between AKT activation and BTG2 expression in genotype dependent manner. BTG2 expression was significantly (P < 0.05) higher in AA and AG carriers compared to GG individuals indicating that various genotypes of AKT differentially regu­late Btg2 gene expression and hence will impart distinct effect of various cellular processes. Discussion CRC is a multifactorial disease. Exposure to envi­ronmental toxins, life style and internal factors in­cluding genetic variations are important factors re­sponsible for CRC development.23 It has been dem­onstrated that lifestyle factors, including diet has a significant association with risk of CRC. Dietary pattern contributes to risk of CRC and mortality among CRC survivors. Higher intake of red and processed meat is associated with increased risk of CRC, while higher intake of vegetables, whole grains, dairy products, and fish show inverse as­sociations with CRC risk.24 In the current research project, vegetable consumption was however, not significantly associated with CRC risk, as nearly all patients were from low economic background 343 who most of the time rely on vegetable sources for their daily diet. Moreover, the smoking behavior in the current population is in general less and hence smoking was also a non-significant contributor to CRC risk in the current model, as most of the pa­tients were non-smokers. The genetic factor involving genes sequence variations have been linked with an increased risk for various types of cancers. AKT has a key role in controlling various cellular functions like cell growth, proliferation, DNA damage repair and cell survival etc.25 Various research based evidences suggest that AKT is activated in various types of cancers.16 Furthermore, genetic variations in AKT are reported to affect the AKT functioning and hence can have a crucial role in tumorigenesis.26 So, we investigated the association between AKT1 rs1130233 polymorphism and colorectal cancer risk in Pashtun population of Khyber Pakhtunkhwa Pakistan. The presence of allele (AG/AA) of AKT1 rs1130233 polymorphism was significantly associ­ated with risk of colorectal cancer. The AA geno­type was found to be more profound risk factor compared to AG. The association was also assessed using dominant and recessive genetic models and mutant polymorphic forms were observed to be the risk factors for colorectal cancer. The allele frequencies of AKT1 rs1130233 differ widely in different ethnic groups. For example, the A allele frequency of 0.300 in Caucasians, 0.051 in Africans and 0.575 in East Asians (consisting of Japanese and Chinese)27 and 0.3438 in Pashtun population of Pakistan, suggesting the population specific sus­ceptibility to cancer. When patients were divided on the basis of age, sex, cancer history and food style, no significant differences in genotype fre­quencies were observed. The association of mutant alleles (AG and AA) with CRC was independent of patients’ age, sex, and life style. A sub group anal­ysis also showed an increased risk both for colon and rectum cancers. AKT1 rs1130233 polymorphism has been ob­served to be associated with bladder cancer in Iranian population16 and head and neck squa­mous cell carcinoma in Northeast Chinese popu­lation.28 AKT1 rs1130233 A/A genotype has also been observed to have a significant impact on drug response. Giovannetti et al. report that AKT1 rs1130233 A/A genotype was associated with shorter time-to-progression (P = 0.04) and overall survival (P = 0.007) among non–small cell lung cancer patients treated with gefitinib.29 Similarly, the AKT1 rs1130233 has been found to play an important role in modulating the acute effects of delta-9- tetrahydrocannabinol-induced medial temporal function during fear processing, with these being associated with the A allele presence.30 Furthermore, AKT1 rs1130233 G/A+A/A genotypes have been observed to favor apoptosis, resulting in the higher risk of muscle atrophy and cachexia and weight loss in human cachexia cancer. The underlying mechanism involves the increased pro­duction of inflammatory cytokines in patients who suffer from tumor induced inflammation.27 Various genetic variations of AKTs, such as sin­gle nucleotide polymorphisms (SNPs), have also been well recognized to modulate gene function. The G to A substitution significantly decreased AKT1 expression and phosphorylation. The AKT1 rs1130233 polymorphism is located in exon 8 and the G.A variation is located at the boundary of exon 8 and intron 7.31 Because of this unique locali­zation the G to A transition interferes the posttran­scriptional modification of AKT1 gene leading to decrease in its expression that in turn causes low AKT1 protein synthesis and activation.32 AKT1 rs1130233 (AG and AA genotypes) was observed to be linked with an increase in DNA damage. There are however, conflicting reports about the role of AKT in DNA damage. The de­regulation of the PI3K-AKT/ mTORC1/ p70S6K pathway has been observed to have profound effects on genome stability via suppression of MRE11 expression leading to escalation of Ras-induced DNA damage.33 Gol et al. has shown that both AKT1 and AKT2 isoforms are involved in ra­diation induced-DNA double strand break repair through homologous recombination in colon can­cer cells.34 Because AG and AA genotypes are char­acterized by a decrease in AKT activation (phos­phorylation), which in turn leads to an increase in genome instability and hence provides a possible link between AG and AA genotypes and associ­ated DNA damage. AKT1 is shown to exert its effects through vari­ous mediators, such as protein kinases and phos­phatases, survival factors, regulators of protein synthesis etc.35 Previously we have reported that AKT increases cells survival and proliferation of cancer cells through downregulation of BTG2 ex­pression.20 The current study shows a strong corre­lation between Btg2 upregulation and a decreased in AKT expression and activation as depicted in AG and AA carriers. Btg2 gene has also been shown to be upregulated in response to DNA damage and hence acts as a marker of DNA damage and repair pathway.36 344 In the current research a decreased in AKT ex­pression and activation is linked with an increase in DNA damage indicating an important mecha­nism for BTG2 upregulation. However, more work is required to underpin this signaling mechanism. The AA genotype of AKT rs1130233 is present at a low frequency, therefore a large set of population is required to further confirm the impact of AA allele in CRC. AKT is widely employed in a number of different types of cancers and it is important to de­termine the association of AKT rs1130233 polymor­phism with other types of cancers also. Moreover, how G to A transition in AKT rs1130233 effects posttranscriptional modification of AKT needs to be addressed. Conclusions The present study concludes the possibly impor­tant role of Akt1 in the development of colorectal cancer. The study determined that AKT1 rs1130233 polymorphism is a risk factor for the development of colon and rectum cancers and is significantly as­sociated with DNA damage. Acknowledgements This research was financially supported by a grant (NRPU 4714) from Higher Education Commission of Pakistan under National Research Program for Universities. 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AKT1 and SELP polymorphisms predict the risk of developing cachexia in pan­creatic cancer patients. PLoS One 2014; 9: e108057. doi: 10.1371/journal. pone.0108057 28. Li Y, Zhu L, Yao H, Zhang Y, Kong X, Chen L, et al. Association of inflammation-related gene polymorphisms with susceptibility and radiotherapy sensitivity in head and neck squamous cell carcinoma patients in northeast China. Front Oncol 2021; 11: 651632. doi: 10.3389/fonc.2021.651632 29. Giovannetti E, Zucali PA, Peters GJ, Cortesi F, D’Incecco A, Smit EF, et al. Association of polymorphisms in AKT1 and EGFR with clinical outcome and toxicity in non–small cell lung cancer patients treated with gefitinib. Mol Cancer Ther 2010; 9: 581-93. doi: 10.1158/1535-7163 30. Blest-Hopley G, Colizzi M, Prata D. Giampietro V, Brammer M, McGuire P, et al. Epigenetic mediation of AKT1 rs1130233’s effect on delta-9-tetrahydro­cannabinol-induced medial temporal function during fear processing. 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Mol Carcinog 2000; 27: 57-64. doi: 10.1038/ng1296-482 research article Real-life long-term outcomes of upfront surgery in patients with resectable stage I-IIIA non-small cell lung cancer Marko Bitenc1, Tanja Cufer2, Izidor Kern3, Martina Miklavcic1, Sabrina Petrovic1, Vida Groznik4,5, Aleksander Sadikov4 1 Surgery Bitenc, Ljubljana, Slovenia 2 Medical Faculty, University of Ljubljana, Ljubljana, Slovenia 3 Laboratory for Cytology and Pathology, University Clinic Golnik, Golnik, Slovenia 4 Faculty of Computer and Information Science, University of Ljubljana, Ljubljana, Slovenia 5 Faculty of Mathematics, Natural Sciences and Information Technologies, University of Primorska, Koper, Slovenia Radiol Oncol 2022; 56(3): 346-354. Received 17 May 2022 Accepted 10 June 2022 Correspondence to: Martina Miklavcic, M.D., Surgery Bitenc, Ljubljana, Slovenia. Email: martina.miklavcic1@gmail.com, tel. +386 41 765 771 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. Treatment of early-stage non-small cell lung cancer (NSCLC) is rapidly evolving. When introducing nov­elties, real-life data on effectiveness of currently used treatment strategies are needed. The present study evaluated outcomes of stage I–IIIA NSCLC patients treated with upfront radical surgery in everyday clinical practice, between 2010–2017. Patients and methods. Data of 539 consecutive patients were retrieved from a prospective hospital-based registry. All diagnostic, treatment and follow-up procedures were performed at the same thoracic oncology centre accord­ing to the valid guidelines. The primary outcome was overall survival (OS) analysed by clinical(c) and pathological(p) TNM (tumour, node, metastases) stage. The impact of clinicopathological characteristics on OS was evaluated using univariable (UVA) and multivariable regression analysis (MVA). Results. With a median follow-up of 53.9 months, median OS and 5-year OS rate in the overall population were 90.4 months and 64.4%. Five-year OS rates by pTNM stage I, II and IIIA were 70.2%, 60.21%, and 49.9%, respectively. Both cTNM and pTNM stages were associated with OS; but only pTNM retained its independent prognostic value (p = 0.003) in MVA. Agreement between cTNM and pTNM was 69.0%. Next to pTNM, age (p = 0.001) and gender (p = 0.004) re­tained their independent prognostic value for OS. Conclusions. The study showed favourable outcomes of resectable stage I–IIIA NSCLC treated with upfront surgery in real-life. Relatively low agreement between cTNM and pTNM stages and independent prognostic value of only pTNM, observed in real-life data, suggest that surgery remains the most accurate provider of the anatomical stage of disease and important upfront therapy. Key words: resectable NSCLC; upfront surgery; real-life data; overall survival; prognostic factors Introduction Lung cancer is a major public health issue world­wide, with an estimated 2.2 million new cases and 1.8 million deaths in 2020 making it the second most common cancer and the leading cause of can­cer death worldwide.1 After decades of poor con­trol of lung cancer, the mortality rates began to de­crease in the last two decades.1 This trend coincides with a slow, but steady increase in lung cancer sur­vival rates, that was up to now mostly noticeable in localized (stage I and II) non-small cell lung cancer 347 (NSCLC). Currently the 5-year net survival of lo­calized lung cancer is around 60%.2,3 Localized lung cancer accounts for around 25% of newly diagnosed lung cancers, with a vast ma­jority of them having NSCLC histology.3 Surgery with curative intent remains fundamental treat­ment for stage I–II and for selected stage IIIA NSCLC patients.4 With the introduction of novel, less invasive surgical techniques, such as video-as­sisted thoracoscopic surgery and improved perio­perative care, the outcomes of patients with resect-able NSCLC improved substantially.3,4 Platinum-based adjuvant chemotherapy, which is nowadays considered as a standard adjuvant treatment of early-stage NSCLC, further improved cure rates.5 With the incorporation of novel targeted therapies and immunotherapy with immune checkpoint in­hibitors (ICIs) additional increase in overall sur­vival is expected. Targeted therapy with osimer­tinib, which led to significant reduction in distant recurrence or death in a prospective phase 3 trial has already been incorporated into treatment rec­ommendations for epidermal growth factor recep­tor (EGFR) positive patients.5 Based on the positive results of some recently published adjuvant trials, it is expected that ICIs will soon become a part of standard adjuvant therapy for early-stage NSCLC as well. There is growing evidence that neoad­juvant treatment with ICI leads to major or even complete pathologic responses in a substantial per­centage of patients without compromising surgery for resectable NSCLC6, thus making neoadjuvant immunotherapy an appealing approach in the fu­ture. It is expected that the percentage of patients di­agnosed with resectable NSCLC will increase in the next years. Several international clinical trials, including the European NELSON study confirmed the efficacy of low-dose CT screening in decreas­ing lung cancer mortality in the high-risk popula­tion of heavy smokers.7,8 With the introduction of screening programs, we expect not only an increase of patients diagnosed with localized NSCLC but it might also become necessary to redefine treatment paradigms for early-stage NSCLC. There is no doubt that major changes in the de­tection and treatment of early-stage NSCLC are expected shortly. To better predict and evaluate the effectiveness of those novel strategies in every­day clinical practice and to develop individualized risk-adjusted treatment strategies for individual patients, more data on clinicopathological charac­teristics and outcomes of early-stage NSCLC pa­tients treated in a real-life before the introduction of those novelties, are needed. The International Association for the Study of Lung Cancer (IASLC) recommendations for TNM classification scheme, based on a database of nearly 90.000 patients9 as well as some IASCL validation studies performed on the Caucasian population10 provide valuable data on survival of patients treated in routine clini­cal practice. Next to the IASLC data, there is almost complete lack of information on the outcomes of the cohorts of resectable stage I–IIIA NSCLC pa­tients, treated in a real-life scenario in the last decade. Most of the real-life observational trials reported recently present data for specific sub­populations of resectable NSCLC, such as patients treated with adjuvant chemotherapy11 or patients with stage IIIA or N2 disease.12-14 Our study aimed to evaluate overall survival of consecutive resecta­ble TNM stage I–IIIA NSCLC patients treated with upfront radical surgery in a real-life practice, us­ing prospectively collected hospital-based registry data. We also assessed the impact of clinicopatho-logical characteristics, particularly TNM stage, on survival. Patients and methods Data source and study population Data were retrieved from the hospital-based lung cancer registry, which prospectively collects de­mographics, clinicopathological, treatment, and survival data for all lung cancer patients diagnosed and treated at the centre. In hospital follow-up data are supplemented with the death certificates pro­vided by the National Health Institute on a regu­lar basis. All data was collected in an anonymised fashion. For the purpose of this study, survival status was updated and the data were retrieved in January 2020. We retrieved the data of consecutive patients with resectable cTNM stage I–III NSCLC, treated with upfront radical surgical resection at a sin­gle thoracic oncology centre in Slovenia, between January 2010 and December 2017. All patients had pathologically confirmed NSCLC. Diagnostic and treatment procedures were performed as recommended by the international guidelines valid at the time.15,16 Lymph nodes showing (18) F-fluorodeoxyglucose (FDG) uptake on preopera­tive PET-CT scans, or their short axis > 1 cm on CT scans were marked as clinically positive. In patients with clinically positive mediastinal lymph nodes endobronchial ultrasound-guided lymph node biopsy (EBUS TBNB) was performed, whenever feasible. For all patients, including those with cN2 disease, the institutional multidisciplinary tumour board concluded that they have resectable NSCLC and were referred to upfront surgery. All patients underwent radical surgical resec­tion (R0) with lobectomy, bilobectomy, or pneu­monectomy with complete lymph node dissec­tion as a standard surgical procedur.16,17 Adjuvant chemotherapy and/or postoperative radiotherapy were performed according to the international guidelines valid at that time.15,16 Patients with neo­adjuvant treatment were not included in the study population. Clinical stage was defined as the last stage deter­mined before surgical resection. All resected tissue including lymph nodes was examined by board certified pathologists. Clinical and pathological stages were assigned based on the 7th edition TNM classification for NSCLC17, valid at the time. Testing for EGFR mutations and anaplastic lymphoma ki­nase (ALK) rearrangements has been introduced gradually as recommended by the international so­cieties.18 Testing was performed on formalin fixed, paraffin embedded tumour tissue specimens or different cytological specimens. For EGFR testing allele-specific PCR method with commercial kits, either Cobas EGFR mutation test (Roche, USA) or Therascreen EGFR PCR Kit (Qiagen, UK). ALK im­munohistochemical detection was based on ALK CDx assay (Ventana, Roche, USA). Patients were followed-up with physical examination and chest CT scan, first biannually and after two years an­nually. The hospital-based registry data collection and all subsequent analyses for academic pur­poses were approved by the Slovenian National Committee for Medical Ethics (approval number 135/07/09 and 40/04/12). All patients consented for data collection and subsequent analyses. Outcome measures and statistical analyses The primary endpoint was overall survival (OS), defined as the time in months from the date of sur­gery until either the date of death from any cause or the date the patient was last known to be alive (censored data). Patient and treatment characteris­tics were analysed using descriptive statistics. The agreement between clinical and pathological TNM staging variables was calculated as simple percent agreement to ease the interpretation of the results. Survival curves were estimated using the Kaplan-Meier estimator. The independent prognostic val­ue of each included characteristic was tested in a Cox proportional hazards regression model. All variables with p = 0.250 in univariable regression analysis (UVA) were considered for and included in the multivariable regression analysis (MVA), ex­cept EGFR and ALK status due to being applicable only to a subset of patients. A p-value below 0.05 was considered statistically significant. All report­ed p-values are two-tailed. All statistical analyses were carried out using IBM SPSS Statistics software (version 21). Results We identified 539 consecutive stage I–IIIA NSCLC patients treated with upfront radical surgery. Demographic, clinicopathological, and treatment characteristics of the study population are present­ed in Table 1. The median age was 64 years (range, 39–83), males accounted for 58.4% of patients. Most patients were current or former smokers, with only 12.7% of never smokers included in the study. Adenocarcinoma appeared most frequently (63.3%), followed by squamous-cell carcinoma (36.2%) and other rare types of NSCLC (0.6%). EGFR mutations and ALK rearrangements were detected in 12.3% and 5.3 % of tested patients, with low completeness of ALK testing due to the intro­duction of testing to routine clinical practice from 2014 onward. Lobectomy was performed in a vast majority of patients, bilobectomy or pneumonecto-my was required in only 5.8% and 9.1% of patients, respectively. Adjuvant platinum doublet chemo­therapy was delivered in 146 (27.1%) of patients, the vast majority of whom had pathologically con­firmed lymph node involvement. Postoperative radiotherapy was used in 36 (6.7%) patients; all of them had pathological N2 disease. PET-CT was performed in 94.8% of patients (511/539). EBUS TBNB was gradually introduced in the routine clinical practice during the study pe­riod and was applied in 112 patients, with cN1 and cN2 disease according to CT and/or PET-CT scan. Lymph node involvement was confirmed in 65.5% of the samples obtained from the patients with cN2 disease. Mediastinoscopy was performed in five patients with cN2 and negative EBUS TNBN of me-diastinal nodes; all lymph node samples obtained by mediastinoscopy were negative. Most patients were diagnosed with clinical stage I (57.3%) or stage II (26.9%). Clinical stage IIIA was determined in 15.8% of patients. All patients had either a single zone cN2 involvement or cT3/T4 disease without 349 TABLE 1. Demographic, clinicopathological and treatment characteristics of study population Age in years: median (range) < 65 years = 65 years Gender Male Female Smoking status (n = 537; completeness = 99.6 %) Current Former Never Histology Adenocarcinoma Squamous-cell carcinoma NSCLC other rare types EGFRa status in non-squamous NSCLC (n = 334; completeness = 99.7%) Positive Negative ALKb status in non-squamous NSCLC (n = 334; completeness = 39.2%) Positive Negative Clinical TNM stagec I II IIIA Clinical T stage T1 T2 T3 T4 Clinical N stage N0 N1 N2 64 (39–83) 271 (50.3) 268 (49.7) 315 (58.4) 224 (41.6) 257 (47.8) 212 (39.5) 68 (12.7) 341 (63.3) 195 (36.2) 3 (0.6) 41 (12.3) 292 (87.7) 7 (5.3) 124 (94.7) 309 (57.3) 145 (26.9) 85 (15.8) 242 (44.9) 193 (35.8) 96 (17.8) 8 (1.5) 393 (72.9) 102 (18.9) 44 (8.2) Pathological TNM stagec (n = 532; completeness = 98.7%) I296 (55.6) II150 (28.2) III 86 (16.2) Pathological T stage (n = 537; completeness = 99.6%) T1223 (41.5) T2248 (46.2) T358 (10.8) T4 8 (1.5) Pathological N stage (n = 534; completeness = 99.1% N0386 (72.3) N181 (15.2) N2 67 (12.5) Surgery type Lobectomy459 (85.2) Bilobectomy31 (5.8) Pneumonectomy 49 (9.1) Adjuvant treatment 146 (27.1) Platinum-based chemotherapy 36 (6.7) Postoperative radiotherapy aEGFR: epidermal growth factor receptor; bALK: anaplastic lymphoma kinase; cstage defined by American Joint Committee on Cancer staging tumour invasion to the adjacent vessels or organs. Postoperative pathological examination and stag­ing also revealed high rate of pathological stage FIGURE 1. Overall survival of patients with completely resected stage I–III A non-small cell lung cancer. I (55.6%) or stage II (28.2%), with low percentage of stage IIIA disease (16.2%). However, the agree­ment between clinical and pathological staging was relatively low. Table 2 shows the comparison between clinical (cTNM) and pathological (pTNM) staging accord­ing to TNM staging categories. The agreement be­tween cTNM and pTNM stages was the highest for stage I (81%) and much lower for stage II (55%) and stage IIIA (49%). Of note, cTNM stage IIIA turned out to be pTNM stage II or stage I in 36% and 14% of patients, respectively. When analysing T and N descriptors separately, the accuracy of cT-descrip­tor decreased with increasing stage while for cN-descriptor the lowest accuracy rate was observed for cN1 stage. The overall agreement between clini­cal and pathological stage were quite similar for all three descriptors, TNM stage, T stage and N stage, i.e., 69.0%, 72.3% and 71.9%, respectively. The median follow-up time was 53.9 (50.9–56.9) months. At the end of follow-up, 177/539 patients (32.8%) died. The median OS (mOS) for the whole cohort of patients was 90.4 months (95% CI calcula­tion unreliable due to few events after mOS), with an estimated 5-year OS rate of 64.4% (Figure 1). The overall survival of patients grouped by cTNM, pTNM, cN and pN stage is depicted in Figure 2. The mOS has not been reached in the majority of the subgroups. The estimated 5-year OS rates for patients with cTNM stage I, stage II, and stage IIIA were 70.6%, 56.9%, and 55.3%; while the estimat­ed 5-year OS rates for patients with pTNM stage I, stage II, and stage IIIA were 70.2%, 60.2%, and 49.9%, respectively, (Figures 2A and 2B). When observing the N status alone, Figures 2C and 2D show that pN stage provides a much clearer sep­aration of survival curves than cN stage – as also demonstrated by UVA below. In UVA the factors significantly associated with shorter overall survival were age = 65 years and male gender. Furthermore, with respect to the anatomical stages, all stage categories, except cN (p = 0.313), were significantly associated with OS in the UVA (Table 3). However, in MVA that included either cTNM or pTNM stage as a deter-minator of the anatomical extent of disease, pTNM retained its significant and independent impact on OS (p = 0.003), next to age and gender, while cTNM stage lost its independent prognostic value (p = 0.092) (Table 4). Of note, TNM stage (clinical or pathological) was always included in the model for multivariate analyses, while the other factors were included in the stepwise procedure (thus only the significant factors are reported in Table 4). Discussion This observational cohort study presents real-life data on long-term survival and the impact of clin­icopathological characteristics on overall survival 351 of resectable stage I–IIIA NSCLC patients, treated with upfront radical surgery at a single thoracic oncology centre in the period 2010–2017. The me­dian OS time of 90.4 months and estimated 5-year survival rate of 64.4% observed in our real-life co­hort of 539 consecutive patients are encouraging. Our data exceed the median OS of 63 months ob­served in a German cohort of patients with radi­cally resected stage I–IIIB NSCLC, treated at a sin­gle academic centre in a very similar period (from 2009 to 2014), which also included patients with a higher stage IIIB disease.10 When comparing by pTNM stage I, II and IIIA, the estimated 5-year sur­vival rates of 70.2%, 60.2% and 49.9%, respectively, observed in our study, correspond very well to the 5-year survival rates in the German study.10 Our findings also slightly exceed the 5-year survival rates of 83%–71%, 57%–49% and 36% for pTNM stage IA–B, II A–B and IIIA, published by IASLC.9 Furthermore, our findings are also in line with 5-year survival rates between 37%–47%, observed in real-life cohorts of patients with resectable stage IIIA–N2 NSCLC, treated with upfront surgery in a similar period.12-14 Thus, our observation sup­ports the idea that selected patients with stage IIIA NSCLC might have a favourable outcome when treated by upfront radical surgery followed by ad-juvant chemotherapy and/or irradiation. As expected, the observed survival rates de­creased with increasing stage of all staging varia­bles (T, N, and TNM). But of note, while significant differences in survival were observed according to both clinical and pathological T and both clinical and pathological TNM stage, clinical N stage (as opposed to pathological N stage) did not prove a significant prognostic factor already in the UVA. Furthermore, in the multivariate analyses in which only TNM stage as a comprehensive denominator of T and N stages was included, only pTNM stage retained its significant and independent impact on overall survival, while cTNM stage failed to do so (likely due to its N stage part). This clearly points towards a much stronger prognostic value of path­ological compared to clinical staging variables in resectable NSCLC. Also, in many previous studies evaluating prognostic impact of clinical and patho­logical TNM or N stage on OS the information on pathological stage improved prognostic value of the model.9,14,17 There is evidence suggesting quite a high rate of disagreement between clinical and pathological staging in operable NSCLC patients treated in everyday practice. Even in studies per­formed after introduction of PET-CT and EBUS TBNB in routine clinical practice, relatively high TABLE 2. Comparison between clinical (c) and pathological (p) TNM staging 2A. Comparison between clinical and pathological TNM stage (n = 532; completeness = 98.7%) p Stage I 246 (81%) 38 (26%) 12 (14%) p Stage II 40 (13%) 79 (55%) 31 (36%) p Stage IIIA 17 (6%) 27 (19%) 42 (49%) Overall agreement: 367 out of 532 cases (69.0%) 2B. Comparison between clinical and pathological T stage (n = 537; completeness = 99.6%) pT1 187 (78%) 24 (13%) 10 (10%) 2 (25%) pT2 46 (19%) 158 (82%) 41 (43%) 3 (37%) pT3 5 (2%) 9 (4%) 42 (44%) 2 (25%) pT4 2 (1%) 2 (1%) 3 (3%) 1 (13%) Overall agreement between: 388 out of 537 cases (72.3%) 2C. Comparison between clinical and pathological N stage (n = 534; completeness = 99.1%) pN0 324 (84%) 49 (48%) 13 (30%) pN1 42 (11%) 34 (33%) 5 (11%) pN2 22 (6%) 19 (19%) 26 (59%) Overall agreement: 384 out of 534 cases (71.9%) rate of disagreement between clinical and patho­logical N and TNM staging was observed. In the Dutch observational study performed in patients with pathological stage IIIA disease, the agreement between clinical and pathological T and N stage was 57.1% and 28.5%, respectively.19 The agree­ment rates observed in our study were relatively high for all three descriptors T, N and TNM stage (72.3%, 71.9% and 69.0%, respectively), but still not optimal. However, EBUS TBNB have only been in­troduced in our everyday clinical practice during the study period. With the incoming era of neoad­juvant systemic therapy, the accurate non-surgical staging of not only mediastinal lymph nodes but also hilar lymph nodes were becoming important. In our study the lowest agreement between clini­cal and pathological N status was observed par­ticularly for cN1 stage (33%). Very interesting and clinically important observation is that almost half (48%) of cN1 patients were down staged to pN0, while upgrading to pN2 was found in a smaller, 19% proportion of patients. With recent dilemmas whether more invasive mediastinal lymph node TABLE 3. Univariate analyses of overall survival Age < 65 = 65 Gender Male Female Smoking status never current or former Histology adenocarcinoma or NOS squamous cell carcinoma EGFR statusa (positive vs negative) negative positive Clinical TNM stage I II IIIA Clinical T stage T1 T2 T3 or T4 Clinical N stage N0 N1 N2 Pathological TNM stage I II IIIA Pathological T stage T1 T2 T3 or T4 Pathological N stage N0 N1 N2 0.002 0.001 0.115 0.111 0.111 0.027* 0.034 0.025 0.001* 0.882 0.001 0.317* 0.958 0.137 0.003* 0.030 0.001 0.007* 0.019 0.004 0.002* 0.054 0.001 1 1.59 (1.18 – 2.15) 1 0.59 (0.43 – 0.81) 1 1.50 (0.91 – 2.47) 1 1.28 (0.95 – 1.73) 1 0.56 (0.27 – 1.14) 1 1.44 (1.03 – 2.02) 1.57 (1.06 – 2.34) 1 0.97 (0.69 – 1.38) 1.86 (1.29 – 2.68) 1 0.99 (0.67 – 1.46) 1.44 (0.89 – 2.34) 1 1.46 (1.04 – 2.06) 1.90 (1.29 – 2.79) 1 1.49 (1.07 – 2.07) 1.92 (1.23 – 2.98) 1 1.48 (0.99 – 2.20) 1.93 (1.29 – 2.87) aonly in non-squamous NSCLC; *for the whole variable staging might change the treatment paradigm and outcomes of NSCLC patients with cN1 disease our data become even more appealing. Notably, the survival rates observed in our cur­rent study far exceed those observed in a retro­spective analysis of NSCLC patients treated at our centre in 2006.20 The latter revealed much shorter median overall survival rates for all clinical TNM stages I, II and IIIA NSCLC with the largest differ­ences observed in stages II–IIIA. In that analysis all consecutive patients were included, regardless of whether they received treatment with curative in­tent or not, which is definitively one of the reasons for worse survival rates. But still, improvement in overall survival achieved over the last years is ob­vious. This can be attributed to major advances in diagnostic procedures, surgical techniques, post­operative care and adjuvant therapies for early NSCLC that we witnessed in the last decade and their rapid transfer into everyday clinical practice at our institution.21 The clinicopathological characteristics of our cohort of patients mirror the typical population of NSCLC patients in our country and region at the beginning of this century, with prevailing smok­ers and squamous-cell histology.21 Next to pTNM stage, age and gender retained their significant and independent prognostic value for OS in MVA; while smoking status and histology failed to show prognostic value already in the UVA. Our results are in concordance with the observations made on a large series of patients with NSCLC confirming older age and male gender as independent prog­nostic factors for worse survival.22,23 Male gender was confirmed as an independent prognostic fac­tor for worse survival in published trials, however this has been seen particularly in patients with advanced NSCLC and adenocarcinomas.23 In our study male gender turned out to be an independent predictor of worse survival in early-stage NSCLC and irrespective of histology, thus suggesting other probable causes of poor survival in male NSCLC patients which need to be further investigated. Our study also provides valuable data on the frequency of EGFR mutations and their prognos­tic value in early-stage NSCLC. The findings are in line with the results of recently published large in­dividual study24 which failed to confirm prognostic impact of EGFR status on survival of patients with resectable NSCLC. There are still uncertainties about the percentage of EGFR mutated tumours in early-stage NSCLC. In our study, EGFR testing performed on a large series of 334 patients with resectable non-squamous cell NSCLC, revealed a 12.3% positivity rate which is quite comparable to the 13.8% positivity rate observed in advanced NSCLC in the countries and the centres which par­ticipated in the INSIGHT registry trial.25 Similarly, ALK positivity rate of 5.3% observed in our series of resectable NSCLC corresponds very well with the positivity rates observed in advanced NSCLC.26 The results of our study should be considered in the context of its strengths and limitations. The study provides a wealth of information on clinico-pathological characteristics and survival outcomes of a large cohort of resectable NSCLC patients, treated with upfront surgery in real-life practice. Additionally, all data were collected prospectively by the hospital-based lung cancer registry. Looking at potential limitations, results from a single cen­tre study might not be generalisable to the overall population in the country or region. However, at our centre more than a half of the country’s newly 353 TABLE 4. Multivariate analyses of overall survival (separate for clinical and for diagnosed resectable NSCLC are treated, thus rep- pathological stage) resenting the entire population quite well. It is also encouraging that the activities on establishing a na­tionwide register of lung cancer patients collecting Age < 65 1 = 65 0.003 1.58 (1.17 – 2.14) Gender Male 1 Female 0.006 0.63 (0.46 – 0.88) Clinical TNM stage 0.092* I 1 II 0.078 1.36 (0.97 – 1.91) IIIA 0.068 1.46 (0.97 – 2.18) detailed data on clinicopathological characteristics and individual treatments at the Cancer Registry of Slovenia are ongoing. Since our hospital-based reg­istry does not capture data on the cause of death, we do not present data on cancer specific survival but on overall survival, which might be influenced by comorbidities and other conditions often pre­sent in fairly old population of patients with re-sectable NSCLC. The hospital registry also does not collect precise data on modality of preopera­tive staging (imaging versus invasive procedures) to determine clinical N stage in each individual pa­tient. Therefore, the data on mediastinal staging by EBUS TNBN and mediastinoscopy were collected retrospectively and might be subject to bias. Our study with a lengthy follow-up, showed a favourable outcome for patients with resectable stage I–IIIA NSCLC treated with upfront surgery in a real-life setting. Particularly encouraging are the survival rates observed in patients with stage IIIA disease indicating that selected patients with N2 disease are candidates for upfront surgery. Relatively low agreement between cTNM and pT-NM stages and the independent prognostic value of pTNM but not cTNM stage observed in our study, suggest that we should aim to further im­prove preoperative staging. Until then we should always weight our decisions about upfront treat­ment of resectable NSCLC very carefully for each individual patient. Currently, surgery remains the most reliable provider of information on anatomi­cal TNM stage as one of the strongest prognostic factors and enables us to make an informed deci­sion on adjuvant systemic treatment in each indi­vidual patient. Finally, it is inspiring to notice a substantial im­provement in overall survival rates of early-stage NSCLC patients treated over the last decades at the same large thoracic oncology centre. With the aim of further improving our results, we are planning an additional study which will strive to evaluate preoperative staging of nodal involvement more profoundly, thus providing for better multimodal­ity treatment selection for each individual patient. Acknowledgement The authors thank all doctors and other staff pro­viding standard care for patients with NSCLC at Age < 65 = 65 0.001 Gender Male Female 0.004 Pathological TNM stage 0.003* I II 0.076 IIIA 0.001 *for the whole variable Surgery Bitenc and at the University Clinic Golnik. Special thanks go to the staff of the hospital-based registry, especially to Ana Herzog and Tjaša Brus Picman, for their dedicated work.This work was supported by the Slovenian Research Agency [grants number J3-4076, J3-7372] by providing the funds for establishment of the hospital-based regis­try. This analysis has received no funding. The raw data underlying this article are avail­able in the article. Due to data privacy, and hospital registry-related restrictions, the clinicopathological data cannot be made public, i.e., accessible to any­one for any purpose without a review process and without putting an agreement in place. Data availability statement and author contribution statement Marko Bitenc: conceptualization, writing – origi­nal draft, formal analysis, writing – review & ed­iting. Tanja Cufer: conceptualization, formal anal­ysis, writing – original draft, writing – review & editing, supervision. Izidor Kern: investigation, writing—original draft, formal analysis. Martina Miklavcic: data curation, investigation, writing – original draft, writing – review & editing. Sabrina Petrovic: data curation, investigation, writing – original draft. Vida Groznik: software, data cura­tion. Aleksander Sadikov: software, formal analy­ 1 1.68 (1.24 – 2.28) 1 0.62 (0.45 – 0.86) 1 1.37 (0.97 – 1.93) 1.95 (1.32 – 2.88) 354 sis, visualization, writing – review & editing, su­pervision. 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BMC Cancer 2018; 18: 269. doi: 10.1186/s12885-018-4023-4 355 research article Identification of women with high grade histopathology results after conisation by artificial neural networks Marko Mlinaric1, Miljenko Krizmaric2, Iztok Takac3,4, Alenka Repse Fokter5 1 Outpatient Clinic for Gynaecology and Obstetrics Marko Mlinaric, Dr. Med., Zagorje ob Savi, Slovenia 2 Faculty of Medicine, University of Maribor, Maribor, Slovenia 3 University Clinic of Gynaecology and Perinatology, University Medical Centre Maribor, Maribor, Slovenia 4 Department of Gynaecology and Perinatology, Faculty of Medicine, University of Maribor, Maribor, Slovenia 5 Department of Pathology and Cytology, General Hospital Celje, Celje, Slovenia Radiol Oncol 2022; 56(3): 355-364. Received 16 January 2022 Accepted 25 April 2022 Correspondence to: Marko Mlinaric, M.D., Outpatient Clinic for Gynaecology and Obstetrics Marko Mlinaric, Dr. Med., Cesta zmage 1, 1410 Zagorje ob Savi, Slovenia. E-mail: info@ginekoloska-ambulanta.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 the study was to evaluate if artificial neural networks can predict high-grade histopathology results after conisation from risk factors and their combinations in patients undergoing conisation because of patho­logical changes on uterine cervix. Patients and methods. We analysed 1475 patients who had conisation surgery at the University Clinic for Gynaecology and Obstetrics of University Clinical Centre Maribor from 1993–2005. The database in different datasets was arranged to deal with unbalance data and enhance classification performance. Weka open-source software was used for analysis with artificial neural networks. Last Papanicolaou smear (PAP) and risk factors for development of cervical dysplasia and carcinoma were used as input and high-grade dysplasia Yes/No as output result. 10-fold cross validation was used for defining training and holdout set for analysis. Results. Baseline classification and multiple runs of artificial neural network on various risk factors settings were per­formed. We achieved 84.19% correct classifications, area under the curve 0.87, kappa 0.64, F-measure 0.884 and Matthews correlation coefficient (MCC) 0.640 in model, where baseline prediction was 69.79%. Conclusions. With artificial neural networks we were able to identify more patients who developed high-grade squa­mous intraepithelial lesion on final histopathology result of conisation as with baseline prediction. But, characteristics of 1475 patients who had conisation in years 1993–2005 at the University Clinical Centre Maribor did not allow reliable prediction with artificial neural networks for every-day clinical practice. Key words: uterine cervical dysplasia; uterine cervical cancer; conisation; artificial neural networks Introduction Cervical cancer is a preventable disease. Effective measures are organised cervical cancer screen­ing programme in combination with vaccination against human papilloma virus (HPV) and treat­ment of precancerous lesions.1 There are many risk factors, which can facilitate development of cervical dysplasia and cancer. Among them are early onset of sexual activity, multiple sex part­ners, parity, marital status, socioeconomic status, factors that influence persistent infection (genetics, sex hormones, immunological impairment as in human immunodeficiency virus (HIV) infection, sexually transmitted diseases (HPV, HIV, Herpes simplex virus [HSV], Chlamydia), factors related to HPV (genotype, numbers of viral copies), long term use of hormonal contraception, smoking and obesity.2-13 HPV is very important risk factor necessary for development of cervical dysplasia and cancer. 14-15 After initiation of sexual activity, almost all women acquire infection with HPV. This infection can only be transitory, clears spontaneously and does not progress to dysplasia.16 Patients aged 30–35 years are tested positive in 13.5% compared to 5.4% pa­tients older than 35 years.17 In computer science artificial neural networks (ANN) are part of artificial intelligence and rep­resent deep machine learning. ANN are nonlinear computational models. They are able to perform tasks, similar to human brain. Just by analysing examples (training set) can perform classification, decision-making, prediction, visualisation, recog­nition and other. The name neural networks came from similarities with structure and behaviour of that of human brain.18 There are many types of dif­ferent ANN. They are very important tool in pro­cessing large amount of data, image processing, image recognition, computer vision and natural language processing. Because of their ability to learn and make prediction make them very use­ful tool in medicine.19,20 They are used in every day clinical practice in cancer diagnostics where they help radiologists to recognise pathological fea­tures, help to predict malignant tumour response to treatment, help in triage and others.21-25 This study has been designed to evaluate if neu­ral networks can help us to identify patients with higher risk for high grade squamous intraepithelial lesion (HSIL) and cervical cancer based only on the evaluation of their risk factors for cervical dyspla­sia and result of the last Papanicolaou smear (PAP). If neural networks are successful in predicting high risk patients, we could use them to identify and take special measures in situation when such pa­tients became non-responders in organised cervi­cal cancer screening programme. With such special attention, we could prevent them from acquiring cervical cancer. Patients and methods Our study has been approved by Medical Ethics Committee of the Republic of Slovenia on 10. 11. 2015, No.: 0120-553/2015-2 KME63/11/15. Data from patients who had conisation in the years 1993–2005 were collected in database: age at the time of surgery, age at first intercourse, number of sexual partners, number of pregnancies (births, spontaneous and legal abortions), socio-economic status, marital status, type of contraception, smok­ing habits, menstrual pain, vaginal discharge, co-agulopathy, colposcopic findings, result of last PAP smear, histopathology of cervical biopsy prior conisation, indication for conisation, additional smears (HPV 16, 18, 31 and 33 and possible other pathogens), vaginal therapy before conisation, type of conisation, data regarding complications after conisation if present, final histopathology and data if margins of the cone were free of disease. Records from database were anonymised and we used only data of suspected risk factors for HSIL regarding age at the time of surgery, age at menarche, age at first intercourse, number of sexual partners, num­ber of deliveries, spontaneous and legal abortions, type of contraception, marital status, socioeconom­ic status, smoking habits, last PAP smear result and final histopathology of the cone. All patients with incomplete data were removed from analysis. The sample is relatively small and is not rep­resentative of the real-life situation because more patients have dysplasia or carcinoma and only smaller portion of patients have low risk squamous intraepithelial lesion (LSIL) or no dysplasia at all. In Slovenia, healthy women without dysplasia rep­resent majority of women who attend organised Cervical cancer screening programme ZORA. In year 2019 in Slovenia, we diagnosed 105 new cases of cervical carcinoma and 1056 cases of HSIL. In the same period, we analysed 220301 PAP smears from 206323 women.26 First line treatment for dys-plastic changes on uterine cervix is conisation or large loop excision of transformation zone (LLETZ) in majority of cases.27 In 2019, we performed 2017 conisation procedures. 1334 (66%) patients had conisation because of HSIL (cervical intraepithe­lial neoplasm [CIN]), 400 (20%) patients because of low-grade squamous intraepithelial lesions (LSIL) and 283 (14%) had no dysplasia.26 In Slovenia number of conisations is decreasing in favour of LLETZ.28 We constructed two basic settings of our data­base. In Raw setting we used previously mentioned risk factors with age in years and last PAP result. For better classification performance we construct­ed another classification (Class) setting in which we grouped patients by Age at the time of surgery in 15 age groups with 5 years interval and divided Last PAP smear result in two groups (high risk PAP smear Yes: PAP III–V and No: PAP I–II). We di­vided Final histopathology result of conisation in two groups (HSIL: CIN 2, 3, CIS [carcinoma in situ], Ca 357 [carcinoma] and NO-HSIL: CIN 1, 1–2 and non-dysplastic changes). In our database are complete data of 1475 pa­tients, 26 (1.8%) without dysplasia on final histo­logical result of conisation, 160 (10.8%) with L-SIL and 1289 (87.4%) with HSIL. Last PAP smear was high risk in 16 patients (61.5%) without dysplasia, 127 patients (79.4%) with LSIL and in 1169 patients (90.7%) with HSIL. Mean age of patients without HSIL was 38.6 years (13–83 years, standard deviation 10.47) and 34.9 (13–81, standard deviation 8.98) in the group of patients with HSIL. Mean age at menarche was 13.7 (10–19, standard deviation 1.84) in group of patients without HSIL and 13.5 (9–20, standard de­viation 1.16) in HSIL patients. Mean age at first in­tercourse was 17.6 (13–25, standard deviation 1.59) in patients without HSIL and 17.4 (12–25, standard deviation 1.66) in patients with HSIL. HSIL and NO-HSIL group of patients were statistically dif­ferent regarding age (p < 0.01), age at 1st intercourse (p < 0.035), number of sex partners (p < 0.004) and high risk PAP smear (p < 0.01). In our group of patients without HSIL 57% tested HPV 16 negative and 27% positive (16% not tested) and in the group of patients with HSIL 54% tested negative and 33% positive (14% not tested). In the NO-HSIL group 65% tested HPV 18 nega­tive, 21% positive (15% not tested) and in HSIL group 60% tested negative and 27% positive (13% not tested). Because many patients did not have HPV test­ing, we decided to remove such patients from anal­ysis. When we analysed removed patients because of no HPV testing (HPV 16, 18, 31 or 33), we dis­covered that numerous patients with HSIL would be missed (Table 1). Chi-square test (. = 1.631, p = 0.202) found no statistically difference of HPV 16, 18 status and presence of HSIL in our group of patients. In this time period we didn’t routinely tested presence of TABLE 1. Final histology of the cone in patients without human papilloma virus (HPV) testing NO dysplasia 9 1.8 CIN 1 26 5.3 CIN 1–2 27 5.4 CIN 2 90 18.1 CIN 2–3 55 11.1 CIN 3 223 45.0 CIS 55 11.1 invasive ca 11 2.2 Total 496 100.0 CIN = cervical intraepithelial neoplasm HPV infection. Because of a chance that we detect­ed transitory infection with HPV testing and that over 400 patients with HSIL would be excluded from analysis because they were not tested against HPV, we decided to exclude HPV from further analysis. HPV 16 and 18 statuses in our patients are presented in Table 2. Human neuron or nerve cell is a cell, which can be electrically or chemically excited. It has body – soma and dendrites – which lead signal to neuron and single axon which lead signal from neuron and interconnects with other neural cells. Information is transferred via electrical or chemical mecha­nism.29 In ANN we have different neurones. There are two main types. Input neurone called perceptron receives information. Output neurone produces final output. All neurones are arranged in layers. First layer is input layer with perceptrons, last lay­er is layer with output neurones. In between there can be one or many hidden layers. Every neuron interconnect with all neurones from previous and TABLE 2. Number and percentage of patients according to human papilloma virus (HPV) 16 and 18 statuses in high grade squamous intraepithelial lesion (HSIL) and NO-HSIL group not performed 17714 29 1617213 27 15 negative 693 54 106 57 775 60 120 65 positive 41932 51 2734227 39 20 Total 1289 100 186 100 1289 100 186 100 next layer.30 Diagram of simple ANN is presented in Figure 1. As in neural cell, artificial neurones in neural networks receive information and became excited. When excitation level (weight) is reached, they promote signal to other neurones. Before weight is reached no output signal is produced. There are many different mathematical functions for neu­ron activation. Activation function of output neu­rons can be different from that of previous layers. Output of the last neuron is numerical value which can range from 0–1. Threshold for classified posi­tive/negative is by default 0.5, meaning that cases with values > 0.5 are classified as positive and cases with value = 0.5 as negative. Threshold value can be changed according to the performance of the al­gorithm and our goals.18 Dataset must be split in two parts-training and holdout set. Training set is used to build model, test relations between input variables and deter­mining weights of the neurones. Algorithms are then tested on holdout set in which are instances unknown to neural network. Training set must be larger than holdout.18 In every classification process, we have actual positive and negative cases, which can be classified correctly as positives or negatives or classified in­correctly. The best way to visualize the situation is to use confusion matrix. Effectiveness of ANN or any other classification system or algorithm can be measured. In our study we used precision (positive predicted value; PPV), recall (sensitivity, true positive rate; TPR), receiver op­erator characteristic curve (ROC curve), area under the ROC curve (AUC).31 F-measure and Matthews correlation coefficient (MCC) are another measure for efficiency. F-measure is combined measure of precision and recall: It ranges between 0 (worst) and 1 (best). MCC ranges between -1 and +1. -1 meaning perfect misclassification, 0 means as expected in random guessing and +1 perfect classification.32 Precision-recall curve (PRC curve) is another measure of clas­sification efficiency. Precision (PPV) is plotted on y-axis and Recall (TPR) on x-axis. It is more inform­ative than ROC Curve in imbalanced data settings because it analyses fraction of true positives among all positive predictions.33 Quality of data is of vital importance – sufficient numbers of instances (collection of attributes in database) and qualitative attributes (features that measure or describe different aspect of instances). Before running classification algorithm, it is neces­sary to run simulation of baseline classification. We can then compare results derived from our model with baseline results and decide how good (or bad) our model is in classification and prediction. Dealing with unbalanced data When we have imbalanced datasets where one of the variables represents only a small proportion of the sample, baseline prediction for majority class is very high. For example – if majority class rep­resents 88% of instances as in our case, baseline prediction is high – 88%. If prediction algorithm predicts with 92% accuracy this is not statistically significant. There are some methods, how to deal with unbalanced data: • Under-sampling: randomised reduction of ma­jority class to match minority class • Over-sampling: n-fold replication of minority class to match majority class • SMOTE: synthetic minority over-sampling tech­nique creates new synthetic instances, which have similar characteristics as original ones in minority class.34,35 Experiment with WEKA Weka (1999–2020 The University of Waikato, Hamilton, New Zealand) is open-source applica­tion for data mining with many other possibilities beside ANN as are Bayesian networks, Logistic re­gression, Classification trees, K-nearest neighbours and others.36 It enables us to test classification al­gorithm on whole dataset, we can split dataset by percentage, test whole dataset against separate training dataset from different dataset which we import in Weka and n-fold cross validation. When 359 we manually or randomly split dataset in training and holdout part, there is always a chance that we collect all important instances in one of the sets, especially if one kind of instances represent small proportion of all instances. N-fold cross validation is powerful option which can minimise the chance of such situation. It divides entire database into n parts. Each n-1 part is used as training and each n part as holdout set. All combinations of n and n-1 parts are then tested against each other and algo­rithm at the end presents the best result of tested combinations. In our experiment, we used 10-fold cross validation.37 Preparation of datasets for analysis We prepared eight data sets: • Raw set: we used as variable original risk factors and as output HSIL_Y/N. • Class set: same as raw set except age groups in­stead of age and PAP_HR_Y/N instead of last PAP. • Raw and class with under-sampling, over-sam­pling and SMOTE method for equalising imbal­anced dataset. Original dataset consisted of 186 No-HSIL and 1289 HSIL patients. To prepare over-sampling dataset we duplicated HSIL negative patients to get 558 No-HSIL and original 1289 HSIL patients. For under-sampling, we randomly selected and deleted HSIL patients to get 272 HSIL and original 186 No-HSIL patients. With SMOTE algorithm, we created data set with original 1289 HSIL patients and 744 No-HSIL patients. Baseline prediction was calculated for each set and results for multi-layer perceptron with 10-fold cross validation was recorded. Results are present­ed in Table 4. Results In first part of analysis, we analysed original da­tabase with artificial neural network, multi-layer perceptron (MLP). We achieved 81.42% correct predictions which is worse than baseline – ZeroR prediction 87.39% (kappa = 0.08 showing no level of agreement between predicted and actual status, AUC 0.594, MCC 0.086, F-Measure 0.806, precision 0.799 and recall 0.814). When we corrected minor­ity class with over-sampling method ZeroR predic­ tion was 69,79%, achieved 79,21% (kappa = 0.523 showing weak level of agreement between pre­dicted and actual status, AUC 0.837, MCC 0.525, TABLE 3. Confusion matrix for classification with all possible outcomes Actual pos (P) True positives (TP) False negatives (FN) Actual neg (N) False positives (FP) True negatives (TN) Neg = negatives; Pos = positives FIGURE 2. Matthews correlation coefficient (MCC) for categorisation squamous intraepithelial lesion (HSIL)-combined for YES and NO prediction for different equalisation methods (no correction of minority class, under-sampling, over­sampling and synthetic minority over-sampling technique [SMOTE]) for both RAW and Class settings. Best performance of multi-layer perceptron (MLP) is on dataset with data organised in classes and over-sampling method for minority class – MCC = 0.64. Lowest performance is with original dataset without correction for minority class – MCC = 0.086. FIGURE 3. True positive and False positive rate for different settings for prediction Yes and No combined and for different equalisation methods (no correction of minority class, under-sampling, over-sampling and synthetic minority over-sampling technique [SMOTE]) for both RAW and Class settings. Best performance model from Figure 2 has 0.842 true positive rate and 0.182 false positive rate. Lowest performance model from Figure 2 has high 0.814 true positive rate which is almost as high as best performance model but also high false positive rate 0.735. Raw = original settings; Class = class setting; FPR = false positive rate; HSIL = high grade squamous intraepithelial lesion; overs = oversampling; TPR = true positive rate; unders = undersampling; SMOTE = synthetic minority over-sampling technique 360 TABLE 4. Results of multi-layer perceptron (MLP) classifications for different settings with baseline prediction – ZeroR, percentage of correct classification and Kappa statistic for all analysis. Results are for prediction high grade squamous intraepithelial lesion (HSIL)-Yes (Y), prediction NO-HSIL (N) and weighted average for whole model (YES and NO combined) – Weighted average (AVG). In bold-type letters are results, where prediction by MLP is better than baseline prediction ZeroR Class_orig–Y 0.751 0.634 0.739 0.751 0.745 0.118 0.567 0.735 Yes 82.10 0.0965 87.39 Class_orig–N 0.366 0.249 0.308 0.366 0.373 0.118 0.567 0.377 No Class_orig–AVG 0.637 0.521 0.633 0.637 0.635 0.118 0.567 0.629 Weighted Avg Class_overs–Y 0.860 0.201 0.908 0.860 0.884 0.640 0.870 0.920 Yes 84.19 0.6376 69.79 Class_overs–N 0.799 0.140 0.712 0.799 0.753 0.640 0.870 0.703 No Class_overs–AVG 0.842 0.182 0.849 0.842 0.844 0.640 0.870 0.855 Weighted Avg Class_SMOTE–Y 0.797 0.274 0.834 0.797 0.815 0.515 0.802 0.850 Yes 77.08 0.5141 63.40 Class_SMOTE–N 0.726 0.203 0.673 0.726 0.699 0.515 0.802 0.669 No Class_SMOTE–AVG 0.771 0.248 0.775 0.771 0.772 0.515 0.802 0.784 Weighted Avg Class_unders–Y 0.669 0.559 0.636 0.669 0.652 0.112 0.542 0.608 Yes 57.64 0.1113 59.39 Class_unders–N 0.441 0.331 0.477 0.441 0.458 0.112 0.542 0.448 No Class_unders–AVG 0.576 0.466 0.572 0.576 0.573 0.112 0.542 0.543 Weighted Avg RAW_orig–Y 0.907 0.828 0.884 0.907 0.895 0.086 0.594 0.905 Yes 81.42 0.0856 87.39 RAW_orig–N 0.172 0.093 0.211 0.172 0.189 0.086 0.594 0.174 No RAW_orig–AVG 0.814 0.735 0.799 0.814 0.806 0.086 0.594 0.813 Weighted Avg RAW_overs–Y 0.825 0.285 0.870 0.825 0.847 0.525 0.837 0.905 Yes 79.21 0.523 69.79 RAW_overs–N 0.715 0.175 0.639 0.715 0.675 0.525 0.837 0.661 No RAW_overs–AVG 0.792 0.252 0.800 0.792 0.795 0.525 0.837 0.831 Weighted Avg RAW_SMOTE–Y 0.800 0.258 0.843 0.800 0.821 0.533 0.814 0.867 Yes 77.87 0.5318 63.4 RAW_SMOTE–N 0.742 0.200 0.681 0.742 0.710 0.533 0.814 0.691 No RAW_SMOTE–AVG 0.779 0.237 0.784 0.779 0.780 0.533 0.814 0.802 Weighted Avg RAW_unders–Y 0.688 0.575 0.636 0.688 0.661 0.115 0.551 0.614 Yes 58.08 0.1144 59.39 RAW_unders–N 0.425 0.313 0.482 0.425 0.451 0.115 0.551 0.466 No RAW_unders–AVG 0.581 0.469 0.573 0.581 0.576 0.115 0.551 0.554 Weighted Avg Raw = original settings; Class= class setting; overs = oversampling; SMOTE = synthetic minority over-sampling technique; unders = undersampling F-Measure 0.795, precision 0.800 and recall 0.792). SMOTE performed inferior than over-sampling with baseline ZeroR 63.40% and achieved 77.87% (kappa = 0.53 showing weak level of agreement between predicted and actual status, AUC 0.814, MCC 0.533, F-Measure 0.780, precision 0.784 and recall 0.779). Under-sampling method performed worse than analysis on original dataset with ZeroR prediction 59.39%, achieved 58.08% (kappa = 0.11 showing no level of agreement between pre­dicted and actual status, AUC 0.551, MCC 0.115, F-Measure 0.576, Precision 0.573 and Recall 0.581). In second part of analysis, we grouped data in classes as described previously. Analysis with MLP on original data achieved 82.10% correct pre­diction which is less than baseline 87.39% ZeroR prediction (kappa = 0.09 showing no agreement between predicted and actual status, AUC 0.567, MCC 0.118, F-Measure 0.635, precision 0.633 and recall 0.637). Performance of MLP was better with over-sampling method, where baseline ZeroR pre­diction was 69.79% and MLP achieved 84.19% cor­rect predictions (kappa = 0.64 showing moderate level of agreement between predicted and actual 361 status, AUC 0.870, MCC 0.640, F-Measure 0.844, precision 0.849 and recall 0.842). With SMOTE method baseline ZeroR prediction was 63,40% and achieved prediction 77,08% (kappa = 0.51 showing weak level of agreement between predicted and actual status, AUC 0.802, MCC 0.515, F-Measure 0.772, precision 0.775 and recall 0.771). Under-sampling method performed worse than analysis on original data with ZeroR prediction 59.39% and 57,64% correct predictions (kappa = 0.11 showing no agreement between predicted and actual status, AUC 0.542, MCC 0.112, F-Measure 0.573, precision 0.572 and recall 0.576). All results are presented in Table 4. MCC for all models is graphically presented in Figure 2 for pre­diction HSIL-Yes and NO combined. True positive rate and false positive rate for all models are graph­ically presented in Figure 3. ROC curve for worst performance model is represented on Figure 4 and for best performance model on Figure 5. Discussion In medicine, we mostly deal with imbalanced classes. In such data sets baseline prediction is high for majority class. In most cases, we have situation in which we must precisely and accurately classify patients from minority class.38 Misclassification of patient with severe disease as negative means that we potentially endanger their health and be­cause of delayed diagnosis, disease can progress to life-threatening situation or death. Such situa­tion endangers only patient involved. In case that we classify patients, for example, who have very contagious disease, misclassification as negative means that such false negative patients will spread the disease and endanger other healthy people. Misclassification of healthy patients as positive results in further diagnostic tests and eventually leads to correct diagnosis. Unnecessary procedures result in greater stress for patient, higher expenses and bigger load for health system. Good classifica­tion algorithms therefore must have very high sen­sitivity and specificity. Cervical cancer is preventable disease.1 Artificial intelligence (AI) and deep learning methods are used for optimisation of screening, diagnostic and treatment procedures and are also present in the field of cervical cancer. Cervical cytology is of vi­tal importance in screening programmes. Mango et Laurie39 published article of computer assisted cervical cancer screening using neural networks in 1993. They used robotic arm for loading and un- FIGURE 5. Receiver operator characteristic (ROC) curve for multi-layer perceptron (MLP) performance on dataset with patients grouping in classes and synthetic minority over-sampling technique (SMOTE) correction for minority class where X axis represent 1- specificity (false positive rate) and Y axis represents sensitivity (true positive rate). Area under the ROC curve (AUC) = 0.802 which is well above classification with random guessing where AUC is 0.5. This Figure represents best performance model of MLP from our study. loading slides of PAP smears from storage contain­er, automated microscope and automated high-definition camera for imaging the slide. Multiple pictures from each slide were recorded. In the re­view station cytologists examined pictures. They used ANN to recognise different cells from images. After training neural network on sample pictures overall ANN sensitivity for all cytologic findings was 96% compared to 81% of that of cytologists.39 Sompawong et al. used ANN on images of liq-uid-based cytology (LBC) PAP smears to detect and analyse features of nucleus of the cervical cell and to screen normal and abnormal morphologi­cal features. In his study they achieved 57.8% mean average precision and 91.7% accuracy, sensitivity and specificity. This could help technicians and cy­tologists in their work.40 Holmstr et al. tested the use of ANN to ana­lyse PAP smears to detect pathological changes in rural Kenya where cervical cancer represent signif­icant health burden with high mortality rate. PAP smears were digitalised with portable scanner, up­loaded to cloud and analysed in regional medical centre. Sensitivity of ANN was 95.7% and specific­ity 84.7% compared to 100% sensitivity and 78.4% specificity of human examinator. AUC for ANN was 0.94. NPV was very high 99–100% particularly for HSIL. They concluded, that such model can be very helpful in cervical cancer screening in areas with low resources of health care professionals.41 Bao et al. 42 and Turic et al. 43 published study of AI assisted cytology in cancer screening programme in China. They digitalised LBC images of cervical smears and analysed them with AI. PAP smears were also analysed by cytologists. Agreement be­tween AI and manual reading was 94.7 with kappa 0.92 which is almost perfect agreement and AI as­sisted cytology was more sensitive for detection CIN2+ lesions than manual reading by 5,8% with slight reduction in specificity. Colposcopy is very important diagnostic proce­dure. Clinical experience is important for accurate colposcopic result.44 With the use of AI - deep con-volutional networks it is possible to analyse colpo­scopic images with higher accuracy than subjec­tive assessment by human. In his study Chandran and colleagues published 92,4% sensitivity, 96.2% specificity and kappa 0.88 which showed strong association between predicted and actual status of colposcopic changes.45 It is important, that wom­en referred for colposcopy are correctly selected to prevent overload in colposcopic clinics. Such overload with improper patients can result in miss diagnostics, unnecessary procedures and can be a threat for subsequent pregnancies.46 Karakitsos et al.47 used learning vector quantizer neural network to identify patients who need referral for colpos-copy. They analysed PAP smear using LBC and several markers of HR-HPV infection. All women had colposcopic directed biopsy performed by ex­perienced colposcopist and histologic result was golden standard to determine if colposcopy was necessary or not. They did not only identified more patients in need for immediate colposcopy with the use of AI but also reduced number of patients with clinical insignificant lesions compared to other methods. Combined sensitivity for training and testing set was 85.16% with specificity 98.01%, PPV 85.71%, NPV 97.92% and overall accuracy of 96.42%. ANN are very good in recognising patho­logical morphological features on images and all parameters are very good in all studies.47 Pouliakis et al. obtained similar results with study of classifi­cation and regression trees (CART) for the triage of women for referral to colposcopy and risk estima­tion for CIN. They used LBC and several markers of HR-HPV infection. This study is important be­cause they used missing data, which can be a prob­lem and most studies exclude them from analysis. CART has 83.28% sensitivity, 94.26% specificity, 79.04 PPV, 95.06 NPV and 100%valid cases while other methods have only 67.75%-96.25% valid cases depending on the method used. CART performed superiorly compared to cytology alone when used ASCUS+ threshold level (p < 0.0001).48 In our study we used MLP, which is back propagation artificial neural network on our data­set of patients, which had conisation surgery in University Gynaecologic clinic Maribor in years 1993–2005. As input layer, we used known risk fac­tors for development of cervical dysplasia and car­cinoma, High-risk dysplasia CIN2+ Yes/No as out­put layer. Risk factors are important and increase risk for development of disease but not all patients with risk factors develop disease.49 All patients with incomplete data were removed from analysis as are in majority of studies. Original dataset was imbalanced and patients without HSIL represent­ed minority class. To our knowledge this is first study with such settings. MLP performed worse on original dataset in comparison with baseline prediction. Such out­come can be expected in dataset where data are im­balanced.36 There are several methods to equalise imbalanced data. We can reduce the majority class by randomly selecting and removing instances from majority class with under-sampling method.34 When we balanced dataset with under-sampling 363 method, prediction did not improve and stayed be­low baseline. Reason for this may be in removing instances with important variables from training and/or testing set. We prepared dataset with un­der-sampling method few more times but with all settings, we could not achieve better performance. MLP correctly classified 57.64% cases which is in­ferior compared to baseline zeroR 59.39% and also kappa statistic 0.1113 showed no agreement be­tween real and predicted status. SMOTE and over-sampling methods improved performance of MLP.35 With over-sampling meth­od we multiplicate instances from minority class to match that of majority class. In this case is al­ways a chance, that we can find equal instances in training and testing set.34 SMOTE method uses k-nearest neighbour algorithm to create new syn­thetic instances which are all unique.35 In best per­formance model where baseline prediction ZeroR was 69,79% MLP correctly classified 84,19% cases and kappa statistic 0.64 showed moderate agree­ment between real and predicted status. In real clinical practice, many patients have multiple risk factors but never develop disease or, many with only a few became ill. It is possible that patients do not tell the truth about risk factors be­cause they are too intimate, they are ashamed or they do not remember. 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E-mail: Matthias.moll@meduniwien.ac.at 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 evaluate the development of treatment of primary high-risk prostate can­cer in regards to biochemical no evidence of disease (bNED), acute and late gastrointestinal (GI) and genitourinary (GU) side effects. Patients and methods. Primary high-risk prostate cancer patients treated between 1994 and 2016 were included. Applied doses ranged from 60 to 80 Gy, with a dose of 1.8 or 2 Gy per fraction. Techniques were either 3D conformal or intensity modulated radiotherapy and volumetric intensity modulated arc therapy. Results. 142 patients were treated with doses up to 70 Gy (median dose 66 Gy; 66 Gy group), 282 with doses be­tween 70 and 76 Gy (median dose 74 Gy; 74 Gy group), and 141 with doses >76 Gy (median dose 78 Gy; 78 Gy group). The median follow-up was 48 months. The bNED rates were 50% after 5 years and 44% after 9 years in the 66 Gy group; 65% and 54%, respectively, in the 74 Gy group; and 83% and 66%, respectively, in the 78 Gy group (p = 0.03 vs. 74 Gy and p < 0.0001 vs. 66 Gy). We found a higher rate of acute GI side effects in the 78 Gy group compared to the other groups, but not in maximum acute GU side effects and late maximum GI and GU effects. Conclusions. High-risk prostate cancer patients treated with doses of 78 Gy had significantly better bNED rates. Compared to the historical 66 Gy group, 50% more patients achieved bNED after a follow-up of 9 years. Key words: biochemical control; gastrointestinal toxicity; genitourinary toxicity; dose escalation Introduction Prostate cancer is the most common cancer in men in the US and central Europe, accounting for 20– 25% of all cases.1-3 One in five of these cases is di­agnosed with high-risk prostate cancer.4 However, prostate cancer is only responsible for cancer mor­tality rates of 6–10%3,5,6 and death from other rea­sons is much more likely after being diagnosed with prostate cancer7 in study conditions. In the last 25 years, many improvements have been introduced in the field of prostate cancer. In regards to diagnostics and staging, comprehen­sive PSA screening1,2, use of ultrasound-guided bi­opsy8, and computed tomography (CT)9, magnetic resonance imagining (MRI)10, and prostate-specific membrane antigen (PSMA) positron emission to­mography (PET)/CT11 have found their way into clinical routine, especially for high-risk prostate cancer. In addition, external beam radiotherapy (EBRT) has taken a leap forward within the last three dec­ades. Starting with 3D conventional radiotherapy12 and the use of lead blocks, and ending with volu­metric intensity modulated arc therapy (VMAT)13, new techniques allow dose escalation to 72 Gy with tolerable side effects14, and even to =74 Gy15-17, while also providing similar results as radical prostatecto-my (RPE)7,14 in localized prostate cancer. This dose escalation has significantly increased the curability of prostate cancer and is, therefore, in our opinion, the most important advancement in the field of prostate cancer radiotherapy over the last quarter century. Although a final conclusion has not yet been reached about the optimal duration18-20, evidence-based androgen deprivation therapy (ADT)2,19-22, especially in high-risk prostate cancer, has im­proved the outcome after radiotherapy. With similar oncological results between RPE and EBRT, the focus of patient decision-making shifts more and more to side effects. Therefore, the goal of our study was not only to evaluate the de­velopment of high-risk prostate cancer treatment over the last 25 years and the resulting biochemical no evidence of disease (bNED), but also to com­pare the gastrointestinal (GI) and genitourinary (GU) side effects of radiotherapy. Patients and methods The study protocol was approved by the ethical re­view board of our medical university according to local laws and regulations (EK Nr: 1291/2020). All patients included in this study were treated at our Department of Radiation Oncology between 1994 and 2016. The inclusion criteria were high-risk prostate cancer as defined by the NCCN clas­sification1 (PSA > 20 ng/ml, Gleason Score 8-10, or T stage = T3). The required staging was localized cancer without evidence of locoregional or distant metastases. The lymph nodes of all patients were staged using CT. Bone scintigraphy and ADT were performed at the discretion of the treating urolo­gist but were recommended for 3 years according to Bolla et al.23 Patients were primarily treated lo­cally with EBRT. The definition of the clinical target volume was determined using CT and, from 1997 onwards, MRI for planning. The total prescribed dose ranged from 60 Gy to 80 Gy, with a dose of 1.8 to 2 Gy per fraction. Pelvic lymph nodes were irradiated with a dose of 1.8 or 2 Gy per fraction up to 45–50.4 Gy. Treatment groups were based on the median dose; 58% of patients in the 66 Gy group received 66 Gy, with a maximum of < 70 Gy, 63% in the 74 Gy group received 74 Gy, with doses between 70 and 76 Gy and 90% in the 78 Gy group received 78 Gy, with doses > 76 Gy. The dose was prescribed to 95% of the planning target volume (PTV) accord­ing to ICRU report 62.24 Clinical target volumes (CTV) were defined as the prostate and the seminal vesicles. If pelvic lymph nodes were treated, the CTV also included the iliac vessels up to the aortic bifurcation. The safety margin around the clinical target volume was 5 mm in all directions with gold marker fiducials, 7 mm in all directions without fi­ducials for the 78 Gy group, and 10 mm in the 74 Gy group for the first 66 Gy and 5 mm dorsally for the last 8 Gy. For 66 Gy, the safety margin varied between 10 and 20 mm. Due to the broad time pe­riod of our study, safety margins varied over time. All patients received a rectal balloon25 as internal immobilization. The irradiation was performed in supine position via either a 3D conformal 4-field box up until January 2013 or intensity-modulated radiation therapy (IMRT) or the VMAT technique from then on. Follow-up was scheduled for 3 and 12 months after treatment, and then yearly thereafter. We de­fined bNED failure using the Phoenix criteria (na­dir + 2 ng/ml).26 Recent PSA values and late GI and GU side effects according to RTOG grading27 were compiled by the physician during each follow-up. Survival data were retrieved from the population census (Statistik Austria). Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, San Diego, USA) and R version 3.6.1 (2019-07-05) with RStudio 1.2.1335 (packages: survival version 2.44-1.1, sur­vminer version 0.4.6). A p-value < 0.05 was con­sidered significant. The bNED and survival rates were estimated using the Kaplan-Meier method. The resulting curves were compared using the log-rank test. Multivariable Cox regression models were created including the initial PSA value (log2 transformed); Gleason score = 6/Histograding 1, 7/Histograding 2, and 8-10/Histograding 3; applied dose in Gy; T stage 1a-c and 2a/X (reference), 2b/c and 3, or 4 according to the NCCN guidelines1; and pelvic irradiation. Side effects were analysed using the Mann-Whitney U test. Results Patient characteristics are provided in Table 1. As our observation period covers decades, irradiation techniques changed. Therefore, almost all patients in the 78 Gy group were treated using IMRT or VMAT. With the implementation of IMRT, we also introduced routine irradiation of the pelvic lymph nodes for high-risk prostate cancer patients. Thus, almost all patients in the 78 Gy group were also irradiated in the region of the pelvic lymph nodes. Exceptions were made for, for example, patients with earlier intestinal surgery. 367 TABLE 1. Patient characteristics Dose distribution in Gy Min 76 70.4 60 Max 8075 70 N with median dose 127 90% 178 63% 83 58% T category T1 43 30% 48 17% 21 15% T2 61 43% 108 38% 53 37% T3 36 26% 121 43% 60 42% T4 1 1% 5 2% 8 6% Gleason score Observed bNED rates for the 66 Gy group were =6 or histological grading 1 20 14% 94 31% 40 11% 50% after 5 years and 44% after 9 years. For the 74 7 or histological grading 2 2921%6620%52 4% Gy group, these values were 65% and 54%, respec- 8–10 or histol. grading 3 92 65% 118 42% 42 30% tively, and for the 78 Gy group, 83% and 66%, re­ X 0 0% 4 1% 8 6% spectively. A significant difference was found when comparing all groups at once (p < 0.0001; Figure 1). iPSA in ng/mlRegarding survival, we detected 7 disease- Median 15.7 20.6 21 specific deaths and 40 other causes of death in Technique the 66 Gy group, 11 and 44, respectively, in the 74 3D-conformal 11 8% 281 100% 142 100% Gy group, and 0 and 7, respectively, in the 78 Gy IMRT or VMAT 13092% 1 0% 0 0% group, respectively. Disease-specific survival rates after 5 years were Inclusion of LN 133 94% 105 37% 15 11% 95% in the 66 Gy group, 97% in the 74 Gy group, ADT 126 89% 259 92% 113 80%and 100% in the 78 Gy group (p = 0.11). The overall Mean in months 21 16 23 survival rates after 5 years were 74%, 82%, and 96% Follow-up in months (p = 0.0002), respectively. Min 323 The results of the multivariable analysis are dis­ Max 116 240 240 played in Table 2. The log2-transformed PSA value has to be interpreted as a twice as high initial PSA Median 4847 59 value leading to a 19% increased risk of bNED fail-Age in yearsure when comparing two patients. Min 49 51 53 Maximum acute GI and GU side effects are pro­ Max 84 86 93 vided in Table 3. Significantly more acute GI side Median 75 73 71 effects occurred in the 78 Gy group compared to the 74 Gy and 66 Gy groups (p < 0.001 and p = 0.02, Gold marker fiducials 53% 1% 0% respectively). No significant differences were ob- ADT = androgen deprivation therapy; iPSA = initial prostate specific antigen, IMRT = intensity served for acute GU side effects (p = 0.19 for 78 vs. modulated radiotherapy, T = Tumour extension; VMAT = volumetric intensity modulated arc therapy; LN = lymph nodes; X = no Gleason score or histological grading available 66 Gy, and p = 0.88 78 vs. 74 Gy). Table 4 provides the maximum late GI and GU side effects. No significant differences were found (GI side effects: p = 0.40 for 78 vs. 66 Gy, and p = We also performed a subgroup analysis and 0.74 for 78 vs. 66 Gy; GU side effects: p = 0.13 and compared the onset of late GU toxicity in patients 0.37, respectively). with irradiated lymph nodes. No significant differ- The onset of RTOG grade 2 or higher is shown ences were found when comparing all dose groups in Figure 2 for late GI side effects and Figure 3 for at once and 78 Gy with 74 Gy (p = 0.15 and 0.17, late GU side effects. No significant difference was respectively). found for late GI side effects (p = 0.96). For late GU One case of RTOG grade 4 acute GU toxicity was side effects, we detected a significant difference (p observed in a patient treated with 74 Gy without = 0.006). irradiation of the pelvic lymph nodes. That patient TABLE 2. Multivariate analysis of potential predictors of biochemical no evidence of disease (bNED) iPSA (log2 transformed) 1.193 1.058–1.345 0.004 Gleason = 6 or Histograding 1 reference Gleason 7 or Histograding 2 1.254 0.797–1.890 0.280 Gleason 8-10 or Histograding 3 1.687 1.132–2.515 0.010 Pelvic irradiation 0.783 0.540–1.135 0.196 T stage = 2a reference T stage 2b/c 1.466 0.950-2.262 0.084 T stage 3/4 1.517 1.054-2.181 0.025 Dose (Gy) 0.928 0.890-0.969 < 0.001 CI =confidence interval; HR = hazard ratio; iPSA = initial PSA; T stage low = T1a-c and 2a/X; intermediate = 2b/c; high = 3 or 4 TABLE 3. Maximum acute side effects 78 Gy 11% 50% 39% 1% 78 Gy 13% 54% 32% 1% 74 Gy 35% 35% 29% 1% 74 Gy 19% 45% 34% 1% 66 Gy 38% 22% 40% 0% 66 Gy 25% 44% 30% 1% GI = gastrointestinal; GU = genitourinary TABLE 4. Maximum late side effects 78 Gy 62% 21% 13% 4% 78 Gy 49% 23% 23% 5% 74 Gy 63% 22% 13% 1% 74 Gy 53% 21% 22% 3% 66 Gy 66% 22% 12% 0% 66 Gy 54% 29% 15% 2% GI = gastrointestinal; GU = genitourinary developed overflow incontinence and required surgery. No other grade 4 side effects were ob­served. Discussion The goal of our study was to evaluate the devel­opment of high-risk prostate cancer treatment over more than two decades in our department. As sur­gery and radiotherapy are comparable treatment alternatives, side effects are an important factor in choosing a therapy based on informed decision­making.1,2,7 FIGURE 3. Onset of RTOG grade = 2 genitourinary (GU) side effects after treatment over a follow-up period of 120 months. Starting in the late 1990s, several important studies regarding dose escalation were initiated. Dearnaley et al.16 showed a 10-year bNED rate of 55% in patients treated with 74 Gy compared to 43% after treatment with 64 Gy. Even higher rates were reported in the M.D. Anderson trial15 and by Peeters et al.17, who escalated the dose from 70 Gy or 68 Gy to 78 Gy. Peeters et al. reported a 5-year bNED rate of approximately 70%, and the M.D. Anderson trial reported 75% after 10 years in high-risk patients. Concerning biochemical control, we are able to reproduce the increased bNED rates by escalat­ing the dose as in the above studies.15-17 Our bNED rates of 54% and 66% after 9 years for 74 Gy and 78 Gy, respectively, are comparable to the 55% bNED for 74 Gy after 10 years16 and 70% after 5 years17 and to the 75% after 10 years for 78 Gy.15 Notably, our mean ADT duration was higher in the 78 Gy group than in the 74 Gy group, possibly shifting 369 the bNED rates additionally in favour of the 78 Gy group.19 Regarding our 78 Gy group, the bNED rate of 83% after 5 years is similar to the 78% de­scribed by Ozyigit et al.28 However, the mean ADT duration was 21 months in the 78 Gy group, which is lower than the suggested 24 to 36 months of ADT2 after Bolla et al.20 showed inferior survival af­ter only 6 months of ADT compared to 36 months. Evidence indicates that 18 months leads to no worse outcomes than 36 months18, possibly reduc­ing the recommended duration of ADT in the fu­ture. Regarding pelvic lymph node irradiation, we were able to detect a tendency of increased bNED rates in our multivariable analysis but no signifi­cance, leaving this question unanswered. Regarding follow-up and survival, as our de­partment has a large catchment area, it is difficult to gather reliable data concerning disease-specific and overall survival, as patients often die in an­other hospital not associated with our digital infra­structure. Therefore, with a median follow-up of 48 to 59 months, we decided to report only 5-year dis-ease-specific and overall survival rates. However, the similar follow-up does not harm the compara­bility between groups. That said, our data suggest great success of high-risk prostate cancer treatment, as 78 Gy provides a 50% increase in bNED rates after 9 years compared to 66 Gy. With absolute bNED rates in the 78 Gy group of 83% and 66% after 5 and 9 years, respec­tively, and a median age of 75 years in that treat­ment group, life-long curation of high-risk prostate cancer can be achieved in many cases. A direct comparison of side effects between our groups is hampered by the fact that our 78 Gy group was almost completely treated using VMAT with reduced margins. Therefore, as IMRT leads to low­er GI toxicity29, caution in making comparisons is advised. However, almost all patients in this group received pelvic lymph node irradiation, which in­creases toxicity30, though only by a small amount. Over time, we were able to detect significantly more late GU side effects with increased dose while seeing no difference in late GI side effects. This is possibly due to smaller safety margins, especially when gold markers were implanted, as well as broader use of the IMRT and VMAT technique. Maximum late GI and GU side effects were not significantly differ­ent when comparing the 78 Gy group to the other groups. However, when defining the onset of late GU side effects = grade 2 as an event, we detected asignificant difference. As the subgroup analysis in­cluding only patients with irradiated lymph nodes did not show a significant difference, the cause for this is more likely in the dose escalation to the pros-tatic urethra and the bottom of the bladder. A limitation of this study is its retrospective nature. In addition, due to the broad time period of the study, not only doses, but also irradiation technique and irradiated volume, varied over the 25-year observation period. Furthermore, our groups varied in regards to the percentage of pa­tients with lymph node irradiation. A strength of our study is that it is monocentric with systematic recording of GI/GU side effects. Thus, it provides consistent acquisition of side ef­fects. This is especially important because of the large difference in reported toxicity by patients and physicians.31 Moreover, we include a large col­lective of only one risk group for which we are able to present the development of daily routine with­out any bias due to study conditions. Over the last quarter century, long-term bNED rates of patients treated with EBRT have increased by 50%. If such success could be achieved by a new drug, it would be all over the news. Sadly, our dis­cipline fails to market this great success according­ly compared to developments in the areas of sur­gery and systemic treatments, especially with new, promising developments in high-risk prostate can­cer treatment, such as simultaneously integrated boosts, as displayed in the FLAME-trial.32 Conclusions Great progress has been made in the treatment of high-risk prostate cancer. Doses of 78 Gy result in significantly higher biochemical control rates and acceptable side effects. 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The influence of a rectal balloon tube as internal immobilization device on variations of volumes and dose-volume histograms during treatment course of conformal radiotherapy for prostate cancer. Int J Radiat Oncol Biol Phys 2002; 52: 91-100. doi: 10.1016/s0360-3016(01)01821-1 26. Roach M, Hanks G, Thames H, Schellhammer P, Shipley WU, Sokol GH, et al. Defining biochemical failure following radiotherapy with or with­out hormonal therapy in men with clinically localized prostate cancer: Recommendations of the RTOG-ASTRO Phoenix Consensus Conference. Int J Radiat Oncol Biol Phys 2006; 65: 965-74. doi: 10.1016/j.ijrobp.2006.04.029 27. Cox JD, Stetz J, Pajak TF. Toxicity criteria of the Radiation Therapy Oncology Group (RTOG) and the European Organization for Research and Treatment of Cancer (EORTC). Int J Radiat Oncol 1995; 31: 1341-6. doi: 10.1016/0360­3016(95)00060-C 28. Ozyigit G, Onal C, Igdem S, Alicikus ZA, Iribas A, Akin M, et al. Treatment outcomes of prostate cancer patients with Gleason score 8–10 treat­ed with definitive radiotherapy: TROD 09-001 multi-institutional study. Strahlentherapie und Onkol 2019; 195: 882-93. doi: 10.1007/s00066-019­01476-z 29. Yu T, Zhang Q, Zheng T, Shi H, Liu Y, Feng S, et al. The effectiveness of intensity modulated radiation therapy versus three-dimensional radiation therapy in prostate cancer: a meta-analysis of the literatures. PLoS One 2016; 11: 1-17. doi: 10.1371/journal.pone.0154499. 30. Dearnaley D, Griffin CL, Lewis R, Mayles P, Mayles H, Naismith OF, et al. Toxicity and patient-reported outcomes of a Phase 2 randomized trial of prostate and pelvic lymph node versus prostate only radiotherapy in advanced localised prostate cancer (PIVOTAL). Int J Radiat Oncol Biol Phys 2019; 103: 605-17. doi: 10.1016/j.ijrobp.2018.10.003 31. Rammant E, Ost P, Swimberghe M, Vanderstraeten B, Lumen N, Decaestecker K, et al. Patient- versus physician-reported outcomes in prostate cancer patients receiving hypofractionated radiotherapy within a randomized controlled trial. Strahlenther Onkol 2019; 195: 393-401. doi: 10.1007/s00066-018-1395-y 32. Kerkmeijer LGW, Groen VH, Pos FJ, Haustermans K, Monninkhof EM, Smeenk RJ, et al. Focal boost to the intraprostatic tumor in external beam radiotherapy for patients with localized prostate cancer: results from the FLAME randomized Phase III trial. J Clin Oncol 2021; 39: 787-96. doi: 10.1200/JCO.20.02873 371 research article Real-world outcomes, treatment patterns and T790M testing rates in non-small cell lung cancer patients treated with first-line first- or second-generation epidermal growth factor receptor tyrosine kinase inhibitors from the Slovenian cohort of the REFLECT study Nina Turnsek1, Rok Devjak1, Natalija Edelbaher2, Ilonka Osrajnik2, Mojca Unk1, Dusanka Vidovic2, Tina Jeric3, Urska Janzic4 1 Department of Medical Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Department of Pulmonary Diseases, University Medical Centre Maribor, Pohorje, Slovenia 3 AstraZeneca UK Limited, Branch office in Slovenia, Ljubljana, Slovenia 4 Department of Medical Oncology, University Clinic Golnik, Golnik, Slovenia Radiol Oncol 2022; 56(3): 371-379. Received 31 01 2022 Accepted 07 04 2022 Correspondence to: Nina Turnšek, M.D., Ph.D., Department of Medical Oncology, Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. E-mail: nturnsek@onko-i.si Disclosure. NT: honoraria (self) from MSD, Boehringer Ingelheim, Roche, Pfizer, AstraZeneca; honoraria (institution) from MSD, Boehringer Ingelheim, Roche, Pfizer, AstraZeneca; RD: consulting fees from Krka, Novartis, Roche; honoraria (self) from Abbott, Amgen, AstraZeneca, Boehringer Ingelheim, Merck Sharp & Dohme, Novartis, Roche, Sanofi Aventis, Takeda; honoraria (institution) from Abbott, Amgen, AstraZeneca, Boehringer Ingelheim, Merck Sharp & Dohme, Novartis, Roche, Sanofi Aventis, Takeda; support for attendings meetings or travel, Merck Sharp & Dohme, Roche; NE: honoraria (self) from AstraZeneca, Chiesi, Pfizer, Roche, Boehringer Ingelheim, Merck Sharp & Dohme, Amgen, Eli Lilly, Novartis, Sapio, GSK, Krka; honoraria (institution) from AstraZeneca, Chiesi, Pfizer, Roche, Boehringer Ingelheim, Sapio, Merck Sharp & Dohme, Amgen, Eli Lilly, Novartis, GSK, Krka; IO: Honoraria (self) from AstraZeneca, Chiesi, Pfizer, Roche, Boehringer Ingelheim, Merck Sharp & Dohme, Amgen, Eli Lilly, Novartis, Sapio, GSK; honoraria (institution) from AstraZeneca, Chiesi, Pfizer, Roche, Boehringer Ingelheim, Sapio, Merck Sharp & Dohme, Amgen, Eli Lilly, Novartis, GSK; MU: honoraria (self) from AstraZeneca, Boehringer Ingelheim, BMS, Roche, Eli Lily, MSD, Lek, Krka, Novartis, TEVA, Pfizer; DV: honoraria (self) from AstraZeneca, Chiesi, Pfizer, Roche, Boehringer Ingelheim, Merck Sharp & Dohme, Amgen, Eli Lilly, Novartis, Sapio, GSK; honoraria (institution): AstraZeneca, Chiesi, Pfizer, Roche, Boehringer Ingelheim, Sapio, Merck Sharp & Dohme, Amgen, Eli Lilly, Novartis, GSK; TJ: employee of AstraZeneca; UJ: honoraria (self) from AstraZeneca, Boehringer Ingelheim, MSD, Roche, Pfizer; honoraria (institution) from AstraZeneca, Boehringer Ingelheim, MSD, Roche, Pfizer, Novartis, BMS This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Background. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are effective treatments for EGFR mutation-positive (EGFRm) non-small cell lung cancer (NSCLC). However, routine clinical practice is different between countries/institutions. Patients and methods. The REFLECT study (NCT04031898) is a retrospective medical chart review that explored real-life treatment and outcomes of EGFRm NSCLC patients receiving first-line (1L) first-/second-generation (1G/2G) EGFR TKIs in 8 countries. This study included adult patients with documented advanced/metastatic EGFRm NSCLC with 1L 1G/2G EGFR TKIs initiated between Jan 2015 – Jun 2018. We reviewed data on clinical characteristics, treat­ments, EGFR/T790M testing patterns, and survival outcomes. Here, we report data from 120 medical charts in 3 study sites from Slovenia. Results. The Slovenian cohort (median age 70 years, 74% females) received 37% erlotinib, 32% afatinib, 31% gefitinib. At the time of data collection, 94 (78%) discontinuations of 1L TKI, and 89 (74%) progression events on 1L treatment were reported. Among patients progressing on 1L, 73 (82%) were tested for T790M mutation yielding 50 (68%) positive results, and 62 (85%) received 2L treatment. 82% of patients received osimertinib. Attrition rate between 1L and 2L was 10%. The median (95% CI) real-world progression free survival on 1L EGFR TKIs was 15.6 (12.6, 19.2) months; median overall survival (95% CI) was 28.9 (25.0, 34.3) months. Conclusions. This real-world study provides valuable information about 1G/2G EGFR TKIs treatment outcomes and attrition rates in Slovenian EGFRm NSCLC patients. The reduced attrition rate and improved survival outcomes empha­size the importance of 1L treatment decision. Key words: real-world study; non-small cell lung cancer; epidermal growth factor receptor; T790M testing, attrition Introduction Lung cancer remains a major public health chal­lenge worldwide, due to its diagnosis in advanced stages and high rate of mortality.1,2 The discovery of sensitizing mutations to epidermal growth fac­tor receptor (EGFR) has changed the treatment paradigm for lung cancer and has allowed for im­proved outcomes in patients with tumours harbor­ing such actionable mutations.3,4 Tyrosine kinase inhibitors (TKIs) targeting EGFR have proven effi­cacy for the treatment of EGFR-mutated (EGFRm) non-small cell lung cancer (NSCLC) and are the treatment of choice when this sensitizing muta­tion is found.1 Several generations of EGFR TKIs have been developed and have become gradually available – from the first-generation erlotinib and gefitinib, to second-generation afatinib and dac­omitinib, and third-generation osimertinib.5 Initial treatment recommendations for metastat­ic EGFRm NSCLC relied on first- and second-gen­eration (1G/2G) EGFR TKIs, but despite promising initial responses to these therapies, the disease in­evitably develops resistance and the progression requires treatment change.6 In approximately half of cases, the resistance is mediated by the EGFR secondary mutation T790M1,7, which is targeted by osimertinib in exon 20.8 Based on AURA3 study results, the standard of care is now testing for the T790M mutation in all patients whose disease has progressed on 1G/2G EGFR TKIs and treatment with osimertinib when the T790M resistance muta­tion is identified.1,7,8 Based on the FLAURA study results, which showed significant survival benefit with osimertinib versus comparator EGFR TKIs, osimertinib received approval by the European Medicines Agency in 2018 and became the pre­ferred first-line (1L) treatment option in advanced or metastatic EGFRm NSCLC.1,9 The implementation of testing and treatment recommendations in clinical practice is not always a simple process. Access to new methods of mo­lecular testing and novel therapies may be affected by lengthy local approvals and reimbursement processes, particularly in Central Eastern Europe (CEE).10,11 Among countries in this region, Slovenia benefits from having a long tradition in cancer care and one of the oldest population-based cancer reg­istries in Europe.12,13 The advantage of having im­plemented a national cancer registry consists in the objective evaluation of the burden of disease and trends over time and is in direct conjunction with adequate setting and resource allocation at institu­tional level.12,14 In Slovenia, the molecular testing of EGFRm is reflex and it was partially covered by pharmaceuti­cal companies until July 2020, when it became fully reimbursed by the public health system.15 However, the reimbursement of innovative anti-cancer thera­pies is still not optimal, and it exceeds 2 years.16 For example, the newly approved osimertinib as 1L therapy was reimbursed only in October 2020. In addition to patient and tumour characteristics, the treatment decisions in real-world (RW) practice are driven by clinical and cost-effectiveness, safety, and availability of treatments.17 As shown by the recent RW experience with 1L 1G/2G EGFR TKIs, the efficacy and safety of these agents proven in registration trials usually translate in real-life prac­tice; yet, the testing rates of the resistance muta­tion T790M are not optimal.18-26 To what extent the same findings apply in the Slovenian population is unknown. For this reason, Slovenia participated in this multinational medical chart review with the overarching goals of understanding the outcomes of EGFRm NSCLC patients initiated on 1L 1G/2G EGFR TKIs, treatment and T790M testing patterns, and attrition rates in various locations from Europe and Israel.27 Here we present the results of the Slovenian patients included in this study. Patients and methods Study design and participants The retrospective medical chart review “Real-world treatment patterns, clinical outcomes, and EGFR / T790M testing practices in EGFR-mutated advanced non–small cell lung cancer patients re­ceiving First-Line EGFR TKI Therapy“ (REFLECT, ClinicalTrials.gov: NCT04031898) was conducted in 7 European countries and Israel. Overall, medi­cal chart review and data collection were carried out in 49 clinical centres from May to December 2019, and 3 comprehensive cancer care centres in Slovenia participated in this study. In Slovenia, data abstraction was conducted from October to December 2019. The study design has been reported elsewhere.27 Briefly, eligible patients for this study were = 18 years of age with a confirmed diagnosis of locally advanced or metastatic EGFRm NSCLC who initi­ated 1L therapy with a 1G/2G EGFR TKI (afatin­ib, gefitinib or erlotinib) between January 1, 2015 and June 30, 2018. At the time of medical chart re­view, patients could have been alive or deceased, provided that the date of last follow-up or death was known. Patients were identified in the chrono­ 373 logical order of initiating 1L EGFR TKIs within the study period of interest (i.e., starting with January 1, 2015) and enrolled consecutively in the elec­tronic data collection form until the site’s quota was reached. Patients enrolled in a clinical trial for experimental treatments related to EGFRm NSCLC and patients receiving systemic treatment for their locally advanced or metastatic NSCLC prior to the 1L EGFR TKIs were excluded. In each participating country, the Institutional Review Boards (IRBs) or Ethics Committees (ECs) approved the protocol and study conduct. This medical chart review did not require informed, written consent from patients who were alive at the time of data collection unless the local IRBs/ECs required otherwise. In Slovenia, the Agency for Medicinal Products and Medical Devices (JAZMP) and the National Medical Ethics Committee (KME) approved the study, and an informed consent waiver was granted. Outcomes and definitions The primary outcome included progression events during treatment with 1L EGFR TKIs and time to progression, defined as time from initiation of 1L 1G/2G EGFR TKI therapy until the earliest sign of progression or death prior to start of a new thera­py line or start of a new therapy line. Progression was defined as radiological progression according to any imaging method, start of new therapy line, death, or other record indicative of progression, such as documented evaluation of the clinician. To differentiate this primary outcome from the progression free survival (PFS) reported in rand­omized clinical trials, we use the term “real-world PFS” (rwPFS). The secondary outcomes of this study included attrition rates and T790M testing rates among pa­tients progressing on 1L 1G/2G EGFR TKIs, types of treatments received in subsequent lines, inci­dence of central nervous system (CNS) metastases and leptomeningeal disease (LMD) and time to their development, overall survival (OS) from the start of 1L EGFR TKI therapy, and OS from first di­agnosis of CNS metastases and/or LMD to the date of death from any cause, with patients last known to be alive censored at the date of last available follow-up. Data collection Patient- and disease-specific data were obtained from the patient’s medical records and registered TABLE 1. Clinical characteristics at the time of initial NSCLC diagnosis Smoking history Current smoker 7 (6) Former smoker 33 (28) Never smoker 76 (63) Unknown 4 (3) ECOG performance status 0 28 (23) 1 62 (52) 2 22 (18) 3 6 (5) 4 1 (1) Unknown 1 (1) Stage at initial diagnosis Early stage (I-II) 13 (11) Limited regional (IIIA) 4 (3) Locally advanced (IIIB) 0 Metastatic (IV) 103 (86) Site of distant metastases Adrenal 12 (10) Bone 54 (45) Brain 33 (28) Liver 19 (16) Lung 60 (50) Lymph nodes 60 (50) Peritoneal 2 (2) Pleura 38 (32) Skin/soft tissue 3 (3) Other* 10 (8) * Other sites of distant metastases included: bone marrow, eye, kidney, spleen, and pericardium. ECOG=Eastern Cooperative Oncology Group by participating investigators in an electronic case report form. Each patient’s case was allocated an anonymized, encrypted identifier. Data were col­lected from the time of initial NSCLC diagnosis un­til death or the last available follow-up at the time of the patient’s inclusion in the study. Statistical analysis Sample size was based on the feasibility informa­tion received from each country, taking into ac­ FIGURE 1. (A) Kaplan-Meier curves for median real-world progression free survival on first-line (1L) epidermal growth factor (EGFR) tyrosine kinase inhibitors (TKIs) therapy. (B) Kaplan-Meier curves for median overall survival from start of 1L EGFR TKI therapy. Censored patients are indicated with a cross. CI = confidence interval; OS = overall survival; rwPFS = real-world progression-free survival count the volume of potentially eligible patients treated with 1L EGFR TKIs in the period of interest for the study. It was anticipated that each partici­pating physician would contribute with 5–30 case records to the study and each country would col­lect data from 50–180 medical records. This study had no formal statistical hypothesis; descriptive statistics were used to assess the demo­graphic and clinical characteristics, treatment pat­terns, and attrition rate. Kaplan-Meier estimators were used to describe median PFS and OS with 95% confidence interval (95% CIs). All analyses were performed in the full analysis set. The strati­fied OS analysis required > 20 number of events and a level of maturity of > 50%. The study was not powered for group comparisons. Results In total, 120 medical charts were included in this medical chart review from 3 study sites in Slovenia. The sites participating in the REFLECT study were also the only centres where lung cancer is being treated in Slovenia: 1 national cancer centre and 2 university hospitals. Demographic, clinical and EGFR mutation characteristics at baseline The median age (range) of patients was 70 (33–93) years, the majority were female (74%) and had nev­er smoked (63%). At the initial diagnosis of NSCLC, adenocarcinoma was the predominant histological subtype (99%), and the majority of patients (86%) had metastatic stage. Most patients (75%) had an Eastern Cooperative Oncology Group (ECOG) performance status of 0–1. The most frequent sites of metastases at the time of initial diagnosis were lung and lymph nodes (50% each), bone (45%), pleura (32%), and brain (28%) (Table 1). The me­dian (range) follow-up time was 24.3 (1.6–57.7) months. EGFR mutation status was determined from tis­sue biopsy (75%) or cytology specimens (25%). The specimen was extracted from the primary tumour in most cases (73%). In 2% of patients, the biopsy site was unknown. The most frequent EGFR muta­tion was exon 19 deletion (58%) followed by exon 21 L858R point mutation (28%); uncommon muta­tions (15%) included G719X, L861Q, S768I, T790M, and exon 20 insertions. First-line EGFR TKI therapy, progression and survival The 1L EGFR TKI therapies initiated during the pe­riod of interest for the study had a balanced distri­bution: 37% of patients received erlotinib, 32% ge­fitinib and 31% afatinib. At the time of data collec­tion, 94 patients (78%) discontinued 1L EGFR TKIs due to progression events or toxicities. Toxicities occurred in 9 cases (8%), with 5 of them (4%) not 375 starting any further treatment line. A number of 26 patients (22%) continued 1L treatment. In total, 89 progression events per protocol were reported: 47 radiological progression events (39%), 22 clinical progression events (18%), 16 deaths (13%) and 4 cases (3%) with start of a new therapy line without documented progression. Median (95% CI) rwPFS was 15.6 (12.6, 19.2) months (Figure 1A). Estimated probabilities for rwPFS (95% CI) at 12, 24 and 36 months were 63% (54%, 71%), 39% (30%, 48%) and 18%% (10%, 28%), respectively. Median (95% CI) OS from start of 1L EGFR TKI was 28.9 (25.0, 34.3) months (Figure 1B). Estimated probabilities for OS (95% CI) at 12, 24 and 36 months were 83% (75%, 89%), 61% (51%, 69%) and 36% (27%, 46%), respectively. T790M mutation testing and osimertinib treatment Of the 89 patients with progression events on 1L EGFR TKI therapy, 73 (82%) were tested for the T790M mutation at any time. Of the 73 patients tested for T790M mutation, the mutation was iden­tified in 50 patients (68%) and the test was nega­tive for 23 patients (32%). Of the 73 patients with disease progression on 1L EGFR TKIs who were tested for the T790M mutation, 62 (85%) received second-line (2L) treatment. In these patients, the 2L included osimertinib (84%), chemotherapy (15%) or targeted therapy (1%). Among the rest of the 16 patients with progres­sion on 1L EGFR TKI therapy who were not tested for T790M mutation, 4 patients (25%) received 2L treatment, with either chemotherapy or osimerti-nib (50% each). Testing for the T790M mutation was performed by using liquid biopsy in most cases (77%), fol­lowed by tissue biopsy (14%) or cytology specimen (9%). Most tests (97%) were based on Cobas® EGFR mutation test (Roche). The mean time (standard deviation) between the initiation of 1L EGFR TKIs and T790M testing was 14.4 (9.0) months. Second and subsequent therapy lines Of the 89 patients with disease progression on 1L EGFR TKIs, 66 (74%) initiated 2L treatment. In the Slovenian cohort of patients, 16 (13%) patients who discontinued 1L died before receiving 2L treat­ment, while 12 (10%) patients alive of the time of 1L discontinuation did not receive any further line. The 2L treatments included osimertinib (82%), chemotherapy (17%) and other targeted therapy FIGURE 2. Treatment patterns patients in with locally advanced or metastatic epidermal growth factor receptor mutated (EGFRm) non-small cell lung cancer (NSCLC) treated with first-line (1L) first-/second-generation (1G/2G) EGFR tyrosine kinase inhibitors (TKIs). Note that multiple treatments could have been administered at each line of treatment. * Targeted therapy besides afatinib, erlotinib, gefitinib and osimertinib (1L: not specified; 3L: crizotinib); 2L = second-line; 3L =third-line; IO = immuno-oncology (1%). At the time of data collection 18 patients (28%) were still receiving 2L treatment (Figure 2). Of the 48 patients discontinuing 2L, 19 (40%) received third-line (3L) treatment, which consisted of chemotherapy (53%), targeted therapy (26%), osimertinib (16%), or immuno-oncological therapy (5%) (Figure 2). At the end of data collection, 1 pa­tient (5%) was still on 3L treatment. Attrition rates on 1L, 2L, and 3L treatment are shown in Figure 3. Of the 18 patients discontinuing 3L, 5 (28%) re­ceived fourth-line (4L) treatment, which consisted of targeted therapy (60%) or osimertinib (40%). All patients discontinued 4L, with one case of death being registered, while the remaining 4 patients received the fifth-line of treatment (5L), which con­sisted of targeted therapy (50%), chemotherapy (25%), and osimertinib (25%) (Figure 2). All pa­tients discontinued 5L treatment. Central nervous system metastases The medical charts of 46 patients (38%) recorded the presence of central nervous system (CNS) me­tastases: in 33 cases (28%) these were present at the start of 1L EGFR TKIs, and in 13 cases (11%) the CNS metastases developed after the start of 1L treatment. In all cases (100%), an imaging examina­tion (computed tomography or magnetic resonance imaging scan) was used for the diagnosis of the CNS metastases, and in 2 cases (4%) tissue biopsy was also performed. Patients with CNS metastases FIGURE 3. Attrition rates at first-line (1L), second-line (2L) and third-line (3L) in patients with locally advanced or metastatic epidermal growth factor receptor mutated (EGFRm) non-small cell lung cancer (NSCLC). had a median age (range) of 67.5 (33.0–87.0) years and most (70%) were female. Treatments applied for CNS metastases included whole brain radiation therapy (63%), targeted therapy (63%), stereotactic radiosurgery (11%) and surgical resection (9%); in 4% of cases no treatment was provided. The median (range) time from the initiation of 1L EGFR TKIs to CNS metastases diagnosed dur­ing 1L or later lines treatment was 19.8 (7.7, 34.6) months. The median (95% CI) OS in patients with CNS metastases at the start of 1L EGFR TKIs was 24.3 (18.4, 41.5) months, with 24 events reported. In the group of patients with CNS metastases devel­oped during treatment, the number of events was too small to allow reporting of OS. Leptomeningeal disease Leptomeningeal disease (LMD) was reported in 4 patients: for 1 patient before and for 3 after the start of 1L EGFR TKI therapy. In all patients the di­agnosis relied on imaging examinations only. The median (range) time from the initiation of 1L EGFR TKIs to LMD diagnosed during treatment was 19.6 (4.5, 28.7) months. The number of events was too small to allow reporting of OS. Discussion This is the first comprehensive analysis of the outcomes, treatment patterns, and testing rates in metastatic EGFRm NSCLC patients who received 1L 1G/2G EGFR TKI therapy in Slovenia over 3.5 years, from 2015 to 2018. This is a nationally rep­resentative dataset for our clinical practice because all 3 large-volume centers from Slovenia that en­sure an integrated oncology care of lung cancer pa­tients, with national coverage, participated in the REFLECT study. Considering the real-life setting, the unselected population of patients with EGFRm NSCLC and the relatively equal distribution of 1G/2G EGFR TKIs (37% erlotinib, 32% afatinib, 31% gefitinib), our findings indicate positive treatment outcomes with 1L EGFR TKIs with a median rwPFS of 15.6 months. In the overall cohort from the REFLECT study (n=896), the median rwPFS was 13.0 (95% CI 12.3, 14.1) months and more patients received afatinib (45%).27 In clinical trials of 1G/2G EGFR TKI therapy, the acquired resistance developed af­ter a median of 9.2–14.7 months of targeted treat­ment.6 Other European RW studies that partially overlap with the limits of the data collection set for the REFLECT study, but with a different distribu­tion of the 1G/2G EGFR TKI therapies have shown PFS ranging from 7.6 to 11.0 months.18-20,22,26 The en­hanced rwPFS outcomes observed in the Slovenian cohort may be the result of more standardized and homogenous cancer care across centers, including established pathways for EGFR and T790M muta­tion testing, as well as effective control policies. Furthermore, in many cases the treatment may have continued beyond radiological progression, a common approach in patients with genetic ac­tionable alterations.1 Another observational study specifically exploring the continuation of EGFR TKIs beyond radiological progression showed that patients continued treatment without clinical de­terioration for a median of 5.1 months and had a median PFS of 15.3 months.28 The median OS from the start of the 1L 1G/2G EGFR TKI therapy was 28.9 months in the Slovenian cohort and 26.2 (95% CI 23.6, 28.4) months in the overall REFLECT study cohort.27 In general, the median OS reported in RW studies with 1L 1G/2G EGFR TKIs varies greatly, due to timelines set for the analysis, factors related to the healthcare system and access to EGFR TKIs, patient characteristics and data quality. Our findings are in line with those of other reports and are relevant for the period under study, when third-generation EGFR TKI osimerti-nib was not yet approved as 1L treatment.18,20,22,24,26 Following osimertinib 1L approval and subsequent market entries, more data on the effectiveness of osimertinib in various geographies are awaited. Upon progression on 1L 1G/2G EGFR TKIs, Slovenian national guidelines for the treatment of NSCLC, in accordance with European guide­lines, recommend testing for resistance mutation 377 T790M and, in patients with positive test results initiation of osimertinib.1,29 In this cohort, 82% of patients were tested for the presence of T790M up­on progression on 1L; the resistance mutation was identified in two-thirds (68%) of these patients, thereby providing an opportunity for treatment that is effective against disease with T790M muta­tion. Expressed at the level of the overall Slovenian cohort (42%), this positive rate of T790M is in line with other RW data from European cohorts.21,23,30,31 Additionally, in most patients (n=66) receiving 2L treatment in our cohort, post-progression treat­ment consisted of osimertinib (82%), preponder­antly in patients with the T790M mutation. These results support a unified approach to T790M test­ing and subsequent treatment at the national level, consistent with guidelines recommendations.1,29 In current local practice, when a clinical progression is suspected (even before radiologic progression), an active search with minimally invasive liquid bi­opsy for the presence of resistance T790M mutation is begun. This approach allows for early initiation of 2L systemic therapy with the goal of improving patient outcomes. Over the course of the lung cancer disease, many patients develop CNS metastases, which confer a poor prognosis and present additional treatment challenges.32 CNS metastases are often identified in patients with adenocarcinoma and molecular alterations, and their incidence is sig­nificantly correlated with the presence of EGFR-activating mutations.1,33,34 In this cohort, 38% of pa­tients had CNS metastases, most of them present at the time of diagnosis of the metastatic stage of lung cancer (28%). In a local retrospective analy­sis exploring the cumulative incidence of brain metastases in 629 patients with adenocarcinomas tested for EGFRm, those with the EGFR activating mutation had a longer time to CNS progression (25.9 vs. 11.9 months, p=0.002).35 In this REFLECT study cohort, the time to CNS progression was 19.8 months, with a median OS of 24.3 months in pa­tients with CNS metastases at the start of 1L 1G/2G EGFR TKI therapy. The difference may be due to advances in radiological techniques used to iden­tify CNS metastases, as well as practice changes. The dynamic landscape of technology, improved local control and reduced morbidity are reflected in the current management of CNS metastases as stereotactic radiosurgery has become the foremost treatment modality in patients with “limited” in-tracranial disease.36 REFLECT was primarily a study of attrition rates between treatment lines. In this cohort, of the 78 patients who started 1L 1G/2G EGFR TKIs and were alive at the time of treatment discontinuation, 12 (15%) did not receive 2L treatment. The trend of not receiving further treatment was sustained in subsequent lines, although the number of pa­tients alive at the time of treatment discontinua­tion progressively decreased. The rate of patients not receiving 2L treatment after the 1L EGFR TKIs was initially reported in clinical trials and it was approximately 35%, whereas in RW studies the rate varies more widely (10–62%).25,37,38 Although the REFLECT study did not explore the reasons why patients did not receive further treatment lines, data reported in the literature suggest vari­ous causes, including lack of genetic testing, low T790M mutation rate, poor performance status and even patient’s preference not to receive the next line of treatment, which would be chemotherapy in many cases.37 In our cohort we noticed that 18% patients progressing on 1L EGFR TKI were not tested for presence of T790M mutation. The ration­ale behind the lack of T790M testing at progression was not investigated, but such finding might be explained by rapid deterioration of clinical status followed by death on 1L EGFR TKI, presence of exon 20 insertion, which is associated with limited efficacy of common EGFR TKIs and unfavorable prognosis or poor performance status at the time of disease progression rendering patient ineligible for any further systemic therapy.37,39,40 Hence, the true T790M positivity rate and proportion of pa­tients eligible for targeted 2L may be different in real-life. Beyond possible differences in healthcare setting and availability of effective treatment op­tions, exploring locally in more depth the reasons behind attrition rates is crucial to further improve patient outcomes. The real-life character of this study confers both strengths and limitations. With a minimal set of in­clusion and exclusion criteria, and a representative dataset for Slovenia, this study allowed for build­ing RW evidence on 1L 1G/2G EGFR TKI therapy at the national level based on a 3.5-year data review (2015–2018). The fact that data collection relied en­tirely on information existing in patients’ records, which sometimes have insufficient or missing data, is a key limitation in such designs. Nevertheless, Slovenia benefited from the participation of all 3 of the country’s institutions in which lung cancer is treated. As a result, data availability was very good, with minimal cases of unknown information in patients’ histories. In general, secondary data collection may be subject to selection bias, includ­ing of sites and patients. To reduce site selection bi­ 378 as and potential patients’ spreading between sites, all 3 Slovenian comprehensive cancer centers were included in the study. To reduce patient selection bias, the ethics review package submitted has re­quested an informed consent waiver, which was granted by the National Ethics Committee. Thus, all medical records of eligible patients were consid­ered, irrespective of the vital status at the time of data collection and patients were enrolled consecu­tively in the electronic data collection form in the chronological order of starting the 1L 1G/2G EGFR TKI therapy. In contrast to clinical trials design, disease progression was not confirmed through a standardized, objective method, and the study definition reflects the RW situation (start of a new line of therapy or any other records indicative of progression, besides radiological tests). Finally, the study was not powered to compare the individual 1G/2G EGFR TKIs, and therefore outcomes could not be further characterized by molecule. Conclusions This real-world study, performed in a representa­tive dataset for Slovenian clinical practice, provides insights into the effectiveness of 1G/2G EGFR TKIs and T790M testing patterns in EGFRm NSCLC pa­tients receiving routine care. The survival outcomes and reduced attrition rate reported in this real-life setting from our country are encouraging. Newer 1L treatment options require follow-up studies to reflect the dynamic changes in clinical practice. Acknowledgments Medical writing support was provided by Ana Maria Iordan (MD, MSc) of MedInteractiv (Bucharest, Romania) and funded by AstraZeneca in accordance with Good Publication Practice (GPP3) guidelines. Statistical analyses were pro­vided by Planimeter Inc (Budapest, Hungary) and were funded by AstraZeneca. The REFLECT study (NCT04031898) was funded by AstraZeneca. 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New York: Academic Press; 1976. p. 45-74. research article Trends in treatment of childhood cancer and subsequent primary neoplasm risk Maja Cesen Mazic1, Raoul C. Reulen2, Janez Jazbec1, Lorna Zadravec Zaletel3 1 University Children’s Hospital Ljubljana, Ljubljana, Slovenia 2 Centre for Childhood Cancer Survivor Studies, Institute of Applied Health Research, Robert Aikten Building, University of Birmingham, Birmingham, United Kingdom 3 Institute of Oncology Ljubljana, Ljubljana, Slovenia Radiol Oncol 2022; 56(3): 380-389. Received 27 Feb 2022 Accepted 10 May 2022 Correspondence to: Maja Cesen Mazic, M.D., University Children’s Hospital Ljubljana, Bohoriceva ulica 20, SI-1000 Ljubljana, Slovenia. E-mail: maja.cesenmazic@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 the study was to investigate long-term risk and spectrum of subsequent neoplasm (SN) in childhood cancer survivors and to identify how trends in therapy influenced cumulative incidence of SN. Patients and methods. The population-based cohort comprises 3271 childhood cancer patients diagnosed in Slovenia aged = 18 years between 1st January 1961 and 31st December 2013 with a follow-up through 31st December 2018. Main outcome measures are standardised incidence ratios (SIRs), absolute excess risks (AERs), and cumulative incidence of SN. Results. After median follow-up time of 21.5 years for 5-year survivors, 230 patients experienced 273 SN, including 183 subsequent malignant neoplasm (SMN), 34 meningiomas and 56 nonmelanoma skin cancers. 10.5% patients received radiotherapy only, 31% chemotherapy only, 26.9% a combination of chemotherapy and radiotherapy and 16.1% surgery only. The overall SIR was almost 3 times more than expected (SIR 2.9), with survivors still at 2-fold increased risk after attained age 50 years. The observed cumulative incidence of SMN at 30-year after diagnosis was significantly lower for those diagnosed in 1960s, compared with the 1970s and the 1980s (P heterogeneity < 0.001). Despite re­duced use of radiotherapy over time, the difference in cumulative incidence for the first 15 years after diagnosis was not significant for patients treated before or after 1995 (p = 0.11). Conclusions. Risks of developing a SMN in this study are similar to other European population-based cohorts. The intensity of treatment peaked later and use of radiotherapy declined slower compared to high income countries, making continuous surveillance even more important in the future. Key words: population-based study, childhood cancer survivors, subsequent neoplasm Introduction Currently, ~ 80% of children with cancer are long-term survivors with possible late seque­lae.1,2 Treatment of childhood cancer depends on surgery, radiotherapy, and chemotherapy de­spite their potential toxicity. Late effects of cancer treatment are important causes of morbidity and mortality in survivors of childhood cancer.3 The burden of therapy was reduced, through clinical trials, in childhood cancers with good or excellent survival.4 However, for many children with cancer relapse of primary disease is still the leading cause of death.4 Death due to subsequent neoplasm (SN) is the most common non-relapse related event.5 Large population-based studies in childhood cancer survivors have been conducted in the Nordic countries and Britain with long and almost 381 complete follow-up.6,7 Cancer registries generally have limited or no information on treatment varia­bles. Multicentre studies conducted in Netherlands and US collect data through questionnaires or hos­pital registries, with up to one third of patients lost to follow up.8-14 However, detailed treatment data extracted from hospital registries provided impor­tant information about the risk factors for SN.8-14 Population based analysis of SN after treatment of childhood cancer in Slovenia was first published in 2004.15 The aims of present analysis were to as­sess long-term risk and spectrum of SN in Slovenia; identify how trends in therapy influenced cumula­tive incidence of SN. Patients and methods Cohort ascertainment and subsequent neoplasm ascertainment The study cohort comprises patients in Slovenia aged = 18 years with childhood cancer diagnosis between 1st January 1961 and 31st December 2013 and a follow-up through 31st December 2018. The cohort was ascertained through the popula­tion-based Cancer Registry of Slovenia (CRS). The registry combines data from University Children Hospital Ljubljana and Institute of Oncology Ljubljana, representing all institutions where child­hood cancer patients are treated and subjected to follow-up.16 Data coverage is estimated to be close to complete. CRS is linked to the Central popula­tion registry for information on vital status and causes of death. Childhood cancers were coded according to International Classification of Diseases for Oncology (ICD, 3rd version).17 For every patient basic treatment information (use of surgery, chem­otherapy, and radiation) and outcome (recurrence of primary cancer, subsequent neoplasms, cause of death) were reported. Subsequent neoplasms (SNs) for the entire co­hort were defined as a neoplasm on new location, which is not a direct spread or metastasis of the pri­mary neoplasm, or neoplasm on the same location with a different histological type (18.). SNs were validated through pathology reports or in some cases with other means through a clinical diagno­sis (e.g., meningioma). SN were classified as subse­quent malignant neoplasm (SMN), having ICD-O behaviour code of 3, meningioma, non-melanoma skin cancer (NMSC). As registration of neoplasms with ICD-O be-haviour code 2 is close to complete in CRS, these were included in SMN (in situ cervical carcinoma, in situ carcinoma of bladder, ductal in situ carcino­ma of breast and in situ melanoma). As registration of meningioma and NMSC is incomplete for gen­eral population, they were reported for our cohort but excluded from further statistical analysis. Statistical analysis Time at risk for SN was set at diagnosis of child­hood cancer (at latest 31st December 2013) and ended at the earliest occurrence of loss of follow up, death or study exit date (31st December 2018). During this period 3350 children were diagnosed with cancer, 79 patients were excluded from analy­sis after reviewing diagnosis (histology missing, benign tumours or Langerhans cell histiocytosis). Standardized incidence ratio (SIR) was calcu­lated as the observed divided by expected number of SMN. The expected number of SMNs were cal­culated by multiplying the number of person-years at risk in the cohort within specific sex, five-year age strata and single calendar year interval by cor­responding neoplasm incidence rates in Slovenian population extracted from CRS. Absolute excess risk (AER) was calculated as observed minus ex­pected number of SMN divided by person-years at risk and multiplied by 1000, unless otherwise spec­ified. AER is the number of extra SMN observed beyond that expected per 1000 persons per year. Meningioma and NMSC were excluded from SIR and AER calculations since their ascertainment is not complete in CRS. SIRs and AERs were stratified by sex, age at di­agnosis of primary cancer, attained age (age of the subjects at the study exit date, death or lost of fol­low up), primary neoplasm type, treatment period of childhood cancer, years from diagnosis of child­hood cancer and childhood cancer therapy. A mul­tivariable Poisson regression model was used to calculate relative risk (RR) and relative excess risk (RER) and analyse the potential simultaneous ef­fect of this explanatory factors on the SIR and AER. Relative risk represents ratio of SIRs adjusted for explanatory factors and RER as ratio of AERs ad­justed for explanatory factors (19.). Results relating to overall SIRs and AERs were only reported in text whenever there were at least 3 observed SMNs. For SIR, AER, RR and RER 95% confidence intervals were estimated (95% CI). The cumulative incidence for the first occur­rence of SN, SMN, NMSC and meningioma was computed as a function of time from childhood cancer diagnosis with death due to any other cause prior to developing SN considered as a competing event. Expected cumulative incidence for SMNs was calculated using the Ederer II method.20 Five-year relative survival following an SN was estimated using the Stata command strs.21All statis­tical analysis were conducted using Stata statistical software, version 17.0. All tests were 2-sided, with p value < 0.05 considered statistically significant. Results Cohort characteristics In this retrospective cohort study 3,271 childhood cancer patients accrued a total of 46,464 person-years of follow-up, with median follow-up time of 21.5 years (range, 5.25–57.8 years) for 5-year sur­vivors. The most common types of childhood can­cer were leukaemia (26.6%), CNS tumours (19.1%), Hodgkin’s lymphoma (9.6%) and non-Hodgkin’s lymphoma (8.5%) (Table 1). In total, 230 patients experienced 273 SN, includ­ing 183 SMN, 34 meningiomas and 56 NMSC. Of all individuals with an SN, 192 had one, 33 two and 5 three SNs. At the study exit date 53% (n = 1744) of patients were alive (Table 2). A total of 10.5% patients received radiotherapy only, 31% chemo­therapy only, 26.9% a combination of chemothera­py and radiotherapy and 16.1% surgery only. The proportion of patients treated with radiotherapy was highest for those diagnosed from 1970 to 1989 (> 50%) and decreased over time (29.7% > 2000). Simultaneously, the number of patients treated with chemotherapy increased from 49.1% in 1970s to 75.2% after year 2000. In the cohort 16% (n = 527) patients had no therapy, of whom 75% were diag­nosed before 1970 and majority died of childhood cancer. After 1980 there is approximately 4% of children with cancer undergoing observation only (e.g., low grade glioma, low risk neuroblastoma) (Table 3). The overall risk of developing an SNs and SMNs The estimated cumulative incidence of developing an SMN in the cohort was 2.8% at 20 years and in­creased to 5.7% at 30 years after childhood cancer diagnosis. The cumulative incidence of SNs and SMNs increased with attained age without pla­teauing (Figure 1). Cumulative incidence of developing an SMN at 40 years after childhood cancer diagnosis was sig­nificantly lower for patients having surgery only (P 383 TABLE 1. Characteristics of all individuals in study and number of subsequent neoplasms TABLE 2. Vital status by decade of childhood cancer diagnosis All survivors 3271 (100%) 183 (100%) 56 (100%) 34 (100%) Gender Male 1830 (55.9%) 77 (42.1%) 30 (54%) 14 (41%) Female 1441 (44.1%) 106 (57.9%) 26 (46%) 20 (59%) Leukaemia 870 (26.6%) 23 (12.6%) 11 (20%) 14 (41%) Hodgkin’s lymphoma 315 (9.6%) 51 (27.9%) 17 (30%) 2 (6%) Non-Hodgkin’s lymphoma 277 (8.5%) 16 (8.7%) 4 (7%) 2 (6%) Central nervous system tumour 625 (19.1%) 25 (13.7%) 12 (21%) 15 (44%) Neuroblastoma 124 (3.8%) 6 (3.3%) 1 (2%) 0 (0%) Retinoblastoma 60 (1.8%) 0 (0%) 0 (0%) 0 (0%) Wilms’ tumour 143 (4.4%) 9 (4.9%) 1 (2%) 1 (3%) Childhood cancer Bone tumour 199 (6.1%) 13 (7.1%) 0 (0%) 0 (0%) type Soft-tissue sarcoma 224 (6.8%) 14 (7.7%) 4 (7%) 0 (0%) Germ cell 168 (5.1%) 8 (4.4%) 3 (5%) 0 (0%) Liver 27 (0.8%) 1 (0.5%) 1 (2%) 0 (0%) Thyroid 86 (2.6%) 8 (4.4%) 1 (2%) 0 (0%) Nasopharyngeal carcinoma 13 (0.4%) 4 (2.2%) 1 (2%) 0 (0%) Melanoma 75 (2.3%) 2 (1.1%) 0 (0%) 0 (0%) Carcinoma 59 (1.8%) 3 (1.6%) 0 (0%) 0 (0%) Other 6 (0.2%) 0 (%) 0 (0%) 0 (0%) Mean 9.4 (6.0) 11.2 (5.7) 11.3(6.0) 7.0 (4.1) Age at childhood cancer diagnosis (years) 0–4 5–9 10–14 1065 (32.6%) 656 (20.1%) 690 (21.1%) 39 (21.3%) 34 (18.6%) 49 (26.8%) 13 (23%) 9 (16%) 13 (23%) 12 (35%) 15 (44%) 5 (15%) 15–19 860 (26.3%) 61 (33.3%) 21 (38%) 2 (6%) < 1970 528 (16.1%) 22 (12.0%) 4 (7%) 3 (9%) Decade of 1970–79 560 (17.1%) 50 (27.3%) 18 (32%) 11 (32%) diagnosis of childhood cancer 1980–89 651 (19.9%) 63 (34.4%) 22 (39%) 16 (47%) 1990–2000 679 (20.8%) 32 (17.5%) 9 (16%) 3 (9%) 2000–2018 853 (26.1%) 16 (8.7%) 3 (5%) 1 (3%) 0–19 1643 (50.2%) 31 (16.9%) 4 (7%) 2 (6%) 20–29 550 (16.8%) 33 (18.0%) 4 (7%) 7 (21%) Attained age (years) 30–39 40–49 494 (15.1%) 353 (10.8%) 59 (32.2%) 33 (18.0%) 20 (36%) 19 (34%) 19 (56%) 5 (15%) 50–59 151 (4.6%) 19 (10.4%) 7 (12%) 0 (0%) 60+ 80 (2.4%) 8 (4.4%) 2 (4%) 1 (3%) No therapy 527 (16.1%) 4 (2.2%) 9 (16%) 2 (6%) Surgery only 506 (15.5%) 24 (13.1%) 3 (5%) 0 (0%) Treatment of childhood cancer Chemotherapy 1014 (31.0%) 40 (21.9%) 17 (30%) 2 (6%) Radiotherapy 345 (10.5%) 44 (24.0%) 27 (48%) 11 (32%) Radiotherapy and chemotherapy 879 (26.9%) 71 (38.8%) 9 (16%) 19 (56%) < 1970 447 81 1970–1979 394 166 1980–1989 309 342 1990–2000 222 457 2000–2013 155 698 Total 1527 1744 heterogeneity < 0.001) (Figure 2). The observed cu­mulative incidence of SMN at 30 years after child­hood cancer diagnosis was significantly lower for those diagnosed in 1960s (P heterogeneity < 0.001) (Figure 3). Despite reduced use of radiotherapy af­ter 1995 difference in cumulative incidence of SMN for the first 15 years after diagnosis was not signifi­cant (Pepe Mori’s test for difference, p = 0.11). The risk of developing any SMN was almost 3-fold (SIR 2.9; 95% CI: 2.5–3.3) in the cohort com­pared with the general population, corresponding to an absolute excess risk of 2.6 per 1000 person-years (95% CI: 2.1–3.2.). Males appeared to be at higher risk than females in terms of the SIR (P het­erogeneity < 0.001). With increasing attained age, the SIR gradually decreased, and AER increased (Table 4), with survivors still at 2-fold increased risk after age 50 years (SIR = 2.0; 95% CI: 1.3–3.1). The risk of an SMN was highest among patients with nasopharyngeal carcinoma (SIR 7.5; 95% CI: 2.8–20.0), neuroblastoma (SIR 5.1; 95% CI: 2.3–11.3) and Hodgkin’s lymphoma (SIR 5.0; 95% CI: 3.8– 6.6) (Table 4). Elevated SIRs and AERs were evident for all childhood cancers, except for retinoblastomas, melanomas, and carcinomas. Not a single retino­blastoma patient in cohort developed SN. Five-year overall survival was estimated for children with different solid tumours through dec­ades to enable interpretation of results. Survival for patients with retinoblastoma was 50%, 56%, 88% and 100% for those diagnosed in 1960s, 1970s, 1980s and after 2000, respectively. Patients with central nervous system (CNS) tumours, sarcomas and Wilms tumours diagnosed in 1970s and 1990s experienced increase of five-year overall survival from 44% to 65%, 46% to 62% and 58% to 76%, re­spectively. Risk of specific subsequent primary neoplasms The most frequent SMNs were those of the thyroid (n = 37), genitourinary (n = 36; 15 cervical carci­noma in situ) and breast (n = 26) carcinoma. The majority of breast (n = 13) and thyroid (n = 19) car­cinoma occurred in Hodgkin’s lymphoma. Most genitourinary cancers occurred among bone and soft tissue sarcoma survivors (n = 12). Seventy per­cent of SN occurred in patients with CNS tumours, leukaemia, and lymphoma (Table 5). The greatest risk for SMN was observed for thy­roid, (SIR 21.6; 95% CI: 15.2–29.7), CNS (SIR 13.4; 95% CI: 7.9–21.2), soft tissue sarcoma (SIR 9.5; 95% CI: 3.1–22.2) and head and neck carcinoma (SIR 6.4; 95% CI: 2.9–12.1). SMNs of the thyroid (AER 76), breast (AER 41) and CNS (AER 36) contributed together almost 60% to the total AER. The distri­bution of observed excess SMN changed with at­tained age. In patients up to 40 years of age thyroid (AER 71), breast (AER 35), CNS tumours (AER 30) and leukaemia (AER 17) represent the major­ity of SMNs. After 40 years of age thyroid (AER 109), genitourinary (AER 87), breast (AER 84), CNS (AER 78) and respiratory (AER 75) tumours were responsible for 80% of the total AER (Table 6). Because follow up commenced at the time of childhood cancer diagnosis all subsequent leukae- TABLE 3. Treatment modality by decade of childhood cancer diagnosis No therapy 399 (75.6%) 38 (6.8%) 27 (4.2%) 30 (4.4%) 33 (3.9%) Surgery only 41 (7.8%) 102 (18.2%) 85 (13.1%) 116 (17.1%) 162 (19.0%) Chemotherapy only 30 (5.7%) 135 (24.1%) 174 (26.7%) 270 (39.8%) 405 (47.5%) Radiotherapy only 49 (9.3%) 145 (25.9%) 88 (13.5%) 46 (6.8%) 17 (2.0%) Radiotherapy and chemotherapy 9 (1.7%) 140 (25.0%) 277 (42.6%) 217 (32.0%) 236 (27.7%) Total 528 (100%) 560 (100%) 651 (100%) 679 (100%) 853 (100%) 385 TABLE 4. Standardized incidence ratios (SIR), absolute excess risks (AER), relative risk (RR) and relative excess risk (RER) for any subequent malignant neoplasm (SMN) Overall All combined 183 2.9 (2.5,3.3) – 2.6 (2.1,3.2) -­ Male 77 4.0 (3.2,5.0) 1.0 (ref.) 2.3 (1.7,3.1) 1.0 (ref.) Sex Female 106 2.4 (2.0,2.9) 0.7 (0.5-1.0) 2.9 (2.1,4.0) 1.4 (0.9-2.1) Pheterogeneity* <0.001 0.03 0.30 0.16 Age at diagnosis of childhood 0–4 5–9 10–14 39 34 49 3.9 (2.8,5.3) 3.3 (2.3,4.6) 3.1 (2.3,4.1) 1.0 (ref.) 0.9 (0.6-1.6) 0.9 (0.5-1.5) 2.0 (1.3,3.1) 2.4 (1.5,4.0) 3.2 (2.1,4.9) 1.0 (ref.) 0.8 (0.4-1.6) 0.7 (0.4-1.5) cancer 15–19 61 2.3 (1.8,2.9) 0.8 (0.5-1.3) 2.7 (1.7,4.3) 0.6 (0.3-1.2) (years) Ptrend* 0.01 0.3 0.23 0.13 < 1970 22 1.4 (1.0,2.2) 1.0 (ref.) 1.2 (0.3,4.8) 1.0 (ref.) Decade of 1970–1979 50 3.4 (2.6,4.5) 1.7 (1.0-3.0) 4.1 (2.8,6.1) 3.4 (1.0-11.9) diagnosis of childhood 1980–1989 1990–2000 63 32 4.0 (3.1,5.1) 2.7 (1.9,3.8) 1.7 (0.9-3.0) 1.1 (0.5-2.1) 3.8 (2.7,5.3) 1.8 (1.0,3.1) 3.5 (1.0-12.5) 2.6 (0.7-9.7) cancer 2000–2018 16 2.7 (1.7,4.4) 0.9 (0.4-2.0) 1.2 (0.5,2.5) 2.5 (0.6-10.4) Ptrend* 0.07 0.3 0.02 0.61 Era diagnosis < 1995 > = 1995 Pheterogeneity* 151 32 2.9 (2.5,3.4) 2.8 (2.0,4.0) 0.88 1.0 (ref.) 0.7 (0.5-1.1) 0.15 3.1 (2.4,3.9) 1.4 (0.8,2.5) 0.01 1.0 (ref.) 1.0 (0.6-1.8) 0.9 < 20 31 10.6 (7.4,15.0) 1.0 (ref.) 1.5 (1.0,2.2) 1.0 (ref.) 20–29 33 2.2 (1.6,3.1) 0.2 (0.1-0.4) 1.4 (0.7,2.5) 1.0 (0.5-2.0) Attained Age (yrs) 30–39 40–49 50–59 59 33 19 3.5 (2.7,4.5) 2.7 (1.9,3.8) 2.0 (1.3,3.1) 0.3 (0.2-0.5) 0.2 (0.1-0.4) 0.2 (0.1-0.4) 5.1 (3.6,7.3) 5.2 (3.0,9.0) 6.6 (2.7,16.3) 3.4 (1.9-6.1) 3.4 (1.5-7.4) 7.5 (2.8-20.4) 60+ 8 1.3 (0.6,2.6) 0.1 (0.1-0.4) 3.7 (0.2,90.4) 10.8 (1.6-74.0) Ptrend* <0.001 <0.001 <0.001 <0.001 Time since diagnosis of 0–9 10–19 20–29 38 37 51 6.0 (4.3,8.2) 2.6 (1.9,3.6) 3.1 (2.4,4.1) 1.0 (ref.) 0.4 (0.3-0.7) 0.4 (0.2-0.6) 1.6 (1.1,2.3) 1.7 (1.0,2.9) 4.3 (2.9,6.5) 1.0 (ref.) 1.1 (0.6-2.0) 2.5 (1.4-4.4) childhood 20–39 36 2.6 (1.9,3.6) 0.3 (0.2-0.6) 5.7 (3.4,9.6) 3.4 (1.7-6.9) cancer (years) 40+ Ptrend* 21 1.7 (1.1,2.5) <0.001 0.2 (0.1-0.4) <0.001 5.3 (1.8,15.5) <0.001 5.2 (2.1-12.4) <0.001 Leukaemia 23 2.7 (1.8,4.0) 1.0 (ref.) 1.6 (0.8,3.0) 1.0 (ref.) Hodgkin’s lymphoma 51 5.0 (3.8,6.6) 2.5 (1.4-4.2) 6.5 (4.6,9.1) 2.8 (1.4-5.7) non-Hodgkin’s lymphoma 16 4.3 (2.7,7.1) 1.7 (0.9-3.3) 3.3 (1.7,6.2) 1.3 (0.5-3.6) Central nervous system tumour 25 2.8 (1.9,4.2) 1.2 (0.7-2.2) 2.1 (1.1,3.8) 1.1 (0.5-2.4) Neuroblastoma 6 5.1 (2.3,11.3) 1.8 (0.7-4.5) 3.2 (1.2,8.6) 1.8 (0.6-5.5) Retinoblastoma 0 0 - 0 - Wilms Tumour 9 3.8 (2.0,7.3) 1.4 (0.6-3.1) 2.6 (1.1,6.3) 1.0 (0.3-3.3) Type of childhood cancer Bone sarcoma Soft-tissue sarcoma 13 14 2.7 (1.6,4.6) 2.6 (1.5,4.4) 1.6 (0.8-3.3) 1.2 (0.6-2.3) 3.3 (1.4,7.8) 2.3 (1.0,5.4) 1.8 (0.6-5.0) 1.1 (0.4-2.9) Germ-cell 8 1.6 (0.8,3.1) 0.9 (0.4-2.1) 1.0 (0.1,6.7) 0.6 (0.1-3.0) Liver 1 7.9 (1.1,56.1) 2.2 (0.3-16.3) 3.2 (0.3,30.4) 2.1 (0.2-19.2) Thyroid 8 2.0 (1.0,4.0) 1.2 (0.5-2.7) 2.3 (0.6,8.9) 0.9 (0.2-4.0) Nasopharyngeal carcinoma 4 7.5 (2.8,20.0) 4.2 (1.4-12.8) 12.6 (4.1,39.1) 6.9 (1.9-24.6) Melanoma 2 0.4 (0.1,1.8) 0.3 (0.1-1.4) 0.1 0 Carcinoma 3 1.1 (0.4,3.4) 0.8 (0.2-2.6) 0.2 0 Pheterogeneity* <0.001 <0.001 <0.001 <0.001 No therapy treatment of childhood 4 0.4 (0.2,1.1) 0.3 (0.1-0.8) 0 - Surgery only 24 1.7 (1.1,2.5) 1.0 (ref.) 1.0 (0.4,2.7) 1.0 (ref.) Treatment of childhood cancer Chemotherapy Radiotherapy 40 44 3.3 (2.4,4.4) 4.4 (3.3,5.9) 1.8 (1.1-3.1) 2.6 (1.6-4.3) 2.2 (1.4,3.4) 5.7 (3.9,8.4) 4.6 (1.0-20.9) 7.3 (1.6-33.5) Radio and chemotherapy 71 4.3 (3.4,5.4) 2.4 (1.5-3.9) 3.8 (2.8,5.2) 7.0 (1.6-30.8) Pheterogeneity* <0.001 <0.001 <0.001 <0.001 * = observed TABLE 5. Number and type of subsequent neoplasms (SN) by childhood cancer type Meningioma 14 2 2 15 0 0 1 0 0 0 0 0 0 0 0 34 NMSC 11 17 4 12 1 0 1 0 4 3 1 1 1 0 0 56 Breast (C50 D05) 1 14 0 0 0 0 1 1 2 2 0 1 1 1 2 26 CNS (C70-C72) 6 0 0 11 1 0 0 0 0 0 0 0 0 0 0 18 Digestive (C15-C26) 1 3 4 0 1 0 0 2 1 0 0 1 0 0 0 13 Genitourinary (C51-C68, D09, D06) 3 3 2 4 0 0 2 5 7 3 1 5 0 1 0 36 Leukaemia (C90-C93) 3 2 1 1 0 0 0 1 1 0 0 0 0 0 0 9 Lymphoma (C81-C85) 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 Melanoma (C43, D03) 0 0 1 1 0 0 1 0 0 1 0 0 0 0 0 4 Bone (C40-C41) 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 2 Head&Neck (C00-C14) 2 1 1 1 0 0 0 2 0 0 0 0 1 0 1 9 Other 1 3 1 1 0 0 2 0 0 0 0 1 0 0 0 9 Respiratory (C30-C39) 0 4 3 0 1 0 0 0 2 1 0 0 1 0 0 12 Soft-tissue (C49) 0 2 0 0 2 0 0 0 0 1 0 0 0 0 0 5 Thyroid (C73) 2 19 3 6 1 0 3 1 1 0 0 0 1 0 0 37 Total 48 70 22 52 7 0 11 13 18 11 2 9 5 2 3 273 ALL/AML = acute lymphoblastic/myelolastic leukaemia; CNS = central nervous system; HL = Hodgkin’s lymphoma; NHL = non-Hodgkin’s lymphoma; NMSC = non-melanoma skin cancer mias (SL) were reported. There were 9 cases of sub­sequent leukaemia. Compared to general popula­tion, childhood cancer survivors had a 6-fold over­all increased risk of leukaemia and an 8-fold (95% CI: 3.5–15.8) increased risk before age 40 (Table 6). Six patients developed SL within first 5 years of childhood cancer diagnosis. Only two out of nine patients survived the disease. Mortality and survival following SMN Fifty-nine patients out of 183 with SMN died within study period (1961 – 2018); 52 due to SMN and 7 of other causes. Five-year relative survival for patients with a SMN was 69 % (95% CI: 61–76). Most deaths were attributed to CNS tumours, SL, gastrointesti­nal, respiratory, head and neck carcinomas. Six pa­tients developed lethal SMNs outside the radiother­apy field or without radiotherapy, two of them were with a known cancer predisposition syndrome. Discussion Main findings Our study reports almost 3-fold increase in SMN among survivors of childhood cancer compared with general population. The SIRs reported by at­tained age are similar to other population-based studies, but somewhat lower than in non-popula­tion-based studies, particularly for those after age 40 years.22 For the first time we provided treatment data for our cohort. Intensive radiotherapy and chemo­therapy started in 1970s, with highest proportion of patients having radiotherapy in 1980s. Low in­tensity treatment in 1960s consequently resulted in only sporadic survival. Childhood cancer patients diagnosed in 1980s had the most intensive cancer treatment (56% radiotherapy and 70% chemo­therapy). In high income countries radiotherapy for childhood cancer was already declining from 75% before 1980 to 43% after 1980 and chemother­apy was given to more than 80% of patients after 1980.11,13,23 The maximum proportion of patients treated with radiotherapy and chemotherapy at any time was lower in our cohort and became com­parable only recently, with approximately 30% of children with cancer having radiotherapy and 75% chemotherapy.11,13 The previous study on our co­hort reported 48 SNs compared to 273 in current study, emphasizing need for continuous follow up despite lower risk.15 This is even more important since use of radiotherapy declined later. Namely, 387 TABLE 6. Standardized incidence ratios and absolute excess risks for specific subsequent malignant neoplasm overall and by attained age (0-39, 40+ years). Absolute excess risks are per 100,000 person-years All sites 183 63.2 2.9 (2.5,3.3) 257 (213,307) 123 34.9 3.5 (2.9,4.2) 216 (173,266) 60 28.2 2.1 (1.6,2.7) 545 (372,770) Head & Neck (C00-C14) 9 1.4 6.4 (2.9,12.1) 16 (7,33) 5 0.3 17.8 (5.8,41.5) 12 (4,28) 4 1.1 3.5 (1.0,9.0) 49 (10,147) Digestive organs (C15-C26) 13 5.5 2.4 (1.3,4.1) 16 (7,32) 7 1.0 6.8 (2.8,14.1) 15 (5,32) 6 4.4 1.4 (0.5,2.9) 27 (2,112) Respiratory organs (C30-C39) 12 2.9 4.2 (2.2,7.4) 20 (9,37) 5 0.2 23.3 (7.6,54.5) 12 (4,28) 7 2.6 2.7 (1.1,5.5) 75 (22,185) Bone (C40-C41) 2 0.4 5.1 (0.6,18.3) 3 (0,14) 2 0.3 5.8 (0.7,21.0) 4 (0,16) 0 0.0 0.0 (.,73.9) 0 Melanoma of skin (C43, D03) 4 4.2 1.0 (0.3,2.4) 0 3 2.2 1.4 (0.3,4.0) 2 (0,13) 1 2.0 0.5 (0.0,2.8) 0 Soft tissue (C49) 5 0.5 9.5 (3.1,22.2) 10 (3,23) 4 0.4 11.2 (3.1,28.7) 9 (2,24) 1 0.2 5.9 (0.1,32.9) 14 (0,90) Breast (C50, D05) 26 6.6 3.9 (2.6,5.7) 41 (25,65) 16 1.6 10.3 (5.9,16.7) 35 (20,59) 10 5.1 2.0 (0.9,3.6) 84 (27,198) Genitourinary (C51-C68, D09, D06) 36 32.2 1.1 (0.8,1.5) 8 (2,21) 22 23.3 0.9 (0.6,1.4) 0 14 8.9 1.6 (0.9,2.6) 87 (28,201) Central nervous system (C70-C72) 18 1.3 13.4 (7.9,21.2) 36 (21,57) 13 0.9 14.3 (7.6,24.4) 30 (15,52) 5 0.4 11.6 (3.8,27.0) 78 (24,190) Thyroid gland (C73) 37 1.7 21.6 (15.2,29.7) 76 (53,105) 30 1.1 27.3 (18.5,39.0) 71 (47,102) 7 0.6 11.3 (4.6,23.4) 109 (42,233) Lymphoma (C81-C85) 3 2.8 1.1 (0.2,3.2) 0 (0,9) 3 2.0 1.5 (0.3,4.4) 3 (0,14) 0 0.8 0.0 (.,4.6) 0 Leukemia (C90-C93) 9 1.5 6.0 (2.8,11.4) 16 (7,32) 8 1.0 8.0 (3.5,15.8) 17 (7,35) 1 0.5 2.0 (0.1,11.2) 9 (0,80) AER = absolute excess risks; Exp = expected; Obs = observed; SIR - Standardized incidence ratios; SMN - subequent malignant neoplasm prophylactic cranial radiotherapy (CRT) in pa­tients with acute lymphoblastic leukaemia (ALL) was gradually omitted in Slovenia after 1995 and for majority of patients after year 2002. Systematic review of randomized trials addressing prophy­lactic CRT in ALL patients conducted between the 1970s and 1990s showed that radiotherapy can generally be replaced by intrathecal therapy.24 There is substantial variation in percentage of ir­radiated patients between different childhood ALL treatment groups, however children from high in­come countries included in randomized trials had prophylactic CRT omitted a decade earlier then our patients.25 How different trends in treatment will correlate with cumulative incidence of SN in our cohort needs longer observation time. Risk of SMNs in retinoblastoma survivors In our cohort, no SNs were observed among retino­blastoma patients, which is likely related to the fact that less than 20% had external beam radiotherapy. In countries using external beam radiotherapy, five-year overall survival of retinoblastoma pa­tients diagnosed in 1966–1970 and 1996–2000 in­creased from 86% to 96%.26 In Slovenia only half of patients with retinoblastoma survived the disease in the 1960s and 1970s. With the use of chemother­apy and modern local therapies, survival increased to 88% in the 1980s and is 100% nowadays.27 The risk for SMN in nonhereditary retinoblastoma pa­tients treated with surgery only, is comparable to general population and only hereditary retinoblas­toma patients treated with radiotherapy have high­er risk for SMN.28 In our study only four long term survivors with probable hereditary retinoblastoma had radiotherapy. Risk of subsequent sarcomas In our study the risk of subsequent soft tissue (SIR 9.5, 95% CI: 3.1–22.2 vs. 15.7 95% CI: 14–17.6) and bone sarcomas (SIR 5.1, 95% CI: 0.6–18.3 vs. 21.65, 95% CI: 18.97–24.6) was significantly lower than in PanCareSurFup cohort, that comprises data from 12 European countries.29,30 The risk of subsequent soft tissue (SIR 12.1, 95% CI: 9.1–16) and bone sarco­ma (SIR 10.1, 95% CI: 7.2–14) is more comparable to Nordic population-based cohort study then British, where highest overall SIR for any specific subse­quent neoplasm was observed for subsequent bone neoplasms (SIR, 30.5; 95% CI, 24.9–37.3).6,7 Again, the greatest risk for subsequent primary sarcomas was observed in survivors of hereditary retino­blastoma treated with radiotherapy, but there are only few of such patients in our cohort. 29,30 Similar trends in survival are seen for other childhood can­cers contributing to subsequent sarcomas, namely patients with CNS tumours, sarcomas, and Wilms tumours.7,29,30 Survival of children diagnosed with CNS tumours, sarcomas, and Wilms tumours in 1970s and 1990s increased from 44% to 65%, 46% to 62% and 58% to 76%, respectively. As radio­therapy and chemotherapy are known risk factors for subsequent sarcomas, we might never see such an increase as in British and PanCareSurFup stud­ies, since less patients were exposed to high-dose, high-volume radiotherapy and chemotherapy at any time. 31,32 As the most intensive treatment in our cohort was implemented later, we might ex­pect increased risk with continued follow up. Risk of subsequent leukaemia (SL) In our cohort risk of SL is somewhat higher (SIR 6.0, 95% CI 2.8–11.4) compared to PanCareSurFup cohort (SIR 3.7, 95% CI 3.1–4.5).33 The risk of SL is estimated for five-year survivors in published studies, making comparison difficult.6,33,34 By stud­ying five-year survivors two thirds of SL in our co­hort would be lost, with majority of patients dead due to high mortality of SL. Determining risk of SL before patients became 5-year survivors may have implications for other studies despite low numbers in our cohort. Mortality and causes of death following SMNs Recurrence of primary cancer is still the leading cause of death in childhood cancer patients up to 15 years after diagnosis, afterwards death due to SMN takes the lead.5,35 Ten percent of patients that died of SMN had either no radiotherapy or SMN outside radiotherapy field. One third had known genetic cancer predisposition syndrome. Even these small numbers could stress the importance of surveillance for patients after radiotherapy or with known genetic predisposition syndromes.22 Clinical implications The fact that the risks of developing an SMN in this study are similar to other European population-based cohorts is important knowledge as it shows that follow-up guidelines for potential surveillance of SMNs developed for European survivors are relevant to the Slovenian childhood cancer sur­vivor population. Follow up provided by a dedi­cated physician applying current guidelines, as in Slovenia, is probably the best care possible for long-term survivors. Study limitations Strength of our study is almost complete follow up in population-based setting with little heterogene­ity in data collection and patient’s management. Potential limitations are the relatively small num­ber of SPNs and unavailable detailed treatment information not allowing for investigations into the risks by specific cumulative radiotherapy and chemotherapy doses. Conclusions Within this population-based study with nearly complete follow we observed almost 3-fold in­creased risk for SMN among childhood cancer survivors. What is new, are treatment data for our cohort, showing that most intensive treatment with radiotherapy and chemotherapy was implemented later in practice and radiotherapy also declined slower compared to high income countries. The evidence assembled in this study stresses the im­portance of continuous surveillance according to European guidelines and further studies to assess whether risk of SMNs in childhood cancers survi­vors in Slovenia will be different in the future. References 1. Steliarova-Foucher E, Stiller C, Kaatsch P, Berrino F, Coebergh JW, Lacour B, et al. Geographical patterns and time trends of cancer incidence and survival among children and adolescents in Europe since the 1970s (the ACCIS project): an epidemiological study. Lancet 2004; 364: 2097-105. doi: 10.1016/S0140-6736(04)17550-8 2. Gatta G, Botta L, Rossi S, Aareleid T, Bielska-Lasota M, Clavel J, et al. EUROCARE Working Group. Childhood cancer survival in Europe 1999-2007: results of EUROCARE-5-a population-based study. Lancet Oncol 2013; 15: 35-47. doi: 10.1016/S1470-2045(13)70548-5 3. Oeffinger KC, Mertens AC, Sklar CA, Kawashima T, Hudson MM, Meadows AT, et al; Childhood Cancer Survivor Study. Chronic health conditions in adult survivors of childhood cancer. N Engl J Med 2006; 355: 1572-82. doi: 10.1056/NEJMsa060185 4. Vassal G, Schrappe M, Pritchard-Jones K, Arnold F, Basset L, Biondi A, et al. The SIOPE strategic plan: A European cancer plan for children and adoles­cents. J Cancer Policy 2016; 8: 17-32. doi: 10.1016/j.jcpo.2016.03.007 5. Reulen RC, Winter DL, Frobisher C, Lancashire ER, Stiller CA, Jenney ME, et al; British Childhood Cancer Survivor Study Steering Group. Long-term cause-specific mortality among survivors of childhood cancer. JAMA 2010; 304: 172-9. doi: 10.1001/jama.2010.923 6. Olsen JH, Mler T, Anderson H, Langmark F, Sankila R, Tryggvadtír L, et al. Lifelong cancer incidence in 47,697 patients treated for childhood cancer in the Nordic countries. J Natl Cancer Inst 2009; 101: 806-13. doi: 10.1093/ jnci/djp104 7. Reulen RC, Frobisher C, Winter DL, Kelly J, Lancashire ER, Stiller CA, et al, British Childhood Cancer Survivor Study Steering Group. Long-term risks of subsequent primary neoplasms among survivors of childhood cancer. JAMA 2011; 305: 2311-19. doi: 10.1001/jama.2011.747 389 8. Neglia JP, Friedman DL, Yasui Y, Mertens AC, Hammond S, Stovall M, et al. Second malignant neoplasms in five-year survivors of childhood cancer: childhood cancer survivor study. J Natl Cancer Inst 2001; 93: 618-29. doi: 10.1093/jnci/93.8.618 9. Meadows AT, Friedman DL, Neglia JP, Mertens AC, Donaldson SS, Stovall M, et al. Second neoplasms in survivors of childhood cancer: findings from the Childhood Cancer Survivor Study cohort. J Clin Oncol 2009; 27: 2356-62. doi: 10.1200/JCO.2008.21.1920 10. Friedman DL, Whitton J, Leisenring W, Mertens AC, Hammond S, Stovall M, et al. Subsequent neoplasms in 5-year survivors of childhood cancer: the Childhood Cancer Survivor Study. J Natl Cancer Inst 2010; 102: 1083-95. doi: 10.1093/jnci/djq238 11. Turcotte LM, Liu Q, Yasui Y, Arnold MA, Hammond S, Howell RM, et al. Temporal trends in treatment and subsequent neoplasm risk among 5-year survivors of childhood cancer, 1970-2015. JAMA 2017; 317: 814-24. doi: 10.1001/jama.2017.0693 12. Turcotte LM, Whitton JA, Friedman DL, Hammond S, Armstrong GT, Leisenring W, et al. Risk of subsequent neoplasms during the fifth and sixth decades of life in the childhood cancer survivor study cohort. J Clin Oncol 2015; 33: 3568-75. doi: 10.1200/JCO.2015.60.9487 13. Teepen JC, van Leeuwen FE, Tissing WJ, van Dulmen-den Broeder E, van den Heuvel-Eibrink MM, van der Pal HJ, et al. DCOG LATER Study Group. Long­term risk of subsequent malignant neoplasms after treatment of childhood cancer in the DCOG LATER Study Cohort: role of chemotherapy. J Clin Oncol 2017; 35: 2288-98. doi: 10.1200/JCO.2016.71.6902 14. Teepen JC, Kremer LC, van der Heiden-van der Loo M, Tissing WJ, van der Pal HJ, van den Heuvel-Eibrink MM, et al. DCOG-LATER Study Group. Clinical characteristics and survival patterns of subsequent sarcoma, breast cancer, and melanoma after childhood cancer in the DCOG-LATER cohort. Cancer Causes Control 2019; 30: 909-22. doi: 10.1007/s10552-019-01204-z 15. Jazbec J, Ecimovic P, Jereb B. Second neoplasms after treatment of child­hood cancer in Slovenia. Pediatr Blood Cancer 2004; 42: 574-81. doi: 10.1002/pbc.20025 16. Jereb B. Model for long-term follow-up of survivors of childhood can­cer. Med Pediatr Oncol 2000; 34: 256-8. doi: 10.1002/(sici)1096­911x(200004)34:4<256::aid-mpo5>3.0.co;2-8 17. International Classification of Diseases for Oncology. Third Edition, First Revision. Fritz A, Percy C, Jack A, Shanmugaratnam K, Sobin L, Parkin DM, et al, editors. Geneva: World Health Organization; 2013. 18. Cancer registration: principles and methods. Jensen OM, Parkin DM, MacLennan R, Muir C, Skeet RG, editors. Lyon, France: World Health Organization, IARC Sci Publ; 1991. p. 95. 19. dos Santos Silva I. 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Systematic review and meta-analysis of randomized trials of central nervous system directed therapy for childhood acute lymphoblastic leukemia. Pediatr Blood Cancer 2013; 60: 185-95. doi: 10.1002/pbc.24228 25. Vora A, Andreano A, Pui CH, Hunger SP, Schrappe M, Moericke A, et al. Influence of cranial radiotherapy on outcome in children with acute lymph-oblastic leukemia treated with contemporary therapy. J Clin Oncol 2016; 34: 919-26. doi: 10.1200/JCO.2015.64.2850 26. Pritchard-Jones K. Childhood cancer in Britain: incidence, survival, and mor­tality. Br J Cancer 2007; 96: 1927. doi: 10.1038/sj.bjc.6603800 27. Abramson DH. Retinoblastoma in the 20th Century: past success and future challenges the Weisenfeld lecture. Invest Ophthalmol Vis Sci 2005; 46: 2684­91. doi: 10.1167/iovs.04-1462 28. Schonfeld SJ, Kleinerman RA, Abramson DH, Seddon JM, Tucker MA, Morton LM. Long-term risk of subsequent cancer incidence among he­reditary and nonhereditary retinoblastoma survivors. Br J Cancer 2021; 124: 1312-19. doi: 10.1038/s41416-020-01248-y 29. Bright CJ, Hawkins MM, Winter DL, Alessi D, Allodji RS, Bagnasco F, et al. PanCareSurFup Consortium. Risk of soft-tissue sarcoma among 69,460 five-year survivors of childhood cancer in Europe. J Natl Cancer Inst 2018; 110: 649–66. doi: 10.1093/jnci/djx235 30. Fidler MM, Reulen RC, Winter DL, Allodji RS, Bagnasco F, Bárdi E, et al. Risk of subsequent bone cancers among 69,460 five-year survivors of childhood and adolescent cancer in Europe. J Natl Cancer Inst 2018; 110: 183-94. doi: 10.1093/jnci/djx165. PMID: 28954302 31. Henderson TO, Whitton J, Stovall M, Mertens AC, Mitby P, Friedman D, et al. Secondary sarcomas in childhood cancer survivors: a report from the Childhood Cancer Survivor Study. J Natl Cancer Inst 2007; 99: 300-8. doi: 10.1093/jnci/djk052 32. Garwicz S, Anderson H, Olsen JH, Dlner H, Hertz H, Jonmundsson G, et al. Second malignant neoplasms after cancer in childhood and adolescence: a population-based case-control study in the 5 Nordic countries. Int J Cancer 2000; 88: 672-8. doi: 10.1002/1097-0215(20001115)88:4<672::aid-ijc24>3.0.co;2-n 33. Allodji RS, Hawkins MM, Bright CJ, Fidler-Benaoudia MM, Winter DL, Alessi D, et al. Risk of subsequent primary leukaemias among 69,460 five-year survivors of childhood cancer diagnosed from 1940 to 2008 in Europe: a cohort study within PanCareSurFup. Eur J Cancer 2019; 117: 71-83. doi: 10.1016/j.ejca.2019.05.013 34. Nottage K, Lanctot J, Li Z, Neglia JP, Bhatia S, Hammond S, et al. Long-term risk for subsequent leukemia after treatment for childhood cancer: a report from the Childhood Cancer Survivor Study. Blood 2011; 117: 6315-8. doi: 10.1182/blood-2011-02-335158 35. Bagnasco F, Caruso S, Andreano A, Valsecchi MG, Jankovic M, Biondi A, et al. OTR-AIEOP Registry. Late mortality and causes of death among 5-year survivors of childhood cancer diagnosed in the period 1960-1999 and regis­tered in the Italian Off-Therapy Registry. Eur J Cancer 2019; 110: 86-97. doi: 10.1016/j.ejca.2018.12.021 research article Abbreviated 13C-mixed triglyceride breath test for detection of pancreatic exocrine insufficiency performs equally as standard 5-hour test in patients after gastrectomy performed for gastric cancer Darko Siuka1, Kristina Kumer2, Borut Stabuc1,3, David Stubljar4, David Drobne1,3, Rado Jansa1,3 1 Department of Gastroenterology, University Medical Centre Ljubljana, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 2 Institute of Clinical Chemistry and Biochemistry, University Medical Centre Ljubljana, Ljubljana, Slovenia 3 Medical Faculty, University of Ljubljana, Ljubljana, Slovenia 4 In-Medico, Research and Development, Metlika, Slovenia Radiol Oncol 2022; 56(3): 390-397. Received 28 March 2022 Accepted 12 June 2022 Correspondence to: Darko Siuka, M.D., Department of Gastroenterology, Japljeva 2, 1000 Ljubljana, University Medical Centre Ljubljana, Ljubljana, Slovenia, E-mail: darko_siuka@yahoo.com, Phone: +386 41 958 682; and Assist. Prof. Rado Janša, M.D, Ph.D, Department of Gastroenterology, Japljeva 2, 1000 Ljubljana, University Medical Centre Ljubljana, Ljubljana, Slovenia, E-mail: rado.jansa@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. 13C-mixed triglyceride breath test (13C-MTGT) is a non-invasive test for the detection of moderate and severe pancreatic exocrine insufficiency (PEI), but it requires prolonged breath sampling. The aim of this study was to determine the diagnostic power of abbreviated 13C-MTGT in detecting PEI in patients after subtotal and total gastrec­tomy performed due to gastric cancer. Subjects and methods. This cross-sectional observational study included 3 groups of subjects; healthy controls, patients with subtotal and patients with total gastrectomy. Demographic and clinical data of patients were collected. Stool samples to determine faecal elastase (Fe-1) and chymotrypsin were collected and measured by ELISA. All sub­jects performed 5-hour 13C-MTGT breath test. The concentration and relative content of 13C in exhaled air was meas­ured by isotope ratio mass spectrometer (IRMS). PEI was confirmed as values of 13C-exhalation < 26.8% after 5 hours. Results. Overall, 65 participants were included into analysis, 22 having PEI (n = 11 after subtotal and n = 11 after total gastrectomy, both performed for gastric cancer). 13C-MTGT breath test showed difference in percent of exhaled 13C between PEI and non-PEI patients already after 60 minutes (p = 0.034). Receiver operating characteristic (ROC) curve analysis showed that cut-off value of 13.74% after 150 minutes is showing equivalent diagnostic power to the longer test with sensitivity and specificity both above 90% for the exclusion of PEI in patients after subtotal and/or total gastrectomy. Conclusions. In this study abbreviated 13C-MTGT test could be shortened from 5 to 2.5 hours without decrease in its diagnostic accuracy for detection of PEI in patients with subtotal or total gastrectomy performed for gastric cancer. This allows significant time savings in the diagnostics of PEI in this subgroup of patients. Key words: abbreviated 13C-mixed triglyceride breath test; pancreatic exocrine insufficiency; gastrectomy; faecal elastase; gastric cancer Introduction vation of pancreatic enzymes and/or bicarbonate in the gastrointestinal tract. This leads to maldiges-Pancreatic exocrine insufficiency (PEI) is a malab-tion, malabsorption, and malnutrition with conse­sorption syndrome caused by deficiency or inacti-quent higher morbidity, higher long-term mortal­ 391 ity, and reduced quality of life.1 We distinguish primary PEI, in which the mechanism is tied to the pancreas itself (various diseases of the pancre­atic parenchyma or pancreatic duct) and second­ary PEI, which is often unrecognized because the mechanisms of PEI are extrapancreatic.1,2 Secondary PEI also includes PEI in patients with altered anatomy due to gastric surgery (subtotal and total gastrectomy). Subtotal and total gas-trectomy are common surgical procedures, very often performed in patients with gastric tumours, mostly with resectable adenocarcinoma.3 Thus, be­sides chronic pancreatitis, diabetes mellitus (DM), coeliac disease, cystic fibrosis, inflammatory bowel disease and pancreatic cancer, the surgical proce­dures are the most common causes of PEI. In these diseases, there is a presence of decreased exocrine secretion.2,4 The pathophysiology of PEI post-gastrectomy is attributed to several factors. Firstly, the loss of the gastric reservoir leads to an absence of the ini­tial mechanical digestion of food and faster transit of osmotically active food particles into the small intestine. The less digested food particles are less potent stimulators of cholecystokinin (CCK), re­sulting in a decrease in endogenous stimulation to release digestive enzymes. Secondly, loss of duode­nal transit of food with reconstructive techniques bypassing the duodenum, such as Billroth-II (B2) and Roux-en Y (RY) reconstructions, leads to less CCK being released in response to the detection of chyme in the duodenum and upper jejunum. Thirdly, the release of pancreatic enzymes is not coordinated with the intestinal transit of food and inadequate mixing occurs (post-cibal asynchrony), leading to ineffective digestion. Finally, truncal vagotomy has been shown to reduce secretin-stimulated pancreatic trypsin and lipase secretion by 50-60%. This is attributed to the interruption of the cephalic phase of pancreatic digestion, during which sensory inputs are transmitted to the exo­crine pancreas through the vagus nerve.5 PEI has great impact on the quality of life, mor­bidity and mortality of these patients also in pa­tients after gastrectomy, especially if the condition remains unrecognised.6 Diagnostic tests for the direct pancreatic func­tion are gold standard as they are most sensi­tive for the detection of PEI but are invasive. The alternative are non-invasive tests such as faecal elastase-I (Fe-1), however with low sensitivity and specificity falls in diagnosing mild to moderate PEI.7,8 The specificity of this test seems to be even reduced after total and subtotal gastrectomy.8-10 Therefore, diagnostics of PEI patients after gastrec­tomy may be difficult, as faecal elastase (Fe-1), the standard of PEI detection, may be of normal range values. The sensitivity of these tests is low due to its extrapancreatic mechanism of PEI in these pa­tients.11 On the other hand, the 13C mixed triglycer­ide breath test (13C-MTGT) is a non-invasive assay that indirectly evaluates pancreatic lipase activity and pancreatic exocrine function. The disadvan­tage of this breath test is the long time required for the test (5-6 hours). This is time consuming for patients and for medical staff, so there is a great need for a test that will be of shorter duration and therefore more patient-friendly.1,2,11-14 Limited number of studies have tested patients with sus­pected PEI who underwent a long 6-hour or modi­fied, shortened 13C-MTGT breath test and showed some good results for shortening the test from 6 to 4 hours.15-17 As mentioned, the long time period of breath sampling and immobilization is a drawback and the period less than 6 hours led to decreased sensitivity of 13C-MTGT. Even though with shorter times (1-5 hours), sensitivity and specificity ranged from 73% to 85% and 83% to 100%, respectively.16 However, none of these studies were performed in a subgroup of patients after subtotal and total gastrectomy with test whether even shorter dura­tion of test can be performed. We hypothesised that in this specific subgroup of post-gastrectomy patients the 13C-MTGT breath test could be signifi­cantly shortened due to the changed anatomy after resection of the stomach. Since this has not been explored before we performed this prospective ob­servational study that specifically focused on pa­tients with resectable gastric cancer. Therefore, the purpose of our study is to deter­mine the diagnostic value of abbreviated 13C mixed triglyceride respiratory test (13C-MTGT) for the evaluation of PEI in patients after subtotal and to­tal gastrectomy performed for gastric cancer. The goal was to determine and confirm the equivalence of the sensitivity of the shortened and standard 13C-MTGT breath test in detecting PEI, while de­termining the optimal required cut-off time of the abbreviated 13C-MTGT breath test with preserved sensitivity and specificity of PEI determination. Subjects and methods Participants The study was designed as a cross-sectional, ob­servational study from a single centre, University Medical Centre of Ljubljana. The subjects were divided into three groups: healthy controls, sub­jects with subtotal gastrectomy and subjects with total gastrectomy. The group of healthy controls served as a base population for better estimation of diagnostic accuracy for abbreviated 13C-MTGT breath test. All subjects were adults, 18 years of age or older. Before voluntary participation all partici­pants needed to give the written informed consent. The study design and execution were approved by National Ethics Committee of Republic of Slovenia for Medical Ethics (registration number 140/02/10). Exclusion criteria for patients in both gastrecto-my groups, as well as in the group of healthy indi­viduals, were conditions often associated with PEI (type 1 and 2 diabetes, celiac disease, acute pancrea­titis, chronic pancreatitis, surgical conditions such as pancreatectomy, pancreatic head tumours, etc.). Since we studied the impact of changed anatomy af­ter gastric surgery on the performance of 13C-MTGT breath test we excluded patients with primary metastatic gastric cancer. The other exclusion crite­ria were other metastatic diseases, liver disease in which bile secretion is impaired, inability to partici­pate in research due to psychiatric illness, pregnan­cy, lactation and allergy to butter or chocolate (these patients are unable to performed the test). Healthy controls were without any clinical signs and symp­toms of gastric diseases, normal pancreatic elastase and without concomitant diseases. Upon inclusion, demographic and clinical data on patients (gender, age, associated diseases, regu­lar therapy, eating habits, smoking, coffee drinking, physical activity, weight, height, calculated body mass index [BMI]) were collected. Gastrointestinal symptoms and the degree of expression (diar­rhoea, steatorrhea, abdominal pain, weight loss, flatulence, anorexia, increased appetite) were also recorded. Blood analyses Moreover, peripheral blood for haemogram, amyl­ase, lipase, CRP, hepatogram (AST, ALT, total and direct bilirubin, AF and GGT, prothrombin time, INR), electrolytes, urea, creatinine, calcium, lipi­dogram was drawn from all subjects in the morn­ing after a 12-hour fast. Subjects submitted the first morning urine for amylase, lipase, glucose and stool to determine faecal elastase and faecal chymo-trypsin. Analysing pancreatic faecal elastase (FE-1) and chymotrypsin levels subjects needed to pass the stool samples. The concentrations were meas­ured by Enzyme-Linked ImmunoSorbent Assay method (ELISA) and detected photometrically. Performance of 13C-mixed triglyceride breath test (13C-MTGT) All subjects underwent our standardized 13C-MTGT breath test that took in total 5 hours (= 300 min). This is in line to already published pro­cedure and is standard of care in our institution.12 The first exhalation is done as a baseline, before eating test meal and then at 30-minute intervals. After the first exhalation the subject ate a test meal consisting of two slices of white bread, each weigh­ing 100 g, with one piece of bread accompanied by a piece of butter weighing 20 g. With another piece of bread, subjects consumed 30 g of choco­late spread (Nutella, Ferrero Rocher, Germany). The chocolate cream was mixed with 250 mg of a substrate of mixed triglycerides (1,3-distearyl-2-octanyl glycerol) labelled with the isotope 13C (Euriso-top, Saarbrken, Germany). After eating the bread, the subjects drank 200 mL of water. They were requested to sit throughout the whole exami­nation. The subjects blew exhaled air into test tubes at intervals of 30 minutes. Based on the difference in concentration between 13C and 12C, the relative isotope ratio (IRMS) mass spectrometer was used to determine the relative 13C content of exhaled CO2. The concentration of 13C in exhaled air and measurement of the ratio of 13C to 12C in exhaled CO2 were analysed. The measured isotope ratio in the samples were expressed as the relative differ­ence (d per mL in %) and subtracted from the base­line. The values of 13C exhalation were compared with the standard parameter of 13C of healthy vol­unteers. PEI was confirmed according to Keller et al. if patients had a ratio of 13C below 26.8%.17 Statistical analysis Statistical analyses were performed using the soft­ware package SPSS 21.0 (IBM Inc., Chicago, USA). Normally distributed variables were expressed as arithmetic mean and standard deviation, and One-Way ANOVA test was used for comparisons be­tween variables. In case of abnormally distributed variables the differences between continuous vari­ables were analysed by the nonparametric Mann-Whitney test. Differences between categorical vari­ables and calculation of the positive/negative pre­dictive values (PPV, NPV) were performed by us­ing the Pearson’s chi-square test. The optimal time of the 13C MTGT breath test was tested with a re­ceiver operating characteristic (ROC) analysis with area under curve (AUC), sensitivity and specificity. Respective cut-off values were performed for each 393 TABLE 1. Basic characteristics of subjects enrolled in the study Sex M/F 38 (58.5%)/27 (41.5%) 7/13 14/9 17/5 0.020 Age [years] 59.3 ± 16.9 43.4 ± 13.4 62.7 ± 13.8 70.2 ± 11.4 < 0.001 Age group < 0.001 18-40 y 8 (12.3%) 7 (35.0%) 1 (4.3%) 0 41-65 y 35 (53.8%) 13 (65.0%) 15 (65.3%) 7 (31.8%) > 65 let 22 (33.8%) 0 7 (30.4%) 15 (62.2%) Weight [kg] 67.9 ± 15.3 66.6 ± 16.0 70.0 ± 13.7 67.0 ± 16.8 0.738 Height [m] 1.7 ± 0.1 1.7 ± 0.1 1.7 ± 0.1 1.7 ± 0.1 0.576 BMI 23.2 ± 4.2 22.3 ± 4.3 24.1 ± 4.0 23.0 ± 4.2 0.333 PEI (< 26.8)# 22 (33.8%) 0 11 (47.8%) 11 (50.0%) 0.001 Pancreatic elastase (mcg/g) 349.8 ± 182.1 440.2 ± 126.3 312.2 ± 188.8 307.0 ± 195.2 0.026 Normal 50 (76.9%) 20 (100%) 16 (69.6%) 14 (63.6%) Mild decrease 7 (10.8%) 0 2 (8.7%) 5 (22.7%) Moderate decrease 2 (3.1%) 0 2 (8.7%) 0 Severe decrease 6 (9.2%) 0 3 (13.0%) 3 (13.6%) Chymotrypsin (U/g) 225.3 ± 160.8 301.7 ± 187.4 181.9 ± 130.3 201.3 ± 144.8 0.033 Normal 52 (80.0%) 20 (100%) 14 (60.9%) 18 (81.8%) PEI 13 (20.0%) 0 9 (39.1%) 4 (18.2%) Smoking 0.445 No 56 (86.2%) 19 (95.0%) 18 (78.3%) 19 (86.4%) < 15 cigarettes /day 4 (6.2%) 0 3 (13.0%) 1 (4.5%) > 15 cigarettes /day 5 (7.7%) 1 (5.0%) 2 (8.7%) 2 (9.1%) Coffee 0.136 No 23 (35.4%) 4 (20.0%) 7 (30.4%) 12 (54.5%) 1-2 cups /day 29 (44.6%) 10 (50.0%) 12 (52.2%) 7 (31.8%) > 2 cups /day 12 (18.5%) 6 (30.0%) 3 (13.0%) 3 (13.6%) Alcohol 0.108 No 47 (72.3%) 12 (60.0%) 15 (65.2%) 17 (77.3%) < 2 units/day 16 (24.6) 8 (40.0%) 5 (21.7%) 3 (13.6%) > 2 units/day 2 (3.1%) 0 0 2 (9.1%) Cholesterol (mmol/l) 4.5 ± 0.8 4.5 ± 0.7 4.4 ± 1.0 4.5 ± 0.7 0.736 HDL_cholesterol 1.5 ± 0.5 1.5 ± 0.4 1.4 ± 0.4 1.5 ± 0.6 0.637 LDL_cholesterol 2.5 ± 0.7 2.5 ± 0.6 2.5 ± 0.8 2.5 ± 0.7 0.921 Triglycerides 1.2 ± 0.8 0.9 ± 0.2 1.2 ± 0.9 1.5 ± 0.9 0.033 # According to the 13C-mixed triglyceride breath test; BMI = body mass index; F = female; M = male; PEI = pancreatic exocrine insufficiency timepoint of 13C measurements, and their diagnos-Results tic accuracy for PEI was calculated. Calculation of statistical power assumed a sample of 20 patients Overall, 65 participants were included into analy-in each group with 80% in order to confirm or re-sis and were then divided into 3 groups: (i) healthy fuse the hypothesis when assuming an error a be-controls (n = 20), (ii) group of patients with subtotal low 0.05. Thus, statistical significance for all tests resection (n = 23) and (iii) group of patients with was determined as p-value below 0.05. total gastrectomy (n = 22). Baseline characteristics TABLE 2. Differences between patient with positive and negative pancreatic exocrine insufficiency (PEI) determined by 13C-mixed triglyceride breath* Sex Male/Female 16/6 22/21 Age [years] 63.5 ± 12.8 57.2 ± 18.5 Weight [kg] 72.8 ± 15.8 65.5 ± 14.7 Height [m] 1.7 ± 0.1 1.7 ± 0.1 BMI 24.4 ± 4.0 22.5 ± 4.1 Smoking No 17 (77.3%) 39 (90.7%) < 15/day 3 (13.6%) (2.3%) 15/day 2 (9.1%) 3 (7.0%) Coffee No 9 (40.9%) 14 (32.6%) 1-2/day 12 (54.5%) 17 (39.5%) > 2/day 1 (4.5%) 11 (25.6%) Alcohol No 14 (63.6%) 33 (76.7%) < 2 units/day 7 (31.8%) 9 (20.9%) > 2 units/day 1 (4.5%) 1 (2.3%) Cholesterol 4.5 ± 0.8 4.4 ± 0.8 HDL_cholesterol 1.3 ± 0.4 1.5 ± 0.5 LDL_cholesterol 2.6 ± 0.8 2.5 ± 0.6 Triglycerides 1.6 ± 1.0 1.0 ± 0.6 0.095 0.157 0.069 0.447 0.082 0.181 0.107 0.527 0.5230.0910.6300.011 * Determined by 13C-mixed triglyceride breath test @300 min: PEI group 19.6 ± 9.5; non-PEI group 40.9 ± 10.4 (< 0.001); BMI = body mass index TABLE 3. Percent of exhaled 13C in patients with pancreatic exocrine insufficiency (PEI) and without PEI at respective timepoint of 13C measurement 30 min 0.29 ± 0.71 0.47 ± 0.62 0.233 60 min 0.97 ± 1.87 2.23 ± 2.00 0.034 90 min 2.05 ± 2.96 5.23 ± 3.24 < 0.001 120 min 3.53 ± 3.94 8.85 ± 4.05 < 0.001 150 min 8.39 ± 6.36 20.88 ± 5.96 < 0.001 180 min 10.12 ± 7.03 25.07 ± 6.63 < 0.001 210 min 12.21 ± 7.37 29.33 ± 7.29 < 0.001 240 min 14.50 ± 7.66 33.52 ± 8.06 < 0.001 270 min 17.10 ± 8.25 37.33 ± 9.09 < 0.001 300 min 17.72 ± 9.44 40.76 ± 10.43 < 0.001 *Determined by Mann-Whitney test of subject at enrolment are presented in Table 1. According to the baseline the groups of patients with gastrectomy differed in age and in gender ra­tio when compared to the healthy controls. Moreover, PEI were identified in patients after subtotal and total gastrectomy with 13C-MTGT breath test, FE-1, and faecal chymotrypsin, but not with 100% coverage. 13C-MTGT breath test after 300 minutes with a score < 26.8% was taken as a reference to determine PEI17,18, and at the end de­termined 22/45 (48,9%) patients with PEI. Approximately half of the patients in group of patients after subtotal and total gastrectomy had PEI confirmed. Meanwhile, no significant differ­ences in patients’ characteristics or habits were de­termined between patients with PEI and patients without PEI (Table 2). After performing 13C-MTGT breath test there has been an observation of difference in percent of exhaled 13C between the patients without PEI and patients with PEI soon after 2 measurements at 60 minutes (Table 3). In later timepoints, after 3rd measurement at 90 minutes the differences were increasing (p < 0.001) confirming that the abbrevi­ated of 13C-MTGT breath test to exclude PEI can be reliably used in patients after gastrectomy. The required test time was not shorter in pa­tients with total gastrectomy than in those after subtotal gastrectomy (Table 4) as no differences were observed in any of the timepoints. The optimal duration of the abbreviated 13C-MTGT breath test was determined by cut-off values and ROC analysis showing that shorten­ing the test to 150 minutes with the cut-off value of 13.74% is showing high sensitivity and specific­ity, both above 90% and high PPV and NPV for the exclusion of PEI in patients after subtotal and/ or total gastrectomy. The reliability of the abbrevi­ated 13C-MTGT breath test showed an equivalence of sensitivity in comparison to the standard, 5-hour 13C-MTGT breath test (Table 5, Figure 1) in this subgroup of patients. Discussion PEI in patients after subtotal and total gastrectomy should be detected as early as possible and with a highly sensitive test as early treatment of PEI im­proves outcome for these patients. Diagnostic of measuring fecal elastase in stool is most commonly performed, but it is important to supplement or even substitute it with more sensitive tests such as the 13C-MTGT breath test as it is crucial to treat PEI 395 TABLE 4. Percent of exhaled 13C according to diagnosis of in respective timepoint of C13 measurement 30 min 0.54 ± 0.74 0.32 ± 0.71 0.38 ± 0.51 0.700 0.936 60 min 2.28 ± 2.32 1.27 ± 1.88 1.92 ± 1.86 0.193 0.534 90 min 5.12 ± 3.76 3.15 ± 3.12 4.32 ± 3.45 0.202 0.495 120 min 8.63 ± 4.76 5.669 ± 4.54 7.04 ± 4.63 0.162 0.593 150 min 20.60 ± 7.09 13.96 ± 9.00 15.87 ± 8.11 0.056 0.714 180 min 24.91 ± 7.86 16.94 ± 10.74 18.77 ± 9.00 0.031 0.790 210 min 29.31 ± 8.61 20.45 ± 12.33 21.51 ± 9.55 0.023 0.937 240 min 33.67 ± 9.26 24.18 ± 13.81 24.14 ± 10.11 0.014 1.000 270 min 37.71 ± 9.82 28.04 ± 15.26 26.47 ± 10.63 0.010 1.000 300 min 41.14 ± 10.40 32.01 ± 17.21 28.51 ± 11.00 0.008 0.658 * Mann-Whitney test; ** Tukey Post-hoc analysis between subtotal and total resection groups early in these patients. However, the execution of this test takes significant time, generally 5-6 hours. The test is feasible as soon as patients are able to eat, and all of our patients have passed the test within 6 months of gastrectomy. All patients underwent a C13 breath test less than 6 months after gastrectomy. Previously it has been shown that the time required for a breath test in patients with fast food passage in the upper gas­trointestinal tract may be shorter16,17, so it is still necessary to determine the most optimal time re­quired for breath test in patients with gastrectomy where changed anatomy impacts test meal transi­tion time even more. The aim of the current study was to find the optimal duration and cut-off value for the 13C-MTGT breath test in secondary PEI at respective timepoints in two groups of patients, namely with subtotal and with total gastrectomy performed for gastric cancer. Because the time after gastrectomy in which patients underwent 13C-MTGT breath test after gastrectomy was too short, laboratory-detectable malnutrition had not yet occurred. If the 13C-MTGT breath test would be performed after a longer period of time, we would expect reduced laboratory nutritional mark­ers at the same time as the pathological 13C-MTGT breath test, which in principle would not affect the 13C-MTGT breath test itself. Other conditions that could simultaneously lead to PEI and consequently a change in the 13C-MTGT breath test could be ruled out by additional investi­gations (DM, etc). Our analysis showed that the diagnostic sensi­tivity and specificity of the abbreviated 13C-MTGT breath test for detection of PEI was equivalent to the sensitivity of the longer 5-hour 13C-MTGT breath TABLE 5. Cut-off values for prediction of non-pancreatic exocrine insufficiency (non-PEI) within respective timepoints in all subjects 60 min 1.16 0.662 0.522-0.801 0.034 67.4% 54.5% 46.2% 74.4% 90 min 3.79 0.776 0.654-0.898 < 0.001 67.4% 81.8% 56.3% 87.9% 120 min 4.71 0.845 0.738-0.952 < 0.001 88.4% 72.7% 76.2% 86.4% 150 min 13.74 0.929 0.853-1.000 < 0.001 93.0% 90.9% 87.0% 95.2% 180 min 16.19 0.938 0.869-1.000 < 0.001 93.0% 90.9% 87.0% 95.2% 210 min 18.64 0.948 0.888-1.000 < 0.001 95.3% 90.9% 90.9% 95.3% 240 min 20.85 0.962 0.902-1.000 < 0.001 97.7% 90.9% 95.2% 95.5% 270 min 25.71 0.962 0.891-1.000 < 0.001 97.7% 95.5% 95.5% 97.7% 300 min 26.95 0.962 0.889-1.000 < 0.001 100% 95.5% 100% 97.7% AUC = area under curve; NPV = negative predictive values; PPV = positive predictive values test in patients after subtotal and total gastrectomy. Two and half hours have been determined as opti­mal to detect patients with PEI with the cut-off val­ue of exhaled 13C at 13.74% after 2.5 hours (Table 5). Taking all that, there was also no difference in the required duration of the 13C-MTGT breath test when comparing patients after total gastrectomy and the duration of the test in patients after sub­total gastrectomy, even though the transit time in the upper gastrointestinal tract depends on the type of gastrectomy and affects the time required for a breath test. Since the number of patients was small, no significant differences occurred, but with a larger number, we would expect a shorter duration time of the 13C-MTGT breath test required when used in patients after total gastrectomy compared to subtotal gastrectomy patients. Our study was the first in this regard to perform the sub-analysis of patients with subtotal and total gastrectomy, and at the same time confirming shortening of the test.16,17 Keller et al. in 2011 indicated that shortening the test to less than 6 hours, decreases the sensitivity, how­ever, even with considerable shorter sampling, the sensitivity and specificity ranged from 73% to 85% and 83% to 100%, respectively, and reached even higher sensitivity and specificity rates in mild to moderate PEI (100% and 92%, respectively).17 They also showed that abbreviated version of the test was promising. Abbreviated test as such makes the examination more acceptable and comfortable in time, both for patients and medical staff. Our study contributes to the innovation in the diagnostics and treatment of patients with PEI after gastrectomy and improves their quality of life, as well as facili­tates the diagnostic process of these patients. This is important as in patients after gastrectomy, fecal elastase in the faeces may be preserved and the sen­sitivity of this test is expected to be low because the mechanism of PEI is extrapancreatic.18,19 Therefore, there is a presence of the risk that patients may be deprived of appropriate treatment with pancreatic enzyme replacement therapy (PERT).4 Because PEI has a strong impact on quality of life, additional tests such as 13C-MTGT breath test in addition to Fe-1 or 13C-MTGT breath test on its own detects more patients. Meanwhile the 13C-MTGT breath test is a non-invasive test that indirectly assesses pancreatic lipase activity and pancreatic exocrine, detects levels of undigested or digested products following gastric resection, so it is appropriate for patients after gastrectomy.11-14,18,20 Other trials have also tested patients with suspected PEI who under­went a modified shortened 13C-MTGT breath test, but some did not include patients after gastrecto­my.15-17 On the other hand, they demonstrated high sensitivity for severe PEI ranging 90% to 100% and specificity ranging 80% to 90%.4,17,21 Our data are though in concordance with these results. The current study showed that our abbrevi­ated version of the 13C-MTGT after 2.5 hours shows valuable diagnostic power. Both sensitivity and specificity exceeded 90% which represent a strong performance and importantly, after 2.5 hours might detect almost all patients with moderate or severe PEI. Similar performance was observed by Keller et al. in two of their previous research with similar cut-off value but rather after 4 hours.13,14 The findings of all studies suggested that for clini­cal purposes the testing period may be shortened. Keller et al. performed their study in 181 patients and revealed that cumulative 13C-exhalation with 13C-MTGT breath test over 4 hours had 88% sensi­tivity and 94% specificity for detection of PEI when compared to the standard 6-hour test.17 This reli­ability has been previously confirmed.16 Thus, the evaluation of pancreatic exocrine function using abbreviated test was in concordance with several studies showing that the abbreviated test might be of diagnostic value and used in clinical practice.16,17 However, due to different optimal timepoint more studies need to be evaluated confirming the exact time point for determine PEI as previous studies showed only minor abbreviation when compared to our data showing the abbreviation of more than 3 hours. Nevertheless, our findings are significant for bringing innovation into clinical practice and the study design encompassed two groups of patients 397 that might develop PEI, our analysis had limita­tions. The sample size that was used is relatively small. Out of 65 subjects, only 22 had PEI. Secondly, the patients were not split by the surgical proce­dure. They had undergone the Roux-en-Y method or the Billroth I (BI) and the types might be associ­ated with differences in fat digestive and absorp­tive function as BI reconstruction was proven to be superior to that after Roux-en-Y reconstruction.13 Furthermore, the basic characteristics of healthy controls did not match in age with patients’ group. Here it must be highlighted that the controls were used only as a baseline group stimulating statisti­cal power of PPV and NPV in subjects. The testing time was not compared to other treatment modali­ties and possible diet was not evaluated to impact the testing results. Finally, in our study gastric emptying was not performed so its influence on the duration of the test or on the rates of abbrevi­ated 13C-exhalation was not covered, despite that gastric emptying parameter was proven similar in patients and controls, and correction for these did not improve accuracy of 13C-MTGT.13 One of the limitations was also not regarding the possible con­comitant adjuvant/ neoadjuvant chemotherapy. The importance of our study is that it demon­strated the possibility of shortening the 13C-MTGT breath test for patients after total and subtotal gas-trectomy, which may make the test less time con­suming and therefore more patient-friendly and medical stuff-friendly and suitable for wider clini­cal use in these two groups of patients for the as­sessment of PEI. Conclusions The negative side of the breath test for detection of PEI is the long 5-hour procedure, which is burden­some for patients and medical personnel. Because of this there is a great clinical need for the test to be shortened. In the study we confirmed that this can be performed in a subgroup of patients with re-sected stomach due to gastric cancer. The abbrevi­ated 13C-MTGT breath test to 2.5 hours performed equally as the standard 5-hour test in this subgroup of patients. The results of study support the use of abbreviated test in patients after gastrectomy. Acknowledgement The study neither its execution did not receive any funding. References 1. Lr M, Oliver M, Frulloni L. Synopsis of recent guidelines on pancreatic exocrine insufficiency. Unit European Gastroenterol J 2013; 1: 79-83. doi: 10.1177/2050640613476500 2. Lr JM, Dominguez-Munoz E, Rosendahl J, Besselink M, Mayerle J, Lerch MM, et al. United European Gatroenterology evidence-based guidelines for the diagnosis and therapy of chronic pacreatitis (HaPanEU). United European Gastroenterol J 2017; 5: 153-99. doi: 10.1177/2050640616684695 3. Kanhere H, Goel R, Finlay B, Trochsler M, Maddern G. Radical gastrectomy: still the cornerstone of curative treatment for gastric cancer in the periop­erative chemotherapy era - a single institute experience over a decade. Int J Surg Oncol 2018; 2018: 9371492. doi: 10.1155/2018/9371492 4. Keller J, Hammer HF, Afolabi PR, Benninga M, Borrelli O, Dominguez-Munoz E, et al. European guideline on indications, performance and clinical impact of 13C-breath tests in adult and pediatric patients: an EAGEN, ESNM, and ESPGHAN consensus, supported by EPC. United European Gastroenterol J 2021; 9: 598-625. doi: 10.1002/ueg2.12099 5. Lee AHH, Ward SM. Pancreatic exocrine insufficiency after total gastrectomy – a systematic review. J Pancreas (Online) 2019; 20: 130-7. 6. Straatman J, Wiegel J, van der Wielen N, Jansma EP, Cuesta MA, van der Peet DL. Systematic review of exocrine pancreatic insufficiency after gas-trectomy for cancer. Dig Surg 2017; 34: 364-70. doi: 10.1159/000454958) 7. Vanga RR, Tansel A, Sidiq S, El-Serag HB, Othman MO. Diagnostic perfor­mance of measurement of fecal elastase-1 in detection of exocrine pancre­atic insufficiency: systematic review and meta-analysis. Clin Gastroenterol Hepatol 2018; 16: 1220-8. doi: 10.1016/j.cgh.2018.01.027 8. Guman MSS, van Olst N, Yaman ZG, Voermans RP, de Brauw ML, Nieuwdorp M, et al. Pancreatic exocrine insufficiency after bariatric surgery. Surg Obes Relat Dis 2021; 18: 445-52. doi: 10.1016/j.soard.2021.12.017 9. Bozzetti F, Ravera E, Cozzaglio L, Dossena G, Agradi E, Bonfanti G, et al. Comparison of nutritional status after total or subtotal gastrectomy. Nutrition 1990; 6: 371-5. PMID: 2134560 10. Yu W, Chung HY. Nutritional status after curative surgery in patients with gastric cancer: comparison of total versus subtotal gastrectomy. J Korean Surg Soc 2001; 60: 297-301. 11. Vujasinovic M, Valente R, Thorell A, Rutkowski W, Haas SL, Arnelo U, et al. Pancreatic exocrine insufficiency after bariatric surgery. Nutrients 2017; 9: 1241. doi: 10.3390/nu9111241 12. Walther B, Clementsson C, Vallgren S, Ihse I, Akesson B. Fat malab­sorption in patients before and after total gastrectomy, studied by the triolein breath test. Scand J Gastroenterol 1989; 24: 309-14. doi: 10.3109/00365528909093052 13. Borbély Y, Plebani A, Krl D, Ghisla S, Nett PC. Exocrine pancreatic insuf­ficiency after Roux-en-Y gastric bypass. Surg Obes Relat Dis 2016; 12: 790-4. doi: 10.1016/j.soard.2015.10.084 14. Nakamura H, Murakami Y, Morifuji M, Uemura K, Hayashidani Y, Sudo T, et al. Analysis of fat digestive and absorptive function after subtotal gastrec­tomy by a 13C-labeled mixed triglyceride breath test. Digestion 2009; 80: 98-103. doi: 10.1159/000220098 15. Takase M, Sumiyama. Y, Nagao J. Quantitative evaluation of reconstruction methods after gastrectomy using a new type of examination: digestion and absorption test with stable isotope 13 C-labeled lipid compound. Gastric Cancer 2003; 6: 134-41. doi: 10.1007/s10120-003-0238-3 16. Keller J, Brkel S, Jahr C, Layer P. A modified 13C-mixed triglyceride breath test detects moderate pancreatic exocrine insufficiency. Pancreas 2011; 40: 1201-5. doi: 10.1097/MPA.0b013e318220ad98 17. Keller J, Meier V, Wilfram KU, Layer P. Sensitivity and specificity of an abbre­viated (13)C-mixed triglyceride breath test for measurement of pancreatic exocrine function. United European Gastroenterol J 2014; 2: 288-94. doi: 10.1177/2050640614542496 18. Chaudhary A, Domínguez-Muz JE, Layer P, Lerch MM. Pancreatic exocrine insufficiency as a complication of gastrointestinal surgery and the impact of pancreatic enzyme replacement therapy. Dig Dis 2020; 38: 53-68. doi: 10.1159/000501675 19. Lindkvist B, Phillips ME, Domínguez-Muz JE. Clinical, anthropometric and laboratory nutritional markers of pancreatic exocrine insufficiency: preva­lence and diagnostic use. Pancreatology 2015; 15: 589-97. doi: 10.1016/j. pan.2015.07.001 20. González-Sánchez V, Amrani R, González V, Trigo C, PicA, deMadaria E. Diagnosis of exocrine pancreatic insufficiency in chronic pancreatitis: 13C-mixed triglyceride breath test versus fecal elastase. Pancreatology 2017; 17: 580-5. doi: 10.1016/j.pan.2017.03.002 21. Hoffmeister A, Mayerle J, Beglinger C, Bhler MW, Bufler P, Dathe K, et al. English language version of the S3-consensus guidelines on chronic pan-creatitis: definition, aetiology, diagnostic examinations, medical, endoscopic and surgical management of chronic pancreatitis. Z Gastroenterol 2015; 53: 1447-95. doi: 10.1055/s-0041-107379 study protocol Treatment of skin tumors with intratumoral interleukin 12 gene electrotransfer in the head and neck region: a first-in-human clinical trial protocol Ales Groselj1,2, Masa Bosnjak3,4, Tanja Jesenko2,3, Maja Cemazar3,5, Bostjan Markelc3,6, Primoz Strojan2,7, Gregor Sersa3,6 1 Department of Otorhinolaryngology and Cervicofacial Surgery, University Medical Centre Ljubljana, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Department of Experimental Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 4 Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia 5 Faculty of Health Sciences, University of Primorska, Izola, Slovenia 6 Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia 7 Department of Radiation Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia Radiol Oncol 2022; 56(3): 398-408. Received 1 April 2022 Accepted 7 April 2022 Ales Groselj and Masa Bosnjak have contributed equally to this work and share first authorship. Correspondence to: Prof. Gregor Serša, Ph.D., Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. E-mail: gsersa@onko-i.si Disclosure: No potential conflicts of interest were disclosed. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Background. Immune therapies are currently under intensive investigation providing in many cases excellent re­sponses in different tumors. Other possible approach for immunotherapy is a targeted intratumoral delivery of inter­leukin 12 (IL-12), a cytokine with anti-tumor effectiveness. Due to its immunomodulatory action, it can be used as an imunostimulating component to in situ vaccinating effect of local ablative therapies. We have developed a phIL12 plasmid devoid of antibiotic resistance marker with a transgene for human IL-12 p70 protein. The plasmid can be delivered intratumorally by gene electrotransfer (GET). Patients and methods. Here we present a first-in-human clinical trial protocol for phIL12 GET (ISRCTN15479959, ClinicalTrials NCT05077033). The study is aimed at evaluating the safety and tolerability of phIL12 GET in treatment of basal cell carcinomas in patients with operable tumors in the head and neck region. The study is designed as an ex­ploratory, dose escalating study with the aim to determine the safety and tolerability of the treatment and to identify the dose of plasmid phIL12 that is safe and elicits its biological activity. Conclusions. The results of this trail protocol will therefore provide the basis for the use of phIL12 GET as an adjuvant treatment to local ablative therapies, to potentially increase their local and elicit a systemic response. Key words: gene therapy; interleukin 12; gene electrotransfer; basal cell carcinoma; head and neck region Introduction Immune therapies are currently under intensive in­vestigation. Immune checkpoint inhibitors became standard of care for variety of tumor types, provid­ing in many cases excellent responses in patients with advanced or progressive disease, unsuitable for treatment with local therapies. However, there are still patients that are non-responders and the reasons for that are still not well understood.1 The other category of immune stimulators are cytokines. These have been extensively investigat­ 399 ed, predominantly in combined treatment schemes. The IL-12 is a soluble cytokine with anti-tumor ef­fectiveness. It stimulates adoptive and natural im­munity in the organism, predominantly through production of interferon gamma (IFN-.).2 Besides immunostimulatory effectiveness it also has anti-angiogenic mode of action.2 Treatment with recom­binant IL-12 has confirmed its anti-tumor effec­tiveness, but due to the high drug concentrations required to induce effect also severe toxicity were observed, which eventually lead to abrogation of IL-12 use.3 Gene therapy provides a new technical ap­proach for more localized delivery of the thera­peutic proteins to tissues, therefore reducing their eventual systemic toxicity. In most of the clinical trials, genes are delivered by viral vectors; how­ever, non-viral gene delivery systems like gene electrotransfer (GET) also provide a safe approach for naked plasmid DNA delivery to tumors.4,5 GET is based on electroporation that uses electric pulses to destabilize the cell membrane for deliv­ery of large and non-permeant molecules into the cells.6 The transport of plasmids through the cell membrane and into the nucleus for transcription is not well understood.7 However, there are several clinical studies providing evidence of feasibility, safety, and effectiveness of this non-viral gene de- livery.4,5,8,9 Studies on murine tumor models have shown that GET with plasmid DNA encoding interleu­ kin 12 (IL-12 GET) is especially successful in the treatment of skin tumors and their metastases.10 Furthermore, the safety and efficacy data of such treatment on skin melanoma metastases were al­ready published.4 In a human clinical study with IL-12 GET of melanoma metastases, local and also systemic effectiveness on the distant non-treated tumors were demonstrated. The study included 24 patients with skin metastatic melanoma.4 The plas­mid encoding human IL-12 under the control of the CMV promoter and with resistance to kanamycin, as the selection gene, was used. Gene therapy was performed three times on each tumor, resulting in promoted local clinical response of treated tu­mors and in systemic anti-tumor effect on distant non-treated nodules in 53% of patients. This treat­ment approach is currently being investigated in the treatment of melanoma, Merkel cell carcinoma, breast cancer8,9,11,12, and also in combination with immune checkpoint inhibitors.13,14 However, despite these encouraging results, IL­12 plasmid used in the described studies contain antibiotic resistance gene serving as a selection marker for the production of plasmid DNA. As the presence of antibiotic-resistance genes raises safety concerns, European Union regulatory re­quirements endorse the use of plasmids without the selective genes. Therefore, we developed the phIL12 plasmid and its clinical grade production process in our previous studies.15,16 In the proposed first-in-human study (EudraCT: 2021-000852-21, ISRCTN15479959, ClinicalTrials NCT05077033) we intend to study the safety and tolerability of phIL12 GET in treatment of basal cell carcinomas in patients with operable tumors in the head and neck region. The study is designed as an explorato­ry, dose escalating study with the aim to determine the safety and tolerability of the treatment and to identify the dose of plasmid phIL12 that is safe and elicits its biological activity. Trial rational Basal cell carcinoma accounts for 80% of all non-melanoma skin cancers and is the most common malignant tumor that occurs on the skin. It grows slowly and rarely metastasizes. However, basal cell carcinoma comprises heterogeneous histolog­ic variants, from highly biologically benevolent, well-limited and superficially growing tumors to aggressive, infiltratively growing or deeply inva­sive lesions. Due to complex anatomical condi­tions in some parts of the head (nose, orbital area, ears) combined with a possibly more aggressive form of growth, an inadequate first treatment may present a serious therapeutic problem. The basic therapeutic options in basal cell carcinoma are surgery, radiotherapy and electrochemotherapy, that result in comparable local control when early lesions are treated.17 Since the baseline tumor as­sessment could be inadequate and the disease un­derestimated, relapses at the treatment site are not uncommon. Repeated treatment, whether surgical or radiotherapeutic, is associated with reduced ef­ficacy and/or functional or cosmetic impairment in the treated area. Therefore, from clinical perspec­tive, searching for new therapeutic alternatives is of high importance. Recently, immunotherapy became important for the treatment of locally ad­vanced or metastatic basal cell carcinoma, with first approved immune checkpoint inhibitor cemi­plimab, which has set the stage for investigation of other immunotherapies.18 Basal cell carcinoma has the highest mutational burden compared to other skin cancers resulting in the activation of the local immune response that FIGURE 1. Clinical trial design. CTCAE v.5 = Common Terminology Criteria for Adverse Events version 5.0; CR = complete response; EORTC QLQ-C30 = European Organization for the Research and Treatment of Cancer Quality of Life Questionnaire C30; PR = partial response; PD = progressive disease; SD = stable disease prevents rapid tumor growth and metastasis.19 Indeed, clinical studies confirmed that electro-chemotherapy is more effective in basal cell car­cinoma than in other histological tumor types.20,21 This can be explained by a higher mutational load and an increased presence of tumor antigens or neoantigens that are triggered after electrochemo-therapy and are required for effective stimulation of immune system and elimination of tumor cells.22 In accordance with these findings we have de­signed the proposed clinical study. It is based on GET of plasmid DNA encoding IL 12 into the tu­mor, aiming to subsequently stimulate local im­mune response that would result in tumor eradi­cation. If this therapeutic approach with IL-12 will prove to be safe and effective, it will be the first “proof of principle” of its kind in clinic and a valu­able addition to the existing therapeutic armamen­tarium. However, in the case that our study will confirm the safety of the proposed therapy, but will not yield expected therapeutic response, the tumors will be treated with standard treatment, i.e. surgery. The prolonged interval to surgery should not affect prognosis of treatment outcome, due to slow course of basal cell carcinoma growth.23 Trial design This is a clinical, interventional, open label, single arm, Phase I trial for intratumoral phIL12 GET. It is intended to treat basal cell carcinomas in patients with operable tumors in the head and neck region. The aim of the study is to evaluate the safety and tolerability of phIL12 GET. The study is designed as an exploratory, dose escalating study with the aim to determine the dose that produces IL-12 expres­sion in the tumors with best biological activity, in­filtration of the immune cells and no toxicity. Study hypothesis: Intratumoral phIL12 GET is safe and tolerable in treatment of skin tumors (Figure 1). The aim will be achieved with i) controlled ap­plication of the treatment, ii) evaluation of trial and obtained clinical results, iii) presentation of the results by providing written and audio-visual ma­terial, participation at and organization of expert meetings and scientific meetings. In the study 3–6 patients per IL-12 dose level will be included; 3 doses, for a total estimated number of 9 patients (depending on the course of the study, from 3 and up to 18 patients will be included). The enrollment of the patients will be staggered: the waiting period will be 30 days after the treatment of previous patient, based on expected duration of acute and subacute toxicity. Consecutive cohorts of 3 to 6 patients will be treated with increasing doses of phIL12 at three dose levels (0.5 mg/ml, 1 mg/ml and 2 mg/ml) according to an adapted 3 + 3 design as described below. There will be no intra-patient dose escalation. The clinical study will be con­ducted in accordance with this protocol and GCP guidelines and in accordance with the regulations. The study is conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Institute of Oncology Ljubljana (protocol code ERIDEK-0086/2020, date of approval 25 November 2020). The study was also approved by the National Ethics Committee of the Republic of Slovenia (0120-524/2020-12) and the Agency for Medicinal Products and Medical Devices of the Republic of Slovenia. The study was registered in the ISRCTN (ISRCTN15479959) and Clinical Trials (NCT05077033) database. 401 TABLE 1. Primary objectives Assessment of the safety of intratumoral phIL12 GET Assessment of adverse events in accordance with the CTCAE v5 criteria From the beginning of therapy until the follow-up examination on day 30 after the treatment (day 1, 3, 8 and 31) Assessment of the tolerability of intratumoral phIL12 GET Assessment of patient reported outcome by the quality of life questionnaire EORTC QLQ-C30 A follow-up examination on day 0, 8 and 31 CTCAE = Common Terminology Criteria for Adverse Events; GET = gene electrotransfer TABLE 2. Secondary objectives Pharmacokinetics and Determination of serum levels of IL-12 cytokine. biodistribution. Determination of tumor IL-12 and IFN-. levels in Pharmacodynamics tumor biopsies. Determination of plasmid DNA in tumor biopsies. Evaluation of the appropriateness and execution of Feasibility of recruitment the treatment and follow up procedures. Measurement of pharmacodynamics data and Determination of selection of the phIL12 dose that produces IL-12 recommended dose for expression in the tumors with best biological activity, confirmatory studies infiltration of the immune cells and no toxicity. A follow-up examination according to clinical trial protocol (day 0, 3, 8 and 31). A follow-up examination according to clinical trial protocol (day 8 and 31). During recruitment, execution of the treatment and follow up. Based on all measurements during follow up. Objectives Primary and secondary objective of the study are presented in Table 1 and Table 2, respectively. Inclusion and exclusion criteria In the study, only patients with confirmed basal cell skin carcinoma of the head and neck will be in­cluded. Their inclusion eligibility will be assessed in accordance with the inclusion and exclusion cri­teria (Table 3). Trial procedures Inclusion of patients Patients with basal cell skin carcinoma of the head and neck, discussed at the multidisciplinary on­cology advisory team meeting where all patients with confirm malignancies in the head and neck are presented and discussed, will be reviewed and informed of their eligibility to participate in the SmartGeneH&N clinical study. They will be pre­sented with all information (in written and oral form) regarding the study and other possible treat­ment options. Prior to inclusion, they will sign an informed consent form. The patients included in the study will be the one that meet all the inclu­sion criteria and will not have any exclusion crite­ria (Table 3). The enrollment of the patients will be staggered. The waiting period between each individual will be 30 days after completion of therapy, based on expected duration of acute and subacute toxicity. Consecutive cohorts of 3 to 6 patients will be treated with increasing doses of phIL12 at three dose levels (0.5 mg/mL, 1 mg/mL and 2 mg/mL) according to the adapted 3 + 3 design (see below). There will be no intra-patient dose escalation. Patients will be assigned to a treatment cohort and will receive phIL12 at a single dose-level. If no dose limiting toxicity (DLTs) are observed during the therapy, until day 30 after the treatment (day 31), in the first 3 patients treated at a dose level, 3 additional patients will be enrolled and treated at the next higher dose level. Doses will be escalated until = 2 of 3 patients (67–100%) in a dose cohort have at least 1 DLT. If exactly 1 of the first 3 patients treated at a dose level experiences at least one DLT, 3 additional patients will be enrolled and treated at that dose level. If none of the additional 3 patients (i.e., 1 of 6 [16.7%] total patients in this dose cohort) experiences at least 1 DLT, dose esca­lation may proceed. If any patient of the additional TABLE 3. Inclusion and exclusion criteria Histologically or cytologically confirmed, previously untreated cutaneous basal cell carcinoma located in the head and neck region Solitary tumors, with largest diameter up to 3 cm, in the region where curative (R0) surgery is feasible Age 18-years or older Life expectancy > 3 months Physical performance in accordance with the Karnofsky scale = 70 or < 2 in accordance with World Health Organization (WHO) scale The patient must be capable of understanding the treatment procedure and possible adverse events, which may arise during treatment The patient must be capable of signing the informed consent to participate in the clinical study (voluntary and conscientious consent after education) Prior to inclusion in the trial, the patient must be presented at a multidisciplinary advisory team meeting Other malignancy at the time of inclusion Lesions not suitable for treatment with GET (invasion into the bone, infiltration of large vessels) A life-threatening infection and/or severe heart failure and/or liver failure and/or other life-threatening systemic diseases Significantly reduced lung function, which requires the determination of DLCO. Patients should not be treated if DLCO is abnormal Treatment with immunosuppressive drugs, steroids and other drugs that would affect poor wound healing Age under 18-years Major disruptions in the coagulation system (who does not respond to the standard therapy – replacement of vitamin K or freshly frozen plasma) A chronic decline in the kidney function (creatinine > 150 µmol/L) Epilepsy Pregnancy and breast-feeding The patient’s incapability of comprehending the purpose or course of the trial, or not agreeing to be included in the trial Patients unwilling or unable to comply with the protocol requirements and scheduled visits DLCO = Diffusing Capacity of the Lungs for carbon monoxide; GET = gene electrotransfer 3 patients (i.e., = 2 of 6 [33.3%] total patients in this cohort) experiences at least 1 DLT, dose escalation will be stopped. A DLT is defined as any grade 4 clinical or bio­logical event related to the study treatment and oc­curring during the first 30 days after the treatment with phIL12 GET. Randomization This is a one arm, open label clinical study without randomization. Treatment The application of the general or local anesthesia or sedation will be selected by the anesthesiologist. The phIL12 will be injected intratumorally, and 5 minutes thereafter the electric pulses will be ap­plied to the tumor for the transfection of tumor cells. Drug dosage and regime was determined based on non-clinical data. A single dose of phIL12 will be administered by intratumoral injection. In the study we will use three doses of phIL12: 0.5 mg/ml, 1 mg/ml, 2 mg/ml, which were determined based on non-clinical data. The lowest dose, 0.5 mg/ml, is the dose that, according to the guidelines of the European Medicines Agency (EMA), elicits a phar­macological effect in the mouse model in preclini-cal testing. The next two doses are also determined in accordance with the guidelines with 2 mg/ml be­ ing the maximum feasible dose (MFD). Preclinical testing of all three doses has shown satisfactory results in mice that received a single pmIL12 GET therapy (mouse orthologue of phIL12).15 An elec­trical pulse generator CLINIPORATORTM (IGEA, s.p.A.) holding authorization for the use in the clinical environment with designation CE, will be used. It generates electric pulses appropriate for phIL12 GET. The electrodes of the same manufac­turer with parallel row needle array will be used. The application of electric pulses will be performed as described in the updated Standard Operating Procedures where a detailed information on elec­trodes and their usage in different clinical aspects is defined.24 At the end of the treatment, we will take care of the lesions with standard wound-dressing techniques and move the patient into the post-anesthetic care for further monitoring and pain management. When patient will recover from anesthesia, treatment toxicity, possible side effects (Common Terminology Criteria for Adverse Events version 5.0 [CTCAE v.5]) and post-procedure pain (visual analog scale [VAS] scale) will be assessed. 403 TABLE 4. Trial procedures Informed consent X Concurrent treatments1 X Clinical examination X X X X Complete blood count, biochemistry, serum cytokines X2 X X X Coagulation profile X2 Digital imaging of the tumor and tumor measurement X3 X X X X Immune profile determination4 X X X X Saliva sample and a skin swab from the location of therapy X X X X X EORTC QLQ-C30 X X X ECOG X Examination prior to anesthesia5 X phIL12 GET X Pain assessment in accordance with the VAS scale X X X X CTCAE v.5 X X X X Punch biopsy X X6 Excision of tumor lesion X7 1 A detailed description of concurrent treatments (name of the medicinal products, dosage and treatment protocol, beginning of the treatment and reason of the treatment). 2 Complete blood count, biochemistry and coagulation profile must be carried out after the inclusion examination, no more than 7 calendar days prior to therapy. 3 Temporary measurement of the size of the tumor and initial imaging must be carried out no more than 7 days prior to therapy. 4 Peripheral blood mononuclear cells will be examined with flow cytometry to determine content of different subgroups. 5 Prior to therapy, patients will be examined and assessed by anesthesiologists in accordance with the ASA scale. 6 Punch biopsy will be performed in case of complete response. 7 Tumor lesion will be excised if tumor will NOT completely respond to the treatment. CTCAE = Common Terminology Criteria for Adverse Events; ECOG = Eastern Cooperative Oncology Group; EORTC QLQ-C30 = European Organization for the Research and Treatment of Cancer Quality of Life Questionnaire C30; GET = gene electrotransfer; VAS scale = visual analog scale scale Post-treatment analgesia will be prescribed by the principal investigator in accordance with standard practice. The principal investigator will decide on the need for post-treatment hospitalization regard­ing the patient’s condition following phIL12 GET therapy and anesthesia in accordance with stand­ard practice following invasive procedures. In this case, hospitalization is not considered as a negative side effect. Course of the study All of the trail procedures are listed in Table 4. Visit 1 (day -6 to 0): Screening and inclusion of the patient into the study Patients that will meet all inclusion/exclusion crite­ria, will be acquainted with the course of the study and will sign the informed consent form. An overview of medical history and treatment (names and dosages of medicinal products, start date and reason for treatment, and therapy dura­ tion) will be done. The investigator will collect the patient’s medical history documents with a special emphasis on oncological diseases, previous treat­ments and response to therapies thus far. A clinical overview will include the measurement of weight, height, blood pressure, pulse. A complete blood count, biochemistry and coagulation profile will be determined at visit 1. Digital imaging of tumor will be performed. The tumor location and its size will be determined. Peripheral blood mononuclear cells will be examined with flow cytometry to de­termine the content of different subgroups. A sa­liva sample and skin swabs from the location of the planned application of plasmid DNA will be col­lected. The quality of life questionnaire (European Organization for the Research and Treatment of Cancer Quality of Life Questionnaire C30, EORTC QLQ-C30) will be completed. EORTC QLQ-C30 is a validated questionnaire for the assessment of qual­ity of life in oncology patients with different cancer types in four areas (physical condition, social/fam­ily condition, psychological condition, functional condition).25 As part of the questionnaire, patients answer on a four-point scale: not at all/a little/quite a bit/very much or with scores from 1–4. Patient performance status will be evaluated in accord­ance with Eastern Cooperative Oncology Group (ECOG) criteria. Patients given a score of 0, 1 or 2 are eligible to participate in the clinical study. In all patients, the eligibility status for anesthe­sia will be evaluated with the standard pre-anes­thetic procedure (ASA scale). Patients not eligible for general anesthesia or sedation, will not be in­cluded in the clinical study. Visit 2 (day 1): Therapy with phIL12 GET No more than 7 calendar days may pass from the visit 1 to the visit 2. On visit 2, a follow-up exami­nation will include: confirmation of the eligibility of the patient to continue treatment in the clinical study, the measurement and digital imaging of the tumor, therapy with phIL12 GET, pain assessment in accordance with Visual Analog Scale (VAS) cri­teria following treatment, collection of saliva sam­ple and skin swabs from the location of plasmid DNA application, monitoring toxicity and adverse events regarding the CTCAE v.5 criteria. Visit 3 (day 3): Follow-up examination 2 days after the treatment On visit 3, a follow-up examination will include: pain assessment in accordance with VAS criteria following treatment, a complete blood count and biochemistry, a blood draw for determining the patient’s immune profile (PBMC and subgroups of lymphocytes T), collection of saliva sample and skin swabs from the location of the application of plasmid DNA, monitoring toxicity and adverse events regarding the CTCAE v.5 criteria, the meas­urement and digital imaging of the tumor. Visit 4 (day 8 after the treatment): Follow-up examination 7–10 days after the treatment Visit 4 may vary ± 3 day from the planned visit. On visit 4, a follow-up examination of the patient will include: a clinical examination, pain assessment in accordance with VAS criteria following treatment, a complete blood count and biochemistry, a blood draw for determining the patient’s immune profile (PBMC and subgroups of lymphocytes T), collec­tion of saliva sample and skin swabs from the loca­tion of the plasmid DNA application, monitoring toxicity and adverse events regarding the CTCAE v.5 criteria, measurement and digital imaging of the tumor, punch biopsy to determine the efficien­cy of transfection. Visit 5 (day 31): Follow-up examination 30 days after treatment Visit 5 may vary ± 3 day from the planned visit. On visit 5, a follow-up examination of the patient will include: a clinical examination, pain assessment in accordance with VAS criteria following treatment, a complete blood count and biochemistry, a blood draw for determining the patient’s immune profile (PBMC and subgroups of lymphocytes T), collec­tion of saliva sample and skin swabs from the loca­tion of the plasmid DNA application, monitoring toxicity and adverse events regarding the CTCAE v.5 criteria, the measurement and digital imaging of the tumor, quality of life questionnaires (EORTC QLQ-C30), preliminary evaluation of treatment ef­fectiveness according to the RECIST v1.1 criteria, punch biopsy for assessment of viability of the tu­mor. Tumors that will not respond to the treatment completely will be excised on day 30 after the treat­ment. Patients will be instructed to immediately report new symptoms to the doctor. Follow-up after the therapy The endpoint of clinical trial is 30 days after the treatment (visit 5). Nevertheless, the patients will be followed up in accordance with national Recommendation for diagnosis, treatment and follow-up of patients with basal cell carcinoma and Guidance on follow up on patients administered with gene therapy medicinal products.26,27 The clin­ical follow up will be performed 3, 6 and 12 months after the treatment. Since the plasmid DNA is as­sociated with low risk of delayed adverse reaction the yearly follow up will be arranged in form of the questionnaire forwarded to the patient (as defined in Guidance on follow up on patients administered with gene therapy medicinal products). Endpoints evaluation criteria Safety of intratumoral phIL12 GET At each visit, the investigators will note all adverse events (Adverse Event - AE and Serious Adverse Event - SAE) in the appropriate Clinical Report Form (CRF), from the inclusion of the patient into the clinical study until the end of patient follow­up. The investigator will evaluate and record: 405 • Symptoms or a diagnosis associated with AE; • The date of the beginning and end of AE; • The severity of symptoms; • A causal link to the disease or therapy in a clinical trial; • A description of measures regarding elimi­nation of AE. Recording adverse reactions of phIL12 GET will be in the scope of the CTCAE v.5 criteria (NIH, 2017). The investigator will evaluate the cause-effect link between the adverse event of phIL12 GET in accordance with the following criteria: • Not linked to the study; • Probably not linked to the study; • Possibly linked to the study; • Probably linked to the study; • Surely linked to the study. The severity of adverse reactions will be evalu­ated based on the level: • Grade 1 – mild; • Grade 2 – moderate; • Grade 3 - a difficult complication requiring medical care and treatment; • Grade 4 - a life-threatening condition re­quiring immediate medical attention; • Grade 5 – death. The investigator will follow-up on patients dur­ing the presence of the adverse reactions. According to the investigator assessment of the patient condi­tion, the latter could be withdrawn from the clini­cal study. The reason for withdrawal of treatment within the clinical study will be recorded on the CRF form for monitoring adverse reactions and on the CRF form upon study completion. All adverse reactions (AR) that might have a link to AE and the clinical study will investigator report to the sponsor. If a serious adverse reaction or a suspicion on it (SAR and SUSAR) is recorded during the study, within 24 hours the principal in­vestigator will in writing inform the study coordi­nator. The latter will further inform the National Centre for Pharmacovigilance of the Agency for Medicinal Products and Medical Devices of the Republic of Slovenia and the Ethics Committee of the Institute of Oncology Ljubljana. The principal investigator will report to the study coordinator within 24 hours on all changes of the condition re­garding a serious adverse reaction. In accordance with the regulation, the sponsor shall also submit regular periodic and annual safety reports. In case of death of a patient, the principal investigator will report on this to the sponsor regardless of whether the death was associated with progressive disease, therapy or the investigational product or with an unrelated. Assessment of the tolerability of intratumoral phIL12 GET The patients will fill out the quality of life question­naire prior to treatment and in determined intervals following the treatment (Table 4). We will monitor the hospitalization duration and medicinal prod­ucts required for pain management during hospi­talization and at each control examination. We will use the following questionnaires: • Quality of life questionnaires EORTC QLQ-C30; • Pain assessment in accordance with VAS criteria. Preliminary evaluation of treatment effectiveness according to the RECIST v1.1 Criteria Preliminary objective tumor response will be as­sessed in accordance with RECIST v1.1. criteria. The responses to be measured are: • Complete Response (CR): Disappearance of target lesion; • Partial Response (PR): At least a 30% de­crease in the sum of diameters of target le­sion, taking as reference the baseline sum diameters; • Progressive Disease (PD): At least a 20% increase in the sum of diameters of target lesion, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm (the appearance of one or more new lesions is also considered progression); • Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient in­crease to qualify for PD, taking as reference the smallest sum diameters while on study. Pharmacokinetics, pharmacodynamics of the treatment Levels of plasmid phIL12 and IL-12 and IFN-. protein levels will be evaluated in tumor biopsies. Serum levels of IL-12 cytokine will be monitored during each visit. As a part of the clinical study, the swabs will be taken prior to treatment, after treat­ment and at control checkups from the location of plasmid DNA application. Furthermore, the saliva samples will be collected at each visit. Saliva sam­ples and skin swabs will be used to determine the shedding of plasmid DNA. From the samples, the presence of plasmid DNA will be evaluated with the quantitative PCR method in real time. Changes in blood parameters will be followed at each visit determining complete blood count, biochemistry and immune subgroups profile. Sample size and statistical analysis The study is exploratory; therefore, no formal sam­ple size calculation was performed. The design (3 + 3 design) and the corresponding sample size are usual for Phase I trials in oncology. All statistical analyses will be descriptive. Discussion The current trial is designed to evaluate the safety and tolerability of the intratumoral phIL12 plasmid DNA gene electrotransfer. The drug dosage that will be established as safe and capable to elicits local immune response will be used for design of next clinical trials. The gene electrotransfer utilizing IL-12 plas­mid DNA was tested extensively in preclinical models and was also used in clinical trials before. To our knowledge, one Phase 1 clinical study (NCT00323206)4 and four Phase 2 clinical stud­ies have been conducted in USA in patients with malignant melanoma, cutaneous lymphoma, squa­mous cell carcinoma of the head and neck, and Merkel cell cancer (NCT01502293, NCT01579318, NCT02345330, NCT01440816).9,28 Further, four new studies (NCT03132675, NCT04526730, NCT03567720, NCT03823131) are active, also in mucosal head and neck squamous cell carcinoma patients, to evaluate a combination of GET of plas­mid DNA encoding IL-12 in combination with pembrolizumab or nivolumab. The above-mentioned Phase 2 clinical studies were performed with tavokinogene telseplasmid (TAVOTM), a plasmid encoding IL-12 produced by OncoSec Medical Incorporated (USA). It is a plasmid that encodes genes for the p35 and p40 subunits of the heterodimeric human IL-12 pro­tein separated by an internal ribosome entry site. Constitutive expression of the subunits is driven by a single cytomegalovirus promoter. Once the plas­mid is introduced into a mammalian cell, a func­tional IL-12 p70 protein is expressed and secreted into the tumor microenvironment. Intratumoral GET of accessible lesions is performed using an in situ EP device.8 The TAVOTM plasmid contains a kanamycin resistance gene as the selection gene for the production process, a feature that is discour­aged by EMA. Plasmid phIL12, like TAVOTM, is a plasmid DNA, encoding for p35 and p40 subunits of the heterodimeric human IL-12 protein. However, the plasmid backbone of phIL12 is different, since we aimed for the plasmid devoid of antibiotic re­sistance marker and for the treatment-inducible, tumor-specific promoter, whereas the transgene product is the same in both plasmids, a functional IL-12 p70 protein. In phIL12 plasmid, operator-re­pressor titration ORT® technology, which is based on providing the titration of repressor of essential gene for propagation of bacteria in plasmid, was used. Multiple operators present on the plasmid titrate the repressor leading to expression of essen­tial gene. In our case, the essential gene in bacteria was dapD, which was under transcriptional control of lac operator/promotor (lacO/P). Genome encod­ed Lacl repressor prevents bacterial growth, unless transformed with plasmid containing the lac op­erator (lacO) that titrates the Lacl repressor from the operator.29 In addition, since the use of phIL12 GET is foreseen as an adjuvant treatment to local ablative therapies, to increase their local and elicit a systemic response, the IL12 coding sequence was placed under the transcriptional control of an in­ducible p21 (or cyclin dependent kinase inhibitor 1A (CDKN1A)) promoter that can be activated by the hypoxic tumor microenvironment and/or by the genotoxic stress induced by local ablative ther­apies, such as tumor irradiation of electrochemo­therapy.16 Based on preclinical data for TAVOTM that are adequately comparable to phIL12 and since the ex­pression product and a mode of action are the same (i.e. protein IL-12), we decided to use the same doses as in the above clinical studies as a basis for selecting a starting dose in non-clinical study and in this first-in-human phase I study. The treatment protocol with TAVOTM was de­signed as a repetitive protocol at days 1, 5 and 8, with possible repetitive cycles at 6- or 12-weeks in­tervals. However, our intent is to use phIL12 as ad-juvant immunotherapeutic to the established local ablative therapies. As already reported, ablation of tumors induces local immune response with im­munogenic cell death.30,31 Therefore, studies com­bining local ablative therapies like radiotherapy or electrochemotherapy can be boosted from local to 407 locoregional or even systemic response by immune References checkpoint inhibitors. The recent study combining electrochemotherapy with pembrolizumab, dem­onstrated that this approach is feasible. The patients treated with pembrolizumab and ECT experienced lower disease progression rates and longer surviv­al than those who received pembrolizumab alone.32 Similar approach could be by immunostimulation using phIL12 intratumoral GET. Further, different studies combining tumor irradiation and immune checkpoint inhibitors demonstrated the same.33–36 Therefore, in the future clinical trials phIL12 is en­visioned as an immunostimulating component to in situ vaccinating effect of local ablative therapies such as electrochemotherapy or radiotherapy. We believe that in the frame of such treatment combi­nation, a single treatment with IL-12 GET would be sufficient to elicit pronounced antitumor effect. Conclusions The designed first-in-human clinical trial is aimed at evaluation of safety and tolerability of phIL12 GET, thus setting the stage for the use of phIL12 GET as an adjuvant treatment to local ablative ther­apies, to increase their local and elicit a systemic response. Author contributions GS, AG, MC, PS, MB, TJ and BM contributed to conception and design of the study. MB, TJ and GS wrote the first draft of the manuscript. AG, MC, PS and BM wrote sections of the manuscript. All au­thors contributed to manuscript revision, read, and approved the submitted version. Acknowledgments The authors acknowledge the financial support from the European Regional Development Fund provided by the Ministry of Education, Science and Sport in the scope of the SmartGene.si pro­ ject (https://www.smartgene.si/). The authors also acknowledge the financial support from the state budget by the Slovenian Research Agency (pro­ gram No. P3-0003). 1 Fares CM, van Allen EM, Drake CG, Allison JP, Hu-Lieskovan S. Mechanisms of resistance to immune checkpoint blockade: why does checkpoint inhibi­tor immunotherapy not work for all patients? Am Soc Clin Oncol Educ Book 2019; 39: 147-64. doi: 10.1200/EDBK_240837 2 Nguyen KG, Vrabel MR, Mantooth SM, Hopkins JJ, Wagner ES, Gabaldon TA, et al. Localized interleukin-12 for cancer immunotherapy. Front Immunol 2020; 11: 575597. doi: 10.3389/fimmu.2020.575597 3 Leonard JP, Sherman ML, Fisher GL, Buchanan LJ, Larsen G, Atkins MB, et al. 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Cancers 2021; 13: 4289. doi:10.3390/cancers13174289 33 Buchwald ZS, Wynne J, Nasti TH, Zhu S, Mourad WF, Yan W, et al. Radiation, immune checkpoint blockade and the abscopal effect: a critical review on timing, dose and fractionation. Front Oncol 2018; 8: 612. doi: 10.3389/ fonc.2018.00612 34 Golden EB, Demaria S, Schiff PB, Chachoua A, Formenti SC. An abscopal response to radiation and ipilimumab in a patient with metastatic non-small cell lung cancer. Cancer Immunol Res 2013; 1: 365-72. doi: 10.1158/2326­6066.CIR-13-0115 35 Formenti SC, Rudqvist NP, Golden E, Cooper B, Wennerberg E, Lhuillier C, et al. Radiotherapy induces responses of lung cancer to CTLA-4 blockade. Nat Med 2018; 24: 1845-51. doi: 10.1038/s41591-018-0232-2 36 Plavc G, Jesenko T, Oražem M, Strojan P. Challenges in combining immuno-therapy with radiotherapy in recurrent/metastatic head and neck cancer. Cancers 2020; 12: 1-25. doi: 10.3390/cancers12113197 Radiol Oncol 2022; 56(3): 267-284. doi: 10.2478/raon-2022-0032 Sevalno zdravljenje možganskih metastaz melanoma. Sistematicni pregled Thompson JF, Williams GJ, Hong AM Izhodišca. Sevalno zdravljenje možganskih metastaz melanoma, ki ga izvajamo v obliki obsevalnega zdravljenja celotnih možganov ali stereotakticne radiokirurgije, je uveljavljena oblika zdravljenja te bo­lezni. Dokazov, ki bi omogocili primerjavo rezultatov, prednosti in slabosti obeh nacinov sevalnega zdra­vljenja pa je malo. Do sedaj je bilo tudi zelo malo randomiziranih kontroliranih raziskav. To je povzrocilo precejšnjo negotovost in nedosledna priporocila v smernicah. V pricujocem sistematicnem pregledu smo obravnavali 112 raziskav, ki so porocale o rezultatih zdravljenja bolnikov z metastazami melanoma v možganih in ki so jih sevalno zdravili. Tri so bile randomizirane kontrolirane raziskave, vendar je bila le ena dovolj obsežna, da jo je bila dovolj povedna. Vecina izsledkov je izvirala iz nerandomiziranih raziskav, kjer so izvajali posebna zdravljenja ali pa so obravnavali kohorte bolnikov z doloceno obliko bolezni. O merilih za izbiro zdravljenja je porocalo le 32 raziskav in kakovost teh raziskav je bila razlicna. Srednje preživetje, merjeno od casa diagnoze možganskih metastaz, je bilo ob obsevanju celotnih možganov kot edinim zdravljenju samo 3,5 meseca (interkvartilni razpon [IQR] 2,4–4,0 meseca), ce pa so bolnike zdravili samo s stereotakticno radiokirurgijo je bilo srednje preživetje 7,5 meseca (IQR 6,7–9,0 meseca). Skupno preži­vetje bolnikov se je s casom povecevalo (od obdobja pred letom 1989 do leta 2015), vendar to ni bilo razvidno v razlicnih skupinah zdravljenja. Zakljucki. Sistematicni pregled preživetja bolnikov z možganskimi metastazami melanoma lahko omo­goci primerjavo z rezultati ucinkovitosti zdravljenja nedavno uvedenih sistemskih zdravljenj, kot so tarcno zdravljenje ali imunoterapija. Radiol Oncol 2022; 56(3): 285-291. doi: 10.2478/raon-2022-0022 Elektrokemoterapija solidnih tumorjev. Pregled literature in predstavitev novega endoskopskega pristopa Schipilliti FM, Onorato M, Arrivi G, Panebianco M, LerinD, Milano A, Roberto M, Capalbo C, Mazzuca F Izhodišca. Elektrokemoterapija (ECT) je minimalno invazivno in varno zdravljenje, s katerim dosegamo dobre in dolgotrajne protitumorske ucinke, ki so zbudili pozornost med znanstvenimi raziskavami. To je lokalno zdravljenje, ki združuje uporabo elektroporacije in citotoksicnih zdravil za povzrocitev celicne smrti v ciljnem tkivu. ECT vecinoma uporabljamo za zdravljenje kožnih in podkožnih sprememb, o dobrih rezultatih pa so porocali tudi pri zdravljenju globokih visceralnih tumorjev. Sodoben pregled literature to potrjuje. Eno od najnovejših zdravljenj visceralnih tumorjev je elektrokemoterapevtsko endoskopsko zdra­vljenje kolorektalnega raka. Predstavljamo primer endoskopske uporabe EKT obstruktivnega raka danke, ker operacija ne bi omogocila dolgotrajnejšega ucinka. Ugotovili smo dober odziv na EKT. Koncentricno razrašcanje tumorja danke se je zmanjšalo, stenoze pa nismo zaznali. Zakljucki. Klinicne raziskave so pokazale, da je EKT zelo ucinkovito zdravljenje tumorjev razlicnih histo­loških tipov in lokalizacij. Endoskopsko zdravljenje raka prebavil je inovativna uporaba ECT. Kombinacija sistemskega zdravljenja in ECT je bila varna in zelo ucinkovita pri zdravljenju raka debelega crevesa in danke, zlasti obstruktivnega, kar je bolniku omogocilo znatno izboljšanje kakovosti življenja. Radiol Oncol 2022; 56(3): 292-302. doi: 10.2478/raon-2022-0024 Portalna hipertenzija lahko vpliva na zaznavo hipointenzivnosti kontrastnega sredstva pri majhnih lezijah hepatocelularnega raka v hepatobiliarni fazi pri MR in uporabi gadokseticne kisline Caparroz C, Forner A, Rimola J, Darnell A, García-Criado A, Ayuso JR, Reig M, Bruix J, Ayuso C Izhodišca. Namen raziskave je bil analizirati povezavo med privzemom gadokseticne kisline v jetrih (Gd-EOB-DTPA) v hepatobiliarni fazi in prisotnostjo klinicno pomembne portalne hipertenzije pri bolnikih s cirozo ter oceniti. kako ta pojav vpliva na zaznavo hepatocelularnega raka. Bolniki in metode. Opravili smo naknadno analizo prospektivne skupine 62 bolnikov z jetrno cirozo, ki smo jim ultrazvocno na novo odkrili nodul velikosti med 1–2 cm (študijska skupina). Kontrolna skupina je predstavljalo 20 zdravih oseb. Kvalitativno in kvantitativno smo analizirali privzem kontrasta v jetrih v he-patobiliarni fazi, ga ocenili z relativnim privzemom kontrasta oz. razmerjem med kontrastnim obarvanjem jeter in vranice, jeter in mišicja ter jeter in ledvic. Opazovali smo tudi indeks privzema kontrasta, indeks jetrnega privzema in biliarno izlocanje kontrasta. Klinicno pomembna portalna hipertenzija smo potrdili invazivno (hepaticni venski tlacni gradient >10 mm Hg) ali z indirektnimi parametri. Analizirali smo zaznavo hepatocelularnega raka v hepatobiliarni fazi. Rezultati. 19 bolnikov (30,6 %) ni imelo klinicno pomembne portalne hipertenzije. Pri 41 bolniki (66,1 %) je bila koncna diagnoza hepatocelularni rak. Vsi indeksi so bili bistveno višji v kontrolni skupini, kar kaže, da je imela kontrolna skupina intenzivnejši jetrni signal v hepatobiliarni fazi kot skupina s cirozo. To smo zaznali, tudi ce smo naredili primerjavo, kjer pri preiskovancih nismo zaznali klinicno pomembno portalno hipertenzijo. Klinicno pomembna portalna hipertenzija je bila povezana z nižjo stopnjo hipointenzivnosti privzema kontrasta pri hepatocelularnem raku v hepatobiliarni fazi preiskave (51,9 % proti 85,7 % brez klinicno pomembne portalne hipertenzije, p = 0,004). Zakljucki. Privzem gadokseticne kisline v jetrih v hepatobialiarni fazi se zmanjša, kadar ima bolnik ci­rozo, tudi ce je delovanje jeter minimalno okvarjeno ter znatno pade pri bolnikih z klinicno pomembno portalno hipertenzijo. Ta pojav ogroža prepoznavanje hipointenzivnih lezij in lahko predstavlja omejitev pri odkrivanju majhnega hepatocelularnega raka, kadar imajo bolniki cirozo in klinicno pomembno portalno hipertenzijo. Radiol Oncol 2022; 56(3): 303-310. doi: 10.2478/raon-2022-0029 Primerjava zgodnje izolirane subarahnoidalne krvavitve in hemoragicnega infarkta pri možganski venski trombozi Kobal J, Cankar K, Ivanušic K, Vudrag B, Šurlan Popovic K Izhodišca. Možganska venska tromboza je redko žilno obolenje. Radiološko in klinicno se zelo razlicno pojavlja. Novejše raziskave so pokazale, da izolirana subarahnoidna krvavitev ob možganski venski trom­bozi ni tako redka in je povezana z dobro napovedjo poteka bolezni. Nasprotno pa je hemoragicni mo-žganski infarkt napovedni dejavnik, ki napoveduje slabši potek bolezni in je vecinoma povezan z okluzijo vec ven ali/in sinusov. Postavili smo hipotezo, da bodo imeli bolniki v raziskavi z izolirano subarahnoidno krvavitvijo boljši klinicni izid bolezni v primerjavi s tistimi, ki so doživeli hemoragicni možganski infarkt. Bolniki in metode. Izbrali smo bolnike hospitalizirane zaradi možganske venske tromboze, ki so imeli izolirano subarahnoidno krvavitev ali hemoragicni infarkt ob sprejemu oziroma v casu 24 ur od sprejema. Kriterijem je ustrezalo 23 bolnikov (10 moških), starih 22-73 let. Podatke smo pridobili iz bolniškega arhiva ter racunalniške in radiološke podatkovne baze. Rezultati. V skupini z izolirano subarahnoidno krvavitvijo je bilo 8 bolnikov (6 moških) starih 49,3 ± 16,2 let in v skupini s hemoragicnim možganskim infarktom 15 bolnikov (4 moških) starih 47,9 ± 16,8 let. Skupina z izolirano subarahnoidno krvavitvijo je imela po 3 mesecih bistveno boljši izid zdravljenja ocenjen z modifi­cirano Rankinivo lestvico kot skupina s hemoragicnim infarktom (Mann-Whitney Rank Sum Test, p = 0,026) kljub znacilno vecjemu številu tromboziranih venskih sinusov in/ali globokih ven (Mann-Whitney Rank Sum Test, p = 0,002). Dodatne spremenljivki z znacilnim vplivom na potek bolezni sta bili tvorba možganskega edema (p = 0,004) in obliteracija sulkusov (p = 0,014). Zakljucki. Bolniki, ki so utrpeli izolirano subarahnoidno krvavitev, so imeli boljši izid bolezni kljub znacilno vecjemu številu tromboziranih sinusov in/ali ven. Možen razlog bi lahko bile prehodne povrhnje komuni­kantne vene. Radiol Oncol 2022; 56(3): 311-318. doi: 10.2478/raon-2022-0019 Varnost in ucinkovitost transarterijske kemoembolizacije z delci pod nadzorom racunalniške tomografije s stožcastim snopom pri bolnikih z jetrnocelicnim rakom v zgodnjem in srednjem stadiju bolezni Koršic S, Levasic N, Dežman R, Lešnik Zupan LA, Trotovšek B, Janša R, Šmid A, Popovic P Izhodišca. Transarterijska kemombolizacija z mikrodelci, ki nase vežejo kemoterapevtik (angl. drug-eluting microspheres transarterial chemoembolization, DEM-TACE), izboljša preživetje bolnikov z jetrno­celicnim rakom v zgodnjem in srednjem stadiju bolezni ter zagotavlja tarcne nadzorovane citotoksicne in ishemicne ucinke zdravljenja. Ustrezen izbor bolnikov in izboljšana tehnika zdravljenja so povezane z daljšim srednjim preživetjem. Namen pricujoce raziskave je bil preveriti varnost in ucinkovitost DEM-TACE pod nadzorom racunalniške tomografije s stožcastim snopom (angl. cone-beam computed tomogra­phy, CBCT) pri bolnikih z jetrnocelicnim rakom v zgodnjem in srednjem stadiju bolezni. Bolniki in metode. Raziskava je bila retrospektivna analiza 144-ih bolnikov (srednja starost 67,9 ± 8,0 let, 127 moških in 17 žensk), ki smo jih zdravili z DEM-TACE z doksorubicinom med februarjem 2010 in decem­brom 2018. Uporabili smo mikrodelce razlicnih dimenzij dveh proizvajalcev (premera 70–150 µm, 100–300 µm ali 300–500 µm in 40-µm, 75-µm ali 100-µm). Mikrodelci so vsebovali 50–150 mg doksorubicina. Beležili smo objektivni odgovor na zdravljenje po kriterijih mRECIST, cas do napredovanja bolezni, neželene ucin­ke zdravljenja in celokupno preživetje. Rezultati. Naredili smo 452 posegov DEM-TACE. Zabeležili smo štiri vecje zaplete (0,9 % od vseh pose-gov). Postembolizacijski sindrom se je pojavil pri 35 % posegov. Ob prvi slikovni kontroli 2–3 mesece po zdravljenju smo videli objektivni odgovor na zdravljenje pri 91 % bolnikov. Srednji cas do napredovanja bolezni je bil 10,2 mesecev (95 % interval zaupanja [IZ]: 8,3–12,1 mesecev). 1- 2-, 3-, 4- in 5-letno celoku­pno preživetje je bilo 85 %, 53 %, 33 %, 20 % in 14 %; srednje preživetje pa 25,8 mesecev (95 % IZ: 22,1–29,5 mesecev). Zakljucki. DEM-TACE z doksorubicinom pod nadzorom CBCT je varna in ucinkovita metoda zdravljenja bolnikov, ki imajo jetrnocelicni rak v zgodnjem in srednjem stadiju bolezni. Dosegli smo dober objektivni odgovor na zdravljenje in primerljivo preživetje glede na objavljene raziskave. Radiol Oncol 2022; 56(3): 319-325. doi: 10.2478/raon-2022-0033 Aplikacije nusinersena pod nadzorom racunalniške tomografije s stožcastim snopom pri odraslih bolniki s spinalno mišicno atrofijo in z zapletenimi anatomskimi predispozicijami. Izkušnje posamicnega terciarnega centra Salapura V, Snoj Ž, Lea Leonardis L, Koritnik B, Kostadinova V Izhodišca. Zaradi zapletenih anatomskih predispozicij ni moc vedno izvesti obicajno lumbalno punkcijo pri odraslih bolnikih s spinalno mišicno atrofijo. Potrebno je vpeljati novo metodo, ki bi omogocala varno aplikacijo zdravila nusinersen. Metoda CT slikanja s stožcastim snopom nam omogoca dober prostorski prikaz predela, kamor želimo aplicirati zdravilo, nacrtovanje pred posegom in nadzor nad položajem igle med uvajanjem. Tako smo v raziskavi ugotavljali tehnicno uspešnost, varnost in izvedljivost lumbalnih intratekalnih aplikacij zdravila nusinersen s pomocjo CT slikanja s stožcastim snopom pri bolnikih s spinalno mišicno atrofijo in zapletenimi anatomskimi predispozicijami. Bolniki in metode. Na Inštitutu za radiologijo, UKC Ljubljana smo zdravili 38 bolnikov s spinalno mišicno atrofijo. Multidisciplinarni tim je izbral za slikovno vodeno aplikacijo nusinersena tiste bolnike, ki so imeli zapletene anatomske predispozicije. Vkljucili smo bolnike z operativnim zdravljenjem hrbtenice zaradi skolioze, bolnike s hudo skoliozo, ki smo jo definirali s Cobbovim kotom > 40°, ter bolnike z indeksom tele­sne mase nad 35. Opravili smo analizo tehnicne uspešnosti, izpostavljenosti sevanju in stranskih ucinkov. Rezultati. V raziskavo smo vkljucili 20 bolnikov in opravili 108 aplikacij zdravila s pomocjo CT slikanja s stožcastim snopom. Vsakemu bolniku smo zdravilo aplicirali vsaj štirikrat. V 82 % smo izvedli transforami­nalni pristop. Tehnicna uspešnost metode je bila 100 %. V prvem poskusu smo uspešno aplicirali zdravilo v 93,5 % primerov. Srednja vrednost sevalne efektivne doze pri vseh aplikacijah zdravila je bila 5 mSv. V raziskavi smo zabeležili zgolj blage stranske ucinke zaradi obravnave bolnikov. Zakljucki. Aplikacija nusinersena pod nadzorom CT slikanja s stožcastim snopom je bila varna in izvedlji­va metoda pri bolnikih s spinalno mišicno atrofijo in zapletenimi anatomskimi predispozicijami. Radiol Oncol 2022; 56(3): 326-335. doi: 10.2478/raon-2022-0028 Nanosekundni elektricni pulzi so enako ucinkoviti v elektrokemoterapiji s cisplatinom kot mikrosekundni pulzi Vižintin A, Markovic S, Šcancar J, Kladnik J, Turel I, Miklavcic D Izhodišca. Uporaba nanosekundnih elektricnih pulzov v elektrokemoterapiji je pokazala spodbudne rezultate, vendar osnovni mehanizmi delovanja še vedno niso raziskani. Namen raziskave je bil povezati kolicino celicnega cisplatina s preživetjem celic, elektroporiranih z nanosekundnimi ali standardnimi 8 × 100 µs pulzi ter preuciti ucinke elektricnih pulzov na strukturo cisplatina. Materiali in metode. Ovarijske celice kitajskega hrcka CHO in celice mišjega melanoma B16F1 smo izpostavili 1 × 200 ns pulzu pri 12,6 kV/cm ali 25 × 400 ns pulzom pri 3,9 kV/cm s ponavljalno frekvenco 10 Hz ali 8 × 100 µs pulzom pri 1,1 kV/cm (CHO) ali 0,9 kV/cm (B16F1) s ponavljalno frekvenco 1 Hz in trem razlicnim koncentracijam cisplatina. Celicno preživetje smo dolocili s testom klonogenosti, kolicino plati­ne v celicnih peletih pa s spektroskopijo z induktivno sklopljeno plazmo. Ucinke na strukturo cisplatina smo spremljali z jedrsko magnetno resonanco in masno spektrometrijo visoke locljivosti. Rezultati. Parametri nanosekundnih pulzov, ekvivalentni 8 × 100 µs pulzom, so bili doloceni in vitro na podlagi permeabilizacije celicne membrane in preživetja celic. Ekvivalentni nanosekundi pulzi so bili pri elektrokemoterapiji enako ucinkoviti pri zmanjševanju preživetja celic in kopicenju cisplatina v celicah kot 8 × 100 µs pulzi. Število znotrajcelicnih molekul cisplatina je mocno korelirano s celicnim preživetjem za celice B16F1, manj pa za celice CHO, kar nakazuje na možno vpletenost drugih mehanizmov v elek­trokemoterapiji. Visokonapetostni elektricni pulzi niso vplivali na strukturo cisplatina. Zakljucki. Ekvivalentni nanosekundni pulzi so enako ucinkoviti pri elektrokemoterapiji kot standardno uporabljeni 8 × 100 µs pulzi. Radiol Oncol 2022; 56(3): 336-345. doi: 10.2478/raon-2022-0031 Vpliv polimorfizma AKT1 na poškodbe DNK, ekspresijo BTG2 in tveganje za razvoj kolorektalnega raka Zubair H, Khan Z, Imran M Izhodišca. AKT, imenovana tudi protein kinaza B, je serin-treonin kinaza, ki deluje kot mediator signalne poti PI3K-Akt-mTOR in ima pomembno vlogo v nizu celicnih procesov. Ugotovili so, da so številni polimor­fizmi enega nukleotida (SNP) v genu AKT povezani z razlicnimi vrstami raka. Namen pricujoce raziskave je bil prouciti povezavo med funkcionalnim SNP rs1130233 v AKT, ki prikazuje prehod G v A, in tveganjem za razvoj kolorektalnega raka. Zato smo ugotavljali aktivacijo AKT, poškodbo DNK in izražanje gena 2 (Btg2) za translokacijo B-celic v zgodnjem odzivu. Bolniki in metode. V raziskavo smo vkljucili 200 bolnikov s kolorektalnim rakom in 197 preiskovancev iz populacije kot kontrolno skupino. Naredili smo genotipizacijo za SNP rs1130233. Izražanje AKT ter akti­vacijo in izražanje BTG2 smo dolocili pri nosilcih genotipa GG, AG in AA. Poškodbe DNK smo dolocili s kometnim testom. Rezultati. Heterozigotni genotip AG (55,67 %) je bil v lokalni populaciji bolj razširjen v primerjavi s homoz­igotnim nemutiranim tipom GG (37,78 %) in homozigotnim mutiranim genotipom AA (6,55 %). Ob tem smo opazili, da alela AG in AA pomembno prispevata (P = 0,01; razmerje obetov [OR] = 1,80; interval za­upanja [CI] = 1,18–2,74 ter P = 0,001; OR = 5,00; CI = 1,90–13,18) k povecanju tveganja za kolorektalnega raka. Analiza imunoblot je pokazala, da je prehod G v A zmanjšal izražanje in aktivacijo AKT. Poleg tega sta genotipa AG in AA AKT1 rs1130233 pokazala znatno povecanje poškodbe DNA in izražanja Btg2. Zakljucki. Raziskava je pokazala, da je zamenjava G z A lahko dejavnik tveganja za razvoj kolorektal­nega raka, ki vkljucuje zmanjšanje izražanja in aktivacije AKT ter povecanje poškodbe DNK. Radiol Oncol 2022; 56(3): 346-354. doi: 10.2478/raon-2022-0030 Rezultati zdravljenja pri bolnikih z zacetno operacijo in resektabilnim nedrobnocelicnim rakom pljuc stadija I–IIIA v vsakodnevni klinicni praksi Bitenc M, Cufer T, Kern I, Miklavcic M, Petrovic S, Groznik V, Sadikov A Izhodišca. Zdravljenje nedrobnocelicnega pljucnega raka v zgodnjem stadiju se hitro razvija. Pri uva­janju novosti so potrebni dejanski podatki o ucinkovitosti obravnave v dosedanji vsakodnevni klinicni praksi. Proucili smo preživetja bolnikov z nedrobnocelicnim pljucnim rakom stadijev I–IIIA, ki smo jih zdravili v obdobju 2010–2017 z zacetno radikalno kirurgijo. Bolniki in metode. Podatke o 539 zaporednih bolnikih smo pridobili iz prospektivnega bolnišnicnega registra. Vse diagnosticne postopke, zdravljenje in sledenje bolnikov smo opravili v istem centru in v skla­du s takrat veljavnimi smernicami. Primarni cilj raziskave je bila analiza celokupnega preživetja glede na klinicni (k) in patološki (p) stadij TNM (tumor, bezgavke, metastaze). Z metodama univariatne in multiva­riatne regresijske analize pa smo analizirali tudi vpliv drugih klinicno patoloških znacilnosti. Rezultati. Po srednji opazovalni dobi 53,9 mesecev je bilo srednje in 5-letno celokupno preživetje celo­tne kohorte 90,4 mesecev in 64,4 %. 5-letno celokupno preživetje bolnikov s stadiji pTNM I, II in IIIA je bilo 70,2 %, 60,21 % in 49,9 %. S celokupnim preživetjem sta bila znacilno povezana stadija kTNM in pTNM, ven­dar pa je samo pTNM v multivariatni analizi ohranil neodvisno napovedno vrednost (p = 0,003). Skladnost med kTNM in pTNM je bila 69,0 %. Poleg stadija pTNM sta starost (p = 0,001) in spol (p = 0,004) ohranila neodvisno napovedno vrednost za celokupno preživetje. Zakljucki. Raziskava je pokazala dobra celokupna preživetja bolnikov z resektabilnim nedrobnoce­licnim pljucnim rakom, ki smo jih zdravili z zacetno operacijo v vsakodnevni klinicni praksi. Nepopolno ujemanje med stadijema kTNM in pTNM in neodvisna napovedno vrednost samo stadija pTNM za celo­kupno preživetje pa kažeta, da zacetna kirurgija še vedno zagotavlja najzanesljivejšo dolocitev anatom­skega stadija in ostaja pomembno zdravljenje zgodnjega nedrobnocelicnega pljucnega raka. Radiol Oncol 2022; 56(3): 355-364. doi: 10.2478/raon-2022-0023 Prepoznavanje žensk s histopatološkimi izvidi visoke stopnje po konizaciji s pomocjo umetnih nevronskih mrež Mlinaric M, Križmaric M, Takac I, Repše Fokter A Izhodišca. Namen raziskave je bil oceniti, ali lahko z uporabo umetnih nevronskih mrež napovemo histo­patološki izvid visoke stopnje pri bolnicah, ki so imele konizacijo zaradi sprememb na maternicnem vratu. Materiali in metode. Analizirali smo 1475 bolnic, pri katerih smo naredili konizacijo na Univerzitetni kliniki za ginekologijo in porodništvo Univerzitetnega klinicnega centra Maribor v letih 1993–2005. Zaradi neuravnoteženega števila bolnic z in brez sprememb ter z namenom izboljšanja klasifikacije smo bazo podatkov uredili v razlicne sklope. Za analizo z umetnimi nevronskimi mrežami smo uporabili odprtokodni program Weka. Za vhodne podatke pa smo uporabili zadnji bris po Papanikolaouju in dejavnike tvega­nja za razvoj patoloških sprememb na maternicnem vratu ter visokorizicno displazijo DA/NE kot izhodni rezultat. 10-kratno navzkrižno preverjanje smo uporabili za definiranje ucnega in testnega seta za analizo. Rezultati. Z nevronskimi mrežami smo izvedli simulacijo bazalne klasifikacije in vec testiranj glede na razlicne dejavnike tveganja. Nevronske mreže so v modelu, v katerem je bila bazalna uspešnost 69,79 %, pravilno razvrstile 84,19 % primerov; podrocje pod krivuljo je bilo 0,87; kappa vrednost 0,64: F-mera 0,884 in Matthewsov korelacijski koeficient (MCC) 0,640. Zakljucki. Z umetnimi nevronskimi mrežami smo uspeli prepoznati vec bolnic, ki bodo imele visoko rizi-cen izvid po konizaciji kot pa z bazalno napovedjo. Kljub temu pa na podlagi znacilnosti 1475 bolnic, ki so imele konizacijo, nevronske mreže niso dosegle zanesljivosti, ki bi bila primerna za vsakodnevno klinicno delo. Radiol Oncol 2022; 56(3): 365-370. doi: 10.2478/raon-2022-0018 Napredek v radioonkološkem zdravljenju raka prostate z visokim tveganjem. Cetrt stoletja dosežkov Moll M, Herrmann H, Zaharie A, Goldner G Izhodišca. Namen raziskave je bil ovrednotiti napredek pri zdravljenju bolnikov, ki so zboleli zaradi pri­marnega raka prostate z visokim tveganjem. Analizirali smo biokemicno odsotnost bolezni ter akutne in pozne gastrointestinalne in genitourinarne stranske ucinke. Bolniki in metode. Vkljucili smo bolnike s primarnim rakom prostate z visokim tveganjem, ki smo jih zdravili v letih 1904–2016. Prejete obsevalne doze so znašale od 60 do 80 Gy, doza na frakcijo pa 1,8 ali 2 Gy. Uporabili smo konformno tehniko 3D ali intenzitetno modulirano radioterapijo ali pa volumetricno intenzitetno modulirano locno terapijo. Rezultati. Bolnike smo razdelili v 3 skupine: 142 bolnikov smo obsevali z dozami do 70 Gy (srednja doza 66 Gy; skupina 66 Gy), 282 z dozami med 70 in 76 Gy (srednja doza 74 Gy; skupina 74 Gy) in 141 z dozami > 76 Gy (srednja doza 78 Gy; skupina 78 Gy). Srednji cas spremljanja je bil 48 mesecev. V skupini 66 Gy je bil delež biokemicne odsotnosti bolezni po petih letih 50 % in po devetih letih 44 %; v skupini 74 Gy 65 % in 54 %; ter v skupini 78 Gy 83 % in 66 % (p = 0,03 proti skupini 74 Gy in p < 0,0001 proti skupini 66 Gy). V skupini 78 Gy smo ugotovili višji delež akutnih gastrointestinalnih stranskih ucinkov kot v drugih skupinah, ne pa tudi razlik v maksimalnih akutnih genitourinarnih stranskih ucinkih in tudi ne v kasnih maksimalnih gastrointestinalnih in genitourinarnih stranskih ucinkih. Zakljucki. Bolniki z rakom prostate z visokim tveganjem, ki so bili zdravljeni z dozami 78 Gy, so imeli sta­tisticno pomembno boljše deleže biokemicne odsotnosti bolezni. V primerjavi s historicno skupino 66 Gy je po devetih letih sledenja 50 % vec bolnikov doseglo biokemicno odsotnost bolezni. Radiol Oncol 2022; 56(3): 371-379. doi: 10.2478/raon-2022-0025 Preživetje, nacini zdravljenja in deleži testiranja T790M pri bolnikih z nedrobnocelicnim rakom pljuc, ki smo jih primarno zdravili z zaviralci tirozinske kinaze receptorja epidermalnega rastnega dejavnika prve ali druge generacije v vsakdanji klinicni praksi. Podatki za bolnike iz slovenske kohorte v klinicni raziskavi REFLECT Turnšek N, Devjak R, Edelbaher N, Osrajnik I, Unk M, Vidovic D, Jeric T, Janžic U Izhodišca. Zaviralci tirozinske kinaze (angl. tyrosine kinase inhibitors; TKI) receptorja epidermalnega ra­stnega dejavnika (angl. epidermal growth factor receptor; EGFR) so ucinkoviti v zdravljenju napredova­lega nedrobnocelicnega raka pljuc s pozitivnimi mutacijami EGFR (EGFRm). Ker se vsakdanje zdravljenje bolnikov razlikuje med posameznimi centri, smo želeli analizirati obravnavo bolnikov v Sloveniji. Bolniki in metode. Retrospektivna raziskava REFLECT (NCT04031898) je analizirala podatke bolnikov z napredovalim EGFRm nedrobnocelicnega raka pljuc, ki so priceli rutinsko zdravljenje med leti 2015 in 2018 in so v 1. redu zdravljenja prejeli 1. ali 2.generacijo EGFR TKI. Pridobili smo podatke o klinicnih zna-cilnostih, nacinu zdravljenja, vzorcih testiranja EGFR/T790M in izhodih zdravljenja. Raziskava je potekala v 8 državah, predstavljamo pa slovenske podatke 120 bolnikov, zdravljenih v 3 onkoloških centrih v državi. Rezultati. Med slovenskimi bolniki je bila srednja starost 70 let, žensk je bilo 74 %, v 1. redu zdravljenja so prejeli: erlotinib (37 %), afatinib (32 %) in gefitinib (31 %). V casu analize je 94 (78 %) bolnikov prenehalo zdravljenje s 1. redom EGFR TKI, 89 (74%) zaradi napredovanja bolezni. Med bolniki z napredovanjem bo­lezni je bilo 73 (82 %) testiranih za mutacijo T790M, pri 50 (68 %) smo to mutacijo odkrili in 62 (85 %) bolnikov je prejelo 2. red zdravljenja, od tega 82% z osimertinibom. Stopnja osipa med 1. in 2. redom zdravljenja je znašala 10 %. Srednje preživetje brez napredovanja bolezni (95 % interval zaupanja) s 1. redom zdra­vljenja je znašalo 15,6 (12,6–19,2) mesecev, srednje celokupno preživetje pa 28,9 (25,0–34,3) mesecev. Zakljucki. Raziskava vsakdanje klinicne prakse ponuja koristne informacije o ucinkovitosti zdravljenja z EGFR TKI pri bolnikih z napredovalim EGFRm nedrobnocelicnega raka pljuc. Stopnja osipa med zdra­vljenjem 1. in 2. reda ter odlicni rezultati preživetja kažejo na pomembnost pricetka zdravljenja z najbolj ucinkovitim EGFR TKI. Radiol Oncol 2022; 56(3): 380-389. doi: 10.2478/raon-2022-0027 Spremembe pri zdravljenju raka v otroštvu in tveganje za sekundarne maligne neoplazme Mazic Cesen M, Reulen CR, Jazbec J, Zadravec Zaletel L Izhodišca. Namen raziskave je oceniti tveganje in opredeliti sekundarne neoplazme po zdravljenju raka v otroštvu v povezavi s spremembami v zdravljenju. Bolniki in metode. V populacijsko raziskavo smo vkljucili 3271 bolnikov, ki smo jih zdravili zaradi raka do dopolnjnega 18. leta starosti v obdobju med letoma 1961 in 2013 in sledili do vkljucno leta 2018. Uporabili smo statisticne metode: standardizirano incidencno stopnjo, absolutno presežno tveganje in kumulativ-no incidenco. Rezultati. V povprecnem casu sledenja 23,2 let smo pri 230 bolnikih zabeležili 273 primerov sekundarnih neoplazem; od tega 183 malignih neoplazem, 34 meningiomov in 56 primerov nemelanomskega raka kože. 10,5 % bolnikov smo zdravili z obsevanjem, 31 % s kemoterapijo, 26,9 % s kombinacijo kemoterapije in obsevanja in 16,1 % izkljucno z operacijo. Tveganje za sekundarno maligno neoplazmo je bilo skoraj 3-krat vecje kot v splošni populaciji (standardizirana incidencna stopnja 2,9) in je ostalo 2-krat vecje tudi po dopolnjenem 50. letu starosti. Kumulativna incidenca za sekundarne maligne neoplazme 30 let po diagnozi otroškega raka je bila pomembno nižja za bolnike, ki smo jih zdravili med leti 1960–1970 v pri­merjavi z bolniki, ki smo jih zdravili med leti 1970–1990 (p < 0.001). Kljub pomembnemu zmanjšanju deleža obsevanih bolnikov po letu 1995, kumulativna incidenca za sekundarne maligne neoplazme prvih 15 let po diagnozi ni pomembno nižja po letu 1995 (p = 0.11). Zakljucki. Tveganje za sekundarne maligne neoplazme pri bolnikih, ki smo jih zdravili zaradi raka v otro­štvu, je v pricujoci raziskavi primerljivo s tveganjem v drugih populacijskih raziskavah. Razlika v intenzivnosti onkološkega zdravljenja, ki je doseglo vrhunec in upad kasneje kot v drugih evropskih državah, pomeni, da se tveganje za sekundarne neoplazme v prihodnosti lahko poveca. Radiol Oncol 2022; 56(3): 390-397. doi: 10.2478/raon-2022-0034 Skrajšani dihalni test z mešanimi trigliceridi C13 je pri odkrivanju eksokrine insuficience trebušne slinavke enako natancen kot standardni 5-urni test pri bolnikih po gastrektomiji zaradi raka želodca Siuka D, Kumer K, Štabuc B, Štubljar D, Drobne D, Janša R Izhodišca. Dihalni test z mešanimi trigliceridi C13 (C13-MTGT) je neinvazivni test za odkrivanje zmerne in hude eksokrine insuficience trebušne slinavke, potrebno pa je dolgotrajno vzorcenje izdihanega zraka. Namen pricujoce raziskave je bil ugotoviti diagnosticno moc skrajšanega C13-MTGT pri odkrivanju ekso­krine insuficience trebušne slinavke pri bolnikih po subtotalni in totalni gastrektomiji, ki je bila narejena zaradi raka želodca. Preiskovanci in metode. V presecno opazovalno raziskavo smo vkljucili 3 skupine preiskovancev: zdrave kontrolne osebe ter bolnike po subtotalni in bolnike po totalni gastrektomiji zaradi raka želod-ca. Zbrali smo demografske in klinicne podatke bolnikov. Vzorce blata za dolocanje fekalne elastaze (Fe-1) in kimotripsina smo izmerili s testom ELISA. Vsi preiskovanci so opravili 5-urni dihalni test C13-MTGT. Koncentracijo in relativno vsebnost C13 v izdihanem zraku smo izmerili z masnim spektrometrom za raz­merje izotopov. Eksokrino insuficienco trebušne slinavke smo potrdili kot vrednosti C13-izdiha < 26,8 % po 5 urah. Rezultati. V analizi smo obravnavali 65 udeležencev, 22 jih je imelo eksokrino insuficienco trebušne slinavke (11 po subtotalni gastrektomiji in 11 po totalni gastrektomiji). Dihalni test C13-MTGT je pokazal razliko v odstotkih izdihanega C13 med bolniki z ali brez eksokrine insuficience trebušne slinavke že po 60 minutah (p = 0,034). Analiza krivulje karakteristike delovanja sprejemnika (ROC) je pokazala, da mejna vrednost 13,74 % po 150 minutah kaže enakovredno diagnosticno moc daljšemu testu z obcutljivostjo in specificnostjo nad 90 % za izkljucitev eksokrine insuficience trebušne slinavke pri bolnikih po subtotalni ali totalni gastrektomiji. Zakljucki. Raziskava je pokazala, da skrajšanje testa C13-MTGT s 5 na 2,5 ure ne zmanjša njegove di­agnosticne natancnosti za odkrivanje eksokrine insuficience trebušne slinavke pri bolnikih po subtotalni ali totalni gastrektomiji, opravljeni zaradi raka želodca, kar lahko omogoca znatne prihranke casa pri diagnostiki te podskupine bolnikov. Radiol Oncol 2022; 56(3): 398-408. doi: 10.2478/raon-2022-0021 Zdravljenje kožnih tumorjev glave in vratu z intratumorskim genskim elektroprenosom gena za interlevkin 12. Protokol prvega klinicnega preskušanja pri ljudeh Grošelj A, Bošnjak M, Jesenko T, Cemažar M, Markelc B, Strojan P, Serša G Izhodišca. Imunske terapije so trenutno predmet mnogih raziskav, saj pogosto zagotavljajo odlicne odgovore na zdravljenje pri razlicnih tumorjih. Možen nacin imunoterapije je ciljna intratumorska dostava interlevkina 12 (IL-12), citokina z znano protitumorsko ucinkovitostjo. Takšen nacin zaradi svojega imuno­modulatornega delovanja omogoci stimulacijo imunskega odziva, ki lahko okrepi ucinek vakcinacije in situ pri lokalnih ablativnih terapijah. Razvili smo plazmid phIL12, ki je brez gena za odpornost proti an-tibiotikom in nosi zapis za humani protein IL-12. Plazmid lahko dostavimo v tumor z uporabo genskega elektroprenosa (GET). Bolniki in metode. Predstavljamo protokol prvega klinicnega preskušanja pri ljudeh za phIL12 GET (ISRCTN15479959, ClinicalTrials NCT05077033). Raziskava je namenjena oceni varnosti in tolerancnosti phIL12 GET pri zdravljenju bazalnocelicnega raka pri bolnikih z operabilnimi tumorji v predelu glave in vratu. Raziskavo smo zasnovali kot I. klinicno fazo, pri kateri smo povecevali odmerek plazmida phIL12 z namenom ugotoviti varnost in tolerancnost zdravljenja. Želeli smo dolociti odmerek plazmida phIL12, ki je varen in izzove biološko aktivnost. Zakljucki. Rezultati pricujocega klinicnega protokola bodo zagotovili osnovo, ki bo omogocila upora­bo phIL12 GET kot dopolnilnega zdravljenja k lokalnim ablativnim terapijam. Namen takšne terapije je povecati lokalni odgovor na zdravljenje in tudi izzvati sistemski odziv. 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 PR/BSI/BEN/2022/017 Za lajšanje bolecine Bistvene informacije iz Povzetka glavnih znacilnosti zdravila Tantum Verde 1,5 mg/ml oralno pršilo, raztopinaTantum Verde 3 mg/ml oralno pršilo, raztopina Sestava: 1,5 mg/ml: indikacije: Samozdravljenje: 2 do 4 razprški 2- do 6-krat na dan. Kontraindikacije: pride do bronhospazma. Pri takih bolnikih je potrebna previdnost. To zdravilo vsebuje 13,6 mg alkohola (etanola) v enem razpršku (0,17 ml), kar ustreza manj kot 0,34 ml piva oziroma 0,14 ml vina. Majhna kolicina alkohola v zdravilu ne bo imela nobenih opaznih ucinkov. To zdravilo vsebuje metilparahidroksibenzoat (E218). Lahko povzroci alergijske reakcije (lahko zapoznele). To zdravilo vsebuje manj kot 1 mmol (23 mg) natrija v enem razpršku (0,17 ml), kar v bistvu pomeni ‘brez natrija’. Zdravilo vsebuje aromo poprove mete z benzilalkoholom, cinamilalkoholom, citralom, citronelolom, geraniolom, izoevgenolom, linalolom, evgenolom in D-limonen, ki lahko povzrocijo alergijske reakcije. Zdravilo z jakostjo 3 mg/ml vsebuje makrogolglicerol hidroksistearat 40. Lahko povzroci želodcne težave in drisko. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Študij medsebojnega delovanja niso izvedli. Nosecnost in dojenje: O uporabi benzidamina pri nosecnicah in dojecih ženskah ni zadostnih podatkov. Uporaba zdravila med nosecnostjo in dojenjem ni priporocljiva. Vpliv na sposobnost vožnje in upravljanja strojev: Zdravilo v priporocenem odmerku nima vpliva na sposobnost vožnje in upravljanja strojev. Neželeni ucinki: Neznana pogostnost (ni mogoce oceniti iz razpoložljivih podatkov): anafilakticne reakcije, preobcutljivostne reakcije, odrevenelost, laringospazem, suha usta, navzea in bruhanje, oralna hipestezija, angioedem, fotosenzitivnost, pekoc obcutek v ustih. Neposredno po uporabi se lahko pojavi obcutek odrevenelosti v ustih in v žrelu. Ta ucinek se pojavi zaradi nacina delovanja zdravila in po kratkem casu izgine. Nacin 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: 05. 04. 2022 Pred svetovanjem ali izdajo preberite celoten Povzetek glavnih znacilnosti zdravila. Samo za strokovno javnost. Datum priprave informacije: april 2022 Odgovoren za trženje: Bonifar d.o.o. Zdravilo LUMYKRAS®. - prvo tarcno zdravljenje za bolnike z mutacijo KRAS G12C1 Peroralni zaviralec je kot monoterapija indiciran za zdravljenje bolnikov z napredovalim nedrobnocelicnim rakom pljuc (NDRP) z mutacijo KRAS G12C, pri katerih je bolezen napredovala po vsaj eni predhodni liniji sistemskega zdravljenja.2 UCINKOVITO: DELUJE HITRO: NUDI DOLGOTRAJEN TARCNO ZDRAVILO: stopnja nadzora nad deluje že ODGOVOR: 1x dnevno jemanje boleznijo je 80,6 %3 v 1,4 meseca3 mediana celokupnega preživetja je in dobro prenašanje zdravila3 12,5 meseca3 Zdravilo LUMYKRAS® še ni krito iz obveznega zdravstvenega zavarovanja. Literatura: 1. Mullard A, et al. Nat Rev Drug Discov 2021;20:496. 2. Povzetek glavnih znacilnosti zdravila LUMYKRAS®, Amgen. 3. Skoulidis F, et al. N Engl J Med 2021;384:2371–81. LUMYKRAS® 120 mg filmsko obložene tablete (sotorasib)SKRAJŠAN POVZETEK GLAVNIH ZNACILNOSTI ZDRAVILA Samo za strokovno javnost. Pred predpisovanjem si preberite celoten Povzetek glavnih znacilnosti zdravila. .Za to zdravilo se izvaja dodatno spremljanje varnosti. Porocati je potrebno ovseh domnevnih neželenih ucinkih zdravila. SESTAVA ZDRAVILA: Ena filmsko obložena tableta vsebuje 120 mg sotorasiba. TERAPEVTSKE INDIKACIJE: Kot monoterapija za zdravljenje odraslih z napredovalim nedrobnocelicnim rakom pljuc (NDRP) z mutacijo KRAS G12C, pri katerih je bolezen napredovala po vsaj eni predhodni liniji sistemskega zdravljenja. ODMERJANJE IN NACIN UPORABE: Zdravljenje mora uvesti zdravnik, ki ima izkušnje z uporabo zdravil za zdravljenje raka. Pred uvedbo zdravljenja je treba z validiranim testom potrditi prisotnost mutacije KRAS G12C. Odmerjanje:Priporoceni odmerek je 960 mg sotorasiba (osem 120mg tablet) enkrat dnevno, vsak dan ob istem casu. Zdravljenje se priporoca do napredovanja bolezni ali pojava nesprejemljive toksicnosti. Izpušceni odmerki ali bruhanje: Ce je od nacrtovanega odmerkapreteklo manj kot 6 ur, mora bolnik vzeti odmerek kot obicajno. Ce je od nacrtovanega odmerka preteklo vec kot 6 ur, bolnik ne sme vzeti odmerka. Zdravljenje mora nadaljevati naslednji dan, kot je prepisano. Ce bolnik po zaužitju zdravila LUMYKRAS bruha, tisti dan ne sme vzeti dodatnega odmerka in mora zdravljenje nadaljevati naslednji dan, kot je predpisano. Prilagajanje odmerka:Odmerjanje je treba prilagajati glede na toksicnost zdravila LUMYKRAS. Za stopnje zmanjšanja odmerka in prila­goditve odmerka zaradi neželenih ucinkov med zdravljenjem glejte celoten Povzetek glavnih znacilnosti zdravila. Ce se pojavijo toksicni ucinki, je odmerek dovoljeno zmanjšati najvec dvakrat. Ce bolniki ne prenašajo najmanjšega odmerka, tj. 240 mg enkrat dnevno, je treba zdravljenje z zdravilom LUMYKRAS prekiniti. Socasna uporaba zdravila LUMYKRAS zzdravili za zmanjševanje kisline: Socasna uporaba zaviralcev protonske crpalke (PPI) ali antagonistov receptorjev H2 z zdravilom LUMYKRAS ni priporocena. Ce je potrebno zdravljenje z zdravilom za zmanjševanje kisline, se lahko uporabi nesistemski antacid, zdravilo LUMYKRAS pa je treba vzetibodisi 4 ure pred uporabo nesistemskega antacida bodisi 10 ur po njem. Posebne populacije:Pri bolnikih, starih 75 let in vec, prilagajanje odmerka ni potrebno. Prilagajanje odmerka pri bolnikih z blago okvaro jeter ni priporoceno. Uporaba sotorasiba pri bolnikih z zmerno in hudo okvaro jeter ni priporocena. Prilagajanje odmerka pri bolnikih z blago okvaro ledvic (CrCl = 60 ml/min) ni priporoceno. Zdravila LUMYKRAS pri bolnikih z zmerno ali hudo okvaro ledvic (CrCl < 60 ml/min) niso preucevali. Pri zdravljenju bolnikov z zmerno ali hudo okvaro in koncno odpovedjo ledvic je zato potrebna previdnost. Zdravilo LUMYKRAS ni namenjeno za uporabo pri pediat­ricni populaciji za zdravljenje nedrobnocelicnega raka pljuc. Nacin uporabe:Zdravilo LUMYKRAS je namenjeno za peroralno uporabo. Tablete je treba pogoltniti cele. Ni podatkov, ki bi podpirali jemanje zdravila LUMYKRAS tako, da se tablete žveci, zdrobi ali razpo­lovi, vendar je tablete mogoce raztopiti v vodi. Tablete se lahko jemljejo s hrano ali brez nje. Za uporabo pri bolnikih, ki težko pogoltnejo zdravila v trdni obliki glejte celoten Povzetek glavnih znacilnosti zdravila. KONTRAINDIKACIJE:Preobcutljivost na ucinkovino ali katero koli pomožno snov. POSEBNA OPOZORILA IN PREVIDNOSTNI UKREPI: Hepatotoksicnost:Sotorasib lahko povzroci hepatotoksicnost, ki lahko privede do z zdravilom povzrocene poškodbe jeter in hepatitisa. Sotorasib je bil povezan s prehodnimi zvišanji ravni transaminaz (ALT in AST) v serumu. Zvišane ravni so se izboljšale ali izzvenele po prilagoditvi odmerka ali trajni prekinitvi zdravljenja in v klinicnih študijah niso privedle do primerov odpovedi jeter ali smrtnih primerov. Pri bolnikih je treba spremljati delovanje jeter (ALT, AST in celokupni bilirubin), in sicer pred zacetkom zdravljenja z zdravilom LUMYKRAS, nato v prvih 3 mesecih zdravljenja vsake 3 tedne, nato pa enkrat mesecno ali kot je klinicno indicirano, preiskave pa je treba opravljati pogosteje pri bolnikih, pri katerih se pojavijo zvišanja ravni transaminaz in/ali bilirubina. Glede na stopnjo resnosti nenormalnih izvidov laboratorijskih preiskav je treba zdravljenje z zdravilom LUMYKRAS prekiniti do izboljšanja na stopnjo = 1 ali na izhodišcno stopnjo ter v skladu s priporocili bodisi prilagoditi odmerek bodisi trajno prekiniti zdravljenje. Intersticijska pljucna bolezen (ILD)/pnevmonitis: Bolnike spremljajte za pojav novih ali poslabšanje obstojecih pljucnih simptomov, ki bi kazali na ILD/pnevmonitis (npr. dispneja, kašelj, vrocina). Nemudoma prekinite zdravljenje z zdravilom LUMYKRAS pri bolnikih z domnevno ILD/pnevmonitisom in trajno prekinite zdravljenje z zdravilom LUMYKRAS, ce ne odkrijete nobenih drugih morebitnih vzrokov za ILD/pnevmonitis. Intoleranca za laktozo:Zdravilo LUMYKRAS vsebuje laktozo. Bolniki z redko dedno intoleranco za galaktozo, odsotnostjo encima laktaze ali malabsorpcijo glukoze/galaktoze ne smejo jemati tega zdravila. Natrij:To zdravilo vsebuje manj kot 1 mmol (23 mg) natrija na tableto, kar v bistvu pomeni „brez natrija“. INTERAKCIJE: Vpliv drugih zdravil na sotorasib: Socasna uporaba sotorasiba s PPI (omeprazolom) ali antagonistom receptorjev H2 (famotidinom) je privedla do zmanjšanja koncentracij sotorasiba. Socasna uporaba PPI in antagonistov receptorjev H2 z zdravilom LUMYKRAS ni priporocena, saj ni znano, kakšenbi bil vpliv na ucinkovitost sotorasiba. Ce je potrebno zdravljenje z zdravilom za zmanjševanje kisline, je zdravilo LUMYKRAS treba vzeti 4 ure pred uporabo nesistemskega antacida ali 10 ur po njem. Socasna uporaba veckratnih odmerkov itrakonazola (mocnega zaviralca CYP3A4 in Pgp) ni povecala izpostavljenosti sotorasibu v klinicno pomembnem obsegu. Prilagoditev odmerka zdravila LUMYKRAS pri socasni uporabi z zaviralci CYP3A4 ni priporocena. Socasna uporaba mocnih induktorjev CYP3A4 (npr. rifampicina, karbamazepina, enzalutamida, mitotana, fenitoina in šentjanževke) z zdravilom LUMYKRAS ni priporocena, saj lahko ta zdravila zmanjšajo izpostavljenost sotorasibu. Vpliv sotorasiba na druga zdravila: Sotorasib je zmeren induktor CYP3A4. Socasna uporaba sotorasiba s substrati CYP3A4 je privedla do zmanjšanja njihovih plazemskih koncentracij, kar bi lahko zmanjšalo ucinkovitost teh substratov. Izogibajte se socasni uporabi zdravila LUMYKRAS s substrati CYP3A4 z ozkimi terapevtskimi indeksi, ki med drugim vkljucujejo alfentanil, ciklosporin, dihidroergotamin, ergotamin, fentanil, hormonske kontraceptive, pimozid, kinidin, sirolimus in takrolimus. Ce se socasni uporabi ni mogoce izogniti, prilagodite odmerek substrata CYP3A4 v skladu s trenutnim Povzetkom glavnih znacilnosti zdravila. Podatki in vitro so pokazali, da bi sotorasib morda lahko induciral CYP2B6, CYP2C8, CYP2C9 in CYP2C19; klinicni pomen teh ugotovitev ni znan. Pri socasni uporabi sotorasiba z drugimi zdravili, ki se presnavljajo s pomocjo teh encimov, se priporoca ustrezno spremljanje. Podatki in vitro so pokazali, da bi sotorasib morda lahko zaviral CYP2D6; klinicni pomen teh ugotovitev ni znan. Pri socasni uporabi zdravila LUMYKRAS s substrati CYP2D6 (npr. flekainidom, propafenonom, metoprololom), se priporoca ustrezno spremljanje. Podatki in vitro so pokazali, da bi sotorasib morda lahko zaviral BCRP; klinicni pomen teh ugotovitev ni znan. Pri socasni uporabi zdravila LUMYKRAS s substrati BCRP (npr. metotreksatom, mitok­santronom, topotekanom in lapatinibom), se priporoca ustrezno spremljanje. Socasna uporaba zdravila LUMYKRAS s substrati Pgp z ozkimi terapevtskimi indeksi ni priporocena. Ce se socasni uporabi ni mogoce izogniti, prilagodite odmerek substrata Pgp v skladu s trenutnim Povzetkom glavnih znacilnosti zdravila. PLODNOST, NOSECNOST IN DOJENJE:Ženskam v rodni dobi je treba svetovati, naj se med zdravljenjem z zdravilom LUMYKRAS izogibajo zanositvi. Bolnice v rodni dobi, ki prejemajo zdravilo LUMYKRAS, morajo med zdravljenjem in še najmanj 7 dni po zadnjem odmerku zdravila LUMYKRAS uporabljati visokoucinkovite metode kontracepcije. Zdravilo LUMYKRAS lahko zmanjša ucinkovitost hormonskih kontraceptivov, zato morajo ženske, ki uporabljajo hormonske kontraceptive, uporabljati tudi dodatno pregradno metodo. Zdravila LUMYKRAS ne uporabljajte pri nosecnicah in pri ženskah v rodni dobi, ki ne uporabljajo ucinkovite kontracepcije. Bolnice je treba seznaniti z morebitnimi tveganji za plod, ce se zdravilo LUMYKRAS uporablja med nosecnostjo ali ce bolnica zanosi med jemanjem zdravila LUMYKRAS. NEŽELENI UCINKI: Zelo pogosti: anemija, glavobol, kašelj, dispneja, driska, navzea, bruhanje, zaprtje, bolecine v trebuhu, artralgija, bolecine v hrbtu, utrujenost, pireksija, zvišane ravni AST, zvišane ravni ALT. Pogosti: z zdravilom povzrocena poškodba jeter, zvišane ravni alkalne fosfataze v krvi, zvišane ravni bilirubina v krvi, zvišane ravni gama glutamiltransferaze. Obcasni: ILD/pnevmonitis. REŽIM IZDAJE: Rp/Spec. IMETNIK DOVOLJENJA ZA PROMET: AmgenEurope B.V., Minervum 7061, 4817 ZK Breda, Nizozemska. Dodatne informacije: Amgenzdravilad.o.o., Ameriška ulica 2, 1000 Ljubljana. DATUM ZADNJE REVIZIJE BESEDILA: Januar2022. DATUM PRIPRAVE INFORMACIJE: Maj 2022. SI-510-0522-00002 Zdravilo Lunsumio je kot monoterapija indicirano za zdravljenje odraslih bolnikov s ponovljenim ali neodzivnim (refraktarnim) folikularnim limfomom (FL), ki so prejeli vsaj dve predhodni sistemski zdravljenji.1 Za to zdravilo se izvaja dodatno spremljanje varnosti, kar oznacuje navzdol obrnjen crn trikotnik. Tako bodo hitreje na voljo nove informacije o njegovi varnosti. Zdravstvene delavce naprošamo, da porocajo o katerem koli domnevnem neželenem ucinku zdravila. Vir: 1 Povzetek glavnih znacilnosti zdravila Lunsumio. Dostopano julij 2022 na https://www.ema.europa.eu/en/documents/product-information/lunsumio-epar-product­ information_sl.pdf Skrajšan povzetek glavnih znacilnosti zdravila Lunsumio Ime zdravila: Lunsumio 1 mg/30 mg koncentrat za raztopino za infundiranje Kakovostna in kolicinska sestava: Lunsumio 1 mg koncentrat za raztopino za infundiranje: Ena viala vsebuje 1 mg mosunetuzumaba v 1 ml, v koncentraciji 1 mg/ml. Lunsumio 30 mg koncentrat za raztopino za infundiranje: Ena viala vsebuje 30 mg mosunetuzumaba v 30 ml, v koncentraciji 1 mg/ml. Mosunetuzumab je humanizirano protitelo (imunoglobulin) polne dolžine, izotipa G1 (IgG1), usmerjeno proti CD20/CD3, pridobljeno iz celic jajcnika kitajskega hrcka s tehnologijo rekombinantne DNA. Terapevtske indikacije: Zdravilo Lunsumio je kot monoterapija indicirano za zdravljenje odraslih bolnikov s ponovljenim ali neodzivnim (refraktarnim) folikularnim limfomom (FL), ki so prejeli vsaj dve predhodni sistemski zdravljenji. Odmerjanje in nacin uporabe: Zdravilo Lunsumio se sme dajati le pod nadzorom zdravnika, usposobljenega za uporabo zdravil za zdravljenje raka, in v okolju, ki omogoca ustrezno zdravstveno obravnavo in obvladanje hudih reakcij, kot je sindrom sprošcanja citokinov (cytokine release syndrome - CRS). Odmerjanje: Profilaksa in premedikacija: Bolniki, ki prejmejo zdravilo Lunsumio, morajo biti dobro hidrirani. Priporocena premedikacija in priporoceni odmerki za vsak 21-dnevni cikel so podrobno prikazani v Povzetku glavnih znacilnosti zdravila. Trajanje zdravljenja: Zdravilo Lunsumio je treba dajati 8 ciklov, razen ce se pri bolniku pojavi nesprejemljiva toksicnost ali bolezen napreduje. Bolniki, ki dosežejo popolni odziv, po 8 ciklih ne potrebujejo nadaljnjega zdravljenja. Bolniki, ki po 8 ciklih zdravljenja z zdravilom Lunsumio dosežejo delni odziv ali imajo stabilno bolezen, morajo dobiti dodatnih 9 ciklov zdravljenja, razen ce se pojavijo nesprejemljiva toksicnost ali bolezen napreduje. Prilagoditev odmerka: Zdravljenje bolnikov, pri katerih se pojavijo ucinki 3. ali 4. stopnje, je treba zacasno prekiniti, dokler simptomi ne minejo. Nacin uporabe: Zdravilo Lunsumio je namenjeno le za intravensko uporabo. Zdravilo Lunsumio je treba razredciti z upoštevanjem asepticnega postopka. Dati ga je treba v intravenski infuziji po namenski infuzijski liniji. Za dajanje zdravila Lunsumio ne uporabljajte linijskega filtra, uporabite pa lahko filtre v kapalni komori. Zdravila Lunsumio se ne sme dati kot hiter intravenski odmerek ali bolus. Kontraindikacije: Preobcutljivost na ucinkovino ali katero koli pomožno snov. Posebna opozorila in previdnostni ukrepi: Sindrom CRS: Pri nekaterih bolnikih, ki so prejemali zdravilo Lunsumio, se je pojavil CRS, vkljucno z življenje ogrožajocimi reakcijami. Z infundiranjem povezane reakcije se lahko kažejo z enako klinicno sliko kot CRS. Premedikacijo morajo bolniki prejeti vsaj od zacetka zdravljenja do konca 2. cikla zdravljenja. Bolnike je treba nadzorovati glede znakov ali simptomov CRS. Narociti jim je treba, naj nemudoma poišcejo zdravniško pomoc, ce se kadar koli pojavijo znaki ali simptomi CRS. V primeru pojava CRS morajo zdravniki uvesti podporno zdravljenje. Resne okužbe: Pri nekaterih bolnikih, ki so prejemali zdravilo Lunsumio, so se pojavile resne okužbe. Po infundiranju zdravila Lunsumio so pri nekaterih bolnikih opažali febrilno nevtropenijo. Bolniki z aktivnimi okužbami ne smejo prejeti zdravila Lunsumio. Zdravilo Lunsumio je treba previdno uporabljati pri bolnikih z anamnezo ponavljajocih se ali kronicnih okužb, s pridruženimi boleznimi, ki lahko povecajo nagnjenost k okužbam, ali z intenzivnim predhodnim imunosupresivnim zdravljenjem. Bolniki morajo dobiti ustrezna profilakticna zdravila. Bolnike je treba nadzorovati glede znakov in simptomov okužbe pred in po dajanju zdravila Lunsumio ter jih ustrezno zdraviti. Zagon tumorja: Pri nekaterih bolnikih, zdravljenih z zdravilom Lunsumio, so porocali o zagonu tumorja. Specificnih dejavnikov tveganja za pojav zagona tumorja niso ugotovili. Vendar pa obstaja tveganje za prizadetost in umrljivost bolnika zaradi ucinka mase tumorja ob zagonu tumorja pri bolnikih z obsežnimi tumorji, ki se nahajajo v neposredni bližini dihalnih poti in/ali vitalnih organov. Pri bolnikih, ki prejemajo zdravilo Lunsumio, je treba nadzorovati in ocenjevati kriticna anatomska mesta glede pojava zagona tumorja. Sindrom razpada tumorja: Pri bolnikih, ki so prejemali zdravilo Lunsumio, so porocali o sindromu razpada tumorja. Bolniki morajo dobiti ustrezno profilakticno zdravljenje. Bolnike je treba nadzorovati glede znakov in simptomov sidroma razpada tumorja; to še zlasti velja za bolnike z velikim tumorskim bremenom ali hitro rastocimi tumorji ter bolnike z zmanjšanim delovanjem ledvic. Spremljati je treba biokemijske izvide bolnikov in v primeru odstopanj takoj ukrepati. Imunizacija: Socasno z zdravilom Lunsumio se ne sme dajati živih in/ali živih oslabljenih cepiv. Kartica za bolnika: Zdravnik, ki zdravilo predpiše, se mora z bolnikom pogovoriti o tveganjih zdravljenja z zdravilom Lunsumio. Bolnik mora dobiti kartico za bolnika in izrecno navodilo, naj jo ima vedno pri sebi. Medsebojno delovanje z drugimi zdravili in druge oblike interakcij: Prehodnega klinicno pomembnega ucinka na substrate CYP450 z ozkim terapevtskim indeksom ni mogoce izkljuciti, saj uvedba zdravila Lunsumio povzroci prehoden porast citokinov, to pa lahko povzroci zavrtje encimov CYP450. Pri uvedbi zdravila Lunsumio bolnikom, ki se zdravijo s substrati CYP450 z ozkim terapevtskim indeksom, je treba razmisliti o spremljanju zdravljenja. Odmerek socasno uporabljanega zdravila je treba ustrezno prilagoditi. Neželeni ucinki: Najpogostejši neželeni ucinki so bili sindrom sprošcanja citokinov, nevtropenija, zvišana telesna temperatura, hipofosfatemija in glavobol. Najpogostejši resni neželeni ucinki so bili CRS, zvišana telesna temperatura in pljucnica. Porocanje o domnevnih neželenih ucinkih: Porocanje o domnevnih neželenih ucinkih zdravila po izdaji dovoljenja za promet je pomembno. Omogoca namrec stalno spremljanje razmerja med koristmi in tveganji zdravila. Od zdravstvenih delavcev se zahteva, da porocajo o katerem koli domnevnem neželenem ucinku zdravila na: Javna agencija Republike Slovenije za zdravila in medicinske pripomocke, Sektor za farmakovigilanco, Nacionalni center za farmakovigilanco, Slovenceva ulica 22, SI-1000 Ljubljana, Tel: +386 (0)8 2000 500, Faks: +386 (0)8 2000 510, e-pošta: h-farmakovigilanca@jazmp.si, spletna stran: www.jazmp.si. Za zagotavljanje sledljivosti zdravila je pomembno, da pri izpolnjevanju obrazca o domnevnih neželenih ucinkih zdravila navedete številko serije biološkega zdravila. Režim izdaje zdravila: H. Imetnik dovoljenja za promet: Roche Registration GmbH, Emil-Barell-Strasse 1, 79639 Grenzach-Wyhlen, Nemcija. Verzija: 1.0/22 Dodatne informacije so na voljo pri: Zdravilo še ni krito iz obveznega zdravstvenega zavarovanja. Roche farmacevtska družba d.o.o., Stegne 13g, 1000 Ljubljana Datum priprave informacije: avgust 2022 Samo za strokovno javnost. M-SI-00000555(v1.0) Zdravilo Lonsurf je indicirano v monoterapiji za zdravljenje odraslih Zdravilo Lonsurf je indicirano v monoterapiji bolnikov z metastatskim kolorektalnim rakom (KRR), ki so bili predhodno za zdravljenje odraslih bolnikov z že zdravljeni ali niso primerni za zdravljenja, ki so na voljo. Ta vkljucujejo metastatskim rakom želodca vkljucno z kemoterapijo na osnovi fluoropirimidina, oksaliplatina in irinotekana, adenokarcinomom gastro-ezofagealnega zdravljenje z zaviralci žilnega endotelijskega rastnega dejavnika prehoda, ki so bili predhodno že zdravljeni (VEGF – Vascular Endothelial Growth Factor) in zaviralci receptorjev za z najmanj dvema sistemskima režimoma epidermalni rastni dejavnik (EGFR – Epidermal Growth Factor Receptor).1 zdravljenja za napredovalo bolezen.1 za vec trenutkov, ki štejejo Skrajšan povzetek glavnih znacilnosti zdravila: Lonsurf 15 mg/6,14 mg filmsko obložene tablete in Lonsurf 20 mg/8,19 mg filmsko obložene tablete SESTAVA*: Lonsurf 15 mg/6,14 mg: Ena filmsko obložena tableta vsebuje 15 mg trifluridina in 6,14 mg tipiracila (v obliki klorida). Lonsurf 20 mg/8,19 mg: Ena filmsko obložena tableta vsebuje 20 mg trifluridina in 8,19 mg tipiracila (v obliki klorida). TERAPEVTSKE INDIKACIJE*: Kolorektalni rak ‡ v monoterapiji za zdravljenje odraslih bolnikov z metastatskim kolorektalnim rakom, ki so bili predhodno že zdravljeni ali niso primerni za zdravljenja, ki so na voljo. Ta vkljucujejo kemoterapijo na osnovi fluoropirimidina, oksaliplatina in irinotekana, zdravljenje z zaviralci žilnega endotelijskega rastnega dejavnika (VEGF ‡ Vascular Endothelial Growth Factor) in zaviralci receptorjev za epidermalni rastni dejavnik (EGFR ‡ Epidermal Growth Factor Receptor). Rak želodca ‡ v monoterapiji za zdravljenje odraslih bolnikov z metastatskim rakom želodca vkljucno z adenokarcinomom gastro-ezofagealnega prehoda, ki so bili predhodno že zdravljeni z najmanj dvema sistemskima režimoma zdravljenja za napredovalo bolezen. ODMERJANJE IN NACIN UPORABE*: Priporoceni zacetni odmerek zdravila Lonsurf pri odraslih je 35 mg/m2/odmerek peroralno dvakrat dnevno na 1. do 5. dan in 8. do 12. dan vsakega 28-dnevnega cikla zdravljenja, najpozneje 1 uro po zakljucku jutranjega in vecernega obroka (20 mg/m2/odmerek dvakrat dnevno pri bolnikih s hudo ledvicno okvaro). Odmerek, izracunan glede na telesno površino, ne sme preseci 80 mg/odmerek. Možne prilagoditve odmerka glede na varnost in prenašanje zdravila: dovoljena so zmanjšanja odmerka na najmanjši odmerek 20 mg/m2 dvakrat dnevno (oz. 15 mg/m2 dvakrat dnevno pri bolnikih s hudo ledvicno okvaro). Potem ko je bil odmerek zmanjšan, povecanje ni dovoljeno. KONTRAINDIKACIJE*: Preobcutljivost na ucinkovini ali katero koli pomožno snov. OPOZORILA IN PREVIDNOSTNI UKREPI*: Supresija kostnega mozga: Pred uvedbo zdravljenja in po potrebi za spremljanje toksicnosti zdravila, najmanj pred vsakim ciklom zdravljenja, je treba pregledati celotno krvno sliko. Zdravljenja ne smete zaceti, ce je absolutno število nevtrofilcev < 1,5 x 109/l, ce je število trombocitov < 75 x 109/l ali ce se je pri bolniku zaradi predhodnih zdravljenj pojavila klinicno pomembna nehematološka toksicnost 3. ali 4. stopnje, ki še traja. Bolnike je treba skrbno spremljati zaradi morebitnih okužb, uvesti je treba ustrezne ukrepe, kot je klinicno indicirano. Toksicnost za prebavila: Potrebna je uporaba antiemetikov, antidiaroikov ter drugih ukrepov, kot je klinicno indicirano. Ce je potrebno, prilagodite odmerke. Ledvicna okvara: Uporaba zdravila ni priporocljiva pri bolnikih s koncno stopnjo ledvicne okvare. Bolnike z ledvicno okvaro je potrebno med zdravljenjem skrbno spremljati; bolnike z zmerno ali hudo ledvicno okvaro je treba zaradi hematološke toksicnosti bolj pogosto spremljati. Jetrna okvara: Uporaba zdravila Lonsurf pri bolnikih z obstojeco zmerno ali hudo jetrno okvaro ni priporocljiva. Proteinurija: Pred zacetkom zdravljenja in med njim je priporocljivo spremljanje proteinurije z urinskimi testnimi listici. Pomožne snovi: Zdravilo vsebuje laktozo. INTERAKCIJE*: Previdnost: Zdravila, ki medsebojno delujejo z nukleozidnimi prenašalci CNT1, ENT1 in ENT2, zaviralci OCT2 ali MATE1, substrati humane timidin-kinaze (npr. zidovudin), hormonski kontraceptivi. PLODNOST*. NOSECNOST IN DOJENJE*: Ni priporocljivo. KONTRACEPCIJA*: Ženske in moški morajo uporabljati zelo ucinkovite metode kontracepcije med zdravljenjem in do 6 mesecev po zakljucku zdravljenja. VPLIV NA SPOSOBNOST VOŽNJE IN UPRAVLJANJA STROJEV*: Med zdravljenjem se lahko pojavijo utrujenost, omotica ali splošno slabo pocutje. NEŽELENI UCINKI*: Zelo pogosti: nevtropenija, levkopenija, anemija, trombocitopenija, zmanjšan apetit, diareja, navzea, bruhanje, utrujenost. Pogosti: okužba spodnjih dihal, febrilna nevtropenija, limfopenija, hipoalbuminemija, disgevzija, periferna nevropatija, dispneja, bolecina v trebuhu, zaprtje, stomatitis, bolezni ustne votline, hiperbilirubinemija, sindrom palmarne plantarne eritrodisestezije, izpušcaj, alopecija, pruritus, suha koža, proteinurija, pireksija, edem, vnetje sluznice, splošno slabo pocutje, zvišanje jetrnih encimov, zvišanje alkalne fosfataze v krvi, zmanjšanje telesne mase. Obcasni: septicni šok, infekcijski enteritis, pljucnica, okužba žolcevoda, gripa, okužba secil, gingivitis, herpes zoster, tinea pedis, okužba s kandido, bakterijska okužba, okužba, nevtropenicna sepsa, okužba zgornjih dihal, konjunktivitis, bolecina zaradi raka, pancitopenija, granulocitopenija, monocitopenija, eritropenija, levkocitoza, monocitoza, dehidracija, hiperglikemija, hiperkaliemija, hipokaliemija, hipofosfatemija, hipernatriemija, hiponatriemija, hipokalciemija, protin, anksioznost, nespecnost, nevrotoksicnost, disestezija, hiperestezija, hipoestezija, sinkopa, parestezija, pekoc obcutek, letargija, omotica, glavobol, zmanjšana ostrina vida, zamegljen vid, diplopija, katarakta, suho oko, vrtoglavica, neugodje v ušesu, angina pektoris, aritmija, palpitacije, embolija, hipertenzija, hipotenzija, vrocinski oblivi, pljucna embolija, plevralni izliv, izcedek iz nosu, disfonija, orofaringealna bolecina, epistaksa, kašelj, hemoragicni enterokolitis, krvavitev v prebavilih, akutni pankreatitis, ascites, ileus, subileus, kolitis, gastritis, refluksni gastritis, ezofagitis, moteno praznjenje želodca, abdominalna distenzija, analno vnetje, razjede v ustih, dispepsija, gastroezofagealna refluksna bolezen, proktalgija, bukalni polip, krvavitev dlesni, glositis, parodontalna bolezen, bolezen zob, siljenje na bruhanje, flatulenca, slab zadah, hepatotoksicnost, razširitev žolcnih vodov, lušcenje kože, urtikarija, preobcutljivostne reakcije na svetlobo, eritem, akne, hiperhidroza, žulj, bolezni nohtov, otekanje sklepov, artralgija, bolecina v kosteh, mialgija, mišicno-skeletna bolecina, mišicna oslabelost, mišicni krci, bolecina v okoncinah, ledvicna odpoved, neinfektivni cistitis, motnje mikcije, hematurija, levkociturija, motnje menstruacije, poslabšanje splošnega zdravstvenega stanja, bolecina, obcutek spremembe telesne temperature, kseroza, nelagodje, zvišanje kreatinina v krvi, podaljšanje intervala QT na elektrokardiogramu, povecanje mednarodnega umerjenega razmerja (INR), podaljšanje aktiviranega parcialnega tromboplastinskega casa (aPTC), zvišanje secnine v krvi, zvišanje laktatne dehidrogenaze v krvi, znižanje celokupnih proteinov, zvišanje C-reaktivnega proteina, zmanjšan hematokrit. Post-marketinške izkušnje: intersticijska bolezen pljuc. PREVELIKO ODMERJANJE*: Neželeni ucinki, o katerih so porocali v povezavi s prevelikim odmerjanjem, so bili v skladu z uveljavljenim varnostnim profilom. Glavni pricakovani zaplet prevelikega odmerjanja je supresija kostnega mozga. FARMAKODINAMICNE LASTNOSTI*: Farmakoterapevtska skupina: zdravila z delovanjem na novotvorbe, antimetaboliti, oznaka ATC: L01BC59. Zdravilo Lonsurf sestavljata antineoplasticni timidinski nukleozidni analog, trifluridin, in zaviralec timidin-fosforilaze (TPaze), tipiracilijev klorid. Po privzemu v rakave celice timidin-kinaza fosforilira trifluridin. Ta se v celicah nato presnovi v substrat deoksiribonukleinske kisline (DNA), ki se vgradi neposredno v DNA ter tako preprecuje celicno proliferacijo. TPaza hitro razgradi trifluridin in njegova presnova po peroralni uporabi je hitra zaradi ucinka prvega prehoda, zato je v zdravilo vkljucen zaviralec TPaze, tipiracilijev klorid. PAKIRANJE*: 20 filmsko obloženih tablet. NACIN PREDPISOVANJA IN IZDAJE ZDRAVILA: Rp/Spec. Imetnik dovoljenja za promet: Les Laboratoires Servier, 50, rue Carnot, 92284 Suresnes cedex, Francija. Številka dovoljenja za promet z zdravilom: EU/1/16/1096/001 (Lonsurf 15 mg/6,14 mg), EU/1/16/1096/004 (Lonsurf 20 mg/8,19 mg). Datum zadnje revizije besedila: december 2020. *Pred predpisovanjem preberite celoten povzetek glavnih znacilnosti zdravila. Celoten povzetek glavnih znacilnosti zdravila in podrobnejše informacije so na voljo pri: Servier Pharma d.o.o., Podmilšcakova ulica 24, 1000 Ljubljana, tel: 01 563 48 11, www.servier.si. LON AD1 C1 2021-22. Samo za strokovno javnost. Datum priprave informacije: september 2021. SEDAJ ODOBRENO PO VSAJ ENI PREDHODNI TERAPIJI NA PODLAGI ANTI-HER21 NEPRIMERLJIVO PREŽIVETJE* POSTAVLJA NOVE STANDARDE ZDRAVLJENJA HER2+ METASTATSKEGA RAKA DOJK2 Pri zdravilu ENHERTU so porocali o primerih intersticijske pljucne bolezni (ILD) in pnevmonitisa. Za diagnozo je kljucno prepoznavanje simptomov. Bolnike je treba spremljati in priceti z zdravljenjem ob prvih znakih ILD.1,2 SKRAJŠAN POVZETEK GLAVNIH ZNA˜ILNOSTI ZDRAVILA Za to zdravilo se izvaja dodatno spremljanje varnosti. Tako bodo hitreje na voljo nove informacije o njegovi varnosti. Zdravstvene delavce naprošamo, da poro˜ajo o katerem koli domnevnem neželenem u˜inku zdravila. ENHERTU 100°mg prašek za koncentrat za raztopino za infundiranje SESTAVA: Ena viala praška za koncentrat za raztopino za infundiranje vsebuje 100.mg trastuzumab derukstekana. Po rekonstituciji ena viala s 5.ml raztopine vsebuje 20.mg/ml trastuzumab derukstekana. Trastuzumab derukstekan je konjugat protitelesa in zdravila, ki vsebuje humanizirano monoklonsko protitelo IgG1 proti HER2.z istim zaporedjem aminokislin, kot ga ima trastuzumab. Proizvajajo ga sesalske celice (ovarij kitajskega hr˜ka) in je prek razcepljivega veznika na tetrapeptidni bazi kovalentno vezan na DXd, ki je derivat eksatekana in zavi­ralec topoizomeraze.I. Na vsako molekulo protitelesa je vezanih približno 8.molekul derukstekana. Pomožne snovi: Lhistidin, Lhistidinijev klorid monohidrat, saharoza, polisorbat.80. TERAPEVTSKE INDIKACIJE: Zdravilo Enhertu kot monoterapija je indicirano za zdravljenje odraslih bolnikov z neresektabilnim ali metastatskim HER2-pozitivnim rakom dojk, ki so pred tem že prejeli eno ali ve˜ shem zdravljenja na podlagi anti-HER2. ODMERJANJE IN NA˜IN UPORABE: Zdravilo Enhertu mora predpisati zdravnik in njegovo dajanje nadzorovati zdravstveni de­lavec, ki sta izkušena v uporabi zdravil proti raku. Za prepre˜itev napak, povezanih z zdravili, je pomembno, da preverite nalepke na vialah in se prepri˜ate, da je zdravilo, ki se pripravlja in daje, res zdravilo Enhertu (trastuzumab derukstekan), in ne trastuzumab ali trastuzumab emtanzin. Zdravilo Enhertu se ne sme zamenjati s trastuzumabom ali trastuzumab emtanzinom. Bolniki, ki se zdravijo s trastuzumab derukstekanom, morajo imeti dokumentiran HER2pozitiven status tumorja, ki je opredeljen kot ocena 3.+ na podlagi imunohistokemije (IHC) ali razmerje ..2,0 na podlagi in°situ hibridizacije (ISH) ali fuorescen˜ne in°situ hibridizacije (FISH), ocenjeno z in°vitro diagnosti˜nim (IVD) medicinskim pripomo˜kom z oznako.CE. ˆe IVD z oznako.CE ni na voljo, je treba status.HER2 oceniti z drugim potrjenim testom. Odmerjanje: Priporo˜eni odmerek zdravila Enhertu je 5,4.mg/kg, ki se daje z intravensko infuzijo enkrat vsake 3.tedne (21dnevni cikel) do napredovanja bolezni ali nesprejemljive toksi˜nosti. Za˜etni odmerek je treba dati z 90-minutno intravensko infuzijo. ˆe bolnik prejšnjo infuzijo dobro prenaša, se lahko naslednji odmerki zdravila Enhertu dajejo kot 30-minutne infuzije. Za proflakso ali obvladovanje se bolniku smejo dati antiemetiki, skladno z lokalno klini˜no prakso in prenašanjem pri bolniku. Hitrost infundiranja zdravila Enhertu je treba zmanjšati ali infundi­ranje prekiniti, ˜e se pri bolniku razvijejo simptomi, povezani z infuzijo. V primeru hudih reakcij na infuzijo je treba zdravilo Enhertu trajno ukiniti. Prilagajanje odmerka: Obvladovanje neželenih u˜inkov lahko zajema za˜asno prekinitev uporabe, zmanjšanje odmerka ali ukinitev zdravljenja z zdravilom Enhertu, skladno s smernicami, podanimi v povzetku glavnih zna˜ilnosti zdravila (preglednici.1 in.2). Po zmanjšanju odmerka zdravila Enhertu se odmerek ne sme ve˜ ponovno pove˜ati. Na˜rt zmanjševanja odmerka: Za˜etni odmerek je 5,4.mg/kg, prvo zmanjšanje odmerka (4,4.mg/kg), drugo zmanjšanje odmerka (3,2.mg/kg), pri potrebi po nadaljnjem zmanjšanju odmerka ukinite zdravljenje. Prosimo, glejte celoten povzetek glavnih zna.ilnosti zdravila Enhertu za prilagajanje odmerka zaradi neželenih u.inkov: intersticijska plju.na bolezen (ILD)/pnevmonitis (asimptomatska ILD/asimptomatski pnevmonitis (stopnja.1), simptomatska ILD/simptomatski pnevmonitis (stopnja.2 ali višja)), nevtropenija (stopnja.3 (manj kot 1,00,5.×.109/l), stopnja.4 (manj kot 0,5.×.109/l)), febrilna nevtropenija (absolutno število nevtroflcev manj kot 1,0.×.109/l in telesna temperatura, višja od 38,3.°C, ali telesna temperatura 38.°C ali višja, ki vztraja ve˜ kot eno uro), zmanjšan iztisni delež levega prekata (LVEF) (LVEF ve˜ kot 45.% in absolutno zmanjšanje glede na izhodiš˜no vrednost za 10.%.do.20.%; LVEF 40.%.do.45.%; LVEF manj kot 40.% ali absolutno zmanjšanje glede na izhodiš˜no vrednost za ve˜ kot 20.%; simptomati˜no kongestivno sr˜no popuš˜anje). Zakasnjen ali izpuš˜en odmerek: ˆe se na˜rtovani odmerek zakasni ali izpusti, ga je treba dati takoj, ko je mogo˜e, brez ˜akanja na naslednji na˜rtovani cikel. ˆasovni na˜rt dajanja je treba prilagoditi, da se ohrani 3-tedenski razmik med odmerki. Infuzijo je treba dati s hitrostjo in odmerkom, ki ga je bolnik prenašal pri zadnji infuziji. Posebne populacije: S tarejši: Pri bolnikih, starih 65.let ali starejših, prilagajanje odmerka zdravila Enhertu ni potrebno. Podatki pri bolnikih, starih ..75.let, so omejeni. Okvara ledvic: P rilagajanje odmerka pri bolnikih z blago (o˜istek kreatinina [CLcr]...60 in <.90.ml/min) ali zmerno (CLcr...30 in <.60.ml/min) okvaro ledvic ni potrebno. Morebitne potrebe po prilagajanju odmerka pri bolnikih s hudo okvaro ledvic ni mogo˜e opredeliti zaradi pomanjkanja podatkov. Pri bolnikih z zmerno okvaro ledvic so opazili višjo incidenco ILD stopnje.1.in.2/pnevmonitisa, ki sta vodila do zve˜anja števila prekinitev zdravljenja. Bolnike z zmerno ali hudo okvaro ledvic je treba natan˜no spremljati glede neželenih u˜inkov, vklju˜no z ILD/pnevmonitisom. Okvara jeter: P ri bolnikih, ki imajo celokupni bilirubin ..1,5kratnik zgornje meje normalnih vrednosti (ZMN), ne glede na vrednost aspartat transaminaze (AST), odmerka ni treba prilagajati. Morebitne potrebe po prilagajanju odmerka pri bolnikih, ki imajo celokupni bilirubin >.1,5kratnik ZMN, ne glede na vrednost AST, ni mogo˜e opredeliti zaradi pomanjkanja podatkov. Zato je treba te bolnike natan˜no spremljati. Na˜in uporabe: Zdravilo Enhertu je za intravensko uporabo. Zdravstveni delavec ga mora rekonstituirati in razred˜iti. Treba ga je dati z intravenskim infundiranjem. Zdravilo Enhertu se ne sme dati kot hitro intravensko injekcijo ali bolus. KONTRAINDIKACIJE: Preob˜ut­ljivost na u˜inkovino ali katero koli pomožno snov. POSEBNA OPOZORILA IN PREVIDNOSTNI UKREPI: Intersticijska plju˜na bolezen/pnevmonitis: Pri zdravilu Enhertu so poro˜ali o primerih intersticijske plju˜ne bolezni (ILD) in/ali pnevmonitisa. Nekateri primeri so bili smrtni. Bolnikom je treba naro˜iti, naj takoj poro˜ajo o kašlju, dispneji, zvišani telesni temperaturi in/ali katerih koli novih dihalnih simptomih ali poslabšanju obstoje˜ih. Bolnike je treba spremljati glede znakov in simptomov ILD/pnevmonitisa. Dokaze za ILD/pnevmonitis je treba takoj prou˜iti. Bolnike s sumom na ILD/pnevmonitis je treba oceniti z radiografskimi posnetki, najbolje z ra˜unalniško tomografjo (CT). Treba je razmisliti o posvetu s pulmologom. Nevtropenija: V klini˜nih študijah z zdravilom Enhertu so poro˜ali o primerih nevtropenije, vklju˜no s pri­meri febrilne nevtropenije. Pred uvedbo zdravila Enhertu in pred vsakim odmerkom ter vsaki˜, ko je klini˜no indicirano, je treba preveriti celotno krvno sliko. Morda bo treba za˜asno prekiniti dajanje zdravila Enhertu ali zmanjšati odmerek, odvisno od tega, kako huda je nevtrope­nija. Zmanjšanje iztisnega deleža levega prekata: P ri zdravljenjih antiHER2 so poro˜ali o zmanjšanem iztisnem deležu levega prekata (LVEF). Pred uvedbo zdravljenja z zdravilom Enhertu in v rednih intervalih med njim (v skladu s klini˜nimi indikacijami) je treba izvesti standardne preiskave delovanja srca (ehokardiografja ali slikanje MUGA) za oceno LVEF. Zmanjšanje LVEF je treba obvladovati s prekinitvami zdravljenja. Zdravljenje z zdravilom Enhertu je treba trajno ukiniti, ˜e se potrdi LVEF manj kot 40.% ali absolutno zmanjšanje glede na izhodiš˜no vrednost za ve˜ kot 20.%. Zdravilo Enhertu je treba trajno ukiniti pri bolnikih s simptomatskim kongestivnim sr˜nim popuš˜anjem. Embrio-fetalna toksi˜nost: Zdravilo Enhertu lahko ima škodljiv vpliv na plod, ˜e se da nose˜nici. Pri ženskah v rodni dobi je treba pred uvedbo zdravljenja z zdravilom Enhertu preveriti status nose˜nosti. Bolnice je treba seznaniti z možnimi tveganji za plod. Ženskam v rodni dobi je treba svetovati, da uporabljajo u˜inkovito kontracepcijo med zdravljenjem in še vsaj 7.mesecev po zadnjem odmerku zdravila Enhertu. Moškim bolnikom s partnerkami v rodni dobi je treba svetovati, da uporabljajo u˜inkovito kontracepcijo med zdravljenjem z zdravilom Enhertu in še vsaj 4.mesece po zadnjem odmerku zdravila Enhertu. Bolniki z zmerno ali hudo okvaro jeter: Zdravilo Enhertu je treba pri bolnikih z zmerno in hudo okvaro jeter dajati previdno. MEDSEBOJNO DELOVANJE Z DRUGIMI ZDRAVILI IN DRUGE OBLIKE INTERAKCIJ: Pri so˜asnem dajanju trastuzumab derukstekana z zdravili, ki so zaviralci CYP3A ali OATP1B ali prenašalcev Pgp, odmerka ni treba prilagajati. P LODNOST, NOSE˜NOST IN DOJENJE: N ose˜nost: D ajanje zdravila Enhertu nose˜nicam se ne priporo˜a. Bolnice je treba seznaniti z možnimi tveganji za plod, preden zanosijo. Ženske, ki zanosijo, se morajo takoj obrniti na zdravnika. ˆe ženska zanosi med zdravljenjem z zdravilom Enhertu ali v obdobju 7.mesecev po zadnjem odmerku zdravila Enhertu, se priporo˜a natan˜no spremljanje. Dojenje: Ni znano, ali se trastuzumab derukstekan izlo˜a v materino mleko. Humani IgG se izlo˜a v materino mleko in potencial za absorpcijo in resne neželene u˜inke na dojen˜ka ni znan. Zato ženske ne smejo dojiti med zdravljenjem z zdravilom Enhertu in še 7.mesecev po zadnjem odmerku. Odlo˜iti se je treba med prenehanjem dojenja in prenehanjem zdravljenja z zdravilom Enhertu, pri ˜emer je treba pretehtati prednosti dojenja za otroka in prednosti zdravljenja za mater. P lodnost: Namenskih študij plodnosti s trastuzumab derukstekanom niso izvedli. Ni znano, ali so trastuzumab derukstekan ali njegovi presnovki prisotni v semenski teko˜ini. Pred za˜etkom zdravljenja je treba moškim bolnikom svetovati, da se posvetujejo o možnosti shranjevanja semena. Moški bolniki v celotnem obdobju zdravljenja in še najmanj 4.mesece po zadnjem odmerku zdravila Enhertu ne smejo zamrzniti ali darovati semena. NEŽELENI U˜INKI: Združeno varnostno populacijo so ocenili pri bolnikih, ki so v klini˜nih študijah dobili vsaj en odmerek 5,4.mg/kg zdravila Enhertu (n.=.573) zaradi razli˜nih vrst tumorjev. Mediani ˜as trajanja zdravljenja v tej združeni populaciji je bil 11,3.meseca (razpon: 0,7-37,9.meseca). Zelo pogosti: okužba zgornjih dihal, nevtropenija, anemija, levkopenija, trombocitopenija, limfopenija, hipokaliemija, zmanjšan apetit, glavobol, omotica, intersticijska plju˜na bolezen, dispneja, kašelj, epistaksa, navzea, bruhanje, driska, bole˜ina v trebuhu, zaprtje, stomatitis, dispepsija, zvišane transaminaze, alopecija, miši˜no-skeletna bole˜ina, utrujenost, pireksija, zmanjšan iztisni delež, izguba telesne mase. Pogosti: plju˜nica, febrilna nevtropenija, dehidracija, disgevzija, zamegljen vid, izpuš˜aj, hiperpigmentacija kože, pruritus, perifermni edem, zvišana alkalna fosfataza v krvi, zvišan bilirubin v krvi, zvišan kreatinin v krvi, reakcije povezane z infuzijo. IMETNIK DOVOLJENJA ZA PROMET Z ZDRAVILOM: Daiichi Sankyo Europe GmbH, Zielstattstrasse 48, 81379 Mchen, Nem˜ija DATUM ZADNJE REVIZIJE BESEDILA: 11.07.2022 (SI-2310) REŽIM PREDPISOVANJA IN IZDAJE: H Prosimo, da pred predpisovanjem preberete celoten povzetek glavnih zna.ilnosti zdravila. Dodatne informacije so na voljo pri podjetju AstraZeneca UK Limited, Podružnica v Sloveniji, Verovškova 55, 1000 Ljubljana, telefon: 01/51 35 600. * zmanjšanje tveganja za napredovanje bolezni ali smrti (PFS) PFS - preživetje brez napredovanja bolezni, mPFS - mediano preživetje brez napredovanja bolezni, T-DM1 - trastuzumab emtazin, BICR - ocena slepega neodvisnega pregleda (blinded independent central review), IZ - interval zaupanja, HR - razmerje ogroženosti Literatura: 1. Povzetek glavnih zna˜ilnosti zdravila ENHERTU, dostopano 8.8.2022 2. J.Cortes et al; Trastuzumab Deruxtecan versus Trastuzumab Emtasine for Breast Cancer; NEJM 2022;386(12):1143-1154 ENHERTU® je registrirana blagovna znamka družbe Daiichi Sankyo Company, Limited. © 2022 Daiichi Sankyo Company, Ltd. in AstraZeneca Ltd. Datum priprave materiala: avgust 2022. Samo za strokovno javnost. SI-2322 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 zna˜ilnosti 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 priporo˜ljivo. Za obvladovanje neželenih u˜inkov je treba uporabo zdravila KEYTRUDA zadržati ali ukiniti, prosimo, glejte celoten Povzetek glavnih zna˜ilnosti zdravila. Kontraindikacije: Preob˜utljivost na u˜inkovino ali katero koli pomožno snov. Povzetek posebnih opozoril, previdnostnih ukrepov, interakcij in neželenih u˜inkov: Imunsko pogojeni neželeni u˜inki (pnevmonitis, kolitis, hepatitis, nefritis, endokrinopatije, neželeni u˜inki na kožo in drugi): Pri bolnikih, ki so prejemali pembrolizumab, so se pojavili imunsko pogojeni neželeni u˜inki, vklju˜no s hudimi in smrtnimi primeri. Ve˜ina imunsko pogojenih neželenih u˜inkov, 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 ve˜ organskih sistemov. V primeru suma na imunsko pogojene neželene u˜inke je treba poskrbeti za ustrezno oceno za potrditev etiologije oziroma izklju˜itev drugih vzrokov. Glede na izrazitost neželenega u˜inka je treba zadržati uporabo pembrolizumaba in uporabiti kortikosteroide – za natan˜na navodila, prosimo, glejte Povzetek glavnih zna˜ilnosti zdravila Keytruda. Zdravljenje s pembrolizumabom lahko pove˜a tveganje za zavrnitev pri prejemnikih presadkov ˜vrstih organov. Pri bolnikih, ki so prejemali pembrolizumab, so poro˜ali o hudih z infuzijo povezanih reakcijah, vklju˜no s preob˜utljivostjo in anaÿlaksijo. Pembrolizumab se iz obtoka odstrani s katabolizmom, zato presnovnih medsebojnih delovanj zdravil ni pri˜akovati. Uporabi sistemskih kortikosteroidov ali imunosupresivov pred uvedbo pembrolizumaba se je treba izogibati, ker lahko vplivajo na farmakodinami˜no aktivnost in u˜inkovitost pembrolizumaba. Vendar pa je kortikosteroide ali druge imunosupresive mogo˜e uporabiti za zdravljenje imunsko pogojenih neželenih u˜inkov. Kortikosteroide je mogo˜e uporabiti tudi kot premedikacijo, ˜e je pembrolizumab uporabljen v kombinaciji s kemoterapijo, kot antiemeti˜no proÿlakso in/ali za ublažitev neželenih u˜inkov, povezanih s kemoterapijo. Ženske v rodni dobi morajo med zdravljenjem s pembrolizumabom in vsaj še 4 mesece po zadnjem odmerku pembrolizumaba uporabljati u˜inkovito kontracepcijo, med nose˜nostjo in dojenjem se ga ne sme uporabljati. Varnost pembrolizumaba pri samostojnem zdravljenju so v klini˜nih študijah ocenili pri 7.631 bolnikih, ki so imeli razli˜ne vrste raka, 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 ˜as opazovanja znašal 8,5 meseca (v razponu od 1 dneva do 39 mesecev), najpogostejši neželeni u˜inki zdravljenja s pembrolizumabom pa so bili utrujenost (31 %), diareja (22 %) in navzea (20 %). Ve˜ina poro˜anih neželenih u˜inkov pri samostojnem zdravljenju je bila po izrazitosti 1. ali 2. stopnje. Najresnejši neželeni u˜inki so bili imunsko pogojeni neželeni u˜inki in hude z infuzijo povezane reakcije. Pojavnost imunsko pogojenih neželenih u˜inkov 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 u˜inkov. Varnost pembrolizumaba pri kombiniranem zdravljenju s kemoterapijo so ocenili pri 3.123 bolnikih z razli˜nimi vrstami raka, ki so v klini˜nih 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 u˜inki naslednji: anemija (55 %), navzea (54 %), utrujenost (38 %), nevtropenija (36 %), zaprtost (35 %), alopecija (35 %), diareja (34 %), bruhanje (28 %) in zmanjšanje apetita (27 %). Pojavnost neželenih u˜inkov 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 %, pri bolnikih s TNBC pri kombiniranem zdravljenju s pembrolizumabom 80 % in pri zdravljenju samo s kemoterapijo 77 % in pri bolnicah z rakom materni˜nega vratu pri kombiniranem zdravljenju s pembrolizumabom 82 % in pri zdravljenju samo s kemoterapijo 75 %. 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 klini˜nih 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 u˜inki diareja (58 %), hipertenzija (54 %), hipotiroidizem (46 %), utrujenost (41 %), zmanjšan apetit (40 %), navzea (40 %), artralgija (30 %), bruhanje (28 %), zmanjšanje telesne mase (28 %), disfonija (28 %), bole˜ine v trebuhu (28 %), proteinurija (27 %), sindrom palmarno-plantarne eritrodizestezije (26 %), izpuš˜aj (26 %), stomatitis (25 %), zaprtost (25 %), miši˜no-skeletna bole˜ina (23 %), glavobol (23 %) in kašelj (21 %). Neželenih u˜inkov 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 u˜inkov od 3. do 5. stopnje med uporabo pembrolizumaba v kombinaciji z lenvatinibom 89 % in med uporabo kemoterapije same 73 %. Za celoten seznam neželenih u˜inkov, prosimo, glejte celoten Povzetek glavnih zna˜ilnosti zdravila. Za dodatne informacije o varnosti v primeru uporabe pembrolizumaba v kombinaciji glejte povzetke glavnih zna˜ilnosti zdravila za posamezne komponente kombiniranega zdravljenja. Na˜in 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, 08/2022; SI-KEY-00481 EXP: 08/2024 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 zna˜ilnosti zdravila Keytruda, ki je na voljo pri naših strokovnih sodelavcih ali na lokalnem sedežu družbe. instructions Instructions for authors The editorial policy Radiology and Oncology is a multidisciplinary journal devoted to the publishing original and high-quality scientific papers and review articles, pertinent to oncologic imaging, interventional radiology, nuclear medicine, radiotherapy, clinical and experimental oncology, radiobiology, medical physics, and radiation protection. Papers on more general aspects of interest to the radiologists and oncologists are also published (no case reports). 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Pri bolniki z metastazami v CŽS ali brez nji SOO.ITE ALK+ mNSCLC Z ZDRAVILOM LORVIQUA BISTVENI PODATKI IZ POVZETKA GLAVNIH ZNA.ILNOSTI ZDRAVILA Za to zdravilo se izvaja dodatno spremljanje varnosti. Tako bodo itreje na voljo nove informacije o njegovi varnosti. Zdravstvene delavce naprošamo, da poro—ajo o kateremkoli domnevnem neželenem u—inku zdravila. Glejte poglavje 4.8 povzetka glavni zna—ilnosti zdravila, kako poro—ati o neželeni u—inki . Sestava n obl ka zdrav la: Ena flmsko obložena tableta vsebuje 25 mg ali 100fmg lorlatiniba in 1,58 mg oz. 4,20 mgflaktoze mono idrata. Ind kac je: Zdravljenje odrasli bolnikov z napredovalim nedrobnoceli—nim rakom plju— (NSCLC – Non-Small Cell Lung Cancer), ki je ALK (anaplasti—na limfomska kinaza) pozitiven in se pred odno niso zdravili z zaviralcem ALK, ter pri bolniki , pri kateri je bolezen napredovala po: zdravljenju z alektinibom ali ceritinibom kot prvim ALK zaviralcem tirozin kinaze (TKI – Tyrosine Kinase In ibitor) ali zdravljenju s krizotinibom in vsaj še 1 drugim ALK TKI. Odmerjanje n na‰ n uporabe: Zdravljenje mora uvesti in nadzorovati zdravnik, ki ima izkušnje z uporabo zdravil za zdravljenje rakavi bolezni. Odkrivanje ALK-pozitivnega NSCLC je potrebno pri izbiri bolnikov, saj so to edini bolniki, pri kateri so dokazali korist. Priporo—eni odmerek je 100 mg peroralno enkrat na dan. Zdravljenje je treba nadaljevati do napredovanja bolezni ali nesprejemljive toksi—nosti. ie bolnik izpusti odmerek, ga mora vzeti takoj, ko se spomni, razen —e do naslednjega odmerka manjka manj kot 4 ure. Bolniki ne smejo vzeti 2 odmerkov krati, da bi nadomestili izpuš—eni odmerek. Prilagajanje odmerkov: Ravni zmanjšanja odmerka: prvo zmanjšanje odmerka: 75 mg peroralno enkrat na dan; drugo zmanjšanje odmerka: 50 mg peroralno enkrat na dan. Zdravljenje je treba trajno prekiniti, —e bolnik ne prenaša odmerka 50 mg peroralno enkrat na dan. Za prilagajanje odmerkov zaradi neželeni u—inkov glejte preglednicof1 v SmPC-ju. Posebne populacije: Starejši bolniki (. 65 let): Zaradi omejeni podatkov priporo—il o odmerjanju ni mogo—e dati. Okvara ledvic: Prilagajanje odmerkov pri bolniki z normalnim delovanjem in blago ali zmerno okvaro [absolutna ocena itrosti glomerulne fltracije (eGFR – estimated Glomerular Filtration Rate): .f 30f ml/min] ni potrebno. Pri bolniki s udo okvaro ledvic (absolutna vrednost eGFRf